Increased Progesterone Secretion Research Articles

Increased progesterone production in cumulus-oocyte complexes of female mice sired by males with the Y-chromosome long arm deletion and its potential influence on fertilization efficiency.

It was revealed previously that B10.BR(Y(del)) females sired by males with the Y-chromosome long arm deletion differ from genetically identical B10.BR females sired by males with the intact Y chromosome. This is interpreted as a result of different epigenetic information which females of both groups inherit from their fathers. In the following study, we show that cumulus-oocyte complexes ovulated by B10.BR(Y(del)) females synthesize increased amounts of progesterone, which is important sperm stimulator. Because their extracellular matrix is excessively firm, the increased progesterone secretion belongs presumably to factors that compensate this feature enabling unchanged fertilization ratios. Described compensatory mechanism can act only on sperm of high quality, presenting proper receptors. Indeed, low proportion of sperm of Y(del) males that poorly fertilize B10.BR(Y(del)) oocytes demonstrates positive staining of membrane progesterone receptors. This proportion is significantly higher for sperm of control males that fertilize B10.BR(Y(del)) and B10.BR oocytes with the same efficiency.

Manipulation of the periovulatory sex steroidal milieu affects endometrial but not luteal gene expression in early diestrus Nelore cows

In beef cattle, the ability to conceive has been associated positively with size of the preovulatory follicle (POF). Proestrus estradiol and subsequent progesterone concentrations can regulate the endometrium to affect receptivity and fertility. The aim of the present study was to verify the effect of the size of the POF on luteal and endometrial gene expression during subsequent early diestrus in beef cattle. Eighty-three multiparous, nonlactating, presynchronized Nelore cows received a progesterone-releasing device and estradiol benzoate on Day–10 (D−10). Animals received cloprostenol (large follicle-large CL group; LF-LCL; N = 42) or not (small follicle-small CL group; SF-SCL; N = 41) on D−10. Progesterone devices were withdrawn and cloprostenol administered 42 to 60 hours (LF-LCL) or 30 to 36 hours (SF-SCL) before GnRH treatment (D0). Tissues were collected at slaughter on D7. The LF-LCL group had larger (P < 0.0001) POF (13.24 ± 0.33 mm vs. 10.76 ± 0.29 mm), greater (P < 0.0007) estradiol concentrations on D0 (2.94 ± 0.28 pg/mL vs. 1.27 ± 0.20 pg/mL), and greater (P < 0.01) progesterone concentrations on D7 (3.71 ± 0.25 ng/mL vs. 2.62 ± 0.26 ng/mL) compared with the SF-SCL group. Luteal gene expression of vascular endothelial growth factor A, kinase insert domain receptor, fms-related tyrosine kinase 1, steroidogenic acute regulatory protein, cytochrome P450, family 11, subfamily A, polypeptide 1, and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 7 was similar between groups. Endometrial gene expression of oxytocin receptor and peptidase inhibitor 3, skin-derived was reduced, and estrogen receptor alpha 2, aldo-keto reductase family 1, member C4, and lipoprotein lipase expression was increased in LF-LCL versus SF-SCL. Results support the hypothesis that the size of the POF alters the periovulatory endocrine milieu (i.e., proestrus estradiol and diestrus progesterone concentrations) and acts on the uterus to alter endometrial gene expression. It is proposed that the uterine environment and receptivity might also be modulated. Additionally, it is suggested that increased progesterone secretion of cows ovulating larger follicles is likely due to increased CL size rather than increased luteal expression of steroidogenic genes.

Influence of gonadotropins on steroidogenesis by large follicle buffalo granulosa cells under in vitro serum-free conditions

The study was made to examine the effects of FSH and LH on estradiol–17β and progesterone production by large follicle derived buffalo granulosa cells cultured under completely serum-free conditions. Granulosa cells (3×105) from large (&gt;9 mm diameter) follicles were cultured for 24 h in 48-well plates coated with 3.3μg/cm2 fibronectin in DMEM: Nutrient mixture F-12 Ham (1:1 ratio) supplemented with 10-7 M androstenedione, 5 mg/ml human apo-transferrin and 0.1% BSA, in the presence or absence of FSH or LH (1 to 64 ng/ml each). FSH had no effect on estradiol-17β output but at 4 and 8 ng/ml increased progesterone secretion by granulosa cells. All doses of LH tested had stimulatory effect on estradiol-17β while progesterone secretion by granulosa cells was unaffected.

Beneficial effect of resveratrol on bovine oocyte maturation and subsequent embryonic development after in vitro fertilization

To analyze the potential beneficial effects and mechanisms of action of resveratrol on the maturation of bovine oocytes that were incubated in different concentrations of resveratrol (0.1, 1.0, or 10.0 μM) as germinal vesicle-stage oocytes. In vitro prospective study. University research laboratory. Animal models for human studies. In vitro culture in the presence of various concentrations of the antioxidant resveratrol. Parameters of hormone levels, oocyte nuclear maturation, cumulus expansion, levels of intracellular glutathione and reactive oxygen species, embryonic cleavage, blastocyst formation, gene expression associated with mature bovine oocytes and cumulus cells, and level of sirtuin 1 gene expression. Resveratrol statistically significantly increased progesterone secretion and decreased estradiol-17β secretion by cumulus cells. The elevated levels of progesterone activated the Mos/MEK/p42 mitogen-activated protein kinase (MAPK) cascade in the oocytes. At a concentration of 1.0 μM, resveratrol statistically significantly improved cumulus expansion, polar body formation, the (hatched) blastocyst rate, and the mean number of cells/blastocysts. Meanwhile, resveratrol statistically significantly reduced the level of reactive oxygen species (ROS) and increased the level of glutathione (GSH). For the first time, the expression of the sirtuin-1 gene was identified in granulosa cells, cumulus cells, oocytes, and blastocysts. Further studies revealed that resveratrol promoted sirtuin-1 gene expression. Resveratrol promoted bovine oocyte maturation and subsequent post-in vitro fertilization embryonic development by inducing progesterone secretion and an antioxidant effect, probably in a manner dependent on sirtuin-1.

Cellular and functional characterization of buffalo (Bubalus bubalis) corpus luteum during the estrous cycle and pregnancy

In the present paper, cellular composition of buffalo corpus luteum (CL) with its functional characterization based on 3β-HSD and progesterone secretory ability at different stages of estrous cycle and pregnancy was studied. Buffalo uteri along with ovaries bearing CL were collected from the local slaughter house. These were classified into different stages of estrous cycle (Stage I, II, III and IV) and pregnancy (Stage I, II and III) based on morphological appearance of CL, surface follicles on the ovary and crown rump length of conceptus. Luteal cell population, progesterone content and steroidogenic properties were studied by dispersion of luteal cells using collagenase type I enzyme, RIA and 3β-HSD activity, respectively. Large luteal cells (LLC) appeared as polyhedral or spherical in shape with a centrally placed large round nucleus and an abundance of cytoplasmic lipid droplets. However, small luteal cells (SLC) appeared to be spindle shaped with an eccentrically placed irregular nucleus and there was paucity of cytoplasmic lipid droplets. The size of SLC (range 12–23μm) and LLC (range 25–55μm) increased (P<0.01) with the advancement of stage of estrous cycle and pregnancy. The mean progesterone concentration per gram and per CL increased (P<0.01) from Stage I to III of estrous cycle with maximum concentration at Stage III of estrous cycle and pregnancy. The progesterone concentration decreased at Stage IV (day 17–20) of estrous cycle coinciding with CL regression. Total luteal cell number (LLC and SLC) also increased (P<0.01) from Stage I to III of estrous cycle and decreased (P<0.05), thereafter, at Stage IV indicating degeneration of luteal cells and regression of the CL. Total luteal cell population during pregnancy also increased (P<0.01) from Stage I to II and thereafter decreased (P>0.05) indicating cessation of mitosis. Increased (P<0.05) large luteal cell numbers from Stage I to III of estrous cycle and pregnancy coincided with the increased progesterone secretion and 3β-HSD activity of CL. Thus, proportionate increases of large compared with small luteal cells were primarily responsible for increased progesterone secretion during the advanced stages of the estrous cycle and pregnancy. Total luteal cells and progesterone content per CL during the mid-luteal stage in buffalo as observed in the present study seem to be less than with cattle suggesting inherent luteal deficiency.

Activation of the LH receptor up regulates the type 2 adiponectin receptor in human granulosa cells

PurposeAdiponectin is a predominantly adipocyte-derived hormone which influences insulin sensitivity and energy homeostasis through at least two receptors, AdipoR1 and AdipoR2. In animal models, adiponectin may regulate ovarian steroidogenesis, folliculogenesis, and ovulation. The receptors AdipoR1 and AdipoR2 are present in the human ovary, but their regulation is unknown. In these studies, we determined the effects of LH receptor activation on the expression and function of the two adiponectin receptors in human granulosa cells.MethodsGranulosa cells were obtained at the time of oocyte retrieval in women undergoing in vitro fertilization (IVF). Cells were isolated and cultured for 48 h in DMEM/F12 medium with 5 % FBS and 50 ug/ml gentamicin. Medium was changed to low serum for 12 h and cells were treated with hCG (100 ng/ml), forskolin (30 μMol/L), or FSH (1 IU/ml) for 24 h for mRNA experiments. mRNA was isolated and RT PCR was performed using Taqman assays and quantification with the delta delta CT method. For immunocytochemistry, cells were grown on chamber slides and treated with hCG for 1 to 24 h and fixed with acetone. ICC was performed with polyclonal rabbit primary antibodies followed by alexa fluor goat anti-rabbit antibody and imaging with a fluorescence microscope and Zeiss software analysis. 3β-hydroxysteroid dehydrogenase (3βHSD) enzyme activity was determined by measuring the progesterone produced when cells were provided with an excess of 22-hydroxy-cholesterol as substrate following an incubation with hCG (1 IU/ml) and/or adiponectin (10 ng/ml). Progesterone content in the media was determined by ELISA.ResultsMessenger RNA for the two Adiponectin receptors is differentially regulated by activation of LHR with hCG treatment. AdipoR2 was increased nearly 4-fold (p < 0.05), whereas AdipoR1 expression was not changed by hCG treatment. Treatment with either FSH or forskolin (an activator of cAMP) had similar effects. Basal AdipoR2 protein was fairly low in granulosa cells in culture however treatment of cells with hCG resulted in a discernible increase in immunodetectable cytoplasmic protein as early as 6 h after treatment and was maintained for at least 24 h. The number of cells positive for AdipoR2 at 6 h increased from a basal of 20 % to almost 60 % (p < 0.05). Adiponectin treatment of hCG-primed cells resulted in increased 3βHSD activity by approximately 60 % over hCG alone and more than 3-fold over basal levels.ConclusionsAdipoR2 is regulated by the LH receptor function via a cAMP dependant mechanism. Increased expression of adipoR2 prior to and following ovulation may contribute to enhanced 3βHSD activity and increased progesterone secretion by the corpus luteum of the ovary. Dysregulation of adiponectin that may occur with PCOS may impair normal progesterone production.

Ovarian Expression of Insulin-Like Peptide 3 (INSL3) and Its Receptor (RXFP2) During Development of Bovine Antral Follicles and Corpora Lutea and Measurement of Circulating INSL3 Levels During Synchronized Estrous Cycles

Insulin-like peptide 3 (INSL3), a major product of testicular Leydig cells, is also expressed by the ovary, but its functional role remains poorly understood. Here, we quantified expression of INSL3 and its receptor RXFP2 in theca interna cell (TIC) and granulosa cell compartments of developing bovine antral follicles and in corpora lutea (CL). INSL3 and RXFP2 mRNA levels were much higher in TIC than granulosa cell and increased progressively during follicle maturation with INSL3 peaking in large (11-18 mm) estrogen-active follicles and RXFP2 peaking in 9- to 10-mm follicles before declining in larger (11-18 mm) follicles. Expression of both INSL3 and RXFP2 in CL was much lower than in TIC. In situ hybridization and immunohistochemistry confirmed abundant expression of INSL3 mRNA and protein in TIC. These observations indicate follicular TIC rather than CL as the primary site of both INSL3 production and action, implying a predominantly autocrine/paracrine role in TIC. To corroborate the above findings, we showed that in vitro exposure of TIC to a luteinizing concentration of LH greatly attenuated expression of both INSL3 and its receptor while increasing progesterone secretion and expression of STAR and CYP11A1. Moreover, in vivo, a significant cyclic variation in plasma INSL3 was observed during synchronized estrous cycles. INSL3 and estradiol-17β followed a similar pattern, both increasing after luteolysis, before falling sharply after the LH surge. Thus, theca-derived INSL3, likely from the dominant preovulatory follicle, is detectable in peripheral blood of cattle, and expression is down-regulated during luteinization induced by the preovulatory LH surge. Collectively, these findings underscore the likely role of INSL3 as an important intrafollicular modulator of TIC function/steroidogenesis, while raising doubts about its potential contribution to CL function.

Inhibition of proteasomal proteolysis affects expression of extracellular matrix components and steroidogenesis in porcine oocyte-cumulus complexes

Porcine oocyte-cumulus complexes (OCCs) form an expanded cumulus extracellular matrix (ECM) in response to gonadotropins during meiotic maturation. Essential components of ECM are hyaluronan (HA), tumor necrosis factor α-induced protein 6 (TNFAIP6) and heavy chains (HC) of interalpha-trypsin inhibitor. To form expanded cumulus ECM, intermediate complexes (TNFAIP6-HC) must bind to HA to allow HC transfer onto HA. Protein turnover by the ubiquitin-proteasome pathway is poorly characterized in this process. It is known that the specific proteasomal inhibitor MG132 prevents cumulus expansion and formation of ECM. To determine whether inhibition of proteasomal proteolysis with MG132 affects cumulus cell steroidogenesis and expression of the cumulus expansion-related components (hyaluronan synthase type 2, HAS2, TNFAIP6) we cultured porcine OCCs and granulosa cells (GCs) in a medium supplemented with FSH/LH. Methods performed included real-time reverse transcription PCR, immunofluorescence and RIAs. The expression of TNFAIP6 and HAS2 transcripts increased significantly after the stimulation of OCCs and GCs with FSH/LH. In contrast, treatment with MG132 reduced the expression of TNFAIP6 and HAS2. Hyaluronan was detected with biotinylated HA-binding proteins within FSH/LH-stimulated expanded OCCs but not in those treated with MG132. Progesterone production, although increased almost three times after OCCs stimulation with FSH/LH, was significantly suppressed by MG132. The FSH/LH-stimulated a 40-fold increase in progesterone secretion by GCs was inhibited in the presence of MG132. In conclusion, MG132 affects progesterone secretion and expression of cumulus expansion-related components by cumulus and GCs, suggesting the requirement of ubiquitin-proteasome pathway–regulated protein turnover for formation of ECM during cumulus expansion in the preovulatory period in the pig.

Effects of unsaturated fatty acids on progesterone secretion and selected protein kinases in goat granulosa cells

Previous studies in cattle have shown influences of dietary unsaturated fatty acid (UFA) supplementation on ovarian function. However, it is unclear whether these UFA exert direct or indirect effects on ovarian steroid production or their mechanisms of action. We have recently shown that 5′AMP-activated protein kinase (AMPK) regulates progesterone secretion through mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK ERK1/2) in rodent granulosa cells. Here, we investigated the effects of 3 UFAs, oleic acid (OA), linoleic acid (LA), and α-linolenic acid (ALA) on progesterone secretion in goat granulosa cells. Finally, we examined the effects of UFAs on MAPK ERK1/2 and AMPK phosphorylation in these granulosa cells. Oleic acid and LA (10 μM each), but not ALA (100 μM), increased progesterone secretion ( P < 0.05) in the presence or absence of insulin-like growth factor (IGF)-1 (10 -8 M) or FSH (5 × 10 −8 M). The different AMPK subunits, except for γ3, are present in the goat ovary. Treatment with metformin (10 mM), an activator of AMPK, increased AMPK phosphorylation ( P < 0.05) and reduced progesterone secretion by 50% ( P < 0.05) in the basal state and in response to IGF-1 or FSH in goat granulosa cells. Oleic acid and LA had no effect on AMPK phosphorylation, whereas they rapidly increased MAPK ERK1/2 phosphorylation ( P < 0.05). Finally, U0126, a MAPK ERK1/2 inhibitor, decreased OA- and LA-induced progesterone secretion ( P < 0.05), suggesting that these UFAs could stimulate progesterone secretion partly through MAPK ERK1/2 in the absence of IGF-1 and FSH in goat granulosa cells. The involvement of AMPK in this process remains to be demonstrated. Taken together, some fatty acids could improve ovarian steroidogenesis through the MAPK ERK1/2 signaling pathway and, consequently, have beneficial effects on goat fertility.

Mixture of dominant PBDE congeners (BDE-47, -99, -100 and -209) at levels noted in human blood dramatically enhances progesterone secretion by ovarian follicles

Polybrominated diphenyl ethers (PBDEs) are used as flame retardants in a wide variety of products. They show an accumulation pattern similar to that of well-known persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs), DDT and others. So far, most of information showing adverse effects of various POPs was based on the exposure to single chemicals. However, in this study we intended to evaluate in vitro effect of the mixture of four most abundant PBDE congeners (PBDE-47, -99, -100 and -209) as usually found in wildlife and humans on the secretion of gonad hormones by ovarian follicles. Cocultures of theca and granulosa cells collected from medium size follicles of cycling pigs were exposed for 24 or 48 h to three different levels of respective PBDE congeners mixture: 1. low level (0.5; 0.25; 0.1; 0.05 ng/ml); 2. medium level (25; 10; 4.0; 0.25 ng/ml) and high level (50; 17.5; 12.5; 0.5 ng/ml). Additionally, to show if the effect on steroid secretion by ovarian follicles is reversible, the cells were cultured for 24 h with the reagents and, after the medium was exchanged, the cells were cultured without reagents for additional 48 h. At the end of each culture procedure, progesterone (P4), testosterone (T) and estradiol (E2) secretion in the culture medium were determined by ELISA. After short term exposure increased secretion of all investigated steroids was noted only under the influence of small dose of mixture. However, after longer time of exposure the stimulation of estradiol and testosterone secretion was observed under the influence of both the low and medium dose of mixture, while 3.0-, 6.8- and 7.2-fold increase in progesterone secretion was noted under the influence of low, medium and high doses of mixture, respectively. Taken together, our findings from this and previous studies allow several interrelated conclusions to be drawn. First, the increase of P4/T ratio with a parallel decrease of T/E2 ratio was found suggesting preterm luteinization in antral follicles followed by the disruption of ovulation and, second, it was not possible to reverse the steroidogenic effects of PBDEs mixture by removing reagents from the cell cultures.

Influence of triiodothyronine (T 3) on secretion of steroids and thyroid hormone receptor expression in chicken ovarian follicles

The present study was designed to (1) assess the role of triiodothyronine (T 3) with regard to in vitro steroid hormone secretion by chicken ovarian follicles; (2) determine whether T 3 influences the in vivo function of the pituitary-ovarian axis in the hen; and (3) detect expression of thyroid hormone receptor (TR) mRNA in chicken ovarian follicles. In the first experiment, laying hens were decapitated 22.5 h before ovulation. White prehierarchical follicles (1–8 mm) and fragments of theca and granulosa layers of the 3 largest yellow preovulatory follicles F3-F1 (22–35 mm) were incubated in a medium supplemented with T 3 (0, 0.1, 1, 10, 100, or 1000 ng/mL) or ovine luteinizing hormone (LH) (10 ng/mL) in combination with doses of T 3 (1, 10, and 100 ng/mL). Triiodothyronine decreased basal and LH-stimulated estradiol secretion by white follicles and the theca layer of all preovulatory follicles. On the other hand, it increased progesterone secretion by F2 and F1 follicles. In the second experiment, hens were injected 1 h after ovulation with saline (control) or T 3 (10 μg/100 g body weight, intraperitoneally). Results indicated that exogenous T 3 decreased plasma concentrations of LH and estradiol and increased plasma concentrations of progesterone. In the third experiment, using reverse transcription polymerase chain reaction (RT-PCR) analysis, expression of thyroid hormone receptor (TRα and TRβ0), mRNA was detected in all of the ovarian compartments. The expression of TRα mRNA was relatively greater in comparison with TRβ0. There were no differences between white ovarian follicles in the expression of TRα and TRβ0 mRNA. A considerably higher TRα and lower TRβ0 expression was detected in the granulosa layer of preovulatory follicles in comparison with the theca layer. In conclusion, the data indicate that thyroid hormones acting via nuclear receptors are involved in regulation of the pituitary-ovarian axis and processes associated with follicle growth and maturation.

SHORT COMMUNICATION: Interleukin‐17 Increased Progesterone Secretion by JEG‐3 Human Choriocarcinoma Cells

Problem JEG‐3 choriocarcinoma cell line has previously been reported to express a receptor for interleukin (IL)‐17. The involvement of IL‐17 in the production of progesterone and human chorionic gonadotropin by placental trophoblast has not been investigated.Method of study The present study investigated the in vitro effect of IL‐17 on progesterone and human chorionic gonadotropin (hCG) secretion by JEG‐3 cells. Both hormones were quantified using enzyme‐linked immunosorbent assays.Results The results showed that IL‐17 significantly increased progesterone secretion at 6 (P &lt; 0.001) and 24 (P &lt; 0.01) hr, while this cytokine had no effect on hCG secretion.Conclusion Interleukin‐17 may regulate the function of JEG‐3 cells through increased progesterone secretion.

Expression of adrenomedullin in human ovaries, ovarian sex cord–stromal tumors and cultured granulosa–luteal cells

The aim of the present study was to characterise the expression pattern of the multifunctional vasoactive peptide adrenomedullin (ADM) in human ovarian tumors, and to find hormonal regulators of ADM expression in human ovaries. The expression of ADM messenger RNA (mRNA) was higher in granulosa cell tumors than in fibrothecomas and normal ovaries, as analysed by Northern blots. In normal ovaries, ADM immunoreactivity was localised in both granulosa and thecal cells. Eight of the 90 granulosa cell tumors (9%) showed moderate and 53 (59%) weak ADM immunoreactivity, whereas 27% (11/41) of the fibrothecomas displayed weak ADM staining. FSH, protein kinase A activator (Bu)2cAMP, prostaglandin E2 (PGE2), activin A and the broad protein kinase regulator staurosporine decreased ADM mRNA accumulation in cultured granulosa–luteal cells time- and dose-dependently. FSH, (Bu)2cAMP and PGE2 increased progesterone secretion and the accumulation of the steroidogenic acute regulatory protein mRNA in these cells. In conclusion, ADM is expressed in normal human ovaries and sex cord–stromal tumors, particularly in those of granulosa cell origin. FSH, PGE2, (Bu)2cAMP and activin A suppress ADM gene expression in granulosa–luteal cells. Expression of ADM in human ovaries and its hormonal regulation in granulosa cells suggests a paracrine role for ADM in ovarian function.

Endocrinology of pregnancy in the dog: A review

Pregnancy regulation in the dog is not yet fully elucidated. Since plasma progesterone concentrations are similar in pregnant versus non-pregnant animals, it is a poor reflection on CL function and progesterone metabolism. Increased progesterone secretion by the CL in pregnant animals follows implantation and relaxin secretion by the feto-placental units. Progesterone is absolutely required to maintain pregnancy and no placental sources of progesterone have been identified. Pregnancy can be artificially maintained by progesterone administration. Prolactin secretion appears to be increased in response to the increase in relaxin production and occurs independent of estrogen production by the CL. The respective roles of LH, FSH and prolactin are still unclear, with considerable conflicting evidence among studies. However, it appears that prolactin is absolutely required, whereas LH is either permissive or facilitates CL function during pregnancy. Pre-implantation events are still poorly defined in the bitch, and no embryonic factors have been isolated or purified, preventing early pregnancy diagnosis. Parturition occurs following luteolysis, which results from the release of prostaglandin F(2alpha), which begins 36h prepartum in a process similar to that observed in other species. The role of estrogens at the time of parturition remains undefined.

Nitrophenols isolated from diesel exhaust particles regulate steroidogenic gene expression and steroid synthesis in the human H295R adrenocortical cell line

Studies of nitrophenols isolated from diesel exhaust particles (DEPs), 3-methyl-4-nitrophenol (PNMC) and 4-nitro-3-phenylphenol (PNMPP) have revealed that these chemicals possess estrogenic and anti-androgenic activity in vitro and in vivo and that PNMC accumulate in adrenal glands in vivo. However, the impacts of exposure to these compounds on adrenal endocrine disruption and steroidogenesis have not been investigated. To elucidate the non-receptor mediated effects of PNMC and PNMPP, we investigated the production of the steroid hormones progesterone, cortisol, testosterone, and estradiol-17β and modulation of nine major enzyme genes involved in the synthesis of steroid hormones ( CYP11A, CYP11B1, CYP17, CYP19, 17βHSD1, 17βHSD4, CYP21, 3βHSD2, StAR) in human adrenal H295R cells supplied with cAMP. Exposure to 10 − 7 to 10 − 5 M PNMC and 1 mM 8-Br-cAMP for 48 h decreased testosterone, cortisol, and estradiol-17β levels and increased progesterone secretion. At 10 − 5 M, PNMC with 1 mM 8-Br-cAMP significantly stimulated expression of the 17βHSD4 and significantly suppressed expression of 3βHSD2. In comparison, 10 − 7 to 2 × 10 − 5 M PNMPP with 1 mM 8-Br-cAMP for 48 h decreased concentrations of estradiol-17β, increased progesterone levels, but did not affect testosterone and cortisol secretion due to the significant suppression of CYP17 and the non-significant but obvious suppression of CYP19. Our results clarified steroidogenic enzymes as candidates responsible for the inhibition or stimulation for the production of steroid hormones in the steroidogenic pathway, thus providing the first experimental evidence for multiple mechanisms of disruption of endocrine pathways by these nitrophenols.

Time-dependent action of DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) and its metabolite DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene) on human chorionic gonadotropin and progesterone secretion

Explants of human placenta were used to study the effects of two isomers of DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) [p,p′-DDT and o,p′-DDT] and their DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene) metabolites [p,p′-DDE and o,p′-DDE] on the secretion of progesterone and human chorionic gonadotropin (hCG). Explants were treated with 1, 10, 100 or 1000 ng/ml of each compound for 24 h or 72 h. We found opposite effects (stimulatory after short-term and inhibitory after long-term exposure) of all compounds on progesterone secretion. However, both short- and long-term exposure to all investigated compounds caused decreased hCG secretion. In conclusion, we suggest the existence of a local axis between steroid hormones and hCG in placenta. DDT (which has estrogenic properties) increases progesterone secretion and consequently decreases hCG secretion. After long-term exposure, the low level of hCG is insufficient to stimulate progesterone.

Over-activation of cAMP signaling increases vulnerability to hydrogen peroxide via actin rearrangement, which is inhibited by prostaglandin E2 in a mouse granulosa cell line.

Apoptosis of granulosa cells (GC) contributes to ovarian follicular atresia, and has been implicated to depend on the oxidant status of GC within follicles. Here, we investigated the effects of cAMP and prostaglandin E 2 (PGE 2 ) on sensitivity to oxidative stress using 4B2 cells with cAMP-dependent, steroidogenic and differentiated properties. This cell line was isolated from mouse GC co-transfected with genes for SV40 large T antigen and Ad4BP/SF1 transcription factor (Kamei et al. 2005). Treatment of serum-starved cells with 8-BrcAMP caused 30 to 40% of the cells to become polygonal within 2 h through actin rearrangement. Interestingly, H2O2 treatment showed that these polygonal cells were vulnerable to oxidative stress that led to cell death, which was inhibited by pretreatment with phalloidin, an F-actin stabilizing agent. Although PGE2 alone had no effect, cotreatment with PGE 2 and 8-Br-cAMP completely inhibited the effects of 8-Br-cAMP on cell shape change and oxidative stress vulnerability through phosphatidylinositol 3-kinase (PI3K)-dependent manner. Notably, PGE 2 and 8-Br-cAMP cooperated additively to increase progesterone secretion. These data suggest that cAMP signaling in GC may enhance oxidative stress risk through actin rearrangement, and PGE2 may reduce such risk through activating PI3K, while cooperating with cAMP signaling in steroidogenesis.

Escherichia coli lipopolysaccharide modulates bovine luteal cell function

<i>Escherichia coli</i> lipopolysaccharide modulates bovine luteal cell function

Progesterone and pregnancy status of buffaloes treated with a GNRH agonist

The primary aim of the present study was to establish whether the treatment with a GnRH agonist on Day 5 after AI may result in the formation of an accessory corpus luteum, greater progesterone secretion, and the increased likelihood of pregnancy success in buffaloes. The study was conducted during a period of increasing daylight length when progesterone secretion is suppressed and embryonic mortality is relatively high in buffaloes. In Experiment 1, treatment with a GnRH agonist (buserelin, 12 μg) on Day 5 after AI induced acute increases in circulating concentrations of LH, FSH and oestradiol-17β. Pregnant buffaloes (n=14) at Day 40 following AI showed an increase (P<0.01) in milk whey progesterone concentration between Day 5 (310±55 pg/ml) and Day 15 (424±50 pg/ml). The non-pregnant buffaloes (n=7) showed a decrease (P<0.01) in progesterone level from Day 5 (410±87 pg/ml) to Day 15 (188±30 pg/ml) following AI. In Experiment 2, the treatment with buserelin (12 μg) on Day 5 after AI induced ovulation in 62% of the buffaloes (31/50) and these buffaloes showed a progressive increase in milk whey progesterone concentration on Day 10, 15 and 20 of pregnancy. Buffaloes that did not ovulate, recorded a relatively constant milk whey progesterone level from Day 10 to Day 20 following AI. Milk whey progesterone concentrations increased after the administration of the GnRH agonist in 97% of the pregnant buffaloes and 68% of the non-pregnant buffaloes. The diameter of the largest follicle in buffaloes that ovulated (ovulated n=31) (8.9±0.04 mm; range 4.2 – 13.0 mm) did not differ significantly from the diameter of the largest follicle in buffaloes that did not ovulate (not ovulate n=19) (8.7±0.04 mm; range: 4.0 – 12.0 mm). The latter observation suggested that notional ovulatory size follicles in buffaloes are heterogeneous with respect to stage of follicle maturation and capacity to respond to plasma LH. The present study showed that treatment with a GnRH agonist on Day 5 following AI provides a strategy to increase progesterone secretion and the likelihood of pregnancy in buffaloes mated during periods of increasing daylight length.

IGF-1 receptor signaling pathways and effects of AMPK activation on IGF-1-induced progesterone secretion in hen granulosa cells

IGF-1 plays a key role in the proliferation and differentiation of granulosa cells. However, the molecular mechanism of IGF-1 action in avian granulosa cells during follicle maturation is unclear. Here, we first studied IGF-1 receptor (IGF-1R) expression, IGF-1-induced progesterone production and some IGF-1R signaling pathways in granulosa cells from different follicles. IGF-1R (mRNA and protein) was higher in fresh or cultured granulosa cells from the largest follicles (F1 or F2) than in those from smaller follicles (F3 or F4). In vitro, IGF-1 treatment (10 −8 M, 36 h) increased progesterone secretion by four-fold in mixed F3 and F4 (F3/4) granulosa cells and by 1.5-fold in F1 granulosa cells. IGF-1 (10 −8 M, 30 min)-induced increases in tyrosine phosphorylation of IGF-1R beta subunit and phosphorylation of ERK were higher in F1 than in F3/4 granulosa cells. Interestingly, IGF-1 stimulation (10 −8 M, 10 min) decreased the level of AMPK Thr172 phosphorylation in F1 and F3/4 granulosa cells. We have recently showed that AMPK (AMP-activated protein kinase) is a protein kinase involved in the steroidogenesis in chicken granulosa cells. We then studied the effects of AMPK activation by AICAR (5-aminoimidazole-4-carboxamide ribonucleoside), an activator of AMPK, on IGF-1-induced progesterone secretion by F3/4 and F1 granulosa cells. AICAR treatment (1 mM, 36 h) increased IGF-1-induced progesterone secretion, StAR protein levels and decreased ERK phosphorylation in F1 granulosa cells. Opposite data were observed in F3/4 granulosa cells. Adenovirus-mediated expression of dominant negative AMPK totally reversed the effects of AICAR on IGF-1-induced progesterone secretion, StAR protein production and ERK phosphorylation in both F3/4 and F1 granulosa cells. Thus, a variation of energy metabolism through AMPK activation could modulate differently IGF-1-induced progesterone production in F1 and F3/4 granulosa cells.