7alpha-hydroxytestosterone affects 1 beta-hydroxysteroid dehydrogenase 1 direction in rat Leydig cells.

The cytochrome P450 2A1 (CYP2A1) is a P450 enzyme that catalyzes the metabolism of testosterone. CYP2A1 has been reported to be present in rat testis. However, its developmental changes and function have not been well characterized. The purpose of this study was to measure the abundance of CYP2A1 (Cyp2a1) mRNA in the developing rat testis and Leydig cells and examine the effects of its product, 7 alpha-hydroxytestosterone (7HT), on an important enzyme, 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1) that interconverts active corticosterone and inactive 11-dehydrocorticosterone. As detected by real-time PCR, Cyp2a1 was found to be present exclusively in the Leydig cell. CYP2A1 activity in adult Leydig cells was 5-fold higher than those in progenitor or immature Leydig cells. 7HT competitively suppressed 11 beta-HSD1 oxidase and reductase activities in rat testis microsome with inhibitory constant of 1.2 and 2.9 mum, respectively. In intact Leydig cells, 7HT did not inhibit 11 beta-HSD1 reductase activity, but it stimulated its reductase activity. Thus, at 100 nm and higher concentrations, 7HT significantly switched 11 beta-HSD1 oxidoreductase activities toward reductase. The present data shows that 7HT, the product formed by CYP2A1 from testosterone, regulates the direction of 11 beta-HSD1 activity in rat Leydig cells.

Erythropoietin attenuates lung injury in lipopolysaccharide treated rats.

BACKGROUND: Erythropoietin (EPO) elicits protective effects in lung ischemia-reperfusion, hyperoxia, acute necrotizing pancreatitis, and some other tissues. In the present study, we investigated the possible protective roles of EPO in the lipopolysaccharide (LPS) induced lung injury. MATERIALS AND METHODS: Male Sprague-Dawley rats were treated with EPO (3000 U/kg, i.p.) or vehicle (saline), 30 min prior to LPS administration (6 mg/kg, i.v.). Four h following LPS injection, samples of pulmonary tissue were collected. Optical microscopy was performed to examine pathological changes in lungs. Validated methods were used to measure wet/dry ratios (W/D), myeloperoxidase (MPO) activity, malondialdehyde (MDA) concentrations, and nitrite/nitrate (NO(2)(-)/NO(3)(-)) levels in lungs. Western blotting was performed to study the pulmonary expression of inducible nitric oxide synthase (iNOS) and nitrotyrosine protein. RESULTS: Pretreatment with EPO led to (1) significant attenuation of endotoxemia induced evident lung histologic injury and edema; (2) inhibition of LPS mediated induction in MPO activity and MDA concentration; (3) inhibition of LPS mediated overproduction of pulmonary NO(2)(-)/NO(3)(-) levels; and (4) marked suppression in endotoxin induced expression of iNOS and nitrotyrosine. CONCLUSIONS: This study provides considerable evidence that EPO has an ability to significantly attenuate endotoxin-induced acute lung injury in rats.

Molecular dissection of an hCG-beta epitope using single-step solid phase radioimmunoassay.

BACKGROUND: Peptides and proteins have both sequence-specific (contiguous) and conformation-specific (discontiguous) epitopes. Sequence-specific epitopes are delineated by peptide approach and other robust methods like competition assays, gene expression assays, synthetic peptide library based assays etc. Available methods for delineation of conformation-specific epitopes are cumbersome (X-ray crystallography etc.), time-consuming and require costly sophisticated equipments. Hence, there is a need to develop a simple method for identification and mapping of conformation-specific epitopes. METHOD: In the single-step solid phase radioimmunoassay (SS-SPRIA), an immunochemical bridge of 'mouse IgG-anti-mouse IgG' was prepared in the polypropylene wells followed by adsorption with hCG specific monoclonal antibody (MAb) G(1)G(10).1. The extent of competitive inhibition in binding ability of (125)IhCG-beta with chemically or enzymatically modified hCG-beta to immobilized MAb G(1)G(10).1 in comparison to hCG-beta standards was utilized to identify the epitopic amino acid involved in epitope-paratope interaction. RESULTS: Data clearly suggest that the epitope under investigation consisted of Arg (94, 95) and Asp (99) at the core region with a Lys (104) and a His (106) in the proximity and absence of chymotrypsin susceptible Phe or Tyr in this region. CONCLUSION: The data of SS-SPRIA revealed the 93-100 loop of amino acid sequence, as the core region of conformation-specific epitope of hCG-beta at or near the receptor-binding region. Hence, SS-SPRIA seems to be a simple method for identification and mapping of conformation-specific epitopes.

Clomiphene citrate inhibits gonadotropin-induced ovulation by reducing cyclic adenosine 3',5'-cyclic monophosphate and prostaglandin E2 levels in rat ovary.

OBJECTIVE: To determine whether clomiphene citrate (CC) inhibited gonadotropin-induced ovulation by reducing cyclic adenosine 3',5'-cyclic monophosphate (cAMP) and prostaglandin E2 (PGE2) levels in ovary. If so, to determine whether E2 coadministration could protect against these effects of CC. DESIGN: A controlled prospective study. SETTING: Laboratory research setting in the department of reproductive biomedicine at a national research institute in India. ANIMAL(S): Sixty sexually immature female rats that were 24-25 days of age. INTERVENTION(S): The sexually immature female rats were given a single injection (10 IU i.m.) of pregnant mare serum gonadotropin. After 48 hours, the rats were given single injections of hCG (10 IU) along with CC, with or without E2, for 16 hours. MAIN OUTCOME MEASURE(S): Number of superovulated COCs, ovary and uterus weight, FSH and LH levels in serum, and cAMP and PGE2 levels in ovary. RESULT(S): The superovulatory dose of gonadotropins significantly increased ovary and uterus weights, cAMP and PGE2 levels in ovary, and serum levels of FSH and LH. Coadministration of CC (10 mg/kg body weight) significantly reduced levels of cAMP, PGE2 in the ovary, ovary and uterus weights, and ovulation rate, whereas FSH and LH levels were not significantly altered. Supplementation of E2 protected against these inhibitory effects of CC and augmented levels of FSH and LH in serum. CONCLUSION(S): Clomiphene citrate inhibited gonadotropin-induced ovulation by reducing cAMP and PGE2 levels in the ovary, and E2 protected against these effects of CC on gonadotropin-induced ovulation in rat.

Extract of Azadirachta indica (neem) leaf induces apoptosis in rat oocytes cultured in vitro.

OBJECTIVE: To determine whether aqueous neem leaf extract (NLE) could induce degeneration of rat oocytes and, if so, whether apoptosis is involved during NLE-induced degeneration of oocytes cultured in vitro. DESIGN: A controlled prospective study. SETTING: Laboratory research setting at Department of Reproductive Biomedicine of the Institute. ANIMAL(S): Fifty-four sexually immature female rats that were 24-25 days of age. INTERVENTION(S): The immature female rats were injected with 10 IU pregnant mare serum gonadotropin for 48 h followed by 10 IU human chorionic gonadotropin (hCG) for 16 h. After 16 h, the rats were killed and ovulated cumulus oocyte complexes were collected from the oviduct. Cumulus-enclosed as well as denuded oocytes were used in the present study. MAIN OUTCOME MEASURE(S): Rates of shrinkage, membrane leakage, degeneration, assessment of morphological apoptotic changes, bax protein expression, and DNA fragmentation. RESULT(S): The NLE induced morphologic apoptotic changes such as shrinkage, membrane leakage, and cytoplasmic fragmentation prior to degeneration of oocytes. The NLE-treated oocytes that had morphologic apoptotic features showed overexpression of bax protein and DNA fragmentation as evidenced by terminal deoxynucleotidyl transferase nick-end labeling-positive staining and DNA ladder pattern. CONCLUSION(S): Neem leaf extract induced apoptosis in rat oocytes prior to degeneration in vitro.

Development of colorimetric enzyme-linked immunosorbent assay for human chorionic gonadotropin.

The present study demonstrated the development of a solid phase competitive enzyme linked immunosorbent assay (ELISA) for direct estimation of human chorionic gonadotropin (hCG) in serum and urine. Polyclonal antisera raised against the beta- subunit of peak-I hCG was used in the assay. The Peak-IA hCG-penicillinase was used as tracer. The performance of this antiserum and tracer was compared against hCG-beta antisera of NIH, USA and penicillinase conjugated to hCG-beta obtained from NIH, respectively. Almost parallel standard curves were obtained in both cases, suggesting that these antisera and enzyme label have much potential for developing ELISA system. To the anti-rabbit gamma globulin (ARGG) coated polystyrene tubes, standard or serum or urine samples (50 microL), 100 microL of hCG-beta antiserum, 100 microL of peak-I(A) hCG-penicillinase conjugate and 350 microL of assay buffer were incubated at 37 degrees C for 2 hours. Bound enzyme activity was measured using Penicilline V as substrate. In this new strategy, locally available polystyrene tubes were ground from inside and coated with ARGG. The sensitivity of the assay was 17 mIU/mL in urine and 18 mIU/mL in serum. The intra-assay and inter-assay coefficients of variation (CVs) appeared to be within acceptable limits of 10%. The serum and urinary hCG values, obtained by this method, correlated well with those obtained by radioimmunoassay (RIA) r = 0.98 (n = 100 for serum samples; n = 250 for urinary samples).

Estradiol protects clomiphene citrate-induced apoptosis in ovarian follicular cells and ovulated cumulus-oocyte complexes.

OBJECTIVE: To determine whether clomiphene citrate (CC) reduces E(2) level in the ovary and circulation and induces apoptosis in ovarian follicular cells and ovulated cumulus-oocyte complexes (COCs). If yes, to determine whether E(2) coadministration could protect against these adverse effects of CC. DESIGN: A controlled prospective study. SETTING: Laboratory research setting. ANIMAL(S): Ninety sexually immature female rats that were 24-25 days of age. INTERVENTION(S): The immature female rats were injected with a single dose of 10 IU of pregnant mare serum gonadotropin. After 48 hours, 10 IU of hCG along with 10 mg of CC per kilogram of body weight, with or without 2.0 mg of E2 per kilogram of body weight were coadministered. After 16 hrs, the rats were killed; COCs were collected from oviduct and ovaries were isolated. MAIN OUTCOME MEASURE(S): Number of superovulated COCs, oocyte morphology, E2 level in ovary and serum, histology of ovary, DNA fragmentation, and bax protein expression in ovary and COCs. RESULT(S): The number of COCs and E2 level in ovary and serum were reduced, whereas membrane blebbing in oocytes, bax protein expression, and DNA fragmentation in ovarian follicular cells and ovulated COCs were induced after CC treatment. These adverse effects of CC were protected against if animals were coadministered with E2. CONCLUSION(S): Clomiphene citrate-induced apoptosis in ovarian follicular cells (probably granulosa cells), thereby reducing E2 level in ovary and circulation that might have resulted in poor development and maturation of oocytes leading to reduced ovulation.

Isolation of hCG and its characterization by radioimmunoassay, enzyme-immunoassay, and radio-receptor assay.

The nature of human chorionic gonadotropin (hCG) molecules present during early pregnancy of Indian women is poorly understood. Therefore, a study has been undertaken to isolate hCG and characterize different forms of hCG from urine. The hCG molecules from urine of pregnant women (45-75 days post LMP) were adsorbed onto kaolin, eluted with ammonium hydroxide, and precipitated using acetone and then lyophilized. The lyophilized extract was subjected to Sephadex G-100 chromatography followed by ion-exchange fractionation. Three major fractions of protein (i.e., Peaks I, II, and III) associated with carbohydrate activity were obtained. Peaks II and III eventually resolved into a single peak I following repeated ion exchange chromatography, which suggested the presence of aggregates of molecules. Further purification on an affinity column resolved all three peak fractions into one unadsorbed and two adsorbed (A and B) fractions. These adsorbed fractions were characterized by radioreceptor assay (RRA), radioimmunoassay (RIA), and enzyme linked immunosorbent assay (ELISA). The activity was standardized against WHO reference preparation 75/589. Peaks I (A and B) were found to have maximum at about 75% of immunologically potent hCG, followed by peaks II (40%) and III (5%). The molecular sizes of peaks I, II, and III on a Sephadex G-200 column corresponded to 27,500D, 66,000D, and 84,000D, respectively. Relative mobilities of all adsorbed fractions in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed the presence of hCG-alpha (mol. wt. 19,539D) and hCG-beta (28,870D) subunits. The presence of both subunits of hCG were also revealed by Western blot analysis. For the first time, we report the low molecular weight hCG molecule, of 27,500D by size exclusion chromatography, which has immunological and biological activity as measured by RIA, ELISA, and RRA.

Isolation of alpha- and beta-subunits of peak-I hCG and generation of highly specific polyclonal antisera.

Development of polyclonal antisera is still a choice in some hard-pressed budget laboratories. In the present study, an attempt was made to isolate alpha- and beta-subunits from peak-I hCG, generation of polyclonal antisera and their characterization. The anti-hCG-a antisera showed titres of 1: 8000 and anti-hCG-beta antisera 1: 16,000 at 50% binding to radiolabelled hCG in RIA. Studies on specificity using anti hCG-beta antiserum demonstrated no cross-reaction with several hormones tested in the present study, except for hCG-beta and hCG, thus eliciting a highly specific hCG-beta antiserum.