Conditional inactivation of Miwi2 reveals that MIWI2 is only essential for prospermatogonial development in mice.

The PIWI-piRNA pathway serves as a critical defense mechanism through which the genome of the male germline is protected from invasion by transposable elements (TEs). MIWI2/PIWIL4, a member of the murine PIWI subclade of the Argonaute family, has been shown to be expressed in primordial germ cells (PGCs) and prospermatogonia in fetal and prepubertal testes. Global inactivation of Miwi2 leads to male sterility due to an early meiotic arrest, which correlates with retrotransposon desuppression. However, it remains unclear whether MIWI2 functions beyond the PGC stage and whether MIWI2 has a role beyond TE suppression during male germ line development. Through conditional inactivation of Miwi2, we demonstrate herein that MIWI2 function is restricted to a narrow time window during male PGC reprograming and that Miwi2 is dispensable for postnatal male germline development and testicular function in mice. Moreover, persistent activation of LINE1 and IAP retrotransposons caused by Miwi2 inactivation is compatible with mitotic cell cycle progression of spermatogonia during the first wave of spermatogenesis, but can cause zygotene to pachytene arrest in early meiosis due to multiple defects including enhanced DNA double-strand breaks, aberrant histone modifications and altered mRNA transcriptome. Our data not only validate those from global Miwi2 KO studies, but also suggest that MIWI2 and MIWI2-associated piRNAs have functions beyond TE suppression.

A deficiency of lunatic fringe is associated with cystic dilation of the rete testis.

Lunatic fringe belongs to a family of beta1-3 N-acetyltransferases that modulate the affinity of the Notch receptors for their ligands through the elongation of O-fucose moieties on their extracellular domain. A role for Notch signaling in vertebrate fertility has been predicted by the intricate expression of the Notch receptors and their ligands in the oocyte and granulosa cells of the ovary and the spermatozoa and Sertoli cells of the testis. It has been demonstrated that disruption of Notch signaling by inactivation of lunatic fringe led to infertility associated with pleiotropic defects in follicle development and meiotic maturation of oocytes. Lunatic fringe null males were found to be subfertile. Here, we report that gene expression data demonstrate that fringe and Notch signaling genes are expressed in the developing testis and the intratesticular ductal tract, predicting roles for this pathway during embryonic gonadogenesis and spermatogenesis. Spermatogenesis was not impaired in the majority of the lunatic fringe null males; however, spermatozoa were unilaterally absent in the epididymis of many mice. Histological and immunohistochemical analysis of these testes revealed the development of unilateral cystic dilation of the rete testis. Tracer dye experiments confirm a block in the connection between the rete testis and the efferent ducts. Further, the dye studies demonstrated that many lunatic fringe mutant males had partial blocks of the connection between the rete testis and the efferent ducts bilaterally.

Expression, action, and regulation of transforming growth factor alpha and epidermal growth factor receptor during embryonic and perinatal rat testis development.

The objective of the current study was to extend previous observations and examine the expression pattern and effects of transforming growth factor alpha (TGFalpha) and epidermal growth factor receptor (EGFR) on embryonic testis morphogenesis and growth. The expression of TGFalpha was determined after morphological sex determination (seminiferous cord formation at embryonic day 13 [ED13]) through perinatal testis development (postnatal day 5 [PD5]) with a quantitative reverse transcription-polymerase chain reaction procedure. Expression of messenger RNA (mRNA) for TGFalpha appeared to be more dynamic during testis development when compared with the expression of mRNA for EGFR. Message for TGFalpha was reduced at ED16 and PD4, and was elevated at PD0 during testis development. In contrast, EGFR mRNA levels were negligible at ED15 and were elevated constitutively from ED16 through PD5. Immunohistochemistry was conducted at ED14, ED16, ED19, PD0, PD3, and PD5 to localize cellular expression of both TGFalpha and EGFR. At ED16, positive staining for EGFR was localized to the cords, and by ED19, was mainly in the cords with slight expression in the interstitium. From PD0 to PD5, positive staining for EGFR was detected in the germ, Sertoli, and interstitial cells. Immunohistochemistry for TGFalpha detected localization at ED14 and ED16 to the Sertoli cells and to specific cells in the interstitium. From ED19 through PD5, TGFalpha was detected in the Sertoli, germ, and interstitial cells, and in endothelial cells within the interstitium. To determine the effects of TGFalpha on embryonic testis growth and seminiferous cord formation, ED13 testis organ cultures were treated with sense and antisense TGFalpha oligonucleotides. Antisense TGFalpha inhibited testis growth by 25%-30% in ED13 testis organ cultures when compared with sense oligonucleotide control pairs. To examine the effects of TGFalpha on perinatal testis growth, PD0 testis cultures were treated with different doses of TGFalpha. TGFalpha increased thymidine incorporation into DNA in PD0 testis cultures. Therefore, TGFalpha appears to have actions on both embryonic and perinatal testis growth. The regulation of TGFalpha and EGFR mRNA levels were examined using PD0 testis cultures treated with hormones that stimulate testis growth. Follicle-stimulating hormone (FSH) stimulated (P < .05) and testosterone tended to stimulate (P < .07) mRNA expression of EGFR. Epidermal growth factor stimulation of PD0 testis cultures did not affect levels of mRNA expression for EGFR, but did suppress expression of mRNA for TGFalpha. These results taken together demonstrate that TGFalpha can act to regulate early embryonic and perinatal testis growth. Furthermore, TGFalpha and EGFR expression can be regulated through growth stimulatory hormones such as FSH and testosterone.

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.

Role of the transcriptional coactivator CBP/p300 in linking basic helix-loop-helix and CREB responses for follicle-stimulating hormone-mediated activation of the transferrin promoter in Sertoli cells.

Sertoli cells are the epithelial cells responsible for the onset of pubertal development and the maintenance of spermatogenesis in the adult. Transferrin is one of the major secretory products expressed by differentiated Sertoli cells. Investigation of the transcriptional control of transferrin gene expression provides insight regarding the regulation of Sertoli cell differentiation. The optimal activation of the mouse transferrin promoter (mTf) by FSH requires the synergistic actions of the cAMP response element-binding protein (CREB) binding to the cAMP response element-like proximal region II (PRII) and the basic helix-loop-helix (bHLH) binding to the E-box. Proximal region II alone is sufficient for cAMP-mediated activation. The proximity of the PRII and E-box (220 base pairs apart) suggests the possibility of interaction between CREB and bHLH proteins. Such an interaction can be mediated by transcriptional integrators such as CREB-binding protein (CBP) and/or p300 and may stabilize the binding of trans-acting factors to their respective cis-elements. Such an interaction may also provide a mechanism for cell-specific promoter activation. The hypothesis tested in this study was that CBP/p300 is required for the synergistic activation of the transferrin promoter involving PRII and E-box through the formation of a ternary complex. In the Sertoli cells, both CBP and p300 proteins are expressed. The effect of CBP/p300 on transferrin promoter activation and, hence, Sertoli cell function was studied by using antisense oligonucleotides (AS-oligo). In the presence of CBP/p300 AS-oligo, activity of the FSH-induced mTf-chloramphenicol acetyl transferase (CAT) was significantly lower as compared to the respective controls. Interestingly, AS-oligo had no effect on cAMP-induced activation of the transferrin promoter reporter construct (mTf-CAT). Mutations in the E-box (EB*) significantly reduced the FSH response. The presence of AS-oligo had no further effect on the FSH-mediated activation of the EB*-mTf-CAT construct but reduced cAMP-mediated activation. Mutations in the CRE-like PRII (PRII*) also significantly reduced the FSH response. Activation of the PRII*-mTf-CAT in response to cAMP was completely abolished. The presence of AS-oligo had no further effect on the FSH- or cAMP-mediated activation of the PRII*-mTf-CAT construct. In Sertoli cells, CBP/p300 was coimmunoprecipitated with CREB and the bHLH protein E47. These observations suggest that CBP/p300 appears to be involved in regulating FSH-mediated activation of the transferrin promoter by linking bHLH and CREB activities.

Actions of the endocrine disruptor methoxychlor and its estrogenic metabolite on in vitro embryonic rat seminiferous cord formation and perinatal testis growth.

The current study examines the actions of methoxychlor and its estrogenic metabolite, 2, 2-bis-(p-hydroxyphenyl)-1, 1, 1-trichloroethane (HPTE), on seminiferous cord formation and growth of the developing rat testis. The developing testis in the embryonic and early postnatal period is likely more sensitive to hormonally active agents than at later stages of development. Embryonic day 13 (E13) testis organ cultures were treated with either 0.2, 2, or 20 microM methoxychlor or 1, 3, 6, 15, 30, or 60 microM HPTE to examine effects on cord formation. No concentration of methoxychlor completely inhibited cord formation. However, cord formation was abnormal with the presence of a reduced number of cords and appearance of "swollen" cords at the 2 and 20 microM concentrations of methoxychlor. The swollen cords were due to an increase in the number of cells in a cord cross section and reduction of interstitial cell numbers between cords. Treatment of embryonic day 13 (E13) testes with HPTE caused abnormal cord formation at the 3 microM and 6 microM concentrations, and completely inhibited cord formation at the 15, 30, and 60 microM concentrations. In addition to the estrogenic metabolite HTPE, methoxychlor can also be metabolized into anti-androgenic compounds. Therefore, to determine the spectrum of potential actions of methoxychlor on testis development, different concentrations of estradiol, testosterone, and an anti-androgen (flutamide) were utilized to determine their effects on E13 testis organ culture morphology. Estradiol (1 microM) and flutamide (0.1microM) both inhibited seminiferous cord formation in E13 testis organ cultures. Therefore, methoxychlor may be acting through the androgen and/or estrogen receptors to elicit its actions on seminiferous cord formation. Reverse transcription polymerase chain reaction (PCR) (RT-PCR) confirmed the presence of estrogen receptor alpha (ERalpha) mRNA from embryonic day 14 (E14) through postnatal day 5 (P5) while estrogen receptor beta (ERbeta) mRNA did not appear until approximately E16 of testis development. Androgen receptor (AR) expression was present from E14 through P5 of testis development, but at apparently reduced levels at E14 and E16. Immunohistochemical analysis localized ERalpha to the cells of the seminiferous cords at E14 though P5 while ERbeta was present in cells of the interstitium at E16 and P0. Androgen receptor was localized to germ and interstitial cells. The effects of methoxychlor, HPTE, estradiol, and testosterone on cell growth of perinatal testes was determined with a thymidine incorporation assay in postnatal day zero (P0) testis cell cultures. Methoxychlor (0.002, 0.02, and 0.2 microM) and HPTE (2 and 20 microM) stimulated thymidine incorporation in P0 testis cell cultures in a similar manner to estradiol (0.01, 0.1, and 1 microM). In addition, testosterone (0.1 microM) also stimulated thymidine incorporation in P0 testis cultures. Observations suggest that methoxychlor and its metabolite HPTE can alter normal embryonic testis development and growth. The actions of methoxychlor and HPTE are likely mediated in part through the steroid receptors confirmed to be present in the developing testis.

Hormonal regulation and differential actions of the helix-loop-helix transcriptional inhibitors of differentiation (Id1, Id2, Id3, and Id4) in Sertoli cells.

The testicular Sertoli cells support spermatogenesis by providing a microenvironment and structural support for the developing germ cells. Sertoli cell functions are regulated by the gonadotropin FSH. Sertoli cells become a terminally differentiated nongrowing cell population in the adult. In response to FSH, the Sertoli cells express a large number of differentiated gene products, such as transferrin, which transports iron to the developing germ cells. Previously, members of the basic helix-loop-helix (bHLH) family of transcription factors have been shown to influence FSH-mediated gene expression in Sertoli cells. The functions of the bHLH proteins are modulated by Id (inhibitor of differentiation) proteins, which lack the DNA-binding basic domain. The Id proteins form transcriptionally inactive dimers with bHLH proteins and thus regulate cell proliferation and differentiation. The current study investigated the expression and function of Id proteins in the postmitotic Sertoli cell. Freshly isolated and cultured Sertoli cells coexpress all four isoforms of Id (Id1, Id2, Id3, and Id4), as determined by immunoprecipitation with isoform-specific anti-Id antibodies, RT-PCR, and Northern blot analysis. Id2 and Id3 expression levels seem higher than Id1. Interestingly, the expression of Id4 in Sertoli cells is only detectable after stimulation with FSH or cAMP. The Id1 expression is down-regulated by FSH and cAMP, whereas Id2 and Id3 levels remain unchanged in response to FSH. In contrast, serum induces the expression of Id1, Id2, and Id3. Treatment of Sertoli cells with serum significantly reduces the expression of the larger 4-kb Id4 transcript and promotes the presence of a novel 1.3-kb transcript of Id4. The regulatory role of FSH in the expression of all four isoforms of Id is mimicked by a cAMP analog, suggesting that the actions of FSH are mediated through the protein kinase A pathway. An antisense approach was used to study the functional significance of Id proteins in Sertoli cells. Antisense to Id1 stimulated transferrin promoter activity in a transient transfection assay. Interestingly, an antisense to Id2 down-regulated transferrin promoter activity. Id3 and Id4 antisense oligonucleotides had no effect on FSH-mediated transferrin promoter activation. Contrary to the hypothesis that Id proteins have redundant functions, the results of the current study suggest that Id1, Id2, Id3, and Id4 are differentially regulated and may have distinct functions. Id1 may act to maintain Sertoli cell growth potential, whereas Id2 and Id4 may be involved in the differentiation and hormone regulation of Sertoli cells.

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.

Cellular interactions that control primordial follicle development and folliculogenesis.

Specific factors that mediate local cell--cell interactions in the ovary related to the initiation and progression of follicle development will be discussed. Recently, several factors produced locally by the primordial follicle have been shown to induce primordial follicle development from a quiescent state to promote follicle development. Kit ligand/stem cell factor (KL/SCF) produced by the immature granulosa cells appears to promote theca cell organization. Basic fibroblast growth factor produced predominately by the oocyte, but by all cells at reduced levels, also was found to induce primordial follicle development similar to KL. It is likely that numerous locally produced factors will mediate cellular interactions and interact between each other to control the induction of primordial follicle development and influence processes such as the onset of puberty and menopause. After follicle development has been induced, theca cells and granulosa cells interact through classical mesenchymal-epithelial type interactions to influence the progression of follicle development. Mesenchymally derived theca cells have been shown to produce transforming growth factor alpha (TGF-alpha), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and transforming growth factor beta to regulate granulosa cell growth and function. The epithelial granulosa cells have been show to produce KL/SCF that can feed back on the theca cells to regulate theca cell growth and stimulate the production of the theca cell factors (TGF-alpha, KGF, and HGF). Therefore, a positive feedback loop between the theca cells and granulosa cells appears to exist to promote the dramatic cell growth required during folliculogenesis. Interestingly, hormones such as estrogen and gonadotropins stimulate the expression of these paracrine growth factors. Therefore, the actions of hormones to stimulate follicle development and growth are mediated in part through altering these local cell--cell interactions. In summary, the locally produced paracrine factors that mediate cell-cell interactions involved in primordial follicle development and the progression of follicle development during folliculogenesis are starting to be elucidated.

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 neurotropin-3 and nerve growth factor in embryonic and early postnatal rat testis development.

The current study examines the expression and potential actions of neurotropin-3 (NT3), nerve growth factor (NGF), and their receptors during morphological sex determination (seminiferous cord formation) and perinatal rat testis development. The expression of neurotropins and their receptors was analyzed with immunohistochemistry. Cellular localization of neurotropin ligand and receptor proteins changed during embryonic testis development. Neurotropin-3 was localized to Sertoli cells at Embryonic Day 14 (E14), was present in gonocytes at Postnatal Day 0 (P0), and after birth became localized to the interstitium and Sertoli cells (P3-P5). The expression of trk C (the high affinity receptor for NT3) was localized to mesonephric ducts and cells surrounding the cords (E14-E18). In addition, Sertoli cells and preperitubular cells surrounding the cords at E14 also stained for trk C. Neurotropin-3 was expressed in gonocytes and Sertoli cells at P0-P5. Nerve growth factor was detected in Sertoli cells at E14, was clearly in Sertoli and interstitial cells at E16 and E18, and in Sertoli, germ, and interstitial cells from P0-P5. The expression of trk A (the high affinity receptor for NGF) was located in Sertoli and interstitial cells at E16-P5. To determine the actions of neurotropins during embryonic and perinatal testis development, experiments were conducted on E13 and P0 testis. Antisense oligonucleotide experiments with NT3 were used on E13 testis organ cultures to determine effects on seminiferous cord formation. Cord formation was inhibited in 40% of the organ cultures treated with the antisense NT3 oligonucleotides, while no inhibition was observed with sense oligonucleotides. In P0 testis cultures, both NT3 and NGF alone and in combination stimulated thymidine incorporation into DNA. Therefore, the neurotropins are involved in embryonic morphological events (cord formation; NT3) and in growth of the perinatal testis (P0; NT3 and NGF). To define further the growth effects of neurotropins on testis development, expression of transforming growth factor alpha and beta (TGF alpha and TGF beta) were examined in response to neurotropins. The P0 testis cultures were treated with neurotropins, and expression of mRNA for TGF alpha and TGF beta was analyzed utilizing a quantitative reverse transcription-polymerase chain reaction assay. Nerve growth factor and NT3 alone or in combination inhibited expression of mRNA for TGF alpha while NT3 increased mRNA expression of epidermal growth factor receptor. The combination treatment of neurotropins inhibited expression of TGF beta 1 and increase expression of TGF beta 3. In summary, observations suggest that NT3, NGF, trk A, and trk C are localized to cells critical to seminiferous cord formation and appear to be important regulators of morphological sex determination. In addition to these morphological effects, both NT3 and NGF stimulate P0 testis growth and may elicit their action through altering the expression of locally produced growth factors such as TGF alpha and TGF beta. Taken together these results suggest that neurotropins are regulators of paracrine cell-cell interactions that result in morphological sex determination and perinatal testis growth.

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.

Characterization of a novel transcript of 14-3-3 theta in Sertoli cells.

The isoforms of the highly conserved and ubiquitously expressed 14-3-3 family of proteins function primarily as adapters that modulate interactions between components of various cellular signaling and cell cycle regulatory pathways. Low levels of 14-3-3 isoforms appear to be expressed in most tissues, but specific isoforms or combinations have been shown to be overexpressed in a cell-specific manner. In the present study we show that the theta isoform of 14-3-3 is expressed in Sertoli cells. Although previous reports have shown the presence of a 14-3-3 theta isoform in mouse testicular germ cells, this report demonstrates the presence of the 14-3-3 theta isoform in rat Sertoli cells. The 14-3-3 theta isoform isolated from rat Sertoli cells appears to have a truncated 3' UTR, which makes the transcript shorter by 244 bp, compared with its brain counterpart. Northern blot analysis suggests that the 14-3-3 theta isoform may also be present in other testicular cell types and tissues. The truncation of the 3' UTR suggests a potential role in regulating cell-specific expression of 14-3-3 theta. The expression of 14-3-3 theta in Sertoli cells was confirmed by Northern blot, polymerase chain reaction, Western blot, and immunocytochemical analysis. The levels of 14-3-3 theta mRNA and protein in Sertoli cells remained unchanged in response to the gonadotropin, FSH. Consistent with the absence of the effect of FSH on the expression of 14-3-3 theta, an antisense oligonucleotide to 14-3-3 theta had no effect on FSH-induced activation of the transferrin promoter in Sertoli cells. The widespread expression of 14-3-3 theta in testis and the lack of effect of FSH on levels of its expression suggest that 14-3-3 theta influences Sertoli cell function in an FSH-independent manner.

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.

Role of winged helix transcription factor (WIN) in the regulation of Sertoli cell differentiated functions: WIN acts as an early event gene for follicle-stimulating hormone.

Members of the winged helix transcription factor family are known to regulate epithelial cell differentiation by regulating cell-specific gene expression. rWIN is a newly discovered member of the winged helix family shown to be present in the adult rat testis. In the testis the human homolog of rWIN, HFH-11, was localized to the germ cells (i.e. spermatocytes and spermatids) undergoing spermatogenesis. In the present study we show that rWIN is also expressed in testicular Sertoli cells. Sertoli cells are the epithelial component of the seminiferous tubule and provide both the cytoarchitectural support and the microenvironment for developing germ cells. The presence of rWIN in Sertoli cells was confirmed by Northern blot and RT-PCR analysis. The rWIN transcript size in the Sertoli cells was different from the germ cell transcript that is probably due to alternative splicing or modifications of the 3'-untranslated region. At least two spliced variants of rWIN were observed in the Sertoli cells corresponding to the deletion of an exon in the DNA-binding region. Long term stimulation of cultured Sertoli cells with the gonadotropin FSH down-regulated rWIN expression. In contrast, short-term stimulation (2 h) transiently up-regulated rWIN expression. The FSH-induced transient stimulation of rWIN precedes expression of the transferrin gene that is a marker of Sertoli cell differentiation. FSH-induced transferrin promoter activity was inhibited when cultured Sertoli cells were treated with an antisense oligonucleotide to rWIN. Interestingly, the constitutive overexpression of the DNA-binding domain of rWIN also down-regulated transferrin promoter activity. Analysis of the transferrin promoter with various deletion mutations suggested that rWIN acts at an upstream gene of the transferrin promoter. The results indicate that a transient up-regulation of rWIN in part mediates the ability of FSH to activate the transferrin promoter, which can be inhibited with a rWIN antisense oligonucleotide or constitutive expression of the rWIN DNA-binding domain. The current study demonstrates that rWIN acts as an early event gene for FSH actions on Sertoli cells and that rWIN appears to have a role in the regulation of Sertoli cell differentiated functions.

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.

Role of transforming growth factor-alpha and the epidermal growth factor receptor in embryonic rat testis development.

Embryonic testis development requires the morphogenesis of cords and growth of all cell populations to allow organ formation. It is anticipated that coordination of the growth and differentiation of various cell types involves locally produced growth factors. The current study was an investigation of the hypothesis that transforming growth factor-alpha (TGF-alpha) is involved in regulating embryonic testis growth. TGF-alpha has previously been shown to function in the postnatal testis. TGF-alpha and other members of the epidermal growth factor (EGF) family act through the epidermal growth factor receptor (EGFR) to stimulate cell proliferation and tissue morphogenesis. To understand the potential actions of TGF-alpha in the embryonic testis, general cell proliferation was investigated. Characterization of cell proliferation in the rat testis throughout embryonic and postnatal development indicated that each cell type has a distinct pattern of proliferation. Germ cell growth was transiently suppressed around birth. Interstitial cell growth was high embryonically and decreased to low levels around birth. A low level of Sertoli cell proliferation was observed at the onset of testis cord formation. Sertoli cell proliferation in early embryonic development was low; the levels were high later in embryonic development and remained high until the onset of puberty. Both TGF-alpha and the EGFR were shown to be expressed in the embryonic and postnatal rat and mouse testis. Perturbation of TGF-alpha function using neutralizing antibodies to TGF-alpha on testis organ cultures dramatically inhibited the growth of both embryonic and neonatal testis. TGF-alpha antibodies had no effect on cord formation. The TGF-alpha antibody was found to be specific for TGF-alpha in Western blots when compared to EGF and heregulin. Testis growth was also inhibited by perturbation of EGFR signaling using an EGFR kinase inhibitor. Therefore, TGF-alpha appears to influence embryonic testis growth but not morphogenesis (i.e., cord formation). Treatment of embryonic testis organ cultures with exogenous TGF-alpha also perturbed development, leading to an increased proliferation of unorganized cells. Testis from EGFR and TGF-alpha knockout mice were analyzed for testis morphology. TGF-alpha knockout mice had no alterations in testis phenotype, while EGFR knockout mice had a transient decrease in the relative amount of interstitial cells before birth. Observations suggest that there may be alternate or compensatory factors that allow testis growth to occur in the apparent absence of TGF-alpha actions in the mutant mice. In summary, the results obtained suggest that TGF-alpha is an important factor in the regulation of embryonic testis growth, but other factors will also be involved in the process.