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Biochemical studies on the male reproductive system of Drosophila melanogaster Ingman-Baker, Jane 1980

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BIOCHEMICAL STUDIES ON THE MALE REPRODUCTIVE SYSTEM OF DROSOPHILA MELANOGASTER \ y JANE INGMAN-BAKER B . S c , The University of Sussex, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE "REQUIREMENTS FOR THE DEGREE DOCTOR OF PHILOSOPHY THE FACULTY OF GRADUATE STUDIES Department of Biochemistry-Faculty of Medicine We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1980 BY © Jane Ingman-Baker, 1980 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department Of Biochemistry The University of British Columbia 2075 «Wesbrook Place Vancouver, Canada V6T 1W5 Date i. Abstract Testes and paragonial glands of Drosophila melanogaster wild type males were labelled in vitro using S-methionine, and the proteins synthesized were analysed by 2-dimensional gel electrophoresis (O'Farrell, 1975). Testes and paragonial glands were also labelled in vivo by feeding male o r larvae on S-labelled yeast and then dissecting the adult males. Approximately 1200 proteins were resolved by autoradiography of the gels. The in vitro method was shown to be more sensitive and to allow faithful synthesis of all proteins produced in vivo. H-proline was also used to label testes, and no significant differences from the pattern obtained with S-methionme were found. Different laboratory stocks were analyzed to examine the degree of genetic heterozygosity of testicular proteins. The variation between patterns was very low, facilitating subsequent studies in which fl ies of defined genetic constitution, but with different genetic backgrounds, were compared. Testes and paragonial glands from X/0 and X/Y/Y •DC males were labelled in vitro with S-methionine, and the proteins synthesized were compared to those produced by wild-type males of identical autosomal background. No differences attributable to the Y chromosome could be detected in the testes or paragonial gland samples. Non-equilibrium pH gradient 2 dimensional gels (O'Farrell et a l . , 1977) were also run on testis proteins from X/0, X/Y and X/Y/Y males. These gels will resolve basic as well as acidic proteins and once again no differences attributable to the Y chromosome were seen. Pure sperm was manually dissected from in vivo labelled males and the proteins analyzed. Ninety-two proteins were detected, and all were synthesized in comparable amounts by X/0, X/Y and X/Y/Y males, ii. showing that the Y chromosome does not code for any of these structural sperm proteins. It is postulated that no Y chromosome products were detected because they are organizational factors, or regulatory proteins only present in very small amounts in the adult testes. S labelled males were also mated to unlabel led females, and the proteins of the transferred sperm were analyzed by 2DPAGE. The contributions of the testes and paragonial gland to the ejaculate were determined. Testes at various stages of development were also cultured in vitro in S-methiomne containing media. A profile of the proteins synthesized during development revealed that the spectrum of proteins synthesized at different stages between third instar larvae and the imago were remarkably similar, despite the morphological changes taking place in the organ. Sperm proteins were localized on the patterns, and the quantitative changes occurring during this period were examined. The basic proteins of the testis were studied in an attempt to biochemically identify a Drosophila protamine. Sperm was isolated by dissection, and the acid soluble proteins were separated on a 15% modified Laemmli SDS gel. No unusually small basic protein was seen upon staining, but a protein was present which comigrated with trout histone H4. This suggests that fj. melanogaster males may retain somatic histones in the nucleus during the condensation of the sperm head. Testes were labelled in  vitro with H-arginine and the basic proteins were analyzed on a 15% modified Laemmli SDS gel. The gel was autoradiographed and a prominent doublet was seen at the front of the gel, suggesting that a small highly basic protein is synthesized in the testis. iii. TABLE OF CONTENTS Page Abstract Table of Contents '. List of Figures Acknowledgement Dedication INTRODUCTION 1 I. The Sequential Events in Spermatogenesis in J}. melanogaster 3 i) Elongation 4 i i ) Individualization .4 i i i ) Entrapment and Coiling 4 iv) Release 4 II. The Genetics of Male Fertil ity in D. melanogaster 9 i) The Y Chromosome 9 i i ) X and Autosomal Linked Mutations 11 i i i ) Male Sterile X-Autosomal Translocations 14 III. The Timing of Transcription in D. melanogaster spermiogenesis 15 IV. Spermatogenesis in £. Hydei 16 V. The Paragonial Glands, Genital Duct and Ejaculatory Pump of D_. melanogaster 18 VI. Patterns of Protein Synthesis During Development 21 VII. The Basic Proteins of Sperm 27 VIII. The Present Investigation 31 MATERIALS AND METHODS 33 I. Materials and Abbreviations 33 a) Materials 33 b) Abbreviations 33 iv. Page II. Drosophila Stocks 34 III. Generation of X/0, X/Y and X/Y/Y Flies 35 IV. 'Organ Culture of Testes and Paragonial Glands 36 a) Media 36 b) Addition of Isotope 36 c) Culture of Adult Testes and Paragonial Glands 37 d) Culture of Larval and Pupal Oregon R Testes 38 V. In vivo Labelling of Flies 40 VI. Isolation of In vivo Labelled Testes, Paragonial Glands and Sperm 41 VII. Mass Isolation of Testes and Ovaries 42 VIII. Sperm Isolation 42 IX. Sample Preparation 43 i) Two Dimensional Gel Electrophoresis 43 i i ) Acid Extraction of Basic Proteins 43 i i i ) Chromatin Preparation 43 iv) Micrococcal Nuclease Digestion of Acid Soluble Material from Oregon R Ovaries 44 v) Delipidation of Ovary Samples 44 X. Gel Electrophoresis of Proteins 45 i) Two-Dimensional Polyacrylamide Gel Electrophoresis 45 i i ) Non-Equilibrium pH Gradient Electrophoresis Combined with SDS Gel Electrophoresis (NEpH2DSDSPAGE) 46 i i i ) SDS Polyacrylamide Gel Electrophoresis 46 iv) Acid/Urea Gel Electrophoresis 47 CHAPTER ONE: Two-dimensional patterns of proteins syn-thesized in the X/0, X/Y and X/Y/Y testis and evidence that the Y chromosome does not code for structural sperm proteins 49 Introduction 49 V. Page Results and Discussion 50 1) Levels of Incorporation 50 i i ) Comparison of the lu_ vivo and ln_ vitro Protein Patterns 51 i i i ) Isotope Comparison 54 iv) Stock Variation 54 v) Sperm Isolation 59 vi) X/0, X/Y and X/Y/Y Testis Patterns on 2DPAGE 59 vi i) X/0, X/Y Patterns on NEpH2DPAGE 62 v i i i ) Evidence that the Y Chromosome Does Not Code for Structural Sperm Proteins 62 CHAPTER TWO: Analysis of proteins in the ejaculate of ID. melanogaster males and of the proteins synthesized in the X/0, X/Y and X/Y/Y paragonial gland 72 Introduction 72 Results and Dicussion 72 i) In vivo Labelling 72 i i ) The Transfer Experiment 73 i i i ) In vitro Labelling 78 iv) Comparison of X/0, X/Y and X/Y/Y Paragonial Glands .. 83 CHAPTER THREE: Changes in protein synthesis patterns during the development of the testis. Evidence that sperm proteins are syn-thesized early in development 85 Introduction 85 Results and Discussion 86 i) The Culturing of Immature Testes 86 i i ) Levels of Incorporation 86 i i i ) The Patterns of Synthesized Proteins 86 CHAPTER FOUR: The basic proteins of the ID. melanogaster testis 100 Introduction 100 vi. Page Results and Discussion 100 i) Acid Soluble Proteins from Unlabelled Testes 100 i i ) Radioactive Labelling of Drosophila Basic Proteins 105 BIBLIOGRAPHY 118 LIST OF FIGURES Figure  INTRODUCTION 1 The Drosophila melanogaster male reproductive system 2 A diagrammatic representation of the events occurring during spermiogenesis in the adult testes CHAPTER I 3 Comparison of testis protein labelling in vivo and iin vitro 53 4 Comparison of testis proteins labelled in vitro with 14C -proline and 35s_methionine 56 5 Comparison of the proteins synthesized by the testis in different _D. melanogaster stocks 58 6 Structural sperm proteins of D_. melanogaster Oregon R strain 61 7 Comparison of proteins synthsized by X/0, X/Y and X/Y/Y testes 64 8 Two-dimensional electrophoretic separation of basic and acidic proteins from X/0 and X/Y testes 66 9 Illustration that the X/0 testis does synthesize the structural sperm proteins detected 68 CHAPTER II 10 Proteins of the D. melanogaster male paragonial glands, anterior ejaculatory duct and ejacu-latory bulb 75 11 The proteins of the D. melanogaster Oregon R male ejaculate 77 12 A difference map of the proteins of the rj. mel anogaster male ejaculate 80 13 Paragonial gland proteins labeled in vitro 82 Page viii. Figure Page  CHAPTER III 14 Proteins synthesized in the testis at the beginning and middle of pupation 88 15 Patterns of proteins synthesized by the developing wild-type testis I 91 16 Patterns of proteins synthesized by the developing wild-type testis II 93 17 Changes in levels of structural sperm proteins synthsized in early third instar larvae and in the adult 95 CHAPTER IV 18 Proteins of sperm, testes and ovaries of Drosophila 103 19 Testis proteins labelled with ^H-arginine and ^H-lysine in vitro 107 20 3n_Arginine labelled proteins of the testis I l l 21 NEpH2DSDSPAGE of 3H-arginine labelled proteins of the testis 115 ix. Acknowledgement I would like to thank my supervisor, Dr. Peter Candido, for his good counsel and friendship during the course of this work. I also want to thank all the people who have given me advice and help during this period, particularly Drs. David Cross and Ray Reeves. I would also like to thank my friends and lovers for helping me through the diff icult periods and for making Graduate School a special time in my l i fe . Lastly, I would like to acknowledge the support I received in the form of a Medical Research Council of Canada Studentship from 1976-1980. X. Dedication To my mother and grandmother whose belief in me made i t al l possible. 1. INTRODUCTION In recent years much has been learned about the molecular biology of the ce l l . The development of DNA cloning and sequencing techniques has led to great progress towards understanding the processing of genetic information. However, we remain ignorant of how the genetic material controls and directs tne determination and differentiation of cells during the development of an organism. It has been said (Keifer, 1973) that there are two approaches to the study of genetic mechanisms that regulate development. The f irst is to elucidate the molecular mechanisms involved in the regulation of information transfer. The second is to study a genetically defined system, which can be perturbed in such a way as to produce specific developmental effects that can then be measured. Unfortunately, the biological systems which have been favoured by molecular biologists have often had very l i t t le genetic definition and conversely, the organisms amenable to genetic analysis are often intractable biochemically. However, it is the combination of the two approaches which offers the most hope for an understanding of cellular aspects of development. Classical embryologists have for decades been trying to understand development by using experimental manipulation, exploiting the concept of producing abnormal events which will help one to understand the normal processes. This concept is also relevant in the study of gene regulation. Not only can genetic manipulation aid the biochemist in a technical way, but the generation of mutants in a particular process can lead to increased understanding of the normal sequence of events. In choosing an organism for study, Drosophila melanogaster has many advantages. It is a complex higher organism, its genome can be readily manipulated and offspring can be produced quickly and in relatively large 2. numbers. The large number of phenotypic markers available on the adult and the banding patterns of the larval salivary gland polytene chromosomes have made the fruit fly very popular with geneticists. The accumulated information available from the many years of genetic analysis has made D.  melanogaster one of the most desirable eucaryotic organisms for many studies. To a developmental biologist it is an attractive organism, since much descriptive embryology has been done (see Fullilove and Jacobson, 1979 for a review) and a great deal is known about the imaginal discs of D.  melanogaster (see Bryant, 1979; Shearn, 1979; Gehring, 1979 and Hadorn, 1979 for reviews). To a biochemist, however, Drosophila has never appeared as a good experimental organism. The time and expense involved in obtaining the quantities of f l ies necessary for conventional biochemical analysis, as well as the problems encountered with digestive enzymes can be major drawbacks. Techniques do exist, however, for efficient mass culture and for large scale tissue isolations (see Boyd, 1979 for a review). Permanent cell lines are available (Schneider and Blumenthal, 1979) and relatively simple organ culture techniques (Martin and Schneider, 1979) are available which facilitate radioactive labelling of cellular substances to high specific activities. Biochemical techniques are also becoming much more refined and the sample size required for many techniques has decreased dramatically. With respect to the choice of a cellular system for analysis, spermatogenesis has many advantages. The morphogenic changes that occur during spermatogenesis are well defined and synchronous, with distinct initial and terminal points. The process is autonomous and isolated from other somatic processes, and the development of the sperm is not complicated by the diversification of cell types occurring in the adult. The process is also temporally isolated from the more complex process of general embryonic 3. development. It is unique for two reasons, 1) in that the end product of differentiation is a highly specialized motile vector for transportation of a haploid nucleus, stripped of nearly all the essentials of a living ce l l , and 2) in that all of the differentiation processes in spermatids are dependent upon specific activities of the diploid spermatocyte nucleus, without appreciable contribution of the spermatid nucleus itself (Lindsley and Grell, 1968). A particular advantage of studying spermatogenesis in D. melanogaster is the ease of isolation of male sterile mutations (Berg, 1937; Lindsley and Lifschytz, 1972; Ayles et al_., 1973; Shellenbarger and Cross, 1979). I. The Sequential Events in Spermatogenesis in D. melanogaster Spermatogenesis in Drosophila has been extensively studied at the level of both the light (Cooper, 1950; Hannah-Alava, 1965) and electron microscope (Bairati, 1967; Shoup, 1967; Tokuyasu e_t _a]_., 1972 a and b; Stanley et a l . , 1972; Hardy e_t aJL, 1979). The testes of D. melanogaster are two blind tubes of about 2 mm in length and 0.1 mm in width (Figure 1). The testis is blind at the apex or apical end and is connected to the rest of the genital tract at the basal end. The testiculodeferential valve at the end of the testis opens into the seminal vesicle where mature sperm is stored. Mitotic division of sperm cells gives rise to primary spermatogonia at the apical end of the testis (Figure 2). These undergo four mitotic divisions as they are displaced towards the basal region to result in sixteen primary spermatocytes, that are connected to each other by intercellular bridges. Primary spermatocytes undergo a twenty-five fold increase in volume as they continue their migration toward the base of the testis. Meiosis occurs when they are about half the distance from the apex to the base of the testis. After meiosis the nuclei of the resulting 64 spermatids are s t i l l embedded in a common cytoplasm, and the ensemble is extruded into the lumen. Here 4. the spermatids within a cyst undergo a process of complex differentiation leading to individualised motile sperm. This process can be divided into four stages (Peacock et_ aK, 1972): i) Elongation The spermatids elongate bidirectionally with the two mitochondrial derivatives extending with the axoneme the whole length of the ta i l . Concurrently the nucleus is transformed to the smaller sperm head. This reduction (1:300) is achieved by an ordered process of condensation of the chromatin into a small region of the nucleus while the rest of the nucleus goes through a series of shape changes which result in the bullet shaped sperm head. i i) Individualization The syncytial membrane is already indented around the sperm head at the beginning of this stage and this invagination continues around each spermatid by an investment cone proceeding the full length of head to ta i l . The investment cone sweeps in front of it unwanted cytoplasmic materials forming a characteristic cystic bulge. The investment cones eventually pass off the caudal tip of the tail forming a huge waste bag containing the cytoplasmic and nucleoplasmic debris. i i i ) Entrapment and Coiling The spermatid heads then become embedded in the terminal epithelial cells. Coiling is initiated in the head region and proceeds with the retraction of the tails from the apical region. iv) Release The motile sperm are then released, s t i l l bundled into the seminal vesicle, leaving the waste bag to be engulfed by the terminal epithelial eel Is. 5. Figure I: The Drosophila melanogaster male reproductive system T = Testis TV = Testiculodifferential valve SV = Seminal vesicle PG = Paragonial gland AED = Anterior Ejaculatory Duct EP = Ejaculatory Pump PG Figure 1 7. Figure 2: A diagramatic representation of the events occurring during  spermiogenesis in the adult testis T) The apical region of the testis where gonial cell division gives rise to primary spermatogonia. 2) A cyst of 16 spermatocytes. 3) A cyst of 64 spermatids which have arisen through meiosis. These spermatids are connected by intercellular bridges and are undergoing elongation. 4) A fully extended sperm bundle with the cystic bulge (CB) in the head region. 5) The cystic bulge proceeds down the sperm bundle and at this stage the basal half of the sperm bundle is now individualized while the caudal half is s t i l l syncytial. 6) A completely individualized sperm bundle with the waste bag (WB) at the far end of the testis. 7) The completed and coiled sperm bundle with the head region embedded in a terminal epithelial cell (TE). Figure 2 9. In adult flies the process of spermatogenesis takes approximately 10 days at 25°C. The premeiotic stages and postmeiotic stages take five days each. Primary spermatogonia! divisions occur in the egg, with secondary spermatogonia appearing init ial ly in the middle of the f irst instar. Primary spermatocyte growth continues throughout the larval stages with meiosis taking place around the time of pupation. Spermiogenesis occurs throughout the pupal period and coiling is initiated just before eclosion. Motile sperm are f irst seen in the seminal vesicles about seven hours after eclosion (Lindsley and Lifschytz, 1972). II. The Genetics of Male Fertility in D. melanogaster i) The Y Chromosome In somatic cells the presence or absence of a Y chromosome has l i t t le effect on phenotype. An extra Y chromosome (X/Y/Y males or X/X/Y females) can have some effects, such as suppression of position-effect variegation (Cooper, 1956) and effects on lethality due to X chromosome aberrations (Lindsley et aj_., 1960); a variety of minor phenotypic changes also occur, such as an increase in the number of stenopleural bristles, and changes in the length of the third longitudinal wing vein (Hannah, 1951). The whole Y chromosome, divided by the centromere into two arms of unequal length, is heterochromatic in somatic cel ls, it is positively heteropycnotic and late replicating (Brown, 1966). There is a region of homology between the short arm of the Y chromosome and the proximal (heterochromatic) part of the X chromosome, and both chromosomes have been shown to carry the nucleolus organizer in this region (Ritossa and Spiegelman, 1965; Ritossa et_ aj_., 1966). A fly requires only one complete nucleolus organizer region on either chromosome in order to be viable. The 10. mature ribosomal RNA's transcribed from the nucleolus organizers on the X and Y chromosomes are very similar i f not identical (Maden and Tartof, 1974). The ribosomal RNA genes, however, are not identical. Ribosomal RNA genes have been shown to carry introns (Tartof and Dawid, 1976) and both X and Y chromosomes carry interrupted ribosomal RNA genes, but both qualitative and quantitative differences with respect to the insertion sequences are found (Wellauer e_t al_., 1978). The only indispensable function of the Y chromosome is in spermiogenesis, where its presence in the male confers fer t i l i ty . X/0 males of D. melanogaster are phenotypically and behaviourally indistinguishable from X/Y males except that they are sterile (Bridges, 1916; Safir, 1920). Early cytological examinations of testes from X/0 males (Safir, 1920) revealed that these testes are normal in size and shape but contain only small amounts of nonmotile sperm. More detailed examinations with the electron microscope (reviewed by Keifer, 1973) reveal that spermiogenesis in the X/0 testis is severely disturbed. The overall appearance is of a disorganised, degenerating sperm mass and it is diff icult to distinguish abnormal development from degradation. At the ultrastructural level, however, all of the necessary structural elements appear to be present in the X/0 testes. A more recent investigation (Lifschytz and Harevan, 1977) has indicated that developmental defects in the X/0 testis can be detected at the primary spermatocyte stage, suggesting an early expression of Y chromosome genes. Genetic analysis of the Y chromosome has shown it carries seven fert i l i ty factors. Brousseau (1960) used X-ray induced Y sterile mutations to show that the Y chromosome carries seven complementation groups involved in spermatogenesis, two on the short arm, Y , and five on the long arm, 11. Y L . All seven of these factors must be present in at least one wild type dose for a male to produce motile sperm. Deficiencies in one or more of these factors results in sperm that reach various stages of elongation and coiling, but then start to degenerate. All mutant Y bearing males show irregularities in axoneme formation, development of mitochondrial derivatives, and individualisation. Although certain mutants may show.more of one abnormality than another, it is difficult to categorize the. mutants even on a quantitative basis, since a single mutant male will show differences in degree of maturation, type of abnormalities found, and degree of degeneration among the spermatids examined (Keifer, 1973). Williamson (1972) further extended the analysis of Brousseau and showed that the seven fert i l i ty factors can be divided into more than one functional subunit. Temperature sensitive mutants can be induced by chemical mutagens, in the male fert i l i ty loci of the Y chromosome (Ayles ejt al_., 1973; Williamson, 1969). Males carrying mutants of this type are fertile when reared at the permissive temperature (20-22°C) and sterile when reared at the restrictive temperature (28-29°C). Ayles et^  aj_. (1973) isolated eight Y chromosome temperature sensitive steriles which mapped to four complementation groups on the long arm of the Y. The temperature sensitive periods of the mutations were determined, and evidence for premeiotic, meiotic and postmeiotic lesions was obtained. i i ) X and Autosomal Linked Mutations While the Y chromosome is necessary for normal and complete spermiogenesis, the genetic information contained in this chromosome is not alone responsible for the major morphogenic events of spermatid differentiation. It appears that the bulk of these processes are under the 12. control of genes located elsewhere in the chromosome complement. These genes could be of two general types. First, they could be structural genes producing a product which makes a direct contribution to the sperm, either structurally or enzymatically; second, they could be involved in the regulation of the f irst group of genes or of those on the Y chromosome. Many male steriles have been detected on the X chromosome and autosomes, and although only a few of these have been examined for morphological defects (Keifer, 1973), no truly specific morphological lesions have been seen. Many of the mutants display a disorganised, degenerating phenotype similar to that seen in the X/0 males. However, some mutations with specific lesions are known. Romrell et_ aj_. (1972 a and b) have made electron microscope observations on two male steriles and have seen specific lesions. The f irst is considered to exhibit a failure of cytokinesis, and the second shows disrupted axonemal complexes and mitochondrial derivatives. Putative male meiotic sterile X linked mutants have been identified (Lifschytz and Meyer, 1977). More recently, it has been proposed (Shellenbarger and Cross, 1979; Lifschytz and Yakabovitz, 1978; Lifschytz, 1978) that there are genes which are both necessary for male fert i l i ty and essential in somatic cel ls, and thus if mutant, would be lethal. This would include genes involved in basic cellular processes such as metabolism or cell division and possibly in specifying the basic structural components of differentiated cells. To test this hypothesis, Shellenbarger and Cross (1979) studied more than 100 temperature sensitive lethal mutations, and found that a high proportion of them were also temperature-sensitive male sterile mutations. These mutant genes had developmental blocks occurring early in spermatogenesis. One of these, l(l)mys , when brooded to steril ity had only spermatogonia! cells and a few immature spermatocyte 13. cysts. Another, 1(1)J1024, contained premeiotic cysts and spermatids arrested in early elongation. This approach appears to be promising for the isolation of specific mutants of spermiogenesis. Two recently reported mutations are also interesting. Geer et 1^_. (1979) reported the isolation of an EMS induced male sterile, MS(1)7. The testis, accessary gland and sperm appear normal by all morphological criteria in MS(1)7 males; sperm is transferred to females on mating at normal frequency, and by labelling the 32 males with P prior to mating, the male pronucleus has been shown to penetrate the egg. Development proceeds no further, indicating that it is the process of nuclear decondensation in the embryo which is abnormal in this stock. Kemphries ejt aj_. (1979) have reported the isolation of a dominant mutation on chromosome III, MS(3)KK .^ The sperm in this stock show a variety of defects. At the ultrastructural level there is general disorganization of the axoneme, abnormal accessory tubules, additional central tubules and abnormal mitochondrial derivatives. The overall morphology of the developing spermatids was found to be always disrupted, but the extent of the abnormalities varied in individual f l ies . In squash preparations, there were no mature spermatozoa in the seminal vesicles, and spermatid bundles appeared to be degenerating at the base of the testes. No spermatid bundles showed full wild-type elongation, meiosis appeared abnormal as shown by the presence of macro- and micronuclei at the round spermatid stage, and mitochondrial aggregates were abnormally large and were associated with more than one nucleus. The above description is very similar to the diverse abnormalities seen in many male steriles, and in the presumptive meiotic mutants; however, the primary lesion in this stock is in the structural gene for a testis-specific e subunit of tubulin. The 14. phenotype of this mutation is interesting, as one would have expected a tubulin mutation to exhibit a specific morphological lesion. This result suggests that searching for specific lesions in sperm structure may be futi le, as the disorganized, degenerating appearance may result from any interference with the normal process of spermatogenesis, i i i ) Male-Sterile X-Autospmal Translocations The correlation between X-autosomal translocations and steril ity is striking. More than 75% of translocations between the X chromosome and either chromosome 2 or 3 are male sterile. The exceptions are those that only interchange the chromosome tips, and some which have one breakpoint in the heterochromatin adjacent to the X centromere. The steri l i ty of X-autosome translocations cannot easily be attributed to the effects of specific genes, and the effect is dominant. Fertility cannot be restored to sterile translocations by duplications covering the areas of the breakpoints (for a review see Lifschytz, 1972), showing that the steril ity is not due to specific gene loss. The non-specific nature of the steri l i ty caused by the translocation, the distribution of fertile and sterile breakpoints, and the dominance of the effect, led Lifschytz and Lindsley (1972) to propose that the cause of steril ity is interference in the normal process of chromosome inactivation. They further extended this hypothesis and proposed that not only in Drosophila but in all male heterogametic organisms, inactivation of the X chromosome in the primary spermatocytes is a prerequisite for normal spermatogenesis. The translocation of part of the X to an autosome leads to a failure of inactivation in that piece, and male steril ity results. Shoup (1967) examined males carrying the translocation T(l;2H)25(20)yl25 and found that the major lesions appeared in the head structures, the substructure of the axial filaments and mitochondrial derivatives being similar to those of 15. wild-type. The mutant sperm head was of abnormal shape and did not complete head elongation, chromatin condensation was incomplete, and the transition from lysine-rich histones to arginine-rich histones as determined by histochemical methods did not take place. This phenotype is consistent with Lifschytz and Lindsley's proposals. However, Keifer (1973) examined males carrying translocation T(1:3)10;93B, and found a very different phenotype. In this stock 50-80% of axonemes are missing the central pair of tubules, no other defects are detectable, and the head structure forms normally. The lack of any further studies on the phenotypes of X-autosome translocation steriles makes generalization impossible; either of the above phenotypes could be due to some other mutated gene present on the chromosome. III.The Timing of Transcription in D. melanogaster Spermiogenesis Lindsley and Grell (1966) observed that a fully viable sperm could be produced containing only the small fourth chromosome. They concluded that chromosomal material was not necessary for a spermatid to develop into a functional spermatozoan. There are four possible explanations for this apparent dispensability of genetic information in sperm development. 1) No postmeiotic translation is required; all molecular components required for spermiogenesis are synthesized prior to meiosis at the diploid stage, and their orderly assembly over the subsequent days takes place in the absence of new genetic instructions. 2) No postmeiotic transcription is required; all RNA synthesis takes place in the primary spermatocyte, and the orderly translation from stable messages takes place in the absence of new genetic instructions. 3) Both transcription and translation are required, but a cyst of 64 spermatids connected by intercellular bridges develops as a unit, with the 16. genes in all nuclei contributing to the process. Thus even though a particular nucleus may be nullosomic, its sister nucleus is likely to be reciprocally disomic and the 64 nucleus heterokaryon collectively euploid, containing 32 X chromosomes, 32 Y chromosomes and 64 each of the chromosomes 2, 3 and 4. 4) The diploid cyst cells surrounding each group of developing spermatids transfer the information required for spermiogeneis to the spermatids. The first alternative is ruled out by the demonstration (Das et a l . , 1964b; Brink, 1968; Gould-Somero and Holland, 1974) that labelled amino acids are incorporated into spermatids in diverse stages of maturation. Olivieri and Olivieri (1965) labelled testes in vivo by injection of H uridine, and failed to find any evidence for RNA synthesis after meiosis. Gould-Somero and Holland (1974) cultured testes in vitro with H-uridine, and again found no evidence of RNA synthesis after meiosis. Using inhibitors, they also found that most if not all of the RNA required for the differentiation and elongation of sperm is synthesized earlier in the primary spermatocytes. The fourth alternative is unlikely. Cross and Shellenbarger (1979) have shown that in tissue culture an individual cyst will develop into 64 elongated, individualized and coiled sperm without interaction with cells outside the cyst. The concensus of opinion is that the majority of RNA synthesis is completed prior to meiosis, and that stable transcription products direct protein synthesis through spermiogenesis. IV. Spermatogenesis in D. hydei Although, genetically, less is known about D. hydei than about D. melanogaster, much work has been done on spermiogenesis in this species. 17. The main reason for this is that nuclei of D. hydei primary spermatocytes have large chromosomal loops, resembling the lampbush chromosome loops in amphibians. Nuclear structures have been detected in 52 Drosophila species (Hess and Meyer, 1968), including D. melanogaster, but they are most easily seen in D. hydei. The loops eminate from the normally inert Y chromosome and in D. hydei there are six loop pairs. These loops are sites of RNA synthesis, and labelling studies show that RNA synthesis is.initiated from one end.of the loop. RNA-DNA hybridization studies (reviewed by Hennig et a l . , 1973) have detected a rapidly labelled RNA in the D. hydei testis which is complementary to Y chromosome DNA. The X/0 male in D. hydei is viable, but sterile. The development of the sperm is arrested much earlier than in D. melanogaster, and does not proceed beyond the stage of the f irst spermatocyte. No Y chromosome loops can be seen in the X/0 spermatocyte. The presence of part of a Y chromosome will allow spermiogenesis to proceed further. Y chromosome deficiencies have been mapped into two groups: those which interfere with differentiation immediately following meiosis (early effects), and those that permit the development of nearly complete sperm in at least some spermatids (late effects). However, none of the deficiencies result in the inability to form any major structural component of the sperm, and all defects appear organizational or regulatory in nature. A duplication of the Y chromosome in D. hydei leads to sperm 13-14 mm long, twice the normal 6 mm. Sperm carrying partial duplications for the Y are of intermediate length. Temperature sensitive mutations on the Y chromosome were isolated from EMS-treated males (Leoncini, 1977). Four mutants in different complementation groups were isolated on the distal half of the long arm, but 18. no changes were seen in the Y chromosome loops. The temperature sensitive periods of the four mutants all occurred in the primary spermatocyte stage and in early spermatid development, while the manifestation of the effect was postmeiotic. One of these is particularly interesting, as the sperm appear morphologically normal in the electron microscope but are not motile. Hennig et aj_. (1973) reported the detection, by Coomassie blue staining of a lof SDS gel, of a high molecular weight protein which was present in the X/Y testis but not the X/0 testis. Preliminary interspecies hybrid studies between D. hydei and D. neohydei indicated that this protein was a Y chromosome product; however, no further information has been published on this protein. Recently, Lifschytz (1979) has reported the cloning of Y specific mid-repetitive sequences from D. hydei. A low yield of Y specific clones was obtained, and only one plasmid, designated pDhYl, was studied extensively. This hybridized to Y DNA and was found to be mid-repetitive in nature. On in situ hybridization to metaphase chromosomes of D. hydei, it was found to hybridize to a specific region of the Y next to the centromere. If these techniques can be extended to isolate more Y chromosome DNA clones the results will be interesting, particularly if Y loop DNA and unique sequence Y DNA can be isolated. Lifschytz considers that such an approach will be more difficult to apply in D. melanogaster due to the possibility that Y chromosome sequences may be duplicated on the autosomes. V. The Paragonial Glands, Genital Duct and Ejaculatory Pump of D.  melanogaster The paragonial or accessory glands of D. melanogaster play a crucial role in the reproductive process of this species (Chen, 1971; Fowler, 1973a). The paragonial glands are a pair of elongate sacs approximately 400 19. pm wide and 130 ym long (Figure I). The wall consists of two types of secretory cells: large binucleate and ovoid cel ls. On top of this there is a covering muscular layer. The ovoid cells contain large vacuoles and project into the lumen. There are about 58 of these cells in D.  melanogaster (Gi l l , 1964). These vacuolate cells are in an active secretory phase and produce the viscous fluid which contains "refractive granules of unequal size" (Nonidez, 1920), the "needle like crystals" of Gill (1964), and the "filamentous bodies" of Bairati (1968). Lefevre and Jonsson (1962) performed repeated mating experiments and showed that an individual male is sterile after 4-5 successive matings. On examination it was found that the male s t i l l contained large numbers of sperm but that the accessory glands were completely exhausted. It thus appears that the paragonial gland secretion is necessary for sperm transfer. The granules or "filamentous bodies" in the paragonial gland secretion have been the object of much interest. Bairati (1968) has shown that these bodies cannot only be detected in the lumen of the paragonial glands, but also along the male genital tract and in the female receptacle following copulation. In the electron microscope, the structures are similar to contractile elements and derive from the epithelial cells of the paragonial glands. Bairati (1968) suggested that these filamentous structures are a component of the ejaculate, which: i) aids in the transfer of sperm along the reproductive tract, i i ) serves as reserve material to be used in some way by the spermatozoa during their long storage in the female receptacles, or i i i ) contains some substance that would activate and support the process during which the spermatozoan penetrates the ovum. Hormone-like activities on the female have also been attributed to this secretion, which is speculated to 20. lower the receptivity of the female towards insemination by other males following an initial mating, and to stimulate egg laying (de Wilde and de Loof, 1974). An ethanolamine containing galactoside of a unique structure has been isolated from the secretion (Chen et al_., 1977). This substance, previously called the sex peptide, is transferred to females during mating but its specific function has not yet been determined. The enzyme L-alanine amino transferase is found in the paragonial glands, and its activity is found to increase following copulation (Chen and Baker, 1976). Von Wyle (1976) reported that the paragonial gland contains 46 proteins, which were detected by Coomassie Blue staining of proteins from whole tissue samples applied to a Laemmli SDS tube gel. On a modified Davis system, he detected 12 bands and at this level of resolution he could find no differences between X/0 and X/Y paragonial glands which could be attributed to the presence of a Y chromosome. Von Wyle and Steiner (1977) investigated the rate of protein synthesis in paragonial glands in vitro. The glands were cultured in simplified Robbs medium containing ^ C amino acids for eight hours. A linear increase in incorporation was observed for two hours under these conditions, and a stimulation of protein synthesis was seen following copulation. Protein synthesis continued for a longer period if Schneider's medium supplemented with 10% fetal calf serum was used. The ejaculatory bulb of D. melanogaster also appears to secrete a substance which is transferred to females upon mating (Fowler, 1973; Bairati, 1968). The ejaculatory bulb, the main function of which is considered to be one of regulating ejaculation, seems to produce a waxy plug or viscous solution which appears in the female tract before sperm. It is not known what function it serves but its appearance in the female before the rest of the ejaculate suggests that it may have a role in ensuring 21. successful sperm storage. Breiger and Butterworth (1970) isolated and characterized the l ip id, cis vaccenyl acetate from D. melanogaster. This lipid was localized to the ejaculatory bulb (Butterworth, 1969). The lipid is found in mated females and X/0 males, but it has not been proven that this is the waxy plug secretion in mated females. Peristaltic movements exhibited by the seminal vesicles and the accessory glands aid in the transfer of spermatozoa from the male to the female during copulation, even though it is the contraction of the ejaculatory duct, particularly the tubular portion, which is primarily responsible for propelling the sperm across the ejaculatory duct at the time of ejaculation. The release of spermatozoa from the seminal vesicles into the anterior ejaculatory duct occurs during mating, the process being regulated by a sphincter which is under neuronal control (Bairati, 1968). VI. Patterns of Protein Synthesis During Development It is a generally accepted concept of developmental genetics that cells in any one developmental state have a different spectrum of gene expression from cells in any other. The expression of differential gene activity is presumed to lie primarily in the synthesis of a particular protein. One-dimensional protein separation techniques were used init ial ly to study the changes occurring during embryogenesis (reviewed by Davidson, 1976). This work showed striking changes in the protein synthesis patterns during early sea-urchin and amphibian embryogenesis occurring in the absence of transcription. In most systems changes occurred by the gastrula stage and in many cases there were differences much earlier. These results indicated the importance of maternal information stored in the egg prior to ferti l ization. These studies have more recently been extended using 22. sensitive two-dimensional protein separation techniques. Brandhorst (1976) investigated the changes in patterns of protein synthesis before and after fertilization in the sea urchin using 2DPAGE. He found that the proteins synthesized during early embryonic development were also synthesized prior to ferti l ization, i.e. the changes in early development were quantitative rather than qualitative. The f irst new proteins in the sea urchin were detected by Brandhorst in gastrulae and were easily seen in neurulae. Brock and Reeves (1978) studied the de novo protein synthesis patterns of early Xenopus laevis embryos using 2DPAGE. Starting with unfertilized eggs the authors studied the proteins synthesized at 6 stages of embryonic development and found reproducible and dramatic changes in protein synthesis patterns. Pattern changes occurred between each stage, with a major change between the gastrula and neurula stages of development. Qualitatively, similar 2DPAGE results for these developmental stages of Xenopus were also found by Bravo and Knowland (1979). On the other hand, Ballantine et a l . (1979) performed essentially the same experiments but started their investigation with the oocyte. They observed no changes in protein synthesis patterns during oocyte maturation, and concluded that all the proteins synthesized in early embryogenesis were also synthesized in the oocyte. The f irst new proteins observed by these authors appeared in gastrulae and became distinct in neurulae. Harsa-King et_ al_. (1979) also studied the Xenopus oocyte using 2DPAGE and found stage specific quantitative changes in the rates of synthesis of 30% of the proteins and found 19 stage specific proteins. Bravo and Knowland (1979) studied the proteins of the Xenopus oocyte and embryo and compared them to the proteins synthesized in various adult tissues. The results suggest that there are distinct categories of proteins which can be detected on 2-dimensional 23. gels. Two such classes are proteins made in all embryonic stages and tadpole stages studied and proteins made at only one embryonic stage or only in differentiated tissues. They also suggest that some proteins synthesized rapidly in the oocyte are likely to be synthesized in differentiated tissues as well, while proteins synthesized for the f irst time at fertilization are much less likely to be synthesised in differentiated tissues. All of these findings for Xenopus embryos are apparently in agreement with recent findings by Dwarkin and David (1980 a,b) who studied the changing populations of embryonic poly(A)+ RNA's during Xenopus early development using a library of recombinant DNA clones for colony hybridization experiments. These authors find that during oogenesis and until the beginning of gastrulation there is a relative constancy of the abundant poly(A)+ RNA population. However, a dramatic change in the pattern of abundant poly(A)+ RNA species occurs between the gastrula and the tailbud stages of development with the new pattern remaining fairly constant for at least 2 days of development up to the late prefeeding tadpole stages. These results suggest that many of the changes observed in protein synthesis patterns during early Xenopus development and oogenesis by 2DPAGE may reflect translational level control of protein synthesis during these stages. Tufaro and Brandhorst (1979) analysed the proteins synthesised by isolated blastomeres of early sea urchin embryos and could not find any qualitative differences between the proteins synthesised. The 16 cell stage blastomeres are committed to different developmental fates and over 1,000 proteins were detected in this study. This result is compatible with the findings of Hutchins ejt aK (1979) who found that the commitment to vegetalised development of the embryo prior to hatching is not accompanied 24. by detectable changes in the patterns of protein synthesis, though the expression of this commitment is accompanied by marked differences when vegetalised embryos are compared to normal embryos. A preliminary investigation of the protein changes during organogenesis in the mouse (Klose and von Wallenberg-Pachaly, 1976) suggests that proteins clearly characteristic of particular organs can be detected only after organogenesis is complete. Bravo et_ aJL (1979) have also suggested that in Xenopus, gene expression characteristic of differentiated tissues does not occur until the specialised cells responsible are fully differentiated. Gutzeit and Gehring (1979) investigated the proteins synthesised in D.  melanogaster oocytes and embryos. No oocyte specific proteins were found but the nurse cells of stages 9, 10 and 12 fol l icles synthesised stage specific proteins. Differences also existed between the proteins synthesised in anterior and posterior fragments of stage 10 fo l l ic les. At the blastoderm stage the posteriorly located pole cells are very active in protein synthesis and the marked differences in synthesis patterns from anterior and posterior halves of the blastoderm embryo were found. Gutzeit e_t aj_. concluded that in D. melanogaster even the earliest stages of determination are reflected by marked changes at the biochemical level. This result differs from the results obtained from sea urchin and amphibian embryos and may reflect the more mosaic pattern of development seen in Drosophila embryos. The best characterized determinant in early development is probably the special cytoplasm, present in many organisms which interacts with cells to specify the primordial germ cel ls. In Drosophila this cytoplasm is characterized by the presence of structures called polar granules. Waring et_ _al_. (1978) have succeeded in isolating by NEpH SDS PAGE the major protein 25. component of these granules. This protein is basic and has a molecular weight of 95,000. The isolation of this protein promises to lead to important findings about the nature of cytoplasmic determination. During embryogenesis in D. melanogaster and other holometabolous insects, the presumptive somatic cells are segregated into two cell lineages; larval and imaginal. After hatching, the differentiated cells of the larvae grow by increasing in cell size and their chromosomes become polytene. In contrast, the imaginal disc cells proliferate through normal mitotic division and remain embryonic in appearance. Each disc in a mature larva may be identified by its location and characteristic morphology. During metamorphosis when most larval tissue degenerates, the imaginal discs give rise to the external structures of the adult, e.g. the head is primarily derived from a pair of eye-antennae discs. Rodgers and Shearn (1977) studied the patterns of protein synthesis in imaginal discs of D. 35 melanogaster labelled in vivo with S, using 2DPAGE. They detected approximately 400 proteins, and 81-84% of these were common to the three disc types studied. The rest were unique to individual disc types or found in pairs of disc types. Seybold and Sullivan (1978) investigated the protein synthetic patterns during differentiation of imaginal discs in vitro. These authors used one dimension SDS PAGE to analyse 35 S-methionine labelled proteins and found that during the culture period profound changes in the protein synthesis patterns of distinct discs were seen. However, a comparison between the three disc types revealed striking simularities in the changes that occurred. In general, it was found that the protein synthesis patterns of different imaginal discs at the same period during differentiation showed greater similarities than the patterns 26. of a single disc type at different periods. Utsumi and Natori (1980) analyzed the imaginal disc proteins of Sarcophaga peregrina by 2DPAGE. This organism has the advantage of being very easy to synchronize at the larval stage and the physiology of the ecdysone response has been studied in depth. Coomassie Blue staining proteins and proteins newly synthesised in  vivo after ecdysone treatment ( 3 5S labelled) were analysed. In this organism no disc specific proteins could be detected in either case. Changes in protein synthesis during myogenesis in D. melanogaster have also been studied. Storti et al_. (1978) and Fyrberg and Donady (1979), studied myogenesis in primary cell cultures. Cultures were labelled with 35 S-methionine and the proteins analysed using 2DPAGE. Changes were seen in the pattern of proteins synthesized as myogenesis proceeded and three classes of proteins were identified. Class A proteins were the most abundant and were synthesised continuously throughout myogenesis. Class B proteins are proteins whose synthesis is initiated during myogenesis and continues throughout development. Class C proteins are those synthesized at specific times during development. Drosophi1 a muscle has been shown to contain three isoelectric forms of actin (Storti et aj_., 1978; Horovitch et al_., 1979; Fyrberg et aj_., 1979) on 2DPAGE which are called forms I, II and III in order of increasing basicity. Isoactins II and III are considered to be the general cellular actins, while isoactin I is considered to be muscle specific. During myogenesis (Storti et_ aj_., 1978 and Fyrberg et a]_., 1979), the synthesis of form I increases relative to that of forms II and III and in fusing myogenic cells approximately 80% of the actin synthesized is isoactin I. Berger and Cox (1979) studied actin synthesis and turnover in cultured cells of D. melanogaster. They found that only form II actually accumulates in the cells with form III being synthesized as a precursor to 27. the more stable isoactin II. The h a l f - l i f e of isoactin III was found to be 50 mins and i t is postulated that a post-translational modification of isoactin III, such as acetylation, leads to the shift in isoelectric point to give isoactin II. VII.The Basic Proteins of Sperm Many organisms undergo qualitative changes in the basic protein complement of sperm chromatin during spermiogenesis. The classical examples of sperm nucleoprotein replacement are in salmon and related f i s h . Instead of histones typical of somatic nuclei, with molecular weights of 11,000-20,000, their sperm contain basic nuclear proteins rich in arginine but much lower in molecular weight (around 3,000-7,000) (for reviews see Ando et aj_., 1973 and Coelingh and Rozyn, 1975). The biological role of the replacement is not entirely clear, since there is variation in the extent of replacement in different animals. Condensation is a characteristic of most sperm heads, and i t is generally accepted that the small basic proteins cause the DNA in the sperm head to be condensed into a tight array. The small volume of the head may confer hydrodynamic advantages to the sperm; however, i t does not appear that the primary structure of the protein alone determines the shape (Bloch, 1969). It has been proposed that tight packaging of the DNA will protect i t from damage. The sperm is transported and stored for a great deal of time in the absence of any DNA repair mechanisms. Tight packing may make the DNA more resistant to damage from such things as water, enzymes and bacteria (Subirana, 1975). Other theories are that i) the change in nucleoprotein erases the developmental history of the c e l l , allowing the highly specialized nucleus to enter the egg in a totipotent state; i i ) that the new nucleoprotein merely inhibits gene 28. activity in the sperm; i i i ) that the new protein may have some role in early embryogenesis; and finally, iv) that since there is so l i t t le conservation in the primary sequence of this class of proteins, they may have no particular function but may have a primitive structural role. The commonly accepted theory is that protamines facilitate nuclear condensation. This could be achieved by increasing the level of somatic histones, but this might lead to problems in the egg after ferti l ization. It is difficult to envisage a mechanism whereby the egg cytoplasm could selectively remove excess somatic histones from the male pronucleus, and it is easier to imagine specific enzymatic removal of a special sperm nucleoprotein. How the process of decondensation actually occurs is unknown. In contrast to the remarkably conservative primary structure of histones throughout a wide range of plants and animals (DeLange and Smith, 1971), the basic proteins of the sperm head are remarkably varied, and attempts have been made to group them into classes (for a review see Coelingh and Rozyn, 1975). Tne simplest molecules, called protamines by Kossel in (1928), are the classic small molecular weight, arginine-rich proteins found in salmon and related fishes. These proteins are easily solubilized, and tend to leach from cytological preparations, making early in situ studies diff icult (Bloch, 1969). The sperm of most mammals contain proteins more complex than the protamines. They have high arginine contents, but also a large amount of sulphur-containing amino acids, giving the potential for extensive cross-linking (Bellve et aj_., 1975). This makes this class very stable to extraction, and they have sometimes been called the stable protamines. Two other classes of protein have been proposed, but are less well characterized. These are the "histone-1ike proteins" found in some echinoderms and anurans (Bols and Kasinski, 1971) and the "intermediate 29. proteins" found in some molluscs, echinoderms, amphibians and fishes, which fall between histones and protamines (Subirana et al.,1973). Das et_ aj_. (1964a) investigated sperm nucleus maturation in D.  melanogaster using histochemical methods. They concluded, using arginine-specific stains, that a shift from lysine-rich to arginine-rich histones occurred during sperm maturation. At this time, a protamine was defined cytochemically as an arginine-rich protein which was labile enough to be leached out of the cell during hydrolysis with hot trichloroacetic acid (TCA). In the case of Drosophila, the replacement protein did not leach out on hot TCA treatment, leading the authors to conclude that D. melanogaster males did not make a protamine. Das et al_. (1964b) further showed by autoradiography that sperm incorporate H-arginine into the heads during maturation. Histochemistry and autoradiography thus point to the appearance of an arginine-rich protein late in sperm development in Drosophila. No biochemical studies have been published on this protein. There is great interest in this area because of the potential for isolating mutants in the sperm head condensation and decondensation process. Such mutants might well help us to understand how and why the process occurs. Speculation already exists that a much studied mutation is in fact a lesion of Drosophi1 a protamine. There is a mutation in D. melanogaster called Segregation Distorter (Sandler et_ al_., 1959). Males heterozygous for this locus (SD/SD+) produce a gross excess of SD bearing offspring. The mechanism of this meiotic drive in heterozygous males is the disfunction of nearly all the SD+ bearing sperm. SD/SD homozygous males are sterile, and this is due to a disfunction of nearly all the sperm (see review by Hartl and Hiraizumi, 1976). The phenomenon is similar to that seen in other 30. species, the best known being the T locus in mouse (reviewed by Bennett, 1975). Electron microscope observations of testes of SD/SD+ males have revealed abnormalities in spermatogenesis in individual sperm on a one to one basis, as expected from the observed progeny (Peacock et. jal_., 1971). SD bearing chromosomes were init ial ly isolated from wild populations of D.  melanogaster, and the locus is a complex one containing at least two components called Sd (for segregation distorter) and Rsp (for responder). The Sd component maps on the left arm of the second chromosome near the locus of purple, which is also in the region of the histone genes (39 D-E of the salivary gland chromosomes). This has led to the speculation that the syndrome of Sd could be due to a lesion in the process of histone replacement in the sperm head. The disfunctioning sperm in SD males do show abnormalities in head condensation, supporting this hypothesis. Kettaneh and Hartl (1976) investigated this hypothesis by making SD homozygotes by combining SD chromosomes isolated from different populations. They then examined, by histochemical methods, whether the transition from lysine-rich to arginine-rich histones occurred in the sperm of these males. No transition occurred in these sterile males, and the authors speculated that SD could therefore be a lesion either in the structural gene coding for the new sperm nucleoprotein, or in a regulatory locus controlling the histone transition. Unfortunately, the authors did not investigate any other male steriles to show that these did undergo a lysine-rich to arginine-rich histone transition. It is possible that an early lesion in sperm development would block the later events in sperm differentiation. It is also possible that the histochemical methods used would fai l to detect arginine-rich histones if they were present in a free or modified form, and not complexed together with the DNA. Furthermore, it is unwise to place too 31. much significance on the position of the Sd locus in the genome. There is no evidence to suggest that genes with similar products must be grouped together in the genome. This is not the case for tRNA's (Steffenson and Wimber, 1971) and the linking of the somatic histones may well be a specialized case arising from the need for coordinate expression (Pardue et a l . , 1977). Shoup (1967) investigated a sterile translocation (X:2) heterozygote which showed a failure of sperm head differentiation. She demonstrated that in this translocation the transition from lysine-rich to arginine-rich histones (as determined histologically) also failed to take place. Since it was assummed that in the translocation no genetic material was lost, this implies that a nonrelated disruption of spermiogenesis can lead to a failure of the cytochemically detectable nucleoprotein transition. VIII.The Present Investigation As we have seen above, the process of spermiogenesis in D. melanogaster is a promising system for the study of differentiation. At the beginning of this study, no biochemical analysis of the process existed. The availability of high resolution two-dimensional protein separation techniques, combined with in vitro organ culture in radioactive media, facilitated the investigation of the proteins synthesized by the testis. In Chapter 1, the problem of whether the Y chromosome codes for structural sperm proteins is investigated. In Chapter 2, data are presented on the proteins synthesized in the non-testicular tissue of the male reproductive system, and the components of the ejaculate are discussed. The question of when structural sperm proteins are f irst synthesized is treated in Chapter 3. Since individual cysts will grow and differentiate normally in culture (Cross and Shellenbarger, 1979) it was possible to culture testes at various 32. stages of development and to study the changes in protein synthesis that occurred during development. With this data, it was possible to deduce the time at which structural sperm proteins are f irst synthesized, and also to look at the qualitative changes in protein synthesis that occur during this period of morphogenetic change. In Chapter 4, the results of an investigation into the basic proteins of the testis are presented. 33. MATERIALS AND METHODS I. Materials and Abbreviations a) Materials All chemicals obtained commercially were of the highest purity or reagent grade. Special reagents were obtained as follows. Acrylamide and bisacrylamide were obtained from Eastman Organic Chemicals; ultrapure urea from Schwartz Mann; ampholines from L.K.B. Produkter Lab; sodium dodecyl sulphate from B.D.H.; Nonidet P40 from Particle Data Labs Ltd. All the components for tissue culture media were obtained from Sigma Chemical Co. (grade one amino acids were used) except the yeastolate which came from Difco; NCS tissue solubilizer and aqueous counting scintil lant, ACS, were obtained from Amersham/Searle; millex f i l ters were obtained from Millipore; microflex tubes and pestles from T.M. Kontes Glass Co.; micrococcal nuclease (E.C. 3.1.47) from Sigma; en Hance from NEM; molecular weight marker kits from Pharmacia; glass fibre fi lters from Reeve Angel. Radioactive Compounds DL-[4,5-3H]lysine (40 Ci/mmole), L[5-3H]arginine (8.9 or 16.8 3 35 Ci/mmole), H-proline (14 Ci/mmole), S-methionine (1065 Ci/mmole), and 35 Carrier-free SO^  were all obtained from Amersham/Searle Corporation. b) Abbreviations Tris: Tris(hydroxymethyl)aminomethane TCA: Trichloroacetic acid SDS: Sodium dodecyl sulphate TEMED: N, N, N', N'-Tetramethylethylenediamine DNA: Deoxyribonucleic acid 34. RNA: ACS: PAGE: PMSF: IEF: 2DPAGE: NEpH2DSDS-PAGE: EMS: Buffer A: Buffer L: Buffer M: Buffer 0: Mr: Ribonucleic acid Aqueous counting solution Polyacrylamide gel electrophoresis Phenylmethylsulphonyl fluoride Isoelectric focussing Two-dimensional polyacrylamide gel electrophoresis Nonequilibrium pH two-dimensional SDS polyacrylamide gel electrophoresis Ethyl methane sulphonate 9.5 M urea, 2% (w/v) NP-40, 2% ampholines (composed of 1.6% pH range 5 to 7 and 0.4% pH range 3 to 10) and 5% B-mercaptoethanol 1.5 M Tris-HCl pH 8.8 and 0.4% SDS 0.5 M Tris-HCl pH 6.8 and 0.4% SDS 10% (w/v) glycerol, 5% v/v B-mercaptoethanol 2.3% (w/v) SDS and 0.0625 M Tris-HCl pH 6.8 molecular weight 11. Drosophila Stocks 1. Oregon-R wild type 2. T(Y:2)CyO, DTS/+ males; +/+ females 3. y 2 / y 2 4. In(l)sc 4sc 8w a B y/B sYy + males; C(1)DX, yf/B sYy + females 5. C(1)RM, yw/0 females; y2su-wawa Ys Y Ly + /0 males Except when noted, all markers used in this experiment are described by Lindsley and Grell, 1968. All stocks were raised at 22°C on standard cornmeal, sugar, agar, yeast medium with methyl parahydroxybenzoate as mould inhibitor. 35. III. Generation of X/0, X/Y and X/Y/Y Flies XO males were generated three ways: (i) Wright and Green (1974) originally constructed the T(Y:2)CyO, DTS chromosome to be used in an automated virgin collection system. Males of the genotype T(Y:2)CyO, DTS will be curly winged if raised at 22°C, but will not emerge if raised at 29°C. In a stock of T(Y:2)CyO, DTS/+ males, +/+ females, straight winged males are found at a frequency of 1 in 100. They are sterile, and are the product of primary nondisjunction in the female, i.e. are X/0 in genotype. These males are easy to find in a stock raised at 29°C despite their low frequency. (ii) If males of genotype In(l)sc 4sc 8w a B y/B sYy + are crossed to +/+ virgin females, one expects to find wild-type females with a slight expression of bar phenotype and males with extreme Bar Stone eye. However, a high frequency of wild-type males and moderate Bar females is produced due to primary nondisjunction in the male. Peacock (1965) showed 4 8 that primary nondisjunction is a common event in the sc sc male, the frequency of which will depend on the Y chromosome used and on the temperature at which the fl ies are raised. In this case 15% nondisjunction was seen, which was sufficiently high to provide adequate numbers of X/0 males for experimentation. ( i i i ) The third method of generating X/0 males also allowed the generation of X/Y/Y males, y /B Yy males (from a cross of y ly 4 8 3 s + males to Insc sc w B y/B Yy females) were crossed to C(1)RM, yw/0 females. This produced y 2 /0 males (X/0) and C(1)RM, yw/BsYy+ females. These females when crossed to y2su-wa wa Ys Y*~y+/0 males produced C(1)RM, yw/0 females and y^su-wa wa Ys Y L y + /B S Yy + males, i.e. X/Y/Y. 36. In all three cases, a sample of the X/0 males generated were tested for steril ity using 5 wild-type virgin females. In no case were the putative X/O's found to be fert i le. IV. Organ Culture of Testes and Paragonial Glands a) Media Modified SheiIds-Sang M3BF medium (SheiIds-Sang, 1977 as modified by Cross and Sang, 1978) without fetal calf serum, was used for the majority of culturing experiments. Medium for labelling studies was made without the amino acid which was to be added later in a radioactive form. Minimal Evagination medium was made as described (Frinstrom e_t aj_., 1973), Mandron's medium was made as described (Mandron, 1971; Martin and Schneider, 1973) with the omission of lysine and arginine. All the above media were made by adding the phosphates only after all the other components were fully dissolved. This prevents the formation of insoluble precipitates. The media were made in 100 ml batches and were sterilized using Millex disposable f i l ters, 0.22 ym (Millipore Ltd.). The media were stored at 4°C, as insoluble precipitates form if they are frozen. Penicillin and streptomycin sulphate were added to all of the above media to give final concentrations of 50 units/ml and 100 mg/litre, respectively. Insect Ringers (Ephrussi and Beadle, 1936) was made in 1 l itre batches. The Ringers used for tissue isolation was not sterilized and was kept at room temperature; for use as an organ culture medium, penicillin (50 units/ml) and streptomycin sulphate (100 mg/litre) were added. 100 ml aliquots were then sterilized using a Millex disposable f i l ter (0.22 yM) and stored at 4°C. b) Addition of Isotope 35 S-methionine in solution was dried down under a gentle stream of nitrogen at 37°C, and then taken up in sterile SheiIds-Sang medium lacking 37. added methionine, to give a final concentration of 1 mCi/ml. The medium was then resterilized using a Millex disposable f i l ter (0.22 pM) and divided into approximately 50 yl aliquots in sterile Microflex tubes which acted as culture vessels and could also be centrifuged and used as homogenizers in sample preparations. 3 3 H-Proline and H-arginine solutions were placed on a sterile watchglass and dried down inside a sterile Petri dish at 60°C. The isotope was then taken up in the sterile medium of choice and divided into aliquots in sterile Microflex tubes without being resterilized. All radioactive media were kept at room temperature for two days before being stored at 4°C. This ensured that any microbial or fungal contamination of the media 3 would become obvious before use. H-Proline was added to SheiIds-Sang 3 media at a concentration of 100 yCi/ml. H-Arginine was added to various media at a concentration of 500 yCi/ml. c) Culture of Adult Testes and Paragonial Glands Newly emerged males were collected and anaesthetized with ether. The genital tissue was removed from the fly in a drop of sterile medium on a siliconized slide, using watch makers' forceps no. 6 Biologie (Dumont). The testes or paragonial glands were then dissected away from the rest of the tissue and collected in a drop of sterile medium on a siliconized slide. This slide was kept in a Petri dish containing sterile water to prevent the collecting drop from drying out during dissection. When all the necessary tissue was in the collecting drop, it was sterilized by passing it through three drops of sterile medium. The tissue was handled with forceps which were flamed before each passage of tissue. The tissue was finally placed in the radioactive culture medium in a Microflex tube capped and placed in the incubator (Fisher Low Temperature Incubator, Model 38. 300). At the end of the culture period the samples were spun down in the Microflex tube, the medium was removed, and the appropriate cold buffer added. The samples were then stored at -70°. d) Culture of Larval and Pupal Oregon R Testes Larvae of two stages were cultured. The earliest stage was a sample of early third instar larvae which were picked by eye from a sample of larvae isolated by sucrose floatation from OrR stock bottles. The testes are visible through the larval wall at this stage, allowing sexing of the sample prior to dissection. The larvae were washed three times with sterile water to remove any contaminating food. The testes at this stage are firmly embedded in fat body and are small, transparent, circular structures. To facilitate handling, the testes were dissected out and transferred to the holding drop with a good deal of fat body s t i l l attached. Only when the sample had been sterilized by washing was the majority of the fat body dissected away from the testis. The testis is very fragile at this stage, which made it impossible to remove all the adhering fat body; some of the fat body was therefore cultured separately as a control. The testes were transferred to the culture vessel using a tungsten loop. This was made a l i t t le larger than the testes so that they were held in the loop by surface tension. Fine tungsten wire can be flame sterilized repeatedly. Wandering third instar larvae were also cultured. At the end of the third instar, just before pupation, larvae leave the food and crawl up the sides of the culture vial looking for a place to pupate. This wandering phase lasts for several hours. Larvae were picked from the walls of the culture vial , sexed and washed with sterile water. The testis is s t i l l embedded in fat body at this stage, so the complex was treated as described above. 39. At the end of the larval period in Drosophila, the larvae stop moving and assume a characteristic barrel shape. These prepupae remain white for one hour at 25°C (Ephrussi and Beadle, 1936). Collection of white prepupae therefore gives a synchronous pupal population. For practical purposes in this study, we have taken the formation of the white prepupa to mark the start of the pupal period. This is technically incorrect, as true pupation (Bodenstein, 1950) starts approximately 24 hours after white prepupa formation. The testes are s t i l l visible through the cuticle of the white prepupa, so they were collected for half hour periods, sexed, and placed in glass vials in an incubator at 22°C. White prepupae were dissected for culture, and the testes were st i l l embedded in fat body; they were therefore handled like the larval samples. Pupal testes were cultured at approximately 24 hour intervals throughout pupation. The pupae were surface sterilized with 70% ethanol for two minutes and then washed with several changes of sterile water before dissection. The pupa was then placed in a drop of medium and cut in the middle with forceps. The testes were then gently teased from the posterior half of the pupa. Handling of the pupal testis can pose some problems. At the start of pupation, the testis is s t i l l round and can be handled with a tungsten loop. However, as development proceeds, the testis elongates to a pear-shaped organ. This stage (approximately 48 hours at 22°C) is extremely difficult to handle as it is a very fragile and sticky piece of tissue. At this stage the tissue was only washed once as the losses on transfer were considerable. A modified tungsten loop or forceps can be used, but neither are completely satisfactory. It is difficult to use a micropipette as the testes are very sticky, and si 1iconization of the micropipettes is only a partial solution. It is also difficult to avoid the transfer of excess medium 40. using micropipettes. The testes remain difficult to handle until they have achieved virtually adult morphology (approximately 90 hours at 22°C), when they can be handled with forceps easily. The analytical system subsequently used allowed small samples to be processed, so cultures with as few as four testes could be studied. At the end of the culture period, the tissue was spun down in the Microflex tube, the medium was removed, and 50-100 ul of cold lysis buffer A was added. The samples were stored at -70°. V. In Vivo Labelling of Flies Yeast was labelled according to the method of Candido and Bail lie (1974). 10 mCi of carrier free S04 was used per culture, and greater than 90 of the isotope was incorporated into TCA precipitable counts. The yeast pellet was stored at -20°C undiluted until used. Labelled yeast was applied to the top of a 1/4" of standard fly food in a Shell v ial , at a concentration of 1 mCi/vial. Wild-type larvae were collected, washed and sexed. Approximately 300 male larvae of second and third instar were then added to the prepared vial . On emergence, the males were allowed to remain for approximately three days on labelled food. They were then placed in a vial of unlabelled food for four hours prior to dissection. These labelling conditions resulted in only a small amount of lethality, where 35 greater amounts of S yeast led to high larval mortality. The adult c males contained 5 x 10 cpm, when solubilized with NCS. These males were fert i le, although progeny were not scored. Due to the nature of this experiment extreme care was taken in handling the radioactive f l ies . CO2 was used to anaesthetize the flies in the vial before any attempt at transfer was made. They were then further anaesthetized with ether before dissection. Unfortunately, wingless fl ies could not be used due to 41. problems of genetic background and because males from this experiment were also used in the mating experiments. VI. Isolation of In Vivo Labelled Testes, Paragonial Glands and Sperm Anaesthetized, labelled males were dissected in Drosophila Ringers (Ephrussi and Beadle, 1936). The genitals were removed and the testes or paragonial glands dissected away from the other tisue. The testes or paragonial glands were washed in two drops of Ringers and then collected in 50-100 yl of cold buffer A and stored at -70° until analyzed. Sperm was isolated from dissected, washed testes in Ringers, by teasing open the seminal vesicle with fine watch makers' forceps (No.. 6 Biologie, Dumont). The sperm that burst out were collected on a fine glass rod. Care was taken not to include any of the yellow wall of the seminal vesicle. The isolated sperm were placed in 50-100 yl of cold lysis buffer A and stored at -70°. Mating Experiment to Detect Ejaculate Proteins On emergence, 3^S labelled adult males were left for three to five days on J 3 S yeast containing food and then transferred to a vial of unlabel led food for one hour to reduce the amount of unincorporated S yeast on or in the f l ies . The males were then transferred to a shell vial containing unlabel led food and twice their number of unlabelled, wild-type virgin females. The flies were left for varying periods of time, and in some cases were left undisturbed for over six hours. This was necessary as the labelled males had to be etherized before they could be transferred, and this is known to reduce mating activity. After several matings had taken place in the culture, the fl ies were treated with C O 2 , etherized and separated. The females were placed in a drop of insect Ringers (Ephrussi and Beadle, 1936) and the reproductive organs dissected. The 42. ovaries and any large pieces of fat were removed and the remaining organs, including the paired spermathecae, the seminal receptacle and uterus, were washed through two drops of Ringers and collected in 100 yl of lysis buffer A (O'Farrell, 1975). Under these conditions any radioactivity detected in the sample of female tissue must have come from the male ejaculate transferred on mating. VII. Mass Isolation of Testes, Ovaries Young Oregon R or y^/0 males were collected, etherized and dissected in Ringers solution. The testes were collected in a drop of Ringers, on a siliconized slide in a moisture chamber. When the dissection was complete, the tissue was examined and any contaminating paragonial glands or malpighian tubules were removed. The tissue was then placed in the appropriate buffer and stored at -70°C. OrR females were collected within 12 hours of emergence at 22°C. The females were aged for 3 days to ensure that they were virgins. The virgins were etherized and the ovaries dissected in Ringers. Once again the tissue was collected in a drop of Ringers, examined for homogeneity, and transferred to the appropriate buffer and stored at -70°C. VIII. Sperm Isolation Males were collected within 12 hours of emergence at 22°C and aged for approximately 5 days without females. Ten males at a time were etherized, and the testes dissected and collected in a holding drop. The seminal vesicles were then burst and the sperm teased out using a fine glass rod. The sperm was then transferred to a drop of 0.4 N h^ SO^  kept on dry ice. At the end of each group of about 100 males, the drop was thawed and refrozen before being placed at -70°C. Sperm was collected from a total of 1,000 males in a final volume of 500 x. 43. IX. Sample Preparation i) Two Dimensional Gel Electrophoresis The samples in lysis buffer A were freeze-thawed five times to disrupt the tissue. They were then homogenized directly in the Microflex tubes with the appropriate pestle. Following this procedure, no intact tissue could be seen. One microlitre aliquots were taken and precipitated with 10£ TCA using 100 yl of 1 mg/ml bovine serum albumin (BSA) as carrier. The precipitate was collected on glass fibre f i l ters. The f i l ters were then washed successively with TCA, ethanol and ether and then dried and counted in ACS liquid scintillation f luid. i i ) Acid Extraction of Basic Proteins Samples in 0.4 N H2S04 were freeze-thawed twice and then homogenized on ice in a small teflon pestle homogenizer. The homogenate was then extracted for 30 mins at 4°C, and the insoluble material removed by centrifugation (3,000 x g for 10 min). The pellet was rehomogenized in 4 volumes of 0.4 N H2S04 and reextracted for 30 mins at 4°C. The insoluble material was removed by centrifugation (3,000 x £ for 10 mins), and the supernatants combined. The supernatant and pellet were then oialyzed overnight against 0.1 M acetic acid, lyophilized and redissolved in an appropriate gel buffer. The level of incorporation in radioactive samples was monitored by TCA precipitation as described above, except that a final concentration of 20 TCA was used to precipitate these samples. i i i ) Chromatin Preparation Testes from 850 young OrR males were dissected and stored at -70°C in Ringers. The tissue was spun down (1,000 g for 10 min) and taken up in four volumes of TMKS pH 7.4 (Tris-HCl 50 mM pH 7.4, 1 mM MgCl2, 25 mM KC1 and 0.25 M Sucrose) with 0.1 mM phenylmethylsulfonyl fluoride (PMSF) added 44. as a protease inhibitor. The tissue was homogenized, and the homogenate spun at 5,000 x £ for 10 min. The pellet, containing nuclei and whole cells, was gently rehomogenized in TMK pH 7.4 (Tris-HCl 50 mM pH 7.4, 1 mM MgCl2, 25 mM KC1). The homogenate was spun (5,000 x a_ for 10 min) and 0.7 mis of 10 mM Tris HC1, pH 7.4, was added to the pellet. The resulting lysed nuclei were layered on top of a sucrose cushion (1 M sucrose, 10 mM Tris HC1, pH 7.4). This was spun at 10,000 x g_ for 20 mins. The resulting chromatin pellet was then acid extracted with 1 ml of 0.4 N H,,S04 for 30 mins and the acid extract dialyzed against 0.1 M acetic acid for 12 hours. iv) Micrococcal Nuclease Digestion of Acid Soluble Material from  Oregon R Ovaries. The acid soluble material from 650 ovaries was suspended in 30 x of buffer (10 mM Tris HC1 pH 7.5, 1 mM Ca 2 + ) . To this 1 unit of micrococcal nuclease was added, and the mixture was incubated at 25°C for 30 mins. The reaction was stopped by the addition of 30 x of ice-cold, two times sample buffer (1.8 N acetic acid, 30# Sucrose). v) Delipidation of Ovary Samples 500 x of ethanol/ether 3:1 (Brown et a]_., 1969) was added to the dried ovary sample, and the sample was thoroughly mixed. It was left once for 1.5 hours, then spun for 1.5 hours at 5,000 x g_. The supernatant was discarded and the pellet reextracted with 500 pi of ethanol/ether 3:1. This process was repeated twice. The pellet was then finally extracted with 2 mis of ether, the sample spun (1.5 hr, 5,000 x g_) and half of the supernatant aspirated off. The pellet was then dried at 37°C under a gentle stream of nitrogen. 45. X. Gel Electrophoresis of Proteins or Samples were analyzed by gel electrophoresis. S labelled testes, paragonial glands, accessory ducts, sperm pumps, sperm and ejaculate were analyzed by two-dimensional electrophoresis (2DPAGE, O'Farrell, 1975). •^S labelled testes were also analyzed by nonequi1ibrium pH gradient electrophoresis combined with SDS gel electrophoresis (NEpHGE-SDS PAGE, O'Farrell e_t aj_., 1977). The latter method allows resolution of basic as well as acidic proteins. Acid extracts and the insoluble pellets from testes and ovaries were analyzed in 15% SDS polyacrylamide slab gels, NEpHGE-SDS gels, and on acid-urea gels. i) Two-Dimensional Polyacrylamide Gel Electrophoresis High resolution two-dimensional electrophoresis was carried out according to the procedure of O'Farrell (1975). The pH range of the f irst dimension gel was the same as that in the original paper. Isoelectric focussing gels were run for 8,000 volt-hours as this was found to maximize resolution. The second dimension was a concave exponential gradient SDS slab gel, with a uniform percentage gel at the top. The slab was formed using a two chambered gradient maker with 13.3 mis of 22% acrylamide solution in buffer L and 75% glycerol in the mixing chamber, and 13.3 mis of 5% acrylamide in buffer L in the feeding chamber. The gradient was poured at a rate of 3 mls/min. Spacers 0.8 mm thick were used and the separating gel had the dimensions of 15 cm x 14 cm. This was left to polymerize for 24 hours. A 2 cm, 4.5% stacking gel in buffer M was then poured on top of the separating gel. The second dimension gels were run without cooling at 220 volts (constant voltage) for 8 hours. Gels were deampholined, rehydrated and then stained with Coomassie Blue in 50 tTCA as described in the original procedure. They were then dried 46. and exposed for varying lengths of time to X-omat R film which was developed according to standard procedures. The autoradiograms were analyzed by inspection. This tedious task was improved by making contact prints of the standard autoradiograms and then laying the autoradiogram to be analyzed directly on top of a contact print. Thus, an original autoradiogram of one gel, with black spots on a clear background, could be easily compared with the contact print of another gel, in which the spots appeared white on a black background. Tritium labelled protein samples were fluorographed according to the method of Laskey and Mills (1975) to facilitate detection. i i) Non-Equilibrium pH Gradient Electrophoresis Combined with SDS Gel Electrophoresis (NEpH2DSDSPAGE). NEpH2DSDSPAGE was carried out as described by O'Farrell et al_. (1977). 2% v/v ampholines of pH range 3-10 were used in the f irst dimension to display the full range of proteins. A pH gradient of 3.6 - 9.1 was measured in the f irst dimension. The first dimension gels were run for 2.5 hours at 400 volts, i .e. a total time of 1,000 volt hours to ensure that no basic proteins were lost from the gel. The second dimension was a uniform 15% modified Laemmli SDS slab gel with a 5% stacking gel, poured as described below. The spacers were 0.8 mm. The second dimension was run at 220 volts, constant voltage, for 5 hours. The gels were deampholined, stained and autoradiographed as described above for 2DPAGE. ii i) .SDS Polyacrylamide Gel Electrophoresis Fifteen percent polyacrylamide SDS slab gels were made using a modified Laemmli procedure. The following volumes of stock solutions were used to prepare the lower 15% SDS separating gel: 15 mis of acrylamide solution (29.2% acrylamide, 0.8% methylene bisacrylamide), 17.5 mis of Tris HC1 47. buffer (1.5 M Tris, pH 8.8), 0.3 mis of 10% SDS solution, and 7.05 ml of distilled water were combined, degassed and 10 yl of TEMED added to initiate polymerization. The gels were overlayed with water until polymerized, after which the overlay was replaced with a mixture of 2.5 mis Tris HC1 buffer (1.5 M Tris, pH 8.8), 100 yl lO^SDS, and 7.4 mis of distilled water, and the gels left overnight. The 5% stacking gel was prepared using 1.66 mis of acrylamide solution (29.2% acrylamide, 0.8 methylene bisacrylamide), 2.5 mis of Tris-HCl buffer (0.5 M Tris pH 6.8), 0.1 mis of 10% SDS solution, 5.7 mis distilled water, and 45 yl of 10% ammonium persulphate. The above were combined, degassed and polymerized after the addition of 5 yl of TEMED. The protein sample was added to a sample buffer containing 0.125 M Tris-HCl pH 6.8, 4 SDS, 10% B-mercaptoethanol and 20% glycerol. After the addition of the running buffer (0.0495 M Tris, 0.384 M glycine and 0.1% SDS), the samples were loaded and the gels were run for 6-7 hours at 130 volts. The gels were stained with 0.25% Coomassie Blue in methanol/acetic acid/water (5:1:5, vol/vol) and destained by diffusion in methanol/acetic acid/water (2:1:5, vol/vol). Samples containing tritium were treated with en Hance, an autoradiographic enhancer, as described in the product information sheet, iv) Acid/Urea Gel Electrophoresis The system of Panyim and Chalkley (1969) was used to separate basic proteins, with the following modifications: the gels were not prerun, and were cast as slabs rather than tubes. To prepare a slab 6.25 mis of TEMED solution (43.2% acetic acid, 4% TEMED) and 25 mis acrylamide solution (30% acrylamide, 0.4% methylene bisacrylamide) were added to 19.8 g of urea (6.25 M final concentration). The solution was stirred over low heat, then 48. made up to a total volume of 49.5 mis with distil led water and fi ltered. The solution was degassed before the addition of 0.625 mis of 10% ammonium persulphate solution, and polymerized in the slab gel apparatus. Samples were dissolved in 20 to 30 yl of sample buffer (0.9 N acetic acid, 157? Sucrose). The samples were subjected to electrophoresis using an acetic acid running buffer (0.9 N acetic acid) for 8 hours at 170 volts, at 4°C. The gels were stained in coomassie blue in methanol stain as above. 49. CHAPTER I Two-dimensional patterns of proteins synthesized in the X/0, X/Y and  X/Y/Y testis and evidence that the Y chromosome does not code for  structural sperm proteins. Introduction The role of Y chromosome function in spermiogenesis has aroused much interest, but virtually no biochemical analysis. To investigate whether the Y chromosome produces a protein product, it was necessary to use the most sensitive protein separation methods available. Two methods were usea: O'Farrell (1975), and O'Farrell et aj_. (1977). Both methods combine two different separation techniques, the f irst dimension being separation based on the intrinsic charge of the protein, and the second dimension exploiting the molecular weight of the individual polypeptide chains. The f irst method (O'Farrell, 1975), 2DPAGE, uses isoelectric focusing in narrow tube gels as a f irst dimension. This provides excellent separation, but is limited in that the pH gradient of the isoelectric focusing gel covers only the range of pH 5 to pH 8, so that only proteins with isoelectric points in this range will enter or remain in the gel. The second method, NEpH2DPAGE, uses an isoelectric focusing gel as the f irst dimension, but proteins are loaded at the basic end of the gradient and are not allowed to reach equilibrium in the gel. This results in proteins of all charges being retained in the gel. Both methods then involve equilibration of the f irst dimension gels with SDS, and electrophoresis of the sample perpendicularly to the f irst dimension, on an SDS slab gel (Laemmli, 1970). Microscopic observations of X/0 testes (reviewed by Keifer, 1973) have shown them to contain disorganized and degenerating sperm. Protein degradation can lead to spurious differences between samples, which are 50. minimized if only newly synthesized material is examined. The in vitro culturing methods available (Cross and Shellenbarger, 1973) can be used to 35 label testis proteins with S-methionine, but it was necessary to show that the proteins synthesized in the isolated testes were the same as those normally synthesized in vivo. This was done by feeding male larvae 35 S-labelled yeast, and then dissecting the testes from the emerging f l ies . Testes were also labelled with ^C-proline and 3H-proline, and the proteins then compared to those labelled with S-methionine. Experiments were also performed to determine what level of genetic heterozygosity existed between common laboratory stocks. It was important that there be not too great a variation between the testicular proteins of different stocks, since if such variations existed it would be extremely difficult to separate these differences from those attributable to the presence or absence of a Y chomosome. X/0 and X/Y males were generated in two different ways, and a set of X/0, X/Y and X/Y/Y fl ies by a third method. The proteins produced by these testes were then separated by 2DPAGE and by NEpH2DPAGE. 3 5S-labelled 35 sperm were hand dissected from the seminal vesicles of S-labelled males, and also examined by 2DPAGE. Results and Discussion i) Levels of Incorporation A maximum incorporation of 1 x 10^  cpm was seen in ^S-methionine labelled cultures containing 40 testes. The level of incorporation varied, and samples of approximately 100,000 cpm were routinely loaded onto gels. Any differences in sample size between gels could be compensated for by changing the exposure time during autoradiography. Lower levels of incorporation were seen in the H-proline labelled cultures, reaching a 51. maximum of approximately 90,000 cpm; the maximum incorporation with 1 4C-proline was approximately 60,000 cpm. No evidence of protein degradation, which is readily detectable in 2DPAGE, was seen in the autoradiographs, even in the intensely labelled ^S-methionine samples. Since we were looking for small differences in patterns between gels, the comparative experiments described below were repeated independently many times to guard against possible spurious differences. The in vivo labelled testes appeared healthy, and contained on the average 10,000 cpm/testis pair. Again, when the proteins were analyzed, there was no evidence of degradation and the confirmation of fert i l i ty in the males strengthened the conclusion that the isotope dosage had few detrimental effects on the testis. ii) Comparison of the In Vivo and In Vitro Protein Patterns It was important to determine whether isolated testes cultured in vitro were synthesizing the same proteins as they would in vivo. Thus, patterns obtained from both labelling methods were compared (Figure 3). It can be seen that the patterns obtained are remarkably similar. All proteins detected in the in vivo labelled testes are present in the in vitro labelled organ, showing that the testes do not contain major protein products which are synthesized in other parts of the f ly . There are more proteins in the in vitro labelled sample, and quantitative differences in individual spots exist. These differences are probably due to the labelling of somatic proteins in the in vivo experiments because of the length of the labelling period, while in the in vitro labelled sample, the bulk of the radioactive precursor will be incorporated into the more rapidly turning over, germ line proteins. It is concluded that the organ culture method is a highly sensitive and reproducible technique for labelling testis proteins in D.  melanogaster. 52. Figure 3. Comparison of testis protein labelling in vivo and in vitro. Autoradiograms of two-dimensional polyacrylamide gels. (A) D. melanogaster Oregon R testis proteins, labelled in vivo with 35s as described in Methods. 137,000 cpm were loaded onto the f i rst dimension gel and the second dimension gel was exposed for 26 days. (b) D. melanogaster Oregon R testis proteins labelled in vitro with ^-methionine as described in Methods. Twenty testes were cultured together for 20 hours at 22°C. 165,000 cpm were loaded onto the f i rst dimension and the second dimension gel was exposed for 24 days. Figure 3 54. i i i ) Isotope Comparison Testes from Oregon R males were labelled with either ^C-proline or 35 S-methionine, and the resulting protein patterns compared (Figure 4). A maximum of 450 proteins were labelled with the 1 4C-proline, and although fluorography (Bonner and Laskey, 1974) increased individual spot size, it did not improve overall resolution. A maximum of 1,200 proteins were labelled with J3S-methionine; however, the patterns of the 2 sets of proteins were remarkably similar, i.e. only 2 spots from the * 4C-proline gel were missing on the S-methionine gel. Quantitative differences in spot size were seen, presumably due to differences in the proline and methionine contents of proteins. The increased level of detection obtained 35 with S-methiomne led to its being used in the majority of subsequent o . incubations. iv) Stock Variation 3 14 3S °H-proline, 1 C-proline and S methionine were used to label the testes of various laboratory stocks in tissue culture, and no major differences between the stocks were found (Figure 5 compares 4 different stocks labelled in vitro with S-methionine). This is in agreement with recent work on the degree of genetic heterozygosity in D. melanogaster (Leigh et aj_., 1979). These authors investigated the degree of variation in proteins between 54 different strains of D. melanogaster, by Coomassie Blue staining of protein patterns from whole f l ies after separation of extracts by 2DPAGE. They concluded that the heterozygosity per locus was approximately 4%, with 6 Loci polymorphic. This is a much lower estimate than those previously reported. The similarity of the patterns of the resolved testicular proteins from different stocks is important, as it makes it possible to directly compare the proteins from different stocks and 55. Figure 4. Comparison of testis proteins labelled in vitro with 14C -proline and 35s_niethionine (~A) Oregon R adult testis proteins labelled with l^C-proline in  vitro. The sample contained approximately 100,000 cpm and was analysed by 2DPAGE. The gel was fluorographed and autoradiographed for 6 days. (B) Oregon R adult testis proteins labelled with 35s-methionine in  vitro. The sample contained approximately 150,000 cpm and it was analysed by 2DPAGE. The dried gel was autoradiographed without fluorography for 14 days. Figure 4 57. Figure 5. Comparison of the proteins synthesized by testes in males from  different D. melanogaster stocks. All the samples, consisted of 40 testes which were cultured in vitro in -^-methionine containing medium. The samples were all analyzed by 2DPAGE. (A) T(Y:2) CyO, DTS/+ males. (B) In sc4Sc«wa B y/BSYy+ males. (C) y2/Y males. (D) vg/vg, e/e males. 58. Figure 5 59. mutants, without the necessity of ensuring a uniformity in genetic background. , v) Sperm Isolation Sperm were dissected from the seminal vesicles of in vivo labelled J 0 S males. The sperm was collected from as many as 100 males for one gel, as the incorporation of 3^S into pure sperm was low. The age of the male determined the success of dissection. In younger males there was very l i t t le sperm in the vesicle, and in males older than 10 days the vesicle was contracted and it was very difficult to release the compact plug of sperm from i t . The optimum age for dissection was 3 to 5 days after emergence. On average, 30,000 cpm were loaded onto a gel and 92 proteins were detected. A typical pattern is seen in Figure 6. Fifteen of the major proteins were detectable by Coomassie Blue staining prior to autoradiography. vi) X/Qj-X/Y and X/Y/Y Testis Patterns on 2DPAGE X/0 males were generated three ways, as it was necessary to ensure that any detected differences were not due to markers used in the selection systems. In X/Y, X/0 pairs I and II, i.e. T(Y:2)Cy0, DTS/+, X/0 and B sYy +/+, X/0, respectively, the autosomal backgrounds were identical, since the X/0's in both cases arose by nondisjunction. In a third experiment, X/Y/Y's were also generated, as it was reasoned that a Y chromosome product might be elevated in this stock, making its detection easier. The males in this experiment, however, did not have autosomal backgrounds identical to that of the X/0 males. From all three sets of experiments, the protein patterns obtained after ^-methionine labelling of the testes, followed by 2DPAGE, were remarkably similar. No differences attributable to the presence or absence of a Y chromosome could be detected, although putative electropohoretic 60. Figure 6. Structural sperm proteins of D. melanogaster OrR strain. Sperm was dissected from males labelled in vivo with 35s as described in Methods. 14,000 cpm was loaded onto the TTrst dimension and the second dimension gel was exposed for 44 days. 62. shifts were seen (Figure 7), confirming the sensitivity of the method. The results from the third experiment, i.e. the analysis of y /0, y /Y and y^ su-wa wa Ys YL y + /B s Yy + testes proteins, are shown in Figure 7. Comparisons are most readily made between gels which have been focused together; however, by locating reference spots and certain spot clusters, gels focused separately can be readily compared. vii) X/0, X/Y patterns on NEpH2D PAGE 7 7 Testes from y^/Y and y /0 males, were labelled in vitro with •3 c; S-methionine and the proteins analyzed on nonequi1ibrium pH gels (O'Farrell et aj_., 1977). As previously stated, these gels have the advantage of including basic as well as acidic proteins in the f irst dimension. To prevent the loss of any highly basic proteins, the f irst dimension was run for only 1,000 volt hours. This meant that the separation achieved was not optimal; however, 450 proteins were easily resolved. A pair of X/Y, X/0 NEpH2DPAGE gels are shown in Figure 8. Once again, no differences attributable to the Y chromosome can be seen. The experiment was repeated several times, as it was anticipated that this system would be less reproducible than the 2DPAGE; although the best comparisons are made between samples run together, it is possible to compare gels run at different times, particularly if the f irst dimensions are run for the same length of time. v i i i ) Evidence That the Y Chromosome Does Not Code for Structural  Sperm Proteins. X/0 testes were examined to see if they were synthesizing structural sperm proteins to the same extent as the X/Y testes. Figure 9 shows BsYy+/X and X/0 testes patterns with the structural sperm proteins labeled on the X/0 gel. The sperm proteins were positively located in both 63. Figure 7. Comparison of proteins synthesized by X/0, X/Y and X/Y/Y testes. Autoradiograms of two dimensional polyacrylamide gels. The testes of different genotypes were labelled in vitro with 35s-methionine as described in Methods. (A) X/Y testes (y 2 /Y). 102,000 cpm were loaded onto the f irst dimension and the second dimension was exposed for 21 days. (B) X/Y/Y testes (y2Su-wa w^ YS yL y + /BsYy + ) . 110,000 cpm were loaded onto the f irst dimension and the second dimension was exposed for 21 days. (C) X/0 testes (y 2/0). 1 5 0 , 0 0 0 cpm were loaded onto the f irst dimension and the second dimension was exposed for 21 days. The arrows indicate proteins which show electrophoretic variance between the genotypes. 64. Figure 7 65. Figure 8 Two dimensional electrophoretic separation of basic and acidic  proteins from X/0 and X/Y testes. [A] 40 y-^ /Y testes were labelled in vitro with 35$_methionine. The sample contained 220,000 cpm and was loaded onto a NEpH gel and run for 1000 volt hours. The second dimension was a 15% acrylamide modified Laemmli SDS gel. The dried gel was autoradiographed for 40 days. (B) 40 y2/0 testes were labelled in vitro with 35s_methionine. The sample contained 300,000 cpm and was run on a NE pH gel for 1000 volt hours in parallel with the y2/Y sample. The second dimension was a 15% acrylamide modified Laemmli SDS slab gel. The dried gel was autoradiographed for 40 days. 66. 67. Figure 9. Illustration that the X/0 testis does synthesize the structural  sperm proteins detected. Autoradiograms of two-dimensional polyacrylamide gels. The proteins of the testes were labelled in vitro with 35s_methionine as described in Methods. (A) Proteins synthesized by the X/Y, BSYy+/+ testis. (B) Proteins synthesized by the X/0 testis. These males had identical autosomal background to the X/Y (A). The squares indicate structural sperm proteins. These were assigned after comparing many gels to compensate for individual differences between gels. In the pair shown here the X/0 autoradiograms were exposed longer than the X/Y testis gel. 1EF—> A B Figure 9 69. the X/0 and X/Y sample, indicating that the Y chromosome does not code for the structural sperm proteins detected in this system. The results presented here provide biochemical evidence to support the morphological observations on the X/0 testis. Whatever the role of the Y chromosome in sperm development, it is a subtle one. The detected proteins synthesized by the X/0 testis are identical to those produced by the wild-type testis, and structural sperm proteins are present in normal amounts. This is compatible with the cytological findings of Patterson and Stone (1952), who found four species of Drosophila where the male is normally X/0. The presence of temperature sensitive sterile mutations on the Y chromosome of D. melanogaster indicates, however, that the Y chromosome does produce protein products. In microorganisms, temperature sensitivity of an allele has been shown to be a consequence of a single amino acid substitution, which results in thermal lability of the gene product. The evidence that EMS induced temperature sensitive mutations in D. melanogaster are in fact point mutations resulting in missense transformations has been discussed elsewhere (Suzuki et_ aj_., 1976). There are several possibilities as to why a Y chromosome product might not Jiave been detected here. Firstly, the protein could have been excluded from the gel. The NEpH2DPAGE results indicate that this is not a problem of isoelectric point. Alternatively, the proteins could have unusual solubility properties, so that they never enter the f irst dimension, or they could be extremely large, leading to exclusion from the gel on the basis of size. Both explanations are possible, but it seems unlikely that all of the proposed Y chromosome products would have such unusual physical parameters. It is possible that all Y chromosome products are methionine-free. H-proline labelled testes from T(Y:2)CoDTS/+ and X/0 males, however, gave 70. identical patterns on 2DPAGE, although the level of resolution with tritium is lower. Thirdly, and most likely, Y chromosome products could be present at a level below the limits of detection of these systems. There is probably no system available to the biochemist that is sensitive enough to detect proteins present in very low numbers per ce l l , for example specific gene regulators, without recourse to some preliminary enrichment step. Lifschytz and Harevan (1977) have postulated that some Y chromosome factors play a role in meiosis, and such a role would be consistent with a small amount of product. The Y chromosome products could also (have an enzymatic function in sperm protein assembly, and so only small amounts of the enzymes would be needed in the testis. Keifer (1973) noted that only 3 of the Y chromosome can be accounted for by the nucleolus organizer and the fert i l i ty factors. The other 97% of the Y chromosome (9.7% of the total genome) appears to be silent DNA or, Keifer suggests, could contain reiterated copies of autosomal or X linked genes which are important in spermatogenesis. Genetically, it would be difficult to detect such genes. The lack of any obvious quantitative differences in sperm protein levels between X/0, X/Y and X/Y/Y gels seems to argue against this idea. Also there are very few multiple spots on either testis or sperm gels. If duplication of structural genes had occurred, then one would expect to see multiple spots reflecting electrophoretic changes in the protein as a result of sequence divergence. In conclusion, the results presented in this chapter show the X/0 testis of D. melanogaster to be biochemically identical to wild-type with respect to detectable proteins synthesized. The isolation of structural sperm proteins confirms the hypothesis that the Y chromosome does not contain genes coding for these proteins. The evidence favours the hypothesis that Y 71. chromosome products are synthesized in very low amounts due to their role as organizers or regulators of sperm production. Consistent with this hypothesis is the idea that structural sperm proteins are common cellular proteins, i.e. it is the relative amounts and organization of these proteins which are controlled in the male to produce a viable sperm. This agrees with the genetic data. Many attempts have been made to screen for male sterile mutations with specific morphological lesions in the sperm, but none have been found. However, recent studies (Lifschytz, 1978; Shellenbarger and Cross, 1979) have shown that some X-linked temperature sensitive lethals are also temperature sensitive male steriles. These results are encouraging, and indicate that further studies of this kind may lead to the isolation of conditional male steriles with specific lesions. 72. CHAPTER II Analysis of proteins in the ejaculate of D. melanogaster males and of  the proteins synthesized-in the X/0, X/Y and X/Y/Y paragonial gland. Introduction The paragonial gland in D. melanogaster males plays an important role in reproduction. Extensive mating of males results in temporary steril ity which is due not to an exhaustion of the supply of sperm, but to a lack of paragonial gland secretion. Other components of the system, the anterior ejaculatory duct and the ejaculatory bulb, may also contribute to the nonsperm components of the ejaculate. Yeast can be grown on sulphate-free •DC medium, supplemented with carrier-free SO ,^ and a large percentage of 35 the SO^  is incorporated by the yeast cells into T.C.A. precipitable counts (Candido and Bail l ie, 1974). Larvae can then be fed on labelled yeast, diluted with unlabelled food, and the. larvae left to pupate. The males which emerge are viable and can be mated to unlabelled females. The mated females can then be dissected, and the proteins of the sperm receiving and storage organs analyzed on 2DPAGE. The proteins subsequently detected by autoradiography are thus derived from the male. By comparing these ejaculate protein patterns to patterns of proteins from the paragonial glands, anterior ejaculatory duct, ejaculatory bulb and to that of structural sperm proteins, one can in theory determine where each component of the ejaculate originated. The results of such an experiment are presented below, as well as a comparison of the proteins synthesized in  vitro by the X/0, X/Y and X/Y/Y paragonial glands. Results and Discussion i) In vivo Labelling 35 Paragonial glands dissected from S-labelled males had an average 73. incorporation of 6,000 cpm/gland. The anterior ejaculatory ducts contained approximately 5,000 cpm/duct, and the ejaculatory bulbs approximately 4,000 cpm each. The labelled males used were fert i le. When the samples were analyzed, over 500 proteins were detected in the paragonial gland sample (Figure 10A), and in the anterior ejaculatory duct and ejaculatory bulb (Figure 10B and IOC). The high resolution 2DPAGE technique is a very sensitive method and readily shows any protein degradation present in the sample. Partially degraded proteins appear as multiple spots in the isoelectric focussing dimension, with streaks in the molecular weight dimension. Degradation was not detected in the in vivo labelled samples, indicating that the f l ies sufferred no detrimental effects from the radioactive labelling. i i) The Transfer Experiment The number of counts transferred to females when mated to radioactive males is very low. An average of 25,000 cpm was detected in samples, with a maximum incorporation of 70,000 cpm in one experiment. The counts varied from one experiment to the next, presumably due to the varying frequency of mating in the cultures. Because of the low frequency of mating, cultures were left for some time before the females were dissected. This in turn leads to a loss in the radioactivity recovered from each mated female, since a mated female appears to store only about 700 sperm, 10 to 20% of the sperm actually received; the rest are lost in the f irst three hours following mating (Fowler, 1973). Another factor which contributed to the low level of soluble radioactivity in the samples was the difficulty encountered in solubilizing the spermathecae. These cuticular structures are very difficult to break open mechanically, and seem to resist freeze thawing techniques. However, the spermatothecae contain only 10 to 20% of the sperm 74. Figure 10. Proteins of the D. melanogaster male paragonial glands, anterior  ejaculatory duct and ejaculatory bu1b7 Autoradiograms of two-dimensional polyacrylamide gels. The proteins were labelled in vivo as described in Methods. (A) In vivo labelled paragonial gland proteins. Approximately 80,000 cpm were loaded onto the f irst dimension and the second dimension was exposed for 26 days. (B) In vivo labelled ejaculatory bulbs. Approximately 40,000 cpm were loaded onto the f irst dimension and the second dimension was exposed for 26 days. (C) In vivo labelled anterior ejaculatory ducts. Approximately 20,000 cpm were loaded onto the f irst dimension and the second dimension was exposed for 28 days. Figure 10 76. Figure 11. The proteins of the D. melanogaster Oregon R male ejaculate. Autoradiogram of a two-dimensional polyacrylamide gel. Approximately 25,000 cpm were loaded onto the f i rst dimension and the second dimension was exposed for 42 days. SDS I 1EF' Figure 11 78. stored by the female; the rest is held in the more fragile seminal receptacle. Figure 11 is a typical autoradiogram of a female sample, i.e. it shows proteins of the male ejaculate. This experiment was repeated many times to eliminate the possibility of artefacts. 122 proteins were detected. By comparing the ejaculate gel to that of structural sperm proteins and that of the paragonial gland sample, one can determine the relative contributions of sperm and ejaculate fluid to the ejaculate (Figure 12). The anterior ejaculatory duct and the ejaculatory bulb do not appear to contribute any major unique proteins to the ejaculate, although one protein seems to be enriched in the ejaculatory bulb. Assigning proteins to either the testis or paragonial gland is not clear cut. Some proteins which are present in the sperm are also synthesized in the paragonial gland. There are some obvious candidates for such common proteins, for example tubulin, actin, and mitochondrial proteins. The paragonial gland contributes a large number of proteins to the ejaculate. This is consistent with the observation that the paragonial gland secretion is necessary for fert i l i ty . One cannot make any conclusions about the relative quantities of sperm and paragonial proteins, however, because of the possibility of differential labelling in the testis and paragonial glands, and of differential storage by the female. i i i ) In Vitro Labelling After 24 hours in organ culture, no obvious signs of cell death could be seen in the paragonial cultures. Occasionally, contractile movement was 35 seen. The levels of S-methionine incorporation into proteins were high, the maximum level being 280,000 cpm/20 paragonial glands, with an average value of approximately 100,000 cpm/culture. The final autoradiograms are shown in Figure 13, and it can be seen that again there is no evidence of 79. Figure 12. A difference map of the proteins of the D. melanogaster male  ejaculate. Sperm proteins are indicated by^rand proteins synthesized by the paragonial gland byO. Common proteins are indicated by^. TEF- > SDS T 0 0 0 o 0 0 o t T T V V • 0 0 0 • T T • • T T • • Figure 12 81. Figure 13. Paragonial gland proteins labelled in vitro. Autoradiograms of two-dimensional polyacrylamide gel electrophoresis. The glands were labelled in culture with 35s-methionine as described in Methods. All gels were exposed for 18 days. (A) X/Y paragonial gland (y 2/Y). Approximately 100,000 cpm were loaded onto the f i rst dimension. (B) X/0 paragonial gland (y 2/0). Approximately 170,000 cpm were loaded onto the f irst dimension. (C) X/Y/Y paragonial gland (y2Su-wawa ys Y Ly +/BSYy +). 80,000 cpm were loaded onto the f irst dimension. The circles indicate a row of spots which is tentatively identified as a glycoprotein (see text). Figure 13 83. protein degradation on the gels. The gel pattern is remarkably similar to that of the in vivo labelled paragonial gland (Figure 10A); however, the level of detection is much greater in the in vitro labelled sample, and some quantitative differences also exist in spot intensity, presumably due to the different labelling periods. The number of proteins which can be detected on these gels is considerably higher than previously reported (von Wyle, 1976), and is more consistent with the acknowledged high synthetic activity of this gland. Anderson and Anderson (1977) showed that glycoproteins migrate on 2DPAGE in a characteristic manner, appearing as rows of spots with varying charge and molecular weight, due to the differing charge and size contributions of attached carbohydrate. The paragonial gels contain some rows of spots which suggest the presence of glycoproteins in the sample (Figure 13). This is consistent with the observation of von Wyle (1976), who stained his paragonial gland gels.with periodic acid Schiff stain for glycoproteins and detected 5 weakly staining bands. Beaulaton and Perrin-Walderner (1975) also located glycoproteins in the secretory granules of the paragonial gland using histochemical techniques, iv) Comparison of X/0, X/Y and X/Y/Y Paragonial Glands The question of whether the Y chromosome codes for a product in the paragonial gland was approached using in vitro labelling techniques, combined with 2DPAGE. The results are shown in Figure 13. It can be seen that even at this high level of resolution no differences were detected in the X/0, X/Y or X/Y/Y gland. This is biochemical confirmation of genetic and embryological data which have shown Y chromosome activity to lie solely in the gonial tissue (Marsh and Wieschaus, 1978; Nissani et_al_., 1979). These experiments are initial steps in defining the biochemistry of an interesting organ. The paragonial gland not only contributes to the male 84. reproductive system by conferring fert i l i ty , but it also secretes substances which act in the female to complete the reproductive cycle. The above experiments show that it is possible to analyze biochemically the ejaculate and to show the contribution of the various organs. The lack of a detectable Y chromosome product in the highly synthetically active gland is consistent with the theory that the Y chromosome only functions in the germinal tissue. The potential for genetic analysis of this gland is great, since by using the powerful selection criterion of fer t i l i ty , one should be able to isolate a series of paragonial gland mutations, which would facilitate analysis of the function of this secretion in the male and its endocrine functions in the female. 85. CHAPTER III Changes in protein synthesis patterns during the development of the  testis. Evidence that sperm proteins are synthesized early in development. Introduction In the study of development it is often assummed that changes in gene activity during development of a tissue are expressed primarily in significant changes in the spectrum of proteins made which ultimately result in morphogenetic changes. The testes of Drosophila are tissues in which we can study the changes in protein patterns during morphogenesis. When the larva hatches the testes are small spherical structures and during the larval period general growth takes place. On pupation, growth accelerates, changes in overall morphology take place, and much cellular differentiation occurs in a similar manner to that of the imaginal discs. The adult male imago is fertile within a few hours of hatching. Due to the highly specialized end. product of differentiation and to the" large morphogenetic changes that take place during development, the growing testis seemed an interesting organ in which to investigate changes in protein synthesis patterns. The results of Cross and Shellenbarger (1979) which showed that the testis could grow and differentiate in culture without the addition of hormones or interaction with other tissues were encouraging. The culture of 35 various ages of testis in vitro with S-methionine in the medium allowed us to study the changes in protein synthesis patterns during development. Results, presented in Chapter I, allowed the detection of structural sperm proteins on the 2DPAGE patterns from the young testes. It was thus possible to study qualitative and quantitative changes in sperm proteins during development. 86. Results and Discussion i) The Culturing of Immature Testes The method used to synchronize pupae worked very well and in one control sample all the male imagos emerged within a 2 hour period. The larval testes used were the least synchronous samples; however, they were all identical in appearance. The fat body control cultures appeared viable and presented no problems except that care was needed in recovering the floating tissue at the end of the culture period. The fat body cultures contained approximately ten times the amount of fat body which was in the testes cultures. All the larval and pupal testes were healthy in appearance at the end of the culture period and no opaque dead testes were seen. No problems were encountered with yeast contamination of cultures, even though some of the mid-pupal samples were only washed once in sterile medium because of handling diff icult ies. i i ) Levels of Incorporation The levels of incorporation of S-methionine into T.C.A. precipitable counts were good, with an average level being approximately 100,000 c.p.m./culture of 20 testes. The cultures with fewer testes had proportionately lower levels of incorporation. The fat body cultures had considerably lower levels of incorporation of isotope, reflecting the lower rate of synthetic activity in this tissue at this stage. i i i ) The Patterns of Synthesized Proteins The testis samples were prepared and electrophoresed on 2DPAGE (O'Farrell, 1975) as described in Materials and Methods. The patterns of proteins synthesized through pupation are remarkably similar. Figure 14 shows some data from an initial experiment. The cultures were maintained for 24 hours at 22°C. At 12 hours of pupation (Fig. 14A) in the staging 87. Figure 14. Proteins synthesized in the testis at the beginning and middle  of pupation. [A] Proteins synthesized in the testes of Oregon R pupae 12 hours after white prepupa formation. 15 testes were dissected from 12 hour pupae and cultured for 24 hours in vitro in medium containing 35s_methionine at 25°C. The sample contained approximately 40,000 cpm and was analyzed by 2DPAGE. (B) Proteins synthesized in the testis of Oregon R pupa, 84 hours after white prepupa formation. 10 testes were dissected and cultured for 24 hours in 35s-methionine containing media at 25°C. The sample contained approximately 75,000 cpm and was analyzed by 2DPAGE. 88. Figure 14 89. system used here, the pupa is just fully coloured and the testes are s t i l l visible as clear spherical structures through the pupal case. On dissection, histolysis can be seen to have started in the pupae and it is possible to tease the degenerating fat body away from the testis. In contrast, at 84 hours (Fig. 14B) the imago is fully formed within the pupal case and the apricot coloured eye is beginning to turn red. The testes are adult in morphology except that they are not yet pigmented. The pair of gels in Figure 14 are nearly identical; there are very few qualitative differences between them. Two faint spots in the 12 hour gel appear to be missing in the 84 hour gel and 5 spots are present in the 84 hour gel which are not detected on the 12 hour gel. However, these small differences could have been artifactual, so a more extensive series of experiments was initiated involving the culture of testes at 10 different stages in development. Shorter labelling times in culture were used and the dried gels were autoradiographed for longer periods in order to detect more proteins and to see if the differences detected init ial ly were truly qualitative changes. The initial results had indicated that the changes in proteins made during pupation were subtle and so younger testes were cultured in the second series in the hope of seeing more dramatic changes. Data from the second series of gels is presented in Figures 15, 16 and 17. The youngest stage cultured was early third instar larva. Fat body controls were necessary at this stage as it is very difficult to obtain the testis completely free of adhering fat without rupturing the testis. The fat body was found to synthesize about 10 major proteins and many minor proteins. Many of the minor fat body proteins comigrate with proteins on the testis gels, inferring that these may be common "household" proteins. The most prominent fat body proteins could not be detected on the corresponding 90. Figure 15. Patterns of proteins synthesized by the developing wild-type  testis I. (A) Thirty testes were dissected from a third instar larva at the wandering stage. The tissue was cultured for 12.5 hours at 21°C in medium containing 35s_m ethionine. The sample contained approximately 300,000 cpm and was analyzed by 2DPAGE. The dried gel was autoradiographed for 44 days. (B) Forty testes were dissected from pupae which were two hours past the white prepupal stage. The tissue was cultured for 12.5 hours at 21°C in medium containing 35s-methionine. The sample contained approximately 110,000 cpm and was analyzed by 2DPAGE. The dried gel was autoradiographed for 44 days. (C) 16 hour old pupae were dissected and three testes were obtained intact. These were cultured in medium containing 35s_methionine for 16.5 hours at 21°C. The sample contained approximately 60,000 cpm and was analyzed by 2DPAGE. The dried gel was autoradiographed for 44 days. (D) Twenty testes were dissected from 24 hour old pupae and cultured in medium containing 35s_methionine for 15 hours at 21°C. The sample contained approximately 220,000 cpm and was analyzed by 2DPAGE. The dried gel was autoradiographed for 44 days. 91. Figure 15 92. Figure 16. Patterns of proteins synthesized by the developing wild type  testes IT. (T) Thirteen testes were dissected from 48 hour pupae and cultured in medium containing 35s-methionine for 14 hours at 21°C. The sample contained approximately 98,000 cpm and was analyzed by 2DPAGE. The dried gel was autoradiographed for 21 days. (F) Four testes were obtained from 70 hour pupae and cultured in medium containing 35s-methionine for 13 hours at 21°C. The sample contained approxiately 101,000 cpm and was analyzed by 2DPAGE. The dried gel was autoradiographed for 21 days. (G) Twelve testes were dissected from 102 hour pupae and cultured in medium containing 35s-methionine for 12.4 hours at 21°C. The sample contained approximately 202,000 cpm and was analyzed by 2DPAGE. The dried gel was autoradiographed for 21 days. (H) Twenty testes were dissected from 131 hour pupae and cultured in medium containing 35s-methionine for 12 hours at 21°C. The sample contained approximately 230,000 cpm and the dried gel was autoradiographed for 21 days. 93. Figure 16 94. Figure 17. Changes in -levels of structural sperm proteins synthesized in  early third instar larvae and in the adult. ("A") Forty testes were dissected from early third instar larvae and cultured in 35s-methionine containing medium for 12 hours at 21°C. The sample contained approximately 7 0 , 0 0 0 cpm and was analyzed by 2DPAGE. The dried gel was autoradiographed for 44 days. The proteins designated numbers 1-8 are major structural sperm proteins. (B) Twenty testes were dissected from newly emerged adult f l ies and cultured in 35s-methionine containing medium for 2 1 . 5 hours at 21°C. The sample contained approxiately 160,000 cpm and was analyzed by 2DPAGE. The dried gel was autoradiographed for 44 days. The proteins labelled numbers 1-8 are major structural sperm proteins. Figure 17 96. testis gel in the early third instar larva, wandering third instar larva, or white prepupa plus 2 hour stages. Jhis indicated that the contaminating fat body made no significant contribution to the testis gel pattern. As can be seen in Figures 15, 16 and 17, the patterns are again very similar. At this level of resolution no consistent qualitative changes could be detected. There are, however, quantitative changes taking place. In Figure 17, an early third instar larva sample, which is the f irst time point taken, is compared to an adult sample. There are some obvious quantitative changes. The two gels were compared to the gel of structural sperm proteins discussed in Chapter I. All the structural sperm proteins detected are synthesized in the early third instar larva and 8 major sperm proteins are indicated (nos. 1-8). These proteins were then identified on the whole series of gels and any changes in relative synthetic rates during the period studied were noted. Proteins 1 and 2 were equal in quantity, and appeared as a doublet in the adult testes; in the early third instar larva they were present in very low levels, and protein 1 appeared to be present in larger amounts than protein 2. However, close examination of other data not shown here, suggests that this may be due to another protein which migrates in approximately the same position as protein 1. This illustrates how even in the two-dimensional protein separation systems, quantitative changes can be due to the comigration of more than one protein species. Proteins 1 and 2 all increase in synthesis around pupation and by 48 hours are synthesized at approximately the adult rate. Protein 3 is present in very small amounts in the early third instar larva testis and increases dramatically in amount through pupation. At 16 hours after pupation it is one of the five most heavily synthesized proteins and continues to increase until it is the most heavily synthesized protein in the adult testis. The area of the gel where 97. proteins 1, 2 and 3 run is where the various subunits of tubulin have been found to migrate (Brock and Reeves, 1978; Paulin e_t aj_, 1979; Lefevre, 1980). Sperm flagellae contain large amounts of tubulin (Fawcett, 1975) and the tails of D. hydei have been shown to contain tubulin. Rungger-Brandle e_t al_. (1978) used the technique of indirect immunofluorescence microscopy to show that the tails of D. hydei sperm reacted with antibody directed against 6S brain tubulin. It seems likely that some or all of the proteins 1 to 3 may be tubulin and the large amounts of these proteins in the adult testes suggests that they might be components of the long tail of Drosophila sperm. It is not possible to correlate the data presented here with that of Kemphries et aj_. (1979), who identified a testis specific tubulin, as the authors did not show whole gels in their paper but showed only the area containing tubulin. Proteins 4 and 5 both increase in synthesis, but differentially: In the early third instar larva, more 5 is synthesized than 4 but during pupation the relative intensities change and in the adult more of protein 4 is made than protein 5. Protein 6 does not appear to change in relative intensity through development and has a consistently high rate of synthesis . Protein 7 increases through development, intensifying noticeably from 70 hours onwards. Protein 8 is a major protein in the third instar larva, and increases in amount somewhat during pupation. This protein probably corresponds to isoactin II. Drosophila flight muscle was dissected from adult f l ies , homogenized in lysis buffer, and run on 2DPAGE (O'Farrell, 1975). In this system myosin does not run on the gel, and the major protein seen is actin. In the flight muscle sample (not shown) actin ran as 3 distinct spots with identical molecular weights but with slightly different isoelectric points. The pi of isoactin II corresponds to that of protein 8, and the nonsperm protein no. 9 comigrates with isoactin III. The 98. identification of proteins 8 and 9 as actin agrees with the data of Fyrberg and Donady (1979), Horovitch et al_. (1979), and Berger and Cox (1979). The absence of protein 9 in sperm and the changes in the relative intensities of proteins 8 and 9 during pupation support the proposal of Berger and Cox (1979), that isoactin III is a precursor to isoactin II. The failure to detect isoactin I (which is very prominent in the thoracic muscle sample) in the testis, also agrees with the findings of Fyrberg and Donady (1979) and Horovitch et_ aj_. (1979), that isoactin I is the muscle form of actin. It is generally accepted that actin filaments are not involved in sperm tail movement (Fawcett, 1975); however, actin has been found in isolated sperm preparations (Tilney, 1975; Clark and Yanagimachi, 1978). It is proposed that actin filaments may serve a role in the acrosomal reaction. Clark and Yanagimachi (1978) showed by indirect immunofluorescence that fixed sperm preparations from eight mammalian species contain intracellular actin as a major component of the post-acrosomal region. The analysis of the proteins synthesized during testis development has revealed a remarkably conservative picture. Structural sperm proteins are synthesized early in organogenesis. This could reflect early expression of differentiated gene products, or could be explained if structural sperm proteins have other roles in the testis. These two possibilities are hard to distinguish until one can find the initial expression of a product known to be unique to sperm. In the absence of qualitative changes in the proteins synthesized during this period of morphological change, quantitative changes in structural sperm protein levels are interesting. However, care must be taken in interpreting changes in protein levels. As we have already noted, comigration of two or more proteins can give misleading information and can occur even in the two-dimensional separation 99. systems. It is also interesting to note the results of Ivarie and O'Farrell (1978), who found that the response of HTC cells to dexamethesone was not completely reproducible. Within the population of responding proteins, these investigators found a subset of proteins whose response varied in subsequent experiments. The rate of synthesis of these proteins was influenced markedly by external factors such as cell density in the cultures and type of medium used. This illustrates how quantitative changes must be interpreted with care. In the experiments presented above, the quantitative changes discussed were those which could be seen as trends through the developmental series. The lack of qualitative changes and the small degree of quantitative change observed was a somewhat surprising result. However, as more systems are examined it appears that conservation is a more expected result, and the changes in protein synthesis during organogenesis are less dramatic than anticipated. Rodgers and Shearn (1978) by means of 2DPAGE studied the proteins synthesized in vitro by different imaginal discs of D.  melanogaster. The authors discussed the differences they observed between the different types of discs. However, O'Farrell (personal communication) recently pointed out that the more remarkable result is how similar the patterns from different disc types are. The experiments presented in this chapter could be extended. It would be interesting to look at younger testes to see when structural sperm proteins are f irst synthesized. This would require large numbers of well-synchronized larvae, as the smaller testes are more difficult to handle. The sample size would have to be increased and it would be desirable to culture the testes for shorter periods. It might also be interesting to run nonequilibrium 2DPAGE to look at the changes in basic proteins during development. However, the decreased resolution of these gels precludes them from being a f irst choice as a separation method. 100. Chapter IV The basic proteins of the D. melanogaster testis  Introduction The best known class of sperm protein is probably the protamines, or sperm specific nucleoproteins, due to their unusual physical properties. There is histochemical and autoradiographic evidence that an arginine rich protein exists in the sperm heads of D. melanogaster, but no biochemical evidence has been published. When the protamines from other species are examined, a general set of properties emerges which can be expected of any protamine: it should be small, highly basic, present only in sperm nuclei, have a high arginine content and be deficient in a certain number of other amino acids, and its quantity in the testis should increase as sperm maturation proceeds. This chapter contains the results of a preliminary investigation of the basic proteins of D. melanogaster testis. Results and Discussion i) Acid Soluble Proteins from Unlabelled Testes One physical characteristic of salmon protamine is that it migrates faster than other protein species when run on a Panyim-Chalkley, acid-urea gel (Panyim and Chalkley 1969) due to its high positive charge. Drosophila testes were dissected and collected in 500 yl of 0.4 N H^SO .^ This sample was then homogenized, dialysed and lyophilyzed as described in Materials and Methods. The small sample size made dialysis of the sample undesirable due to the inevitable losses incurred; however, the presence of even small amounts of sulphuric acid were found to interfere with sample migration during electrophoresis. Ovaries were chosen as control material. They were considered the most suitable control due to their high content of ribosomal proteins and histones, these being the major, nonprotamine, acid 101. soluble proteins in the testis. Ovaries are relatively easy to obtain, and due to the nature of this experiment only virgin females were used. The ovaries were treated in the same way as the testes and the samples were run alongside each other on Panyim-Chalkley acid-urea gels with salmon protamine and trout histones as markers. The testis acid soluble proteins gave a distinct banding pattern; however, the ovary sample gave an intensely staining smear in which the background was so high that only major bands could be distinguished. This background was presumed to be due to yolk components interfering with migration. Ovary samples were then delipidated (Brown et a l . 1969), which only improved the resolution slightly. Since nucleic acids can also interfere with electrophoresis, the ovary samples were digested with micrococcal nuclease as described in Materials and Methods, but again this did not improve the separation. No band was seen in the testis sample which migrated at the same place as salmon protamine, but there were several bands running faster than the histone standards. Since the ovary sample also had a smear in this region, testis and ovary samples were analyzed by SDS PAGE. 15% SDS gels were run on the acid-soluble and the non acid-soluble pellet proteins of the testes and ovary as described in Materials and Methods. Several differences in banding patterns of the acid-soluble proteins were seen. Sperm was then dissected from the seminal vesicle of over 1000 males, and the acid-soluble and non acid-soluble proteins were run on a 15% gel, (Figure 18). A protein with an apparent molecular weight of 55,000 is the major component of sperm and this is presumed to be tubulin, the major structural protein of the long sperm tai ls . No prominent low molecular weight, acid-soluble protein is seen in the pure sperm; however, an acid-soluble protein is seen in the sperm which co-migrates with trout histone H4. H4 is the most highly conserved of the 102. Figure 18: Proteins of sperm, testes and ovaries of Drosophila. Tissues were acid extracted, dialyzed and lyophilized as described Materials and Methods and run on a 15% acrylamide modified Laemmli SDS 1 = Trout histone markers 2 = Acid soluble testis proteins 3 = Acid soluble ovary proteins 4 = Acid insoluble sperm proteins 5 = Acid soluble ovary proteins 6 = Acid soluble sperm proteins 7 = Acid soluble testis proteins 8 = Acid soluble ovary proteins 9 = Acid insoluble testis proteins 10 = Acid insoluble ovary proteins 103. Figure 18 104. histone species, and has one of the most conserved sequences of any protein known so far (Allfrey, V.G., 1977). Labelling studies, described in more detail below, also show that the low molecular weight, acid-soluble proteins 3 in the sperm comigrate with proteins that label with H-lysine and 3 H-argimne in vitro. These experiments suggest that Drosophi la sperm heads may retain somatic histones in the nucleus during chromatin condensation. In the cricket, no somatic histones are retained in the sperm head, and the condensed chromatin contains a small, highly basic protein (McMaster-Kaye and Kaye, 1976). However, it appears that there is l i t t le correlation between the type of organism and the proteins of the sperm head. Kennedy and Davies (1980) recently showed that in the Winter Flounder, sperm retain somatic histones and do not contain a protamine. However, a class of nuclear proteins called High Mobility Group proteins is lost, and a group of large acid-soluble proteins appears in the sperm. This result is surprising when one considers that salmonids totally replace their histones with protamine. However, Carp is reported to also retain histones in the nucleus (Subirana et a l . 1975). It would be of interest to show conclusively that Drosophila sperm do retain somatic histones, and to investigate what happens to the High Mobility Group proteins during sperm head condensation. This would require the isolation and characterization of pure Drosophila nuclear proteins. However, the difficulties of obtaining large quantities of pure sperm would make experiments other than electrophoretic characterization very diff icult . The possibility does exist of using fluorescent antibodies to nuclear proteins to show their presence in sperm heads; however, care must be taken with negative results, as a failure to cross react with antibody could be due to the antigen being too tightly complexed in the head. 105. i i) Radioactive Labelling of Drosophila Basic Proteins Electrophoretic analysis of testis and sperm proteins indicated no obvious small basic proteins. This may be due to difficulties in seeing such a protein in whole extracts. In other systems, the detection of protamine by staining requires large amounts of material and some specific enrichment (McMaster-Kaye and Kaye, 1976). As a high arginine content is a general feature of protamines and of the large sperm protein found in the Winter Flounder (Kennedy and Davies, 1980), testes were cultured in vitro with radioactive basic amino acids. Pilot studies were performed using a "3 O mixture of H-lysine and H-arginine in Shields-Sang1s medium made without added arginine or lysine, as described in Materials and Methods. Twenty testes were cultured in the labelled medium (125 yCi H-lysine, 125 yCi JH-arginine in 100 yl of medium) for 17 hours. The reaction was stopped with 0.4 N H 2S0 4, 500 unlabelled testes were added as carrier, and the acid-soluble proteins extracted. The sample was dialyzed, lyophilized, and run on a 15% SDS gel. The gel was treated with enhance, dried and autoradiographed. The results are seen in Figure 19. The percentage of labelled precursors incorporated into acid soluble, T.C.A. precipitable counts was very low, although a good pattern was obtained after a long exposure period, indicating that the testes had remained viable in culture and that degradation had not occurred. The low level of incorporation obtained with both of the basic amino acids in the medium was disappointing and was attributed to the presence of unlabelled arginine and lysine in the yeastolate added to the medium which diluted the added radioactive amino acids. A series of experiments was then undertaken to see if other Drosophila culture media gave better results. The media used were chosen for the following reasons. Ringer's supplemented with penicillin and 106. Figure 19. Testis proteins labelled with ^H-arginine and -^-lysine in vitro. Two hundred testes were cultured in vitro in -^H-arginine and 3H-lysine containing medium. 500 carrier testes were added and the sample acid extracted,sdialyzed and lyophilized as described in Materials and Methods. The sample was applied to a 15% acrylamide modified Laemmli SDS slab gel. (A) Protein staining pattern of the gel. 1) = Trout hi stone standards 2) = Acid soluble ovary prteins 3) = Acid soluble testis proteins (B) Autoradiogram of the gel. The stained gel was enhanced, dried and autoradiographed for 60 days. During this process the gel swelled and cracked. The acid soluble fraction of the labelled testis sample contained 60,000 counts and the whole sample was applied to the gel. 107. Figure 19 108. streptomycin is the simplest medium available, contains no nutrients, and can be used for short incubation periods. Minimal evagination medium was used as it is again a simple medium not normally containing basic amino acids, but it has been shown to support the complex process of disc evagination in vitro (Fristrom et_ al_., 1973). Mandron's medium was chosen as it is a complex medium which supports extensive growth of Drosophi1 a cells and the evagination of imaginal discs; it is a defined medium which does not contain yeastolate, but is supplemented instead with vitamins. Mandron's medium (Mandron, 1971), was prepared as described, without the addition of arginine. Arginine free SheiIds-Sang's medium was also tested. Test cultures, containing 40 adult testes in 100 yl of test medium with 50 yCi of H-arginine, were made. Testes were cultured for either 5 hours in Ringer's, minimal evagination medium and Mandron's medium for 12 hours in minimal evagination medium and Mandron's medium, or for 24 hours in Mandron's medium, Mandron's medium with arginine, and SheiIds-Sang's medium. The greatest incorporation of label into T.C.A. precipitable material, approximately 150,000 cpm, was obtained with SheiIds-Sang's medium. This was an order of magnitude greater than the incorporation in any of the other cultures except for the two 24 hour cultures in Mandron's medium. Of these, the 24 hour culture with no arginine had high levels of incorporation approximately 100,000 cpm, while the one containing unlabelled arginine at the concentration normally used in the medium had only a third of the incorporation. This indicated that the specific activity of H arginine in the cultures was sufficient to allow amino acid transport into the cells. The levels of incorporation with cultures of less then 24 hours were considerably lower, indicating that 24 hours is not too long a period and that the low levels of incorporation of H-arginine may reflect a low 109. synthetic rate of basic proteins in the testis. Arginine free SheiIds-Sang's medium was used for further culturing experiments, although arginine free Mandron's medium would make an acceptable alternative. During this period it was realized that the type of dialysis tubing used during the sample preparation was cr i t ica l . Up until this point, conventional Fisher dialysis tubing had been used. This is adequate for most purposes, and retains histones; however, the pores will only retain effectively molecules with molecular weights larger than 13,000. Dr. Bhullar in our laboratory suggested the use of Spectrapor membrane tubing no. 3 (Spectrum Medical Industry, Inc.), which retains molecules which have a molecular weight above 3,000, and this was used for the remainder of the experiments described here. 3 H-Arginine labelled testes were mixed with carrier where necessary, and the acid-soluble proteins were extracted and analyzed using 15% acrylamide SDS PAGE as described in Materials and Methods. On staining with Coomassie blue, a band was seen migrating at the front of the gel. This 3 band seemed to diffuse rapidly. When the gel was treated with en Hance, dried and autoradiographed, a faint band was seen at the front of the gel which had not been seen previously. The other bands on the autoradiograms corresponded well with those in Figure 19, with changes in the relative intensities of the bands. The experiment was then repeated and care was 3 taken to stain, destain, treat with en Hance and dry the gel as quickly as possible; the result is seen in Figure 20. The prominent band on the autoradiogram was barely stained with Coomassie blue on the original gel; however, the gel had not destained completely before it was treated with en^Hance. The other faint bands on the autoradiograms again correspond to those in Figure 19. The heavy band at the front is a doublet with the larger member of the pair running behind the front. This doublet is almost 110. Figure 20. H^—Amginine labelled proteins of the testis. Two-hundred and eighty-six testes were cultured in vitro in medium containing 3H-arginine. The sample was acid extracted, dialyzed in spectrum 3 tubing, lyophilized and analyzed on a 15% acrylamide modified Laemmli SDS gel. 36,000 TCA precipitable cpm were in the acid soluble testis protein sample with 30,000 cpm in the acid insoluble protein sample. The sample was stained, destained and then en3Hanced, dried and autoradiographed for 15 days. (A) Protein staining pattern. 1) = Acid soluble testis proteins 2) = Acid soluble ovary proteins 3) = Acid insoluble testis proteins (B) Autoradiogram of the above gel. 1) = 3H-arginine labelled acid soluble testis proteins 2) = 3h-arginine labelled acid insoluble testis proteins Figure 20 112. certainly not free arginine, since the sample had been dialyzed for 15 hours in Spectrapor 3 dialysis tubing against 2 changes of 0.1 N Acetic acid, which would allow any free arginine to leave the sample. However, to test this hypothesis, free arginine in SDS loading buffer was applied to a 22^ SDS slab gel which was made with DATD. DATD is a crosslinker that can be cleaved by digestion with a 2% solution of periodic acid. The digested gel slices can then be counted in A.C.S. After running, the 22% gel was sliced and digested, but no evidence of radioactivity was found in the gel. The possibility exists that the doublet consists of degradation products. This seems unlikely because of the clear banding pattern which is seen, and because of the distinct doublet nature of the band. If degradation had occurred it is likely that evidence of it would be seen in the protein stained pattern, particularly as no cold carrier testes were added to this sample. It is also unlikely that degradation products would show so heavily on the autoradiogram and yet fail to stain with Coomassie blue. An important criterion for any protamine is its presence in chromatin, so testes were labelled with ^C-arginine, mixed with cold carrier testes, and used to make chromatin as described in Materials and Methods. This simple method did not give good results and many proteins were lost, suggesting that other isolation methods would be more suitable for Drosophi1 a chromatin preparation. One further experiment was performed. JH-Arginine labelled testes in 0.4 N H2S04 were homogenized, dialyzed, etc. but the supernatant and pellet were not separated. The sample was dried and taken up in O'Farrell's lysis buffer A, and analyzed on NEpH2DSDSPAGE. A sample of unlabelled virgin ovaries was treated in the same manner as a control. NEpH2DSDSPAGE was chosen as it could potentially indicate the degree of basicity of the protein seen in the 15% SDS gels. 113. The result is seen in Figure 21, and no low molecular weight arginine containing protein is seen on this gel. If such a protein had been present in the starting material it is possible that it did not enter the I.E.F. gel; however, controls run with trout histones showed that highly basic proteins will enter these gels. Alternatively, the protein might have run off the I.E.F. gel. Inspection of the ovary control sample which was run at the same time shows a class of proteins which migrated very rapidly through the I.E.F. gel which may be ribosomal proteins. It is thus possible that a protein with a very high arginine content could have migrated through the gel. Free arginine was also run on this system and no radioactivity was detected on the final gel. The sample- in Figure 21 does not appear to be degraded although non equilibrium two dimensional gels are less sensitive to this than the equilibrium gels. Of interest are the 4 prominent proteins in the 30,000 molecular weight range. If this gel is compared to that of testis proteins labelled with S methionine run on NEpH2DSDSPAGE, Figure 3 35 8, the patterns of H-arginine and S-methionine labelled proteins are quite similar, with major differences in the intensities of various spots. The four prominent proteins on the JH-arginine gel in the 30,000 molecular weight range appear to be present in the 3^S labelled sample, which argues against the possibility that they are histones. The low molecular weight or protein which has a prominent tail in the S gel, also appears to be present on the JH gel, but it is not particularly rich in arginine. The unlabel led sperm protein sample, Figure 18, does appear to have a partially acid soluble component in the 30,000 molecular weight range. The evidence presented in this chapter suggests that the sperm of D.  melanogaster does not lose all somatic histones during head condensation. 114. •5 Figure 21. NEpH2DSDSPAGE of 3H-arginine labelled proteins of the testis. Three-hundred and seventy testes were cultured in vitro in medium containing ^H-arginine. The sample was treated with acid but the supernatant and pellet were not separated. The sample was dialyzed using spectrum 3 tubing, lyophilized, taken up in lysis buffer A, and applied to a NEpH gel. The gel was focussed for 1000 volt hours which was then placed on a 15% acrylamide modified Laemmli SDS gel. The gel was stained, destained, en3Hanced and the dried gel autoradiographed for 10 days. The sample contained approximately 250,000 cpm. Figure 21 116. Data are presented which suggests that a low molecular weight protein rich in arginine is also present in testes. However, we cannot at this stage consider it a Drosophila protamine. It would be interesting to continue this study as some experiments are immediately obvious. First, it would be 3 interesting to analyze acid soluble, H-arginine labelled testis proteins on a 15% acrylamide Panyim-Chalkley acid-urea gel, taking care to run the gel for a short period of time. The best control for this system would 3 probably be H-arginine labelled Drosophila tissue culture cells. It would also be worthwhile to pursue the question of whether the protein is a component of testis chromatin, and again tissue culture cells could provide either cold carrier material and/or labelled controls. It would be difficult to prove definitely that such a protein is a component of sperm heads in Drosophila owing to the problems of obtaining sufficient pure sperm. Another convincing argument, often used in such research, is to show that the quantity of this protein increases as sperm matures. This can be done relatively easily in species where the sperm all develop in one • synchronous burst as, for example, in salmon. In Drosophila such an experiment would involve following the protein through development using 3 H-arginine labelling. However, as we have seen already, the early stages at which structural sperm proteins are made in Drosophila would necessitate making cultures of very young testes. 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