Artemisinins ameliorate polycystic ovarian syndrome by mediating LONP1-CYP11A1 interaction.

Polycystic ovary syndrome (PCOS), a prevalent reproductive disorder in women of reproductive age, features androgen excess, ovulatory dysfunction, and polycystic ovaries. Despite its high prevalence, specific pharmacologic intervention for PCOS is challenging. In this study, we identified artemisinins as anti-PCOS agents. Our finding demonstrated the efficacy of artemisinin derivatives in alleviating PCOS symptoms in both rodent models and human patients, curbing hyperandrogenemia through suppression of ovarian androgen synthesis. Artemisinins promoted cytochrome P450 family 11 subfamily A member 1 (CYP11A1) protein degradation to block androgen overproduction. Mechanistically, artemisinins directly targeted lon peptidase 1 (LONP1), enhanced LONP1-CYP11A1 interaction, and facilitated LONP1-catalyzed CYP11A1 degradation. Overexpression of LONP1 replicated the androgen-lowering effect of artemisinins. Our data suggest that artemisinin application is a promising approach for treating PCOS and highlight the crucial role of the LONP1-CYP11A1 interaction in controlling hyperandrogenism and PCOS occurrence.

Synergistic cooperation between the ß-catenin and SF1 regulates progesterone synthesis in laying hen ovarian granulosa cells.

The development of ovarian follicles in poultry is a key factor affecting the performance of egg production. Ovarian follicle development is regulated via the Wnt/ß-catenin signaling pathway, and ß-catenin, encoded by CTNNB1, is a core component of this pathway. In this study, using ovary GCs from laying hens, we investigated the regulatory role of CTNNB1 in steroid synthesis. We found that CTNNB1 significantly regulates the expression of StAR and CYP11A1 (key genes related to progesterone synthesis) and the secretion of progesterone (P4). Furthermore, simultaneous overexpression of CTNNB1 and SF1 resulted in significantly higher levels of CYP11A1 and secretion of P4 than in cells overexpressing CTNNB1 or SF1 alone. We also found that in GCs overexpressing SF1, levels of CYP11A1 and secreted P4 were significantly greater than in controls. Silencing of CYP11A1 resulted in the inhibition of P4 secretion while overexpression of SF1 in CYP11A1-silenced cells restored P4 secretion to normal levels. Together, these results indicate that synergistic cooperation between the ß-catenin and SF1 regulates progesterone synthesis in laying hen ovarian hierarchical granulosa cells to promote CYP11A1 expression.

Prenatal diethylhexylphthalate exposure disturbs adult Leydig cell function via epigenetic downregulation of METTL4 expression in male rats.

Prenatal exposure to diethylhexyl phthalate (DEHP) has been linked with a decline in testosterone levels in adult male rats, but the underlying mechanism remains unclear. We investigated the potential epigenetic regulation, particularly focusing on N6-methyladenosine (m6A) modification, as a possible mechanism. Dams were gavaged with DEHP (0, 10, 100, and 750 mg/kg/day) from gestational day 14 to day 21. The male offspring were examined at the age of 56 days. Prenatal DEHP administration at 750 mg/kg/day caused a decline in testosterone concentrations, an elevation in follicle-stimulating hormone, a downregulated expression of CYP11A1 HSD3B2, without affecting Leydig cell numbers. Interestingly, Methyltransferase Like 4 (METTL4), an m6A methyltransferase, was downregulated, while there were no changes in METTL3 and METTL14. Moreover, CYP11A1 showed m6A reduction in response to prenatal DEHP exposure. Additionally, METTL4 expression increased postnatally, peaking in adulthood. Knockdown of METTL4 resulted in the downregulation of CYP11A1 and HSD3B2 and an increase in SCARB1 expression. Furthermore, the increase in autophagy protection in adult Leydig cells induced by prenatal DEHP exposure was not affected by 3-methyladenosine (3MA) treatment, indicating a potential protective role of autophagy in response to DEHP exposure. In conclusion, prenatal DEHP exposure reduces testosterone by downregulating CYP11A1 and HSD3B2 via m6A epigenetic regulation and induction of autophagy protection in adult Leydig cells as a response to DEHP exposure.

Changes on corpus luteum structure and progesterone synthesis pathway after hCG or GnRH treatment during the early luteal phase in sheep.

This study investigated the effect of hCG or GnRH on structural changes of the corpora lutea (CL) and the regulation of the expression of steroidogenic enzymes involved in P4 secretion in post-ovulatory (po-CL) and accessory CL (acc-CL). Sixty-four ewes were assigned to three groups receiving: 300 IU of hCG (hCG) or 4 µg Buserelin (GnRH) or 1 mL of saline solution (Control) on Day (d) 4 post artificial insemination (FTAI). Laparoscopic ovarian were performed on d 4, 14 and, 21 post-FTAI to determine the numbers of CL. Blood samples were collected for serum LH and P4 analysis. On d 14 post-FTAI, both CL were removed from the ovary to determine large luteal cell (LLC) number and to evaluate the expression of steroidogenic enzymes (HSD3B1, STAR, CYP11A1). Only hCG and GnRH treated ewes generated acc-CL. The LLC in both po- and acc-CL were significantly greater in the hCG group compared to GnRH and Control groups (P<0.05). Overall, hCG group showed the greatest immunodetection of HSD3B1and STAR in both po- and acc-CL (P<0.05). rnRNA expression of HSD3B1, STAR and CYP11A1 in the acc-CL tended to be greater in hCG group than in GnRH group (P<0.1). The LH concentration was increased in GnRH group (P<0.05) and P4 concentration was greater in hCG group compared to the other groups (P<0.05). In conclusion, administration of hCG has a notably impact on acc-CL development and the expression of steroidogenic enzymes compared to GnRH treatment in ewes. This leads to elevated P4 concentration and improved luteal function.

Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2 155 expressing mammalian steroidogenesis system.

BACKGROUND: Pregnenolone and progesterone are the life-important steroid hormones regulating essential vital functions in mammals, and widely used in different fields of medicine. Microbiological production of these compounds from sterols is based on the use of recombinant strains expressing the enzyme system cholesterol hydroxylase/C20-C22 lyase (CH/L) of mammalian steroidogenesis. However, the efficiency of the known recombinant strains is still low. New recombinant strains and combination approaches are now needed to produce these steroid hormones. RESULTS: Based on Mycolicibacterium smegmatis, a recombinant strain was created that expresses the steroidogenesis system (CYP11A1, adrenodoxin reductase, adrenodoxin) of the bovine adrenal cortex. The recombinant strain transformed cholesterol and phytosterol to form progesterone among the metabolites. When 3-methoxymethyl ethers of sterols were applied as bioconversion substrates, the corresponding 3-ethers of pregnenolone and dehydroepiandrosterone (DHEA) were identified as major metabolites. Under optimized conditions, the recombinant strain produced 85.2 ± 4.7 mol % 3-methoxymethyl-pregnenolone within 48 h, while production of 3-substituted DHEA was not detected. After the 3-methoxymethyl function was deprotected by acid hydrolysis, crystalline pregnenolone was isolated in high purity (over 98%, w/w). The structures of steroids were confirmed using TLC, HPLC, MS and 1H- and 13C-NMR analyses. CONCLUSION: The use of mycolicybacteria as a microbial platform for the expression of systems at the initial stage of mammalian steroidogenesis ensures the production of valuable steroid hormones-progesterone and pregnenolone from cholesterol. Selective production of pregnenolone from cholesterol is ensured by the use of 3-substituted cholesterol as a substrate and optimization of the conditions for its bioconversion. The results open the prospects for the generation of the new microbial biocatalysts capable of effectively producing value-added steroid hormones.

Enhancement of progesterone biosynthesis via kisspeptin stimulation: Upregulation of steroidogenic transcripts and phosphorylated extracellular signal-regulated kinase (p-ERK1/2) expression in the buffalo luteal cells.

The presence of Kisspeptin (Kp) and its receptors in the corpus luteum (CL) of buffalo has recently been demonstrated. In this study, we investigated the role of Kp in the modulation of progesterone (P4) synthesis in vitro. The primary culture of bubaline luteal cells (LCs) was treated with 10, 50, and 100 nM of Kp and Kp antagonist (KpA) alongside a vehicle control. The combined effect of Kp and KpA was assessed at 100 nM concentration. Intracellular response to Kp treatment in the LCs was assessed by examining transcript profiles (LHR, STAR, CYP11A1, HSD3B1, and ERK1/2) using quantitative polymerase chain reaction (qPCR). In addition, the immunolocalization of ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) in the LCs was studied using immunocytochemistry. Accumulation of P4 from the culture supernatant was determined using enzyme-linked immunosorbent assay (ELISA). The results indicated that LCs had a greater p-ERK1/2 expression in the Kp treatment groups. A significant increase in the P4 concentration was recorded at 50 nM and 100 nM Kp, while KpA did not affect the basal concentration of P4. However, the addition of KpA to the Kp-treated group at 100 nM concentration suppressed the Kp-induced P4 accumulation into a concentration similar to the control. There was significant upregulation of ERK1/2 and CYP11A1 expressions in the Kp-treated LCs at 100 nM (18.1 and 37fold, respectively, p < 0.01). However, the addition of KpA to Kp-treated LCs modulated ERK1/2, LHR, STAR, CYP11A1, and HSD3B1 at 100 nM concentration. It can be concluded that Kp at 100 nM stimulated P4 production, while the addition of KpA suppressed Kp-induced P4 production in the buffalo LCs culture. Furthermore, an increment in p-ERK1/2 expression in the LCs indicated activation of the Kp signaling pathway was associated with luteal steroidogenesis.

Effect of quercetin on granulosa cells development from hierarchical follicles in chicken.

1. The bioflavonoid quercetin is a biologically active component, but its functional regulation of granulosa cells (GCs) during chicken follicular development is little studied. To investigate the effect of quercetin on follicular development in laying hens, an in vitro study was conducted on granulosa cells from hierarchical follicles treated with quercetin.2. The effect of quercetin on cell activity, proliferation and apoptosis of granulosa cells was detected by CCK-8, EdU and apoptosis assays. The effect on progesterone secretion from granulosa cells was investigated by enzyme-linked immunosorbent assay (ELISA). Expression of proliferating cell nuclear antigen (PCNA) mRNA and oestrogen receptors (ERs), as well as the expression of steroid acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA during progesterone synthesis, were measured by real-time quantitative polymerase chain reaction (RT-qPCR). PCNA, StAR and CYP11A1 protein expression levels were detected using Western blotting (WB).3. The results showed that treatment with quercetin in granulosa cells significantly enhanced cell vitality and proliferation, reduced apoptosis and promoted the expression of gene and protein levels of PCNA. The levels of progesterone secretion increased significantly following quercetin treatment, as did the expression levels of StAR and CYP11A1 using the Western Blot (WB) method.4. The mRNA expression levels of ERα were significantly upregulated in the 100 ng/ml and 1000 ng/ml quercetin-treated groups, while there was no significant difference in expression levels of ERß mRNA.

Effect of cadmium on the regulatory mechanism of steroidogenic pathway of Leydig cells during spermatogenesis.

Cadmium is a male reproductive toxicant that interacts with a variety of pathogenetic mechanisms. However, the effect of cadmium on the regulatory mechanism of the steroidogenic pathway of Leydig cells during spermatogenesis is still ambiguous. Light microscopy, Western blot, immunohistochemistry, immunofluorescence, and quantitative polymerase chain reaction were performed to study the regulatory mechanism of the steroidogenic pathway of Leydig cells during spermatogenesis. The results indicated that in the control group, Leydig cells showed dynamic immunoreactivity and immunosignaling action with a strong positive significant secretion of 3ß-hydroxysteroid hydrogenase (3ß-HSD) in the interstitial compartment of the testis. Leydig cells showed a high active regulator mechanism of the steroidogenic pathway with increased the proteins and genes expression level of steroidogenic acute regulatory protein (STAR), cytochrome P450 cholesterol (CYP11A1), cytochrome P450 cholesterol (CYP17A1), 3ß-hydroxysteroid hydrogenase (3ß-HSD) 17ß-hydroxysteroid hydrogenase (17ß-HSD), and androgen receptor (AR) that maintained the healthy and vigorous progressive motile spermatozoa. However, on treatment with cadmium, Leydig cells were irregularly dispersed in the interstitial compartment of the testis. Leydig cells showed reduced immunoreactivity and immunosignaling of 3ß-HSD protein. Meanwhile, cadmium impaired the regulatory mechanism of the steroidogenic process of the Leydig cells with reduced protein and gene expression levels of STAR, CYP11A1, CYP17A1, 3ß-HSD, 17ß-HSD, and AR in the testis. Additionally, treatment with cadmium impaired the serum LH, FSH, and testosterone levels in blood as compared to control. This study explores the hazardous effect of cadmium on the regulatory mechanism of the steroidogenic pathway of Leydig cells during spermatogenesis.

The Role of DNMT1 and C/EBPα in the Regulation of CYP11A1 Expression During Syncytialization of Human Placental Trophoblasts.

Progesterone synthesized in the placenta is essential for pregnancy maintenance. CYP11A1 is a key enzyme in progesterone synthesis, and its expression increases greatly during trophoblast syncytialization. However, the underlying mechanism remains elusive. Here, we demonstrated that passive demethylation of CYP11A1 promoter accounted for the upregulation of CYP11A1 expression during syncytialization with the participation of the transcription factor C/EBPα. We found that the methylation rate of a CpG locus in the CYP11A1 promoter was significantly reduced along with decreased DNA methyltransferase 1 (DNMT1) expression and its enrichment at the CYP11A1 promoter during syncytialization. DNMT1 overexpression not only increased the methylation of this CpG locus in the CYP11A1 promoter, but also decreased CYP11A1 expression and progesterone production. In silico analysis disclosed multiple C/EBPα binding sites in both CYP11A1 and DNMT1 promoters. C/EBPα expression and its enrichments at both the DNMT1 and CYP11A1 promoters were significantly increased during syncytialization. Knocking-down C/EBPα expression increased DNMT1 while it decreased CYP11A1 expression during syncytialization. Conclusively, C/EBPα plays a dual role in the regulation of CYP11A1 during syncytialization. C/EBPα not only drives CYP11A1 expression directly, but also indirectly through downregulation of DNMT1, which leads to decreased methylation in the CpG locus of the CYP11A1 promoter, resulting in increased progesterone production during syncytialization.

Effects of insulin-like growth factor-1 on the mRNA expression of estradiol receptors, steroidogenic enzymes, and steroid production in bovine follicles.

Insulin-like growth factor-1 (IGF-1) plays a crucial role in follicular growth and stimulates steroid hormone production in bovine follicles. Steroid hormones are synthesized through the actions of steroidogenic enzymes, specifically STAR, CYP11A1, HSD3B, and CYP19A1 in both theca cells (TCs) and granulosa cells (GCs), under the influence of gonadotropins. Particularly, estradiol 17ß (E2) assumes a central role in follicular development and selection by activating estrogen receptors ß (ESR2) in GCs. We assessed ESR2 mRNA expression in GCs of developing follicles and investigated the impact of IGF-1 on the mRNA expression of ESR2, CYP19A1, FSHR, and LHCGR, STAR, CYP11A1, and HSD17B in cultured GCs and TCs, respectively. Additionally, we assessed the influence of IGF-1 on androstenedione (A4), progesterone (P4), and testosterone (T) production in TCs. Small-sized follicles (< 6 mm) exhibited the highest levels of ESR2 mRNA expression, whereas medium-sized follicles (7-8 mm) displayed higher levels than large-sized follicles (≥ 9 mm) (P < 0.05). IGF-1 increased the mRNA expression of ESR2, CYP19A1, and FSHR in GCs of follicles of both sizes, except for FSHR mRNA in medium-sized follicles (P < 0.05). IGF-1 significantly elevated mRNA expression of LHCGR, STAR, CYP11A1, and CYP17B in TCs of small- and medium-sized follicles (P < 0.05). Moreover, IGF-1 augmented the production of A4 and P4 but had no impact on T production in TCs of small- and medium-sized follicles. Taken together, our findings indicate that IGF-1 upregulates steroidogenic enzymes and steroid hormone production, underscoring the crucial role of IGF-1 in follicle development and selection.

Expression of cholesterol synthesis and steroidogenic markers in females of the Chinese brown frog (Rana dybowskii) during prespawning and prehibernation.

The oviduct of the Chinese brown frog (Rana dybowskii) expands in prehibernation rather than in prespawning, which is one of the physiological phenomena that occur in the preparation for hibernation. Steroid hormones are known to regulate oviductal development. Cholesterol synthesis and steroidogenesis may play an important role in the expansion of the oviduct before hibernation. In this study, we investigated the expression patterns of the markers that are involved in the de novo steroid synthesis pathway in the oviduct of R. dybowskii during prespawning and prehibernation. According to histological analysis, the oviduct of R. dybowskii contains epithelial cells, glandular cells, and tubule lumens. During prehibernation, oviductal pipe diameter and weight were significantly larger than during prespawning. 3-Hydroxy-3-methylglutaryl CoA reductase (HMGCR), low-density lipoprotein receptor (LDLR), steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and steroidogenic factor 1 (SF-1) were detected in epithelial cells in prehibernation and glandular cells during prespawning. HMGCR, LDLR, StAR, and P450scc protein expression levels were higher in prehibernation than during prespawning, but the SF-1 protein expression level did not significantly differ. HMGCR, LDLR, StAR, P450scc (CYP11A1), and SF-1 (NR5A1) mRNA expression levels were significantly higher in prehibernation compared with prespawning. The transcriptome results showed that the steroid synthesis pathway was highly expressed during prehibernation. Existing results indicate that the oviduct is able to synthesize steroid hormones using cholesterol, and that steroid hormones may affect the oviductal functions of R. dybowskii.

Effects of exposure to the explosive and environmental pollutant 2,4,6-trinitrotoluene on ovarian follicle development in rats.

Although 2,4,6-trinitrotoluene (TNT) is a dangerous carcinogen in environmental pollution, information on the reproductive effects of TNT explosive contamination is limited. To explore the possible ovarian effects, TNT explosive-exposed rat models were established, and Wistar female rats were exposed to low and high TNT (40 g and 80 g, air and internal) explosives. After a month of exposure, the estrous cycle, ovarian histopathology, and follicle counting were conducted. Serum hormones follicle-stimulating hormone (FSH), luteinizing hormone (LH), anti-Müllerian hormone (AMH), progesterone, testosterone, and estradiol were detected, and the mRNA and protein expression of steroidogenic enzymes were measured. The results showed that the diestrus phase duration was significantly (P < 0.05) increased in the high TNT-exposed groups. In addition, the proportions of preantral follicles were significantly (P < 0.05) decreased in the high TNT-exposed groups, as well as the proportions of atretic follicles. The serum estradiol levels were significantly (P < 0.05) increased, and the follicle-stimulating hormone and luteinizing hormone levels were significantly (P < 0.05) decreased in the high TNT-exposed groups. The mRNA levels of steroidogenic acute regulatory protein (Star), cytochrome P450 cholesterol side chain cleavage (Cyp11a1, Cyp17a1 and Cyp19a1), hydroxysteroid dehydrogenase 3b (Hsd3b) and steroidogenic factor-1 (SF-1) were significantly (P < 0.05) increased in the TNT-exposed groups. The protein levels of Star, Cyp11a1 and Hsd3b were increased (P < 0.05) in the TNT-exposed groups. These results indicate that the exposure of rats to TNT explosive can subsequently affect ovarian follicle development, suggesting that the mechanism may involve disrupting steroidogenesis.

Exploring the impact of CYP11A1's missense SNPs on the interaction between CYP11A1 and cholesterol: A comprehensive structural analysis and MD simulation study.

The process of steroidogenesis plays a vital role in human physiology as it governs the biosynthesis of mineralocorticoids, glucocorticoids, and androgens. These three classes of steroid hormones are primarily produced in the adrenal and gonadal glands through steroidogenesis pathways. Initiated by the side chain cleavage of cholesterol (CLR), this process leads to the conversion of cholesterol into pregnenolone and isocaproic aldehyde. The enzyme CYP11A1, encoded by the CYP11A1 gene, plays a key role in catalyzing the side chain cleavage of CLR. Several single nucleotide polymorphisms (SNPs) have been identified in the CYP11A1 gene, which may predispose carriers to disorders associated with abnormal steroidogenesis. Specifically, missense SNPs in the CYP11A1 gene have the potential to negatively impact the interaction between CYP11A1 and CLR, thus affecting the overall metabolome of steroid hormones. In this computational study, we focused on a specific set of missense SNPs reported in the CYP11A1 gene, aiming to identify variants that directly impact the interaction between CYP11A1 and CLR. The three-dimensional structure of the CYP11A1-CLR complex was obtained from the RCSB Protein Data Bank, while missense SNPs in the CYP11A1 gene were retrieved from Ensembl. To predict the most deleterious variants, we utilized the ConSurf server, SIFT, and PolyPhen. Furthermore, we assessed the impact of induced amino acid (AA) substitutions on the CYP11A1-CLR interaction using the PRODIGY server, PyMol, and Ligplot programs. Additionally, molecular dynamics (MD) simulations were conducted to analyze the effects of deleterious variants on the structural dynamics of the CYP11A1-CLR complex. Among the 8096 retrieved variants, we identified ten missense SNPs (E91K, W147G, R151W, R151Q, S391C, V392M, Q395K, Q416E, R460W, and R460Q) as deleterious for the interaction between CYP11A1 and CLR. MD simulations of the CYP11A1-CLR complexes carrying these deleterious AA substitutions revealed that Q416E, W147G, R460Q, and R460W had the most pronounced impacts on the structural dynamics of the complex. Consequently, these missense SNPs were considered the most deleterious ones. Further functional tests are recommended to assess the impact of these four missense SNPs on the enzymatic activity of CYP11A1. Moreover, Genome-Wide Association Studies (GWAS) should be conducted to determine the significance of their association with abnormal steroidogenesis diseases in various patient groups.

Mitochondrial cytochrome P450 1B1 is involved in pregnenolone synthesis in human brain cells.

Neurosteroids, which are steroids synthesized by the nervous system, can exert neuromodulatory and neuroprotective effects via genomic and nongenomic pathways. The neurosteroid and major steroid precursor pregnenolone has therapeutical potential in various diseases, such as psychiatric and pain disorders, and may play important roles in myelination, neuroinflammation, neurotransmission, and neuroplasticity. Although pregnenolone is synthesized by CYP11A1 in peripheral steroidogenic organs, our recent study showed that pregnenolone must be synthesized by another mitochondrial cytochrome P450 (CYP450) enzyme other than CYP11A1 in human glial cells. Therefore, we sought to identify the CYP450 responsible for pregnenolone production in the human brain. Upon screening for CYP450s expressed in the human brain that have mitochondrial localization, we identified three enzyme candidates: CYP27A1, CYP1A1, and CYP1B1. We found that inhibition of CYP27A1 through inhibitors and siRNA knockdown did not negatively affect pregnenolone synthesis in human glial cells. Meanwhile, treatment of human glial cells with CYP1A1/CYP1B1 inhibitors significantly reduced pregnenolone production in the presence of 22(R)-hydroxycholesterol. We performed siRNA knockdown of CYP1A1 or CYP1B1 in human glial cells and found that only CYP1B1 knockdown significantly decreased pregnenolone production. Furthermore, overexpression of mitochondria-targeted CYP1B1 significantly increased pregnenolone production under basal conditions and in the presence of hydroxycholesterols and low-density lipoprotein. Inhibition of CYP1A1 and/or CYP1B1 via inhibitors or siRNA knockdown did not significantly reduce pregnenolone synthesis in human adrenal cortical cells, implying that CYP1B1 is not a major pregnenolone-producing enzyme in the periphery. These data suggest that mitochondrial CYP1B1 is involved in pregnenolone synthesis in human glial cells.

Perfluorooctane sulfonic acid modulates expression of placental steroidogenesis-associated genes and hormone levels in pregnant rats.

Perfluorooctane sulfonate (PFOS) is a widespread and persistent chemical in the environment. Reports show that PFOS is a potential endocrine disruptor; however, the possible effects of PFOS on placental endocrine function are unclear. This study aimed to investigate the endocrine-disrupting effects of PFOS on the placenta in pregnant rats and its potential mechanism. Pregnant rats from gestational days 4-20 were exposed to 0, 10, and 50 µg/mL PFOS through drinking water followed by analysis of various biochemical parameters. PFOS dose-dependently decreased fetal and placental weight in both sexes, with a specific decrease in weight of labyrinth but not junctional layer. Plasma progesterone (↑166%), aldosterone (↑201%), corticosterone (↑205%), testosterone (↑45%), luteinizing hormone (↑49%) levels were significantly increased, while estradiol (↓27%), prolactin (↓28%) and hCG (↓62%) levels were reduced in groups exposed to higher doses of PFOS. Real-time quantitative reverse transcriptase-polymerase chain reaction analysis revealed a significant increase in mRNA levels of placental steroid biosynthesis enzymes, including Cyp11A1 and 3ß-HSD1 in male placenta and StAR, Cyp11A1, 17ß-HSD1 and 17ß-HSD3 in female placenta of PFOS dams. Cyp19A1 expression in ovaries was significantly decreased in PFOS dams. mRNA levels for placental steroid metabolism enzyme UGT1A1 increased in male but not in female placenta of PFOS dams. These results suggest that the placenta is a target tissue of PFOS and PFOS-induced dysregulation in steroid hormone production might be related to the altered expression of hormone biosynthesis and metabolism enzyme genes in the placenta. This hormone disruption might affect maternal health and fetal growth.

Spheroid formation and luteinization of granulosa cells of felids in a long-term 3D culture.

In the present study, granulosa cells (GCs) from domestic cats and Persian leopard were cultured and characterized from selected days. The culture period was divided into two phases: maintenance, which lasted for 7 days, and luteinization, which followed for up to 11 days. Luteinization was performed on ultra-low attachment plates, supporting the formation of spheroids in a medium supplemented with insulin, forskolin, and luteinizing hormone (LH). GCs of domestic cat produced estradiol (E2) and progesterone (P4) during the maintenance phase. The gene expressions of some proteins involved in steroidogenesis were stable (STAR, HSD3B1) or decreased over time (CYP11A1, HSD17B1, CYP17A1, and CYP19A1), which was similar to the expressions of gonatropin receptors (LHCGR and FSHR). During the luteinization phase, P4 concentration significantly increased (P < 0.05), and E2, in contrast to the proliferation phase, was below detection range. The expression of genes of proteins involved in steroidogenesis (STAR, CYP11A1, HSD3B1, HSD17B1, CYP17A1, and CYP19A1) and of gonadotropin receptors (LHCGR and FSHR) significantly increased during the luteinization period, but some expressions exhibited a decrease at the end of the phase (LHCGR, FSHR, HSD17B1, CYP19A1). The morphology of the luteinized GCs of domestic cat resembled large luteal cells and had numerous vacuole-like structures. Also, the GCs of Persian leopard underwent luteinization, shown by increasing P4 production and HSD3B1 expression. This study confirms that GCs from felids can be luteinized in a 3D spheroid system which can be a basis for further studies on luteal cell function of felids. Additionally, we could show that the domestic cat can serve as a model species for establishing cell culture methods which can be transferred to other felids.

Differential tissue expression of sex steroid-synthesizing enzyme CYP11A1 in male Tibetan sheep (Ovis aries).

Steroid metabolism is a fundament to testicular development and function. The cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) is a key rate-limiting enzyme for catalyzing the conversion of cholesterol to pregnenolone. However, despite its importance, what expression and roles of CYP11A1 possesses and how it regulates the testicular development and spermatogenesis in Tibetan sheep remains largely unknown. Based on this, we evaluated the expression and localization patterns of CYP11A1 in testes and epididymides of Tibetan sheep at three developmental stages (three-month-old, pre-puberty; one-year-old, sexual maturity and three-year-old, adult) by quantitative real-time PCR (qPCR), western blot and immunofluorescence. The results showed that CYP11A1 mRNA and protein were expressed in testes and epididymides throughout the development stages and obviously more intense in one- and three-year-old groups than three-month-old group (except for the caput epididymidis). Immunofluorescence assay showed that the CYP11A1 protein was mainly located in Leydig cells and epididymal epithelial cells. In addition, positive signals of CYP11A1 protein were observed in germ cells, epididymal connective tissue and sperms stored in the epididymal lumen. Collectively, these results suggested that the CYP11A1 gene might be mainly involved in regulating spermatogenesis and androgen synthesis in developmental Tibetan sheep testis and epididymis.

Cadmium-induced preeclampsia-like phenotype in the rat is related to decreased progesterone synthesis in the placenta.

Cadmium (Cd) is a toxic element, which is associated with preeclampsia (PE).We treated pregnant rats with cadmium chloride from gestational days (GDs) 9-12 to introduce the PE-like animal model. Maternal systolic blood pressures (SBPs) and body weights were measured on GDs 0, 5, 10, 15, and 20. Foetuses were delivered by Caesarean section on GD20. Then, the dams were sacrificed and the specimens were obtained. The morphological analysis of the placentae was carried out by haematoxylin and eosin staining examination and immunohistochemistry assay.Our study showed that Cd-treated rats developed PE-like phenotypes, such as hypertension, albuminuria, and foetal growth restriction. Moreover, Cd-injected rats displayed abnormal placental angiogenesis and lower progesterone (P4) levels. We further demonstrated that Cd also inhibited the mRNA and protein expressions of steroidogenic acute regulatory protein, cytochrome P450 cholesterol side-chain cleavage enzyme (CYP11A1), and 3ß-hydroxysteroid dehydrogenase 1 (3ß-HSD1), which are involved in P4 synthesis in the rat placenta.Our study demonstrates that maternal Cd exposure disrupts the local synthesis of P4 in the placenta, which contributes to the onset of PE in pregnant rats. Supplementing P4 at the early gestational stage may be a promising therapeutic strategy to prevent PE, which requires further investigation.

Local adrenomedullin gene delivery inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo.

Adrenomedullin (ADM) has beneficial effects on Leydig cells under pathological conditions, including lipopolysaccharide (LPS)-induced orchitis. Our previous studies demonstrated that ADM exerts a restorative effect on steroidogenesis in LPS-treated primary rat Leydig cells by attenuating oxidative stress, inflammation and apoptosis. In this study, we aim to investigate whether ADM inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo. Rats were administered with LPS and injected with Ad-ADM, an adeno-associated virus vector that expressed ADM. Then, rat testes were collected for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) immunofluorescence staining. Steroidogenic enzymes or steroidogenic regulatory factors or protein, including steroidogenic factor-1 (SF-1), liver receptor homologue-1 (LRH1), Nur77, steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc), 3ß-HSD, cytochrome P450 17α-hydroxylase/17, 20 lyase (CYP17) and 17ß-hydroxysteroid dehydrogenase (17ß-HSD), were detected via gene expression profiling and western blot analysis. Plasma testosterone concentrations were measured. Results showed that ADM may inhibit Leydig cell dysfunction by rescuing steroidogenic enzymes and steroidogenic regulatory factors in vivo. The reduction in the number of Leydig cells after LPS exposure was reversed by ADM. ADM rescued the gene or protein levels of SF-1, LRH1, Nur77, StAR, P450scc, 3ß-HSD, CYP17 and 17ß-HSD and plasma testosterone concentrations. To summarize ADM could rescue some important steroidogenic enzymes, steroidogenic regulatory factors and testosterone production in Leydig cells in vivo.

Evidence of a role for cAMP in mitochondrial regulation in ovarian granulosa cells.

In the ovary, proliferation and differentiation of granulosa cells (GCs) drive follicular growth. Our immunohistochemical study in a non-human primate, the Rhesus monkey, showed that the mitochondrial activity marker protein cytochrome c oxidase subunit 4 (COX4) increases in GCs in parallel to follicle size, and furthermore, its intracellular localization changes. This suggested that there is mitochondrial biogenesis and trafficking, and implicates the actions of gonadotropins, which regulate follicular growth and ovulation. Human KGN cells, i.e. granulosa tumour cells, were therefore used to study these possibilities. To robustly elevate cAMP, and thereby mimic the actions of gonadotropins, we used forskolin (FSK). FSK increased the cell size and the amount of mitochondrial DNA of KGN cells within 24 h. As revealed by MitoTracker™ experiments and ultrastructural 3D reconstruction, FSK treatment induced the formation of elaborate mitochondrial networks. H89, a protein kinase A (PKA) inhibitor, reduced the network formation. A proteomic analysis indicated that FSK elevated the levels of regulators of the cytoskeleton, among others (data available via ProteomeXchange with identifier PXD032160). The steroidogenic enzyme CYP11A1 (Cytochrome P450 Family 11 Subfamily A Member 1), located in mitochondria, was more than 3-fold increased by FSK, implying that the cAMP/PKA-associated structural changes occur in parallel with the acquisition of steroidogenic competence of mitochondria in KGN cells. In summary, the observations show increases in mitochondria and suggest intracellular trafficking of mitochondria in GCs during follicular growth, and indicate that they may partially be under the control of gonadotropins and cAMP. In line with this, increased cAMP in KGN cells profoundly affected mitochondrial dynamics in a PKA-dependent manner and implicated cytoskeletal changes.