Growth factors--the new regulators of reproduction

Experimental data on the role of growth factors in the processes of oocyte maturation and of proliferation and differentiation in the mammalian ovaries are reviewed. Evidence is provided that theca and granulosa cells can synthesize epidermal, fibroblast, insulin-like and transforming growth factors and have receptors to all of them. The regulation of oocyte maturation induction by the growth factors is analysed. A possible physiological influence of growth factors and inhibin on regulation of folliculogenesis and gametogenesis and also on selection of the dominant follicle in the mammalian ovaries is discussed.     

Characterization of integrin expression in the mouse ovary

Integrin alpha:beta heterodimers mediate cell contacts to the extracellular matrix and initiate intracellular signaling cascades in response to a variety of factors. Integrins interact with many determinants of cellular phenotypes and play roles in controlling the development, structural integrity, and function of every type of tissue. Despite their importance, little is known about the regulation of integrin subunits in the mammalian ovary and how they function in folliculogenesis. To determine their relevance to ovarian physiology, we have studied the expression of integrin subunit mRNAs by Northern blot analysis and in situ hybridization in ovaries of wild-type, growth differentiation factor 9 (Gdf 9) knockout, FSHbeta (Fshb) knockout, and inhibin alpha (Inha) knockout mice. Integrin alpha6 mRNA is expressed in oocytes and granulosa cells of single-layer follicles and in oocytes and theca cells of multilayer follicles. Integrin alpha6 is highly expressed in Gdf 9 knockout ovaries, which are enriched in oocytes and primary (single layer) follicles because of a block at this stage of follicular development. Integrin alpha(v) mRNA is most highly expressed in the granulosa cells of multilayer growing follicles, and therefore only low levels of expression are detectable in the Gdf 9 knockout ovaries. Integrin beta1 mRNA exhibits a broad expression pattern in ovaries, including oocytes, granulosa cells, theca cells, and corpora lutea. Integrin beta3 mRNA is expressed in theca and interstitial cells and is upregulated in corpora lutea. It is nearly undetectable in ovaries of Fshb knockout mice, which develop preantral follicles but have no luteal cells. Integrin beta5 mRNA is predominantly expressed in granulosa cells of multilayer follicles. It is expressed at high levels in the Fshb knockout mice and in a compartmentalized manner in the granulosa cell/Sertoli cell tumors that develop in the Inha knockout mice. Specific integrins are associated with ovarian cellular phenotypes in mice, which raises intriguing possibilities as to integrin functions in oocyte competence, follicular development, luteinization, and granulosa cell proliferation.     

Quantitative analysis of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) gene expression in calf and adult bovine ovaries

Background  

It has been reported that calf oocytes are less developmentally competent than oocytes obtained from adult cows. Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) play critical roles in folliculogenesis, follicular development and ovulation in mammalian ovaries. In the present study, we attempted to compare the expression patterns of BMP15 and GDF9 in the cells of calf and cow ovaries to determine a relationship between the level of these genes and the low developmental competence of calf oocytes.     

Role of keratinocyte-derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes

Melanocytes characterized by the activities of tyrosinase, tyrosinase-related protein (TRP)-1 and TRP-2 as well as by melanosomes and dendrites are located mainly in the epidermis, dermis and hair bulb of the mammalian skin. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from embryonic neural crest cells. Because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes, the mechanism of the regulation of the proliferation and differentiation of epidermal melanocytes should be clarified. The regulation by the tissue environment, especially by keratinocytes is indispensable in addition to the regulation by genetic factors in melanocytes. Recent advances in the techniques of tissue culture and biochemistry have enabled us to clarify factors derived from keratinocytes. Alpha-melanocyte-stimulating hormone, adrenocorticotrophic hormone, basic fibroblast growth factor, nerve growth factor, endothelins, granulocyte-macrophage colony-stimulating factor, steel factor, leukemia inhibitory factor and hepatocyte growth factor have been suggested to be the keratinocyte-derived factors and to regulate the proliferation and/or differentiation of mammalian epidermal melanocytes. Numerous factors may be produced in and released from keratinocytes and be involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes through receptor-mediated signaling pathways.     

Regulation of oocyte and cumulus cell interactions by intermedin/adrenomedullin 2

Ovarian folliculogenesis has been studied as a model of hormonal regulation of development and differentiation, cell death, and cell-cell communication. In addition to gonadotropins from the pituitary and follicular paracrine factors, oocyte secreted factors have been shown to play critical roles in the regulation of follicular cell functions. Except for the well characterized BMP family proteins, including GDF9 and BMP15, oocytes are known to secrete oocyte secreted factors that are important for the regulation of cumulus cell survival and the maintenance of tertiary structure of cumulus cell-enclosed oocyte complexes (COCs). Based on genomic screening and studies of COCs cultured in vitro, we showed that intermedin (IMD)/adrenomedullin 2 (ADM2) is a novel oocyte-derived ligand important for the regulation of cell interactions in COCs that functions, in part, by suppressing cumulus cell apoptosis. Consistently, we showed that suppression of IMD/ADM2 signaling in growing rat ovaries in vivo leads to oocyte atresia and aberrant cell cycle progression in follicular cells. Together, our studies indicated that mammalian oocytes deploy a G protein-coupled receptor ligand to coordinate normal interactions of oocytes and cumulus cells and provided a better understanding of how the tertiary structure of a COC is maintained as follicles undergo exponential growth during the late stages of folliculogenesis.     

Role of keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes

Melanocytes characterized by the activities of tyrosinase, tyrosinase‐related protein (TRP)‐1 and TRP‐2 as well as by melanosomes and dendrites are located mainly in the epidermis, dermis and hair bulb of the mammalian skin. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from embryonic neural crest cells. Because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes, the mechanism of the regulation of the proliferation and differentiation of epidermal melanocytes should be clarified. The regulation by the tissue environment, especially by keratinocytes is indispensable in addition to the regulation by genetic factors in melanocytes. Recent advances in the techniques of tissue culture and biochemistry have enabled us to clarify factors derived from keratinocytes. Alpha‐melanocyte‐stimulating hormone, adrenocorticotrophic hormone, basic fibroblast growth factor, nerve growth factor, endothelins, granulocyte‐macrophage colony‐stimulating factor, steel factor, leukemia inhibitory factor and hepatocyte growth factor have been suggested to be the keratinocyte‐derived factors and to regulate the proliferation and/or differentiation of mammalian epidermal melanocytes. Numerous factors may be produced in and released from keratinocytes and be involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes through receptor‐mediated signaling pathways.     

Changes of ovarian interstitial cell hormone receptors and behavior of resident mesenchymal cells in developing and adult rats with steroid-induced sterility

In the present paper, we report that injection of testosterone propionate (500 microg) during the critical window of rat development (postnatal day 5) induces temporary appearance of aged interstitial cells in developing ovaries (days 7 and 10). Aged interstitial cells showed large size (> or = 12 microm), enhanced androgen receptor (AR) and low estrogen (ER) and luteinizing hormone receptor (LHR) expression. Although normal mature interstitial cells (large size and strong ER and LHR expression) appeared later (day 14), and ovaries of androgenized rats were similar to normal ovaries between days 14 and 35, ovaries of adult androgenized females showed only aged and no mature interstitial cells. Androgenization on day 10 caused the development of aged interstitial cells on day 14, but adult ovaries were normal. Long lasting postnatal estrogenization (estradiol dipropionate for four postnatal weeks) caused in developing and adult ovaries a lack of interstitial cell development beyond the immature state. Immature interstitial cells were characterized by a small size (< or = 7 microm) and a lack of AR, ER and LHR expression. Because the critical window for steroid-induced sterility coincides with the termination of immune adaptation, we also investigated distribution of mesenchymal cells (Thy-1 mast cells and pericytes, ED1 monocyte-derived cells, CD8 T cells, and cells expressing OX-62 of dendritic cells) in developing and adult ovaries. Developing ovaries of normal, androgenized and estrogenized females were populated by similar mesenchymal cells, regardless of differences in the state of differentiation of interstitial cells. However, mesenchymal cells in adult ovaries showed distinct behavior. In normal adult ovaries, differentiation of mature interstitial cells was accompanied by differentiation of mesenchymal cells. Aged interstitial cells in ovaries of androgenized rats showed precipitous degeneration of resident mesenchymal cells. Immature interstitial cells in ovaries of estrogenized rats showed a lack of differentiation of resident mesenchymal cells. These observations indicate that an alteration of interstitial cell differentiation during immune adaptation toward the aged phenotype results in precipitous degeneration of resident mesenchymal cells and premature aging of ovaries in adult rats, and alteration toward immature phenotype results in a lack of differentiation of mesenchymal cells and permanent immaturity of ovaries in adult females.     

From growth arrest to growth suppression.

Since the introduction of the cell cycle concept two approaches to study growth regulation of cells have been proposed. One claims that cells are naturally quiescent, requiring a stimulatory encouter with growth factors for induction of cell division. The other considers cellular multiplication as the natural steady-state; cessation of multiplication is thus a restriction imposed on the system. In the latter case emphasis is mainly on the signals involved in arrest of multiplication. This Prospect focuses on specific events occurring in mammalian cells at growth arrest, senescence, and terminal differentiation, specifically emphasizing the growth inhibitory factors, tumor suppressor genes, and other signals for growth suppression.     

Immunohistochemical Localization of Laminin and Cytokeratin in Embryonic Alligator Gonads

Gonadal sex differentiation is temperature-dependent in Alligator mississippiensis; testis differentiation occurs in embryos incubated at 33°C and ovary differentiation occurs in embryos incubated at 30°C. Laminin and cytokeratin were examined immunohistochemically in the gonads of alligator embryos incubated at these temperatures. The aim of this study was to determine whether these structural proteins show the same sex-specific expression patterns reported for mammalian embryos, and to assess their usefulness as early markers of gonadal differentiation in species with temperature-dependent sex determination. Laminin delineated enlarged seminiferous cords in differentiating testes from developmental stage 23 to hatching. Laminin distribution was more diffuse and revealed smaller cords of cells in differentiating ovaries. Cytokeratin was also detected in developing gonads of both sexes. Cytokeratin became concentrated in the basal cytoplasm of differentiating Sertoli cells in developing testes. In developing ovaries, prefollicular cells of the ovarian cortex and cell cords in the medulla stained strongly for cytokeratin. Cytokeratin did not show the same basal distribution in female medullary cord cells as seen in the Sertoli cells of testes, however. These sex-specific patterns of laminin and cytokeratin distribution in embryonic alligator gonads may serve as early markers of sexual differentiation.     

Activin/EDF as an inhibitor of neural differentiation

Activin/EDG, a stimulator of the secretion of follicle stimulating hormone (FSH) from pituitary gland and an inducer of erythroid differentiation for Friend leukemia cells, has since been implicated in a variety of biological roles. Here, we show some novel effects of activin on murine embryonal carcinoma cells (EC cells). First, activin acts as a growth factor on undifferentiated P19 cells, a well characterized EC cell line for the study of mammalian development. Second, activin inhibits the retinoic acid (RA) induced differentiation of P19 cells to neurons and glial cells. The inhibitory effect of activin on neural differentiation, which has yet to be proved in other physiological peptides, is confirmed also on the differentiation of various neuroblastoma cell lines. Our results suggest a possible role of activin as a negative regulator of neural differentiation in mammalian development.     

Apoptosis of granulosa cells and female infertility in achondroplastic mice expressing mutant fibroblast growth factor receptor 3G374R

Fibroblast growth factors play an important role in the control of ovarian folliculogenesis, but the complete repertoire of ovarian receptors which can transduce the fibroblast growth factor signals and their precise localization in the ovary have not yet been characterized. The most common form of inherited human dwarfism results from a point mutation in the transmembrane region of fibroblast growth factor receptor 3. A mouse model for achondroplasia was generated by introducing the human mutation (glycine 380-arginine) into the mouse fibroblast growth factor receptor 3 (G374R) by a "knock-in" approach using gene targeting leading to a constitutively active receptor. This resulted in the development of dwarf mice that share many features with human achondroplasia. Here we report that female (fibroblast growth factor receptor 3 G374R) dwarf mice become infertile. While no significant changes were observed in the anatomical and histological appearance of ovaries of 3-wk-old dwarf mice, a dramatic difference was observed in ovaries of 3-month-old mice. The normal ovary consists mainly of healthy corpora lutea and follicles at different stages of development, whereas the ovaries of the dwarf mice remain small and contain mainly follicles with a progressive apoptosis in the granulosa cells, and no corpora lutea could be observed. The levels of LH, FSH, and progesterone were lower by 72.3%, 38.0%, and 40.0%, respectively, in the blood of the dwarf mice compared with normal mice, and the total bioactivity of pituitary FSH and LH was lower by 65.6% and 79.6%, respectively, in the dwarf mice compared with normal mice. However treatment with PMSG and human CG of the dwarf mice led to rapid follicular development and formation of corpora lutea. Interestingly, the expression of the tumor suppressor gene p53 was increased dramatically in ovaries of the dwarf mice. The presence of the fibroblast growth factor receptor 3 cellular receptors in both normal and dwarf animals was demonstrated by Western blot and immunostaining. However, the distribution of the fibroblast growth factor receptors in the two strains shows significant differences. In the normal ovaries fibroblast growth factor receptor 3 was homogeneously distributed on the cell membrane of the granulosa cells and was absent in theca as well as corpora lutea cells, whereas in dwarf mice ovaries it was highly clustered on granulosa cells and very often appears in endocytic vesicles. Aged oocytes were more frequently observed in preantral follicles of ovaries of the dwarf mice. Nevertheless, oocytes isolated from antral follicles resume their meiotic division at a high percentage, similar to oocytes obtained from normal ovaries. The results imply fibroblast growth factor receptor 3 involvement in the control of follicular development through regulation of granulosa cell growth and differentiation, and that unovulation in the dwarf mice could be overcome in part by administration of exogenous gonadotropins. Moreover, it is suggested that the infertile phenotype is partially due to defects in the pituitary-gonadal axis.     

TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary

Although it is well established that both follicular assembly and the initiation of follicle growth in the mammalian ovary occur independently of pituitary hormone support, the factors controlling these processes remain poorly understood. We now report that neurotrophins (NTs) signaling via TrkB receptors are required for the growth of newly formed follicles. Both neurotrophin-4/5 (NT-4) and brain-derived neurotrophic factor (BDNF), the preferred TrkB ligands, are expressed in the infantile mouse ovary. Initially, they are present in oocytes, but this site of expression switches to granulosa cells after the newly assembled primordial follicles develop into growing primary follicles. Full-length kinase domain-containing TrkB receptors are expressed at low and seemingly unchanging levels in the oocytes and granulosa cells of both primordial and growing follicles. In contrast, a truncated TrkB isoform lacking the intracellular domain of the receptor is selectively expressed in oocytes, where it is targeted to the cell membrane as primary follicles initiate growth. Using gene-targeted mice lacking all TrkB isoforms, we show that the ovaries of these mice or those lacking both NT-4 and BDNF suffer a stage-selective deficiency in early follicular development that compromises the ability of follicles to grow beyond the primary stage. Proliferation of granulosa cells-required for this transition-and expression of FSH receptors (FSHR), which reflects the degree of biochemical differentiation of growing follicles, are reduced in trkB-null mice. Ovaries from these animals grafted under the kidney capsule of wild-type mice fail to sustain follicular growth and show a striking loss of follicular organization, preceded by massive oocyte death. These results indicate that TrkB receptors are required for the early growth of ovarian follicles and that they exert this function by primarily supporting oocyte development as well as providing granulosa cells with a proliferative signal that requires oocyte-somatic cell bidirectional communication. The predominance of truncated TrkB receptors in oocytes and their developmental pattern of subcellular expression suggest that a significant number of NT-4/BDNF actions in the developing mammalian ovary are mediated by these receptors.     

Polycystic ovary syndrome: interaction of follicle stimulating hormone and polypeptide growth factors in oestradiol production by human granulosa cells

The mechanism of the ovarian dysfunction in polycystic ovary syndrome, the most common cause of anovulatory infertility, remains obscure. Clinical data suggest that follicle stimulating hormone (FSH) action may be inhibited at the ovarian level by paracrine factors derived, presumably, from interstitial cells. The greater responsiveness to FSH of granulosa cells isolated from polycystic ovaries (PCO) compared with that seen in cells derived from normal ovaries, provides some support for this hypothesis and we present data which suggests that epidermal growth factor, or more likely transforming growth factor alpha, could be a candidate for this inhibitor. It should be emphasized, however, that the cardinal biochemical feature of the PCO is hypersecretion of androgens by interstitial cells. Stromal tissue from the PCO will secrete significant quantities of androstenedione in response to LH, whereas there is a negligible response in stroma from normal ovaries. It remains to be determined whether androgens have a direct inhibitory effect on FSH-induced oestradiol production in the human follicle, or whether they might exert an indirect effect by activating inhibitory polypeptide growth factors.     

Differentiation of mesenchymal tissues during phallic morphogenesis with emphasis on the os penis: Roles of androgens and other regulatory agents

This article reviews various aspects of differentiation and growth of phallic mesodermal tissues with special reference to the os penis. In many species of certain mammalian orders the penile interior contains an os penis or baculum with bona fide bone. Mechanisms of phenotypic sex differentiation and the androgenic regulation of morphogenesis of genitourinary tracts of both sexes are first overviewed. Thereafter the various mesodermal tissues in fully developed penes and clitorides are discussed. The developmental fate of mesenchymal cells in the fetal genital tubercles is then considered in detail, including consideration of epithelial-mesenchymal interactions. The review concludes with a discussion of the possible roles of certain polypeptide growth factors acting in concert with androgenic steroids. Special emphasis is placed on the potential role of bone morphogenetic proteins in formation of the os penis in a restricted number of eutherian mammalian taxa.