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      Analysis of 20alpha-hydroxysteroid dehydrogenase expression in the corpus luteum of the buffalo cow: effect of prostaglandin F2-alpha treatment on circulating 20alpha-hydroxyprogesterone levels

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          Abstract

          Background

          During female reproductive cycles, a rapid fall in circulating progesterone (P4) levels is one of the earliest events that occur during induced luteolysis in mammals. In rodents, it is well recognized that during luteolysis, P4 is catabolized to its inactive metabolite, 20alpha-hydroxyprogesterone (20alpha-OHP) by the action of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) enzyme and involves transcription factor, Nur77. Studies have been carried out to examine expression of 20alpha-HSD and its activity in the corpus luteum (CL) of buffalo cow.

          Methods

          The expression of 20alpha-HSD across different bovine tissues along with CL was examined by qPCR analysis. Circulating P4 levels were monitored before and during PGF2alpha treatment. Expression of 20alpha-HSD and Nur77 mRNA was determined in CL at different time points post PGF2alpha treatment in buffalo cows. The chromatographic separation of P4 and its metabolite, 20alpha-OHP, in rat and buffalo cow serum samples were performed on reverse phase HPLC system. To further support the findings, 20alpha-HSD enzyme activity was quantitated in cytosolic fraction of CL of both rat and buffalo cow.

          Results

          Circulating P4 concentration declined rapidly in response to PGF2alpha treatment. HPLC analysis of serum samples did not reveal changes in circulating 20alpha-OHP levels in buffalo cows but serum from pseudo pregnant rats receiving PGF2alpha treatment showed an increased 20alpha-OHP level at 24 h post treatment with accompanying decrease in P4 concentration. qPCR expression of 20alpha-HSD in CL from control and PGF2alpha-treated buffalo cows showed higher expression at 3 and 18 h post treatment, but its specific activity was not altered at different time points post PGF2alpha treatment. The Nur77 expression increased several fold 3 h post PGF2alpha treatment similar to the increased expression observed in the PGF2alpha-treated pseudo pregnant rats which perhaps suggest initiation of activation of apoptotic pathways in response to PGF2alpha treatment.

          Conclusions

          The results taken together suggest that synthesis of P4 appears to be primarily affected by PGF2alpha treatment in buffalo cows in contrast to increased metabolism of P4 in rodents.

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          Most cited references 43

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          Requirement for the orphan steroid receptor Nur77 in apoptosis of T-cell hybridomas.

          Apoptosis is a phenomenon observed during development of many cell types in many organisms. It is an internal, programmed cell death characterized by DNA fragmentation into nucleosome-size pieces. Anti-CD3-induced apoptosis in T-cell hybridomas and immature thymocytes requires new gene transcription and may be related to negative selection during T-cell development. Using subtractive hybridization, we isolated a complementary DNA clone encoding the orphan steroid receptor Nur77 (refs 7-9). It shows different patterns of messenger RNA induction between apoptotic and stimulated T cells. We report here the use of gel shift analysis to demonstrate that the Nur77 protein is present at high levels in apoptotic T-cell hybridomas and apoptotic thymocytes, but not in growing T cells or stimulated splenocytes. A Nur77 dominant negative protected T-cell hybridomas from activation-induced apoptosis. Hence Nur77 is necessary for induced apoptosis in T-cell hybridomas.
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            Mechanisms controlling the function and life span of the corpus luteum.

            The primary function of the corpus luteum is secretion of the hormone progesterone, which is required for maintenance of normal pregnancy in mammals. The corpus luteum develops from residual follicular granulosal and thecal cells after ovulation. Luteinizing hormone (LH) from the anterior pituitary is important for normal development and function of the corpus luteum in most mammals, although growth hormone, prolactin, and estradiol also play a role in several species. The mature corpus luteum is composed of at least two steroidogenic cell types based on morphological and biochemical criteria and on the follicular source of origin. Small luteal cells appear to be of thecal cell origin and respond to LH with increased secretion of progesterone. LH directly stimulates the secretion of progesterone from small luteal cells via activation of the protein kinase A second messenger pathway. Large luteal cells are of granulosal cell origin and contain receptors for PGF(2alpha) and appear to mediate the luteolytic actions of this hormone. If pregnancy does not occur, the corpus luteum must regress to allow follicular growth and ovulation and the reproductive cycle begins again. Luteal regression is initiated by PGF(2alpha) of uterine origin in most subprimate species. The role played by PGF(2alpha) in primates remains controversial. In primates, if PGF(2alpha) plays a role in luteolysis, it appears to be of ovarian origin. The antisteroidogenic effects of PGF(2alpha) appear to be mediated by the protein kinase C second messenger pathway, whereas loss of luteal cells appears to follow an influx of calcium, activation of endonucleases, and an apoptotic form of cell death. If the female becomes pregnant, continued secretion of progesterone from the corpus luteum is required to provide an appropriate uterine environment for maintenance of pregnancy. The mechanisms whereby the pregnant uterus signals the corpus luteum that a conceptus is present varies from secretion of a chorionic gonadotropin (primates and equids), to secretion of an antiluteolytic factor (domestic ruminants), and to a neuroendocrine reflex arc that modifies the secretory patterns of hormones from the anterior pituitary (most rodents).
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              Apoptotic signals delivered through the T-cell receptor of a T-cell hybrid require the immediate-early gene nur77.

              Engagement of the T-cell antigen receptor (TCR) on immature thymic T cells induces death by apoptosis. Although several lines of evidence indicate that apoptosis requires de novo gene expression, little is known about the molecular pathways that mediate this response. Here we show that nur77 (refs 4-7), a zinc-finger transcription factor, is expressed in response to TCR engagement in immature T cells and T-cell hybrids. Antisense inhibition of nur77 expression prevents apoptosis in TCR-stimulated cells. nur77 is also expressed in response to mitogens, but in this case transcription is regulated by 5' upstream elements that are distinct from those used for induction of apoptosis. In addition, polyadenylation is only observed on nur77 transcripts found in condemned cells. These data support a role for nur77 in cell death that may be distinct from that of activation.
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                Author and article information

                Journal
                Reprod Biol Endocrinol
                Reprod. Biol. Endocrinol
                Reproductive Biology and Endocrinology : RB&E
                BioMed Central
                1477-7827
                2013
                11 December 2013
                : 11
                : 111
                Affiliations
                [1 ]Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India
                Article
                1477-7827-11-111
                10.1186/1477-7827-11-111
                3878843
                24330451
                Copyright © 2013 Sudeshna et al.; licensee BioMed Central Ltd.

                This is an open access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Categories
                Research

                Human biology

                corpus luteum, p4, 20α-hsd, buffalo cow, pgf2α, 20α-ohp, nur77

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