TGF-β1-induced collagen promotes chicken ovarian follicle development via an intercellular cooperative pattern

Follicle development is a complex process under strict regulation of diverse hormones and cytokines including transforming growth factor β (TGF-β) superfamily members. TGF-β is pivotal for the regulation of ovarian functions under physiological and pathological conditions. In this study, effect of TGF-β1 on chicken follicle development was examined through investigating the accumulation and action of collagen, an indispensable member of the extracellular matrix (ECM) involved in this process. The granulosa cells (GCs) and theca cells (TCs) were separated from growing follicles of the laying chicken for treatment of TGF-β1 and analysis of expression of ECM components and key proteins in intracellular signaling pathways. Results showed that collagen was mainly distributed in the follicular theca layer and was produced with the formation of the granulosa layer during ovarian development. Collagen accumulation increased with follicle growth and treatment of GCs with TGF-β1 elicited an increased expression of collagen. After production from GCs, collagen was transferred to the neighboring TCs to promote cell proliferation and inhibit apoptosis. Treatment of collagen remarkably increased expression of p-ERK, mitogen-activated protein kinase (MAPK), and p-MAPK, but treatment with hydroxylase inhibitor (to break collagen structure) reversed these alterations. In conclusion, during follicle growth collagen was secreted by GCs under TGF-β1 stimulation and was subsequently collaboratively transferred to neighboring TCs to increase cell proliferation and thus to promote follicle development via an intercellular cooperative pattern during development of chicken growing follicles.     

Changes in the organization of the extracellular matrix in ovarian follicles during the preovulatory phase and atresia. An immunofluorescence study

The distribution of laminin, type IV collagen and fibronectin was studied by immunofluorescence in rat, pig and cow ovarian follicles. The results obtained in the three species investigated were similar. In all the follicles, laminin and type IV collagen were identically localized in the basal lamina (BL) separating the granulosa and the theca layers. In addition, these two proteins were also distributed in the wall of blood vessels of the thecae and ovarian stroma. The staining showed that the BL of primordial and growing follicles was regular and continuous, but underwent striking modifications during ovulation and atresia. In fact, in preovulatory follicles the BL appeared thinner and discontinuous, whereas it was much thickened and ruptured in atretic follicles. Fibronectin was localized mainly in inner granulosa cells of small and medium-sized growing follicles, and as a broad and irregular layer around the cavity of the degenerated follicles. The results show that each stage of follicular growth and involution is associated with a precise and peculiar pattern of distribution of laminin, type IV collagen and fibronectin. The possibility that these proteins play a role in the local control of ovarian follicular dynamics is advanced.     

Role of ovarian theca and granulosa cell interaction in hormone productionand cell growth during the bovine follicular maturation process

We have investigated the possible role of theca and granulosa cell interaction in the control of the hormone-producing activity and growth of granulosa and theca cells during bovine ovarian follicular development, using a coculture system in which granulosa and theca cells were grown on opposite sides of a collagen membrane. When follicular cells were isolated from small follicles (3-5 mm), theca cells reduced estradiol, progesterone, and inhibin production by granulosa cells to 14 +/- 5%, 64 +/- 6%, and 27 +/- 4%, respectively, of the production by granulosa cells cultured alone. On the other hand, when the cells were isolated from large follicles (15-18 mm), theca cells increased these levels to 253 +/- 34%, 156 +/- 24%, and 287 +/- 45%, respectively. Theca cells did not affect the growth of granulosa cells. Androstenedione production by theca cells was augmented by granulosa cells to 861 +/- 190% (in small follicles) and 1298 +/- 414% (in large follicles), respectively. The growth of theca cells was also augmented by granulosa cells (small follicle, 210 +/- 43%, and large follicle, 194 +/- 24%, respectively). These results indicate that theca cells secrete factor(s) inhibiting the differentiation of immature while promoting that of matured granulosa cells; they also suggest that granulosa cells secrete factor(s) promoting both the differentiation and growth of theca cells throughout the follicular maturation process.     

Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary

The aim of the study was to determine the expression of proliferating cell nuclear antigen protein (PCNA) in the pig ovary. The localization of PCNA was demonstrated in paraffin sections of pig ovarian tissue using primary mouse monoclonal anti-PCNA antibody. In primordial follicles, no remarkable staining for PCNA either in granulosa cells or in the oocytes was observed. In primary to secondary follicles, positive staining in oocytes and in some granulosa cells was detected. The advanced preantral and particularly actively growing small to large antral follicles showed extensive PCNA labeling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. PCNA labeling was expressed in nuclei of oocytes in preantral and small antral follicles. In atretic follicles, the level of PCNA protein expression was dependent on the stage of atresia. Follicles demonstrating advanced atresia showed only limited or no PCNA labeled granulosa and theca cells. The results of the study demonstrate that follicular growth and development in pig ovary may be effectively monitored by determining the granulosa cell expression of PCNA.     

Dynamics of extracellular matrix in ovarian follicles and corpora lutea of mice

Despite the mouse being an important laboratory species, little is known about changes in its extracellular matrix (ECM) during follicle and corpora lutea formation and regression. Follicle development was induced in mice (29 days of age/experimental day 0) by injections of pregnant mare’s serum gonadotrophin on days 0 and 1 and ovulation was induced by injection of human chorionic gonadotrophin on day 2. Ovaries were collected for immunohistochemistry (n=10 per group) on days 0, 2 and 5. Another group was mated and ovaries were examined on day 11 (n=7). Collagen type IV α1 and α2, laminin α1, β1 and γ1 chains, nidogens 1 and 2 and perlecan were present in the follicular basal lamina of all developmental stages. Collagen type XVIII was only found in basal lamina of primordial, primary and some preantral follicles, whereas laminin α2 was only detected in some preantral and antral follicles. The focimatrix, a specialised matrix of the membrana granulosa, contained collagen type IV α1 and α2, laminin α1, β1 and γ1 chains, nidogens 1 and 2, perlecan and collagen type XVIII. In the corpora lutea, staining was restricted to capillary sub-endothelial basal laminas containing collagen type IV α1 and α2, laminin α1, β1 and γ1 chains, nidogens 1 and 2, perlecan and collagen type XVIII. Laminins α4 and α5 were not immunolocalised to any structure in the mouse ovary. The ECM composition of the mouse ovary has similarities to, but also major differences from, other species with respect to nidogens 1 and 2 and perlecan.     

Expression of extracellular matrix components is disrupted in the immature and adult estrogen receptor β-null mouse ovary

Within the ovary, Estrogen Receptor β (ERβ) is localized to the granulosa cells of growing follicles. 17β-estradiol (E2) acting via ERβ augments the actions of follicle stimulating hormone in granulosa cells, leading to granulosa cell differentiation and formation of a preovulatory follicle. Adult ERβ-null females are subfertile and possess ovaries with reduced numbers of growing follicles and corpora lutea. Because the majority of E2 production by granulosa cells occurs once puberty is reached, a role for ERβ in the ovary prior to puberty has not been well examined. We now provide evidence that lack of ERβ disrupts gene expression as early as post-natal day (PND) 13, and in particular, we identify a number of genes of the extracellular matrix (ECM) that are significantly higher in ERβ-null follicles than in wildtype (WT) follicles. Considerable changes occur to the ECM occur during normal folliculogenesis to allow for the dramatic growth, cellular differentiation, and reorganization of the follicle from the primary to preovulatory stage. Using quantitative PCR and immunofluorescence, we now show that several ECM genes are aberrantly overexpressed in ERβ-null follicles. We find that Collagen11a1, a protein highly expressed in cartilage, is significantly higher in ERβ-null follicles than WT follicles as early as PND 13, and this heightened expression continues through PND 23-29 into adulthood. Similarly, Nidogen 2, a highly conserved basement membrane glycoprotein, is elevated in ERβ-null follicles at PND 13 into adulthood, and is elevated specifically in the ERβ-null focimatrix, a basal lamina-like matrix located between granulosa cells. Focimatrix laminin and Collagen IV expression were also higher in ERβ-null ovaries than in WT ovaries at various ages. Our findings suggest two novel observations: a) that ERβ regulates granulosa cell gene expression ovary prior to puberty, and b) that ERβ regulates expression of ECM components in the mouse ovary.     

Requirement for ADAMTS-1 in extracellular matrix remodeling during ovarian folliculogenesis and lymphangiogenesis

Murine ovarian folliculogenesis commences after birth involving oocyte growth, somatic cell differentiation and structural remodeling of follicle stromal boundaries. The extracellular metalloproteinase ADAMTS-1 has activity against proteoglycans and collagen and is produced by the granulosa cells of ovarian follicles. Mice with ADAMTS-1 gene disruption are subfertile due to an unknown mechanism resulting in severely reduced ovulation. Here we show that ADAMTS-1 is necessary for structural remodeling during ovarian follicle growth. A significant reduction in the number of healthy growing follicles and corresponding follicle dysmorphogenesis commencing at the stage of antrum formation was identified in ADAMTS-1-/- ovaries. Morphological analysis and immunostaining of basement membrane components identified stages of follicle dysgenesis from focal disruption in ECM integrity to complete loss of follicular structures. Cells expressing the thecal marker Cyp-17 were lost from dysgenic regions, while oocytes and dispersed cells expressing the granulosa cell marker anti-mullerian hormone persisted in ovarian stroma. Furthermore, we found that the ovarian lymphatic system develops coincidentally with follicular development in early postnatal life but is severely delayed in ADAMTS-1-/- ovaries. These novel roles for ADAMTS-1 in structural maintenance of follicular basement membranes and lymphangiogenesis provide new mechanistic understanding of folliculogenesis, fertility and disease.     

Expression of follicle stimulating hormone and luteinizing hormone receptors during follicular growth in the domestic cat ovary

In order to better understand the pituitary regulation of follicular growth in the domestic cat, follicle stimulating hormone (FSH) and luteinizing hormone (LH) receptors (R) were localized and quantified in relation to follicle diameter and atresia using in situ ligand binding on ovarian sections. Expression of FSHR was homogeneous and restricted to follicle granulosa cells from the early antral stage onwards, whereas expression of LHR was heterogeneous on theca cells of all follicles from the early antral stage onward, and homogeneous on granulosa cells of healthy follicles larger than 800 microm in diameter and in corpora lutea. LHR were also widely expressed as heterogeneous aggregates in the ovarian interstitial tissue. Atretic follicles exhibited significantly reduced levels of both FSHR and LHR on granulosa cells, compared with healthy follicles whatever the follicular diameter, whereas levels of LHR on theca cells were lower only for atretic follicles larger than 1,600 microm in diameter. In healthy follicles, levels of FSHR and LHR in all follicular compartments increased significantly with diameter. Although generally comparable to that observed in other mammals, the expression pattern of gonadotropin receptors in the cat ovary is characterized by an early acquisition of LHR on granulosa cells of growing follicles and islets of LH binding sites in the ovarian interstitial tissue.     

Messenger RNA and protein expression analysis of betaglycan in the pituitary and ovary of the domestic hen

Betaglycan was originally characterized as the type III receptor for TGFbeta, yet recent research has indicated that betaglycan can serve as an accessory receptor for inhibin. To understand better the action of inhibin in avian follicular development, we have investigated the expression of betaglycan in the pituitary gland and ovary of the hen. In experiments 1 and 2, betaglycan mRNA was detected at 6 kilobases (kb) by Northern blot analysis (n = 5) in chicken pituitary, granulosa, and theca layers and whole ovary. Expression of betaglycan was greatest in the pituitary gland in experiment 1 and greater in the granulosa layer of small yellow follicles (SYF) compared with the granulosa layer of larger follicles. In experiment 2, betaglycan mRNA was more abundantly expressed in the theca layer compared with the granulosa layer for all follicle sizes, although there was no significant difference in betaglycan expression in the theca layer among follicle sizes. In experiment 3, immunohistochemical analysis revealed betaglycan protein in the anterior pituitary as well as in the ovary (n = 4) and SYF (n = 4). Colocalization studies revealed a high abundance of cells within the anterior pituitary expressing both betaglycan and FSH (n = 4). Betaglycan protein was found in the granulosa layer; however, markedly enhanced staining was observed in the theca layer of ovarian follicles. Our results provide evidence for expression of betaglycan mRNA and protein colocalization with FSH in the anterior pituitary, consistent with known inhibin effects. Ovarian localization of betaglycan, particularly in the theca layer, suggests a paracrine role for inhibin in the hen.     

Transforming growth factor-alpha in the adult bovine ovary: identification in growing ovarian follicles

The pituitary gonadotropins and gonadal steroids are required for normal follicular growth and development but neither has been shown to act directly as a granulosa cell mitogen in vitro. A number of polypeptide growth factors, however, are known to have pronounced mitogenic effects on the cells of the follicle. We have localized transforming growth factor-alpha (TGF-alpha), a potent mitogen, in bovine thecal cells via immunoperoxidase staining using a monoclonal antibody for TGF-alpha that does not cross-react with epidermal growth factor. TGF-alpha staining is most intense in the theca of follicles at the discrete physiological stages known to show rapid granulosa cell growth (small follicles of 0.7-2.0 mm diameter). Staining intensity for TGF-alpha declines in large preovulatory follicles, coincident with the known decline in granulosa cell mitosis. These studies provide further evidence for paracrine interactions in the ovary and show that TGF-alpha may play an important role in the regulation of follicular development in the adult bovine ovary.     

Follicle-restricted compartmentalization of transforming growth factor beta superfamily ligands in the feline ovary

Ovarian follicular development, follicle selection, and the process of ovulation remain poorly understood in most species. Throughout reproductive life, follicle fate is balanced between growth and apoptosis. These opposing forces are controlled by numerous endocrine, paracrine, and autocrine factors, including the ligands represented by the transforming growth factor beta (TGFbeta) superfamily. TGFbeta, activin, inhibin, bone morphometric protein (BMP), and growth differentiation factor 9 (GDF-9) are present in the ovary of many animals; however, no comprehensive analysis of the localization of each ligand or its receptors and intracellular signaling molecules during folliculogenesis has been done. The domestic cat is an ideal model for studying ovarian follicle dynamics due to an abundance of all follicle populations, including primordial stage, and the amount of readily available tissue following routine animal spaying. Additionally, knowledge of the factors involved in feline follicular development could make an important impact on in vitro maturation/in vitro fertilization (IVM/IVF) success for endangered feline species. Thus, the presence and position of TGFbeta superfamily members within the feline ovary have been evaluated in all stages of follicular development by immunolocalization. The cat inhibin alpha subunit protein is present in all follicle stages but increases in intensity within the mural granulosa cells in large antral follicles. The inhibin betaA and betaB subunit proteins, in addition to the activin type I (ActRIB) and activin type II receptor (ActRIIB), are produced in primordial and primary follicle granulosa cells. Additionally, inhibin betaA subunit is detected in the theca cells from secondary through large antral follicle size classes. GDF-9 is restricted to the oocyte of preantral and antral follicles, whereas the type II BMP receptor (BMP-RII) protein is predominantly localized to primordial- and primary-stage follicles. TGFbeta1, 2, and 3 ligand immunoreactivity is observed in both small and large follicles, whereas the TGFbeta type II receptor (TGFbeta RII) is detected in the oocyte and granulosa cells of antral follicles. The intracellular signaling proteins Smad2 and Smad4 are present in the granulosa cell cytoplasm of all follicle size classes. Smad3 is detected in the granulosa cell nucleus, the oocyte, and the theca cell nucleus of all follicle size classes. These data suggest that the complete activin signal transduction pathway is present in small follicles and that large follicles primarily produce the inhibins. Our data also suggest that TGFbeta ligands and receptors are colocalized to large antral follicles. Taken together, the ligands, receptors, and signaling proteins for the TGFbeta superfamily are present at distinct points throughout feline folliculogenesis, suggesting discrete roles for each of these ligands during follicle maturation.     

Gene expression of endothelin-1 in the porcine ovary: follicular development

We have investigated which follicular compartment and stage of follicular development are associated with endothelin-1 (ET-1) gene expression in the porcine ovary. The localization of mature ET-1 peptide and of its mRNA was determined by immunohistochemistry and by in situ hybridization. Stage of follicular development associated with ET-1 expression was investigated in terms of follicular class and occurrence of atresia. The latter was investigated by determining the occurrence of DNA fragmentation in apoptotic cells on adjacent sections to those used for ET-1 gene expression. Fifteen ovaries from 10 prepubertal pigs stimulated with gonadotropin were collected; a total of 1050 follicles were examined. Specific ET-1 immunoreactivity was restricted to the ovarian vasculature and to the granulosa cell compartment of antral follicles. The pattern of ET-1 mRNA expression was similar to that found for ET-1 immunoreactivity. Primordial, primary, and most secondary follicles did not express ET-1. The theca cell layer did not express ET-1 regardless of follicle developmental stage. ET-1 expression occurred with a significantly greater probability (P < 0.001 by the likelihood ratio test) in the granulosa cell compartment of antral follicles than in any other follicle class. Furthermore, in antral follicles, ET-1 expression occurred with a greater likelihood in large antral follicles than in small antral follicles (P < 0.001 by the likelihood ratio test). In small antral follicles, only 16.8% expressed ET-1; in contrast, 66.7% of large antral follicles exhibited ET-1 expression. It is interesting that in follicles in which ovulation had already occurred, intense ET-1 expression was found only in the prominent developing vasculature, the other cells present in the luteinized follicle did not display any ET-1 expression. The pattern of ET-1 gene expression observed in this study would be in agreement with our previous suggestion of a plausible physiological role for ET-1 in preventing premature progesterone production by granulosa cells of an antral follicle. The occurrence of atresia and expression of ET-1 in the same follicle was rare. Small and large antral follicles constituted 5.1% and 5.6%, respectively, of the examined follicles in this category. The majority of atretic follicles did not express ET-1 and, conversely, follicles that expressed ET-1 were not atretic. To the best of our knowledge, this is the first report in which large, nonatretic follicles are clearly identified as the population of follicles expressing ET-1. The results of this study delineate the follicular developmental stage and the compartment of when and where ET-1 may be physiologically meaningful.     

Immunohistochemical localization of tissue-type plasminogen activator in ovaries before and after induced and spontaneous ovulation in the rat

Summary The observation that tissue-type plasminogen activator (tPA) activity increased dramatically in preovulatory follicles has led to the hypothesis that plasminogen activation is causally related to follicle rupture. With immunohistochemistry, we have studied the appearance of tPA in ovaries of immature rats induced to ovulate and in adult cycling rats. Treatment of immature female rats with a single dose of pregnant mare serum gonadotropin (PMSG) induced follicular maturation. A subsequent human chorionic gonadotropin (hCG) injection resulted in follicle rupture 12–14 h later. PMSG treatment alone did not induce appearance of tPA-immunoreactive cells in any ovarian compartment. After hCG stimulation, however, theca cells, granulosa cells, and oocytes of pre- and postovulatory follicles displayed distinct tPA immunoreactivity. Fibroblastlike cells in the theca layers and tunica albuginea of the follicle apex also demonstrated localized cytoplasmic tPA reactivity. In addition to tPA synthesis in preovulatory follicles, hCG also induced tPA staining in the theca (but not granulosa) layers of non-ovulatory follicles. At 24 h after hCG treatment, there was a marked tPA staining in developing corpora lutea, ovulated ova, and oviductal epithelium. Ovaries from regularly cycling adult rats displayed a similar ovulation-related pattern of tPA immunostaining. The appearance of tPA in different cell types of the preovulatory follicle and in the fibroblast-like cells at the follicle apex, strengthens the hypothesis of a direct involvement of tPA in follicle rupture. Presence of tPA in postovulatory oocytes, cumulus cells, and surrounding oviductal epithelium may also indicate a role for tPA in the transfer of eggs in the oviduct.This work was supported by NIH Research Grants HD-14084; 12303     

Kit ligand actions on ovarian stromal cells: effects on theca cell recruitment and steroid production

Factors that control recruitment of theca cells from ovarian stromal-interstitial cells are important for early follicle development in the ovary. During recruitment, theca cells organize into distinct layers around early developing follicles and establish essential cell-cell interactions with granulosa cells. Recruitment of theca cells from ovarian stromal stem cells is proposed to involve cellular proliferation, as well as induction of theca cell-specific functional markers. Previously, the speculation was made that a granulosa cell-derived "theca cell organizer" is involved in theca cell recruitment. Granulosa cells have been shown to produce kit-ligand/stem cell factor (KL). KL is known to promote stem cell proliferation and differentiation in a number of tissues. Therefore, the hypothesis was tested in the current study that granulosa cell-derived KL may help recruit theca cells from undifferentiated stromal stem cells during early follicle development. The actions of KL were examined using adult bovine ovarian fragment organ culture and isolated ovarian stromal-interstitial cells. In organ culture KL significantly increased the number of theca cell layers around primary follicles. Experiments using purified stromal-interstitial cell cultures showed that KL stimulated ovarian stromal cell proliferation in a dose-dependent manner. Stromal cell differentiation into theca cells was analyzed by the induction of theca cell functional markers (i.e., androstenedione and progesterone production). Bovine ovarian stromal cells produced low levels of androstenedione (5-40 ng/microg DNA) and progesterone (5-30 ng/microg DNA) in vitro that were approximately 20-fold lower than theca cells under similar conditions. Treatment with KL did not affect ovarian stromal cell androstenedione or progesterone production. Interestingly, hormones such as estrogen and hCG did stimulate stromal cell steroid production. The results in this study suggest that granulosa cell-derived KL appears to promote the formation of theca cell layers around small (i.e., primary) ovarian follicles. KL directly stimulated ovarian stromal cell proliferation but alone did not induce functional differentiation (i.e., high steroid production). Therefore, KL is proposed to promote early follicle development by inducing proliferation and organization of stromal stem cells around small follicles. Observations suggest that KL may act as a granulosa-derived "theca cell organizer" to promote stem cell recruitment of ovarian stromal cells in a manner similar to the way that KL promotes hematopoietic and lymphoid stem cells in bone marrow and the thymus.     

Apelin and APJ receptor expression in granulosa and theca cells during different stages of follicular development in the bovine ovary: Involvement of apoptosis and hormonal regulation

In the mammalian ovary, the microvasculature in the thecal layer of follicles is associated with follicular development. Apelin and its receptor, APJ, are expressed in the tissues and organs which include the vasculature. The aims of the present study were to examine the mRNA expression of apelin and the APJ receptor in granulosa cells and theca tissue of bovine follicles and the effects of steroid hormone and gonadotrophins on the expression of these genes in cultured granulosa cells and theca cells. The expression of apelin mRNA was not found in the granulosa cells of bovine follicles. The expression of the APJ gene was increased in granulosa cells of estrogen-inactive dominant follicles. The expression of apelin mRNA increased in theca tissues of estrogen-inactive dominant follicles. APJ expression in theca tissues increased with follicle growth. Progesterone stimulated the expression of APJ mRNA in the cultured granulosa cells. FSH stimulated the expression of APJ mRNA in the cultured granulosa cells. LH induced the expression of apelin and APJ receptor mRNAs in cultured theca cells. Taken together, our data indicate that the APJ receptor in granulosa cells and both apelin and the APJ receptor in theca tissues are expressed in bovine ovary, that APJ in granulosa cells may be involved in the appearance of the cell apoptosis, and that LH stimulates the expression of apelin and APJ genes in theca cells.     

Immunolocalization of GnRHRI,gonadotropin receptors,PGR, and PGRMCI during follicular development in the rabbit ovary

The aim of this study was to investigate the presence and localization of gonadotropin-releasing hormone receptor-I (GnRHRI), gonadotropin receptors (FSHR, LHR), progesterone receptor (PGR), and progesterone receptor membrane-binding component-I (PGRMCI) in the different developmental stages of the rabbit follicle. The ovaries were collected from four healthy New Zealand white rabbits, and the mRNA expression and protein levels of GnRHRI, FSHR, LHR, PGR, and PGRMCI were examined with real-time PCR and immunohistochemistry. The results showed that GnRHRI, FSHR, LHR, PGR, and PGRMCI mRNA was expressed in the ovary; furthermore, we show cell-type specific and follicular development stage-specific expression of these receptors at the protein level. Specifically, all of the receptors were detected in the oocytes from the primordial to the tertiary follicles and in the granulosa and theca cells from the secondary and tertiary follicles. In the mature follicles, all receptors were primarily localized in the granulosa and theca cells. In addition, LHR was also localized in the granulosa cells from the primordial and primary follicles. With follicular development, the expression level of all of the receptors, except GnRHRI, in the follicles showed a tendency to decrease because the area of the follicle increased sharply. The expression level of GnRHRI, FSHR, and PGR in the granulosa and theca cells showed an increasing trend with ongoing follicular development. Interestingly, the expression level of FSHR in the oocytes obviously decreased from the primary to the tertiary follicles, whereas LHR in the oocytes increased from the secondary to tertiary follicles. In conclusion, the expression of GnRHRI, the gonadotropin receptors, PGR, and PGRMCI decreased from the preantral follicles (primordial, primary, and secondary follicles) to the tertiary follicles. The expression of GnRHRI and LHR in the oocytes increased from the secondary to the tertiary follicles, whereas FSHR decreased from the primary to the tertiary follicles. The expression of GnRHRI and PGR in the granulosa and theca cells increased from the secondary to the mature follicles. These observations suggest that these receptors play roles in follicular development and participate in the regulation of follicular development.