Most dairy cows develop a dominant follicle within two weeks postpartum, but 60% of these follicles fail to ovulate. In a previous study, we determined that cows destined to ovulate have higher LH pulse frequency and circulating estradiol. The latter characteristic provided a method for distinguishing ovulatory from nonovulatory follicles during development and we found that nonovulatory follicles have lower estradiol and androstenedione in their follicular fluid. We hypothesized that lower LH pulse frequency impairs androgen production by theca cells of nonovulatory cows, reducing their ability to make estradiol. In the present study, we applied our method for predicting follicle fate to collect dominant follicles from predicted ovulatory (n = 7) and nonovulatory (n = 3) follicles. Theca and granulosa cells were separated and cultured in the absence or presence of LH, FSH, and/or testosterone for three days, with daily collection of culture medium for steroid RIAs. Estradiol and progesterone production by granulosa cells were not different between ovulatory and nonovulatory follicles. By contrast, overall androstenedione production by theca cells from ovulatory follicles was significantly higher compared with nonovulatory follicles on all three days of culture and, as culture progressed, theca from nonovulatory follicles had increasingly poorer responses to LH. In the same cultures, the progesterone production by theca cells was similar in ovulatory and nonovulatory groups. In support of our hypothesis, the results show that estradiol production by granulosa cells from nonovulatory follicles is robust when androgen substrate is present, but that thecal androgen production in response to LH is impaired. This suggests that the initial defect in steroidogenesis in dominant follicles that fail to ovulate postpartum is lower production of androgen by theca cells.

In cattle and other species, the fetal ovary is steroidogenically active before follicular development commences, and there is evidence that estradiol and progesterone inhibit follicle formation and activation. Estradiol levels decline sharply around the time of follicle formation. In the present study, we hypothesized that FGF10 and FGF18, which inhibit estradiol secretion from granulosa cells of antral follicles, also regulate fetal ovarian steroid production. Fetuses were collected at local abattoirs, and age determined by crown‐rump length measurements. Real‐time polymerase chain reaction assays with RNA extracted from whole ovaries revealed that the abundance of CYP19A1 messenger RNA (mRNA) decreased from 60 to 90 days of gestation, which is consistent with the decline in estradiol secretion previously observed. Immunohistochemistry revealed the presence of FGF18 in ovigerous cords in early gestation and in oocytes later in fetal age (≥150 days). The abundance of FGF18 mRNA increased after Day 90 gestation. Addition of recombinant FGF18 to fetal ovarian pieces inhibited estradiol and progesterone secretion in vitro, whereas FGF10 was without effect. Consistent with these results, FGF18 decreased levels of mRNA for CYP19A1 and CYP11A1 in ovarian pieces in vitro. These data suggest that FGF18 may be an intraovarian factor that regulates steroidogenesis in fetal ovaries.

Ultrasound imaging has shown that cattle exhibit 2 or 3 waves of follicular development during an oestrous cycle. The waves consist of the contemporaneous appearance, about every 7 days, of a group of follicles > or = 5 mm in diameter. One follicle gradually becomes larger than the rest (i.e. dominant). There are several lines of evidence suggesting that the waves occur regularly under conditions of basal LH and FSH. (1) Cycles with 3 waves of follicular development are longer and have longer luteal phases than do cycles with 2 waves, indicating that the number of waves in a cycle is determined by the time of luteal regression. (2) Cycles with 4 or 5 waves of follicular development can occur when the luteal phase is artificially prolonged with exogenous progesterone. (3) Waves of follicular development occur during pregnancy. However, the secondary surge of FSH may be important in initiating new follicular recruitment after ovulation, since suppression of the secondary surge delays the first wave of follicular development. Follicles are functionally dominant (capable of ovulating after luteal regression) while they are still growing and early during their plateau in growth. Functional dominance is lost some time between the early and late plateau phases, while the follicle is still morphologically dominant (i.e. the largest follicle). The factors that lead to dominance of one follicle and the mechanisms that suppress the growth of subordinate follicles are not well understood. When the luteal phase is artificially extended with low doses of exogenous progesterone, the normal pattern of follicular development is altered and the ovulatory follicle grows for a prolonged period of time. This finding indicates that subtle changes in the hormonal milieu can dramatically alter follicular dynamics and that the experimental model of prolonged dominance may be useful in studying the mechanisms of follicular dominance. In contrast, patterns of follicular development in sheep must be assessed in more indirect ways, but sheep offer the advantage of breeds that differ in ovulation rate. Correlation of the endocrine environment with ovulation rate in this species provides a valuable approach to understanding the mechanisms controlling follicle selection and ovulation rate. It has been suggested that in some species a high ovulation rate is achieved by increased recruitment, whereas in others there is increased selection. There is evidence that oestradiol is involved in regulating the number of dominant follicles in sheep. Follicular recruitment requires the presence of gonadotrophins, particularly FSH. In general, the mechanisms that regulate follicular selection and dominance in domestic ruminants are not well understood. Further experiments may determine the relative roles of paracrine factors and ovarian-pituitary-hypothalamic interactions in regulation of follicular selection and dominance in cattle and sheep.

Mammalian follicles begin to form an antral cavity at 0.2–0.4 mm (diameter). Once follicles reach a particular, species-specific size, they become atretic (die) or are recruited into a wave (cohort) of follicles that grow larger. A species-specific number of dominant follicles is selected from the cohort for further growth and potential ovulation. Follicular dynamics in various species and the mechanisms that subserve the recruitment of follicular waves and selection of follicles for dominance are reviewed in this article. As dominant follicles develop towards ovulation, their increasingly greater secretion of estradiol induces the gonadotropin surge that triggers maturation of the oocyte and ovulation.

Mammalian ovarian follicles synthesize all three classes of sex steroids: progestins, androgens, and estrogens. During the follicular phase of estrous or menstrual cycles, developing preovulatory follicles secrete increasingly greater quantities of estradiol. In mammalian species that have been studied, estradiol is produced via interactions between the two follicular endocrine cell types, theca and granulosa cells. Theca cells respond to luteinizing hormone (LH) by producing androgens, but they lack the aromatase enzyme necessary to convert them to estrogens. Granulosa cells do not synthesize androgens, but they can convert thecal androgens to estradiol under the influence of follicle stimulating hormone (FSH), which induces aromatase.

STUDY QUESTION Does anti-Müllerian hormone (AMH) inhibit activation (initiation of growth) of primordial follicles and attenuate the growth of primary follicles in cattle, an excellent animal model for human ovarian follicular development? SUMMARY ANSWER AMH inhibited activation of bovine primordial follicles and attenuated the growth of activated follicles in vitro. WHAT IS KNOWN ALREADY In mice null mutant for AMH, the pool of primordial follicles is depleted prematurely and AMH inhibits follicle activation in vitro. Results of studies with human ovarian tissue in vitro were inconsistent. Our previous work provided indirect evidence that AMH inhibits follicle activation in bovine ovaries. STUDY DESIGN, SIZE, DURATION Pieces of fetal bovine ovarian cortex (2 pieces/culture well), obtained during mid or late pregnancy, were cultured in control medium or with graded doses of AMH for 2, 10 or 12 days. Effects of treatment on follicle activation and growth were determined by histological morphometry; follicles in every 20th histological section were staged (primordial or primary), counted, and measured. In addition, AMH was immunolocalized in bovine ovaries obtained at various times during pregnancy (n = 20 ovaries). PARTICIPANTS/MATERIALS, SETTING, METHODS Bovine fetal ovaries at mid or late gestation were obtained at a commercial abattoir. Pieces of ovarian cortex were cultured without or with AMH and fixed for histological morphometry on Day 0 and at the end of culture. Treatments were applied to duplicate cultures from each of two or three fetuses. In 12-day cultures, addition of AMH was delayed until the third day. Histological analysis provided information about the types, numbers and sizes of follicles in cortical pieces before and after treatments. Ovaries obtained during the second and third trimesters were assessed for the presence of AMH by immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE AMH (100-500 ng/ml) inhibited follicle activation in response to an activator (insulin) in ovarian cortical pieces from fetal ovaries in late gestation. Dose-dependent inhibitory effects on the diameters of primary follicles and their oocytes were also observed. These results were obtained only when AMH was added to cultures in advance of insulin (presumably because it penetrates tissue more slowly than insulin). Results of experiments with cortical pieces from fetal ovaries at mid-gestation, when follicles are forming, showed that AMH did not inhibit the formation of follicles. Immunohistochemical localization of AMH showed that it is not present in fetal ovaries until the third trimester, when it was localized to the granulosa cells of secondary and small antral follicles. LIMITATIONS REASONS FOR CAUTION The experiments were performed with fetal ovaries because follicles form and follicle activation begins during fetal life in cattle (as it does in humans), so fetal ovarian cortex of later gestation provides tissue rich in primordial follicles. We assume, but have no experimental evidence, that our findings also apply to post-natal ovaries. WIDER IMPLICATIONS OF THE FINDINGS Although circulating AMH is used as an indication of the follicular reserve in women, little is known about AMH in human ovaries. Cattle are an excellent non-primate model for human ovarian follicular development and, hence, the findings suggest similar roles for AMH in human follicular development. LARGE SCALE DATA Not applicable. STUDY FUNDNG/COMPETING INTEREST(S) This research was supported by National Research Initiative Competitive Grants no. 00-35203-9151, 2003-35203-13532, and 2008-35203-05989) from the U.S. Dept. of Agriculture's National Institute of Food and Agriculture to JEF and by an NIH National Research Service Award (F32 HD08264) to RAC. There are no conflicts of interest or competing interests.

Little is known about the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of extracellular proteases in ovarian follicles of non-rodent species, particularly in theca cells. In the present study, temporal changes in the abundance of mRNA encoding four ADAMTS subtypes and hormonal regulation of mRNA encoding two subtypes were investigated in theca interna cells during the periovulatory period in cattle. Gonadotropin-releasing hormone (GnRH) was injected into animals to induce a luteinizing hormone (LH)/follicle-stimulating hormone (FSH) surge, and follicles were obtained at 0 hr post-GnRH (preovulatory) or at 6, 12, 18, or 24 hr (periovulatory). ADAMTS1, -2, -7, and -9 transcript abundance was then determined in the isolated theca interna. ADAMTS1 and -9 mRNA levels were up-regulated at 24 hr post-GnRH, whereas ADAMTS2 mRNA was higher at 12-24 hr post-GnRH and ADAMTS7 mRNA increased transiently at 12 hr post-GnRH compared to other time points. Subsequent in vitro experiments using preovulatory theca interna (0 hr post-GnRH) showed that application of LH in vitro can mimic the effects of the gonadotropin surge on mRNAs encoding ADAMTS1 and -9 and that progesterone/progesterone receptor and/or prostaglandins may regulate the levels of mRNA encoding ADAMTS1 and -9 in theca interna, downstream of the LH surge. Time- and subtype-specific changes in ADAMTS mRNA abundance in vivo, and their regulation in vitro by hormones, indicate that ADAMTS family members produced by theca cells may play important roles in follicle rupture and the accompanying tissue remodeling in cattle. Mol. Reprod. Dev. 84: 55-66, 2017. © 2016 Wiley Periodicals, Inc.

In cattle, primordial follicles form before birth. Fetal ovarian capacity to produce progesterone and estradiol is high before follicle formation begins and decreases around the time follicles first appear (around 90 days of gestation). However, mechanisms that regulate steroid production during this time remain unclear. We hypothesized that LH stimulates progesterone and androgen production and that FSH stimulates aromatization of androgens to estradiol. To test this, we cultured pieces from fetal bovine ovaries for 10 days without or with exogenous hormones and then measured the accumulation of steroids in the culture medium by RIA. LH (100 ng/mL) alone increased the accumulation of progesterone, androstenedione, testosterone and estradiol. FSH (100 ng/mL) alone increased both progesterone and estradiol accumulation, but had no effect on androgens. Exogenous testosterone (0.5 µM) alone greatly increased estradiol accumulation and the combination of testosterone + FSH, but not testosterone + LH, increased estradiol relative to testosterone alone. Interestingly, exogenous testosterone and estradiol decreased progesterone accumulation in a dose-dependent manner. Because the highest dose of estradiol (0.5 µM) decreased progesterone accumulation, but increased both pregnenolone and androstenedione in the same cultures, endogenous estradiol may be a paracrine regulator of steroid synthesis. Together, these results confirm our initial hypotheses and indicate that LH stimulates androgen production in fetal bovine ovaries via the Δ5 pathway, whereas FSH stimulates aromatization of androgens to estradiol. These results are consistent with the two-cell, two-gonadotropin model of estradiol production by bovine preovulatory follicles, which suggests that the mechanisms regulating ovarian steroid production are established during fetal life.

The signals that regulate activation, a key transition in ovarian follicular development, are still not well understood, especially in nonrodent species. To gain insight into the regulation of this transition in cattle, we combined a microarray approach with an in vitro system in which ovarian cortical pieces cultured in control medium are enriched for primordial follicles, whereas pieces cultured with insulin are enriched for primary follicles. Total RNA was extracted from cultured cortical pieces, and then transcripts were identified and analyzed using the Affymetrix Bovine Genome GeneChip array. Around 65% of the transcripts in the bovine GeneChip were detected in cultured cortical pieces. Comparison between pieces cultured with or without insulin generated 158 differentially expressed transcripts. Compared with controls, 90 transcripts were upregulated and 68 were downregulated by insulin. These transcripts are involved in many biological processes and functions, but most are associated with cellular growth or cell cycle/cell death. The transcript encoding ubiquitin-conjugating enzyme E2C (UBE2C) was significantly upregulated during follicle activation, and Ingenuity Pathways Analysis revealed that UBE2C can interact with the tumor suppressor phosphatase and tensin homolog (PTEN). Both PTEN mRNA and protein were lower in cortical pieces cultured with insulin than in controls. In addition, FOXO3a, a downstream effector of PTEN signaling, underwent nuclear-cytoplasmic shuttling during primordial to primary follicle development in bovine fetal ovaries, further suggesting the involvement of the PTEN pathway in follicle activation in cattle. Genes and pathways identified in this study provide interesting candidates for further investigation of mechanisms underlying follicle activation.

Purpose The objective of this study is to characterize the impact of exposure to cryoprotectants followed by vitrification on primordial follicle survival and activation using a fetal bovine model. Methods In the first study, fetal bovine cortical pieces were exposed to cryoprotectants with or without sucrose and cultured up to 7 days in the presence or absence of insulin. In the second study, cortical pieces were exposed to cryoprotectants with or without sucrose, vitrified, and cultured up to 7 days after warming in the presence or absence of insulin. Viability and morphology of follicles, as well as proliferation and/or DNA repair in ovarian tissue were analyzed. Results When compared to non-exposed controls, normal follicular morphology was affected in groups exposed to cryoprotectants only immediately post-exposure and after 1 day of culture, but improved by day 3 and did not significantly differ by day 7. Similarly, normal follicular morphology was compromised in vitrified groups after warming and on day 1 compared to controls, but improved by days 3 and 7. Proliferation and/or DNA repair in ovarian tissue was not affected by vitrification in this model. Cryoprotectant exposure and vitrification of ovarian tissue did not impair the activation of primordial follicles in response to insulin, although activation was delayed relative to non-exposed controls. Interestingly, sucrose had no noticeable protective effect. Conclusion Vitrified fetal bovine ovarian tissue has the intrinsic capacity to mitigate the immediate damage to primordial follicles’ morphology and retains the capacity to activate. These findings provide a basis for a successful cryopreservation protocol for ovarian cortical tissue in other species including humans.

The ovarian follicular reserve has been linked to fertility in cattle. Young adult cattle with low vs. high numbers of antral follicles ≥ 3 mm in diameter in follicular waves also have fewer preantral follicles and decreased fertility. This underscores the importance of understanding the factors that regulate early follicular development and establish the ovarian follicular reserve, but little is known about how the follicular reserve is first established. In ruminants and humans, follicles form during fetal life, but there is a gap (about 50 d in cattle) between the appearance of the first primordial follicles and the first growing, primary follicles. In this review we present evidence that in cattle, fetal ovarian steroids (i.e., estradiol and progesterone) are negative regulators of both follicle formation and of the acquisition by newly formed follicles of the capacity to activate (i.e., initiate growth). The results indicate that capacity to activate is linked to the completion of meiotic prophase I by the oocyte. The inhibitory effects of estradiol on follicle activation were found to be reversible and correlated with inhibition of the progression of meiotic prophase I. Fetal bovine ovaries produce steroid hormones and production varies considerably during gestation and in a pattern consistent with the hypothesis that they inhibit follicle formation and capacity of newly formed follicles to activate in vivo. However, little was known about how steroid production is regulated. In our studies, both LH and FSH stimulated progesterone and estradiol production by ovarian pieces in vitro. The addition of testosterone to the culture medium enhanced estradiol production, especially when FSH was also present, but inhibited progesterone production, even in the presence of gonadotropins. Evidence is also presented for effects of maternal nutrition and health and for potential effects of estrogenic endocrine-disrupting chemicals on the size of the ovarian follicular reserve established during fetal life. In summary, fetal ovarian steroids may be important regulators of the early stages of follicular development in cattle. Therefore, external factors that alter steroid production or action may affect the size of the ovarian follicular reserve.

The present study evaluated whether the gonadotrophin surge modulates components of the renin-angiotensin system and whether angiotensin II (Ang II) plays a role in the production of hormones by follicular cells during the ovulatory process. In Experiment 1, cows were ovariectomised at various times (0, 3, 6, 12 and 24 h) after GnRH injection to obtain preovulatory follicles. The concentration of Ang II in follicular fluid increased after GnRH and reached a peak at 24 h, concomitant with the peak of angiotensinogen (AGT) mRNA expression in granulosa cells. AGT mRNA was not expressed in theca cells. Ang II receptor type 2 and angiotensin-converting enzyme mRNA levels were transiently upregulated in theca cells. In Experiment 2, an in vitro culture was used to determine whether Ang II could modulate hormone production by healthy dominant follicles. In the absence of LH, Ang II did not alter hormonal production by either theca or granulosa cells. Ang II plus LH increased progesterone and prostaglandin secretion by granulosa cells. In summary, the renin-angiotensin system is actively controlled during the preovulatory period and Ang II amplifies the stimulatory effects of LH on the secretion of progesterone and prostaglandins by granulosa cells.

The formation of the primordial follicle pool, a process essential to female reproductive capacity, begins around day 90 of bovine gestation (gestational length= 281 days). Previous results indicate that bovine fetal ovaries produce steroids and that ovarian estradiol (E2) and progesterone (P4) decline around the time of follicle formation. Furthermore, we showed that in vitro E2 and P4 can inhibit follicle formation and prevent primordial follicles from acquiring the capacity to activate (initiate growth). Although it is known that the fetal ovary is capable of producing steroids and that E2 and P4 can affect the formation and development of follicles, little is known about the mechanisms that regulate fetal steroid production in vivo. To begin to investigate the regulation of fetal steroid production, pairs of fetal ovaries (n=4, 81-97 days) were collected at an abattoir and cut into pieces (1 mm3). Ovarian pieces were cultured in 24-well culture plates (2 pieces/well) and treatments were applied to duplicate wells within each experiment (fetus). Medium was collected and replaced every 2 days for 10 days and measured for E2 and P4 by radioimmunoassay. To test the hypothesis that gonadotropins can stimulate steroid production, ovarian pieces were cultured in the presence or absence of LH (luteinizing hormone) and/or FSH (follicle-stimulating hormone) at 100 ng/ml. Average cumulative levels of E2 and P4 in control cultures over 10 days of culture were 0.40 and 0.72 ng/well, respectively. LH, FSH, and LH+FSH stimulated E2 production, whereas only LH+FSH stimulated P4 accumulation above control levels (P < 0.05). The combination of LH+FSH enhanced E2 levels more than FSH alone (P < 0.05). To test the hypothesis that availability of androgen substrate limits fetal ovarian E2 production, ovarian pieces were cultured with testosterone (T, 0.5 μM) in the presence or absence of LH and/or FSH (n=4 fetus). Estradiol secretion was stimulated by T (18-fold), LH+T (24-fold) and FSH+T (35-fold), compared with control medium and FSH+T increased E2 values more than T alone (P < 0.05). The high levels of E2 observed in the presence of exogenous T support our hypothesis that androgen is limiting to E2 synthesis in vitro. Unexpectedly, T decreased P4 production by 98% compared to control medium (P < 0.05). Testosterone was also inhibitory to P4 production in the presence of gonadotropins (P < 0.05). To determine if T inhibits P4 directly or indirectly through aromatization to E2, ovarian pieces (n=3 fetuses) were cultured in control medium or with T or E2 (0.5 μM). The average cumulative level of P4 in control medium was 1.18 ng/well. Treatment with T or E2 decreased P4 accumulation by 92 and 81%, respectively, relative to control medium (P < 0.05), suggesting that the effects of T on progesterone production are exerted, at least in part, by its aromatization to E2. Further studies are needed to elucidate the mechanism(s) of steroidal inhibition of P4 production and the physiological role of this inhibition. Together these results show that fetal bovine ovaries are responsive to both LH and FSH, that ovarian production of androgens may be limiting to E2 synthesis and that steroid hormones may play a role in regulating fetal steroid production. (This project was supported by National Research Initiative Competitive Grant no. 2008-35203-05989 from the USDA National Institute of Food and Agriculture).

Signals regulating follicle formation are not well understood, especially in larger mammals where the process occurs during fetal development. Bovine primordial follicles first appear around Day 90 of gestation (gestation length = ~280 days). In previous studies, secretion of estradiol (E2) by ovaries from 70- to 140-day-old bovine fetuses declined with age and E2 inhibited follicle formation in vitro in ovarian cortical pieces obtained during the time of bovine follicle assembly (Day 91-120), suggesting a role for declining E2 in follicle formation. However, these data were derived in different experiments from different fetal ovaries. Therefore, the first objective of the present study was to further test the relationship between declining E2 and follicle formation by culturing pieces of ovarian cortex (4 pieces/treatment/fetus) obtained from fetuses (n=3) just prior to follicle formation (Day 80-90) for 6 days, to determine morphology and accumulation of E2 and progesterone (P4) in the culture medium (collected and replaced every 2 days). Since mRNA for aromatase was localized primarily to the medulla around the time of bovine follicle formation, we hypothesized that isolating the ovarian cortex (where follicles form) from the medulla would reduce intra-cortical E2 concentrations (which would be reflected in lower E2 in the culture medium) and allow follicle assembly to begin. Histological morphometry showed that on day 0 cortical pieces had many oogonia and oocytes, but only rare follicles, whereas after 6 days of culture, there was a 2.1-fold increase (P < 0.05) in primordial follicles, compared with day 0 and a few primary follicles were present. These data suggest that follicle formation occurred in vitro and some follicles became capable of activating during culture. Interestingly, as the number of follicles in cortical pieces increased in vitro, E2 accumulation in culture medium decreased gradually over 6 days of culture with the lowest accumulation between day 4-6 of culture (0.66 ± 0.2, 0.56 ± 0.1, and 0.34 ± 0.1 ng/culture ± SEM on days 0-2, 2-4 and 4-6; P < 0.05). This inverse correlation between E2 accumulation in the medium and follicle formation is consistent with our previous evidence for negative regulation of follicle formation by E2. In contrast, P4 increased ~6-fold (P < 0.05) during the same period. To begin to study the regulation of steroid production by fetal ovaries, ovarian pieces from 90- to110-day-old bovine fetuses were cultured for 12 days with graded doses of FGF-10 or FGF-18 (50, 100 and 200 ng/ml). We have detected FGF-10 and -18 in bovine fetal ovaries and showed previously that they inhibit steroidogenesis by granulosa cells from antral follicles. E2 accumulation was decreased, in a dose-dependent fashion, by as much as 70% in cultures treated with FGF-18 (P < 0.05). After day 4 of culture, FGF-18 (200 ng/ml) similarly reduced P4 accumulation in the medium (P < 0.05). In contrast, FGF-10 had no effect on P4 secretion and only occasional, inconsistent effects on E2 accumulation. Taken together, the results show that reduced intra-cortical E2 in vitro is accompanied by accelerated follicle formation and activation and that FGF-18 can regulate steroid production in vitro, suggesting a role for this growth factor in follicle formation and activation.

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.

Endocrine disrupting chemicals (EDCs) include phytoestrogens (biologically active natural substances commonly found in forage) and environmental estrogens found in pesticides, plastics, and industrial chemicals. Normal levels of estrogen are essential for reproduction, but EDCs can mimic or disrupt estrogen action. The formation of ovarian follicles and initiation of follicle growth (activation) are two processes that determine reproductive life span since they affect the number of follicles in the ovaries. In cattle, follicle formation begins around Day 90 of gestation and activation around Day 140 (gestation = 280 days). We recently showed that estrogen can inhibit follicle formation and activation in vitro in ovaries from 91- to 140-day-old bovine fetuses. In recent years there is increasing concerns about the potential harmful effects of EDCs on animal and human health and reproduction. Cattle are not only an important food source, but also are a good model for ovarian follicular development and function in women. Therefore, in the present study we used cattle as an experimental model to examine whether exposure of fetal bovine ovarian cortex to phytoestrogens or environmental estrogens in vitro has adverse effects on activation of ovarian follicles or follicle health during culture. Ovaries were obtained from bovine fetuses (n=2) estimated to be Day 120-130 of gestation (crown-rump length). Cortical pieces were dissected and then cultured (4 pieces/treatment/fetus) for 10 days with control medium or with 1 µM phytoestrogens (equol, coumestrol, genistein, and daidzein) or environmental estrogens (BPA and octylphenol). Cortical pieces were then fixed and embedded in plastic for serial sectioning and morphometric analysis. On day 0, cortical pieces contained mostly primordial follicles, whereas after 10 days in culture, most primordial follicles had activated, differentiating into primary follicles as expected. Neither phytoestrogens nor environmental estrogens affected the number of primordial follicles compared with controls. However, genistein, coumestrol and octylphenol decreased the number of primary follicles (0.75 ± 0.18, 1.6 ± 0.49 and 0.38 ± 0.04 follicles/section, respectively vs. 4.33 ± 0.59 in controls; P < 0.05). Moreover, there were significantly fewer total follicles (primordial + primary) in cortical pieces cultured with genistein and octylphenol than in controls (1.65 ± 0.2 and 0.76 ± 0.08 respectively vs. 5.62 ± 0.9; P < 0.05). The decreases in the numbers of primary and total follicles suggest that genistein, coumestrol and octylphenol induce the death of growing follicles. In conclusion, exposure to phytoestrogens (genistein and coumestrol) and environmental estrogen (octylphenol) has adverse effects on early follicular development in cattle in vitro. (poster)

The establishment of a stockpile of non-growing, primordial follicles and its gradual depletion through activation of primordial follicles are essential processes for female fertility. However, the mechanisms that regulate follicle formation, the activation of primordial follicles to begin growth and the primary-to-secondary follicle transition are poorly understood, especially in domestic animals and primates. The authors' laboratory is engaged in studying early stages of follicular development in cattle and this review summarises the progress to date. Bovine follicles begin to form in fetal ovaries around the beginning of the second trimester of pregnancy (about Day 90), but the first activated, primary follicles do not appear until after Day 140. Bovine fetal ovaries produce steroids and production is highest during the first trimester. In vitro, oestradiol and progesterone inhibit follicle formation and acquisition by newly formed follicles of the capacity to activate. Meiotic arrest of the oocyte in the diplotene stage of first prophase does not occur until after follicle formation and is correlated with acquisition of the capacity to activate. This may explain the gap between follicle formation and appearance of the first activated follicles. Once capacity to activate has been acquired, it seems likely that activation in vivo is controlled by the balance between stimulators and inhibitors of activation. Insulin and kit ligand stimulate and anti-Müllerian hormone (AMH) inhibits activation in vitro. Few bovine follicles transition from the primary to the secondary stage in vitro, but this transition is increased by medium supplements, testosterone and vascular endothelial growth factor (VEGF).

The formation of primordial follicles to establish a reservoir of resting follicles and the gradual depletion of that reservoir to provide a succession of growing follicles are key to female fertility, but little is known about the regulation of these early stages of follicular development. This review summarizes the efforts of our laboratory to elucidate these critical processes in cattle. Primordial follicles first appear in fetal ovaries around the end of the first trimester of pregnancy (Day 90), during a decline in fetal ovarian production of estradiol and progesterone. In ovarian cortical pieces from 90 to 120-day-old fetuses, follicles form in vitro and estradiol or progesterone inhibits follicle formation, whereas the non-aromatizable androgen 5alpha-dihydrotestosterone (DHT) does not. Newly formed bovine follicles are not capable of activating within 2 days in vitro, but they can acquire the capacity to activate during a longer culture; estradiol and progesterone inhibit the acquisition of their capacity to activate. When primordial follicles first form in cattle, their oocytes are not yet in meiotic arrest and acquisition of competence to activate is correlated with their progression to meiotic arrest at the diplotene stage of first prophase. After they acquire the competence to activate, bovine primordial follicles can be stimulated to activate in vitro by insulin or kit ligand, whereas anti-Mullerian hormone (AMH) is inhibitory. Although few follicles progress to the secondary stage in vitro, addition of testosterone or vascular endothelial growth factor (VEGF) dramatically increased the incidence of that transition. Regulation of the earliest stages of follicular development is complex and far from understood; better understanding could lead to new interventions to enhance fertility.

BACKGROUND Female cancer patients are offered 'banking' of gametes before starting fertility-threatening cancer therapy. Transplants of fresh and frozen ovarian tissue between healthy fertile and infertile women have demonstrated the utility of the tissue banked for restoration of endocrine and fertility function. Additional methods, like follicle culture and isolated follicle transplantation, are in development. METHODS Specialist reproductive medicine scientists and clinicians with complementary expertise in ovarian tissue culture and transplantation presented relevant published literature in their field of expertise and also unpublished promising data for discussion. As the major aims were to identify the current gaps prohibiting advancement, to share technical experience and to orient new research, contributors were allowed to provide their opinioned expert views on future research. RESULTS Normal healthy children have been born in cancer survivors after orthotopic transplantation of their cryopreserved ovarian tissue. Longevity of the graft might be optimized by using new vitrification techniques and by promoting rapid revascularization of the graft. For the in vitro culture of follicles, a successive battery of culture methods including the use of defined media, growth factors and three-dimensional extracellular matrix support might overcome growth arrest of the follicles. Molecular methods and immunoassay can evaluate stage of maturation and guide adequate differentiation. Large animals, including non-human primates, are essential working models. CONCLUSIONS Experiments on ovarian tissue from non-human primate models and from consenting fertile and infertile patients benefit from a multidisciplinary approach. The new discipline of oncofertility requires professionalization, multidisciplinarity and mobilization of funding for basic and translational research.