Reduced Serum Testosterone Levels Research Articles

The protective effect of Ozone on the mice testicular damage induced by methotrexate.

Methotrexate (MTX) is widely administered for the treatment of various cancers. However, MTX induces male reproductive toxicity. In the current study, the effect of ozone therapy (OT) on reducing the toxic effects of MTX in the mouse testicles has been investigated. Twenty-four mice were divided into four groups: control, OT (4 mg/kg ozone), MTX (20 mg/kg), and MTX + OT. Testosterone levels, histological changes, and oxidative stress biomarkers were assessed to evaluate the protective effects of OT. The results demonstrated that MTX disrupted germinal epithelium, reduced serum testosterone levels, and enhanced oxidative stress in testicular tissue. However, treatment with OT attenuated these adverse effects. OT effectively restored the levels of antioxidant enzymes, such as catalase (CAT), glutathione (GSH), and superoxide dismutase (SOD). OT reduced lipid peroxidation, as indicated by decreased malondialdehyde (MDA) levels. OT preserved normal spermatogenesis, improved morphometric parameters, and reduced histological changes by MTX. Moreover, OT effectively restored testosterone levels. OT protects against MTX-induced testicular damage by suppressing oxidative stress.

EFFECT OF AFLATOXIN B1 ON THE HAEMATOLOGY, TESTES, AND LUNGS OF ADULT MALE RATS: A PHYSIOLOGICAL AND HISTOLOGICAL STUDY

Background: Aflatoxins are food-contaminating metabolites that can intoxicate and damage multiple organs. This study compared the effects of aflatoxin powder (AFB) and aflatoxin liquid (AFS) administrations on the testes and lungs of rats and elaborated their potential activity mechanisms.Methods: Fifteen rats were equally divided into 3 groups. The AFB and AFS groups were orally administered with aflatoxin B1 powder (0.5 mg/kg) and aflatoxin B1 liquid (0.5 mg/kg) for 21 days. The control group was fed on a basal pellet diet and water. The body weights of the rats were measured at the beginning of the trial and on the 7th, 14th, and 21st days. Blood samples were withdrawn at the end of the experiments and examined for complete blood count (CBC) [red blood cells (RBCs), hemoglobin content, mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), white blood cells (WBCs), and platelet, neutrophil, monocyte, lymphocyte, basophil, and eosinophil counts]. The levels of oxidative stress biomarkers, e.g. malonaldehyde (MDA) and nitric oxide (NO), and serum testosterone were determined. Testes and lungs were examined under a light microscope to assess the aflatoxin B1-induced histopathological changes.Results: The weight gain and its percentages were significantly high in the AFS group than AFB group (P <0.050). Neutrophil, lymphocyte, monocyte, and basophil counts were also significantly increased in AFB and AFS groups in comparison to the controls. A significant increase in eosinophil count was noted in the AFB group as compared to controls whereas lymphocyte count remained significantly high in the AFS group than AFB group. Testosterone serum levels significantly decreased in both groups (AFB and AFS) as compared to the control group. MDA and NO serum levels were also significantly high in the AFS group than the AFB group whereas the levels of both groups (AFB and AFS) were higher than in controls. Histological examinations of the testes revealed severe testicular damage, loss of the spermatogenesis process, necrosis, degeneration, shrinkage, and atrophy of seminiferous tubules in both AFB and AFS groups. Histological observations of the lungs depicted large numbers of inflammatory cells, mast cell infiltration, dilated alveolar sacs with exudates, and marked hemorrhages in both groups (AFB and AFS). The histological damages of the testes and lungs were more prominent in the AFS group than AFB group.Conclusion: The results demonstrated elevated oxidative stress markers and reduced serum testosterone levels in AFB and AFS-treated rats. Both studied organs (testes and lung) were severely damaged with more obvious changes in AFS treated group than in AFB treated group.

The protective effect of crocin against testicular toxicity induced by ionizing radiation via AKT/FOXO pathway.

Crocin, a pharmacologically active component of Crocus sativus L. (saffron), has been informed to be beneficial in the treatment of stress-related oxidative impairment. In the present study, we examined the protective role of crocin against testicular damage induced by radiation (acute and fractionated) and the alteration of the AKT/FOXO signaling pathway. Male Wister albino rats were exposed to acute dose of 6 Gy and a fractionated dose of gamma radiation (2 Gy every 2 days up to 6 Gy total doses). Rats were pretreated intraperitoneally with crocin in a dose of 50 mg/kg for seven consecutive days prior to exposure to irradiation at a level of 6 Gy and during the fractionated irradiation of rats. Control groups were run concurrently. Ionizing radiation caused changes in the level of oxidative stress biomarkers manifested as elevation of thiobarbituric acid reactive substance, total nitrate/nitrite and reactive oxygen species (ROS) associated with a decrease in catalase as well as in the level of inflammatory parameters (decrease in expression of Nrf2 which was related to a significant increase in expression of NF-κB p65). Irradiation produced cellular damage characterized by an increase in serum lactate dehydrogenase. These findings were aligned with increased expression of the forkhead box O-1 (FOXO-1) and activation of protein kinase B (AKT) pathway. Irradiation of rats led to reduction in serum testosterone level and testicular weights. Pretreatment with the indicated dose of crocin shielded against the changes in all the evaluated parameters. Administration of crocin can be introduced as a novel preclinical approach for regulation of testicular damage induced by radiation; via controlling the ongoing oxidative stress and inflammatory reaction as well as activation FOXO/AKT signaling pathway.

A mixture of glyphosate and 2,4-D herbicides enhances the deleterious reproductive outcomes induced by Western diet in obese male mice.

The consumption of Western diet (WD) - enriched in fats and sugars - is associated with overweight, obesity and male reproductive disorders. In addition to WD intake, crops and dairy products display residues of herbicides, including glyphosate and 2,4-D that are widely applied worldwide. The concomitant exposure to WD and herbicides - mimicking contemporary scenarios - is not fully investigated. Thus, we evaluated the effects of glyphosate and 2,4-D, alone or in mixture, on WD-induced alterations in the male genital system. Male C57BL6J mice were submitted to WD (chow containing 20% lard, 0.2% cholesterol, 20% sucrose, and high sugar solution with 23.1 and 18.9 g/L of D-fructose and D-glucose) for 6 months. Concomitantly to WD, the animals received glyphosate (0.05, 5, or 50 mg/kg/day), 2,4-D (0.02, 2 or 20 mg/kg/day) or their mixture (0, 05 + 0.02, 5 + 2, or 50 + 20 mg/kg/day) by intragastrical administration (5×/week). Doses were based on Acceptable Daily Intake (ADIs) or No Observed Adverse Effect Level (NOAEL) values. Herbicide exposure did not alter the WD-induced obesity, hypercholesterolemia and hyperglycemia. WD induced sperm cell abnormalities, reduced the number, volume and area of Leydig cells, enhanced the frequency of epididymal abnormalities, decreased the proliferation in both germinal and epididymal epithelia, and reduced the number of androgen receptor (AR) positive epididymal cells. Remarkably, the herbicide mixtures promoted such WD-induced effects: increased the frequency of sperm cell and epididymal abnormalities (absence of sperm, cytoplasmic vacuoles, and clear cell hypertrophy) (5 + 2 and 50 + 20 doses); decreased Leydig cell nuclei volume and area (5 + 2 and 50 + 20 doses), reduced epididymal cell proliferation (all mixtures), and AR expression (50 + 20 dose). In addition, herbicide mixtures reduced serum testosterone levels (5 + 2 and 50 + 20 doses). Our findings indicate that the mixture of glyphosate and 2,4-D herbicides, mimicking environmentally relevant scenarios, promotes WD-induced changes in the male genital system.

Obesity May Impairs Mice Testicular Structure by STAT3/CYP19A1/Testosterone

Obesity is an increasing serious global public health issue and a risk factor for male infertility. Aromatase expression level is elevated in obese men, and this high level is as-sociated with diminished serum testosterone levels. We explored the underlying mechanism of infertility in obese men. Genome-wide association study datasets were used to predict the causal relationship between obesity and male infertility. An obese male mouse model was generated by feeding mice a high-fat diet while control model was generated by feeding mice a normal chow diet. Serum indicators including free fatty acids, insulin, Follicle-stimulating hormone, and testosterone were measured using ELISA, and impaired fertility in mice was demonstrated using immunohistochemistry. The expression levels of STAT3 and CYP19A1 were measured by RT-qPCR and western blotting, respectively. Simultaneous bioinformatics analysis of publicly available data was performed using the R programming language and JASPAR database. Obesity might lead to male infertility from a genomic perspective. Obese mice displayed reduced serum testosterone and follicle stimulating hormone levels along with structural abnormalities, ectopic lipid deposition, and chronic inflammation in the testes. Upregulation of STAT3/CYP19A1 expression in obese mice was linked to reduced serum testosterone levels and impaired fertility. Our observation may support the hypothesis that since the elevation of STAT3 expression in obesity, the promoted expressed CYP19A1 could increase a shift from testosterone to estrogens. The persistently elevated level of estrogen will further stimulate STAT3, which may constitute a positive feedback loop. These findings provide new insights into the mechanisms of obesity-mediated male fertility impairment.

Leydig cell development in pubertal male rats is blocked by perfluorotetradecanoic acid through decreasing AMPK-mTOR-autophagy pathway

Perfluorotetradecanoic acid (PFTeDA) is a type of perfluoroalkyl acid that has been linked to various health effects in animals and humans. The study aimed to investigate the potential impact of PFTeDA exposure on Leydig cell development in rats during puberty. Understanding the effects of PFTeDA on Leydig cells is crucial as these cells play a significant role in male reproductive function. Male Sprague-Dawley rats were gavaged with PFTeDA at doses of 0, 1, 5, and 10 mg/kg/day from postnatal day 35–56. The serum hormone levels were measured and testicular transcriptome changes were analyzed by RNA-seq and verified by qPCR, and the levels of steroidogenesis-related proteins and energy regulators were measured. PFTeDA significantly reduced serum testosterone levels while slightly increasing LH levels. RNA-seq and qPCR analysis showed that genes responsive to oxidative phosphorylation (Naufa1 and Ndufs6) and steroidogenesis (Ldlr, Star, Cyp11a1) were markedly downregulated at ≥ 5 mg/kg, while those related to ferroptosis (Alox15) and cell senescence (Map2k3 and RT1-CE3) were significantly upregulated. PFTeDA markedly reduced SIRT1 (silent information regulator 1) /PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1α) and AMPKA (AMP activated kinase A), LC3B and Beclin1 (biomarkers for autophagy) levels while increasing phosphorylated mTOR. In vitro treatment of PFTeDA at 5 μM significantly reduced androgen output of Leydig cells from 35-day-old male rats while ferrostatin 1 (10 μM) reversed PFTeDA-mediated inhibition. In conclusion, the inhibitory effects of PFTeDA on pubertal rat Leydig cell development are possibly regulated by inducing ferroptosis thereby downregulating SIRT1/AMPKA/ autophagy pathways, eventually resulting in reduced steroidogenesis.

Melatonin Ameliorates Cyclophosphamide-Induced Spermatogenesis Disorder by Reducing Pyroptosis

As a chemotherapeutic drug, cyclophosphamide (CP) has a negative impact on male fertility due to its reproductive toxicity. Melatonin (Mel) promotes the male reproductive system and increases testosterone synthesis. This study is aimed at exploring the molecular mechanism of Mel as a protector of male fertility against CP-induced cytotoxicity. A CP toxicity model was established in adult ICR male mice by intraperitoneal injection of 100 mg/kg CP every other day for a week. Protective effects of Mel on the testis from CP-induced damage were evaluated using four groups of ICR male mice that received intraperitoneal injections of normal saline, 100 mg/kg CP, 10 mg/kg Mel, or the same dosage of CP and Mel, respectively. Testis morphology was observed by hematoxylin and eosin (HE) staining. Sperm quality parameters were evaluated, and sperm function was studied by in vitro fertilization (IVF). Proliferation, meiosis, and pyroptosis markers were examined by western blot. Results showed that CP treatment induced testis toxicity in a time-dependent manner with the most severe damage to the testis at two weeks post CP treatment. CP-treated mice showed reduced testicular weight and impaired spermatogenesis by downregulating PCNA and SYCP3, reduced serum testosterone levels, decreased sperm counts and motility, increased seminiferous tubule vacuolization, and oxidative damage to spermatogenic cells. All these effects, apart from testicular weight, could be ameliorated by Mel administration. The IVF experiment revealed that CP treatment reduced the rates of sperm fertilization and blastocyst development, which were also enhanced by Mel. Mel-treated mice also showed increased expression of proliferation-associated protein PCNA and meiosis-associated proteins REC8, STRA8, and SYCP3, which were all reduced by CP. Furthermore, Mel inhibited the pyroptosis of spermatogenic cells by reducing GSDMD and IL18 expression. In conclusion, this study indicated that Mel might protect the testis from CP-induced DNA damage to germ cells through the alleviation of pyroptosis.

Androgen Deprivation Therapy in Current Clinical Practice: Challenges and Future Perspective

Androgen deprivation therapy (ADT) has been used for many years for treating advanced prostate cancer (PCa) and remains the backbone of treatment. Luteinising hormone-releasing hormone (LHRH) receptor agonists are the most widely used ADT drugs. However, newer options, including gonadotropin releasing hormone (GnRH) receptor antagonists such as degarelix and relugolix, may be clinically more beneficial for some patients. GnRH antagonists reduce serum testosterone levels more rapidly than LHRH agonists, without an initial testosterone surge or subsequent microsurges. This article summarises a symposium delivered on 11th March 2023 at the 38th European Association of Urology (EAU) Annual Congress in Milan, Italy, where speakers from three different disciplines described challenges and future perspectives for ADT in current clinical practice. Kurt Miller, Urologist, Charité University Hospital, Berlin, Germany, described the evolution of ADT in the treatment of PCa, from early reports of the benefits of surgical castration to the recent development of oral treatment for chemical castration. Miller explained the acceleration in progress in ADT research over recent years, with the development of novel drugs, drug sequences, and combinations, which have transformed outcomes in PCa. Alberto Bossi, Radiation Oncologist, Amethyst Group, Institut Gustav Roussy (IGR), Paris, France, next described current challenges with ADT management, including outstanding questions about the personalisation of ADT. Finally, Patrick Davey, Consultant Cardiologist, Northampton General Hospital, UK, spoke about ways to maintain a healthy heart on hormone treatment, and noted that cardiovascular safety is a major challenge in the use of ADT. The meeting was chaired by Heather Payne, Consultant Clinical Oncologist, University College Hospital, London, UK, who introduced the speakers and co-ordinated a question-and-answer session at the end of the symposium.

Long COVID and risk of erectile dysfunction in recovered patients from mild to moderate COVID-19

Patients with coronavirus disease 2019 (COVID-19) were shown to have reduced serum testosterone levels compared to healthy individuals. Low testosterone levels are linked with the development of erectile dysfunction (ED). In this case-controlled study, 20 healthy controls and 39 patients with ED 3 months after recovering from mild-to-moderate COVID-19 pneumonia were studied. The patients ranged in age from 31 to 47 years. To identify early and late COVID-19 infections, real-time polymerase-chain reaction (RT-PCR) and COVID-19 antibody testing were done. The levels of luteinizing hormone (LH), follicular stimulating hormone (FSH), total testosterone (TT), free testosterone (FT), free androgenic index (FAI), and sex hormone-binding globulin (SHBG) were measured. The sexual health inventory for patients (SHIM) score was used to measure the erectile function of the patients and controls. When compared to the controls, the TT serum level in long COVID-19 (LC) patients with ED was low (p = 0.01). In contrast to controls, FT and FAI were both lower in LC patients with ED. (p = 0.001). FSH serum levels did not significantly differ (p = 0.07), but in ED patients, LH serum levels were elevated. SHIM scores were associated with low TT (p = 0.30), FT (p = 0.09), and high LH (p = 0.76) in LC patients with ED. Male patients with decreased serum levels of LH and testosterone may have hypothalamic-pituitary–gonadal axis dysfunction, which could lead to the development of LC-induced ED. Therefore, an in-depth research is necessary to confirm the causal link between COVID-19 and ED in LC patients.

Remote limb ischemic postconditioning attenuates myocardial dysfunction induced by testicular torsion/detorsion in rats.

Torsion of the spermatic cord is a urological emergency that must be treated immediately with surgery, yet detorsion of the testis can cause testicular tissue damage because of ischemia‒reperfusion (I/R) injury. I/R injury is a complex pathophysiological process that may affect the functions of distant organs. Here, we examined whether testicular torsion/detorsion causes myocardial dysfunction. We next investigated the potential beneficial effect and underlying mechanisms of remote ischemic postconditioning (RIPost) on cardiac function after testicular torsion/detorsion. Male Sprague Dawley rats were assigned to three different sets of experimental groups. Testicular I/R was induced by rotating the right testis to 1080 degrees clockwise for 3 hours followed by 3 h of detorsion. RIPost was induced at the onset of testicular detorsion by four cycles of 5-min bilateral femoral artery occlusion with 5-min reperfusion. Cardiac function was determined postdetorsion, and the cardioprotective effect of RIPost was examined. Testicular torsion/detorsion-treated rats had reduced serum testosterone levels, impaired systemic hemodynamics, elevated systemic inflammatory responses and increased serum levels of LDH, CK-MB, α-HBDH and cTnI. Healthy rats transfused with testicular torsion/detorsion-treated plasma presented impaired cardiac function. However, RIPost attenuated remote heart dysfunction induced by testicular torsion/detorsion. Furthermore, RIPost enhanced the phosphorylation of ventricular STAT-3, which is a key component of the SAFE signaling pathways. Inhibition of STAT-3 with Ag490 abolished the RIPost-induced cardioprotection and STAT-3 phosphorylation. Testicular torsion followed by detorsion resulted in impairment of cardiac function. RIPost effectively attenuates this remote cardiac dysfunction. RIPost-induced protective effects may be mediated by the STAT-3 signaling pathway.

Melatonin therapy reverses lead exposure-induced testicular damage in rats despite the lack of effect on serum testosterone levels.

Lead (Pb) exposure induces testicular damage and infertility. The aim of this study was to analyze and compare the therapeutic effects of antioxidants or vitamin D and calcium, which have previously been shown to reduce the toxic effects of Pb co-exposure, in rats. Rats were exposed to Pb for 28 days and subsequently treated with antioxidant (melatonin, silymarin), vitamin D and calcium (VitDCa) or a combination (melatonin or silymarin with VitDCa) for 28 days. Control groups included untreated rats (no Pb exposure or therapy), rats exposed only to melatonin or silymarin and rats exposed to Pb without post exposure therapy. Pb exposure induced testicular damage, increased blood lead level (BLL) and reduced serum testosterone level (STL). Rats exposed to Pb and left untreated for 28 days showed persistent pathological testicular alterations. The two treatments that were most effective in reversing pathological testis damage and restoring spermatogenesis were melatonin and silymarin. However, silymarin and melatonin treatment resulted in significantly different serum testosterone levels in rats. Whereas melatonin therapy reduced serum testosterone to levels lower than those in control rats, silymarin increased serum testosterone to levels higher than those in controls. Our pathological analysis of testes revealed that melatonin promoted spermatogenesis and regression of Pb exposure-induced degenerative changes, despite the associated reduction in serum testosterone levels. This result suggests that circulating testosterone may not have an important role in spermatogenesis. Collectively, our results suggest that melatonin and silymarin are effective therapies against the toxic effects Pb exposure in the male reproductive system.

Bone morphogenetic protein 4 inhibits rat stem/progenitor Leydig cell development and regeneration via SMAD-dependent and SMAD-independent signaling

Bone morphogenetic protein 4 (BMP4) is an important member of the transforming growth factor-β superfamily. BMP4 is expressed in the Leydig cell lineage. We hypothesized that BMP4 might regulate the development of stem/progenitor Leydig cells. The BMP4 receptors, BMPR1A, BMPR1B, and BMPR2 were found to be expressed in progenitor Leydig cells of prepubertal testis and isolated cells. BMP4 at 1 and 10 ng/mL significantly reduced androgen production and down-regulated steroidogenesis-related gene and protein expression possibly by activating the SMAD signaling pathway (increasing SMAD1/5 phosphorylation and SMAD4) at 24 h treatment. BMP4 at 0.1 ng/mL and higher concentrations markedly reduced the EdU labeling index of CD90+ stem Leydig cells after 24 h treatment and significantly reduced the number of EdU+ stem Leydig cells on the surface of seminiferous tubules after 7 days of culture. BMP4 at 0.01 ng/mL and higher concentrations significantly blocked the differentiation of stem Leydig cells into adult cells, as shown by the reduction of testosterone secretion and the downregulation of Lhcgr, Scarb1, Cyp11a1, Hsd11b1, and Insl3 and their function after 3D seminiferous tubule culture for 3 weeks, and this effect was reversed by co-treatment with the BMP4 antagonists noggin and doxomorphine. In addition, BMP4 also blocked stem Leydig cell differentiation through SMAD-independent signaling pathways (ERK1/2 and AMPK). Ethanedimethane sulfonate (EDS) single injection can result in reduction of testosterone, restoration can happen post treatment. In an in vivo model of Leydig cell regeneration following EDS treatment, intratesticular injection of BMP4 from day 14 to day 28 post-elimination significantly reduced serum testosterone levels and down-regulated the expression of Scarb1, Star, Hsd11b1, and Insl3 and its proteins, possibly through SMAD-dependent and SMAD-independent (ERK1/2 and AMPK) signaling pathways. In conclusion, BMP4 is expressed in cells of the Leydig cell lineage and blocks entry of stem/progenitor Leydig cells into adult Leydig cells through SMAD-dependent and SMAD-independent signaling pathways.

In utero di-(2-ethylhexyl) phthalate-induced testicular dysgenesis syndrome in male newborn rats is rescued by taxifolin through reducing oxidative stress

Testicular dysgenesis syndrome in male neonates manifests as cryptorchidism and hypospadias, which can be mimicked by in utero phthalate exposure. However, the underlying phthalate mediated mechanism and therapeutic effects of taxifolin remain unclear. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundantly used phthalate and can induce testicular dysgenesis syndrome in male rats. To explore the mechanism of DEHP mediated effects and develop a therapeutic drug, the natural phytomedicine taxifolin was used. Pregnant Sprague-Dawley female rats were daily gavaged with 750 mg/kg/d DEHP or 10 or 20 mg/kg/d taxifolin alone or in combination from gestational day 14 to 21, and male pup's fetal Leydig cell function, testicular MDA, and antioxidants were examined. DEHP significantly reduced serum testosterone levels of male pups, down-regulated the expression of SCARB1, CYP11A1, HSD3B1, HSD17B3, and INSL3, reduced the cell size of fetal Leydig cells, decreased the levels of antioxidant and related signals (SOD2 and CAT, SIRT1, and PGC1α), induced abnormal aggregation of fetal Leydig cells, and stimulated formation of multinucleated gonocytes and MDA levels. Taxifolin alone (10 and 20 mg/kg/d) did not affect these parameters. However, taxifolin significantly rescued DEHP-induced alterations. DEHP exposure in utero can induce testicular dysgenesis syndrome by altering the oxidative balance and SIRT1/PGC1α levels, and taxifolin is an ideal phytomedicine to prevent phthalate induced testicular dysgenesis syndrome.

Eugenol reduces serum testosterone levels and sperm viability in adult Wistar rats

Eugenol is the main constituent of clove extract. It is a remarkably versatile molecule incorporated as a functional ingredient in several food products and widely applied in the pharmaceutical industry. Men consume natural products enriched with eugenol for treating sexual disorders and using as aphrodisiacs. Nevertheless, there is no information about the impact of eugenol intake on the reproductive parameters of healthy males. Therefore, we provided 10, 20, and 40 mg kg-1 pure eugenol to adult Wistar rats for 60 days. Testis, epididymis, and spermatozoa were analyzed under microscopic, biochemical, and functional approaches. This phenolic compound did not alter testicular and epididymal biometry and microscopy. However, 20 and 40 mg kg-1 eugenol reduced serum testosterone levels. The highest dose altered lactate and glucose concentrations in the epididymis. All the eugenol concentrations diminished CAT activity and MDA levels in the testis and increased FRAP and CAT activity in the epididymis. Epididymal sperm from rats receiving 10, 20, and 40 mg kg-1 eugenol presented high Ca2+ ATPase activity and low motility. In conclusion, eugenol at low and high doses negatively impacted the competence of epididymal sperm and modified oxidative parameters in male organs, with no influence on their microscopy.

Triphenyltin disrupts the testicular microenvironment and reduces sperm quality in adult male rats.

Triphenyltin (TPT) is organotin that is widely used as an anti-fouling agent and has been determined to have male reproductive toxicity. The objective of this study was to investigate the effects of TPT on the testicular microenvironment and sperm quality in male rats. Adult male Sprague Dawley rats were daily gavaged with TPT (0, 0.5, 1, and 2mg/kg body weight) for 28 days. The results showed that TPT dose-dependently decreased sperm count and sperm motility, interfered with sperm histone-protamine replacement process, and significantly increased sperm deformity rate, but did not affect sperm DNA integrity. TPT at 2mg/kg significantly decreased the gene and protein expressions of testis PCNA and Ki67, and dose-dependently decreased the number of PCNA-positive cells and Ki67-positive cells. TPT at 1mg/kg and/or 2mg/kg down-regulated the expression of StAR, SF1, P450scc, FSHR, WT1, DDX4 and PLZF, and up-regulated SOX9 expression. Simultaneously, TPT reduced serum testosterone levels at each dose and dose-dependently decreased the expression of Leydig cells regulators (INSL3, IGF1, inhibin B) and Sertoli cells regulators (GDNF, FGF2, CXCL12, ETV5), altered testicular microenvironment. Further, in vitro, we treated TM3 (Leydig cells), TM4 (Sertoli cells) and GC-1 (spermatogonia) cells with 1-100nM TPT for 24h. 100nM TPT significantly down-regulated the expression of the above indicators in TM3 and TM4 cells but did not directly affect the cell proliferation ability of GC-1. However, after co-culturing TPT-treated TM3 or TM4 cells with GC-1cells, it was found that TPT-treated TM3 or TM4 cells dose-dependently reduced the gene and protein expression levels of PCNA and Ki67 and increased cytotoxicity in GC-1cells. In conclusion, TPT impairs the proliferative ability of spermatogonia by disrupting the microenvironment of Leydig cells and Sertoli cells, which in turn leads to low sperm quality in adult male rats.

Dimethylbisphenol A inhibits the differentiation of stem Leydig cells in adult male rats by androgen receptor (NR3C4) antagonism

Dimethylbisphenol A (DMBPA) is a novel alternative to bisphenol A. Whether short-term exposure to DMBPA affects Leydig cell regeneration remains unknown. The Leydig cell regeneration model was generated by intraperitoneal injection of 75 mg/kg ethane dimethane sulfonate (EDS) to adult male Sprague-Dawley rats. Leydig cell regeneration began on day 14 after EDS. Rats were gavaged with 0, 10, 50, or 200 mg/kg DMBPA from days 14–28 post-EDS, and Leydig cell regeneration was assessed on days 28 and 56 post-EDS. DMBPA significantly reduced serum testosterone levels on days 28 and 56 at 10 mg/kg and higher doses and sperm count in the caudal epididymis on day 56 at 200 mg/kg, without affecting estradiol, luteinizing hormone, and follicle-stimulating hormone. DMBPA had no effect on Leydig cell number but significantly down-regulated Scarb1 expression at ≥ 10 mg/kg on day 28, Cyp17a1 expression on day 28 at 200 mg/kg and on day 56 at ≥ 10 mg/kg. DMBPA markedly upregulated Srd5a1 expression at doses of 50 and 200 mg/kg on day 56 after EDS. DMBPA significantly down-regulated the expression of Sod1 and Nr3c4 at a dose of 200 mg/kg on day 28. Further semi-quantitative immunohistochemistry showed that DMBPA reduced NR3C4 levels in Leydig and Sertoli cells at 50 and 200 mg/kg. In vitro DMBPA treatment of immature Leydig cells for 24 h showed that it significantly reduced testosterone production at 10 and 50 μM, and further mechanistic studies showed that an NR3C4 agonist 7α-methyl-19-nortestosterone significantly reversed DMBPA-mediated suppression on testosterone output, but the estrogen receptor antagonist ICI 182,780 and G-coupled estrogen receptor 1 agonist G15 had no effect. In conclusion, DMBPA delays Leydig cell regeneration after short-term exposure during early Leydig cell regeneration via NR3C4 antagonism.

Bisphenol F blocks Leydig cell maturation and steroidogenesis in pubertal male rats through suppressing androgen receptor signaling and activating G-protein coupled estrogen receptor 1 (GPER1) signaling

Bisphenol F (BPF) is a new analog of bisphenol A (BPA). BPA has deleterious effects on the male reproductive system, but the effect of BPF has not been studied in detail. In this study we focus on the effect of BPF on Leydig cell maturation. Male Sprague-Dawley rats were gavaged with 0, 1, 10, or 100 mg/kg BPF from postnatal days 35–56. BPF significantly reduced serum testosterone levels and sperm count in cauda epididymis at dose ≥1 mg/kg. It significantly down-regulated the expression of steroidogenic enzymes, while increasing FSHR and SOX9 levels at 10 and 100 mg/kg. Further studies showed that BPF reduced NR3C4 expression in Leydig and Sertoli cells without affecting its levels in peritubular myoid cells. BPF markedly increased GPER1 in Leydig cells at 100 mg/kg, and it significantly reduced SIRT1 and PGC1α levels in the testes at 100 mg/kg. BPF significantly inhibited testosterone production by immature Leydig cells at 50 μM after 24 h of treatment, which was completely reversed by NR3C4 agonist 7α-methyl-19-nortestosterone and partially reversed by GPER1 antagonist G15 not by ESR1 antagonist ICI 182,780. In conclusion, BPF negatively affects Leydig cell maturation in pubertal male rats through NR3C4 antagonism and GPER1 agonism.

Preconception Antidiabetic Drugs in Men and Birth Defects in Offspring: A Nationwide Cohort Study

(Abstracted from Ann Intern Med 2022;175:665–673) Diabetes mellitus is known to compromise sperm quality and is associated with impaired male fertility. Metformin is a first-line oral diabetes medication that has been shown to improve markers of semen quality in obese men but reduces serum testosterone levels independently of glycemic control.

Immobilization stress exacerbates arsenic-induced reprotoxic effects in adult rats.

The central objective of this study was to investigate the cumulative effects restraint stress and sodium arsenite on reproductive health in male rats. Healthy male Wistar rats were allocated into 4 groups (n=8). Animals in group 1 served as controls and did not subjected to any stress. Rats in groups 2, 3, and 4 were subjected to either restraint stress (5h/day) or maintained on arsenic (25ppm) via drinking water or both for 65days. After completion of the experimental period, all the rats were analyzed for selected reproductive endpoints. Restraint stress or sodium arsenite treatment increased serum corticosterone levels, reduced testicular daily sperm count, epididymal sperm viability, motility, membrane integrity, and decreased testicular steroidogenic enzymes such as 3β- and 17β-hydroxysteroid dehydrogenases associated with reduced serum testosterone levels, deteriorated testicular architecture, and reduced activity levels of testicular superoxide dismutase and catalase accompanied by elevated lipid peroxidation levels. In rats subjected to restraint stress and sodium arsenite, a significant decrease in selected sperm qualitative and quantitative parameters, serum testosterone levels were observed as compared with rats subjected to sodium arsenite alone. A significant increase in the levels of lipid peroxidation with a concomitant decrease in the activities of antioxidant enzymes was observed in the testis of rats subjected to both restraint stress and sodium arsenite treatment as compared with sodium arsenite alone intoxicated rats. Surprisingly, serum corticosterone levels were significantly elevated in rats following both stressors as compared with arsenic alone treated rats. Analysis of atomic absorption spectroscopy revealed that the accumulation of arsenic in the testis of arsenic-treated and arsenic plus immobilization stress groups was significant as compared with controls. Based on the findings, it can be concluded that deterioration of male reproductive health could be accelerated in arsenic intoxicated rats following restraint stress.

Outcomes of treatment for LOH syndrome assessed by free testosterone

ABSTRACT Objective Late-onset hypogonadism (LOH) is defined by reduced serum testosterone levels, and androgen replacement therapy (ART) is widely used. While serum total testosterone (TT) is used as an indicator for diagnosis and treatment for LOH in the United States and Europe, serum free testosterone (FT) is used in Japan. Patients with low FT levels (less than 8.5 pg/mL) are to be indicated for ART as LOH, and patients with moderate FT levels (between 8.5 pg/mL and 11.8 pg/mL) are also to be considered for ART as LOH borderline. In this study, we examined the outcomes of LOH and LOH borderline patients who were treated by ART. Methods We examined Aging Male Symptom (AMS) scale scores of LOH (n=76) and LOH borderline patients (n=16) who were treated with ART for at least 6 months at our hospital. Results In LOH patients, AMS score decreased from 52.1±12.3 to 41.5±12.2 (p<0.05). On the other hand, in borderline cases AMS score also decreased from 55.8±10.2 to 36.5±8.45 (p<0.05). There was no significant difference between two groups. Conclusion ART improves AMS score both in LOH and LOH borderline patients, but there were no significant differences between two groups in this study. Disclosure Work supported by industry: no.