Raymond J. Rodgers

Formation of the ovarian follicular antrum and follicular fluid.

The formation of the follicular antrum and follicular fluid has received scant attention from researchers, yet both are important processes in follicular development. The central hypothesis on follicular fluid formation suggests that production by granulosa cells of hyaluronan and the chondroitin sulfate proteoglycan versican generates an osmotic gradient. This gradient draws in fluid derived from the thecal vasculature. Inter-alpha-trypsin inhibitor is also present in follicular fluid at least in species with large follicles, and inter-alpha-trypsin inhibitor and versican could additionally bind or cross-link with hyaluronan, resulting in the retention of these molecules within the follicular antrum. Barriers to the movement of fluid across the membrana granulosa are apparently minimal, as even relatively large serum proteins are present in follicular fluid. Despite the relative permeability of the follicular wall, aquaporins are present in granulosa cells and could be actively involved in the transport of water into the follicle. The formation of an antrum also requires movement of granulosa cells relative to each other to allow the fluid to accumulate. This presumably involves remodeling of cell-cell junctions and in species with small follicles may involve death of centrally located granulosa cells. Remodeling of the stroma and thecal layers also accompanies growth and expansion of the antrum and presumably involves similar processes that accompany growth of other glands.

Immunohistochemical localization of basic fibroblast growth factor in bovine ovarian follicles

Basic fibroblast growth factor (bFGF, FGF2) controls cell proliferation and differentiation in many organs and tissues. In the ovary, cells proliferate and differentiate during folliculogenesis and during formation of the corpus luteum. While previous studies have inferred a role for bFGF in these processes, the precise contribution of bFGF to follicular activation or recruitment has not been established. For this reason, bFGF was immunolocalized in bovine follicles, using anti-bFGF immunoglobulin specific for the 1-24-amino acid terminus of the 18-kDa peptide. Basic FGF was immunolocalized to the cytoplasm of oocytes from bovine primordial and primary follicles. Strong immunostaining was also observed in corpora lutea, the ovarian surface epithelium, and smooth muscle cells surrounding blood vessels, while substantial levels of immunostaining were also present in cells of the theca interna. In most of the healthy antral follicles examined, the three or so layers of granulosa cells which were closest to the basement membrane were also stained, with greatest levels of staining at the most basal region of each cell. Atretic antral follicles had significant and uniform levels of immunostaining throughout the theca interna and the membrana granulosa. Immunostaining as described above was reduced to background levels when the primary specific immunoglobulin was preabsorbed with a 350 molar excess of peptide comprising the NH2-terminal 24 amino acids of bFGF. Based upon our previous observations and those reported here, we propose that basic fibroblast growth factor is synthesized by immature oocytes, especially those from primordial and primary follicles, and that bFGF has a potential role in activating follicle growth via stimulation of granulosa cell proliferation and follicular basement membrane synthesis.

Dynamics of the membrana granulosa during expansion of the ovarian follicular antrum

As an endocrine organ, the ovary has some unique characteristics. The formation, the maturation and the regression of the hormone producing cells really determine the timing, the amount and the type of hormone secreted. Here, we focus on the granulosa cells of ovarian follicles which express 17beta-hydroxysteroid dehydrogenase type 1 and cytochrome P450 aromatase. Follicles only produce estradiol late in follicular development before either ovulation or atresia ensues. We discuss the evidence that the membrana granulosa has many characteristics in common with other epithelia, including that it arises from stem cells. The corollary of this is that individual cells within the membrana granulosa are of different ages or stages of specialization. This is evident as regional differences across the membrana granulosa in terms of cell ages, shapes, gene expression, and even behaviour on cell death. We discuss theoretical considerations of the effects of antrum formation on the behavior of the membrana granulosa, and show evidence for differences between follicles in cell shapes, basal lamina phenotypes and location of younger cells, which we speculate is due to different rates of antrum expansion. Clearly, the membrana granulosa is dynamic, and this could explain much about the differences in the behaviors of cells from within the membrana granulosa, and between ovarian follicles.

Inhibin mRNAs in ovine and bovine ovarian follicles and corpora lutea throughout the estrous cycle and gestation.

Follicles and corpora lutea were dissected from ovine and bovine ovaries and the RNA extracted and subjected to Northern blot analyses for alpha- and beta A-inhibin mRNAs, using bovine cDNA and cRNA probes. A cDNA probe detecting mRNA for cholesterol side-chain cleavage cytochrome P-450 (P-450scc) was used as a positive control. In cattle, alpha- and beta A-inhibin mRNAs were not detected in ovarian stroma, which could potentially have contained follicles up to 0.5 mm in diameter. Inhibin-alpha and -beta A mRNAs were detected in bovine antral follicles but after ovulation, the relative levels of alpha- and beta A-inhibin mRNAs declined and were undetectable in mature fully developed cyclic corpora lutea and in pregnancy corpora lutea from early to late gestation of the cow. In sheep, alpha- and beta A-inhibin mRNAs were detected in a pool of antral follicles but not in cyclic or pregnancy corpora lutea, which did contain P-450scc mRNA. It is concluded that in cattle and sheep, follicles and not mature corpora lutea are the ovarian source of inhibin.

Secretion and gene expression of inhibin, oxytocin and steroid hormones during the in vitro differentiation of bovine granulosa cells.

Bovine granulosa cells were cultured under defined conditions to examine (1) their secretion of immunoreactive inhibin, oxytocin, progesterone and oestradiol during differentiation in vitro; (2) their expression, by Northern analysis, of specific mRNAs for inhibin and oxytocin as compared with uncultured cells; (3) possible interrelationships between the four secreted hormones; and (4) the hypothesis that androgens and steroidogenesis influence the secretion of inhibin. The secretion of inhibin and oestradiol fell rapidly over the first few days of culture but remained at detectable levels for at least 7 days. Conversely, the secretion of oxytocin and progesterone rose steadily as culture progressed. These changes occurred spontaneously (i.e. without gonadotrophin treatment) and were not dependent on the addition of serum to the culture medium. Messenger RNAs for the inhibin alpha- and beta A-subunits were present in uncultured cells but barely detectable or undetectable in cells cultured for 4 days. Conversely, the mRNA for oxytocin, which was not detectable in uncultured cells, was present in cultured cells and increased in quantity as culture progressed. Treatment of cells with testosterone (5 nM-5 microM), in the presence or absence of serum (10% FCS), had no effect on the secretion of inhibin but stimulated the declining oestradiol secretion. Treatment with ascorbic acid (0.5 mM) increased the secretion of oxytocin and progesterone, as previously described, but not that of inhibin. Treatment with aminoglutethimide (0.5 mM), an inhibitor of steroidogenesis, substantially inhibited progesterone secretion and the response of oestradiol secretion to testosterone, but had no effect on the secretion of either inhibin or oxytocin. We conclude that bovine granulosa cells differentiate spontaneously in defined culture in a manner that, as defined by the secretion of steroid and peptide hormones, closely resembles their luteinization in vivo. The switch in protein hormone secretion from inhibin to oxytocin is accompanied by a corresponding change in mRNA expression. The changes in steroid and peptide hormone secretions that take place in culture appear to occur independently of one another although their absolute cause remains to be determined. In contrast to previous studies, we could find no evidence for the regulation of inhibin secretion by either androgens or steroidogenesis.

Development of the ovarian follicular epithelium.

A lot is known about the endocrine control of the development of ovarian follicles, but a key question now facing researchers is which molecular and cellular processes take part in control of follicular growth and development. The growth and development of ovarian follicles occurs postnatally and throughout adult life. In this review, we focus on the follicular epithelium (membrana granulosa) and its basal lamina. We discuss a model of how granulosa cells arise from a population of stem cells and then enter different lineages before differentiation. The structure of the epithelium at the antral stage of development is presented, and the effects that follicle growth has on the behavior of the granulosa cells are discussed. Finally, we discuss the evidence that during follicle development the follicular basal lamina changes in composition. This would be expected if the behavior of the granulosa cells changes, or if the permeability of the basal lamina changes. It will be evident that the follicular epithelium has similarities to other epithelia in the body, but that it is more dynamic, as gross changes occur during the course of follicle development. This basic information will be important for the development of future reproductive technologies in both humans and animals, and possibly for understanding polycystic ovarian syndrome in women.

Dynamic Changes in the Expression of Relaxin-Like Factor (Insl3), Cholesterol Side-Chain Cleavage Cytochrome P450, and 3β-Hydroxysteroid Dehydrogenase in Bovine Ovarian Follicles During Growth and Atresia

Abstract Relaxin-like factor (RLF) is a new member of the insulin-relaxin gene family known to be expressed in the ovarian follicular thecal cells of ruminants. To investigate the pattern of RLF expression in development and atresia of bovine follicles, antisera were raised in rats and rabbits to recombinantly expressed bovine pro-RLF and to chemically synthesized ovine RLF B chain, respectively. On dot blotting analysis, the rat anitserum bound to pro-RLF and less strongly to a synthetic mature ovine RLF lacking the C-domain, whereas the rabbit antiserum bound the mature form of ovine RLF. These antisera were used to immunostain bovine ovarian follicles of differing sizes and stages of health and atresia. 3β-Hydroxysteroid dehydrogenase was colocalized with pro-RLF (n = 86 follicles), and cholesterol side-chain cleavage cytochrome P450 was localized in another section of many of the same follicles (n = 66). Not all follicles expressed pro-RLF in the theca interna, so the results are presented as the proportion of follicles expressing pro-RLF. Both mature and pro-RLF were immunolocalized to steroidogenic thecal cells of healthy follicles. As follicles enlarged to >5 mm, the proportion expressing pro-RLF declined (19/19 for <5 mm and 18/26 for >6 mm). Atresia was divided into antral (antral granulosa cells dying first) or basal (basal cells dying first) and further divided into early, middle, and late. For antral atresia of small follicles (2–5 mm), no decline in the proportion expressing pro-RLF was observed (early 6/6, middle 2/2) until the late stages (1/4). For basal atresia, which only occurs in small follicles (2–5 mm), the proportion expressing pro-RLF declined in the middle (2/5) and late (0/8) stages. In larger follicles (>6 to <10 mm), the proportion expressing pro-RLF also declined with atresia (1/13). These declines in RLF expression with atresia or increasing size were not accompanied by a decline in the expression of steroidogenic enzymes in the theca interna. A significant (P < 0.001) inverse relationship in the expression of pro-RLF and 3β-hydroxysteroid dehydrogenase in the membrana granulosa was observed. We conclude that the expression of pro-RLF in the theca interna is switched off as follicles enlarge or enter atresia, whereas the expression of steroidogenic enzymes is maintained in the theca interna.

A New Model of Development of the Mammalian Ovary and Follicles

Ovarian follicular granulosa cells surround and nurture oocytes, and produce sex steroid hormones. It is believed that during development the ovarian surface epithelial cells penetrate into the ovary and develop into granulosa cells when associating with oogonia to form follicles. Using bovine fetal ovaries (n = 80) we identified a novel cell type, termed GREL for Gonadal Ridge Epithelial-Like. Using 26 markers for GREL and other cells and extracellular matrix we conducted immunohistochemistry and electron microscopy and chronologically tracked all somatic cell types during development. Before 70 days of gestation the gonadal ridge/ovarian primordium is formed by proliferation of GREL cells at the surface epithelium of the mesonephros. Primordial germ cells (PGCs) migrate into the ovarian primordium. After 70 days, stroma from the underlying mesonephros begins to penetrate the primordium, partitioning the developing ovary into irregularly-shaped ovigerous cords composed of GREL cells and PGCs/oogonia. Importantly we identified that the cords are always separated from the stroma by a basal lamina. Around 130 days of gestation the stroma expands laterally below the outermost layers of GREL cells forming a sub-epithelial basal lamina and establishing an epithelial-stromal interface. It is at this stage that a mature surface epithelium develops from the GREL cells on the surface of the ovary primordium. Expansion of the stroma continues to partition the ovigerous cords into smaller groups of cells eventually forming follicles containing an oogonium/oocyte surrounded by GREL cells, which become granulosa cells, all enclosed by a basal lamina. Thus in contrast to the prevailing theory, the ovarian surface epithelial cells do not penetrate into the ovary to form the granulosa cells of follicles, instead ovarian surface epithelial cells and granulosa cells have a common precursor, the GREL cell.

Transcriptome profiling of granulosa cells from bovine ovarian follicles during atresia.

BackgroundThe major function of the ovary is to produce oocytes for fertilisation. Oocytes mature in follicles surrounded by nurturing granulosa cells and all are enclosed by a basal lamina. During growth, granulosa cells replicate and a large fluid-filled cavity (the antrum) develops in the centre. Only follicles that have enlarged to over 10 mm can ovulate in cows. In mammals, the number of primordial follicles far exceeds the numbers that ever ovulate and atresia or regression of follicles is a mechanism to regulate the number of oocytes ovulated and to contribute to the timing of ovulation. To better understand the molecular basis of follicular atresia, we undertook transcriptome profiling of granulosa cells from healthy (n = 10) and atretic (n = 5) bovine follicles at early antral stages (< 5 mm).ResultsPrincipal Component Analysis (PCA) and hierarchical classification of the signal intensity plots for the arrays showed primary clustering into two groups, healthy and atretic. These analyses and size-frequency plots of coefficients of variation of signal intensities revealed that the healthy follicles were more heterogeneous. Examining the differentially-expressed genes the most significantly affected functions in atretic follicles were cell death, organ development, tissue development and embryonic development. The overall processes influenced by transcription factor gene TP53 were predicted to be activated, whereas those of MYC were inhibited on the basis of known interactions with the genes in our dataset. The top ranked canonical pathway contained signalling molecules common to various inflammatory/fibrotic pathways such as the transforming growth factor-β and tumour necrosis factor-α pathways. The two most significant networks also reflect this pattern of tissue remodelling/fibrosis gene expression. These networks also contain molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor-β signalling and were up regulated.ConclusionsSmall healthy antral follicles, which have a number of growth outcomes, exhibit greater variability in gene expression, particularly in genes associated with cell division and other growth-related functions. Atresia, on the other hand, not only involves cell death but clearly is an active process similar to wound healing.

Identification and Immunolocalization of Decorin, Versican, Perlecan, Nidogen, and Chondroitin Sulfate Proteoglycans in Bovine Small-Antral Ovarian Follicles

Abstract 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 35SO4 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.