Autoradiographic analysis of follicle-stimulating hormone and human chorionic gonadotropin receptors in the ovary of immature rats treated with equine chorionic gonadotropin.

The gonadotropin-primed immature rat has become the most common model for the study of follicular development and ovulation. In this study, prepubertal female rats, 23 and 24 days old, were injected s. c. with 5 IU eCG, and ovaries were collected for topical autoradiography of FSH and hCG receptors at 48 or 24 h post-eCG, respectively (i.e., Day 25). In a baseline group, on Day 25 (before eCG), even the smallest preantral follicles with 1 layer of granulosa cells (GCs; primary follicles) possessed FSH receptors, but hCG receptors were found only on the theca of follicles with 2 or more layers of GCs. Human CG receptors were especially prominent in the interstitium that intimately surrounds preantral follicles without any distinction between theca and interstitial cells. There was a discrete theca surrounding antral follicles. Occasionally antral follicles had hCG receptors in the interstitium, but the adjacent theca was negative, suggesting that these follicles might be destined for atresia. By 24 h post-eCG, a now-discrete theca layer with hCG receptors surrounded all preantral follicles except for the primary follicles, which never responded to eCG. The interstitium was hypertrophied and epithelioid, as was the theca surrounding nonatretic preantral and antral follicles. Increased mitotic activity characterized the growing preantral follicle, and for the first time, FSH binding in GCs of antral follicles was greater than in the preantral population. By 48 h post-eCG, the primary follicles were still unresponsive to eCG. FSH receptors were even more pronounced in the GCs of large antral follicles, although hCG receptors were present in the GCs of only one third of the antral follicles, reflecting the small dose of eCG administered. By 48 h post-eCG, receptors in the interstitium were barely detectable. Using this model, the following study considers the functional in vitro changes in steroidogenesis in follicles from the smallest preantral follicles to the largest antral follicles.

In vitro steroidogenesis by dissociated rat follicles, primary to antral, before and after injection of equine chorionic gonadotropin.

Prepubertal female rats were injected s.c. with 5.0 IU eCG, and ovaries were collected 24 and 48 h post-eCG, on Day 25, as well as from an untreated group also on Day 25. Large antral follicles were manually dissected, and the ovarian remnants were incubated with collagenase overnight to liberate preantral follicles from adhering stromal cells. The viability of the follicles was established by normal histology and lack of pyknotic granulosa cells (GCs) and by their ability to secrete steroids. After a 1-h baseline incubation, either 10 ng LH or 100 ng FSH was added for an additional hour, and the media-before and after gonadotropin administration-were used to measure progesterone, androstenedione, and estradiol by RIA. A distinct hierarchy existed in steroid synthesis, with the maximal production by the largest (700 microm) antral follicles. The major steroid that had accumulated after addition of LH at 48 h post-eCG was androstenedione (1099 pg/follicle per hour), followed by equal amounts of progesterone (155 pg/follicle per hour) and estradiol (191 pg/follicle per hour). There was a precipitous drop in steroid production by 550-microm and 400-microm antral follicles, especially in estradiol for the latter-sized follicles (0.08 pg/follicle per hour). Preantral follicles also produced progesterone and androstenedione after addition of LH. For example, follicles 222 microm in diameter with 4-5 layers of GCs and well-developed theca responded to LH at 48 h post-eCG by accumulating androstenedione (37 pg/follicle per hour) and progesterone (6 pg/follicle per hour) but negligible estradiol. The smallest follicles secreting steroids, 110-148 microm in diameter, had 2-4 layers of GCs. However, primary follicles (1 layer of GCs and no theca) did not synthesize appreciable amounts of any steroid. Although small preantral follicles were consistently stimulated by LH, FSH was ineffective. This result differs from findings in the hamster showing that intact preantral follicles with 1-4 layers of GCs and no theca respond to FSH by secreting progesterone in vitro (Roy and Greenwald, Biol Reprod 1987; 31:39-46). The technique developed to collect intact rat follicles should be useful for numerous investigations.

Apoptosis during spontaneous luteolysis in the cyclic golden hamster: biochemical and morphological evidence.

The corpora lutea (CL) of the cyclic hamster are destroyed between Days 2 and 3 of the 4-day estrous cycle so that only one set is ever present (Day 1 = estrus, Day 4 = proestrus). The possibility that luteal cell death in the cyclic hamster is attributable to apoptosis was explored. The earliest histological signs of structural luteolysis were detected at 0600 h of Day 3 as evidenced by a few scattered luteal cells displaying the characteristic morphology of apoptotic cells and by a massive infiltration of neutrophils. The peaks of neutrophil influx and luteal apoptosis were reached on Day 3, 1200 h, and Day 3, 2400 h, respectively. Thus, the increase in neutrophils occurs before the major onset of luteolysis. By Day 3, 2400 h, the CL had already shrunken one third by weight, and they virtually vanished by the next Day 1. Apoptosis ultimately destroyed luteal endothelial cells, luteal cells, and neutrophils. Electrophoretic analysis of low-molecular weight DNA in luteal cell lysates revealed a definite ladder pattern of oligonucleosomal-length DNA fragments--characteristic of apoptosis--on Day 3 beginning at 1200 h. The pattern was not detectable in CL collected on Day 2. Comparing Day 3 CL collected at 0900-1200 h with those at 1500-1800 h showed that only the latter group exhibited inter-nucleosomal cleavage activity. The minimal number of CL on Day 3, 1500 h, needed to demonstrate DNA laddering was six. In summary, the electrophoretic separation of oligonucleosomal fragments and histology indicated that apoptosis occurs during spontaneous luteal regression on Day 3 of the hamster cycle. The initiation of apoptosis is not apparent until several hours after the onset of functional luteolysis. The rapidity with which apoptosis eliminates the CL over a very precise time schedule makes the cyclic hamster an ideal model to analyze the factors involved in structural luteolysis.

Progesterone production in vitro by mouse luteal cells: response to follicle-stimulating hormone, luteinizing hormone, and prolactin.

The purpose of this study was to determine the effects of ovine follicle-stimulating hormone (FSH), luteinizing hormone (LH); prolactin, and recombinant FSH and a protein kinase C activator (phorbol 12-myristate 13-acetate [PMA]) on progesterone production by dispersed luteal cells (large + small) from Day 4 pregnant mice. Corpora lutea (CL) were collected on Day 4 of pregnancy (Day 1 = sperm positive smear), and dispersed luteal cells were isolated using collagenase. After overnight incubation, the luteal cells were incubated with or without FSH, LH, prolactin, or recombinant human FSH or PMA for 4 hr or an additional 24 hr at 37 degrees C; media were collected and progesterone was determined by RIA. Ten nanograms and 100 ng of ovine FSH, LH and prolactin were all equally effective in stimulating progesterone synthesis in media recovered after 24 hr of incubation. Moreover, the combination of all three gonadotropins yielded maximum levels of progesterone indicating a luteotrophic complex in vitro, paralleling previous in vivo findings. Recombinant human FSH-devoid of LH contamination-at doses of 10 and 100 ng also significantly stimulated progesterone synthesis, which strongly suggests that FSH has luteotropic activity in the mouse, thus agreeing with our previous in vitro results with CL of the pregnant hamster and rat. One hundred nanomolar PMA by itself did not affect progesterone production but significantly decreased dibutyrl cAMP-, forskolin-, FSH-, and LH-induced progesterone production, suggesting that activation of protein kinase C may block the luteotropic effects of LH and FSH during murine pregnancy.

Temporal changes in inhibin, steroid hormones, and steroidogenic enzymes during induced follicular atresia in the hypophysectomized cyclic hamster.

The time course for loss of ability of Graafian follicles to secrete inhibin and estradiol was investigated during induced follicular atresia. Cyclic hamsters were hypophysectomized on Day 1 (estrus) and injected s.c. with 30 IU eCG. Thereafter, these animals were given a single i.p. injection of antiserum to eCG on the morning of Day 4 to induce follicular atresia in a rapid and predictable manner. A drastic fall in plasma levels of estradiol and testosterone was noted within 1 h, whereas relatively high levels of plasma inhibin were maintained until 12 h, followed by an abrupt decline by 24 h. The first histological signs of pyknosis in granulosa cells appeared by 4 h, and breakdown of the mural granulosa layer was observed in most follicles by 8-12 h after immunoneutralization of circulating eCG. According to immunohistochemical analysis, inhibin activity was unchanged in granulosa cells at 12 h followed by a slight decline by 24 h, whereas positive reaction for aromatase in these cells rapidly declined by 8 h. Immunoreactivity of 17alpha-hydroxylase/C17,20-lyase (CYP 17) was also reduced in theca cells by 8 h. These results indicate that granulosa cells continue to secrete inhibin during the process of follicular atresia, although these cells quickly shut off the secretion of estradiol, and theca cells shut off the secretion of testosterone. The present results indicate as well that a rapid decline of estradiol and testosterone in plasma is an early sign of atresia in antral follicles. These results, therefore, suggest that the loss of enzymatic activity of aromatase in granulosa cells and CYP 17 in theca cells is a part of the process of follicular atresia.

A comprehensive management system for heart failure improves clinical outcomes and reduces medical resource utilization.

The effectiveness of heart failure management in clinical practice is limited by physicians' suboptimal utilization of effective medications, patients' poor adherence to dietary sodium limitation and optimal drug therapy, and the lack of systematic monitoring of patients after hospitalization. The present study evaluated the feasibility and safety of MULTIFIT, a physician-supervised, nurse-mediated, home-based system for heart failure management that implements consensus guidelines for pharmacologic and dietary therapy using a nurse manager to enhance dietary and pharmacologic adherence and to monitor clinical status by frequent telephone contact. Fifty-one patients with the clinical diagnosis of heart failure were followed for 138 +/- 44 days. Daily dietary sodium intake fell by 38%, from 3,393 to 2,088 mg (p = 0.0001); average daily medication doses increased significantly (lisinopril: 17 to 23 mg, p <0.001; hydralazine: 140 to 252 mg, p = 0.01). Functional status and exercise capacity improved significantly (p = 0.01). Compared with the 6 months before enrollment and normalized for variable follow-up, the frequency of general medical and cardiology visits declined by 23% and 31%, respectively (both p <0.03); emergency room visits for heart failure and for all causes declined 67% and 53%, respectively (both p <0.001). Hospitalization rates for heart failure and for all causes declined 87% and 74%, respectively (p = 0.001), compared with the year before enrollment. The MULTIFIT system enhanced the effectiveness of pharmacologic and dietary therapy for heart failure in clinical practice, improving clinical outcomes and reducing medical resource utilization.

Methods of separation and in-vitro culture of pre-antral follicles from mammalian ovaries.

A variety of procedures to isolate intact pre-antral follicles have been developed in our laboratories based on different concentrations of collagenase and DNase in a simple salt solution containing glucose as an energy substrate. The enzyme mixture effectively separates follicles of different sizes derived from ovaries of hamster, mouse, rat, pig and human. While no damage of the basal lamina is apparent for the hamster and human follicles, some loss is present in the other species. Follicles were classified into several stages or classes based on their diameter, the number of granulosa cell layers and the presence of an antral cavity. Pre-antral hamster and human follicles cultured in the presence of follicle stimulating hormone for up to 168 h developed an antral cavity with intact germinal vesicle oocytes.

Follicular development through preantral stages: signalling via growth factors.

Although gonadotrophins, particularly FSH, are the primary pituitary regulators of ovarian folliculogenesis, the involvement of ovarian-derived growth factors in follicular growth and maturation has become increasingly apparent over the past decade. Regulators of ovarian somatic cells can be broadly divided into (1) mitogenic factors, and (2) differentiation-induction factors, based on two primary cellular requirements, that is, proliferation and differentiation that are fundamental to folliculogenesis and ovarian functions. In this article, direct as well as indirect evidence is presented that highlights the roles of epidermal growth factor--a mitogen, and transforming growth factor beta--a differentiation-induction factor, in modulating gonadotrophin action in the ovary, particularly in the preantral follicles. An exquisitely timed and regionalized expression of these two types of peptide factor, along with their membrane receptors, may determine the differential success of follicle development, hence, allowing selection of the best oocytes for fertilization and subsequent development.

Attenuated mutants of Venezuelan equine encephalitis virus containing lethal mutations in the PE2 cleavage signal combined with a second-site suppressor mutation in E1.

The PE2 cleavage signal in a full-length cDNA clone of the alphavirus Venezuelan equine encephalitis virus (VEE) was ablated by site-directed mutagenesis. RNA transcripts derived from the resulting plasmids programmed the production of nonviable particles upon transfection of baby hamster kidney (BHK) cells. However, the mutant RNAs also gave rise to a small proportion of viable revertants. Analysis of these biological revertants and their molecularly cloned homologs demonstrated that second-site suppressor mutations at either E2 position 243 or E1 position 253 were able to restore viability to PE2 cleavage signal mutants. The viable revertants incorporated unprocessed PE2 into particles which showed normal infectivity for BHK cells, but reduced ability to grow in C6/36 mosquito cells. A mutant carrying a lethal PE2 cleavage signal mutation in combination with a suppressor at E1 253 was either avirulent or highly attenuated in adult mice when inoculated by the subcutaneous, intracerebral, or intranasal route and conferred complete protection against both intraperitoneal and intranasal challenge with virulent VEE. These results indicate the close functional association of the E2 and E1 proteins in the alphavirus spike. They also have implications for the design of recombinant live virus vaccines for VEE, for other alphaviruses, and for other viruses that use a similar mechanism for glycoprotein maturation.

In vitro effects of interactions of follicle-stimulating hormone, luteinizing hormone, and prolactin on progesterone synthesis by rat luteal cells during pregnancy.

The in vitro ability of ovine (o) follicle-stimulating hormone (FSH), (o)luteinizing hormone (LH), (o)prolactin (PRL), and recombinant human FSH (rhFSH) to stimulate progesterone (P4) synthesis by rat corpora lutea on Day 4 of pregnancy was investigated. Dispersed luteal cells (large + small cells) were incubated in the presence of the gonadotropins (1-100 ng) alone or in various combinations (10 ng each) for 4 or 24 hr. Given alone, all the ovine preparations stimulated P4 in a dose-dependent manner with even 1 ng of each hormone significantly enhancing P4 production. Significantly, rhFSH--which is devoid of LH contamination--at 10 and 100 ng also stimulated P4 production, thus clearly establishing for the first time that FSH is a luteotropic hormone in the rat. The combination of oFSH + LH + PRL (10 ng each) significantly stimulated P4 synthesis to a greater extent than the combination of any two hormones or individual hormones at both 4 hr or an additional 24 hr of incubation (P < 0.05). This verified in vitro a previously established in vivo luteotropic complex. One hundred nanamolars of phorbol 12-myristate 13-acetate (PMA) did not affect basal P4 secretion but inhibited cAMP, oFSH, and oLH stimulation of P4. Thus, the luteotropic effects of FSH, LH, and activators of protein kinase A are antagonized by the protein kinase C pathway.

In vitro and in vivo evidence on the site of neutralization of equine chorionic gonadotrophin (eCG) by an eCG antiserum.

This study was designed to determine whether the major site of eCG neutralization by an antiserum to the hormone is at the peripheral or ovarian level. Hamsters hypophysectomized at oestrus were injected s.c. with 25 iu eCG. Three days later, preovulatory follicles were dissected and cultured for 5 h and the medium was changed every hour. At the end of the first hour of incubation, oestradiol and androstenedione accumulation was high, with a sharp drop over the next 4 h, whereas progesterone concentrations did not change over the entire period. Addition of eCG antiserum to the incubated follicles did not affect steroidogenesis. Addition of 1.0 iu eCG in the second hour or every hour sustained oestradiol production at supraphysiological amounts. However, addition of eCG plus eCG antiserum every hour eliminated the stimulatory effects of eCG on oestradiol production. In another experiment, hamsters injected with eCG were treated 3 days later by i.p. injection of eCG antiserum and groups of animals were killed over the next 8 h. Serum samples before and after injecting eCG antiserum were incubated overnight with a goat anti-rabbit immunoglobulin to separate free, unbound eCG from bound eCG. At time zero (before injecting the antiserum) free eCG was increased, but within 1 h after eCG antiserum there was an eightfold decrease of the hormone, and these concentrations were maintained over the next 7 h. The fall in unbound eCG in vivo coincided with the decay in serum oestradiol and androstenedione.(ABSTRACT TRUNCATED AT 250 WORDS)

Follicle-stimulating hormone, human chorionic gonadotropin, and prolactin receptors in hamster corpora lutea or dispersed luteal cells during pregnancy.

In vitro progesterone (P4) production by hamster luteal cells is stimulated throughout pregnancy by FSH and LH. Prolactin (PRL) by itself, however, increases P4 synthesis only on Day 12; on Day 4, FSH+LH+PRL induces optimal P4 secretion [Biol Reprod 1994; 51:43-49]. In light of these findings, in this study we investigated FSH, hCG, and PRL receptors in hamster CL or dispersed luteal cells on Days 4, 8, and 12 of pregnancy. Scatchard analysis of hamster CL on Days 4 and 8 showed considerably more unoccupied hCG receptors than FSH receptors: on Day 4, there was 9.5 fmol/mg protein for FSH binding sites vs. 1741 fmol/mg protein for hCG binding. Moreover, the binding affinity of hCG was greater than for FSH: the Day 4 Kd was 0.136 nM for hCG vs. 0.308 for FSH. Similar differences were observed on Day 8. Dispersed luteal cells (large+small cells) were incubated for 24 h with or without 10 ng of ovine FSH, LH, and PRL or human recombinant FSH (r-hFSH), alone or in different combinations. The cells were then washed and incubated for 4 h with iodinated hCG, FSH, or PRL with or without 100-fold excess of unlabeled hormones. The number of binding sites per 200,000 luteal cells did not change appreciably for FSH and hCG on Days 4 and 12 of pregnancy, whereas PRL binding sites significantly increased on Day 12.(ABSTRACT TRUNCATED AT 250 WORDS)

Luteotropic effects of follicle-stimulating hormone (FSH): II. FSH luteinizing hormone, and prolactin effects on second messenger systems in the corpus luteum of the pregnant hamster.

We have recently shown that FSH, LH, and prolactin (PRL)--alone or combined--act as luteotropins when incubated with luteal cells from pregnant hamsters (Yuan and Greenwald, Biol Reprod 1994; 51:43-49). The purpose of the present study was to determine which second messenger systems are affected by these hormones with progesterone (P4) synthesis as the principal endpoint after 4 h of incubation with 100,000 luteal cells. Luteal cells on Days 4, 10, or 12 of pregnancy were incubated with the following reagents: 10 ng of recombinant human FSH (r-hFSH), ovine (o) FSH, oLH, oPRL, forskolin, db-cAMP, protein kinase A inhibitor (PKI), protein kinase C activator (phorbol 12-myristate 13-acetate; PMA), or various combinations of the reagents. Forskolin and db-cAMP each stimulated P4 in a dose-dependent manner, while PKI significantly inhibited forskolin-, r-hFSH-, oFSH-, and oLH-stimulated P4 on Day 4 of pregnancy. PMA (0.001-1.0 microM) did not affect basal P4 on Day 4, 10, or 12 of pregnancy; however, 100 nM PMA inhibited db-cAMP-, forskolin-, oFSH-, and oLH-stimulated P4 synthesis on Days 4 and 12. The antagonistic effects of PMA were reversed in all cases by concurrent incubation with a PKC inhibitor, H-7. On Day 4 of pregnancy, P4 was stimulated by oFSH and oLH with the highest levels observed in medium stimulated by the luteotropic complex of oFSH, oLH, and oPRL. Recombinant hFSH enhanced P4 production in a dose-dependent manner; doses of 10 ng and above resulted in statistically significant differences from the control values (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Abortifacient effects in the pregnant hamster of an antibody to progesterone are reversed by exogenous prolactin.

A single i.p. injection of 10 nmol of a monoclonal antibody to progesterone (mAb-P4) on Day 4 of pregnancy (day of nidation) interrupts pregnancy by Day 8 (Day 1 = sperm-positive smear) in 75% of treated hamsters (n = 8). This correlates with structural and functional luteolysis, significantly (p < 0.05) reduced serum prolactin (PRL), and a nonsignificant trend for reduced FSH (which constitute the minimal luteotropic complex of the hamster), but LH is unchanged. Embryos implant and develop normally for a while, but by Day 8 the uterus is distended with the resorbing products of conception. The abortifacient effects of passive immunization against progesterone are reversed 100% by s.c. injection of 100 micrograms PRL daily on Days 4-7; deferring PRL treatment until Days 6-7 maintained pregnancy in 75% of the animals, still significantly different from the untreated mAb-P4 group. Injection of 50 micrograms PRL on Days 4-7 maintains pregnancy in 50% of the mAb-P4-treated hamsters (not significantly different), whereas 10 micrograms PRL on Days 4-7 is wholly ineffective. No dose of FSH (0.1-10.0 micrograms) or LH (0.4-2.0 micrograms) on Days 4-7 reversed the effects of mAb-P4; neither did 10 micrograms PRL plus 0.4 or 2 micrograms of FSH. The maintenance of pregnancy after 100 micrograms PRL on Days 4-7 is associated with normal serum levels of PRL, FSH, and LH and no change in the serum concentration of the mAb-P4 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Luteotropic effects of follicle-stimulating hormone (FSH): I. FSH has in vitro luteotropic and synergistic effects with luteinizing hormone and prolactin on progesterone production by hamster luteal cells during pregnancy.

This study was designed to evaluate the in vitro ability of FSH, LH, and prolactin (PRL) to stimulate progesterone (P4) production by enzymatically dispersed CL cells from pregnant hamsters. In light of previous in vivo findings [1], we were especially interested in determining whether FSH is a luteotropin. The CL were collected and pooled on Days 4, 8, 12, and 16 of gestation (Day 1 = sperm-positive vaginal smear). After enzymatic dissociation, combined large and small luteal cells (LC+SC) were incubated in the presence of 10 ng ovine (o) FSH, oLH, and oPRL, alone or in various combinations, for a total of 144 h with the first medium change at 24 h and other changes every 48 h thereafter. FSH and LH alone significantly increased P4 production on Days 4, 8, and 12, while PRL alone increased P4 only on Day 12 (p < 0.05). The combination oFSH+oLH+oPRL significantly stimulated P4 production on Day 4 to a greater extent than the combination of any two hormones (p < 0.05). Ovine FSH+oLH enhanced P4 production on Days 12 and 16 at 48, 96, and 144 h of incubation, to an extent greater than either hormone alone (p < 0.05). When recombinant human FSH (r-hFSH), which is devoid of LH activity, was added (1-100 ng) to dispersed luteal cells from Day 4 pregnant hamsters, a dose-response increase in P4 was evident (p < 0.05); even 1 ng r-hFSH stimulated P4 production at 96 h (p < 0.05). On Day 2 of the cycle, oFSH or oLH, but not oPRL, also significantly stimulated P4 production (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

A molecular genetic approach to the study of Venezuelan equine encephalitis virus pathogenesis.

Viral pathogenesis can be described as a series of steps, analogous to a biochemical pathway, whose endpoint is disease of the infected host. Distinct viral functions may be critical at each required step. Our genetic approach is to use Venezuelan equine encephalitis virus (VEE) mutants blocked at different steps to delineate the process of pathogenesis. A full-length cDNA clone of a virulent strain of VEE was used as a template for in vitro mutagenesis to produce attenuated single-site mutants. The spread of molecularly cloned parent or mutant viruses in the mouse was monitored by infectivity, immunocytochemistry, in situ hybridization and histopathology. Virulent VEE spread through the lymphatic system, produced viremia and replicated in several visceral organs. As virus was being cleared from these sites, it began to appear in the brain, frequently beginning in the olfactory tracts. A single-site mutant in the E2 glycoprotein appeared to block pathogenesis at a very early step, and required a reversion mutation to spread beyond the site of inoculation. The feasibility of combining attenuating mutations to produce a stable VEE vaccine strain has been demonstrated using three E2 mutations.

Synergistic effects of steroids with FSH on folliculogenesis, steroidogenesis and FSH- and hCG-receptors in hypophysectomized mice.

Injection of ovine FSH (4 micrograms day-1) for 4 days into hypophysectomized mice does not restore folliculogenesis to normal cyclic values. This may be due to insufficient production of oestradiol. The present study was designed to determine whether FSH- and LH-induced oestradiol was critical for growth and differentiation of follicles at all stages. Twelve days after hypophysectomy, mice were injected s.c. with 10, 50 or 250 micrograms oestradiol cyclopentylpropionate daily with or without ovine FSH (4 micrograms day-1) for 1-4 days. One ovary from each animal was used for histology. From the second ovary, follicles were isolated at different stages and incubated with [3H]thymidine for 3 h to determine the rate of DNA synthesis. Incubation medium and serum were used for steroid determinations. After oestradiol treatment alone, there was a dose-dependent response in serum oestradiol, but ovarian and uterine weights did not increase further with the increasing doses of oestradiol administered. This finding was consistent with an increase in the number of preantral follicles and small antral follicles but excluding the development of preovulatory follicles. Treatment with 10 and 50 micrograms of oestradiol cyclopentylpropionate did not prevent antral follicles from undergoing atresia. The higher dose (250 micrograms day-1) of oestradiol cyclopentylpropionate delayed atresia of antral follicles and maintained more large healthy antral follicles. After concurrent injection of oestradiol and FSH, ovarian weight was 2-3 times greater than with either FSH or oestradiol alone; the number of follicles and follicular DNA synthesis at all stages of development increased without any signs of atresia; the in vitro accumulation of oestradiol also increased. Oestradiol alone induced FSH receptors in granulosa cells, but did not induce hCG receptors in any ovarian compartment; FSH alone induced FSH and hCG receptors in granulosa cells but not in thecal-interstitial tissues, whereas, oestradiol plus FSH enhanced FSH receptors in granulosa cells and LH/hCG receptors in granulosa and thecal-interstitial tissues. The synergistic effect of oestradiol with FSH was mimicked by the same dose of diethylstilboestrol, testosterone or dihydrotestosterone, but the latter steroids increased only the number of antral follicles, presumably because of their shorter half-lives. These results indicate that in mice oestradiol stimulates the growth of preantral and antral follicles and delays follicular atresia; oestrogens and androgens act synergistically with FSH to enhance follicular proliferation and differentiation, and prevent follicles from undergoing atresia.

Human chorionic gonadotropin or human recombinant follicle-stimulating hormone (FSH)-induced ovulation and subsequent fertilization and early embryo development in hypophysectomized FSH-primed mice.

Adult hypophysectomized FSH-primed mice were used to study ovulation, fertilization, and preimplantation embryo development. Twelve days after hypophysectomy, animals were injected sc with oFSH (4 micrograms/day) twice a day for 3 days. This resulted in large preovulatory follicles that did not secrete estrogen. Concurrent with the last FSH injection, either hCG (5 IU) or human recombinant FSH (10 IU) was injected sc to induce ovulation. Animals were mated or not and killed 1-4 days later. The ovulation rate was similar for both the hCG-induced group (FH) and the FSH-induced group (FF), 97% and 90%, respectively. About 45% of the FH mice mated successfully with 56% of the eggs fertilized compared to only 25% of the FF mice with 45% of the eggs fertilized. However, only 5% of ovulated eggs developed to four-cell stages in vivo by day 3 for the FH animals and none in the FF group. To determine the reasons for the in vivo retarded embryo development, embryos at the one- or two-cell stage were collected on day 2 from the FH group. After 96 h of culture, 22% of two-cell embryos were converted to blastocysts, and 11% of one-cell eggs divided to the four-cell stage. In contrast, 80% of two-cell embryos from normal mice develop into blastocysts by 72 h of culture. The ovarian incubation medium and serum were used to measure progesterone (P4), androstenedione (A), and estradiol (E2). The patterns of serum and in vitro production of steroids were parallel. In FH mice, P4 increased immediately on the day after hCG injection (day 1) and decreased progressively on days 2 and 3; A and E2 levels increased on day 2, A decreased on day 3, and E2 decreased on day 4. When human recombinant FSH was used to induce ovulation, there were no significant changes in serum P4 and A; E2 levels were about 4 times higher on day 1 than in the FSH-primed control, then dropped to baseline levels on days 2 and 3. However, on day 3 in both the FH and FF groups, FSH receptors were still present on the granulosa cells of antral follicles, and LH/hCG receptors were present on the granulosa cells of large antral follicles and newly formed corpora lutea.(ABSTRACT TRUNCATED AT 400 WORDS)