Xiaokang Liuwei Dihuang decoction ameliorates the immune infertility of male rats induced by lipopolysaccharide through regulating the levels of sex hormones, reactive oxygen species, pro-apoptotic and immune factors.

Male immune infertility is a kind of disease that damages family life and happiness. The development of novel methods treating male immune infertility is of great significance. This study aimed to investigate the therapeutic effects of Chinese medicine Xiaokang Liuwei Dihuang decoction on immune infertility of male rats and explored the involved mechanisms. Model rats were established by lipopolysaccharide (LPS) injection. Anti-sperm antibody (AsAb) was detected by ELISA assay and testicular cell apoptosis was evaluated by TUNEL staining to verify the successful model establishment and screen suitable doses of Xiaokang Liuwei Dihuang decoction. Thirty rats were then divided into five groups (n = 6 per group): Control, LPS, Xiaokang Liuwei Dihuang decoction (15.12 g/kg, 30.24 g/kg and 45.36 g/kg). Results of HE staining showed that compared with LPS group, Xiaokang Liuwei Dihuang decoction treatments gradually improved the morphology of seminiferous tubules and elevated the number of spermatogenic cells as the doses increased. The sperm number and the levels of testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) in the serum of 15.12 g/kg, 30.24 g/kg and 45.36 g/kg Xiaokang Liuwei Dihuang decoction groups were much higher than those in LPS group. Results of TUNEL staining, ELISA assay and western blot showed that compared with LPS group, the testicular cell apoptosis and the levels of interleukin 1ß (IL-1ß), tumor necrosis factor α (TNF-α), AsAb, malondialdehyde (MDA) and toll-like receptor 2 (TLR2) in the testicular tissue significantly decreased in three Xiaokang Liuwei Dihuang decoction groups. Compared with LPS group, Bax expression in the 30.24 g/kg and 45.36 g/kg Xiaokang Liuwei Dihuang decoction groups was significantly down-regulated as well. In conclusion, Xiaokang Liuwei Dihuang decoction might ameliorate the immune infertility of male rats induced by LPS through regulating the levels of sex hormones, reactive oxygen species, pro-apoptotic and immune factors.

Impact of chronic vitamin A excess on sperm morphogenesis in mice.

BACKGROUND: The increasing availability of fortified foods and supplements has caused an overconsumption of vitamin A (VA), above the recommended level. To date, the effects of chronic VA excess (VAE) on spermatogenesis remain unclear. OBJECTIVE: This study aims to investigate the long-term excessive intake of VA effects on spermatogenesis in mice. MATERIALS AND METHODS: Dams were initially fed a control diet (4 IU/g) or a VAE diet (250 IU/g), 4 weeks prior to mating and during pregnancy. Dams and their male pups continued this diet regimen until the offspring reached 12 weeks of age. At 12 weeks of age, epididymis caudal spermatozoa and testes were collected. For histological analysis, sections were stained with periodic acid-Schiff-hematoxylin, and quantitative PCR was used to detect changes in gene expression in the testes of the VAE mice. Sperm motility and morphology were evaluated to detect the endpoint of VAE toxicity. RESULTS: Body weights were not significantly different between the control and VAE groups. Testicular cross-sections from the control and VAE mice contained a normal array of germ cells, and the daily sperm production was similar between the two groups. However, the percentage of seminiferous tubules in stages VII and VIII was significantly lower in the VAE mice than in the control. In addition, significant changes in the expression of genes involved in retinoid metabolism, spermatogenesis, and spermiogenesis were detected in the testes of the VAE mice. Consistently, sperm motility and head morphology were significantly impaired in the VAE mice. DISCUSSION AND CONCLUSION: Our findings suggest that long-term dietary intake of VAE was able to influence both pre- and post-meiotic spermatogenesis. As a result of testicular toxicity, we demonstrated, to the best of our knowledge, for the first time that long-term VAE caused sperm-head abnormalities.

Green tea consumption increases sperm concentration and viability in male rats and is safe for reproductive, liver and kidney health.

Green tea is a popularly consumed beverage worldwide and contains polyphenols, whose antioxidant activities could improve sperm parameters and fertility thereof. We investigated the effect of green tea on the male rat reproductive system as well as its safety. Male Wistar rats were administered 2 and 5% aqueous extract of green tea for 52 days' ad libitum, while the control group received tap water. Total polyphenol, flavanol, flavonol and soluble solids significantly increased in a concentration-dependent manner in vitro (P < 0.01). Weights of body, testis, epididymis, prostate gland, seminal vesicles, and liver, serum levels of testosterone, ferric reducing antioxidant power, creatinine, and sperm motility, remained unchanged (P > 0.05). Kidney weight, sperm concentration and vitality, spontaneous acrosome reaction increased (P < 0.05), while alanine transaminase and aspartate transaminase levels decreased (P < 0.05). Catalase, superoxide dismutase, glutathione and lipid peroxidation remained unchanged in the testes, liver and kidney (P > 0.05). Histological sections of testis, epididymis, kidney and liver showed no conspicuous alteration. Diameter and epithelial height of seminiferous tubule decreased, while caudal epididymis epithelial height increased (P < 0.01). Consumption of green tea in the conditions used in the present study seems to be safe and improved sperm parameters. However, subtle structural changes observed in the decreased diameter and epithelial height of the seminiferous tubule and increased acrosome reaction needs further investigation.

Chestnut polysaccharides benefit spermatogenesis through improvement in the expression of important genes.

Recently there has been a continuing worldwide decrease in the quality of human spermatozoa, especially in spermatozoa motility and concentration. Many factors are involved in this decline, and great efforts have been made to rescue spermatogenesis; however, there has been little progress in the improvement of sperm quality. Chestnuts are used in traditional Chinese medicine; their major active components are chestnut polysaccharides (CPs). CPs have many biological activities but their effects on spermatogenesis are unknown. The current investigation was designed to explore the impact of CPs on spermatogenesis and the underlying mechanisms. We demonstrated that CPs significantly increased sperm motility and concentration (4-fold and 12-fold, respectively), and improved seminiferous tubule development by increasing the number of germ cells after busulfan treatment. CPs dramatically rescued the expression of important genes and proteins (STRA8, DAZL, SYCP1, SYCP3, TNP1 etc.) in spermatogenesis. Furthermore, CPs increased the levels of hormone synthesis proteins such as CYP17A1 and HSD17ß1. All the data suggested that CPs improved the testicular microenvironment to rescue spermatogenesis. With CPs being natural products, they may be an attractive alternative for treating infertile patients in the future. At the same time, the deep underlying mechanisms of their action need to be explored.

Oral supplementation of fucoxanthin-rich brown algae extract ameliorates cisplatin-induced testicular damage in hamsters.

Oxidative stress is recognized as a common pathology that affects up to half of all men infertile. Fucoxanthin possesses antioxidant activity, and several investigators have reported anti-inflammatory action. This study extracted powder of Sargassum glaucescens by acetone to obtained fucoxanthin rich-brown algae extract (FXE). The objective of this study was to evaluate the ameliorative effects of fucoxanthin extract from Sargassum glaucescens on lipopolysaccharide-induced inflammation in RAW264.7 macrophage cells and its protective effects of against Cisplatin (CP)-induced reproductive damage in hamsters. Eighty male Syrian hamsters were injected with and without CP, then daily oral gavage with various concentrations of fucoxanthin for 5 days. Treatment with FXE reduced the level of reactive oxygen species and malondialdehyde in RAW 264.7 cells and the rats' testis as well as protective effects on mitochondrial membrane potential. The FXE administration also improved testosterone level and alpha-glucosidase activity. The sperm count also increased after treated with FXE, whereas sperm abnormality was reduced. Histopathological analysis showed that FXE successfully improved the seminiferous tubules morphology. According to these findings, fucoxanthin extract from Sargassum glaucescens can be used as an alternative for the treatment of testicular damage.

Vitamin E but Not GSH Decreases Reactive Oxygen Species Accumulation and Enhances Sperm Production during In Vitro Maturation of Frozen-Thawed Prepubertal Mouse Testicular Tissue.

Freezing-thawing procedures and in vitro culture conditions are considered as a source of stress associated with increased reactive oxygen species (ROS) generation, leading to a damaged cell aerobic metabolism and consequently to oxidative stress. In the present study, we sought to investigate whether vitamin E (Vit E) or reduced glutathione (GSH) enhances sperm production by decreasing ROS accumulation during in vitro maturation of prepubertal mice testes. Testes of prepubertal mice were cryopreserved using a freezing medium supplemented or not supplemented with Vit E and were cultured after thawing. In presence of Rol alone in culture medium, frozen-thawed (F-T) testicular tissues exhibited a higher ROS accumulation than fresh tissue during in vitro culture. However, Vit E supplementation in freezing, thawing, and culture media significantly decreased cytoplasmic ROS accumulation in F-T testicular tissue during in vitro maturation when compared with F-T testicular tissue cultured in the presence of Rol alone, whereas GSH supplementation in culture medium significantly increased ROS accumulation associated with cytolysis and tissue disintegration. Vit E but not GSH promoted a better in vitro sperm production and was a suitable ROS scavenger and effective molecule to improve the yield of in vitro spermatogenesis from F-T prepubertal mice testes. The prevention of oxidative stress in the cytoplasmic compartment should be regarded as a potential strategy for improving testicular tissue viability and functionality during the freeze-thaw procedure and in vitro maturation.

Rosa damascena Mill. Essential Oil Has Protective Effect Against Testicular Damage in Diabetic Rats.

This study investigates the protective effect of Rosa damascena essential oil on diabetes-induced testicular damage in rats. Thirty-six male Wistar rats were randomly divided into 6 equal groups: Group I: negative control (no treatment); Group II: positive control (diabetic by alloxan injection); Groups III-VI that rendered diabetic and received, respectively, 50, 100, 200, and 400 µg/kg/day rose oil, orally for 28 days. Rose oil did not significantly change body weight and blood glucose level as compared to positive control. Serum testosterone level of rose oil-treated rats remained statistically the same with both negative and positive control groups (Groups I and II). Rats treated with rose oil especially at 2 higher dosages (Groups V and VI) had higher sperm count and increased diameters of seminiferous tubules as compared to Group II. Rose oil even at the lowest dosage significantly increased cell count of spermatogonia, primary spermatocytes, Sertoli cells, and Leydig cells, with better outcomes for higher dosages. It appears that short-term repeated dose administration of rose oil can dose-dependently improve structural deteriorations of testes and epididymal sperm count in diabetic rats.

Reproductive toxicity of melamine against male mice and the related mechanism.

Melamine is a nitrogen-containing heterocyclic organic compound with a triazine skeleton, which has been widely applied in industrial and chemical fields. Previous toxicity studies of melamine mainly focused on renal toxicity and hepatic pathological changes, but its toxicity against the reproductive system has seldom been assessed. We investigated the effects of melamine on the reproductive system of male mice. Forty healthy male Kunming mice were randomly divided into a normal saline negative control group, a low-dose melamine group, a medium-dose melamine group and a high-dose melamine group (n = 10). The mice were administered for five consecutive days and killed on the 35th day after first administration. In melamine administration groups, seminiferous tubules had disordered, loose arrangement, and spermatogenic cells at all levels obviously decreased. The sperm count and motility decreased significantly, and the sperm deformity rate increased significantly. Melamine induced apoptosis of testicular spermatogenic cells. To further explore the mechanism, we detected metabolism-related enzymes sorbitol dehydrogenase (SDH) and lactate dehydrogenase (LDH) as well as oxidative stress indices superoxide dismutase (SOD) and malondialdehyde (MDA). The activities of SDH, LDH and SOD in melamine treatment groups decreased significantly, and the MDA level increased obviously. The expressions of apoptosis-related proteins Bcl-2, Bax and caspase-3 were detected by immunohistochemistry. The expression of Bcl-2 significantly increased, but those of Bax and caspase-3 significantly reduced (p < 0.05). In conclusion, melamine damaged the reproductive system of mice via the oxidative stress pathway and by inducing cell apoptosis.

Evaluation of apoptotic- and autophagic-related protein expressions before and after IVM of fresh, slow-frozen and vitrified pre-pubertal mouse testicular tissue.

STUDY QUESTION: Do freezing and in vitro culture procedures enhance the expression of proteins involved in apoptotic or autophagic pathways in murine pre-pubertal testicular tissue? SUMMARY ANSWER: IVM strongly modified apoptosis- and autophagy-related relative protein levels in mice testicular tissue whereas the impact of cryopreservation procedures was minimal at the end of the culture. WHAT IS KNOWN ALREADY: In vitro spermatogenesis remains a challenging technical issue as it imposes to find a very close balance between survival and death of germ cell natural precursors (i.e. gonocytes and spermatogonia), which will eventually undergo a complete spermatogenesis close to in vivo conditions. The establishment of efficient culture conditions coupled with suitable cryopreservation procedures (e.g. controlled slow freezing [CSF] and solid surface vitrification [SSV]) of pre-pubertal testicular tissue is a crucial step in the fields of fertility preservation and restoration to improve the spermatic yield obtained in vitro. STUDY DESIGN, SIZE, DURATION: Here, we study cryopreservation procedures (i.e. CSF or SSV) and the impact of culture media compositions. A first set of 66 mouse pre-pubertal testes were directly cultured during 30, 36, 38 and 60 days (D) from 2.5 to 6.5-day-old CD-1 mice to evaluate the impact of time-aspect of culture and to endorse the reverse phase protein microarrays (RPPM) technique as an adapted experimental tool for the field of in vitro spermatogenesis. Ninety others fresh, slow-frozen and vitrified pre-pubertal testes were cultured during 30 days for the principal study to evaluate the impact of cryopreservation procedures before and after culture. Thirty-four testes dissected from 2.5, 6.5, 36.5, 40.5, 42.5 and 62.5 days postpartum (dpp) mice, corresponding to the time frames of spermatogenesis orchestrated in vitro, were used as in vivo controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: After in vitro culture, testicular tissue samples originated from 2.5 or 6.5-day-old CD-1 male mice were analyzed using RPPM. This targeted proteomic technique allowed us to assess the expression level of 29 apoptosis- and autophagy-related factors by normalizing blank-corrected signal values. In addition, morphological analyses (e.g. HES, PAS, TRA98 and CREM) and DNA fragmentation in intra-tubular cells (i.e. terminal deoxynucleotidyl transferase dUTP nick end labeling; TUNEL) were assessed for the distinct experimental conditions tested as well as for in vivo control mouse testes. MAIN RESULTS AND THE ROLE OF CHANCE: A validation of the RPPM procedure in the field of in vitro spermatogenesis was completed with assay and array robustness before a principal study concerning the evaluation of the impact of in vitro culture and cryopreservation procedures. The proportion of elongated spermatids and the total cell number per seminiferous tubule tended to be very different between the in vivo and in vitro conditions (P < 0.05), suggesting the presence of a beneficial regulation on the first spermatogenesis wave by intrinsic apoptosis (Caspase_9) and autophagy (Atg5) factors (P < 0.0003 and r2 = 0.74). Concerning the impact of culture media compositions, a basic medium (BM) composed of αMEM plus 10% KnockOut™ serum replacement and gentamicin supplemented with retinol (Rol) and vitamin E (Vit. E) was selected as the best culture medium for fresh 6.5 dpp tissue cultured during 30D with 27.7 ± 8.10% of seminiferous tubules containing elongated spermatids. Concerning the impact of cryopreservation procedures, SSV did not have any impact on the morphological parameters evaluated after culture in comparison to fresh tissue (FT) controls. The proportion of tubules with elongated spermatids on testicular explants cultured with BMRol+Vit. E was not different between SSV (6.6 ± 1.6%) and CSF (5.3 ± 1.9%); however, round spermatids were observed more frequently for SSV (19 ± 6.2%) than CSF (3.3 ± 1.9%, P = 0.0317). Even if the proportion of TUNEL-positive cells for BMRol+Vit. E was higher at D30 after SSV (4.12 ± 0.26%) than CSF (1.86 ± 0.12%, P = 0.0022) and FT (2.69 ± 0.33%, P = 0.0108), the DNA damages observed at the end of the culture (i.e. D30) were similar to respective 6.5 dpp controls. In addition, the relative protein level expression ratio of an apoptotic factor, the phosphorylated FADD on Fas, was reduced by 64-fold in vitrified testes cultured with BMRol+Vit. E. Furthermore, we found in this study that the StemPro®-34 SFM culture medium supplemented with growth factors (e.g. EGF, bFGF, GDNF and LIF) prevented the differentiation of spermatogonial stem cells in favor of a significant proliferation with a better architectural pattern than in vivo 6.5 dpp controls with an increase of seminiferous tubules area for FT (P = 0.0357) and CSF (P = 0.0317). LIMITATIONS REASONS FOR CAUTION: Despite our promising results, the evaluation of apoptotic- and autophagic-related proteins was studied for a limited amount of proteins and on global testicular tissue. WIDER IMPLICATIONS OF THE FINDINGS: The data presented herein will help to improve apoptotic and autophagic understanding during the first spermatogenic wave. Moreover, our findings illustrate for the first time that, using finely-tuned experimental conditions, a testicular in vitro culture combined with proteomic technologies may significantly facilitate the study of cryopreservation procedures and in vitro culture evaluations. This study may also contribute to improve work on testicular tissues from pre-pubertal and adolescent cancer survivors. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by a Ph.D. grant from the Rouen Normandie Université and a financial support from 'la Ligue nationale contre le cancer' (both awarded to L.D.), funding from Institute for Research and Innovation in Biomedicine (IRIB), Agence de la Biomédecine, and co-supported by European Union and Région Normandie. Europe gets involved in Normandie with European Regional Development Fund (ERDF). The authors declare that there is no conflict of interest.

Chronic consumption of Hypericum humifusum leaf extracts impairs epididymis spermatozoa characters in association with oxidative stress in adult male Wistar rats.

Recently, there has been increasing interest in Hypericum (Hypericaceae) genus. The first part of the present study focused on the phytochemical analysis of the methanolic and aqueous extracts of Hypericum humifusum leaves. The second part of the study investigated the effect of Hypericum humifusum leaf extracts on male reproductive parameters. 30 male rats were grouped into control (1mL/rat, distilled water), treated by 200mg/kg body weight (bw) aqueous extract (A200), 400mg/kg bw aqueous extract (A400), 10mg/kg bw methanolic extract (M10) and 20mg/kg bw methanolic extract (M20) groups. The phytochemical analysis revealed the presence of tannins, flavonoids, steroids, carbohydrates, and phenolic compounds. After thirty-day treatment, body and reproductive organs were weighed. Testes in all rat groups were processed for biochemical assays and histopathological examinations. Epididymis sperm analyses were also performed. Testicular tissue homogenate samples were used for Malondialdehyde (MDA), catalase and superoxide dismutase (SOD) measurements. We showed that Hh extracts induced a severe seminiferous tubular damage with an increase in the percentage of empty seminiferous tubules. Epididymis sperm analysis revealed a significant reduction in density and viability of sperm with alteration of spermatozoa morphology. Also, we found that Hh leaf extracts decreased plasma total cholesterol, HDL-cholesterol and triglycerides levels. These results were associated with an increase of MDA levels and a decrease of catalase and SOD activities in testis tissues. Our finding revealed that chronic consumption of Hh extracts induces disruption of normal spermatogenesis by alteration of sperm density, viability, and morphology. This action may be due to an inhibition of the antioxidant-defense system.

In vitro differentiation of rat spermatogonia into round spermatids in tissue culture.

STUDY QUESTION: Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? SUMMARY ANSWER: We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. WHAT IS KNOWN ALREADY: Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in vitro or testosterone production was observed. LIMITATIONS, REASONS FOR CAUTION: The human testis is very different in physiology from the rat testis, further investigations are still needed to optimize the organ culture system for future use in humans. WIDER IMPLICATIONS OF THE FINDINGS: The successful differentiation of undifferentiated spermatogonia using the testis explant culture system might be employed in future to produce sperm from human spermatogonia as a clinical tool for fertility preservation in boys and men suffering infertility. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: This work was supported financially by the Frimurare Barnhuset in Stockholm, the Paediatric Research Foundation, Jeanssons Foundation, Sällskåpet Barnåvard in Stockholm, Swedish Research Council/Academy of Finland, Emil and Wera Cornells Foundation, Samariten Foundation, the Swedish Childhood Cancer Foundation as well as through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. All authors declare no conflicts of interests.

Effects of Dietary Selenium Supplementation on Seminiferous Tubules and SelW, GPx4, LHCGR, and ACE Expression in Chicken Testis.

We investigated the effects of dietary selenium (Se) supplementation on the development of chicken testis and the expression of selenoprotein W (SelW), glutathione peroxidase4 (GPx4), luteinizing hormone/choriogonadotropin receptor (LHCGR), and angiotensin converting enzyme (ACE). Sixty roosters were assigned randomly into the control group fed with a basic diet (containing 0.3 mg Se/kg) and the experimental group fed with a diet (containing 0.6 mg Se/kg). The testes were collected individually at age of 6, 9, and 12 weeks. Se was supplemented in chicken feed for 15 days before sampling. The results indicated that dietary Se affected the number of cells in the seminiferous tubules and viability of Sertoli cells in vitro culture. SelW and GPx4 expression in the testes increased significantly in the experimental group compared to that in the control group. LHCGR expression in the testes increased significantly in the experimental group after 12 weeks compared to that in the control group. In contrast, ACE expression was inhibited in the experimental group compared to that in the control group. These results suggest that dietary supplementation with Se improved development of the seminiferous tubules at the cellular level and that SelW, GPx4, LHCGR, and ACE are involved.

Protective effects of grape seed extract on cadmium-induced testicular damage, apoptosis, and endothelial nitric oxide synthases expression in rats.

This study aims to evaluate the protective effect of grape seed proanthocyanidin extract (GSPE) on cadmium (Cd)-induced testicular apoptosis, endothelial nitric oxide synthases (eNOS) expression, and toxicity in rats. A total of 24 male Wistar rats were divided into four groups, namely, control, Cd (2.5 mg/kg), Cd + GSPE (100 mg/kg/day), and GSPE. Spermatogenesis and mean seminiferous tubule diameter were significantly decreased in the Cd groups. Furthermore, the GSPE-treated animals showed an improved histological appearance in the Cd group. The immunoreactivity of eNOS and the number of apoptotic cells were increased in Cd group. Our data indicate a significant reduction of terminal deoxynucleotide transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end-labeling staining and a decrease in the expression of eNOS in the testes tissue of the Cd group treated with GSPE therapy. Therefore, our results suggest that GSPE acts as a potent protective agent against Cd-induced testicular toxicity in rats.

LH-Independent Testosterone Secretion Is Mediated by the Interaction Between GNRH2 and Its Receptor Within Porcine Testes.

Unlike classic gonadotropin-releasing hormone 1 (GNRH1), the second mammalian isoform (GNRH2) is an ineffective stimulant of gonadotropin release. Species that produce GNRH2 may not maintain a functional GNRH2 receptor (GNRHR2) due to coding errors. A full-length GNRHR2 gene has been identified in swine, but its role in reproduction requires further elucidation. Our objective was to examine the role of GNRH2 and GNRHR2 in testicular function of boars. We discovered that GNRH2 levels were higher in the testis than in the anterior pituitary gland or hypothalamus, corresponding to greater GNRHR2 abundance in the testis versus the anterior pituitary gland. Moreover, GNRH2 immunostaining was most prevalent within seminiferous tubules, whereas GNRHR2 was detected in high abundance on Leydig cells. GNRH2 pretreatment of testis explant cultures elicited testosterone secretion similar to that of human chorionic gonadotropin stimulation. Treatment of mature boars with GNRH2 elevated testosterone levels similar to those of GNRH1-treated males, despite minimal GNRH2-induced release of luteinizing hormone (LH). When pretreated with a GNRHR1 antagonist (SB-75), subsequent GNRH2 treatment stimulated low levels of testosterone secretion despite a pattern of LH release similar to that in the previous trial, suggesting that SB-75 inhibited testicular GNRHR2s. Given that pigs lack testicular GNRHR1, these data may indicate that GNRH2 and its receptor are involved in autocrine or paracrine regulation of testosterone secretion. Notably, our data are the first to suggest a biological function of a novel GNRH2-GNRHR2 system in the testes of swine.

Role of Se+Zn in regeneration (Ki-67) following Pb toxicity (p53andcad) in the germinal epithelium of adult Wistar rats.

The germinal epithelium is the delicate epithelial lining of the seminiferous tubule lying on the blood-testes barrier; formed by the sustenacular cells of Sertoli and the adjoining basement epithelium this study addresses the effect of lead (Pb) toxicity on the epithelium and the proliferative effect of Zinc (Zn) and Selenium (Se) administered in trace concentration. Sixty F1 generation adult male Wistar rats were divided into four groups of 15 animals each. Group 1 received normal saline, group 2: 100 mg kg(-1) of lead acetate, group 3: 100 mg kg(-1) of lead acetate then 2.25 mg kg(-1) each of Zinc (Chelated zinc) and Selenium (Sodium Selenium) and group 4: 2.25 mg kg(-1) of zinc and selenium (Se+Zn). The duration of treatment was 56 days following which the animals were sacrificed on the 57th day and the testes harvested and fixed in Bouin's fluid. Pb induced toxicity can follow a mitochondria pathway involving Cathepsin D (CAD) or a cytoplasmic pathway involving p53 (protein 53; a 53 KDa nucleolase), the most predominant form of cell death is apoptosis which can result from both pathways. Se+Zn treatment improves proliferation and counters Pb toxicity by substitution, activation of enzymes (radical scavengers and vitamins), growth factors, activation of endothelial factors and activation of oxygen radical scavengers.

Oral administration of ethyl acetate-soluble portion of Terminalia chebula conferring protection from streptozotocin-induced diabetic mellitus and its complications.

Terminalia chebula has been widely used in India as a folk medicine. This study investigated the in vivo anti-hyperglycemia and anti-diabetic complication effects of the EtOAc-soluble portion of ethanolic extract of T. chebula fruit (EETC) containing 29.4% chebulic acid. Rats were divided into non-diabetic, untreated diabetic and diabetic groups. Streptozotocin (40 mg/kg body weight (BW))-induced diabetic rats were orally administered the aminoguanidine (100 mg/kg BW), high dose (500 mg/kg BW; HEETC) and low dose (100 mg/kg BW; LEETC) for 13 weeks. HEETC administration reduced the levels of blood glucose and serum lipids, decreased malondialdehyde concentrations of serum and thoracic aorta in diabetic rats, and significantly improved serum biochemical values and the pathomorphological changes of the liver and kidney in diabetic rats. Also, HEETC decreased the advanced glycation end products (AGEs) distribution in testis seminiferous tubules. Therefore, HEETC has a merit to be a potent candidate to control glycemic and diabetic complications.

Antioxidant protective effect of honey in cigarette smoke-induced testicular damage in rats.

Cigarette smoke (CS) can cause testicular damage and we investigated the possible protective effect of honey against CS-induced testicular damage and oxidative stress in rats. CS exposure (8 min, 3 times daily) and honey supplementation (1.2 g/kg daily) were given for 13 weeks. Rats exposed to CS significantly had smaller seminiferous tubules diameter and epithelial height, lower Leydig cell count and increased percentage of tubules with germ cell loss. CS also produced increased lipid peroxidation (TBARS) and glutathione peroxidase (GPx) activity, as well as reduced total antioxidant status (TAS) and activities of superoxide dismutase (SOD) and catalase (CAT). However, supplementation of honey significantly reduced histological changes and TBARS level, increased TAS level, as well as significantly restored activities of GPx, SOD and CAT in rat testis. These findings may suggest that honey has a protective effect against damage and oxidative stress induced by CS in rat testis.

Toxicant-induced leakage of germ cell-specific proteins from seminiferous tubules in the rat: relationship to blood-testis barrier integrity and prospects for biomonitoring.

Evaluation of testicular toxicity during drug development is currently based on histopathological evaluation. A sensitive biomarker for testicular toxicology could provide an in-life and "early warning" measurement. Previous studies suggested that disruption of spermatogenesis induced leakage of germ cell proteins from seminiferous tubules (STs) into interstitial fluid (IF); such proteins have potential for use as biomarkers. To investigate this possibility further, adult male rats were treated with three testicular toxicants thought to have differing sites of action; cadmium chloride affects the blood-testis barrier (BTB), methoxyacetic acid (MAA) disrupts pachytene spermatocytes, and 1,3-dinitrobenzene (DNB) targets Sertoli cells. IF proteins were assessed by Coomassie-based dye-stained gels. Immunostaining was used to identify toxicant-induced damage (DAZL) and BTB integrity (ZO-1, occludin, N-cadherin, and ß-catenin) and function (biotin). Cadmium chloride induced dose-dependent leakage of proteins from STs into IF coincident with loss of integrity and function of the BTB. Two of the "leaked" proteins were identified on Westerns as being germ cell specific, namely VASA and fatty acid-binding protein 9 (FABP9). In contrast, similar protein leakage was not evident after either MAA-induced or DNB-induced disruption of spermatogenesis and neither of these treatments affected BTB integrity or function. These results suggest that loss of BTB integrity is required for germ cell-specific proteins to leak from STs into IF, implying that use of such biomarkers has very limited potential for noninvasive monitoring of compound-induced disruption to spermatogenesis.

Expression of Pkd2l2 in testis is implicated in spermatogenesis.

Pkd2l2 is a novel member of the polycystic kidney disease (PKD) gene family in mammals. Prominently expressed in testis, this gene is still poorly understood. In this study, reverse transcription polymerase chain reaction (RT-PCR) results showed a time-dependent expression pattern of Pkd2l2 in postnatal mouse testis. Immunohistochemical analysis revealed that Pkd2l2 encoded a protein, polycystin-L2, which was predominantly detectable in the plasma membrane of spermatocytes and round spermatids, as well as in the head and tail of elongating spermatids within seminiferous tubules in mouse testis tissue sections of postnatal day 14 and adult mice. A green fluorescent fusion protein of Pkd2l2 resided in the plasma membrane of HEK 293 and MDCK cells, suggesting that it functions as a plasma membrane protein. Overexpression of Pkd2l2 increased the intracellular calcium concentration of MDCK cells, as detected by flow cytometry. Collectively, these data indicated that Pkd2l2 may be involved in the mid-late stage of spermatogenesis through modulation of the intracellular calcium concentration.

Gonadotropin-inhibitory hormone and its receptor in the avian reproductive system.

Many hormones that are classified as neuropeptides are synthesized in vertebrate gonads in addition to the brain. Receptors for these hormones are also expressed in gonadal tissue; thus there is potential for a highly localized autocrine or paracrine effect of these hormones on a variety of gonadal functions. In the present study we focused on gonadotropin-inhibitory hormone (GnIH), a neuropeptide that was first discovered in the hypothalamus of birds. We present different lines of evidence for the synthesis of GnIH and its receptor in the avian reproductive system including gonads and accessory reproductive organs by studies on two orders of birds: Passeriformes and Galliformes. Binding sites for GnIH were initially identified via in vivo and in vitro receptor fluorography, and were localized in ovarian granulosa cells along with the interstitial layer and seminiferous tubules of the testis. Furthermore, species-specific primers produced clear PCR products of GnIH and GnIH receptor (GnIH-R) in songbird and quail gonadal and other reproductive tissues, such as oviduct, epididymis and vas deferens. Sequencing of the PCR products confirmed their identities. Immunocytochemistry detected GnIH peptide in ovarian thecal and granulosa cells, testicular interstitial cells and germ cells and pseudostratified columnar epithelial cells in the epididymis. In situ hybridization of GnIH-R mRNA in testes produced a strong reaction product which was localized to the germ cells and interstitium. In the epididymis, the product was also localized in the pseudostratified columnar epithelial cells. In sum, these results indicate that the avian reproductive system has the capability to synthesize and bind GnIH in several tissues. The distribution of GnIH and its receptor suggest a potential for autocrine/paracrine regulation of gonadal steroid production and germ cell differentiation and maturation.