Progesterone Biosynthesis Research Articles

New Classification and Management of Abetalipoproteinemia and Related Disorders

New Classification and Management of Abetalipoproteinemia and Related Disorders

Metformin attenuates steroidogenesis in ovarian follicles of the broiler breeder hen.

The follicular hierarchy in broiler breeder chicken ovary is often deranged due to excessive ovarian follicular recruitment, resulting in a condition that resembles polycystic ovary syndrome (PCOS) in women. Metformin is widely prescribed to correct PCOS and has been shown to affect granulosa cell functions in humans and rodent models. The objectives of this study are to determine the effects of metformin on signal transduction pathways, gene expression related to steroidogenesis, and progesterone secretion from granulosa cells isolated from the most recently recruited preovulatory and prehierarchical follicles of broiler breeder chickens. Granulosa cells were treated with 0, 1, 10, or 20 mM of metformin in the presence of FSH. The abundance of pAMPK, pACC, pERK, and pAkt was determined by Western blotting. The expression of genes related to progesterone biosynthesis was quantified by qPCR. Progesterone concentrations in culture media were quantified by ELISA. Metformin treatment did not have an effect on the abundance of pAMPK and pACC in prehierarchical follicles but significantly decreased the abundance of pERK and pAkt in a dose-dependent manner in preovulatory and prehierarchical follicles. The expression of genes related to steroidogenesis such as FSHR, STAR, CYP11A1, HSD3B, and progesterone secretion was significantly decreased in response to metformin treatment in a dose-dependent manner. Our data suggest that metformin treatment attenuates progesterone secretion via AMPK-independent pathways in granulosa cells of prehierarchical and preovulatory follicles of broiler breeder hens. Further studies are required to determine if metformin administration could ameliorate ovarian dysfunction in obese broiler breeder hens.

Structural and functional analysis of the inhibition of equine glutathione transferase A3-3 by organotin endocrine disrupting pollutants.

Organotin compounds are highly toxic environmental pollutants with neurotoxic and endocrine-disrupting effects. They are potent inhibitors of glutathione transferases (GSTs), thus impeding their detoxication and antioxidant functions. Several GSTs, including equine GST A3-3 (EcaGST A3-3), exhibit steroid double-bond isomerase activity and are involved in the biosynthesis of testosterone and progesterone. We have performed enzyme kinetics analyses of the inhibition of EcaGST A3-3 by organotin compounds. We have also solved crystal structures of EcaGST A3-3 in complexes with glutathione, and with glutathione together with covalently bound triethyltin. Our structural data indicate that the tin atom forms strong bonds with a covalent character not only with the glutathione, but also with a tyrosyl residue of the enzyme itself, thereby preventing the release of the glutathione-organotin adduct and completely blocking the enzyme function. This work presents a structural basis for the general mechanism of GST inhibition by organotin compounds and contributes to the understanding of their neurotoxic and endocrine disrupting effects.

Bisphenol A decreases progesterone synthesis in human ovarian granulosa cells.

Evidences indicate that the decline of female fertility is becoming a common problem over the past few decades. Environmental exposure of Bisphenol A (BPA) has been considered as an endocrine-disrupting chemical deleteriously affecting human reproductions. To better understand the effect of BPA exposure on human ovarian granulosa cells, we treated human ovarian granulosa cell line (KGN) with increasing concentrations (0.1, 1, 10, and 100 μM) of BPA for 24 hr. About 0.1, 1, and 10 μM BPA did not significantly affect the viability of KGN while 100 μM of BPA caused a statistically significant decrease in the viability of KGN. Treatment KGN with 10 μM BPA resulted in a significant decrease in progesterone biosynthesis. The treatment also significantly increased the expression of ATP binding cassette subfamily A member 1 (ABCA1) and steroidogenic acute regulatory protein (STAR). In the current study, exposure to BPA could lead to decreased progesterone production probably through the upregulation of ABCA1 in human granulosa cells.

Changes in the localization of ovarian visfatin protein and its possible role during estrous cycle of mice

Visfatin is a crucial adipokine, which also regulates ovarian functions in many animals. Mice estrous cycle is characterized by a dynamic complex physiological process in the reproductive system. Expression of various factors changes during the estrous cycle in the ovary. To the best of our knowledge, no previous study has been conducted on the expression of visfatin in mice ovaries during the estrous cycle. Therefore, we investigated the localization and expression of visfatin protein in the ovary of mice during the estrous cycle. Western blot analysis showed the elevated expression of visfatin in proestrus and lowest in diestrus. Immunohistochemical localization of visfatin showed intense staining in the corpus luteum of proestrus and diestrus ovaries. Thecal cells, granulosa cells, and oocytes also showed the presence of visfatin. Expression of ovarian visfatin was correlated to BCL2 and active caspase3 expression and exhibited a significant positive correlation. Furthermore, in vivo inhibition of visfatin by FK866 in the proestrus ovary down-regulated active caspase3 and PCNA expression, and up-regulated the BCL2 expression. These results suggest the role of visfatin in the proliferation and apoptosis of the follicles and specific localization of visfatin in the corpus luteum also indicate its role in corpus luteum function, which may be in progesterone biosynthesis and regression of old corpus luteum. However, further study is required to support these findings. In conclusion, visfatin may also be regulating follicular growth during the estrous cycle by regulating proliferation and apoptosis.

PREDICTIVE VALUE OF FOLLICULAR FLUID GHRELIN IN WOMEN WITH POLYCYSTIC OVARIAN DISEASE WHO UNDERGO INTRACYTOPLASMIC SPERM INJECTION

Ghrelin’s role in reproduction was first suggested by its wide expression in many human reproductive tissues including its immunohistochemical expression in the human ovary. Ghrelin expression is identified in the seminiferous tubuli of the interstitial Leyding and Sertoli cells in male testicles, whereas it is identified in the ovarian hilus interstitial cells and mature corpus lutea in females.It is postulated that ghrelin plays a local regulatory role in the male and female gonads. In females, Ghrelin acts centrally by suppressing hypothalamic GnRH release and GnRH-induced gonadotropin secretion by the pituitary; and in gonads it acts by increasing luteolytic factors such as PGF2 alfa, and ghrelin inhibiting estradiol and progesterone biosynthesis in granulose-lutein cells as well as granulosa cell (GC) proliferation.This potential regulatory effect on steroidogenesis and ovarian cellular function led to the hypothesis that ghrelin may mediate the relationship between metabolism and oocyte quality in addition to possible role in the regulation of embryo development in as the cleavage development before the six-cell stage of a preimplantation human embryo depends solely on maternally inherited material. Ghrelin levels can, therefore, be an indicator of an underlying pathology as in humans in states of chronic malnutrition such as anorexia nervosa, serum ghrelin levels may be increased, while in other conditions it could be associated with decreased ghrelin levels such as obesity and polycystic ovarian syndrome (PCOS).

Aldosterone enhances progesterone biosynthesis regulated by bone morphogenetic protein in rat granulosa cells

Aldosterone (Aldo) is involved in various cardiovascular diseases such as hypertension and heart failure. Aldo levels are known to be increased in patients with polycystic ovary syndrome, and expression of the mineralocorticoid receptor (MR) has also been detected in the ovary. However, the effect of Aldo on reproductive function has yet to be elucidated. Here, we examined the effects of Aldo on follicular steroidogenesis using primary culture of rat granulosa cells by focusing on the ovarian bone morphogenetic protein (BMP) system acting as a luteinizing inhibitor. We found that Aldo treatment increased FSH-induced progesterone production in a concentration-responsive manner. Consistent with the effects on steroidogenesis, Aldo increased mRNA levels of progesterogenic factor and enzymes including StAR and P450scc, whereas Aldo failed to change FSH-induced estradiol and cAMP synthesis or P450arom expression by granulosa cells. Progesterone production and StAR expression induced by FSH and Aldo were reversed by co-treatment with spironolactone, suggesting the involvement of geonomic MR action. Aldo treatment attenuated Smad1/5/9 phosphorylation and Id1 transcription induced by BMP-6. Furthermore, Aldo enhanced the expression of inhibitory Smad6 in the presence of BMP-6. In addition, BMP-6 downregulated MR expression, while Aldo modulated the mRNA levels of endogenous BMP-6 and BMP type-II receptors, indicating the existence of a feedback loop between the BMP system and MR in granulosa cells. Collectively, the results indicated that Aldo predominantly enhances FSH-induced progesterone production by inhibiting BMP-Smad signaling, suggesting a novel role of Aldo in ovarian steroidogenesis and a functional link between MR and BMP pathways in granulosa cells.

Effect of dietary n-3 polyunsaturated fatty acid supplementation on the expression of genes involved in progesterone biosynthesis in the corpus luteum of goat (Capra hircus).

Emerging evidence indicates that dietary n-3 polyunsaturated fatty acids (PUFA) alter the fatty acid composition of corpus luteum (CL) and directly affect the luteal function in the cow, which is independent of the inhibitory effect on the endometrial PGF2α production. The present study, thus, investigated the effects of n-3 PUFA rich fish oil (FO) supplementation on the transcriptional modulation of genes involved in the biosynthesis of progesterone (P4 ) in the CL collected during the luteolytic phase of oestrous cycle in the goat. On the day of synchronized oestrus, goats (n=6/group) were fed an isocaloric diet supplemented with either FO or palm oil (PO). The dose of oil supplementation was 0.6mlkg-1 body weight, and the duration was 55-57days. The FO provided 156mgkg-1 body weight of n-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The CL was collected by laparotomy on day 16 post-oestrus, and the relative abundance of P450 side-chain cleaving enzyme, steroid acute regulatory protein (StAR) and 3β-hydroxy steroid dehydrogenase (3β-HSD) genes was quantitated by real-time PCR. The results indicated that the dietary FO significantly upregulated the expression of 3β-HSD by 1.13-fold and downregulated StAR by ~2-fold as compared to PO group (p<.05). It is concluded that dietary FO differently affected the expression of genes involved in P4 synthesis in the CL during the luteolytic window of the oestrous cycle in the goat.

RNAi-Mediated Silencing of Catalase Gene Promotes Apoptosis and Impairs Proliferation of Bovine Granulosa Cells under Heat Stress.

Simple SummaryReduced fertility of modern-day dairy cattle across the world is implicated by the global warming phenomenon. Heat stress (HS) is well-known for compromising the normal physiological functions of granulosa cells (GCs) by promoting reactive oxygen species (ROS), which subsequently induce apoptosis, impair the biosynthesis of estrogen and progesterone, and disrupt the mitochondrial membrane potential. The catalase (CAT) enzyme serves a key antioxidant role in catalyzing the HS-induced ROS, thereby preventing the adverse effects of oxidative stress on cells. Therefore, the regulation of the CAT gene under HS has been the subject of increasing interest among researchers. However, no researches till date performed a functional validation of the CAT gene in bovine GCs under HS. For instance, we silenced the CAT gene using siRNA in GCs and found that the silencing of CAT aggravated the HS-induced damages to these cells, depicting a protective role of the CAT gene under HS. Thus, the regulation of CAT under HS could be used as an effective molecular marker to enhance the fertility in heat-stressed dairy cows.Heat stress in dairy cattle is recognized to compromise fertility by altering the functions of ovarian follicle-enclosed cells, e.g., oocyte and granulosa cells (GCs). Catalase is an antioxidant enzyme that plays a significant role in cellular protection against oxidative damage by the degradation of hydrogen peroxide to oxygen and water. In this study, the role and mechanism of CAT on the heat stress (HS)-induced apoptosis and altered proliferation of bovine GCs were studied. The catalase gene was knocked-down successfully in bovine GCs at both the transcriptional and translational levels. After a successful knockdown using siRNA, GCs were divided into HS (40 °C + NC and 40 °C + CAT siRNA) and 38 °C + NC (NC) groups. The GCs were then examined for ROS, viability, mitochondrial membrane potential (MMP), cell cycle, and biosynthesis of progesterone (P4) and estrogen (E2) hormones. The results indicated that CAT silencing promoted ROS production and apoptosis by up-regulating the Bcl-2-associated X protein (BAX) and Caspase-3 genes both at the transcriptional and translational levels. Furthermore, the knockdown of CAT markedly disrupted the MMP, impaired the production of P4 and E2, altered the progression of the G1 phase of the cell cycle, and decreased the number of cells in the S phase. This was further verified by the down-regulation of proliferating cell nuclear antigen (PCNA), CyclinB1, steroidogenic acute regulatory protein (STAR), and cytochrome P450 family 11 subfamily A member 1 (Cyp11A1) genes. Our study presented a novel strategy to characterize how CAT can regulate cell proliferation and apoptosis in GCs under HS. We concluded that CAT is a broad regulatory marker in GCs by regulating apoptosis, cellular progression, and simultaneously by vital fluctuations in hormonal signaling. Our findings infer a crucial evidence of how to boost the fertility of heat-stressed cows.

ALTERATION OF MITOCHONDRIAL MEMBRANE POTENTIAL (ΔΨM): POTENTIAL ROLE IN THE MECHANISM OF MEHP‐INDUCED INHIBITION OF STEROID BIOSYNTHESIS IN MA‐10 LEYDIG CELLS

INTRODUCTION AND OBJECTIVESMono‐(2‐ethylhexyl) phthalate (MEHP) is the active metabolite of di‐(2‐ethylhexyl) phthalate (DEHP), a plasticizer with endocrine disruptor activity that is widely used in the manufacturing industry. MEHP inhibits luteinizing hormone (LH)‐stimulated steroid biosynthesis in Leydig cells by molecular mechanisms that remain unclear. In the present study, using MA‐10 mouse tumor Leydig cells, we examined changes in mitochondrial function caused by MEHP exposures in relationship to possible link to alteration of progesterone biosynthesis.METHODSThe effect of MEHP on LH‐stimulated progesterone production was assessed after treating MA‐10 cells with (0–300 μM) MEHP for 24 hrs. Progesterone production was quantified by enzyme‐linked immunosorbent assay (ELISA). To measure oxygen consumption, cells were treated with MEHP (0–300 μM) for 24 hrs, and oxygen consumption was monitored using a Clarke‐type oxygen electrode. Mitochondrial membrane potential (ΔΨm) was assessed using the JC‐1 dye mitochondrial membrane potential indicator. Mitochondria‐derived superoxide was measured using the MitoSOX™ red mitochondrial superoxide indicator.RESULTSExposures of MA‐10 cells to 0–300 μM MEHP caused inhibition LH‐stimulated progesterone biosynthesis in a dose‐dependent manner. The decrease in the Leydig ability to produce progesterone correlated with a loss in the Leydig cell mitochondrial membrane potential (ΔΨm), and was linked to perturbation of the capacity to energize the mitochondrial membrane. MEHP exposure was suggested to cause a disruption in the electron flow through the electron transport chain, evidenced by a decrease in the ability to consumption molecular oxygen and an increase in the generation of superoxide in the mitochondria.CONCLUSIONSTogether, these observations suggest that MEHP inhibits steroid biosynthesis in MA‐10 cells via a mechanism involving a loss of the mitochondrial membrane potential (ΔΨm) and an increase in the oxidative stress level, two crucial changes known to affect mitochondrial function and cause inhibition of STAR‐mediated cholesterol transport and steroid biosynthesis.Support or Funding InformationThis material is based upon work supported by NIH grant SC2GM099578 and by the North Carolina Biotechnology Center, NCBC grant #2019‐IDG‐1017.

Luteinizing hormone regulates the phosphorylation and localization of the mitochondrial effector dynamin-related protein-1 (DRP1) and steroidogenesis in the bovine corpus luteum.

The corpus luteum is an endocrine gland that synthesizes and secretes progesterone. Luteinizing hormone (LH) activates protein kinase A (PKA) signaling in luteal cells, increasing delivery of substrate to mitochondria for progesterone production. Mitochondria maintain a highly regulated equilibrium between fusion and fission in order to sustain biological function. Dynamin-related protein 1 (DRP1), is a key mediator of mitochondrial fission. The mechanism by which DRP1 is regulated in the ovary is largely unknown. We hypothesize that LH via PKA differentially regulates the phosphorylation of DRP1 on Ser616 and Ser637 in bovine luteal cells. In primary cultures of steroidogenic small luteal cells (SLCs), LH, and forskolin stimulated phosphorylation of DRP1 (Ser 637), and inhibited phosphorylation of DRP1 (Ser 616). Overexpression of a PKA inhibitor blocked the effects of LH and forskolin on DRP1 phosphorylation. In addition, LH decreased the association of DRP1 with the mitochondria. Genetic knockdown of the DRP1 mitochondria receptor, and a small molecule inhibitor of DRP1 increased basal and LH-induced progesterone production. Studies with a general Dynamin inhibitor and siRNA knockdown of DRP1 showed that DRP1 is required for optimal LH-induced progesterone biosynthesis. Taken together, the findings place DRP1 as an important target downstream of PKA in steroidogenic luteal cells.

Suppression of Notch Signaling Stimulates Progesterone Synthesis by Enhancing the Expression of NR5A2 and NR2F2 in Porcine Granulosa Cells

The conserved Notch pathway is reported to be involved in progesterone synthesis and secretion; however, the exact effects remain controversial. To determine the role and potential mechanisms of the Notch signaling pathway in progesterone biosynthesis in porcine granulosa cells (pGCs), we first used a pharmacological γ-secretase inhibitor, N-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester (DAPT), to block the Notch pathway in cultured pGCs and then evaluated the expression of genes in the progesterone biosynthesis pathway and key transcription factors (TFs) regulating steroidogenesis. We found that DAPT dose- and time-dependently increased progesterone secretion. The expression of steroidogenic proteins NPC1 and StAR and two TFs, NR5A2 and NR2F2, was significantly upregulated, while the expression of HSD3B was significantly downregulated. Furthermore, knockdown of both NR5A2 and NR2F2 with specific siRNAs blocked the upregulatory effects of DAPT on progesterone secretion and reversed the effects of DAPT on the expression of NPC1, StAR, and HSD3B. Moreover, knockdown of NR5A2 and NR2F2 stimulated the expression of Notch3. In conclusion, the inhibition of Notch signaling stimulated progesterone secretion by enhancing the expression of NPC1 and StAR, and the two TFs NR5A2 and NR2F2 acted as downstream TFs of Notch signaling in regulating progesterone synthesis.

In utero and lactational exposure to triclocarban: reproductive effects on female rat offspring.

Triclocarban (TCC) is an antimicrobial compound widely used in personal care products such as soaps, toothpaste, and shampoo. This agent is incompletely removed by wastewater treatment and represents an environmental contaminant. Recent studies have shown that TCC is associated with some endocrine disruptions. The aim of the present study was to evaluate if TCC exposure during critical periods of development (gestation and lactation) could lead to adverse effects on reproductive and behavior parameters of female offspring. Pregnant female Wistar rats were divided into four groups (n = 8-11/group): Control; TCC 0.3 mg/kg (TCC 0.3); TCC 1.5 mg/kg; TCC 3.0 mg/kg (TCC 3.0); and treated daily by oral gavage from gestational day 0 to lactational day 21. The female pups (F1 generation) were weaned on post-natal day 21 and included in the study. No litter-mates were used for the same group. There was a decrease in estradiol levels in the TCC 0.3 and TCC 3.0 groups. Moreover, there was a decrease in progesterone levels and an increase in pre-implantation loss in the TCC 3.0 group in adulthood. It is suggested, in this study, that the decrease in progesterone biosynthesis could interfere with implantation process. The exposure window to TCC is an important factor, as we found alterations only in the offspring.

Maternal cadmium exposure during late pregnancy causes fetal growth restriction via inhibiting placental progesterone synthesis

Cadmium (Cd) is a major environmental pollutant. Maternal Cd exposure throughout pregnancy caused fetal growth restriction (FGR). However, the pivotal time window of Cd-evoked FGR and its mechanism are unknown. Here, we will establish a murine model to explore the effects of maternal Cd exposure at different stages of gestation on fetal growth and placental progesterone biosynthesis. Pregnant mice were randomly divided into four groups. For Cd groups, mice were given with CdCl2 (150 mg/L) through drinking water at early (GD0-GD6), middle (GD7-GD12) and late (GD13-GD17) gestation, respectively. The controls received reverses osmosis (RO) water. Results showed that maternal cadmium exposure only in late gestation lowered fetal weight and length. Correspondingly, placental Cd level in late gestational Cd exposure is the highest among three different gestational stages. Although gestational Cd exposure had few adverse effects in the weight and diameter of mouse placenta, placental vascular development, as determined by H&E staining and cluster of differentiation-34 (CD-34) immunostaining, was impaired in mice exposed to Cd during late pregnancy. Additionally, late gestational exposure to cadmium markedly reduced progesterone level in maternal serum and placenta. In line, the expression of key progesterone synthetases, including steroidogenic acute regulatory protein (StAR) and 3β-hydroxyl steroid dehydrogenase (3β-HSD), was obviously downregulated in placenta from mice was exposed Cd during late pregnancy. These data suggest that maternal Cd exposure during late pregnancy, but not early and middle pregnancy, induces fetal growth restriction partially via inhibiting placental progesterone synthesis.

Pentachloronitrobenzene alters progesterone production and primordial follicle recruitment in cultured granulosa cells and rat ovary†.

Pentachloronitrobenzene (PCNB) is an organochlorine fungicide widely used for crop production and has become an environmental concern. Little is known about the effect of PCNB on ovarian steroidogenesis and follicular development. We found that PCNB stimulated Star expression and progesterone production in cultured rat granulosa cells in a dose-dependent manner. PCNB activated mitogen-activated protein kinase (MAPK3/1) extracellulat regulated kinase (ERK1/2), thus inhibition of either protein kinase A (PKA) or MAPK3/1 signaling pathway significantly attenuated progesterone biosynthesis caused by PCNB, suggesting that PCNB induced progesterone production by activating the cyclic adenosine monophosphate (cAMP/PKA) and MAPK3/1 signaling pathways. Further investigation demonstrated that PCNB induced Star expression and altered MAPK3/1 signaling in ovary tissues of immature SD rats treated with PCNB at the dose of 100, 200, or 300mg/kg by daily gavage for 7days, while serum progesterone level was dose-dependently decreased. We demonstrated that PCNB exposure accelerated the recruitment of primordial follicles into the growing follicle pool in ovary tissues, accompanied by increased levels of anti-Mullerian hormone (AMH) in both ovary tissues and serum. Taken together, our data demonstrate for the first time that PCNB stimulated Star expression, altered MAPK3/1 signaling and progesterone production in vivo and in vitro, and accelerated follicular development with a concomitant increase in AMH in ovary tissues and serum. Our findings provide novel insight into the toxicity of PCNB to animal ovary function.

Inhibition of progesterone biosynthesis induced by deca-brominated diphenyl ether (BDE-209) in mouse Leydig tumor cell (MLTC-1)

Polybrominated Diphenyl Ethers (PBDEs) have been extensively applied as flame retardants in different polymeric materials since the 1970s, which have become a group of long-lasting environmental pollutants. They have been reported from previous studies to accumulate and then disrupt the endocrine system in humans. However, the mechanisms are still little known. In the present study, mouse Leydig tumor cells were utilized to investigate steroidogenic activity influenced by deca-brominated diphenyl ether (BDE-209). Our data showed that BDE-209 did not change intracellular cAMP level in the presence of human Chorionic Gonadotropin (hCG), cholera toxin (CT), and forskolin, which indicated that reduction of progesterone may not be related to the hCG-cAMP signal pathway in MLTC-1 cells. Furthermore, the reduction of progesterone generation was not shifted by 8-Br-cAMP, an analog of cAMP, indicating that BDE-209 may inhibit post-cAMP sites. In addition, mRNA expression levels of P450 side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) presented a concentration-dependent decrease. In conclusion, this study suggested that BDE-209 may attenuate the progesterone secretion mainly through lowering the expression of these two enzymes.

Cardiotonic steroid ouabain stimulates steroidogenesis in Leydig cells via the α3 isoform of the sodium pump

Cardiotonic steroids such as ouabain are potent inhibitors of the sodium pump and have been widely used for centuries in the treatment of congestive heart failure. In recent decades, however, they have also been identified as hormone-like molecules that trigger signaling cascades of physiological relevance by using the various sodium pump α subunit isoforms as receptors. The murine Leydig cell line MLTC-1 expresses both the ubiquitous, relatively ouabain-insensitive α1 isoform of the sodium pump and the ouabain-sensitive α3 isoform that is normally found in neuronal cells. The physiological relevance of the simultaneous presence of the two isoforms in Leydig cells has not been previously addressed.MLTC-1 Leydig cells contain lipid droplets (LDs) and are capable of progesterone biosynthesis when stimulated by luteinizing hormone (LH). When exposed to low nanomolar concentrations of ouabain, they respond with stimulation of Erk1/2, CREB, and ATF-1 phosphorylation, LD enlargement, and perilipin2 mobilization to the LDs. As a result, progesterone biosynthesis is augmented. Abrogation of α3 isoform expression by siRNA prevents all of the above responses, indicating that it is the hormone/receptor-like interaction of ouabain exclusively with this isoform that triggers the signaling events that normally occur when LH binds to its receptor.Considering that ouabain is produced endogenously and is found in seminal fluid, one can speculate that effects of this substance on germ and somatic cells of the testis might play a role in male reproductive physiology.

Raf-ERK1/2 signalling pathways mediate steroid hormone synthesis in bovine ovarian granulosa cells.

Steroid hormones are required for normal reproductive function of female. The aim of this study was to investigate the role of Raf-ERK1/2 on steroid hormone synthesis in bovine ovarian granulosa cells. Immunohistochemistry assay showed that both B-Raf and C-Raf were expressed in granulosa cells, theca cells and Sertoli cells. The protein expression of Raf or ERK1/2 was clearly decreased by Raf inhibitor GSK2118436 or ERK1/2 inhibitor SCH772984, respectively (p<0.05). In addition, western blotting was performed for investigating the crosstalk between Raf and ERK1/2, the data showed that Raf positively regulated ERK1/2, whereas ERK1/2 had a negative feedback effect on Raf. The biosynthesis of oestradiol or testosterone was significantly decreased by treatment with GSK2118436 or SCH772984 (p<0.05). Conversely, the progesterone biosynthesis was clearly increased by treatment with those inhibitors (p<0.05). Furthermore, the mRNA expression of STAR, aromatase and CYP17 was blocked by Raf-ERK1/2 signalling inhibition, which oppositely induced the mRNA expression of CYP11. Together, these findings suggested that Raf-ERK1/2 signalling pathways mediate steroid hormone synthesis via affecting the expression of steroidogenic enzymes.

Changes in content of steroid regulators during cold hardening of winter wheat - Steroid physiological/biochemical activity and impact on frost tolerance

The purpose of experiments was to describe the alterations of content of steroid regulators (brassinosteroids, progesterone) during cold hardening of winter wheat. Further we studied physiological and biochemical changes induced by these steroids in cold hardened winter wheat together with estimation of plant frost tolerance. The endogenous brassinosteroid content was elevated in winter wheat during cold hardening while level of progesterone was lowered. A higher content of brassinosteroids (but not progesterone) was connected to better frost tolerance of winter wheat cultivars. Plant supplementation with brassinosteroid (24-epibrassinolide) and progesterone before cold hardening reduced frost damage. Tests with the inhibitors of the biosynthesis of brassinosteroids and progesterone suggested that these steroids are one of players in regulating the antioxidant system in winter wheat during cold hardening. Their role in regulating the expression of Rubisco or the Rubisco activase gene was less clear. Steroid regulators did not affect the content of the stress hormone ABA. Model studies of the membranes, made on a Langmuir bath, showed an increase in the value of the parameter describing differences in membrane compressibility (resulting from stronger interactions among the molecules in the monolayers). This suggests that 24-epibrassinolide and progesterone enter into the lipid layer and - in a similar way to sterols – stabilise the interaction among lipids. It may be significant step for better frost tolerance. The use of steroid regulators (especially brassinosteroids) as agrochemicals improving frost tolerance of winter cereals will be discussed.

BDE-47 Decreases Progesterone Levels in BeWo Cells by Interfering with Mitochondrial Functions and Genes Related to Cholesterol Transport.

Polybrominated diphenyl ethers (PBDEs) have been reported to exert reproductive endocrine toxicity, but the mechanisms for this process remain unclear. Currently available studies have concentrated on the enzymatic reactions during steroidogenesis, but the results are not consistent. In this study, we explored the effects of 2,2',4,4'-tertrabromodiphenyl ether (BDE-47) on progesterone biosynthesis and the potential mechanisms in human placental choriocarcinoma cells. The results showed that BDE-47 decreased progesterone production in a dose-dependent manner but had no effect on key enzymes (Cyp11a1 and 3β-HSD). BDE-47 exposure depolarized the mitochondrial membrane potential and downregulated adenosine triphosphate levels. The gene expression levels of Mfn2, Tspo, Atad3, Vdac1, Fis1, and Drp1, which are involved in mitochondrial dynamics and cholesterol transport, were disturbed. The demethylation of some CpG loci of mitochondrial biomarkers (Drp1, Opa1, Vdac2, and Atad3) was induced in the 1 μM BDE-47 exposure group, but no methylation change was observed with 50 μM treatment. Our findings unveiled that the reduction of progesterone synthesis induced by BDE-47 might be associated with cholesterol transportation, mitochondrial dynamics, and mitochondrial functions. These findings provide substantial data on the reproductive endocrine toxicity of PBDEs.