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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The objective of this study was to examine the effects of different culture media used for maturation of bovine oocytes on in vitro embryo development following in vitro fertilization. Oocytes were aspirated from 2-5 mm follicles of ovaries collected at a local abattoir. The oocyte-cumulus complexes (OCCs) were cultured for 23-25 h in one of seven commercially available media supplemented with 6 mg/ml bovine serum albumin (BSA), 0.25 mM pyruvate, 10 micrograms/ml luteinizing hormone (LH), 0.5 microgram/ml follicle-stimulating hormone (FSH), and 1 microgram/ml estradiol. After maturation for 23-25 h, all eggs were subjected to the same in vitro fertilization protocol using modified TALP medium and subsequently cultured in the same serum-free embryo culture medium (HECM-1/BSA) for 8 days, after which embryo development was assessed. Five media (SFRE, MEM alpha, TCM199, MEM alpha/+, RPMI:MEM alpha) better supported normal oocyte maturation as determined by embryo development to the two-cell (76-82%), morula/blastocyst (25-32%), and blastocyst (12-19%) stages. Oocytes that were matured in Waymouth's medium MB 752/l or Ham's F-12 had a significantly reduced incidence of cleavage to the two-cell stage (52% and 37%, respectively), which was not attributed to failure of fertilization. Of the eggs that did cleave to the two-cell stage in these two media, 27% and 9% developed to morulae/blastocysts but only 6% and 3%, respectively, developed into blastocysts.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1992 Jan
PMID:Effect of oocyte maturation medium on in vitro development of in vitro fertilized bovine embryos. 156 30

The priming effect of LHRH in vitro (which results in increased responsiveness of gonadotropes to both LHRH receptor-mediated and receptor-independent stimuli) is brought about by an unknown mechanism. The present results indicate that induction of the LHRH priming effect is inhibited in a concentration-dependent manner by the protein kinase C (PKC) inhibitors staurosporine, K252a, H7 and by the novel highly-selective PKC inhibitor, Ro 31-8220. In contrast, a range of other compounds that are relatively selective inhibitors of other kinases such as tyrosine kinases and Ca2+/calmodulin-dependent kinases were unable to prevent priming. The PKC inhibitors prevented priming without affecting initial LHRH-induced gonadotropin secretion. Thus, the priming-elicited increment in secretion was selectively removed, restoring hormone release to the level measured during an initial response to LHRH. Similar results were obtained on different days of the estrous cycle where the magnitude of the priming effect varies. Experiments on the time course of PKC inhibitor action revealed that the critical period was in the induction of the priming effect, not its expression. The PKC inhibitors had neither acute nor delayed effects on gonadotropin secretion induced by ionomycin. Staurosporine, K252a and Ro 31-8220 inhibited LHRH priming with identical potencies to their inhibition of phorbol ester-induced gonadotropin secretion. The reduced potency of H7 seen on LHRH priming compared to phorbol ester-induced gonadotropin release parallels results seen with this inhibitor on phorbol ester-induced secretion of growth hormone (Johnson and Mitchell (1989) Biochem. Soc. Trans. 17, 751-752) and on the pharmacological characteristics of PKCs partially purified from anterior pituitary tissue. In all aspects of this study, effects on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion appeared to be entirely similar.
Mol Cell Endocrinol 1992 Jun
PMID:The priming effect of luteinizing hormone-releasing hormone (LHRH) but not LHRH-induced gonadotropin release, can be prevented by certain protein kinase C inhibitors. 163 16

In Sertoli cells from 21-day-old rats, the expression of the mRNA encoding the alpha-subunit of inhibin, and the production of immunoreactive inhibin are stimulated by follicle-stimulating hormone (FSH). In contrast, the amount of beta B-subunit mRNA is not increased after FSH treatment of the cells, and the ratio between bioactive and immunoactive inhibin decreases after stimulation with FSH. These data suggest that the beta B-subunit is the limiting factor in the production of bioactive inhibin. The aim of the present experiments was to investigate the effect of changes in the amount of beta B-subunit mRNA on the production of bioactive and immunoreactive inhibin. During early postnatal testicular development, the relative amounts of the 4.2 kb and 3.5 kb mRNAs encoding the beta B-subunit of inhibin changed markedly. The meaning of this changing ratio between beta B-subunit mRNAs is not clear, since both mRNAs are actively translated, as demonstrated by polysomal analysis. The total amount of beta B-subunit mRNA correlated with the in vitro production of bioactive inhibin as published earlier. Prolonged stimulation of cultured Sertoli cells from 14-day-old rats with 4 beta-phorbol 12-myristate 13-acetate (PMA) caused a decreased expression of the beta B-subunit mRNAs, presumably by down-regulation of protein kinase C. A similar effect was obtained after addition of the calcium ionophore A23187. Concomitantly, a decreased production of bioactive inhibin was observed. Furthermore, Western blotting revealed that secretion of the 32 kDa inhibin alpha beta-dimer was decreased, whereas secretion of the combination of the C-terminal part with the pro-region of the alpha-subunit was increased. It is concluded that the level of the beta B-subunit of inhibin is rate-limiting for the production of bioactive inhibin in cultured Sertoli cells, and that its expression can be influenced by modulation of protein kinase C, and/or intracellular calcium levels.
Mol Cell Endocrinol 1992 Jun
PMID:Regulation of inhibin beta B-subunit mRNA expression in rat Sertoli cells: consequences for the production of bioactive and immunoreactive inhibin. 163 19

Receptor activated adenylate cyclase acts as a major transmembrane signalling system. It is widely accepted that upon binding to its receptor, follicle-stimulating hormone (FSH) activates the cAMP-dependent pathway which in turn mediates FSH-induced estradiol production in Sertoli cells. Studies utilizing several chemically derived variants of FSH have demonstrated that these variants bind to the FSH receptors with equal avidity but differ in their ability to activate cAMP-dependent pathways. Since cAMP is believed to be the second messenger responsible for FSH signal transduction, we tested two hypotheses: (1) that the effects of different oFSH variants on cAMP production and aromatase induction (as measured by estradiol production) would be in parallel; and (2) that deglycosylated ovine FSH (DG-oFSH) would antagonize the ability of intact oFSH to stimulate aromatase induction, similar to its reported antagonistic effect on cAMP production. Immature rat (7- to 10-day-old) Sertoli cells were cultured and the effects of several different oFSH variants on cAMP production and/or aromatase induction were tested. The variants tested were native oFSH, DG-oFSH, asialo oFSH (AS-oFSH), a recombinant of intact LH alpha and FSH beta (alpha + beta) and a recombinant of deglycosylated LH alpha and intact FSH beta (DG alpha + beta). Both native oFSH and alpha + beta recombinant at relatively large doses (10 ng) elicited a significant increase in extracellular cAMP accumulation as well as total cAMP production. In contrast, DG-oFSH did not produce an increase in cAMP even at 10-fold higher doses than native oFSH. Intracellular cAMP concentrations did not increase following stimulation with native oFSH, DG-oFSH or DG alpha + beta. In contrast to the divergent effects of oFSH and DG-oFSH on cAMP production all variants of oFSH stimulated estradiol production from Sertoli cells albeit with varying potencies. The sensitivity (minimal effective dose) and ED50 (dose at which half maximal response is achieved) of the estradiol (E2) response curve to increasing concentrations of native oFSH were 0.025 +/- 0.01 and 0.33 +/- 0.05 ng, respectively. Asialo-oFSH (AS-oFSH) increased E2 production with a potency (comparative dose required for effect) similar to that of native oFSH.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Cell Endocrinol 1991 Aug
PMID:Follicle-stimulating hormone signal transduction: role of carbohydrate in aromatase induction in immature rat Sertoli cells. 165 61

Recent evidence has been presented that follicle-stimulating hormone (FSH) stimulates the induction of granulosa cell c-fos protooncogene mRNA in vivo (Pennybacker and Herman (1989) J. Cell Biol. 109, 151A; Delidow et al. (1990) Endocrinology 126, 2302-2306), yet the mechanisms by which FSH induces c-fos mRNA expression have not been delineated. To elucidate the mechanisms of FSH-dependent c-fos mRNA expression, we measured the time and dose dependence of c-fos mRNA levels using Northern blot analysis in intact ovaries and cultured granulosa cells in response to FSH. In intact ovaries, FSH-induced c-fos mRNA expression was time dependent with maximal expression at 90 min post FSH injection, while in cultures of granulosa cells obtained from estrogen-primed immature female rats, c-fos mRNA levels were highest after 30 min exposure to FSH and at a concentration of 100 ng/ml. Neither 8-bromo adenosine 3',5'-cyclic monophosphate (8-br-cAMP), at doses ranging from 0.1 to 10 mM, nor 100 microM forskolin (in the presence or absence of 200 microM isobutyl-methylxanthine) or luteinizing hormone (LH, 100 ng/ml) were able to mimic FSH-induced c-fos mRNA expression in granulosa cell cultures. However, tetradecanoyl-13-phorbol acetate (TPA, 200 nM) was able to induce c-fos mRNA expression. The protein kinase C (PKC) inhibitors H-7 (0.3-30 microM) and staurosporine (0.75 micrograms/ml) blocked FSH-induced c-fos mRNA expression in cultured granulosa cells while HA 1004, an inhibitor of cGMP- and cAMP-dependent protein kinases at 30 microM had no effect on TPA-induced c-fos expression, and only minimally inhibited FSH-induced c-fos expression. Both FSH (100 ng/ml) and forskolin (3 microM) increased progesterone production in cultured granulosa cells. These data support the hypothesis that FSH specifically induces c-fos mRNA expression by a PKC-dependent mechanism and that the cAMP arm of the FSH response pathway is operant in these cells.
Mol Cell Endocrinol 1991 Sep
PMID:Follicle-stimulating hormone increases c-fos mRNA levels in rat granulosa cells via a protein kinase C-dependent mechanism. 165 43

The gene for the beta subunit of porcine LH (LH-beta) was cloned from a genomic library constructed in EMBL3. The nucleotide sequence was determined for the entire gene transcriptional unit of porcine LH-beta in addition to 1277 and 372 bp of the 5'- and 3'-flanking regions respectively. Southern blot analysis of the porcine genomic DNA indicated that the LH-beta gene is present as a single copy. The transcriptional unit of porcine LH-beta spanned 1107 bp and contained three exons interrupted by two introns of 326 and 289 bp. The short untranslated sequence in the first exon and the location of the exon/intron junctions at amino acid residues -16/-15 and +41/+42 were highly conserved in the rat, human and bovine LH-beta genes. In the 5'-flanking region, one TATA box and two CCAAT boxes were present. The steroid-responsive element was not found up to 1277 bases upstream of the transcription start site. The potential AP-2 factor-responsive elements appeared nine times within the sequence that was determined, and four of them were located in the 5'-flanking region. Two distal AP-2 elements were arranged in an inverted repeat forming a 16 bp palindromic sequence. This feature suggested that hypothalamic gonadotrophin-releasing hormone stimulates expression of the LH-beta gene, predominantly by a signal-transduction system with the protein kinase C cascade and a mediator, the AP-2 factor. A further characteristic feature of the porcine LH-beta gene was the presence of clusters of GC boxes and CACCC elements in the 5'-flanking region and the downstream sequence. Co-existence of these regulatory elements with other elements, such as the AP-2 element or CCAAT box, was also found. The porcine LH-beta gene shows a structure distinct from the porcine FSH-beta and common alpha genes, which are counterparts of the LH-beta gene, reflecting differential control of their synthesis during gametogenesis.
J Mol Endocrinol 1990 Oct
PMID:The gene for the beta subunit of porcine LH: clusters of GC boxes and CACCC elements. 170 Oct 88

The conformation of the common alpha-subunit of human glycoprotein hormones, luteinizing hormone (hLH), follicle-stimulating hormone (hFSH), thyroid-stimulating hormone (hTSH) and chorionic gonadotropin (hCG) was probed using a highly specific polyclonal antiserum against the alpha-subunit of hCG and several monoclonal antibodies (MAbs) produced against hCG which recognized the alpha-subunit in free and combined form. The alpha-subunit was found to be conformationally altered (compared to its conformation in the isolated state) when it was in combination with various beta-subunits as indicated by shifts in the displacement curves of binding of [125I]hCG alpha to the polyclonal antiserum. The extent of the change was dependent on the beta-subunit present with minimum change being observed with hLH beta, intermediate with hCG beta and maximum change with hFSH and TSH beta-subunits. However, the affinity constants of this antiserum for all four hormones were nearly similar. Further, it was also found that binding of any one of the glycoprotein hormones to this antibody could be completely inhibited by any other hormone suggesting that the conformation of the alpha-subunit in all the four hormones is probably very similar. This was further investigated using five hCG MAbs capable of recognizing the alpha-subunit, but with different epitope specificities. All these MAbs could recognize all the four hormones suggesting the presence of the epitopes in these proteins. These epitopes were conformation specific since the MAbs did not bind reduced and carboxymethylated alpha-subunit. Displacement analysis using [125I]hCG as the tracer showed that two epitopes have nearly the same conformation in all the four hormones, while two were partially modified depending on the beta-subunit present. Based on these results, it is concluded that the alpha-subunit of glycoprotein hormones has nearly the same conformation, though subtle differences do exist.
Mol Cell Endocrinol 1990 Jul 30
PMID:Conformation of the alpha-subunit of glycoprotein hormones: a study using polyclonal and monoclonal antibodies. 170 93

While the regulation of gonadotrophin secretion by gonadotrophin-releasing hormone (GnRH) has been well documented in both rats and sheep, its role in the synthesis of gonadotrophin subunits remains unclear. We have investigated the effects of the specific inhibition of GnRH by a GnRH agonist on the expression of gonadotrophin subunit genes and the subsequent storage and release of both intact hormones and free alpha subunit. Treatment with GnRH agonist for 6 weeks abolished pulsatile LH secretion, reduced plasma concentrations of FSH and prevented GnRH-induced release of LH and FSH. This was associated with a reduction of pituitary LH-beta mRNA and FSH-beta mRNA levels (to 5 and 30% of luteal control values respectively), but not alpha mRNA which was significantly increased (75% above controls). While there was a small decrease in the pituitary content of FSH (30% of controls), there was a drastic reduction in LH pituitary content (3% of controls). In contrast to the observed rise in alpha mRNA, there was a decrease in free alpha subunit in both the pituitary and plasma (to 30 and 80% of control levels). These results suggest that, while GnRH positively regulates the expression of both gonadotrophin beta-subunit genes, it can, under certain circumstances, negatively regulate alpha-subunit gene expression. Despite the complete absence of LH and FSH in response to GnRH, there remained a basal level of beta-subunit gene expression and only a modest reduction (50%) in the plasma levels of both FSH and LH, suggesting that there is a basal secretory pathway.(ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Endocrinol 1991 Aug
PMID:Gonadotrophin-releasing hormone modulation of gonadotrophins in the ewe: evidence for differential effects on gene expression and hormone secretion. 171 38

The effects of dibutyryl cyclic AMP [Bu)2cAMP) and phorbol ester (TPA), in the absence or presence of follicle-stimulating hormone (FSH) and/or testosterone, on the development of tight junctions by immature rat Sertoli cells (Sc) were investigated in vitro using the two-compartment culture system. The tight junction status was evaluated by repeated measurements of transepithelial electrical resistance (TER). Untreated cell monolayers developed stable TER of approximately 120 omega cm2 during 3 days of culture. Continuous presence of FSH (200 ng/ml) from day 1 onward significantly increased the TER up to approximately 300 omega cm2 after a transient (24-36 h) delay. The initial delay was prolonged to 3-4 days by the addition of 1-methyl-3-isobutylxanthine (MIX) (0.2 mM), whereas the subsequent increase of TER was significantly potentiated by the concomitant presence of testosterone (10 microM). Cholera toxin (CHT; 10 ng/ml) and forskolin (FR; 50 microM) mimicked these FSH effects. (Bu)2cAMP, at concentrations which maximally stimulated immunoactive inhibin secretion (100-500 microM), inhibited the initial TER increase and significantly decreased the TER level when added on days 1 and 5 of culture, respectively. In contrast, low concentrations of (Bu)2cAMP (4-20 microM) consistently stimulated the TER development, mimicking the stimulatory phase of FSH action. TPA (100 nM) alone had no effect on TER development, but potentiated the stimulatory effect of testosterone in a manner similar to FSH, CHT, FR or low concentrations of (Bu)2cAMP. These results demonstrate, for the first time, a concentration-dependent, dual effect of exogenous cAMP on the Sc function.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Endocrinol 1991 Nov
PMID:Effects of cyclic AMP and phorbol ester on transepithelial electrical resistance of Sertoli cell monolayers in two-compartment culture. 172 79

The regulation by FSH (follitropin; follicle-stimulating hormone) of FSH receptor mRNA and protein (FSH binding) was studied using cultured Sertoli cells isolated from 21-day-old rats. FSH induced a dose-dependent and almost complete down-regulation of receptor mRNA at 4 h after addition of the hormone. At subsequent time points (16 h and later) the FSH receptor mRNA levels had returned close to control values. The effect of FSH was mimicked by dibutyryl cyclic AMP (dbcAMP) and forskolin, and the phosphodiesterase inhibitor methyl-isobutylxanthine (MIX) prolonged the FSH action. These findings indicate that the effect of FSH on its receptor mRNA was mediated by cAMP. A down-regulatory effect of FSH and dbcAMP on FSH receptor mRNA was also observed in the presence of the protein synthesis inhibitor cycloheximide, suggesting a direct effect of FSH/dbcAMP on the expression of the FSH receptor gene. Transcriptional run-on experiments revealed that FSH did not inhibit initiation of the FSH receptor gene; hence a post-transcriptional mechanism is involved. Binding of 125I-FSH to the cultured Sertoli cells was rapidly (4 h) decreased when the cells were incubated with FSH or FSH in combination with MIX. This effect can be explained by ligand-induced receptor sequestration. In contrast, incubation of Sertoli cells with dbcAMP had no effect on binding of 125I-FSH after 4 h, but resulted in a 60% loss of FSH binding sites after 24 h, probably caused by decreased mRNA expression. In conclusion, FSH receptor down-regulation in Sertoli cells is effected not only by the well-documented ligand-induced loss of receptors from the plasma membrane, but also involves a cAMP-mediated decrease of FSH receptor mRNA through a post-transcriptional mechanism.
Mol Cell Endocrinol 1991 Jul
PMID:Follitropin receptor down-regulation involves a cAMP-dependent post-transcriptional decrease of receptor mRNA expression. 172 86


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