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The relative effectiveness of exogenous testosterone and 5n-dihydrotestosterone (DHT) in causing uterine growth in intact vs ovariectomized immature rats has been investigated to determine the extent to which the uterotrophic responses to these androgens depends upon their aromatization in the ovaries.Ovariectomy depressed the abilityof testosteroneto a much greaterextent than that of DHT to induce uterine growth, especiallyhypertrophy of the uterineluminal epithelial cells. This ability was not furtherdecreased by adrenalectomy. Exogenous estradiol-17(3 administration was aseffectiveinstimulatinguterinegrowth and increased luminal epithelial cellheightin ovariectomized rats treated with DHT as in those treated with testosterone, indicating that the decreased effectiveness of DHT to induce these changes was not the result of greater anti-estrogenic activity of DHT than of testosterone. Ovarian contents of immunoreactive estradiol-1 713increased markedly, and approximately in parallel with increased ovarian contents of immunoreactive testosterone + DHT 6 and 12 h after s.c. injection of testosterone;by 24 h, ovarian contents of both steroids declined approximately to pre-injection levels. In contrast, s.c. injection of DHT was followed by significantly decreased ovarian contents of estradiol-1 7(3 at all 3 time intervals. It is concluded that increased ovarian formation of estradiol-1 7j3 occurs when ovarian contents of testosterone are elevated by administration of high dosages of exogenous testosterone to immature rats, and that this estrogen contributes significantly to the uterine growth and histologic changes which follow testosterone administration. The lack of such changes as a result of 01-IT administration attests to the inability of this androgen to undergo aromatization in vivo; the decreased ovarian content of estradiol-1713 following DHT administration suggests that this steroid, in addition to being ineffectual as an estrogen precursor, may actually play a role in inhibition of ovarian estrogen biosynthesis.

An intra-ovarian role for oestrogens in the control of steroid production was investigated using dispersed thecal cells obtained from porcine follicles. Thecal cells were incubated for 14 h at 37 degrees C and the media subsequently assayed for androstenedione, progesterone and cyclic AMP. LH caused a dose-dependent stimulation of both steroids and the addition of oestradiol at doses of 10 ng-10 micrograms/ml significantly (P less than 0.01) inhibited both basal and LH-stimulated steroid production from doses of 500 ng/ml and upwards. Of other oestrogens investigated, oestrone and oestriol were somewhat less potent than oestradiol in inhibiting steroid synthesis, whereas the synthetic oestrogen diethylstilbestrol (DES) was more potent. The presence of oestradiol at doses of 10 ng-10 micrograms/ml had no significant effect (P less than 0.05) on either basal or LH-stimulated cAMP suggesting that the oestradiol inhibition does not involve inhibition of LH receptor-linked adenylate cyclase. These results demonstrate that physiological doses of oestrogen can act by local negative feedback to control the synthesis of its own precursor and thus regulate intrafollicular steroidogenesis.

The biosynthesis of non-aromatic 19-norsteroids has been studied using primary cultures of porcine granulosa cells. Formation of 5(10)-estrene-3 beta,17 beta-diol, a novel 19-norsteroid, from androstenedione and 19-hydroxyandrostenedione by porcine granulosa cells is reported for the first time. The structure was deduced from (i) comparison of its elution times on C18 reverse phase HPLC with authentic 5(10)-estrene-3 beta,17 beta-diol (ii) identification with 5(10)-estrene-3 beta,17 beta-diol-diacetate after acetylation (iii) oxidation/acid catalysed isomerization to 19-norandrostenedione. Serum or serum plus FSH significantly stimulated (seven fold increase) formation of 5(10)-estrene-3 beta,17 beta-diol from androstenedione and 19-hydroxyandrostenedione. Formation of 5(10)-estrene-3 beta,17 beta-diol from both substrates was significantly (p less than 0.01) reduced by the aromatase inhibitors 4-hydroxyandrostenedione (15 microM) and aminoglutethimide phosphate (10(-4)M). These results suggest that 5(10)-estrene-3 beta,17 beta-diol (and 19-norandrostenedione) may be formed by enzymes similar to the aromatase complex required for estradiol-17 beta biosynthesis. 5(10)-Estrene-3 beta,17 beta-diol is converted by granulosa cells to four metabolites. 19-Norandrostenedione was identified by crystallization to constant specific activity; 19-nortestosterone is a minor product. Production of 19-norandrostenedione and 19-nortestosterone indicates that granulosa cells possess the enzymes necessary for the transformation of 5(10)-estrene-3 beta,17 beta-diol and other 3-hydroxy-5(10)-estrenes to 19-nor-4-ene-3-ketosteroids. The formation of 5(10)-estrene-3 beta,17 beta-diol and 19-norandrostenedione as substantial metabolites of androstenedione suggest a physiological role for these 19-norsteroids in ovarian follicular development.

Current understanding of the physiology of human lactation is derived largely from principles drawn from animal experiments, tissue explant data and acute human studies of varying physiological states. Extrapolation of controlling mechanisms from these sources must be viewed with some caution. As plasma profiles of hormones and other appropriate compounds can be artificially influenced by factors unrelated to mammary gland lactogenesis, analysis of breast fluid and milk might serve to identify prior synthetic events.

Thecal preparations from medium-sized procine ovarian follicles (3.5-5 mm diameter) were incubated for 4 h in a chemically defined medium in the presence or absence of highly purified luteinizing hormone (LH) and/or estradiol-17 beta (estradiol). LH (1 microgram/ml) stimulated the thecal production of testosterone (T) and dihydrotestosterone (DHT) by 2- to 3-fold. Although estradiol (10 microgram/ml) alone had only a slight but non-significant inhibitory effect on basal testosterone production, it significantly inhibited the production of both T and DHT as well as decreasing the DHT/T ratio in a dose-related manner in the presence of LH. Production of cyclic adenosine 3',5'-monophosphate (cAMP) and progesterone by the thecal cells was stimulated 50-to 200-fold and 2.5-fold, respectively, by LH. Estradiol had no significant effect on thecal cAMP and progesterone production in the presence or absence of the gonadotropin. These findings are consistent with the concept that estradiol produced by granulosa cells following hormonal stimulation may serve as a local negative feedback mechanism to control thecal androgen production.

Thecal and granulosa cells isolated from human ovarian follicles were incubated separately for various time intervals (0, 0.5, 1, and 2 h) in the presence or absence of hCG or highly purified FSH. At the end of the incubation period, absolute ethanol was added to the culture tubes and androgen and cAMP production were determined by RIA and protein-binding assay, respectively. In the absence of gonadotropin, human thecal cells produced androgen and cAMP in vitro. While FSH (0.01-10 μg/ml) failed to exert any significant effect on this androgen and cAMP production during a 2-h incubation, synthesis of androgen (2- to 5-fold) and the nucleotide (5- to 50-fold) was markedly increased by hCG (1 IU/ml). These responses of the theca to the gonadotropin were dose dependent, and as little as 0.1 IU hCG/ml medium was effective in enhancing androgen production by 2- to 3-fold. A temporal relationship between the hCG-stimulated androgen and cAMP synthesis seems to exist, with significant increases in the production o...

Rat oocytes from preantral follicles have been shown to grow and acquire meiotic competence in vitro. Follicles were isolated by enzymatic digestion of ovaries from infant (10- or 11-day-old) Wistar rats. Follicles were cultured for up to 20 days in Minimum Essential Medium (MEM) supplemented with 2 mM hypoxanthine to maintain meiotic arrest. When cultures were begun, oocytes were in midgrowth phase (40-45 microns diameter), and were incapable of undergoing meiotic maturation when placed in hypoxanthine-free MEM. Oocytes grew and acquired meiotic competence during culture for 20 days attaining mean diameters of 62.6 +/- 0.6 microns and 61.1 +/- 0.6 microns in two experiments. Germinal vesicle breakdown (GVB) occurred in 60-70% of oocytes when transferred to MEM without hypoxanthine. Concomitant with oocyte growth and maturation were spontaneous increases in follicular production of progestins, androgens and estrogens. When oocytes grown and matured in this system were inseminated in vitro with epididymal sperm, 36.1% and 25.8% were penetrated by one or more sperm in two experiments. However, fertilization was not generally normal with multiple penetrations and abnormal numbers of pronuclei (one or three) being common, suggesting that in these oocytes cytoplasmic maturation was incomplete or abnormal. In the two experiments, normal fertilization (two pronuclei and one sperm tail in the vitellus) occurred in 34.6% and 47.1% of penetrated oocytes with development of these apparently normal zygotes to two-cell embryos being 66.7% and 62.5%, respectively.

Experiments were conducted to determine the relative importance of the decline in circulating estrogen levels, as opposed to the increase in progesterone levels, in the hormonal control of the loss of luminal fluid from the uterus of the rat, an event which usually occurs early on the morning of estrus. Silastic capsules containing crystalline estradiol-17beta (E2) were implanted subcutaneously in ovariectomized immature rats. Approximately 64 h later, the capsules were either removed (E2 withdrawal) to mimic the fall in peripheral estrogen levels occurring at proestrus, or left in place (continuous E2), and, at the same time, progesterone or oil was administered. Fifteen hours later, irrespective of whether E2 was withdrawn or not, 2 mg progesterone reduced uterine luminal fluid accumulation to levels usually seen in intact animals on the morning of estrus; however, the loss of fluid occurred slightly sooner if E2 was withdrawn when progesterone was administered. By itself, E2 withdrawal resulted in only a small decrease in uterine luminal fluid by 15 h, even though serum E2 levels had fallen to less than 1.5 pg/ml by this time. This loss was brought about by escape of the fluid through the cervix rather than by its reabsorption from the lumen. The dose-response relationships between progesterone and uterine luminal fluid accumulation indicated that when E2 was withdrawn, a smaller amount of progesterone brought about the loss of uterine luminal fluid accumulation than when E2 was continuous. These results suggest that increased progesterone secretion on the afternoon of proestrus is essential for the loss of uterine luminal fluid, and that the decline in estrogen secretion at this time may be of importance by allowing the progesterone to be more effective.

The female reproductive system is exposed to a great variety of environmental stresses. These include many noxious chemicals consumed either intentionally in the form of therapeutic and recreational drugs, or unwittingly as residues in the food we eat or pollutants in the air we breathe. These stresses and noxious agents influence ovarian function through actions at a number of sites and by diverse mechanisms. Sites of action include: the hypothalamo-hypophyseal system, resulting in disruption of the normal pattern of gonadotropin secretion; the ovary, resulting in direct destruction of the oocyte (ovotoxicity) or genetic damage (mutagenicity); and other organs, leading indirectly to altered ovarian function, e.g., through metabolic alterations that change the balance of feedback control of the hypothalamo-pituitary-ovarian system. Susceptibility of the ovaries to the different classes of agents depends on the stage of development at which exposure occurs. Consequences may be temporary and reversible when the source of the "stress" is removed, or permanent if exposure occurs at a "critical stage" in ovarian or hypothalamic differentiation.

Granulosa cells have been isolated from ovaries of estrogen-treated immature intact and hypophysectomized rats, and have been maintained in culture in a chemically-defined medium. Progesterone secretion by these cells was stimulated by the addition of FSH to the medium. In the presence of 0.5 uM testosterone or 17β-OH-5α-androstan-3-one (DKT), progesterone secretion was low or undetectable. However, the addition of testosterone or DHT together with FSH caused a dramatic 8- to 19-fold increase over that caused by FSH alone. On the other hand, luteinizing hormone (LH) alone had no effect on progesterone secretion, but produced a small stimulation when added together with testosterone. These results demonstrate synergism between androgens and FSH in the control of progesterone secretion by granulosa cells in culture.

Goats in Group A were pretreated for 9 days with a synthetic progestagen, administered via intravaginal sponge, and 1000 i.u. PMSG s.c. on Day 12 of the oestrous cycle. Goats in Group B had the same PMSG treatment, but not the progestagen pretreatment. Group C goats received a s.c. twice daily injection of a porcine FSH preparation (8 mg on Day 12, 4 mg Day 13, 2 mg Day 14 and 1 mg Day 15). Oestrus was synchronized in all animals by 50 micrograms cloprostenol, 2 days after the start of gonadotrophin treatment. The vaginal progestagen sponges were removed from Group A at the same time. Mean ovulation rate was slightly higher in FSH-treated than in the PMSG-treated animals, whereas the incidence of large follicles that failed to ovulate was significantly elevated in PMSG-treated animals in Group B. More goats in Groups A and B than in Group C exhibited premature luteal failure. Progestagen pretreatment appeared to suppress both follicular and luteal activity, as indicated by numbers of large non-ovulating follicles and by the magnitude and duration of elevated plasma oestradiol levels following PMSG stimulation, and by decreased plasma progesterone levels before and after PMSG treatment. Oestrogenic response to FSH was considerably less than that to PMSG, as indicated both by a considerably shorter duration of elevation of circulating oestradiol levels during the peri-ovulatory period, and by lower maximal oestradiol levels. Differences in the ovarian responses to PMSG and FSH may be attributed primarily to differences in the biological half-life of each preparation.

Rates of 3α-, 5α- and 20α-reduction of progesterone have been measured in homogenates of rat uterus at varying stages of gestation. The activity of 5α-reductase was low at days 14-16 and increased 2.5-fold before the end of pregnancy. The activity of 20α-hydroxysteroid dehydrogenase increased 3.5-fold during the latter half of gestation. Removal of endometrium by scraping indicated this increase was limited to the endometrium.(Endocrinology92: 624, 1973)

Cycling rats hypophysectomized during metestrus or diestrus and injected with 5 μg of ovine NIH-LH in saline twice daily for 4 days developed cornified vaginal smears. Bovine LH, 5 μg twice daily for 4 days, did not stimulate vaginal cornification although histological sections of this tissue showed stratified epithelia. Both treatments caused increased uterine weights. Bovine LH, 50 μg daily for 4 days administered in a depot vehicle (95 % sesame oil: 5 % beeswax), stimulated cornification of the vagina shown by vaginal smears and confirmed by subsequent histological sections. Cycling rats hypophysectomized and pituitary-autotransplanted during metestrus exhibited evidence of progesterone secretion and, when injected with 50 μg bovine NIH-LH daily for 4 days, had significantly (p

The effects of estrone (lμg/day) and prolactin (200 μg/day), alone and in combination, on deciduomal growth in response to uterine trauma and on uptake and metabolism of pi'ogesterone by nontraumatized uteri have been investigated in ovariectomized, progesteronetreated (2 mg/day) rats. Estrone significantly increased the size of traumatized uteri, whether administered during the 3-day period of uterine sensitization, immediately before traumatization, or during the 3-day period of deciduomal growth, immediately after traumatization. Prolactin significantly increased the size of traumatized uteri in the absence of estrone, but, when administered in the presence of estrone, significantly decreased the stimulatory effect normally produced by estrone. Both estrone and estradiol increased the uptake of progesterone by isolated uterine strips, but these increases could be accounted for entirely on the basis of the uterotrophic effects of the steroids and were not evident when expressed per mg uterine weight. Ne...

Traumatization of uteri of rats with ovaries luteinized by the PMS-HCG priming procedure of Parlow fails to elicit formation of deciduomata when performed 2, 3, 4, 5, 6 or 7 days after HCG treatment. Since progesterone synthesis and secretion by such ovaries has been demonstrated, it is concluded that the lack of deciduomal response is due to secretion of excessive amounts of an inhibitor of this response. Neither prolactin nor luteinizing hormone overcomes the failure of the deciduomal response. Evidence of increased estrogen secretion (uterine enlargement and edema, increased stratification and mucification of vaginal epithelium) follows treatment of PMS-HCG primed rats with luteinizing hormone. Equine LH produces such effects whether administered in an aqueous vehicle twice daily, once daily, or once at the beginning of a 5-day period. Ovine LH is effective if administered in an aqueous vehicle twice daily, but not if administered less frequently. Bovine LH is ineffective even when given twice daily in...

1. The specific radioactivities of non-esterified and esterified cholesterol, progesterone and 20alpha-hydroxypregn-4-en-3-one were determined in slices of superovulated rat ovary after incubation with [1-(14)C]acetate in vitro for various times. The specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal, and (during the fourth hour of incubation) exceeded those of the non-esterified cholesterol and the esterified cholesterol by factors of 2.8 and 7.6 respectively. 2. After separation of homogenates of superovulated rat ovary slices previously incubated with [(14)C]acetate into subcellular fractions by differential centrifugation, the specific radioactivities of non-esterified cholesterol in the cytosol, mitochondria, lipid-containing storage granules and microsomal fraction were 1220, 1510, 1420 and 4020d.p.m./mumol respectively; the corresponding values for the specific radioactivity of the esterified cholesterol were 600, 700, 730 and 760d.p.m./mumol. The specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal in all fractions; the corresponding mean specific radioactivity of progesterone+20alpha-hydroxypregn-4-en-3-one was 6150d.p.m./mumol. 3. By using glutamate dehydrogenase and cytochrome (a+a(3)) as mitochondrial markers, the presence of cholesterol side-chain cleavage enzyme was demonstrated in microsomal fraction free of mitochondrial contamination. 4. The specific radioactivities of ovarian non-esterified and esterified cholesterol, progesterone and 20alpha-hydroxypregn-4-en-3-one were determined up to 8h after the intravenous injection of [4-(14)C]cholesterol into superovulated rats. At all times the specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal to the specific radioactivity of non-esterified cholesterol and exceeded, by up to 3.3-fold, that of the esterified cholesterol. 5. It is concluded that non-esterified cholesterol formed from [(14)C]acetate in the endoplasmic reticulum equilibrates slowly with non-esterified cholesterol in other subcellular fractions, and is preferentially converted into steroids. Such a mechanism presupposes the operation of a microsomal cholesterol side-chain cleavage enzyme using non-esterified cholesterol as its substrate. Unrelated evidence is presented in support of the existence of such an enzyme. The results are discussed in the light of other biochemical and electron-microscopic findings relating to the compartmentation of cholesterol in steroidogenic tissues.

Ovarian tissue cholesterol, in rats with luteinized ovaries, has been labeled by intravenous injection of cholesterol-7-3H. The effect of luteinizing hormone (LH) in vivo and in vitro on the incorporation of cholesterol-7-3H into progesterone and 20α-hydroxypregn-4-en- 3-one in these ovaries has been observed. Increased progestin synthesis in vitro resulted from iv administration of LH to rats 30 min before removing ovaries for incubation, as well as from direct addition of LH to the incubation medium. A corresponding increase was found in circulating progestins after LH administration in vivo. The incorporation of radioactivity from the cholesterol- 7-3H increased to approximately the same extent as synthesis of the progestins, so that the specific activities of the progesterone and 20α-hydroxypregn- 4-en-3-one synthesized were essentially the same in tissue stimulated by LH as in unstimulated tissue. It was concluded that LH, in vivo and in vitro, stimulated the conversion of intracellular cholesterol t...

Cholesterol ester-storage granules were isolated from luteinized rat ovary and rabbit ovarian interstitial tissue by centrifugal flotation and were investigated with regard to their structure and function. Cholesterol ester, protein, phospholipid and unesterified cholesterol accounted for the dry weight of granules from luteinized rat ovary. The protein and the phospholipid were resistant to removal by washing. Substrate specificities of nucleotide phosphatase and specific radioactivities of lipid-soluble P (determined after administration of [32P]Piin vivo) were the same in granules and in a microsomal fraction from the same tissue. After administration of [32P]Piin vivo, luteinizing hormone increased the specific radioactivity of lipid-soluble P in granules, mitochondria and the microsomal fraction. Since granules did not swell in hypo-osmotic media, whereas microsomal particles did, it is suggested that adherent phospholipid and protein in granule suspensions is unlikely to result from contamination with endoplasmic reticulum. Luteinizing hormone administered in vivo increased the phospholipid and unesterified cholesterol contents of isolated granules relative to their cholesterol ester content, and also tended to raise their protein content. This treatment decreased the ability of isolated granules to act as a substrate for cholesterol esterase in vitro and increased the activity of cholesterol esterase. Cycloheximide in vivo also raised the unesterified cholesterol/cholesterol ester ratio of isolated granules, and when administered with luteinizing hormone acted synergistically to bring about a further increase. These results are considered compatible with evidence obtained by microscopy which suggests that granules may be surrounded by a membrane, that they arise by pinching off from the endoplasmic reticulum, and that they shrink on trophic stimulation of the tissue.

Indomethacin, an inhibitor of prostaglandin synthesis, blocks ovulation in immature rats pre-treated with pregnant mare serum gonadotropin (PMS), when given either at 0800, 1200 or 1600 hours on the second day after PMS treatment (the equivalent of proestrus in normally cycling adult rats). The drug also blocked ovulation in response to exogenous luteinizing hormone, whether the latter was administered 30 minutes before or 30 minutes after the inhibitor. Luteinization of follicles, and signs of pre-ovulatory progesterone secretion (loss of uterine lumen fluid) were not prevented when the inhibitor was given at 1600 hours, or when exogenous LH was administered in addition to the inhibitor, indicating that the luteinizing and steroidogenesis actions of LH upon the ovary were not completely blocked. When the drug was administered before the “critical period” for LH secretion, follicular luteinization and signs of progesterone secretion were also prevented, suggesting an additional action of indomethacin at the level of the hypothalamic-pituitary axis, inhibiting LH secretion.

A method involving the use of isolated cholesterol ester-storage granules as substrate is described for the assay of cholesterol esterase in rabbit ovarian tissues. Activities of cholesterol esterase 100–200-fold higher than those previously reported in ovarian tissues were measured by using this method. In addition to that of cholesterol esterase, activities of cholesterol ester synthetase, cholesterol side-chain cleavage enzyme and 3β-hydroxy steroid dehydrogenase were determined in rabbit ovarian interstitial tissue and corpora lutea. Activities of these enzymes are in general compatible with the flows through them measured under a variety of conditions both in vivo and in vitro. It is concluded that, in the rabbit ovarian tissues investigated, these enzymes are capable of catalysing the conversions usually attributed to them.

Superovulated, immature rats were administered luteinizing hormone (LH) and/or aminoglutethimide phosphate (AGP), an inhibitor of cholesterol side-chain cleavage, to ascertain whether the steroidogenic action could be separated from the cholesterol-depleting action of LH. The injection of AGP [Formula: see text] and [Formula: see text] before sacrifice significantly reduced tissue levels of progesterone and 20α-dihydroprogesterone to 25–30% of normal, and increased tissue levels of free cholesterol in the absence and presence of LH given [Formula: see text] or 4 h before sacrifice. Cholesterol ester concentration was increased twofold after [Formula: see text] AGP, whereas LH injection [Formula: see text] after AGP and 4 h before sacrifice reduced the tissue concentration to the control level but not as low as that observed when only LH was given 4 h before sacrifice (50% of control level). The cholesterol ester depletion induced by LH, even though steroidogenesis was inhibited, indicates that these may represent separate events in the action of LH on luteal tissue.

1. Experimental evidence is presented for a role of progesterone and 20alpha-hydroxypregn-4-en-3-one as inhibitors of cholesterol ester synthetase in the acute depletion of ovarian cholesterol ester after trophic stimulation. 2. Luteinizing hormone in vitro decreased by 84% the rate of esterification of cholesterol with added [(14)C]oleate by slices of rabbit ovarian interstitial tissue; this effect was mimicked by cyclic AMP (adenosine 3':5'-cyclic monophosphate) in vitro, and occurred without large changes in precursor pool sizes or membrane permeability. 3. Cyclic AMP was shown to have no direct effect on cholesterol ester synthetase or cholesterol esterase in cell-free extracts of rabbit ovarian interstitial tissue, but decreased the activity of cholesterol ester synthetase (not that of cholesterol esterase) in extracts prepared from slices previously incubated with it. 4. The inhibitory effect of cyclic AMP on esterification of cholesterol with added [(14)C]-oleate was prevented by both cycloheximide and aminoglutethimide phosphate (which also inhibited steroid synthesis in response to cyclic AMP). 5. Cyclic AMP raised the intracellular concentrations of progesterone and 20alpha-hydroxypregn-4-en-3-one in incubated slices by factors of 2.8 and 3.9 respectively. 6. Cycloheximide and aminoglutethimide phosphate administered in vivo blocked cholesterol ester depletion in response to luteinizing hormone in rats; in these ovaries cycloheximide and aminoglutethimide phosphate decreased the concentrations of progesterone and 20alpha-hydroxypregn-4-en-3-one and luteinizing hormone raised them. 7. Progesterone and 20alpha-hydroxypregn-4-en-3-one added to cell-free extracts of rabbit ovarian interstitial tissue in vitro (at concentrations comparable with those found in incubated slices) inhibited cholesterol ester synthetase by up to 85%. 8. The results are discussed with reference to the acute control of cholesterol ester concentrations in the ovary and adrenal cortex.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine secreted by lymphohaemopoietic and other cell lineages, is known to influence ovarian cyclicity and embryo development. The aim of this study was to examine the effect of GM-CSF on ovarian follicular cell function using GM-CSF-deficient (GM -/-) mice. Immature GM -/- and GM +/+ mice were stimulated with eCG, and cumulus-oocyte complexes and mural granulosa cells were collected 48 h later. Expression of GM-CSF receptor (GM-CSFR) alpha and beta mRNA subunits by cumulus-oocyte complexes and mural granulosa cells was examined using RT-PCR. Cumulus-oocyte complexes from both genotypes were found to express mRNA for the GM-CSFRalpha-subunit only, while the mural granulosa cells expressed both the alpha and beta receptor subunits. Cumulus-oocyte complexes recovered from GM -/- mice had approximately twice the number of cumulus cells per cumulus-oocyte complex than did those of GM +/+ mice (P < 0.05), even though the growth-promoting activity of denuded GM -/- oocytes was found to be equivalent to that of wild-type oocytes. GM-CSF deficiency was associated with marginally increased DNA synthesis in cumulus cells and significantly (P < 0.05) lower progesterone production by mural granulosa cells recovered from GM -/- compared with those recovered from GM +/+ mice. The addition of rec-mGM-CSF in vitro did not affect DNA synthesis in either cell type or progesterone production by mural granulosa cells, irrespective of GM-CSF status. There was no effect of GM-CSF deficiency on the capacity of FSH and insulin-like growth factor I to stimulate DNA synthesis in cumulus-oocyte complexes (approximately 15- and threefold, respectively) and in mural granulosa cells (approximately two- and threefold, respectively). Taken together, these data show that GM-CSF influences events associated with follicular maturation in mice. The effects of GM-CSF are not exerted directly in granulosa or cumulus cells, but appear to be mediated indirectly, perhaps through the agency of steroidogenesis-regulating secretions of local macrophage populations residing in the theca.

Porcine granulosa cells convert androstenedione (A) to 19-norandrostenedione (19-norA) and 19-hydroxyandrostenedione (19-OHA) in the presence of FSH and 10% porcine serum; 19-norA is also formed from independent incubations with 19-OHA. 19-NorA and 19-OHA formation from A, and 19-norA from 19-OHA, is blocked by 4-hydroxyandrostenedione, an irreversible inhibitor of aromatase.

Implantation of blastocysts in the rat is initiated by estrogen. Delay of blastocyst implantation is obtained if the pituitary is removed the day after vaginal sperm are observed and progesterone is provided exogenously or endogenous progesterone secretion is maintained by prolactin injection or by prolactin secreted from a pituitary autograf t. This study shows that luteinizing hormone (LH) will induce blastocyst implantation when administered at any time up to 12 days after breeding. Follicle-stimulating hormone and prolactin were without effect. This supports the hypothesis that LH stimulates secretion of physiological quantities of estrogen from luteinized ovaries with or without the capability to secrete progesterone. (Endocrinology 80: 172, 1967)