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UBC Theses and Dissertations

Effector mechanisms in the endocrine control of steroidogenesis Rodway, Marie R.


Production of hormones in the ovary is controlled by endocrine, paracrine, autocrine and intracrine influences. Similar controls may exist in the placenta. I wished to investigate the involvement of second messengers in the action of hormones in control of hormonogenesis in rat ovary and human placenta. The second messengers involved in the action of gonadotropin-releasing hormone (GnRH) and prostaglandin (PG) F₂[formula omitted] were investigated in rat granulosa and luteal cells. As well, the endocrine role of GnRH in the placenta and the possible second messengers involved were investigated. Monolayer cultures of rat granulosa and luteal cells and human placental cells were prepared. Rat granulosa cells were mechanically dispersed; rat luteal cells were enzymatically dispersed with collagenase and DNase. Rat granulosa cells were treated during the first 24 hours in culture; rat luteal cells were treated up to 3 days after dispersion. Radioimmunoassay of medium was used to determine the effect of treatments on hormone production. Studies which examined the effect of hormones on the intracellular free calcium concentration ([Ca²⁺]i) in single cells using the calcium sensitive fluorescent dye, Fura-2, were done in monolayer rat granulosa and luteal cell cultures. Human placental cells, from first trimester and term placentae, were dispersed using trypsin-DNase or collagenase-DNase. Cells were cultured for 2 days prior to treatment. The effects of treatments on production of steroid (progesterone and estrogen), glycoprotein (human chorionic gonadotropin; hCG) and protein (human placental lactogen; hPL) hormones were determined by radioimmunoassay of the medium. In rat granulosa and luteal cell cultures, I examined the effect of a number of hormones and second messengers. Effects of follicle-stimulating hormone (FSH), luteinizing hormone (LH), cyclic adenosine monophosphate (cAMP), GnRH and PGF₂[formula omitted] on ovarian hormonogenesis have been previously reported. Changes in cytosolic free calcium concentrations ([Ca²⁺]i) in response to PGF₂[formula omitted] were measured in single rat granulosa and luteal cells. I found that in 34% of granulosa cells, and 53% of luteal cells, there was a 3 to 4 fold increase in resting [Ca²⁺]i within 30 seconds of administration of PGF₂[formula omitted]. Many cells which responded to PGF₂[formula omitted] also responded to GnRH (39% of granulosa cells; 67% of luteal cells). The immediate source of the increased [Ca²⁺]i appeared to be common intracellular stores. No change in hormone production in response to GnRH in placental cell cultures was seen. Trypsin dispersion may have damaged cell surface receptors, therefore the effect of second messengers on hormone production in these cultures was examined. In term and first trimester trophoblast cultures, I observed the following effects with 8-bromo-cyclic adenosine monophosphate (8-br-cAMP): inhibited estrogen production from the supplied androgen precursors; stimulated hCG production; stimulated hPL production in first trimester placental cell cultures (hPL was not measured in enough term cultures to determine the effect of 8-br-cAMP), and stimulated progesterone production. I also investigated the effects of activators and inhibitors of the phosphoinositide (PtdIns(4,5)P₂) breakdown second messenger pathway (TPA, A23187, arachidonic acid); no effects of these agents were seen. Other hormones suspected of having endocrine, paracrine or autocrine effects in the placenta were tested without effect. I conclude that GnRH and PGF₂[formula omitted] cause increases in [Ca²]i in rat ovarian cells, from common intracellular stores of calcium, and that the production of hormones by the human placenta may be under regulation of an agent or agents which induce production of cAMP.

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