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Biomedical subjects

A J Rao

Publications and source records attributed to A J Rao.

At least 19 recordsLinked to original sources
PubMed2003

Evaluation of relative roles of LH and FSH in regulation of differentiation of Leydig cells using an ethane 1,2-dimethylsulfonate-treated adult rat model.

The relative role of LH and FSH in regulation of differentiation of Leydig cells was assessed using an ethane 1,2-dimethylsulfonate (EDS)-treated rat model in which endogenous LH or FSH was neutralized from day 3 to day 22 following EDS treatment. Serum testosterone and the in vitro response of the purified Leydig cells to human chorionic gonadotropin (hCG) was monitored. In addition RNA was isolated from the Leydig cells to monitor the steady-state mRNA levels by RT-PCR for 17alpha-hydroxylase, side chain cleavage enzyme, steroidogenic acute regulatory protein (StAR), LH receptor, estrogen receptor (ER-alpha) and cyclophilin (internal control). Serum testosterone was undetected and the isolated Leydig cells secreted negligible amount of testosterone on stimulation with hCG in the group of rats that were treated with LH antiserum following EDS treatment. RT-PCR analysis revealed the absence of message for cholesterol side chain cleavage enzyme and 17alpha-hydroxylase although ER-alpha and LH receptor mRNA could be detected, indicating the presence of undifferentiated precursor Leydig cells. In contrast, the effects following deprival of endogenous FSH were not as drastic as seen following LH neutralization. Deprival of endogenous FSH in EDS-treated rats led to a significant decrease in serum testosterone and in vitro response to hCG by the Leydig cells. Also, there was a significant decrease in the steady-state mRNA levels of 17alpha-hydroxylase, cholesterol side chain cleavage enzyme, LH receptor and StAR as assessed by a semiquantitative RT-PCR. These results establish that while LH is obligatory for the functional differentiation of Leydig cells, repopulation of precursor Leydig cells is independent of LH, and also unequivocally establish an important role for FSH in regulation of Leydig cell function.

Animals↗
PubMed2001

Regulation of telomerase during human placental differentiation: a role for TGFbeta1.

The transient tumor-like attributes of the first-trimester placenta anchor the developing embryo to the uterine wall thus establishing a vital link between the mother and the fetus. Dysregulation of this invasive behavior and/or controlled proliferation of the placenta is associated with abnormal pregnancies. Several of these diseased states also exhibit aberrant telomerase activity, among other pathophysiological manifestations. Considering the strong correlation between telomerase activity and tumorigenesis, it was of interest to see whether the crucial processes of trophoblast proliferation and differentiation were brought about through the modulation of telomerase. Using two in vitro model systems of trophoblast differentiation, we demonstrate here that telomerase activity is negatively regulated during placental differentiation. We further show that this modulation is at the level of transcription of hTERT. We also propose a role for TGF beta1 in regulating telomerase activity in differentiating trophoblasts by down-regulating the expression of hTERT at the transcriptional level.

Blotting, Northern↗
PubMed2001

Embryo implantation and GnRH antagonists: the search for the human placental GnRH receptor.

Hypothalamic gonadotrophin-releasing hormone (GnRH) plays a major role in the endocrine control of reproduction. Acting through its high affinity receptors on pituitary gonadotrophs, it regulates the secretion of gonadotrophins. In addition, GnRH also functions as a local regulator in a number of other cell lines and tissues, including the placenta. In a manner analogous to hypothalamic GnRH stimulation of LH and FSH from the anterior pituitary, GnRH was found to cause a dose-dependent release of human chorionic gonadotrophin (HCG) from the placenta. So began the search for a putative GnRH receptor in the human placenta. Although early radio-receptor studies reported specific binding, the properties of these 'putative' GnRH binding sites were found to differ significantly from those of their pituitary counterparts in several important respects. This was followed by a series of contradictory reports that led to more questions and opened up avenues for further investigations. Even after nearly two decades of research, the human placental GnRH receptor has not been characterized beyond all reasonable doubt. This review recalls the discovery, the controversies and unanswered questions concerning the human placental GnRH receptor.

Binding Sites↗
PubMed2001

Primate epididymis-specific proteins: characterization of ESC42, a novel protein containing a trefoil-like motif in monkey and human.

Epididymal secreted proteins promote sperm maturation and fertilizing capacity by interacting with sperm during passage through the epididymis. Here we investigate the molecular basis of sperm maturation by isolating cDNA clones for novel epididymis-specific expressed sequences. Thirty-six novel cDNAs were isolated and sequenced from a subtracted Macaca mulatta epididymis library. The clones encode proteins with a range of motifs characteristic of protein-modifying enzymes, protease inhibitors, hydrophobic ligand-binding and transport proteins, extracellular matrix-interacting proteins, and transcription regulatory factors. The full length coding sequences were obtained for 11 clones representing a range of abundance levels. Expression of each is regionally localized and androgen regulated. The most abundant, ESC42, contains a cysteine-rich region similar to the signature binding domain of the trefoil family of motogenic wound repair proteins. The monkey and human proteins are nearly 90% identical. Immunohistochemical staining revealed that the protein is most abundant in the epithelium of the caput and is also present in the lumen and bound to sperm. The ESC42 gene, located on chromosome 20q11, contains two exons encoding two nearly identical predicted signal peptides and a third exon encoding the rest of the protein.

Amino Acid Sequence↗
PubMed2000

Effect of deprival of LH on Leydig cell proliferation: involvement of PCNA, cyclin D3 and IGF-1.

The levels of proliferating cell nuclear antigen (PCNA) and cyclin D3 which are known markers of cellular proliferation were monitored by immunoblotting in progenitor Leydig cells (PLC), immature Leydig cells (ILC) and adult Leydig cells (ALC) isolated from 21, 35 and 90 day old rats, respectively which represent the Leydig cells at different stages of development. The levels of PCNA and cyclin D3 were highest in PLC, intermediate in ILC and lowest in ALC. Following administration of an antiserum to LH to deprive endogenous LH in 21 day old rats, a significant decrease in the levels of PCNA and Cyclin D3 were observed suggesting the involvement of Lutenizing hormone (LH) in PLC proliferation. In support of this observation, Bromodeoxyuridine (BrdU) incorporation was highest in PLC when compared with ILC and ALC, and administration of LH antiserum to 21 day old rats led to a total absence of BrdU incorporation by the isolated PLC. Also, there was a decrease in the level of IGF-1 and IGF-1 receptor mRNA levels by 55 and 35%, respectively as assessed by semi-quantitative RT-PCR. In addition, the PLC isolated from rats deprived of endogenous LH incorporated much less BrdU following addition of IGF-1. These results, which are obtained using an in vivo model system establish that LH has a very important role in Leydig cell proliferation in immature rats.

Animals↗
PubMed2000

Cloning and characterization of an apoptosis-associated gene in the human placenta.

Placenta is a transient feto-maternal association that develops during mammalian pregnancies. Human placental tissue during the first trimester of pregnancy is an actively dividing and differentiating tissue, while near term, it represents a fully differentiated unit performing many life-sustaining functions for the fetus. Previous studies have demonstrated that the percentage of placental cells that undergo apoptosis is greater at full term as compared to the first trimester of pregnancy. In this study, we undertook a study aimed at gaining an insight into the kind of genes expressed in the two developmentally distinct stages of gestation ie, the first trimester and term using Differential Display RT-PCR. Cloning and sequencing of one of the differentially expressed cDNAs from term placental tissue revealed that it is a novel gene, referred to as T-18 in the text. In this study, we also examined the regulation of this gene during apoptosis in the human placenta. A model for analysis of placental apoptosis was established by incubating placental villi in serum-free culture medium. It was observed that apoptosis occurred rapidly following incubation of placental villi without tropic support, and the proposed free-radical scavenger, superoxide dismutase (SOD) suppressed apoptosis in the placenta. Interestingly, the levels of T-18 mRNA increased significantly during spontaneous induction of apoptosis and decreased when apoptosis was blocked by SOD. These data clearly suggest that there is a strong correlation between the expression of T-18 and placental apoptosis and that T-18, may play a significant role in this process. Furthermore, the establishment of a defined in vitro explant culture model should facilitate elucidation of factors, which regulate apoptosis in human placenta.

Apoptosis↗
PubMed2000

Stabilization and solidification of metal-laden wastes by compaction and magnesium phosphate-based binder.

Bench-scale and full-scale investigations of waste stabilization and volume reduction were conducted using spiked soil and ash wastes containing heavy metals such as Cd, Cr, Pb, Ni, and Hg. The waste streams were stabilized and solidified using chemically bonded phosphate ceramic (CBPC) binder, and then compacted by either uniaxial or harmonic press for volume reduction. The physical properties of the final waste forms were determined by measuring volume reduction, density, porosity, and compressive strength. The leachability of heavy metals in the final waste forms was determined by a toxicity characteristic leaching procedure (TCLP) test and a 90-day immersion test (ANS 16.1). The structural composition and nature of waste forms were determined by X-ray diffraction (XRD) and scanning electron microscopy (SEM), respectively. CBPC binder and compaction can achieve 80-wt% waste loading and 39-47% reduction in waste volume. Compressive strength of final waste forms ranged from 1500 to 2000 psi. TCLP testing of waste forms showed that all heavy metals except Hg passed the TCLP limits using the phosphate-based binder. When Na2S was added to the binder, the waste forms also passed TCLP limits for Hg. Long-term leachability resistance of the final waste forms was achieved for all metals in both soil and ash wastes, and the leachability index was approximately 14. XRD patterns of waste forms indicated vermiculite in the ash waste was chemically incorporated into the CBPC matrix. SEM showed that waste forms are layered when compacted by uniaxial press and are homogeneous when compacted by harmonic press.

Hazardous Waste↗
PubMed2000

Cloning and characterization of bonnet monkey GnRH receptor.

Gonadotrophin releasing hormone (GnRH) plays an important role in the reproductive processes of both males and females. It is synthesized by the hypothalamus and binds to a specific receptor on the pituitary to bring about the release of the gonadotrophins, lutineizing hormone and follicle stimulating hormone, which in turn bring about the release of the gonadal steroids. Although the structure of the GnRH receptor (GnRHR) has been elucidated from a number of sources, no information is available about the receptor from the non-human primate species. Here we report the cloning and characterization of the receptor from the pituitary of the bonnet monkey. Antiserum to a bacterially expressed recombinant fragment was used in Western blot analysis and fluorescence microscopy to demonstrate the presence of GnRHR in both human and monkey placentae and pituitary.

Amino Acid Sequence↗
PubMed1999

Regulation of an 8-kDa peptide involved in testosterone production by luteinizing hormone in rat Leydig cells.

Results of Western blot analysis carried out with an interstitial cell extract from male guinea pig and ovarian extract from immature female rats administered equine chorionic gonadotropin (eCG) provide supportive evidence to our earlier suggestion that an 8-kDa peptide is involved in acquisition of steroidogenic capacity by the rat Leydig cells. It was found that though the signal was observed in other tissues such as liver, kidney and lung which do not produce gonadal hormones, the peptide was modulated only by lutenizing hormone (LH) in the rat Leydig cells.

Animals↗
PubMed1999

Progesterone receptor expression in the human placenta.

The presence of progesterone receptors (PR) in the human placenta has been demonstrated using the reverse transcriptase-polymerase chain reaction technique. It was observed that the amount of PR in the human placenta is less during late gestation. Electrophoretic mobility shift assays with nuclear extract isolated from the first trimester and term revealed three complexes when incubated with [32P]dCTP-labelled progesterone response element, and, in competition with unlabelled progesterone response element, the formation of all three complexes was inhibited. When supershift analysis of these complexes was carried out using antibodies which cross-react with both the A and B types of the PR or only with the B type receptor, only the A-form of PR was detected in the human placenta.

Antibodies, Monoclonal↗
PubMed1999

Studies on LH modulated 8kDa peptide involved regulation of testosterone production in rat Leydig cells.

AIM: To demonstrate the role of the 8 kDa peptide in regulation of testosterone production by rat Leydig cells. METHODS: A peptide similar to 8 kDa peptide purified from immature rat Leydig cells was isolated and purified from rat lung cytosol. Immunological and structural similarity between the peptides purified from lung and Leydig cells was established by Western blot and tryptic map comparison respectively. RESULTS: Addition of the 8 kDa peptide 10, 50, 100, and 150 microg decreased the production of testosterone in Leydig cells dose-dependently. But the addition of the peptide 150 microg along with hCG had no effect on hCG-stimulated increase in testosterone production. CONCLUSION: In vitro addition of the peptide purified from lung cytosol to adult rat Leydig cells resulted in a concentration-dependent decrease in basal testosterone production although it had no effect on hCG-stimulated testosterone production.

Animals↗
PubMed1999

Ethane dimethylsulphonate selectively destroys Leydig cells in the adult bonnet monkeys (Macaca radiata).

AIM: To study the effect of intratesticular administration of ethane-1,2-dimethylsulphonate (EDS) which has been extensively used to selectively destroy Leydig cells in rats and study the role of gonadotropin in regulation of differentiation of Leydig cells (LC) in the adult male bonnet monkey. METHODS AND RESULTS: In vitro studies with cultured interstitial cells isolated from monkey testis revealed an inhibitory effect of EDS on LC as assessed by decrease in testosterone production. Intratesticular administration of EDS (5, 10, 20, 50 mg/testis) resulted in a dose-dependent rapid decrease in serum testosterone levels, with a 65% decrease with 5 mg of EDS by the 3rd day, which returned to control levels by the 45th day. EDS treatment resulted in a significant decrease in testicular testosterone. In addition a significant decrease in [125I]hCG binding and phenylesterase activity in the interstitial cells was noticed. Histological analysis of the testes on the 5th day after administration of EDS revealed an interstitium devoid of LC indicating the destructive action of EDS. CONCLUSION: The monkey LC are sensitive to destructive action of EDS.

Animals↗
PubMed1998

Regulation of low density lipoprotein receptor mRNA levels by estradiol 17beta and chorionic gonadotropin in human placenta.

Inhibition of synthesis of estradiol 17beta by the addition of inhibitors of aromatase, a key enzyme in the biosynthesis of estradiol 17beta, or addition of tamoxifen - an estrogen receptor antagonist, to human placental minces resulted in an increase in the level of LDL-receptor mRNA. This increase could be blocked by the simultaneous addition of estradiol 17beta. A concentration dependent effect of estradiol 17beta on the level of LDL-receptor mRNA was seen both in first trimester, and term placenta. Addition of human chorionic gonadotropin (hCG) to term placental minces also increased the LDL-receptor mRNA levels. When hCG and cycloheximide were added together, an additive effect was observed. The results obtained in this study suggest that the LDL-receptor mRNA levels in the human placenta are regulated by estradiol 17beta and hCG.

Aminoglutethimide↗
PubMed1997

Expression of progesterone receptor mRNA in the first trimester human placenta.

The human placenta produces large quantities of progesterone, the function and target tissues of action of which during pregnancy are not completely understood. Although it has been suggested that placenta itself could be a potential target tissue for progesterone action, there is no conclusive evidence for the presence of progesterone receptors in the human placenta. We provide evidence for the expression of progesterone receptor mRNA in the human placenta by reverse transcription coupled to polymerase chain reaction (RT-PCR). This was further confirmed by Southern hybridization, restriction analysis and sequencing of the PCR amplified fragment.

Base Sequence↗
PubMed1997

Regulation of progesterone biosynthesis in the human placenta by estradiol 17 beta and progesterone.

Ex vivo addition of estradiol 17 beta to first trimester or term human placental minces caused a significant increase in the quantity of progesterone produced. Addition of an aromatase inhibitor, CGS 16949 A, or the estrogen receptor antagonist, ICI 182780, significantly inhibited progesterone production confirming the role of estradiol 17 beta in the regulation of progesterone synthesis in human placenta. RU 486 and ZK 98299, which are antagonists of progesterone receptor, significantly modulated progesterone synthesis in the human placenta but exhibited paradoxical effects on the first trimester and term placenta. We conclude that progesterone synthesis in the human placenta is regulated by estradiol 17 beta and progesterone. This is the first report providing evidence for autoregulation of progesterone synthesis in the human placenta.

Estradiol↗
PubMed1997

Identification of actin as an estradiol 17-beta-stimulated protein in the human placenta.

Addition of estradiol 17-beta to first trimester human placental minces resulted in an increased synthesis of a protein of apparent molecular weight 45 kDa. The specific involvement of estrogen in the stimulation of this protein was established by demonstrating a reduction in the level of this protein by the addition of CGS 16949 A, an inhibitor of aromatase, a key enzyme in the biosynthesis of estradiol 17-beta and ICI 182,780, an estrogen receptor antagonist. The protein was purified to homogeneity and N-terminal sequencing of two of the internal peptides obtained by enzymatic digestion of the protein, as well as the absence of a free N-terminal indicated that it could be actin. This was confirmed by Western blotting using commercially available actin antiserum. The role of estradiol 17-beta in the stimulation of actin synthesis in human placenta was also established by monitoring the quantitative inhibition of DNase I by actin.

Actins↗