Establishment of an organ culture system to induce Sertoli cell differentiation from undifferentiated mouse gonads.

Organ culture systems are useful for elucidating the process of testicular differentiation from mammalian undifferentiated genetically male gonads, as they permit various experiments, including experiments involving the control of gene expression. However, without addition of testicular differentiation-related factors, it is difficult to induce the formation of testis cord from immature gonads by a time point earlier 12 tail somites (ts) that corresponding to 11.0 days post coitum (dpc). In this study, we attempted to establish an organ culture system that induces testis formation from immature gonads (around 8 ts: 10.5 dpc) just before Sry (sex-determining region of the Y chromosome) expression. A paired gonad-mesonephros complex of around 8 ts was placed in the groove of an agarose gel block and put the semi-cylindrical piece of agarose gel to maintain the gonad morphology. The gonads were cultured in the gas phase for 96 hr. As a result, testis cord-like structures appeared in many genetically male gonads. Cells expressing the Sertoli cell markers Sox9 (SRY-box 9) and Amh (anti-Müllerian hormone) were observed, while granulosa cell marker Foxl2 (forkhead box L2) was not detected. In addition, Sox9- and Amh-expressing cells were observed throughout the entire gonad in many individuals. Amh mRNA expression was also upregulated. Surprisingly, formation of a partial testicular structure was observed from more immature gonads (6 ts). These results show that our gonadal organ culture system is useful for elucidating the regulation mechanism of Sry expression in undifferentiated bipotential gonads.

Role of Ezrin Phosphorylation in HIV-1 Replication.

Host-cell expression of the ezrin protein is required for CXCR4 (X4)-tropic HIV-1 infection. Ezrin function is regulated by phosphorylation at threonine-567. This study investigates the role of ezrin phosphorylation in HIV-1 infection and virion release. We analyzed the effects of ezrin mutations involving substitution of threonine-567 by alanine (EZ-TA), a constitutively inactive mutant, or by aspartic acid (EZ-TD), which mimics phosphorylated threonine. We also investigated the effects of ezrin silencing on HIV-1 virion release using a specific siRNA. We observed that X4-tropic HIV-1 vector infection was inhibited by expression of the EZ-TA mutant but increased by expression of the EZ-TD mutant, suggesting that ezrin phosphorylation in target cells is required for efficient HIV-1 entry. Expression of a dominant-negative mutant of ezrin (EZ-N) and ezrin silencing in HIV-1 vector-producing cells significantly reduced the infectivity of released virions without affecting virion production. This result indicates that endogenous ezrin expression is required for virion infectivity. The EZ-TD but not the EZ-TA inhibited virion release from HIV-1 vector-producing cells. Taken together, these findings suggest that ezrin phosphorylation in target cells is required for efficient HIV-1 entry but inhibits virion release from HIV-1 vector-producing cells.

Contribution of the coelomic epithelial cells specific to the left testis in the chicken embryo.

BACKGROUND: The left male gonad in the chicken embryo has a thickened cortical layer, but it eventually becomes flattened after the onset of testicular development. Because the destination of the cortical cells migrating from the left gonad remains unclear, we examined this issue herein. RESULTS: The testis-inducing gene doublesex- and mab-3-related transcription factor 1 (DMRT1) was detected in a proportion of the columnar and cubic epithelial cells in the cortex of the left testis as well as Sertoli cells in both testes. Interestingly, some of the DMRT1-expressing cortical cells were contiguous with Sertoli cells in the testis cord. Some cortical cells exhibited a vimentin-positive cytoplasm that was elongated all the way to the medulla. In addition, a desmosome-like structure was observed between the elongated cytoplasm in these cells and the adjacent Sertoli cell. After the organ culture, a few cells labeled with a fluorescent dye that stained only the cortical cells at the beginning of the culture were located in the testis cord of the left testis. CONCLUSIONS: Some cortical cells expressing DMRT1 were suggested to contribute to the Sertoli cells in the testis cord only after the onset of testicular development and only in the left testis. Developmental Dynamics 246:148-156, 2017. © 2016 Wiley Periodicals, Inc.

Ontogenic and morphological study of gonadal formation in genetically-modified sex reversal XY(POS) mice.

Mammalian sexual fate is determined by the presence or absence of sex determining region of the Y chromosome (Sry) in the "bipotential" gonads. Recent studies have demonstrated that both male and female sexual development are induced by distinct and active genetic pathways. Breeding the Y chromosome from Mus m. domesticus poschiavinus (POS) strains into C57BL/6J (B6J) mice (B6J-XY(POS)) has been shown to induce sex reversal (75%: bilateral ovary, 25%: true hermaphrodites). However, our B6N-XY(POS) mice, which were generated by backcrossing of B6J-XY(POS) on an inbred B6N-XX, develop as males (36%: bilateral testis with fertility as well as bilateral ovary (34%), and the remainder develop as true hermaphrodites. Here, we investigated in detail the expressions of essential sex-related genes and histological features in B6N-XY(POS) mice from the fetal period to adulthood. The onsets of both Sry and SRY-box 9 (Sox9) expressions as determined spatiotemporally by whole-mount immunohistochemistry in the B6N-XY(POS) gonads occurred 2-3 tail somites later than those in B6N-XY(B6) gonads, but earlier than those in B6J-XY(POS), respectively. It is possible that such a small difference in timing of the Sry expression underlies testicular development in our B6N-XY(POS). Our study is the first to histologically show the expression and ectopic localization of a female-related gene in the XY(POS) testes and a male-related gene in the XY(POS) ovaries. The results from these and previous experiments indicate that the interplay between genome variants, epigenetics and developmental gene regulation is crucial for testis development.

Immunohistochemical analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity on the developmental dentate gyrus and hippocampal fimbria in fetal mice.

Dioxins are widespread persistent environmental contaminants with adverse impacts on humans and experimental animals. Behavioral and cognitive functions are impaired by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. TCDD exerts its toxicity via the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. The hippocampus, which plays important roles in episodic memory and spatial function, is considered vulnerable to TCDD-induced neurotoxicity, because it contains the AhR. We herein investigated the effects of TCDD toxicity on hippocampal development in embryonic mice. TCDD was administered to dams at 8.5 days postcoitum with a single dose of 20, 200, 2,000 and 5,000 ng/kg body weight (groups T20, T200, T2000 and T5000, respectively), and the brains were dissected from their pups at embryonic day 18.5. Immunohistochemical analysis demonstrated that the Glial Fibrillary Acidic Protein (GFAP) immunoreactivities in the dentate gyrus (DG) were reduced in the T5000 group. Granular GFAP immunoreactivity was observed in the hippocampal fimbria, and the number of immunoreactive fimbria was significantly decreased in the T5000 group. The number of Proliferating Cell Nuclear Antigen (PCNA)-positive cells was decreased in all TCDD-exposed groups and significantly reduced in the T20, T200 and T5000 groups. Together, these results demonstrate that maternal TCDD exposure has adverse impacts on neural stem cells (NSCs), neural precursor cells (NPCs) and granular cells in the DG and disrupts the NSC maintenance and timing of differentiation in the hippocampal fimbria, which in turn interrupt neuronal development in future generations of mice.

The combined effect of clothianidin and environmental stress on the behavioral and reproductive function in male mice.

Neonicotinoids, some of the most widely used pesticides in the world, act as agonists to the nicotinic acetylcholine receptors (nAChRs) of insects, resulting in death from abnormal excitability. Neonicotinoids unexpectedly became a major topic as a compelling cause of honeybee colony collapse disorder, which is damaging crop production that requires pollination worldwide. Mammal nAChRs appear to have a certain affinity for neonicotinoids with lower levels than those of insects; there is thus rising concern about unpredictable adverse effects of neonicotinoids on vertebrates. We hypothesized that the effects of neonicotinoids would be enhanced under a chronic stressed condition, which is known to alter the expression of targets of neonicotinoids, i.e., neuronal nAChRs. We performed immunohistochemical and behavioral analyses in male mice actively administered a neonicotinoid, clothianidin (CTD; 0, 10, 50 and 250 mg/kg/day), for 4 weeks under an unpredictable chronic stress procedure. Vacuolated seminiferous epithelia and a decrease in the immunoreactivity of the antioxidant enzyme glutathione peroxidase 4 were observed in the testes of the CTD+stress mice. In an open field test, although the locomotor activities were not affected, the anxiety-like behaviors of the mice were elevated by both CTD and stress. The present study demonstrates that the behavioral and reproductive effects of CTD become more serious in combination with environmental stress, which may reflect our actual situation of multiple exposure.

Insight into the mechanism of reproductive dysfunction caused by neonicotinoid pesticides.

Neonicotinoids, which were developed in the 1990 s as an insecticide having selective toxicity, were later found to cause reproductive abnormalities in experimental animals. In Japan there is an attempt to preserve endangered animals, including the Japanese crested ibis, and there is a question of whether neonicotinoids affect the reproduction of this bird, since they are used in its habitat. Hence, we investigated whether the daily oral administration of the neonicotinoid clothianidin (CTD) has any deleterious effects on the reproductive function of mature male only or both young male and female quails as experimental animals. Vacuolization and the number of germ cells having fragmented DNA in seminiferous tubules, as well as the number and size of vacuoles in hepatocytes, increased dose-dependently. The ovaries showed abnormal histology in the granulosa cells, which produce progesterone. There were significant differences in egg-laying rates and embryo weights between the groups. Glutathione Peroxidase 4 (GPx4) and Manganese Superoxide Dismutase (Mn-SOD), which protect the organism from oxidative damage, showed a dose-dependent decrease. Thus, it is possible neonicotinoids affect the bird's reproductive system through oxidative stress, reflecting an imbalance between the production of reactive oxygen species (ROS) and a biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting damage. Responding to our study, Sado Island has since succeeded in breeding Japanese crested ibis in the wild without the use of neonicotinoids.

Unpredictable chronic stress-induced reproductive suppression associated with the decrease of kisspeptin immunoreactivity in male mice.

Environmental stress affects various parts of mammals typically through the circulation of stress hormones. It has been identified as one of the possible reasons for male reproductive difficulties, but the complex mechanisms responsible for stress-induced reproductive suppression are poorly understood. Here, we examined the relationship between chronic environmental stress and hypothalamic kisspeptin, a recently discovered upstream regulator of the reproductive endocrine feedback system. We studied male mice under an unpredictable chronic stress procedure to replicate the situation of animals under chronic stress. Histological and immunohistochemical analyses were performed focusing on kisspeptin neurons in the arcuate hypothalamic nucleus (ARC) and DNA fragmented cells in seminiferous tubules. Although the ARC was not morphologically altered in either the stressed or non-stressed group, granular kisspeptin immunoreactivities decreased slightly in the stress group. In the testes of the stress group, several signs of testicular degeneration were observed, including increased numbers of ssDNA-positive cells per seminiferous tubule, thinning, vacuoled seminiferous epithelia and multinucleated giant cells. The decreases in kisspeptin in the stress group might be due to other hypothalamic peptides, such as corticotropin-releasing hormone and leptin, whose receptors are known to coexpress in the ARC. In addition, environmental stress directly and indirectly affects testicular function through stress hormones and gonadotropins. In summary, our findings enhance the understanding of stress-induced reproductive suppression possibly mediated by kisspeptin in the ARC.