Basal Progesterone Research Articles

Luteinizing Hormone and GATA4 Action in the Adrenocortical Tumorigenesis of Gonadectomized Female Mice

Background/Aims: Physiological role of luteinizing hormone (LH) and its receptor (LHCGR) in adrenal remains unknown. In inhibin-α/Simian Virus 40 T antigen (SV40Tag) (inhα/Tag) mice, gonadectomy-induced (OVX) elevated LH triggers the growth of transcription factor GATA4 (GATA4)-positive adrenocortical tumors in a hyperplasia-adenoma-adenocarcinoma sequence. Methods: We investigated the role of LHCGR in tumor induction, by crossbreeding inhα/Tag with Lhcgr knockout (LuRKO) mice. By knocking out Lhcgr and Gata4 in Cα1 adrenocortical cells (Lhcgr-ko, Gata4-ko) we tested their role in tumor progression. Results: Adrenal tumors of OVX inhα/Tag mice develop from the hyperplastic cells localized in the topmost layer of zona fasciculata. OVX inhα/Tag/LuRKO only developed SV40Tag positive hyperplastic cells that were GATA4 negative, cleaved caspase-3 positive and did not progress into adenoma. In contrast to Lhcgr-ko, Gata4-ko Cα1 cells presented decreased proliferation, increased apoptosis, decreased expression of Inha, SV40Tag and Lhcgr tumor markers, as well as up-regulated adrenal- and down-regulated sex steroid gene expression. Both Gata4-ko and Lhcgr-ko Cα1 cells had decreased expression of steroidogenic genes resulting in decreased basal progesterone production. Conclusion: Our data indicate that LH/LHCGR signaling is critical for the adrenal cell reprogramming by GATA4 induction prompting adenoma formation and gonadal-like phenotype of the adrenocortical tumors in inhα/Tag mice.

60 EFFECTS OF CONCANAVALIN A ON THE PROGESTERONE PRODUCTION BY BOVINE STEROIDOGENIC LUTEAL CELLS IN VITRO

The corpus luteum is a temporary organ that is responsible for progesterone (P4) secretion and is essential for the establishment and maintenance of pregnancy in cattle. Concanavalin A (CONA) is a lectin that was originally extracted from the Jack bean (Canavalia ensiformis) and that interacts with several kinds of cells, including immune cells and luteal cells. The aim of the present study was to evaluate the effects of CONA on the P4 production by bovine steroidogenic luteal cells (LC) in vitro. Luteal cells were collected during the mid-luteal stage (at 10–12 days following ovulation) and processed in the laboratory. Luteal cells were grown for 7 days in a humid atmosphere with 5% CO2, with or without 10% fetal bovine serum (FBS), and were subjected to the following treatments: control: no treatment; CONA (10 μg mL−1); LH (100 μg mL−1); CONA+LH; LH (100 μg mL−1) + prostaglandin F2α (PGF2α; 10 ng mL−1); CONA+LH+PGF2α. Samples of the culture media were collected on Day 1 and Day 7 for P4 quantification. The cells were counted on Day 7 of culture. Differences between treatments were considered statistically significant at P < 0.05. The P4 concentration in the culture media was numerically greater on Day 1 (558.0 ng mL−1) than on Day 7 (25.4 ng mL−1). The P4 concentration in the culture media was numerically greater for treatments with 10% FBS than for the FBS-free treatments, and the presence of CONA decreased LC P4-secreting capacity. This effect required more than 24 h of exposure to CONA to be fully manifested. On Day 1 of culture, CONA had no effect on P4 production of LC cultured in serum-free medium (P > 0.05).The suppressive action of CONA was more pronounced for cultures without FBS. By Day 7 of culture, the effects of CONA on P4 production were readily apparent. In the absence of serum, CONA had a highly significant (P < 0.01) inhibitory effect on basal progesterone production, as well as in the presence of LH or LH + PGF. In the presence of FBS, there was a tendency for decreased P4 in response to CONA in the LH- and the LH + PGF-treated cells (P = 0.090 and 0.085, respectively). The number of the cells present on Day 7 was not affected by the treatments tested (P > 0.05). More studies are required to better understand the effect of CONA on the P4 production of bovine LC. Financial support from FAPESP is acknowledged: grant no. 2013/00992–3, grant no. 2013/07439–8, and grant no. 2015/01940–2.

Clinical Value of Basal Serum Progesterone Prior to Initiate Ovarian Hyper-Stimulation with GnRH Antagonists: A Retrospective Cohort Study

Background: The routine assessment of day 3 serum progesterone prior to initiation of ovarian hyper-stimulation with the use of GnRH antagonists is under debate. In this study, we evaluated the clinical utility of this policy. Methods: Retrospective cohort study of women undergoing in vitro fertilization (IVF) with the use of GnRH antagonists aimed at determining the frequency of cases with progesterone levels exceeding the recommended threshold of 1,660 pg/ml and at evaluating whether this assessment may be predictive of pregnancy. Results: Serum progesterone exceeded the recommended threshold in one case (0.3%, 95% CI 0.01-1.5). The median (interquartile range) basal progesterone in women who did (n = 95) and did not (n = 217) become pregnant were 351 (234-476) and 380 (237-531) pg/ml, respectively (p = 0.28). The 90th percentile of the basal progesterone distribution in women who became pregnant was 660 pg/ml. Cases with serum progesterone exceeding this threshold in successful and unsuccessful cycles were 10 (10%) and 30 (14%), respectively (p = 0.47). The capacity of basal progesterone to predict pregnancy was evaluated using receiver operating characteristic curve (area under the curve = 0.54, 95% CI 0.47-0.61, p = 0.28). No graphically evident threshold emerged. Conclusion: Routine day 3 serum progesterone assessment in IVF cycles with the use of GnRH antagonists is not justified. Further evidence is warranted prior to claiming its systematic use.

Stimulatory effects of combined endocrine disruptors on MA-10 Leydig cell steroid production and lipid homeostasis.

Previous work in our laboratory demonstrated that in-utero exposure to a mixture of the phytoestrogen Genistein (GEN), and plasticizer DEHP, induces short- and long-term alterations in testicular gene and protein expression different from individual exposures. These studies identified fetal and adult Leydig cells as sensitive targets for low dose endocrine disruptor (ED) mixtures. To further investigate the direct effects and mechanisms of toxicity of GEN and DEHP, MA-10 mouse tumor Leydig cells were exposed in-vitro to varying concentrations of GEN and MEHP, the principal bioactive metabolite of DEHP. Combined 10μM GEN+10μM MEHP had a stimulatory effect on basal progesterone production. Consistent with increased androgenicity, the mRNA of steroidogenic and cholesterol mediators Star, Cyp11a, Srb1 and Hsl, as well as upstream orphan nuclear receptors Nr2f2 and Sf1 were all significantly increased uniquely in the mixture treatment group. Insl3, a sensitive marker of Leydig endocrine disruption and cell function, was significantly decreased by combined GEN+MEHP. Lipid analysis by high-performance thin layer chromatography demonstrated the ability of combined 10μM combined GEN+MEHP, but not individual exposures, to increase levels of several neutral lipids and phospholipid classes, indicating a generalized deregulation of lipid homeostasis. Further investigation by qPCR analysis revealed a concomitant increase in cholesterol (Hmgcoa) and phospholipid (Srebp1c, Fasn) mediator mRNAs, suggesting the possible involvement of upstream LXRα agonism. These results suggest a deregulation of MA-10 Leydig function in response to a combination of GEN+MEHP. We propose a working model for GEN+MEHP doses relevant to human exposure involving LXR agonism and activation of other transcription factors. Taken more broadly, this research highlights the importance of assessing the impact of ED mixtures in multiple toxicological models across a range of environmentally relevant doses.

Aging influences steroid hormone release by mink ovaries and their response to leptin and IGF-I.

ABSTRACTThe aim of our study was to understand whether ovarian steroid hormones, and their response to the metabolic hormones leptin and IGF-I leptin, could be involved in the control of mink reproductive aging via changes in basal release of ovarian progesterone and estradiol. For this purpose, we compared the release of progesterone and estradiol by ovarian fragments isolated from young (yearlings) and old (3-5 years of age) minks cultured with and without leptin and IGF-I (0, 1, 10 or 100 ng/ml). We observed that isolated ovaries of older animals produced less progesterone but not less estradiol than the ovaries of young animals. Leptin addition stimulated estradiol release by the ovarian tissue of young animals but inhibited it in older females. Leptin did not influence progesterone output by the ovaries of either young or older animals. IGF-I inhibited estradiol output in young but not old animals, whereas progesterone release was inhibited by IGF-I irrespective of the animal age. Our observations demonstrate the involvement of both leptin and IGF-I in the control of mink ovarian steroid hormones release. Furthermore, our findings suggest that reproductive aging in minks can be due to (a) reduction in basal progesterone release and (b) alterations in the response of estradiol but not of progesterone to leptin and IGF-I.

Organophosphate Flame Retardants Act as Endocrine-Disrupting Chemicals in MA-10 Mouse Tumor Leydig Cells.

The organophosphate flame retardants (OPFRs) have emerged as alternatives to banned brominated flame retardants but little is known about their possible activity as endocrine disruptors. Our goal was to compare the effects of 7 commonly used OPFRsin vitroon MA-10 mouse Leydig tumor cells to those of a major brominated flame retardant, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47). The effects of OPFRs and BDE-47 on mitochondrial activity, cell counts, oxidative stress, steroid secretion and gene expression were investigated. BDE-47 and all 7 OPFRs tested significantly reduced MA-10 cell mitochondrial activity (concentrations ≥50 μM) and cell number (concentrations ≥10 μM). All of the OPFRs significantly increased (10 μM, 1.7-4.4-fold) superoxide production whereas BDE-47 had no significant effect. Basal progesterone production was significantly increased (10 μM, 1.5 to 3-fold) by 2-ethylhexyl diphenyl phosphate, isodecyl diphenyl phosphate, isopropylated triphenyl phosphate, tert-butylphenyl diphenyl phosphate, and tricresyl phosphate, while BDE-47, triphenyl phosphate and tri-o-cresyl phosphate had no effect. Interestingly, isopropylated triphenyl phosphate enhanced dbcAMP-stimulated steroid production (∼2-fold), while tri-o-cresyl phosphate decreased (∼2/3) LH-stimulated steroid production. Several OPFRs affected the expression of genes involved in the biosynthesis of progesterone. In conclusion, all the OPFRs tested affected mitochondrial activity, cell survival, and superoxide production. Basal or stimulated steroid secretion was affected by all of the OPFRs except triphenyl phosphate; BDE-47 had no effect. Hence, the OPFRs currently used as alternatives affect Leydig cells to a greater extent than the brominated flame retardants that they have replaced.

Effects of dbcAMP on progesterone synthesis by cultured goat luteal cell subpopulations isolated from early and late luteal stage corpora lutea

This research aimed to investigate the effects of dbcAMP on steroid accumulation by culturing two distinct luteal cell subpopulations isolated from early and late luteal stage corpora lutea. Cells were isolated from corpora lutea collected from eight Angora goats on either the 5th or 15th days of their estrous cycles. Cell isolation was performed by enzymatic digestion using collagenase and DNase. Isolated cells were separated into two distinct subpopulations enriched with small and large luteal cells by percoll density-gradient centrifugation. Isolated cells were stained in order to detect 3β-hydroxysteroid dehydrogenase (3β-HSD). Cells stained positively for 3β-HSD activity (5 x 104 cell⁄well) were incubated with dbcAMP in the absence or presence of 22(R)-hydroxycholesterol (22R-HC) for periods of up to 7 days. Large luteal cell enriched subpopulations produced more basal progesterone (P < 0.05) than did the small luteal cell enriched subpopulations. Treatment of cells with 22R-HC alone induced 4.00 to 11.60 times increase in steroid synthesis depending on type of cells incubated, luteal age and days of incubation. Incubation of cells with 1 mM dbcAMP in the absence or presence of 22R-HC induced in a significant increase (P < 0.01) in steroid accumulation in all treated groups. In contrast, when cells are treated with low dose dbcAMP (0.1 mM), treatment induced stimulation failed to reach significant level in most treated groups. In conclusion, although treatment of goat luteal cells with dbcAMP induces an increase in steroid accumulation, a high dose is necessary to reach significant levels. Stimulatory effect of dbcAMP on steroidogenesis maintains during long life culturing.

Effect of Anti-Müllerian hormone (AMH) and bone morphogenetic protein 15 (BMP-15) on steroidogenesis in primary-cultured human luteinizing granulosa cells through Smad5 signalling.

To determine if there is any effect of AMH and BMP-15 on estradiol and progesterone production from primary-cultured human luteinizing granulosa cells, to delineate what is the effect of FSH on their actions and which are the possible mechanisms involved. Luteinizing granulosa cells (GCs), obtained from follicular fluid of 30 women undergoing in vitro fertilization, were cultured, after a short 24-h preincubation period, in serum-free medium for 24 or/and 48 h in the presence/absence of various concentrations of AMH, BMP-15 and FSH alone or in combinations. Estradiol and progesterone production, SMAD5 phosphorylation and StAR expression were studied in parallel. Steroids were measured in culture-supernatant using enzyme-immunoassays, while Smad5-signaling pathway activation and StAR protein expression were assessed immunocytochemically. We found that the treatment of AMH in GCs for 24/48 h attenuated FSH-induced estradiol production (p < 0.001), had no effect on basal estradiol levels, decreased basal progesterone production (p < 0.001) and FSH-induced StAR expression (p < 0.001). On the other hand, BMP-15 decreased basal estradiol levels (p < 0.001) and attenuated FSH-induced estradiol production (p < 0.001). Furthermore, BMP-15 reduced progesterone basal secretion (p < 0.001), an effect that was partially reversed by FSH (p < 0.01), probably via increasing StAR expression (p < 0.001). FSH-induced StAR expression was also attenuated by BMP-15 (p < 0.001). FSH, AMH and BMP-15 activated Smad-signaling pathway, as confirmed by the increase of phospo-Smad5 protein levels (p < 0.001 compared to control). AMH and BMP-15 by interacting with FSH affect the production of estradiol and progesterone from cultured luteinizing-granulosa cells possibly via Smad5-protein phosphorylation.

Distribution and function of 3′,5′-Cyclic-AMP phosphodiesterases in the human ovary

The concentration of the important second messenger cAMP is regulated by phosphodiesterases (PDEs) and hence an attractive drug target. However, limited human data are available about the PDEs in the ovary. The aim of the present study was to describe and characterise the PDEs in the human ovary. Results were obtained by analysis of mRNA microarray data from follicles and granulosa cells (GCs), combined RT-PCR and enzymatic activity analysis in GCs, immunohistochemical analysis of ovarian sections and by studying the effect of PDE inhibitors on progesterone production from cultured GCs. We found that PDE3, PDE4, PDE7 and PDE8 are the major families present while PDE11A was not detected. PDE8B was differentially expressed during folliculogenesis. In cultured GCs, inhibition of PDE7 and PDE8 increased basal progesterone secretion while PDE4 inhibition increased forskolin-stimulated progesterone secretion. In conclusion, we identified PDE3, PDE4, PDE7 and PDE8 as the major PDEs in the human ovary.

Different Types of Luteal Sub-Function in Relation to Pregnancy RateIn Nelore Cattl

This study was carried out to determine different types of luteal sub-function in relation to pregnancy rate in Nelore cattle. Fifty seven cows were used. Blood samples were collected twice per week for determining progesterone concentration within estrous cycle. Seven progesterone profiles were found normal progesterone profiles, delayed progesterone profiles, insufficient progesterone profiles, short luteal phase, long luteal phase, basal progesterone profile, and non-response to prostaglandin F2 treatment. Normal progesterone profiles (52.8 %) were higher than abnormal progesterone profiles. Therefore, pregnancy rate was significant higher in normal progesterone profile (45.6 %) compare to other abnormal progesterone profiles. In conclusion, Different progesterone profiles were observed in individual animal after AI. In addition to, it was observed that the post insemination progesterone profile has an effect on the pregnancy rates in Nelore cattle. Keywords: progesterone profiles, pregnancy rate, artificial insemination, Nelore cattle

Effect of growth hormone on basal and LH-stimulated steroid secretion by chicken yellow ovarian follicles. An in vitro study.

The aim of the present study was to determine the effect of growth hormone (GH) on basal and LH-regulated steroid secretion by yellow hierarchical follicles before and after maturation, and the granulosa and theca layers of the largest preovulatory follicles during the ovulatory cycle in the chicken. In the first experiment, whole yellow follicles (8-12 mm, 12-18 mm, 18-24 mm and 24-30 mm) isolated from 15 and 17-18 week-old chickens were used. In the second experiment, the granulosa and theca layers of the 3 largest yellow preovulatory follicles (F3<F2<F1) were isolated from the ovary of the laying hen (age 21 weeks) at stages 22 h and 3 h before F1 ovulation. The collected follicles and fragments of the granulosa and theca layers were randomly assigned to Eagle's medium containing 0 (control), chicken GH (cGH; 10 ng/ml), ovine LH (oLH; 10 ng/ml) or cGH+oLH (10 ng/ml+10 ng/ml) and incubated for 24 h at 38 degrees C. Following incubation, the progesterone and estradiol levels were determined in collected medias by RIA. It was found that cGH (1) elevated basal progesterone secretion by all classes of yellow follicles, (2) decreased estradiol secretion by the largest (18-30 mm) follicles and (3) attenuated to the control level the LH-stimulated estradiol secretion by the theca layer of F3 and F2 follicles at 22 h before ovulation. These results suggest that GH directly stimulates progesterone secretion and inhibits estradiol secretion by yellow hierarchical ovarian follicles in chicken. However, the effect of GH on separated granulosa and theca layers of the preovulatory follicles is not as pronounced as on whole follicles. It cannot be excluded that GH temporarily modulates LH action in estradiol production by the theca cells of chicken preovulatory ovarian follicles.

Basal progesterone levels represent the only independent risk factor prior to initiation of stimulation for elevated progesterone on the day of hCG

The detrimental role of progesterone elevation (PE) on the day of human chorionic gonadotrophin (hCG) for pregnancy achievement after a fresh IVF cycle has been confirmed by many studies. The aim of this study is to evaluate whether there are early markers that could identify women at risk for PE prior to the completion of their stimulation.

Basal progesterone level as the main determinant of progesterone elevation on the day of hCG triggering in controlled ovarian stimulation cycles

Modest increases of serum progesterone at human chorionic gonadotrophin (hCG) administration in controlled ovarian hyperstimulation (COH) cycles have been shown to have a negative impact on pregnancy outcomes. The aim of this study was to identify early predictors of progesterone elevation at hCG. Pregnancy outcome of 303 consecutive patients undergoing COH and fresh day-3 embryo transfer was analysed. Considering the non-linear relationship between progesterone at hCG triggering and pregnancy outcomes, partial area under the curve (pAUC) analysis was used to implement marker identification potential of receiver operating characteristic (ROC) curve analysis. Multivariate logistic analysis was then performed to identify predictors of progesterone rise. Pregnancy outcomes could be predicted by pAUC analysis (pAUC = 0.58, 95 % CI 0.51-0.66, p = 0.02) and a significant detrimental cut-off could be calculated (progesterone at hCG > 1.35 ng/ml). Total dose of rFSH administered, E2 level at hCG but mostly basal progesterone level (OR = 12.21, 95 % CI 1.82-81.70) were predictors of progesterone rise above the cut-off. Basal progesterone is shown to be the main prognostic factor for progesterone elevation. This observation should be taken into consideration in the clinical management of IVF/ICSI cycles to improve pregnancy outcomes.

Constitutive and follicle-stimulating hormone-induced action of somatostatin receptor-2 on regulation of apoptosis and steroidogenesis in bovine granulosa cells

In the present study, we employed primary bovine culture of granulosa cells (GCs) as a cellular model to study the potential involvement of somatostatin receptor 2 (SSTR2) in ovarian function. The results showed that bovine GCs expressed SST2 receptor and further found that SSTR2 was possibly regulated by follicle-stimulating hormone (FSH), as a significant increase in protein level of SSTR2 was observed in FSH-treated GCs. For further analysis, endogenous SSTR2 expression was disrupted using small inhibitory RNA (siRNA) and the efficacy of differential silencing of endogenous SSTR2 expression was measured both at transcriptional and translational levels. Transient blockage of SSTR2 evidenced its constitutive action on GCs, as it significantly increased level of cAMP (2.4-folds) and basal progesterone production (∼2-fold, P<0.05) with significant increase (P<0.05) in mRNA levels of StAR and P450ssc without altering estradiol concentration and aromatase mRNA expression. Furthermore, silencing of SSTR2 reduced GCs apoptosis (52.5%, P<0.05) and increased cell proliferation, which was further corroborated by up-regulation in protein expressions of B-cell leukemia/lymphoma 2 (Bcl-2), inhibition of caspase3 and mRNA level of bcl2-associated-X protein (Bax). These results provide evidence that SSTR2 subtype controls GCs apoptosis, proliferation and hormonal secretions through selective constitutive action, independently of somatostatin (SST). Given the local inhibitory actions of SSTR2 on the gonads, we further found that apoptosis in ssRNAi-2 transfected cells decreased (6.8% vs 1.9%, P<0.05) more strongly on FSH treatment. Apoptotic protein expressions and steroid hormone mRNA levels were correlated with a relative decrease in apoptosis and increase in progesterone production. Our results suggest that SSTR2 may play a crucial role as a local inhibitor of FSH action on GCs apoptosis and steroidogenesis.

Editorial:Molecular EndocrinologyArticles in the Spotlight for December 2013

Welcome to the December issue of Molecular Endocrinology. Among this month’s papers you’ll find the following research to be of great interest, particularly to those interested in reproductive biology. “ATF3 Expression in the Corpus Luteum: Possible Role in Luteal Regression” by Mao et al shows that the luteolytic hormone prostaglandin F2 (PGF2 ) selectively stimulated the expression of activating transcription factor 3 (ATF3) mRNA and ATF3 protein in large steroidogenic cells of the bovine corpus luteum in vivo and in vitro. The action of PGF2 was associated with the rapid activation of stress-activated protein kinases and the induction of ATF3 in large luteal cells. Furthermore, expression of ATF3 in large or small steroidogenic cells reduced LH-stimulated cAMP response element (CRE)mediated transcription downstream of protein kinase A (PKA) signaling and inhibited progesterone synthesis at a step before conversion of cholesterol to progesterone. Therefore, ATF3 may be primarily responsible for the PGF2 -dependent reduction in steroid synthesis that occurs during luteal regression. In “COUP-TFII Regulates Human Endometrial Stromal Genes Involved in Inflammation,” Li et al demonstrate that chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) was spatially and temporally expressed in endometrial stroma cells during the menstrual cycle in women. Furthermore, COUP-TFII was found to be reduced in stroma cells of ectopic lesions in endometriosis patients. Cistromic and transcriptomic analyses demonstrated that COUP-TF regulated processes critical for human endometrial stroma cell decidualization in vitro. Specifically, COUP-TFII was responsible for the down-regulation of inflammatory genes during decidualization. This may contribute to the function of decidual stroma cells in pregnancy and the pathology observed in endometriosis. In “BMP15 Suppresses Progesterone Production by Down-regulating StAR via ALK3 in Human Granulosa Cells” (freely available at http://mend.endojournals.org/), Chang et al found that bone morphogenetic protein 15 (BMP15), but not growth differentiation factor 9 (GDF9), reduced basal progesterone production by suppressing the expression of the rate-limiting steroidogenic regulatory (StAR) protein. Neither affected the expression of cytochrome P450 side chain cleavage (P450scc) enzyme or 3 -hydroxysteroid dehydrogenase (3 -HSD). The authors also showed that knockdown of ALK2 dramatically decreased basal StAR levels, indicating a positive role for ALK2 in maintaining StAR expression. Therefore, these findings suggest that oocyte-derived BMP15 plays a role in preventing premature luteinization by modulating progesterone production during the late stage of follicle development. Congratulations to all the authors in this issue for their fine work and research. Donald B. DeFranco, PhD Editor-in-Chief, Molecular Endocrinology

Endocrine monitoring of the ovarian cycle in captive female Amazonian manatees (Trichechus inunguis)

The Amazonian manatee (Trichechus inunguis; Mammalia: Sirenia), a threatened aquatic mammal endemic to the Amazon basin, is the only sirenian that lives exclusively in fresh water. Information about the reproductive endocrinology of the Amazonian manatee is scarce; therefore, the aim of this study was to monitor salivary progesterone and estradiol patterns during the ovarian cycle in T. inunguis. Salivary samples were collected daily during a 12-week period of two consecutive years from two captive adult females. The salivary estradiol and progesterone were measured by enzyme immunoassay. The results were analyzed in an iterative process of excluding values that were higher than the mean plus 2 standard deviations until the basal values were determined. The interval between two peaks of salivary estradiol followed by a rise of progesterone was considered as one complete cycle for the calculation of the cycle length. We observed only three complete cycles in all samples analyzed. The cycle length ranged from 42 to 48 days (mean of 44.67 days). We also observed two distinct salivary estradiol peaks during all cycles analyzed, with the first peak occurring before the rise in salivary progesterone and the second occurred followed by a return to basal progesterone levels. This is the first in-depth study of the ovarian cycle in Amazonian manatees. Our results demonstrate that salivary samples can be a useful tool in the endocrine monitoring of this species and suggest that T. inunguis shows a peculiar hormonal pattern during the ovarian cycle, a finding that may have physiological and ecological significance in the reproductive strategy of these animals.

Swainsonine differentially affects steroidogenesis and viability in caprine luteal cells in vitro

Plants containing swainsonine (SW) have been reported to impair reproductive function and fertility after long-term ingestion by livestock. However, direct effects of SW on luteal cell steroidogenesis remain unclear. In this study, primary and transfected luteal cells were used to investigate the effects of SW on progesterone secretion and cell viability and the mechanisms involved in these processes. After treatment with various concentrations of SW for 24 or 48 hours, progesterone production and the number of living cells were assessed using radioimmunoassay and trypan blue dye exclusion assay, respectively. Lower concentrations of SW enhanced basal, 22R-hydroxycholesterol- or pregnenolone-stimulated progesterone secretion (P < 0.05), whereas higher concentrations of SW inhibited progesterone secretion (P < 0.05). Lower concentrations of SW promoted expression of P450 side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase, two key enzymes involved in luteal cell steroidogenesis, at mRNA and protein levels (P < 0.05), but did not affect expression of steroidogenic acute regulatory protein and cell proliferation. In contrast, higher concentrations of SW inhibited luteal cell proliferation by inducing growth phase 1/quiescent state cell cycle arrest and apoptosis (P < 0.05). Taken together, these results demonstrated that lower concentrations of SW induced progesterone production through upregulation of P450 side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase without affecting cell viability, whereas higher concentrations of SW induced cell cycle arrest and apoptosis and impaired steroidogenesis. These findings provided new insights into understanding the effect of SW on luteal cell steroidogenesis.

Anticonvulsive effect of vitamin C on pentylenetetrazol-induced seizures in immature rats

Vitamin C helps to prevent brain oxidative stress and participate in the synthesis of progesterone. It also possesses a progesterone-like effect and acts synergistically with progesterone on the brain. Progesterone and its metabolites, but also vitamin C have been associated with anticonvulsant effects. We evaluated the progesterone concentration 30 min and 24 h after the last administration of vitamin C (500 mg/kg, i.p. for five days). We also evaluated how vitamin C altered pentylenetetrazol (PTZ)-induced seizures by measuring the onset latency of seizures, percentage of incidence and mortality as well as amino acid levels after seizures. Vitamin C treatment alone increased basal progesterone concentrations to 531% after 30 min compared to 253% after 24 h. Furthermore, vitamin C significantly increased the latency to the first myoclonic, clonic and tonic seizure induced by PTZ (80 mg/kg, i.p.) and decreased the percentage of incidence of clonic and tonic seizures as well as the mortality rate. Changes in tissue concentration of amino acids were primarily observed at 24 h after vitamin C treatment. Our results suggest that vitamin C together with progesterone and/or its metabolites are involved in the protection against PTZ-induced seizures in immature rats.

Growth Differentiation Factor 9 Reverses Activin A Suppression of Steroidogenic Acute Regulatory Protein Expression and Progesterone Production in Human Granulosa-Lutein Cells

Abstract Background: We have reported that growth differentiation factor (GDF) 9 can enhance activin A (βAβA)-induced inhibin B (αβB) secretion in human granulosa-lutein (hGL) cells, but its effects on steroidogenic acute regulatory protein (StAR), ovarian steroidogenic enzymes, and progesterone production are unknown. We undertook this study to further evaluate GDF9 in this regard. Methods: hGL cells from women undergoing in vitro fertilization treatment were cultured with and without small interfering RNA (siRNA) transfection targeted at inhibin α-subunit or GDF9 before treatment with GDF9, activin A, FSH, or combinations. We compared StAR, P450 side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase expression in hGL cells and progesterone levels in culture media after these treatments. mRNA, protein, and hormone levels were assessed with real-time RT-PCR, immunoblotting, and ELISA, respectively. Data were analyzed by ANOVA followed by Tukey’s test. Results: Activin A alone reduced basal and FSH-induced progesterone production by decreasing the expression of StAR protein, which regulates the rate-limiting step in steroidogenesis but not P450 side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase. GDF9 attenuated these activin A effects on StAR and progesterone. After transfection of a-subunit siRNA, activin A level increased (P &amp;lt; 0.001), whereas basal and activin A-induced inhibin B levels (with and without GDF9) decreased. Furthermore, the effects of GDF9 in reversing activin A suppression of progesterone production were attenuated (P &amp;lt; 0.001). Transfection of GDF9 siRNA decreased GDF9 as expected and led to lower StAR expression and progesterone secretion than those observed with activin A treatment alone. Conclusion: GDF9 attenuates the suppressive effects of activin A on StAR expression and progesterone production by increasing the expression of inhibin B, which acts as an activin A competitor.

High glucose‐induced changes in steroid production in adrenal cells

Increased activity of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in enhanced adrenocorticotropin (ACTH) and serum glucocorticoid levels, has been described in patients with diabetes mellitus and in animal models of this disease; however, altered steroid production by adrenocortical cells could result from local changes triggered by increased reactive oxygen species (ROS), induced in turn by chronic hyperglycaemia. Experiments were designed (1) to analyse the effects of incubating murine adrenocortical cells in hyperglycaemic media on the generation of oxidative stress, on steroid synthesis and on its modulation by the activity of haeme oxygenase (HO); and (2) to evaluate the effect of antioxidant treatment on these parameters. Y1 cells were incubated for 7 days with either normal or high glucose (HG, 30 mmol/L) concentrations, with or without antioxidant treatment. Parameters of oxidative stress and expression levels of haeme oxygenase-1 (HO-1), nitrite levels, L-arginine uptake and progesterone production were determined. HG augmented ROS and lipoperoxide production, decreasing glutathione (GSH) levels and increasing antioxidant enzymes and HO-1 expression. Basal progesterone production was reduced, while a higher response to ACTH was observed in HG-treated cells. The increase in HO-1 expression and the effects on basal steroid production were abolished by antioxidant treatment. Inhibition of HO activity increased basal and ACTH-stimulated steroid release. Similar results were obtained by HO-1 gene silencing while the opposite effect was observed in Y1 cells overexpressing HO-1. HG induces oxidative stress and affects steroid production in adrenal cells; the involvement of HO activity in the modulation of steroidogenesis in Y1 cells is postulated.