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Background: Inflammation-induced testicular damage is a significant contributing factor to the increasing incidence of infertility. Traditional treatments during the inflammatory phase often fail to achieve the desired fertility outcomes, necessitating innovative interventions such as cell therapy., Methods: We explored the in vivo properties of intravenously administered Sertoli cells (SCs) in an acute lipopolysaccharide (LPS)-induced inflammatory mouse model. Infiltrating and resident myeloid cell phenotypes were assessed using flow cytometry. The impact of SC administration on testis morphology and germ cell quality was evaluated using computer-assisted sperm analysis (CASA) and immunohistochemistry., Results: SCs demonstrated a distinctive migration pattern, importantly they preferentially concentrated in the testes and liver. SC application significantly reduced neutrophil infiltration as well as preserved the resident macrophage subpopulations. SCs upregulated MerTK expression in both interstitial and peritubular macrophages. Applied SC treatment exhibited protective effects on sperm including their motility and kinematic parameters, and maintained the physiological testicular morphology., Conclusion: Our study provides compelling evidence of the therapeutic efficacy of SC transplantation in alleviating acute inflammation-induced testicular damage. These findings contribute to the expanding knowledge on the potential applications of cell-based therapies for addressing reproductive health challenges and offer a promising approach for targeted interventions in male infertility., (© 2024. The Author(s).)

Infertility is a frequent long-term adverse effect of cancer therapy for children. Compromised testicular functions in adolescence are frequent observations after chemotherapy and there are currently no well-established alternatives to avoid this damage. Antimetabolites such as 6-mercaptopurine (6-MP) are used to treat a variety of cancer; however, its treatment-associated adverse effects on the male reproductive functions are overlooked. Here, the molecular processes underlying 6-MP-induced male germ cell damage in juvenile Sprague-Dawley (SD) rats (3 weeks) have been investigated. Rats were administered with low (5 mg/kg), medium (10 mg/kg), and high (20 mg/kg) doses of 6-MP per orally either singly (1 week × 1 cycle) or intermittently (1 week treatment followed by 1 week remission period × 3 cycles). The toxicity was evaluated in terms of genotoxicity and testes- and sperm-related cellular and molecular parameters. Single cycle of exposure either produced mild or no toxic manifestations at the end of the 6th week. Intermittent cycles of exposure, particularly at the 10 and 20 mg/kg, decreased body and organ weights, increased micronucleated cells in the peripheral blood, induced oxidative/nitrosative stress, altered sperm chromatin quality, reduced serum testosterone and follicle stimulating hormone (FSH) levels, increased testicular structural aberrations, DNA damage, and apoptosis, and upregulated TNF-α expression and downregulated p-AMPK and β-catenin expressions. Conclusively, intermittent cycles of exposure at 10 and 20 mg/kg doses to the juvenile rats significantly induced oxidative stress, genotoxicity, and cellular and molecular perturbations in the testes and sperm of adult rats., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The spatiotemporal expression patterns of genes are crucial for maintaining normal physiological functions in animals. Conditional gene knockout using the cyclization recombination enzyme ( Cre )/locus of crossover of P1 ( Cre / LoxP ) strategy has been extensively employed for functional assays at specific tissue or developmental stages. This approach aids in uncovering the associations between phenotypes and gene regulation while minimizing interference among distinct tissues. Various Cre -engineered mouse models have been utilized in the male reproductive system, including Dppa3 - MERCre for primordial germ cells, Ddx4 - Cre and Stra8 - Cre for spermatogonia, Prm1 - Cre and Acrv1 - iCre for haploid spermatids, Cyp17a1 - iCre for the Leydig cell, Sox9 - Cre for the Sertoli cell, and Lcn5/8/9 - Cre for differentiated segments of the epididymis. Notably, the specificity and functioning stage of Cre recombinases vary, and the efficiency of recombination driven by Cre depends on endogenous promoters with different sequences as well as the constructed Cre vectors, even when controlled by an identical promoter. Cre mouse models generated via traditional recombination or CRISPR/Cas9 also exhibit distinct knockout properties. This review focuses on Cre -engineered mouse models applied to the male reproductive system, including Cre -targeting strategies, mouse model screening, and practical challenges encountered, particularly with novel mouse strains over the past decade. It aims to provide valuable references for studies conducted on the male reproductive system.

Fumonisins (FBs) are widespread Fusarium toxins commonly found in corn. This study aimed to establish the mechanism of oxidative stress via the Nrf2 signaling pathway associated with FB 1 -induced toxicity in mice testis. Male mice were fed with 5 mg/kg FB 1 diet for 21 or 42 days, the expression of inflammatory related genes, apoptosis related genes and Nrf2 pathway genes were detected by RT-qPCR, Western blot and immunohistochemical. Furthermore, Sertoli cell was treatment with FB 1 . Cell viability was measured by CCK8 assay, ROS level and apoptosis related genes were detected by immunofluorescence staining. The results showed that FB 1 had toxic effects on testis, which could increase the ROS level of Sertoli cells, affect the Keap1-Nrf2 pathway related factors, destroy the oxidative balance of testis, lead to the occurrence of inflammation and the initiation of apoptosis, and finally destroy the testicular tissue structure and affect the formation of sperm., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Integrins are transmembrane receptors expressed in all nucleated mammalian cells, critically involved in cell-matrix adhesion and cell-cell interactions that modulate many signalling cascades. It is assumed that integrins also provide essential functions of the reproductive system. In this study, we describe the detailed localization and distribution of αV integrin in the plasma membrane of bull sperm head and tail. Integrin αV was observed in the area of forming acrosome in developing sperm since the stage of round spermatids and persists in the acrosome during epididymal maturation and ejaculation till the acrosomal exocytosis. We detected CD9 and CD81 tetraspanins as the potential partners of αV integrin. Their similar staining pattern in testicular tissue suggested the involvement of these molecules in the tetraspanin web of "testisomes". Moreover, the complex of αV with β1 and β3 integrin subunits cannot be excluded at least in sperm. The presented findings contribute to understanding the mutual action of integrins and tetraspanins during sperm development and maturation., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Anogenital distance is considered a non-invasive measure to assess the development and functionality of sexual organs in different animal species. Hence, this measurement could potentially be used during the selection of non-human primates for reproductive biotechnology programs. The aim of this study was to assess the correlation between anogenital distance and reproductive parameters in captive Saimiri collinsi., Methods: Eight mature S. collinsi males were evaluated. Body weight, reproductive hormone levels, testicular volume, and seminal parameters were determined, and their relationship with anogenital distance measurements was assessed., Results: Anogenital distance was correlated with seminal volume, sperm motility, vigor, and plasma membrane integrity, but not with body weight, reproductive hormones, and testicular volume., Conclusion: The determination of anogenital distance is a non-invasive method to predict seminal quality. This procedure has the advantage of providing andrologic information without a negative impact on animal welfare., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Diabetes is a chronic metabolic disorder characterized by hyperglycemia and associated with many health complications due to the long-term damage and dysfunction of various organs. A consequential complication of diabetes in men is reproductive dysfunction, reduced fertility, and poor reproductive outcomes. However, the molecular mechanisms responsible for diabetic environment-induced sperm damage and overall decreased reproductive outcomes are not fully established. We evaluated the effects of type 2 diabetes exposure on the reproductive system and the reproductive outcomes of males and their male offspring, using a mouse model. We demonstrate that paternal exposure to type 2 diabetes mediates intergenerational and transgenerational effects on the reproductive health of the offspring, especially on sperm quality, and on metabolic characteristics. Given the transgenerational impairment of reproductive and metabolic parameters through two generations, these changes likely take the form of inherited epigenetic marks through the germline. Our results emphasize the importance of improving metabolic health not only in women of reproductive age, but also in potential fathers, in order to reduce the negative impacts of diabetes on subsequent generations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zatecka, Bohuslavova, Valaskova, Margaryan, Elzeinova, Kubatova, Hylmarova, Peknicova and Pavlinkova.)

Improved knowledge about reproductive patterns and potential in male wolves (i.e., testicular development and size relative to age, pubertal age, and seasonal effects) is needed for evaluation and monitoring of reproductive outcomes in populations. Reproductive organs from 215 male wolves, culled as a result of licensed hunting, protective culling or from carcasses found were examined. The testes and epididymis were weighed and measured. There were biopsy samples collected from the testes and the cauda epididymis for histological determinations if there were spermatozoa in tissues collected. There were reproductive tissue analyses of 197 males while there were separate evaluations of tissues from ten cryptorchid animals. Juvenile wolves (< 1 year, n = 47) had a lesser body mass and mean testes mass than subadult (1-2 years, n = 71) and adult (>2 years, n = 79) males. Season also affected testicular characteristics of structures evaluated with subadult and adult males having a lesser mass during summer months (May-August). Of the 197 males, 70 % had spermatozoa in the seminiferous tubules and the cauda epididymis and were classified as being 'potentially fertile' when tissues were collected, while 22 % were classified as being non-fertile (no spermatozoa, including males that were pre-pubertal) and tissues of 8% could not be evaluated. When testes mass was greater, there was a greater likelihood that spermatozoa were present. There were seven of the ten cryptorchid males of the unilateral type. These testicular and epididymal findings will be useful for evaluating the reproductive potential and management of wolves in Scandinavia., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Heat stress is a major concern in animal reproduction, as testicular temperature must be 3-5 °C below body core temperature for production of motile and fertile sperm in mammals. Although recent studies concluded that increased temperature per se was the underlying pathophysiology of testicular impairment, more studies are required to better understand the mechanisms. Therefore, our objective was to investigate the impacts of mild acute heat stress on sperm and testes, and based on mRNA, elucidate involvement of StAR, Trp53 and Trp53-dependent intrinsic and extrinsic apoptotic pathways in pathophysiology of testicular heat stress. Forty-eight C57 BCL6 elite male mice were equally allocated into six groups, anesthetized and the distal third of their body immersed in a water-bath at 40 or 30 °C (heat treatment and control, respectively) for 20 min. Intervals from heat exposure (Day 0) to euthanasia were: 8 and 24 h and 7, 14 and 21 d (plus a control group at 14 d). The epididymides were excised, minced and placed in Tyrode albumin lactate pyruvate hepes (TALPH) at 37 °C for 15 min to recover sperm. Based on computer assisted sperm analysis (CASA), heat treatment reduced total and progressive motility ∼40% (P < 0.05) on Days 14 and 21. Furthermore, percentage morphologically normal sperm was significantly decreased on Day 7, with greater reductions on Days 14 and 21, mostly due to increased midpiece defects. Acrosome integrity (FITC PSA) was decreased ∼35% at 8 h (P < 0.05) and reached a nadir on Day 14. There were decreases (P < 0.05) in seminiferous tubule diameter and testicular weight (relative to body weight) on Day 14. Testicular RNA was extracted, reverse-transcribed and cDNA used for PCR. Expression of genes Hspa1b (Hsp70) and Gpx1 had 7- and 10-fold increases (P < 0.001 for each) at 8 and 24 h, respectively, with Hspa1b remaining upregulated at 24 h, whereas StAR peaked at Day 14 (15-fold, P < 0.0001) and had returned to baseline on Day 21. Both Trp53 and Casp8 were upregulated (P < 0.05) on Day 14, whereas Bcl-2 was decreased (P < 0.05) on Days 7 and 14. In conclusion, acute mild heat stress severely reduced sperm quality and based on mRNA, there was upregulation of chaperone and antioxidant systems and Trp53-dependent intrinsic and extrinsic apoptotic pathways, with deleterious effects on sperm, spermatocytes and spermatids. These findings provided insights into the pathophysiology of heat stress and should contribute to development of evidence-based approaches to mitigate effects of testicular heating., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Many aspects of primate sperm physiology and reproductive behavior have been influenced by sexual selection, especially in taxa exposed to sperm competition where females mate with multiple partners. Primate sperm diversity reflects therefore the evolutionary divergences of the different primate species and the impact of a combination of variables exerting selection pressures on sperm form, function, and competition. Thereby, mating systems, life cycle or ecological variables are some of the important factors driving sperm diversity and explaining variation in terms of sperm morphology, parameters or male sexual characters. Here, we address primate sperm diversity through a compilation of all data available in the literature concerning primate sperm parameters and relationships between them. We also review the factors that can influence primate sperm diversity (e.g. mating systems, trade-off between investments in precopulatory and postcopulatory sexual traits, male and female sexual behaviors, seasonality, social constraints, testosterone levels), and discuss also their relevance to our understanding of human reproduction., (Copyright © 2020 Elsevier B.V. All rights reserved.)

SARS-CoV-2 infection and pregnancy has been the topic of hundreds of publications over the last several months; however, few studies have focused on the implications of infection in early pregnancy and reproductive tissues. Here, we analyzed available evidence pertaining to SARS-CoV-2 infection, in early pregnancy, and in reproductive tissues. We searched PubMed and Embase databases in accordance with guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) for publications from inception to June 4, 2020. Four reviewers screened titles and abstracts and obtained full-text articles for analysis. Sixty-two studies were included in the review. Biological plausibility for infection with SARS-CoV-2 exists in testis, ovaries, and placenta as they express ACE2 receptor activity. In males, SARS-CoV-2 infection could lead to functional abnormalities leading to spermatogenic failure and male infertility. In females, an alteration of the ACE2 cascade via SARS-CoV-2 infection could lead to impairment in important follicular and luteal processes. There is also evidence of significant placental pathology in SARS-CoV-2 infection, but it is unclear what effects there may be for early pregnancy, though available data suggest less severe effects compared to other respiratory virus outbreaks. Further investigation is needed regarding SARS-CoV-2 in reproductive function and early pregnancy., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The roles of androgens in male reproductive development and function in zebrafish are poorly understood. To investigate this topic, we employed CRISPR/Cas9 to generate cyp11c1 (11β-hydroxylase) mutant zebrafish lines. Our study confirms recently published findings from a different cyp11c1-/- mutant zebrafish line, and also reports novel aspects of the phenotype caused by loss of Cyp11c1 function. We report that Cyp11c1-deficient zebrafish display predominantly female secondary sex characteristics, but may possess either ovaries or testes. Moreover, we observed that cyp11c1-/- mutant male zebrafish are profoundly androgen- and cortisol-deficient. These results provide further evidence that androgens are dispensable for testis formation in zebrafish, as has been demonstrated previously in androgen-deficient and androgen-resistant zebrafish. Herein, we show that the testes of cyp11c1-/- mutant zebrafish exhibit a disorganised tubular structure; and for the first time demonstrate that the spermatic ducts, which connect the testes to the urogenital orifice, are severely hypoplastic in androgen-deficient zebrafish. Furthermore, we show that spermatogenesis and characteristic breeding behaviours are impaired in cyp11c1-/- mutant zebrafish. Expression of nanos2, a type A spermatogonia marker, was significantly increased in the testes of Cyp11c1-deficient zebrafish, whereas expression of markers for later stages of spermatogenesis was significantly decreased. These observations indicate that in zebrafish, production of type A spermatogonia is androgen-independent, but differentiation of type A spermatogonia is an androgen-dependent process. Overall, our results demonstrate that whilst androgens are not required for testis formation, they play important roles in determining secondary sexual characteristics, proper organisation of seminiferous tubules, and differentiation of male germ cells.

Ketamine is increasingly used in clinical practice, and ketamine addiction is common in young individuals. There are limited reviews on the chronic effects of ketamine on the testes. Three groups of rats received saline or ketamine 50 mg/kg/day intraperitoneally for 6 weeks with or without a subsequent 4-week drug-free period. Serum follicle-stimulating hormone, luteinizing hormone, prolactin, and testosterone levels, as well as testicular malondialdehyde concentrations, were measured. Epididymal sperm parameters were assessed. Testicular tissues were examined by hematoxylin and eosin staining and immunohistochemical staining using caspase-3 and vimentin antibodies. Chronic ketamine injection significantly decreased the levels of the examined hormones and adversely affected sperm parameters. Testicular tissue showed a significant increase in caspase-3 expression. In addition, Sertoli cell shape and position were disrupted. These effects disappeared 4 weeks after drug withdrawal. Chronic ketamine treatment has revisable hazardous effects on the rat reproductive function. There is a need to increase the knowledge of physicians and the public regarding these harmful effects of ketamine.

This study was aimed at determining the effects of corn and wheat gluten, used as dietary protein sources, on live weight gain, sperm quality and the histology of the testes and accessory glands in male rats. For this purpose, 20-day-old 24 male Wistar albino rats were randomly assigned to three groups. Group 1 (Control), Group 2 and Group 3 were fed on a basal ration supplemented with high levels of soybean meal, corn gluten and wheat gluten, respectively, as a protein source. At the end of the study, when compared to Group 1, live weight values were determined to have increased in Group 3 and to have decreased in Group 2 (p < .05). Furthermore, sperm density, sperm motility, the dead/ live sperm ratio and testes weight were determined to have significantly decreased in Group 2, in comparison to Groups 1 and 3 (p < .05). The percentages of abnormal spermatozoon, and head, acrosome, mid-piece and tail abnormalities were high in Group 2 (p < .05). Histological examination demonstrated that, in Group 2, the diameter of the Tubulus Seminiferous Contortus (TSC) and the size of the Tubular Epithelial Cells (TE) were small, and the tubular and anatomical structure of the testes were shrunken and altered. Group 2 also presented with connective tissue increase and alveolar lumen enlargement in the prostate gland, and with connective tissue thickening, muscle tissue increase and secretory capacity decrease in the seminal vesicle (p < .05). Moreover, in Group 2, the Gl. Bulbourethral (Cowper's gland) presented with a decreased size and dilatations in the mucous structures. In a result, based on the findings obtained in this study, it is suggested that high levels of dietary corn gluten adversely affect live weight, sperm quality, and the testes and accessory glands., (© 2020 Blackwell Verlag GmbH.)

Estrogens are steroid hormones that affect a wide range of physiological functions. The effect of estrogens on male reproductive tissues and sperm cells through specific receptors is essential for sperm development, maturation, and function. Although estrogen receptors (ERs) have been studied in several mammalian species, including humans, they have not yet been described in bull spermatozoa and reproductive tissues. In this study, we analyzed the presence of all types of ERs (ESR1, ESR2, and GPER1) in bull testicular and epididymal tissues and epididymal and ejaculated spermatozoa, and we characterize them here for the first time. We observed different localizations of each type of ER in the sperm head by immunofluorescent microscopy. Additionally, using a selected polyclonal antibody, we found that each type of ER in bull sperm extracts had two isoforms with different molecular masses. The detailed detection of ERs is a prerequisite not only for understanding the effect of estrogen on all reproductive events but also for further studying the negative effect of environmental estrogens (endocrine disruptors) on processes that lead to fertilization., Competing Interests: The authors declare no conflict of interest.

Equine chorionic gonadotrophin (eCG) is a hormone having FSH/LH effects. It can be used to enhance sperm quality in male goats (bucks) during the non-breeding season. In a previous study carried out during the non-breeding season, we treated ten bucks with eCG (leaving nine untreated animals as control). Over a 20-day period, the treated bucks received an initial dose of 800 IU of eCG, followed by four doses of 500 IU. We found eCG enhanced semen quality, however, as also happens in female goats (does), eCG also induced a high titer of anti-eCG antibodies. In does, this lowers fertility. The aim of the present study was to determine if the eCG treatment carried out on bucks during the non-breeding season had any negative effects on their reproductive status during the following breeding season. We measured serum concentration of testosterone and anti-eCG antibody, as well as key testicular and seminal characteristics. This study commenced 91 days after the final dose of eCG in the previous study. The anti-eCG titer was higher in the treatment bucks than in untreated ones (181.7 ± 61.3 ng/μL vs 31.1 ± 10.7 ng/μL; P < 0.05). However, there were no significant differences between treated and untreated bucks in testosterone concentration, scrotal circumference, testes pixel intensity, fresh and thawed semen characteristics, or sperm cryoresistance. So, although the eCG-treated bucks had greater titers of anti-eCG antibodies, their reproductive pattern was unaffected.

Zika virus (ZIKV) infection in pregnant women is a serious threat to the development and viability of the fetus. The primary mode of ZIKV transmission to humans is through mosquito bites, but sexual transmission has also been well documented in humans. However, little is known of the short- and long-term effects of ZIKV infection on the human male reproductive system. This study examines the effects of ZIKV infection on the male reproductive organs and semen and the immune response of the olive baboon ( Papio anubis ). Nine mature male baboons were infected with ZIKV (French Polynesian strain) subcutaneously. Six animals were euthanized at 41 days, while three animals were euthanized at 10 or 11 days postinfection (dpi). Viremia and clinical evidence of infection were present in all nine baboons. ZIKV RNA was present in the semen of five of nine baboons. ZIKV was present in the testes of two of three males euthanized at 10 or 11 dpi, but in none of six males at 41 dpi. Immunofluorescence of testes suggested the presence of ZIKV in sperm progenitor cells, macrophage penetration of seminiferous tubules, and increased tumor necrosis factor alpha (TNF-α), particularly in vascular walls. These data demonstrate that male olive baboons approximate the male human ZIKV response, including viremia, the adaptive immune response, and persistent ZIKV in semen. Although gross testicular pathology was not seen, the demonstrated breach of the testes-blood barrier and targeting of spermatogenic precursors suggest possible long-term implications in ZIKV-infected primates. IMPORTANCE Zika virus (ZIKV) is an emerging flavivirus spread through mosquitoes and sexual contact. ZIKV infection during pregnancy can lead to severe fetal outcomes, including miscarriage, fetal death, preterm birth, intrauterine growth restriction, and fetal microcephaly, collectively known as congenital Zika syndrome. Therefore, it is important to understand how this virus spreads, as well as the resulting pathogenesis in translational animal models that faithfully mimic ZIKV infection in humans. Such models will contribute to the future development of efficient therapeutics and prevention mechanisms. Through our previous work in olive baboons, we developed a nonhuman primate model that is permissive to ZIKV infection and transfers the virus vertically from mother to fetus, modeling human observations. The present study contributes to understanding of ZIKV infection in male baboon reproductive tissues and begins to elucidate how this may affect fertility, reproductive capacity, and sexual transmission of the virus., (Copyright © 2019 American Society for Microbiology.)

Spermatogenesis and spermatozoa ultrastructure of the amphibian leech Batracobdella algira Moquin-Tandon, 1846 (Hirudinida: Glossiphoniidae) have been investigated by means of electron and fluorescent microscopy. In B. algira, there are seven pairs of testisacs (testes) that are located latero-ventrally throughout the body. Each testis contains numerous cysts with developing germ cells. The germ cells in a given cyst are in the same developmental stage (i.e., there are spermatogonial, spermatocytic, and spermatid cysts); however, there is no developmental synchrony between the cysts, and therefore, all of the developmental stages occur simultaneously in the same testis. In the cysts, each germ cell is connected to acentral cytoplasmic mass, the cytophore, by one intercellular bridge. The spermatozoa of the studied species conform to the general organization plan that is known for Hirudinida: they are filiform cells that are formed in sequence by an elongated and twisted acrosome that consists of an anterior and posterior acrosome, a fully condensed and helicoid nucleus, a midpiece composed of a single and twisted mitochondrion that is characteristically surrounded by an electron-dense sheath, and a flagellum with the conventional 9 × 2 + 2 axonemal pattern. Using a comprehensive approach, we compared our findings with the ultrastructural data that had been obtained from the spermatozoa of the other hirudinids that have been studied to date. Only minor differences in the length and shape of the studied organelles were found which seems to be connected with the different ways of insemination, specific properties of female reproductive tracts, and physiology of fertilization. Additionally, we studied the organization of the microtubular cytoskeleton in male germline cysts at consecutive stages of spermatogenesis using fluorescent and electron microscopy. By comparing the present data with those from Oligochaeta, Branchiobdellida, and Acanthobdellida, we found that only the presence of an anterior acrosome characterizes the true leeches and that, at present, should be regarded as an autapomorphic character of Hirudinida. Our results showed that the arrangement of the microtubules changed dynamically during spermatogenesis.

The present study aimed to evaluate the protective role of Selenium (Se) (0.1 ppm) on the male reproductive system of the catfish Clarias gariepinus exposed to sublethal doses of Mercury (Hg) (0.04 and 0.12 ppm) for 30 days. Indicators of seminal and gonadal hormone disruption (testosterone, estradiol and 11 keto testosterone), antioxidants (total antioxidant capacity (TAO), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)), oxidative stress biomarkers (lipid peroxidation (LPO), percentage of DNA fragmentation, carbonylated proteins (CP) and nitric oxide (NO)) and histopathological alterations in testicles of Clarias gariepinus were determined. The exposure to Hg resulted in a high accumulation of residues of this metal in testicular tissues. The results showed a significant decrease in sperm count, activity and motility and in all gonadal hormones in Hg exposed groups. Hg exposure also induced a decline in TAO, SOD, CAT and GPx, whereas LPO, DNA fragmentation, CP and NO significantly increased in testicles of C. gariepinus respect to the control group. Although exposure to Se did not reduce the degree of mercury bioconcentration in the testicles, the sperm quality parameters were recovered. Moreover, TAO levels and GPx activity significantly increased after fish exposure to Se, whereas CP levels decreased. LPO, NO, CAT and SOD were also partially normalized when compared with the groups exposed to only Hg. In conclusion, the results showed that Hg, even in the small doses is capable to induce reproductive toxicity in the male catfish. Se exposure partially restored the values of biochemical parameters and sperm quality in Hg-treated fish suggesting protective effects against Hg reproductive toxicity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The effect of glyphosate, both pure and formulated (Roundup Ultramax®), was evaluated on males of the estuarine crab Neohelice granulata, by means of both in vivo and in vitro assays. The in vivo assays comprised the exposure for 30 d to 1 mg/L of the herbicide, until finally assessing weight gain, levels of energy reserves, sperm number per spermatophore, proportion of abnormal spermatophores, and sperm viability. At the end of this assay, significant (p < 0.05) decrease in weight gain and muscle protein levels was detected by effect of both pure and formulated glyphosate. In spermatophores from the vas deferens, a significant (p < 0.05) decrease of the sperm count was observed by effect of Roundup, while a significant incidence (p < 0.05) of abnormal spermatophores was observed either with glyphosate or with Roundup treatment. No changes were seen in the spermatophore area or in vas deferens secretions. Since no sperm mortality was induced by the formulated herbicide, we propose a probable inhibiting effect on spermatogenesis might explain the observed sperm count decrease. In this sense, an in vitro assay was designed by incubating testes and vasa deferentia with Roundup, in order to corroborate the possible interference of glyphosate with the secretion of the androgenic gland hormone that controls the spermatogenesis, in the presence or absence of the androgenic gland. Although the herbicide per se was able to reduce the sperm count to some extent, the increase in the number of spermatozoa/spermatophore produced by the co-incubation with the androgenic gland was completely reverted by the addition of Roundup (1 mg/L of glyphosate a.e.), suggesting that an inhibition on the secretion and/or transduction of the androgenic gland hormone could be taking place., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Studying reproductive trait allometries can help to understand optimal male investment strategies under sexual selection. In promiscuous mating systems, studies across several taxa suggest that testes allometry is usually positive, presumably due to strong selection on sperm numbers through intense sperm competition. Here, we investigated testes allometry in a bush-cricket species, Metaplastes ornatus, in which females mate promiscuously, but where sperm removal behaviour by males likely drastically reduces realised sperm competition level., Results: As hypothesised, we found evidence for negative testes allometry and hence a fundamentally different male investment strategy compared to species under intense sperm competition. In addition, the mean relative testes size of M. ornatus was small compared to other species of bush-crickets. Surprisingly, the spermatophore gland, a potential alternative trait that males could invest in instead of testes, also did not show positive allometry, but was approximately isometric. We further observed the expected pattern of negative allometry for the male morphological structure responsible for sperm removal in this species, the subgenital plate, supporting the one-size-fits-all hypothesis for intromittent genitalia., Conclusion: Our findings suggest that the evolution of sperm removal behaviour in M. ornatus was a key adaptation for avoiding sperm competition, with important consequences for reproductive trait allometries. Nevertheless, they also imply that it does not pay for larger males to invest disproportionately in nuptial gift production in this species.

In both plants and animals, male gametogenesis is particularly sensitive to heat stress, to the extent that a single hot or cold day can compromise crop productivity or population persistence. In animals, heat stress during development can impact a male's ability to secure copulations and/or his post-copulatory fertility. Despite such observations, relatively few studies have examined the consequences of developmental temperature on the reproductive behaviour and physiology of individuals. Here, we report for the first time the effects of developmental temperature on the phenotypic expression of both apyrene and eupyrene sperm and the copulatory behaviour of the Indian meal moth, Plodia interpunctella. We show that the length of both apyrene and eupyrene sperm decreases with increasing developmental temperature and that males are less likely to engage in copulation when reared at the highest and lowest temperatures. Where copulation occurred, the duration of copula decreased as male developmental temperature increased. We argue that identification of the mechanisms and consequences of reproductive failure in animals facing heat stress will help understand how wild and domesticated populations will respond to global climate change. We also contend that such studies will help elucidate long-standing evolutionary questions around the maintenance of genetic variation in traits highly relevant to fitness and the role of phenotypic plasticity in driving the evolution of novel traits., (© 2019 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2019 European Society For Evolutionary Biology.)

The exposure and health effects of fluoride are an ongoing topic that has attracted worldwide attention. Fluoride exposure disturbs the testicular development, sexual hormone levels and spermatogenesis. However, as to whether fluoride interferes with acrosome formation which is essential for production of capable spermatozoa during spermatogenesis still remains unclear. The objective was to determine the effects of fluoride on the acrosome formation and to further elucidate the potential mechanism of impaired reproductive function. For this, forty adult rats were assigned into four groups. The control group received distilled water, while the other three groups were treated with 25, 50 and 100 mg NaF/L via drinking water for 56 d, respectively. Testes were processed for total RNA extraction and western blot analysis. Three samples of each group were fixed in 2.5% glutaraldehyde solution for transmission electron microscopy analysis. From the results, we first found that fluoride decreased the expression of mRNA and protein levels of Zpbp1, Spaca1 and Dpy19l2 of seven markers during acrosome biogenesis in testes. Furthermore, fluoride damaged not only the acrosome structure, but also the structure of the nuclear lamina which was observed to be discontinuous and partially missing by transmission electron microscopy. Moreover, the results indicated that the altered structure in nuclear lamina maybe due to reduced LMNB2 expression in testis induced by fluoride. In a nutshell, fluoride exposure could restrain acrosome biogenesis during spermatogenesis and contribute to the elucidation of the underlying mechanisms of fluoride-induced male reproductive toxicity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Insulin-degrading enzyme (IDE) is a zinc metalloprotease responsible for degrading and inactivating several bioactive peptides, including insulin. Individuals without this enzyme or with a loss-of-function mutation in the gene that codifies it, present hyperinsulinemia. In addition, impairment of IDE-mediated insulin clearance is associated with the development of metabolic diseases, namely prediabetes. Although insulin regulates male fertility, the role of IDE on male reproductive function remains unknown. We proposed to study the influence of IDE in the reproductive potential of males. As insulin mediates key events for the normal occurrence of spermatogenesis, we hypothesized that IDE functioning might be linked with sperm quality. We used C57BL/6N mice that were divided in three groups according to its genotype: wild type (WT), heterozygous and knockout (KO) male mice for Ide. Spermatozoa were collected from the cauda of epididymis and sperm parameters were evaluated. Testicular tissue morphology was assessed through hematoxylin and eosin stain. Mitochondrial complex protein levels and lipid peroxidation were also evaluated in the testicular tissue. Our results show that KO mice present a 50% decrease in testes weight compared to WT mice as well as a decrease in seminiferous tubules diameter. Moreover, KO mice present impaired sperm quality, namely a decrease in both sperm viability and morphology. These results provide evidence that IDE plays an important role in determining the reproductive potential of males., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Crab shell (CS), which contains antioxidant compounds, has been used as a promising nutritional and medical compound in traditional medicine. Oxidative stress plays a critical role in the development of diabetes. This study aims to investigate the effects of CS extract (CSE) on sperm parameters, antioxidant status, and histopathology changes of testes in diabetic rats. In this experimental study, 40 Wistar male rats were investigated in five groups (n = 8/group): diabetic rats treated with different CS concentrations (i.e., 100, 200, and 400 mg/kg), diabetic group, and nondiabetic control. To induce diabetes, a single dose (60 mg/kg) of streptozotocin (STZ) was injected intraperitoneally; three days later, treatment with CSE was begun and conducted for 14 days. The fasting blood glucose, testes weight, and viability, number, and motility of sperm were assessed. In addition, the levels of ferric reducing antioxidant power (FRAP) and nitric oxide (NO) were estimated in the testes. Testes were examined using histological analysis. The results of this study revealed that sperm number, motility, and serum testosterone levels of CSE-treated diabetic rats increased significantly (p = .000) compared with the untreated diabetic group in a dose-depended manner while the number of immotile sperm decreased significantly (p = .017). CS also reduced the testicular level of nitric oxide and fasting blood glucose; however, it led to significant growth in the FRAP levels of testes (p = .002). Our results suggest that CSE improves sperm parameters and protects the testicular tissue against the oxidative stress damage induced by diabetes.

The organochlorine pesticides aldrin (0.14 μg/L) and methoxychlor (0.23 μg/L) were both present in the Albasini Dam, Limpopo Province, South Africa, during a field survey in 2014. The use of aldrin has been banned in the USA since 1987 and restricted in South Africa since 1992. The use of methoxychlor, however, remains undefined with little information available about its registration in South Africa despite being banned in Europe (2002) and USA (2003). The aim of this study was to determine the potential effects of environmentally relevant concentrations of aldrin and methoxychlor on the reproductive system of male catfish, Clarias gariepinus. Males were exposed for 96 h to the two pesticides under controlled laboratory conditions. Following exposure, each fish was weighed and measured, and a necropsy performed to determine any macroscopic abnormalities and the general health of the fish. The fish were killed and dissected and the testes removed, weighed and measured to determine the gonadosomatic index (GSI). The right testis of each fish was sectioned for histopathological assessment and to calculate the testes index (I T ). The left testis was used for computer-assisted sperm analysis (CASA). The histopathological assessment of the testes showed histopathological changes such as of melano-macrophage centres (MMCs) and vacuolation of spermatogonia and spermatocytes. However, the classification of these changes indicated that the testes tissue structure was normal with slight histological changes. No statistically significant differences (p > 0.05) were found in the CASA parameters between exposure groups. The results of this study showed that the environmentally relevant concentrations of aldrin and methoxychlor did not have a negative effect on the motility of the mature sperm, but adverse effects were noted in the early stages of spermatogenesis, indicating possible effects over longer exposure periods.

The growing patronage on herbal remedies and formulations from natural products in most developing countries has warranted research into certain health challenges including their antifertility effects. This study assessed the effects of boiled Telfairia occidentalis (TO) seed-supplemented diets on the level of testosterone and semen quality in Wistar rats. Boiled TO seed diets at 10%, 15% and 30% were given to rats for 60 days. Our study showed that sperm quality was impaired as evidenced by the decreased number of motile spermatozoa, epididymal sperm numbers, percentage live/dead ratio and increased numbers of abnormal spermatozoa comparable to control values (p < .05). Feeding of rats with 10% and 15% TO seed-supplemented diets increased testosterone levels nonsignificantly, while in the 30% TO seed diet animals, the level of serum testosterone was found to decrease significantly compared to control values. Furthermore, TO diet caused a nonsignificant increase in the activity of superoxide dismutase and the concentrations of reduced glutathione and malondialdehyde except for the significant increase in malondialdehyde level in the testes of the 10% TO diet group. A nonsignificant decrease in myeloperoxidase activity was also observed in the 10% and 15% but not 30% TO diet group. Histological damages characterised by severe loss of germ cells were more pronounced in the 10% TO diet group. Fourier transform infrared spectroscopy analysis of boiled TO seeds revealed the presence of esters, alkenes, hydroxyl and alcohol functional groups. Thus, boiled TO seed-supplemented diet evoked antifertility effects in rats, and the effects on the toxicity end points investigated were not dose-dependent., (© 2018 Blackwell Verlag GmbH.)

The toxicity of metallic nanoparticles is a growing concern due to its application in industries and homes. We investigated the toxicity of cerium oxide nanoparticles (CeO 2 NPs) on reproductive system in male balb/c mice. Twenty mice were divided into four groups of five animals each and treated thus: normal saline (control), 100, 200 and 300 μg/kg CeO 2 NPs (i.p.,) thrice in a week for five consecutive weeks. Results showed that CeO 2 NPs significantly reduced the levels of haemoglobin, PCV and RBC count relative to controls. In addition, luteinising and follicle-stimulating hormones (FSH and LH) and prolactin were significantly reduced in the mice. Specifically, CeO 2 NPs at 100 μg/kg decreased testosterone by 23%, while CeO 2 NPs at 200 μg/kg decreased FSH, LH and prolactin by 25%, 26% and 13%, respectively. Testicular malondialdehyde was increased by 103%, 106% and 135% in mice treated with 100, 200 and 300 μg/kg CeO 2 NPs, respectively. CeO 2 NPs caused a significant reduction in activities of antioxidant enzymes and levels of reduced glutathione and total nitric oxide. Moreso, CeO 2 NPs decreased sperm motility and count and increased total sperm abnormality in mice. Histology revealed congestion and degeneration of seminiferous tubules. Overall, CeO 2 NPs induces testicular dysfunction via disruption of antioxidant/oxidant balance and endocrine suppression., (© 2017 Blackwell Verlag GmbH.)

The H19/insulin-like growth factor 2 (Igf2) gene plays vital roles during development, but their expression and methylation status have not been elucidated in male cloned animals. In this study, we investigated the methylation status of imprinting control region (ICR) and differentially methylated region (DMR) of the H19/Igf2 locus in the sperm and testes of naturally reproduced Saanen dairy goats (NG, control), transgenic cloned dairy goats (CG), and the first generation of mating the CGs with the female NGs (FG). Bisulfite sequencing polymerase chain reaction revealed that the H19 ICR was highly methylated in the sperm of all testing groups (97.07% ± 0.47%, 93.73% ± 0.85%, and 96.67% ± 0.54%). Notably, the methylation level of H19 ICR was decreased in the testes of CG goats compared with that of NG goats (73.83% ± 3.28% vs. 97.27% ± 0.71%, p < 0.05), while the Igf2 DMR2 was highly methylated in the sperm of these three groups (89.57% ± 1.74%, 87.23% ± 1.11%, and 83.2% ± 1.27%). However, the Igf2 DMR2 was abnormally hypomethylated in the testes of CG goats (27.33% ± 1.005%, p < 0.001), and moderately methylated in FG goats (53.1% ± 1.47%, p < 0.01) compared with that in NG goats (82.67% ± 0.70%). Furthermore, we found significantly decreased expression of H19 (p < 0.01) and increased expression of Igf2 (p < 0.01) in CG goats. Thus, we infer that somatic cell nuclear transfer has clear effect on genomic imprinting in the testes, but not sperm of cloned goats.

Background/aims: Changes in the external mechanical field result in cytoskeleton reorganization and the formation of adaptive patterns in different types of cells, including somatic cells and sex cells. The aim of this research was to study the protein and mRNA content of cytoskeletal and sperm-specific genes in the sperm and testis cells of mice., Methods: Mice were subjected to 30 days of antiorthostatic suspension to simulate weightlessness, followed by 12 h of recovery, while receiving essential phospholipids at a dosage of 500 mg/kg/day (30HSE and 30HSE+12h groups) or a similar dosage of a placebo (30HS and 30HS+12h groups). Accordingly, reference groups (CE group and C group) were formed. The total number and the percentage of motile spermatozoa were calculated using a Makler chamber. To analyze the number of viable spermatozoa and the permeability of their membranes, eosin staining was used as well as Diff-Quick for a morphological evaluation. Relative protein and mRNA content was estimated in a western blot and quantitative PCR assay, respectively., Results: The relative protein expression levels of actin (beta and gamma) and two alpha-actinin isoforms (1 and 4) remained constant in the sperm of all study groups, except for the 30HS+12h group, where the alpha-actinin-4 level was 13% higher than in the reference group (p < 0.1). In the testis cells, the relative actin isoform content was equivalent to that in the spermatozoa. However, in the testis cells, the ACTN1 mRNA content was 17% higher in the 30HS group than in the C group (p < 0.05), and decreased after 12 h of recovery. In contrast, the ACTN4 mRNA content was 20% lower in the 30HS group than in the reference group (p < 0.05) and increased after the 12-h recovery period. At the same time, in the group administered the essential phospholipids, the relative ACTN1 and ACTN4 mRNA content did not differ from those of the reference group. The relative beta-tubulin content was similar in the reference C group and the reference CE group, which was administered the essential phospholipids. In the 30HS and 30HS+12h groups, the beta-tubulin content decreased by 19% and 22% (p < 0.05), respectively, and they also decreased in the groups administered the essential phospholipids (30HSE and 30HSE+12h groups, by 27% and 33%, respectively, p < 0.05). In the testis tissue, the relative tubulin content did not change in any of the experimental groups. At the same time, the relative mRNA content of the genes encoding the studied cytoskeletal proteins increased, which may indicate the protein content was regulated mainly at the translational level., Conclusion: The spermogram parameters and the content of the sperm-specific proteins and the associated mRNAs revealed a decrease in the number of mature spermatozoa in mice suspended under conditions of weightlessness. Moreover, the decrease was prevented by the administration of essential phospholipids., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Previous studies have indicated that fluoride exposure damaged the male reproductive function; however, the cellular mechanism of fluoride-induced testicular toxicity is still unclear. In this study, twenty-two female pregnant Wistar rats were allotted randomly to two groups: control (deionized water) and sodium fluoride (NaF, contain F - : 67.86 mg/L) groups. After delivery, the dosage was continued for 15 weeks for puppies. Twelve rats in each group were tested at 6 and 9 (pubertal); 12 and 15 (mature) weeks of age. Our results suggested that organ coefficient of epididymis was significantly decreased in the mature (12 and 15 week-old) rats. Epididymal sperm abnormality and femur fluoride concentration were increased with the concomitant decrease in sperm motility and concentration in these experimental periods. Compared to the control, in the NaF group, the seminiferous tubules of each age were reduced in terms of diameter and thickness. The sperm cells were lost and shedding and finally disappeared after 9 weeks. mRNA and protein levels of HSP27 and 90 were decreased with a concomitant increase in HSP70 and HSF mRNA and protein levels in NaF exposed rats. The mRNA and protein levels of HSP27 and HSF (only mRNA) were significantly increased in NaF treated rats at 9 and 15 weeks of age, respectively. In summary, these results emphasize that NaF induces testicular and sperm abnormalities through the involvement of HSPs especially during the pubertal period., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Onabotulinumtoxin A (onabotA) is gaining wide medical use in children. The present study was planned to investigate the influence of its injection on the maturing testicular structures in rats. Immature rats were injected in the bilateral cremaster muscles by onabotA with three doses of (10, 20, and 40 U/kg) three times in a 2-week interval. The effect of these injections on fertility indices was examined. Levels of antisperm antibodies and several apoptosis parameters were also investigated. DNA content in form of ploidy and histopathological alterations were assessed. OnabotA-injected groups showed decreased sperm count and semen quality, while sperm vitality, morphology, and testosterone levels were not significantly affected. Furthermore, DNA flow cytometric analysis confirmed delayed sperm maturation. Apoptosis markers were significantly increased by the injections. In conclusion, onabotA injection in growing rats adversely affected sperm count and maturation. OnabotA testicular effects are mediated, at least partly, by apoptosis., (© 2016 Wiley Periodicals, Inc.)

Objectives: Exposure to 2,5-hexanedione (2,5-HD) is well known to be associated with reproductive dysfunctions in both humans and animals. However, the role of oxidative stress in 2,5-HD-induced toxicity in testes and sperm has not yet been studied., Methodology: The present study investigated the influence of 2,5-HD on antioxidant systems in the testes and epididymal sperm of rats following exposure to 0, 0.25, 0.5, and 1% 2,5-HD in drinking water for 21 consecutive days., Results: Administration of 0.5% 2,5-HD significantly (P < 0.05) decreased epididymis weight, whereas 1% 2,5-HD-treated rats showed significantly decreased body weight, testis, and epididymis weights compared with the control group. Exposure to 2,5-HD caused a significant dose-dependent increase in the activities of superoxide dismutase, catalase, and glutathione peroxidase in both testes and sperm compared with the control group. Moreover, 2,5-HD-exposed rats showed significant decrease in glutathione-S-transferase activity and glutathione level with concomitant significant elevation in the levels of hydrogen peroxide and malondialdehyde in both testes and sperm. Testicular and epididymal atrophy with significant, dose-dependent, decrease in epididymal sperm number, sperm motility, and viability were observed in 2,5-HD-treated rats., Conclusion: 2,5-HD exposure impaired testicular function and sperm characteristics by disruption of the antioxidant systems and consequently, increased oxidative stress in the treated rats.

The main objective of this study was to evaluate the efficacy of an additional cryoprotectant in 10% dimethyl sulfoxide (DMSO) on cryopreserving germ cells from stallions at different reproductive stages. Testicular samples were obtained from pre-pubertal (1-1.5 yr, n=6) and post-pubertal (3-7 yr, n=5) stallions. Germ cells were isolated using a two-enzyme digestion procedure and cryopreserved in minimal essential medium alpha containing 10% fetal bovine serum and 10% DMSO with or without addition of trehalose (50, 100, or 200mM) or polyethylene glycol (PEG, 2.5, 5, or 10%). Viability, cell population, and viable population were assessed after 1 and 3 months of cryopreservation. The viable UTF1-positive population of pre-pubertal stallion germ cells was also measured using immunocytochemistry after 1 and 3 months of cryopreservation. As expected, the viability, cell population, and viable cell population were significantly reduced after 1 and 3 months of cryopreservation. At the pre-pubertal stage, the addition of trehalose or PEG to 10% DMSO did not show any effect on the viability, cell population, viable cell population, or viable UTF1-positive germ cells at either 1 or 3 months after cryopreservation. However, at the post-pubertal stage, the viable population was significantly higher in germ cells that were cryopreserved with 5% or 10% PEG, than in the cells cryopreserved with 10% DMSO only. In conclusion, PEG at 5% or 10% added to 10% DMSO serves as an optimal cryoprotectant agent for the cryopreservation of germ cells from post-pubertal stallions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

It is known that during spermatogenesis, pluripotent germ cells differentiate to become efficient delivery vehicles to the oocyte of paternal DNA, and the process is easily damaged by external poison. In this study, the effects of fluoride on the body weight, fluoride content in femur, testosterone levels in serum and testis, sperm quality, and the expressions of Y chromosome microdeletion genes and protein levels were examined in testes of Kunming male mice treated with different concentrations of 0, 25, 50, 100 mg/L of NaF in drinking water for 11 weeks, respectively. The results showed that compared with the control group, fluoride contents in three treatment groups were significantly increased and the structure of testes was seriously injured. The testosterone contents and the sperm count were decreased. Sperm malformation ratio was distinctly elevated. The expressions of Sly and HSF2 mRNA were markedly reduced in 100 mg/L NaF group and Ssty2 mRNA expression was dramatically decreased in 50 and 100 mg/L NaF groups. Meanwhile, the protein levels of Ssty2 and Sly were significantly reduced in 50 and 100 mg/L NaF groups and HSF2 protein levels were significantly decreased in 100 mg/L NaF group. These studies indicated that fluoride had toxic effects on male reproductive system by reducing the testosterone and sperm count, and increasing the sperm malformation ratio, supported by the damage of testicular structure, as a consequence of depressed HSF2 level, which resulted in the down-regulation of Ssty2 and Sly mRNA and protein., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Valproic acid (VPA) is a drug widely use for the treatment of epilepsy in both children and adults. Evidence suggests that long-term use of VPA may lead to an impairment in the male reproductive function. Oxidative stress is considered to play a major role in VPA associated toxicity. In the present work, we demonstrated that the natural antioxidant compound resveratrol (RSV) can be use to prevent VPA oxidative damage. Wistar rats treated with VPA (400mgkg(-1)) by gavage for 28days showed decrease in sperm motility accompanied by increase in oxidative damage to lipids and proteins. Additionally, VPA administration leaded to depletion of reduced glutathione and decrease in total antioxidant potential in testes and epididymides of Wistar rats. The co-administration of RSV (10mgkg(-1)) efficiently prevented VPA pro-oxidant effects. In summary, RSV was shown to protect the reproductive system from the damage induced by VPA. Altogether, our data strongly suggests that RSV administration might be a valuable strategy to minimize reproductive impairment in patients requiring long-term VPA treatment., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Copper overload can cause sperm cell damage by inducing oxidative stress. On the other hand, cumin has a good antioxidant potential. Therefore, the aim of this study was to evaluate the effects of cumin on sperm quality and testicular tissue following experimentally induced copper poisoning in mice. Forty-eight mature male mice were divided into four equal groups as follows: group Cu which received 0.1 ml copper sulphate at dose of 100 mg kg(-1) , group Cc which received Cuminum cyminum at dose of 1 mg kg(-1) , treatment group which received copper sulphate (100 mg kg(-1) ) and treated with Cuminum cyminum (1 mg kg(-1) ), and control group which received the same volume of normal saline. Six mice in each group were sacrificed at week 4 and week 6. The results showed that sperm concentration, motility and viability in group Cu were significantly decreased at weeks 4 and 6, and severe degenerative changes were observed in testicular tissues in comparison with the control group. In treatment group, significant improvement in the sperm count, motility and viability, and normal architecture in most seminiferous tubules with organised epithelium was observed compared to the group Cu. The sperm quality parameters in the treatment group approached those of the control group., (© 2015 Blackwell Verlag GmbH.)

Study Hypothesis: We hypothesize that fertility disorders in patients with aberrant expression of Cysteine-RIch Secretory Protein 2 (CRISP2) could be linked to the proposed functional role of this protein in fertilization., Study Finding: Our in vivo and in vitro observations reveal that Crisp2-knockout mice exhibit significant defects in fertility-associated parameters under demanding conditions, as well as deficiencies in sperm fertilizing ability, hyperactivation development and intracellular Ca(2+) regulation., What Is Known Already: Testicular CRISP2 is present in mature sperm and has been proposed to participate in gamete fusion in both humans and rodents. Interestingly, evidence in humans shows that aberrant expression of CRISP2 is associated with male infertility., Study Design, Samples/materials, Methods: A mouse line carrying a deletion in the sixth exon of the Crisp2 gene was generated. The analyses of the reproductive phenotype of Crisp2(-/-) adult males included the evaluation of their fertility before and after being subjected to unilateral vasectomy, in vivo fertilization rates obtained after mating with either estrus or superovulated females, in vitro sperm fertilizing ability and different sperm functional parameters associated with capacitation such as tyrosine phosphorylation (by western blot), acrosome reaction (by Coomassie Blue staining), hyperactivation (by computer-assisted sperm analysis) and intracellular Ca(2+) levels (by flow cytometry)., Main Results and the Role of Chance: Crisp2(-/-) males presented normal fertility and in vivo fertilization rates when mated with estrus females. However, the mutant mice showed clear defects in those reproductive parameters compared with controls under more demanding conditions, i.e. when subjected to unilateral vasectomy to reduce the number of ejaculated sperm (n = 5; P< 0.05), or when mated with hormone-treated females containing a high number of eggs in the ampulla (n ≥ 5; P< 0.01). In vitro fertilization studies revealed that Crisp2(-/-) sperm exhibited deficiencies to penetrate the egg vestments (i.e. cumulus oophorus and zona pellucida) and to fuse with the egg (n ≥ 6; P< 0.01). Consistent with this, Crisp2-null sperm showed lower levels of hyperactivation (n = 7; P< 0.05), a vigorous motility required for penetration of the egg coats, as well as a dysregulation in intracellular Ca(2+) levels associated with capacitation (n = 5; P< 0.001)., Limitations, Reasons for Caution: The analysis of the possible mechanisms involved in fertility disorders in men with abnormal expression of CRISP2 was carried out in Crisp2 knockout mice due to the ethical and technical problems inherent to the use of human gametes for fertilization studies., Wider Implications of the Findings: Our findings in mice showing that Crisp2(-/-) males exhibit fertility and fertilization defects under demanding conditions support fertilization defects in sperm as a mechanism underlying infertility in men with aberrant expression of CRISP2. Moreover, our observations in mice resemble the situation in humans where fertility disorders can or cannot be detected depending on the accumulation of own individual defects or the fertility status of the partner. Finally, the fact that reproductive defects in mice are masked by conventional mating highlights the need of using different experimental approaches to analyze male fertility., Study Funding and Competing Interests: This study was supported by the World Health Organization (H9/TSA/037), the National Research Council of Argentina (PIP 2009-290), the National Agency for Scientific and Technological Promotion of Argentina (PICT 2011, 2023) and the Rene Baron Foundation to P.S.C. and by the MEXT of Japan to M.I. The authors declare that there are no conflicts of interest., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The aim of the present study was to investigate the effects of a crude extract of Garcinia kola on male sexual function after subchronic and chronic treatment periods at different sublethal doses. Adult male Wistar rats were treated orally with 100, 200 and 400 mg kg(-1) of a 70% ethanolic extract of G. kola daily for 56 days. Sexual behaviour studies were performed on days 28 and 50. At termination on day 56, organ weights, sperm count, reproductive hormone levels and testicular histology were assessed. Subchronic and chronic treatment of normal male rats with G. kola extract resulted in overall increase in components of libido, erection and ejaculation in treated rats - with lower doses being more efficient than the higher dose. There was a slight reduction in some components of sexual behaviour with prolonged time of treatment. G. kola treatment at all doses resulted in increased testicular weights, increased sperm count with no change in motility and increased serum testosterone levels with no change in gonadotropin levels. Gross testicular histology was not affected by treatment. We conclude that G. kola seed extract possesses potent aphrodisiac activity in male albino rats with resultant increase in sperm count and testosterone levels., (© 2015 Blackwell Verlag GmbH.)

A study by fluorescence microscopy has been carried out on male gametes from testicular follicles, seminal vesicles, spermatophores, and seminal receptacles of the bush-cricket Tylopsis liliifolia, focusing the attention on localization and movements of F-actin and α-tubulin during sperm differentiation, since data in this respect are lacking in the Orthoptera. F-actin and α-tubulin positivity was detected in the testicular follicles, in particular at the bridges connecting spermatids of a same clone and around their nucleus, during the first differentiation stages. During the following differentiation stages in the testes, F-actin was found at one of the spermatid poles and then, during nucleus elongation, at the whole acrosomal region. A peculiar F-actin-positivity was found at the flagellum, more markedly immediately posterior to the nucleus, at the basal body region of the gametes from the testicular follicles and from the other examined districts. Other interesting data from our investigations concerns the α-tubulin displacements during the differentiation stages of the spermatid and a constant absence of α-tubulin-positivity where the centrioles are located. No positivity was also found for both α-tubulin and nuclear markers at the anterior region of the gamete, where the acrosomal wings are localized. Our results, compared with what is so far known in literature for the insects, lead us to assert that microfilaments and microtubules undergo gradual displacements, markedly in the testicular follicles, during the morphogenesis of the male gamete of T. liliifolia aimed to its organization and motility and probably also to its interaction with the female gamete., (© 2015 Wiley Periodicals, Inc.)

Androgens and the androgen receptor (AR) are of great importance to spermatogenesis and male fertility. AR knockout (ARKO) mice display a complete insensitivity to androgens and male infertility; however, the exact molecular mechanism for this effect remains unclear. In this study, we found that the expression levels of Prmt6 mRNA and protein were significantly up-regulated in the testes of ARKO mice compared to wild type (WT) mice. PRMT6 was principally localized to the nucleus of spermatogonia and spermatocytes by immunofluorescence staining. Furthermore, luciferase assay data showed that AR together with testosterone treatment suppressed Prmt6 transcription via binding to the androgen-responsive element (ARE) of the Prmt6 promoter. Moreover, knockdown of Prmt6 suppressed germ cells migration and promoted apoptosis. In addition, both of these cellular activities could not be enhanced by testosterone treatment. Taken together, these data indicate that PRMT6, which was down-regulated by AR and influenced cell migration and apoptosis of germ cells, could play a potentially important role in spermatogenesis.

The a2 isoform of vacuolar-ATPase (ATP6V0A2, referred to as a2V) is required for normal spermatogenesis and maturation of sperm. Treatment of male mice with anti-a2V disturbs the testicular cytokine/chemokine balance and leads to severe deficiencies of spermatogenesis. The aim of the present study was to investigate the role of a2V in male fertility and in the regulation of apoptotic pathways required for normal spermatogenesis in mice. To study the role of a2V single dose of anti-a2V monoclonal antibody or mouse IgG isotype (3μg/animal) was injected i.p. into males on alternate days for 10 days. The expression of sperm maturation-related molecules and pro-apoptotic molecules was measured by real-time PCR or immunohistochemistry in control and anti-a2V-treated testes. The caspase levels and their activity were measured by western blot and fluorometry. We found that the expression of the sperm maturation-related molecules SPAM1, ADAM1, and ADAM2 was significantly decreased in testes from anti-a2V-treated males. The expression of pro-apoptotic molecules (Bax, p53, and p21) and molecules involved in the intrinsic pathway of apoptosis (caspase-9, caspase-3, and PARP), which are crucial for normal spermatogenesis was significantly reduced in testes from anti-a2V-treated males compared with the control. The total ATP level was significantly lower in anti-a2V-treated testes. The data provide novel evidence showing that a2V can regulate the apoptotic pathways, an essential testicular feature, and is necessary for efficient spermatogenesis., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Alterations of sperm quality were studied in tench (Tinca tinca) exposed to sub-lethal doses of 17-alpha-ethynylestradiol-EE2-(50, 100 and 500μg/kg t.w) under semi-static conditions for 30 days. Thus, different biomarkers of sperm quality were assessed: concentration and volume of ejaculate, total number of spermatozoa, percentage of motile spermatozoa, sperm motility and percentage of live and dead spermatozoa. Sperm motility was examined by computer-assisted image analysis and the viability of spermatozoa was assessed through flow cytometry. The most relevant alterations observed were significant reductions in the reproductive parameters such as testicular somatic index, spermatozoa concentration, straight line velocity, curvilinear velocity, average path velocity and wobble in tench exposed to 50μg/kg t.w of EE2. Our study about the effects of EE2 on the sperm quality in tench provides new evidences which strengthen the fact that this synthetic estrogen is included in the list of non-monotonic dose response compounds in animal studies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Exposure to benzo[a]pyrene (B[a]P) is well reported to be associated with neurological and reproductive dysfunctions. The present study investigated the influence of kolaviron, an isolated biflavonoid from the seed of Garcinia kola, on functional alterations along the brain-pituitary-gonadal axis in male rats exposed to B[a]P. Benzo[a]pyrene was orally administered at a dose of 10mg/kg alone or orally co-administered with kolaviron at 100 and 200mg/kg for 15 consecutive days. Administration of B[a]P significantly (p<0.05) decreased plasma levels of pituitary hormones namely follicle-stimulating hormone (FSH) and prolactin but increased luteinizing hormone (LH) by 47%, 55% and 20.9%, respectively, when compared with the control. The significant decrease in gonadosomatic index (GSI) was accompanied by significant decrease in testosterone production and sperm functional parameters in the B[a]P-treated rats. Moreover, B[a]P-treated rats showed significant elevation in the circulatory concentrations of pro-inflammatory cytokines and oxidative stress indices in the brain, testes and sperm of B[a]P-treated rats. Light microscopy revealed severe necrosis of the Purkinje cells in the cerebellum, neuronal degeneration of the cerebral cortex, neuronal necrosis of the hippocampus and testicular atrophy in B[a]P-treated rats. Kolaviron co-treatment significantly ameliorated B[a]P mediated damages by suppressing pro-inflammatory mediators and enhancing the antioxidant status, neuroendocrine function, sperm characteristics and improving the architecture of the brain and testes in B[a]P-treated rats. The findings in the present investigation highlight that kolaviron may be developed to novel therapeutic agent against toxicity resulting from B[a]P exposure., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Lead (Pb) is one of the most toxic environmental pollutants and known to exert multiple toxic effects including gonadotoxic and spermiotoxic effects. In order to understand toxic mechanisms of lead (Pb) on the testes and the accessory glands of crabs, we investigated Pb accumulation in testes and accessory glands and the survival rate of sperms of freshwater crab, Sinopotamon henanense. The tissue damaging effects of Pb was also investigated by histopathological examination and analyses of antioxidant enzymes as well as lipid peroxidation. Crabs were exposed to different Pb concentrations (0, 3.675, 7.35, 14.7, 29.4 and 58.8 mg/L) for 3, 5 and 7 days. The results showed that Pb levels in testes and accessory glands increased significantly following Pb exposure for 5 and 7 days in almost all treated groups, and survival rate of sperm decreased with increasing Pb concentrations at 5 and 7 days. Morphological changes identified histologically were discovered in testes, including a disordered arrangement of germ cells, a decreased number of sperm in the lumina of the seminiferous tubules, extensive necrosis in the germinal layer of the seminiferous tubules, etc. At the same time, histological abnormalities were discovered in accessory glands, the wall cells were separated from the basement membrane, and wall cells were missing partly. The activities of SOD, GPx and CAT in testes showed no statistically significant changes compared to the control for 3 days, and initially increased and subsequently decreased with increasing Pb concentrations at 5 and 7 days. The antioxidant enzyme activities in accessory glands initially increased and subsequently decreased with increasing Pb concentrations and Pb exposure. This was accompanied with an increase in malondialdehyde (MDA) content in a concentration-dependent manner. These results showed that acute Pb exposure led to a reduction of survival rate of sperm and harmful effects at the cellular level of crab testes and accessory glands, which are most likely linked to Pb-induced oxidative stress., (Copyright © 2015 Elsevier Inc. All rights reserved.)

This study investigates the ameliorative potential of Launea taraxacifolia (LT) aqueous leaf extract on cisplatin-induced testicular dysfunction in Wistar rats. Thirty rats were randomly divided into six groups (A-F) of 5 rats each: Group A which served as control received water; Group B was intraperitoneally (ip) injected 10 mg kg(-1) body wt cisplatin on day 21; Groups C and D were given 100 and 400 mg of LT via oral administration, respectively, for 21 days while Groups E and F received similar treatment as Groups C and D, respectively, and then exposed to ip administration of 10 mg kg(-1) body weight cisplatin on the 21st day. Exclusively, Cisplatin-exposed Group B rats showed reduced sperm characteristics and increased sperm morphological abnormalities; distorted histological architecture of seminiferous tubules; significantly increased lipid peroxidation (LPO) and decreased activities of superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH)levels in the testes. These parameters in LT alone treated Groups C and D were not markedly different compared with the control group. The rats with the combined treatment in Groups E and F showed significantly improved sperm parameters, testicular histo-architecture and antioxidant enzymatic activities. Conclusively, aqueous extract of L. taraxacifolia has protective potential against cisplatin damage., (© 2014 Blackwell Verlag GmbH.)

This paper presents the process of spermatogenesis in the leech Hirudo troctina Johnson, 1816 using light, fluorescent and transmission electron microscopy. At the onset of spermatogenesis in testes, the pear-shaped spermatogonia divide mitotically without full cytokinesis and as a result isogenic groups are formed (clusters, clones) with 2, 4, 8, 16, 32, 64, 128 spermatogonia and, finally, 256 primary spermatocytes occur. The final meiotic divisions of spermatocytes give rise to clones with 1024 spermatids. There are hundreds of developing germ-line clones in each testis. In each clone, the male germ cells divide in full synchrony and they are in the same phase of spermatogenesis. During complex spermiogenesis each spermatid becomes a filiform spermatozoon with a helicoid nucleus, which is characterized by the presence of a long acrosome with two regions - anterior and posterior, which are followed by a helicoid nucleus, a midpiece with only one mitochondrion and a long flagellum. Our results were compared to those on other clitellate annelids that have been studied to date, especially to sperm formation in Hirudo medicinalis Linnaeus, 1785. Only minor differences were found in the length and the diameter of different organelles and the number of spermatids in germ-line clones., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Isoniazid is reported to be the most reliable and cost-effective remedy for tuberculosis treatment and prophylaxis among first line anti-tuberculosis drugs. Conventionally, the most common and best studied adverse effect of isoniazid is hepatotoxicity, but as for testicular toxicity the problem has not yet explored extensively. The aim of the study was to identify in vivo influence of isoniazid on induction of testicular cytochrome Р-450 2Е1 (CYP2E1) mRNA expression and enzymatic activity, testes DNA fragmentation, serum total testosterone level, and spermatogenesis indices. The significant induction of CYP2E1 was demonstrated in rat's testes following isoniazid administration, specifically CYP2E1 mRNA expression and p-nitrophenolhydroxylase activity was increased in 28 and 7 times as compared with control, respectively. These changes were accompanied by activating of testicular GST in 32%, changing in levels and character of DNA fragmentation, as well as damaging of the spermatogenic epithelium, decreasing in serum testosterone content (1.62 fold), sperm count (19%), and losing of fertility in comparison with untreated males. We assume that in testes of isoniazid-treated rats CYP2E1 may act as a trigger in generating of reactive oxygen species and other toxic metabolites which subsequently mediates DNA damage, spermatogenesis disturbances, and altered male fertilizing capacity., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Knowledge of the reproductive biology of game species is vital for sustainable management. In moose (Alces alces), research in reproductive characteristics has focused on the female, whereas there are few studies in male moose. The aim of the present study was to investigate sperm morphology and chromatin integrity (SCSA), and their relationships with testicular and epididymal features, as well as temporal aspects with respect to the hunting season. In total, 143 male moose aged 1.5-11.5 years were sampled from 2008 to 2011. The proportion of normal spermatozoa (PNS) ranged from 1.5% to 82.0%, with a mean of 51%, and the %DFI (DNA fragmentation index) ranged from 2.5% to 36.7% (mean 9.5). PNS decreased temporally, and was positively associated with carcass and testes weight. Body weight and testes weight had positive effect on PNS regardless of age. No effect of any explanatory variables was observed on the DFI. The testis/body weight ratio of moose (0.033%) is among the lowest reported among mammals, indicating a less polygynous mating system than in roe deer and red deer. For reproduction success in moose, a high body weight in males is favorable, as is a balanced sex ratio. Thus, males should not be harvested prior to the time when the majority of females have passed their first oestrus of the season., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Context: Cytarabine (Ara-C) is an effective chemotherapeutic drug for the treatment of acute leukaemias. It inhibits the DNA synthesis and repair, thereby causes cytotoxicity in the proliferating cells., Objective: This study was aimed to investigate the effects of pre-pubertal exposure of Ara-C on testesticular development in juvenile SD rats and their function at puberty., Materials and Methods: Ara-C was injected at the doses of 50, 100 and 200 mg/kg/day from postnatal day (PND) 29-42 (14 days) by intraperitoneal (i.p.) route. Half of the animals were sacrificed on PND 43 and remaining on PND 70. End points of the evaluation included gross pathological examination, histomorphometric analysis, sperm count and sperm head morphology, cell proliferation and DNA damage as well as apoptosis analysis., Results: Ara-C treatment significantly decreased food and water intake, weight gain, testes and epididymis weight and increased histological alterations in the seminiferous tubule. Furthermore, Ara-C treatment significantly decreased the PCNA-positive cells and sperm count in a dose-dependent manner. Ara-C treatment also increased the DNA damage and apoptosis in testes and sperm as evident from the comet and TUNEL assays results., Discussion: The present study results clearly indicated that Ara-C treatment impaired spermatogenesis and adversely affects the testicular development and its function in rats by reducing the germ cell proliferation and the inducing DNA damage and apoptosis.