Kentaro Suzuki

The corpus cavernosum, a key erectile structure within the penis, plays a central role in male reproductive function. While its prenatal development is known to be androgen‐dependent, the temporal dynamics of postnatal growth remain poorly understood. To delineate the postnatal developmental dynamics of the penis and corpus cavernosum, we established a standardized measurement framework and analyzed their developmental trajectories in mice. Penile and corpus cavernosum growth exhibited a bimodal pattern, with distinct peaks corresponding to the early postnatal period, known as minipuberty, and puberty. The proximal region containing the corpus cavernosum showed more pronounced growth than the distal region mainly composed of os penis, particularly during this early postnatal period. This bimodal growth pattern closely mirrors that of human penile development, highlighting the conserved androgen‐responsive developmental programs between mice and humans. Furthermore, early postnatal androgen exposure was sufficient to induce masculinization including increased external genital size and the formation of a corpus cavernosum‐like structure in females, whereas later exposure had limited effects. Our findings demonstrate that the early postnatal period, minipuberty, is a critical window for masculinization of external genitalia in mice. These results suggest that the mouse is a valuable model for understanding the developmental mechanisms of the human penis and corpus cavernosum in response to androgens, offering insights into erectile tissue formation and the etiology of congenital penile disorders such as micropenis.

The expression of hormonal receptors in pain-processing regions complicates understanding the hormonal effects on pain mechanisms. This study investigates androgen receptor (AR) involvement in pain sensitivity and sex differences in pain perception. Mechanical pain thresholds were higher in normal male mice compared to gonadectomized (GDX) male and normal female mice, correlating with serum testosterone levels. In the dorsal root ganglia (DRG), AR was expressed in normal males but undetectable in GDX males and normal females. Androgen receptor overlapped with NeuN, a neuronal nuclei marker, indicating androgen signaling activation in sensory neurons. In male sensory neuron–selective AR conditional knockout (AR-cKO) mice, mechanical pain thresholds were significantly lower than in wild-type males, with the greatest AR depletion in calcitonin gene–related peptide (CGRP) ⁺ neurons. Electrophysiological analyses revealed increased excitability of spinal dorsal horn neurons in both GDX males and AR-cKO males. In female mice, administration of testosterone propionate or dihydrotestosterone significantly raised mechanical pain thresholds, accompanied by increased AR expression in the DRG. This effect was abolished in AR-cKO females, where AR depletion was most prominent in CGRP ⁺ neurons, consistent with male findings. These results indicate that primary sensory neurons, particularly CGRP ⁺ neurons, are critical targets of androgen in regulating mechanical pain sensitivity. Therefore, manipulating androgen signaling in sensory neurons may offer a promising approach to managing mechanical pain.

Recent studies have revealed marked sex differences in pathophysiological roles of spinal microglia in neuropathic pain, with microglia contributing to pain exacerbation exclusively in males. However, the characteristics of pain-enhancing microglia, which are more prominent in males, remain poorly understood. Here, we reanalyzed a previously published single-cell RNA sequencing dataset and identified a microglial subpopulation that significantly increases in the spinal dorsal horn (SDH) of male mice following peripheral nerve injury. CC-chemokine ligand 4 (CCL4) was highly expressed in this subpopulation and its mRNA levels were increased in the SDH after partial sciatic nerve ligation (PSL) only in male mice. Notably, CCL4 expression was reduced in male mice following microglial depletion, indicating that microglia are the primary source of CCL4. Intrathecal administration of maraviroc, an inhibitor of the CCL4–CC-chemokine receptor 5 (CCR5) signaling pathway, after PSL, significantly suppressed mechanical allodynia only in male mice. Furthermore, intrathecal administration of CCL4 induced mechanical allodynia in both sexes, accompanied by increased expression of c-fos, a neuronal excitation marker, in the SDH. These findings highlight a sex-biased difference in the gene expression profile of spinal microglia following peripheral nerve injury, with elevated CCL4 expression in male mice potentially contributing to pain exacerbation.

It is well‐established that spinal microglia and peripheral macrophages play critical roles in the etiology of neuropathic pain; however, growing evidence suggests sex differences in pain hypersensitivity owing to microglia and macrophages. Therefore, it is crucial to understand sex‐ and androgen‐dependent characteristics of pain‐related myeloid cells in mice with nerve injury‐induced neuropathic pain. To deplete microglia and macrophages, pexidartinib (PLX3397), an inhibitor of the colony‐stimulating factor 1 receptor, was orally administered, and mice were subjected to partial sciatic nerve ligation (PSL). Following PSL induction, healthy male and female mice and male gonadectomized (GDX) mice exhibited similar levels of spinal microglial activation, peripheral macrophage accumulation, and mechanical allodynia. Treatment with PLX3397 significantly suppressed mechanical allodynia in normal males; this was not observed in female and GDX male mice. Sex‐ and androgen‐dependent differences in the PLX3397‐mediated preventive effects were observed on spinal microglia and dorsal root ganglia (DRG) macrophages, as well as in expression patterns of pain‐related inflammatory mediators in these cells. Conversely, no sex‐ or androgen‐dependent differences were detected in sciatic nerve macrophages, and inhibition of peripheral CC‐chemokine receptor 5 prevented neuropathic pain in both sexes. Collectively, these findings demonstrate the presence of considerable sex‐ and androgen‐dependent differences in the etiology of neuropathic pain in spinal microglia and DRG macrophages but not in sciatic nerve macrophages. Given that the mechanisms of neuropathic pain may differ among experimental models and clinical conditions, accumulating several lines of evidence is crucial to comprehensively clarifying the sex‐dependent regulatory mechanisms of pain.

Nuclear receptor subfamily 5 group A member 1 (NR5A1) is expressed in the pituitary gonadotrope and regulates their differentiation. Although several regulatory regions were implicated in Nr5a1 gene expression in the pituitary gland, none of these regions have been verified using mouse models. Furthermore, the molecular functions of NR5A1 in the pituitary gonadotrope have not been fully elucidated. In the present study, we generated mice lacking the pituitary enhancer located in the 6th intron of the Nr5a1 gene. These mice showed pituitary gland-specific disappearance of NR5A1, confirming the functional importance of the enhancer. Enhancer-deleted male mice demonstrated no defects at fetal stages. Meanwhile, androgen production decreased markedly in adult, and postnatal development of reproductive organs, such as the seminal vesicle, prostate, and penis was severely impaired. We further performed transcriptomic analyses of the whole pituitary gland of the enhancer-deleted mice and controls, as well as gonadotropes isolated from Ad4BP-BAC-EGFP mice. These analyses identified several genes showing gonadotrope-specific, NR5A1-dependent expressions, such as Spp1, Tgfbr3l, Grem1, and Nr0b2. These factors are thought to function downstream of NR5A1 and play important roles in reproductive organ development through regulation of pituitary gonadotrope functions.

Many industrial chemicals have been reported as antiandrogenic substances. Exposure to these substances represents a potential risk to human health, particularly to the development of reproductive organs such as embryonic external genitalia (eExG). Currently, there is a need for more assay systems that can elucidate the toxicological actions and mechanisms of endocrine-disrupting chemicals. In this study, we show that the eExG slice culture assay is useful for the evaluation of the differing modes of action of endocrine-disrupting chemicals on urethra formation. We assessed the possible endocrine-disrupting activity of 3 chemicals with reported antiandrogenic function, diazinon, dibutyl phthalate, and fenitrothion (FNT) on eExG slices. Exposure to FNT, but not diazinon and dibutyl phthalate, induced defects of androgen-induced urethral masculinization and reduced expression of the androgen-target gene Mafb. Live imaging analyses showed that FNT treatment inhibited androgen-dependent MAFB induction within 12 h. Furthermore, FNT-treated tissue slices showed reduced expression of the androgen receptor. These results indicate that FNT disrupts androgen signaling by reduction of androgen receptor expression during androgen-induced eExG masculinization. This study thus highlights the importance of animal models, which allow for the effective assessment of tissue-specific endocrine-disrupting activity to further reveal the etiology of chemical-induced congenital anomalies.

The murine penile erectile tissues including corpus cavernosum (CC) are composed of blood vessels, smooth muscle, and connective tissue, showing marked sexual differences. It has been known that the androgens are required for sexually dimorphic organogenesis. It is however unknown about the features of androgen signaling during mouse CC development. It is also unclear how androgen-driven downstream factors are involved such processes. In the current study, we analyzed the onset of sexually dimorphic CC formation based on histological analyses, the dynamics of androgen receptor (AR) expression, and regulation of cell proliferation. Of note, we identified Dickkopf-related protein 2 (Dkk2), an inhibitor of beta-catenin signaling, was predominantly expressed in female CC compared with male. Furthermore, administration of androgens resulted in activation of beta-catenin signaling. We have found the Sox9 gene, one of the essential markers for chondrocyte, was specifically expressed in the developing CC. Hence, we utilized CC-specific, Sox9(CreERT2), beta-catenin conditional mutant mice. Such mutant mice showed defective cell proliferation. Furthermore, introduction of activated form of beta-catenin mutation (gain of function mutation for Wnt/beta-catenin signaling) in CC induced augmented cell proliferation. Altogether, we revealed androgen-Wnt/beta-catenin signal dependent cell proliferation was essential for sexually dimorphic CC formation. These findings open new avenues for understanding developmental mechanisms of androgen-dependent cell proliferation during sexual differentiation.