Male fertility depends on normal pubertal development. Di(2-ethylhexyl) phthalate (DEHP) is a potent antiandrogen chemical, and exposure to DEHP during peripuberty can damage the developing male reproductive system, especially the testis. However, the specific cellular targets and differentiation processes affected by DEHP, which lead to testicular toxicity, remain poorly defined. Herein, we presented the first single-cell transcriptomic profile of the pubertal mouse testis following DEHP exposure. To carry out the experiment, two groups (n = 8 each) of three-week old male mice were orally administered 0.5% carboxymethylcellulose sodium salt or 100 mg/kg body weight DEHP daily from postnatal day 21 to 48, respectively. Using single-cell RNA sequencing, a total of 31 distinct cell populations were identified, notably, Sertoli and Leydig cells emerged as important targets of DEHP. DEHP exposure significantly decreased the proportions of Sertoli cell clusters expressing mature Sertoli markers (Sox9 and Ar), and selectively reduced the expression of testosterone synthesis genes in fetal Leydig cells. Through cell-cell interaction analyses, we observed changed numbers of interactions in Sertoli cells 1 (SCs1), Leydig cells 1 (LCs1) and interstitial macrophages (ITMs), and we also identified cell-specific ligand gene expressions in these clusters, such as Inha, Fyn, Vcam1, and Apoe. Complementary in vitro assays confirmed that DEHP directly reduced the expression of genes related to Sertoli cell adhesion and intercellular communication. In conclusion, peripubertal DEHP exposure reduced the number of mature Sertoli cells and may disrupt testicular steroidogenesis by affecting the testosterone synthesis genes in fetal Leydig cells rather than adult Leydig cells.

Leydig cells are essential components of testicular interstitial tissue and serve as a primary source of androgen in males. A functional deficiency in Leydig cells often causes severe reproductive disorders; however, the transcriptional programs underlying the fate decisions and steroidogenesis of these cells have not been fully defined. In this study, we report that the homeodomain transcription factor PBX1 is a master regulator of Leydig cell differentiation and testosterone production in mice. PBX1 was highly expressed in Leydig cells and peritubular myoid cells in the adult testis. Conditional deletion of Pbx1 in Leydig cells caused spermatogenic defects and complete sterility. Histological examinations revealed that Pbx1 deletion impaired testicular structure and led to disorganization of the seminiferous tubules. Single-cell RNA-seq analysis revealed that loss of Pbx1 function affected the fate decisions of progenitor Leydig cells and altered the transcription of genes associated with testosterone synthesis in the adult testis. Pbx1 directly regulates the transcription of genes that play important roles in steroidogenesis (Prlr, Nr2f2 and Nedd4). Further analysis demonstrated that deletion of Pbx1 leads to a significant decrease in testosterone levels, accompanied by increases in pregnenolone, androstenedione and luteinizing hormone. Collectively, our data revealed that PBX1 is indispensable for maintaining Leydig cell function. These findings provide insights into testicular dysgenesis and the regulation of hormone secretion in Leydig cells.

Insulin-like peptide 3 (INSL3) is a circulating biomarker for Leydig cell functional capacity in men, also indicating Leydig Cell Insufficiency (LCI) and potential primary hypogonadism. Using results from large cohort studies we explore sources of biological and technical variance, and establish a reference range for adult men. It is constitutively secreted with little within-individual variation and reflects testicular capacity to produce testosterone. The main INSL3 assays available indicate good concordance with low technical variance; there is no effect of ethnicity. INSL3 declines with age from 35 years at about 15% per decade. Like low calculated free testosterone, and to a lesser extent low total testosterone, reduced INSL3 is significantly associated with increasing age-related morbidity, including lower overall sexual function, reflecting LCI. Consequently, low INSL3 (≤0.4 ng/ml; ca. <2 SD from the population mean) might serve as an additional biochemical marker in the assessment of functional hypogonadism (late-onset hypogonadism, LOH) where testosterone is in the borderline low range. Excluding individuals with low LCI (INSL3 ≤ 0.4 ng/ml) leads to an age-independent (> 35 years) reference range (serum) for INSL3 in the eugonadal population of 0.4 - 2.3 ng/ml, with low INSL3 prospectively identifying individuals at risk of increased future morbidity.

Spermatogonial stem cell (SSC) acquisition of meiotogenetic state during puberty to produce genetically diverse gametes is blocked by drugs collectively referred as \'puberty blocker\' (PB). Investigating the impact of PB on juvenile SSC state and function is challenging due to limited tissue access and clinical data. Herein, we report largest clinically annotated juvenile testicular biorepository with all children with gender dysphoria on chronic PB treatment highlighting shift in pediatric patient demography in US. At the tissue level, we report mild-to-severe sex gland atrophy in PB treated children. We developed most extensive integrated single-cell RNA dataset to date (>100K single cells; 25 patients), merging both public and novel (52 month PB-treated) datasets, alongside innovative computational approach tailed for germ cells and evaluated the impact of PB and aging on SSC. We report novel constitutional ranges for each testicular cell type across the entire age spectrum, distinct effects of treatments on prepubertal vs adult SSC, presence of spermatogenic epithelial cells exhibiting post-meiotic-state, irrespective of age, puberty status, or PB treatment. Further, we defined distinct effects of PB and aging on testicular cell lineage composition, and SSC meiotogenetic state and function. Using single cell data from prepubertal and young adult, we were able to accurately predict sexual maturity based both on overall cell type proportions, as well as on gene expression patterns within each major cell type. Applying these models to a PB-treated patient that they appeared pre-pubertal across the entire tissue. This combined with the noted gland atrophy and abnormalities from the histology data raise a potential concern regarding the complete \'reversibility\' and reproductive fitness of SSC. The biorepository, data, and research approach presented in this study provide unique opportunity to explore the impact of PB on testicular reproductive health.

In the domain of medical advancement, nanotechnology plays a pivotal role, especially in the synthesis of biocompatible materials for therapeutic use. Superparamagnetic Iron Oxide Nanoparticles (SPIONs), known for their magnetic properties and low toxicity, stand at the forefront of this innovation. This study explored the reproductive toxicological effects of Sodium Citrate-functionalized SPIONs (Cit_SPIONs) in adult male mice, an area of research that holds significant potential yet remains largely unknown. Our findings reveal that Cit_SPIONs induce notable morphological changes in interstitial cells and the seminiferous epithelium when introduced via intratesticular injection. This observation is critical in understanding the interactions of nanomaterials within reproductive biological systems. A striking feature of this study is the rapid localization of Cit_SPIONs in Leydig cells post-injection, a factor that appears to be closely linked with the observed decrease in steroidogenic activity and testosterone levels. This data suggests a possible application in developing nanostructured therapies targeting androgen-related processes. Over 56 days, these nanoparticles exhibited remarkable biological distribution in testis parenchyma, infiltrating various cells within the tubular and intertubular compartments. While the duration of spermatogenesis remained unchanged, there were many Tunel-positive germ cells, a notable reduction in daily sperm production, and reduced progressive sperm motility in the treated group. These insights not only shed light on the intricate mechanisms of Cit_SPIONs interaction with the male reproductive system but also highlight the potential of nanotechnology in developing advanced biomedical applications.

Cryptorchidism is the condition in which one or both testes have not descended adequately into the scrotum. The congenital form of cryptorchidism is one of the most prevalent urogenital anomalies in male newborns. In the acquired form of cryptorchidism, the testis that was previously descended normally is no longer located in the scrotum. Cryptorchidism is associated with an increased risk of infertility and testicular germ cell tumors. However, data on pubertal progression are less well-established because of the limited number of studies. Here, we aim to review the currently available data on pubertal development in boys with a history of non-syndromic cryptorchidism-both congenital and acquired cryptorchidism. The review is focused on the timing of puberty, physical changes, testicular growth, and endocrine development during puberty. The available evidence demonstrated that the timing of the onset of puberty in boys with a history of congenital cryptorchidism does not differ from that of non-cryptorchid boys. Hypothalamic-pituitary-gonadal hormone measurements showed an impaired function or fewer Sertoli cells and/or germ cells among boys with a history of cryptorchidism, particularly with a history of bilateral cryptorchidism treated with orchiopexy. Leydig cell function is generally not affected in boys with a history of cryptorchidism. Data on pubertal development among boys with acquired cryptorchidism are lacking; therefore, more research is needed to investigate pubertal progression among such boys.

Cadmium (Cd) is a common heavy metal pollutant in the environment, and the widespread use of products containing Cd compounds in industry has led to excessive levels in the environment, which enter the animal body through the food chain, thus seriously affecting the reproductive development of animals. Related studies have reported that Cd severely affects spermatogonia development and spermatogenesis in animals. In contrast, the reproductive toxicity of Cd in males and its mechanism of action have not been clarified. Therefore, this paper reviewed the toxic effects of Cd on germ cells, spermatogonia somatic cells and hypothalamic-pituitary-gonadal axis (HPG axis) of male animals and its toxic action mechanisms of oxidative stress, apoptosis and autophagy from the perspectives of cytology, genetics and neuroendocrinology. The effects of Cd stress on epigenetic modification of reproductive development in male animals were also analyzed. We hope to provide a reference for the in-depth study of the toxicity of Cd on male animal reproduction.

Although several testicular alterations promoted by coronavirus infection have been demonstrated, the extent, causes, and players of testicular pathogenesis are not totally understood. The present study aimed to investigate the short-term effects on male fertility of intranasally administered murine hepatitis virus strain 3 (MHV-3), a member of the genus Betacoronavirus, which causes a severe systemic acute infection. This mouse model might be used as a in vivo prototype for investigating the impact of betacoronavirus on the endocrine and exocrine testicular functions with the advantage to be performed in a biosafety level 2 condition. Herein, we performed virological, histopathological, and molecular studies regarding the testicular spermatogenesis and the spermatic quality analyses in an MHV-3-infected C57BL/6 mice. The main outcomes showed that MHV-3 infects mouse testis and induces a testicular inflammatory state, impairing the steroidogenic pathway. The infection led to several alterations in the testicular parenchyma, such as: seminiferous epithelium sloughing, retention of residual bodies, germ cell apoptosis, alterations in intercellular junction proteins, and worse spermatogenic parameters. Moreover, the levels of plasmatic testosterone as well as the quality of sperm production reduced. Therefore, the present data suggest that the viral/inflammatory impairment of the steroidogenic pathway and the consequent imbalance of androgen levels is critical in testicular pathology, disturbing the SC barrier function and the germ cell differentiation. Our study is important for comprehending the effects of beta coronavirus infections on testis function in order to develop treatments that could prevent virus-mediated male infertility.

Di (2-ethylhexyl) phthalate (DEHP), identified as an endocrine-disrupting chemical, is associated with reproductive toxicity. This association is particularly noteworthy in newborns with incompletely developed metabolic functions, as exposure to DEHP can induce enduring damage to the reproductive system, potentially influencing adult reproductive health. In this study, we continuously administered 40 μg/kg and 80 μg/kg DEHP to postnatal day 5 (PD5) mice for ten days to simulate low and high doses of DEHP exposure during infancy. Utilizing single-cell RNA sequencing (scRNA-seq), our analysis revealed that varying concentrations of DEHP exposure during infancy induced distinct DNA damage response characteristics in testicular Undifferentiated spermatogonia (Undiff SPG). Specifically, DNA damage triggered mitochondrial dysfunction, leading to acetyl-CoA content alterations. Subsequently, this disruption caused aberrations in histone acetylation patterns, ultimately resulting in apoptosis of Undiff SPG in the 40 μg/kg DEHP group and autophagy in the 80 μg/kg DEHP group. Furthermore, we found that DEHP exposure impacts the development and functionality of Sertoli and Leydig cells through the focal adhesion and PPAR signaling pathways, respectively. We also revealed that Leydig cells regulate the metabolic environment of Undiff SPG via Ptn-Sdc4 and Mdk-Sdc4 after DEHP exposure. Finally, our study provided pioneering evidence that disruptions in testicular homeostasis induced by DEHP exposure during infancy endure into adulthood. In summary, this study elucidates the molecular mechanisms through which DEHP exposure during infancy influences the development of testicular cell populations.

Glyphosate is an endocrine disruptor and can act on the activity of certain enzymes of metabolism subsequently altering some functions such as reproduction. The goal of the present study is to evaluate the involvement of glyphosate based-herbicide (GBH) in spermatogenesis disruption and to investigate which cells of the adult Wistar rat testis are most affected by short-term exposure to GBH. Treated groups received a diluted solution of GBH orally for 21 days (D1: 102.5 mg/Kg; D2: 200 mg/Kg; D3: 400 mg/Kg). The control group (C) received water in the same manner. Hormone levels, oxidative stress markers were evaluated, histological and morphometric analysis were performed, AR and p53 expression was conducted. Seminiferious epithelium sloughing associated to erosion of Sertoli and spermatogonia from the basement of the seminiferous tubules, with intraluminal exfoliated cells among with immature spermatids were observed. A significant change in morphometric measurement and significant decrease in AR expression in Sertoli cells were noted for all treated groups. A significant increase in NO level and p53 expression in Leydig cells were showed for animals treated with 200 and 400 mg/kg BW/day. These data demonstrate that short-term exposure to high doses of GBH has led to a disruption of certain parameters that could disturb spermatogenesis. The treatment showed that both Leydig and Sertoli cells are affected in the same manner by GBH, the activation of p53 expression in both Leydig cells and peritubular myloid cells nuclei, and the reduction in AR expression in Sertoli cells, which resulted in important testicular damage.