Development of sexual dimorphism of skeletal muscles through the adrenal cortex, caused by androgen-induced global gene suppression.

The zona fasciculata (zF) in the adrenal cortex contributes to multiple physiological actions through glucocorticoid synthesis. The size, proliferation, and glucocorticoid synthesis characteristics are all female biased, and sexual dimorphism is established by androgen. In this study, transcriptomes were obtained to unveil the sex differentiation mechanism. Interestingly, both the amount of mRNA and the expressions of nearly all genes were higher in females. The expression of Nr5a1, which is essential for steroidogenic cell differentiation, was also female biased. Whole-genome studies demonstrated that NR5A1 regulates nearly all gene expression directly or indirectly. This suggests that androgen-induced global gene suppression is potentially mediated by NR5A1. Using Nr5a1 heterozygous mice, whose adrenal cortex is smaller than the wild type, we demonstrated that the size of skeletal muscles is possibly regulated by glucocorticoid synthesized by zF. Taken together, considering the ubiquitous presence of glucocorticoid receptors, our findings provide a pathway for sex differentiation through glucocorticoid synthesis.

Attenuated cerebellar phenotypes in Inpp4a truncation mutants with preserved phosphatase activity.

Phosphoinositides (PIPs) act as intracellular signaling molecules that regulate various cellular processes. Abnormalities in PIP metabolism cause various pathological conditions, including neurodegenerative diseases, cancer and immune disorders. Several neurological diseases with diverse phenotypes, such as ataxia with cerebellar atrophy or intellectual disability without brain malformation, are caused by mutations in INPP4A, which encodes a phosphoinositide phosphatase. We examined two strains of Inpp4a mutant mice with distinct cerebellar phenotypes: the Inpp4aΔEx1,2 mutant exhibited striatal degeneration without cerebellar atrophy, and the Inpp4aΔEx23 mutant exhibited a severe striatal phenotype with cerebellar atrophy. Both strains exhibited reduced expression of Inpp4a mutant proteins in the cerebellum. N-terminal-truncated Inpp4a proteins were expressed from the Inpp4aΔEx1,2 allele by alternative translation initiation and had phosphatase activity for PI(3,4)P2, whereas the Inpp4a mutant protein encoded by Inpp4aΔEx23 completely lacked phosphatase activity. Our results indicate that the diverse phenotypes observed in Inpp4a-related neurological diseases could be due to the varying protein expression levels and retained phosphatase activity in different Inpp4a variants. These findings provide insights into the role of INPP4A mutations in disease pathogenesis and may help to develop personalized therapy.

Tmsb10 triggers fetal Leydig differentiation by suppressing the RAS/ERK pathway.

Leydig cells in fetal testes play crucial roles in masculinizing fetuses through androgen production. Gene knockout studies have revealed that growth factors are implicated in fetal Leydig cell (FLC) differentiation, but little is known about the mechanisms regulating this process. We investigate this issue by characterizing FLC progenitor cells using single-cell RNA sequencing. The sequence datasets suggest that thymosin ß10 (Tmsb10) is transiently upregulated in the progenitors. While studying the function of Tmsb10, we reveal that platelet-derived growth factor (PDGF) regulates ciliogenesis through the RAS/ERK and PI3K/AKT pathways, and thereby promotes desert hedgehog (DHH)-dependent FLC differentiation. Tmsb10 expressed in the progenitor cells induces their differentiation into FLCs by suppressing the RAS/ERK pathway. Through characterizing the transiently expressed Tmsb10 in the FLC progenitors, this study unveils the molecular process of FLC differentiation and shows that it is cooperatively induced by DHH and PDGF.

GC-MS-based metabolic signatures reveal comparative steroidogenic pathways between fetal and adult mouse testes.

BACKGROUND: Previous studies on gonadal steroidogenesis have not compared metabolic pathways between fetal and adult mouse testes to date. OBJECTIVES: To evaluate comparative metabolic signatures of testicular steroids between fetus and adult mice using gas chromatography-mass spectrometry (GC-MS)-based steroid profiling. MATERIALS AND METHODS: GC-MS with molecular-specific scan modes was optimized for selective and sensitive detection of 23 androgens, 7 estrogens, 14 progestogens, and 13 corticoids from mouse testes with a quantification limit of 0.1-5.0 ng/mL and reproducibility (coefficient of variation: 0.3%-19.9%). Based on 26 steroids quantitatively detected in testes, comparative steroid signatures were analyzed for mouse testes of 8 fetuses on embryonic day 16.5 and 8 adults on postnatal days 56-60. RESULTS: In contrast to large amounts of steroids in adult testes (P < .0002), all testicular levels per weight unit of protein were significantly increased in fetal testes (P < .002, except 6ß-hydroxytestosterone of P = .065). Both 11ß-hydroxyandrostenedione and 7α-hydroxytestosterone were only measurable in fetal testes, and metabolic ratios of testosterone to androstenediol and androstenedione were also increased in fetal testes (P < .05 for both). DISCUSSION AND CONCLUSION: Testicular steroid signatures showed that both steroidogenic Δ4 and Δ5 pathways in the production of testosterone were activated more during prenatal development. Both 7α- and 11ß-hydroxylations were predominant, while hydroxylations at C-6, C-15, and C-16 of testosterone and androstenedione were decreased in the fetus. The present GC-MS-based steroid profiling may facilitate understanding of the development of testicular steroidogenesis.

Gene expression and functional abnormalities in XX/Sry Leydig cells.

The SRY gene induces testis development even in XX individuals. However, XX/Sry testes fail to produce mature sperm, due to the absence of Y chromosome carrying genes essential for spermatogenesis. XX/Sry Sertoli cells show abnormalities in the production of lactate and cholesterol required for germ cell development. Leydig cells are essential for male functions through testosterone production. However, whether XX/Sry adult Leydig cells (XX/Sry ALCs) function normally remains unclear. In this study, the transcriptomes from XY and XX/Sry ALCs demonstrated that immediate early and cholesterogenic gene expressions differed between these cells. Interestingly, cholesterogenic genes were upregulated in XX/Sry ALCs, although downregulated in XX/Sry Sertoli cells. Among the steroidogenic enzymes, CYP17A1 mediates steroid 17α-hydroxylation and 17,20-lyase reaction, necessary for testosterone production. In XX/Sry ALCs, the latter reaction was selectively decreased. The defects in XX/Sry ALCs, together with those in the germ and Sertoli cells, might explain the infertility of XX/Sry testes.

Identification of reference genes for quantitative PCR analyses in developing mouse gonads.

Stable reference genes are important for gene expression analyses such as quantitative PCR. The stability of 15 candidate reference genes that can be used to developing mouse gonads was thoroughly verified using combinations of multiple algorithms. The expression of these genes fluctuated greatly depending on the analysis period and/or gender. Peptidylprolyl isomerase A (Ppia) and polymerase (RNA) II (DNA directed) polypeptide A (Polr2a) were the reference genes that were used stably for a wide analysis period in developing mouse gonads. Furthermore, the stable reference genes corresponding to the analysis period and/or gender were ranked. These results are useful for the selection of the optimal reference gene required for high-precision measurements.

Peripubertal exposure to the neonicotinoid pesticide dinotefuran affects dopaminergic neurons and causes hyperactivity in male mice.

Although neonicotinoid pesticides are expected to have harmful influence on mammals, there is little animal experimental data to support the effect and mechanisms. Since acetylcholine causes the release of dopamine, neonicotinoids may confer a risk of developmental disorders via a disturbance in the monoamine systems. Male mice were peripubertally administered dinotefuran (DIN) referring to no observed effect level (NOEL) and performed behavioral and immunohistological analyses. In an open field test, the total locomotor activity was increased in a dose-dependent manner. The immunoreactivity of tyrosine hydroxylase in the substantia nigra was increased in DIN-exposed mice. These results suggest that exposure to DIN in peripubertal male mice causes hyperactivity and a disturbance of dopaminergic signaling.

Verification of the causal relationship between subchronic exposures to dinotefuran and depression-related phenotype in juvenile mice.

It has been suggested that an increase in the use of pesticides affects neurodevelopment, but there has been no animal experiment showing a causal relation between neonicotinoid pesticides (NNs) and depression. We examined whether dinotefuran (DIN), the most widely used NN in Japan, induces depression. Male mice were administered DIN between 3 and 8 weeks of age, referring to the no-observed-effect level (NOEL). The mice were then subjected to a tail suspension test (TST) and a forced swimming test (FST). After these tests, their brains were dissected for immunohistochemical analyses of serotonin (5-HT). Antidepressant activity in TST and no decrease in 5-HT-positive cells were observed. The subchronic exposure to DIN alone in juvenile male mice may not cause depression-like indication.

NOAEL-dose of a neonicotinoid pesticide, clothianidin, acutely induce anxiety-related behavior with human-audible vocalizations in male mice in a novel environment.

Neonicotinoids are novel systemic pesticides acting as agonists on the nicotinic acetylcholine receptors (nAChRs) of insects. Experimental studies have revealed that neonicotinoids pose potential risks for the nervous systems of non-target species, but the brain regions responsible for their behavioral effects remain incompletely understood. This study aimed to assess the neurobehavioral effects of clothianidin (CTD), a later neonicotinoid developed in 2001 and widely used worldwide, and to explore the target regions of neonicotinoids in the mammalian brain. A single-administration of 5 or 50mg/kg CTD to male C57BL/6N mice at or below the no-observed-adverse-effect level (NOAEL) induced an acute increase in anxiety during the elevated plus-maze test. In addition, mice in the CTD-administered group spontaneously emitted human-audible vocalizations (4-16kHz), which are behavioral signs of aversive emotions, and showed increased numbers of c-fos immunoreactive cells in the paraventricular thalamic nucleus and dentate gyrus of the hippocampus. In conclusion, mice exposed to NOAEL-dose CTD would be rendered vulnerable to a novel environment via the activation of thalamic and hippocampal regions related to stress responses. These findings should provide critical insight into the neurobehavioral effects of neonicotinoids on mammals.

Prenatal and early postnatal NOAEL-dose clothianidin exposure leads to a reduction of germ cells in juvenile male mice.

Neonicotinoids are pesticides used worldwide. They bind to insect nicotinic acetylcholine receptors (nAChRs) with high affinity. We previously reported that clothianidin (CTD), one of the latest neonicotinoids, reduced antioxidant expression and induced germ cell death in the adult testis of vertebrates. Here, we investigated the male reproductive toxicity of prenatal and early postnatal exposure to CTD, because it is likely that developmental exposure more severely affects the testis compared to adults due to the absence of the blood-testis barrier. Pregnant C57BL/6 mice were given water gel blended with CTD (0, 10 or 50 mg/kg/day; no-observed-adverse-effect-level [NOAEL for mice]: 47.2 mg/kg/day) between gestational day 1 and 14 days post-partum. We then examined the testes of male offspring at postnatal day 14. The testis weights and the numbers of germ cells per seminiferous tubule were decreased in the CTD-50 group, and abnormal tubules containing no germ cells appeared. Nevertheless, the apoptotic cell number and proliferative activity were not significantly different between the control and CTD-exposed groups. There were no significant differences in the androgen-related parameters, such as the Leydig cell volume per testis, the Sertoli cell number and the tubule diameter. The present study is the first demonstration that in utero and lactational exposures to CTD at around the NOAEL for mice reduce the germ cell number, but our findings suggest that these exposures do not affect steroidogenesis in Leydig cells during prenatal or early postnatal life.

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.

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.

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.