Early-life exposure to acephate inhibits sexual development and induces testicular and ovarian toxicity in mice.

Acephate is an organophosphate insecticide that exerts its toxicity by acting on the nervous system of insects. In addition to its action on the mammalian nervous system, acephate can also induce endocrine disruption of the reproductive system in mammals. However, the effects of acephate on sexual maturation and ovary development remain unclear. This study evaluated whether early-life exposure to acephate negatively impacts the male and female sexual maturation process and mature reproductive tissues. C57BL/6N mice were exposed to acephate (0, 0.3, 300 ppm) in drinking water from embryonic day 11.5 to ablactation, when the pups were four weeks old. Both sexes in the high-dose group experienced an early postnatal growth deficit, while females in the low-dose group continued to gain weight until 10 weeks of age. Exposure to acephate altered the anogenital index in females. Furthermore, preputial separation and vaginal opening were delayed in the high-dose group. At maturity, the weight of the seminal vesicles was decreased in the high-dose group. All treated groups exhibited increased vacuolation, accumulation of residual bodies, and degeneration in the testes. Furthermore, follicle regression was observed, and the healthy follicle number at each developmental stage was decreased in all treated groups. These results are probably due to the inhibition of the regulation by the hypothalamic-pituitary-gonadal axis and direct toxicity to reproductive organs. In conclusion, our study demonstrates that early-life exposure to acephate in mice may disrupt normal postnatal development, postpone puberty onset, and adversely affect reproductive functions during the reproductive period in both sexes.

Effects of early-life tosufloxacin tosilate hydrate administration on growth rate, neurobehavior, and gut microbiota at adulthood in male mice.

Reportedly, antibiotics, which are frequently prescribed in children, have long-term effects owing to gut microbiota dysregulation. Tosufloxacin tosilate hydrate (TFLX) is the first orally administered new quinolone with high efficacy and broad-spectrum action approved as an antibacterial agent for pediatric use in Japan. However, studies on the effects of its early-stage administration are limited. Therefore, we aimed to analyze the later effects of its developmental administration by monitoring growth rate, neurobehavior, and gut microbiota in mice. The TFLX was administered via drinking water at a dose of up to 300 mg/kg for two consecutive weeks during the developmental period (4-6 weeks of age) or adulthood (8-10 weeks of age). Thereafter, the body weights of the mice were measured weekly to monitor growth rate. Behavioral tests were also conducted on 11-12-week-old mice to examine the neurobehavioral effects of the treatment. Further, to examine the effects of the treatment on microbiota, fecal samples were collected from the rectum of mice dissected at 12 weeks of age, and 16s rRNA analysis was conducted. Our results showed increased body weights after TFLX administration, without any long-term effects. Behavioral analysis suggested alterations in anxiety-like behaviors and memory recall dysregulation, and gut microbiota analysis revealed significant differences in bacterial composition. These findings indicated that TFLX administration during the developmental period affects mice growth rate, neurobehavior, and gut microbiota structure. This is the first study to report that TFLX is potentially associated with the risk of long effects.

Testicular histone hyperacetylation in mice by valproic acid administration affects the next generation by changes in sperm DNA methylation.

Various studies have described epigenetic inheritance through sperms. However, the detailed mechanisms remain unclear. In this study, we focused on DNA methylation in mice treated with valproic acid (VPA), an inducer of epigenomic changes, and analyzed the treatment effects on the sperm from the next generation of mice. The administration of 200 mg/kg/day VPA to mice for 4 weeks caused transient histone hyperacetylation in the testes and DNA methylation changes in the sperm, including promoter CpGs of genes related to brain function. Oocytes fertilized with VPA-treated mouse sperm showed methylation fluctuations at the morula stage. Pups that were fathered by these mice also showed behavioral changes in the light/dark transition test after maturation. Brain RNA-seq of these mice showed that the expression of genes related to neural functions were altered. Comparison of the sperm DNA methylation status of the next generation of mice with that of the parental generation revealed the disappearance of methylation changes observed in the sperm of the parental generation. These findings suggest that VPA-induced histone hyperacetylation may have brain function-related effects on the next generation through changes in sperm DNA methylation.

High concentration of dopamine treatment may induce acceleration of human sperm motility.

Purpose: In humans, catecholamines (including dopamine) have been identified in semen and fallopian tubes, while dopamine D2 receptors (D2DR) are found in the sperm midpiece region. How dopamine dose affects human sperm function and whether dopamine treatment is useful in assisted reproductive technology is unclear. Methods: Sperm samples were obtained from patients with normal semen parameters undergoing fertility treatment. We investigated the effects of dopamine treatment on tyrosine phosphorylation and sperm motility. Sperm motility was analyzed using the computer-assisted sperm analysis (CASA) system. Results: This study revealed that various dopamine concentrations (0.1-100 µM) did not increase sperm tyrosine phosphorylation. Progressive motility increased substantially when treated with high concentrations of dopamine (10 and 100 µM) and was blocked by raclopride (a D2DR antagonist). After 24-h sperm culture, the addition of 10 µM dopamine significantly increased curvilinear velocity and amplitude of lateral head displacement, which are indicators of hyperactivation. Conclusion: Dopamine did not affect tyrosine phosphorylation, but increased sperm motility. High concentrations of dopamine were more effective to accelerate sperm motility in cases where sperm motile capacity was low.

Exogenous gamma-aminobutyric acid addition enhances porcine sperm acrosome reaction.

The widely used porcine artificial insemination procedure involves the use of liquid-stored semen because it is difficult to control the quality of frozen-thawed porcine sperm. Therefore, there is a high demand for porcine semen. The control and enhancement of sperm function are required for the efficient reproduction of pigs. We previously reported that gamma-aminobutyric acid (GABA) enhanced sperm capacitation and acrosome reaction in mice. In this study, we demonstrated the presence of GABAA receptors in porcine sperm acrosome. Furthermore, we investigated the GABA effects on porcine sperm function. We did not detect any marked effect of GABA on sperm motility and tyrosine phosphorylation of sperm proteins. However, GABA promoted acrosome reaction, which was suppressed by a selective GABAA receptor antagonist. GABA binds to GABAA receptors, resulting in chloride ion influx. We found that treatment with 1 µM GABA increased the intracellular concentration of chloride ion in the sperm. In addition, the GABA concentration effective in the acrosome reaction was correlated with the porcine sperm concentration. These results indicate that GABA and its receptors can act as modulators of acrosome reaction. This study is the first to report the effects of GABA on porcine sperm function.

Characteristics of alpha smooth muscle actin-positive peritubular cells in prepubertal bovine testes.

Characteristics of peritubular myoid cells (PMCs) in sexually immature cattle remain largely unknown. Here, we report the character and behavior of peritubular cells expressing alpha smooth muscle actin (αSMA), a marker of PMCs in adult testes, in prepubertal testes procured from 5-months-old bulls. Elastin distribution around αSMA + PMCs was dim and discontinuous in prepubertal testes, but strong and continuous in adult testes. Fibroblast-specific protein 1 (FSP1) was rarely expressed in αSMA + PMCs of prepubertal testes, while in adult testes, majority of αSMA + PMCs were FSP1+. Moreover, αSMA + PMCs in prepubertal testes proliferated more actively than those in adult testes. In vitro culture of isolated seminiferous tubules from prepubertal testes revealed that αSMA + PMCs migrate from peritubular to interstitial area. Hence, in prepubertal bovine testes, (1) PMCs rarely portray fibroblast-like properties, (2) PMCs exhibit heterogeneity in FSP1 expression, (3) PMCs proliferate more actively than those in adult testes, and (4) PMCs have a potential to migrate to the interstitium. Our observations help to understand the maturation of PMCs and their involvement in bovine testicular function.

Loss of Axdnd1 causes sterility due to impaired spermatid differentiation in mice.

Purpose: Spermiogenesis, the process of deformation of sperm head morphology and flagella formation, is a phenomenon unique to sperm. Axonemal dynein light chain proteins are localized to sperm flagella and are known to be involved in sperm motility. Here, we focused on the gene axonemal dynein light chain domain containing 1 (Axdnd1) with the aim to determine the function of its protein product AXDND1. Methods: To elucidate the role of AXDND1 in spermatogenesis, we generated Axdnd1 knockout (KO) mice using the CRISPR/Cas9 system. The generated mice were subjected to fertility tests and analyzed by immunohistochemistry. Result: The Axdnd1 KO mouse exhibited sterility caused by impaired spermiogenesis during the elongation step as well as abnormal nuclear shaping and manchette, which are essential for spermiogenesis. Moreover, AXDND1 showed enriched testicular expression and was localized from the mid-pachytene spermatocytes to the early spermatids. Conclusion: Axdnd1 is essential for spermatogenesis in the mouse testes. These findings improve our understanding of spermiogenesis and related defects. According to a recent report, deleterious heterozygous mutations in AXDND1 were found in non-obstructive azoospermia (NOA) patients. Therefore, Axdnd1 KO mice could be used as a model system for NOA, which will greatly contribute to future NOA treatment studies.

Potential of sperm small non-coding RNAs as biomarkers of testicular toxicity in a doxorubicin-induced mouse model.

Testicular toxicity is a major concern in cancer chemotherapy and drug development as it can result in infertility; however, there are no effective biomarkers for this adverse effect. To identify new biomarkers, we investigated the expression of small non-coding RNAs (sncRNAs) in a mouse model of doxorubicin (DXR)-induced testicular toxicity. First, we performed small RNA-seq analysis of sperm from DXR-treated or control mice and observed differential expression of many genome-derived sequences. We then performed real-time RT-PCR validation of these sequences and discovered that sncRNA detected by one primers, dxRN_3, showed similar differential expression as that seen in the RNA-seq experiment. These findings suggest that the sncRNAs present in sperm have potential as clinically acceptable biomarkers for testicular toxicity.

Persistence of undifferentiated spermatogonia in aged Japanese Black cattle.

Aging is a major risk factor for spermatogenesis deterioration. However, the influence of age on spermatogenic stem cells and their progenitors in bulls is largely unknown. Here, we report age-related changes in undifferentiated and differentiating spermatogonia in Japanese Black cattle with nearly constant sperm output, by using spermatogonial markers. The numbers of differentiating spermatogonia and more differentiated spermatogenic cells were significantly decreased in aged bovine testes compared with those in young testes. In contrast, the number of undifferentiated spermatogonia was maintained, and their proliferative activity did not differ significantly between young and aged bovine testes. Although severe calcification was only observed to a small extent in aged testes, fewer Sertoli cells and interstitial fibrosis were observed in noncalcified testicular regions. These results suggest that, even in old bulls with nearly constant sperm output, testicular spermatogenic activity declined whereas undifferentiated spermatogonia numbers were maintained. Thus, we propose that undifferentiated spermatogonia may be resistant to age-related changes in bovine testes. Because undifferentiated spermatogonia may contain stem cell activity, our findings highlight the potential utility of undifferentiated spermatogonia as an agricultural resource to produce spermatozoa beyond the natural bovine lifetime through transplantation and in vitro spermatogenesis in future animal production.

Behavioural effects in mice orally exposed to domoic acid or ibotenic acid are influenced by developmental stages and sex differences.

The structure of the brain is dramatically altered during the critical period. Physiological substances (neurotransmitters, hormones, etc.) in the body fluctuate significantly before and after sexual maturation. Therefore, the effect of chemical exposure on the central nervous system often differs depending on the developmental stage and sex. We aimed to compare the behavioural effects that emerged from the administration of chemicals to mice of different life stages (immature or mature) and different sex (male or female). We administered mice with domoic acid (DA), a marine poison, and ibotenic acid (IA), found in poisonous mushrooms. These excitatory amino acids act as agonists for glutamate and are potent neurotoxins. Interestingly, the behavioural effects of these chemicals were completely different. Following DA administration, we observed memory deficits only in groups of male mice treated at maturity. Following IA administration, we observed deviations in emotional behaviour in groups of male mice treated at both immaturity and maturity. In contrast, few characteristic changes were detected in all groups of females. Our results support the theory that the behavioural effects of chemical administration vary considerably with developmental stages and sex. In conclusion, our findings promote better understanding of individual differences in excitatory chemical-induced neurotoxicity and provide evidence for future risk strategies and treatments.

Caffeine induces sperm detachment from sperm head-to-head agglutination in bull.

Sperm head-to-head agglutination is a well-known known phenomenon in mammalian and non-mammalian species. Although several factors have been reported to induce sperm agglutination, information on the trigger and process of sperm detachment from the agglutination is scarce. Since hyperactivated motility is involved in bovine sperm detachment from the oviduct, we focused on caffeine, a well-known hyperactivation inducer, and aimed to determine the role of caffeine in sperm detachment from agglutination. Agglutination rate of bovine sperm was significantly decreased upon incubation with caffeine following pre-incubation without caffeine. Additionally, we observed that bovine sperm were detached from agglutination only when the medium contained caffeine. The detached sperm showed more asymmetrical flagellar beating compared to the undetached motile sperm, regardless of whether before or after the detachment. Intriguingly, some sperm that detached from agglutination re-agglutinated with different sperm agglutination. These findings indicated caffeine as a trigger for sperm detachment from the agglutination in bull. Furthermore, another well-known hyperactivation inducer, thimerosal, also significantly reduced the sperm agglutination rate. Overall, the study demonstrated the complete process of sperm detachment from sperm head-to-head agglutination and proposed that hyperactivated motility facilitates sperm detachment from another sperm. These findings would provide a better understanding of sperm physiology and fertilization process in mammals.

Expression and Possible Role of Nicotinic Acetylcholine Receptor ε Subunit (AChRe) in Mouse Sperm.

The nicotinic acetylcholine receptor (nAChR) is one of the receptors of acetylcholine (ACh), and nicotine (NIC) acts as an agonist of this receptor. Among the nAChR subunits, we found that the ε subunit (AChRe) had approximately 10 to 1000 times higher level of mRNA expression in mouse testes than the other subunits. In this study, we aimed to elucidate the expression and localization of AChRe in the testes and spermatozoa of mice and clarify the effect of AChRe on sperm function. Immunocytochemistry showed that AChRe was expressed in the murine testes and spermatozoa. We found that AChRe was localized only in elongated spermatids from step 12 onwards in the testes. In spermatozoa, AChRe was localized in the head, especially in the anterior region of the acrosome, but only approximately 50% of spermatozoa showed this immunoreactivity. Additionally, we analyzed the effects of ACh and NIC on sperm acrosome reaction (AR) and found that both ACh and NIC suppressed the AR rate, which was restored by an AChRe-specific antagonist. These results suggest that AChRe may be a regulator of mammalian sperm AR.

Expression and localization of alpha-tubulin N-acetyltransferase 1 in the reproductive system of male mice.

The structure of microtubules is essential for the fertilizing ability of spermatozoa. Acetylation of α-tubulin plays an important role in flagellar elongation and spermatozoa motility. Previous reports have suggested that alpha-tubulin N-acetyltransferase 1 (ATAT1) is the main acetyltransferase involved in the acetylation of α-tubulin. Although ATAT1 is reported to express in the testis, no information is available regarding its expression in elongated spermatids, epididymis, and mature spermatozoa. Hence, it remains unclear whether ATAT1 is involved in spermatozoa maturation and capacitation. Therefore, we evaluated the expression of ATAT1 in the mouse male reproductive system using immunostaining and western blotting. Our results showed that ATAT1 was expressed in spermatids during spermiogenesis in mouse testes, but its expression varied according to the seminiferous tubule stage. We observed ATAT1 in the cytoplasm of round spermatids, the flagella of elongated spermatids, and in the cytoplasm of step 16 spermatids, just before its release into the lumen. In addition, ATAT1 was expressed in epithelial cells of the epididymis. In spermatozoa of the cauda epididymis, ATAT1 expression was primarily observed in the midpiece of the spermatozoa. The localization of ATAT1 protein in the male germline was observed during spermiogenesis as well as during spermatozoa maturation. Our results suggest that ATAT1 may be involved in the formation of flagella and in the acetylation process, which has attracted attention in recent years regarding male infertility.

Effect of vitamin E deficiency on spermatogenesis in mice and its similarity to aging.

Vitamin E (VE) plays numerous important roles in mammals because of its antioxidant activity. As a result, VE deficiency (VED) leads to the dysfunction of central nervous, reproductive, and immune systems. However, few studies have reported the effects of VED on the male reproductive system. In this study, we investigated the effects of VED on male reproductive function and examined its relationship to involution in the male reproductive system with aging. We fed a VED or control diet to 4-week-old mice for 12 or 24 weeks. Following the histopathological analysis of reproductive organs, we found seminiferous tubules with exfoliation in the VED groups, and its frequency was significantly increased compared with the controls. Additionally, in the epididymis, a decrease in spermatozoa and an increase in apoptotic germ cells were observed in the VED groups compared with the controls. By Papanicolaou staining, we also found an increase in the proportion of sperm with abnormal morphology in the VED groups compared with the controls. These reproductive effects induced by VED were highly similar to one aspect of those observed in aged mice. Our findings demonstrate that the aging of the male reproductive system may be accelerated because of the impaired in vivo antioxidant capacity induced by VED.

Characteristics and Possible Role of Bovine Sperm Head-to-Head Agglutination.

Although sperm head-to-head agglutination has been reported in many mammalian species, the biological significance of this unique sperm-sperm interaction remains largely unknown. Here, we aimed to examine the functional characteristics of agglutinated bovine sperm to determine the possible role of sperm agglutination in the fertilization process. We initially examined temporal changes to the degree of head-to-head agglutination in culture, and found that bovine sperm agglutinated despite the lack of sperm agglutination inducers in medium. Sperm viability and motility were evaluated by SYBR14/PI and JC-1 staining, respectively, to identify the relationship between sperm agglutination and fertilizing ability. Agglutinated sperm had increased motility, viability, and intact mitochondrial function compared with unagglutinated sperm. Furthermore, we found that heparin significantly increased the percentage of unagglutinated sperm, but did not affect viability of both agglutinated and unagglutinated sperm, suggesting that sperm agglutination dictated the viability. In conclusion, agglutinated bovine sperm maintained viability and motility for a longer time than unagglutinated sperm. Thus, we propose that the head-to-head agglutination is a crucial sperm-sperm interaction to ensure the fertilizing ability of sperm.

Effect of neurotensin on cultured mouse preimplantation embryos.

Previously, we revealed that neurotensin (NTS) derived from the oviduct and uterus can function during fertilization. However, little is known about NTS actions on the pre-implantation embryo after fertilization. Here, we found that pro-Nts mRNA is expressed in the oviduct and uterus during when preimplantation embryos develop and an increase in mRNA level in the uterus is induced by human chorionic gonadotropin (hCG) treatment. Expression of mRNA for two NTS receptors, Ntr1 and Ntr3, was found throughout these stages, whereas Ntr2 mRNA was not detected, suggesting that NTS signaling occurred through NTR1 and NTR3. Supplementation of 1, 10, 100 or 1000 nM NTS to embryo culture medium after fertilization showed that 100 nM NTS significantly improved the blastocyst formation. In comparison, the total number of cells and inner cell mass ratio of blastocysts was not significant different between the 0 nM and 100 nM NTS treatment groups. These results indicate that NTS has a positive effect upon preimplantation embryo development in vitro.

Stromal cell-derived factor 1 regulates in vitro sperm migration towards the cumulus-oocyte complex in cattle.

Sperm migration towards an oocyte in the female reproductive tract is an important step for successful fertilization. Although several sperm-chemotactic factors have been identified in mammals, it is unclear whether these chemoattractants contribute to sperm migration towards an oocyte that is the final destination for sperm. Furthermore, chemoattractants for bovine sperm are still undiscovered even though the follicular fluid attracts sperm in cattle. Here, we demonstrated that a single bovine cumulus-oocyte complex (COC) had the ability to attract sperm, suggesting that the COC secreted sperm chemoattractants. We identified stromal cell-derived factor 1 (SDF1), which was expressed in COCs, and its receptor CXCR4 in sperm, as a candidate. Our results showed that bovine sperm preferentially migrated to the area with a high SDF1 concentration and occasionally showed turn movements by asymmetric flagellar bends during the migration. We also demonstrated that increasing the intracellular Ca2+ concentration via Ca2+ channels was related to SDF1-induced sperm chemotaxis. Finally, a CXCR4 inhibitor significantly suppressed the in vitro bovine sperm migration towards a COC. Taken together, we propose that SDF1 is a chemotactic factor for bovine sperm to regulate their migration towards an oocyte via the CXCR4 receptor.

Differences in resistance against osmotic challenge among C57BL/6, DBA/2 and their hybrid mice metaphase II (MII) stage oocytes.

Oocytes of B6D2F1 (BDF1) mice are often used as recipients for intracytoplasmic sperm injection because of their cell membrane resistance against capillary penetration. It is assumed that oocytes of BDF1 mice have superior traits because of their hybrid vigour. However, the mechanisms of hybrid vigour are unclear. In this study, we focused on the membrane resistance of MII stage oocytes against changes in extracellular osmotic pressure. As a result, MII stage oocytes of inbred C57BL/6 and DBA/2 mice showed high tolerance in either a hypertonic or a hypotonic environment. Conversely, MII stage oocytes of hybrid BDF1 and D2B6F1 mice showed high tolerance in both hypertonic and hypotonic environments. Therefore, it is considered that MII stage oocytes of hybrid mice have superior traits than those of inbred mice. Our findings demonstrated that the hybrid vigour exists in the form of resistance to extracellular osmotic environment in hybrid MII stage oocytes.

Early-life exposure to low levels of permethrin exerts impairments in learning and memory with the effects on neuronal and glial population in adult male mice.

Permethrin, a pyrethroid chemical, is widely used as a pesticide because of its rapid insecticidal activity. Although permethrin is considered to exert very low toxicity in mammals, the effects of early, low-level, chronic exposure on the adult central nervous system are unclear. In this study, we investigated the effects of low-level, chronic permethrin exposure in early life on the brain functions of adult mice, using environmentally relevant concentrations. We exposed mice to the acceptable daily intake level of permethrin (0.3 ppm) in drinking water during the prenatal and postnatal periods. We then examined the effects on the central nervous system in adult male offspring. In the permethrin group, we detected behavior that displayed incomplete adaptation to a novel environment, as well as an impairment in learning and memory. In addition, immunohistochemical analysis revealed an increase in doublecortin- (an immature neuron marker) positive cells in the hippocampal dentate gyrus in the permethrin exposure group compared with the control group. Additionally, in the permethrin exposure group there was a decrease in astrocyte number in the hilus of the dentate gyrus, and remaining astrocytes were often irregularly shaped. These results suggest that exposure to permethrin at low levels in early life affects the formation of the neural circuit base and behavior after maturation. Therefore, in the central nervous system of male mice, low-level, chronic permethrin exposure during the prenatal and postnatal periods has effects that were not expected based on the known effects of permethrin exposure in mature animals.

Mammalian germ cell migration during development, growth, and homeostasis.

BACKGROUND: Germ cells represent one of the typical cell types that moves over a long period of time and large distance within the animal body. To continue its life cycle, germ cells must migrate to spatially distinct locations for proper development. Defects in such migration processes can result in infertility. Thus, for more than a century, the principles of germ cell migration have been a focus of interest in the field of reproductive biology. METHODS: Based on published reports (mainly from rodents), investigations of germ cell migration before releasing from the body, including primordial germ cells (PGCs), gonocytes, spermatogonia, and immature spermatozoon, were summarized. MAIN FINDINGS: Germ cells migrate with various patterns, with each migration step regulated by distinct mechanisms. During development, PGCs actively and passively migrate from the extraembryonic region toward genital ridges through the hindgut epithelium. After sex determination, male germline cells migrate heterogeneously in a developmental stage-dependent manner within the testis. CONCLUSION: During migration, there are multiple gates that disallow germ cells from re-entering the proper developmental pathway after wandering off the original migration path. The presence of gates may ensure the robustness of germ cell development during development, growth, and homeostasis.