Regulation of folliculogenesis and the determination of ovulation rate in ruminants.

The paper presents an update of our 1993 model of ovarian follicular development in ruminants, based on knowledge gained from the past 15 years of research. The model addresses the sequence of events from follicular formation in fetal life, through the successive waves of follicular growth and atresia, culminating with the emergence of ovulatory follicles during reproductive cycles. The original concept of five developmental classes of follicles, defined primarily by their responses to gonadotrophins, is retained: primordial, committed, gonadotrophin-responsive, gonadotrophin-dependent and ovulatory follicles. The updated model has more extensive integration of the morphological, molecular and cellular events during folliculogenesis with systemic events in the whole animal. It also incorporates knowledge on factors that influence oocyte quality and the critical roles of the oocyte in regulating follicular development and ovulation rate. The original hypothetical mechanisms determining ovulation rate are retained but with some refinements; the enhanced viability of gonadotrophin-dependent follicles and increases in the number of gonadotrophin-responsive follicles by increases in the throughput of follicles to this stage of growth. Finally, we reexamine how these two mechanisms, which are thought not to be mutually exclusive, appear to account for most of the known genetic and environmental effects on ovulation rate.

Glucose uptake and lactate production by the autotransplanted ovary of the ewe during the luteal and follicular phases of the oestrous cycle.

Two experiments were carried out on ewes with ovarian autotransplants to estimate the ovarian uptake of glucose and production of lactate. The first was carried out in the luteal phase of the oestrous cycle. Samples of carotid arterial, ovarian venous and jugular venous blood were collected simultaneously for glucose analysis. The arterial concentration of glucose (58.0 +/- 5.0mg/dL; Mean+/-SEM) was significantly higher than the ovarian venous concentration (42.3+/-2.4 mg/dL; P<0.001). Next, a second more complete experiment was carried out in the luteal and follicular phases of the oestrous cycle. The oestrous cycle was synchronised and samples of carotid arterial, ovarian venous and jugular venous blood were collected simultaneously for glucose and lactate analysis. There were significant positive arterio-venous differences in the concentration of glucose in the luteal (5.6+/-1.2mg/dL, mean+/-SEM; P=0.001), early (3.1+/-0.82 mg/d; P=0.003) and late follicular (6.4+/-1.3mg/dL; P=0.001) phases of the oestrous cycle. There was a significant negative arterio-ovarian venous difference in the concentration of lactate in only the luteal phase (-2.2+/-0.96 mg/dL; P=0.043). The results show significant removal of glucose from the arterial circulation during its passage through the ovary in the luteal, early follicular and late follicular phases of the oestrous cycle. Furthermore, there was lactate production in the luteal phase but not in the follicular phase suggesting that in the luteal phase of the oestrous cycle, ovarian metabolism can be anaerobic.

Effect of direct ovarian infusion of bone morphogenetic protein 6 (BMP6) on ovarian function in sheep.

Bone morphogenetic protein 6 (BMP6) has been suggested as an important local factor capable of modulating the stimulatory actions of follicle-stimulating hormone in granulosa cells in vitro. The objective of this experiment was to determine the effect of direct ovarian infusion of BMP6 (2 microg/h) on ovarian function in ewes with an autotransplanted ovary. Treated ewes (n = 6) and vehicle-treated controls (n = 6) were infused during the early follicular phase, between 12 and 24 h after luteal regression, and ovarian response was determined by collection of samples of ovarian venous blood and transdermal ultrasound. In the absence of any change in circulating gonadotropins or in the antral follicle population, BMP6 infusion resulted in acute but transient increases in ovarian inhibin A, androstenedione, and estradiol secretion (P < 0.05) during the second half of the infusion period. Thereafter, treated animals had an advance in the time of the LH surge by around 10 h (43.3 +/- 2.8 h in treated vs. 53.3 +/- 2.7 h in controls; P < 0.05) and smaller preovulatory follicles (4.1 +/- 0.2 mm in treated vs. 5.3 +/- 0.1 mm in controls; P < 0.01), which gave rise to smaller corpora lutea (9.5 +/- 0.8 mm in treated vs. 11.7 +/- 0.6 mm in controls; P < 0.05). There was, however, no effect of infusion on ovulation rate. Despite the changes in the size of the ovulatory follicles, when the hormonal data were aligned to the time of the luteinizing hormone surge, there were no differences in preovulatory estradiol, androstenedione, or inhibin A between groups. This study therefore provides strong in vivo evidence to support the hypothesis that BMP6 is an important local regulator of ovarian function and that alterations in BMP6 cellular signaling may explain some of the effects of the FecB mutation in inducing precocious maturation of ovulatory follicles.

Advances in recombinant DNA technology: corifollitropin alfa, a hybrid molecule with sustained follicle-stimulating activity and reduced injection frequency.

BACKGROUND: Recombinant DNA technologies have been used to develop longer-acting therapeutic proteins. One approach is to introduce sequences containing additional glycosylation sites. Using this technique, a new chimeric gene has been developed containing the coding sequences of the FSH beta-subunit and the C-terminal peptide of the hCG beta-subunit, which bears four O-linked oligosaccharide binding sites. Co-expression of the alpha-subunit and the chimeric FSH beta-subunit produces a new recombinant molecule, named corifollitropin alfa, with a prolonged elimination half-life and enhanced in vivo bioactivity compared with wild-type FSH. METHODS: Medline searches by subject and additional searching by hand. RESULTS: Initial studies in pituitary suppressed female volunteers confirmed the extended half-life of the compound. Phase II studies have shown that corifollitropin alfa is able to induce and sustain multi-follicular growth for an entire week in women undergoing ovarian stimulation using GnRH antagonist co-treatment for IVF. Corifollitropin alfa regimens have been developed with dosages of 100 and 150 microg, for patients with body weight 60 kg, respectively. CONCLUSIONS: Corifollitropin alfa is the first long-acting hybrid molecule with sustained follicle-stimulating activity developed for the induction of multi-follicular growth along with GnRH antagonist co-treatment for IVF. This new treatment option may be simpler and more convenient for patients compared with conventional long protocols of daily FSH injections in combination with GnRH agonist co-treatment. The safety and efficacy of such regimens is currently being evaluated in large comparative phase III clinical trials. The development of corifollitropin alfa is the first step towards a new generation of recombinant gonadotrophins.

Ovarian cryopreservation for fertility preservation: indications and outcomes.

Female fertility preservation provides significantly different challenges to that for the male, with the only established method being cryopreservation of embryos thus necessitating the involvement of a male. Other, experimental, options include oocyte or ovarian tissue cryopreservation. The latter has been regarded as a potential method for more than a decade, but has resulted in the birth of only five babies. It is not possible to be certain how many women have had ovarian tissue cryopreserved. Oocyte cryopreservation also remains experimental, but approximately 100-fold more babies have been born through this technique over the last two decades. Ovarian tissue cryopreservation has the potential advantages of preservation of a large number of oocytes within primordial follicles, it does not require hormonal stimulation when time is short and indeed may be appropriate for the pre-pubertal. Disadvantages include the need for an invasive procedure, and the uncertain risk of ovarian contamination in haematological and other malignancies. We here review this approach in the context of our own experience of 36 women, highlighting issues of patient selection especially in the young, and uncertainties over the effects of cancer treatments on subsequent fertility. Of these 36 women, 11 have died but 5 have had spontaneous pregnancies. So far, none have requested reimplantation of their stored ovarian tissue. Ovarian cryopreservation appears to be a potentially valuable method for fertility preservation, but the indications and approaches best used remain unclear.

A novel estrogen-free oral contraceptive pill for women: multicentre, double-blind, randomized controlled trial of mifepristone and progestogen-only pill (levonorgestrel).

BACKGROUND: The acceptability and continuation rate of oral contraceptive steroids are limited by unpredictable bleeding and the fear of long-term risks such as breast cancer. By inhibiting ovulation and by altering the receptivity of the endometrium, antagonists of progesterone, such as mifepristone, could be developed as estrogen-free novel contraceptives. METHODS: Multicentre, double-blind, randomized controlled trial comparing frequency of amenorrhoea (primary outcome), bleeding patterns, side effects and efficacy in women taking daily 5 mg mifepristone (n = 73) or 0.03 mg levonorgestrel (progestogen-only pill; POP, n = 23) for 24 weeks. RESULTS: More women were amenorrhoeic while taking mifepristone than POP (49 versus 0% P < 0.001), and fewer women bled or spotted for >5 days per month (4 versus 39% P < 0.001). Forty-eight percent of women who took mifepristone for 6 months had cystic glandular dilatation of the endometrium but none showed hyperplasia or atypia. There were no pregnancies in 356 months of exposure in women who used only mifepristone for contraception. Two pregnancies occurred in women taking mifepristone who were also using condoms for dual protection. CONCLUSIONS: Daily mifepristone (5 mg) is an effective oral contraceptive pill which has a better pattern of menstrual bleeding than an existing POP (levonorgestrel).

Mifepristone induced progesterone withdrawal reveals novel regulatory pathways in human endometrium.

In women, a single dose of the antiprogestin mifepristone (RU486) in the secretory phase rapidly renders the endometrium unreceptive and is followed by endometrial breakdown and menstruation within 72 h. This model provides a system to identify progesterone-regulated genes, which may be involved in endometrial receptivity and the induction of menstruation. We used cDNA microarrays to monitor the response of the endometriuim over 24 h following administration of mifepristone in the mid-secretory phase. We identified 571 transcripts whose expression was significantly altered, representing 131 biochemical pathways. These include new progesterone regulated members of the Wnt, matrix metalloproteinase (MMP), prostaglandin (PG) and chemokine regulatory pathways. Transcripts involved in thyroid hormone metabolism and signalling such as type II iodothyronine deiodinase and thyroid receptors were also found to be highly regulated by progesterone antagonism in the endometrium. Transcripts required for thyroid hormone synthesis such as thyroid peroxidase (TPO) and thyroglobulin (TG) were also expressed, indicating that the endometrium may be a site of thyroxin production. These results add to the existing knowledge of the role of the Wnt, chemokine, MMP and PG pathways in receptivity and early menstrual events. They provide in vivo evidence supporting direct or indirect regulation of many new transcripts by progesterone. We have also identified for the first time the very early transcriptional changes in vivo in response to progesterone withdrawal. This greatly increases our understanding of the pathways leading to menstruation and may provide new approaches to diagnose and treat menstrual disorders.

The effect of the presence and pattern of luteinizing hormone stimulation on ovulatory follicle development in sheep.

The aim of this study was to examine the role of LH on the growth of the large preovulatory follicle and its secretion of hormones in sheep. Ewes with ovarian autotransplants were treated with GnRH-antagonist at the time of luteal regression and different LH regimes applied for 60-66 h before administration of an ovulatory stimulus (hCG). In Experiment 1 (N = 24; n = 8), ewes received either no LH or constant or pulsatile infusion of LH at the same dose (1.25 microg/h). In Experiment 2 (N = 12, n = 6), LH was constantly infused at a rate of 1.25 microg or 2.5 microg oLH/h. In Experiment 1, animals receiving either pulsatile or constant LH exhibited increases in estradiol and inhibin A secretion (P < 0.001) and a depression in FSH (P < 0.001) that resembled the normal follicular phase. Similarly in Experiment 2, doubling the dose of LH resulted in a two-fold increase in ovarian estradiol secretion (P < 0.05) but no other changes. All animals receiving LH, regardless of the pattern of stimulation, ovulated and established a normal luteal phase. In contrast, no LH treatment resulted in constant immuno-active LH without pulses, unchanged FSH and inhibin A concentrations (P < 0.05), and basal estradiol secretion (P < 0.001). Morphologically normal large antral follicles were observed in this group and although corpora lutea formed in response to hCG, progesterone profiles were abnormal. In conclusion, these results suggest that LH is an essential requirement for normal ovulatory follicle development and subsequent luteal function and show that a pulsatile mode of LH stimulation is not required by ovulatory follicles.

Enhanced response of granulosa and theca cells from sheep carriers of the FecB mutation in vitro to gonadotropins and bone morphogenic protein-2, -4, and -6.

The FecB (Booroola) mutation, which leads to increased ovulation rates and multiple births in sheep, is now known to occur in the signaling domain of the bone morphogenic protein (BMP)-1B receptor. We examined the effect of the mutation on the responsiveness of granulosa (GC) and theca cells (TC) to BMPs and other local regulators using tissue from animals with (Fec(B/B)) and without (Fec(+/+)) the FecB mutation. Experiments examined the effect of BMP-2, -4, and -6 (0.005-50 ng/ml), and their interaction with IGF-I (0.1-10 ng/ml LR3 analog) and gonadotropins, on the proliferation and differentiation of GCs and TCs isolated from small (<2 mm) antral follicles and maintained in serum-free culture for up to 8 d. Dose-finding studies using ovaries from wild-type sheep obtained from the abbattoir showed no difference among the different BMPs in stimulating (P < 0.001) estradiol (E2) production by GCs cultured with FSH (10 ng/ml), but there was a clear interaction (P < 0.001) with IGF-I. BMPs had no effect on GC proliferation or the sensitivity of GCs to FSH. In contrast, higher doses of BMPs (5-50 ng/ml) inhibited LH-stimulated androstenedione production by TCs, whereas lower doses (0.005-0.05 ng/ml) stimulated TC proliferation (P < 0.01). Regardless of dose of IGF-I, at the end of culture (96-192 h) hormone production by GCs (E2, inhibin A) and TCs (androstenedione) was 4- to 5-fold greater (P < 0.001) by cells from Fec(B/B), compared with Fec(+/+) ewes exposed to the same dose of gonadotropin. In the presence of low concentrations of IGF-I (0.1 ng/ml), the maximum increase in the production of E2 and inhibin A by GCs from FF ewes in response to BMPs was observed at doses that were 3- to 10-fold lower (3-10 ng/ml) than ++ (30 ng/ml; P < 0.001). Low doses of BMPs stimulated proliferation of TCs from ++ (P < 0.01) but not FF ewes. Immunohistochemistry confirmed BMP-6 protein expression in the oocyte, granulosa, and thecal layers of antral follicles from both genotypes. These results confirm a major role for BMPs in controlling ovarian somatic cell function in sheep and provide evidence to support the hypothesis that the FecB mutation increases the BMP response of somatic cells when stimulated to differentiate by gonadotropins.

Fertility and ageing.

The late 20th century trend to delay birth of the first child until the age at which female fecundity or reproductive capacity is lower has increased the incidence of age-related infertility. The trend and its consequences have also stimulated interest in the possible factors in the female and the male that may contribute to the decline in fecundity with age; in the means that exist to predict fecundity; and in the consequences for pregnancy and childbirth. In the female, the number of oocytes decreases with age until the menopause. Oocyte quality also diminishes, due in part to increased aneuploidy because of factors such as changes in spindle integrity. Although older male age affects the likelihood of conception, abnormalities in sperm chromosomes and in some components of the semen analysis are less important than the frequency of intercourse. Age is as accurate as any other predictor of conception with assisted reproductive technology. The decline in fecundity becomes clinically relevant when women reach their mid-30s, when even assisted reproduction treatment cannot compensate for the decline in fecundity associated with delaying attempts at conceiving. Pregnancies among women aged >40 years are associated with more non-severe complications, more premature births, more congenital malformations and more interventions at birth.

Corpora lutea induced by gonadotrophin-releasing hormone treatment of anoestrous Welsh Mountain ewes: reduced sensitivity to luteinizing hormone in vivo and to chorionic gonadotrophin in vitro.

Seasonally anoestrous Welsh Mountain ewes received 250 ng gonadotrophin-releasing hormone (GnRH) every 2 h, with (Group 1; n=13) or without (Group 2; n=14) progesterone priming for 48 h. Fourteen control ewes (Group 3) were studied during the luteal phase in the breeding season. Animals in Group 4 (n=12) received progesterone priming followed by 250 ng GnRH at increasing frequency for 72 h, while ewes in Group 5 (n=13) were given three bolus injections of 30 microg GnRH at 90-min intervals. All treatment regimens induced ovulation. However, only corpora lutea (CL) from ewes in Group 3 (breeding season) or Group 4 exhibited normal luteal function. Luteal luteinizing hormone (LH) receptor levels were significantly higher on day 12 than day 4, and CL from groups with adequate CL (3 and 4) had significantly higher (125)I-human chorionic gonadotrophin (hCG)-binding levels than the three groups with inadequate CL on day 12. LH-binding affinity was unchanged. Exogenous ovine LH (10 microg) in vivo on days 3 or 11 after ovulation induced a pulse of progesterone in ewes with adequate CL: however, ewes in Groups 1, 2 and 5 showed no significant response. Basal progesterone secretion in vitro was significantly greater on day 4 than on day 12. Maximal steroidogenic responses of adequate and inadequate CL to hCG and to dibutyryl cyclic-3',5'-AMP were similar at both stages of the luteal phase. However, the EC50 for hCG on days 4 and 12 was 10-fold lower for groups with an adequate CL (0.1 IU hCG/ml) than for inadequate-CL groups (1 IU hCG/ml; P <0.05). Thus, in addition to the well-characterized premature sensitivity of GnRH-induced inadequate CL to endometrial luteolysin, we have shown (1) a marked decrease in total number of cells in the CL, a profound reduction in vascular surface area, and a decrease in mean large luteal cell volume (with no change in large luteal cell numbers), (2) decreased luteal LH receptor and progesterone content compared with adequate CL and (3) that CL that were becoming, or were destined to become, inadequate failed to respond to ovine LH in vivo and were 10-fold less sensitive to hCG in terms of luteal progesterone secretion in vitro.

Reduced LH sensitivity in vivo and in vitro of corpora lutea induced during anoestrus by GnRH, and during the late breeding season, in Scottish Blackface ewes.

Scottish Blackface ewes were synchronised in mid-breeding (November; group 1; n=12 ewes) or late-breeding season (March; group 2; n=16). Anoestrous ewes (May) were treated with progestagen sponges for 7 days and then given 250 ng GnRH 3-hourly for 24 h, 2-hourly for 24 h and hourly for a further 24 h (group 3; n=12). A second group of anoestrous ewes (group 4, n=19) received three bolus injections (30 microg) of GnRH at 90-min intervals without progestagen pretreatment. After ovulation, ewes were bled twice daily until slaughter (day 4 or day 12: oestrus=day 0). Mid-breeding season (group 1) and anoestrous ewes in group 3 formed 'adequate' corpora lutea (CL) with high plasma progesterone levels (3-4 ng/ml) maintained for at least 12 days, and responded in vivo to ovine LH (oLH) (10 microg) with a rise in plasma progesterone on day 11 (group 3, but not group 1, ewes also responded on day 3). CL minces from these ewes responded to human chorionic gonadotrophin (hCG) in vitro with a dose-dependent increase in progesterone secretion. Ewes in group 4 had a foreshortened luteal phase (8-10 days) and low plasma progesterone levels (approximately 1 ng/ml), consistent with formation of inadequate CL. LH injection failed to induce a significant plasma progesterone increase. Furthermore, although progesterone secretion in vitro in response to maximally stimulating doses of hCG or dibutyryl cAMP (dbcAMP) was similar to that in adequate CL, the sensitivity of these CL to hCG (EC (effective concentration)50, 1 IU hCG/ml) was reduced 10-fold compared with adequate CL (EC50, 0.1 IU hCG/ml; P<0.01). Ewes that ovulated in the late breeding season (group 2) had high plasma progesterone, although levels began to decrease after day 10. Injection of oLH in vivo increased plasma progesterone. However, sensitivity to hCG in vitro (EC50, 0.5 IU hCG/ml) was intermediate between that of adequate luteal tissue (groups 1 and 3; EC50, 0.1 IU/ml) and that of group 4 ewes (EC50, 1 IU hCG/ml). Our data demonstrate a markedly reduced luteal sensitivity to LH in vivo and hCG in vitro in Scottish Blackface ewes with inadequate CL, and suggest that a similar loss of sensitivity to LH may occur in the late breeding season.

Direct in vivo effects of leptin on ovarian steroidogenesis in sheep.

Leptin, the metabolic fat hormone, has been shown to have effects on reproduction in mice and to modulate steroid production by cultured ovarian somatic cells in a number of species. However, a direct role of leptin on normal ovarian function in vivo has not been shown. In this paper the effect of passive immunisation against leptin (experiment 1; 20 ml antiserum or non-immune plasma i.v.; n = 6/treatment) and direct ovarian infusion of leptin (experiment 2; 0, 2 or 20 mug recombinant ovine leptin; n = 4/treatment) during the early follicular phase was investigated in sheep with ovarian autotransplants, which allow recovery of ovarian venous blood and regular non-invasive scanning of the ovary. Passive immunisation against leptin resulted in an acute increase (P < 0.05) in ovarian oestradiol secretion but had no effect on gonadotrophin concentrations, ovulation or subsequent luteal function. Conversely, direct ovarian arterial infusion of the low dose of leptin resulted in an acute decline (P < 0.05) in ovarian oestradiol secretion whereas the high dose, which resulted in supra-physiological leptin concentrations, had no effect on oestradiol production compared with the controls. Neither dose of leptin had any effect on gonadotrophin concentrations or ovulation but both doses resulted in an increase (P < 0.05) in progesterone concentrations over the subsequent luteal phase. In conclusion, together these data provide strong in vivo evidence that leptin can modulate ovarian steroidogenesis directly and acutely in ruminants and suggest that leptin is an alternate regulatory system whereby nutritional status can regulate reproductive activity.

Spontaneous conception in a young woman who had ovarian cortical tissue cryopreserved before chemotherapy and radiotherapy for a Ewing's sarcoma of the pelvis: case report.

We report the case of a 14 year old girl who presented with a non-metastatic Ewing's sarcoma involving her superior pubic ramus. She received 14 courses of alkylating agent-based chemotherapy and direct radiation to her hemi-pelvis (55 Gy) and is alive and disease-free 8 years later. Multiple biopsies of ovarian cortical tissue were cryopreserved, with her written consent, before treatment began. Ovarian failure was confirmed on completion of treatment with cessation of menses and persistently elevated serum gonadotrophin and low estradiol levels on repeated measurement over 2 years. HRT was initiated. Irregular vaginal bleeding occurred due to radiation vaginitis. Reimplantation of ovarian cortical tissue was considered at 19 years as fertility was desired, but the decision deferred. A spontaneous conception occurred 1 year later and a healthy boy (birthweight 2.9 kg, 3rd-10th centile) was delivered at term by elective Caesarean section. This is the first case of a spontaneous conception occurring in a young woman with documented ovarian failure in whom ovarian cortical tissue had been cryopreserved. Clinicians should be aware of the possibility of spontaneous conception despite confirmed ovarian failure in young women successfully treated for cancer.

Mechanisms of action of the principal prolific genes and their application to sheep production.

The prolificacy variation in sheep makes it an excellent animal model to understand the mechanisms regulating ovulation rate. Identification of mutations responsible for the increased prolificacy of the Inverdale, Booroola, Javanese, Cambridge and Belclare sheep open new avenues of investigation for the paracrine control of folliculogenesis. To date, all known mutations are in genes from ligands or receptors of the transforming growth factor beta superfamily, and point to the bone morphogenetic protein family of peptides as local regulators of ovarian follicle growth. The mechanism of action of the mutated genes is not fully understood, but results in the ovulation of a higher number of follicles with smaller diameter and fewer granulosa cells than that of the wildtype, thus speeding the differentiation of ovulatory follicles. Comparisons of the performance of Booroola-crossed flocks in different countries showed that carriers of the prolificacy mutation have higher ewe productivity but also higher perinatal mortality and lighter weight lambs. Their economic impact on the sheep industry depends on farm environment and management. Nevertheless, the diagnostic tests now available to identify the genetic mutations resulting in increased ovulation rate, will simplify the introduction of these mutations and their monitoring in flocks for research and commercial purposes.

Systemic and intraovarian effects of dominant follicles on ovine follicular growth.

The objective was to study the endocrine activity in sheep with large ovarian follicles and the effects of dominant follicles on other follicles, looking for possible intraovarian differences. Induction of dominant follicles was achieved using controlled exogenous LH pulses every 90 min over 14 days in eight Scottish Blackface ewes. During this period, follicular development was assessed by daily transrectal ultrasonography and jugular venous blood samples were collected every 12 h for FSH, LH inhibin and oestradiol assay. The exogenous LH pulses caused the appearance of large follicles in all the ewes, which reached a maximum mean diameter of 7.2 +/- 0.5 mm on Day 5.5 +/- 2.6 after first detection. In the presence of a dominant follicle, no other follicle grew to a diameter larger than 4 mm and there was a decrease in the number of new growing follicles (P < 0.05) and in the number of smaller follicles (P < 0.01). This effect of dominance was mediated by changes in FSH concentration, since FSH level decreased (P < 0.05) as dominant follicles grew and the decrease in FSH levels was related to a decline in the number of remaining follicles (P < 0.05). However, the greatest decrease in the number of small follicles growing to larger sizes was observed in the ovary ipsilateral to the dominant follicle (P < 0.05). These data confirm that the presence of a large follicle depresses the recruitment and growth of other follicles by systemic factors and provide some evidence of local inhibitors blocking the final development of other putative large follicles.

Depot testosterone with etonogestrel implants result in induction of azoospermia in all men for long-term contraception.

BACKGROUND: Combined testosterone and progestogen preparations are a promising approach to male hormonal contraception. We investigated the effect of s.c. etonogestrel with depot testosterone on spermatogenesis in normal men over a period of 48 weeks. METHODS: Fifteen healthy men received three s.c. 68 mg etonogestrel implants. Testosterone pellets (400 mg) were administered at 12 weekly intervals. RESULTS: Nine men completed 48 weeks of treatment. Four subjects chose to discontinue after 6 months, one man withdrew from the study early for personal reasons and one was withdrawn due to illness. Sperm concentrations of <1 x 10(6)/ml were achieved in all men by 16 weeks of treatment. All men became azoospermic, although the time to achieve this varied from 8 to 28 weeks. Azoospermia was maintained in eight of the nine men treated for 48 weeks, one subject showing partial recovery from 40 weeks. Testosterone levels remained in the physiological range throughout. Treatment did not result in weight gain, change in body composition or decline in high-density lipoprotein cholesterol concentrations. CONCLUSIONS: The combination of three etonogestrel implants with depot testosterone results in rapid and consistent suppression of spermatogenesis. This can be maintained for up to 1 year and may therefore be a suitable approach for a long-acting male hormonal contraceptive.

The Booroola (FecB) mutation is associated with smaller adrenal glands in young adult ewes.

The Booroola (FecB) phenotype is associated with a mutation in the bone morphogenetic protein (BMP) receptor 1B. The BMP action is important during development; surprisingly the only differences so far observed in adult animals are restricted to the ovaries where precocious development of the antral follicles and increased ovulation rate of mutant ewes is observed. The internal organs of 17 ewes homozygous for the mutation (BB) and 18 wild-type ewes (++) were macroscopically examined and weighed. No macroscopic differences were found, and the weight of the heart, liver, lungs, kidneys, and spleen were similar for both genotypes (P > 0.05). In contrast, the adrenals of BB ewes were lighter than those of ++ ewes (P < 0.05). The effect of the mutation on the adrenal function of cortisol secretion was measured at basal level and after an adrenocorticotrophic hormone challenge, before and after dexamethasone suppression. The Booroola mutation had no effect (P > 0.05) in any of these conditions. These findings indicate that the Booroola mutation also affects the size of the adrenal glands and suggest that the mutated gene could be important in the development of other organs in addition to the ovary. However, in the mutant ewes the function of the adrenal glands is not compromised or it is compensated.