Expression and action of transforming growth factor beta (TGFbeta1, TGFbeta2, TGFbeta3) in normal bovine ovarian surface epithelium and implications for human ovarian cancer.

The majority of ovarian tumors are derived from the single layer of epithelial cells on the surface of the ovary termed the ovarian surface epithelium (OSE). Stromal cell-OSE interactions are postulated to be an important aspect of normal OSE biology and the biology of ovarian cancer. Transforming growth factor beta (TGFbeta) has been shown to often be a mesenchymal cell-derived growth factor that mediates stromal cell-epithelial cell interactions in a variety of different tissues. The current study investigates the expression and action of TGFbeta isoforms (TGFbeta1, TGFbeta2, and TGFbeta3) in OSE and the underlying stroma in both normal bovine and human tumor tissues. Normal bovine ovaries are similar to human ovaries and are used as a model system to investigate normal OSE and stromal cell functions. All three TGFbeta isoforms and their receptor, transforming growth factor beta receptor type II (TGFbetaRII), proteins were found to be detected in the OSE from normal bovine ovaries using immunohistochemistry. Ovarian stromal tissue also contained positive immunostaining for TGFbeta isoforms and TGFbetaRII. RNA was collected from normal bovine OSE and ovarian stromal cells to examine TGFbeta gene expression. TGFbeta1, TGFbeta2, and TGFbeta3 transcripts were detected in both freshly isolated and cultured bovine OSE and stromal cells by a sensitive quantitative polymerase chain reaction assay. TGFbeta1 and TGFbeta2 mRNA levels were found to be present at similar levels in freshly isolated OSE and stroma. Interestingly, TGFbeta3 mRNA levels were significantly higher in freshly isolated OSE than stromal cells. All but TGFbeta3 mRNA in OSE increased when the cells were cultured. Observations indicate that normal bovine OSE and stroma cells express the three TGFbeta isoforms in vivo and in vitro. Human ovarian tumors from stage II, stage III and stage IV cases were found to express TGFbeta1, TGFbeta2, TGFbeta3 and TGFbetaRII protein primarily in the epithelial cell component by immunohistochemistry analysis. The stromal cell component of the human ovarian tumors contained little or no TGFbeta or TGFbetaRII immunostaining. TGFbeta actions on bovine OSE and stromal cells were also investigated. TGFbeta was found to inhibit the growth of OSE, but not stromal cells. To further examine the actions of TGFbeta on OSE, the expression of two growth factors previously shown to be expressed by OSE were analyzed. TGFbeta1 was found to stimulate the expression of both keratinocyte growth factor (KGF) and kit ligand/stem cell factor (KL) by bovine OSE. Therefore, TGFbeta actions on OSE will likely promote a cascade of cell-cell interactions and cellular responses involving multiple growth factors. The effects of regulatory agents on TGFbeta expression by the bovine OSE were examined. Transforming growth factor alpha (TGFalpha) stimulated TGFbeta1 expression, TGFbeta1 stimulated TGFbeta2 expression, and follicle stimulating hormone (FSH) stimulated TGFbeta3 expression. These results demonstrate that TGFbeta isoforms are regulated differently by the regulatory agents tested. In summary, all the TGFbeta isoforms are differentially expressed by the OSE and TGFbeta appears to have an important role in regulating OSE and possibly stromal-OSE interactions. A complex network of endocrine and paracrine interactions appears to influence the expression and actions of TGFbeta on OSE. Abnormal expression and/or action of TGFbeta is postulated to in part be involved in the onset and progression of ovarian cancer.

Stromal-epithelial interactions in the progression of ovarian cancer: influence and source of tumor stromal cells.

Stromal cells are essential for the progression of many cancers including ovarian tumors. Stromal cell-epithelial cell interactions are important for tumor development, growth, angiogenesis, and metastasis. In the current study, the effects of normal ovarian bovine stromal cells on ovarian tumor progression was investigated. The hypothesis tested is that ovarian stromal cells will alter the onset and progression of ovarian tumors. Conditioned medium from normal bovine ovarian surface stromal cells was found to stimulate the growth of normal ovarian surface epithelium and had no effect on the growth of human tumor cell lines SKOV3 and OCC1. Human ovarian cancer cell lines, SKOV3 and OCC1, were injected subcutaneously into nude mice to examine tumor progression. Tumor growth in the nude mice was dramatically reduced when normal ovarian surface stromal cells were co-injected with SKOV3 or OCC1 cells. Similar results were obtained with normal bovine or human ovarian stromal cells. In contrast, irrelevant testicular stromal cells and epithelial cells had no effect on tumor growth in the nude mouse. Histological examination of these tumors revealed a characteristic stromal cell component adjacent to epithelial cell colonies. Sections of these tumors were hybridized with species specific genomic probes using fluorescence in situ hybridization to identify cell populations. Epithelial cells were shown to be of human origin (i.e. SKOV3 or OCC1), but stromal cells were found to be primarily murine in origin (i.e. host tissue). No detectable bovine cells were observed in the tumors after one week post-injection. Results suggest that stromal cells are an essential component of ovarian tumors. Interestingly, normal ovarian stromal cells had the ability to inhibit tumor growth, but were not able to survive long-term incubation at the tumor site. The developing tumor appears to recruit host (i.e. murine) stromal cells to invade the tumor and support its growth. In summary, normal ovarian stromal cells can inhibit ovarian tumor progression and the developing tumors recruit adjacent host stroma to become "tumor stroma". The tumor stroma likely develop an altered phenotype that cooperates with the tumorigenic epithelial cells to help promote the progression of ovarian cancer.

Basic fibroblast growth factor induces primordial follicle development and initiates folliculogenesis.

The recruitment of primordial follicles to initiate folliculogenesis determines the population of developing follicles available for ovulation and directly regulates female reproductive efficiency. In the current study, a floating organ culture system was used to examine the progression of primordial (stage 0) follicles to developing (stages 1-4) follicles in 4-day-old pre-pubertal rat ovaries. Basic fibroblast growth factor (bFGF) was found to induce primordial follicle development similar to what has been demonstrated for kit ligand/stem cell factor (KL). The bFGF-treated ovaries contained 85% developing follicles compared with 50% developing follicles for control untreated organ cultures. Correspondingly, the number of primordial follicles in bFGF-treated ovaries decreased to 15% of the total compared with 45% for controls. A bFGF neutralizing antibody was found to decrease the small amount of spontaneous follicle development that occurs during the organ culture. Basic FGF was localized to primordial and early developing follicles by immunocytochemistry and was primarily observed in the oocytes. Treatment of bovine ovarian theca cells and stroma cells with bFGF was found to promote cell growth. Basic FGF produced by the oocyte in early stage follicles appears to act on adjacent somatic cells to promote cell growth and development. Basic FGF, like KL, appears to be a primordial follicle-inducing factor. In summary, bFGF can regulate primordial follicle development that directly influences female reproductive efficiency.

Expression and actions of both the follicle stimulating hormone receptor and the luteinizing hormone receptor in normal ovarian surface epithelium and ovarian cancer.

The ability of gonadotropins to act on and regulate normal ovarian surface epithelial (OSE) cells and ovarian cancer cells was investigated. Bovine OSE was used as a model to study normal OSE. Results demonstrate that follicle stimulating hormone (FSH) and the luteinizing hormone (LH) like molecule, human chorionic gonadotropin (hCG), can both stimulate (3H)-thymidine incorporation into DNA in normal OSE cells. Similar results were obtained using either purified hormones or recombinant human hormones. A human ovarian cancer cell-line OCC1 was also stimulated to grow in response to FSH and hCG, but the growth of a different human ovarian cancer cell-line SKOV3 was not affected. In addition to effects on cell growth, gonadotropins also stimulated growth factor expression. Both FSH and hCG stimulated steady state levels of keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and kit ligand (KL) mRNA in OSE cells. Previously, KGF, HGF, and KL have been shown to stimulate OSE growth. Both follicle stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) were observed in OSE cells by Northern blot analysis. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed on fresh and cultured OSE cells. Normal OSE was found to express FSHR and LHR both in vivo and in vitro. The PCR reaction products were sequenced and found to provide a 100% homology with the bovine gonadotropin receptor sequences previously reported. FSHR and LHR transcripts were also detected in gonadotropin responsive OCC1 cells, but not in the gonadotropin insensitive SKOV3 cells. Observations support the hypothesis that gonadotropins may influence some ovarian cancers. In summary, the current study demonstrates the novel observation that both the FSHR and LHR are expressed by bovine OSE and selected ovarian cancers. Interestingly, the actions of FSH and LH to promote OSE growth may in part be mediated indirectly through an elevation in the expression of autocrine growth factors (KGF, HGF, and KL). Ovarian cancer is more common in conditions with elevated gonadotropins such as post-menopausal women. Therefore, gonadotropin actions on the OSE are postulated to be a potential factor in the onset and progression of some ovarian cancers.

Direct effects of nerve growth factor on thecal cells from antral ovarian follicles.

TrkA, the nerve growth factor (NGF) tyrosine kinase receptor, is expressed not only in the nervous system, but also in nonneural cells, including discrete cellular subsets of the endocrine and immune system. In the rat ovary, trkA receptor abundance increases strikingly in thecal-interstitial cells during the hours preceding the first ovulation. Blockade of either trkA transducing capacity or NGF biological activity inhibited ovulation, suggesting a role for NGF in the ovulatory process of this species. To identify some of the processes that may be affected by trkA activation in the thecal compartment, we used purified thecal cells/thecal fibroblasts from bovine ovaries (heretofore referred to as thecal cells). Ribonuclease protection assays employing bovine-specific cRNA probes demonstrated the presence of the messenger RNAs (mRNAs) encoding NGF and its receptors, p75 NTR and trkA, in the thecal compartment of small, medium, and large antral follicles and showed that trkA mRNA is also expressed in granulosa cells. In situ hybridization and immunohistochemical examination of intact ovaries confirmed these cellular sites of NGF and trkA synthesis. TrkA mRNA, but not NGF mRNA, was lost within 48 h of placing thecal cells in culture. Thus, to study trkA-mediated actions of NGF on these cells we transiently expressed the receptor by transfection with a vector containing a full-length rat trkA complementary DNA under transcriptional control of the cytomegalovirus promoter. Because ovulation is preceded by an LH-dependent increase in androgen and progesterone production, the ability of NGF to modify the release of these steroids was determined in freshly plated cells still containing endogenous trkA receptors and in cells undergoing luteinization in culture that were transiently transfected with the trkA-encoding plasmid. NGF stimulated both androgen and progesterone release in freshly plated thecal cells, but not in luteinizing cells provided with trkA receptors. As ovulation in rodents requires an increased formation of PGE2 and has been shown to be antedated by proliferation of thecal fibroblasts, we determined the ability of NGF to affect these parameters in trkA-transfected thecal cells. The neurotrophin rapidly stimulated PGE2 release and amplified the early steroidal response to hCG in trkA-expressing cells, but not in cells lacking the receptor. Likewise, NGF stimulated [3H]thymidine incorporation into trkA-containing cells, but not into cells that had lost the receptor in culture. Induction of ovulation in immature rats by gonadotropin treatment verified that an increased cell proliferation in the thecal compartment, determined by the incorporation of bromodeoxyuridine into cell nuclei, occurs 4-5 h before ovulation in this species. These results suggest that the contribution of NGF to the ovulatory process includes a stimulatory effect of the neurotrophin on steroidogenesis, PGE2 formation, and proliferative activity of thecal compartment cells.

Expression and action of keratinocyte growth factor (KGF) in normal ovarian surface epithelium and ovarian cancer.

The current study investigates the expression and action of keratinocyte growth factor (KGF) in normal ovarian surface epithelium (OSE) and ovarian cancer tissues. Ovarian tumors are primarily derived from the OSE. KGF is a mesenchymal cell-derived growth factor that mediates stromal cell-epithelial cell interactions in a variety of different tissues. Human ovarian tumors from borderline, stage I and stage III cases were found to express KGF protein in the epithelial cell component by immunocytochemical analysis. The stromal cell component of human ovarian tumors contained little or no KGF immunostaining. Normal bovine ovaries have similarities to human ovaries and are used as a model system to investigate normal OSE functions. KGF protein was detected in the OSE from normal human and bovine ovaries by immunocytochemistry. Ovarian stromal tissue contained light but positive KGF immunostaining. RNA was collected from normal bovine OSE and ovarian stromal cells to examine KGF gene expression. KGF transcripts were detected in cultured OSE and stromal cells by Northern blot analysis. In order to examine and quantitate KGF gene expression in freshly isolated versus cultured tissues, a sensitive quantitative RT-PCR assay for KGF was utilized. KGF gene expression was found to be high in freshly isolated OSE, but very low in freshly isolated stroma. Levels of KGF gene expression after culture of OSE and stromal cells increased. Observations indicate that normal OSE express high levels of KGF in vivo and in vitro. Expression of KGF by normal epithelial cells versus stromal cells was unexpected and suggests KGF may be an important autocrine stimulator of OSE. KGF actions on bovine OSE cells were investigated. KGF was found to stimulate the growth of normal OSE cells to the same level as epidermal growth factor. Two ovarian cancer cell lines, SKOV3 and OCC1, were also stimulated to proliferate in response to KGF. Current results demonstrate the production and action of KGF on normal OSE cells and ovarian cancer cells. Observations can be interpreted to suggest that KGF may in part be involved in the growth of ovarian tumors. This appears to be one of the first reports of KGF production by an epithelial cell. The autocrine stimulation of OSE growth by the local production and action of KGF provides insight into how the OSE may develop abnormal growth characteristics involved in the onset and progression of ovarian cancer.

Expression and action of transforming growth factor alpha in normal ovarian surface epithelium and ovarian cancer.

Greater than 95% of ovarian cancers originate in the epithelial cells on the surface of the ovary. The current study investigates the expression and action of transforming growth factor alpha (TGFalpha) in ovarian surface epithelium (OSE) and the underlying stroma in both normal and tumorigenic ovarian tissues. Normal bovine ovaries are used in the current study as a model system to investigate normal OSE functions. Transforming growth factor alpha and its receptor, the epidermal growth factor receptor (EGFR), were detected in the OSE from normal ovaries by immunocytochemistry (ICC). Ovarian stromal tissue also contained reduced but positive TGFalpha and EGFR immunostaining. To examine TGFalpha and EGFR gene expression, RNA was collected from normal bovine OSE and ovarian stromal cells. The TGFalpha and EGFR transcripts were detected in both fresh and cultured OSE and stromal cells by a sensitive quantitative reverse transcription polymerase chain reaction (QRT-PCR) assay. Transforming growth factor alpha gene expression was found to be high in freshly isolated OSE, but low in freshly isolated stroma. In contrast, EGFR expression was higher in the stroma compared to the OSE. Both the ICC and QRT-PCR indicate that normal OSE express high levels of TGFalpha in vivo and in vitro. In vitro, normal ovarian stromal cells develop the capacity to express high levels of EGFR. Human ovarian tumors from stage II, stage III, and stage IV ovarian cancer cases were found to express TGFalpha and EGFR protein in the epithelial cell component of the tumor by ICC analysis. The stromal cell component of human ovarian tumors contained little or no TGFalpha/EGFR immunostaining. Observations suggest that tumor progression may in part require autocrine stimulation of the epithelia. Transforming growth factor alpha was found to stimulate the growth of normal bovine OSE and stroma cells to the same level as epidermal growth factor. Two ovarian cancer cell lines, SKOV3 and OCC1, were also stimulated to proliferate in response to TGFalpha. Transforming growth factor alpha was also found to stimulate the expression of two growth factors previously shown to be produced by OSE. Transforming growth factor alpha stimulates both kit ligand/stem cell factor and keratinocyte growth factor production by OSE. The effect of hormones on TGFalpha and EGFR expression by the OSE was also examined. Human chorionic gonadotropin stimulated TGFalpha expression, but not FSH. Both hCG and FSH stimulated EGFR expression by OSE. Combined observations suggest a role of systemic hormones and a locally produced growth factor, TGFalpha, in OSE biology. Insight is also provided into how the OSE may develop abnormal growth characteristics involved in the onset and progression of ovarian cancer.

Autocrine interactions of keratinocyte growth factor, hepatocyte growth factor, and kit-ligand in the regulation of normal ovarian surface epithelial cells.

Ovarian tumors are primarily derived from the layer of epithelium surrounding the ovary termed the ovarian surface epithelium (OSE). Although extensive research has focused on established ovarian tumors, relatively little is known about the normal biology of the OSE that gives rise to ovarian cancer. The local expression and actions of growth factors are likely involved in both normal and tumorigenic OSE biology. The current study investigates the expression and action of keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and kit-ligand (KL) in normal ovarian surface epithelium (OSE). The actions of various growth factors on KGF, HGF, and KL expression are examined. Observations indicate that freshly isolated normal OSE express the genes for KGF, HGF, and KL and expression is maintained in vitro. KGF messenger RNA expression in OSE was found to be stimulated by KGF and HGF, but not KL. HGF expression in OSE was found to be stimulated by KGF, HGF, and KL. KL expression in OSE was also found to be stimulated by KGF, HGF, and KL. Therefore, the various growth factors can regulate the mRNA expression of each other in OSE. Effects of growth factors on OSE growth were examined. KGF, HGF, and KL stimulated OSE growth to similar levels as the positive control epidermal growth factor. Observations suggest that KGF, HGF, and KL interact to promote OSE growth and growth factor expression. The ability of these growth factors to interact in a positive autocrine feedback loop is postulated to be important for normal OSE biology. Paracrine interactions with the adjacent stromal cells will also be a factor in OSE biology. Abnormal interactions of these growth factors may be involved in the onset and progression of ovarian cancer.

Expression and action of kit ligand/stem cell factor in normal human and bovine ovarian surface epithelium and ovarian cancer.

Greater than 95% of ovarian cancers originate from the epithelial cells on the surface of the ovary termed ovarian surface epithelium (OSE). A normal aspect of OSE function is repeated proliferation after ovulation, and this is postulated to be involved in part in the onset of ovarian cancer. The hypothesis tested is that locally produced growth factors have an important role in controlling OSE proliferation. The current study investigates the potential role of the growth factor kit ligand (KL)/stem cell growth factor and its receptor c-kit in normal OSE biology and ovarian cancer. Human tumors from borderline, stage I, and stage III cases of ovarian cancer were found to express KL and c-kit protein in the epithelial cell component by ICC analysis. The stromal cell component of human ovarian tumors contained little immunostaining. Bovine ovarian physiology and endocrinology are similar to the human such that cow ovaries were used as a model system to investigate normal OSE functions. KL and c-kit proteins were detected in the OSE from both normal human and bovine ovaries. Adjacent ovarian stromal tissue contained less intense but positive KL and c-kit immunostaining. To extend the ICC results, RNA was collected from normal bovine OSE and ovarian stromal cells to examine KL gene expression. KL transcripts were detected in cultured OSE and stromal cells by Northern blot analysis. KL gene expression was found to be high in freshly isolated OSE but low in freshly isolated stroma using a quantitative polymerase chain reaction procedure. Levels of KL gene expression in cultured OSE and stroma increased to high levels. Observations indicate that normal OSE expresses high levels of KL in vivo and in vitro. The actions of KL on the growth of both normal OSE cells and ovarian cancer cells was investigated. KL was found to stimulate the growth of normal OSE cells in a similar manner to epidermal growth factor. Observations demonstrate the production and action of KL by normal OSE cells and ovarian cancer cells. Coexpression of KL and c-kit by normal OSE suggests that KL can act as an autocrine factor for OSE. The local production and action of KL on OSE provides insight into normal OSE biology, and a factor that may be involved in the onset and progression of ovarian cancer.

Expression and action of hepatocyte growth factor in human and bovine normal ovarian surface epithelium and ovarian cancer.

More than 95% of ovarian cancers originate from the epithelial cells on the surface of the ovary, which are termed ovarian surface epithelium (OSE). These OSE cells are modified peritoneal mesothelial cells separated from underlying ovarian surface stromal tissue by a basal lamina of dense collagenous connective tissue. Mesenchymal-epithelial cell interactions between stromal cells and OSE cells are postulated to be important for normal OSE biology and for the onset of ovarian cancer. Hepatocyte growth factor (HGF) is a mesenchymal-derived growth factor that mediates mesenchymal-epithelial cell interactions in a number of different tissues. The current study was an investigation of the expression and actions of HGF in normal OSE and ovarian cancer. Human epithelial cells from borderline and stage III ovarian cancer cases were found to express HGF protein in the epithelial cell component by immunocytochemistry analysis. The stromal cell component of human ovarian tumors contained little or no HGF immunostaining. Normal bovine ovaries have a similar physiology and endocrinology to human ovaries and are used as a model system to investigate normal OSE functions. HGF protein was detected in the OSE from both normal human and bovine ovaries. Adjacent ovarian stromal tissue contained light but positive HGF immunostaining. RNA was collected from normal bovine ovarian stromal cells to examine HGF gene expression. HGF transcripts were detected in cultured OSE and stromal cells by Northern blot analysis. Using a quantitative reverse transcription-polymerase chain reaction procedure, HGF gene expression was found to be high in freshly isolated OSE but low in freshly isolated stroma. Levels of HGF gene expression after culture of stroma increased. Observations indicate that normal OSE express high levels of HGF in vivo and in vitro. Expression of HGF by normal epithelial cells versus stromal cells was unexpected and suggests that HGF may be important in an autocrine regulation of OSE. HGF actions on normal OSE cells and ovarian cancer cells were investigated. HGF was found to stimulate the growth of normal OSE cells in a manner similar to such growth stimulated by epidermal growth factor. Two ovarian cancer cell lines, SKOV3 and OCC1, were also stimulated to grow in response to HGF. This observation suggests that HGF may be involved in sustaining growth of ovarian tumors. These results are the first to demonstrate the production and action of HGF in normal OSE cells and ovarian cancer cells. This appears to be an example of HGF production by an epithelial cell, such that a mesenchymal-epithelial mixed phenotype is present. The autocrine stimulation of OSE growth by the local production and action of HGF provides insight into how the OSE may develop abnormal growth characteristics involved in the onset and progression of ovarian cancer.

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

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

Kit-ligand/stem cell factor induces primordial follicle development and initiates folliculogenesis.

Initiation of folliculogenesis through the induction of primordial follicle development in the ovary has an important role in determining the fertility and reproductive fitness of most mammalian species. The factors that control this critical process are largely unknown. The hypothesis tested in the current study was that kit-ligand/stem cell factor (KL) promotes the initiation and progression of primordial follicle development in the ovary. Ovaries from 4-day-old rats were maintained in organ culture for 5 and 14 days and treated with no factor (control), recombinant kit-ligand (KL), or gonadotropins (FSH and hCG). Follicles in ovarian sections were counted and histologically classified as primordial (stage 0), early primary (stage 1), primary (stage 2), transitional (stage 3), or preantral (stage 4). Fresh ovaries from 4-day-old rats contained 68% primordial follicles (stage 0) and 32% developing follicles (stages 1-4) per section. After 5 and 14 days in culture, section from control ovaries contained approximately 41% and 55%, respectively, developing follicles (stage 1-4) per section due to spontaneous development of primordial follicles. Spontaneous primordial follicle development was completely blocked by ACK-2, a c-kit antibody that blocks KL actions. This observation suggests that endogenous KL is necessary for primordial follicle development in vitro. After 14 days of KL treatment, sections from ovaries contained 17% primordial follicles (stage 0) and 83% developing follicles (stage 1-4) per section demonstrating a dramatic induction of primordial follicle development by KL. Gonadotropins (FSH and hCG) did not induce primordial follicle development but did increase the percentage of preantral follicles (stage 4) per section. This small increase in preantral follicles in response to gonadotropins was blocked by ACK-2 suggesting that KL may in part mediate gonadotropin actions after the initiation of primordial follicle development. Ovaries contained an average of 309+/-10 follicles per section. The total number of follicles per section did not significantly vary between treatments suggesting that the effects of KL were not due to an alteration in follicle number (i.e. survival). KL appears to be one of the first factors identified to be involved in the promotion of primordial follicle development. Results suggest that KL is necessary and sufficient to induce primordial follicle development and initiate folliculogenesis.

Developmental and hormonal regulation of hepatocyte growth factor expression and action in the bovine ovarian follicle.

Ovarian hormones (i.e., estrogen and LH) may promote folliculogenesis by regulating the local production of mesenchymal "inducer proteins" that mediate theca cell-granulosa cell interactions. Theca cells produce hepatocyte growth factor (HGF) that can stimulate granulosa cell growth. In order to investigate the physiological role of HGF in the ovarian follicle, the developmental and hormonal regulation of HGF was examined during follicular development in the bovine ovary. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to examine HGF expression in theca cells and the HGF receptor (HGFR or c-met) in granulosa cells. Both HGF and HGFR were detected throughout follicular development in small (< 5 mm)-, medium (5-10 mm)-, and large (> 10 mm)-sized follicles. Steady-state levels of HGF and HGFR mRNAs were determined using sensitive quantitative RT-PCR assays. Developmental regulation of HGF in theca cells and HGFR in granulosa cells was analyzed in freshly isolated small-, medium-, and large-sized follicles. Observations demonstrate that expression of HGF (in theca cells) and HGFR (in granulosa cells) was highest in large-sized follicles. Hormonal regulation of HGF was analyzed in hormone-treated theca cell cultures. Steady-state levels of HGF mRNA in theca cells were increased by treatment with hCG (an LH agonist), but estradiol had no effect. These results suggest that LH may promote ovarian follicular growth (i.e., granulosa cell proliferation) in part by stimulating the local production of HGF by theca cells. Effects of HGF on granulosa cell differentiated functions were examined. Treatment with HGF reduced basal and FSH-stimulated levels of aromatase activity in bovine and rat granulosa cells. In addition, HGF inhibited the ability of hCG to stimulate progesterone production by granulosa cells. The inhibition of granulosa cell steroid production by HGF is proposed to be the indirect effect of promoting cellular proliferation. Therefore, HGF directly stimulates granulosa cell proliferation and indirectly inhibits granulosa cell differentiated functions. The developmental and hormonal regulation of HGF and HGFR during folliculogenesis provides evidence that HGF may be important for hormone-induced granulosa cell proliferation. As a result, HGF may be essential for establishing the granulosa cell population and microenvironment required for oocyte maturation in the female.

Thecal cell-granulosa cell interactions involve a positive feedback loop among keratinocyte growth factor, hepatocyte growth factor, and Kit ligand during ovarian follicular development.

Interactions between mesenchymal-derived thecal cells and epithelial-derived granulosa cells are essential for follicular development in the ovary. These mesenchymal-epithelial cell interactions are in part mediated by keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and Kit ligand (KL). This study investigates the hypothesis that thecal cell-derived growth factors (e.g. KGF and HGF) regulate granulosa cell function, and granulosa cell-derived growth factors (e.g. KL) regulate thecal cell function. Gonadotropin regulation of this cell-cell interaction is also examined. Sensitive quantitative RT-PCR assays were used to analyze gene expression of KGF, HGF, and KL in the ovary. Thecal cell-derived KGF and HGF stimulated KL expression in bovine granulosa cells. Granulosa cell-derived KL stimulated KGF and HGF expression in bovine thecal cells. These results suggest that thecal and granulosa cells interact in a positive feedback loop mediated by KGF, HGF, and KL. Previous studies have suggested that gonadotropins (i.e. FSH and LH) regulate locally produced growth factor expression in the ovary. Treatment of bovine granulosa cells with FSH and hCG (a LH agonist) directly stimulated KL expression. The LH agonist hCG was also found to stimulate both KGF and HGF expression in thecal cells. The actions of gonadotropins on follicular development may in part be indirectly regulated by KL, KGF, and HGF expression. A novel positive feedback loop was identified between thecal cells and granulosa cells that is mediated by KL, KGF, and HGF. Thecal cell-derived KGF and HGF can stimulate granulosa cell-derived KL expression, and KL, in turn, can stimulate thecal cell-derived KGF and HGF expression. Combined observations support the hypothesis that mesenchymal-epithelial cell interactions between thecal and granulosa cells can play a significant role during ovarian follicular development and mediate gonadotropin actions.

Developmental and hormonal regulation of keratinocyte growth factor expression and action in the ovarian follicle.

The developing ovarian follicle is one of the most rapidly proliferating normal tissues in vivo. Mesenchymal-epithelial cell interactions between theca cells and granulosa cells are essential for this follicular expansion. Ovarian hormones (i.e. estrogen and LH) may promote follicular development by regulating the local production of mesenchymal inducer proteins that mediate theca cell-granulosa cell interactions. Recently, theca cells were shown to produce keratinocyte growth factor (KGF) that can act in a paracrine manner to stimulate granulosa cell growth. In this study, the developmental and hormonal regulation of KGF was examined during follicular development in the bovine ovary. Expression of KGF in theca cells and the KGF receptor (KGFR, or splice variant of the fibroblast growth factor family receptor family, FGFR-2) in granulosa cells was examined using RT-PCR. Both KGF and KGFR were detected throughout follicular development in small (<5 mm), medium (5-10 mm), and large (>10 mm) follicles. Quantitative RT-PCR assays were used to determine steady-state levels of KGF and KGFR messenger RNAs. Developmental regulation of KGF and KGFR was analyzed in freshly isolated theca cells and granulosa cells from small, medium, and large follicles. Observations demonstrated that expression of KGF (in theca cells) and KGFR (in granulosa cells) was highest in large follicles. These results suggest that KGF actions are important for the rapid proliferation of granulosa cells in large follicles. Estrogen and LH are the primary endocrine hormones that regulate theca cell function in vivo. Therefore, hormonal regulation of KGF was analyzed by treating serum-free theca cell cultures with estrogen and human CG (hCG, an LH agonist). Results showed that both estrogen and hCG stimulated KGF gene expression in theca cells. These results suggest that estrogen and LH may promote follicular growth (i.e. granulosa cell proliferation), in part, by stimulating the local production of KGF. Effects of KGF on granulosa cell differentiated functions were examined. Treatment with KGF reduced basal levels and FSH-stimulated levels of aromatase activity in bovine and rat granulosa cells. In addition, KGF inhibited the ability of hCG to stimulate progesterone production by granulosa cells. The inhibition of granulosa cell steroid production by KGF was likely the indirect effect of promoting cellular proliferation. Therefore, KGF directly stimulates granulosa cell proliferation and indirectly inhibits granulosa cell differentiated functions. Combined results suggest that theca cell production of KGF may be important for ovarian folliculogenesis. This is the first report of the regulation of KGF expression in the ovary. The developmental and hormonal regulation of KGF and KGFR during folliculogenesis provides evidence that KGF may be important for hormone-induced granulosa cell proliferation. As a result, KGF may be essential for establishing the microenvironment required for oocyte maturation in the ovary.

Direct actions of kit-ligand on theca cell growth and differentiation during follicle development.

The direct actions of kit-ligand/stem cell factor (KL) in developing ovarian follicles were investigated. Previous studies have shown that granulosa cells express KL that can support oocyte development. The current study demonstrates that KL can also act directly on theca cells to promote cellular growth and differentiation. Through RT-PCR analysis it was shown that bovine granulosa cells express KL, and theca cells express the receptor c-kit. Bovine theca interna cells were isolated and cultured in serum-free conditions to study KL actions. KL stimulated theca cell growth in a dose-dependent manner as measured by [3H]thymidine incorporation into DNA when cells were cultured under subconfluent conditions. KL had no effect on theca cell androstenedione or progesterone production under these growth-permissive conditions. In contrast, KL stimulated theca cell androstenedione production but had no effect on progesterone production when theca cells were cultured under confluent (non-growth-permissive) conditions. Estradiol (10(-7) M) and human CG (100 ng/ml) were used as controls and regulated theca cell steroid production at any cell density. These results demonstrate that KL can directly stimulate theca cell growth and steroid production during follicular development. The observation that KL stimulated androstenedione production but not progesterone production suggests that KL promotes a follicular phase differentiated state in theca cells. The potential regulation of KL and c-kit expression during follicular development was studied using a specific quantitative RT-PCR procedure. Total RNA from granulosa cells (for KL) and theca cells (for c-kit) was examined from small (<5 mm), medium (5-10 mm), and large (>10 mm) size follicles. Steady state levels of KL messenger RNA were highest in granulosa cells from large size follicles and lowest in small and medium size follicles. No differences were observed in the steady state levels of c-kit messenger RNA in theca cells from small, medium, or large size follicles. The observation that KL expression is highest in large size follicles suggests that KL may be important for increased growth and steroid production in large and dominant follicles. Observations demonstrate that KL can dramatically alter theca cell function and support the hypothesis that local granulosa-theca cell interactions play an important role in regulating cellular function within ovarian follicles. This study identifies KL as the first granulosa cell-derived growth factor that can directly stimulate theca cell growth and androstenedione production in the absence of gonadotropins.

Involvement of nerve growth factor in the ovulatory cascade: trkA receptor activation inhibits gap junctional communication between thecal cells.

Activation of trkA, the nerve growth factor (NGF) tyrosine kinase receptor, has been recently implicated in the process of mammalian ovulation. During the hour preceding follicular rupture, a marked increase in trkA and NGF gene expression occurs in thecal-interstitial cells of the ovary. Immunoneutralization of NGF actions or pharmacological blockade of trkA transducing activity inhibits ovulation, suggesting that activation of the NGF-trkA complex in nonneural cells of the periovulatory follicle is a physiological component of the ovulatory cascade. As thecal cells of Graafian follicles are functionally coupled by gap junctions, and the ovulatory rupture requires dissociation of thecal cell-cell communication, we sought to determine whether NGF affects the integrity of this communication. We now report that NGF-induced activation of trkA receptors in isolated ovarian thecal cells disrupts cell to cell communication by affecting the functional integrity of gap junctions. Bovine thecal cells expressing trkA receptors, but not cells lacking the receptors, respond to NGF with a reduction in the transfer of calcein, a fluorescent dye that passes through gap junctions. This effect was associated with a rapid (10-30 min) increase in serine phosphorylation of connexin-43, the main protein constituent of gap junctions in the ovary. The reduction in dye transfer was not observed when the cells were exposed to epidermal growth factor or other neurotrophins, including neurotrophin 3, neurotrophin 4, and brain-derived neurotrophic factor. Thus, cell-specific activation of trkA receptors in periovulatory follicles may provide one of the signals involved in inducing the cellular dissociation of the follicular wall that precedes ovulatory rupture.

Mesenchymal-epithelial interactions in the ovarian follicle involve keratinocyte and hepatocyte growth factor production by thecal cells and their action on granulosa cells.

Mesenchymal-epithelial cell interactions between thecal and granulosa cells in bovine ovarian follicles were investigated. Experiments were designed to examine the local production and action of two mesenchymal (stromal)-derived growth factors, keratinocyte and hepatocyte growth factors (KGF and HGF). Using reverse transcription-polymerase chain reaction, gene expression for KGF and HGF was detected in the mesenchymal-derived thecal cells, but not in the epithelial granulosa cells. The bovine polymerase chain reaction products for KGF and HGF were sequenced and found to be similar to known mouse, rat, and human sequences. The bovine KGF sequence was found to have a high degree of identity (86-95%) with the other species, whereas bovine HGF has a lesser degree of identity (60-63%). Immunoprecipitation of radiolabeled thecal cell secreted proteins with a KGF antibody demonstrated production of the 28-kilodalton (kDa) KGF protein. An immunoblot of thecal cell secreted proteins with HGF antibodies detected the 87-kDa HGF as well as relevant 69- and 34-kDa subunits. Therefore, thecal cells were found to express the genes and secrete the proteins for KGF and HGF. Granulosa cells had no detectable KGF or HGF expression. Treatment with recombinant KGF or HGF stimulated the proliferation of granulosa cells, but not thecal cells. Therefore, the actions of KGF and HGF in the ovarian follicle appear to be restricted to granulosa cells. The combined results indicate that KGF and HGF are produced locally in the bovine ovarian follicle by thecal cells, and that both of these growth factors can act on granulosa cells to influence cell proliferation. These observations demonstrate that KGF and HGF can mediate mesenchymal-epithelial cell interactions between thecal and granulosa cells. The potential importance that the mesenchymal derived thecal cells may have in ovarian follicle development is discussed.

Extrahepatic expression of fibrinogen by granulosa cells: potential role in ovulation.

Granulosa cells from ovarian follicles were shown to express and secrete fibrinogen under the control of FSH. Conditioned medium was collected from granulosa cell cultures and found to contain FSH-dependent 50-kilodalton (kDa) and 93- to 95-kDa proteins. N-Terminal microsequence analysis identified these proteins as fibrinogen beta- and gamma-chains, respectively. Proteins migrating at 93 and 95 kDa contain identical gamma-chain sequences at the N-terminal, suggesting differential processing of fibrinogen. These fibrinogen chains were specifically detected with antifibrinogen antibodies in immunoblot and immuno-precipitation analysis. Fibrinogen gamma-chain mRNA was detected in granulosa cells by polymerase chain reaction analysis, confirming fibrinogen gene expression by these cells. Fibrinogen secretion by granulosa cells was measured by a competitive enzyme-linked immunosorbent assay. Granulosa cells treated with FSH (100 ng/ml) secreted 2-3 times more fibrinogen than untreated cells. These data show that fibrinogen, a major product of the liver, is also a secretory product of granulosa cells. This provides a novel extrahepatic site of fibrinogen expression. As hepatic parenchymal cells normally maintain high circulating levels of fibrinogen, the local production of fibrinogen in the ovary is anticipated to have specialized functions. Locally produced fibrinogen may be important in the clotting process following tissue rupture at ovulation. In addition, fibrinogen fragments may be involved in the mechanism of ovulation by increasing the activity of tissue-type plasminogen activator to control the proteolytic activity required for ovulation.

Analysis of the steroid binding domain of rat androgen-binding protein.

The site-directed photoaffinity ligand [3H]17 beta-hydroxy-4,6-androstadien-3-one (delta 6-testosterone) was used to label the steroid binding domain of rat androgen-binding protein (rABP). After digestion with trypsin, the major radiolabeled peptide was isolated by reverse phase chromatography. The peptide was found to have the following amino acid sequence: Ile Ala Leu Gly Gly Leu Leu Leu Pro Thr Ser. Gaps in the sequence that one would anticipate if delta 6-testosterone formed an adduct with a single amino acid were not encountered. Several different amino acids appear to have been labeled as expected given the free radical nature of photoactivated delta 6-testosterone. The sequence obtained corresponded to a tryptic peptide (amino acids 171-181) of the rABP precursor. The only other protein having this amino acid sequence was human sex hormone binding globulin. The binding domain lies in a hydrophobic pocket that contains a predicted beta-sheet and turn secondary structure, as would be anticipated given the hydrophobic nature of the steroid molecule. A hydropathy and secondary structure analysis of rABP was performed as a basis for discussing the results of the current study in relation to previous studies on the steroid binding domain on human sex hormone binding globulin.