Remodeling of extracellular matrix at ovulation of the bovine ovarian follicle

Using immunohistochemistry and RNA analyses we examined the fate of components of a newly identified matrix that develops between granulosa cells (focimatrix, abbreviated from focal intraepithelial matrix) and of the follicular basal lamina in ovulating bovine ovarian follicles. Pre- and postovulatory follicles were generated by treatment with estradiol (Day 1), progesterone (Days 1-10), and prostaglandin analogue (Day 9) with either no further treatment (Group 1, n = 6) and or with 25 mg porcine LH (Day 11, Group 2, n = 8 or Day 10, Group 3, n = 8) and ovariectomy on Day 12 (12-14 hr post LH in Group 2, 38-40.5 hr in Group 3). In the time frame examined no loss of follicular basal lamina laminin chains beta2 and gamma1 or nidogen 1 was observed. In the follicular basal lamina collagen type IV alpha1 and perlecan were present prior to ovulation; after ovulation collagen type IV alpha1 was discontinuously distributed and perlecan was absent. Versican in the theca interna adjacent to the follicular basal lamina in preovulatory follicles was not observed post ovulation, however, the granulosa cells then showed strong cytoplasmic staining for versican. Expression of versican isoforms V0, V1, and V3 was detected at all stages. Focimatrix was observed in preovulatory follicles. It contained collagen type IV alpha1, laminins beta2 and gamma1, nidogen 1 and perlecan and underwent changes in composition similar to that of the follicular basal lamina. In conclusion focimatrix and the follicular basal lamina are degraded at ovulation. Individual components are lost at different times.     

Ultrastructure of the basal lamina of bovine ovarian follicles and its relationship to the membrana granulosa

Different morphological phenotypes of follicular basal lamina and of membrana granulosa have been observed. Ten preantral follicles (< 0. 1 mm), and 17 healthy and six atretic antral follicles (0.5-12 mm in diameter) were processed for light and electron microscopy to investigate the relationship the between follicular basal lamina and membrana granulosa. Within each antral follicle, the shape of the basal cells of the membrana granulosa was uniform, and either rounded or columnar. There were equal proportions of follicles 相似文献   

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.     

Identification and immunolocalization of decorin, versican, perlecan, nidogen, and chondroitin sulfate proteoglycans in bovine small-antral ovarian follicles

Proteoglycans (PGs) consist of a core protein and attached glycosaminoglycans (GAGs) and have diverse roles in cell and tissue biology. In follicles PGs have been detected only in follicular fluid and in cultured granulosa cells, and the composition of their GAGs has been determined. To identify PGs in whole ovarian follicles, not just in follicular fluid and granulosa cells, small (1-3-mm) bovine follicles were harvested. A proportion of these was incubated with (35)SO(4) for 24 h to incorporate radiolabel into the GAGs. The freshly harvested and cultured follicles were sequentially extracted with 6 M urea buffer, the same buffer with 0.1% Triton X-100 and then with 0.1 M NaOH. Proteoglycans were subjected to ion-exchange and size-exclusion chromatography. The GAGs were analyzed by chemical and enzymic digestion, and on the basis of their composition, we chose a list of known PGs to measure by ELISA analyses. Versican, perlecan, decorin, but not aggrecan or biglycan, were identified. These, excluding decorin for technical reasons, as well as a basal lamina glycoprotein, nidogen/entactin, were immunolocalized. Versican was localized to the thecal layers, including externa and the interna particularly in an area adjacent to the follicular basal lamina. Perlecan and nidogen were localized to the follicular basal lamina of antral follicles, both healthy and atretic, but not to that of preantral follicles. Both were localized to subendothelial basal laminas, but the former was not readily detected in arteriole smooth muscle layers. This study has confirmed the presence of versican and perlecan, but not the latter as a component of follicular fluid, and identified decorin and nidogen in ovarian antral follicles.     

Cholesterol side-chain cleavage cytochrome P450 and 3beta-hydroxysteroid dehydrogenase expression and the concentrations of steroid hormones in the follicular fluids of different phenotypes of healthy and atretic bovine ovarian follicles

Bovine ovarian antral follicles exhibit either one or the other of two patterns of granulosa cell death in atresia. Death can commence either from the antrum and progress toward the basal lamina (antral atresia) or the converse (basal atresia). In basal atresia, the remaining live antrally situated cells appeared to continue maturing. Beyond that, little is known about these distinct patterns of atresia. Healthy (nonatretic) follicles also exhibit either one or the other of two patterns of granulosa cell shape, follicular basal lamina ultrastructure or location of younger cells within the membrana granulosa. To examine these different phenotypes, the expression of the steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (SCC) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in granulosa cells and concentrations of steroid hormones in follicular fluid were measured in individual histologically classified bovine antral follicles. Healthy follicles first expressed SCC and 3beta-HSD in granulosa cells only when the follicles reached an approximate threshold of 10 mm in diameter. The pattern of expression in antral atretic follicles was the same as healthy follicles. Basal atretic follicles were all <5 mm. In these, the surviving antral granulosa cells expressed SCC and 3beta-HSD. In examining follicles of 3-5 mm, basal atretic follicles were found to have substantially elevated progesterone (P < 0.001) and decreased androstenedione and testosterone compared to healthy and antral atretic follicles. Estradiol was highest in the large healthy follicles, lower in the small healthy follicles, lower still in the antral atretic follicles, and lowest in the basal atretic follicles. Our findings have two major implications. First, the traditional method of identifying atretic follicles by measurement of steroid hormone concentrations may be less valid with small bovine follicles. Second, features of the two forms of follicular atresia are so different as to imply different mechanisms of initiation and regulation.     

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.     

Theca interna: the other side of bovine follicular atresia

Currently, histological classifications of ovarian follicular atresia are almost exclusively based on the morphology of the membrana granulosa without reference to the theca interna. Atresia in the bovine small antral ovarian follicle has been redefined into antral or basal atresia where cell death commences initially within antral or basal regions of the membrana granulosa, respectively. To examine cell death in the theca interna in the two types of atretic follicles, bovine ovaries were collected and processed for immunohistochemistry and light microscopy. Follicles were classified as healthy, antral atretic, or basal atretic. Follicle diameter was recorded and sections stained with lectin from Bandeiraea simplicifolia to identify endothelial cells or with an antibody to cytochrome P450 cholesterol side-chain cleavage to identify steroidogenic cells and combined with TUNEL labeling to identify dead cells. The numerical density of steroidogenic cells within the theca interna was significantly reduced (P < 0.001) in basal atretic follicles in comparison with other follicles. Cell death was greater in both endothelial cells (P < 0.05) and steroidogenic cells (P < 0.01) of the theca interna of basal atretic follicles compared with healthy and antral atretic follicles. Thus, we conclude that the theca interna is susceptible to cell death early in atresia, particularly in basal atretic follicles.     

Expression of retinol-binding protein and cellular retinol-binding protein in the bovine ovary

Retinol (vitamin A) is essential for reproduction, and retinoids have been suggested to play a role in ovarian steroidogenesis, oocyte maturation, and early embryonic development. Retinol is transported systemically and intercellularly by retinol-binding protein (RBP). Within the cell, cellular retinol-binding protein (CRBP) functions in retinol accumulation and metabolism. Since the actions of retinoids are mediated, in part, by retinoid-binding proteins, the objective of this study was to investigate cell-specific expression of RBP and CRBP in the bovine ovary. Immunocytochemical analysis (ICC) localized RBP to the thecal and granulosa cell layers of antral and preantral follicles with the most intense staining in the cells of large, healthy follicles. The tunica adventitia of arterial blood vessels also exhibited RBP staining. Immunostaining of CRBP was most intense in the granulosa cells of preantral follicles and present, but diminished, in thecal and granulosa cells of antral follicles. Within the corpus luteum, both proteins were observed in large luteal cells, but only RBP was observed in small luteal cells. Northern blot analysis demonstrated that thecal and granulosa cells from antral follicles and luteal tissue expressed RBP and CRBP mRNA. Synthesis and secretion of RBP by thecal cells, granulosa cells, and luteal cells were demonstrated by immune-complex precipitation of radiolabeled RBP from the medium of cultured cells or explants, followed by SDS-PAGE and fluorography. Follicular fluid was collected from small (<5 mm) and large (8-14 mm) follicles, pooled according to follicular size, and analyzed for retinol, RBP, estradiol-17beta, and progesterone. Concentrations of retinol, RBP, and estradiol were greater in the fluid of large follicles. Results demonstrate retinoid-binding protein expression by bovine ovaries and provide physical evidence that supports the concept that retinoids play a role in ovarian function.     

The fine structure of the cumulus oophorus during follicular development in sheep

Summary The cumulus and membrana granulosa of non-atretic ovarian follicles from primordial up to a stage shortly before ovulation were studied by electron microscopy.The follicular cells of primordial follicles were undifferentiated and rested on a thick basal lamina. In secondary follicles the endoplasmic reticulum had proliferated forming an anastomosing network. In early antral and antral follicles (0.5–2.0 mm dia.) the ER was composed of short cisternae, the mitochondria had elongated and gap junctions were first observed. In late antral follicles (3.0–5.9 mm dia.) gap junctions were frequent. In the cumulus the glycogen was associated with electron lucent areas whereas in the granulosa it was invariably associated with membranes. In large antral follicles large membrane bound bodies were present in the basal cells of the cumulus. At early oestrus a distinctive mitochondrial morphology was noted in the granulosa but not elsewhere in the follicles. At mid oestrus numerous annular nexuses were present in the granulosa but not in the cumulus. At late oestrus numerous lipid droplets were formed in both cumulus and granulosa, the boundary with theca interna became indistinct and the basal lamina became incomplete.Deceased     

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.     

Role of Thy-1+ and Ia+ cells in ovarian function

Cryostat sections of anovulatory ovaries from persistent estrous rats following a single postnatal dose of testosterone and from persistent diestrous rats following long-term postnatal estradiol treatment were investigated. The indirect immunoperoxidase technique was used to localize ovarian Thy-1 and Ia glycoproteins, as well as several other cell surface markers, and the results were compared with those obtained in normal ovaries of cycling females. Folliculogenesis in persistent estrous rats proceeded up to cystic antral follicles and was associated with the occurrence of Thy-1+ stromal cells under follicular basal lamina. In contrast to normal ovaries, Thy-1+ material did not invade the basal layers of granulosa cells. There was also no association of Ia+ cells with follicular basal lamina, but Ia+ cells were usually found associated with some thecal vessels. In persistent diestrous rats folliculogenesis was significantly retarded in both advanced antrum formation and thecal development. Thy-1+ cells were usually present in theca. No Thy-1+ material was found among basal layers of granulosa cells and the depletion of thecal Ia+ cells was almost complete. We suggest that normal follicular development may be dependent on the correct effects of Thy-1+ and Ia+ cells in addition to appropriate gonadotropin and steroid stimulation. On the other hand, anovulatory syndromes following postnatal androgen or estrogen treatment might be induced by temporary direct ovarian effects disturbing the establishment of the normal relationship between follicular structures and the immune system.     

IGF-Ⅰ及其受体、IGF结合蛋白-2和LH受体mRNA在卵泡中的表达

利用原位杂交和原位DNA－3’末端标记的方法研究了胰岛素样生长因子河（IG－I）、IGF－I受体、IGF结合蛋白－2、和促性腺激素受体的信使核糖核酸（mRNA）在不同生长与闭锁阶段的大鼠卵巢卵泡中表达的变化。结果表明：IGF－I主要在正常生长的初级卵泡、窦前卵泡和小窦状卵泡中表达。在各生长与成熟阶段的卵泡中都检测到IGF－I受体mRNA,闭锁卵泡的IGF－I受体表达降低。窦前与窦状的生长和闭锁卵泡均表达IGFBP－2。促卵泡激素（FSH）受体在窦前和小窦状卵泡的表达水平比其在大卵泡中的高。窦前与小窦状卵泡仅在膜细胞中表达黄体生成素（LH）受体mRNA,大卵泡的膜细胞与颗粒细胞均表达LH受体,在闭锁卵泡中仅在膜细胞中观察到LH受体的信号。综上结果,提示IGF－I,IGF－I受体和FSH受体在窦前和小窦状卵泡中的协同表达对卵泡的早期发育有重要作用。LH受体mRNA特异地在大卵泡的颗粒细胞中表达可能与优势卵泡选择相关。     

A novel basal lamina matrix of the stratified epithelium of the ovarian follicle.

Basal laminas are important sheets of specialized extracellular matrix that underlie and surround groups of cells, such as epithelia or endothelia, enabling the cells to orientate their basal/apical polarity and creating a microenvironment for them. Basal laminas can also individually encapsulate whole cells, such as muscle cells, thereby forming a microenvironment but not polarizing the enclosed cells. Other mesenchymal or stromal cells exist with no basal lamina. In the course of studying the bovine follicular basal lamina which underlies the multilayered epithelium of the ovarian follicle, we identified a developmentally regulated novel extracellular matrix (which we call focimatrix for focal intra-epithelial matrix). Focimatrix is composed of basal lamina-like material deposited as plaques or aggregates between the multilayers of the epithelial granulosa cells. The focimatrix does not encapsulate individual or groups of cells and therefore does not form a microenvironment for them. Focimatrix contains collagen type IV subunits alpha1 and alpha2 (but not alpha3-alpha6), and laminin chains alpha1, beta2 and gamma1 (but not alpha2 or beta1), and nidogen-1 and perlecan (but not versican). The amount of focimatrix increases with increasing follicular size, and its appearance precedes the expression by granulosa cells of the enzymes for steroid hormone synthesis, cholesterol side-chain cleavage cytochrome P450 (SCC) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD), in the days preceding ovulation. The expression in granulosa cells of two components examined, nidogen-1 and perlecan, also increases substantially when follicles enlarge to a sufficient size capable of ovulating. Following ovulation the follicular basal lamina is degraded, and presumably focimatrix is too since it is not detected in corpora lutea that develop from the ovulating follicles. During this development the granulosa cells undergo an epithelial-mesenchymal transition (EMT) into luteal cells following ovulation, and substantially increase their expression of steroidogenic enzymes in the process. During EMT epithelial cells lose polarity. Since focimatrix exists on more than one side of the granulosa cells, we propose that it disrupts the polarity induced by the follicular basal lamina in the lead up to ovulation. Hence focimatrix maybe a key part of the follicular/luteal EMT.     

An enzymatic method for dissociation of intact follicles from the hamster ovary: histological and quantitative aspects

An enzymatic method was developed to collect intact follicles at different stages of development from cyclic hamsters to study ovarian folliculogenesis under various circumstances. Ovaries from 6 adult hamsters on each day of the cycle (Day 1 = ovulation) were collected, corpora lutea and large preantral and antral follicles were dissected, and follicles saved. Minced ovaries were then incubated with a mixture of collagenase, DNAse and pronase at 37 degrees C for 20 min to disperse intact follicles. Histological studies with 2191 isolated follicles revealed 10 different stages of follicular development (depending on the number of granulosa cell layers surrounding the oocyte and development of the antrum). Of the total follicular population, 14% showed signs of atresia, with 50% of those having 1-3 layers of granulosa cells (Stages 1-3); a second peak of 18% was observed in antral follicles (Stages 8-10). No signs of thecal cells were evident until the follicles reached Stage 6 (7-8 layers of granulosa cells), which possibly accounts for reduced atresia in this class and beyond. Ultrastructural study revealed that there were no signs of morphological damage to the basement membrane or to other subcellular organelles in the small preantral follicles. The presence of subnuclear lipid droplets in follicles with 3 layers of granulosa cells provided evidence for potential steroidogenesis by small follicles. The number of Stage 1-10 follicles was remarkably constant throughout the estrous cycle (460 +/- 34 per animal on Day 1 vs. 492 +/- 66 on Day 4). The usefulness of this method in analyzing follicular kinetics is illustrated in experiments involving hypophysectomy and the effects of unilateral ovariectomy. This procedure offers an improved method to study the factors responsible for the growth and the differentiation of small preantral follicles in the mammalian ovary.     

Ontogeny and cellular localization of 125I-labeled basic fibroblast growth factor and 125I-labeled epidermal growth factor binding sites in ovaries from bovine fetuses and neonatal calves.

We have recently reported that although specific 125I-FSH receptors are present in granulosa cells from primary and secondary follicles, gonadotropin responsiveness is very low in ovaries from bovine fetuses, which consist mainly of preantral follicles with few early antral follicles. It is well established that a number of polypeptide growth factors show pronounced mitogenic effects on follicular cells. Therefore, we have compared autoradiographically the ontogeny and cellular localization of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) binding activities to assess their possible involvement in the regulation of early follicular growth in fetuses and neonatal calves. Follicular growth was initiated around Day 180 of gestation in fetuses. 125I-bFGF binding values were high in granulosa cells from preantral follicles (mean +/- SEM, 7.8 +/- 1.1-9.8 +/- 0.7 grains/cell, 0.05-0.15-mm diam.) but decreased in early antral follicles (0.16-3.0 mm) to a constant level (5.7 +/- 1.2 grains/cell). Specific 125I-EGF binding values were low in preantral follicles but showed a 2.5- and 5.0-fold increase in both granulosa cells and the theca interna from antral I (0.16-0.5 mm) and antral II follicles (0.6-3.0 mm), respectively. In atretic follicles, 125I-bFGF specific binding values were high (10.4 +/- 0.8 grains/cell), whereas 125I-EGF binding levels were significantly reduced or absent. None of the radioligands tested bound significantly to primordial follicles. There was no age-related difference in any ligand binding to follicles of comparable size. These results provide novel evidence that bFGF, a potent mitogen, is involved in the regulation of granulosa cell function as early as the preantral stage in cattle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth differentiation factor 9 is antiapoptotic during follicular development from preantral to early antral stage

Ovarian follicular atresia represents a selection process that ensures the release of only healthy and viable oocytes during ovulation. The transition from preantral to early antral stage is the penultimate stage of development in terms of gonadotropin dependence and follicle destiny (survival/growth vs. atresia). We have examined whether and how oocyte-derived growth differentiation factor 9 (GDF-9) and FSH regulate follicular development and atresia during the preantral to early antral transition, by a novel combination of in vitro gene manipulation (i.e. intraoocyte injection of GDF-9 antisense oligos) and preantral follicle culture. Injection of GDF-9 antisense suppressed basal and FSH-induced preantral follicle growth in vitro, whereas addition of GDF-9 enhanced basal and FSH-induced follicular development. GDF-9 antisense activated caspase-3 and induced apoptosis in cultured preantral follicles, a response attenuated by exogenous GDF-9. GDF-9 increased phospho-Akt content in granulosa cells of early antral follicles. Although granulosa cell apoptosis induced by ceramide was attenuated by the presence of GDF-9, this protective effect of GDF-9 was prevented by the phosphatidylinositol 3-kinase inhibitor LY294002 and a dominant negative form of Akt. Injection of GDF-9 antisense decreased FSH receptor mRNA levels in cultured follicles, a response preventable by the presence of exogenous GDF-9. The data suggest that GDF-9 is antiapoptotic in preantral follicles and protects granulosa cells from undergoing apoptosis via activation of the phosphatidylinositol 3-kinase/Akt pathway. An adequate level of GDF-9 is required for follicular FSH receptor mRNA expression. GDF-9 promotes follicular survival and growth during the preantral to early antral transition by suppressing granulosa cell apoptosis and follicular atresia.     

Comparative immunohistochemical distribution of connexin 37 and connexin 43 throughout folliculogenesis in the bovine ovary

Among gap junctional proteins previously identified in the mouse ovary, connexins (Cx) Cx37 and Cx43 appeared to be essential for normal follicular growth. The aim of this work was to detect Cx37 expression in the bovine ovary, then to quantify and compare its follicular distribution pattern with that of Cx43 using quantitative analysis of immunofluorescently labeled ovary sections viewed with a confocal laser scanning microscope. Cx37 immunoreactivity was detected in bovine ovarian follicles and was predominantly localized at preantral stages. Unlike follicular Cx43 expression which was restricted to granulosa cells, Cx37 staining was observed in both oocyte and granulosa cell compartments. While no changes were seen during early follicular growth, the level of Cx37 expression decreased significantly at the onset of antral cavity formation (P 相似文献   

BMPRIB and BMPRII mRNA expression levels in goat ovarian follicles and the in vitro effects of BMP-15 on preantral follicle development

This study evaluated the levels of bone morphogenetic protein receptors BMPRIB and BMPRII mRNA in goat follicles and the effects of bone morphogenetic protein-15 (BMP-15) on the in vitro development of cultured preantral follicles. Real-time polymerase chain reaction (PCR) was used to analyze the levels of BMPRIB and BMPRII mRNA in caprine preantral follicles and in small and large antral follicles. Preantral follicles (≥150 μm) were also isolated from goat ovaries and cultured for 18 days in α-MEM(+) supplemented with or without BMP-15 (10, 50, or 100 ng/ml). At the end of culture, some follicles were fixed for ultrastructural evaluation. Real-time PCR showed a reduction in BMPRII mRNA levels from the primary to secondary follicles. Higher levels of BMPRIB mRNA were observed in granulosa/theca cells from large antral follicles compared with small antral follicles. Moreover, BMPRII mRNA was expressed to a greater extent in cumulus-oocyte complexes from large antral follicles than in their respective granulosa/theca cells. In culture, 50 ng/ml BMP-15 positively influenced antral cavity formation and follicle growth after 18 days and also maintained follicular integrity. Thus, BMPRIB and BMPRII mRNAs are present in all follicular categories. BMP-15 (50 ng/ml) stimulates growth, antrum formation and the ultrastructural integrity of isolated caprine preantral follicles after 18 days of culture.     

Distribution of extracellular matrix proteins type I collagen, type IV collagen, fibronectin, and laminin in mouse folliculogenesis

The extracellular matrix (ECM) plays a prominent role in ovarian function by participating in processes such as cell migration, proliferation, growth, and development. Although some of these signaling processes have been characterized in the mouse, the relative quantity and distribution of ECM proteins within developing follicles of the ovary have not been characterized. This study uses immunohistochemistry and real-time PCR to characterize the ECM components type I collagen, type IV collagen, fibronectin, and laminin in the mouse ovary according to follicle stage and cellular compartment. Collagen I was present throughout the ovary, with higher concentrations in the ovarian surface epithelium and follicular compartments. Collagen IV was abundant in the theca cell compartment with low-level expression in the stroma and granulosa cells. The distribution of collagen was consistent throughout follicle maturation. Fibronectin staining in the stroma and theca cell compartment increased throughout follicle development, while staining in the granulosa cell compartment decreased. Heavy staining was also observed in the follicular fluid of antral follicles. Laminin was localized primarily to the theca cell compartment, with a defined ring at the exterior of the follicular granulosa cells marking the basement membrane. Low levels of laminin were also apparent in the stroma and granulosa cell compartment. Taken together, the ECM content of the mouse ovary changes during follicular development and reveals a distinct spatial and temporal pattern. This understanding of ECM composition and distribution can be used in the basic studies of ECM function during follicle development, and could aid in the development of in vitro systems for follicle growth.