Early Stages Of Follicular Development Research Articles

Development of the human ovary: Fetal through pubertal ovarian morphology, folliculogenesis and expression of cellular differentiation markers

A definition of normal human fetal and early postnatal ovarian development is critical to the ability to accurately diagnose the presence or absence of functional ovarian tissue in clinical specimens. Through assembling an extensive histologic and immunohistochemical developmental ontogeny of human ovarian specimens from 8 weeks of gestation through 16 years of postnatal, we present a comprehensive immunohistochemical mapping of normal protein expression patterns in the early fetal through post-pubertal human ovary and detail a specific expression-based definition of the early stages of follicular development. Normal fetal and postnatal ovarian tissue is defined by the presence of follicular structures and characteristic immunohistochemical staining patterns, including granulosa cells expressing Forkhead Box Protein L2 (FOXL2). However, the current standard array of immunohistochemical markers poorly defines ovarian stromal tissue, and additional work is needed to identify new markers to advance our ability to accurately identify ovarian stromal components in gonadal specimens from patients with disorders of sexual differentiation.

AOP key event relationship report: Linking decreased androgen receptor activation with decreased granulosa cell proliferation of gonadotropin-independent follicles

We recently proposed to formally recognize Key Event Relationships (KERs) as building blocks of Adverse Outcome Pathways (AOPs) that can be independently developed and peer-reviewed. Here, we follow this approach and provide an independent KER from AOP345, which describes androgen receptor (AR) antagonism leading to decreased female fertility. This KER connects AR antagonism to reduced granulosa cell proliferation of gonadotropin-independent follicles (KER2273). We have developed both the KER and the two adjacent Key Events (KEs). A systematic approach was used to ensure that all relevant supporting evidence for KER2273 was retrieved. Supporting evidence for the KER highlights the importance of AR action during the early stages of follicular development. Both biological plausibility and empirical evidence are presented, with the latter also assessed for quality. We believe that tackling isolated KERs instead of whole AOPs will accelerate the AOP development. Faster AOP development will lead to the development of simple test methods that will aid screening of chemicals, endocrine disruptor identification, risk assessment, and subsequent regulation.

Assessment of Cortisol Hormonal Level and Relation With Age in Infertile Women With ICSI Outcome

In human-assisted reproduction both partners need to be evaluated during counseling because causes of infertility are due to male factors and female factors. some body hormones play important role in fertile women and cortisol one of these hormones which is essential for normal ovulation, cortisol usually increases during the follicular phase of the menstrual cycle, play a role in the early stages of follicular development by their interaction with aromatase activity and granulose cell functions. To investigate the correlation of cortisol hormonal level in serum and in follicular fluid that relationship with age in women under Intracytoplasmic sperm injection outcome. Its randomized prospective clinical trial study performance conducting in the Higher Institute for Infertility Diagnosis and Assisted Reproductive Technologies / AlNahrain University, Baghdad-Iraq, during the period from November2021to June 2022. a total of Sixty-three Infertile Iraqi women, who planned to inter intrasytoplasmic sperm injection program. The blood sample and follicular fluid sample obtained from all infertile women inter intracytoplasmic sperm injection program to measure cortisol hormonal level at specific days of this study. There was increase in cortisol level in women aged between (30-40) years, So there was significant finding between cortisol level and this age group and there was increase in cortisol level in women aged < 30 years at the same day and no significant difference in cortisol level in women age more than 40. Significant differences in cortisol level appear in group of women age between (30-40) years, also the same result appears in group of women age more than > 40 years and no significant differences in cortisol level in women aged < 30. No significant differences in cortisol level in all age women group (30-40) years, <30 years ,and the women age > 40 years

Impaired leptin signaling causes subfertility in female zebrafish

Reproduction is an energetically costly event across vertebrates and tightly linked to nutritional status and energy reserves. In mammals, the hormone leptin is considered as a link between energy homeostasis and reproduction. However, its role in fish reproduction is still unclear. In this study, we investigated the possible role of leptin in the regulation of reproduction in zebrafish, using a loss of function leptin receptor (lepr) strain. Impaired leptin signaling resulted in severe reproductive deficiencies in female zebrafish. lepr mutant females laid significantly fewer eggs, with low fertilization rates compared to wild-type females. Folliculogenesis was not affected, but oocyte maturation and ovulation were disrupted in lepr mutants. Interestingly, the expression of luteinizing hormone beta (lhb) in the pituitary was significantly lower in mutant females. Analysis of candidate genes in the ovaries and isolated fully grown follicles revealed differential expression of genes involved in steroidogenesis, oocyte maturation and ovulation in the mutants, which are known to be regulated by LH signaling. Moreover, subfertility in lepr mutants could be partially restored by administration of human chorionic gonadotropin. In conclusion, our results show that leptin deficiency does not affect early stages of follicular development, but leptin might be essential in later steps, such as in oocyte maturation and ovulation. To our knowledge, this is the first time that leptin is associated to reproductive deficiencies in zebrafish.

Empty Zona Pellucida Only Case: A Critical Review of the Literature.

The presence of empty zona pellucida (EZP) in oocytes following oocyte retrieval (OR) during an in vitro fertilization (IVF) cycle presents a major clinical and laboratory challenge in assisted reproduction. It has been attributed to several factors such as the ovarian stimulation protocol employed, the damaging of the follicles during oocyte retrieval (OR) mainly through the high aspiration pressure, during the denudation technique, and the degeneration of oolemma within the zona pellucida (ZP) through apoptosis. The role of ZP is pivotal from the early stages of follicular development up to the preimplantation embryo development and embryo hatching. Polymorphisms or alterations on the genes that encode ZP proteins may contribute to EZP. We present a critical review of the published literature hitherto on EZP and available options when encountered with the phenomenon of EZP. Concerning the former, we found that there is rare data on this phenomenon that merits documentation. The latter includes technical, genetic, and pathophysiological perspectives, along with specific treatment options. In conclusion, we identify the lack of a definitive management proposal for couples presenting with this phenomenon, we underline the need for an algorithm, and indicate the questions raised that point towards our goal for a strategy when addressing a previous finding of EZP.

The earliest stages of follicular development: Follicle formation and activation

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 5a-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.

Quantitative proteomic profiling of bovine follicular fluid during follicle development.

Bovine follicular fluid (FF) constitutes the microenvironment of follicles and includes various biologically active proteins. We performed a study involving 18 healthy nonlactating Holstein cows to determine the protein expression profile of FF at key stages of follicular development. Follicles were individually aspirated in vivo at predeviation (F1 ∼ 7.0 mm), deviation (F1 ∼ 8.5 mm), postdeviation (F1 ∼ 12.0 mm), and preovulatory stages of follicle development, which were confirmed by measurement of follicular estradiol and progesterone concentrations. The FFs from nine cows were selected for proteomic analysis. After albumin depletion, triplicates of pooled FF were reduced, alkylated, and digested with trypsin. The resulting peptides were labeled with TMTsixplex and quantified using liquid chromatography-mass spectrometry/mass spectrometry. A total of 143 proteins were identified and assigned to a variety of biological processes, including response to stimulus and metabolic processes. Twenty-two differentially (P<0.05) expressed proteins were found between stages indicating intrafollicular changes over development, with expected deviation time critical to modulate the protein expression. For instance, high concentrations of follistatin, inhibin, serglycin, spondin-1, fibrinogen, and anti-testosterone antibody were found during early stages of follicular development. In contrast, apolipoprotein H, alpha-2-macroglobulin, plasminogen, antithrombin-III, and immunoglobulins were increased after deviation. Among the differentially abundant proteins, 19 were found to be associated with steroidogenesis. Pathway analysis identified proteins that were mainly associated with the acute phase response signaling, coagulation system, complement system, liver/retinoid X receptor activation, and biosynthesis of nitric oxide and reactive oxygen. The differentially expressed proteins provide insights into the size-dependent protein changes in the ovarian follicle microenvironment that could influence follicular function.

Anti-Müllerian hormone: A brief review of the literature

Anti-Müllerian hormone (AMH) is a dimeric glycoprotein, which was originally identified because of its role in male sexual differentiation. In fact, AMH is expressed by the Sertoli cellsof the foetal testes and induces the regression of the Müllerian ducts. In the absence of AMH, the Müllerian ducts evolve in the uterus, fallopian tubes and in the upper part of the vagina. In women AMH is produced by granulose cells, pre-antral and antral follicles and its major physiological role in the ovaries seems to be limited to inhibiting the development of the earliest stages of follicular development. AMH has been studied because of its potential clinical utility: the diagnosis of intersexuality in children, the diagnosis of criptorchism and anorchia, the diagnosis of early or delayed puberty, the diagnosis of granulosa cell tumours, ovarian function in women who have undergone chemotherapy, the determination of female ovary function and the diagnosis and follow-up of polycystic ovarian disease. The objective of this brief review is to ascertain the current status of studies on AMH and potential future prospects. Review the most recent literature. There are still several points that need to be clarified with regard to all of the activities of AMH. The techniques for handling serum samples and hormone assays need to be made even more uniform. In recent years, it has been found that AMH is not only a male hormone, but it is being proven to be a valuable tool to better understand ovarian function, both in young people and in adult females. At the moment, the clinical use of serum assays for AMH still faces technical problems.

Administration of DHEA augments progesterone production in a woman with low ovarian reserve being transplanted with cryopreserved ovarian tissue

In recent years cryopreservation of ovarian tissue prior to potentially gonadotoxic treatment for malignant disease has proved to be an option for preservation of the reproductive function including fertility [1]. Following successful cancer treatment women have chosen to have transplantation of their frozen-thawed tissue if ovarian failure occurred as a consequence of cancer treatment. The autotransplanted tissue has in various centers resulted in the re-establishment of ovarian function leading to the return of fertility and the birth of healthy children [2, 3]. However, the ovarian reserve in the transplanted tissue typically is very low as currently only a limited number of follicles survive the entire procedure including freezing, thawing and transplantation [4]. This diminished reserve is reflected by persistent low levels of AMH [4], limiting the efficacy of controlled ovarian stimulation (COS) with exogenous FSH. In terms of fertility treatment, the women who have had frozen/thawed ovarian tissue transplanted often resemble poor responder patients and patients of advanced reproductive age. Attempts to augment the number of developing follicles by administration of oral DHEA for some months in the latter category of women have been reported [5–7], there being a growing literature describing a higher oocyte yield following COS and oocytes showing a better quality and thereby improving the reproductive outcome [5–7]. The particular mechanism by which DHEA may enhance follicle survival and growth has not yet been fully elucidated, but conversion of DHEA to androgens and an androgen priming effect on early stages of follicular development leading to an increased FSH receptor expression is one possible mechanism [8]. Although the vast majority of DHEA in the body is produced by the adrenals one obstacle in defining a more precise function of DHEA in improving follicle recruitment is the fact that the circulating DHEA is the sum of the local productions in the ovaries and those of adrenal glands [9]. In this study we observed the ovarian and endocrine events following DHEA administration in a woman, who had normal adrenal function but with very low ovarian reserve consequent to transplantation of frozen/thawed ovarian tissue, being postmenopausal at the time of transplantation. Only a fraction of frozen/thawed tissue from one ovary had been grafted with subsequent low ovarian reserve and evidenced by few follicles. This situation allowed a more direct evaluation of the endocrinological effect of DHEA administration on the synthesis of sex steroids.

TRIENNIAL REPRODUCTION SYMPOSIUM: The ovarian follicular reserve in cattle: What regulates its formation and size?1,2

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.

Characterization of Wnt2 Overexpression in a Rat Granulosa Cell Line (DC3): Effects on CTNNB1 Activation1

WNTs comprise a family of secreted glycoproteins that are essential for normal embryonic development of the female reproductive system. The functional role that WNTs play in the postnatal ovary is poorly defined. We have shown previously that Wnt2 and Fzd4 mRNAs are expressed in granulosa cells of the postnatal rat ovary. Here we examine the effects of Wnt2 overexpression in a rat granulosa cell line (DC3) that displays characteristics of granulosa cells at an early stage of follicular development. We show that DC3 cells express a 7.7-kb Fzd4 mRNA transcript similar in size to that detected in the rat and human ovary. Our results demonstrate that Wnt2 overexpression in DC3 promotes cytosolic and nuclear accumulation of beta-catenin (CTNNB1), but does not stimulate CTNNB1/TCF-dependent (pGL3-OT) transcriptional activity. We show that chibby (CBY1), a nuclear CTNNB1-associated antagonist of the WNT pathway, is expressed in DC3 cells and associates with CTNNB1 in the presence and absence of Wnt2 overexpression, suggesting that Cby1 contributes to suppression of CTNNB1/TCF-dependent transcription in these cells. Our results show that Wnt2 overexpression in DC3 cells increases follistatin (Fst) mRNA expression and promotes resistance to activin-induced cell deletion. Taken together, our results suggest that WNT2 opposes activin activity in granulosa cells by up-regulating expression of the activin antagonist Fst in a CTNNB1/TCF-independent manner, and that rat granulosa cells express factors, including Cby1, that suppress CTNNB1/TCF-dependent signal transduction in the presence of a WNT signal.

In vitro and in vivo regulation of follicular formation and activation in cattle

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 earliest stages of follicular development: Follicle formation and activation

The earliest stages of follicular development: Follicle formation and activation

Distribution of androgen and progesterone receptors in the spiny mouse (Acomys cahirinus) ovary during postnatal life

This study describes the localization of androgen (AR) and progesterone (PR) receptors in the developing ovary in the spiny mouse. The immunohistochemical analysis showed for the first time the expression of AR and PR proteins in the ovary as early as in one day-old females. Both AR and PR were present in germinal epithelium cells, stromal cells as well as in the granulosa and theca layer of ovarian follicles. On days 7, 14, 21, 30, 60 and 90, the distribution of AR and PR depended on the stage of follicular development rather than on the animal's age. A novel observation was that PR protein was detected not only in granulosa cells of preovulatory follicles, but also in the growing and early antral follicles. It was demonstrated that there is a different pattern of AR and PR immunoexpression throughout folliculogenesis. In contrast to AR, whose expression decreased during follicular development, the PR immunostaining increased during this time. It is concluded that androgens and progesterone may play an important role in the early stage of follicular development in the spiny mouse.

Yolk testosterone and corticosterone in hierarchical follicles and laid eggs of Japanese quail exposed to long-term restraint stress

Environmental and behavioural stimuli experienced by egg-laying female birds contribute to intra- and inter-female differences in hormones in the egg yolk with consequences for offspring development. The understanding of physiological mechanisms underlying yolk hormone deposition can aid progress in this field. In our study, we measured the concentration of testosterone and corticosterone in hierarchical follicles and egg yolks of Japanese quail in control and chronic stress conditions. Experimental females were reared under hypodynamia, a model situation for restraint stress, from day 3 to 63 days of age. For yolk hormone analysis, four largest follicles of ovarian hierarchy (F1–F4), eggs present in the oviduct and eggs laid on the day before were collected. In chronically stressed birds, yolk testosterone concentrations decreased from F2 onwards, while yolk corticosterone content was increased from the beginning to the end of egg formation. The follicular profile of hormones suggested testosterone transfer into the yolk directly from granulosa and theca cells, with the highest accumulation during a period 48–72 h before laying the egg. Yolk corticosterone was accumulated from maternal plasma preferentially in early stages of follicular development under control conditions and also in last stages of egg formation under stress conditions. These specific patterns of hormone deposition indicate periods when stimuli experienced by female can substantially modify hormonal content of eggs. Lower testosterone and increased corticosterone yolk concentrations in stressed quail may represent signals mediating information about adverse environmental conditions from the mother to progeny.

Oocyte-granulosa-theca cell interactions during preantral follicular development

The preantral-early antral follicle transition is the penultimate stage of follicular development in terms of gonadotropin dependence and follicle destiny (growth versus atresia). Follicular growth during this period is tightly regulated by oocyte-granulosa-theca cell interactions. Formation of the theca cell layer is a key event that occurs during this transitional stage. Granulosal factor(s) stimulates the recruitment of theca cells from cortical stromal cells, while oocyte-derived growth differentiation factor-9 (GDF-9) is involved in the differentiation of theca cells during this early stage of follicular development. The preantral to early antral transition is most susceptible to follicular atresia. GDF-9 promotes follicular survival and growth during transition from preantral stage to early antral stage by suppressing granulosa cell apoptosis and follicular atresia. GDF-9 also enhances preantral follicle growth by up-regulating theca cell androgen production. Thecal factor(s) promotes granulosa cell proliferation and suppress granulosa cell apoptosis. Understanding the intraovarian mechanisms in the regulation of follicular growth and atresia during this stage may be of clinical significance in the selection of the best quality germ cells for assisted reproduction. In addition, since certain ovarian dysfunctions, such as polycystic ovarian syndrome and gonadotropin poor-responsiveness, are consequences of dysregulated follicle growth at this transitional stage, understanding the molecular and cellular mechanisms in the control of follicular development during the preantral-early antral transition may provide important insight into the pathophysiology and rational treatment of these conditions.

Effects of IGF-I bioavailability on bovine preantral follicular development in vitro

The aim of this study was to determine the effect of regulation of IGF-I bioavailability on preantral follicle development in vitro. Bovine preantral follicles were cultured for 6 days in serum-free medium with increasing doses of Long R3 (LR3) IGF-I (an analog with low affinity for IGF-binding proteins (IGFBPs)), or human recombinant IGF-I (hrIGF-I). Follicle diameter and estradiol production were measured every second day. On day 6, ratios of oocyte/follicle diameter and oocyte morphology were assessed by histological examination, and IGFBP-2 and -3 were detected by immunocytochemistry and in situ hybridization respectively. Both types of IGF-I increased follicle diameter in a dose-dependent manner (P < 0.05) and increased estradiol production over control levels (P < 0.05). However, follicles treated with LR3 IGF-I and the highest concentration of hrIGF-I (1000 ng/ml) had smaller oocyte/follicle ratios, and increased oocyte degeneration, compared with controls or follicles treated with physiological concentrations of hrIGF-I (P < 0.05). IGFBPs were detected in cultured preantral follicles, indicating a requirement for regulation of IGF bioavailability during the early stages of follicular development. Specifically, IGFBP-3 mRNA was found to be expressed in oocytes, and IGFBP-2 immunoreactivity was detected in oocytes and granulosa cells of cultured follicles. In summary, the regulation of IGF-I bioavailability by IGFBPs is necessary for the co-ordination of oocyte and follicle development in vitro.

Role of granulosa and theca cell interactions in ovarian follicular maturation

We developed a culture system in which two types of ovarian follicular cells were allowed to attach to opposite sides of a collagen membrane. Using this in vitro cell culture system, we studied the effects of granulosa- and theca-cell interaction on the morphology, structure, and function of bovine ovarian follicular cells. In the first part of the study, we explored how the interaction between theca and granulosa cells affects the morphology and structure of the cells. This study was done using follicular cells collected from bovine ovarian follicles at the early developmental stage. Granulosa cells cultured alone were flattened, and formed a monolayer sheet. By contrast, granulosa cells cultured with theca cells were convex, and formed multilayer sheets. Theca cells cultured alone were thin, flat, and spindle-shaped. Theca cells cultured with granulosa cells were also spindle-shaped; however, they appeared convex and more densely packed when compared with theca cells cultured alone. In the second part of the study, the possible role of the cellular interaction in the control of differentiation and growth of granulosa and theca cells was investigated. When follicular cells were isolated from the early stage of follicular development, theca cells reduced progesterone and inhibin production by granulosa cells and augmented the growth of granulosa cells. When the cells were isolated from the late stage of follicular development, by contrast, theca cells augmented hormonal production by granulosa cells, and did not affect the growth of granulosa cells. The growth and androstenedione production by theca cells were increased by the presence of granulosa cells, irrespective of the origin of follicular cells. These results demonstrated that communication between two types of follicular cells results in reciprocal modulation of their morphology, structure, growth, and function. Cellular interactions seem to be one of the major factors controlling the differentiation and growth of the follicular cells during the follicular maturation process. In contrast to granulosa and theca cells cultured alone, cells in the coculture seemed to possess morphological and functional characteristics more similar to those of cells in the growing follicular wall in vivo. Thus, we speculate that the interaction between these two types of follicular cells is essential for the maintenance of original structure and function of the bovine follicular wall.

Stem cell factor modulates the expression of steroidogenesis related proteins and FSHR during ovarian follicular development

Stem cell factor (SCF) is essential for the development of primordial follicles. By using cultured ovaries from neonatal rats, the effect of SCF on early follicular development was investigated. Steroidogenesis is a hallmark of follicular development. Expressions of three key protein factors in steroidogenesis, SF-1, StAR, and P450arom, were investigated using immunohistochemistry and in situ hybridization. SF-1 and StAR proteins were expressed in all ovarian cells. P450arom mRNA was localized exclusively in oocytes implying that estrogen might be synthesized by oocytes at this stage. SCF up-regulated the mRNA and protein expression of these proteins, suggesting SCF might promote the production of estrogen during this period of time. To study the differentiation status of follicular cells, the expression of FSHR and its response to SCF treatment was examined by using semi-quantitative RT-PCR. The results showed that SCF inhibited the expression of FSHR mRNA. It was also observed that SCF stimulated the expression of basic fibroblast growth factor (bFGF) in oocytes. Inactivation of bFGF by its neutralizing antibody resulted in a reversal of the inhibitory effect of SCF on the expression of FSHR. Therefore, the FSHR inhibitory effect of SCF could be mediated by bFGF. In summary, it seems that, at the early stages of follicular development, SCF might stimulate oocytes to produce estrogen while it inhibits the differentiation of granulosa cells that are the major sources of estrogen at the late stages of follicular development.

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.