cAMP-induced apoptosis in granulosa cells is associated with up-regulation of P53 and bax and down-regulation of clusterin.

Evidence indicates that cAMP induces apoptosis in granulosa cells of rat and human ovary. The mechanism by which cAMP induces apoptosis is not known. This study was carried out to evaluate changes in expression of cell death promoters, P53 and bax, and cell death repressor, bcl-2, in cAMP-treated granulosa cells. Treatment of granulosa cells with forskolin (FSK), or 8-bromo-cAMP induced apoptosis as evidenced by internucleosomal DNA fragmentation and chromatin condensation as revealed by gel electrophoresis and fluorescent DAPI staining, respectively. The apoptotic effect of cAMP was accompanied by an increase in the expression of P53 and bax proteins as evaluated by Western blot and immunocytochemistry. No change in bcl-2 protein level was observed in cAMP-treated granulosa cells as compared to control. These data suggest that cAMP may activate apoptosis in granulosa cells by shifting the ratio of the death promoter to death repressor genes via alteration of P53 and bax expression. cAMP was also shown to inhibit gene expression of clusterin, an apoptosis-associated protein, suggesting a role for this protein in cAMP-induced apoptosis in granulosa cells. The data of the present study provide a basis for future studies to elucidate the molecular mechanism of follicular atresia and regulation of apoptotic cell death in ovarian follicles.

Clusterin protects granulosa cells from apoptotic cell death during follicular atresia.

Clusterin expression is associated with programmed cell death (apoptosis) in many cell types but its exact role has not yet been defined. This study was carried out to determine the cellular localization of clusterin in the ovary and its functional role in the apoptotic cell death of ovarian follicles. A homogenous population of healthy and atretic follicles was obtained by treating immature rats with pregnant mare serum gonadotropin (PMSG). Apoptotic cell death was evaluated by TUNEL. Clusterin expression in the healthy and atretic follicles was examined by immunohistochemical and Western blot analyses, and gene expression was examined by Northern blot analysis. Clusterin protein and its mRNA are only expressed in granulosa cells of atretic follicles obtained from PMSG-treated rats on day 5 of the treatment. Healthy follicles from PMSG-treated rats on day 2 of the treatment do not express clusterin. Theca and stroma cells of both healthy and atretic follicles showed no signs of apoptosis and did not express clusterin. Withdrawal of trophic support from granulosa cells in cultures to induce apoptosis resulted in a dramatic increase in the levels of clusterin and its mRNA compared to cells cultured in serum-supplemented medium. In an attempt to establish the functional role of clusterin in the apoptotic cell death of ovarian follicles, the biosynthesis of clusterin in granulosa cells of healthy follicles was blocked by treatment of cells with antisense oligonucleotide to its cDNA. Treatment of granulosa cells with the antisense oligonucleotide resulted in an increase in the apoptotic cell death compared to the control. These findings indicate that depletion of clusterin can lead to the programmed cell death in ovary, suggesting a functional role for this protein in follicular atresia.

The insulin-like growth factor system in the GT1-7 GnRH neuronal cell line.

Evidence suggests that insulin-like growth factors (IGFs; IGF-I and IGF-II) are involved in the regulation of reproductive function including the development of the gonadotropin-releasing hormone (GnRH) neuronal system and the modulation of GnRH secretory activities. To further characterize the regulatory role of the IGF system on GnRH neuronal function, we have examined the gene expression of IGF-I, IGF-II, IGF-I receptor (IGF-IR), and IGF-binding proteins (IGFBPs) in a GnRH neuronal cell line (GT1-7 cells). The relative effects of IGFs and insulin on GnRH secretion by these cells was also investigated. RT-PCR analysis demonstrated IGF-I, IGF-II and IGF-IR mRNAs in GT1-7 cells. The mRNAs for IGFBP-2, -3, -4, -5 and -6 but not IGFBP-1 were also detected. Immunoreactive protein bands for IGFBP-2, -4 and -5 but not for other IGFBPs were demonstrated by Western blot with IGFBP-5 appearing to be the most abundant IGFBP secreted by GT1-7 cells. IGFBP-5 production by GT1-7 cells was stimulated by both IGF-I and IGF-II in a dose-dependent manner with approximately equal potency, whereas insulin caused no significant effect. GnRH secretion by GT1-7 cells treated with IGF-I or IGF-II but not insulin showed an increase (80-100%) at 2 h of treatment followed by a decrease (46%) at 6 h that continued up to 24 h. We conclude that the expression of IGFs, IGF-IR and IGFBPs and their interactions in the regulation of GnRH secretion by GT1-7 cells as demonstrated by our study provide a basis for an autocrine regulatory role for the IGF system in GnRH neuronal secretory activities.

Neurosteroidogenesis in astrocytes, oligodendrocytes, and neurons of cerebral cortex of rat brain.

The brain is a steroidogenic organ that expresses steroidogenic enzymes and produces neurosteroids. Although considerable information is now available regarding the steroidogenic capacity of the brain, little is known regarding the steroidogenic pathway and relative contributions of astrocytes, oligodendrocytes, and neurons to neurosteroidogenesis. In the present study, we investigated differential gene expression of the key steroidogenic enzymes using RT-PCR and quantitatively evaluated the production of neurosteroids by highly purified astrocytes, oligodendrocytes, and neurons from the cerebral cortex of neonatal rat brains using specific and sensitive RIAs. Astrocytes appear to be the most active steroidogenic cells in the brain. These cells express cytochrome P450 side-chain cleavage (P450scc), 17alpha-hydroxylase/C17-20-lyase (P450c17), 3beta-hydroxysteroid dehydrogenase (3betaHSD), 17beta-hydroxysteroid dehydrogenase (17betaHSD), and cytochrome P450 aromatase (P450arom) and produce pregnenolone (P5), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), estradiol, and estrone. Oligodendrocytes express only P450scc and 3betaHSD and produce P5, P4, and A4. These cells do not express P450c17, 17betaHSD, or P450arom or produce DHEA, T, or estrogen. Neurons express P450scc, P450c17, 3betaHSD, and P450arom and produce P5, DHEA, A4, and estrogen, but do not express 17betaHSD or produce T. By comparing the ability of each cell type in the production of neurosteroids, astrocytes are the major producer of P4, DHEA, and androgens, whereas oligodendrocytes are predominantly the producer of P5 and neurons of estrogens. These findings serve to define the neurosteroidogenic pathway, with special emphasis on the dominant role of astrocytes and their interaction with oligodendrocytes and neurons in the genesis of DHEA and active sex steroids. Thus, we propose that neurosteroidogenesis is accomplished by a tripartite contribution of the three cell types in the brain.

Dehydroepiandrosterone: biosynthesis and metabolism in the brain.

Dehydroepiandrosterone (DHEA) is abundantly found in brain tissues of several species, including human. However, the cellular origin and pathway by which DHEA is synthesized in brain are not yet known. We have, therefore, initiated pilot experiments to explore gene expression of cytochrome P450 17alpha-hydroxylase (P450c17), the key steroidogenic enzyme for androgen synthesis, and evaluate DHEA production by highly purified astrocytes, oligodendrocytes, and neurons. Using RT-PCR, we have demonstrated for the first time that astrocytes and neurons in the cerebral cortex of neonatal rat brain express P450c17. The presence of P450c17 in astrocytes and neurons was supported by the ability of these cells to metabolize pregnenolone to DHEA in a dose-dependent manner as determined by RIA. These data were further confirmed by production of androstenedione by astrocytes using progesterone as a substrate. However, cortical neurons express a low transcript of P450c17 messenger RNA and produce low levels of DHEA and androstenedione compared with astrocytes. Oligodendrocytes neither express the messenger RNA nor produce DHEA. The production of DHEA by astrocytes is not limited to cerebral cortex, as hypothalamic astrocytes produce DHEA at a level 3 times higher than that produced by cortical astrocytes. Cortical and hypothalamic astrocytes also have the capacity to metabolize DHEA to testosterone and estradiol in a dose-dependent manner. However, hypothalamic astrocytes were 3 times more active than cortical astrocytes in the metabolism of DHEA to estradiol. In conclusion, our data presented evidence that astrocytes and neurons express P450c17 and synthesize DHEA from pregnenolone. Astrocytes also have the capacity to metabolize DHEA into sex steroid hormones. These data suggest that as in gonads and adrenal, DHEA is biosynthesized in the brain by a P450c17-dependent mechanism.

Corticotropin-releasing factor inhibits luteinizing hormone-stimulated P450c17 gene expression and androgen production by isolated thecal cells of human ovarian follicles.

Recently, we reported that the thecal compartment of the human ovary contains a CRF system replete with gene expression and protein for corticotropin-releasing factor (CRF), CRF-Receptor 1 (CRF-R1), and the blood-derived high affinity CRF binding protein (CRF-BP). Granulosa cells are devoid of the CRF system. The parallel increases in intensity of CRF, CRF-R1, and 17 alpha-hydroxylase messenger ribonucleic acid (mRNA) and proteins in thecal cells with follicular maturation suggest that the intraovarian CRF system may play an autocrine role regulating androgen biosynthesis, with a downstream effect on estrogen production by granulosa cells. The functionality of the ovarian CRF system may be conditioned by the relative presence of plasma-derived CRF-BP by virtue of its localization of protein, but not transcript in thecal cells and its ability to compete with CRF for the CRF receptor. To further these findings, in the present study we have examined the effect of CRF on LH-stimulated 17 alpha-hydroxylase (P450c17) gene expression and androgen production by isolated thecal cells from human ovarian follicles (11-13 mm). During the 48-h culture, addition of LH (10 ng/mL) to the medium increased by 5- and 6-fold dehydroepiandrosterone and androstenedione production by thecal cells. Remarkably, the LH-stimulated, but not basal, androgen production was inhibited by CRF in a time- and dose-dependent manner. The half-maximal (ID50) effect dose of CRF occurred at 5 x 10(-8) mol/L, and at a maximal concentration of 10(-6) mol/L, CRF completely inhibited LH-stimulated androgen production. This inhibitory effect of CRF became evident at 12 h (45%), and by 24 h the effect was more pronounced, with a 70% reduction from baseline. As determined by Northern analyses, CRF dose dependently decreased LH-stimulated P450c17 mRNA levels, with a maximal inhibition of 85% P450c17 gene expression at a CRF concentration of 10(-6) mol/L. With the addition of 10(-6) mol/L of the antagonist alpha-helical CRF-(9-41), the inhibitory effect of CRF was partially reversed for both P450c17 mRNA (75%) and androgen production (50%), indicating the CRF-R1-mediated event. In conclusion, the present study demonstrated a potent inhibitory effect of CRF on LH-stimulated dehydroepiandrosterone and androstenedione production that appears to be mediated through the reduction of P450c17 gene expression. Thus, the ovarian CRF system may function as autocrine regulators for androgen biosynthesis in the thecal cell compartment to maintain optimal substrate for estrogen biosynthesis by granulosa cells. Further studies to define the role of CRF-BP in the endocrine modulation of the intraovarian CRF system are needed.

Expression of genes encoding corticotropin-releasing factor (CRF), type 1 CRF receptor, and CRF-binding protein and localization of the gene products in the human ovary.

Recently, the presence of immunoreactive corticotropin-releasing factor (IrCRF) in the thecal-stromal cells of the human ovary and the ability of CRF to suppress estrogen production by human granulosa cells in vitro have been reported. To understand the functional role of ovarian CRF requires characterization of the human ovarian CRF system, which includes CRF, type 1 CRF receptor (CRF-R1), and the high affinity CRF-binding protein (CRF-BP). Accordingly, we have examined the ovarian CRF system and the cellular distribution of these proteins and their messenger ribonucleic acids (mRNAs) using immunohistochemistry and in situ hybridization, respectively. Normal ovaries from 10 premenopausal women undergoing hysterectomy with ovariectomy were used in the analyses. IrCRF and its mRNA were localized in thecal cells of small antral and mature follicles. A low abundance of IrCRF and mRNA was also detected in stromal cells of both stages of follicles. Expression of the gene encoding CRF was more prominent in mature follicles than in small antral follicles. CRF-R1 mRNA signal was found exclusively in thecal cells of mature follicles and moderately in small antral follicles. Granulosa cells were devoid of CRF and CRF-R1 mRNAs and proteins. The IrCRF-BP, but not its transcript, was detected in thecal cells and luman of capillary vessels of the thecal/stromal compartment of mature follicles. The absence of CRF-BP gene transcript in human ovarian follicles was confirmed by reverse transcription-PCR, indicating that the IrCRF-BP detected is not derived from the ovarian transcript and suggesting that the presence of IrCRF-BP and luman of capillary vessels in the thecal compartment originates from the peripheral circulation. Thecal cells of mature follicles, relative to those of small antral follicles, exhibited an intensive immunostaining and mRNA signal for 17 alpha-hydroxylase (P450c17) indicative of androgen biosynthesis. We conclude that the thecal compartment of the human ovary contains a CRF system endowed with CRF and CRF-R1 and the blood-derived CRF-BP. Granulosa cells are devoid of the CRF system. The parallel increases in intensity of CRF, CRF-R1, and 17 alpha-hydroxylase proteins and gene expression with follicular maturation suggest that the intraovarian CRF system may play an autocrine role in androgen biosynthesis with a downstream effect on estrogen production by the granulosa cells. The functionality of the ovarian CRF system may be conditioned by the relative presence of circulating CRF-BP by virtue of its ability to compete with CRF for the CRF receptor.

Astrocytes cultured in vitro produce estradiol-17beta and express aromatase cytochrome P-450 (P-450 AROM) mRNA.

Aromatase cytochrome P-450 (P-450AROM) is an enzyme that catalyzes the conversion of androgen to estrogen. Estrogen plays an important role in the neuronal function by promoting the formation of dendrites and may be involved in protecting the neurons in the cerebral cortex against specific pathological conditions such as Alzheimer's disease. However, the cellular origin of estrogen in the brain is not known. The present study demonstrated for the first time the production of estradiol-17beta and expression of P-450AROM mRNA in astrocytes isolated from the cerebral cortex of neonatal rats. Immunocytochemical studies using a monospecific antibody against rat P-450AROM has shown that this enzyme was localized in the cytoplasm of astrocytes. Interleukin-1 (IL-1) has been shown to stimulate the proliferation and differentiation of astrocytes and to affect the aromatase activity in non-neuronal cells such as Sertoli, Leydig, and placental cells. Treatment of astrocytes with IL-1beta induced a dose-dependent inhibition of estradiol production. This inhibitory action of IL-1beta can be reversed by the addition of anti-IL-1beta antibody. Since astrocytes are involved in the synaptic reorganization in the brain by removing cellular debris and by providing the necessary biological factors for neuronal growth, the ability of astrocytes to produce estradiol-17beta and express P-450AROM mRNA in vitro suggests a new role for these cells in protecting and supporting neurons.

Rat seminiferous tubular culture medium contains a biological factor that inhibits Leydig cell steroidogenesis: its purification and mechanism of action.

Seminiferous tubules prepared from adult rats cultured for 48 h in serum-free conditions produce multiple biological factors that modulate Leydig cell steroidogenic function in vitro. Using gel filtration chromatography, it was shown that seminiferous tubular culture medium (STCM) contained at least three inhibitory activities designated AI, AII, and AIII that inhibited testosterone production by purified Leydig cells. The factor that induced AIII activity, designated Leydig cell inhibitor (LCI), was further purified to apparent homogeneity by sequential HPLC using gel permeation, C8-, C18-, C2/C18-reversed-phase, and microbore anion exchange columns. When this batch of purified factor was resolved by SDS-PAGE under reducing conditions, only a single silver stained band with an apparent M(r) of 21,000 was detected. Protein sequence analysis using about 100 pmol of purified LCI revealed that its N-terminus was blocked. Incubation of this highly purified factor with Percoll gradient purified Leydig cells induced a dose-dependent inhibition of hCG-stimulated testosterone production. LCI inhibited the basal testosterone production and hCG-stimulated cAMP production by Leydig cell dose-dependently. It also inhibited the forskolin- and cholera toxin-stimulated testosterone and cAMP production but had no apparent effect on the binding of 125I-labeled hCG to LH receptors. These data suggest that this LCI exerts its inhibitory action at steps beyond the LH receptors but prior to the cAMP formation by affecting the adenylate cyclase activity directly or indirectly through inhibition of the stimulatory G-protein (Gs-protein); however, it is also possible that it decreases the coupling of the receptors to the Gs-protein. LCI also inhibited the conversion of exogenously added 22R-hydroxycholesterol, pregnenolone, progesterone, and 17 alpha-hydroxyprogesterone to testosterone. However, it had no effect on the conversion of dehydroepiandrostenedione and androstenedione to testosterone. These data strongly suggest that LCI affects the steroidogenic enzymes metabolizing cholesterol to testosterone, the cytochrome P-450 side-chain cleavage (P-450SCC), and cytochrome P-450 17 alpha-hydroxylase/17,20-lyase (P-450C17). However, it has no effect on the 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) enzyme activities. Based on the results of the present study, it is apparent that this LCI is distinct from other known potent Leydig cells inhibitors such as interleukin-1 (IL-1) and transforming growth factor-beta (TGF-beta). The LCI appears to involve in the paracrine regulation of Leydig cell function.

Regulation of clusterin secretion and mRNA expression in astrocytes by cytokines.

Clusterin is an authentic Sertoli cell secretory product initially identified in the ram and rat testis. Subsequent studies have shown that this protein is present in almost all organs and in multiple species. Its mRNA increases in the brain undergoing degeneration as a result of infection, brain injury, and other pathological conditions such as Alzheimer's disease. However, its site(s) of synthesis and modulator(s) in the brain are not known. The objectives of this study were to determine if astrocytes could synthesize and secrete clusterin in vitro and to investigate the effects of various cytokines on the secretion and the mRNA expression of clusterin in the primary cultures of astrocytes. Astrocytes were isolated from cerebral cortices of neonatal rats and enriched to a purity of greater than 95% as judged by immunocytochemical staining using antibody against glial fibrillary acidic protein (GFAP), a specific marker of astrocytes. Using immunoprecipitation techniques, we have demonstrated that astrocytes actively synthesize and secrete clusterin in vitro. Immunocytochemical staining using a monospecific antibody against clusterin showed that this protein is localized in the entire cytoplasm and the processes of astrocytes. Treatment of astrocytes with either interleukin-1 beta, or interleukin-2, induced a significant increase in the production and the mRNA levels of clusterin, whereas other cytokines including interleukin-3, interleukin-6, and interferon-gamma had no apparent effect. The results of this study suggest that clusterin may be a marker to study the immune response in the brain.

Response of alpha 2-macroglobulin messenger ribonucleic acid expression to acute inflammation in the testis is different from the response in the liver and brain.

Recent studies from this laboratory have shown that Sertoli cells derived from 20-day-old rats and cultured in vitro synthesize and secrete a nonspecific protease inhibitor that is structurally and immunologically similar to serum alpha 2-macroglobulin (alpha 2-MG). In contrast to its serum homologue, the testicular alpha 2-MG is not an acute-phase protein in the rat since its protein concentration in the rete-testis fluid does not increase in response to inflammation. In the present study we examined the expression of alpha 2-MG mRNA in the rat testis in comparison to that in the brain and liver following induced inflammation. alpha 2-MG mRNA in the testis did not respond to induced inflammation, whereas its protein concentration in serum and its mRNA level in the brain and liver increased significantly in 20-day-old inflamed rats. In 8-day-old rat testis, where the blood-testis barrier is not yet formed, alpha 2-MG mRNA expression also did not respond to induced inflammation. The mRNA expression of clusterin, another authentic Sertoli cell protein whose secretion appears to be closely related to cell-cell interactions in the seminiferous epithelium, was shown to be unaffected by induced inflammation in the testis, brain, and liver. In view of the unexpected differential expression of alpha 2-MG mRNA to induced inflammation in the testis and liver, we sought to examine whether Sertoli cell alpha 2-MG would respond to FSH and testosterone (T), the major regulators of testicular function. Interestingly, expression of alpha 2-MG and clusterin mRNA in the Sertoli cell was not regulated by FSH, T, or a combination of FSH and T. Since there is an intimate morphological relationship between Sertoli cells and germ cells, we next examined the effect of germ cell-conditioned medium (GCCM) on Sertoli cell alpha 2-MG and clusterin mRNA expression. It was noted that GCCM caused a dose-dependent stimulation of alpha 2-MG and inhibition of clusterin mRNA expression in Sertoli cells, respectively. Therefore, our studies have shown that the regulatory mechanism that modulates the expression of alpha 2-MG mRNA in the rat testis is different from its counterpart in the brain and liver.

Chronic inflammatory response in the rat can be blocked by bindarit.

In the rat, injection of Freund's complete adjuvant was accompanied by a significant increase in concanavalin A (Con A)-reactivity of selected plasma proteins along with an increase in concentrations of selected proteins known as acute phase proteins. We have evaluated the effect of bindarit, (2-[(1-benzyl-indazol-3-yl)methoxy]-2-methyl propionic acid), on the expression of alpha 2-macroglobulin, a known acute-phase protein in the rat. This compound has previously been shown to inhibit heat-induced denaturation of rat serum albumin and to strongly reduce the secondary phase response of adjuvant induced arthritis. Adult rats were induced with chronic inflammation by injection with Freund's complete adjuvant. Bindarit was administered to the chronic inflamed rats as a 0.5% medicated diet. Indomethacin, given by gavage daily at a dose of 1 mg/kg body weight, was used as a reference drug. Qualitative and quantitative changes of Con A-reactive proteins and alpha 2-macroglobulin were examined by lectin- and immuno-blots, and by radioimmunoassay. It was noted that the concentration of alpha 2-macroglobulin increased in rats with adjuvant induced arthritis. The addition of bindarit and indomethacin were able to reduce the concentration of alpha 2-macroglobulin as well as the Con A-reactivity of various proteins to normal level 37 days following treatment. We have also examined the effects of chronic inflammation on the levels of rat clusterin, a testicular and serum glycoprotein related to programmed cell death, tissue regression, and complement cascade reaction; and testibumin, a testicular FSH and testosterone-responsive protein with unknown function. It was noted that chronic inflammation did not induce significant changes in both the clusterin and testibumin concentrations in these experimental groups. The involvement of protein glycosylation and denaturation in the production of new antigenic determinants, their role in the development of chronic inflammatory disease and the potential use of bindarit to investigate the relationship between abnormal glycosylation and autoimmune disease were discussed.

Regulation of Sertoli cell alpha 2-macroglobulin and clusterin (SGP-2) secretion by peritubular myoid cells.

alpha 2-Macroglobulin and clusterin are two putative Sertoli cell secretory products; however, the regulator(s) modulating their secretion by Sertoli cells is not known. Recent studies from this laboratory have shown that the testicular alpha 2-macroglobulin, unlike its liver homologue, is not an acute-phase reactant and its concentration is not affected by acute inflammation. We sought to determine whether FSH, testosterone, and other biomolecules would affect the secretion of alpha 2-macroglobulin and clusterin by Sertoli cells as well as whether peritubular myoid cells would affect the secretion of these proteins by Sertoli cells. It was noted that Sertoli cells cultured in vitro secreted increasing amounts of alpha 2-macroglobulin and clusterin as a function of time. FSH (50-1000 ng/ml) and testosterone (10(-11)-10(-5) M) had no apparent effect on the secretion of alpha 2-macroglobulin and clusterin by Sertoli cells. Addition of interleukin-6 to Sertoli cell-enriched cultures, in doses known to stimulate alpha 2-macroglobulin secretion by hepatocytes, did not affect the alpha 2-macroglobulin secretion. However, dexamethasone at 10(-7)-10(-5) M stimulated alpha 2-macroglobulin secretion by Sertoli cells dose-dependently while the addition of interleukin-6 had no synergistic effect on dexamethasone-stimulated alpha 2-macroglobulin secretion. These findings suggest that the synthesis and/or secretion of alpha 2-macroglobulin by Sertoli cells is regulated by a mechanism distinct from that of the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal glycosylation of hemopexin in arthritic rats can be blocked by bindarit.

Induction of arthritis in rats with Freund's complete adjuvant was accompanied by a distinctive alteration of concanavalin A (Con-A) reactivity in their serum proteins in which the concentrations of selected Con-A reactive proteins were significantly higher when compared to healthy rats. To assess if the observed increase in Con-A reactivity of specific serum proteins reflects an increase in carbohydrate moieties in these proteins in addition to an increase in their protein concentrations, a heme binding serum glycoprotein, hemopexin, also an acute phase reactant, was selected as a marker protein. Hemopexin was purified to apparent homogeneity from pools of serum samples derived from rats with yeast induced inflammation, a monospecific polyclonal antibody was prepared and was used for immunoblot analysis. It was noted that the concentration of hemopexin increased in rats with adjuvant induced arthritis; however, its concentration fell to normal levels after administration with a newly synthesized drug, bindarit, (2-[(1-benzyl-indazol-3-yl)methoxy]-2-methyl propionic acid, C19H20N2O3. Hemopexin was micropurified individually from healthy rats, adjuvant induced arthritic rats, and adjuvant arthritic rats treated with bindarit, cleaved with a Glu-C endopeptidase, Staphylococcus aureus protease V8, and the resultant peptide fragments resolved by SDS-PAGE and examined by silver staining, Coomassie blue staining, and lectin blots using Con-A. It was subsequently noted that hemopexin isolated from adjuvant induced arthritic rats showed a significant increase in Con-A reactivity in selected peptide fragments and that such an increase in glycosylation could be reversed to a pattern similar to healthy rats following treatment with bindarit.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat epididymal retinoic acid-binding protein: development of a radioimmunoassay, its tissue distribution, and its changes in selected androgen-dependent organs after orchiectomy.

Rat epididymal retinoic acid-binding protein (EP-RABP) has been purified to apparent homogeneity from extracts of the epididymis by HPLC. N-Terminal amino acid sequence analysis revealed that the HPLC-purified protein consisted of two molecular variants, in that one has three extra N-terminal amino acids of NH2-TEG. These two molecular variants were subsequently separated by high performance electrophoresis chromatography. A specific and sensitive RIA has been developed to quantify this protein in various organ extracts of both male and female rats. Rat EP-RABP is a male-specific protein, since it was detected in male organ extracts, including epididymis, testis, prostate, seminal vesicles, liver, spleen, and brain, with a negligible concentration in the female liver and spleen. It was noted that this protein was absent in the systemic circulation of both male and female rats. It was first detected in the epididymis and testis of rats at 22 and 27 days of age, respectively. Both the concentrations (micrograms per g tissue) and the organ content (micrograms per organ pairs) of this protein in the testis and epididymis increased progressively with an increase in age and reached the highest levels at 60 and 120 days of age, respectively. At 120 days of age, its concentrations in all organs examined, with the exception of the epididymis, showed a dramatic decrease compared to levels in rats at 60 days of age. Orchiectomy decreased its concentrations in the caput, corpus, and cauda epididymis and in the ventral prostate, but had no apparent effect on seminal vesicles. Administration of dihydrotestosterone to castrated rats restored only 30% of the level of this protein in the caput epididymis compared to that in intact animals, but had no apparent effect on the corpus, cauda epididymis, or ventral prostate. These observations suggest that this protein is under multihormonal control in the epididymis and selected androgen-dependent organs.

Identification of an inhibitory factor from a Sertoli clonal cell line (TM4) that modulates adult rat Leydig cell steroidogenesis.

Sertoli cell produces several biological factors that modulate Leydig cell steroidogenic function by either stimulating or inhibiting its testosterone production. We have evaluated the effect of an inhibitory factor in the spent media of a Sertoli clonal cell line (TM4) which inhibits Leydig cell steroidogenesis. The presence of such an inhibitory factor in TM4 media was bioassayed using Percoll purified Leydig cells isolated from adult rats with purity of greater than 95%. TM4 media inhibited both human chorionic gonadotropin (hCG)-stimulated testosterone and cAMP production by purified Leydig cells dose-dependently but had no apparent effect on 8-bromo-cAMP- and forskolin-stimulated testosterone production. Also it did not interfere with the binding of [125I]hCG to Leydig cells. TM4 media inhibited cholera toxin-stimulated testosterone production as well as forskolin- and cholera toxin-stimulated cAMP production. The mechanism of action of this factor in TM4 media appears to be different from transforming growth factor (TGF-beta) which inhibited both 8-bromo-cAMP- and forskolin-stimulated testosterone production and inhibited the binding of [125I]hCG binding to Leydig cells. The inhibitory factor contained in TM4 media has been partially purified by sequential preparative anion exchange and C-18 reversed-phase high-performance liquid chromatography. In summary, the Sertoli TM4 cell line produces at least one potent inhibitory factor which decreases the responsiveness of purified Leydig cells to hCG stimulation with a dramatically different mechanism from other currently known Leydig cell inhibitory factors; this protein may serve as a valuable tool to study testicular paracrine regulation.

Diverse secretory patterns of clusterin by epididymis and prostate/seminal vesicles undergoing cell regression after orchiectomy.

Nucleotide sequence analysis of the complimentary DNAs (cDNA) and N-terminal amino acid sequence analysis have shown that clusterin is equivalent to sulfated glycoprotein-2 (SGP-2), testosterone-repressed prostate protein-2 (TRPP-2), and androgen-repressed protein (ARP) in the rat, as well as serum/seminal plasma protein, SP-40,40, in the human. In view of its widespread presence in various species, a specific RIA was established to quantify the tissue distribution of this protein. Rat clusterin is present in almost all organ tissues examined, including testis, epididymis, serum, liver, prostate, seminal vesicles, and uterus. Displacement curves generated using cytosols prepared from these organs were parallel to those obtained using purified rat clusterin and crude Sertoli cell-enriched culture medium. Immunoreactive clusterin was also visualized in these organ extracts by immunoblots. Studies on the tissue distribution of immunoreactive clusterin using RIA revealed that the concentration of clusterin in the epididymis of adult rats was 6- and 10-fold higher than that in the serum and testis, respectively and is 50- to 100-fold higher in the liver, spleen, kidney, brain, ventral prostate, seminal vesicles, and uterus. A study of the distribution of clusterin in various compartments of the epididymis indicated its concentration in the caput epididymis was almost 3-fold higher than that in the corpus and cauda epididymis. After orchiectomy, the concentrations of clusterin in the ventral prostate and seminal vesicles increased as much as 100- and 10-fold and peaked at day 4 after surgery, respectively; daily injection of dihydrotestosterone (DHT) beginning at day 3 after orchiectomy reduced the concentrations of clusterin and restored them to a normal level. A different pattern was noted in the epididymis after orchiectomy; the concentration of clusterin in the caput epididymis decreased with time; however, daily injection of DHT beginning at day 3 increased the caput epididymal clusterin concentration and restored it to a normal level. The concentration of clusterin was not altered in the corpus or cauda epididymis after castration and/or DHT administration. Also, the serum and liver clusterin levels did not change with time after orchiectomy. These observations suggest that clusterin will be a valuable marker to monitor the diverse effects of androgen withdrawal in the male reproductive tract. We conclude that clusterin may be a multifunctional protein in view of its broad tissue distribution and association with numerous physiological and pathological conditions.

Androgen and 19-norandrogen aromatization by equine and human placental microsomes.

The ability of equine and human placental microsomes to aromatize testosterone and 19-nortestosterone was studied. When 3 microM [1 beta,2 beta-3H]testosterone was used as substrate, the specific activity of equine placental microsomal aromatase was 2.5 times higher than that of the human microsomal enzyme. Although 19-nortestosterone was aromatized 67 times more rapidly by equine than by human aromatase, we found that equine aromatase exhibited a markedly weaker affinity for this substrate than did the human enzyme. Competitive inhibition of testosterone aromatization by 19-nortestosterone occurred with both equine and human aromatases. While having no effect on mare placental microsomes, Na+ and K+ (500 mM) stimulated testosterone aromatization by human placental microsomes by 73 and 52% respectively. If indeed a single enzyme is responsible for the aromatization of testosterone and 19-nortestosterone, which seems to be the case in both equine and human placental aromatase, our results show that differences in the structure of the active sites exist between equine and human aromatases.

Synthesis and aromatization of 19-norandrogens in the stallion testis.

The results of the measurement of 19-nortestosterone in the testiscular artery and vein of the stallion, the very low levels of this steroid in the peripheral blood of geldings and the similar patterns of increase in the peripheral levels of 19-nortestosterone and testosterone after hCG stimulation, show that 19-nortestosterone, like testosterone, is essentially synthesized in the testis. This testicular origin was confirmed by the ability of testicular tissue to synthesize 19-norandrogens from [4-14C]androgens in vitro. 19-Nortestosterone was 50% conjugated in the peripheral blood and almost entirely conjugated after biosynthesis in vitro. The sequence of appearance of steroids in the peripheral blood after a single injection of 10,000 IU hCG suggests that, in the equine testis, 19-norandrogens are produced by a specific C10-19 desmolase (estrene synthetase), stimulable by hCG. 19-Nortestosterone was aromatized into estradiol-17 beta by stallion testicular microsomes. The affinity of the aromatase for 19-nortestosterone was very low compared to that for testosterone. At low and presumably physiological levels, and at a high testosterone/19-nortestosterone ratio, testosterone did not inhibit 19-nortestosterone aromatization by more than 53%. Thus, 19-nortestosterone may be aromatized in vivo in the testis in spite of the endogenous concentrations of androgens. However, the low velocity of 19-nortestosterone aromatization by testicular microsomes at roughly physiological concentrations suggests that 19-norandrogen aromatization may only participate slightly in the testicular estrogen production. These results suggest that in the equine testis, two aromatizing enzyme systems may exist: one which aromatizes both androgens and 19-norandrogens, and a minority system more specific for 19-norandrogens.

Androgen and oestrogen response to a single injection of hCG in cryptorchid horses.

Androgen (testosterone and androstenedione) and oestrogen (oestradiol -17 beta and oestrone) concentrations were measured by radio-immunoassay in the peripheral plasma of two geldings (five-years-old), three bilateral cryptorchids (two, two and a half, and five-years-old) and three normal intact stallions (four, five and five and a half-years-old) before and after a single injection of 10,000 iu human chorionic gonadotrophin (hCG). In the stallions, hCG administration resulted in an immediate sharp increase of conjugated oestrogens and a more gradual increase of unconjugated androgens. In the cryptorchids, the unconjugated androgens increased following a similar pattern to that observed in the stallions, but reached lower peak values, whereas the conjugated oestrogens showed only a very slight increase. In the stallions and cryptorchids, the maximum oestrogen levels were reached two days after injection, whereas the maximal levels for androgens were reached a day later. In the geldings, hCG injection had no effect on plasma steroid levels. It is suggested that the measurement of unconjugated androgens (testosterone or/and androstenedione) before and three days after intravenous injection of 10,000 iu hCG may prove useful for the diagnosis of cryptorchidism or exploration of testicular function in stallions.