Jeff A. Parrott

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 a...

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.

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/μg DNA) and progesterone (5–30 ng/μg 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. Mol. Reprod. Dev. 55:55–64, 2000.

Expression and Action of Kit Ligand/Stem Cell Factor in Normal Human and Bovine Ovarian Surface Epithelium and Ovarian Cancer

Abstract 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 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 hepatocyte growth factor in human and bovine normal ovarian surface epithelium and ovarian cancer.

Abstract 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.

Expression and Action of Transforming Growth Factor Alpha in Normal Ovarian Surface Epithelium and Ovarian Cancer

Abstract 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 (TGFα) 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 TGFα and EGFR immunostaining. To examine TGFα and EGFR gene expression, RNA was collected from normal bovine OSE and ovarian stromal cells. The TGFα 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 TGFα 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 TGFα 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 TGFα/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 TGFα. 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 TGFα and EGFR expression by the OSE was also examined. Human chorionic gonadotropin stimulated TGFα 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, TGFα, 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.

Expression and action of transforming growth factor beta (TGFβ1, TGFβ2, TGFβ3) in normal bovine ovarian surface epithelium and implications for human ovarian cancer

Abstract 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 (TGFβ) 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 TGFβ isoforms (TGFβ1, TGFβ2, and TGFβ3) 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 TGFβ isoforms and their receptor, transforming growth factor beta receptor type II (TGFβRII), proteins were found to be detected in the OSE from normal bovine ovaries using immunohistochemistry. Ovarian stromal tissue also contained positive immunostaining for TGFβ isoforms and TGFβRII. RNA was collected from normal bovine OSE and ovarian stromal cells to examine TGFβ gene expression. TGFβ1, TGFβ2, and TGFβ3 transcripts were detected in both freshly isolated and cultured bovine OSE and stromal cells by a sensitive quantitative polymerase chain reaction assay. TGFβ1 and TGFβ2 mRNA levels were found to be present at similar levels in freshly isolated OSE and stroma. Interestingly, TGFβ3 mRNA levels were significantly higher in freshly isolated OSE than stromal cells. All but TGFβ3 mRNA in OSE increased when the cells were cultured. Observations indicate that normal bovine OSE and stroma cells express the three TGFβ isoforms in vivo and in vitro. Human ovarian tumors from stage II, stage III and stage IV cases were found to express TGFβ1, TGFβ2, TGFβ3 and TGFβRII protein primarily in the epithelial cell component by immunohistochemistry analysis. The stromal cell component of the human ovarian tumors contained little or no TGFβ or TGFβRII immunostaining. TGFβ actions on bovine OSE and stromal cells were also investigated. TGFβ was found to inhibit the growth of OSE, but not stromal cells. To further examine the actions of TGFβ on OSE, the expression of two growth factors previously shown to be expressed by OSE were analyzed. TGFβ1 was found to stimulate the expression of both keratinocyte growth factor (KGF) and kit ligand/stem cell factor (KL) by bovine OSE. Therefore, TGFβ 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 TGFβ expression by the bovine OSE were examined. Transforming growth factor alpha (TGFα) stimulated TGFβ1 expression, TGFβ1 stimulated TGFβ2 expression, and follicle stimulating hormone (FSH) stimulated TGFβ3 expression. These results demonstrate that TGFβ isoforms are regulated differently by the regulatory agents tested. In summary, all the TGFβ isoforms are differentially expressed by the OSE and TGFβ 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 TGFβ on OSE. Abnormal expression and/or action of TGFβ is postulated to in part be involved in the onset and progression of ovarian cancer.