Stages Of Ovarian Follicular Development Research Articles

Comparative transcriptome profile analysis of granulosa cells from porcine ovarian follicles during early atresia

At various stages of ovarian follicular development, more than 99% of follicles will be eliminated through a degenerative process called atresia. The regulatory mechanisms of atresia have been elucidated to some extent, involving hormones, growth factors, cytokines, and other factors. However, the stimuli initiating atresia in follicular granulosa cells remain unknown. In this study, we isolated the granulosa cells from porcine ovarian follicles (3–5 mm diameter) divided into healthy follicles (HFs) and early atretic follicles (EAFs). We applied high-throughput RNA sequencing to identify and compare differentially expressed genes (DEGs) between HFs and EAFs. A total of 31,694 genes were detected, of which 21,806 were co-expressed in six samples, and 243 genes (p < 0.05; FDR < 0.05) were differentially expressed (DEGs), including 123 downregulated and 120 upregulated in EAFs. GO analysis highlighted hormone metabolism, plasma membrane localization, and transporter activity. The pathway analysis indicated that 51 DEGs, involved in steroidogenesis, cell adhesion molecules, and TGF-beta signaling pathways, were highly related to atresia. Additionally, the interaction network of DEGs (p < 0.01; FDR < 0.05) using STRING highlighted LHR, ACACB, and CXCR4 as central nodes. In summary, this transcriptome analysis enriched our knowledge of the shifted mechanisms in granulosa cells during early atresia and provided novel perspectives into the atresia initiation.

Activin Stimulates Follicle Formation and Activation and Modulates Steroidogenesis in Fetal Bovine Ovarian Tissue in Vitro 1

The earliest stages of ovarian follicular development, follicle formation and development of the capacity to activate, are critical to the size of the ovarian reserve. In primates and most domestic animals, these stages occur during fetal life and thus, are difficult to study. Our laboratory has used cattle, an excellent model for human ovarian development, to elucidate the regulation of these early stages in fetal bovine ovarian tissue in vitro. The experiments described here were designed to determine if fetal bovine ovaries have activin A and its receptors and if activin A affects follicular formation and/or activation to begin growth. During the second trimester, when follicles first form, activin increased the total number follicles and the number of primary (activated) follicles in cultured ovarian cortical pieces, compared to control medium; its effects were similar to the positive control, insulin. During the second trimester, activin decreased primordial follicle numbers and increased primary and secondary follicles, again mimicking the positive control. Our previous experiments showed that fetal ovaries produce ovarian steroids, particularly during the second trimester, and that estradiol and progesterone inhibit follicle formation and activation. In control and gonadotropin-treated ovarian pieces, activin A increased progesterone, but decreased androstenedione and estradiol accumulation in the medium. Messenger RNA for activin and its type II receptors was detected in fetal bovine ovaries, with ACVR2A receptor mRNA more abundant than ACVR2B. Immunohistochemistry localized mRNA for activin A and ACVR2A to germ cells of all stages, granulosa cells, and theca cells of early secondary follicles. Staining for ACVR2B was weaker and mostly confined to germ calls. Taken together, these results strongly suggest that activin A of fetal ovarian origin 3 plays a role in ovarian development by promoting follicle formation and activation and modulating ovarian steroidogenesis.

Expression of Angiopoietin 2 in ovarian follicles of crossbred Malabari goat

Angiopoietin-2 (Ang-2) is a local ovarian growth factor belonging to the angiopoietin/Tie (tyrosine kinase with Ig and EGF homology domains) signalling pathway, one of the main pathways involved in ovarian follicular angiogenesis. The main objective of this study is to assess the differential protein expression of Ang-2 in the developmental phases of ovarian follicles of crossbred Malabari goats employing an immunohistochemical technique. Ang-2 protein expression was found in all developmental phases of ovarian follicles, including primordial, primary, small, and large antral ovarian follicles. Ang-2 was found in the granulosa cells, theca interna cells, follicular fluid (granulosa cells in the FF), endothelial cells of capillaries and germinal epithelium of the goat ovaries. The Quantitative Method-Percentage Positivity of Ang-2 expression revealed that it was higher in the initial stages of ovarian follicular development and decreased with later stages of follicular development in cross-bred Malabari goats. Moreover, the protein expression of Ang2 was found to be higher in granulosa cells (GC) than in theca interna (TI) cells of antral follicles as evident from the higher staining intensity in GC cells compared to TI cells. It is therefore concluded that the Ang-2 protein expression in ovarian follicular cells decreased with an increase in follicle size and development and based on the combined scores for immunostaining (staining intensity and the number of positive staining cells), the Ang-2 was found to be expressed more in the granulosa cell layer compared to the theca interna cells of the antral follicle in crossbred Malabari goats.

Composition of Royal Jelly (RJ) and Its Anti-Androgenic Effect on Reproductive Parameters in a Polycystic Ovarian Syndrome (PCOS) Animal Model

Royal jelly (RJ) has been shown to contribute its positive effects upon imbalance in the reproductive system. However, it remains unknown as to whether RJ has an anti-androgenic effect on reproductive parameters in a polycystic ovarian syndrome (PCOS) animal model. Composition of RJ was assessed by phytochemical screening and the LC–MS method. Forty immature female rats (3 weeks, 40–50 g) were randomly divided into five groups (n = 8 per group), i.e., control, testosterone (T), T+100RJ (100 mg/kg/day), T+200RJ (200 mg/kg/day RJ), and T+400RJ (400 mg/kg/day RJ) groups. Hyperandrogenism was induced by daily subcutaneous injection of T propionate for 3 weeks, followed by oral RJ for 4 weeks. The T+200RJ group had a significantly higher follicle-stimulating hormone level, and significantly lower luteinizing hormone, testosterone, and estradiol levels in comparison to the T group. Malondialdehyde level and glutathione peroxidase activity were significantly lower, while total antioxidant capacity level was significantly higher in the T+200RJ group compared to the T group. Histologically, the T+200RJ group showed recovery of various stages of ovarian follicular development. RJ at 200 mg/kg/day for 4 weeks significantly improved reproductive parameters in PCOS rats partly due to its anti-androgenic effect through antioxidant action and probably due to modulation on estrogenic activity, which needs further study to evaluate its exact mechanism of action.

Transcriptome studies of granulosa cells at different stages of ovarian follicular development in buffalo

The normal maturation and ovulation from ovarian follicles is important in ensuring conception and improving fertility of buffalo. The molecular regulation mechanism of buffalo follicles growth, however, remains unknown. This study analyzed the gene expression profiles associated with buffalo ovarian follicle growth. According to the analysis of RNA sequencing, 17,700 unigenes and 13,672 differentially expressed genes (DEGs) were detected. A total of 30 common DEGs were identified during four stages of follicle growth, and the expression patterns are basically synchronized, suggesting the products as a result of expressions of these genes may cooperate to regulate follicular development. Furthermore, GO and KEGG enrichment analyses revealed that the majority of DEGs in early stage of follicular growth were enriched in ribosomal and oxidative phosphorylation signaling pathways, and the expression patterns of these DEGs are basically up-regulated at the beginning of follicular growth (<8 mm, diameter), and then down-regulated (8–12 mm) in the following stages of follicular development. The pathway of immune signaling, including allograft rejection, chemokine signaling pathway, natural killer cell mediated cytotoxicity, phagosome, and antigen processing and presentation, was significantly enriched in the last stage of follicular development (>12 mm), which indicates that the immune system has an important role in the last stage of follicular maturation and ovulation. This study provided a gene expression profile of buffalo follicle growth, and provided an insight into biological processes associated with molecular regulation of ovarian follicle growth.

Monitoring health and reproductive status of olms (Proteus anguinus) by ultrasound.

The olm (Proteus anguinus) is a troglomorphic, neotenous amphibian with extraordinary life expectancy and unique adaptations that deserve further investigation. A low reproductive rate and habitat decline render it threatened by extinction. Establishing captive populations for maintenance and artificial breeding may one day become crucial to the species. Longitudinal, in-vivo assessment of inner organs is invaluable to our understanding of reproductive physiology, health, and behavior. Using ultrasound, we measured heart rate and assessed health and reproductive status of 13 captive olms at Zagreb Zoo. Heart rate averaged 42.9 ± 4.6 bpm (32–55 bpm), as determined via pulsed-wave Doppler at 4–12 MHz. By using frequencies of up to 70 MHz (ultrasound biomicroscopy), inner organs were visualized in detail. Assessment of the gastrointestinal tract provided insights into feeding status and digestive processes. Several subclinical pathologies were detected, including biliary sludge, subcutaneous edema, ascites, and skin lesions. Detection of skin lesions by ultrasound was more sensitive than visual adspection. Olms with ultrasonographically detected skin lesions tested positive for Saprolegnia and were treated. Three of the four affected individuals survived and subsequently tested negative for Saprolegnia. Sex was reliably determined; only one individual proved male. The reason for this extreme female-biased sex-ratio remains unknown. However, as most of the individuals were flushed from the caves by strong currents in spring, the sample may not be representative of natural populations. In female olms, different stages of ovarian follicular development were observed with diameters ranging between 0.1 and 1.1 mm. Results were confirmed by comparing ultrasound, necropsy, and histological findings of one dead specimen. In summary, ultrasound proved a valuable tool to support conservation and captive breeding programs by allowing non-invasive assessment of physiological parameters, clinical condition, and reproductive status in olms.

Localization of IGF proteins in various stages of ovarian follicular development and modulatory role of IGF-I on granulosa cell steroid production in water buffalo (Bubalus bubalis)

The present study aimed to determine the expression of insulin like growth factor (IGF) genes in the bubaline ovarian follicles and modulatory role of IGF-I on progesterone production from granulosa cells (GC) of pre-ovulatory follicle in vitro. According to size, follicles were classified into four groups: GI (small), GII (medium), GIII (large) and GIV (preovulatory). All IGF genes were expressed in both GC and theca interna (TI) cells. The relative expression of IGF-I and IGF receptor I (IGFR-I) genes increased with follicle size and was greatest in the pre-ovulatory follicle (P<0.05). Expression of IGF-II and IGFR-II genes was minimal in GC but was readily detected in TI cells. In TI cells, the gene expression was greater in medium and large as compared to small and pre-ovulatory follicles. The expression of all binding protein (IGFBP) genes was detected in both GC and TI cells. Expression of IGFBP-3 gene increased with follicle size and was greatest in pre-ovulatory follicles (P<0.05). The expression of IGFBP-2 and IGFBP-4 was less in pre-ovulatory follicles but expression of IGFBP-5 and IGFBP-6 genes were greater at this stage. The GC culture was conducted for three time durations and with three doses of IGF-I. Expression of steroidogenic genes (StAR, CYP11A1, HSD3B) and progesterone concentration were increased in a dose and time dependent fashion. The present study, therefore, provided evidence of an autocrine/paracrine role of IGFs in follicular development and a stimulatory role of IGF1 in steroid production in GC of preovulatory follicles in the bubaline species.

The microRNAs important for ovarian and early embryonic development in cattle

Recent progress in genomic analysis and other biochemical methods has led to the discovery of a large population of microRNAs (miRNAs), which have been demonstrated to play important roles in diseases and a wide range of developmental processes. Characterization of miRNA expression profiles in different stages of ovarian follicular development and early embryogenesis has suggested the potential roles of miRNAs in follicular development, maturation of oocytes, and preimplantation embryonic development. This review focuses on the current studies of miRNAs involved in ovarian and early embryonic development in cattle.

Either Kras activation or Pten loss similarly enhance the dominant-stable CTNNB1-induced genetic program to promote granulosa cell tumor development in the ovary and testis.

WNT, PI3K or RAS signaling pathways control specific stages of ovarian follicular development. To analyze the functional interactions of these pathways in granulosa cells during follicular development in vivo, we generated specific mutant mouse models. Stable activation of the WNT signaling effector beta-catenin (CTNNB1) in granulosa cells results in the formation of premalignant lesions that develop into granulosa cell tumors (GCTs) spontaneously later in life or following targeted deletion of the tumor suppressor gene Pten. Conversely, expression of oncogenic KRASG12D dramatically arrests proliferation, differentiation and apoptosis in granulosa cells, and consequently, small abnormal follicle-like structures devoid of oocytes accumulate in the ovary. Because of the potent anti-proliferative effects of KRASG12D in granulosa cells, we sought to determine if KRASG12D would block precancerous lesion and tumor formation in follicles of the CTNNB1 mutant mice. Unexpectedly, transgenic Ctnnb1;Kras mutant mice exhibited increased GC proliferation, decreased apoptosis and impaired differentiation and developed early-onset GCTs leading to premature death in a manner similar to the Ctnnb1;Pten mutant mice. Microarray and RT-PCR analyses revealed that gene regulatory processes induced by CTNNB1 were mostly enhanced by either KRAS activation or Pten loss in remarkably similar patterns and degree. The concomitant activation of CTNNB1 and KRAS in Sertoli cells also caused testicular granulosa cell tumors that showed gene expression patterns that partially overlapped those observed in GCTs of the ovary. Although the mutations analyzed herein have not yet been linked to adult GCTs in humans, 1) other components of these pathways may be altered or mutated, 2) these mutations may relate to juvenile GCTs or 3) they may occur in tumors of other tissues where CTNNB1 is mutated. Importantly, our results provide strong evidence that CTNNB1 is the driver in these contexts and that KRASG12D and Pten loss promote the program set in motion by the CTNNB1.

Inhibition of Activation of Newly Formed Bovine Primordial Follicles by Estradiol Is Reversible and Associated with Inhibition of Progression of Meiotic Prophase I.

In some mammals, like cattle, sheep, and humans, the pool of resting, primordial follicles is formed during fetal life and little is known about the regulation of the early stages of ovarian follicular development. Bovine primordial and primary follicles first appear around day 90 and 140 of gestation, respectively (gestation length = 280 days). Estradiol inhibits activation (i.e. initiation of growth) of newly formed bovine primordial follicles in vitro and secretion of estradiol by bovine fetal ovaries from around 83 to 140-day-old fetuses declines precipitously with age, suggesting an inhibitory role for estradiol in follicle activation in vivo. If inhibition of activation of newly formed bovine primordial follicles by estradiol in vitro mimics a normal developmental mechanism, we would expect the inhibition to be reversible. Therefore, the first objective of the present study was to test the hypothesis that the inhibitory effect of estradiol on follicle activation is reversible. Pieces of fetal ovarian cortex (4 pieces/treatment/fetus; n = 2 fetuses; day 100 and 120 of gestation) were cultured in serum-free medium supplemented with ITS+ (control medium) for 10 or 14 days (controls), with estradiol (10-6 M) for 10 days, or with estradiol for 10 days and then in control medium for 4 more days. On day 0 (time 0 controls) and after culture, cortical pieces were fixed and embedded in paraffin for serial sectioning and sections were stained with H&E for morphmetric analysis. On day 0, cortical pieces contained mostly primordial follicles (99.95%), whereas after culture the percentages of primary follicles in all cultures, except cultures treated with estradiol, were significantly higher than in day 0 controls (P < 0.05), suggesting that follicle activation occurred in vitro. There was a higher percentage of primary follicles in day 14 controls than in day 10 controls (75.4 ± 4% vs. 41.7 ± 8.3%; P < 0.05), suggesting that the number of follicles capable of activating increased with time in culture. The percentage of primary follicles in cortical pieces cultured with estradiol was lower compared with day 10 or 14 controls, (12 ± 4.6 vs. 41.7 ± 8.3 and 75.4 ± 4%, respectively; P < 0.05), consistent with our previous results showing that estradiol inhibits follicle activation. The inhibitory effects of estradiol on activation were reversed by culturing cortical pieces in control medium from day 10-14 (12 ± 4.6 vs. 66.9 ± 13.8% primary follicles; P < 0.05). Since we showed previously that the capacity of bovine primordial follicles to activate is associated with the achievement of meiotic arrest in their oocytes at the diplotene stage of prophase I, we next examined whether the inhibition of follicle activation by estradiol is associated with inhibition of the progression of meiotic prophase I. The results showed a higher percentage of primordial follicles with oocytes in pre-meitotic arrest (pre-diplotene) stages in cultures treated with estradiol than in day 10 or day 14 controls (92.5 ± 7 vs. 43.2 ± 5 or 13.5 ± 13, respectively; P < 0.05). In summary, the capacity of newly formed bovine primordial follicles to activate increases with time in culture. Inhibition of activation of newly formed bovine follicles by estradiol is reversible and is associated with inhibition of the progression of meiotic prophase I.

The Capacity of Primordial Follicles in Fetal Bovine Ovaries to Initiate Growth In Vitro Develops During Mid-Gestation and Is Associated with Meiotic Arrest of Oocytes1

In cattle and other species in which the pool of resting, primordial follicles is formed during fetal life, little is known about the regulation of the early stages of ovarian follicular development. We used histological morphometry and a combination of observations in vivo and experiments in vitro to study the timing and regulation of follicle formation and the acquisition of the capacity of primordial follicles to initiate growth in cattle. In vivo, primordial, primary, and secondary follicles were first observed around Days 90, 140, and 210 of gestation, respectively. The long interval between the first appearance of primordial and primary follicles suggests that primordial follicles are not capable of activating when they are first formed, or they are inhibited from activating. This hypothesis was confirmed by the finding that most primordial follicles in pieces of ovarian cortex obtained from fetal ovaries older than 140 days activated (i.e., initiated growth) after 2 days in vitro, whereas follicles in cortical pieces from 90- to 140-day-old fetal ovaries did not. We tested the hypothesis that the oocytes of newly formed primordial follicles are not in meiotic arrest and found that before Day 141, most oocytes ( approximately 73%) were in prediplotene stages of prophase I, whereas after Day 140, the majority of oocytes ( approximately 85%) had arrested at the diplotene stage. This observation was further confirmed by the finding that levels of mRNA for YBX2, a protein associated with meiotic arrest, were 2.3 times higher in ovarian cortical pieces isolated after versus before Day 141. Primordial follicles in cortical pieces from 90- to 140-day-old fetal ovaries did activate during a longer, 10-day culture, but activation could be inhibited by adding estradiol or progesterone, but not dihydrotestosterone (all at 10(-6) M). Fetal ovaries secreted estradiol in vitro, and secretion by ovaries from 83 to 140-day-old fetuses declined precipitously ( approximately 30-fold) with age, consistent with the hypothesis that estradiol inhibits activation of newly formed primordial follicles in vivo. In summary, the results show that newly formed primordial follicles do not activate in vivo or within 2 days in vitro and that capacity to activate is correlated with achievement of meiotic arrest by the oocyte and can be inhibited by estradiol, which fetal ovaries actively produce around the time of follicle formation.

Effects of oral contraceptives administered at defined stages of ovarian follicular development

To elucidate the effects of initiating oral contraceptives (OC) at defined stages of ovarian follicle development. Prospective longitudinal study. Healthy volunteers in an academic research environment. Forty-five healthy women between the ages of 18 and 35 years, randomized to initiate OC when a follicle diameter of 10, 14, or 18 mm was first detected. The OC administration at defined stages of dominant follicle development. Fates of all dominant follicles and serum concentrations of E(2)-17beta, LH, and P before and after initiating OC. No ovulations (0/16) were observed when OC use was initiated at a follicle diameter of 10 mm, 4/14 (29%) follicles ovulated when OC were initiated at 14 mm, and 14/15 (93%) ovulated when OC were initiated at 18 mm. When ovulation did not occur, follicles regressed or became anovulatory cysts. Peak LH and E(2) levels were lowest in the 10-mm group, moderate in the 14-mm group, and greatest in the 18-mm group. Peak endocrine levels in all treatment groups were lower than the historic reference group. Follicular development, ovulation, and endocrine concentrations were not suppressed effectively when OC were initiated at late stages of dominant follicle development.

Characterization of the mRNA expression of StAR and steroidogenic enzymes in zebrafish ovarian follicles

The objective of this study was to investigate the levels of expression of steroid biosynthetic enzymes and steroidogenic acute regulatory protein (StAR) at different stages of ovarian follicular development in zebrafish (Danio rerio), and to investigate the sites within the steroid biosynthetic pathway that may be regulated by gonadotropins. Ovarian follicles of sexually mature fish were separated into primary, previtellogenic, vitellogenic, and mature stages and the expression of StAR, P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450 hydroxylase/lyase (P450c17), 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), 17beta-hydroxysteroid dehydrogenase type 3 (17beta-HSD3), and P450 aromatase (P450aromA) was determined by Real time RT-PCR. The expression of all genes changed significantly as follicles grew, with a decrease in the expression of StAR, P450scc, 3beta-HSD and P450c17 with maturation, and an increase in the expression of 17beta-HSD3 during vitellogenesis and 17beta-HSD1 and P450aromA during previtellogenesis. In vitro incubation of vitellogenic follicles demonstrated that the expression of StAR, 17beta-HSD3, and P450aromA increased in response to hCG, and decreased in the absence of hCG. In contrast, the expression of P450scc, 3beta-HSD, P450c17, and 17beta-HSD1 remained constant between treatments and over time. Testosterone and estradiol production in the culture medium was stimulated by human chorionic gonadotropin (hCG). These experiments aid in the characterization of the roles and regulation of steroids throughout ovarian development, and suggest that gonadotropins play a key role in the regulation of StAR, 17beta-HSD3, and P450aromA in zebrafish.

Dynamic Anchoring of PKA Is Essential during Oocyte Maturation

In the final stages of ovarian follicular development, the mouse oocyte remains arrested in the first meiotic prophase, and cAMP-stimulated PKA plays an essential role in this arrest. After the LH surge, a decrease in cAMP and PKA activity in the oocyte initiates an irreversible maturation process that culminates in a second arrest at metaphase II prior to fertilization. A-kinase anchoring proteins (AKAPs) mediate the intracellular localization of PKA and control the specificity and kinetics of substrate phosphorylation. Several AKAPs have been identified in oocytes including one at 140 kDa that we now identify as a product of the Akap1 gene. We show that PKA interaction with AKAPs is essential for two sequential steps in the maturation process: the initial maintenance of meiotic arrest and the subsequent irreversible progression to the polar body extruded stage. A peptide inhibitor (HT31) that disrupts AKAP/PKA interactions stimulates oocyte maturation in the continued presence of high cAMP. However, during the early minutes of maturation, type II PKA moves from cytoplasmic sites to the mitochondria, where it associates with AKAP1, and this is shown to be essential for maturation to continue irreversibly.

The orphan nuclear receptors NURR1 and NGFI-B modulate aromatase gene expression in ovarian granulosa cells: a possible mechanism for repression of aromatase expression upon luteinizing hormone surge.

Ovarian granulosa cells play pivotal roles in many aspects of ovary functions including folliculogenesis and steroidogenesis. In response to FSH and LH, the elevation of intracellular cAMP level in granulosa cells leads to activation of multiple ovarian genes. Here, we report findings from a genome-wide study of the cAMP-responsive gene expression profiles in a human granulosa-like tumor cell line, KGN. The study identified 140 genes that are either activated or repressed by 2-fold or greater after stimulation by the adenylyl cyclase activator forskolin. The induction patterns of some cAMP-responsive genes were further analyzed by quantitative real-time PCR. Consistent with previous observations, the LH-responsive genes, such as the nuclear receptor 4A subfamily (NURR1, NGFI-B, and NOR-1), were rapidly but transiently induced, whereas the FSH-responsive gene CYP19 encoding aromatase was induced in a delayed fashion. Interestingly, ectopic expression of NURR1 or NGFI-B severely attenuated the cAMP-responsive activation of the ovary-specific aromatase promoter. Reduction of the endogenous NURR1 or NGFI-B by small interfering RNA significantly elevated aromatase gene expression. The cis-elements responsible for NURR1/NGFI-B-mediated repression were mapped to the minimal aromatase promoter sequence that confers camp responsiveness. Furthermore, the DNA-binding domain of NURR1 was required for the repression. Taken together, these results strongly suggest a causal relationship between the rapid decline of aromatase mRNA and induction of nuclear receptor subfamily 4A expression, which concomitantly occur upon LH surge at the later stages of ovarian follicular development.

Individual variation in plasma estradiol-17β and androgen levels during egg formation in the European starling Sturnus vulgaris: implications for regulation of yolk steroids

While it is clear that maternal transfer of steroids to egg yolk can have significant effects on offspring phenotype, an unresolved question is whether females can facultatively adjust yolk hormone levels independently of their own plasma levels or whether yolk steroid levels are simply a direct consequence of temporal variation in the female’s hormonal status. In part, this is because we lack detailed information about the day-to-day pattern of changes in plasma hormone levels during the laying cycle for non-domesticated birds. Here, we describe changes in plasma estradiol-17β (E2) and androgens, throughout laying in relation to specific stages of ovarian follicular development in the European starling ( Sturnus vulgaris). Plasma E2 levels increased rapidly from the onset of rapid yolk development (RYD) to reach maximum levels in birds with a complete follicle hierarchy (⩾4 yolky follicles). However, levels decreased linearly throughout the later stages of follicle development returning to pre-breeding values before the final yolky follicle was ovulated. In females with ⩾4 yolky follicles there was 10-fold variation in plasma E2 levels among individual females, but this was not related to plasma levels of the main yolk precursor vitellogenin or to the total mass of yolky follicles developing at the time of blood sampling. In contrast to E2, plasma androgen levels showed only a very gradual linear decline throughout the laying cycle from pre-RYD to clutch completion. Furthermore, androgen levels showed less individual variability: 4-fold variation among females with ⩾4 yolky follicles, although this was also independent of our measures of reproductive function. Data on inter- and intra-individual variation in female hormone levels are important to set-up a priori predictions for, and interpretation of, studies of yolk hormone levels.

The early stages of follicular development: activation of primordial follicles and growth of preantral follicles

Although enormous progress has been made in understanding the events and regulation of the later stages of ovarian follicular development, the early stages of development, to a large extent and particularly in large mammals, remain a mystery. Mechanisms that regulate the initiation of follicular growth (follicle activation) and the ensuing growth and differentiation of preantral follicles are of considerable interest, since their elucidation is a prerequisite to use of the primordial pool to enhance reproductive efficiency in domestic animals, humans, and endangered species. This review is an attempt to summarize the approaches that have been taken to further this goal and the results thus far of these efforts. Preantral follicular development can be divided into three stages: activation of primordial follicles, the primary to secondary follicle transition, and the development of secondary follicles to the periantral stage. The activation of primordial follicles in vitro has been achieved thus far in rodents, cattle, and primates, where it occurs spontaneously without the addition of growth factors or hormones. The ovaries of rodents are small enough to be cultured intact and, in that experimental situation, some follicles activate, while many remain in the resting pool, and the addition of specific factors can increase or decrease the number of follicles that leave the resting pool in vitro. In contrast, follicular activation in cattle and primates has been studied by culturing small pieces of the ovarian cortex, rich in primordial follicles, and the great majority of the primordial follicles activate in that situation, suggesting the importance of inhibitory factors to the normal, gradual exit of follicles from the resting pool. In cultured rodent ovaries, follicles appear to pass easily and spontaneously from the primary to the secondary stage, whereas few of the activated follicles in cultured cortical pieces from cattle or primates progress from the primary to the secondary stage. Understanding the requirements for the primary to secondary transition is critical for growing follicles activated in vitro to the late preantral and antral stages. In contrast, the requirements for the continued growth of larger preantral follicles, which can be isolated for in vitro studies, have been extensively explored in rodents and to a lesser extent in domestic species. A number of hormones and factors have been implicated and will be discussed. Taken together, the results highlight the need for a better understanding of the earliest stages of follicular development in domestic ruminants, particularly follicle activation and the primary to secondary follicle transition.

Localization of species conserved zona pellucida antigens in mammalian ovaries

The mammalian zona pellucida (ZP) consists of three glycoproteins (ZP1, ZP2 and ZP3), which are variably conserved among species at the genomic and amino acid levels. In order to evaluate the expression of ZP during ovarian development, a population of antibodies was selected that recognize species conserved antigenic domains of the three ZP proteins. Domain specific antibodies were selected from sera of rabbits immunized with all three native pig ZP proteins by elution of antibodies bound to each of the three human ZP recombinant proteins expressed from cDNAs, using the baculovirus expression system in insect cells. Immunoblot analysis was used to characterize the specificity of the antibodies and immunohistochemistry was used to evaluate the stage specific expression of ZP proteins during ovarian follicular development of the mouse, baboon and human. This study demonstrates that the conserved domains of all three ZP proteins are localized in the oocyte extracellular ZP matrix as well as in a subset of granulosa cells. However, this expression does vary among species with respect to the stage and cell type during early stages of ovarian follicular development. These antibodies should serve as excellent markers for evaluating early stages of human ovarian follicular development and in the development of contraceptive agents.

Distribution of P2X receptor subtypes in the rat female reproductive tract at late pro-oestrus/early oestrus.

Using immunohistochemistry techniques, we have examined the female reproductive organs of the rat in late pro-oestrus/early oestrus for the presence of purine nucleotide P2X1-7 receptors. In contrast to the male genital organs and the urinary tract, P2X1 receptors were present weakly, if at all, on smooth muscle membranes, except in blood vessels, whereas P2X2 immunoreactivity in smooth muscle was present in ovary and uterus as well as in blood vessels. Neither P2X1 nor P2X2 receptors were present in fallopian tubes. P2X5 receptors were seen in the differentiating cell layers of the stratified squamous vaginal epithelium and also in the very early stages of ovarian follicular development; P2X6 receptors were present in secondary follicles. P2X7 receptors, markers for programmed cell death, were present in the keratinised vaginal epithelium and also in the exfoliating superficial endometrial cells. The possible biological significance of these signalling molecules in the female reproductive tract is discussed.

Up-regulation of urokinase plasminogen activator messenger ribonucleic acid and protein in hen granulosa cells by transforming growth factor alpha in vitro during follicular development.

The aim of the present study was to examine the regulatory role of transforming growth factor alpha (TGF alpha) on urokinase plasminogen activator (uPA) gene expression and protein levels in hen granulosa cells from different stages of ovarian follicular development in vitro. Granulosa cells from the first (F1), the second and third (F2-3), and the fourth, fifth, and sixth (F4-6) largest preovulatory follicles were cultured for 21 h in the absence and presence of TGF alpha (10 ng/ml). The uPA mRNA abundance and protein content were determined by Northern and Western blot analysis, respectively. Cell-associated and secreted PA activity was measured by a fibrinolysis assay and characterized by zymography. Hen granulosa cells produce a uPA with a molecular mass of about 35 kDa and a transcript size of approximately 2.5 kb. Basal uPA mRNA abundance, protein content, and activity were highest in granulosa cells from F4-6 follicles and decreased with follicular maturation. Granulosa cell uPA mRNA levels, protein content, and activity were increased in the presence of TGF alpha, reaching maximal levels in granulosa cells from less mature follicles, although the percentage of stimulation was higher in cells from late stages of follicular development. These findings clearly demonstrate specific expression of uPA in proliferatively active granulosa cells and responsiveness of uPA to TGF alpha at both transcriptional and translational levels. They support the concept that PA of the urokinase type plays an important role in extracellular matrix remodeling during TGF alpha-induced granulosa cell proliferation and ovarian follicular growth.