Irregularly shaped inclusion cysts display increased expression of Ki67, Fas, Fas ligand, and procaspase-3 but relatively little active caspase-3.

Human ovarian cancers are thought to arise from sequestered ovarian surface epithelial (OSE) cells that line the wall of inclusion cysts. Nevertheless, the early events toward neoplasia are not well understood. In this study, immunoreactivity for apoptotic proteins in human OSE of control and tumor ovarian sections was examined. Ki67, a marker for cell proliferation, was generally absent in the flat-to-cuboidal OSE cells on the ovarian surface and in regularly shaped inclusion cysts. Fas, Fas ligand, and caspase-3, components of the apoptotic pathway, were also largely absent. Ki67, Fas, Fas ligand, and procaspase-3 expression, though not active caspase-3 expression, was more frequently observed in epithelial cells lining irregularly shaped inclusion cysts, particularly in the columnar and Müllerian-like OSE cell types that resembled ovarian tumor OSE cells. Immunoreactivity for these factors as well as active caspase-3 was found frequently in ovarian tumors. We postulate that the appearance of the Fas system and its related proteins in sequestered columnar OSE cells of irregularly shaped inclusion cysts may contribute to balance cell growth with cell death, although little active caspase-3 expression was observed. Further studies are required to identify whether inhibition of apoptosis in inclusion cysts is an early event in ovarian carcinogenesis.

Luteinizing hormone inhibits Fas-induced apoptosis in ovarian surface epithelial cell lines.

Gonadotrophins including LH have been suggested to play an important role in the etiology of epithelial ovarian cancers. The goal of the present study was to obtain more insight in the mechanism of gonadotrophin action on ovarian surface epithelium (OSE) cells. As the Fas system is known to be a major player in the regulation of the process of apoptosis in the ovary, we investigated whether LH interfered with Fas-induced apoptosis in the human OSE cancer cell lines HEY and Caov-3. Activation of Fas receptor by an agonistic anti-Fas receptor antibody induced apoptosis, as was evaluated by caspase-3 activation, poly(ADP-ribose) polymerase fragmentation, phosphatidylserine externalization and morphological changes characteristic of apoptosis. Co-treatment with LH reduced the number of apoptotic cells following activation of Fas in a transient manner, while LH by itself did not affect apoptosis or cell proliferation. The anti-apoptotic effect of LH could be mimicked by the membrane-permeable cAMP analog 8-(4-chlorophenylthio) cAMP (8-CPT-cAMP), and blocked by H89, a specific inhibitor of protein kinase A (PKA). In conclusion, these findings suggest that LH protects HEY cells against Fas-induced apoptosis through a signaling cascade involving PKA. Although it is plausible that in vivo LH might also enhance OSE tumor growth through inhibition of apoptosis, further research is necessary to confirm this hypothesis.

Identification of markers for precursor and leydig cell differentiation in the adult rat testis following ethane dimethyl sulphonate administration.

Administration of ethane dimethane sulphonate (EDS) to adult rats results in the destruction of all Leydig cells, followed by a complete regeneration. We investigated this regeneration process in more detail, using different markers for precursor and developing Leydig cells: the LH receptor, 3beta-hydroxysteroid dehydrogenase (3beta-HSD), transforming growth factor alpha (TGFalpha), and a new marker for Leydig cell maturation, relaxin-like factor (RLF). LH receptor immunoreactivity was found in Leydig cell-depleted testes at 3 and 8 days after EDS administration. The positive (precursor) cells had a mesenchymal-like morphology. The number of LH receptor-positive cells 8 days after EDS administration was 15 +/- 4 per 500 Sertoli cell nuclei. Fifteen days after EDS administration, the first new Leydig cells could be observed. These cells stained positively with both the antibodies against the LH receptor and 3beta-HSD, while some cells also stained positively for TGFalpha. After EDS administration, RLF mRNA disappeared from the testis and reappeared again at the time of the appearance of the first Leydig cells. Concomitant with the increase in the number of Leydig cells, the number of RLF-expressing cells increased. The observations of the present study give further support to the hypothesis that Leydig cell development in the prepubertal testis, and in the adult testis following EDS administration, takes place along the same cell lineage and suggest, therefore, that the adult EDS-treated rat can serve as a model for studying the adult-type Leydig cell development that normally occurs in the prepubertal rat testis.

Etiology of ovarian cancer: a proposed mechanism.

The majority of ovarian cancers are derived from ovarian surface epithelial cells that are sequestered in inclusion cysts within the ovarian stroma. We propose that the Fas/Fas ligand system is responsible for the normal elimination of these inclusion cysts through apoptosis, thereby removing potential sites of ovarian tumors. Furthermore, we hypothesize that the failure of the Fas/Fas ligand's apoptotic signaling mechanism leads to the persistence of these inclusion cysts in the stroma, and the onset of ovarian tumorigenesis.

Transforming growth factor-beta inhibits DNA synthesis in immature rat Leydig cells in vitro.

TGFbeta isoforms and its receptors are present in the testis and regulate in vitro function of various testicular cells. We have investigated the effects of TGFbeta on basal and mitogen stimulated in vitro proliferation of immature rat Leydig cells. Leydig cells were cultured with TGFbeta1, either alone or in combination with hCG, steroidogenesis-inducing protein (SIP), interleukin-1beta (IL-1beta), insulin or TGFalpha, and the incorporation of [3H]thymidine into DNA was determined. TGFbeta1 blocked the stimulatory effects of hCG, SIP, IL-1beta, insulin and TGF-alpha on DNA synthesis. Since G1- to S-phase transition depends upon cyclins and their associated kinases (cdks), we investigated the effects of TGFbeta on cdks. Immunoreactive levels of cdc2 (or cdk1) and cdk2 were significantly decreased in Leydig cells treated with TGFbeta1. We conclude that TGFbeta1 inhibits proliferation of immature rat Leydig cells and this effect may be mediated, at least in part, through down-regulation of cdc2 and cdk2 synthesis.

Activation of Fas ligand/receptor system kills ovarian cancer cell lines by an apoptotic mechanism.

The majority of ovarian cancers originate from the surface epithelium of the ovary and from inclusion cysts derived from the epithelium that becomes sequestered in the stroma. To identify naturally occurring ligands that could activate mechanisms by which these ovarian neoplasms could be eliminated, we have examined the ability of anti-Fas mAb to induce apoptosis in two cell lines, HEY and Caov-3, derived from ovarian carcinomas of surface epithelial origin. Treatment of each cell line with anti-Fas mAb caused chromatin condensation, nuclear segmentation, and apoptotic body formation, indicative of apoptosis. Furthermore we have shown that anti-Fas mAb activates the sphingomyelin-ceramide signal transduction pathway. Sphingomyelin levels were measured by normal-phase high-performance liquid chromatography interfaced with electrospray mass spectrometry. The six most abundant sphingomyelin species identified in Caov-3 cells were 34:1 (d18:1/16:0), 36:1 (d18:1/18:0), 40:1 (d18:1/22:0), 41:1 (d18:1/23:0), 42:1 (d18:1/24:0), and 42:2 (d18:1/24:1). Treatment of Caov-3 cells for 30 min caused a 40% decrease in the total sphingomyelin content. Specifically three of these species, 34:1, 40:1, and 42:2, were reduced to 44, 70, and 54% of control values, respectively. The decrease was attributed to the hydrolysis of sphingomyelin. Treatment of these cell lines with ceramide, a product of sphingomyelin hydrolysis, using a cell-permeable synthetic ceramide analogue C2-ceramide, also caused the above cells to undergo apoptosis. Thus, the Fas ligand/receptor system, acting through the sphingomyelin-ceramide pathway, provides a mechanism by which ovarian surface epithelial cancer cells can be induced to undergo apoptosis.

Regulation of transforming growth factor alpha gene expression in an ovarian surface epithelial cell line derived from a human carcinoma.

The surface epithelium plays an important role in normal ovarian physiology: the cells proliferate in the vicinity of the developing preovulatory follicle to accommodate the increase in follicular size, and to repair the surface after ovulation. These bouts of mitotic activity in vivo must be strictly regulated by the activity of growth factors and their receptors. Since transforming growth factor alpha (TGF alpha) has been identified as a growth-promoting factor for normal surface epithelial cells from human ovaries and ovarian surface epithelial cell lines, we have examined the regulation of the TGF alpha gene in HEY cells, a surface epithelial cell line derived from a human ovarian carcinoma. Treatment of HEY cells for 60 h with estradiol-17 beta, dihydrotestosterone, or progesterone at concentrations ranging from 5 x 10(-8) to 5 x 10(-6) M did not influence the level of the 4.5-kb transcript for TGF alpha. Treatment of HEY cells with TGF alpha increased the steady-state levels of TGF alpha mRNA, indicating that an autoregulatory mechanism could result in overexpression of TGF alpha. TGF beta, a known growth inhibitor of ovarian surface epithelial cells, decreased the steady-state levels of TGF alpha mRNA, suggesting a mechanism by which the levels of TGF alpha and mitotic activity could be regulated. HEY cells, like the human surface epithelial cells from which they were derived, were found by quantitative polymerase chain reaction (PCR) to contain TGF beta 1 mRNA. The TGF beta 1 mRNA was translated into immunoreactive TGF beta 1, indicating that TGF beta can act in an autocrine manner. By use of quantitative PCR, HEY cells were shown to express the genes for the TGF beta receptor II, betaglycan and endoglin. By cross-linking, these components of the TGF beta receptor system were found to bind TGF beta 1. This is the first demonstration of expression of functional TGF beta receptors in HEY cells and represents the first demonstration in an ovarian cell system. In summary, our findings suggest that the levels of TGF alpha and the cell growth of normal and transformed surface epithelial cells from human ovaries may be regulated by the interaction of autoregulatory mechanisms involving TGF alpha and TGF beta ligand-receptor systems.

Immunolocalization of transforming growth factor alpha and luteinizing hormone receptor in healthy and atretic follicles of the adult rat ovary.

At the onset of the LH surge in the rat, the proliferation of granulosa cells in preovulatory follicles is inhibited, aromatase activity is attenuated, and the cells enter a further phase of differentiation to form the CL. Whereas the LH surge induces these changes in the granulosa cells, it is possible that the actions of LH are mediated or modulated by paracrine and/or autocrine factors. A factor that may modulate the actions of LH is transforming growth factor alpha (TGF alpha). TGF alpha has the ability to inhibit granulosa cell growth and induce a change in the pattern of steroidogenesis from estrogen to progesterone synthesis, a change characteristic of early luteinization. To investigate whether there is a temporal relationship between the appearance of LH receptors and TGF alpha in the adult rat ovary, we localized LH receptors and TGF alpha during follicular development, atresia, and CL formation. In the interstitial compartment, the pattern of immunostaining for LH receptors and TGF alpha remained constant and was not affected by the stage of follicular development. As thecal cells differentiated from the stroma and formed several layers around the secondary follicle, the thecal cells stained positively for both LH receptors and TGF alpha. This immunostaining in thecal cells persisted in preantral, antral, and preovulatory follicles, even in follicles that became atretic. Granulosa cells did not contain detectable levels of LH receptor until the large antral stage of follicular development.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor alpha localization and role in surface epithelium of normal human ovaries and in ovarian carcinoma cells.

Transforming growth factor alpha (TGF alpha) has been localized by immunohistochemistry in the ovarian surface epithelial (OSE) cells of sections from normal human ovaries and in epithelial cells of surface crypts. An ovarian cancer cell line (HEY) derived from the surface epithelium of a human ovary also exhibited intense staining for the TGF alpha peptide. Using Northern analysis, HEY cells were shown to express a 4.5-kb transcript of TGF alpha, indicating that the TGF alpha peptide was synthesized by these cells and not taken up from the serum in the culture medium and sequestered by the cells. This was confirmed using a radioimmunoassay, which showed that HEY cells in culture secrete TGF alpha peptide, both as a soluble (0.12 +/- 0.02 ng/mg protein) and as a membrane-anchored (0.06 +/- 0.006 ng/mg protein) form. In both normal OSE cells and HEY cells, TGF alpha acted as a growth promoter: TGF alpha significantly stimulated [3H]thymidine incorporation into DNA of both primary cultures of normal OSE cells (2.7-fold) and of HEY cells (2-fold). This study provides the first demonstration of TGF alpha immunostaining in normal surface epithelial cells and in HEY cells, and suggests that TGF alpha, localized in normal and transformed OSE, is an autocrine growth promoter for these cells.

Immunohistochemical localization of 3 beta-hydroxysteroid dehydrogenase in the rat ovary during follicular development and atresia.

In the adult ovary, cohorts of growing follicles are continuously generated, from which dominant follicles are selected during each estrous cycle. To compensate for the rapid proliferation of follicular cells in the growing pool of follicles, follicles are eliminated by atresia, thereby maintaining ovarian tissue mass. Estrogens and androgens have been implicated as intraovarian regulators of follicular growth and atresia, suggesting that the fate of an individual follicle to develop to the preovulatory stage or to undergo atresia is associated with distinct profiles of steroid production. We therefore have localized 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), an enzyme required for the biosynthesis of all major steroid hormones, in ovaries of immature and adult rats during follicular development, atresia, and corpus luteum formation. The pattern of immunostaining for 3 beta-HSD remained constant in the interstitial cell compartment and was not affected by the age of the rats nor the stage of the estrous cycle. As thecal cells differentiated from the surrounding stroma and restructured around the secondary follicle, they expressed intense staining for 3 beta-HSD. This staining persisted in preantral, antral, and preovulatory follicles. Granulosa cells in primary, secondary, and antral follicles did not contain detectable levels of 3 beta-HSD and did not stain positively until the follicle reached the preovulatory stage of development. A novel finding presented in this paper is that 3 beta-HSD persisted in the thecal cells of follicles throughout the entire process of atresia, suggesting that during atresia the potential for the synthesis of androgens is retained.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions between FSH, estradiol-17 beta and transforming growth factor-beta regulate growth and differentiation in the rat gonad.

Estradiol-17 beta (E2) is a mitogen in vivo for the proliferation of granulosa cells in the rat ovary. E2 is synthesized by the preovulatory follicle through a series of gonadotrophin-dependent events: LH stimulates thecal cells to synthesize androgens (androstenedione and testosterone) which are substrates for FSH-induced aromatization to estrogens in granulosa cells. More recently, we have found that transforming growth factor-beta (TGF-beta) stimulates DNA synthesis in rat granulosa cells in vitro and this effect is augmented by FSH. Since E2 is a mitogen in vivo and TGF-beta is the only known growth factor to stimulate proliferation in vitro, the possible link between the actions of E2 and TGF-beta were examined. E2 stimulated the secretion of a TGF-beta-like factor by rat granulosa cells in culture, and with time DNA synthesis was stimulated. The mitogenic action of E2 was enhanced in the presence of FSH, and attenuated by a neutralizing antibody to TGF-beta. The latter observations have identified TGF-beta as the "missing-link" in the mitogenic actions of E2 on rat granulosa cells. In addition to the growth-promoting actions of TGF-beta plus FSH, TGF-beta enhanced FSH-induced aromatase activity. Consequently, FSH plus TGF-beta stimulates both the proliferation and aromatization capacity of rat granulosa cells. We propose that interactions between FSH, E2 and TGF-beta lead to the exponential increase in serum E2 levels that occurs during the follicular phase of the cycle. Similarly, FSH stimulates the aromatization of exogenous androgens to estrogen by Sertoli cells isolated from immature rat testes, and there is a correlation between FSH-induced aromatization and mitotic activity. We have shown that FSH plus TGF-beta stimulates DNA synthesis in Sertoli cells. Since E2 increases the secretion of TGF-beta by Sertoli cells, interactions between FSH, E2 and TGF-beta may provide the mitogenic stimulus for Sertoli cells during the prepubertal period. In summary, our findings suggest that the estrogen-induced growth of rat granulosa cells is mediated through the production of TGF-beta, which acts as an autocrine regulator of proliferation. We also propose that the growth-promoting actions of FSH on Sertoli cells may depend upon a cascade series of events involving estrogens and TGF-beta.

Transforming growth factor-alpha: identification in bovine corpus luteum by immunohistochemistry and northern blot analysis.

Transforming growth factor-alpha (TGF-alpha), a product of the thecal cells, has potent mitogenic and steroidogenic influences on cells within the ovarian follicle. Whether TGF-alpha continues to be produced in those follicles that go on to ovulate and form a corpus luteum is currently under investigation. In the present study, TGF-alpha was localized in the bovine corpus luteum by means of immunoperoxidase staining using a monoclonal antibody for TGF-alpha that does not cross-react with epidermal growth factor. In corpora lutea from the mid-luteal phase of the cycle TGF-alpha staining was found predominantly in the large luteal cells. Northern blot analysis using a human TGF-alpha cDNA probe hybridized to the 4.5-4.8 kb TGF-alpha transcript in RNA from the corpus luteum. These studies provide new evidence that TGF-alpha, a potent paracrine regulator within the ovarian follicle, continues to be expressed in the corpus luteum.

Localization of transforming growth factor beta 1 and beta 2 during testicular development in the rat.

The transforming growth factor beta s (TGF beta s) affect the metabolic activities of the somatic cells of the testis. Sertoli cells, peritubular/myoid cells, and germ cells contain mRNA for TGF beta 1 and/or TGF beta 2. We have used immunohistochemical techniques to determine, in vivo, when TGF beta 1 and TGF beta 2 are present in the rat testis during development and have identified the precise localization of these growth factors. The most pronounced changes in TGF beta immunoreactivity occurred during spermatogenesis. TGF beta 1 predominated in spermatocytes and early round spermatids, but as the spermatids elongated around stages VIII-IX of the cycle, the TGF beta 1 levels declined. TGF beta 2 was undetectable in spermatocytes and early round spermatids, but as spermiogenesis progressed, around stages V-VI, the spermatids rapidly acquired TGF beta 2. The intense staining for TGF beta 2 was maintained as the spermatids elongated. TGF beta 1 immunoreactivity was detected in Sertoli cells throughout testicular development. TGF beta 2 was found in fetal Sertoli cells, but became undetectable rapidly after birth. In fetal animals the Leydig cells contained TGF beta 1 and TGF beta 2; after birth TGF beta 1 persisted whereas TGF beta 2 became undetectable in the Leydig cells. Prior to puberty, TGF beta 1 and TGF beta 2 were absent in a portion of the Leydig cells; when the adult stage was reached, TGF beta 1 was no longer detectable and TGF beta 2 staining was faint to absent. In conclusion, our novel findings show that TGF beta 1 and TGF beta 2 are present in vivo in testicular cells at clearly defined stages of their differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Steroidogenesis-inducing protein stimulates protein-tyrosine kinase activity in rat Leydig cells.

Steroidogenesis-inducing protein (SIP) isolated from human ovarian follicular fluid stimulates steroid production in Leydig cells, human luteal cells, and rat adrenal cells. In addition, SIP is a potent mitogen that stimulates the proliferation of Leydig cells from immature rats to a greater extent than do LH/hCG and other known growth factors. We have shown previously that the actions of SIP on Leydig cells are independent of the adenyl cyclase-cAMP pathway. In the present study we have explored the possibility that SIP, like many growth factors, may exert its effects by activation of tyrosine kinase(s). Stimulation of Leydig cells isolated from immature rats with SIP resulted in an increase in the tyrosine phosphorylation of proteins that were detected with phosphotyrosine-specific antibodies. The phosphorylation of a 90-kilodalton (kDa) protein band, a 65-kDa protein band, and a doublet at 140 kDa was apparent after 5 min. After 30 min, additional SIP-induced phosphotyrosine proteins were detected at 42, 44, 50, 80, 100, and 150 kDa. In addition to phosphorylation at tyrosine residues, all of the proteins isolated from SIP-stimulated cells were phosphorylated at threonine and serine residues. SIP-induced phosphoproteins recovered with phosphotyrosine-specific antibodies were found to have associated protein-tyrosine kinase activity. The major substrate for this kinase activity in vitro was a 140-kDa protein, similar to one of the major phosphotyrosine-containing proteins induced by SIP treatment of intact cells. These observations suggest that SIP influences gonadal cell steroidogenesis and proliferation, presumably by activating cellular protein-tyrosine kinase(s) as part of a phosphorylation-based signalling pathway.

Steroidogenesis-inducing protein interacts with transforming growth factor-beta to stimulate DNA synthesis in rat granulosa cells.

We have examined the effects of steroidogenesis-inducing protein (SIP), previously isolated from human follicular fluid, on the synthesis of DNA by granulosa cells isolated from diethylstilbestrol-primed immature rats. SIP alone had no effect but in conjunction with transforming growth factor-beta (TGF-beta) there was an increase in [3H]thymidine incorporation into granulosa cell DNA. The increase in [3H]thymidine into DNA was due to an increase in the number of labeled granulosa cells as assessed by autoradiography. Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) interfered with the ability of SIP and TGF-beta to promote DNA synthesis. Previously, we reported that the growth-promoting action of follicle-stimulating hormone (FSH) on rat granulosa cells in vitro was dependent on TGF-beta, and EGF inhibited the actions of FSH plus TGF-beta on [3H]thymidine incorporation into DNA. Since the dependency of SIP on its interactions with TGF-beta and the ability of EGF to interfere with the process were similar to the properties reported for FSH, this raised the possibility that the actions of SIP were mediated through the accumulation of intracellular cAMP. However, when the hypothesis was tested, SIP had no effect on cAMP levels in the presence or absence of TGF-beta, under conditions in which FSH stimulated cAMP accumulation. In conclusion, DNA synthesis in rat granulosa cells is dependent on the presence of TGF-beta. In the presence of TGF-beta, FSH or SIP, acting through cAMP-dependent and cAMP-independent mechanisms respectively, can recruit more cells to enter the cell cycle and initiate DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 stimulates deoxyribonucleic acid synthesis in immature rat Leydig cells in vitro.

The number of interstitial macrophages in the testis fluctuates according to age, increasing gradually during prepubertal development to reach 15-20% in the interstitial compartment in the adult rat. These macrophages are in close morphological association with Leydig cells. Macrophage products, interleukin-1 (IL-1) and tumor necrosis factor alpha stimulate and/or inhibit steroid production in cultures of Leydig cells. We have studied the effects of macrophage products on DNA synthesis in rat Leydig cells to investigate a possible paracrine role of testicular macrophage products on the proliferation of Leydig cells. Leydig cells isolated from 10-, 20-, and 70-day-old rats were cultured for 48 h in serum-free medium, washed, and treated with different cytokines for 18 h. The medium was then removed, fresh medium containing 0.5 microCi [3H]thymidine was added, and cells were incubated for 4 h prior to determining the incorporation of [3H]thymidine into DNA. Human recombinant IL-1 beta caused a dose-dependent stimulation in the incorporation of [3H]thymidine into DNA in the Leydig cells from 10- and 20-day-old rats but had no effect on DNA synthesis in interstitial cells from adult rats. Maximum stimulation of DNA synthesis in immature Leydig cells was observed with 1-2 ng/ml IL-1 beta. Autoradiography after incubation with [3H]thymidine showed a dramatic increase in the number of labeled Leydig cells after treatment with IL-1 beta (19.27 +/- 3.77% vs. 1.44 +/- 0.52% in control cultures) indicating that IL-1 beta recruited more cells to enter the cell cycle and initiate DNA synthesis. Human recombinant IL-1 alpha and tumor necrosis factor alpha also caused significant stimulation of DNA synthesis in Leydig cells but these cytokines were much less potent (1-10%) than IL-1 beta. IL-1 beta enhanced the effects of maximally effective concentrations of growth-promoting agents previously known to stimulate DNA synthesis in immature rat Leydig cells, i.e. human CG, steroidogenesis-inducing protein, and transforming growth factor alpha plus insulin. On the basis of these results it is concluded that IL-1 may play an important role in the proliferation of Leydig cells during prepubertal development in immature rats.

Immunohistochemical localization of transforming growth factor-beta 1 and -beta 2 during follicular development in the adult rat ovary.

The transforming growth factors-beta (TGF-beta) affect the metabolic activities of each of the cell types in the ovary. In vitro studies using immature rat ovaries have shown the expression of TGF-beta 1 and/or TGF-beta 2 mRNA in thecal/interstitial cells and in granulosa cells (Mulheron and Schomberg, 1990; Mulheron et al., 1991). To obtain information on the localization of TGF-beta 1 and TGF-beta 2 in the rat ovary in vivo, we have examined the immunohistochemical staining using antibodies specific for either TGF-beta 1 or TGF-beta 2. In the adult ovary the immunostaining for TGF-beta 1 was intense, whereas the staining for TGF-beta 2 was faint. The pattern of immunostaining for TGF-beta 1 and TGF-beta 2 remained constant in the interstitial cell compartment and was not affected by the stage of the oestrous cycle. Since the interstitium surrounds follicles at all stages of development we conclude that TGF-beta is not actively involved in regulating the progression of follicles at discrete stages. At the time of antrum formation in the follicle, intense staining for TGF-beta 1 was observed in thecal cells. Around the preovulatory stage of development, TGF-beta 1 and TGF-beta 2 immunoreactivity was also found in the granulosa cells. In the corpus luteum, intense staining for TGF-beta 1 was found in some areas, whereas other areas were negative. Weak to moderate staining for TGF-beta 2 was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal growth factor influences growth and differentiation of rat granulosa cells.

The interactions of epidermal growth factor (EGF) with transforming growth factor beta (TGF beta), insulin-like growth factor-I (IGF-I), and FSH in the modulation of DNA synthesis and differentiated functions were examined in cultures of granulosa cells isolated from the ovaries of immature rats primed with diethylstilbestrol. EGF alone or in the presence of FSH had no effect on [3H]thymidine incorporation into the DNA of granulosa cells; however, EGF inhibited FSH plus TGF beta-induced DNA synthesis. In contrast, when FSH was omitted from the culture medium, EGF acted in concert with TGF beta, and TGF beta plus IGF-I, to promote DNA synthesis. EGF therefore has opposing actions on DNA synthesis; it inhibits or stimulates depending upon the presence or absence of FSH and consequently upon the endocrine environment in the follicle. As shown previously EGF alone had no effect on basal aromatase activity. EGF however inhibited FSH-induced and FSH plus IGF-I-induced aromatase activity. In this paper we show that EGF also inhibited the FSH-induced aromatase activity in the presence of TGF beta, which augmented FSH action on this system. The action of EGF on 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was different from the effect of EGF on aromatase. In the absence of FSH, EGF induced 3 beta-HSD activity in the presence or absence of TGF beta. EGF augmented the action of FSH on 3 beta-HSD, and this interaction was further enhanced by TGF beta. These observations emphasize the multifunctional nature of EGF in influencing the growth and differentiation of immature rat granulosa cells. EGF can inhibit or stimulate growth and differentiated functions (aromatase and 3 beta-HSD), the response depending on the context of the signals that the cell receives from its endocrine and microenvironment.

Immunohistochemical detection of transforming growth factor-alpha in Leydig cells during the development of the rat testis.

In this paper the localization of transforming growth factor alpha (TGF-alpha) is described in the rat testis at various stages throughout development, e.g. neonatal, prepubertal, and adult, in order to examine somatic cells and germinal cells at different stages of differentiation. This was done by immunoperoxidase staining using a monoclonal antibody that does not cross-react with epidermal growth factor (EGF). In sections of testes from neonatal rats, intense staining was present in Leydig cells. In the cells of the seminiferous tubules the staining was faint or undetectable. At the time when many mesenchymal cells differentiate into Leydig cells in the 21-day-old rat, TGF-alpha was visualized in most but not all of the identifiable Leydig cells. In interstitial cell cultures derived from 21-day-old rats, the majority of the Leydig cells contained TGF-alpha, but in a proportion of the Leydig cells TGF-alpha was undetectable. No staining was apparent in Sertoli cells and germ cells in seminiferous tubules or in Sertoli cell cultures derived from 21-day-old rats. Under these in vitro conditions it was found that peritubular-myoid cells also possessed TGF-alpha immunoreactivity. In the adult testis all Leydig cells stained positively for TFG-alpha, whereas no staining was found in the cells of the seminiferous tubules. Treatment of adult rats with ethylene-1,2-dimethane-sulfonate (EDS) resulted in the destruction of Leydig cells and the loss of all positively stained for TGF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

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.