A Novel Model Using AAV9-Cre to Knockout Adult Leydig Cell Gene Expression Reveals a Physiological Role of Glucocorticoid Receptor Signalling in Leydig Cell Function

Glucocorticoids are steroids involved in key physiological processes such as development, metabolism, inflammatory and stress responses and are mostly used exogenously as medications to treat various inflammation-based conditions. They act via the glucocorticoid receptor (GR) expressed in most cells. Exogenous glucocorticoids can negatively impact the function of the Leydig cells in the testis, leading to decreased androgen production. However, endogenous glucocorticoids are produced by the adrenal and within the testis, but whether their action on GR in Leydig cells regulates steroidogenesis is unknown. This study aimed to define the role of endogenous GR signalling in adult Leydig cells. We developed and compared two models; an inducible Cre transgene driven by expression of the Cyp17a1 steroidogenic gene (Cyp17-iCre) that depletes GR during development and a viral vector-driven Cre (AAV9-Cre) to deplete GR in adulthood. The delivery of AAV9-Cre ablated GR in adult mouse Leydig cells depleted Leydig cell GR more efficiently than the Cyp17-iCre model. Importantly, adult depletion of GR in Leydig cells caused reduced expression of luteinising hormone receptor (Lhcgr) and of steroidogenic enzymes required for normal androgen production. These findings reveal that Leydig cell GR signalling plays a physiological role in the testis and highlight that a normal balance of glucocorticoid activity in the testis is important for steroidogenesis.     

Aspartame Consumption,Mitochondrial Disorder-Induced Impaired Ovarian Function,and Infertility Risk

Frequent consumption of diet drinks was associated with oocyte dysmorphism, decreased embryo quality, and an adverse effect on pregnancy rate. We investigated the harmful effects of aspartame and potential mechanisms through which it increases infertility risk through clinical observations and in vivo and in vitro studies. Methods: We established a cohort of 840 pregnant women and retrospectively determined their time to conceive. We assessed the estrus cycle, the anti-Mullerian hormone level, ovarian oxidative stress, and ovarian mitochondrial function in an animal study. We also evaluated mitochondria function, mitochondrial biogenesis, and progesterone release with in vitro studies. Aspartame consumption was associated with increased infertility risk in the younger women (Odds ratio: 1.79, 95% confidence interval: 1.00, 3.22). The results of the in vivo study revealed that aspartame disrupted the estrus cycle and reduced the anti-Mullerian hormone level. Aspartame treatment also suppressed antioxidative activities and resulted in higher oxidative stress in the ovaries and granulosa cells. This phenomenon is caused by an aspartame-induced decline in mitochondrial function (maximal respiration, spare respiratory capacity, and ATP production capacity) and triggered mitochondrial biogenesis (assessed by examining the energy depletion signaling-related factors sirtuin-1, phosphorylated adenosine monophosphate-activated protein kinase, peroxisome proliferator-activated receptor-gamma coactivator-1α, and nuclear respiratory factor 1 expression levels). Aspartame may alter fertility by reserving fewer follicles in the ovary and disrupting steroidogenesis in granulosa cells. Hence, women preparing for pregnancy are suggested to reduce aspartame consumption and avoid oxidative stressors of the ovaries.     

Preliminary Investigation on the Involvement of Cytoskeleton-Related Proteins,DAAM1 and PREP,in Human Testicular Disorders

Herein, for the first time, the potential relationships between the cytoskeleton-associated proteins DAAM1 and PREP with different testicular disorders, such as classic seminoma (CS), Leydig cell tumor (LCT), and Sertoli cell-only syndrome (SOS), were evaluated. Six CS, two LCT, and two SOS tissue samples were obtained during inguinal exploration in patients with a suspect testis tumor based on clinical examination and ultrasonography. DAAM1 and PREP protein levels and immunofluorescent localization were analyzed. An increased DAAM1 protein level in CS and SOS as compared to non-pathological (NP) tissue was observed, while LCT showed no significant differences. Conversely, PREP protein level increased in LCT, while it decreased in CS and SOS compared to NP tissue. These results were strongly supported by the immunofluorescence staining, revealing an altered localization and signal intensity of DAAM1 and PREP in the analyzed samples, highlighting a perturbed cytoarchitecture. Interestingly, in LCT spermatogonia, a specific DAAM1 nuclear localization was found, probably due to an enhanced testosterone production, as confirmed by the increased protein levels of steroidogenic enzymes. Finally, although further studies are needed to verify the involvement of other formins and microtubule-associated proteins, this report raised the opportunity to indicate DAAM1 and PREP as new potential markers, supporting the cytoskeleton dynamics changes occurring during normal and/or pathological cell differentiation.     

Equine Chorionic Gonadotropin as an Effective FSH Replacement for In Vitro Ovine Follicle and Oocyte Development

The use of assisted reproductive technologies (ART) still requires strategies through which to maximize individual fertility chances. In vitro folliculogenesis (ivF) may represent a valid option to convey the large source of immature oocytes in ART. Several efforts have been made to set up ivF cultural protocols in medium-sized mammals, starting with the identification of the most suitable gonadotropic stimulus. In this study, Equine Chorionic Gonadotropin (eCG) is proposed as an alternative to Follicle Stimulating Hormone (FSH) based on its long superovulation use, trans-species validation, long half-life, and low costs. The use of 3D ivF on single-ovine preantral (PA) follicles allowed us to compare the hormonal effects and to validate their influence under two different cultural conditions. The use of eCG helped to stimulate the in vitro growth of ovine PA follicles by maximizing its influence under FBS-free medium. Higher performance of follicular growth, antrum formation, steroidogenic activity and gap junction marker expression were recorded. In addition, eCG, promoted a positive effect on the germinal compartment, leading to a higher incidence of meiotic competent oocytes. These findings should help to widen the use of eCG to ivF as a valid and largely available hormonal support enabling a synchronized in vitro follicle and oocyte development.     

LncGSAR Controls Ovarian Granulosa Cell Steroidogenesis via Sponging MiR-125b to Activate SCAP/SREBP Pathway

Long non-coding RNAs (lncRNAs) have been shown to play important roles in livestock fecundity, and many lncRNAs that affect follicular development and reproductive diseases have been identified in the ovary. However, only a few of them have been functionally annotated and mechanistically validated. In this study, we identified a new lncRNA (lncGSAR) and investigated its effects on the proliferation and steroidogenesis of ovine granulosa cells (GCs). High concentrations of glucose (add 33.6 mM glucose) caused high expression of lncGSAR in GCs by regulating its stability, and lncGSAR overexpression promoted GCs proliferation, estrogen secretion, and inhibited progesterone secretion, whereas interference with lncGASR had the opposite effect. Next, we found that the RNA molecules of lncGSAR act on MiR-125b as competitive endogenous RNA (ceRNA), and SREBP-cleavage-activating protein (SCAP) was verified as a target of MiR-125b. LncGASR overexpression increased the expression of SCAP, SREBP, and steroid hormone-related proteins, which can be attenuated by MiR-125b. Our results demonstrated that lncGSAR can act as a ceRNA to activate SCAP/SREBP signaling by sponging MiR-125b to regulate steroid hormone secretion in GCs. These findings provide new insights into the mechanisms of nutrient-regulated follicle development in ewes.     

Neuromedin S Regulates Steroidogenesis through Maintaining Mitochondrial Morphology and Function via NMUR2 in Goat Ovarian Granulosa Cells

Neuromedin S (NMS) plays various roles in reproductive regulation, while the mechanism by which NMS regulates ovarian steroidogenesis remains unclear. In the current study, we confirmed the enhancement role of NMS in steroidogenesis in goat ovarian granulosa cells (GCs). To further explore the specific mechanism, we conducted a knockdown of NMUR2 in GCs followed by treatment with NMS and determined the effects of NMS treatment on mitochondrial morphology and function. The results found that NMS treatment increased the production of estrogen and up-regulated the expression of STAR, CYP11A1, 3BHSD, and CYP19A1, while the effects of NMS treatment were blocked by the knockdown of NMUR2 in goat GCs. Moreover, NMS treatment enhanced the fusion of mitochondria and up-regulated the expression of OPA1, MFN1, and MFN2, and increased mitochondrial membrane potential, the activity of respiratory chain enzymes and ATP production by maintaining a low expression level of mitochondrial unfolded protein response markers. The effects of NMS treatment on mitochondria were reversed by NMUR2 knockdown and NMS cotreatment. The possible mechanism of the results above was revealed by NMS treatment activating the Hippo pathway effector YAP1 and then managing the expression of phosphorylation PPARGC1A (Ser571). Together, these data showed that NMS promoted the fusion of mitochondria and protected mitochondrial function from mitochondrial unfolded protein response possibly via the NMUR2/YAP1/PPARGC1A pathway, thereby affecting the steroidogenesis of goat GCs. By elaborating the potential mechanism of NMS in regulating estrogen production in goat GCs, our results can serve as the mechanism reference for follicular growth and development.     

Spontaneous Remission of Primary Aldosteronism with Mineralocorticoid Receptor Antagonist Therapy: A Review

In this review, we describe previous basic and clinical studies on autonomous aldosterone production. Over the past decades, mineralocorticoid receptor antagonists (MRAs) have been found to concentration-dependently inhibit steroidogenesis in different degrees. However, many studies have proven the suppressive effects of MRAs on the activities of hormone synthase. The probable factors of cytochrome P-450 reduction, both in microsomes and mitochondria, have also been considered: (1) one of the spironolactone metabolite forms had destructive function, except canrenone, (2) 7α-thio-spironolactone was an obligatory intermediate in the spironolactone-induced CYP450 decrease, and (3) the contributing steroids should have 7α-methylthio or 7α-methylsulfone groups. In previous clinical research, spironolactone-body-containing cells showed a type II pattern of enzyme activity (i.e., enhanced 3β-hydroxysteroid dehydrogenase, glucose-6-phosphate, and NADP-isocitrate dehydrogenase activities and weaken succinate dehydrogenase activity), and the subcapsular micronodules composed of spironolactone-body-containing cells also exhibited a type II pattern and excess aldosterone secretion, indicating that the subcapsular micronodules might be the root of aldosterone-producing adenoma. Moreover, combined with the potential impeditive function to aldosterone secretion, a few cases of spontaneous remission of primary aldosteronism, with normal ranges of blood pressure, plasma potassium, plasma renin activity, and aldosterone renin ratio, have been reported after long-term treatment with MRAs.     

Effects of estradiol on bovine thecal cell function in vitro: dependence on insulin and gonadotropins

The objective of this study was to evaluate the influence of estradiol (E2) on proliferation and steroid production by thecal cells obtained from large (≥8 mm) follicles of cattle. Five experiments evaluated the effect of various doses of E2 during a 2-d exposure in serum-free medium on hormone-induced steroidogenesis and cell proliferation. In LH-treated thecal cells of experiment 1, 300 ng/mL of E2 decreased progesterone production by 30% and increased androstenedione production to 5.8-fold of controls. In the absence of LH, both 3 and 300 ng/mL of E2 increased progesterone production. In experiment 2, in the presence of insulin and LH, 3, 30, and 300 ng/mL of E2 decreased progesterone production (by 17 to 36%), whereas 3 ng/mL of E2 decreased and 300 ng/mL of E2 increased androstenedione production. Doses of LH (3 to 30 ng/mL) tested in experiment 3 increased (to as much as 3.7-fold) progesterone production by thecal cells and E2 attenuated this stimulatory effect by 40%. In contrast, E2 amplified the stimulatory effect of LH on androstenedione production in experiment 3. In experiment 4, E2 (300 ng/mL) decreased IGF-I- and insulin-induced thecal cell progesterone production by 70 to 77%, whereas E2 increased basal, IGF-I, and insulin-induced androstenedione production. In experiment 5, in the presence of insulin, 10 to 1000 ng/mL of E2 had no effect on [¹²⁵I]-IGF-I binding to thecal cells, whereas 10 and 100 ng/mL of E2 increased and 1000 ng/mL of E2 decreased progesterone production by thecal cells. Estradiol had no consistent effect on thecal cell numbers among the 5 experiments. These results support the hypothesis that E2 may act as a paracrine factor to directly regulate hormone-induced steroid production by thecal cells without affecting cell numbers or numbers of insulin-like growth factor type I receptors.     

Diversity in steroidogenesis of symbiotic microorganisms from planthoppers

Ergosta-5,7,24(28)-trien-3-ol (trienol)6 was isolated from the intracellular symbiotes (symbiotic microorganisms) of the planthoppers,Nilaparvata lugens andLaodelphax striatellus. The steroidogenic end product of the symbiotes was found to depend on the environmental conditions, i.e., although trienol6 was produced under symbiotic conditions, ergosterol4 was formed in aerobic culture. When the normal diet was replaced by a steroiddeficient artificial one, the ratio of 24-methylenecholesterol5 to total insect sterols was significantly increased. The above study offers further corroborating evidence for our assumption that the host insects require 24-methylenecholesterol5 as an alternate source for cholesterol1 under certain environmental conditions. In our previous biotransformation experiments, 24methylenecholesterol5 was shown to be an immediate precursor of cholesterol1. However, the complexity of the insects' vital functions hindered all subsequent attempted transformations into cholesterol1 of sitosterol2, trienol6, and ergosterol4, under injection conditions.