Stimulated Testosterone Secretion Research Articles

Trace elements alone or in mixtures associated with unconventional natural gas exploitation affect rat fetal steroidogenesis and testicular development in vitro

Biomonitoring studies have shown that pregnant women living in regions of unconventional natural gas (UNG) exploitation have higher levels of trace elements. Whether developmental endocrine disruption can be expected at these exposure levels during pregnancy is unclear. In this study, we aimed to test the impact of five trace elements alone or in mixtures using in vitro cell- and tissue-based assays relevant to endocrine disruption and development. Manganese, aluminum, strontium, barium, and cobalt were tested at concentrations including those representatives of human fetal exposure. Using transactivation assays, none of the tested elements nor their mixture altered the human estrogen receptor 1 or androgen receptor genomic signalling. In the rat fetal testis assay, an organ culture system, cobalt (5 μg/l), barium (500 μg/l) and strontium (500 μg/l) significantly increased testosterone secretion. Cobalt and strontium were associated with hyperplasia and/or hypertrophy of fetal Leydig cells. Mixing the five elements at concentrations where none had an effect individually stimulated testosterone secretion by the rat fetal testis paralleled by the significant increase of 3β-hydroxysteroid dehydrogenase protein level in comparison to the vehicle control. The mechanisms involved may be specific to the fetal testis as no effect was observed in the steroidogenic H295R cells. Our data suggest that some trace elements in mixture at concentrations representative of human fetal exposure can impact testis development and function. This study highlights the potential risk posed by UNG operations, especially for the most vulnerable populations, pregnant individuals, and their fetus.

α-Tocotrienol in rice bran enhances steroidogenesis in mouse Leydig cell via increased gene expression of steroidogenic acute regulatory protein and induction of its mitochondrial translocation.

Rice is a staple food in the Asian region and one of the world's major energy sources. Testosterone is a steroid hormone that maintains physical, sexual, and cognitive ability, and its decline causes health problems like late-onset hypogonadism. Evaluation of various grain extracts showed rice bran to stimulate testosterone secretion from Leydig model cells. α-Tocotrienol was found as a bioactive compound in rice bran, and mechanistic analysis showed the stimulation of steroid hormone synthesis through enhanced gene expression of steroidogenic acute regulatory protein as well as inducing mitochondrial localization of the protein. Preliminary study showed an increasing trend in serum testosterone levels in mice by oral intake of α-tocotrienol. These results suggest that α-tocotrienol intake may be effective in preventing symptoms caused by low testosterone levels.

Novel Potency Assay for MSC Secretome-Based Treatment of Idiopathic Male Infertility Employed Leydig Cells and Revealed Vascular Endothelial Growth Factor as a Promising Potency Marker.

Idiopathic male infertility is a highly prevalent diagnosis in developed countries with no specific treatment options. Although empirical medical treatment is widely used to restore male fertility, its efficacy remains limited and inconclusively proven. Therefore, the development of novel therapeutic approaches in this field is a high-priority task. Since the failure of testicular microenvironment components might be involved in the pathogenesis of idiopathic male infertility, application of mesenchymal stromal cells (MSCs) as well as the MSC secretome is worth considering. Previously, we showed that the intratesticular injection of MSCs or the MSC secretome led to the recovery of spermatogenesis at least through replenishing the testicular microenvironment and its maintenance by MSC-secreted paracrine factors. However, the clinical use of such products has been limited to single trials to date. This may be due to the lack of relevant potency tests reflecting mechanisms of action of the MSC secretome in male infertility models. Based on the presumptive MSC secretome mode of action on the testicular microenvironment, we suggest a novel approach to test the potential efficacy of the MSC secretome for idiopathic male infertility treatment. It represents a potency assay based on evaluation of testosterone production by isolated Leydig cells. We demonstrated that the MSC secretome stimulated testosterone secretion by Leydig cells in vitro. We then hypothesized that among the major factors of the MSC secretome, vascular endothelial growth factor (VEGF) could be responsible for the observed effects, which we confirmed by the revealed correlation between the extent of stimulated testosterone production and VEGF concentration in the MSC secretome. The pilot results obtained from the doxorubicin-induced male infertility murine model also indicate the important impact of VEGF in the MSC secretome’s regenerative effects. Utilizing VEGF as a surrogate factor, a novel approach to study the potency of MSC secretome-based products for idiopathic male infertility treatment is suggested. Further validation is required for its implementation into the biopharmaceutical manufacturing process.

The cold-soaking extract of Chinese yam (Dioscorea opposita Thunb.) protects against erectile dysfunction by ameliorating testicular function in hydrocortisone-induced KDS-Yang rats and in oxidatively damaged TM3 cells

Ethnopharmacological relevanceClinical applications and pharmacological research suggest that Dioscorea opposita Thunb. (Chinese yam), a well-known traditional Chinese medicine which has been used for more than 2000 years to nourish kidney-yang and protect the male reproductive system, might be efficacious for the treatment of erectile dysfunction (ED). Aim of the studyThis study aimed to investigate the active component extract of Chinese yam, determine its effectiveness in hydrocortisone-induced “kidney-yang deficiency syndrome” (KDS-Yang) rats and in oxidatively damaged TM3 cells and explore the underlying mechanism on restoring erectile function. Materials and methodsWe clarified the Chinese yam cold-soaking extract (CYCSE) as the main active extract of Chinese yam by a CCK8 assay and further identified its composition. The KDS-Yang rats were induced by intragastric administration of hydrocortisone. After 10 d of CYCSE intervention, cavernous and testis morphology were stained with hematoxylin and eosin. Inducible nitric oxide synthase (iNOS), cyclic guanosine monophosphate (cGMP), testosterone, 8-hydroxy-2-deoxyguanosine (8-OHdG) and superoxide dismutase (SOD) levels were detected by enzyme-linked immunosorbent assay kits. Leydig cells were performed using immunohistochemistry. Reactive oxygen species were measured using a DCFH-DA fluorescent probe, and testicular collagenous fibers were stained with a Masson kit. Detection of testicular apoptosis was performed by a TUNEL assay. Nrf2 and NQO1 mRNA expression levels were measured by qRT-PCR. The protein expression levels of Nrf2, HO-1, TGF-β1 and SMAD2/3 were analyzed by Western blot. ResultsWe demonstrated in KDS-Yang rats and oxidatively damaged TM3 cells that CYCSE successfully restored erectile function through ameliorating testicular function. Our data suggested that CYCSE can stimulate the NO/cGMP pathway and restore the cavernous morphology to protect against KDS-Yang-induced ED. It also protected testis morphology, increased Leydig cell proliferation and stimulated testosterone secretion. In the damaged testes, excessive increases in 8-OHdG and inhibition of SOD activity were ameliorated, and the Nrf2/HO-1 signaling pathway was enhanced after treatment with CYCSE, indicating that the antioxidant defense system was activated. These findings were also validated in vitro. Additionally, fibrosis of the testes and TM3 cells was reversed by CYCSE through the TGF-β1/SMAD2/3 pathway. ConclusionCYCSE has a therapeutic effect on KDS-Yang-induced ED, and the mechanism includes stimulation of testosterone secretion, resistance to oxidative stress and prevention of fibrosis. These findings provide a new scientific verification for the application of Chinese yam in the treatment of KDS-Yang-induced ED.

Investigation of the Properties and Effects of Salvia Officinalis L. on the Viability, Steroidogenesis and Reactive Oxygen Species (ROS) Production in TM3 Leydig Cells in Vitro.

The aim of our study was to reveal the in vitro effects of Salvia officinalis L. (37.5, 75, 150, 200, 250, 300 and 600 µg/ml) extract on the TM3 Leydig cell viability, membrane integrity, steroidogenesis and reactive oxygen species production after 24 h and 48 h cultivation. For the present study, the extract prepared from Salvia officinalis L. leaves was analysed by high performance liquid chromatography (HPLC) for selected flavonoids and phenolic acids followed by a determination of its free radicals scavenging activity (DPPH). Furthermore, Leydig cell viability was assessed by the mitochondrial toxicity assay (MTT), while the membrane integrity was evaluated by 5- carboxyfluorescein diacetate-acetoxymethyl ester (5-CFDA-AM). The level of steroid hormones was performed by enzyme-linked immunosorbent assay (ELISA) from the culture media, while the superoxide radical generation was measured by the nitroblue tetrazolium chloride (NBT) assay. The results show that experimental concentrations did not damage the cell membrane integrity and viability when present at below 300 µg/ml, it was only at 600 µg/ml that a significant (P<0.05) cell viability decline was observed after a 48 h cultivation. A significant (P<0.05) stimulation of testosterone secretion was recorded at 250 µg/ml for 24 h, while the prolonged cultivation time significantly (P<0.05) increased the testosterone and progesterone production at 150, 200, 250 and 300 µg/ml. Furthermore, none of the selected doses exhibited significant ROS-promoting effects however, the highest dose of Salvia initiated the free radical scavenging activity in cultured mice Leydig cells.

Administration of equine Chorionic Gonadotrophin (eCG) to rams to improve the reproductive performance during the non-breeding season

In most ram breeds, there are annual changes in testosterone serum concentration and semen quality. This study aimed to determine if eCG administration to rams during the non-breeding season improves the reproductive activity. In the first study, we compared the effects of administering three doses of eCG (0, 400 or 700 IU). In the second experiment we compared the effects of administering eCG to rams from two breeds originated in different latitudes, and thus, with different expected reproductive seasonal pattern (Highlander; HL and Texel; TEX). The first study was performed during the mid non-breeding season with three groups of rams: treated with 400 IU (group eCG400) or 700 IU of eCG (group eCG700), or untreated controls (group CON). Treated rams received three doses of eCG administered every 6 days. Testosterone concentration, testicular traits (scrotal circumference, testicular and sexual skin flush, and ultrasonographic testes pixel color intensity) and seminal characteristics were determined from five days before the first eCG administration until Day 24. Testosterone concentration was greater in eCG700 than in eCG400 (P <0.001) and CON (P <0.0001) rams, and in eCG400 than in CON rams (P = 0.0002). However, the treatment did not modify the testicular traits and seminal quality. The second study was performed with 15 HL and 17 TEX rams; while 8 HL and 9 TEX rams received two doses of 1000 IU of eCG (Days 0 and 5), the other rams remained as untreated controls. Scrotal circumferences, seminal traits (fresh and thawed semen, and cryopreservation ratio –CR-) as well as the sexual behavior were determined, twice before eCG administration and during 3 weeks. The treatment did not modify the scrotal circumference nor the fresh sperm traits evaluated. However, the CR of motile and progressive motile sperm were greater in eCG treated rams than in control rams on Day 7 (P = 0.01 for both). Treated rams had their second and third ejaculations earlier than control rams (P = 0.001 and P = 0.02; respectively). In conclusion, the administration of eCG during the non-breeding season stimulated testosterone secretion without modifying the main reproductive outcomes of rams. Reducing the interejaculatory intervals could be considered for intensive semen collection programs.

Role of the superior ovarian nerve in the regulation of follicular development and steroidogenesis in the morning of diestrus 1.

Little is known about the role of the superior ovarian nerve (SON) in follicular development during the estrus cycle. The aim of the present study was to analyze the role of neural signals arriving through the SON at the ovaries in the regulation of follicular development and ovarian steroid secretion in diestrus 1 of cyclic rats. Cyclic rats were subjected to left, right, or bilateral SON sectioning or to unilateral or bilateral laparotomy at diestrus 1 at 11:00h. Animals were sacrificed 24h after surgery. Compared to laparotomized animals, unilateral SON sectioning decreased the number of preovulatory follicles, while bilateral SON sectioning resulted in a decreased number of atretic preantral follicles. An important observation was the presence of invaginations in the follicular wall of large antral and preovulatory follicles in animals with denervation. Furthermore, left SON sectioning increased progesterone levels but decreased testosterone levels, which are effects that were not observed in animals that were subjected to right denervation. At 11:00h of diestrus 1, the SON was found to stimulate follicle development, possibly via neural signals, such as noradrenaline and/or vasoactive intestinal peptide, and this stimulation induced the formation of follicle-stimulating hormone receptors. The role of the SON in the regulation of ovarian steroid secretion is asymmetric: the left SON inhibits the regulation of progesterone and stimulates testosterone secretion, and the right nerve does not participate in these processes.

Local expressions and function of Kiss1/GPR54 in goats' testes.

Kisspeptin, encoded by the Kiss1 gene, and its receptor GPR54 have a central regulatory role in the male reproduction. However, their functions in peripheral tissues, such as testes, remain unclear. This study investigated the local expressions and function of Kiss1/GPR54 in goats' testes. The mRNA expression of Kiss1/GPR54 in pubertal goat Leydig cells was detected through reverse transcriptase PCR (RT-PCR), and its protein expression in Leydig cells or the testis was examined by immunohistochemistry and Western blot analysis. Isolated and cultured Leydig cells were treated with different concentration of kisspeptin (0, 1, 10 and 100μM) and kisspeptin antagonist for 4, 24 or 72h. The direct effect of kisspeptin on testosterone secretion and Kiss1/GPR54 mRNA expression was evaluated by ELISA and RT-PCR. Kiss1/GPR54 mRNA and protein were expressed in Leydig cells and spermatids, and GPR54 were expressed in Sertoli cells. Kisspeptin treatment significantly stimulated testosterone secretion in Leydig cells, with the highest levels found under 24h of treatment with 10μM kisspeptin. Treatment with kisspeptin+kisspeptin antagonist significantly reduced the kisspeptin-stimulated testosterone secretion in Leydig cells. Kisspeptin treatment significantly enhanced the expression of Kiss1/GPR54 mRNA in Leydig cells. These data suggest the local expressions of Kiss1/GPR54 in goats' testes and its autocrine role in Leydig cells, which is helpful in understanding the regulation role of kisspeptin/GPR54 system in other peripheral tissues.

Treatments with eCG and courtship behaviour in rams during the breeding and the non-breeding seasons

Equine chorionic gonadotrophin (eCG or PMSG) is a hormone with FSH/LH effect that has been widely used in ewes and cows to induce follicle development. However, the possible positive effects of its administration to males have been rarely studied. ECG stimulates testosterone secretion and increases rams’ ability to stimulate ovulation in anoestrous ewes (the ‘ram effect’), but it is still not known if this effect is at least partially explained by an enhancement of a ram’s sexual behaviour. Thus, the aim was to determine if the administration of eCG induces an increase in testosterone concentration, and thus improves sexual behaviour of Saint Croix rams during both the breeding and the non-breeding seasons. Two doses of 1000 IU of eCG were administered separated by 4 days to 10 rams during the breeding and five rams during the non-breeding seasons, with the same number of animals as untreated controls. Administration of eCG increased testosterone concentration during the breeding (P = 0.008) and the non-breeding (P = 0.0009) seasons. The number of ano-genital sniffings, lateral approaches, as well as total courtship behaviours were greater in treated than untreated rams during the breeding season. Treated rams also tended to mount and mate more during the breeding season. During the non-breeding season, treated rams tended to display more ano-genital sniffs, lateral approaches and total courtship behaviours than untreated rams 6 days after the first dose. Overall, eCG increased testosterone concentrations the breeding and the non-breeding seasons, and was associated with a greater display of sexual behaviours during the former.

Lipophagy Contributes to Testosterone Biosynthesis in Male Rat Leydig Cells

In recent years, autophagy was found to regulate lipid metabolism through a process termed lipophagy. Lipophagy modulates the degradation of cholesteryl esters to free cholesterol (FC), which is the substrate of testosterone biosynthesis. However, the role of lipophagy in testosterone production is unknown. To investigate this, primary rat Leydig cells and varicocele rat models were administered to inhibit or promote autophagy, and testosterone, lipid droplets (LDs), total cholesterol (TC), and FC were evaluated. The results demonstrated that inhibiting autophagy in primary rat Leydig cells reduced testosterone production. Further studies demonstrated that inhibiting autophagy increased the number and size of LDs and the level of TC, but decreased the level of FC. Furthermore, hypoxia promoted autophagy in Leydig cells. We found that short-term hypoxia stimulated testosterone secretion; however, the inhibition of autophagy abolished stimulated testosterone release. Hypoxia decreased the number and size of LDs in Leydig cells, but the changes could be largely rescued by blocking autophagy. In experimental varicocele rat models, the administration of autophagy inhibitors substantially reduced serum testosterone. These data demonstrate that autophagy contributes to testosterone biosynthesis at least partially through degrading intracellular LDs/TC. Our observations might reveal an autophagic regulatory mode regarding testosterone biosynthesis.

Effects of long-term feeding of the Obudu natural honey and table sugar-sweetened diets on sex hormones of male and female albino Wistar rats

Aim: This study was done to assess the impact of long-term consumption of table sugar and natural honey on the sex hormones and the biochemical implication of this differential effect. Materials and Methods: Fifty male and female rats (25 rats per sex) weighing 100–120 g were used for the experiment. They were housed in standard laboratory cages at room temperature (25°C), humidity of 50%–64%, and maintained on a 12:12 hour light/dark cycle in the animal house. After a 29-week period of feeding of equivalent quantities of natural honey and table sugar separately, body weight and plasma hormones were quantified in them. The diets were formulated to simulate average sugar composition of common sweetened foods and beverages. Results: The growth pattern observed showed that the diets significantly induced a gain in body weight with alteration of the serum levels of sex hormones. In the females, sugar-sweetened diets inhibited follicle stimulating hormone (FSH) and luteinizing hormone (LH) secretion at high dose but increased the levels of progesterone and estradiol while the consumption of honey-based diets inhibited progesterone and estradiol secretion but stimulated testosterone secretion at a low percentage. In female animals fed with high sugar concentration diet, the serum levels of LH and FSH were observed to be decreased with a decrease in the level of testosterone and an increase in estradiol level. The level of estradiol was found to be significantly higher in sugar-fed animals than the honey-fed groups in the females (p < 0.05). In the male, intake of the sugar-based diet increased the levels of LH, FSH, and testosterone and the consumption of honey at high concentration stimulated FSH and LH secretion while honey at low concentration stimulated testosterone secretion. Conclusion: This study has shown that excess and extended intake of table sugar alters the relationship between gonadotropins (LH, FSH), oestradiol, and testosterone in the female rats more than the Obudu natural honey. This exerts adverse effects on the reproductive function which is graver in females than the males.

AB080. Annexin A5 regulates Leydig cell testosterone production via ERK1/2 pathway

This study was to investigate the effect of annexin A5 on testosterone secretion from primary rat Leydig cells and the underlying mechanisms. Isolated rat Leydig cells were treated with annexin A5. Testosterone production was detected by chemiluminescence assay. The protein and mRNA of steroidogenic acute regulatory (StAR), P450scc, 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD) and 17α-hydroxylase were examined by western blotting and RT-PCR, respectively. Annexin A5 significantly stimulated testosterone secretion from rat Leydig cells in dose- and time-dependent manners and increased mRNA and protein expression of StAR, P450scc, 3β-HSD and 17β-HSD but not 17α-hydroxylase. Annexin A5 knockdown by siRNA significantly decreased the level of testosterone and protein expression of P450scc, 3β-HSD and 17β-HSD. The significant activation of ERK1/2 signaling was observed at 5, 10, and 30 min after annexin A5 treatment. After the pretreatment of Leydig cells with ERK inhibitor PD98059 (50 µmol/L) for 20 min, the effects of annexin A5 on promoting testosterone secretion and increasing the expression of P450scc, 3β-HSD and 17β- HSD were completely abrogated (P<0.05). Thus, ERK1/2 signaling is involved in the roles of annexin A5 in mediating testosterone production and the expression of P450scc, 3β-HSD and 17β-HSD in Leydig cells.

Obestatin Modulates Ghrelin’s Effects on the Basal and Stimulated Testosterone Secretion by the Testis of Rat: an In Vitro Study

The functional antagonism between obestatin and ghrelin in the testis is under investigation. We investigated the ability of obestatin to counteract the inhibitory effect of ghrelin on basal and stimulated testosterone (T) secretion in vitro. Testicular strips from adult rats were incubated with 10 ng/ml and 100 ng/ml of obestatin alone, ghrelin alone and obestatin + ghrelin. Obestatin modulation of stimulated T secretion was evaluated by incubation of testicular samples with 10 ng/ml and 100 ng/ml obestatin, ghrelin and obestatin + ghrelin in the absence and presence of 10 IU of human chorionic gonadotrophin (hCG). T concentrations in the hCG treated groups were significantly (P<0.0001) higher than those in the control groups. Obestatin caused a significant increase in basal T secretion in a dose-dependent manner; however, obestatin at the both 10 ng/ml and 100 ng/ml significantly (P<0.0001) increased hCG-stimulated T secretion. In contrast, ghrelin in a dose-dependent manner significantly (P<0.001) decreased both basal and hCG-induced T secretion by testicular slices. Obestatin opposed the inhibitory effect of ghrelin on T secretion under both basal and hCG-stimulated conditions at all doses tested. In conclusions, administration of obestatin was able to antagonize the inhibitory effect of ghrelin on testosterone secretion in vitro.

Taurine Improves Sexual Function in Streptozotocin-Induced Diabetic Rats.

Previous studies have identified that diabetic erectile dysfunction is associated with androgen and nitric oxide deficiency resulting from hyperglycemia. It has been demonstrated that taurine can stimulate testosterone secretion, increase nitric oxide synthase (NOS) activity and nitric oxide (NO) production, and reduce blood glucose levels in the diabetic animals. Furthermore, recent studies have found that taurine relaxes both the corpus cavernosum and the vasculature. Accordingly, we hypothesized that taurine might exert beneficial effects on erectile function of the diabetic rats. Here, we assessed the effects of taurine on sexual function in streptozotocin (STZ) -induced diabetic male rats. We observed that taurine treatment could markedly increase sexual response and mating ability of STZ-diabetic rats. The serum concentration of gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone (T) were also significantly increased by taurine administration. Importantly, taurine supplementation notably increased mRNA levels and activity of endothelial NOS (eNOS) and neuronal NOS (nNOS), as well as NO and cGMP content, in the corpus cavernosum of the diabetic rats. In conclusion, the present data indicate that taurine can increase sexual function of STZ-induced diabetic male rats mainly by correcting the diabetes, increasing sexual desire, which is implicated in ameliorating the hypothalamic-pituitary-testicular axis function, and by improving penile erection, which requires increased signaling from the penile endothelial- and neuronal-dependent NO-cGMP pathway.

The potential impact of 4-octylphenol on the basal and stimulated testosterone formation by isolated mice Leydig cells

Octylphenol is biodegradation product of alkylphenolethoxylates frequently used in detergents, paints and other industrial applications. This compound is classified as an endocrine disruptor. Recent studies have hypothesized that occupational exposure to octylphenol poses adverse effects on reproductive system of humans and wildlife species. Enzymes involved in the steroid biosynthesis pathway are really sensitive targets for the action of various endocrine-disrupting chemicals. Aim of in vitro study was determined the effect of 4-octylphenol on basal and human chorionic gonadotropin stimulated testosterone formation of ICR mice Leydig cells. On the other hand, was classified potential impact of mentioned endocrine disruptor on Leydig cell viability after 44 h of cultivation. Cell suspension was cultured with addition of 0.04; 0.2; 1.0; 2.5 and 5.0 μg*mL-1 of 4-octylphenol and compared to the control. Hormone quantification from the medium was performed by enzyme-linked immunosorbent assay. Viability of cell suspension was determined by the metabolic activity assay. Unstimulated testosterone production significantly (P˂0.001) increased with 2.5 and 5.0 μg*mL-1 4-octylphenol. Cell viability was also significantly (P˂0.001; P˂0.05) stimulated by 4-octylphenol. Although human chorionic gonadotropin stimulated testosterone secretion was significantly (P˂0.05) affected by the lowest concentration (0.04 μg*mL-1) in the cell viability was recorded significantly (P˂0.001; P˂0.05) higher mitochondrial activity (1.0; 2.5 and 5.0 μg*mL-1). Considerably more detailed and systematic research in this area is required for a better understanding of potential risk to humans or animals.

Environmental chemicals impact dog semen quality in vitro and may be associated with a temporal decline in sperm motility and increased cryptorchidism.

Adverse temporal trends in human semen quality and cryptorchidism in infants have been associated with exposure to environmental chemicals (ECs) during development. Here we report that a population of breeding dogs exhibit a 26 year (1988–2014) decline in sperm quality and a concurrent increased incidence of cryptorchidism in male offspring (1995–2014). A decline in the number of males born relative to the number of females was also observed. ECs, including diethylhexyl phthalate (DEHP) and polychlorinated bisphenol 153 (PCB153), were detected in adult dog testes and commercial dog foods at concentrations reported to perturb reproductive function in other species. Testicular concentrations of DEHP and PCB153 perturbed sperm viability, motility and DNA integrity in vitro but did not affect LH stimulated testosterone secretion from adult testis explants. The direct effects of chemicals on sperm may therefore contribute to the decline in canine semen quality that parallels that reported in the human.

Continuous light after a long-day treatment is equivalent to melatonin implants to stimulate testosterone secretion in Alpine male goats

In rams, artificial long days followed by continuous light stimulate testosterone secretion during the non-breeding season. The objective of this study was to determine whether artificial long days followed by continuous light could stimulate testosterone secretion in Alpine bucks as well as in those exposed to long days followed by a melatonin treatment. All bucks were kept in shaded open pens. Control males were exposed to natural photoperiod conditions (n=5). Males of the two experimental groups were exposed to 2.5 months of long days from 1 December (n=5 each). On 16 February, one group of males was exposed to 24 h of light per day until 30 June; the other group was exposed to natural variations of photoperiod and received two s.c. melatonin implants. Testicular weight was determined every 2 weeks, and the plasma testosterone concentrations once a week. In the control and the two photoperiodic-treated groups, a treatment×time interaction was detected for testicular weight and plasma testosterone concentrations (P<0.001). In control bucks, testicular weight increased from January and peaked in June, whereas in both photoperiodic-treated groups, this variable increased from January, but peaked in April, when the values were higher than in controls (P<0.05). In the control group, plasma testosterone concentrations remained low from January to June, whereas in both photoperiodic-treated groups, this variable remained low from January to March; thereafter, these levels increased in both photoperiodic-treated groups, and were higher than controls in April and May (P<0.05). We conclude that continuous light after a long-day treatment stimulate testosterone secretion in Alpine male goats during the non-breeding season as well as the long days followed by a melatonin treatment. Therefore, continuous light could replace the implants of melatonin.

Ucinok 4-octylphenolu na baz�lnu a stimulovan� produkciu testoster�nu my��ch Leydigov�ch buniek

Octylphenol is biodegradation product of alkylphenolethoxylates frequently used in detergents, paints and other industrial applications. This compound is classified as an endocrine disruptor. Recent studies have hypothesized that occupational exposure to octylphenol poses adverse effects on reproductive system of humans and wildlife species. Enzymes involved in the steroid biosynthesis pathway are really sensitive targets for the action of various endocrine-disrupting chemicals. Aim of in vitro study was determined the effect of 4-octylphenol on basal and human chorionic gonadotropin stimulated testosterone formation of ICR mice Leydig cells. On the other hand, was classified potential impact of mentioned endocrine disruptor on Leydig cell viability after 44 h of cultivation. Cell suspension was cultured with addition of 0.04; 0.2; 1.0; 2.5 and 5.0 μg*mL-1 of 4-octylphenol and compared to the control. Hormone quantification from the medium was performed by enzyme-linked immunosorbent assay. Viability of cell suspension was determined by the metabolic activity assay. Unstimulated testosterone production significantly (P˂0.001) increased with 2.5 and 5.0 μg*mL-1 4-octylphenol. Cell viability was also significantly (P˂0.001; P˂0.05) stimulated by 4-octylphenol. Although human chorionic gonadotropin stimulated testosterone secretion was significantly (P˂0.05) affected by the lowest concentration (0.04 μg*mL-1) in the cell viability was recorded significantly (P˂0.001; P˂0.05) higher mitochondrial activity (1.0; 2.5 and 5.0 μg*mL-1). Considerably more detailed and systematic research in this area is required for a better understanding of potential risk to humans or animals.

Ricinus communis L. stem bark extracts regulate ovarian cell functions and secretory activity and their response to Luteinising hormone.

Ricinus communis L. has ethnopharmacological contraceptive reputation but its stem bark has unexplored mechanisms of action in female reproductive system. In the present study, the effect of methanolic and aqueous extracts from the stem bark of the plant was examined on basic porcine ovarian granulosa cell functions and its response to Luteinising hormone (LH)-the upstream hormonal regulator. Systemic treatment of methanolic and aqueous extracts stimulated cell proliferation (proliferating cell nuclear antigen, PCNA) and also promoted cell apoptosis (caspase-3). Aqueous extract has inverted the stimulatory effect of LH on PCNA but not on caspase-3. Methanolic extract stimulated as well as inhibited progesterone release and stimulated testosterone secretion. Whereas aqueous extract inhibited both steroid releases and suppressed the stimulatory effect of LH on progesterone release and promoted the inhibitory effect of LH on testosterone release. In conclusion, the present study unveils the mechanism of action of R. communis stem bark in in vitro condition. These suggest its possible contraceptive efficacy by exerting its regulatory role over LH and on basic ovarian cell functions and secretion activity.

Annexin A5 regulates Leydig cell testosterone production via ERK1/2 pathway.

This study was to investigate the effect of annexin A5 on testosterone secretion from primary rat Leydig cells and the underlying mechanisms. Isolated rat Leydig cells were treated with annexin A5. Testosterone production was detected by chemiluminescence assay. The protein and mRNA of Steroidogenic acute regulatory (StAR), P450scc, 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), and 17α-hydroxylase were examined by Western blotting and semi-quantitative RT-PCR, respectively. Annexin A5 significantly stimulated testosterone secretion from rat Leydig cells in dose- and time-dependent manners and increased mRNA and protein expression of StAR, P450scc, 3β-HSD, and 17β-HSD but not 17α-hydroxylase. Annexin A5 knockdown by siRNA significantly decreased the level of testosterone and protein expression of P450scc, 3β-HSD, and 17β-HSD. The significant activation of ERK1/2 signaling was observed at 5, 10, and 30 min after annexin A5 treatment. After the pretreatment of Leydig cells with ERK inhibitor PD98059 (50 μmol l−1) for 20 min, the effects of annexin A5 on promoting testosterone secretion and increasing the expression of P450scc, 3β-HSD, and 17β-HSD were completely abrogated (P < 0.05). Thus, ERK1/2 signaling is involved in the roles of annexin A5 in mediating testosterone production and the expression of P450scc, 3β-HSD, and 17β-HSD in Leydig cells.