Progesterone Accumulation Research Articles

The Human Gonadotropin-Inhibitory Hormone Ortholog RFamide-Related Peptide-3 Suppresses Gonadotropin-Induced Progesterone Production in Human Granulosa Cells

RFamide-related peptide-3 (RFRP-3), a mammalian ortholog of avian gonadotropin-inhibitory hormone, has pronounced inhibitory effects on reproduction in a number of species. RFRP-3 suppresses gonadotropin release at the hypothalamic and/or pituitary levels; however, increasing evidence also suggests putative functions within the ovary. We have now demonstrated the expression of both RFRP and its receptor (GPR147) in primary cultures of human granulosa-lutein cells. Immunohistochemical analysis of normal human ovaries from premenopausal women showed that RFRPs and GPR147 were primarily localized in the granulosa cell layer of large preovulatory follicles as well as in the corpus luteum. Treatment of human granulosa-lutein cells with RFRP-3 reduced FSH-, LH- and forskolin-stimulated progesterone production and steroidogenic acute regulatory protein expression but did not affect basal or 8-bromoadenosine 3'5'-cyclic monophosphate stimulated levels. In addition, RFRP-3 inhibited gonadotropin- and forskolin-induced intracellular cAMP accumulation, and these effects were abolished by pretreatment with an inhibitor of inhibitory G(i/o) proteins (pertussis toxin). Importantly, the effects of RFRP-3 on FSH-, LH-, and forskolin-induced cAMP and progesterone accumulation were completely eliminated by cotreatment with the bifunctional GPR147/GPR74 antagonist RF9 or by pretreatment with GPR147 small interfering RNA. These results suggest that RFRP-3 is expressed in human granulosa cells in which it acts via its receptor, GPR147, to inhibit gonadotropin signaling at the level of adenylyl cyclase via activation of a pertussis toxin-sensitive Gα(i/o) protein. This leads to reduced gonadotropin-stimulated cAMP accumulation and progesterone synthesis, likely via reduced steroidogenic acute regulatory protein expression. Thus, ovarian RFRP-3/GPR147 signaling could contribute to normal ovarian function.

Co-culture of buffalo (Bubalus bubalis) preantral follicles with antral follicles: a comparative study of developmental competence of oocytes derived from in vivo developed and in vitro cultured antral follicles

The present study was undertaken to examine whether the presence of antral follicles (AFs) affects the survival, growth and steroidogenesis of preantral follicles (PFs) and compare the maturation and developmental competence of buffalo oocytes derived from in vivo developed and in vitro cultured AFs. Two experiments were carried out. In experiment I, PFs (200-250 μm) were isolated and cultured with or without AFs (3-5 mm) in TCM-199 medium that contained 10% fetal bovine serum (FBS), 1% insulin transferin selenium (ITS), 20 ng/ml epidermal growth factor (EGF), 0.5 μg/ml follicle-stimulating hormone (FSH) and 100 ng/ml insulin-like growth factor (IGF)-I. In experiment II, in vitro developmental competence was compared for the cumulus-oocyte complexes (COCs) recovered from in vivo developed and in vitro cultured AFs. Survival, growth, development of antrum, accumulation of estradiol and progesterone was (P < 0.05) higher when PFs were co-cultured with AFs. Developmental competence of both types of follicular oocytes did not differ significantly in terms of maturation and cleavage rate, but morula and blastocyst production rate were (P < 0.05) higher with in vivo developed AFs as compared with the in vitro cultured antral follicular oocytes. In conclusion, co-culture of PFs with AFs supports long-term survival and growth of buffalo PFs and this co-culture system plays a dual role for in vitro production of embryos as well as understanding the relationship between developing PFs and AFs.

Activated Steroidogenic Factor 1 Expression Induces Estrogen Synthesis in Benign Prostate Hyperplasia Cell Line BPH-1.

Prostate growth in development and disease depends on androgens synthesized by the testis. Thus, treatment of prostate cancer targets androgen production and activity at several fronts. Initially, prostate tumors subject to androgen deprivation therapy regress, but inevitably, rogue cancer cells prevail and an aggressive cancer returns that is unresponsive to this therapy. There are several theories to explain this aggressive resurgence, one of which suggests that prostate adenocarcinoma cells acquire machinery for de novo steroidogenesis. It has long been established that Steroidogenic Factor 1 (NR5A1, ADBP4, SF1) is essential for regulating steroidogenesis in normal endocrine tissues. While normal prostate tissue lacks SF1 expression and is nonsteroidogenic, the presence of SF1 in diseased tissue of the prostate has not been documented, presenting an unexplored potential driver of aberrant steroidogenesis in prostate cancer. We hypothesize then that SF1 is present in prostate cancer and that SF1 is necessary for steroidogenesis to occur in prostate epithelial cells. To test the first part of our hypothesis, we examined SF1 expression in a panel of both benign and cancerous prostate cell lines. All prostate cancer cell lines examined expressed SF1, while a benign prostate hyperplasia cell line did not. We then performed immunohistochemistry on prostate biopsy samples from human patients with benign prostate hyperplasia or recurrent prostate cancer following androgen deprivation therapy. Similar to our results from prostate cell lines, we detected positive nuclear staining for SF1 in prostate adenocarcinoma but not benign prostate hyperplasia specimens. To functionally link SF1 to steroidogenic production in prostate epithelial cells, we tested whether ectopic expression of SF1 was sufficient to induce steroid production in a benign prostate cell line (BPH-1). BPH-1 cells were transfected with expression vectors encoding the mCherry fluorescent protein (negative control), wild type SF1, or a constitutively active mutant of SF1 (SF1ΔLBD). After 48 hours in culture, media was collected and subject to steroid measurement by ELISA. Whereas accumulation of progesterone and testosterone were not detected, estradiol concentrations were increased in BPH-1 cells transfected with wild type SF1 and even more so with transfected SF1ΔLBD. Together, these data indicate that SF1 is induced both in prostate cancer cell lines and in human prostate adenocarcinoma biopsy specimens, and suggest that activated SF1 is sufficient for steroidogenesis in a benign prostate cell line. Our findings present a potential mechanism and target for therapy of androgen deprivation resistant prostate cancers. Future experiments are planned to elucidate the stimulatory signals that induce SF1 within these aggressive prostate tumor cells leading to aberrant steroid production that fuels growth of deadly prostate cancer. This work was supported by The University of Wisconsin-Madison and NIH 5T32-HD041921-07. (platform)

Steroidogenesis and steroidogenic gene expression in postnatal fetal rat Leydig cells

We studied steroidogenesis and the regulation of Leydig cell-specific gene expression in primary cultures of highly purified postnatal fetal Leydig cells (PFLCs). PFLCs activated by hCG and (Bu) 2cAMP demonstrated transient capacity to produce testosterone (T) in vitro. A time dependent decline in T production by (Bu) 2cAMP-stimulated PFLCs was observed and associated with the accumulation of progesterone in the culture media and complete suppression of P450c17 expression at the translational but not transcriptional level. PFLCs was found to lose their capacity to express Leydig cell-related genes (e.g., 3βHSD, P450c17, Insl3), which was restored by treatment with (Bu) 2cAMP. It was also found that PDGFα alone and in combination with (Bu) 2cAMP significantly stimulated proliferation of the isolated PFLCs in vitro. Our data indicate that cAMP-activated signaling pathway(s) play an important role in the regulation of PFLC differentiation and function.

Localization and Expression of Follicle‐Stimulating Hormone Receptor Gene in Buffalo (Bubalus bubalis) Pre‐Antral Follicles

Follicle-stimulating hormone (FSH) stimulates antral follicles to grow, but its role in earlier stages (pre-antral) of follicle development, if any, is obscure. Aim of this study was to study the expression of follicle-stimulating hormone receptor (FSHR) gene in different sizes of pre-antral follicles (PFs) (<150, 200, 250, 300, 350, 400 μm) and to find out an optimum dose of FSH for better growth, development and steroidogenesis of PFs in vitro. Buffalo ovaries were collected from a local abattoir, and PFs were isolated by mechanical method. A semi-quantitative RT-PCR amplification strategy was used for mRNA expression, while FSHR protein was localized by immunohistochemistry. Isolated pre-antral follicles (80-85 μm) were cultured in TCM-199 supplemented with 10% foetal bovine serum, 1% ITS and 30 ng/ml EGF served as control medium. Addition of three different doses of FSH (0.5, 1.0, 2.0 μg/ml) in control medium was considered as treatment groups. A single 2.184-kb receptor mRNA transcript was present in all sizes (<150-400 μm) of follicles. Follicle-stimulating hormone receptor was also localized immunohistochemically in granulosa cells of all sizes of follicles. Survival and growth rate of follicles significantly (p<0.05) increased following supplementation of FSH at a concentration of 1.0 μg/ml and the culture medium also showed a significantly (p<0.05) greater accumulation of oestradiol and progesterone. In conclusion, FSHR is expressed in all sizes of PFs and in vitro survival, growth and steroidogenesis of follicles are optimally stimulated by 1.0 μg/ml FSH. These findings demonstrate that FSH has an important role during the recruitment, growth and development of buffalo ovarian PFs.

Effects of Cholesterol on Progesterone Production by Goat Luteal Cell Subpopulations at Two Different Stages of the Luteal Phase

The aim of the present study was to evaluate the effects of cholesterol on progesterone production during long-term culturing of luteal cell subpopulations at early and late luteal stages of the goat corpora lutea. Corpora lutea were collected from Angora goats on days 5 and 15 of the oestrous cycle. Luteal cells were isolated by collagenase digestion. The cells were separated into two distinct subpopulations by Percoll density-gradient centrifugation. Both subpopulations of luteal cells staining positively for 3β-HSD activities (5 × 10(4) cell/well) were cultured with or without 22(R)-hydroxycholesterol (22R-HC) in serum-free culture medium for periods of up to 7 days. Cells were incubated with serum (10%) for the first 18 h of incubation followed by serum-free medium. Cell treatment (10 and 20 μg/ml) was performed on days 1, 3 and 5. Treatment of cells with both concentrations of 22R-HC resulted in significant (p < 0.01) and dose-dependent stimulation (p > 0.05) on progesterone production in both fractions of cells throughout 7 days of incubation. Treatment of the cells with cholesterol resulted in 2.5- and 9.0-fold increases in progesterone accumulation on day 3 of incubation. Steroid production was maintained throughout the incubations when cells are incubated in serum-free media treated with cholesterol and ITS premix. Cells collected from higher density of percoll layers produced 2.82 and 2.32 times more progesterone, in comparison to the lover density percoll layer, on days 5 and 15 of the oestrous cycle in untreated cell groups, respectively. Progesterone accumulation was decreased as incubation time advanced in all groups of untreated cells. These results demonstrated that goat luteal cell subpopulations secrete substantial amounts of progesterone in response to cholesterol treatment at least for 7 days, and cholesterol is required as progesterone precursor for maintaining a high-level steroidogenesis during long-life culturing of both cell subpopulations.

Association Between Leptin, LH and its Receptor and Luteinization and Progesterone Accumulation (P4) in Bovine Granulosa Cell In Vitro

A full understanding of the cellular events that occur during in vitro luteinization of bovine granulosa cells, stimulated by LH and by leptin, is a complex goal that has not been completely achieved. The aim of this work was to study the effects of leptin, LH and leptin + LH on progesterone accumulation (P4) and on the expression of LH receptors (LHR) in bovine granulosa cells in culture. The results confirm that this in vitro model is representative of functional and morphological luteinization/differentiation. The pattern of expression of LHR with time of incubation was an important marker of in vitro luteinization, with 50-90% of cells expressing LHR by 96 h in culture. Cytoplasmic lipidic droplets were highly abundant in granulosa cells, suggesting a sufficient source of precursors for steroid hormone synthesis: P4 accumulation ranged between 40 and 550 ng/ml. In addition, a positive correlation (r = 0.58, p < 0.05) between the expression of LHR and accumulation of P4 throughout the time of incubation was observed. The expression of LHR was inhibited by LH and leptin + LH treatment. In conclusion, we found an inverse modulation between the expression of LHR and the concentration of LH, and the expression of LHR could be regulated by P4 produced by the luteinized granulosa cells. These findings are contributing to elucidate further the panoply of interactions during the differentiation of granulosa cells into luteal cells in vitro.

Progesterone levels in cesarean and normal delivered term placentas

One of the most important hormones synthesized by the placenta during pregnancy is progesterone. The regulating mechanisms of progesterone synthesis and the mechanism responsible for the spontaneous onset of labor in women are still not fully understood. Progesterone is thought to have been involved in human parturition. The objective of this study was to compare the levels of progesterone in the human placentas, at the end of the gestation (37-41 weeks) in vaginal versus cesarean deliveries, and to evaluate the pattern of progesterone accumulation, instantly following its synthesis by the human placenta at the end of the pregnancy. Progesterone levels in human placental tissue were determined by immunochemiluminescent analysis, following tissue homogenization. Progesterone secretion and accumulation pattern in the placental tissue was demonstrated using the ex vivo, closed, dual perfusion system of isolated human placental cotyledon. Immunochemiluminescent analysis of progesterone levels in human normal and cesarean-delivered placentas showed that placentas following normal vaginal delivery store higher concentrations of progesterone, and produce progesterone more intensively. Results obtained from 120-min perfusions (of vaginal and cesarean-delivered placentas) showed that progesterone tended to accumulate in the maternal rather than the fetal compartment. These data indicate that progesterone levels continuously rise till the end of pregnancy, with no apparent drop in progesterone levels during the labor process. In addition, progesterone is released from the syncytiotrophoblast preferably into the maternal component of the placental tissue.

Differentiation-dependent progesterone synthesis and metabolism in NT2-N human neurons

Human embryonic teratocarcinoma-derived Ntera2/cl.D1 (NT2) cells recapitulate many features of embryonic neuronal progenitor cells. Upon retinoic acid (RA) treatment they terminally differentiate into post-mitotic neuron-like cells (NT2-N), akin to human fetal neurons, thus representing an in vitro model of human neuron terminal differentiation. Experimental evidence also indicate NT2-N cultures as a potential source for cell transplantation therapy. The neurosteroids progesterone and its metabolite 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) promote neurogenesis and show anti-neurodegenerative properties. This study's aim was to assess the neurosteroidogenic competence of NT2 cells during RA-induced neuronal differentiation. Radioimmunoassay measurements revealed progesterone only in NT2-N cultures (4 week RA). Accordingly, progesterone synthesis from 3H-pregnenolone was absent in NT2 cells and increased during RA exposure, being highest in NT2-N. [ 3H]-pregnenolone metabolism, yielding [ 3H]-progesterone and [ 3H]-5α-dihydroprogesterone ([ 3H]-5α-DHP), was time-dependent and inhibited by trilostane, a 3β-hydroxysteroid-dehydrogenase (3β-HSD) inhibitor. Conversely, 3H-progesterone metabolism, which yielded [ 3H]-5α-DHP > [ 3H]-3β,5α-THP > [ 3H]-3α,5α-THP, occurred at all time points examined, though showing a nadir in cultures treated with RA for 1 and 2 weeks. The differentiation-dependent increase of progesterone accumulation matched 3β-HSD type I mRNA expression and 3β-HSD immunoreactivity, that co-localized with Map2a/b- and GAD67 in NT2-N. Hence, in vitro differentiated human neurons, while retaining progesterone metabolic activity, also become competent in progesterone synthesis. These findings suggest an autocrine/paracrine role of neuronal progesterone, either on its own or through its 5α-reduced metabolites, in fetal brain development and allow speculation that NT2-N-produced neurosteroids may contribute to the encouraging results of NT2-N transplants in animal models of neurodegenerative diseases.

Survival and developmental competence of buffalo preantral follicles using three-dimensional collagen gel culture system

The aim of the present study was to develop a three-dimensional (3D) collagen gel culture system for the in vitro growth and survival of buffalo preantral follicles with or without growth factors. Buffalo ovaries were collected from a local abattoir and preantral follicles were isolated through microdissection. Isolated preantral follicles were put either in collagen gel coated culture dish or embedded in a microdrop of collagen gel. The culture medium was TCM-199 fortified with fetal calf serum (10%), insulin transferin selenium solution (ITS, 1%), epidermal growth factor (EGF, 20 ng/ml) and follicle stimulating hormone (FSH, 0.5 μg/ml). Follicles were divided into three groups and cultured in the medium described above (group a, control), with addition of insulin like growth factor (IGF-I, 100 ng/ml, group b), or with addition of IGF-I and basic fibroblast growth factor (bFGF, 10 ng/ml, group c). Preantral follicles were incubated at 38.5 °C in 5% CO 2 and maximum humidity. Culture medium was replenished after every 72 h and spent medium was stored at −30 °C for hormone analysis. We found that the extracellular matrix of collagen gel maintained follicle viability and growth by providing surface interaction and increasing attachment of follicles. Preantral follicles embedded in collagen gel droplets had better antrum formation and development as compared to the whole surface coated culture method. Follicles cultured with IGF-I on collagen gel matrix showed a significantly ( P < 0.05) higher survival rate and larger mean diameter of follicles on day 10 of culture with improved growth and mucification as compared to the control group. However, follicles cultured in the combination of IGF-I with bFGF had decreased survival rate and smaller mean follicles diameter than the IGF-I group (b). Progesterone (P 4) accumulation was greater on day 9 of culture in follicles cultured in IGF-I as compared to control; whereas, P 4 was markedly decreased in the combination of IGF-I with bFGF. Follicles of the control group could survive for up to 10–15 days before degenerating, but follicles cultured with growth factors were able to survive up to 20 days and showed signs of early antrum formation. In summary, we have shown that collagen gel was a novel and efficacious 3D microenvironment for the extended culture of buffalo preantral follicles. Supplementation of culture medium with growth factors was found to be essential for antrum formation.

GATA4 Reduction Enhances 3′,5′-Cyclic Adenosine 5′-Monophosphate-Stimulated Steroidogenic Acute Regulatory Protein Messenger Ribonucleic Acid and Progesterone Production in Luteinized Porcine Granulosa Cells

Previous studies with cultured granulosa cells implicated GATA4 in gonadotropin regulation of the steroidogenic acute regulatory protein (STAR) gene. Caveats to these prior studies exist. First, GATA4 levels are reduced in granulosa-luteal cells after the LH surge when GATA6 expression is relatively high. Second, STAR mRNA expression is negligible in granulosa cells until after the LH surge. Both exogenous GATA4 and GATA6 can transactivate STAR gene promoter constructs. We used an RNA interference (RNAi) approach to determine the contributions of GATA4 and GATA6 to cAMP analog regulation of the endogenous STAR gene in luteinizing granulosa cells. STAR mRNA was stimulated by cAMP under control RNAi conditions. Surprisingly, GATA4 reduction by its respective RNAi approximately doubled the cAMP induction of STAR mRNA. At 24 h cAMP treatment, this augmentation was abolished by co-down-regulation of GATA4+GATA6. GATA6 down-regulation by itself did not alter STAR mRNA levels. GATA4+GATA6 co-down-regulation elevated basal CYP11A mRNA at 24 h treatment but did not affect its induction by cAMP. Basal levels of HSD3B mRNA were reduced by GATA4 RNAi conditions leading to a greater fold induction of its mRNA by cAMP. Fold cAMP-stimulated progesterone production was enhanced by GATA4 down-regulation but not by GATA4+GATA6 co-down-regulation. These data implicate GATA6 as the facilitator in cAMP-stimulated STAR mRNA and downstream progesterone accumulation under reduced GATA4 conditions. Data also demonstrate that basal levels of GATA4/6 are not required for cAMP induction of the STAR gene. The altered ratio of GATA4 to GATA6 after ovulation may allow GATA6 to enhance STAR mRNA accumulation.

Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for appropriate expression of steroidogenic and ovulation-related genes that impact oocyte maturation in vivo and in vitro

In this study, we collected follicular fluid, granulosa cells, and cumulus cells from antral follicles at specific time intervals following equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) treatment of gilts. The treatment with eCG increased the production of estrogen coordinately with up-regulated proliferation of granulosa and cumulus cells. eCG also induced the expression of LHCGR and PGR in cumulus cells and progesterone accumulation was detected in follicular fluid prior to the LH/hCG surge. Moreover, progesterone and progesterone receptor (PGR) were critical for FSH-induced LHCGR expression in cumulus cells in culture. The expression of LHCGR mRNA in cumulus cells was associated with the ability of LH to induce prostaglandin production, release of epidermal growth factor (EGF)-like factors, and a disintegrin and metalloprotease with thrombospondin-like repeats 1 expression, promoting cumulus cell oocyte complexes (COCs) expansion and oocyte maturation. Based on the unique expression and regulation of PGR and LHCGR in cumulus cells, we designed a novel porcine COCs culture system in which hormones were added sequentially to mimic changes observed in vivo. Specifically, COCs from small antral follicles were pre-cultured with FSH and estradiol for 10 h at which time progesterone was added for another 10 h. After 20 h, COCs were moved to fresh medium containing LH, EGF, and progesterone. The oocytes matured in this revised COC culture system exhibited greater developmental competence to blastocyst stage. From these results, we conclude that to achieve optimal COC expansion and oocyte maturation in culture the unique gene expression patterns in cumulus cells of each species need to be characterized and used to increase the effectiveness of hormone stimulation.

Assessment of neuroactive steroid formation in diabetic rat spinal cord using high-performance liquid chromatography and continuous flow scintillation detection

The combination of pulse-chase experiments with high-performance liquid chromatography and continuous flow scintillation detection was used successfully to determine the effects of chronic diabetes on neurosteroid production in the adult rat spinal cord. The long-term diabetes was induced by treatment of adult rats with streptozotocin. In the first part, the review provides an extensive description of the HPLC combined with continuous flow scintillation detection method, its advantages and appropriateness for the question investigated. Afterwards, the paper shows that progesterone formation is up-regulated in the spinal cord of diabetic rats while the biosynthesis of tetrahydroprogesterone decreased. The down-regulation of tetrahydroprogesterone appeared as a mechanism facilitating progesterone accumulation in the spinal cord of streptozotocin-treated rats. Progesterone is well known to be a potent neuroprotective steroid. Enhancement of its biosynthesis may be an endogenous mechanism triggered by neural cells in the spinal tissue to cope with degenerative effects provoked by chronic diabetes. Since steroid metabolism in the spinal cord is pivotal for the modulation of several neurobiological processes including sensorimotor activities, the data analyzed herein may constitute useful information for the development of efficient strategies against deleterious effects of diabetes on the nervous system.

Carbaryl inhibits basal and FSH-induced progesterone biosynthesis of primary human granulosa-lutein cells

Carbaryl is known to impede female reproductive function, however, the mechanisms through which the adverse effects are mediated are not clearly elucidated. In order to get insight into the mechanisms, this study was conducted to raise fresh concerns about the potential effects of carbaryl on steroidogenesis by primary human granulosa-lutein cells (hGLCs) and explore the possible nature of this action. hGLCs were co-incubated with various concentrations of carbaryl at 0, 1, 5, 25, 125 μmol/L for 24 h to examine effects of this carbamate pesticide on progesterone accumulation. We observed that the carbaryl inhibited basal and FSH-induced progesterone production in a dose-dependent manner. We also investigated the effects of carbaryl on 22( R)-hydroxycholesterol (22 R-HC)-stimulated progesterone yield, basal and FSH-stimulated StAR gene expression and cyclic adenosine monophosphate (cAMP) production, as well as forskolin (non-specific activator of adenylyl cyclase)-induced progesterone and cAMP production of hGLCs. We found that the decreased progesterone biosynthesis was accompanied with a reduced cAMP abundance on both basal and FSH-induced condition. Furthermore, our results demonstrated that the 22 R-HC could remove the carbaryl-induced restraint of progesterone biosynthesis, suggesting that carbaryl caused a disruption of cholesterol transport across mitochondrial membranes, which was further confirmed by the observation that carbaryl inhibited the gene expression of steroidogenic acute regulatory protein (StAR). In addition, the inhibitory effects of carbaryl on progesterone and cAMP production were completely reversed by addition of forskolin to the cell culture, which indicated a repaired site on the upstream components of adenylate cyclase or adenylate cyclase per se by carbaryl in the cAMP-mediated signal pathway. All the effects mentioned above were not due to a detrimental action of carbaryl on cell viability by MTS assay. In conclusion, carbaryl may inhibit steroidogenesis, at least in part, by obstructing the delivery of cholesterol over mitochondrial membranes and attenuating cAMP generation.

Ganglionic Adrenergic Action Modulates Ovarian Steroids and Nitric Oxide in Prepubertal Rat

Both peripheral innervation and nitric oxide (NO) participate in ovarian steroidogenesis. The purpose of this work was to analyse the ganglionic adrenergic influence on the ovarian release of steroids and NO and the possible steroids/NO relationship. The experiments were carried out in the ex vivo coeliac ganglion-superior ovarian nerve (SON)-ovary system of prepubertal rats. The coeliac ganglion-SON-ovary system was incubated in Krebs Ringer-bicarbonate buffer in presence of adrenergic agents in the ganglionic compartment. The accumulation of progesterone, androstenedione, oestradiol and NO in the ovarian incubation liquid was measured. Norepinephrine in coeliac ganglion inhibited the liberation of progesterone and increased androstenedione, oestradiol and NO in ovary. The addition of alpha and beta adrenergic antagonists also showed different responses in the liberation of the substances mentioned before, which, from a physiological point of view, reveals the presence of adrenergic receptors in coeliac ganglion. In relation to propranolol, it does not revert the effect of noradrenaline on the liberation of progesterone, which leads us to think that it might also have a "per se" effect on the ganglion, responsible for the ovarian response observed for progesterone. Finally, we can conclude that the ganglionic adrenergic action via SON participates on the regulation of the prepubertal ovary in one of two ways: either increasing the NO, a gaseous neurotransmitter with cytostatic characteristics, to favour the immature follicles to remain dormant or increasing the liberation of androstenedione and oestradiol, the steroids necessary for the beginning of the near first estral cycle.

Participation of specific PKC isozymes in the inhibitory effect of ET-1 on progesterone accumulation in cells isolated from early- and mid-phase corpora lutea

Expression of PKC α, β I, β II, ɛ and μ has been demonstrated in the whole bovine CL with PKC ɛ being differentially expressed as a function of development. In experiment 1 we have investigated the amount of mRNA encoding PKC ɛ at different stages of luteal development (days 1, 4, 10 and 17). In experiment 2, the cellular source of luteal PKC isozymes was determined. Enriched steroidogenic (SC) and endothelial (EC) cells from day-10 CL were used to examine this question by Western blot analysis and immuno-histochemistry. In experiment 3, Western blot analysis was used to examine the ability of ET-1 to activate luteal PKC isozymes in day-10 CL. In experiment 4, the role of luteal PKC isozymes in the ET-1 mediated inhibition of P 4 accumulation in steroidogenic cell cultures from day-4 and day-10 CL was examined. Abundance of PKC ɛ mRNA gradually increased from day-1 to -10 with no further increase on day-17. In experiment 2, PKC ɛ was exclusively detected in SC (LLC and SLC). In contrast, PKC α, β I and β II were detected in both SC and EC, with EC expressing higher amounts of PKC isozymes. In day-10 CL, ET-1 induced cellular redistribution of PKC α, β I, ɛ but not β II. Inhibitors specific for conventional PKC isozymes as well as PKC ɛ were able to negate the inhibitory effects of ET-1 on P4 accumulation in the day10 CL. In the day-4 CL, the inhibitory effect of ET-1 might be mediated via conventional PKC. Thus, an exclusive presence of PKC ɛ in luteal steroidogenic cells, its higher expression along with the ability of ET-1 to stimulate its activation in day-10 CL strongly suggests that this PKC isoform may play an important regulatory role in decreasing P 4 during luteal regression. Inhibition of P 4 by ET-1 in the early CL may be mediated via conventional PKC isozymes.

Effect of Grazing and Fat Supplementation on Production and Reproduction of Holstein Cows

The objective of this trial was to investigate the effects of feeding a soybean oil refining by-product (SORB), made up mainly of sodium salts of long-chain fatty acids, on reproductive performance and productivity of 36 early lactation Holstein cows managed in a free-stall barn or on annual rye-ryegrass pasture. In this 2×2 factorial arrangement of treatments, cows consumed 0 or 0.5kg/d of SORB as part of a total mixed ration for barn cows or as part of a grain supplement fed to cows on intensively, rotationally stocked pasture. Blood was sampled 3 times weekly and plasma was measured for progesterone to assess ovarian activity. Estrus activity was recorded using the HeatWatch estrus detection system. Although average 14-wk milk production (37.2kg/d) was not different among treatments, barn cows had more persistent lactations than did grazing cows. Cows housed in the barn lost less body weight and returned to initial body weight sooner and had lower mean concentrations of plasma nonesterified fatty acids (464 vs. 261mEq/L) than those managed on pasture. The milk fat of cows on pasture contained greater proportions of conjugated linoleic acid and linolenic acid but a corresponding 0.22 percentage unit decrease in milk fat concentration (3.39 vs. 3.16%). Cows managed on pasture had greater peak concentrations of plasma progesterone during the first estrous cycle. Cows managed on pasture and fed SORB had the greatest accumulation of plasma progesterone over the 14 wk of the study (SORB×housing interaction). These cows experienced the most mounts during their first estrus (9.3) and pregnancy rate was also greatest for this treatment (62.5%). Feeding SORB did not affect production of milk, fat, or protein. Loss of body condition was less in cows fed SORB. Ruminal fluid concentration of propionate increased and ruminal pH decreased in cows fed SORB. A lower proportion of fatty acids less than 18 carbons in length was found in the milk fat of cows fed SORB, thus indicating lower de novo synthesis of fatty acids. Higher proportions of C18:2n-6 and conjugated C18:2 were found in the milk fat of cows fed SORB. Based on concentrations of plasma progesterone, cows fed SORB experienced their first ovulation earlier (26.7 vs. 42.4 d postpartum) than did cows not supplemented with SORB. Neither housing system nor SORB supplementation influenced detection of first estrus (50.5 d) or the mean length of each estrus period (447min).

A study to look at hormonal absorption of progesterone cream used in conjunction with transdermal estrogen

Natural progesterone creams are gaining popularity as a possible treatment for menopausal symptoms, and many women may be using them with estrogen. We planned to evaluate, using an open plan study, the systemic absorption of a combination of transdermal estrogen and progesterone. Women applied transdermal progesterone 40 mg and transdermal estrogen 1 mg daily over 48 weeks. Women were assessed at intervals of 12 weeks. Significant increases in plasma levels of progesterone and estradiol were seen after 12 weeks, although only low plasma progesterone levels were found (median 2.5 nmol/l) and no further increase was noted over the remainder of the study period. A significant correlation was found between plasma levels of the two hormone (r = 0.315, p = 0.045). Women reported significant reductions in menopausal symptoms, as measured by the Green Climacteric Scale, after 24 and 48 weeks of combined treatment. There may be similar mechanisms of absorption of the two hormones, although the doses used in our study produced sub-luteal levels of progesterone. There was no evidence of accumulation of progesterone with time, and further study is needed to assess the efficacy and safety of this combination of hormones.

Effect of streptozotocin-induced diabetes on the gene expression and biological activity of 3β-hydroxysteroid dehydrogenase in the rat spinal cord

Abnormal secretion of steroids by the adrenals and gonads is one of the disturbances occurring in diabetics but the impact of diabetes on steroid formation in the nervous system has never been studied. However, it is well known that numerous actions of peripheral steroids on the nervous system require their conversion into neuroactive metabolites within the neural tissue. As this in situ steroid synthesis/metabolism is crucial for the control of several neurobiological functions, we investigated the effects of streptozotocin-induced diabetes on the gene expression and activity of 3β-hydroxysteroid dehydrogenase in the spinal cord, a pivotal structure involved in sensorimotor and neurovegetative mechanisms. 3β-Hydroxysteroid dehydrogenase is a key enzyme which participates to the biosynthesis of all classes of steroids by converting Δ 5-3β-hydroxysteroids such as pregnenolone and dehydroepiandrosterone into Δ 4-3-ketosteroids as progesterone and androstenedione, respectively. Reverse transcription coupled with quantitative real-time polymerase chain reaction revealed that 3β-hydroxysteroid dehydrogenase gene was over-expressed in the spinal cord of streptozotocin-treated rats compared with controls. Pulse-chase experiments combined with high performance liquid chromatography and continuous flow detection of newly-synthesized steroids showed an increase of 3β-hydroxysteroid dehydrogenase activity responsible for a hyper-production of progesterone in the spinal cord of diabetic rats. This up-regulation of progesterone biosynthesis was concomitant with a decrease of its transformation into tetrahydroprogesterone, a process which facilitated progesterone accumulation in the spinal cord of streptozotocin-treated rats. Since progesterone is a potent neuroprotective steroid, increase of its production appeared as an endogenous molecular and biochemical mechanism triggered by spinal nerve cells to cope with degenerative effects of streptozotocin-induced diabetes. Our results constitute the first direct evidence showing an impact of diabetes on steroid biosynthetic and metabolic pathways in the nervous system. The data open new perspectives for the modulation of deleterious effects of diabetes by neuroprotective steroids.

Effects of Selective Protein Kinase C Isozymes in Prostaglandin2α-Induced Ca2+ Signaling and Luteinizing Hormone-Induced Progesterone Accumulation in the Mid-Phase Bovine Corpus Luteum1

A single-cell approach for measuring the concentration of cytoplasmic calcium ions ([Ca(2+)](i)) and a protein kinase C-epsilon (PKCepsilon)-specific inhibitor were used to investigate the developmental role of PKCepsilon in the prostaglandin F(2alpha)(PGF(2alpha))-induced rise in [Ca(2+)](i) and the induced decline in progesterone accumulation in cultures of cells isolated from the bovine corpus luteum. PGF(2alpha) increased [Ca(2+)](i) in Day 4 large luteal cells (LLCs), but the response was significantly lower than in Day 10 LLCs (4.3 +/- 0.6, n = 116 vs. 21.3 +/- 2.3, n = 110). Similarly, the fold increase in the PGF(2alpha)-induced rise in [Ca(2+)](i) in Day 4 small luteal cells (SLCs) was lower than in Day 10 SLCs (1.6 +/- 0.2, n = 198 vs. 2.7 +/- 0.1, n = 95). A PKCepsilon inhibitor reduced the PGF(2alpha)-elicited calcium responses in both Day 10 LLCs and SLCs to 3.5 +/- 0.3 (n = 217) and 1.3 +/- 0.1 (n = 205), respectively. PGF(2alpha) inhibited LH-stimulated progesterone (P(4)) accumulation only in the incubation medium of Day 10 luteal cells. Both conventional and PKCepsilon-specific inhibitors reversed the ability of PGF(2alpha) to decrease LH-stimulated P(4) accumulation, and the PKCepsilon inhibitor was more effective at this than the conventional PKC inhibitor. In conclusion, the evidence indicates that PKCepsilon, an isozyme expressed in corpora lutea with acquired PGF(2alpha) luteolytic capacity, has a regulatory role in the PGF(2alpha)-induced Ca(2+) signaling in luteal steroidogenic cells, and that this in turn may have consequences (at least in part) on the ability of PGF(2alpha) to inhibit LH-stimulated P(4) synthesis at this developmental stage.