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The pig is an important model for studying human diseases and is also a significant livestock species, yet its testicular development remains underexplored. Here, we employ single-cell RNA sequencing to characterize the transcriptomic landscapes across multiple developmental stages of Bama pig testes from fetal stage through infancy, puberty to adulthood, and made comparisons with those of humans and mice. We reveal an exceptionally early onset of porcine meiosis shortly after birth, and identify a distinct subtype of porcine spermatogonia resembling transcriptome state 0 spermatogonial stem cells identified in humans, which were previously thought to be primate specific. We also discover the persistent presence of proliferating progenitors for myoid cells in postnatal testes. The regulatory roles of Leydig cell steroidogenesis and estrogen synthesis in supporting cell lineages are also explored, including the potential impact of estrogen on Sertoli cell maturation and spermatogenesis. Overall, this study offers valuable insights into porcine testicular development, paving the way for future research in reproductive biology, advancements in agricultural breeding, and potential applications in translational medicine. A study using single-cell RNA sequencing to map transcriptomic landscapes across Bama pig testes development reveals early onset meiosis, a unique spermatogonia subtype, and persistent myoid progenitors. [ABSTRACT FROM AUTHOR]

Heavy metals like cadmium (Cd) are one of the main environmental pollutants, with no biological role in the human body. Cd has been well-documented to have disastrous effects on both plants and animals. It is known to accumulate in kidneys, lungs, liver, and testes and is thought to affect these organs' function over time, which is linked to a very long biological half-life and a very poor rate of elimination. According to recent researches, the testes are extremely vulnerable to cadmium. The disruption of the blood–testis barrier, seminiferous tubules, Sertoli cells, and Leydig cells caused by cadmium leads to the loss of sperm through various mechanisms, such as oxidative stress, spermatogenic cell death, testicular swelling, dysfunction in androgen-producing cells, interference with gene regulation, disruption of ionic homeostasis, and damage to the vascular endothelium. Additionally, through epigenetic control, cadmium disrupts the function of germ cells and somatic cells, resulting in infertile or subfertile males. A full grasp of the mechanisms underlying testicular toxicity caused by Cd is very important to develop suitable strategies to ameliorate male fertility. Therefore, this review article outlines cadmium's impact on growth and functions of the testicles, reviews therapeutic approaches and protective mechanisms, considers recent research findings, and identifies future research directions. [ABSTRACT FROM AUTHOR]

Aims: STK11 adnexal tumour is a rare, recently described malignant neoplasm that is associated with Peutz–Jeghers syndrome. [Correction added on 3 October 2024, after first online publication: 'ST11' in preceding sentence has been corrected to 'STK11' in this version.] It predominantly originates from the para‐adnexal soft tissues and often shows secondary involvement of the fallopian tube and ovary. STK11 adnexal tumours have a broad differential diagnosis due to their variable morphology and non‐specific immunoprofile, and diagnostic confirmation currently requires sequencing to identify an STK11 mutation. We investigate the diagnostic utility of STK11 (LKB1) immunohistochemistry (IHC) in a cohort of STK11 adnexal tumours and morphological mimics. Methods and results: IHC for STK11 was performed on 122 tumours, including 17 STK11 adnexal tumours and 105 morphological mimics (10 female adnexal tumours of Wolffian origin, 22 adult granulosa cell tumours, 10 juvenile granulosa cell tumours, four Sertoli–Leydig cell tumours, two Leydig cell tumours, one Sertoli cell tumour, one steroid cell tumour, four extra‐ovarian sex cord‐stromal tumours, 16 ovarian endometrioid carcinomas, eight tubo‐ovarian high‐grade serous carcinomas, five ovarian mesonephric‐like adenocarcinomas, 14 ovarian carcinosarcomas, five peritoneal malignant mesotheliomas, two pelvic plexiform leiomyomata and one ovarian solid pseudopapillary tumour). All STK11 adnexal tumours showed complete loss of cytoplasmic staining for STK11. All other tumour types showed retained cytoplasmic staining, except for one endometrioid carcinoma with mucinous differentiation which showed complete loss of STK11 expression and a high‐grade serous carcinoma with subclonal loss. Conclusions: STK11 is a highly sensitive and specific immunohistochemical marker for distinguishing STK11 adnexal tumour from its histological mimics, and can obviate the need for confirmatory molecular studies in the appropriate morphological context. [ABSTRACT FROM AUTHOR]

Testicular tumors represent the most common malignancy among young men. Nevertheless, the pathogenesis and molecular underpinning of testicular tumors remain largely elusive. We aimed to delineate the intricate intra‐tumoral heterogeneity and the network of intercellular communication within the tumor microenvironment. A total of 40,760 single‐cell transcriptomes were analyzed, encompassing samples from six individuals with seminomas, two patients with mixed germ cell tumors, one patient with a Leydig cell tumor, and three healthy donors. Five distinct malignant subclusters were identified in the constructed landscape. Among them, malignant 1 and 3 subclusters were associated with a more immunosuppressive state and displayed worse disease‐free survival. Further analysis identified that APP–CD74 interactions were significantly strengthened between malignant 1 and 3 subclusters and 14 types of immune subpopulations. In addition, we established an aberrant spermatogenesis trajectory and delineated the global gene alterations of somatic cells in seminoma testes. Sertoli cells were identified as the somatic cell type that differed the most from healthy donors to seminoma testes. Cellular communication between spermatogonial stem cells and Sertoli cells is disturbed in seminoma testes. Our study delineates the intra‐tumoral heterogeneity and the tumor immune microenvironment in testicular tumors, offering novel insights for targeted therapy. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Background: Non-obstructive azoospermia (NOA) is a major contributor of male infertility. Herein, we used existing datasets to identify novel biomarkers for the diagnosis and prognosis of NOA, which could have great significance in the field of male infertility. Methods: NOA datasets were obtained from the Gene Expression Omnibus (GEO) database. CIBERSORT was utilized to analyze the distributions of 22 immune cell populations. Hub genes were identified by applying weighted gene co-expression network analysis (WGCNA), machine learning methods, and protein–protein interaction (PPI) network analysis. The expression of hub genes was verified in external datasets and was assessed by receiver operating characteristic (ROC) curve analysis. Gene set enrichment analysis (GSEA) was applied to explore the important functions and pathways of hub genes. The mRNA–microRNA (miRNA)–transcription factors (TFs) regulatory network and potential drugs were predicted based on hub genes. Single-cell RNA sequencing data from the testes of patients with NOA were applied for analyzing the distribution of hub genes in single-cell clusters. Furthermore, testis tissue samples were obtained from patients with NOA and obstructive azoospermia (OA) who underwent testicular biopsy. RT-PCR and Western blot were used to validate hub gene expression. Results: Two immune-related oxidative stress hub genes (SHC1 and FGFR1) were identified. Both hub genes were highly expressed in NOA samples compared to control samples. ROC curve analysis showed a remarkable prediction ability (AUCs > 0.8). GSEA revealed that hub genes were predominantly enriched in toll-like receptor and Wnt signaling pathways. A total of 24 TFs, 82 miRNAs, and 111 potential drugs were predicted based on two hub genes. Single-cell RNA sequencing data in NOA patients indicated that SHC1 and FGFR1 were highly expressed in endothelial cells and Leydig cells, respectively. RT-PCR and Western blot results showed that mRNA and protein levels of both hub genes were significantly upregulated in NOA testis tissue samples, which agree with the findings from analysis of the microarray data. Conclusion: It appears that SHC1 and FGFR1 could be significant immune-related oxidative stress biomarkers for detecting and managing patients with NOA. Our findings provide a novel viewpoint for illustrating potential pathogenesis in men suffering from infertility. [ABSTRACT FROM AUTHOR]

Cordycepin (COR), a pure compound of Cordyceps, is known as an adenosine analog that exerts many beneficial effects on human health. The steroidogenesis mediated by ovarian granulosa cells is pivotal in maintaining normal female reproductive function. The steroidogenic acute regulatory protein (STAR) regulates the rate-limiting step in steroidogenesis. COR has been shown to stimulate STAR expression in mouse Leydig cells, the steroidogenic cells in the testes. However, the effect of COR on STAR expression in ovarian granulosa cells remains undetermined. In the present study, we show that treatment with COR downregulates STAR expression in a steroidogenic human granulosa-like tumor cell line, KGN, and primary culture of human granulosa-lutein (hGL) cells obtained from patients undergoing in vitro fertilization. We used specific adenosine receptor (AR) antagonists, and our results reveal that the inhibitory effect of COR on STAR expression is mediated by AR-A1, AR-A2A, and AR-A3. In both KGN and primary hGL cells, COR activates ERK1/2 and AKT signaling pathways, but only activation of ERK1/2 is required for the COR-induced downregulation of STAR expression. In addition, our results demonstrate that COR downregulates STAR expression by reducing the expression of the SP1 transcription factor. These results provide a better understanding of the biological function of COR on STAR expression in the ovary, which may lead to the development of alternative therapeutic approaches for female reproductive disorders. [ABSTRACT FROM AUTHOR]

This study aimed to analyze the molecular characteristics of insulin-like growth factor 1 (IGF1) gene in the testes of Tibetan sheep and its role in the testosterone synthesis and cell development. First, we cloned IGF1 gene for bioinformatics analysis, and the primary Leydig cells (LCs) of Tibetan sheep were isolated to explore its effect on the proliferation, apoptosis and function of LCs. Finally, the specific regulatory mechanism of IGF1 on LCs was analyzed by transcriptome sequencing. Results showed that overexpression of IGF1 increased the proliferation rate and decreased apoptosis of LCs. In addition, overexpression of IGF1 altered expression of genes related to testosterone synthesis and transformation and significantly increased amount of the final product testosterone. Mechanistically, IGF1 stimulated the expression of the proliferating cell nuclear antigen and IGF1R and promoted the proliferation of LCs via the PI3K/Akt signaling pathway. Collectively, what should be clear from the results reported here is that IGF1 might play roles in the proliferation or differentiation and testosterone synthesis of LCs. These findings add to our understanding on the regulation of testosterone synthesis in sheep and other mammals. [ABSTRACT FROM AUTHOR]

Teleost testis development during the annual cycle involves dramatic changes in cellular compositions and molecular events. In this study, the testicular cells derived from adult black rockfish at distinct stages - regressed, regenerating and differentiating - were meticulously dissected via single-cell transcriptome sequencing. A continuous developmental trajectory of spermatogenic cells, from spermatogonia to spermatids, was delineated, elucidating the molecular events involved in spermatogenesis. Subsequently, the dynamic regulation of gene expression associated with spermatogonia proliferation and differentiation was observed across spermatogonia subgroups and developmental stages. A bioenergetic transition from glycolysis to mitochondrial respiration of spermatogonia during the annual developmental cycle was demonstrated, and a deeper level of heterogeneity and molecular characteristics was revealed by re-clustering analysis. Additionally, the developmental trajectory of Sertoli cells was delineated, alongside the divergence of Leydig cells and macrophages. Moreover, the interaction network between testicular micro-environment somatic cells and spermatogenic cells was established. Overall, our study provides detailed information on both germ and somatic cells within teleost testes during the annual reproductive cycle, which lays the foundation for spermatogenesis regulation and germplasm preservation of endangered species. [ABSTRACT FROM AUTHOR]

Background: Androgens are essential hormones for testicular development and the maintenance of male fertility. Environmental factors, stress, aging, and psychological conditions can disrupt androgen production, impacting the androgen signaling pathway and consequently spermatogenesis. Within the testes, testosterone is produced by Leydig cells and acts on Sertoli cells by activating the androgen receptor (AR), which then translocates to the nucleus to function as a transcription factor. Despite clinical correlations between low testosterone levels and diminished sperm quality, the precise mechanism remains unclear. Methods: This study explores the hypothesis that reduced androgen levels impair Sertoli cell function by disrupting AR transcriptional regulation. Using an androgen blockade model with enzalutamide, we investigated the impact of low androgen levels on AR target genes in Sertoli cells through ChIP-seq and RNA-seq assays. Results: Our results reveal that androgen blockage increases AR enrichment on the promoter region of Wee1, promoting Wee1 expression, while decreasing binding to the promoter region of Lfng, inhibiting its expression. Increased WEE1 protein inhibits Sertoli cell proliferation, whereas reduced LFNG affects Notch modification, leading to decreased production of glial cell line-derived neurotrophic factor (GDNF), a key growth factor for spermatogonial stem cell self-renewal. Conclusions: These findings provide new insights into the molecular mechanisms by which low androgen levels interfere with Sertoli cell functions, offering novel perspectives for the clinical treatment of male reproductive disorders. [ABSTRACT FROM AUTHOR]

Background: Non-obstructive azoospermia (NOA) is a major contributor of male infertility. Herein, we used existing datasets to identify novel biomarkers for the diagnosis and prognosis of NOA, which could have great significance in the field of male infertility. Methods: NOA datasets were obtained from the Gene Expression Omnibus (GEO) database. CIBERSORT was utilized to analyze the distributions of 22 immune cell populations. Hub genes were identified by applying weighted gene co-expression network analysis (WGCNA), machine learning methods, and protein-protein interaction (PPI) network analysis. The expression of hub genes was verified in external datasets and was assessed by receiver operating characteristic (ROC) curve analysis. Gene set enrichment analysis (GSEA) was applied to explore the important functions and pathways of hub genes. The mRNA-microRNA (miRNA)-transcription factors (TFs) regulatory network and potential drugs were predicted based on hub genes. Single-cell RNA sequencing data from the testes of patients with NOA were applied for analyzing the distribution of hub genes in single-cell clusters. Furthermore, testis tissue samples were obtained from patients with NOA and obstructive azoospermia (OA) who underwent testicular biopsy. RT-PCR and Western blot were used to validate hub gene expression. Results: Two immune-related oxidative stress hub genes (SHC1 and FGFR1) were identified. Both hub genes were highly expressed in NOA samples compared to control samples. ROC curve analysis showed a remarkable prediction ability (AUCs > 0.8). GSEA revealed that hub genes were predominantly enriched in tolllike receptor and Wnt signaling pathways. A total of 24 TFs, 82 miRNAs, and 111 potential drugs were predicted based on two hub genes. Single-cell RNA sequencing data in NOA patients indicated that SHC1 and FGFR1 were highly expressed in endothelial cells and Leydig cells, respectively. RT-PCR and Western blot results showed that mRNA and protein levels of both hub genes were significantly upregulated in NOA testis tissue samples, which agree with the findings from analysis of the microarray data. Conclusion: It appears that SHC1 and FGFR1 could be significant immunerelated oxidative stress biomarkers for detecting and managing patients with NOA. Our findings provide a novel viewpoint for illustrating potential pathogenesis in men suffering from infertility. [ABSTRACT FROM AUTHOR]

Background: Furan is an organic compound that occurs as a result of heat treatment during the processing and cooking of many food products. Furthermore, the environment contains furan in tobacco smoke and vehicle exhaust gases, and it serves as an intermediate molecule in the synthesis of various pharmaceutical and chemical agents, pesticides, and stabilizers. Studies on the male reproductive system have not been able to elucidate the pathway through which furan exerts its negative effects. Methods and results: In this study, the TM3 Leydig cell line was exposed to various furan concentrations (0.03, 0.3, and 3 mM) for 24 h. In order to assess the cytotoxic effects of furan on Leydig cells, we examined cell viability, cell proliferation, and lactate dehydrogenase enzyme levels. To investigate the detrimental effects of furan on testosterone biosynthesis, quantitative analyses were conducted on cAMP and testosterone levels, as well as the expression levels of key genes and transcription factors implicated in the steroidogenic pathway. The results indicate that furan inhibited the viability and proliferation of Leydig cells and enhanced the activity of lactate dehydrogenase. Leydig cells administered to furan exhibited notable reductions in cAMP and testosterone levels. Additionally, while the expression levels of steroidogenic genes were downregulated, significant changes were detected in the expression levels of the transcription factors responsible for the regulation of these genes. Conclusions: Consequently, our findings suggest that furan exerts inhibitory effects on steroidogenesis in Leydig cells through multiple mechanisms, ultimately leading to infertility by inducing dysfunction in Leydig cells. [ABSTRACT FROM AUTHOR]

Lectin is often used to investigate the differentiation and maturation of spermatogenic cells in the testes of several wild animals, facilitating the screening of mature individuals. Therefore, this study aimed to determine spermatogenesis activity using lectin histochemistry in water monitor lizards (Varanus salvator) harvested from the wild. Testes of 43 comprehensively examined water monitor lizards were collected from slaughterhouses in Palembang (Sumatra) and Banten (West Java), Indonesia, with snout-vent lengths (SVL) ranging from 39 to 96 cm. Additionally, histologic, histomorphometric, periodic acid-Schiff (PAS) staining, and lectin histochemistry (Con A, RCA, WGA, PNA, VVA, DBA, UEA-I) observations were performed. The results showed a positive correlation between SVL, morphometry, and histomorphometry at p < 0.05 with Pearson's correlation analysis. All specimens presented a spermatogenesis process with different activities among various SVL sizes, with larger animals having higher spermatogenic activity. Moreover, PAS-positive reactions in the testes were mainly observed in the cytoplasmic residues of spermatogenic cells, connective tissue, Leydig cells, and blood vessels. The most dominant-reacting lectin bindings were ConA, RCA, and WGA, distributed in nearly the entirety of the testes, including in spermatids and spermatozoa phases. PNA and VVA bindings were localized in Golgi and Cap stage spermatids, while UEA-I and DBA lectins showed no binding reaction. Although the larger animals had more spermatogenic cells, the lectin-binding patterns of all specimens were similar. The presence of lectin binding of PNA, VVA, and WGA in spermatids and spermatozoa in spermatogenic cells signified that male monitor lizards harvested from the wild exhibited active spermatogenic processes and were able to produce mature spermatozoa. [ABSTRACT FROM AUTHOR]

The Banded krait is a species of venomous snakes that ranks among the top 5 in Thailand, with a significance role as a predator for controlling other animals. Despite numerous studies on the ecology, the microanatomy of body systems has still not reported clearly. The present study was to investigate the microanatomy of the testes and male genital ducts of Banded krait that collected from Phatthalung Province, Thailand. The snakes were collected after anesthetized, and their testes and genital ducts were fixed in Bouin's fixative. The reproductive organs were collected during July to October 2022, and were sectioned of 5-µm-thick pieces for staining with Harris's hematoxylin and eosin (H&E), periodic acid Schiff's (PAS), alcian blue (AB) pH 2.5 and pH 1.0, bromophenol blue (BB) and Masson's trichrome (MT). The testes contained the seminiferous tubules with 7 stages of germ cells: (I) type A spermatogonium, (II) type B spermatogonium, (III) primary spermatocyte, (IV) secondary spermatocyte, (V) round spermatid, (VI) elongated spermatid and (VII) spermatozoa. Besides, Sertoli and Leydig's cells were appeared in the seminiferous tubules and the interstitial area, respectively. During the sampling period, the spermatozoa were observed in the seminiferous tubules and genital ducts. The extra-testicular rete testis, ductuli efferentes and ductus epididymis were embedded in the epididymal sheath. The epithelial cells of rete testis, ductus epididymis, as well as ductus deferens possessed microvilli, whereas those of the ductuli efferentes composed of cilia. The epithelial cells of all portions showed the positive staining with PAS and BB due to the presence of neutral glycoproteins and proteins, respectively, and the negative staining for AB pH 2.5 and pH 1.0 because of the absence of acidic glycoproteins. [ABSTRACT FROM AUTHOR]

Diabetes leads to testicular damage and infertility. Mesenchymal stem cells and their secretory trophic factors have shown potential as regenerative therapies for diabetes and its associated complications. This study examined the effects of conditioned medium derived from Wharton's jelly mesenchymal stem cells (WJMSCs‐CM) on sperm parameters, reproductive hormones, biochemical parameters, and histological changes in the testes of diabetic rats. Fifty‐six male Sprague–Dawley rats (250–300 g) were assigned to eight groups: control, diabetes, and six diabetic groups receiving early or late treatments with WJMSCs‐CM (D‐CME, D‐CML), insulin (D‐INSE, D‐INSL), or DMEM (D‐DME, D‐DML). In the early treatment groups, insulin (3 U/day, subcutaneously) and WJMSCs‐CM (10 mg/week, intraperitoneally) were administered immediately after diabetes induction; in the late treatment groups, these interventions began 30 days postinduction. Blood glucose and insulin levels, along with sperm parameters, were assessed. Sex hormones, testicular antioxidant enzyme activity, malondialdehyde (MDA), and glutathione (GSH) concentrations were measured using colorimetric methods. Real‐time PCR detected Bax, Bcl‐2, and tumor necrosis factor‐alpha (TNF‐α) gene expression. Our results showed that diabetes increased blood glucose levels, decreased insulin and sex hormone levels, induced testicular oxidative stress and apoptosis, and reduced sperm parameters compared to the control. WJMSCs‐CM significantly ameliorated hyperglycemia, increased insulin and sex hormone levels, and improved sperm quality. In WJMSCs‐CM‐treated diabetic rats, MDA levels were reduced, while GSH and antioxidant enzyme activity increased. Furthermore, WJMSCs‐CM decreased the testicular Bax/Bcl‐2 ratio and TNF‐α expression, as well as enhanced spermatogenic, Sertoli, and Leydig cells. In conclusion, WJMSC‐CM administration effectively mitigated diabetes‐induced testicular damage by reducing oxidative stress, inflammation, and apoptosis. Early treatment with WJMSCs‐CM was more effective than late treatment for diabetes‐induced reproductive dysfunction. [ABSTRACT FROM AUTHOR]

STUDY QUESTION What is the molecular landscape underlying the functional decline of human testicular ageing? SUMMARY ANSWER The present study provides a comprehensive single-cell transcriptomic atlas of testes from young and old humans and offers insights into the molecular mechanisms and potential targets for human testicular ageing. WHAT IS KNOWN ALREADY Testicular ageing is known to cause male age-related fertility decline and hypogonadism. Dysfunction of testicular cells has been considered as a key factor for testicular ageing. STUDY DESIGN, SIZE, DURATION Human testicular biopsies were collected from three young individuals and three old individuals to perform single-cell RNA sequencing (scRNA-seq). The key results were validated in a larger cohort containing human testicular samples from 10 young donors and 10 old donors. PARTICIPANTS/MATERIALS, SETTING, METHODS scRNA-seq was used to identify gene expression signatures for human testicular cells during ageing. Ageing-associated changes of gene expression in spermatogonial stem cells (SSCs) and Leydig cells (LCs) were analysed by gene set enrichment analysis and validated by immunofluorescent and functional assays. Cell–cell communication analysis was performed using CellChat. MAIN RESULTS AND THE ROLE OF CHANCE The single-cell transcriptomic landscape of testes from young and old men was surveyed, revealing age-related changes in germline and somatic niche cells. In-depth evaluation of the gene expression dynamics in germ cells revealed that the disruption of the base-excision repair pathway is a prominent characteristic of old SSCs, suggesting that defective DNA repair in SSCs may serve as a potential driver for increased de novo germline mutations with age. Further analysis of ageing-associated transcriptional changes demonstrated that stress-related changes and cytokine pathways accumulate in old somatic cells. Age-related impairment of redox homeostasis in old LCs was identified and pharmacological treatment with antioxidants alleviated this cellular dysfunction of LCs and promoted testosterone production. Lastly, our results revealed that decreased pleiotrophin signalling was a contributing factor for impaired spermatogenesis in testicular ageing. LARGE SCALE DATA The scRNA-seq sequencing and processed data reported in this paper were deposited at the Genome Sequence Archive (https://ngdc.cncb.ac.cn/), under the accession number HRA002349. LIMITATIONS, REASONS FOR CAUTION Owing to the difficulty in collecting human testis tissue, the sample size was limited. Further in-depth functional and mechanistic studies are warranted in future. WIDER IMPLICATIONS OF THE FINDINGS These findings provide a comprehensive understanding of the cell type-specific mechanisms underlying human testicular ageing at a single-cell resolution, and suggest potential therapeutic targets that may be leveraged to address age-related male fertility decline and hypogonadism. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Key Research and Development Program of China (2022YFA1104100), the National Natural Science Foundation of China (32130046, 82171564, 82101669, 82371611, 82371609, 82301796), the Natural Science Foundation of Guangdong Province, China (2022A1515010371), the Major Project of Medical Science and Technology Development Research Center of National Health Planning Commission, China (HDSL202001000), the Open Project of NHC Key Laboratory of Male Reproduction and Genetics (KF202001), the Guangdong Province Regional Joint Fund-Youth Fund Project (2021A1515110921, 2022A1515111201), and the China Postdoctoral Science Foundation (2021M703736). The authors declare no conflict of interest. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Do testis-specific cells have a normal karyotype in non-mosaic postpubertal Klinefelter syndrome (KS) patients with focal spermatogenesis and in non-mosaic prepubertal KS boys? SUMMARY ANSWER Spermatogonia have a 46, XY karyotype, and Sertoli cells surrounding these spermatogonia in postpubertal patients also have a 46, XY karyotype, whereas, in prepubertal KS boys, Sertoli cells surrounding the spermatogonia still have a 47, XXY karyotype. WHAT IS KNOWN ALREADY A significant proportion of patients with non-mosaic KS can have children by using assisted reproductive techniques thanks to focal spermatogenesis. However, the karyotype of the cells that are able to support focal spermatogenesis has not been revealed. STUDY DESIGN, SIZE, DURATION Testicular biopsy samples from non-mosaic KS patients were included in the study. Karyotyping for sex chromosomes in testis-specific cells was performed by immunohistochemical analysis of inactive X (Xi) chromosome and/or fluorescent in situ hybridization (FISH) analysis of chromosomes 18, X, and Y. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 22 KS patients (17 postpubertal and 5 prepubertal) who were non-mosaic according to lymphocyte karyotype analysis, were included in the study. After tissue processing, paraffin embedding, and sectioning, the following primary antibodies were used for cell-specific analysis and Xi detection; one section was stained with MAGE A4 for spermatogonia, SOX9 for Sertoli cells, and H3K27me3 for Xi; the other one was stained with CYP17A1 for Leydig cells, ACTA2 for peritubular myoid cells, and H3K27me3 for Xi. Xi negative (Xi−) somatic cells (i.e. Sertoli cells, Leydig cells, and peritubular myoid cells) were evaluated as having the 46, XY karyotype; Xi positive (Xi+) somatic cells were evaluated as having the 47, XXY. FISH stain for chromosomes 18, X, and Y was performed on the same sections to investigate the karyotype of spermatogonia and to validate the immunohistochemistry results for somatic cells. MAIN RESULTS AND THE ROLE OF CHANCE According to our data, all spermatogonia in both postpubertal and prepubertal non-mosaic KS patients seem to have 46, XY karyotype. However, while the Sertoli cells surrounding spermatogonia in postpubertal samples also had a 46, XY karyotype, the Sertoli cells surrounding spermatogonia in prepubertal samples had a 47, XXY karyotype. In addition, while the Sertoli cells in some of the Sertoli cell-only tubules had 46, XY karyotype, the Sertoli cells in some of the other Sertoli cell-only tubules had 47, XXY karyotype in postpubertal samples. In contrast to the postpubertal samples, Sertoli cells in all tubules in the prepubertal samples had the 47, XXY karyotype. Our data also suggest that germ cells lose the extra X chromosome during embryonic, fetal, or neonatal life, while Sertoli cells lose it around puberty. Peritubular myoid cells and Leydig cells may also be mosaic in both postpubertal patients and prepubertal boys, but it requires further investigation. LIMITATIONS, REASONS FOR CAUTION The number of prepubertal testicle samples containing spermatogonia is limited, so more samples are needed for a definitive conclusion. The fact that not all the cell nuclei coincide with the section plane limits the accurate detection of X chromosomes by immunohistochemistry and FISH in some cells. To overcome this limitation, X chromosome analysis could be performed by different techniques on intact cells isolated from fresh tissue. Additionally, there is no evidence that X chromosome inactivation reoccurs after activation of the Xi during germ cell migration during embryogenesis, limiting the prediction of X chromosome content in germ cells by H3K27me3. WIDER IMPLICATIONS OF THE FINDINGS Our findings will lay the groundwork for new clinically important studies on exactly when and by which mechanism an extra X chromosome is lost in spermatogonia and Sertoli cells. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by The Scientific and Technological Research Council of Türkiye (TUBITAK) (2219 – International Postdoctoral Research Fellowship Program for Turkish Citizens) and the Strategic Research Program (SRP89) from the Vrije Universiteit Brussel. The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Testosterone deficiency in humans can be caused by depressive symptoms; however, the causes of this deficiency are incompletely understood. This study demonstrates that male mice with depression-like symptoms due to chronic unpredictable mild stress (CUMS) show reduced serum testosterone levels and disrupted sexual behaviors. However, the observed testosterone reductions were not caused by apoptosis of Leydig cells. Oil red O staining revealed that lipid droplets were dramatically decreased in Leydig cells, suggesting that defects in cholesterol uptake might be related to testosterone deficiency in depression-like mice. To investigate the potential mechanism, lipid homeostasis was examined by liquid chromatography-tandem mass spectrometry. The results revealed that higher levels of sphingomyelins (SM 8:0;2O/28:1, 18:0;2O/22:2, 33:0;3O, 33:1;2O) were linked to decreased cholesterol levels. Further investigation indicated that testosterone biosynthesis from cholesterol in Leydig cells was impaired by the downregulation of Ldlr, Srb1, Lhr, and P450scc. Elevated levels of interferon signaling-associated pathways in depression-like mice testes may also contribute to decreased testosterone levels. Taken together, these findings provide a novel understanding of male reproductive problems under psychological stress and suggest that cholesterol uptake might be a causal factor in reduced testosterone production in depression-like mice. [ABSTRACT FROM AUTHOR]

Di-(2-ethylhexyl) phthalate (DEHP), a prevalent plasticizer, is known to have endocrine-disrupting effects on males and cause reproductive toxicity. There were causal effects of DEHP on testosterone levels in the real world by Mendelian randomization analysis. Exposure to DEHP during the preadult stage might lead to premature testicular senescence, but the mechanisms responsible for this have yet to be determined. In this study, we administered DEHP (300 mg/kg/day) to male C57BL/6 mice from postnatal days 21 to 49. The mice were kept for 6 months without DEHP. RNA sequencing was conducted on testicular tissue at PNM6. The results indicated that DEHP hindered testicular development, lowered serum testosterone levels in male mice, and induced premature testicular senescence. TM3 Leydig cells were exposed to 300 μM of mono(2-ethylhexyl) phthalate (MEHP), the bioactive metabolite of DEHP, for 72 h. The results also found that DEHP/MEHP induced senescence in vivo and in vitro. The mitochondrial respiratory chain was disrupted in Leydig cells. The expression and stability of STAT5B were elevated by MEHP treatment in TM3 cells. Furthermore, p-ERK1/2 was significantly decreased by STAT5B, and mitochondria-STAT3 (p-STAT3 ser727) was significantly decreased due to the decrease of p-ERK1/2. Additionally, the senescence level of TM3 cells was decreased and treated with 5 mM NAC for 1 h after MEHP treatment. In conclusion, these findings provided a novel mechanistic understanding of Leydig cells by disrupting the mitochondrial respiratory chain through STAT5B-mitoSTAT3. [ABSTRACT FROM AUTHOR]

Technological advances have led to a modern-day lighting and smartphone revolution, with artificial light exposure at night increasing to levels never before seen in the evolutionary history of living systems on Earth. Light as a pollutant, however, remains largely unrecognized, and the reproductive effects of light pollution are mostly if not entirely unconsidered. This is despite the reproductive system being intricately linked to metabolism and the circadian system, both of which can be disturbed even by low levels of light. Here, we aim to change this perspective by reviewing the physiological and pathophysiological mechanisms by which light exposure alters the intricate hormonal, metabolic and reproductive networks that are relevant to reproductive toxicology. Nascent human studies have recently identified the photoreceptors responsible for the light dose relationship with melatonin suppression and circadian re-entrainment, directly shown the association between the alignment of light-dark cycles with activity-rest cycles on metabolic health and provided proof-of-principle that properly timed blue light-enriched and blue light-depleted delivery can accelerate circadian re-entrainment. With these advances, there is now a need to consider testicular effects of light pollution. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule that belongs to the group of gasotransmitters along with nitric oxide (NO) and carbon monoxide (CO). H2S plays a pivotal role in male reproductive processes. It is produced in various tissues and cells of the male reproductive system, including testicular tissue, Leydig and Sertoli cells, epididymis, seminal plasma, prostate, penile tissues, and sperm cells. This review aims to summarize the knowledge about the presence and effects of H2S in male reproductive tissues and outline possible therapeutic strategies in pathological conditions related to male fertility, e. g. spermatogenetic disorders and erectile dysfunction (ED). For instance, H2S supports spermatogenesis by maintaining the integrity of the blood-testicular barrier (BTB), stimulating testosterone production, and providing cytoprotective effects. In spermatozoa, H2S modulates sperm motility, promotes sperm maturation, capacitation, and acrosome reaction, and has significant cytoprotective effects. Given its vasorelaxant effects, it supports the erection of penile tissue. These findings suggest the importance and therapeutic potential of H2S in male reproduction, paving the way for further research and potential clinical applications. [ABSTRACT FROM AUTHOR]

Spermatogenesis is a complex biological process crucial for male reproduction and is characterized by intricate interactions between testicular somatic cells and germ cells. Due to the cellular heterogeneity of the testes, investigating different cell types across developmental stages has been challenging. Single-cell RNA sequencing (scRNA-seq) has emerged as a valuable approach for addressing this limitation. Here, we conducted an unbiased transcriptomic study of spermatogenesis in sexually mature 4-month-old Hezuo pigs using 10× Genomics-based scRNA-seq. A total of 16,082 cells were collected from Hezuo pig testes, including germ cells (spermatogonia (SPG), spermatocytes (SPCs), spermatids (SPTs), and sperm (SP)) and somatic cells (Sertoli cells (SCs), Leydig cells (LCs), myoid cells (MCs), endothelial cells (ECs), and natural killer (NK) cells/macrophages). Pseudo-time analysis revealed that LCs and MCs originated from common progenitors in the Hezuo pig. Functional enrichment analysis indicated that the differentially expressed genes (DEGs) in the different types of testicular germ cells were enriched in the PI3K–AKT, Wnt, HIF-1, and adherens junction signaling pathways, while the DEGs in testicular somatic cells were enriched in ECM–receptor interaction and antigen processing and presentation. Moreover, genes related to spermatogenesis, male gamete generation, sperm part, sperm flagellum, and peptide biosynthesis were expressed throughout spermatogenesis. Using immunohistochemistry, we verified several stage-specific marker genes (such as UCHL1, WT1, SOX9, and ACTA2) for SPG, SCs, and MCs. By exploring the changes in the transcription patterns of various cell types during spermatogenesis, our study provided novel insights into spermatogenesis and testicular cells in the Hezuo pig, thereby laying the foundation for the breeding and preservation of this breed. [ABSTRACT FROM AUTHOR]

Melatonin, a hormone synthesized in various tissues, plays a crucial role in modulating sperm characteristics, yet its protective function on boar sperm remains poorly understood. This study aimed to investigate the expression and localization of melatonin-related proteins (AANAT, ASMT, MT1, MT2, and NQO2) in pig tissues, assess the impact of melatonin on pig sperm motility parameters and quality, and elucidate the underlying molecular mechanisms. Our results revealed widespread expression of AANAT, ASMT, MT1, MT2, and NQO2 proteins in pig tissues, particularly in the testis. Specific localization patterns were observed in Leydig cells, reproductive epithelium, and columnar epithelium cells in the testis and cauda epididymis. Additionally, melatonin membrane receptors MT1 and MT2 were detected in boar sperm. Melatonin treatment significantly enhanced boar sperm motility parameters and quality, particularly with 10 nM melatonin treatment. Inhibition of the MT1 receptor, but not the MT2 receptor, resulted in decreased sperm motility, highlighting the pivotal role of the MT1 receptor in mediating melatonin's effects on boar sperm. Metabolomic analysis revealed significant alterations in sperm metabolites following melatonin supplementation, particularly in amino acid metabolism. Overall, our findings provide comprehensive insights into melatonin's mechanisms in improving boar sperm quality, suggesting its potential as a therapeutic agent for enhancing male fertility. • Comprehensive study shows melatonin boosts boar sperm motility and quality, mainly via the MT1 receptor. • Melatonin-related proteins in pig tissues suggest roles in testicular physiology and sperm protection. • Melatonin alters seminal plasma metabolites, showing diverse impacts on sperm function and metabolism. [ABSTRACT FROM AUTHOR]

Background: Spermatogonial stem cells (SSCs) are essential for the maintenance and initiation of male spermatogenesis. Despite the advances in understanding SSC biology in mouse models, the mechanisms underlying human SSC development remain elusive. Results: Here, we analyzed the signaling pathways involved in SSC regulation by testicular somatic cells using single-cell sequencing data (GEO datasets: GSE149512 and GSE112013) and identified that Leydig cells communicate with SSCs through pleiotrophin (PTN) and its receptor syndecan-2 (SDC2). Immunofluorescence, STRING prediction, and protein immunoprecipitation assays confirmed the interaction between PTN and SDC2 in spermatogonia, but their co-localization was observed only in approximately 50% of the cells. The knockdown of SDC2 in human SSC lines impaired cell proliferation, DNA synthesis, and the expression of PLZF, a key marker for SSC self-renewal. Transcriptome analysis revealed that SDC2 knockdown downregulated the expression of GFRA1, a crucial factor for SSC proliferation and self-renewal, and inhibited the HIF-1 signaling pathway. Exogenous PTN rescued the proliferation and GFRA1 expression in SDC2 knockdown SSC lines. In addition, we found downregulation of PTN and SDC2 as well as altered localization in non-obstructive azoospermia (NOA) patients, suggesting that downregulation of PTN and SDC2 may be associated with impaired spermatogenesis. Conclusions: Our results uncover a novel mechanism of human SSC regulation by the testicular microenvironment and suggest a potential therapeutic target for male infertility. [ABSTRACT FROM AUTHOR]

Background Objectives Results Discussion Conclusion Testicular germ cell tumors (TGCTs), derived from primordial germ cells, are rare malignancies with high curative potential. However, the emergence of new evidence indicating that 15% of patients experience tumor progression, leading to death, underscores the need for innovative therapeutics.This review aimed to explore the immune status in maintaining testicular health and the immune‐related aspects of malignancy. Furthermore, it presents an overview of current data on the use of immunotherapy for TGCT patients.Recent advances in immunology have opened a promising avenue for studying diseases and highlighted its role in treating diseases. While the immunopathological facets of TGCTs are not fully understood, investigations suggest a complex interplay among testis‐resident immune cells, testis‐specific cells (i.e., Sertoli cells (SCs) and Leydig cells (LCs)), and immune‐regulating mediators (e.g., sex hormones) in the normal testicle that foster the testicular immune privilege (TIP). Although TIP plays a crucial role in sperm production, it also makes testis vulnerable to tumor development. In the context of cancer‐related inflammation, disruption of TIP leads to an imbalanced immune response, resulting in chronic inflammation that can contribute to testicular tissue dysfunction or loss, potentially aiding in cancer invasion and progression.Comparing the immune profiles of normal and malignant testes is valuable and may provide insights into different aspects of testicular immunity and immune‐based treatment approaches. For patients resistant to chemotherapy and with a poor prognosis, immunotherapy has shown promising results. However, its effectiveness in treating resistant TGCTs or preventing tumor recurrence is still uncertain. [ABSTRACT FROM AUTHOR]

Ultrasound is the most used interdisciplinary non-ionizing imaging technique in clinical pathologies of the testis. The testis may be affected by a plethora of different disorders such as vasculopathies, trauma, infections and manifestations of primary and secondary malignant masses. Conventional ultrasound represents the basic imaging modality of choice to assess scrotal disorders. Contrast-enhanced ultrasound (CEUS) can provide further information to distinguish between benign and malignant testicular mass lesions. The recent introduction of Super Resolution CEUS Micro-Vascular Imaging (MVI SR) and Time of Arrival (TOA SR) parametric mapping compliments the information provided by conventional CEUS, since these two new post-processing techniques improve the visualization of microvascular structures with slow blood flow and provide high-resolution images of the peak contrast enhancement and temporal perfusion patterns. This paper gives a comprehensive overview of differential diagnoses of the testicular disorder and their corresponding sono-morphologic correlates based on representative cases of the Interdisciplinary Ultrasound Center of the University Hospital Munich. [ABSTRACT FROM AUTHOR]

Background: The mammalian testicular interstitial cells are not well-defined. The present study characterized the interstitial cell types and their turnover dynamics in adult rats. Additionally, the heterogeneity of the mesenchymal population and the effects of Leydig cell elimination on interstitial homeostasis were further analyzed by scRNA-seq datasets and immunocytochemical techniques. Methods: Interstitial cells were defined at the transcriptomic level by scRNA-seq and then confirmed and quantified with protein markers. The dividing activity of the major cell types was determined by continuous EdU labeling of the animals for one week. Some of the rats were also treated with a dose of ethylenedimethylsulfonate (EDS) to examine how the loss of Leydig cells (LCs) could affect interstitial homeostasis for three weeks. Results: Seven interstitial cell types were identified, including cell types (percentage of the whole interstitial population) as follows: Leydig (44.6%), macrophage and dendritic (19.1%), lymphoid (6.2%), vascular endothelial (7.9%), smooth muscle (10.7%), and mesenchymal (11.5%) cells. The EdU experiment indicated that most cell types were dividing at relatively low levels (<9%) except for the mesenchymal cells (MCs, 17.1%). Further analysis of the transcriptome of MCs revealed 4 subgroups with distinct functions, including 1) glutathione metabolism and xenobiotic detoxification, 2) ROS response and AP-1 signaling, 3) extracellular matrix synthesis and binding, and 4) immune response and regulation. Stem LCs (SLCs) are primarily associated with subgroup 3, expressing ARG1 and GAP43. EDS treatment not only eliminated LCs but also increased subgroup 3 and decreased subgroups 1 and 2 of the mesenchymal population. Moreover, EDS treatment increased the division of immune cells by more than tenfold in one week. Conclusion: Seven interstitial cell types were identified and quantified for rat testis. Many may play more diversified roles than previously realized. The elimination of LCs led to significant changes in MCs and immune cells, indicating the importance of LCs in maintaining testicular interstitial homeostasis. [ABSTRACT FROM AUTHOR]

Leydig cell failure (LCF) caused by gene mutations leads to testosterone deficiency, infertility and reduced physical function. Adeno‐associated virus serotype 8 (AAV8)‐mediated gene therapy shows potential in treating LCF in the Lhcgr‐deficient (Lhcgr−/−) mouse model. However, the gene‐treated mice still cannot naturally sire offspring, indicating the modestly restored testosterone and spermatogenesis in AAV8‐treated mice remain insufficient to support natural fertility. Recognizing this, we propose that enhancing gene delivery could yield superior results. Here, we screened a panel of AAV serotypes through in vivo transduction of mouse testes and identified AAVDJ as an impressively potent vector for testicular cells. Intratesticular injection of AAVDJ achieved markedly efficient transduction of Leydig cell progenitors, marking a considerable advance over conventional AAV8 vectors. AAVDJ‐Lhcgr gene therapy was well tolerated and resulted in significant recovery of testosterone production, substantial improvement in sexual development, and remarkable restoration of spermatogenesis in Lhcgr−/− mice. Notably, this therapy restored fertility in Lhcgr−/− mice through natural mating, enabling the birth of second‐generation. Additionally, this treatment led to remarkable improvements in adipose, muscle, and bone function in Lhcgr−/− mice. Collectively, our findings underscore AAVDJ‐mediated gene therapy as a promising strategy for LCF and suggest its broader potential in addressing various reproductive disorders. [ABSTRACT FROM AUTHOR]

Leydig cells are the primary source of testosterone or androgen production in male mammals. The blood–testis barrier (BTB) maintains structural integrity and safeguards germ cells from harmful substances by blocking their entry into the seminiferous tubules. L-cysteine is essential to the production of glutathione, a powerful antioxidant crucial to protecting against oxidative stress-induced damage. Animal studies have demonstrated the protective effect of L-cysteine in preventing testicular damage caused by chemicals or radiation. This study examines whether L-cysteine enhances the expression of testosterone biosynthesis and the BTB genes in human Leydig cells and THP-1 monocytes. The Leydig cells and THP-1 monocytes were treated with L-cysteine for 24 h. RNA was extracted following treatment, and the gene expression was analyzed using quantitative RT-PCR. Testosterone levels in the cell supernatant were measured using an ELISA kit. L-cysteine treatment in Leydig cells significantly upregulated the expression of CYP11A1 (p = 0.03) and the BTB genes CLDN1 (p = 0.03), CLDN11 (p = 0.02), and TJP1 (p = 0.02). Similarly, L-cysteine significantly upregulated the expression of CYP11A1 (p = 0.03) and CYP19A1 (p < 0.01), and the BTB genes CLDN1 (p = 0.04), CLDN2 (p < 0.01), CLDN4 (p < 0.01), CLDN11 (p < 0.01), and TJP1 (p = 0.03) in THP-1 monocytes. Further, L-cysteine supplementation increased the testosterone secretion levels in human Leydig cells. The findings suggest that L-cysteine supplementation could be used as an adjuvant therapy to promote the integrity of the BTB genes, testosterone biosynthesis and secretion, and the maintenance of testicular functions, which in turn mitigates the risk of male infertility. [ABSTRACT FROM AUTHOR]

Leydig cells (LCs) in the testes produce the male sex hormone testosterone (T). Several xenobiotics, including clinical drugs, supplements, and environmental chemicals, are known to disrupt T homeostasis. Notably, some of these xenobiotics are known to activate the pregnane X receptor (PXR), a ligand-dependent nuclear receptor. However, it is currently unknown whether PXR is expressed in LCs and whether PXR activation alters T synthesis in rodent LCs. Therefore, in this study, we sought to determine whether PXR is expressed in rodent LCs and whether pregnenolone 16-alpha carbonitrile (PCN), the prototype agonist of rodent PXR, regulates T biosynthesis in rodent LCs. Hormonal as well as protein and gene expression analyses were conducted in rat primary LCs and MA-10 mouse Leydig cells. Results showed that PXR was expressed at the mRNA and protein level in both rat primary LCs and MA-10 cells. Incubation of rat primary LCs with PCN resulted in a significant decrease in T secretion. This PCN-induced decrease in T secretion was associated with decreased protein expression of key steroidogenic enzymes such as 3β-HSD and CYP17A1. RNA-seq results from MA-10 cells showed that PCN down-regulated the transcripts of steroidogenic enzymes and proteins involved in the T synthesis pathway. Together, these results suggest that PCN, an agonist of rodent PXR, can regulate T biosynthesis in rodent LCs by down-regulating the expression of the steroidogenic enzymes involved in T biosynthesis. Our results are significant as they provide a potential novel mechanism for disruption of testosterone homeostasis by a variety of xenobiotics. [ABSTRACT FROM AUTHOR]

Smoking habits (from classic cigarettes to e-cigarettes and heated tobacco) are a relatively common finding in the medical histories of couples referred to fertility centers. Tobacco smoke and e-cigarettes may deliver many substances with known harmful effects on both general and reproductive health, including nicotine. Nicotinic Acetylcholine receptors (nAChRs) form a heterogeneous family of ion channels that are differently expressed in different tissues. According to the homomeric or heteromeric combination of at least five different subunits (named from α to ε), they have peculiar pharmacological and biophysical properties. nAChRs respond to the neurotransmitter acetylcholine, which influences a number of physiological functions not restricted to neurons and plays an important role in the structure and function of non-neuronal tissues such as the testis. nAChRs are also the target of Nicotine, the active element responsible for tobacco addiction. This review summarizes recent findings on the involvement of nAChRs in testicular physiology, highlighting the effects of nicotine exposure observed in animal studies and clinical settings. We will discuss the latest data on fertility outcomes and the implications for understanding nAChR functions in reproductive health. [ABSTRACT FROM AUTHOR]

Simple Summary: This study presents data on the immunohistochemical expression of key iron metabolism proteins in non-neoplastic and neoplastic canine testes. Iron is crucial for spermatogenesis, and its regulation in testicular cells is essential for normal function. This study confirms that Sertoli cells and Transferrin Receptor 1 (TfR1) play significant roles in iron uptake in dogs, aligning with findings in humans and mice. However, it highlights a unique expression of nuclear receptor coactivator 4 (NCOA4) in canine Sertoli cells, suggesting a specific role in iron recycling through ferritinophagy. Differences in iron metabolism were observed in various canine testicular tumors. Higher TfR1 and NCOA4 expressions were noted in Leydig cell tumors and diffuse seminomas, indicating a reliance on iron for tumor growth. The altered expression of iron-related proteins in tumors underscores their potential as therapeutic targets. Targeting TfR1 and utilizing iron-modulating therapies shows promise. Further research is needed to explore the therapeutic potential of modulating iron metabolism in canine testicular cancers. Iron is a key element in spermatogenesis; its metabolic pathway in the testis is strictly regulated. Alterations in iron metabolism are linked to various diseases, including cancer, and changes in iron metabolism-related proteins have been observed in multiple human, mouse and canine tumors. There is limited knowledge about iron metabolism in canine non-neoplastic and neoplastic testes. This study aimed to explore the immunohistochemical expression of molecules involved in iron uptake and storage [Transferrin Receptor 1 (TfR1), ferritin (FTH1), nuclear receptor coactivator 4 (NCOA4)] and PCNA in canine non-neoplastic and neoplastic testicular samples. Non-neoplastic testes showed moderate TfR1 expression in developing germ cells and Sertoli cells, high NCOA4 cytoplasmic immunostaining in the Sertoli cells and occasional cytoplasmic immunopositivity for FTH1 in the spermatogonia and Sertoli cells. In contrast, Leydig cell tumors (LCTs) and Diffuse Type Seminoma (DSEM) exhibited increased expression of TfR1, along with higher PCNA expression, suggesting a higher iron need for proliferation. Intratubular Type Seminoma (ITSEM) showed a higher FTH1 expression, indicating greater iron storage, while the increased NCOA4 expression in the LCTs and DSEM suggested ferritinophagy to release iron for proliferation. Sertoli cell tumors (SCTs) showed only NCOA4 expression. These preliminary findings highlight potential molecular targets for developing new anti-neoplastic treatments in canine testicular tumors. [ABSTRACT FROM AUTHOR]

Simple Summary: Glyoxalase domain-containing protein 4 (GLOD4), a member of the glyoxalase gene family, is widely expressed in Leydig cells and its primary function within the Leydig cells remains unknown. GLOD4 expression in other species is often associated with functions such as cell proliferation, cell cycle, and detoxification. We therefore explored the relevant elements of Leydig cells. We speculate that GLOD4 may regulate testicular development and spermatogenesis by increasing the number of Leydig cells and regulation of testosterone secretion. The expression pattern of GLOD4 in the testis and its regulatory effect on testicular cells was explored in goats to enhance our understanding of spermatogenesis and improve reproduction in breeding rams. In this study, we demonstrated the localization of GLOD4 in testicular cells using immunohistochemistry and subcellular localization analyses. Subsequently, we analyzed the GLOD4 expression pattern in four age-based groups (0, 6, 12, and 18 months old) using real-time quantitative polymerase chain reaction (qRT-PCR) and protein blotting. Finally, we performed GLOD4 silencing and overexpression studies in Leydig cells (LCs) and explored the effects on cell proliferation, the cell cycle, steroid hormone secretion and the expression of candidate testosterone hormone-regulated genes. GLOD4 was mainly expressed in Leydig cells, and the subcellular localization results showed that the GLOD4 protein was mainly localized in the cytoplasm and nucleus. Silencing of GLOD4 significantly suppressed the mRNA expression levels of the testosterone secretion-related genes CYP11A1, 3β-HSD, and CYP17A1 and the mRNA expression levels of cell cycle-related genes CDK6, PCNA, and Cyclin E. Moreover, the cell cycle was blocked at the G2/M phase after GLOD4 silencing, which significantly suppressed testosterone secretion. In contrast, GLOD4 overexpression significantly increased the mRNA expression levels of the testosterone secretion-related genes CYP11A1, 3β-HSD, and CYP17A1 and increased the expression of the cell cycle-related genes CDK6, PCNA, and Cyclin E. Moreover, GLOD4 overexpression promoted the cell cycle from G0/G1 phases to enter the S phase and G2/M phases, promoted the secretion of testosterone. Taken together, our experimental results indicate that GLOD4 may affect the development of cells in Qianbei Ma goats of different ages by influencing the cell cycle, cell proliferation, and testosterone hormone synthesis. These findings enhance our understanding of the functions of GLOD4 in goats. [ABSTRACT FROM AUTHOR]

Clinical and epidemiological studies indicated that there is an increased incidence of male infertility, with many cases that do not respond well to treatments. This study is aimed at evaluating the therapeutic effects of verapamil and tamoxifen against infertility, which may provide an essential strategy for the treatment of this disorder. A casecontrol study was conducted on 64 male albino rats, divided into four groups (16 for each); group A (control group); group B (infertile group that is induced by the ornidazole); and groups C and D, which are pretreated with verapamil and tamoxifen, respectively, in combination with ornidazole every day for four weeks. Serum levels of testosterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), glutathione (GSH), nitric oxide (NO), malondialdehyde (MDA), and superoxide dismutase (SOD) were assessed, and sperm parameters, including sperm count, sperm motility percentage, sperm morphology percentage, and sperm viability, were investigated. Furthermore, Motic Image Plus 2.0 ML was used to measure the mean diameter of the seminiferous tubules (SNT), and a morphometric analysis of 20 tubules per rat was also performed. The results revealed that both verapamil and tamoxifen provide a significant improvement in sperm count, motility, morphology, and dead sperm, with a significant improvement in testosterone, FSH, LH, GSH, NO, MDA, and SOD levels compared with the infertile group. Histophotometric results showed that verapamil and tamoxifen improved the number and size of SNT, Leydig cells, and Sertoli cell counts, which may indicate the promise of using these agents in the treatment of male infertility. [ABSTRACT FROM AUTHOR]

Testicular sex cord-stromal tumors are clonal neoplasms, with the majority being of Leydig cell followed by Sertoli cell origins. In Leydig cell tumors, adipocytic differentiation has been previously reported as a possible distinguishing feature, which has not been reported in other sex cord-stromal tumors. Herein, we report a case of a 48-year-old man who presented with an incidentally discovered 1.1 cm testicular mass, for which he underwent partial orchiectomy. Microscopically, the tumor showed features consistent with sex cord-stromal tumor with strong and diffuse nuclear and cytoplasmic reaction for B-catenin immunohistochemistry, supporting the diagnosis of Sertoli cell tumor. A novel adipocytic differentiation, reported previously in Leydig cell tumors, was present in this tumor. [ABSTRACT FROM AUTHOR]

This article discusses the use of gonadotropin treatment as an alternative to surgery for adults with congenital hypogonadotropic hypogonadism (CHH) and cryptorchidism. CHH is a condition where there is a deficiency of certain hormones, leading to absent or incomplete puberty. Cryptorchidism, or undescended testis, is more common in individuals with CHH. The article presents three case studies of adult males with CHH and persistent cryptorchidism who were successfully treated with gonadotropin therapy. The findings suggest that this treatment can be a safe and effective alternative to surgery for correcting cryptorchidism in young adult males with CHH. Further research is needed to explore this treatment in larger patient groups. [Extracted from the article]

Over the years, the origin of ovarian Leydig cells has been, and still is, a topic subject to deep debate. Seven years ago, we proposed that this origin resided in intraneural elements that came from a possible reservoir of neural crest cells, a reservoir that may be located in the ganglia of the celiac plexus. We believe we have found the evidence necessary to prove this hypothesis. [ABSTRACT FROM AUTHOR]

Testosterone derived from testicular Leydig cells (LCs) is important for male sheep, and the testis is susceptible to external temperature. The present study aimed to explore the alleviating effect of selenomethionine (Se‐Met) on heat‐induced injury in Hu sheep LCs. Isolated LCs were exposed to heat (41.5°C, heat exposure, HE) or not (37°C, nonheat exposure, NE), and cells in NE and HE were treated with 0 (C) or 8 μmol/L (S) Se‐Met for 6 h. Cell viability, testosterone level, and the expression of GPX1, HSD3B, apoptosis‐related genes and p38 mitogen‐activated protein kinase (p38MAPK)/heat shock protein beta‐1 (HSPB1) pathway were examined. The results showed that Se‐Met increased GPX1 expression (NE‐S vs. NE‐C: 2.28‐fold; HE‐S vs. HE‐C: 2.36‐fold, p < 0.05) and alleviated heat‐induced decrease in cell viability (HE‐S vs. HE‐C: 1.41‐fold; HE‐C vs. NE‐C: 0.61‐fold, p < 0.01), although the viability was still lower than that in the NE‐C cells (HE‐S vs. NE‐C: 0.85‐fold) and Se‐Met‐treated cells (HE‐S vs. NE‐S: 0.81‐fold). Se‐Met relieved heat‐induced decrease in testosterone level (HE‐S vs. HE‐C: 1.84‐fold, p < 0.05) and HSD3B expression (HE‐S vs. HE‐C: 1.67‐fold, p < 0.05). Se‐Met alleviated heat‐induced increase in Bcl2‐associated protein X (BAX) expression (HE‐C vs. HE‐S: 2.4‐fold, p < 0.05), and decrease in B‐cell lymphoma‐2 (BCL2) expression (HE‐S vs. HE‐C: 2.62‐fold, p < 0.05), resulting in increased BCL2/BAX ratio in the HE‐S cells (HE‐S vs. HE‐C: 5.24‐fold, p < 0.05). Furthermore, Se‐Met alleviated heat‐induced activation of p‐p38MAPK/p38MAPK (HE‐C vs. HE‐S: 1.79‐fold, p < 0.05) and p‐HSPB1/HSPB1 (HE‐C vs. HE‐S: 2.72‐fold, p < 0.05). In conclusion, p38MAPK/HSPB1 might be involved in Se‐Met‐mediated alleviation of heat‐induced cell apoptosis, cell viability and testosterone secretion impairments in sheep LCs. [ABSTRACT FROM AUTHOR]

Leydig cells (LCs) in the testes produce the male sex hormone testosterone (T). Several xenobiotics, including clinical drugs, supplements, and environmental chemicals, are known to disrupt T homeostasis. Notably, some of these xenobiotics are known to activate the pregnane X receptor (PXR), a ligand-dependent nuclear receptor. However, it is currently unknown whether PXR is expressed in LCs and whether PXR activation alters T synthesis in rodent LCs. Therefore, in this study, we sought to determine whether PXR is expressed in rodent LCs and whether pregnenolone 16-alpha carbonitrile (PCN), the prototype agonist of rodent PXR, regulates T biosynthesis in rodent LCs. Hormonal as well as protein and gene expression analyses were conducted in rat primary LCs and MA-10 mouse Leydig cells. Results showed that PXR was expressed at the mRNA and protein level in both rat primary LCs and MA-10 cells. Incubation of rat primary LCs with PCN resulted in a significant decrease in T secretion. This PCN-induced decrease in T secretion was associated with decreased protein expression of key steroidogenic enzymes such as 3β-HSD and CYP17A1. RNA-seq results from MA-10 cells showed that PCN down-regulated the transcripts of steroidogenic enzymes and proteins involved in the T synthesis pathway. Together, these results suggest that PCN, an agonist of rodent PXR, can regulate T biosynthesis in rodent LCs by down-regulating the expression of the steroidogenic enzymes involved in T biosynthesis. Our results are significant as they provide a potential novel mechanism for disruption of testosterone homeostasis by a variety of xenobiotics.

Taste receptor type 1 subunit 3 (T1R3) is a G protein-coupled receptor encoded by the TAS1R3 gene that can be specifically activated by certain sweeteners or umami agents for sweet/umami recognition. T1R3 is a potential target for regulating male reproduction. However, studies on the impact of non-nutritive sweeteners on reproduction are limited. In the present study, we evaluated the impact of the non-nutritive sweeteners (saccharin sodium, sucralose and acesulfame-K) on testosterone synthesis in testicular Leydig cells of Xiang pigs by comparing the relative abundance of mRNA transcripts and protein expression of T1R3, steroidogenic related factors, and intracellular cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), as well as testosterone levels using Western blotting, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). To clarify the specific mechanism, a dual luciferase assay was used to uncover the relationship between the transcription factors and steroidogenic enzyme. The acute intratesticular injection of a typical non-nutritive sweeteners was conducted to verify this impact in mouse. The results showed that saccharin sodium not only enhanced T1R3 expression in Leydig cells of Xiang pigs, but also caused significant increases in testosterone, cAMP, PKA, phosphorylation of specificity protein 1 (p-SP1), total protein of specificity protein 1 (SP1), steroidogenic acute regulatory protein (StAR), and 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1) (P < 0.05). Similarly, treatment of Leydig cells with sucralose and acesulfame-K also increased testosterone level, protein expression of T1R3, 17-α-hydroxylase/17, 20-lyase (CYP17A1), and 3β-HSD1 (P < 0.05). Treatment with SQ22536 (an adenylate cyclas inhibitor) or H89 (a PKA inhibitor) significantly reduced saccharin sodium-induced protein levels of p-SP1, StAR, CYP17A1, and 3β-HSD1 (P < 0.05). In addition, a dual luciferase assay further demonstrated that SP1 significantly increased the promoter activity of CYP17A1 (P < 0.05). When mouse testes were injected with saccharin sodium, T1R3, p-SP1, CYP17A1, and 3β-HSD1 were upregulated, leading to a significant testicular increase in testosterone and cAMP levels (P < 0.05). These results suggest a mechanism by which the taste receptor T1R3 regulates testosterone production, and this mechanism may be linked to the cAMP-PKA pathway. Understanding the interrelationship between T1R3 and the cAMP-PKA-SP1 pathway contributes to clarify the regulatory mechanisms of male reproduction. [ABSTRACT FROM AUTHOR]

Reproductive performance is a crucial aspect of poultry production and is carefully controlled by endocrine, paracrine, and autocrine factors. This study aimed to investigate the effect of lycopene on testosterone synthesis in Leydig cells of laying breeder roosters, clarify the mechanism of lycopene improving Leydig cells function and promoting testosterone production, and explore the role of related signal transduction pathways in testosterone synthesis. A total of 96 healthy 55-week-old breeding roosters were randomly assigned to one of five dietary treatments. They were provided with a corn-soybean meal-based diet containing different levels of lycopene: 0 mg/kg (control), 50 mg/kg, 100 mg/kg, or 200 mg/kg. The experiment lasted for 6 weeks. With the increase in lycopene levels, the testosterone content in the plasma was significantly higher than in the control group. Testicular Leydig cells were isolated and cultured from fresh testicular tissue of 45-wk-old to 60-wk-old breeding roosters. Various doses of lycopene were administered to Leydig cells, and subsequently, cells were collected for the detection of cell viability and testosterone content. The optimal concentration of lycopene to be added was determined, and changes in mRNA expression and protein levels of key proteins involved in testosterone synthesis were investigated. The results showed that lycopene treatment significantly increased testosterone secretion, mRNA expression, and protein levels of steroid-producing enzymes. Cells were collected to measure the activity of antioxidant enzymes, the mRNA transcription level of apoptotic factors, and the protein expression of apoptotic factors after treatment with lycopene. The results showed that lycopene significantly increased the activities of antioxidant enzymes, and the ability to inhibit oxygen radicals, and decreased the content of malondialdehyde. Apoptosis was inhibited by regulating the expression of apoptosis-inducing and anti-apoptosis factors. After that, the MAPK signaling pathway and downstream SF-1, Nrf2 gene, and protein expression levels were detected. The results showed that lycopene treatment significantly increased the gene and protein expression of JNK , SF-1 , and Nrf2 , and significantly decreased the gene and protein expression of p38. Lycopene treatment could promote testosterone synthesis of testicular Leydig cells by activating MAPK-SF-1 (increasing steroid-producing enzyme level) and MAPK-Nrf2 pathways (resisting oxidative damage). • Lycopene treatment increases testosterone secretion in Leydig cells in aged breeder roosters. • Lycopene promotes testosterone secretion by activating the MAPK-SF-1 and MAPK-Nrf2 signaling pathways. • Lycopene reduces oxidative stress markers and inhibits apoptosis in Leydig cells. [ABSTRACT FROM AUTHOR]

Testosterone is secreted by Leydig cells (LCs), which play an important physiological role in preserving male secondary sex characteristics, protecting male reproductive function, and establishing the blood-testis barrier. Studies have shown that autophagy is particularly active in LCs; however, its involvement in testosterone synthesis in porcine LCs has not been fully explored. Therefore, this experiment aimed to investigate the influence of autophagy on testosterone secretion in porcine LCs and its potential regulatory mechanism. Our results demonstrated that both testicular autophagy and serum testosterone levels increased in piglets during postnatal development from 4 to 18 weeks. In addition, autophagy was found to degrade the Na+/H+ exchange regulatory factor 2 (NHERF2), leading to the up-regulation of scavenger receptor class B type 1 (SRB1). This process resulted in increased cholesterol intake and enhanced testosterone production. The observable level of sirtuin 1 (SIRT1) was directly proportional to the level of autophagy. In vitro investigations have shown that SIRT1 can affect the level of autophagy, cholesterol uptake as well as testosterone release. In conclusion, testosterone synthesis during pig development is regulated by SIRT1. SIRT1 mediates the degradation of NHERF2 through autophagy, thereby weakening its negative regulatory effect on the high-density lipoprotein receptor SRB1 in Leydig cells. This process increases cholesterol uptake and enhances testosterone synthesis. • Autophagy levels match testosterone secretion by Leydig cells in piglets from 4 to 18 weeks. • Autophagy facilitates cholesterol uptake in Leydig cells by degrading NHERF2, influencing testosterone production. • SIRT1 activates autophagy by promoting LC3 expression, thereby participating in testosterone synthesis. [ABSTRACT FROM AUTHOR]

Anti-Müllerian hormone (AMH) has become a pivotal subject in the study of testicular descent, maturation, integrity, and male fertility. Recent studies explored its roles and implications across various domestic species. A prominent approach involved the understanding of the modulation of AMH in reproductive disorders, including cryptorchidism. While substantial findings have been reported in dogs, ruminants, swine, and horses, data on AMH in feline cryptorchidism remains limited. Here, we aimed to bridge this gap by comparing AMH serum levels among cryptorchid, healthy intact, and castrated tomcats, employing an enzyme-linked immunosorbent assay (ELISA) kit for quantification. In addition, AMH expression in retained and descended testes was evaluated through immunohistochemistry, with positive staining quantified via pixel analysis in two distinct regions of interest: the seminiferous tubule and the interstitial space. Furthermore, tissue samples were subjected to histological evaluation and morphometric analysis, which included the calculation of seminiferous tubule areas (STA) and assessment of Johnsen scores. Thus, the relationship between AMH expression, altered testicular histology, and impaired spermatogenesis could be examined. The expression of AMH in retained and descended testes, was investigated, and the relationship between AMH expression, altered testicular histology, and impaired spermatogenesis was examined. Mean serum AMH levels differed significantly (P < 0.001) across the different groups being 15.35 ± 4.66 ng/mL (mean ± SD) in healthy intact tomcats (n = 15), 25.55 ± 2.86 ng/mL (mean ± SD) in cryptorchids (n = 10) and below 0.015 ng/mL in castrated cats (n = 10). STAs and Johnsen scores were significantly reduced in retained testes when compared to descended gonads (P < 0.01). Furthermore, serum AMH was negatively correlated with both the STA (ρ = −0.725, P < 0.001) and the Johnsen scores (ρ = −0.699, P < 0.001), suggesting its potential value for tissue integrity and spermatogenesis evaluation. In addition, positive immunostaining was significantly higher in retained testes (P < 0.05), especially in the interstitial space (P < 0.01), suggesting an involvement of the Leydig cells. Additionally, the increased interstitial expression was linked to the degree of tissue degeneration and the impaired spermatogenesis being negatively corelated with both Johnsen scores (ρ = −0.309, P < 0.01) and STA (ρ = −0.208, P < 0.05). Our findings underscore the potential of AMH in assessing testicular health and reveal possible interspecific differences, stressing the need for further investigation in cats. • Cryptorchid tomcats have higher serum AMH levels than castrated or intact tomcats, but more research is needed to detect age-related thresholds. • Elevated AMH levels correlate with reduced seminiferous tubule area and lower Johnsen scores, indicating testicular degeneration. • Retained feline testicles show increased AMH in interstitial space, suggesting a species-specific expression pattern. • High AMH in retained testicles suggests increased Leydig cell receptor interaction, possibly altering steroidogenic activity. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID-19) reportedly affects male reproductive function by causing spermatogenesis dysfunction and suppressing testosterone secretion. However, the relationship between COVID-19 and impaired reproductive function, such as whether these effects on reproductive function are a direct effect of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection in male reproductive organs or an indirect effect of high fever, is not known. Here, we examined whether the cell entry molecules of SARS-CoV-2, namely, ACE2, NRP1, TMPRSS2, and FURIN, are expressed in the male reproductive organs using the testes and accessory gonads of macaques during the breeding season. RT-PCR expression analysis showed that the testes alone expressed all four molecules. Immunohistochemical staining of testis tissue sections revealed that ACE2 is expressed in Leydig cells and the apical region of Sertoli cells, whereas NRP1 is expressed in the cell bodies surrounding the Leydig and Sertoli cell nuclei. FURIN is mainly expressed in Leydig cells, secondary spermatocytes, and spermatids. However, TMPRSS2 immunopositive cells were not observed. Therefore, it was not possible to observe cells expressing all four molecules in the gonads and accessory gonads of male primates. These results suggest that SARS-CoV-2 is unlikely to directly affect spermatogenesis in primates or proliferate in cells of the seminiferous tubules and undergo release into the semen through the previously known ACE2-mediated infection route. However, the expression of three molecules, including ACE2, was observed in Leydig cells, suggesting that testosterone synthesis and secretion may be affected when primates, including humans, are infected with SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Abstract Background Exploring the molecular mechanisms of primordial germ cell (PGC) migration and the involvement of gonadal somatic cells in gonad development is valuable for comprehending the origins and potential treatments of reproductive-related diseases. Methods Diaphanous related formin 1 (Diaph1 , also known as mDia1) was screened by analyzing publicly available datasets (ATAC-seq, DNase-seq, and RNA-seq). Subsequently, the CRISPR-Cas9 technology was used to construct Diaph1 knockout mice to investigate the role of Diaph1 in gonad development. Results Based on data from public databases, a differentially expressed gene Diaph1, was identified in the migration of mouse PGC. Additionally, the number of PGCs was significantly reduced in Diaph1 knockout mice compared to wild type mice, and the expression levels of genes related to proliferation (Dicer1, Mcm9), adhesion (E-cadherin, Cdh1), and migration (Cxcr4, Hmgcr, Dazl) were significantly decreased. Diaph1 knockout also inhibited Leydig cell proliferation and induced apoptosis in the testis, as well as granulosa cell apoptosis in the ovary. Moreover, the sperm count in the epididymal region and the count of ovarian follicles were significantly reduced in Diaph1 knockout mice, resulting in decreased fertility, concomitant with lowered levels of serum testosterone and estradiol. Further research found that in Diaph1 knockout mice, the key enzymes involved in testosterone synthesis (CYP11A1, 3β-HSD) were decreased in Leydig cells, and the estradiol-associated factor (FSH receptor, AMH) in granulosa cells were also downregulated. Conclusions Overall, our findings indicate that the knockout of Diaph1 can disrupt the expression of factors that regulate sex hormone production, leading to impaired secretion of sex hormones, ultimately resulting in damage to reproductive function. These results provide a new perspective on the molecular mechanisms underlying PGC migration and gonadal development, and offer valuable insights for further research on the causes, diagnosis, and treatment of related diseases.

This research was designed to study the effect of silymarin dosage on the histological structure of the testicle in rabbits and the impact it has on the level of sperm production in these animals. Thirty adult male rabbits were used to complete this work. Their ages ranged between 5 to 6 months, while their weight ranged between 1,500 to 1,700 grams. The animals were divided into three groups based on the amount of dose of silymarin given as follows (C- 10 rabbits given free diet served as a control group: T1-10 rabbits treated with an oral daily dose of 0.5 ml per kg B.W. T2-10 rabbits treated with orally daily dose 1 ml per kg B.W.). The various types of cells were identified in this study based on several criteria, the most important of which are the external appearance of these cells during the series of divisions, the size of the cell, and the shape of the nucleus. The results showed an improvement in the architecture of seminiferous tubule epithelium, the disappearance of vacuolar degenerations in more than 95% of this epithelium, and an increase in the number of mature sperm in the center of each tubule in addition to increasing all parameters of Leydig cells. In conclusion, the testicles in rabbits exposed to silymarin are positively affected, leading to improved sperm production.

Background: Platelet-derived growth factor receptor alpha (PDGFRα) is essential for various biological processes, including fetal Leydig cell differentiation. The PDGFRαEGFP mouse model, which expresses an eGFP fusion gene under the native Pdgfrα promoter, serves as a valuable resource for exploring PDGFRα’s expression and function in vivo. This study investigates PDGFRα expression in adult testicular cells using PDGFRαEGFP mouse model. Methods: Genotyping PCR and gel electrophoresis were used to confirm the zygosity of PDGFRαEGFP mice. Histological examination and fluorescence imaging were used to identify PDGFRα expression within testicular tissue. Immunohistochemical analysis assessed the co-expression of PDGFRα with c-Kit, ANO-1, and TASK-1 in testicular cells. Results: Genotyping confirmed the heterozygous status of the mice, which is crucial for studies due to the embryonic lethal phenotype observed in homozygotes. Histological and fluorescence imaging revealed that PDGFRα+ cells were primarily located in the interstitial spaces of the testis, specifically within Leydig cells and peritubular myoid cells (PMCs). Immunohistochemical results showed PDGFRα co-localization with c-Kit and ANO-1 in Leydig cells and a complete co-localization with TASK-1 in both Leydig cells and PMCs. Conclusions: The findings demonstrate specific expression of PDGFRα in Leydig cells and PMCs in adult testicular tissue. The co-expression of PDGFRα with c-Kit, ANO-1, and TASK-1 suggests complex regulatory mechanisms, possibly influencing testicular function and broader physiological processes.

Background: Stathmin, a cytosolic microtubule-destabilizing phosphoprotein involved in the regulation of mitosis, is widely expressed in various malignancies and acts as an adverse prognostic factor. Our research analyzed its immunohistochemical expression on a large cohort of ovarian sex cord-stromal tumors, evaluating its potential utility in differential diagnosis, prognosis, and therapeutic application. Methods: We examined 390 cases of ovarian sex cord-stromal tumors including 281 adult granulosa cell tumors (AGCT), 5 juvenile granulosa cell tumors (JGCT), 33 Sertoli-Leydig cell tumors (SLCT), 50 fibromas/thecomas (F/T), 11 Leydig cell tumors/steroid cell tumors (LCT/SterCT), 5 sex-cord stromal tumors NOS (SCST-NOS), 3 Sertoli cell tumors (SCT), and 2 sclerosing stromal tumors (ScST). Immunohistochemical analysis was performed using TMAs. Results: Strong expression (> 50%) was observed in all cases of AGCT, JGCT, SLCT, SCST-NOS, SCT and 1 ScST. The other case of ScST exhibited mild expression (5–10%). The negative cases included exclusively F/T and LCT/SterCT, with F/T showing 24% of negative cases and LCT/SterCT comprising 64% of negative cases. Conclusion: The results of our study indicate that stathmin is neither a prognostic marker nor suitable for the differential diagnosis of challenging cases of ovarian sex cord-stromal tumors. However, its predictive value may be theoretically significant, as a decrease in stathmin expression potentialy influences response to chemotherapy treatment. [ABSTRACT FROM AUTHOR]

Fine particulate matter (PM 2.5), a significant component of air pollution particulate matter, is inevitable and closely associated with increasing male reproductive disorder. However, the testicular targets of PM 2.5 and its toxicity related molecular mechanisms are still not fully understood. In this study, the conditional knockout (cKO) mice and primary Leydig cells were used to explore the testicular targets of PM 2.5 and the related underlying mechanisms. First, apparent the structure impairment of seminiferous tubules, Leydig cells vacuolization, decline of serum testosterone and sperm quality reduction were found in male wild-type (WT) and Sirt1 knockout mice after exposure to PM 2.5. Enrichment analyses revealed that differentially expressed genes (DEGs) were enriched in steroid hormone biosynthesis, ferroptosis, and HIF-1 signaling pathway in the mice testes after exposure to PM 2.5 , which were subsequently verified by the molecular biological analyses. Notably, similar enrichment analyses results were also observed in primary Leydig cells after treatment with PM 2.5. In addition, Knockdown of Sirt1 significantly increased PM 2.5 -induced expression and activation of HIF-1α, which was in parallel to the changes of cellular iron levels, oxidative stress indicators and the ferroptosis markers. In conclusion, this highlights that PM 2.5 triggers ferroptosis via SIRT1/HIF-1α signaling pathway to inhibit testosterone synthesis in males. These findings provide a novel research support for the study that PM 2.5 causes male reproductive injury. [Display omitted] • PM 2.5 induced impairment of testicular structures and sperm parameters in mice. • Ferroptosis involved in PM 2.5 -induced male reproductive toxicity. • PM 2.5 inhibited testosterone synthesis-related enzymes. • PM 2.5 activated ferroptosis through SIRT1/HIF-1α signaling pathway. [ABSTRACT FROM AUTHOR]

Bee-derived pharmaceutical products, including propolis (PRO) and royal jelly (ROJ), possess outstanding pharmacological properties. However, their efficiency in counteracting the deleterious influences of cadmium (Cd) in testes and the relevant mechanisms entail further investigations. Therefore, this study sheds light on the therapeutic efficacy of PRO and ROJ against testicular dysfunction and infertility induced by Cd. Toward this end, 30 mature male Wistar albino rats were randomly divided into six groups (5 animals/group), including (I) control, (II) Cd, (III) PRO, (IV) ROJ, (V) PRO + Cd, and (VI) ROJ + Cd groups. Furthermore, antioxidant factors, semen quality, hormonal levels, steroidogenic enzymes, and genotoxicity were assessed. Moreover, histopathological and ultrastructural attributes and offspring rates were investigated. The Cd-treated group revealed marked reductions in reduced glutathione (GSH), total antioxidant capacity (TAC), and superoxide dismutase (SOD) with an amplification of lipid peroxidation in testes, indicating disruption of the antioxidant defense system. Furthermore, myeloperoxidase (MPO) activity and DNA damage were significantly heightened, implying inflammation and genotoxicity, respectively. Moreover, steroidogenic enzymes, including 17β-Hydroxy Steroid Dehydrogenase 3 (HSD17b3), 3β-Hydroxy Steroid Dehydrogenase 2 (HSD3b2), 17α-hydroxylase/17,20-lyase (CYP17A1), and steroid 5α-reductase 2 (SRD5A2) were markedly diminished accompanied with disorders in luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone. Besides, spermatozoa quality was reduced, associated with a diminution in the diameter of seminiferous tubules. By contrast, PRO or ROJ significantly protected and/or counteracted the Cd-induced pathophysiological consequences, ameliorating antioxidant and inflammatory biomarkers, steroidogenic enzymes, hormonal levels, and sperm properties, along with lessening DNA impairments. Critically, histological and ultrastructural analyses manifested several anomalies in the testicular tissues of the Cd-administered group, including the Leydig and Sertoli cells and spermatozoa. Conversely, PRO or ROJ sustained testicular tissues' structure, enhancing spermatozoa integrity and productivity. Interestingly, treatment with PRO or ROJ improved fertility indices through offspring rates compared to the Cd-animal group. Our data suggest that PRO is a more effective countermeasure than ROJ against Cd toxicity for securing the delicate testicular microenvironment for spermatogenesis and steroidogenesis. [ABSTRACT FROM AUTHOR]

D-aspartate (D-Asp) is an amino acid found in high concentrations in the testis and pituitary gland. Increasing evidence suggests that D-Asp promotes spermatogenesis by activating testosterone production in the Leydig cells via LH release from the pituitary gland. In vitro studies indicate that D-Asp may also influence steroidogenesis and spermatogenesis through autocrine and paracrine signals. D-Asp enhances StAR and steroidogenic enzyme expressions, facilitating testicular cell proliferation via the GluR/ERK1/2 pathway. Moreover, it supports spermatogenesis by enhancing the mitochondrial function in spermatocytes, aiding in the metabolic shift during meiosis. Enhanced mitochondrial function, along with improved MAM stability and reduced ER stress, has been observed in Leydig and Sertoli cells treated with D-Asp, indicating potential benefits in steroidogenesis and spermatogenesis efficiency. Conversely, D-Asp exerts a notable anti-apoptotic effect in the testis via the AMPAR/AKT pathway, potentially mediated by antioxidant enzyme modulation to mitigate testicular oxidative stress. This review lays the groundwork for future investigations into the molecules promoting spermatogenesis by stimulating endogenous testosterone biosynthesis, with D-amino acids emerging as promising candidates. [ABSTRACT FROM AUTHOR]