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beta-D-Mannosidase activity in selected normal adult, neonatal and foetal goat tissues and in tissues from animals affected with caprine beta-mannosidosis was examined with the use of 4-methylumbelliferyl beta-D-mannopyranoside as substrate. The enzyme in normal adult thyroid, kidney and brain exhibited a sharp unimodal pH optimum at pH 5.0, whereas the enzyme in both normal adult and mutant liver exhibited broad pH ranges of activity (pH 4.5-8.0). No residual enzyme was detectable in mutant kidney or brain; in contrast, residual activity in mutant liver was 52% of that in a neonatal control. Concanavalin A-Sepharose 4B (Con A-Sepharose) fractionation of normal adult liver beta-D-mannosidase resolved the enzyme into an unbound (non-lysosomal) from (52%) with a broad pH range of activity (pH 4.5-8.0) and a bound (lysosomal) form (48%) with a sharp pH optimum of 5.5. The enzyme in mutant liver consisted entirely of the unbound (non-lysosomal) form. Beta-D-Mannosidase activity in normal adult thyroid, kidney and brain was resolved by chromatofocusing into two major isoenzymes, with pI 5.5 and 5.9, and traces of a minor isoenzyme, with pI 5.0. In normal adult liver the enzyme was also resolved into three isoenzymes with similar pI values; however, that with pI 5.0 predominated. The predominant form of the enzyme in 60-day-foetal liver was bound by Con A, exhibited a unimodal pH optimum (5.0) and was resolved into two isoenzymes, with pI 5.4 and 5.8; only traces of an isoenzyme with pI 5.0 were detectable. Total hepatic beta-D-mannosidase activity increased progressively towards adult values during the last 90 days of gestation as a result of increasing non-lysosomal isoenzyme activity (pI 5.0). Lysosomal beta-D-mannosidase was shown to occur in all normal goat tissues studied as multiple isoenzymes, which are genetically and developmentally distinct from the non-lysosomal isoenzyme occurring predominantly, if not exclusively, in liver.

Prepubertal gifts were treated with 750 IU pregnant mare serum gonadotropin (PMSG) and 72 h later with 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. Laparotomies were carried out on groups of four or five gilts 24, 48, 72, 76, 88, 96, 102, or 108 h after PMSG treatment, and follicular fluid (FF) was aspirated from developing follicles. In most cases FF collected from each ovary of individual gilts was pooled, and the concentration of estrone (E1), estradiol-17β(E2), androstenedione (A), testosterone + 5α-dihydrotestosterone (T+DHT), and progesterone (P) in each sample was determined by radioimmunoassay. The FF concentrations of A and T+DHT remained relatively constant throughout the preovulatory development of the Graafian follicle. The FF concentrations of E1, E2, and P increased until 72 h after administration of PMSG. Following hCG treatment the FF concentrations of E1and E2declined rapidly to levels below those observed 24 h after PMSG treatment and remained at these levels, whereas the levels of P remained relatively constant until 30 h after hCG was given and then increased sharply. Resumption of meiosis was first evident by 16 h after injection of hCG and coincided with the decline in FF estrogen concentrations. These results indicate that the microenvironment of the Graafian follicle during preovulatory growth and development undergoes an orderly sequence of changes in steroid hormone concentrations which appear to be modulated by exposure of the developing follicle to gonadotropic stimuli.

Experiments were carried out to determine the effects of inhibition of synthesis of prostaglandins by indomethacin on ovulation, oocyte maturation, luteinization and luteal function in prepubertal gilts treated with PMSG and hCG to induce follicular growth and ovulation. Indomethacin suppressed ovulation and the preovulatory rise in follicular fluid levels of prostaglandin E and prostaglandin F when administered at 20, 24 or 32 h after hCG injection. Oocyte maturation was not inhibited by indomethacin but progressive degeneration of the cumulus cell mass surrounding the oocyte occurred with time. Unruptured follicles in indomethacin treated animals progressively increased in size, blood and fibrin filled the central cavity and there was thickening and extreme vasodilatation of the thecal sheath; lutein cells lining the cavity appeared normal. Groups of PMSG/hCG treated gilts, treated with indomethacin 20 or 32 h after hCG treatment, had plasma progesterone profiles which were similar to those of untreated controls and indicated normal luteal function. These results support the hypothesis that ovarian synthesis of prostaglandin is a prerequisite in the sequence of events leading to follicle rupture and ovulation but do not implicate prostaglandins in oocyte maturation, or in development of normal luteal function.

Prepubertal (28–30 days old) female rats were infused s.c. over a 60-h period with a purified porcine pituitary follicle-stimulating hormone (FSH) preparation having FSH specific activity 8.4 times that of NIHFSH-S1 and luteinizing hormone (L11) specific activity <0.005 times that of N1H-LH-S1, based on radioreceptor assays. When the FSH infusion rate of this preparation was increased over the range of 0.5–2 units/day (mg NIH-FSH-S1 equivalent), an all-or-none response was observed, with the threshold dose for superovulation being between 1 and 2 units/day. Eleven of twelve rats receiving the 2 units/day dose ovulated a mean ± SEM of 67 ± 8 oocytes on the morning of the third day after the beginning of FSH infusion. Addition of human chorionic gonadotrophin (hCG), as a source of LH activity, to a subthreshold (1 U/day) FSH infusion rate resulted in 20% of rats ovulating at an hCG dosage of 50 mIU/day; increasing the hCG infusion to 200 mlUlday concomitant with the subthreshold FSH infusion rate increased ovulation rate to a mean of 69 ± 8/rat, with 100% of rats ovulating.To determine the effect of varying both FSH infusion rates and LH:FSH ratios, FSH was infused at several rates, with hCG added to give varying hCG:FSH ratios for each FSH infusion rate. Administration of hCG alone was ineffective in causing ovulation except at the highest infusion rates. Adding hCG to FSH to reach a ratio of 0.2 IU hCG/U FSH significantly increased the superovulatory response to an intermediate, 1 U/day FSH dose, but not to the low, 0.5 U/day dose. A threefold increase in ratio to 0.66 IU hCG/U FSH increased the ovulatory response to the lowest FSH dose (0.5 U/day), caused no change in the response to the intermediate FSH dose, and significantly decreased the response to the highest FSH dose (2 U/day). A further threefold increase in hCG:FSH ratio to 2 IU/U significantly decreased the responses to all three FSH doses. Administration of the androgen antagonist, hydroxyflutamide (5 mg/day, s.c.), to rats infused with 1 U/day FSH supplemented with varying doses of hCG was without significant cffect on ovulatory responses at hCG: FSH ratios of 0 and 0.2 IU hCG/U FSH, but completely reversed the inhibition of ovulation seen at an hCG: FSH ratio of 2 IU/U. From the results of the research, it is evident that maximal superovulatory responses in FSH-infused rats occur over an optimal range of LH:FSH activity ratios, with decreased responses resulting at inadequate or excessive LH:FSH ratios. The ability of an anti-androgen to overcome the inhibitory effect of high LH:FSH ratios suggests that androgen-mediated follicular atresia contributes to the reduced ovulation rate resulting from excessive LH stimulation during follicular growth.

Immature female rats were infused s.c. continuously over a 60-h period with a partially purified porcine pituitary follicle-stimulating hormone (FSH) preparation having FSH activity 4.2 × N1H-FSH-S1 and luteinizing hormone (LH) activity 0.022 × NIH-LH-S1. High rates of superovulation were observed in rats receiving 1 U FSH/day, with 69 ± 11 oocytes/rat recovered as cumulus-enclosed oocytes from oviducts on Day 1 (equivalent to the day of estrus). Addition of LH to the FSH, at dosages equivalent to 2.5–100 μg/day NIH-LH-S1 equivalents (2.5–100 mU) resulted in a dose-related inhibition of superovulation, reaching a nadir of 15 ± 7 oocytes recovered from rats receiving 50 mU LH/day together with 1 U FSH/day. At the two highest LH doses, 50 and 100 mU/day, ovulation was advanced so that 12 ± 3 and 15 ± 4 oocytes, respectively, were recovered from oviducts of these rats flushed on the morning of Day 0, compared to none in rats infused with FSH alone.Ovarian steroid concentrations (ng/mg) observed on the morning of Day 0 in rats infused with FSH alone were progesterone, 0.50 ± 0.13; testosterone, 0.16 ± 0.08; androstenedione, 0.06; and estradiol, 0.23 ± 0.05. On the morning of Day 1, ovarian progesterone concentrations in rats infused with FSH alone had risen to 3.30 ± 0.33 ng/mg, whereas concentrations of testosterone, androstenedione, and estradiol, had fallen to essentially undetectable levels. Addition of LH to the FSH infusion resulted in dose-related increases, on Day 0, of all four steroids up to a dosage of 25 mU LH/day. At higher LH dosages, Day 0 ovarian concentrations of androgens and estradiol fell markedly, while progesterone concentrations continued to increase.Histological examination of ovaries revealed increases in the extent of luteinization of granulosa cells in follicles with retained oocytes on both Days 0 and 1 in rats infused with 25 and 50 mU LH/day together with 1 U FSH/day, compared to those observed in rats receiving FSH alone. These findings indicate that the elevated progesterone levels on Day 0 and inhibition of ovulation observed at these LH doses were due to premature luteinization of follicles, thus preventing ovulation. At lower LH doses, no sign (based on histologic or steroidogenic criteria) of premature luteinization was evident, suggesting that the decreased superovulation in these rats was due to decreased follicular maturation and/or increased atresia rather than to luteinization of follicles without ovulation. High androgen:estrogen ratios observed in rats receiving increasing LH infusion rates from 2.5 to 25 mU/day together with 1 U FSH/day suggest that decreased rate of superovulation in response to FSH treatment together with these LH infusion rates is due in part to follicular atresia associated with elevated ovarian androgen levels resulting from excessive LH stimulation.

Immature rat follicular oocytes were cultured either with cumulus cells intact (CI) or cumulus-free (CF) in bovine serum albumin (BSA)- or serum-supplemented medium under conditions in which meiotic maturation occurs spontaneously. After 12 h of culture to permit in vitro maturation (IVM), the cumulus cells were stripped from the CI group. Control oocytes recovered 2–4 h after ovulation from oviducts of pregnant mare’s serum gonadotropin (PMSG)-treated rats were similarly stripped of cumulus cells. Half the oocytes in each group had holes “drilled” in their zonae pellucidae by topical application of acid Tyrode’s solution with a micropipette to enable bypass of the zona barrier to penetration. They were cultured for a further 14–16 h with epididymal sperm and then were assessed for sperm penetration and pronuclear formation. In a preliminary study using various concentration of sperm, 50,000 sperm/ml was identified as an appropriate concentration and was used in all subsequent experiments. For oocytes matured in serum-supplemented medium, penetration rates of non-drilled oocytes-expressed as a percentage of oocytes exposed to sperm for CF, CI, and ovulated oocytes were 10%, 34%, and 80%, respectively (p<0.01). Drilling significantly increased the penetration rates of both IVM groups (CF: 40%, CI: 77%) but not of ovulated oocytes (78%). Forty-one percent of non-drilled CF oocytes failed to form normal pronuclei after penetration. This was significantly higher than either the CI (0%) or ovulated (1%) groups (p<0.001). Drilling increased the incidence of failure to form normal pronuclei in penetrated oocytes of the CF group (64%) but not of the CI or ovulated groups. In contrast, incidence of polyspermy in drilled oocytes was not increased in CF oocytes but was markedly increased in CI (44%) and ovulated (38%) groups. Similar results were obtained with oocytes matured in BSA-supplemented medium. After fertilization, drilled and non-drilled IVM oocytes were transferred to the oviducts of mated recipient rats. Drilling significantly reduced the ability of JVM oocytes to develop to Day 20 fetuses (31% vs. 51%).These results indicated that the presence of cumulus cells during in vitro maturation enhances subsequent fertilization in vitro by increasing the penetrability of the zona pellucida to sperm and by conferring on the ooplasm the capacity to induce normal pronuclear formation.

Immature oocytes were collected from immature female rats (60−65 g) 40 h after injection with 6 IU pregnant mare’s serum gonadotropin (PMSG). Oocytes were matured cumulus-intact (CI) or cumulus-free (CF) in medium supplemented with 0.5% bovine serum albumin (BSA) or 5−20% serum for periods of up to 24 h. After assessment for nuclear maturation, the oocytes were exposed to epididymal sperm for fertilization in vitro. In vitro-matured and ovulated oocytes undergoing fertilization were transferred to unilaterally pregnant recipients for embryonic and fetal development.The presence of cumulus cells and serum shortened (by 2 h) the time required for polar body emission by in vitro-matured oocytes and also helped to increase significantly the penetrability of the oocytes by spermatozoa. A high proportion (45.6%) of fertilized oocytes showed evidence of abnormal fertilization following maturation in the absence of cumulus cells. Oocytes matured CI before fertilization were able to develop to viable fetuses (57.8%) in proportions similar to ovulated oocytes (55.0%) after in vitro fertilization. These findings indicate an essential role for cumulus cells in promoting normal cytoplasmic maturation of oocytes necessary for pronuclear formation and subsequent developmental capability.

Immature female rats were infused s.c. continuously over a 60-b period with partially purified porcine pituitary follicle-stimulating hormone (FSH) preparations differing in degree of purity and having widely divergent luteinizing hormone (LH):FSH potency ratios as defined by radioreceptor assays. Rats infused with the more purified FSH preparation (FSH-A) ovulated a mean of 60–85 oocytes per rat on the morning of the third day (Day 1) after FSH infusion was begun (on Day -2). The same total dose of FSH administered as a single s.c. injection or as twice daily injections over the same 60-h period resulted in ovulation in only a minority of treated rats (3/16), with none achieving ovulation rates approaching those of rats infused continuously.High fertilization rates (80% of ovulated oocytes) were observed in superovulated rats joined with fertile males on the evening of the second day of infusion (Day 0). Of the 67 ± 7fertilized ova per rat retrieved from oviducts flushed on Day 1, 52 ± 8, or 80%, were accounted for as morulae or blastocysts recovered when oviducts and uteri were flushed on the morning of Day 5, demonstrating essentially normal developmental rates and high survival rates in reproductive tracts of superovulated females during the preimplantation period.Infusion of rats with the same dose of a less well-purified FSH preparation (FSH-E) containing 20 times as much LH activity, or injection of rats with a superovulatory dose of pregnant mare’s serum gonadotropin (PMSG) (40 IU), were much less effective in causing superovulation, with ovulation rates of 17 ± 6 and 34 ± 8 oocytes/rat, respectively, compared to 79 ± 9 oocytes/rat infused with the FSH preparation (FSH-A) containing lower LH activity.The high rates of ovulation and of embryo development and recovery following infusion with a partially purified FSH preparation low in LH activity offers a convenient and effective alternative to PMSG as a means of superovulation of rats for the production of high yields of normal oocytes or embryos. The substantially lower ovulation rates observed after injection of PMSG or infusion of less purified FSH suggest a detrimental effect of high LH activity of gonadotropin used for superovulation of immature rats.

Prostaglandin production in vitro by theca and granulosa cells isolated from prepubertal pig ovaries was quantified in order to investigate the role of prostaglandins in intrafollicular function. Prepubertal gilts were slaughtered without treatment (O h, control) or treated with 1000 IU pregnant mare’s serum gonadotropin (PMSG) and slaughtered at 36 or 72 h, or at 75 h following treatment with 500 IU of hCG at 72 h. Theca and granulosa cells were isolated from preovulatory follicles and cultured for 24 h alone or with follicle-stimulating hormone (FSH) or luteinizing hormone (LH). In vitro accumulation of 6-keto-prostaglandin F1α(6-keto-PGF1α), prostaglandin E2(PGE2) and prostaglandin F2α(PGF2α) was measured by radioimmunoassay. On a per follicle basis theca produced more of each prostaglandin (approx. 10-fold) than granulosa at each stage of follicular development; production by each tissue type increased with development of the follicle, responding to administration of gonadotropin (PMSG) in vivo. Neither tissue type was generally responsive to further gonadotropin stimulation in vitro. However, production of PGE2by granulosa cells was increased by addition of gonadotropin, particularly LH, in vitro, with the greatest response observed in tissue obtained at 36 and 72 h after PMSG. There were no functional correlates between prostaglandin production and steroidogenesis by either tissue type and we conclude that prostaglandins do not have an obligatory role in follicular steroidogenesis. However, these data provide additional circumstantial evidence for a role of PGE2in granulosa cell luteinization, and possibly in ovulation. The data also indicate that prostaglandins derived from thecal tissue in relatively large quantities may play an important role in ovulation.

The ability of prolactin and progesterone to inhibit ovulation was utilized to investigate the relationship between ovulation and luteolysis. The first estrus and ovulation were synchronized in 27-day-old, prepubertal rats by the subcutaneous (sc) administration of 4 IU of pregnant mane serum gonadotropin (PMS). Pseudopregnancy was induced by the sc administration of prolactin (100 µg, NIH-P-B2administered twice daily) beginning on the day of ovulation (Day 0) and maintained through Day 2. If prolactin therapy was discontinued on Day 2, a second injection of PMS on Day 3 resulted in ovulation accompanied by involution of the original corpora lutea. Continuous prolactin treatment from Day 0 through Day 6 not only prevented ovulation as a result of PMS administration on Day 3, but prevented luteolysis as well. Progesterone (8 mg/day), if administered on Days 3 through 6, inhibited ovulation in response to PMS administered on Day 3. It did not, however, prevent the luteolysis as a result of the ovulatory stimulus provided by PMS. Progesterone, in the absence of PMS, did not affect luteal weight. The dissociation of luteolysis from ovulation demonstrated that actual ovulation was not a requirement for luteolysis to occur, but leaves open the possibility that an ovulatory stimulus may be an effective luteolytic stimulus.

Serum testosterone levels were found to be significantly higher in male rats aged 83 days compared to rats aged 37 days, whereas serum luteinizing hormone (LH) levels in the same samples were not different. Serum testosterone levels in 90-day-old male rats were elevated by 30 min after the ip administration of 10 μg LH/100 g body wt and remained elevated through 150 min. In a similar experiment with 31-day-old males, testosterone levels were elevated at 90 min but not at 30 or 180 min, and with 37-day-old rats testosterone levels were elevated by 60 min and remained elevated through 150 min. One hundred minutes after receiving 1 μg LH/100 g body wt testosterone levels in 36-day-old rats were slightly elevated, 10 μg raised their testosterone levels to the normal adult male range, but 100 μg caused no further increase. Seventy-six-day-old males did not respond to 1 μg LH/100 g body wt but testosterone levels were greatly elevated by 10 μg/l00 g body wt. The serum testosterone level attained by the adults in response to 10 μg LH/100 g body wt (31.7 ± 7.34 ng/ml) was significantly higher than the level attained by the immature rats (5.85 ± 0.89 ng/ml) in response to the same LH dose. The significantly higher serum testosterone levels found in the adult rats receiving 10 μg LH/100 g body wt indicate that changes in the responsiveness of the testis to LH may be an important part of puberty in the male rat.

Sertoli cells isolated from testes of 20-day-old rats and maintained in primary culture synthesized estradiol-17beta [1,3,5(10)-estratriene-3,17beta-diol] (measured by specific radioimmunoassay) when testosterone (17beta-hydroxy-4-androsten-3-one) 0.5 muM, was added to the culture medium. No detectable estradiol synthesis occurred when cells were incubated in medium containing pregnenolone (3beta-hydroxypregn-5-en-20-one), 0.5 muM, or containing no added steroid substrate. Follicle-stimulating hormone (FSH) (NIH-FSH-S10, 5 mug/ml) stimulated estradiol synthesis 12- to 80-fold when added to medium containing testosterone, but not when added to medium containing pregnenolone or no exogenous steroid substrate. A highly purified FSH preparation, with FSH potency 50 times that of the NIH-FSH, caused a similar stimulation at a concentration of 0.25 mug/ml of medium, whereas luteinizing hormone (NIH-LH-S18, 5 MUG/ML) Caused only marginal stimulation. Dibutyryl-adenosine 3':5' cyclic monophosphate, 0.1 mM, caused a 30-fold increase in estradiol synthesis by Sertoli cells cultured in medium containing testosterone. These studies provide direct demonstration of estradiol-17beta production by Seroli cells from normal animals, and offer evidence that the synthesis of this steroid is regulated at the level of the aromatizing enzyme system by FSH and adenosine 3':5' cyclic monophosphate.

The involvement of androgens in the control of ovulation has been assessed by administration of the androgen antagonist, hydroxyflutamide, to prepubertal rats treated with pregnant mare's serum gonadotropin (PMSG) to induce first estrus and ovulation. Without human chorionic gonadotropin (hCG) injection, only 46% of rats that received six 5-mg, s.c. injections of hydroxyflutamide at 12-h intervals, beginning an hour before s.c. injection of 4 IU PMSG on Day-2 (Day 0 = the day of proestrus), had ovulated a mean of 1.3 ± 0.4 oocytes per rat when killed on the morning of Day 1, whereas 92% of sesame oil-treated controls had ovulated a mean of 6.9 ± 0.6 oocytes. After i.p. injection of hCG at 1600 h on Day 0, 92% of hydroxyflutamide-treated rats ovulated a mean of 8.3 ± 1.2 oocytes compared to 100% of controls, which ovulated 7.3 ± 0.4 oocytes per rat: these groups were not significantly different from each other, nor from control rats that received no hCG. Thus, exogenous hCG completely overcame the inhibitory effect of hydroxyflutamide on ovulation. Rats treated with PMSG and hydroxyflutamide without hCG were killed either on the morning of Day 0 to determine serum and ovarian steroid levels or on the afternoon of Day 0 to determine serum LH levels. Serum levels of estradiol-17β and testosterone in hydroxyflutamide-treated rats were significantly higher (178% and 75%, respectively; p<0.01) than levels observed in controls on the morning of Day 0. Ovarian concentrations of the steroids were also elevated in hydroxyflutamide-treated rats (p<0.01 for testosterone only). Serum luteinizing hormone (LH) levels observed at 1800 b on pro estrus were elevated approximately 650% above basal levels, indicating a spontaneous LH surge in control animals. This elevation was not seen in hydroxyflutamide-treated rats at the same time on proestrus. These studies suggest that the blockade of ovulation by hydroxyflutamide is due to interference with the spontaneous LH surge, and that the blockade is not due to a lack of ovarian steroidogenic response to the PMSG treatment. This indicates an involvement of receptor-mediated action(s) of androgen in the positive feedback system leading to the proestrous LH surge in the rat.

Immature rats superovulated with pregnant mare’s serum gonadotropin (PMSG) were ovariectomized at various times after mating and received various steroid replacement regimes in order to investigate the role of ovarian steroids in the etiology of the infertility which occurs in superovulated rats.Control (4 IU PMSG) and superovulated (40 IU PMSG) rats were allowed to mate on the night of Day 1 and were killed on Day 9, or allowed to mate on the night of Day 2 and were killed on Day 10. All ovariectomized rats received a daily regime of steroid injections which permitted a high rate of pregnancy on Day 10 in control rats ovariectomized at 1000 – 1200 h on Day 3.Implantation failed to occur in all superovulated rats which were not ovariectomized. Five of 11 superovulated rats which mated on the night of Day 1 and were ovariectomized at 1000 – 1200 h on Day 2 (Day 1 of pregnancy) were pregnant on Day 9 (7.2 ± 2.4 implantation sites, weighing 21.6 ± 1.3 mg in pregnant animals). Similarly, 12 of 25 superovulated rats which mated on the night of Day 2 and were ovariectomized at 1000 – 1200 h on Day 3 (Day 1 of pregnancy) were pregnant on Day 10 (10.3 ± 2.3 implantation sites, weighing 26.2 ± 0.8 mg in pregnant animals). Delaying the time of ovariectomy in superovulated rats which mated on the night of Day 2 until 2200 - 2400 h on Day 3 or 1000 – 1200 h on Day 4 decreased the pregnancy rate (8/15 and 2/17, respectively) and the number of implantation sites and implantation site weight in pregnant animals (4.4 ± 0.6 and 1.5 ± 0.5;and 17.2 ± 1.2 mg and 13.7 ± 1.5 mg, respectively).Administration of estradiol at the time of ovariectomy and at 12 h intervals over the next 24 h reversed the beneficial effect of ovariectomy in a dose-related manner, both on the number of animals which remained pregnant and the number of implantation sites per pregnant animal.The results show that at least a large proportion of the embryos present in superovulated rats during the first 36 h after the time of mating are normal, and that the consistent failure of implantation in intact rats results from abnormal ovarian hormone secretion after the time of fertilization, most probably from the hypersecretion of estrogens.

Prepubertal gilts were treated with 1000 IU PMSG and slaughtered at 0, 36, or 72 h, or at 75 h following treatment with 500 IU of hCG at 72 h. Theca and granulosa were isolated from preovulatory follicles and cultured for 24 h alone or with FSH or LH and in combination with or without testosterone. In vitro accumulation of estradiol-17β(E2), progesterone (P), testosterone (T), and androstenedione (A) was measured for each culture. On a per follicle basis, theca produced E2in quantities comparable to granulosa, but granulosa required an exogenous aromatizable substrate (T) whereas theca did not. Theca was the sole source of androgen, predominantly A, and produced significant quantities of P, though granulosa was the primary producer of P. Theca was responsive to LH but not FSH, particularly in the early stages of follicular development, in production of all steroids except E2. The overall thecal production of all steroids increased with development of the follicle to 72 h, and in vivo hCG treatment further stimulated the production of P, A, and T but inhibited E2biosynthesis. In culture in the absence of exogenous steroid, there was a net depletion of A and T by granulosa, matching the accumulation of E2. Granulosa cell androgen content prior to culture was correlated with thecal androgen synthetic activity, providing indirect evidence that thecal androgen is transferred to granulosa cells in vivo. In the presence of T, granulosa production of E2declined with development of the follicle but was enhanced 3 h after hCG treatment in vivo; there was no effect of gonadotropin on in vitro E2synthesis. The granulosa biosynthesis of P increased with follicular development, and was highly responsive to both LH and FSH in the early stages; P production was unaffected by addition of T. We conclude that, in the porcine preovulatory follicle, theca is an important source of E2and the sole source of androgen. After transfer from theca to granulosa cells, A is converted to T and thence to E2. Granulosa is also the predominant site of P synthesis.

Reduced fertility is frequently associated with excessive follicle stimulation following the use of exogenous gonadotropins to induce ovulation. To investigate the mechanisms involved, a superovulatory dose of pregnant mare serum gonadotropin (PMSG, 40 IU) was administered to seventysix 30-day-old rats (0800–0900 h, Day 0). Sixty-eight control rats received 4 IU, a dose which induces normal numbers of ovulations followed by normal pregnancy. Some rats were killed at∿ 1200 h on Day 2. The remainder were caged with mature fertile males on Day 2, scored for the occurrence of mating on the morning of Day 3, and killed at about 1200 h on Days 3, 4, 5, or 7 (Days 1, 2, 3, or 5 of pregnancy, respectively). Corpora lutea (CL) and large, nonluteinized follicles were counted, and oviducts and uteri flushed to determine number, location, and stage of development of embryos. Ovarian and uterine weight, the accumulation of fluid in the uterus and ampulla, the ovarian content of estradiol, and serum levels of estradiol and progesterone were also recorded.Mating and fertilization occurred in most superovulated and control rats. Mean numbers of CL observed in control rats killed on Days 2, 3, 4, 5, and 7 were 0, 10.7, 10.5, 11.6, and 11.5, respectively. The corresponding means for CL plusluteinized follicles in superovulated rats were 55, 65, 85, 85, and 112, respectively. Ovaries from superovulated rats also contained, on average, 20.8, 27.5, 18.5, 13.6, and 12.3 large, nonluteinized follicles on Days 2, 3, 4, 5, and 7, respectively; 10.4 such follicles were observed in controls on Day 2 only. Mean numbers of ova or embryos recovered on Days 2, 3, 4, 5, and 7, respectively, from oviducts of controls were 0, 8.3 (1-cell embryos), 8.7 (2-cell embryos), 8.9 (2-, 3-, and 4-cell embryos), and 0, and from the oviducts of superovulated rats, 15.1 (unfertilized ova), 38.4 (1-cell embryos plusdegenerate ova), 17.9 (2-cell embryos plusdegenerate ova), 6.6 ( 2-, 3-, and 4-cell embryos plusdegenerate ova), and 0. Flushings of uteri yielded 9.4 blastocysts from controls on Day 7 only, and only an occasional embryo from superovulated rats on Days 4 and 5, but none on Day 7. Superovulated rats ovulated over a prolonged period, presumably on the nights of Days 1 and 2. Mean serum estradiol levels (pg/ml ± SEM) observed in control and superovulated rats on Days 2, 3, 4, 5, and 7, respectively, were 93 ± 5 vs 230 ± 10, 34 ± 3 vs 134 ± 19, 31 ± 2 vs 201 ± 33, 34 ± 2 vs 98 ± 10, and 37 ± 3 vs 49 ± 3. The corresponding serum progesterone levels (ng/ml ± SEM) were 2 ± 1 vs 29 ± 3, 9 ± 3 vs 74 ± 5, 22 ± 3 vs 152 ± 11, 43 ± 3 vs 200 ± 15, and 61 ± 4 vs 220 ± 18. respectively. The results suggest that embryo loss following superovulation is due to excessive estrogenic stimulation of the genital tract, probably arising from large follicles which persist after normal ovulation occurs.

Immature Sprague-Dawley rats were maintained with lights off between 1900 and 0500 h and treated with pregnant mare serum gonadotropin (PMSG) at 30 days of age (0800–0900 h, Day 0). One hundred and ninety-eight animals were caged individually with mature fertile males at 1500 h on Day 2, scored for the occurrence of mating on the morning of Day 3, and killed on Day 22. Rats received 4, 8, 16, or 40 IU PMSG; a high proportion of those receiving each dose mated. Of those which mated, almost all receiving 4 or 8 IU, 76% of those receiving 16 IU, and none receiving 40 IU were pregnant on Day 22. The mean numbers of live fetuses in pregnant rats that received 4, 8, and 16 IU PMSG were 8.8, 12.9, and 14.2, respectively. The corresponding means for numbers of implanted embryos were 9.7, 15.4, and 18.8; for numbers of corpora lutea of pregnancy (CLP), 10.8, 20.8, and 48.1; for fetal weight, 2.25, 1.65, and 1.38 g; for placental weight, 0.47, 0.38, and 0.36 g; for peripheral serum estradiol concentration, 104, 125, and 112 pg/ml; and for peripheral serum progesterone concentration, 91, 112, and 164 ng/ml. Serum estradiol levels were more correlated to numbers of live fetuses than to numbers of CLP (r = 0.32, 0.01

In immature female rats, ovarian levels of estradiol-17βappeared to be higher at 8 days than at 15 or 22 days of age. Four h following a single s.c. injection of testosterone (100 μg/g b.w.), ovarian estradiol-17βlevels were significantly elevated at all ages, compared to oil-treated control animals, with the highest levels being reached in 8-day-old animals. We have previously reported that the ovary contributes significantly to the increased ovarian concentration of estrogen when ovarian androgen contents are elevated by administration of high dosages of exogenous testosterone. Taken together, these results suggest that ovarian aromatization is greater in early post natal life and declines with age towards puberty.Ovarian responsiveness to luteinizing hormone (LH) in the formation of endogenous substrate for estrongen production was tested in another experiment. Ovaries from 8-day-old rats failed to respond to LH (NIH-LH-B8, 0.125 μg/g b.w., i.p.) in the production of either testosterone or estradiol-17β. It is suggested that follicle stimulating hormone (FSH), rather than LH, may be responsible for the high aromatization activity occurring in the young rats.

Subcutaneous administration of estradiol-17β(1 mg) to intact, immature female rats for 3 days significantly decreased ovarian androgen levels from 34 ± 7 pg/mg in oil treated control rats to 13 ± 2 pg/mg ovarian tissue. It inhibited ovarian responsiveness to LH, as measured by ovarian androgen, but not as measured by ovarian progesterone concentrations. In oil treated control animals, LH caused an approximately 5-fold increase in ovarian androgen levels, whereas ovarian androgen levels were not stimulated at all by LH in the estrogen treated rats. This inhibitory effect on ovarian androgen response was also demonstrable after 6 h of estrogen treatment.Administration of estradiol-l7βto hypophysectomized immature rats for 3 consecutive days suppressed both ovarian androgen and progesterone levels in response to LH. The cause of the discrepency in the progesterone response between intact and hypophysectomized rats is not clear. However, with regard to the androgen response, it is unlikely that the inhibitory action of estrogen is indirectly mediated by a suppression of endogenous gonadotropin release, since concomitant administration of exogenous gonadotropins failed to alter the estrogen effect. Instead, a direct action on the ovary is suggested by the demonstration that unilateral ovarian estrogen implants exert an inhibitory effect on LH-induced androgen levels in the ipsilateral, but not contralateral, ovary in vivo.These results suggest that in the ovary there exists a local negative feedback loop for the control of testosterone production, in which elevated ovarian estrogen levels suppress ovarian responsiveness to LH stimulation, thereby lowering testosterone production and subsequently decreasing estrogen biosynthesis.

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.

1. Experimental evidence is presented for a role of progesterone and 20α-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 [14C]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 [14C]-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 20α-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 20α-hydroxypregn-4-en-3-one and luteinizing hormone raised them. 7. Progesterone and 20α-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.

1. The specific radioactivities of non-esterified and esterified cholesterol, progesterone and 20α-hydroxypregn-4-en-3-one were determined in slices of superovulated rat ovary after incubation with [1-14C]acetate in vitro for various times. The specific radioactivities of progesterone and 20α-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 [14C]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./μmol respectively; the corresponding values for the specific radioactivity of the esterified cholesterol were 600, 700, 730 and 760d.p.m./μmol. The specific radioactivities of progesterone and 20α-hydroxypregn-4-en-3-one were equal in all fractions; the corresponding mean specific radioactivity of progesterone+20α-hydroxypregn-4-en-3-one was 6150d.p.m./μmol. 3. By using glutamate dehydrogenase and cytochrome (a+a3) 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 20α-hydroxypregn-4-en-3-one were determined up to 8h after the intravenous injection of [4-14C]cholesterol into superovulated rats. At all times the specific radioactivities of progesterone and 20α-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 [14C]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.

X-irradiation of the immature rat ovary luteinized by exogenous gonadotropins (PMS—HCG) caused destruction of most remaining follicles within 8 days, but the morphological integrity of lutein tissue was maintained. X-irradiated ovaries of seven rats exposed to four daily injections of NIH-LH-S11 (50 μg/rat/day) failed to secrete estrogen in physiologically detectable quantities as indicated by the absence of a uterine growth response. The same regimen of LH caused a significant (p < .01) increase in uterine weight in seven animals with luteinized, sham-irradiated ovaries. Total synthesis of progestin (progesterone and 20α-hydroxypregn-4-en-3-one) in vitroby four tissue pools consisting of X-irradiated ovaries from eight rats was quantitatively comparable to that synthesized by a similar number of sham-irradiated ovarian tissue pools. In this species, lutein tissue remains viable and capable of secreting progestins after exposure to X-irradiation in sufficient dosage to cause destruction of follicles. The inability of the luteinized, X-irradiated ovary to secrete estrogen in response to LH may be attributable to the absence of follicles and/or X-ray-induced destruction of estrogen-producing cells within the lutein tissue.