Testosterone Release Research Articles

Sirtuin 1-mediated autophagy regulates testosterone synthesis in Leydig cells of piglets

Testosterone is secreted by Leydig cells (LCs), which play an important physiological role in preserving male secondary sex characteristics, protecting male reproductive function, and establishing the blood-testis barrier. Studies have shown that autophagy is particularly active in LCs; however, its involvement in testosterone synthesis in porcine LCs has not been fully explored. Therefore, this experiment aimed to investigate the influence of autophagy on testosterone secretion in porcine LCs and its potential regulatory mechanism. Our results demonstrated that both testicular autophagy and serum testosterone levels increased in piglets during postnatal development from 4 to 18 weeks. In addition, autophagy was found to degrade the Na+/H+ exchange regulatory factor 2 (NHERF2), leading to the up-regulation of scavenger receptor class B type 1 (SRB1). This process resulted in increased cholesterol intake and enhanced testosterone production. The observable level of sirtuin 1 (SIRT1) was directly proportional to the level of autophagy. In vitro investigations have shown that SIRT1 can affect the level of autophagy, cholesterol uptake as well as testosterone release. In conclusion, testosterone synthesis during pig development is regulated by SIRT1. SIRT1 mediates the degradation of NHERF2 through autophagy, thereby weakening its negative regulatory effect on the high-density lipoprotein receptor SRB1 in Leydig cells. This process increases cholesterol uptake and enhances testosterone synthesis.

REGULATION OF THE HYPOTHALAMIC-PITUITARY-ADRENAL AXIS BY PHYTOSTEROL: ACTIONS AND MECHANISM OF ACTION

Phytosterols (PS), commonly referred to as plant sterols, are naturally occurring chemicals found in plants that resemble cholesterol in structure but have an additional methyl (campesterol) or ethyl (β-sitosterol) group in the side chain. The present review was based on the regulation of the hypothalamic-pituitary-adrenal axis by phytosterol: actions and mechanism of action. PS have been shown to have endocrine-disrupting properties, and studies on humans, rats, mice, goldfish (Carassius auratus), and Japanese quail have all reported experiencing this effect. Dietary PS has been shown to accumulate in the brain lately. Dietary β-sitosterol has the ability to cross the blood–brain barrier (BBB) and build up permanently in the membranes of brain cells. The stress response, which consists of a complex interplay between elements of the central nervous system (CNS) and peripheral systems such the endocrine, immunological, and cardiovascular systems, is triggered by exposure to environmental, physical, or physiological stressors. Under stressful circumstances, the HPA axis mediates the physiological maintenance of homeostasis. In conclusion, adult male quails fed PS alone had lower testosterone concentrations in their plasma and pituitary without appreciably changing their levels of LH. Furthermore, LH release from the pituitary gland and testosterone release from the testes were both markedly increased by cGnRH-1 stimulation. In contrast to control animals, quails fed PS had lower amounts of testosterone and LH.

Involvement of circular RNAs in the control of porcine ovarian cell functions: Upregulation by ciR-00596 and downregulation by ciR-00646

The current understanding of the role of circular RNAs (circRNAs) in regulating ovarian functions is inadequate. To assess the impact of ciR-00596 and ciR-00646 on the regulation of basic porcine ovarian granulosa cell functions, we conducted upregulation (utilizing overexpressing vectors) and downregulation (utilizing shRNA vectors) of these circRNAs. The relative expression of both circRNAs, cell viability and proliferation (accumulation of PCNA, cyclin B1, and XTT-positive cells), cytoplasmic (accumulation of bax and caspase-3) and nuclear (DNA fragmentation) apoptosis, and the release of progesterone, testosterone, estradiol, IGF-I, and oxytocin were evaluated. Transfection of cells with the ciR-00596 overexpression vector resulted in increases in cell viability and proliferation and the release of progesterone and IGF-I, while it decreased the cytoplasmic and nuclear apoptosis, testosterone, estradiol, and oxytocin output. CiR-00596 inhibition had the opposite effects. The overexpression of ciR-00646 decreased cell viability and proliferation, and the release of progesterone, IGF-I, and oxytocin, while increasing cytoplasmic and nuclear apoptosis and the output of testosterone and estradiol. Our findings are the first to show the stimulatory action of ciR-00596 and the inhibitory effect of ciR-00646 on ovarian cell functions, including the cell cycle, apoptosis, and secretory activity.

Central MOTS-c infusion affects reproductive hormones in obese and non-obese rats

MOTS-c, a mitochondrial-derived peptide, acts as a systemic hormone and MOTS-c level is inversely correlated with markers of obesity. Obesity is a risk factor for male reproductive physiology and is expressed as an important cause of infertility. In this study, we aimed to determine the effects of MOTS-c, which has been proven in the hypothalamus and testicles, on the actors involved in the reproductive axis.In the study, 80 male Wistar-Albino rats were divided into two main groups, obese and non-obese (n = 40). Rats in the first main group were fed with fatty diet feed and obesity was induced. The second main group was fed with normal diet feed. Each main group was divided into 4 subgroups (Control, Sham, 10 and 100 µM MOTS-c). The lateral ventricles of the animals in the treatment groups were infused with 10 and 100 µM MOTS-c (solvent in Sham group) for 14 days. At the end of the experiment, hypothalamic Gonadotropin-Releasing Hormone (GnRH) gene expression level, serum testosterone, Luteinizing hormone (LH) and Follicle stimulating hormone (FSH) levels were determined.MOTS-c infusion caused an increase in GnRH mRNA, protein expression levels and serum testosterone, LH and FSH levels in obese and non-obese rats (p < 0.05). MOTS-c administration more significantly upregulated hormone levels in non-obese rats (p < 0.05). MOTS-c administration increases these hormones, suggesting that MOTS-c may stimulate the reproductive axis.Our results reveal that MOTS-c plays a role in the central regulation of reproduction, as well as causes increased LH, FSH and testosterone release.

Inhibition of vinculin activity has an adverse effect on porcine ovarian cells

The existing knowledge of the involvement of vinculin (VCL) in the control of ovarian cell functions is insufficient. To understand the role of VCL in the control of basic porcine ovarian granulosa cell functions, we decreased VCL activity by small interfering RNA (VCL siRNA). The expression of VCL, accumulation of VCL protein, cell viability, proliferation (accumulation of PCNA and cyclin B1), proportion of proliferative active cells, apoptosis (accumulation of bax, caspase 3, p53, antiapoptotic marker bcl2, and bax/bcl-2 ratio), DNA fragmentation, and release of steroid hormones and IGF-I were analyzed by RT‒qPCR, Trypan blue exclusion test, quantitative immunocytochemistry, XTT assay, TUNEL assay, and ELISA. The suppression of VCL activity inhibited cell viability, the accumulation of the proliferation-related proteins PCNA and cyclin B1, the antiapoptotic protein bcl2, and the proportion of proliferative active cells. Moreover, VCL siRNA inhibited the release of progesterone, estradiol, and IGF-1. VCL siRNA increased the proportion of the proapoptotic proteins bax, caspase 3, p53, the proportion of DNA fragmented cells, and stimulated testosterone release. Taken together, the present study is the first evidence that inhibition of VCL suppresses porcine granulosa cell functions. Moreover, the results suggest that VCL can be a potent physiological stimulator of ovarian functions.

Sodium arsenite exposure enhances H3K14 acetylation and impairs male spermatogenesis in rat testes

Histone modifications play important roles in the epigenetic regulation of spermatogenesis via mediating gene transcription. Steroidogenic regulatory enzymes control testosterone biosynthesis, which are essential for spermatogenesis. Arsenic exposure inhibits the expression of steroidogenic genes by significantly increasing tri-methylation of H3K9 (H3K9me3) level in rat testis, finally diminishes testosterone release and lowers the rat sperm quality. Acetylation of H3K14 (H3K14ac) is associated with testosterone production and spermatogenesis. Co-occurrence of H3K9me3/H3K14ac has been identified previously by mass spectrometry in histone H3 isolated from different human cell types. H3K9me3/H3K14ac dually marked regions are in a poised inactive state to inhibit the gene expression. Whereas, whether inorganic arsenic exposure affects spermatogenesis and steroidogenic regulatory enzymes via mediating H3K14ac level has not been studied. Thereupon, the male Sprague-Dawley (SD) rats were exposed to (NaAsO2) for 6 weeks, then the sperm density and motility, testosterone level in serum, arsenic in rat testis were detected. mRNA expression of steroidogenic regulatory enzymes Star, Cyp11a1, Hsd3b and Hsd17b were determined by RT-PCR. H3K14ac level and the expression of histone acetylases of H3K14 (KAT2A and EP300), histone deacetylases of H3K14 (HDAC6 and HDAC3), the reader of H3K14ac (BAZ2A) were determined. The results suggested arsenic enhances H3K14ac in rat testis, which was associated with repression of steroidogenic regulatory genes expression, further reduced testosterone production, and impaired the spermatogenesis.

CircEpha5 regulates the synthesis and secretion of androgen in mouse preantral follicles by targeting miR-758-5p

Circular RNAs are involved in the pathogenesis of various diseases, although its expression pattern and role in polycystic ovary syndrome (PCOS), characterised by hyperandrogenism, are not very clear. This article assessed the circRNAs expression profile in the ovaries of PCOS mice by circRNAs high-throughput sequencing and explored the role of circEpha5 in hyperandrogenism. The results showed that the overexpression of circEpha5 in mouse preantral follicles could increase the expression of Cyp17a1, an androgen synthesis-related gene, which resulted in a higher serum level of testosterone. Dual-luciferase reporter gene studies identified miR-758-5p as a direct target of circEpha5. Consequently, miR-758-5p expression was downregulated upon circEpha5 overexpression. Ectopically expressed miR-758-5p reversed the stimulation effects of circEpha5 on steroidogenesis-related gene expression and testosterone release. Therefore, circEpha5 could sponge miR-758-5p to regulate the expression of Cyp17a1, thereby promoting the synthesis and secretion of androgen in the preantral follicles. This work is contributed to the understanding of the pathogenesis of hyperandrogenemia and lays the foundation for the development of therapeutic targets of PCOS hyperandrogenism.

(101) Ovarian Hyperthecosis as a Cause of Hypersexualization in a Post-menopausal Female

Abstract Introduction Ovarian Hyperthecosis (OH) is a rare benign virilizing ovarian syndrome characterized by androgen overproduction due to stromal and thecal hyperplasia resulting in hirsutism and virilization(1). It is believed to be similar to polycystic ovarian syndrome (PCOS) however, presents with more robust androgen overproduction and an ovarian vein ratio of androstenedione/testosterone less than one, compared with values around 20 in pre-menopausal females and patients with PCOS(2). Specifically in post-menopausal women, the phenotypical presentation is due to unmasking of the increased thecal cell testosterone release which results from lack of granulosa cell mediated aromatization of testosterone to estradiol.(3) Some have described treatment of OH with medications that inhibit ovarian production of estrogen and progesterone especially in pre-menopausal women. However, results are mixed and oophrectomy seems to be the most effective treatment modality for OH(3). Objective Here we describe a case of OH resulting in elevated androgen levels and distressing hypersexualization in a post-menopausal female who was effectively treated with bilateral oophorectomy resulting in normalization of laboratory values and resolution of hypersexualization. Methods The patient is a 75-year-old female with past medical history significant for bipolar disorder, Sjogren’s syndrome and hypothyroidism who presented to the office for evaluation of recurrent vulvovaginitis and urinary tract infections related to vigorous and frequent sexual activity. She reported symptoms of hypersexuality developing a few years prior to presentation at which time she began engaging in sexual activity 2-3 times daily and masturbating with a variety of objects. She had also noted new heavy facial hair and scalp hair loss. There were no changes in her medical history at the time. She was bothered by the increased need for sexual stimulation and orgasm, prompting further evaluation and treatment. Her exam was notable for vulvar contact dermatitis, mild vulvovaginal atrophy, and no prolapse or urinary incontinence. Vaginal infection testing was negative. Results of an endocrine work up are summarized in Table 1. Abdominal imaging showed normal adrenal glands and ovaries. An MRI of the brain showed a normal pituitary gland. She had been started on Prozac by her psychiatrist, which reduced her libido but worsened her mania and therefore was discontinued. A diagnosis of OH was suspected and bilateral oophorectomy was planned. Results The patient underwent a laparoscopic bilateral salpingo-ophrectomy with surgical pathology confirming stromal hyperplasia and hyperthecosis. Post-operatively, her labs rapidly normalized. She noted normalization of her libido, significant improvement in vulvar contact dermatitis and increased scalp hair growth. Conclusions Bilateral oophorectomy in a post-menopausal woman with OH was an effective treatment to normalize androgen levels and restore normal libido resulting in resolution of vulvar contact dermatitis from excessive masturbation. Although an uncommon diagnosis, this disease is an important consideration in the workup of excess androgen production in post-menopausal women. Disclosure No

Rooibos (Aspalathus linearis) and its constituent quercetin can suppress ovarian cell functions and their response to FSH

Rooibos (Aspalathus linearis Brum. f) can directly influence female reproduction, but whether rooibos can influence the response of ovarian cells to FSH and whether the rooibos effects are due to the presence of quercetin remain unknown. We compared the influence of rooibos extract and quercetin (both at 10 µg/ml-1) on porcine ovarian granulosa cells cultured with and without FSH (0, 1, 10 or 100 ng/ml(-1)). The expression of intracellular proliferation (PCNA, cyclin B1) and apoptosis (bax, caspase 3) markers in the cells was detected by immunocytochemistry. The release of progesterone (P), testosterone (T) and estradiol (E) were evaluated with ELISAs. Administration of both rooibos and quercetin reduced the accumulation of proliferation markers and promoted the accumulation of apoptosis markers and the release of T and E. Rooibos stimulated, but quercetin inhibited, P output. Administration of FSH increased the accumulation of proliferation markers, decreased the accumulation of apoptosis markers, promoted the release of P and T, and had a biphasic effect on E output. The addition of both rooibos and quercetin mitigated or prevented the main effects of FSH. The present observations suggest a direct influence of both rooibos and quercetin on basic ovarian functions – proliferation, apoptosis, steroidogenesis and response to FSH. The similarity in the major effects of rooibos and its constituent quercetin indicates that quercetin could be the molecule responsible for the main rooibos effects on the ovary. The potential anti-reproductive effects of rooibos and rooibos constituent quercetin, should be taken into account in animal and human nutrition.

Chia (Salvia hispanica L.) can directly suppress basic ovarian cell functions in two farm animal species and protect ovarian cells from the proliferation-stimulating influence of xylene.

The influence of the functional food plant chia (Salvia hispanica L.) on reproduction functions and its ability to prevent the negative effects of environmental contaminants has not yet been studied. Our study aimed to examine the effect of chia seed extract alone and in combination with xylene on the markers of proliferation, apoptosis and hormones release by cultured bovine and porcine ovarian granulosa cells. The extract of chia reduced all of the measured parameters in bovine and porcine ovarian cells but had no effect on the proliferation of porcine cells. Xylene, stimulated proliferation and IGF-I release and inhibited the release of progesterone and testosterone but not apoptosis of bovine granulosa cells. It promoted proliferation, apoptosis and progesterone output by porcine cells. Chia mitigated the stimulatory effect of xylene on proliferation but not on other parameters in both species. The present results are the first demonstration of a direct effect of chia on basic ovarian cell functions. They confirmed a direct influence of xylene on these functions and found a similar stimulatory action of xylene on bovine and porcine ovarian cell proliferation. The present observations demonstrated species-specific differences in the characteristics of xylene influences on ovarian cell apoptosis and secretory activity. Finally, the present results indicate that chia can be a natural protector against the proliferation-stimulating effects of xylene on ovarian cells in both species.

Testosterone and cortisol responses to acute and prolonged stress during officer training school

Prolonged or severe stress has been found to inhibit the hypothalamic-pituitary-gonadal axis (HPG) and its testosterone release. In contrast, acute stress, including competition, social evaluation, or physical challenges, shows more inconsistent response patterns. This study examined changes in cortisol and testosterone across different types and durations of stress in the same individuals. We further explored the influence of baseline levels on hormonal stress responses. Sixty-seven male officer cadets in the Swiss Armed Forces (mean age 20.46 years ± 1.33) were assessed during two different acute stressors—the Trier Social Stress Test for Groups (TSST-G) and a brief military field exercise—and in the long-term during the 15-week officer training school. Several saliva samples were collected before and after the acute stressors for cortisol and testosterone. Morning testosterone was assessed four times during officer training school. There were significant increases in cortisol and testosterone during the TSST-G and the field exercise. Baseline levels of testosterone were negatively associated with acute cortisol response during the field exercise but not during the TSST-G. Morning saliva testosterone decreased during the first 12 weeks of officer training school and increased again in week 15, with no differences to baseline levels. The findings suggest that group stress tests such as the TSST-G or field exercises in groups may be particularly challenging for young men. The results also point to an adaptive role of testosterone during acute challenges during prolonged stress.

Quercetin can affect porcine ovarian cell functions and to mitigate some of the effects of the environmental contaminant toluene

The present in vitro experiments aimed to examine the effects of the plant polyphenol quercetin and the environmental contaminant toluene on basic ovarian cell functions, including the ability of quercetin to be a natural protector against the adverse effects of toluene. The influence of toluene, quercetin, and their combination on proliferation (accumulation of PCNA), apoptosis (accumulation of bax) and release of progesterone, testosterone and insulin-like growth factor I (IGFI) by cultured porcine ovarian granulosa cells was investigated. Toluene stimulated cell proliferation and inhibited progesterone, IGF-I and testosterone release but did not affect apoptosis. Quercetin, when administered alone, inhibited cell proliferation, apoptosis, IGF-I and testosterone release and stimulated progesterone output. When administered in combination with toluene, quercetin mitigated toluene's effects on proliferation and on progesterone release and induced toluene to exhibit a pro-apoptotic effect. These observations demonstrate the direct effects of both quercetin and toluene on basic ovarian functions and a protective effect of quercetin against the effects of toluene. Therefore, quercetin-containing plants could be regulators of porcine reproduction and natural protectors against the adverse effects of the environmental contaminant toluene.

Effect of High-Fructose Diet-Induced Metabolic Syndrome on the Pituitary-Gonadal Axis in Male Rats.

Plasma testosterone levels have been found to decrease in older insulin-resistant male patients. Both lower total testosterone levels and a higher incidence of metabolic syndrome have also been reported. The aim of this study was to investigate the effects of high-fructose diet-induced diabetes on both the testosterone release by Leydig cells and the activity of the hypothalamus-pituitary-gonadal (HPG) axis in male rats. Male rats were fed with either standard chow (control group) or a high-fructose diet (fructose-fed group) for 21 weeks. Hyperglycemia, hyperinsulinemia, and hypertension were observed in the fructose-fed group. Moreover, plasma testosterone and LH levels decreased in the fructose-fed group compared to the control group. Sperm motility was also reduced by 15% in the fructose-fed rats. In contrast, the basal release of testosterone from rat Leydig cells was not altered by fructose feeding. Moreover, in vitro studies showed that the testosterone release, in response to different stimulants, including forskolin (an adenylyl cyclase activator, 10-5 M), 8-Br-cAMP (a permeable analog of cAMP, 10-5 M), A23187 (a calcium ionophore, 10-5 M), or 25-hydroxy-cholesterol (water-soluble cholesterol, 10-5 M), did not significantly differ between the fructose-fed and control groups. Interestingly, the release of testosterone in response to human chorionic gonadotropin (hCG, 0.05 IU/mL) was enhanced by eightfold in the control group, but elevenfold in the fructose-fed group. LH receptor expression in rat Leydig cells was also increased. Moreover, LH secretion from the anterior pituitary was altered in the fructose diet-fed group. These results suggest that fructose diet-fed rats have lower plasma testosterone levels, which can lead to a higher sensitivity of hCG in Leydig cells.

Re-stimulation of testicular function in GnRH-vaccinated stallions by daily GnRH agonist treatment

In stallions temporarily not intended for breeding, reversible suppression of testicular function by vaccination against GnRH can be of interest. In the present study, effects of GnRH agonist treatment on the resumption of testicular function after GnRH vaccination were investigated. Testis size, testosterone release, semen characteristics and behavior were evaluated. We hypothesized that GnRH agonist treatment would restore testicular function. Shetland stallions were assigned to an experimental and a control group (n = 6 each). Experimental stallions were GnRH-immunized twice, four weeks apart. Ejaculates for semen analysis and blood for analysis of testosterone concentration and GnRH antibody titers were collected. Each experimental stallion was hemicastrated together with an age-matched control animal when testosterone concentration decreased below 0.3 ng/mL. Three weeks thereafter, daily treatment with the GnRH agonist buserelin was initiated (4 μg/day for 4 weeks followed by 8 μg/day). The remaining testicle was removed when testosterone concentration exceeded 0.5 ng/mL in vaccinated stallions. Time from exposure to a mare until mounting increased in GnRH-vaccinated stallions and decreased with buserelin treatment. Total sperm count decreased after vaccination but increased only slightly in response to buserelin. Sperm motility and percentage of membrane-intact spermatozoa decreased after vaccination and returned to pre-vaccination values with buserelin treatment. Testosterone concentration and testis volume decreased after GnRH vaccination and started to increase with buserelin treatment. In conclusion, the downregulation of testicular function by GnRH vaccination can be counteracted with buserelin. This approach may be useful in GnRH-vaccinated stallions with prolonged suppression of testicular function.

P-791 In vitro production of mouse sperm on artificial testis engineered by 3D printing of extracellular matrix

Abstract Study question Are testicular extracellular matrix (T-ECM)-based scaffolds fully capable of supporting mouse in vitro spermatogenesis? Summary answer In the future, 3D printing using T-ECM could be used to provide mature sperm for fertility restoration in new infertility treatments. What is known already Since autotransplantation of spermatogonial stem cells (SSCs) obtained from malignant samples is more likely to result in cancer recurrence, in vitro sperm production is considered to be a safer method of fertility preservation. Given the difficulty of evaluating the process of SSCs differentiation in organ culture and the fact that cell culture on a two-dimensional substrate cannot create niche-like conditions for SSCs, the use of T-ECM-based 3D substrates was considered with the goal of preserving the natural morphology of cells in vivo. Study design, size, duration In this study, the effects of hybrid (alginate-gelatin) and composite (T-ECM-alginate-gelatin) printed scaffolds on the proliferation (two weeks) and differentiation (three weeks) of murine SSCs were investigated at the molecular, cellular, morphological, and functional levels. Participants/materials, setting, methods A hypertonic solution-triton was used to decellularize testicular tissue. The extracted ECM was employed as a bio-ink to print in combination with alginate-gelatin. Pre-meiotic and post-meiotic cells in the studied groups (as testicular suspension and co-culture with Sertoli cells) were validated by real-time PCR, flow cytometry, and immunocytochemistry. Morphology of cells was evaluated using TEM, H&amp;E, toluidine blue, and Giemsa staining. The ability of Leydig and Sertoli cells to secrete hormones determined their functionality. Main results and the role of chance The results demonstrated that decellularization of testicular tissue fragments was successful, with cellular debris effectively eliminated while ECM components were retained. High cell viability, colonization, and enhanced expression of Plzf, Id4, and Gfra1 pre-meiotic markers in cultured testicular cells on ECM-enriched scaffolds were used to determine the effects of proliferation (group 4) (P ≤ 0.05). Also, after 3 weeks of culture in the differentiation medium, cell viability and expression of Sycp3, Acr and Prm1 markers in group 4 were higher than in other groups (P ≤ 0.05). The structure of these cells in group 4 was remarkably comparable to mature sperm with a specialized tail structure, according to morphological analysis. The release of testosterone and inhibin B by Leydig and Sertoli cells was also verified by the hormonal assay. Limitations, reasons for caution We were unable to assess epigenetic alterations and the fertility of mature sperm obtained in this system due to the delay in preparing the material for culture, the long set-up time, the fabrication of hydrogel scaffolds using the 3D printing approach, and the long-term culture in the laboratory. Wider implications of the findings This research demonstrated the rapid generation of morphologically normal sperm in the lab and the simple extraction of mature sperm from a T-ECM-based printing system, paving the path for new infertility and sperm production treatments in azoospermic patients and cancer survivors. Trial registration number N/A

Hydrogen Peroxide Stimulates Dihydrotestosterone Release in C2C12 Myotubes: A New Perspective for Exercise-Related Muscle Steroidogenesis?

Skeletal muscle is a tissue that has recently been recognized for its ability to produce androgens under physiological conditions. The steroidogenesis process is known to be negatively influenced by reactive oxygen species (ROS) in reproductive Leydig and ovary cells, while their effect on muscle steroidogenesis is still an unexplored field. Muscle cells are continuously exposed to ROS, resulting from both their metabolic activity and the surrounding environment. Interestingly, the regulation of signaling pathways, induced by mild ROS levels, plays an important role in muscle fiber adaptation to exercise, in a process that also elicits a significant modulation in the hormonal response. The aim of the present study was to investigate whether ROS could influence steroidogenesis in skeletal muscle cells by evaluating the release of testosterone (T) and dihydrotestosterone (DHT), as well as the evaluation of the relative expression of the key steroidogenic enzymes 5α-reductase, 3β-hydroxysteroid dehydrogenase (HSD), 17β-HSD, and aromatase. C2C12 mouse myotubes were exposed to a non-cytotoxic concentration of hydrogen peroxide (H2O2), a condition intended to reproduce, in vitro, one of the main stimuli linked to the process of homeostasis and adaptation induced by exercise in skeletal muscle. Moreover, the influence of tadalafil (TAD), a phosphodiesterase 5 inhibitor (PDE5i) originally used to treat erectile dysfunction but often misused among athletes as a “performance-enhancing” drug, was evaluated in a single treatment or in combination with H2O2. Our data showed that a mild hydrogen peroxide exposure induced the release of DHT, but not T, and modulated the expression of the enzymes involved in steroidogenesis, while TAD treatment significantly reduced the H2O2-induced DHT release. This study adds a new piece of information about the adaptive skeletal muscle cell response to an oxidative environment, revealing that hydrogen peroxide plays an important role in activating muscle steroidogenesis.

Potential protective effect of puncturevine (Tribulus terrestris, L.) against xylene toxicity on bovine ovarian cell functions.

The action of the medicinal plant Tribulus terrestris (TT) on bovine ovarian cell functions, as well as the protective potential of TT against xylene (X) action, remain unknown. The aim of the present in vitro study was to elucidate the influence of TT, X and their combination on basic bovine ovarian cell functions. For this purpose, we examined the effect of TT (at doses of 0, 1, 10, and 100 ng/mL), X (at 20 ?g/mL) and the combination of TT + X (at these doses) on proliferation, apoptosis and hormone release by cultured bovine ovarian granulosa cells. Markers of proliferation (accumulation of PCNA), apoptosis (accumulation of Bax) and the release of hormones (progesterone, testosterone and insulin-like growth factor I, IGF-I) were analyzed by quantitative immunocytochemistry and RIA, respectively. TT addition was able to stimulate proliferation and testosterone release and inhibit apoptosis and progesterone output. The addition of X alone stimulated proliferation, apoptosis and IGF-I release and inhibited progesterone and testosterone release by ovarian cells. TT was able to modify X effects: it prevented the antiproliferative effect of X, induced the proapoptotic action of X, and promoted X action on progesterone but not testosterone or IGF-I release. Taken together, our observations represent the first demonstration that TT can be a promoter of ovarian cell functions (a stimulator of proliferation and a suppressor of apoptosis) and a regulator of ovarian steroidogenesis. X can increase ovarian cell proliferation and IGF-I release and inhibit ovarian steroidogenesis. These effects could explain its anti-reproductive and cancer actions. The ability of TT to modify X action on proliferation and apoptosis indicates that TT might be a natural protector against some ovarian cell disorders associated with X action on proliferation and apoptosis, but it can also promote its adverse effects on progesterone release.

Testosterone Sustained Release Microspheres for the Treatment of Fecal Incontinence

The objective of this study was to develop a testosterone sustained release formulation for the treatment of fecal incontinence. To suppress the initial burst release of testosterone, which can lead to systemic toxicity, we incorporated a washing step using an aminoalkyl methacrylate copolymer E solution or propylene glycol solution into a typical o/w emulsion method to prepare polylactic-co-glycolic acid microspheres. We used this method to develop a polylactic-co-glycolic acid microsphere formulation that shows sustained release of testosterone for up to one month. Not only did this formulation show a sustained release profile after administration into the intersphincteric groove in minipigs, it also increased both the anal pressure and mass of the external anal sphincter, while keeping systemic testosterone exposure low. Thus, this formulation successfully affected both the internal and external anal sphincters with a sufficient safety profile, deeming it a promising candidate treatment strategy for fecal incontinence.

Leptin secreted from testicular microenvironment modulates hedgehog signaling to augment the endogenous function of Leydig cells

Although testosterone deficiency (TD) may be present in one out of five men 40 years or older, the factors responsible for TD remain largely unknown. Leydig stem cells (LSCs) differentiate into adult Leydig cells (ALC) and produce testosterone in the testes under the pulsatile control of luteinizing hormone (LH) from the pituitary gland. However, recent studies have suggested that the testicular microenvironment (TME), which is comprised of Sertoli and peritubular myoid cells (PMC), plays an instrumental role in LSC differentiation and testosterone production under the regulation of the desert hedgehog signaling pathway (DHH). It was hypothesized that the TME releases paracrine factors to modulate LSC differentiation. For this purpose, cells (Sertoli, PMCs, LSCs, and ALCs) were extracted from men undergoing testis biopsies for sperm retrieval and were evaluated for the paracrine factors in the presence or absence of the TME (Sertoli and PMC). The results demonstrated that TME secretes leptin, which induces LSC differentiation and increases testosterone production. Leptin’s effects on LSC differentiation and testosterone production, however, are inversely concentration-dependent: positive at low doses and negative at higher doses. Mechanistically, leptin binds to the leptin receptor on LSCs and induces DHH signaling to modulate LSC differentiation. Leptin-DHH regulation functions unidirectionally insofar as DHH gain or loss of function has no effect on leptin levels. Taken together, these findings identify leptin as a key paracrine factor released by cells within the TME that modulates LSC differentiation and testosterone release from mature Leydig cells, a finding with important clinical implications for TD.

Preparation and In Vitro and In Vivo Evaluation of a Testosterone Film Forming Gel for the Treatment of Hypoactive Sexual Desire Disorder in Women.

Hypoactive sexual desire disorder (HSDD) is one of the most common sexual complaints in women. Currently, there is an unmet need for a drug treatment for this disorder. The purpose of this study was to develop a testosterone (TS) film forming gel used for women to treat HSDD by measuring the tackiness, peel adhesion force, tensile strength, and elasticity of the formulation. Diethylene glycol monoethyl ether (Transcutol P), an efficient penetration enhancer, was added to the optimized formulation and the transdermal permeation characteristics in vitro were studied using Franz-diffusion cells. The quantitative determination of TS was performed by high-performance liquid chromatography (HPLC). After 24 h, Transcutol P at 3% had the largest cumulative amount of drug and enhancement ratio of TS of 75.14 μg/cm2 and 2.82, respectively. After the screening of film forming polymers and penetration enhancers, the optimal formulation was as follows: glycerol (1%, w/w); 12.5% sodium carboxymethylcellulose (CMC-Na) aqueous solution (0.5%, w/w); 2.5% Carbomer ethanol solution (0.5%, w/w); Transcutol P ethanol solution (3%, w/w) containing 0.5% TS; and 8% Poly vinyl alcohol (PVA) aqueous solution (30%, w/w). The optimized film forming gel had good uniformity and the release of TS in vitro was close to 100% within 24 h. In vivo studies showed the formulations had optimal area under blood drug concentration curve values in the order of 3% Transcutol P > 1% Transcutol P > 5% Transcutol P > control preparation. The formulation with 3% Transcutol P provided the highest permeation effect both in vitro and in vivo. The safety of this formulation was further evaluated with a skin irritation test. It could effectively improve the rabbit skin irritation observed with a marketed transdermal patch Androderm®. The present study provides a promising approach for the development of a novel TS film forming gel for the treatment of HSDD in women.