This study aims to explore the relationship between altered vitamin D (VitD3) status and ovarian steroidogenesis in muskrats during the breeding and non-breeding seasons. During the breeding season, the ovaries of muskrats were observably enlarged and increased in weight, accompanied by elevated serum and ovarian VitD3 status. Vitamin D receptor (VDR), VitD3 metabolic molecules (CYP2R1, CYP27B1, and CYP24A1), and steroidogenic enzymes were immunolocalized in the ovarian cells of muskrats. The mRNA levels of VDR, CYP2R1, CYP27B1, and steroidogenic enzymes were considerably higher during the breeding season compared to the non-breeding season. RNA-seq analysis revealed a prominent enrichment of vitamin-related and ovarian steroidogenesis pathways. Furthermore, the addition of 1,25(OH)2D3 to the muskrat granulosa cells in vitro increased VDR and steroidogenic enzymes mRNA levels and enhanced the 17β-estradiol level. Overall, these findings supported that VitD3 promotes the secretion of steroid hormones, thereby affecting seasonal changes in ovarian function in the muskrats.

OBJECTIVE: To determine the influence of the environmental endocrine disruptor bisphenol A (BPA) on germ cell cyst breakdown and explore the possible mechanisms regulating this activity.
METHODS: BPA (2 μg/kg/d or 20 μg/kg/d) or tocopherol-stripped corn oil (vehicle control) was administered to pregnant mice by gavage at gestational day 11, and the offspring (prenatally treated mice) were sacrificed and ovariectomized at postnatal day (PND) 4 and PND22. Ovarian morphology was documented in the first filial (F1) generation female offspring, and the follicles were analyzed and classified morphologically on PND 4. To discover differentially expressed genes and associated target pathways, we used RNA-seq, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and Gene Ontology (GO) analysis. The mRNA expression of key steroid hormone synthesis-related genes was evaluated by Q-PCR in forskolin-induced KGN cells. Western blotting (WB) and qRTPCR were used to determine the protein and gene expression levels of brain-derived neurotrophic factor (BDNF).
RESULTS: BPA, a typical endocrine disrupting chemical (EDC), decreased the expression of the key steroid hormone synthesis-related genes P450scc and aromatase, while the expression of Star increased significantly and caused no significant difference in the expression of Cyp17a1 or HSD3β in forskolin-induced KGN cells. Moreover, we confirmed that in utero exposure to environmentally relevant concentrations of BPA (2 μg/kg/d and 20 μg/kg/d) could significantly disrupt germ cell cyst breakdown, leading to the generation of fewer primordial follicles than in the control group. The factors mediating the inhibitory effects included the PI3K-Akt signaling pathway and a significant downregulation of BDNF.
CONCLUSIONS: These findings indicate that in utero exposure to BPA at low doses, which are lower than recommended as \'safe\' dosages, may influence the formation of primordial follicles by inhibiting the expression of steroid hormone synthesis-related genes and partly by regulating the BDNF-mediated PI3K/Akt pathway.

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 3β-hydroxysteroid dehydrogenase (3β-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), 3β-HSD, cytochrome P450 17α-hydroxylase/17, 20 lyase (CYP17) and 17β-hydroxysteroid dehydrogenase (17β-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, 3β-HSD, CYP17 and 17β-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.

The purpose of this study was to explore the variations in the circulating leptin concentrations of the wild ground squirrels in relation to seasonal changes in testicular activities. Hematoxylin-eosin staining showed all types of elongated spermatids and spermatogenic cells existed in the testis in April, while the primary spermatocytes and spermatogonia were most advanced stages of germ cells in June. In addition, the primary spermatocytes, secondary spermatocytes, and spermatogonia were most advanced stages of germ cells in September. The highest circulating leptin concentration was consistent with the maximum body weight results from accumulation of adipose tissue in September. The mRNA expression level of leptin receptor (Ob-R) and STAT3 was lowest in June, raised in September, and remained increased in April. Ob-R and STAT3 were stronger staining in the Leydig cells in July. Moreover, the concentrations of testosterone (T) showed the maximum values in April, the minimum values in June, and significant increases in September. Furthermore, it is worth noting that the levels of T increased with the mRNA levels of Ob-R, STAT3, StAR, and testicular steroidogenic enzymes (3β-HSD, P450c17, and P450scc). Moreover, RNA-seq analyses of testis during the different periods showed that a total of 4209 genes were differentially expressed genes (DEGs); further analysis revealed that DEGs related with the Jak/STAT pathways and reproduction were altered. Taken together, the results suggested that the leptin regulated testicular function through the Jak/STAT pathways and testicular steroidogenic factor expressions.

Sugar cane extract (SCE) is the end product of glucose, fructose, and sucrose elimination in molasses. SCE has various biological effects, such as anti-inflammation and antioxidation, and it is commonly found in animal feed. The present research is aimed at investigating the reproductive endocrine influence of SCE in male Japanese quails (Coturnix japonica) by feeding SCE containing food. In addition, in vitro Leydig cell culture was conducted to clarify the mechanism of SCE\'s influence. Our results showed that SCE feed extended the latency to the first neck grab, decreased male quail testis and epididymis weights, cloaca gland size, and reduced serum testosterone concentrations. Steroidogenic enzymes 3βHSD, 17βHSD, P450c17, and P450scc gene expression in the testis were decreased in the SCE groups. Western blot analysis showed decreased 3βHSD in the testis after feeding SCE. Isolated testicular interstitial cells cultured with SCE and ovine-LH suppressed testosterone secretion and 3βHSD gene expression. In conclusion, SCE as a feed additive has an impact on the sexual behavior and reproductive function of male Japanese quail, with the suppression of steroidogenesis in the Leydig cell. Our results may provide beneficial information to the livestock management and the poultry industry.

Trimethyltin (TMT) is widely used as a plastic heat stabilizer and can cause severe toxicity. Here, the effects of TMT on testosterone production by adult Leydig cells and the related mechanisms of action were investigated. Eighteen adult male Sprague Dawley rats (56 days old) were randomly divided into 3 groups and given intraperitoneal injection of TMT for 21 consecutive days at the doses of 0 (vehicle control), 5, or 10 mg/kg/d. After treatment, trunk blood was collected for hormonal analysis. In addition, related gene and protein expression in testes was detected. At 10 mg/kg, TMT significantly reduced serum testosterone levels but increased serum luteinizing and follicle-stimulating hormone levels. The messenger RNA and protein levels of luteinizing hormone/chorionic gonadotropin receptor, steroidogenic acute regulatory protein, cytochrome P450 17-hydroxylase/17,20-lyase, follicle-stimulating hormone receptor, and SRY box 9 were significantly lower in the TMT-treated testes than in controls. Immunohistochemical study showed that TMT decreased adult Leydig cell number. In conclusion, these findings indicate that TMT reduced adult Leydig cell testosterone production in vivo by directly downregulating the expression of steroidogenic enzymes and decreasing adult Leydig cell number in the testis.

Nicotine is consumed largely as a component of cigarettes and has a potential effect on pubertal development of Leydig cells in males. To investigate its effects, 49-day-old male Sprague Dawley rats received intraperitoneal injections of nicotine (0.5 or 1 mg/kg/day) for 2 weeks and immature Leydig cells were isolated from the testes of 35-day-old rats and treated with nicotine (0.05-50 μM). Serum hormones, Leydig cell number and related gene expression levels after in vivo treatment were determined and medium androgen levels were measured and cell cycle, apoptosis, mitochondrial membrane potential (△Ψm), and reactive oxygen species (ROS) of Leydig cells after in vitro treatment were measured. In vivo exposure to nicotine lowered serum luteinizing hormone, follicle stimulating hormone, and testosterone levels and reduced Leydig cell number and gene expression levels. Nicotine in vitro inhibited androgen production in Leydig cells by downregulating the expression levels of P450 cholesterol side cleavage enzyme, 3β-hydroxysteroid dehydrogenase 1, and steroidogenic factor 1 at different concentration ranges. In conclusion, nicotine disrupts Leydig cell steroidogenesis during puberty possibly via down-regulating some key steroidogenic enzyme expressions.

To explore the role of the testicular leptin and JAK-STAT[leptin (LEP)-JAK-STAT] pathway in testosterone biosynthesis during juvenile stages and exercise for weight loss, male C57BL/6J mice were randomly divided into normal-diet and high-fat diet groups. After 10 wk, mice in the high-fat diet-fed group were further divided randomly into obese control, obese moderate-volume exercise, and obese high-volume exercise groups. Mice in the obese moderate-volume exercise group were provided with 2 h/day, 6 days/wk swimming exercise for 8 wk, and mice in the obese high-volume exercise group underwent twice the amount of daily exercise intervention as the obese moderate-volume exercise group. The results showed that a high-fat diet causes obesity, leptin resistance, inhibition of the testicular LEP-JAK-STAT pathway, decreased mRNA and protein expression of steroidogenic factor-1, steroidogenic acute regulatory protein, and the P-450 side-chain cleavage enzyme, a decrease in the serum testosterone-to-estradiol ratio, and declines in sperm quality parameters. Both moderate and high-volume exercise were able to reduce body fat and increase the mRNA and protein expression of LEP-JAK-STAT, but only moderate exercise significantly increased the mRNA and protein expression of steroidogenic factor-1, steroidogenic acute regulatory protein, and P-450 side-chain cleavage enzyme and significantly reversed the serum testosterone-to-estradiol ratio and sperm quality parameters. These findings suggest that by impairing the testicular LEP-JAK-STAT pathway, early-stage obesity inhibits the biosynthesis of testosterone and sexual development and reduces male reproductive potential. Long-term moderate and high-volume exercise can effectively reduce body fat and improve obesity-induced abnormalities in testicular leptin signal transduction, whereas only moderate-volume exercise can reverse the negative impacts of obesity on male reproductive function.

The steroid hormone ecdysone, which controls insect molting and metamorphosis, is synthesized in the prothoracic gland (PG), and several steroidogenic enzymes that are expressed specifically in the PG are involved in ecdysteroidogenesis. In this study, we identified new regulators that are involved in the transcriptional control of the silkworm steroidogenic enzyme genes. In silico analysis predicted several potential cis-regulatory elements (CREs) for the homeodomain transcription factors Antennapedia (Antp) and POU-M2 in the proximal promoters of steroidogenic enzyme genes. Antp and POU-M2 are expressed dynamically in the PG during larval development, and their overexpression in silkworm embryo-derived (BmE) cells induced the expression of steroidogenic enzyme genes. Importantly, luciferase reporter analyses, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays revealed that Antp and POU-M2 promote the transcription of the silkworm steroidogenic enzyme gene Phantom (Phm) by binding directly to specific motifs within overlapping CREs in the Phm promoter. Mutations of these CREs in the Phm promoter suppressed the transcriptional activities of both Antp and POU-M2 in BmE cells and decreased the activities of mutated Phm promoters in the silkworm PG. In addition, pulldown and co-immunoprecipitation assays demonstrated that Antp can interact with POU-M2. Moreover, RNA interference-mediated down-regulation of either Antp or POU-M2 during silkworm wandering not only decreased the ecdysone titer but also led to the failure of metamorphosis. In summary, our results suggest that Antp and POU-M2 coordinate the transcription of the silkworm Phm gene directly, indicating new roles for homeodomain proteins in regulating insect ecdysteroidogenesis.

Ectopic cortisol-producing adrenocortical adenomas (CPA) are extremely rare, and only four cases have previously been reported so far but the tumors were not ultrastructurally studied. Presented in this paper is the fifth case with ectopic CPA which was extensively examined to gain deeper insights in terms of the histopathological features and steroidogenic enzyme profile of the tumor. A 53-year-old woman complained of accidental discovery of left renal mass. She had a 5-year history of hypertension, weight gain, moon face, thin skin and systemic edema. These symptoms completely relieved after the tumor removal. Two years later, the above symptoms recurred, and a recurrent tumor was revealed in left renal hilum. The tumor was removed completely with relief of her symptoms of Cushing\'s syndrome. Histologically and ultrastructurally, the tumor was composed of compact cells and clear cells, and the former was prominent, suggesting an active secretory function of the tumor. The adenoma tissue showed a strong immunostaining for Melan-A, 3beta-hydroxysteroid dehydrogenase (HSD3B2) and 17alpha-hydroxylase1 (CYP17A1). Expression pattern for 11beta-hydroxylase 1 (CYP11B1), 11beta-hydroxylase 2 (CYP11B2), CYP17 and HSD3B2 mRNA in ectopic CPA was similar to that in the adrenal CPA. In conclusion, in terms of histopathological characteristic and steroidogenic enzyme profile, ectopic CPA is similar to adrenal CPA, suggesting that they are of identical cell origin.