Oocyte Competence of Prepubertal Sheep and Goat Oocytes: An Assessment of Large-Scale Chromatin Configuration and Epidermal Growth Factor Receptor Expression in Oocytes and Cumulus Cells.

The oocyte competence of prepubertal females is lower compared to that of adults, mainly because they originate from small follicles. In adult females, the germinal vesicle (GV) and epidermal growth factor receptor (EGFR) have been associated with oocyte competence. This study aimed to analyze GV chromatin configuration and EGFR expression in prepubertal goat and sheep oocytes obtained from small (<3 mm) and large (≥3 mm) follicles and compare them with those from adults. GV chromatin was classified from diffuse to condensed as GV1, GVn, and GVc for goats and NSN, SN, and SNE for sheep. EGFR was quantified in cumulus cells (CCs) by Western blotting and in oocytes by immunofluorescence. Oocytes from prepubertal large follicles and adults exhibited highly condensed chromatin in goats (71% and 69% in GVc, respectively) and sheep (59% and 75% in SNE, respectively). In both species, EGFR expression in CCs and oocytes was higher in prepubertal large follicles than in small ones. In adult females, EGFR expression in oocytes was higher than in prepubertal large follicles. In conclusion, GV configuration and EGFR expression in CCs and oocytes were higher in the large than small follicles of prepubertal females.

Pseudo-Starvation Driven Energy Expenditure Negatively Affects Ovarian Follicle Development.

In the present investigation, we examined whether a change in whole body energy fluxes could affect ovarian follicular development, employing mice ectopically expressing uncoupling protein 1 in skeletal muscle (UCP1-TG). Female UCP1-TG and wild-type (WT) mice were dissected at the age of 12 weeks. Energy intake and expenditure, activity, body weight and length, and body composition were measured. Plasma insulin, glucose, leptin, plasma fibroblast growth factor 21 (FGF21) and plasma insulin-like growth factor 1 (IGF1) levels were analyzed and ovarian follicle and corpus luteum numbers were counted. IGF1 signaling was analyzed by immunohistochemical staining for the activation of insulin receptor substrate 1/2 (IRS1/2) and AKT. UCP1-TG female mice had increased energy expenditure, reduced body size, maintained adiposity, and decreased IGF1 concentrations compared to their WT littermates, while preantral and antral follicle numbers were reduced by 40% and 60%, respectively. Corpora lutea were absent in 40% of the ovaries of UCP1-TG mice. Phospho-IRS1, phospho-AKT -Ser473 and -Thr308 immunostaining was present in the granulosa cells of antral follicles in WT ovaries, but faint to absent in the antral follicles of UCP1-TG mice. In conclusion, the reduction in circulating IGF1 levels due to the ectopic expression of UCP1 is associated with reduced immunostaining of the IRS1-PI3/AKT pathway, which may negatively affect ovarian follicle development and ovulation.

Gynecologic assessment of 19 adult females with cartilage-hair hypoplasia - high rate of HPV positivity.

BACKGROUND: Patients with cartilage-hair hypoplasia (CHH), a rare metaphyseal chondrodysplasia, manifest severe growth failure, variable immunodeficiency and increased risk of malignancies. The impact of CHH on gynecologic and reproductive health is unknown. Vulnerability to genital infections may predispose CHH patients to prolonged human papillomavirus (HPV) infections potentially leading to cervical, vaginal and vulvar cancer. METHODS: We carried out gynecologic evaluation, pelvic ultrasound and laboratory assessment in 19 women with genetically confirmed CHH. All patients were clinically examined and retrospective data were collected from hospital records. RESULTS: The women ranged in age from 19.2 to 70.8 years (median 40.8 years) and in height from 103 to 150 cm (median 123 cm). All women had undergone normal pubertal development as assessed by breast development according to Tanner scale and by age of menarche (mean 12.5 yrs., range 11-14 yrs). Despite significant short stature and potentially small pelvic diameters, a well-developed uterus with fairly normal size and shape was found by pelvic ultrasound in most of the patients. Ovarian follicle reserve, assessed by ultrasound was normal in relation to age in all premenopausal women it could be assessed (12 cases). Anti-Müllerian hormone was normal in relation to age in 17 women (89%). HPV was detected in 44% (8/18) and three women carried more than one HPV serotype; findings did not associate with immunological parameters. Three patients had a concurrent cell atypia in Pap smear. CONCLUSIONS: Pubertal development, reproductive hormones and ovarian structure and function were usually normal in women with CHH suggesting fairly normal reproductive health. However, the immunodeficiency characteristic to CHH may predispose the patients to HPV infections. High prevalence of HPV infections detected in this series highlights the importance of careful gynecologic follow up of these patients.

Doxorubicin-induced female reproductive toxicity: an assessment of ovarian follicular apoptosis, cyclicity and reproductive tissue histology in Wistar rats.

Doxorubicin is a widely used chemotherapeutic agent for various cancers, particularly for the female breast cancer patients. Although the rate of young female cancer patients is increasing every year, conversely the lack of knowledge of adverse effects of doxorubicin on female reproductive system insisted us to assess the toxic effects of doxorubicin on the female reproductive tissue histoarchitecture, cyclicity, and mammary glands in Wistar rats. The rats were divided into two groups depending on the treatment period, i.e., 24 h and 28 d and further subdivided into three subgroups and administered with doxorubicin at 3 mg/kg bw (subgroup I), 6 mg/kg bw (subgroup II), and equal volume of normal saline (subgroup III) intraperitoneally once during the whole treatment period. We observed a significantly altered estrous cycle with a prolonged diestrous and short proestrous in higher dose group and dose-dependent significant changes in the uteri and mammary gland histoarchitecture in 28 days treated rats as compared to control. Moreover, the micronuclei and chromosomal aberration frequency were increased significantly in both treatment groups. A significant increase in follicular atresia in ovaries of the 28 days treated rats was observed. The immunohistochemical analysis of ovarian tissues showed an increased p53 and caspase 3 expression and apoptosis in primordial follicles of treated rats. The results suggest that though doxorubicin is a potential chemotherapeutic drug for many tumors, but the risk of adverse effects on the female reproductive system is there even at low doses.

A putative role for anti-Müllerian hormone (AMH) in optimising ovarian reserve expenditure.

The mammalian ovary has a finite supply of oocytes, which are contained within primordial follicles where they are arrested in a dormant state. The number of primordial follicles in the ovary at puberty is highly variable between females of the same species. Females that enter puberty with a small ovarian reserve are at risk of a shorter reproductive lifespan, as their ovarian reserve is expected to be depleted faster. One of the roles of anti-Müllerian hormone (AMH) is to inhibit primordial follicle activation, which slows the rate at which the ovarian reserve is depleted. A simple interpretation is that the function of AMH is to conserve ovarian reserve. However, the females with the lowest ovarian reserve and the greatest risk of early reserve depletion have the lowest levels of AMH. In contrast, AMH apparently strongly inhibits primordial follicle activation in females with ample ovarian reserve, for reasons that remain unexplained. The rate of primordial follicle activation determines the size of the developing follicle pool, which in turn, determines how many oocytes are available to be selected for ovulation. This review discusses the evidence that AMH regulates the size of the developing follicle pool by altering the rate of primordial follicle activation in a context-dependent manner. The expression patterns of AMH across life are also consistent with changing requirements for primordial follicle activation in the ageing ovary. A potential role of AMH in the fertility of ageing females is proposed herein.

Direct vitamin D3 actions on rhesus macaque follicles in three-dimensional culture: assessment of follicle survival, growth, steroid, and antimüllerian hormone production.

OBJECTIVE: To investigate the direct actions of active 1,25-dihydroxy vitamin D3 (VD3) upon primate follicular development at specific stages of folliculogenesis. DESIGN: Secondary preantral follicles were isolated from rhesus monkeys ovaries, encapsulated in alginate, and cultured for 40 days. Follicles were randomly assigned to experimental groups of control, low-dose VD3 (LVD3; 25 pg/mL), and high-dose VD3 (HVD3; 100 pg/mL). SETTING: National primate research center. ANIMAL(S): Adult, female rhesus macaques (Macaca mulatta). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Follicle survival and growth, as well as oocyte size, were assessed. Progesterone (P4), androstenedione (A4), E2, and antimüllerian hormone (AMH) concentrations in culture media were measured. RESULT(S): Compared with the control group, LVD3 increased preantral follicle survival at week 2 by >66%, while HVD3 increased antral follicle diameters at week 5. Follicles with diameters ≥500 µm at week 5 were categorized as fast-growing follicles. Higher percentages of fast-growing follicles were obtained after HVD3 treatment. Although P4, A4, and E2 production by antral follicles was not altered by VD3, AMH concentrations were 36% higher in the LVD3 group relative to controls at week 5. Oocytes with larger diameters were retrieved from antral follicles developed in both LVD3 and HVD3 groups compared with controls. CONCLUSION(S): The addition of LVD3 increased preantral follicle survival and maintained AMH production by antral follicles, while HVD3 improved antral follicle growth. VD3 supplement promoted oocyte growth in in vitro-developed follicles. Direct actions of VD3 on the primate follicle appear to be both dose and stage dependent.

An assessment of anti-Müllerian hormone in predicting mating outcomes in female hamsters that have undergone natural and chemically-accelerated reproductive aging.

In mammals, female fertility declines with age due in part to a progressive loss of ovarian follicles. The rate of follicle decline varies among individuals making it difficult to predict the age of onset of reproductive senescence. Serum anti-Müllerian hormone (AMH) concentrations correlate with the numbers of ovarian follicles, and therefore, AMH could be a useful predictor of female fertility. In women and some production animals, AMH is used to identify which individuals will respond best to ovarian stimulation for assisted reproductive technologies. However, few studies have evaluated AMH's predictive value in unassisted reproduction, and they have yielded conflicting results. To assess the predictive value of AMH in the context of reproductive aging, we prospectively measured serum AMH in 9-month-old Siberian hamsters shortly before breeding them. Female Siberian hamsters experience substantial declines in fertility and fecundity by 9months of age. We also measured serum AMH in 5-month-old females treated with 4-vinylcyclohexene diepoxide (VCD), which selectively destroys ovarian follicles and functionally accelerates ovarian aging. Vehicle-treated 5-month-old females served as controls. AMH concentrations were significantly reduced in VCD-treated females yet many females with low AMH reproduced successfully. On average, both young and old hamsters that littered had higher AMH concentrations than females that did not. However, some females with relatively high AMH concentrations failed to litter, whereas several with low AMH succeeded. Our results suggest that mean AMH concentration can predict mating outcomes on a population or group level, but on an individual basis, a single AMH determination is less informative.

The effects of insulin and follicle-simulating hormone (FSH) during in vitro development of ovarian goat preantral follicles and the relative mRNA expression for insulin and FSH receptors and cytochrome P450 aromatase in cultured follicles.

The actions of different concentrations of insulin alone or in combination with follicle-stimulating hormone (FSH) were evaluated by in vitro follicular development and mRNA expression of cytochrome P450 aromatase (CYP19A1) and as receptors for insulin (INSR) and FSH (FSHR) from isolated, cultured goat preantral follicles. Goat preantral follicles were microdissected and cultured for 18 days in the absence or presence of insulin (5 and 10 ng/ml or 10 µg/ml) alone or in combination with FSH. After 18 days, the addition of the maximum concentration of insulin to the culture medium reduced follicular survival and antrum formation rates significantly compared to the other treatments. However, when FSH was added to the culture medium, no differences between these two parameters were observed. Preantral and antral follicles from the fresh control as well as from all cultured follicles still presented a normal ultrastructural pattern. In medium supplemented with FSH, only insulin at 10 ng/ml presented oocytes with higher rates of meiosis resumption compared to control, as well as oocytes in metaphase II. Treatment with insulin (10 ng/ml) plus FSH resulted in significantly increased levels of INSR and CYP19A1 mRNA compared to that with other treatments. In conclusion, 10 ng/ml insulin associated with FSH was more efficient in promoting resumption of oocyte meiosis, maintaining survival, stimulating follicular development, and increasing expression of the INSR and CYP19A1 genes in goat preantral follicles.

Ovarian aging: latest thoughts on assessment and management.

PURPOSE OF REVIEW: In the past few decades, women have been intentionally delaying pregnancy and ovarian aging has become one of the most detrimental factors of pregnancy achievement. This review will discuss contemporary methods of ovarian aging assessment and present an overview of current management strategies. RECENT FINDINGS: Antimullerian hormone (AMH) and antral follicle count (AFC) seem to be the most reliable predictors of ovarian aging appraisal. Nevertheless, they have not been shown to predict pregnancy achievement in assisted reproduction. Heritability has a high impact on ovarian aging. Employing several genetic approaches, it is now being widely investigated, but the task is far from being accomplished. Although multivariate models have not been proven to be superior to AFC, new data support the notion that chronological age and genetic markers inclusion may increase their reliability. Several strategies have been suggested to treat ovarian aging in assisted reproductive technology (ART) settings. None of the stimulation protocol manipulations have been found to be advantageous and individualization of treatment is still recommended. Ovarian priming by different androgen preparations has been shown to be promising but more randomized controlled trials are needed to support these findings. Except for oocyte donation other ART strategies have not shown a convincing benefit for ovarian aging. The new development of oocyte vitrification may well introduce opportunities for fertility preservation to women at risk of ovarian aging. SUMMARY: Proper assessment and detection of ovarian aging, employing current or developing predictors of ovarian reserve, especially genetic tests, may enable health providers to recommend, at appropriate biological time, early pregnancy achievement or fertility preservation in women at risk.

Genomic assessment of follicular marker genes as pregnancy predictors for human IVF.

Embryo selection efficiency in human IVF procedure is still suboptimal as shown by low pregnancy rates with single embryo transfer (SET). Bidirectional communication between the oocyte and follicular cells (FC) is essential to achieve developmental competence of the oocyte. Differences in the gene expression profile of FCs from follicles leading to pregnancy could provide useful markers of oocyte developmental competence. FCs were recovered by individual follicle puncture. FC expression levels of potential markers were assessed by Q-PCR with an intra-patient and an inter-patient analysis approach. Using gene expression, a predictive model of ongoing pregnancy was investigated. Using intra-patient analysis, four candidate genes, phosphoglycerate kinase 1 (PGK1), regulator of G-protein signalling 2 (RGS2), regulator of G-protein signalling 3 (RGS3) and cell division cycle 42 (CDC42) showed a difference between FCs from follicles leading to a pregnancy or developmental failure. The best predictors for ongoing pregnancy were PGK1 and RGS2. Additionally, inter-patient analysis revealed differences in FC expression for PGK1 and CDC42 between follicles leading to a transferred embryo with positive pregnancy results and those with negative results. Both inter-patient and intra-patient approaches must be taken into consideration to delineate gene expression variations in the context of follicular competence. A predictor model using biomarkers could improve the efficiency of predicting developmental competence of oocytes. These new approaches provide useful tools in the context of embryo selection and in the improvement of pregnancy rates with SET.

Assessment of estrogen receptors and apoptotic factors in cryopreserved human ovarian cortex.

The aims of this study were: (i) to examine frozen-thawed ovarian tissues for features of follicular health and atresia by histology; (ii) to assess the expression of estrogen receptors alpha (ERalpha) and beta (ERbeta) by real-time PCR; (iii) to evaluate the Bax/Bcl-2 ratio, as an apoptotic index, in the ovarian tissues before and after cryopreservation. Ovarian cortical biopsies were obtained from 11 patients. The fragments were subdivided into two groups, fresh (control tissues) and cryopreserved tissues obtained by direct plunging into liquid nitrogen. Both tissue groups were subjected to a histological evaluation of the healthy and atretic follicles, immunohistochemical localization of the ER, and a real-time PCR (qPCR) to evaluate the expression of ER, Bax, Bcl-2 as well as beta-actin, as control gene. Damage was observed in 31% of primordial, 45% of primary, and 75% of secondary follicles in the cryopreserved tissue group. The qPCR analysis showed that the level of ERbeta was greater in fresh than cryopreserved tissues, whereas the ERalpha expression and Bax/Bcl-2 ratio were similar in both tissue groups. A significant inverse association was observed between ERalpha mRNA levels in the fresh tissue group and subjects' ages. The results show that cryopreservation and thawing of human ovarian tissue does not affect the morphology of primordial or primary follicles and that cryopreservation does not affect apoptosis. However, cryopreservation seems to have an inhibitory effect on the level of ERbeta. Additional studies are needed to evaluate the differential effects of freezing follicles at different stages of follicular development and ovarian steroidogenesis.

Pituitary-ovarian axis during lactational amenorrhoea. I. Longitudinal assessment of follicular growth, gonadotrophins, sex steroids and inhibin levels before and after recovery of menstrual cyclicity.

BACKGROUND: Comparisons of follicular development and hormonal profile in the same women during and after lactational amenorrhoea (LA) are scarce. We report follicular growth, pituitary and ovarian hormone serum levels in the same women during LA and in follicular phases after resumption of menstrual cyclicity. METHODS: Serum samples were obtained from 10 women during LA between days 60 and 89 post-partum and between days 1 and 4 (early follicular phase; EFP) and 7 and 10 (mid-follicular phase; MFP) of the second and third cycles after LA. RESULTS: The number of follicles >3 mm and diameter of the largest follicle were significantly higher during LA when compared to EFP and MFP. Serum levels of inhibin B were similar in LA and EFP and increased significantly in MFP. Pro-alphaC was significantly higher in EFP than in LA and MFP. Estradiol was similar during all stages. In comparison with EFP and MFP, LA is associated with higher prolactin levels, normal or slightly elevated gonadotrophins and increased number and size of follicles without a parallel increase in estradiol, inhibin B and Pro-alphaC. CONCLUSIONS: During LA, there is a profound dissociation between follicular growth and follicular endocrine activity, which suggests an alteration in the stimulus-response relationship at the follicular level.

Proliferating cell nuclear antigen--a marker for ovarian follicle counts.

Enumerating ovarian follicles is an effective way to estimate the extent of ovarian toxicity in female rodents exposed to xenobiotics. Differential follicle counts are useful in safety assessment bioassays and in interspecies extrapolation of ovarian toxicity. Counting the follicles in H&E-stained sections is labor intensive, tedious, and costly. In the present study we demonstrated that in rat formalin-fixed, paraffin-embedded ovary sections follicles of all degrees of maturity can be visualized by the use of antibody directed against proliferating cell nuclear antigen (PCNA). Follicles are easily distinguished from ovarian background with the ability to detect and identify primordial follicles being enhanced. This translates into a significant decrease in variability of follicle counts, labor, and cost. Specifically, variability dropped from 11% to 0.2%, the counting time was reduced by 46%, and the cost by 48%.

High reproducibility of serum anti-Mullerian hormone measurements suggests a multi-staged follicular secretion and strengthens its role in the assessment of ovarian follicular status.

BACKGROUND: Our aim was to compare the intercycle reproducibility of serum anti-Mullerian hormone (AMH) measurements with that of other markers of ovarian follicular status. METHODS: Forty-seven normo-ovulatory, infertile women underwent serum AMH, inhibin B, estradiol and FSH measurements and early antral follicle (2-12 mm in diameter) counts by transvaginal ultrasound on cycle day 3 during three consecutive menstrual cycles. Reproducibility of measurements was estimated using intra-class correlation coefficient (ICC) calculation. We also assessed the number of replicate measurements theoretically needed to reach satisfactory reliability of results. RESULTS: Serum AMH showed significantly higher reproducibility (ICC, 0.89; 95% confidence interval, 0.83-0.94) than inhibin B (0.76; 0.66-0.86; P < 0.03), estradiol (0.22; 0.03-0.41; P < 0.0001) and FSH levels (0.55; 0.39-0.71; P < 0.01), and early antral follicle counts (0.73; 0.62-0.84; P < 0.001), and reached satisfactory reliability with a single measurement. CONCLUSIONS: The improved cycle-to-cycle consistency of AMH as compared with other markers of ovarian follicular status is in keeping with its peculiar production by follicles at several developmental stages and further supports its role as a cost-effective, reliable marker of ovarian fertility potential.

Morphological assessment of the effect of growth hormone on preantral follicles from 11-day-old mice in an in vitro culture system.

The present study was carried out to verify that the cells attached to the outside of the basement membrane of mechanically isolated follicles are theca cells and to evaluate the effect of growth hormone (GH) on these cells. Preantral follicles, 100-140 micrometer in diameter, were mechanically isolated from 11-day-old BDF1 hybrid immature mice, divided randomly into two groups, and cultured in vitro. One group was treated with 0.1% collagenase immediately after mechanical isolation in an attempt to remove theca cells attached to the outside of the basement membrane. The other group was untreated. Morphological examination revealed that 86.1% of mechanically isolated follicles before collagenase treatment had at least one theca cell around the basement membrane on the single section. However, after collagenase treatment no theca cells remained on the basement membrane of the follicles. Androstenedione secretion as a result of stimulation by 100 ng/ml hCG was significantly higher in the culture medium of the follicles with theca cells than in those of collagenase-pretreated follicles (p < 0.0001), indicating that the cells attached to the outside of the basement membrane were actually functional theca cells, not interstitial cells. To elucidate the effect of GH on theca cells, preantral follicles cultured in the presence of 1.0 mIU/ml GH were morphologically examined. Preantral follicles mechanically isolated from immature mice showed significant proliferation of not only granulosa cells but also theca cells in the presence of GH. In particular, theca cells, which remained dotted on the basement membrane in a small number just after isolation, proliferated and finally formed complete layers after the culture with GH. This is the first report that GH induced the proliferation of theca cells to form morphologically complete layers around the preantral follicle from 11-day-old mice.

Assessment of the role of tissue inhibitor of metalloproteinase-1 (TIMP-1) during the periovulatory period in female mice lacking a functional TIMP-1 gene.

Tissue inhibitor of metalloproteinase (TIMP)-1 is a multifunctional peptide that has been implicated in the ovulatory process. To assess the function of TIMP-1 during the periovulatory period in vivo, mice incapable of expressing the TIMP-1 gene product were utilized. Twenty-three-day-old TIMP-1-deficient (n = 59) and wild-type (n = 61) female mice were injected with 5 IU eCG, followed 48 h later by an ovulation-inducing dose of hCG (5 IU). Animals were killed at the time of hCG injection (0-h hCG), at 12 h (12-h hCG), or at 24 h post-hCG (24-h hCG) administration. Serum was collected for the assessment of estradiol-17beta (0-h hCG groups) or progesterone content (12- and 24-h hCG groups), while ovaries were removed for either histological preparation or Northern analysis of TIMP-1, TIMP-2, and TIMP-3. The number of healthy and atretic follicles was determined in the 0-h hCG groups, as was the number of oocytes released in the 24-h hCG group. TIMP-1-deficient females in the 0-h hCG group showed reduced levels of ovarian TIMP-2 (0.29-fold decrease, p < 0.05) and TIMP-3 (3.0-fold decrease, p < 0.05) expression compared to wild-type counterparts. No significant difference was detected between genotypes in the 0-h hCG group for number of healthy or atretic follicles or for serum estradiol-17beta concentrations. Additionally, no significant differences were detected between genotypes in the 12- and 24-h hCG groups for serum progesterone concentrations, ovarian TIMP-2 and TIMP-3 expression, or number of oocytes released (24-h hCG group). To assess the effect of TIMP-1 on steroidogenesis in vitro, granulosa cells were obtained from 23-day-old, eCG-primed TIMP-1-deficient and wild-type females. Addition of recombinant human TIMP-1 significantly increased conditioned media estradiol-17beta concentrations in cell cultures from both mutant (1.32-fold over controls; p = 0.02; n = 4) and wild-type females (1.16-fold over controls; p = 0.04; n = 3). It is concluded from this study that TIMP-1 may modulate ovarian TIMP-2 and TIMP-3 mRNA expression during folliculogenesis. In addition, TIMP-1 exhibits steroidogenic activity in vitro, but no evidence was found for regulation of steroidogenesis in vivo.

Assessment of the need for follicle stimulating hormone in early preantral mouse follicle culture in vitro.

In two consecutive controlled experiments 160 early preantral follicles were cultured in order to evaluate effects of recombinant follicle stimulating hormone (r-FSH) on survival, differentiation, oestradiol and inhibin secretion, cumulus mucification and cumulus-corona-oocyte detachment by human chorionic gonadotrophin (HCG) stimulation. Nuclear maturation in oocytes was also assessed following addition of HCG. A histological analysis of cultured follicles was carried out on semi-thin sections at various culture stages. Addition of r-FSH was essential for follicle survival for 16 days: without r-FSH only 11% of the follicles survived for 12 days (with r-FSH: 79%) and none of these mucified after the HCG stimulus. r-FSH promoted granulosa cell proliferation and antral-like cavity formation. Without r-FSH, histology of the cultures demonstrated degeneration and reduced granulosa cell proliferation; oestradiol and inhibin production were reduced. This study illustrates the essential role of FSH in promoting the in-vitro growth of early preantral mouse ovarian follicles and in maintaining the oocyte under meiotic arrest.