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Analysis of EMS-induced temperature-sensitive sterility mutants of the Y chromosome of Drosophila melanogaster Ayles, George Burton 1969

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ANALYSIS OF EMS-INDUCED TEMPERATURE-SENSITIVE STERILITY MUTANTS OF THE Y CHROMOSOME OF DROSOPHILA MELANOGASTER by GEORGE BURTON AYLES B.Sc, University of B r i t i s h Columbia, 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the department of ZOOLOGY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada ABSTRACT Heterochromatin can be described c y t o l o g i c a l l y as those chromosomes or parts of chromosomes which remain heteropycnotic, or dark staining, through most of the c e l l cycle. Genetically and biochemically heterochromatic regions generally seem to be inert and i t has been suggested that many heterochromatic l o c i are duplicated several times. In micro-organisms, genetic and biochemical analyses have been greatly f a c i l i t a t e d by the use of conditional l e t h a l s which survive under "permissive" conditions but die under " r e s t r i c t i v e " conditions. Temperature-sensitive ethyl methanesulfonate-induced l e t h a l mutations (such mutants r e s u l t i n s u r v i v a l at 22°C but death at 29°C) have previously been used in Drosophila melanogaster for preliminary studies of development. In the present study 8 temperature-sensitive (ts) s t e r i l e mutations (males are f e r t i l e at 22°C but s t e r i l e at 29°C) were induced on the Y chromosome of D. melanogaster. The ts mutants were mapped genetically on the long arm of the Y chromosome and they were found to involve a minimum of 4 d i f f e r e n t l o c i . The Y chromosome of D. melanogaster i s e n t i r e l y heterochromatic and i t i s necessary for male f e r t i l i t y but the exact function of the Y chromosome i s uncertain. The recovery of point mutations (ethyl methanesulfonate-induced temperature-sensitive mutations are presumed to be point mutations) on the Y chromosome indicates that there are l o c i on the Y represented by a single copy. A determination of the s p e c i f i c developmental effects of the ts s t e r i l e mutations, was also attempted. By exposing mutant males to a 48 hour period under the r e s t r i c t i v e conditions (29°C) and observing t h e i r f e r t i l i t y for several days, the stage i n the production of mature sperm during which the ts mutants were having an effect, was determined. i v TABLE OF CONTENTS Page INTRODUCTION 1 METHODS AND MATERIALS 6 RESULTS 15 DISCUSSION 23 SUMMARY 35 BIBLIOGRAPHY 55 LIST OF TABLES Table Page 1. Relative f e r t i l i t y of 10 d i f f e r e n t wild type 36S stocks at 22°C and at 29°C. 2. The re s u l t s of tests to l o c a l i z e ts Y s t e r i l i t y 37 mutants on YS or yL. 3. Results of complementation tests at 28°C between 38 ts Y s t e r i l e chromosomes and Y chromosomes deleted f o r 1 or more known f e r t i l i t y f actors. 4. Results of complementation tests between 2 39 di f f e r e n t ts Y s t e r i l e chromosomes at 28oc. 5. Temperature-sensitive period of s t e r i l i t y . 40 v i LIST OF FIGURES Figure Page 1. Screening protocol for the detection of 41 temperature-sensitive mutations on the Y chromosome. 2. Protocol for the l o c a l i z a t i o n of ts s t e r i l e s 42 on YL-»or Y S. 3. Protocol for complementation tests between ts 43 Y s t e r i l e s and Y chromosomes deleted for one or more known f e r t i l i t y f a c t o r s. 4. Protocol for complementation tests between 44 d i f f e r e n t ts s t e r i l e Y chromosomes. 5. Daily f e r t i l i t y of ts Y s t e r i l e stock A12 45 following a 2 day heat shock at 29°C. 6.3 Daily f e r t i l i t y of ts Y s t e r i l e stock B119 46 following a 2 day heat shock at 29°C. 7. Daily f e r t i l i t y of ts Y s t e r i l e stock H39 47 following a 2 day heat shock at 29°C. 8. Daily f e r t i l i t y of ts Y s t e r i l e stock A141 48 following a 2 day heat shock at 29°C. 9. Daily f e r t i l i t y of ts Y s t e r i l e stock E91 49 following a 2 day heat shock at 29°C. 10. Daily f e r t i l i t y of ts Y s t e r i l e stock A66 50 following a 2 day heat shock at 29°C. 11. Daily f e r t i l i t y of ts Y s t e r i l e stock A82 51 following a 2 day heat shock at 29°C. 12. Daily f e r t i l i t y of ts Y s t e r i l e stock A145 52 following a 2 day heat shock at 29°C. 13. CDaily f e r t i l i t y of Amherst control stock 53 following a 2 day heat shock. 14. Photograph of non-motile sperm of a ts Y s t e r i l e 54 male raised at 29°C. 15. Photograph of motile sperm of a ts Y s t e r i l e 54 male raised at 'RT.. X ACKNOWLEDGEMENTS I would l i k e to thank Dr. D. T. Suzuki for his advice and patient assistance during the course of t h i s work. I would ..also l i k e to thank Dr. Leonie Piternick for her enthusiastic encouragement during the e a r l i e r stages of the study. I am also deeply indebted to a l l those, especially H. A. M., who were w i l l i n g to l i s t e n to discussions of the r e s u l t s even when undoubtedly bored. 1 INTRODUCTION Heitz i n 1928 was the f i r s t to detect differences i n t h e i r state of condensation, between chromosomes or parts of chromosomes at various stages in the c e l l cycle. Such chromosomes, or parts, which seemed to maintain t h e i r compact-ness i n the nucleus (especially during interphase and telophase), stained very darkly and for t h i s reason they were described as being "heterochromatic" or "heterochromatin" as opposed to "euchromatin" which did not have t h i s property. Heterochromatic elements are widespread throughout the plant and animal kingdoms. Supernumerary chromosomes, which are often e n t i r e l y heterochromatic chromosomes, have been observed in many species of Hemiptera and Orthoptera. The supernumeraries of plants, or B chromosomes as they are call e d , are found in as many as f i v e to ten percent of flowering plants. They vary i n number in d i f f e r e n t individuals of a species or even within c e l l s of a single i n d i v i d u a l . At meiosis they do not p a i r with the other standard chromosomes nor i s there any crossing over between supernumeraries and standard chromosomes. Their presence or absence seems to have no influence on the esse n t i a l functions of the plant. In female mammals only one of the two X chromosomes i s euchromatic, the other becomes highly condensed and heteropycnotic and i s commonly referred to as the Barr body. Russel and Lyon (reported i n Mittwoch, 1967) integrated several observations into an hypothesis which states that the heterochromatic X i s 2 ge n e t i c a l l y i n e r t . In male coccids the paternally derived chromosome set becomes heterochromatic during development and i s also g e n e t i c a l l y inert (Brown and Nelson-Rees, 1961). In Drosophila, large blocks of heterochromatin are l o c a l i z e d adjacent to a l l of the centromeres but few, i f any, mutant genes appear to be located in heterochromatin (Muller and Painter, 1932). Autoradiographic studies (reviewed in Brown, 1968) indicate that associated with the condensation and genetic inertness of heterochromatin are l a t e r e p l i c a t i o n of DNA and a f a i l u r e to metabolize RNA. The inevitable conclusion i s that heterochromatin i s not involved i n a genetic function when i t i s in that p a r t i c u l a r state. However, Cooper (1959) states that i t i s not possible to i d e n t i f y any heterochromatic region that i s never euchromatic and thus supposedly never functional. The problem then i n a study of a heterochromatic segment i s to determine when and in what tissue the genes are a c t u a l l y functioning. The Y chromosome of Drosophila melanogaster i s e n t i r e l y heterochromatic during mitosis (Heitz, 1933 reported i n Cooper, 1959). It i s a metacentric chromosome and has an average length at metaphase I of meiosis, of about 2.3 microns, the short arm (Y5) being approximately one h a l f to two thirds the length of the long arm (Y L) (Cooper, 1959). Males that have no Y chromosome (X/0 males) appear to develop normally (Bridges, 1916) but t h e i r sperm do not mature and they are s t e r i l e (Safir, 1930). The Y chromosome can be considered 3 as a s p e c i a l example of heterochromatin as i t s action i s r e s t r i c t e d to a very p a r t i c u l a r function (production of f e r t i l e sperm) and tissue (the t e s t i s ) . Because of these r e s t r i c t i o n s the technical problems that would occur when looking for mutant a l l e l e s in the heterochromatin of the X or the autosomes, are very much reduced. In 1929 Stern (reported in Brosseau, I960) demonstrated that each arm of the Y chromosome ca r r i e s factors necessary for male f e r t i l i t y . Neuhaus (1939) studied the f e r t i l i t y regions of the Y chromosome more completely by inducing a series of Y-4 translocations that produced only s t e r i l e males in the absence of a free Y. He'concluded that there were at L S l e a s t 4 f e r t i l i t y genes on Y and 5 only on Y . Brosseau 8 (I960) used X-rays to induce deletions i n a sc «Y chromosome (a Y chromosome carrying a duplication of the t i p of the X which contains the gene y +) and carried out an extensive genetic study of the s t e r i l e males produced. On the basis of complementation tests between each of the s t e r i l e Y's he L concluded that there were 5 f e r t i l i t y factors on Y and 2 on S Y . He suggested that the difference between hi s conclusions and those of Neuhaus was a r e f l e c t i o n of the d i f f e r e n t methods used and of inconsistencies i n Neuhaus's tests. There are several drawbacks to the type of analysis that Brosseau has employed for studying the Y chromosome: 8 ' 1) the sc *Y i s not a normal Y chromosome and the r e s u l t s may i n some way be affected by the euchromatic X duplication attached to i t . 4 2) the complete s t e r i l i t y of the male means that the mutant Y must be maintained i n males carrying a normal Y chromosome as we l l . 3) the use of X-rays as the mutagen means that blocks of heterochromatin rather than just a single locus may be involved. Williamson (1968) has induced s t e r i l e Y chromosomes with a chemical mutagen (ethyl methanesulfonate) known to produce single base changes in microorganisms (Krieg, 1964) but he also used a Y chromosome carrying small duplications of the X chromosome. 4) Y chromosome mutations which always r e s u l t in completely s t e r i l e males are useful for studies of transmission or c l a s s i c a l Mendelian genetics, but do not provide information on the time or tissue s p e c i f i c i t y of that gene function. Conditional mutations on the other hand, which are expressed under " r e s t r i c t i v e " conditions but not expressed under "permissive" conditions, have greatly f a c i l i t a t e d morphogenetic and biochemical analyses in microorganisms (Epstein, et a L l . , 1963). Temperature-sensitive (ts) l e t h a l s belong to one class of conditional mutants that has been recovered from ethyl methanesulfonate-induced le t h a l s in D. melanogaster (Suzuki, et a L l . , 1967). The most useful aspect of such mutants i s that they may be maintained under permissive conditions, while t h e i r b i o l o g i c a l effects can be 5 studied at r e s t r i c t i v e temperatures. This suggests a method to f a c i l i t a t e a more extensive analysis of the f e r t i l i t y factors of the Y chromosome: Temperature-sensitive (ts) s t e r i l e s which are f e r t i l e under the permissive conditions (22°C) and s t e r i l e under the r e s t r i c t i v e conditions (29°C) could be used. The temperature-sensitive period (TSP) can be determined and some interpretation of the time of function of the gene product can be made. During the TSP, c e l l s requiring the Y chromosome function should be affected by high temperature, and determination of the time lapse from the period under r e s t r i c t i v e conditions to the time when the males are actually s t e r i l e , should indicate the time of formatin or function of the gene product in the t e s t i s . A c o r r e l a t i o n of t h i s information with information already available about the Y chromosome should aid i n determining the tissue i n which the f e r t i l i t y genes are functioning. Thus employing temperature-sensitive mutants i n a study of the Y chromosome presents a unique opportunity for the induction of s p e c i f i c heterochromatic mutations and an examination of t h e i r tissue and temporal s p e c i f i c i t y . 6 METHODS AND MATERIALS (a) Screening protocol for detection of temperature-se n s i t i v e f e r t i l i t y factors of the Y chromosome of Drosophila  melanogaster. Temperature-sensitive (ts) mutations i n Drosophila melanogaster which are l e t h a l at 29°C but survive at 17°C or 22°C have now been extensively analyzed (Suzuki, ^ t _al., 1967; B a i l l i e , Suzuki and Tarasoff, 1968; Suzuki and Procunier, 1969). Normal development of wild type stocks i s also disrupted at 29°C as indicated by t h e i r s t e r i l i t y when raised from egg to adult at th i s temperature. Consequently, ten d i f f e r e n t wild type stock were tested for f e r t i l i t y at 29°C (Table I ) . A wild type stock (Amherst, 1967) received from P.T. Ives, Amherst College, was found to produce a few progeny at 29°C and from these offspring, a temperature-resistant stock was derived (Am t r). I t has now been maintained in mass culture continuously at 29°C for over 30 generations and the males show l i t t l e d i f f e r e n t i a l temperature e f f e c t as regards to f e r t i l i t y . Am t r males were placed for 24 hours in h a l f - p i n t milk bo t t l e s containing a pad of f i l t e r paper saturated with 1 ml. of 0.025% ethyl methanesulfonate (EMS) dissolved in a solution of 1% sucrose (Suzuki, et a l . , 1967). The treated males were then mated in quarter-pint milk b o t t l e s containing standard Drosophila medium, at 22 + 2°C (RT i . e . room temperature) to t r w i ld type (Am ) v i r g i n females (20 males and 50 females per 7 per bottle) . Individual F-^  males carrying a paternally derived EMS-treated Y chromosome were then mated i n s h e l l v i a l s to 1-3 Am t r v i r g i n females for 4 days at RT. The adult f l i e s in each v i a l were then transferred without etherization to fresh v i a l s which were kept at 29°C. After being allowed to lay eggs for four more days, the parents were discarded (and the culture maintained at 29°C). The F2 progeny hatching at RT and at 29°C were then transferred i n mass without etheri z a t i o n to fresh v i a l s and allowed to lay eggs for 4-5 days at the same respective temperatures ,as before. After 10 days, v i a l s kept at 29°C were screened for s t e r i l i t y on the basis of an absence of progeny. The corresponding RT cultures from which such s t e r i l e 29°C F2 cultures were detected, were then examined for the presence of progeny. Those cultures y i e l d i n g f e r t i l e progeny at RT, but s t e r i l e f l i e s at 29°C, were designated as "Putative ts s t e r i l e s " . Autosomal dominant s t e r i l i t y factors would not be detected i n th i s protocol since t h e i r segregation from wild type a l l e l e s would y i e l d some f e r t i l e o f f s p r i n g in each cross. Since males alone carry the same treated Y chromosome, s t e r i l i t y mutations on the Y chromosome are automatically selected. The entire screening protocol i s outlined i n Figure 1. For confirmation of temperature-sensitivity of s t e r i l i t y , 10 males of each Putative ts s t e r i l e stock from the RT culture ( a l l of which carried the treated Y chromosome) were i n d i v i d u a l l y mated to 3 v i r g i n Am t r females and steps 3 and 4 (Figure 1) were 8 repeated. The absence of progeny from a l l of the 10 culture v i a l s at the end of step 4 was taken as confirmation of ts s t e r i l i t y and the stock was then maintained at RT and designated as a "Confirmed ts s t e r i l e " . As controls, untreated Am t r males were also screened for ts s t e r i l i t y as outlined i n Figure 1. (b) L o c a l i z a t i o n of ts s t e r i l e s on Y L or Y S. In order to determine whether the ts s t e r i l e mutation was located on the long or short arm of the Y chromosome, each mutated chromosome was tested for f e r t i l i t y in males carrying an X chromosome to which the long arm of the Y chromosome was attached as a second arm (X____0 (for a complete desc r i p t i o n of the X'Y1* and X-Y S chromosomes and a l l other stocks mentioned below see Lindsley and G r e l l , 1967). A l l of the stocks used i n tes t i n g the ts s t e r i l e Y chromosomes (designated henceforth as Y*) were s t e r i l e at 29°C, but were found to be f e r t i l e at 28 + .25°C (28°C hereafter), although males carrying Y* remained s t e r i l e at th i s temperature. Consequently a l l further genetic analyses of the Y* stocks were carri e d out at 28°C. Approximately 20 X- Y L males were crossed in h a l f - p i n t milk b o t t l e s to an equal number of attached *X females carry-ing the mutated Y chromosome (XX/Y*) and the parents were allowed to lay eggs for 4 days at RT. The adults were then transferred, without etherization, to fresh bottles placed at 28°C and discarded af t e r 4 more days. Ten of the F i male 9 progeny (X»Y /Y*) recovered at each temperature were then crossed in fresh b o t t l e s to 15 v i r g i n females at the same temperature as the males had been cultured. After 4 days, the adults were discarded. Ten days la t e r , each culture was scored for the presence or absence of progeny. Presence of progeny at RT but t h e i r absence at 28°C indicated that the long arm of the Y chromosome f a i l e d to complement with the mutant Y chromosome, thereby in d i c a t i n g the location of the g mutant on Y . On the other hand, complementation or presence of F2 progeny at 28°C and RT showed that the ts s t e r i l e was on Y L. Similar tests of each stock were also carried out with s X*Y chromosomes, i n order to confirm the tests with Y L. The entire t e s t i n g procedure for l o c a l i z a t i o n of each mutant i s outlined i n Figure 2. (c) Complementation tests with Y chromosomes deleted  for one or more known f e r t i l i t y factors (Brosseau, 1960). Once each mutant was l o c a l i z e d on a Y arm, i t was tested for complementation with Y chromosomes deleted for d i f f e r e n t known f e r t i l i t y factors (Brosseau, I960; the normal a l l e l e s for factors on Y L are k l - l + , k l - 2 + , etc., those on Y S are k s - l + , and ks-2 +, the corresponding mutant a l l e l e s would be k l - l " , kl-2~, k s - l ~ , ks-2~, e t c . ) . Seven d i f f e r e n t Y chromosome deleted for one or more of the f e r t i l i t y factors were ava i l a b l e . Each deleted Y carried a duplication of Y* attached to i t s short arm. The chromosomes used (followed by the f e r t i l i t y segment deleted) were: S4 (ks-1), S5 (ks-2), 10 L13 (kl-1), L37 (kl-2) , L l l (kl-3) , L38 (kl-3-4) , and L3 (kl-5) . For tests of each s t e r i l e Y stock, 5 females homozygous for the mutant yellow (y) and carrying one of the deleted Y chromosomes (y/y/y +Y^~) , were mated at RT in..'shell v i a l s to 5 males which car r i e d the inverted X chromosome , In(1) sc^ sc^, ysc (hereafter c a l l e d sc sc ) and a s t e r i l e Y chromosome (sc sc /Y*). After 4 days, the adults were transferred, without etherization, to fresh v i a l s kept at 28°C, and then discarded 4 days l a t e r . Wild type males ( y + s c + ) were selected. These males were presumed to carry the X and chromosomes from t h e i r mother and the Y* chromosome from t h e i r father + k- ° (y/y Y /Y*). Five of such males from each of the RT and 28 C cultures were then mated in s h e l l v i a l s at t h e i r respective temperatures, to v i r g i n y/y females for 4 days and the parents were discarded. Complementation of the mutant Y chromosome with the deleted Y was shown by the presence of progeny at both RT and 28°C. Absence of progeny at 28°C suggested that the EMS-induced mutation was located i n the region deleted from the tester Y. The crosses involved in t h i s l o c a l i z a t i o n are outlined i n Figure 3. Tests of each stock were made at t r l e a s t twice as a control, and males of the Am stock were tested i n the same manner. Males of each of the Y deletion stocks (y/y Y ) were also tested for s t e r i l i t y at RT and at 28°C. (d) Complementation tests between d i f f e r e n t Y* chromosomes. 11 In order to determine whether d i f f e r e n t s t e r i l i t y -mutants were a l l e l i c , males carrying two d i f f e r e n t Y* chromosomes were tested for f e r t i l i t y at 28°C and at RT.' There are two major problems associated with these t e s t s . The f i r s t problem i s to generate an X/X/Y* female so that males carrying a Y* chromosome from each parent can be produced. The other d i f f i c u l t y i s i n distinguishing males carrying two Y* chromosomes from those carrying only one. The second d i f f i c u l t y can be obviated by use of Y chromosomes marked by a duplication of the t i p of the X chromosome (sc • Y) which produces hairs on the second posterior c e l l of the wing when i n 2 doses (Brosseau, I960;) . Unfortunately two normal Y chromosomes have no e a s i l y detectable phenotype. However, the presence of an extra Y chromosome strongly suppresses the variegation of the eyes of f l i e s hemizygous or homozygous for the X chromosome inversion In(l)w m^ (Cooper, 1956; henceforth c a l l e d w m 4) . Thus W^VY* males can be distinguished from w /Y*/Y* males, on the basis of the degree of pigmentation of th e i r eyes. The former problem was solved by u t i l i z i n g the high rate of non-disjunction in males carrying the inversion A O sc sc and a Y chromosome (Sandler and Braver, 1954). Approximately 50 females homozygous for yellow body color (y/y) were crossed ( a l l crosses unless otherwise mentioned were i n quarter-pint milk bottles) to about 30 s c 4 sc^/B sYy + males and discarded a f t e r several days. V i r g i n F^ females that were Bar eyed and had wild type body color (y/sc 4 sc^/B sYy +) were selected and mated to wm4/Y males for several days. V i r g i n F2 females that were Bar eyed (y/w m 4/B sYy + and s c 4 s c 8 / w m 4/B sYy +) were then selected and mated to w^/Y males. F3 females were mated sin g l y and those which were w Yy + V 4 0 . were selected and mated to sc sc°/Y* males. From t h i s cross, w m 4 / s c 4 sc 8/Y* females which were detectable by t h e i r wild type eyes, were mated i n v i a l s at RT to s c 4 sc 8/Y* males carrying d i f f e r e n t Y* chromosomes. After 4-5 days, the adults were transferred without etherization to fresh v i a l s at 28°C, and discarded 4-5 days l a t e r . Wild type male progeny which were presumed to be & r m 4/Y*^Y*^ were then selected at each temperatur and mated to y/y v i r g i n females for 4 days at th e i r respective temperatures. Ten days l a t e r each v i a l was scored for the presence of progeny. The presence of progeny in the 28°C culture indicated complementation between the d i f f e r e n t Y* chromosomes and showed that the mutants contained on each Y chromosome were not a l l e l i c . As a control, each ts s t e r i l e Y was tested with i t s e l f ( i . e . , w^/Y*1 /Y* ) in order to rule out any possible e f f e c t of dosage on f e r t i l i t y . In addition, each ts stock was tested with the wild type chromosome of the t r Am stock to show that the presence of two Y chromosomes does not have a detrimental e f f e c t on f e r t i l i t y at d i f f e r e n t temperatures. The entire protocol i s outlined in Figure 4. A l l tests were car r i e d out at lea s t twice for each cross. (d) Temperature-sensitive period of s t e r i l i t y . Conditional s t e r i l i t y permits a determination of the 13 approximate gonadal stage affected by high temperature, by co r r e l a t i n g the i n t e r v a l between an exposure to high temperature and the onset of s t e r i l i t y . Twenty males of each mutant stock and of the Am t r stock were mated i n d i v i d u a l l y i n v i a l s to 7-10 v i r g i n y/y females for one day and then transferred d a i l y , without etherization, to fresh v i a l s containing new v i r g i n females, for twelve consecutive days, (using surplus (7-10) females and remating the males d a i l y should completely exhaust the testes of sperm produced over a p a r t i c u l a r 24 hour period (Hannah-Alava, personal communication)). The f i r s t 24 hour cross was carried out at RT, the next two 24 hour periods were at 29°C and a l l subsequent periods were at RT. Females i n each 24 hour culture were discarded three days af t e r transfer of the male. A l l of the v i a l s were maintained at RT. Cultures i n v i a l s 2 and 3 were i n i t i a t e d at 29°C and then placed at RT after transfer of the male. A l l adult F 2 f l i e s were counted and the mean number of progeny per male per day was calculated for each stock. Because the f e r t i l i z e d females were discarded af t e r four days the counts do not represent the t o t a l o f f s p r i n g the males would have had, but i t was f e l t that the of f s p r i n g counted from each 24 hour period would represent a s i m i l a r f r a c t i o n of the t o t a l possible. Since many males were l o s t or died during the twelve days of the experiment, ten males that remained f e r t i l e at the end of the experiment were selected for each stock and were then used i n determining the mean number of progeny, (males that produced 14 progeny on at l e a s t two of the l a s t three days of the experiment were considered to be f e r t i l e , and a l l of the stocks seemed to have recovered equally by the l a s t three days). (f) Determination of sperm m o t i l i t y at 28°C. Males which had been raised at 28°G and carried a mutant Y chromosome and were therefore s t e r i l e , were examined for m o t i l i t y of sperm i n the event that differences i n effects of the Y* chromosomes might be demonstrable. Normally, sperm m o t i l i t y cannot be detected in the t e s t i s i t s e l f , but i s e a s i l y observed i n sperm coming from the seminal v e s i c l e (Lefevre and Jonsson, 1962). Consequently, only the seminal v e s i c l e s and vase d e f f e r e n t i a were dissected, squashed on a s l i d e and then examined under phase contrast microscope. Lefevre and Jonsson (1962) reported that Beadle and Ephrussi Drosophila ringer solution (see Lockwood, 1961 for chemical constituents) provides optimal conditions for sperm m o t i l i t y and a l l dissections and examinations were carried out i n that medium. Phase contrast photographs were taken with a Zeiss Photomicroscope with a 40 power objective and they were magnified approximately 1300 times. 15 RESULTS (a) Detection of ts s t e r i l i t y . Thirty-four Putative ts s t e r i l e mutants were recovered from 1680 Y chromosomes tested. Subsequent tests to confirm the ts s t e r i l i t y produced 7 strains (A12, A66, A141, A145, B119, E91, H39), which were completely s t e r i l e at 29°C. One other s t r a i n had 1-3 progeny in the t o t a l of 10 v i a l s (A82) while s t i l l another s t r a i n (E64) had under 5 progeny in 2-3 v i a l s . The l a t t e r two strains were te n t a t i v e l y classed as s l i g h t l y leaky and were maintained with the o r i g i n a l seven while the remaining 25 strains were discarded. When the 9 strains were tested at 28°C, 8 of the 9 retained t h e i r s t e r i l i t y but E64 was quite f e r t i l e and was discarded. When tested at 27.25 + .25°C only A141 and E91 remained s t e r i l e . These tests show the extremely c r i t i c a l nature of the temperature range governing s t e r i l i t y . The 7 strains remained t o t a l l y s t e r i l e at 28°C throughout the duration of the study and the one mutant (A82) that had been classed as s l i g h t l y leaky was also found to be completely s t e r i l e i n the l a t e r tests. (b) L o c a l i z a t i o n of the ts s t e r i l i t y mutants on either L S Y or Y . Results of the complementation tests between the L S d i f f e r e n t ts Y chromosomes and the Y and Y arms are presented in Table 2. I t can be seen that males carrying any of the ts s t e r i l e Y chromosomes and raised at 28°C, were f e r t i l e when a 16 w i l d type Y fragment was present but s t e r i l e in the presence S of the Y arm. Not a l l the stocks were as f e r t i l e as the Am t r control i n the presence of the Y L and occasionally some crosses were s t e r i l e . This i s probably a r e f l e c t i o n of poor v i a b i l i t y at the high temperature. However, the complementation tests unambiguously reveal the p o s i t i o n of a l l of the induced mutants on (c) Results of complementation tests with Y chromo- somes deleted for one or more known f e r t i l i t y factors. The results of complementation tests of the ts Y chromosomes and Y's known to be deleted for one or more f e r t i l i t y factors, are shown in Table 3. As can be seen, some of the r e s u l t s are d i f f i c u l t to interpret i n the l i g h t of the S L previous tests involving Y and Y chromosomes. Stock B119 did not complement with chromosomes deleted for k s - l + or ks-2 +, yet i t did complement with a complete Y^ (see Table 2) which carried neither ks-1 nor ks-2 ^ , Possible reasons for t h i s w i l l be considered i n the Discussion. I t i s also evident from Table 3 that the complementation tests with deletion Y's were not as decisive as was hoped. None of the stocks tested, including the Amtr control, complemented -(- 4- «. with the Y chromosome deleted for kl-2 even though the Am L stock was i t s e l f f e r t i l e at 28°C and none of the stocks of mutant Y's complemented with the Y chromosome deleted for k l - 3 + - 4 + . Williamson has indicated (personal communication) that the former deletion stock was weak and did not give 17 consistent r e s u l t s , and Lucchesi (personal communication) found s i m i l a r problems with the l a t t e r stock. Further consideration of these problems w i l l be given i n the Discussion. The above results make i t impossible to determine the complete complementation pattern. The location of a mutation on factor k l - 2 + or k l - 4 + cannot be determined. Those stocks that did not complement with Y chromosomes deleted for k l - l + , k l - 2 + , and k l - 3 + - 4 + (A141, E91), were assumed to be on k l - l + . Those that did not complement with kl-5~ k l - 2 ~ and kl-3~-4~ (A12, B119, H39) were assumed to be on k l - 5 + . A l l the stocks tested complemented with kl-3~, thus showing that there were no mutations on kl-3 . At 22 C a l l of the complementation crosses yielded f e r t i l e progeny. Brosseau (I960) found that of 13 X-ray-induced s t e r i l e s Q on the sc -Y chromosome that involved a single f e r t i l i t y factor, 5 were mutant i n k l - l + , 1 in k l - 2 + , 4 in k l - 3 + and 3 in k l - 5 + while none involved only k l - 4 + . Williamson (1968) found that with EMS treatment, mutations were detected in k l - 3 + more frequently than in any other factor. Unfortunately, any comparison between the 3 sets of results i s probably without s i g n i f i c a n c e because of the small number of mutations examined, the d i f f e r e n t Y chromosomes involved, the d i f f e r e n t mutagens used, and the difference in screening techniques. Brosseau (1960.) , Lucchesi (1965) and Williamson (personal communication) have found that males carrying a Y chromosome d e f i c i e n t for k l - 3 + , or k l - 3 + - 4 + occasionally have a low l e v e l of f e r t i l i t y . This complication does not appear 18 to have contributed to the ambiguities i n the above results since males carrying any mutated Y chromosome and a Y chromosome and raised at 28°C were quite f e r t i l e and complement-ation was not registered on the basis of 2 or 3 f e r t i l e males. In addition, tests at 28°C involving a small number of males of each k l stock (15-20) revealed no f e r t i l e males among those that carried only a deleted Y chromosome. (d) Complementation tests between d i f f e r e n t ts Y  chromosomes. In determining a l l e l i s m between d i f f e r e n t s t e r i l i t y mutants, only those chromosomes which gave s i m i l a r patterns i n the deletion mapping tests (Table 3) were tested for complementation. Therefore, mutants A141 and E91 ( k l - l ~ ) were tested against each other, A145, A66 and A82 (kl-2~ or kl-4~) were tested, and A12, B119, and H39 (kl-5~) were tested. The re s u l t s are shown in Table 4. Males raised at 28°C and carrying 2 i d e n t i c a l mutated Y chromosomes were always s t e r i l e . 2w.tr Males that carried a mutated Y, and exther a Y or a Y mutated in a d i f f e r e n t f e r t i l i t y factor were usually, but not always, f e r t i l e . The s t e r i l i t y of some of the males may have resulted from mistaken c l a s s i f i c a t i o n of w^ /^Y* males as m4 w /Y*/Y* males or i t may have been due to the problem of genetic balance discussed below. In general, a l l Y* bearing males were f e r t i l e at 28 C when carrying an Am Y chromosome. A l l males were f e r t i l e when raised at RT. The most s i g n i f i c a n t information can be gained from 19 crosses that show complementation ( i . e . males that are o f e r t i l e at 28 C) since s t e r i l i t y may be an indicat i o n that the two mutants are a l l e l e s , or i t may be simply a r e f l e c t i o n of the weakness of that p a r t i c u l a r combination at 28°C. On the other hand, f e r t i l i t y at 28°C is a d e f i n i t e indication that the mutants are not i n the same gene. It can be seen (Table 4) that only the mutations assumed to be on k l - 5 + showed any complementation. In the other two groups, a l l crosses inter se yielded s t e r i l e males. However, f e r t i l i t y factor k l - 5 + appears to contain at le a s t 2 n o n - a l l e l i c s i t e s with mutant H39 i n one s i t e and mutants A12 and B119 i n the other. (e) Temperature-sensitive period of s t e r i l i t y . The results of the tests to determine the temperature-sensi t i v e period are shown in Table 5. Mutated stocks that gave s i m i l a r patterns in deletion mapping tests (see table 3), and thus were assumed to have mutations involving the same f e r t i l i t y factor, are grouped together. Figures 5 to 1.3 represent the mean number of progeny yielded per male at successive day intervals, one day p r i o r to, during, and after, the two day heat shock. In general, maximum f e r t i l i t y was reached by the t h i r d day of the experiment, which was the second day at 29°C. F e r t i l i t y then decreased r a p i d l y to almost complete s t e r i l i t y by the seventh and eighth days and the males were completely f e r t i l e again by the eleventh and twelfth days, although the number of progeny was lower than the y i e l d at the beginning 20 of the experiment. The Aiir-r control also showed maximum f e r t i l i t y on days 2 and 3 and i n succeeding days f e r t i l i t y dropped s l i g h t l y (Figure 1,3) . Kvelland (1965) using Canton-S males and employing mating methods si m i l a r to those outlined above, also observed that the average number of o f f s p r i n g produced per male per 24 hour period, was highest when the males were 3 days old. F e r t i l i t y then st e a d i l y decreased u n t i l the end of the experiment, when the 12 day old males produced approximately one t h i r d as many progeny as they had when 3 days o l d . Determining exactly when the temperature has i t s maximum s t e r i l i t y e f f e c t i s complicated by the design of the o experiment. The 28 C temperature shock was prolonged over a 48 hour i n t e r v a l . S t e r i l i t y could be induced by an e f f e c t of high temperature within a short i n t e r v a l within the 48 hours. Therefore, i f a stock was s t e r i l e during day 7 of the experiment, we can reason that the e f f e c t of the temperature i s on sperm recovered a minimum of 4 and a maximum of 6 days aft e r the temperature shock. For a l l 8 stocks, maximum s t e r i l i t y was noted either by day 7 or day 8 and usually during both days. The temperature thus had i t s major e f f e c t a minimum of 4 days and a maximum of 7 days aft e r the temperature shock. Reduced f e r t i l i t y was usually s t i l l evident as l a t e as day 9, and while there was some reduced f e r t i l i t y as early as day 5, the f e r t i l i t y of the test stocks was not s i g n i f i c a n t l y d i f f e r e n t from the f e r t i l i t y of the control u n t i l day 6. Thus temperature i s e f f e c t i v e a maximum of 3 to 8 days following the temperature shock. However, since the treatment was for 48 hours the proper way to examine the e f f e c t of the temperature i s to examine the cultures of 48 hour periods. The period of maximum s t e r i l i t y occurring on days 7 and 8 of the experiment i s 5 days aft e r the treatment with the high temperature on days 2 and 3. The s i g n i f i c a n t decreases i n f e r t i l i t y occurring on days 6 and 9 indicate that there i s also an e f f e c t 4 and 6 days aft e r the heat shock. (f) Determination of sperm m o t i l i t y at 28°C. Males carrying a ts Y chromosome and raised for t h e i r entire l i v e s at high temperature and which were thus s t e r i l e were found to have only non-motile sperm while Am t r males which were f e r t i l e at 28°C usually had motile sperm. Males having s i m i l a r genetic constitutions, but raised at RT usually had motile sperm. Any non-motility at RT was probably a r e s u l t of the preparation procedure since a small number of Am t r males also appeared to have non-motile sperm. The non-motility at 28°C was assumed to be related to the s t e r i l i t y of the • mutated stocks at that temperature. With the exception of mo t i l i t y , spermiogenesis appeared c y t o l o g i c a l l y normal. Individual sperm seemed to have elongated and separated from the sperm bundles in the same manner as motile sperm (see Figures _4.\and 15) . Males that had no Y chromosome and there-fore were s t e r i l e seemed to present a s l i g h t l y less normal pic t u r e . There were fewer sperm separated from the sperm bundles and there were fragments of sperm t a i l s present as though sperm di s i n t e g r a t i o n was occurring. Brosseau (196.0) also observed that spermiogenesis of x/0 males did not appear to proceed as far as did spermiogenesis in males that c a r r i e d at l e a s t part of the Y chromosome. Hess and Meyer (1967) suggested that i n X/0 males of Drosophila melanogaster, spermiogenesis does not go past stage 4 or 5 and the spermatids remain s y n c y t i a l l y connected. Present observations indicate that the ts s t e r i l e males d e f i n i t e l y produce spermatids that go past stage 5, as sy n c y t i a l connections have broken down. 23 DISCUSSION This study demonstrates that temperature-sensitive (ts) mutations which cause male s t e r i l i t y can be induced on the Y chromosome of Drosophila melanogaster by ethyl methanesulfonate. Strong circumstantial evidence that ts mutations represent point mutants supports Cooper's (1959) contention that heterochromatin of the Y chromosome i s not a fundamentally d i s t i n c t form of chromatin which i s only capable of mutation by gross chromosomal changes. The evidence suggesting that EMS-induced ts mutants i n D. melanogaster are p r i m a r i l y point mutants i s : 1) Among l e t h a l s induced by Y-rays and Mitomycin-C. (which are known to cause chromosome breakage and DNA degeneration) only 3-4% were ts whereas 11-12% of a l l l e t h a l s induced by EMS (which produces point mutations, s p e c i f i c a l l y missence mutations (Krieg, 196<|) were ts (Suzuki, _et _al„, 1967; B a i l l i e , Suzuki and Tarasoff, 1968). 2) Of 200 sex-linked recessive l e t h a l s induced by the chemical mutagen ICR-170 (which i s believed to cause frame-shift mutants) none were found to be temperature-se n s i t i v e (Carlson, _et al., 1967). 3) A l l of the chemically induced ts l e t h a l s have been mapped gen e t i c a l l y as points within single regions (Suzuki and Duck, 1967; Suzuki, unpublished). 24 4) In micro-organisms, temperature-sensitivity of an a l l e l e i s a good c r i t e r i o n for the missence nature of the mutant (Edgar, et a l . , 1967; Jockusch, 1966). Thus, i f ts male s t e r i l i t y mutations induced on the Y chromosome are indeed the consequence of single base changes, i t argues that heterochromatin of the Y chromosome and euchromatin are mutable i n a s i m i l a r manner. The ts s t e r i l i t y also suggests that, i n order to permit recovery of recessive mutants, unless the ts a l l e l e s recovered are, i n fact, dominant mutations, at l e a s t some l o c i on the Y are not highly redundant. The paucity of dominant ts l e t h a l s recovered on the X chromosomes and autosomes (Suzuki, et a l . , 1968; Holden and Suzuki, 1968; Suzuki and Procunier, 1969) argues against the p o s s i b i l i t y of dominants. The i s o l a t i o n of point mutations on the Y chromosome permits a genetic analysis of factors a f f e c t i n g male f e r t i l i t y . The demonstration of " f e r t i l i t y factors" by Neuhaus (1939) and Brosseau (19,6.0) was based on analyses of gross chromosomal aberrations (translocations and deletions) and therefore cannot shed l i g h t on the nature of single l o c i . Their manipulations of blocks of Y chromosome heterochromatin was suggestive of large genetic regions with duplicate functions, as possibly supported by recent studies of DNA/RNA hybridization i n D. hydei (Hennig, 1968). This i s not incompatible with our suggestion that there also e x i s t Y l o c i which are represented by few or even single copies. Several d i f f i c u l t i e s were encountered i n the i s o l a t i o n 25 and t e s t i n g of the ts Y s t e r i l i t y mutants. Some of them undoubtedly r e s u l t from a high temperature which i s very close to' that which s t e r i l i z e s most wild type stocks. More-over, neither the o r i g i n a l temperature-resistant s t r a i n selected nor the off s p r i n g of mutagenized males were at a l l isogenic, and t h i s may lead to inconsistant responses to the high temperature. Unfortunately, the standard stocks containing s p e c i f i c Y chromosomal deletions gave very e r r a t i c results i n tests of complementation. In some of the stocks, crossovers between the deleted Y and the normal Y can generate a complete Y chromosome (Lucchesi, personal communication); at RT several of the known deletion stocks are leaky (Brosseau,, . 196b; Williamson, personal communication; Lucchesi, 196 5) and although tests indicated that they are not leaky at 28°C there are other p e c u l i a r i t i e s at thi s temperature. In spite of these d i f f i c u l t i e s , the Y s t e r i l e s were mapped to some degree. The apparent i n s t a b i l i t y of the mutants (only 8 out of 34 Putative t s s t e r i l e s were confirmed as ts upon retesting) may be p a r t l y a t t r i b u t a b l e to gonadal mosaicism (Jenkins, 1967), but i t i s f e l t that t h i s i s not the major cause of the v a r i a b i l i t y . The loss of apparent ts mutations has been repeatedly observed for sex-linked and autosomal- recessive l e t h a l s i n Drosophila (Suzuki, et al„> 1967; B a i l l i e , Suzuki and Tarasoff, 1968) and i n Habrobracon (Smith, 1968). I t may be that the conditional functioning of missense proteins can be modified by subtle.changes i n background genotype which are 26 enough to ensure s u r v i v a l . In an extensive study of 13 ts mutations Hayashi (M.Sc. thesis) reported s t r i k i n g modifications of l e t h a l i t y upon outcrossing known ts l e t h a l s . Such s e n s i t i v i t y to genotypic changes may explain some of the re s u l t s obtained i n attempts to locate the mutants on the Y chromosome. Although tests for complementation between two d i f f e r e n t ts mutant Y chromosomes cannot be used to determine gene order, they are more precise than tests involving chromosomes containing deletions i n determining whether two mutants are true functional a l l e l e s . Complementation between two mutants which were both l o c a l i z e d to kl-5 strongly suggests that t h i s factor i s , in fact, composed of two n o n - a l l e l i c s i t e s instead of one as Brosseau (I960) concluded. This conclusion must be tempered by the p o s s i b i l i t y that kl-5 i s a c t u a l l y a sing l e s i t e in which i n t r a c i s t r o n i c complementation can occur. The apparent r e s t r i c t i o n of ts s t e r i l e s to Y L i s probably just chance d i s t r i b u t i o n within a small sample, as Brosseau (1961) discovered 13 out of 57 Y s t e r i l e s on Y , and Williamson (personal communication) has reported the recovery S of ts s t e r i l e s on Y . I t may, however, be a true indica t i o n T C of the r e l a t i v e s u s c e p t i b i l i t i e s of factors on Y and Y to temperature-sensitive mutation. Indeed, Edgar, ^ t jal., (1964) deduced that s i t e s capable of mutating to temperature-s e n s i t i v i t y i n T4 0 were only a small proportion of the t o t a l genome and furthermore that the s i t e s so revealed were not 27 uniformly d i s t r i b u t e d genetically. I t i s possible that t h i s may be the case in higher organisms, but extensive studies of ts l e t h a l mutations i n Drosophila do not indicate i t (Suzuki, unpublished). As well as permitting a precise genetic analysis of the Y chromosome the ts s t e r i l e s can provide information on gene function. An analysis of the temperature-sensitive period of the d i f f e r e n t ts s t e r i l e s can aid in determining the time and tissue i n which the Y chromosomal f e r t i l i t y genes are active. The following interpretations are predicted on the assumption that the TSP corresponds to the time of b i o l o g i c a l a c t i v i t y of the product of the ts locus. There are several d i f f e r e n t p o s s i b i l i t i e s that must be considered when tr y i n g to determine the s p e c i f i c e f f e c t of the genes involved. 1) Mutations are known which r e s u l t in abnormal mating behavior i n Drosophila; such a mutant i s f r u i t y , an autosomal recessive which is,expressed in homozygous males by a p r e f e r e n t i a l courting of other males, consequently insemination of a female occurs only r a r e l y ( G i l l , 1963). However, the production of non-motile sperm at 28°C and the observation that mating does occur at the r e s t r i c t i v e temperature, indicate that something other than a behavioral factor i s involved i n s t e r i l i t y of the ts Y mutations. 2) A more l i k e l y s i t e of action of the Y f e r t i l i t y genes than abnormal behavior, i s in some c e l l s of the t e s t i s because of the observations of non-motile and hence immature 28 sperm. The testes are c o i l e d elongated tubes approximately 2 mm. long and 100 microns in diameter (Lefevre and Jonsson, 1962). In the anterior end of the t e s t i s i s the apical complex containing the a p i c a l c e l l s and the spermatogonia which are destined to divide m i t o t i c a l l y and d i f f e r e n t i a t e into spermatocytes (Hannah-Alava, 1965). Spermatogonia divide in synchrony through 4 mitoses to give a cluster of 16 c e l l s . Meiosis then produces a bundle of 64 sperm of common o r i g i n embedded in a n u t r i t i v e c e l l . Maturing sperm pass into the seminal v e s i c l e s where they appear as a mass of densely packed in d i v i d u a l sperm no longer grouped in bundles. At i t s proximal end, each v e s i c l e tapers into a narrow tube that connects with the ejaculatory duct. At roughly the same l e v e l , the paragonia or accessory glands empty into the ejaculatory duct. The accessory glands secrete a viscous, granular substance containing a sex-specific ninhydrin-positive compound, (Chen and Diem, 1961, reported in Lefevre and Jonsson, 1962). The accessory gland plays an important role i n sperm transfer (Lefevre and Jonsson, 1962). Acton (1966) has shown the presence of filamentous structures in the lumen of the gland which he suggests aid in the transfer of sperm along the female reproductive t r a c t . Preliminary studies by Acton have shown that these structures are s t i l l present in the accessory glands of ts mutant males raised at 28°C. Rapoport (1962) has shown that d i methyl phosphate fed to D. melanogaster males r e s u l t s in motile but non functional sperm. He suggested that the p h y s i o l o g i c a l f a i l u r e of the sperm is due to blockage of one of the enzymes (usually phosphatases in other species) perhaps li b e r a t e d from the accessory glands and necessary for the f e r t i l i t y of the sperm. I t i s c e r t a i n l y conceivable that the Y chromosome may a f f e c t the accessory gland, but E M studies indicate that the Y must also be involved in sperm organization before the sperm come into contact with the paragonial f l u i d (Kiefer, 1966, 1969). At t h i s point a possible influence of the Y chromosome on paragonial f l u i d must be considered although there i s no evidence for such a r o l e . 3) Another p o s s i b i l i t y arises from Stern and Hadorn's observations (reported i n Cooper, 1959) that the n u t r i t i v e c e l l s degenerate as the sperm within them mature, and end up as mere nu c l e i adhering to the packets of mature spermatozoa. They suggested that the n u t r i t i v e c e l l s play a r o l e in sperm m o t i l i t y . There i s an actual fusion of the cytoplasm of the spermatids with the n u t r i t i v e c e l l s (Hess and Meyer, 1967) so i t i s possible that mRNA or proteins could be transferred from the n u t r i t i v e c e l l to the maturing spermatid. However, i t has been shown that the n u t r i t i v e c e l l s do not synthesize RNA ( O l i v i e r i and O l i v i e r i , 1966), an indi c a t i o n that the n u t r i t i v e c e l l s are not the p r i n c i p a l s i t e of Y chromosome function. However, i t i s possible that small amounts of a c t i v a t i n g or inducing substances are produced in the n u t r i t i v e c e l l s . 30 4) I t i s possible that the ts s t e r i l e s do, i n fact, act in.the germ c e l l s . Hannah-Alava (1968) has obtained a record of the time i n t e r v a l between any given germ c e l l stage and i t s recovery as a mature sperm i n an insemination. Thus, a determination of the i n t e r v a l between an exposure to high temperature and the onset of s t e r i l i t y may permit an estimation of the gonadal c e l l stage affected by high temperature. There has been considerable controversy concern-ing the methods used in, and the conclusions drawn from,, experiments designed to determine the spermatogenic time-scale (for a review of some of the problems involved see Hannah-Alava, 1965). On the basis of two i r r a d i a t i o n experiments involving an analysis of frequency d i s t r i b u t i o n of cross-overs recovered in d a i l y samples of sperm up to 8 days aft e r treatment, Hannah-Alava concluded that the 6-8 day broods were derived largely, i f not exclusively, from c e l l s that had been primary spermatocytes at the time of i r r a d i a t i o n . The fourth and f i f t h day broods were derived from c e l l s that were primary spermatocytes andi some that were spermatids at the time of treatment. There was considerable overlap in sampling of spermatids and spermatocytes since the time i n t e r v a l from metaphase I to an early spermatid stage i s only a few hours (Abro, 1964). The 1-3 day broods were derived from c e l l s that were sperm and spermatids at the time of i r r a d i a t i o n . On the basis of chromosome labeling studies, Chandley and Bateman (1962) and O l i v i e r i and O l i v i e r i (1965) concluded that the growth stage of the primary spermatocytes requires a period of about 4 days. The results of the tests to determine the temperature sens i t i v e period of the ts s t e r i l e s indicate that the maximum e f f e c t i s evident 5 days af t e r the treatment and there are s i g n i f i c a n t effects on the fourth and sixth days as w e l l . I t would thus appear that the temperature has i t s e f f e c t during the l a t t e r stages of the growth stage of the primary spermatocytes. High temperature speeds up the development time of growing f r u i t f l i e s (Tarasoff, M.Sc. thesis) and i t i s probable that i t also speeds up such processes as spermatogenesis. I t i s possible that the mutants may act u a l l y have an e f f e c t s l i g h t l y e a r l i e r than these studies indicate. These studies on the temperature-sensitive period of mutants on the Y chromosome support previous c y t o l o g i c a l , genetic, and biochemical observations on the functioning of the Y chromosome i n primary spermatocytes. O l i v i e r i and O l i v i e r i (1965) found that the spermatid nuclei were s y n t h e t i c a l l y inactive but RNS synthesis did occur in the spermatogonia and young spermatocytes; using RNA-DNA hybri d i z a t i o n techniques, Hennig (1968) showed that there was a s p e c i f i c non-ribosomal, Y chromosomal RNA produced i n the spermatocytes of D. hydei. Structural d i f f e r e n t i a t i o n of the Y chromosome has been observed in the primary spermatocytes of many Drosophila species (Meyer, Hess and Beerman, 1961; Hess and Meyer, 1967). In D. melanogaster, several Y chromosome structures are 32 evident i n primary spermatocytes, the most conspicuous being t u b u l i with a diameter of 300-400 A°, r e t i c u l a r elements with a diameter of 100-200 A° and basophilic granules with a diameter of 400-700 A° (Meyer, et a l . , 1961). These intranuclear formations of Drosophila spermatocytes have been homologized with the lampbrush chromosomes of amphibian oocytes (Beerman, Hess and Meyer, 1963). Various experiments indicate that the Y chromosome structures contain proteins and are involved i n DNA-dependant RNA synthesis (Hess and Meyer, 1967) and that about 50% of the t o t a l non-ribosomal RNA of the spermatocyte nucleus i s synthesized in the d i f f e r e n t Y structures (Hennig, 1968). In D. hydei the various loop forming s i t e s have been located on the Y by using d i f f e r e n t translocated Y fragments, and i t was found that a l l of the structures must be present to obtain normal spermiogenesis and f e r t i l i t y (Hess, 1967). In addition, point mutants have been found that a l t e r the shape of cert a i n of the structures (Hess and Meyer, 1967). The experiments with the ts Y s t e r i l i t y mutations point to a TSP coinciding with the formation of primary spermatocytes, and i t i s known that normal spermiogenesis i s dependant upon the appearance of intranuclear structures associated with the Y chromosome i n the primary spermatocytes, and also associated with a Y s p e c i f i c RNA produced at the same time. I t i s concluded, therefore, that the most l i k e l y s i t e of function of the ts s t e r i l e s i s , i n fact, the primary spermatocytes. The exact function of the f e r t i l i t y genes i s uncertain, 33 but they do not appear to code for the major s t r u c t u r a l components of the sperm. Kiefer (1966) observed that non-motile sperm are produced by X/0 males and they do contain the major components of the sperm t a i l (the a x i a l filament complex, and the Nebenkern d e r i v a t i v e ) ; however the Nebenkern derivative f a i l s to develop properly and the a x i a l filament complex does not become properly organized. K i e f e r (1969) and Meyer (1969) concluded that Y chromosomal factors are involved i n the coordination of the synthetic and morphogenetic processes leading to the formation of functional sperm, though these factors do not themselves contribute information on the molecular l e v e l to the sperm structure. I t i s not possible at t h i s time to propose p r e c i s e l y how the ts s t e r i l e s might a l t e r these morphogenetic processes, i f i n f a c t they do so. The use of ts mutations of the Y chromosome should aid i n elucidating the nature o f ; i t s functions. Although DNA-RNA hybridization studies remain controversial and crude, Hennig's (1968) demonstration of t e s t i s - s p e c i f i c Y chromosome messenger UNA lends support to the use of annealing studies of the ts mutants. A number of experiments u t i l i z i n g competition between RNA's of d i f f e r e n t ts stocks could reveal relationships between times of t r a n s c r i p t i o n of d i f f e r e n t l o c i . Association of ts s t e r i l i t y with a s p e c i f i c s t r u c t u r a l malformation i n primary spermatocytes would provide very convincing proof of the functions of the Y locus. Dr. Barry Kiefer i s now carrying out analyses of the ts s t e r i l e s 34 with the electron microscope to determine whether there are any u l t r a s t r u c t u r a l abnormalities i n developing sperm under either the permissive or r e s t r i c t i v e conditions. The induction of ts mutations i n Y heterochromatin, which appears to contain many duplicated sequences (Hennig, 1968; Kiefer, 1969) may encourage other workers to investigate other segments of heterochromatin that are also suggested to be redundant (Berendes, 1967). 35 SUMMARY EMS induced temperature-sensitive s t e r i l e mutations were recovered in the heterochromatic Y chromosome of Drosophila melanogaster. A l l eight of the mutants were mapped on the long arm.of the Y chromosome. The recovery of point mutants (EMS induced ts mutants are presumed to be point mutants) indicates that there are l o c i on the Y (and hence i n heterochromatin) which are represented by single copies. The temperature-sensitive periods (TSP) of the mutants were determined. The res u l t s support the suggestion that the Y chromosomal f e r t i l i t y factors', function in the growth stage of the primary spermatocytes. Table 1. Relative f e r t i l i t y of 10 d i f f e r e n t wild type stocks at 22 C and at 29 C. * Number of progeny produced from 25 males and 25 females which were allowed to lay eggs for 2 days. ** Urbana S produced no progeny at 29 C as a l l of f s p r i n g died i n l a t e t h i r d instar or early pupal stages. 36 S t o c k s t e s t e d 22 C * F 1 a t 29 C* P 2 flt 2 9 - C * Amherst ] Q6'7 Canton C D-6 Lausanne 8 Samarkand Samarkand 204 S w e d i s h C T e n n e p s e e 1966 Urbena S 436 309 310 337 42 k 670 344 370 490 325 225 108 I83 19 8 275 226 237 0** 10 0 0 0 Table 2. The re s u l t s of tests to l o c a l i z e ts Y s t e r i l i t y S L mutants on Y or Y . * number of progeny per b o t t l e . 37 t s Y s t e r i l e stock X-YS* X«Y L* Al? o 97 A66 0 115 AT kl 0 101 A 1 4 5 0 88 B 1 1 9 0 67 E91 o 57 H39 0 89 A m t r ( c o n t r o l ) 126 1 5 8 Table '.3;;- Results of complementation tests at 28 C between ts Y s t e r i l e chromosomes and Y chromosomes deletedulfor 1 or more known,:fertility factors. + indicates f e r t i l i t y at 28 C hence complementation. - indicates s t e r i l i t y at 28 C hence no complementation. 38 ts Y sterile Y chromosomes deleted for stock various f e r t i l i t y factors k l - l " kl-2~ k l - 3 " kl-V-k- k l - 5 ~ ks-l~ ks - 2 " A12 + - + + + A66 + - + + + + A82 + - + - + + + A141 + + + + Al>5 + - + + + + B119 + - + - -E91 + - + + + H39 + - + + + A m t r (control) + - + + +. + + Table h. Results of complementation:: tests between 2 different tts Y sterile chromosomes at 28 C. + indicates f e r t i l i t y at 28 C hence complementation. - indicates s t e r i l i t y at 28 C hence no complementation. Mutants mopping on k l - 1 9 6* E 9 1 A l 4 l E91 - --M u t a n t s nwnpinpr on k l - 2 or k l - 4 $ 3 £ A66 A.82 A145 A66 - - -A82 - - -A145 - - -Mutants mapping on k l-5 A12 B119 H39 A l ? - - + E119 - - + H 3 ° + -Table 5 . Temperature-sensitive period of s t e r i l i t y . t s Y s t e r i l e F e r t i l i t y Mean number of progeny/male/day stock f a c t o r Day 1 2 3 4 5 6 7 8 9 10 11 12 A141 68 92 59 32 22 9 3 1 5 28 36 30 k l - 1 " E91 80 73 71 46 34 32 14 4 23 40 44 58 A66 81 79 68 55 46 26 2 1 11 19 24 34 k l - 2 ~ A82 or 79 86 67 59 31 14 14 8 27 43 45 39 k l - 4 A145 65 85 71 48 21 1 0 0 1 9 26 31 A12 52 65 54 51 37 17 0 1 7 36 36 34 B119 k l - 5 ~ 75 74 98 85 15 6 5 6 16 33 34 37 H39 67 81 48 47 30 10 1 0 4 30 47 43 Am t r complete 48 71 68 45 42 57 46 40 51 48 51 46 Figure 1. Screening protocol for the detection of temperature-sensitive mutations on the Y chromosome. 41 Step 1. +/Y < W (0.025 EMS) X +/+ +/Y* dVf X +/+ 4?? Rt for 4 days then transfer parents to a fresh v i a l RT F i progeny transfer parents to fresh v i a l s then discard after 4 days Fi progeny score for presence of F 2 progeny 28° C score for absence of F 2 progeny repeat steps 3 and 4 gure 2. Protocol for the l o c a l i z a t i o n of ts s t e r i l on Y1* or Y S. Step 1. X»YL/Y or " X°YS/Y X XX/Y* RT for 4 days then transfer parents to a fresh v i a l RT X°YL/Y* or, £6 X-Y7Y* X +/+ X X •YVY* or. X"Y°/Y* id 28°C score for presence of progeny score for absence of progeny Figure 3. Protocol for complementation tests between ts Y s t e r i l e s and Y chromosomes deleted for one or more known f e r t i l i t y f a c t o r s. 43 Step 1. 4 8 sc sc /Y* <?<? Rt for 4 days then transfer parents to a fresh v i a l 2. y/y+YK /Y* X y/y x y / y ^ ' / Y * RT 28°C 3. score for presence of progeny score for absence of progeny Figure 4. Protocol for complementation tests between d i f f e r e n t ts s t e r i l e Y chromosomes. s c 4 sc 8/B SYy+ 44 v y/y y / s c 4 sc 8/B sYy + iri4 _ w /Y <f «f y/w m 4/B sYy + and s c 4 sc 8/w m 4/BSYy+ s c 4 s^/Y* 1 <T<T v w m4/w xn4/B sYy + o$ s c 4 s c 8 / Y * 2 V w /w /Y*"1-RT for 4 days then transfer parents to a fresh v i a l v ^ A ^ A * 2 ^ x y/y x wm4/Y**/Y*2 rf* RT 28° C score for presence of progeny score for absence of progeny Figure 5. Daily f e r t i l i t y of ts Y s t e r i l e stock A12 following a 2 day heat shock at 29°C. Days 1 and 4-12 are at RT, days 2-3 at 29° C. # indicates mean of 10 males and the 5% confidence l i m i t s . V M E A N NO. OF P R O G E N Y / M A L E - A W O J ^ O l t D s l O D l D O - 4 1 ^ o o o o o o o o o o o o - i 1 T i 1 r i i n 1 1 1 Figure 6. Daily f e r t i l i t y of ts s t e r i l e stock B119 following a 2 day heat shock at 29°C. Days 1 and 4-12 are at RT days 2-3 at 29°C. indicates mean of 10 males and the 5% confidence l i m i t s . M E A N NO. O F P R O G E N Y / M A L E O O O O O O O O O O O o , . , 1 j r i 1 1 1 1 1 1 Figure 7. Daily f e r t i l i t y of ts s t e r i l e stock H39 following a 2 day heat shock at 29°C. Days 1 and 4-12 are at RT, days 2-3 at 29°C. indicates mean of 10 males and the 5% confidence l i m i t s . 4? L J 10 8 9 10 11 12 DAYS Figure 8. Daily f e r t i l i t y of ts s t e r i l e stock A141 following a 2 day heat shock at 29°C. Days 1 and 4-12 are at RT, days 2-3 at 29°C. indicates mean of 10 males and the 5% confidence l i m i t s . 48 120 r LU HO -< 100 -LU 10 5 6 7 8 9 10 11 12 DAYS Figure 9. Daily f e r t i l i t y of ts s t e r i l e stock E91 following a 2 day heat shock at 29°C. Days 1 and 4-12 are at RT, days 2-3 at 29°C. indicates mean of 10 males and the 5% confidence l i m i t s . 49 7 8 9 10 11 12 DAYS Figure 10. Daily f e r t i l i t y of ts s t e r i l e stock A66 following a 2 day heat shock at 29°C. Days 1 and 4-12 are at RT, days 2-3 at 29°C. indicates mean of 10 males and the 5% confidence l i m i t s . 1 2 3 4 5 6 7 B 9 10 11 12 DAYS Figure 11. Daily f e r t i l i t y of ts s t e r i l e stock A82 following a 2 day heat shock at 29°C. Days 1 and 4-12 are at RT, days 2-3 at 29°C. indicates mean of 10 males and the 5% confidence l i m i t s . 51 D A Y S Figure 12. Daily f e r t i l i t y of ts s t e r i l e stock A145 following a 2 day heat shock at 29°C. Days 1 and 4-12 are at RT, days 2-3 at 29°C. indicates mean of 10 males and the 5% confidence l i m i t s . 52 1 2 0 r L U 1 1 0 -_J < 1 0 0 -_> 9 0 ->-__: 8 0 -L U 7 0 -O O 6 0 -o_ £ L 5 0 -LL 4 0 -O O 3 0 -z z 2 0 -< L U 10 -Figure 13. Daily f e r t i l i t y of Amherst control stock following a 2 day heat shock at 29°C. Days 1 and 4-12 are at RT, days 2-3 at 29°C. indicates mean of 10 males and the 5% confidence l i m i t s . 53 120 r LU 110 -< 100 ^ 9 0 -z 8 0 -o Q_ LL O 4 0 -0 z 6 0 -5 0 -30 Y 2 0 UJ 10 1 2 3 4 5 6 7 8 9 10 11 12 D A Y S Figure 14. Phase-contrast photograph of non-motile sperm of a ts Y s t e r i l e male raised at 2 9 ° C Objective lens 40 power. Magnification approximately 1300 times. Figure 15. Phase-contrast photograph of motile sperm of a ts Y s t e r i l e male raised at RT. Objective lens 40 power. Magnification approximately 1300 times. 55 BIBLIOGRAPHY Abro, A., 1964. Cytological observations on spermiogenesis in Drosophila melanogaster. Arbok Univ. Bergen, Mat. Nat. Ser. 1964, 1-12. Acton, A.B., 1966. An unusual ciliumlike process. J. Cell B i o l . 29; 366-369. B a i l l i e , D., D.T. Suzuki, and M. Tarasoff, 1968. Temperature-sensitive mutations in Drosophila melanogaster. II. Frequency among second chromosome recessive lethals induced by ethyl methanesulfonate. Can. J. 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A thes submitted in p a r t i a l f u l f i l m e n t of the requirements for the degree of Master of Science in the Department of Zoology, the University of B r i t i s h Columbia. Williamson, J.H., 1968. The induction of s t e r i l e Y chromosomes i n Drosophila melanogaster with et h y l -methane sulphonate. Genetics 60: 238. 

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