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.

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.

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.

Male infertility is a global health concern. However, its underlying pathophysiology remains unclear. Taste receptor type 1 member 3 (TAS1R3) is highly expressed in the testes, indicating its potential involvement in male fertility. Using wild-type and Tas1r3 knockout (KO) mice, we investigated whether TAS1R3 modulates male reproductive function. Tas1r3 KO mice exhibited reduced male fertility compared to WT mice, with fewer live pups per litter and a delayed first litter. Testicular transcriptome analysis indicated suppressed PKA/CREB/StAR signaling-mediated testosterone synthesis in Tas1r3 KO mice. In silico single-cell RNA sequencing revealed considerably higher Tas1r3 expression in Leydig cells than in other testicular cell subtypes. An in vitro study validated that Tas1r3 knockdown downregulated the expression of Creb1 and steroidogenic genes in Leydig cells. Our results suggest that testicular TAS1R3 is intricately involved in male reproduction via the PKA/CREB/StAR signaling pathway, highlighting its potential as a promising target for addressing male infertility.

The increasing life expectancy observed in recent years has resulted in a higher prevalence of late-onset hypogonadism (LOH) in older men. LOH is characterized by the decline in testosterone levels and can have significant impacts on physical and mental health. While the underlying causes of LOH are not fully understood, there is a growing interest in exploring the role of inflammaging in its development. Inflammaging is a concept that describes the chronic, low-grade, systemic inflammation that occurs as a result of aging. This inflammatory state has been implicated in the development of various age-related diseases. Several cellular and molecular mechanisms have been identified as contributors to inflammaging, including immune senescence, cellular senescence, autophagy defects, and mitochondrial dysfunction. Despite the extensive research on inflammaging, its relationship with LOH has not yet been thoroughly reviewed in the literature. To address this gap, we aim to review the latest findings related to inflammaging and its impact on the development of LOH. Additionally, we will explore interventions that target inflammaging as potential treatments for LOH.

Children undergoing cancer treatments are at risk for impaired fertility. Cryopreserved prepubertal testicular biopsies could theoretically be later matured in vitro to produce spermatozoa for assisted reproductive technology. A complete in vitro spermatogenesis has been obtained from mouse prepubertal testicular tissue, although with low efficiency. Steroid hormones are essential for the progression of spermatogenesis, the aim of this study was to investigate steroidogenesis and steroid signaling in organotypic cultures. Histological, RT-qPCR, western blot analyses, and steroid hormone measurements were performed on in vitro cultured mouse prepubertal testicular tissues and age-matched in vivo controls. Despite a conserved density of Leydig cells after 30 days of culture (D30), transcript levels of adult Leydig cells and steroidogenic markers were decreased. Increased amounts of progesterone and estradiol and reduced androstenedione levels were observed at D30, together with decreased transcript levels of steroid metabolizing genes and steroid target genes. hCG was insufficient to facilitate Leydig cell differentiation, restore steroidogenesis, and improve sperm yield. In conclusion, this study reports the failure of adult Leydig cell development and altered steroid production and signaling in tissue cultures. The organotypic culture system will need to be further improved before it can be translated into clinics for childhood cancer survivors.

To investigate the role of the nuclear receptor NR5A1 in the testis after sex determination, we analyzed mice lacking NR5A1 in Sertoli cells (SCs) from embryonic day (E) 13.5 onwards. Ablation of Nr5a1 impaired the expression of genes characteristic of SC identity (e.g. Sox9 and Amh), caused SC death from E14.5 onwards through a Trp53-independent mechanism related to anoikis, and induced disorganization of the testis cords. Together, these effects caused germ cells to enter meiosis and die. Single-cell RNA-sequencing experiments revealed that NR5A1-deficient SCs changed their molecular identity: some acquired a \'pre-granulosa-like\' cell identity, whereas other reverted to a \'supporting progenitor-like\' cell identity, most of them being \'intersex\' because they expressed both testicular and ovarian genes. Fetal Leydig cells (LCs) did not display significant changes, indicating that SCs are not required beyond E14.5 for their emergence or maintenance. In contrast, adult LCs were absent from postnatal testes. In addition, adult mutant males displayed persistence of Müllerian duct derivatives, decreased anogenital distance and reduced penis length, which could be explained by the loss of AMH and testosterone synthesis due to SC failure.

Klinefelter\'s syndrome (KS) was once considered infertile due to congenital chromosomal abnormalities, but the presence of focal spermatozoa changed this. The key to predict and promote spermatogenesis is to find targets that regulate focal spermatogenesis.
To explore the trend of fertility changes in KS patients at different ages and identify potential therapeutic targets.
Bibliometric analysis was used to collect clinical research data on KS from the Web of Science Core Collection (WoSCC) from 1992 to 2022. A cross-sectional study was conducted on 75 KS patients who underwent microscopic testicular sperm extraction (mTESE) from 2017 to 2022 in the real world. The reproductive hormones, testicular histopathology, androgen receptors, insulin-like factor 3 (INSL3) receptors and sperm recovery rate (SRR) were analyzed.
Male infertility, dysplasia, Sertoli cells, Leydig cells, testosterone and spermatogenesis were the research focuses related to KS. Luteinizing hormone (LH), testosterone, and INSL3 were evaluation indicators of Leydig cell function that fluctuate with age. Testosterone and LH peaked at ages 13-19 and 30-45, while INSL3 only peaked at ages 13-19. 27 patients (27/75) recovered sperm through mTESE and experienced SRR peaks at the ages of 20, 28, 34, and 37. The SRR of fibrosis patients was 46.15%, fatty degeneration was 7.14%, and melanosis was 40.00%. The INSL3 and androgen receptors were highly expressed and roughly balanced in focal spermatogenesis.
Abnormal metabolism of Leydig cells led to imbalanced expression of INSL3 and androgen receptors, which might be a potential target for spermatogenesis in KS.

The objective of this review was to summarize the applications of sonoelastography in testicular tumor identification and inquire about their test performances. Two authors independently searched English journal articles and full conference papers from CINAHL, Embase, IEEE Xplore®, PubMed, Scopus, and Web of Science from inception and organized them into a PIRO (patient, index test, reference test, outcome) framework. Eleven studies (n = 11) were eligible for data synthesis, nine of which (n = 9) utilized strain elastography and two (n = 2) employed shear-wave elastography. Meta-analyses were performed on the distinction between neoplasm (tumor) and non-neoplasm (non-tumor) from four study arms and between malignancy and benignity from seven study arms. The pooled sensitivity of classifying malignancy and benignity was 86.0% (95%CI, 79.7% to 90.6%). There was substantial heterogeneity in the classification of neoplasm and non-neoplasm and in the specificity of classifying malignancy and benignity, which could not be addressed by the subgroup analysis of sonoelastography techniques. Heterogeneity might be associated with the high risk of bias and applicability concern, including a wide spectrum of testicular pathologies and verification bias in the reference tests. Key technical obstacles in the index test were manual compression in strain elastography, qualitative observation of non-standardized color codes, and locating the Regions of Interest (ROI), in addition to decisions in feature extractions. Future research may focus on multiparametric sonoelastography using deep learning models and ensemble learning. A decision model on the benefits-risks of surgical exploration (reference test) could also be developed to direct the test-and-treat strategy for testicular tumors.

Testicular Leydig cells (LCs) are the primary known source of testosterone, which is necessary for maintaining spermatogenesis and male fertility. However, the isolation, identification, and functional analysis of testosterone in duck LCs are still ambiguous. The aim of the present study was to establish a feasible method for isolating highly purified primary duck LCs. The highly purified primary duck LCs were isolated from the fresh testes of 2-month-old ducks via the digestion of collagenase IV and Percoll density gradient centrifugation; hematoxylin and eosin (H&E), immunohistochemistry (IHC) staining, ELISA, and radioimmunoassay were performed. Results revealed that the LCs were prominently noticeable in the testicular interstitium of 2-month-old ducks as compared to 6-month-old and 1-year-old ducks. Furthermore, IHC demonstrated that the cultured LCs occupied 90% area of the petri dish and highly expressed 3β-HSD 24 h after culture (hac) as compared to 48 and 72 hac. Additionally, ELISA and radioimmunoassay indicate that the testosterone level in cellular supernatant was highly expressed in 24 and 48 hac, whereas the testosterone level gradually decreased in 72 and 96 hac, indicating the primary duck LCs secrete testosterone at an early stage. Based on the above results, the present study has effectively developed a technique for isolating highly purified primary duck LCs and identified its biological function in synthesizing testosterone.