Human oocyte maturation is dependent on LH-stimulated accumulation of the epidermal growth factor-like growth factor, amphiregulin.

BACKGROUND: The LH surge promotes ovulation via activation of multiple signaling networks in the ovarian follicle. Studies in animal models have shown the importance of LH-induced activation of the epidermal growth factor (EGF)signaling network in critical peri-ovulatory events. We investigated the biological significance of regulatory mechanisms mediated by EGF-like growth factors during LH stimulation in humans. METHODS: We characterized the EGF signaling network in mature human ovarian follicles using in vivo and in vitro approaches. Amphiregulin (AREG) levels were measured in 119 follicular fluid (FF) samples from IVF/ICSI patients. Biological activity of human FF was assessed using in vitro oocyte maturation, cumulus expansion and cell mitogenic assays. RESULTS: AREG is the most abundant EGF-like growth factor accumulating in the FF of mature follicles of hCG-stimulated patients. No AREG was detected before the LH surge or before hCG stimulation of granulosa cells in vitro, demonstrating that the accumulation of AREG requires gonadotrophin stimulation. Epiregulin and betacellulin mRNA were detected in both human mural and cumulus granulosa cells, although at significantly lower levels than AREG. FF from stimulated follicles causes cumulus expansion and oocyte maturation in a reconstitution assay. Immunodepletion of AREG abolishes the ability of FF to stimulate expansion (P < 0.0001) and oocyte maturation (P < 0.05), confirming the biological activity of AREG. Conversely, mitogenic activity of FF remained after depletion of AREG, indicating that other mitogens accumulate in FF. FF from follicles yielding an immature germinal vesicle oocyte or from an oocyte that develops into an aberrant embryo contains lower AREG levels than that from follicles yielding a healthy oocyte (P = 0.008). CONCLUSIONS: EGF-like growth factors play a role in critical peri-ovulatory events in humans, and AREG accumulation is a useful marker of gonadotrophin stimulation and oocyte competence.

Binding of fatty acids facilitates oxidation of cysteine-34 and converts copper-albumin complexes from antioxidants to prooxidants.

As a transition metal capable of undergoing one-electron oxidation-reduction conversions, copper (Cu) is essential for life and fulfills important catalytic functions. Paradoxically, the same redox properties of copper can make it extremely dangerous because it can catalyze production of free radical intermediates from molecular oxygen. Factors involved in regulation of redox activity of albumin-bound copper have not been well characterized. In the present study, effects of modification of the albumin cysteine-34 (Cys-34) and binding of nonesterified fatty acids on the redox-cycling activity of the complex of copper with human serum albumin (Cu/HSA) were studied. Because ascorbate is the most abundant natural reductant/scavenger of free radicals in blood plasma, the electron paramagnetic resonance assay of ascorbate radical formation was used as a method to monitor Cu/HSA redox-cycling activity. At Cu/HSA ratios below 1:1, the bound Cu was virtually redox inactive, as long as Cys-34 was in reduced state (Cu/HSA-SH). Alkylation, nitrosylation, or oxidation of Cu/HSA resulted in the appearance of redox-cycling activity. Experiments with ultrafiltration of Cu/HSA alkylated with N-ethylmaleimide (Cu/HSA-NEM) showed that at Cu/HSA-NEM ratios below 1:1, the ascorbate radicals were produced by Cu tightly bound to HSA rather than by Cu released in solution. The rate of ascorbate radical production in HSA-NEM and S-nitrosylated HSA (HSA-NO) was, however, more than one order of magnitude lower than that in a solution containing equivalent concentration of free copper ions. While Cu/HSA-SH was redox inactive, binding of oleic or linoleic acids induced Cu-dependent redox-cycling with maximal activity reached at a fatty acid to protein molar ratio of 3:1 for oleic acid and 2:1 for linoleic acid. Binding of fatty acids caused profound conformational changes and facilitated oxidation of the Cys-34 SH-group at essentially the same ratios as those that caused redox-cycling activity of Cu/HSA. We conclude that fatty acids regulate anti-/prooxidant properties of Cu-albumin via controlling redox status of Cys-34.

Developmental gonadal expression of the transcription factor SET and its target gene, P450c17 (17alpha-hydroxylase/c17,20 lyase).

Cytochrome P450c17 catalyzes the 17alpha-hydroxylase/17,20 lyase activity needed for sex steroid synthesis. We recently characterized the nuclear phosphoprotein SET as a novel transcriptional regulator that binds to the -447/-399 region of the rat P450c17 gene, along with the transcription factors COUP-TF II, NGF-IB, and SF-1. Gel shift studies localized SET binding to nucleotides -410/-402. We have shown that SET activates transcription of the rat P450c17 gene in neuronal precursor cells and now show that it also activates transcription from the -418/-399 region of the rat P450c17 gene in mouse Leydig MA-10 cells. Studying the ontogenic expression of SET and P450c17 in the rodent gonad, we found that SET expression preceded P450c17 expression in the embryonic genital ridge, suggesting that SET may be important for initiating P450c17 expression in this region. Expression of SET also preceded P450c17 expression in the testis and ovary, and its expression was much greater during embryogenesis than in the adult gonad. In the adult rat testis, P450c17 was expressed only in Leydig cells, while SET was expressed in Leydig cells and in spermatocytes. In the adult rat ovary, P450c17 was expressed only in theca cells, while SET was expressed in theca cells and also in oocytes. Because SET is expressed early in development in the genital ridge and in the testis and ovary, and because SET has many functions in addition to its activity as a transcription factor, we determined whether SET acts a transcription factor in oocytes. The SET protein was detected by Western blots in Xenopus oocytes from stages II through VI and in mature oocytes. Using extracts of Xenopus oocytes in gel shift assays, we detected a protein that bound to the -418/-399 region of the rat P450c17 gene, to which SET binds. Nuclear injection of either a -418/-399TK32LUC wildtype reporter construct or a construct containing a mutant SET site into Xenopus oocytes from stages III through VI resulted in activation of luciferase activity with the wildtype but not the mutant construct in all stages. These data suggest that Xenopus SET is able to bind to specific DNA sequences to activate transcription at all stages of Xenopus oogenesis. These data indicate that SET is an evolutionarily conserved transcription factor that participates in the early ontogenesis of the gonadal system, regulates P450c17 gene transcription in Leydig cells, and may also activate other genes expressed in immature oocytes, thus playing a role in oocyte development.

Histocompatibility leukocyte antigen-G is not expressed by endometriosis or endometrial tissue.

OBJECTIVE: The immunological mechanisms that support persistence and proliferation of ectopic endometrial implants within the peritoneal cavity of women with endometriosis are unknown. Inhibition of natural killer (NK) and cytotoxic T-cell function has been proposed as a mechanism. We tested the hypothesis that expression of a nonclassical major histocompatibility antigen, HLA-G, might explain the local immunosuppression associated with ectopic endometrium. DESIGN: Nested case-control study of women with and without laparoscopic evidence of endometriosis. SETTING: Reproductive endocrinology clinic at a university hospital. PATIENT(S): Peritoneal fluid specimens from 10 women with revised AFS stage I-IV endometriosis and from 10 age-matched normal controls without laparoscopic evidence of endometriosis were tested for the presence of HLA-G protein. Endometriosis and normal endometrial biopsies from four patients were used to prepare stromal cell cultures directly evaluated for HLA-G protein. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The expression of HLA-G in peritoneal fluid, tissue, and cell cultures was determined by immunoblotting with a specific monoclonal antibody. RESULT(S): HLA-G protein was not detectable in peritoneal fluid specimens of endometriosis patients or controls. Moreover, ectopic and normal endometrial tissues and stromal cells did not express HLA-G. CONCLUSION(S): Immune cell inhibition in endometriosis must be mediated by factors other than HLA-G.

Purification and characterization of an immunomodulatory endometrial protein, glycodelin.

Human glycodelin is synthesized by endometrial cells in the late secretory phase and early pregnancy under hormonal regulation. Whereas the precise physiological functions of glycodelin are unknown, its expression during embryonic nidation and its inhibition of T cell proliferation suggest an immunomodulatory role. We purified human glycodelin from first trimester human decidual cytosol by using a rapid two-step high-performance liquid chromatography method and investigated its effects on human monocyte migration. Human U937 cells were used as a model of monocyte chemotaxis in Boyden chamber migration assays. N-Formyl-Met-Leu-Phe and the beta-chemokine RANTES (regulated on activation normal T cell expressed and secreted) were used as monocyte chemoattractants. Purified glycodelin inhibited monocyte migration in a dose-dependent fashion (IC50 = 550 nm). Glycodelin activity was totally reversed by heat inactivation (95 degrees C x 15 min) and neutralized by pretreatment with specific anti-glycodelin antibodies. Deglycosylated glycodelin was equipotent to intact glycodelin in the monocyte migration assay. 125I-Glycodelin binding to whole U937 cells revealed a single, saturable site with a Kd = 48 +/- 21 nm by Scatchard analysis. Cross-linking studies indicated that glycodelin binds to a high molecular mass (approximately 250 kDa) protein complex at the monocyte cell surface. Our findings support the hypothesis that glycodelin reduces the local maternal inflammatory response toward the implantation of a semiallogeneic conceptus.

Regulation of vascular endothelial growth factor (VEGF) gene transcription by estrogen receptors alpha and beta.

Vascular endothelial growth factor (VEGF) mediates angiogenic activity in a variety of estrogen target tissues. To determine whether estrogen has a direct transcriptional effect on VEGF gene expression, we developed a model system by transiently transfecting human VEGF promoter-luciferase reporter constructs into primary human endometrial cells and into Ishikawa cells, derived from a well-differentiated human endometrial adenocarcinoma. In primary endometrial epithelial cells, treatment with 17beta-estradiol (E(2)) resulted in a 3.8-fold increase in luciferase activity, whereas a 3. 2-fold induction was demonstrated for stromal cells. Our Ishikawa cells had less than 100 functional estrogen receptors (ER)/cell and were therefore cotransfected with expression vectors encoding either the alpha- or the beta-form of the human ER. In cells cotransfected with ERalpha, E(2) induced 3.2-fold induction in VEGF-promoter luciferase activity. A 2.3-fold increase was observed in cells cotransfected with ERbeta. Through specific deletions, the E(2) response was restricted to a single 385-bp PvuII-SstI fragment in the 5' flanking DNA. Cotransfection of this upstream region with a DNA binding domain ER mutant, or site-directed mutagenesis of a variant ERE within this fragment, resulted in the loss of the E(2) response. Electromobility shift assays demonstrated that this same ERE sequence specifically binds estradiol-ER complexes. These studies demonstrate that E(2)-regulated VEGF gene transcription requires a variant ERE located 1.5 kb upstream from the transcriptional start site. Site-directed mutagenesis of this ERE abrogated E(2)-induced VEGF gene expression.

Novel role for the nuclear phosphoprotein SET in transcriptional activation of P450c17 and initiation of neurosteroidogenesis.

Neurosteroids are important endogenous regulators of gamma-aminobutryic acid (GABA(A)) and N-methyl-D-aspartate (NMDA) receptors and also influence neuronal morphology and function. Neurosteroids are produced in the brain using many of the same enzymes found in the adrenal and gonad. The crucial enzyme for the synthesis of DHEA (dehydroepiandrosterone) in the brain is cytochrome P450c17. The transcriptional strategy for the expression of P450c17 is clearly different in the brain from that in the adrenal or gonad. We previously characterized a novel transcriptional regulator from Leydig MA-10 cells, termed StF-IT-1, that binds at bases -447/-399 of the rat P450c17 promoter, along with the known transcription factors COUP-TF (chicken ovalbumin upstream promoter transcription factor), NGF-IB (nerve growth factor inducible protein B), and SF-1 (steroidogenic factor-1). We have now purified and sequenced this protein from immature porcine testes, identifying it as the nuclear phosphoprotein SET; a role for SET in transcription was not established previously. Binding of bacterially expressed human and rat SET to the DNA site at -418/-399 of the rat P450c17 gene transactivates P450c17 in neuronal and in testicular Leydig cells. We also found SET expressed in human NT2 neuronal precursor cells, implicating a role in neurosteroidogenesis. Immunocytochemistry and in situ hybridization in the mouse fetus show that the ontogeny and distribution of SET in the developing nervous system are consistent with SET being crucial for initiating P450c17 transcription. SET's developmental pattern of expression suggests it may participate in the early ontogenesis of the nervous, as well as the skeletal and hematopoietic, systems. These studies delineate an important new factor in the transcriptional regulation of P450c17 and consequently, in the production of DHEA and sex steroids.

Effects of progestins and relaxin on glycodelin gene expression in human endometrial cells.

OBJECTIVE: Glycodelin is an endometrial protein proposed to play an important role in embryonic implantation. We examined the effects of progestins and relaxin on glycodelin transcription, synthesis, and secretion. STUDY DESIGN: Northern blotting, metabolic labeling, and fluorography were used to assess glycodelin messenger ribonucleic acid and protein synthesis in endometrial tissue and cells. Luciferase reporter constructs transfected into endometrial adenocarcinoma cells (Ishikawa cells) were used to determine whether progestins or relaxin could activate the glycodelin gene promoter. RESULTS: Progestins but not relaxin stimulated glycodelin secretion in primary epithelial cell cultures. A 452-base pair fragment of the glycodelin gene promoter was activated 4.3 +/- 0.7 times normal by 10-nmol/L promegestone; however, addition of relaxin to the same construct repressed progestin-stimulate promoter activation by >30%. CONCLUSION: Glycodelin transcription, synthesis, and secretion by endometrial epithelial cells were stimulated by progestins. However, relaxin failed to stimulate production of this immunomodulatory protein and, in fact, repressed progestin-stimulated activation of the glycodelin gene promoter.

Glycodelin: a pane in the implantation window.

Glycodelin is an endometrial protein with proposed immunomodulatory activity during human embryonic nidation. In this review we describe the effects of ovarian hormones on glycodelin transcription, synthesis, and secretion by human epithelial cells and focus on the importance of glycodelin in implantation. We demonstrate that glycodelin transcription, synthesis, and secretion by human epithelial cells are stimulated by progestins and antiprogestins but not by estrogen. Sequences localized within a 403-base-pair region flanking the 5' human glycodelin gene promoter appear to be responsible for transcriptional activation of this gene mediated by progesterone receptor-ligand complexes. Relaxin, purported to enhance glycodelin production in vivo and in prior in vitro studies, had no stimulatory effect on the expression of this gene in vitro in our models.

Promegestone (R5020) and mifepristone (RU486) both function as progestational agonists of human glycodelin gene expression in isolated human epithelial cells.

One of the most abundant protein products of human secretory endometrium is glycodelin, a glycoprotein previously referred to as PP14. Although the precise function of this protein is unknown, its unique glycosylation pattern is believed to affect immunomodulatory activity during human embryonic implantation and inhibition of sperm-egg binding after ovulation. Having confirmed the expression of glycodelin in secretory endometrial glands, we used purified endometrial epithelial cell cultures to demonstrate the hormonal regulation of glycodelin synthesis and secretion. The findings were corroborated by transiently transfecting glycodelin gene promoter-reporter constructs into human epithelioid HeLa and Ishikawa cells. Our results indicate that glycodelin protein production by endometrial epithelial cells is directly up-regulated 4- to 9-fold by progestins and antiprogestins in vitro. Transcriptional regulation of the glycodelin gene promoter expressed in HeLa cells is progesterone receptor-dependent. As observed in the primary endometrial cells, progestins and antiprogestins both act as agonists on the in vitro expression of this endometrial gene. Our findings provide insight into the regulation of this abundant endometrial protein and raise interesting questions about the physical nature of the interaction of agonist- and antagonist-bound progesterone receptors with the glycodelin gene promoter.

Vectorial secretion of vascular endothelial growth factor by polarized human endometrial epithelial cells.

OBJECTIVE: To analyze directional vascular endothelial growth factor (VEGF) secretion in polarized human endometrial epithelial cell cultures. VEGF has distinct distribution patterns in human endometrium. Stromal cells are diffusely positive for VEGF messenger RNA and protein, whereas glandular epithelium shows focal VEGF immunostaining at the apical surface. The epithelial distribution suggests that VEGF is secreted into gland lumina, potentially influencing the nutrition and/or apposition of the developing blastocyst. DESIGN: Controlled in vitro study of protein secretion by polarized endometrial epithelial cells established on polyethylene filters. SETTING: University hospital. PATIENT(S): Endometrial biopsies were obtained from healthy, ovulatory women undergoing elective surgery. INTERVENTION(S): Primary endometrial epithelial cells were cultured. MAIN OUTCOME MEASURE(S): VEGF mRNA and protein production were measured in polarized cells. The vectorial secretion of VEGF was determined. RESULT(S): VEGF production by endometrial epithelial cells was verified by Northern blotting and immunoassays of conditioned media. The mean (+/-SD) apical secretion of VEGF was 3.9 +/- 1.4 ng per 10(5) cells every 48 hours and the mean (+/-SD) basal secretion was 0.8 +/- 0.2 ng per 10(5) cells every 48 hours. In contrast, the apical and basal secretion of a soluble cellular isoform of fibronectin were 2.76 +/- 0.96 ng per 10(5) cells every 48 hours and 2.64 +/- 1.79 ng per 10(5) cells every 48 hours, respectively. CONCLUSION(S): VEGF is secreted preferentially into the lumina of endometrial glands. Apical VEGF may be an endometrial signal for blastocyst development or implantation.

Eicosanoid secretion by human endothelial cells exposed to normal pregnancy and preeclampsia plasma in vitro.

Eicosanoids play an important role in the pathogenesis of preeclampsia. The major eicosanoid metabolite reported to be secreted by endothelial cells, the vasodilator prostacyclin, is generally reduced in preeclampsia. By contrast, it was shown previously that prostacyclin secretion by cultured human umbilical vein endothelial (HUVE) cells is increased significantly after exposure to blood from preeclamptic women. In the current study, eicosanoid profiles in conditioned media from HUVE cells incubated with pregnancy plasma were analyzed by high-performance liquid chromatography, thin layer chromatography and quantitative radio- and enzyme immunoassays. More prostaglandin F2alpha, prostacyclin and 8-isoprostane were secreted after exposure to plasma from preeclamptic women than plasma from matched, normal pregnant patients. Predominant secretion of the vasoconstrictor prostaglandin F2alpha by HUVE cell cultures and a stimulatory effect of preeclampsia plasma on eicosanoid biosynthesis underscore the importance of bioactive lipids in the vasospasm associated with clinical preeclampsia.

Polyribonucleotide phosphorylase is a double-stranded DNA-binding protein.

Polyribonucleotide phosphorylase (PNPase) is one of the critical components of the E. coli RNA degradosome, which consists of both PNPase and endoribonuclease RNase E. The function of this complex is to control the rate of mRNA degradation. The PNPase possesses two enzymatic activities, namely 3'-5' processive exoribonuclease activity and 5'-3' RNA polymerase activity. In the present study, we used conventional chromatography to purify an E. coli protein that binds to a specific double-stranded DNA sequence. Microsequencing of the purified protein showed that this DNA-binding protein was PNPase. Our data further demonstrate that PNPase binds to DNA in a sequence-specific manner. These data suggest that PNPase may have previously unappreciated DNA-related functions in addition to its known role in mRNA degradation.

Elevated nonesterified fatty acid concentrations in severe preeclampsia shift the isoelectric characteristics of plasma albumin.

We previously hypothesized that the endothelial cell dysfunction observed in women with preeclampsia might be caused by an imbalance between circulating very low density lipoproteins and a cytoprotective pI 5.6 isoform of albumin, referred to as toxicity preventing albumin (TxPA). An accurate simplified method was developed to quantify TxPA in small volumes of pregnancy plasma by gel electrofocusing. This assay revealed that circulating TxPA concentrations in women with severe preeclampsia were significantly reduced compared to those in normal pregnant women and women with benign transient hypertension of pregnancy. Nonesterified fatty acids (NEFA) and triglycerides were elevated in plasma from women with severe preeclampsia compared to those in plasma from the two control groups. The inverse correlation between TxPA and NEFA values led us to analyze the NEFA bound to plasma albumin. Gas chromatography and mass spectrometry demonstrated no qualitative differences in the specific fatty acids bound to plasma albumin in severe preeclamptic and normal pregnant women. However, the quantity of NEFA bound to albumin was greater in preeclampsia plasma (2.5 mol NEFA/mol albumin) compared to that in normal pregnancy plasma (0.8 mol NEFA/mol albumin), accounting for the acidic pI shift observed in albumin from the former patients. Functional assays demonstrated that human very low density lipoprotein particles were toxic to human umbilical vein endothelial cells in vitro, but this toxicity was prevented by the addition of TxPA albumin to the culture medium.

Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells.

Retrograde menstruation is postulated as the initiating event in the histogenesis of endometriosis; however, subsequent steps in the pathogenesis of this common disorder remain poorly characterized. The ip accumulation of activated leukocytes and the infiltration of endometriosis lesions by macrophages and T cells are cytological markers of the inflammatory nature of this syndrome. The apparent recruitment of these leukocytes prompted us to search for chemokine expression by endometriosis cells. We reported previously that pelvic fluid RANTES (regulated upon activation, normal T cell expressed and secreted) concentrations correlated with the stage of endometriosis. In the current study, RANTES messenger ribonucleic acid (mRNA) was identified in normal endometrium and endometriosis lesions, and techniques were developed to localize RANTES protein within these tissues. Using isolated endometrial and endometriosis cell cultures, we demonstrated that RANTES mRNA and protein can be induced by the proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma in endometrial stromal, but not in epithelial or adenocarcinoma cells. Immunocytochemical studies confirmed the biochemical findings. Metabolic labeling experiments verified that nascent RANTES secreted by cytokine-stimulated endometriosis stromal cells was the mature, 8-kDa protein predicted by the mRNA encoding this chemokine. The results indicate that RANTES is a normal constituent of the eutopic endometrium. We propose that secretion of RANTES by ectopic endometriosis implants provides a mechanism for peritoneal leukocyte recruitment.

Characterization of bovine ovarian surface epithelium and stromal cells: identification of secreted proteins.

The majority of ovarian cancers are derived from a single layer of epithelial cells that covers the surface of the ovary termed the ovarian surface epithelium (OSE). Ovarian surface stromal cells underlie the OSE and have a morphology distinct from that of the interstitial stromal cells between follicles. Because of the similarities between bovine and human ovarian physiology, and to allow an adequate supply of tissue and cells, bovine OSE and stromal cell cultures were established to investigate the cell biology of these cell types. Morphological analysis of bovine ovarian sections revealed that several layers of ovarian surface stromal cells can be identified and are structurally distinct from interstitial stromal cells. Both OSE and stromal cells can be isolated and cultured for weeks in the absence of presence of serum. The cell populations were found, through use of a keratin immunocytochemical stain for OSE, to be highly purified. To investigate the functional properties of the two cell types, radiolabeled secreted proteins were collected and electrophoretically analyzed. The radiolabeled secreted protein profiles of OSE and stromal cells were found to be distinct with a discrete number of secreted proteins. Major OSE secretory products were obtained from serum-free concentrated conditioned medium, electrophoretically separated, blotted, and sequenced. Two OSE secretory products of 28 kDa and 40 kDa were sequenced and found to match a sequence in the computerized database. The 28-kDa OSE protein was identified as a tissue inhibitor of metalloproteinase, TIMP. The 40-kDa OSE protein was identified as the insulin-like growth factor (IGF) binding protein-2 (IGFBP2).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Regulation of Sertoli cell differentiation by the testicular paracrine factor PModS: analysis of common signal transduction pathways.

In the testis the mesenchymally derived peritubular cells produce a paracrine factor, PModS, that mediates mesenchymal-epithelial interactions and modulates Sertoli cell functions essential for the process of spermatogenesis. PModS has a more dramatic effect on Sertoli cell differentiated functions in vitro than any regulatory agent previously shown to influence the cells, including FSH. The current study initiates an investigation of the pharmacology of PModS through an analysis of several common signal transduction pathways. PModS was found to stimulate cGMP levels in Sertoli cells and maintain elevated levels for up to 5 days in culture. PModS had no influence on cAMP levels. In contrast, FSH stimulated cAMP, but had no influence on cGMP levels. For comparison, an agent known to influence cGMP levels, atrial naturetic factor (ANF), was used to treat Sertoli cells. ANF caused a dramatic increase in Sertoli cell cGMP levels within minutes of treatment, but did not maintain elevated cGMP levels after a 72-h treatment. Although ANF increased guanylate cyclase in whole Sertoli cell homogenates and particulate fractions, PModS did not directly influence guanylate cyclase activity. As previously shown, PModS stimulates transferrin expression as a marker of Sertoli cell differentiated function. Agents that elevate cellular cGMP, including ANF, sodium nitroprusside, and 8-bromo-cGMP, did not influence Sertoli cell transferrin expression. In addition, these agents did not influence the actions of PModS or FSH. Therefore, cGMP does not appear to directly mediate the actions of PModS. As an alternative signal transduction pathway, calcium mobilization and inositol phosphate (IP) metabolism were examined. PModS did not alter calcium uptake or intracellular calcium mobilization. PModS also did not influence the levels of inositol mono-, bis-, or trisphosphates, whereas calf serum did stimulate levels of all three IP metabolites in Sertoli cells. Therefore, PModS does not appear to act through a mobilization of calcium or increased metabolism of IP. A final signal transduction pathway involving phosphorylation was also examined. PModS treatment was found to increase tyrosine phosphorylation of specific proteins in a crude Sertoli cell cytosol preparation. Genistein is an inhibitor of tyrosine kinases and was found to reduce PModS actions at a 3.7-microM concentration of genistein and inhibit PModS actions at a 37-microM concentration of genistein. Therefore, PModS may act through a tyrosine phosphorylation event that remains to be elucidated. Combined observations indicate that PModS does not use cyclic nucleotides, calcium mobilization, or IP metabolism as a signal transduction pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Steroidogenic adrenocortical cell lines produced by genetically targeted tumorigenesis in transgenic mice.

Studies of adrenal steroidogenesis have been facilitated by the availability of immortalized mouse adrenocortical Y-1 cells. We sought to make new, alternative mouse steroidogenic cell lines by genetically targeted tumorigenesis. Transgenic mice were constructed expressing both the SV40 T-antigen and a bacterial neomycin-resistance gene under the control of the promoter for the human P450 cholesterol side-chain cleavage (P450scc) gene, which encodes the first and rate-limiting enzyme in steroidogenesis. Two female transgenic mice expressed T-antigen in various nonsteroidogenic tissues but generated tumors only in the adrenals, suggesting adrenal tumor formation was an early event. Ovarian tissues, which, unlike the adrenal, do not make steroids in fetal or early postnatal life, did not develop tumors. Cell lines derived from the adrenal tumors were resistant to the neomycin analog G418. Clonal sublines are stable, growing easily in monolayers with a doubling time of 24-60 h. The cell lines secrete progesterone and 11-deoxycorticosterone, indicating these cells express the P450scc system, 3 beta-hydroxysteroid dehydrogenase, and 21-hydroxylase activity. However the 21-hydroxylase activity was not mediated by P450c21, as the cells lacked P450c21 mRNA. The cells did not secrete any 11-hydroxylated steroids, although they contained P450c11 beta mRNA. Both the secretion of progesterone and the abundance of P450scc mRNA increase in response to 8-bromo-cAMP, but not to ACTH or angiotensin II. In addition to expression of steroidogenic enzyme mRNAs, one cell line also expresses mouse renin-1 mRNA, making these cells useful for studies of the role of adrenal renin in regulating adrenal steroidogenesis. These findings represent an approach in transgenic mice to develop highly differentiated adrenal cell lines.

A mechanism for virilization of female spotted hyenas in utero.

Female spotted hyenas exhibit male-like genitalia and dominance over males. Hyena ovarian tissues incubated in vitro produced large quantities of the steroid hormone precursor androstenedione. The activity of aromatase, which converts androstenedione to estrogen, was one-twentieth as great in hyena versus human placental homogenates. In comparison, the activity of 17 beta-hydroxysteroid dehydrogenase, which converts androstenedione to testosterone, was equal in the two homogenates. The limited aromatase activity may allow the hyena placenta to convert high circulating concentrations of androstenedione to testosterone, which results in virilization of the fetal external genitalia and possibly destruction of fetal ovarian follicles. Androstenedione production by residual ovarian stromal cells during reproductive life accounts for the epigenetic transmission of virilization in female spotted hyenas.