The Effects of Separate and Combined Treatment of Male Rats with Type 2 Diabetes with Metformin and Orthosteric and Allosteric Agonists of Luteinizing Hormone Receptor on Steroidogenesis and Spermatogenesis

In men with type 2 diabetes mellitus (T2DM), steroidogenesis and spermatogenesis are impaired. Metformin and the agonists of luteinizing hormone/human chorionic gonadotropin(hCG)-receptor (LH/hCG-R) (hCG, low-molecular-weight allosteric LH/hCG-R-agonists) can be used to restore them. The aim was to study effectiveness of separate and combined administration of metformin, hCG and 5-amino-N-tert-butyl-2-(methylsulfanyl)-4-(3-(nicotinamido)phenyl)thieno[2,3-d]pyrimidine-6-carboxamide (TP3) on steroidogenesis and spermatogenesis in male rats with T2DM. hCG (15 IU/rat/day) and TP3 (15 mg/kg/day) were injected in the last five days of five-week metformin treatment (120 mg/kg/day). Metformin improved testicular steroidogenesis and spermatogenesis and restored LH/hCG-R-expression. Compared to control, in T2DM, hCG stimulated steroidogenesis and StAR-gene expression less effectively and, after five-day administration, reduced LH/hCG-R-expression, while TP3 effects changed weaker. In co-administration of metformin and LH/hCG-R-agonists, on the first day, stimulating effects of LH/hCG-R-agonists on testosterone levels and hCG-stimulated expression of StAR- and CYP17A1-genes were increased, but on the 3–5th day, they disappeared. This was due to reduced LH/hCG-R-gene expression and increased aromatase-catalyzed estradiol production. With co-administration, LH/hCG-R-agonists did not contribute to improving spermatogenesis, induced by metformin. Thus, in T2DM, metformin and LH/hCG-R-agonists restore steroidogenesis and spermatogenesis, with metformin being more effective in restoring spermatogenesis, and their co-administration improves LH/hCG-R-agonist-stimulating testicular steroidogenesis in acute but not chronic administration.

Review on Bee Products as Potential Protective and Therapeutic Agents in Male Reproductive Impairment

Bee products are sources of functional food that have been used in complementary medicine to treat a variety of acute and chronic illnesses in many parts of the world. The products vary from location to location as well as country to country. Therefore, the aim of this review was to identify various bee products with potential preventive and therapeutic values used in the treatment of male reproductive impairment. We undertook a vigorous search for bee products with preventive and therapeutic values for the male reproductive system. These products included honey, royal jelly, bee pollen, bee brood, apilarnil, bee bread, bee wax, and bee venom. We also explained the mechanisms involved in testicular steroidogenesis, reactive oxygen species, oxidative stress, inflammation, and apoptosis, which may cumulatively lead to male reproductive impairment. The effects of bee pollen, bee venom, honey, propolis, royal jelly, and bee bread on male reproductive parameters were examined. Conclusively, these bee products showed positive effects on the steroidogenic, spermatogenic, oxidative stress, inflammatory, and apoptotic parameters, thereby making them a promising possible preventive and therapeutic treatment of male sub/infertility.

Prohibitin-1 Transgenic Mice Revealed an Important Role of Prohibitin in Testicular Steroidogenesis

Testosterone, the male sex hormone, plays an important role in the sexual development and fertility. Consequently, its deficiency causes infertility, obesity, osteoporosis and cardiovascular diseases. Leydig cells (LCs) are the testicular interstitial cells responsible for the biosynthesis of testosterone in response to luteinizing hormone (LH) from the pituitary. Cholesterol is the essential substrate for steroidogenesis which is translocated from the cytosol to the mitochondria where it gets converted to pregnenolone (by P450 side chain cleavage enzyme). Subsequently, pregnenolone translocate to endoplasmic reticulum where action of various enzymes results in the biosynthesis of testosterone. Prohibitin-1 (PHB1) is an evolutionary conserved ubiquitously expressed protein with cell compartment and cell-type specific functions. Mitochondrial function of PHB1 has been widely studied but its role in testicular steroidogenesis is unexplored. Recently, we have reported two transgenic mice models of PHB1, PHB-Tg and mutant-PHB-Tg (mPHB-Tg), expressing PHB1 or Y114F (mutant PHB1) respectively under the control of Fabp-4 gene promoter. During phenotypic characterization of these mice models, we observed a drastic size/weight difference in the testis of PHB-Tg and mPHB-Tg when compared with wild type mice. The mPHB-Tg mice testis was significantly smaller than the PHB-Tg and wild type mice. Further analysis of mPHB-Tg testis revealed wider testicular interstitium with LC hyperplasia and elongated seminiferous tubules. Ultrastructure investigation revealed that LCs of mPHB-Tg mice have prominent nucleus with increased number of mitochondria and lipid droplets. In addition, electron microscopic images of mPHB-Tg mice LCs revealed a sign of lipophagy and mitophagy. This prompted us to measure testosterone levels in these mice; surprisingly mPHB-Tg mice showed significantly higher testosterone levels as compared to PHB-Tg and wild type mice. Furthermore, testicular lysates and primary LCs cell lysates from transgenic mice models revealed that overexpression of PHB/mPHB in LCs inversely effect expression levels of steroidogenic acute regulatory protein (StAR). Moreover, co-immunoprecipitation of PHB1 displayed an interaction with StAR, P450scc and LC3 further revealing a key role of PHB1 in cholesterol translocation, testicular steroidogenesis and autophagy. Taken together, this finding suggests that PHB1 plays a multifaceted role in testicular steroidogenesis from determining testis size to the translocation of cholesterol into the mitochondria, in maintaining lipid homeostasis and biosynthesis of testosterone. Implications of our findings are broad because cholesterol translocation to the mitochondria and its subsequent utilization for steroidogenesis is conserved in all steroidogenic tissues.

Testicular steroidogenesis is suppressed during experimental autoimmune encephalomyelitis in rats

Multiple sclerosis (MS) is an autoimmune disease that usually occurs during the reproductive years in both sexes. Many male patients with MS show lower blood testosterone levels, which was also observed in male rats during experimental autoimmune encephalomyelitis (EAE), an animal model of MS. To better understand the causes of decreased testosterone production during EAE, we investigated the expression status of genes and proteins associated with steroidogenesis in the testes. No changes in the number of interstitial cells were observed in EAE animals, but the expression of the insulin-like 3 gene was reduced at the peak of the disease, implying that the Leydig cell functional capacity was affected. Consistent with this finding, the expression of most steroidogenic enzyme genes and proteins was reduced during EAE, including StAR, CYP11A1, CYP17A1 and HSD3B. No signs of testicular inflammation were observed. Recovery of steroidogenesis was observed after injection of hCG, the placental gonadotropin, or buserelin acetate, a gonadotropin-releasing hormone analogue, at the peak of EAE. Together, our results are consistent with the hypothesis that impaired testicular steroidogenesis originates upstream of the testes and that low serum LH is the main cause of decreased testosterone levels during EAE.

FOXA3, a Negative Regulator of Nur77 Expression and Activity in Testicular Steroidogenesis

Biosynthesis of testosterone occurs mainly in the testicular Leydig cells. Nur77, an orphan nuclear receptor that is expressed in response to the luteinizing hormone/cyclic adenosine monophosphate (LH/cAMP) signaling pathway, is one of the key factors that regulate steroidogenesis in Leydig cells. The function of Nur77 is modulated through interaction with other proteins. FOXA3, a transcription factor that is crucial for male fertility, is also expressed in Leydig cells. Here, we sought to elucidate the role of FOXA3 in testicular steroidogenesis by focusing on its interaction with Nur77. LH/cAMP signaling induces the onset of steroidogenesis in Leydig cells but has a repressive effect on the expression of FOXA3. Overexpression of FOXA3 in MA-10 Leydig cells repressed cAMP-induced expression of Nur77 and its target steroidogenic genes (StAR, P450c17, and Hsd3β). Furthermore, FOXA3 suppressed Nur77 transactivation of the promoter of steroidogenic genes. In mouse primary Leydig cells, adenovirus-mediated overexpression of FOXA3 had similar effects and resulted in decreased production of testosterone. Taken together, these results suggest the role of FOXA3 in the regulation of steroidogenic genes in Leydig cells and fine-tuning steroidogenesis in the testis.

Testicular STAC3 regulates Leydig cell steroidogenesis through potentiating mitochondrial membrane potential and StAR processing

SH3 and cysteine-rich protein 3 (STAC3), a small adapter protein originally identified as a core component of excitation–contraction coupling machinery, regulates the voltage-induced Ca2+ release in skeletal muscle. However, the possibility of additional, as yet unknown, non-muscle effects of STAC3 cannot be ruled out. Herein, we provide the evidence for the expression and functional involvement of STAC3 in spermatogenesis. STAC3 expression was localized in the testicular interstitium of rodent and human testes. By using the cytotoxic drug ethylene dimethane sulfonate (EDS), STAC3 expression was observed to be decreased sharply in rat testis after selective withdrawal of Leydig cells (LCs), and reappeared immediately after LCs repopulation, indicating that testicular expression of STAC3 mainly stems from LCs. From a functional standpoint, in vivo lentiviral vector–mediated suppression of STAC3 resulted in a significant decrease in testosterone production, and thereafter caused impairment of male fertility by inducing oligozoospermia and asthenospermia. The indispensible involvement of STAC3 in testicular steroidogenesis was validated using the in vivo knockdown model with isolated primary LCs as well as in vitro experiments with primary LCs. By generating the TM3Stac3−/− cells, we further revealed that STAC3 depletion attenuated mitochondrial membrane potential and StAR processing in db-cAMP-stimulated LCs. Thus, the inhibitory effect of STAC3 deficiency on testicular steroidogenesis may be ascribed to a disturbed mitochondrial homeostasis. Collectively, the present results strongly suggest that STAC3 may function as a novel regulator linking mitochondrial homeostasis and testicular steroidogenesis in LCs. Our data underscore an unexpected reproductive facet of this muscle-derived factor.

PSVII-27 Early plasma androstenone concentrations may indicate extent of boar taint at slaughter

Castration is a highly invasive procedure performed on male pigs within the first few days after birth. Castration reduces aggressive and sexual behaviours and, more importantly, eliminates the incidence of a meat quality issue called boar taint. Androstenone, one of the boar taint causing compounds, is a steroid hormone produced during puberty in boars, and also during a spike of testicular steroidogenesis at 21 days of age. This peak is thought to mature the hypothalamic-pituitary-gonadal axis; however, 21-day steroid concentrations have not previously been linked to the extent of boar taint development at slaughter. The objective of this research is to determine if androstenone concentrations at 21 days of age can predict boar taint development at slaughter. Crossbred [(YorkshireXLandrace)XDuroc] boars (n = 36) were raised in pens of two females and two males to average market slaughter weight. Blood was taken at 21 days and slaughter, backfat was collected at slaughter. Plasma and fat androstenone concentrations were measured by androstenone-specific ELISA. Data was analyzed using Pearson correlation and ANOVA. Boars >120kg at slaughter showed positive correlation (R=0.54, P =.007) between 21-day plasma androstenone and fat androstenone concentrations at slaughter, and tended to correlate (R=0.40, P =.056) between 21-day plasma androstenone and plasma androstenone concentrations at slaughter. Boars yet to reach 120kg by slaughter did not show correlation (R=0.2, P = .2) between 21-day plasma androstenone and slaughter androstenone concentrations. There was no correlation between age and weight at slaughter, and plasma and fat androstenone concentrations were not different (P >.05) between groups above and below 120kg at slaughter. These results suggest that 21-day plasma androstenone concentrations may be indicative of androstenone accumulation in the fat and plasma if pigs are over 120kg at slaughter. This can aid with identifying boars at risk of developing boar taint early so that solutions such as immunocastration can be implemented.