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Vertebrate spermatogonial stem cells maintain sperm production over the lifetime of an animal, but fertility declines with age. Although morphological studies have informed our understanding of typical spermatogenesis, the molecular and cellular mechanisms underlying the maintenance and decline of spermatogenesis are not yet understood. We used single-cell RNA sequencing to generate a developmental atlas of the aging zebrafish testis. All testes contained spermatogonia, but we observed a progressive decline in spermatogenesis that correlated with age. Testes from some older males only contained spermatogonia and a reduced population of spermatocytes. Spermatogonia in older males were transcriptionally distinct from spermatogonia in testes capable of robust spermatogenesis. Immune cells including macrophages and lymphocytes drastically increased in abundance in testes that could not complete spermatogenesis. Our developmental atlas reveals the cellular changes as the testis ages and defines a molecular roadmap for the regulation of spermatogenesis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Contemporary societies exhibit delayed reproductive age and increased life expectancy. While the male reproductive system demonstrates relatively delayed aging compared to that of females, increasing age substantially impacts its function. A characteristic manifestation is age-induced testosterone decline. Testosterone, a crucial male sex hormone, plays pivotal roles in spermatogenesis and sexual function, and contributes significantly to metabolism, psychology, and cardiovascular health. Aging exerts profound effects on the hypothalamic-pituitary-gonadal axis and Leydig cells, precipitating testosterone reduction, which adversely affects male health. Exogenous testosterone supplementation can partially ameliorate age-related testosterone deficiency; however, its long-term safety remains contentious. Preserving endogenous testosterone production capacity during the aging process warrants further investigation as a potential intervention strategy., Competing Interests: Declarations Ethics approval and consent to participate Not Applicable. Consent for publication Not Applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Due to global warming seasonal heat stress is an increasing problem in temperate zones. Heat stress not only decreases fertility in females, but can also be detrimental to male fertility., Objectives: We studied the effects of natural summer heat stress during spermatogenesis in Holstein bulls on semen quality parameters and on fertilization performance in vitro and possible intergenerational transmission of effects on the next male generation., Materials and Methods: Semen samples from young Holstein breeding bulls, referred to as F0 founders during this study, were collected during summer (F0 "summer" semen) and the following winter (F0 "winter" semen). Parameters such as ejaculate volume, sperm density, motility, thermoresistance, and in vitro blastocyst rates from these F0 semen samples were determined. In addition, after generation of offspring by artificial insemination, semen samples from F1 male offspring were collected and tested for the same quality and performance parameters to capture intergenerational effects. F1 bulls were raised together under identical conditions and semen was collected at about 1 year after birth., Results: The data showed that in vitro blastocyst rates of F0 "summer" semen samples were lower compared with "winter" semen, whereas blastocyst rates of F1 semen samples did not show significant differences. However, whereas F0 semen samples did not indicate significantly different quality parameters we found that motility of F1 semen samples showed significant differences with higher values when collected from bulls generated with F0 "winter" semen., Discussion and Conclusion: From our data, we conclude that (i) natural summer heat stress during spermatogenesis can affect in vitro fertility parameters and (ii) the observed effects on sperm motility of F1 semen samples suggest intergenerational paternal transmission., (© 2024 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

Photoperiod is a pivotal factor in affecting testicular function and spermatogenesis in seasonal-breeding animals. Mitophagy is essential for spermatogenesis, but its association with seasonal photoperiods has not been studied extensively. To explore this, we exposed male Brandt's voles (Lasiopodomys brandtii) to long-photoperiod (LP, 16 h/day) and short-photoperiod (SP, 8 h/day) conditions from their embryonic stages. Our results indicated that testis weight, volume, and relative testes weight were all significantly increased in LP compared to SP. Additionally, blood testosterone levels were markedly higher in LP than SP. Histological examination revealed that seminiferous diameter and epithelium thickness were greater in LP, with an increased abundance of germ cell types and cell numbers compared to SP. RT-qPCR analysis showed that mitophagy-promoting genes, such as Pink1, Prkn, Tomm7, Mnf2, Lc3, Optn, Gabarap, and Nbr1 were all upregulated in LP. Fluorescence in situ hybridization indicated that Pink1 expression was present in spermatogonia in SP, while in LP, Pink1 expression extended to almost all germ cell types with significantly higher mean optical density. Prkn expression was found in all germ cell types in both LP and SP, with a significantly higher mean optical density of 10-week-old LP males. Transmission electron microscopy showed normal mitochondrial morphology with clear membranes in SP, while the LP group had reduced cristae in mitochondria and damaged mitochondria undergoing autophagy. This study suggests that mitophagy may be involved in the photoperiodic spermatogenesis in Brandt's voles, providing insights into the role of photoperiod in seasonal reproduction in wild animals., (© 2024 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.)

Background: As a component of the nucleosome remodeling and deacetylating (NuRD) complex, metastasis-associated protein 1 (MTA1) has been reported to be abundant in male reproductive system and might participate in spermatogenesis and sperm maturation, whereas the precise functional role of MTA1 in these processes is still undetermined., Objective: To investigate the effect and potential function of MTA1 in male fertility., Materials and Methods: Mta1 knockout mice (Mta1 -/- ) were employed to detect their reproductive phenotype. The pH value of Mta1 -/- epididymal luminal fluid was measured, and the potential mechanism of MTA1 involved in regulating luminal acidification was detected in vivo and in vitro. A vasectomy model with abnormal pH of epididymal lumen was established to further detect the effect of MTA1 on epididymal luminal microenvironment., Results: Mta1 -/- mice were fertile without any detectable defects in spermatogenesis or sperm motility while the deficiency of MTA1 could acidify the initial segment of epididymis to a certain extent. MTA1 could interact with estrogen receptor alpha (ERα) and inhibit the transcription of ERα target gene, hydrogen exchanger 3 (NHE3), and ultimately affect the epididymal luminal milieu. After vasectomy, the Mta1 -/- mice presented a more acidic epididymal lumen which was closer to the normal state compared to the wild-type model., Discussion and Conclusion: MTA1 is dispensable for male fertility in mice, but plays a potentially important function in regulating luminal acidification of the epididymis., (© 2024 American Society of Andrology and European Academy of Andrology.)

Background: Extreme ambient temperature has been linked to decline in males' semen quality. Although the temperature-semen quality association has been examined in certain cities of South China, how the effect size of the extreme temperature may lag over critical windows in spermatogenesis and whether the strength of association may vary in North China have yet been adequately explored., Objectives: To quantify the association between air temperature and semen quality, and identify critical exposure windows in a Northern Peninsular Province, China., Materials and Methods: Data on semen quality in 2014-2019 were collected from the Human Sperm Bank of Institute of Women, Children and Reproductive Health, Shandong University, China. Daily meteorological data (0.01°×0.01°) were assigned to each subject's residential address. The linear mixed-effect model combined with the distributed lag nonlinear model was used to estimate the effect of ambient temperature over critical spermatogenesis windows., Results: The temperature-semen quality association was inverted V-shaped, with the maximum lag being 0-45 days before ejaculation and the threshold being 9.2°C. Progressively and total motile sperm number, and total sperm number declined more substantially than other semen quality parameters. Semen quality was more sensitive to cold exposure during the epididymal storage period than the sperm motility development period. By contrast, semen quality was insensitive to heat exposure during both critical spermatogenesis windows. Impairment of certain semen quality parameters was more obvious for males with higher educational attainment and those aged over 35 years., Discussion and Conclusion: Exposure to non-optimal temperature is associated with decreased semen quality in North China, with the epididymal storage and sperm motility development periods more sensitive to cold exposure than heat. Older males and those with higher educations may need particular awareness., (© 2024 American Society of Andrology and European Academy of Andrology.)

Spermatogenesis in eukaryotes is a process that occurs within a very narrow temperature threshold, typically not exceeding 36 °C. SPO11 was isolated from the temperature-sensitive mutant receptor of Saccharomyces cerevisiae and is thought to be the only protein that functions during meiosis. This suggested that SPO11 may be the key protein that influenced the temperature of spermatogenesis not exceeding 36 °C. Elevated temperatures typically damage the spermatogenic cells. Birds have a core body temperature of 41-42 °C, and their testis are located inside their bodies, providing an alternative perspective to investigate the potential impact of temperature threshold on spermatogenesis. The objective of this study was to ascertain whether elevated ambient temperatures affect spermatogenesis in birds and whether SPO11 is the key gene affecting the temperature threshold for spermatogenesis. STRA8, SCP3, SPO11, γ-H2AX, and RAD51 were all crucial components in the process of meiotic initiation, synapsis, DNA double-strand break (DSB) induction, homologous chromosome crossover recombination, and repair of DSB. In this study, 39-day-old Japanese quail were subjected to heat stress (HS) at 38 °C for 8 h per day for 3 (3d HS) and 13 (13d HS) consecutive days and analyzed the expression of meiotic signaling molecules (STRA8, SCP3, SPO11, γ-H2AX, and RAD51) using molecular biology techniques, including Immunohistochemistry (IHC), Western Blot (WB), and Real-time Quantitative Polymerase Chain Reaction (qRT-PCR). We found that spermatogenesis was normal in both groups exposed to HS. Meiotic signaling molecules were expressed normally in the 3d HS group. All detected signaling molecules were normally expressed in the 13d HS group, except for SPO11, which showed a significant increase in expression, indicating that SPO11 was temperature-sensitive. We examined the localized expression of each meiotic signaling molecule in quail testis, explored the temperature sensitivity of SPO11, and determined that quail testis can undergo normal spermatogenesis at ambient temperatures exceeding 36 °C. This study concluded that SPO11 is not the key protein influencing spermatogenesis in birds. These findings enhance our understanding of avian spermatogenesis., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Posttranscriptional regulation of gene expression by RNA-binding proteins can enhance the speed and robustness of cell state transitions by controlling RNA stability, localization, or if, when, or where mRNAs are translated. The RNA helicase YTHDC2 is required to shut down components of the mitotic program to facilitate a proper switch from mitosis to meiosis in mouse germ cells. Here, we show that YTHDC2 has a second essential role in promoting meiotic progression in late spermatocytes. Inducing conditional knockout of Ythdc2 during the first wave of spermatogenesis, after initiation of meiotic prophase, allowed YTHDC2 -deficient germ cells to advance to the pachytene stage and properly express many meiotic markers. However, the YTHDC2 -deficient spermatocytes mis-expressed a number of genes, some up-regulated and some down-regulated, failed to transition to the diplotene stage, and then quickly died. Coimmunoprecipitation experiments revealed that YTHDC2 interacts with several RNA-binding proteins in early or late spermatocytes, with many of the interacting proteins, including MEIOC, localizing to granules, similar to YTHDC2. Our findings suggest that YTHDC2 collaborates with other RNA granule components to facilitate proper progression of germ cells through multiple steps of meiosis via mechanisms influencing posttranscriptional regulation of RNAs., Competing Interests: Competing interests statement:The authors declare no competing interest.

Sertoli cells, omnipresent, somatic cells within the seminiferous tubules of the mammalian testis are essential to male fertility. Sertoli cells maintain the integrity of the testicular microenvironment, regulate hormone synthesis, and of particular importance, synthesize the active derivative of vitamin A, all trans retinoic acid (atRA), which is required for germ cell differentiation and the commitment of male germ cells to meiosis. Stages VIII-IX, when atRA synthesis occurs in the testis, coincide with multiple germ cell development and testicular restructuring events that rely on Sertoli cell gene products to proceed normally. In this study, we have synchronized and captured the mouse testis at four recurrent points of atRA synthesis to observe transcriptomic changes within Sertoli cells as mice age and the Sertoli cells are exposed to increasingly developed germ cell subtypes. This work provides comprehensive, high-resolution characterization of the timing of induction of functional Sertoli cell genes across the first wave of spermatogenesis, and outlines in silico predictions of germ cell derived signaling mechanisms targeting Sertoli cells. We have found that Sertoli cells adapt to their environment, especially to the needs of the germ cell populations present and establish germ-Sertoli cell and Sertoli-Sertoli cell junctions early but gain many of their known immune-regulatory and protein secretory functions in preparation for spermiogenesis and spermiation. Additionally, we have found unique patterns of germ-Sertoli signaling present at each endogenous pulse of atRA, suggesting individual functions of the various germ cells in germ-Sertoli communication., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cytoplasmic dynein participates in transport functions and is essential in spermatogenesis. KM23 belongs to the dynein light chain family. The TGFβ signaling pathway is indispensable in spermatogenesis, and Smad2 is an important member of this pathway. We cloned PTKM23 and PTSMAD2 from Portunus trituberculatus and measured their expression during spermatogenesis. PTKM23 may be related to cell division, acrosome formation, and nuclear remodeling, and PTSMAD2 may participate in regulating the expression of genes related to spermatogenesis. We assessed the localization of PTKM23 with PTDHC and α-tubulin, and the results suggested that PTKM23 functions in intracellular transport during spermatogenesis. We knocked down PTKM23 in vivo, and the expression of p53, B-CATAENIN and CYCLIN B decreased significantly, further suggesting a role of PTKM23 in transport and cell division. The localization of PTDIC with α-tubulin and that of PTSMAD2 with PTDHC changed after PTKM23 knockdown. We transfected PTKM23 and PTSMAD2 into HEK-293 T cells and verified their colocalization. These results indicate that PTKM23 is involved in the assembly of cytoplasmic dynein and microtubules during spermatogenesis and that PTKM23 mediates the participation of cytoplasmic dynein in the transport of PTSMAD2 during spermatogenesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The actions of spermatogenic stem cells (SSCs) provide the foundation for continual spermatogenesis and regeneration of the cognate lineage following cytotoxic insult or transplantation. Several decades of research with rodent models have yielded knowledge about the core biology, morphological features, and molecular profiles of mammalian SSCs. Translation of these discoveries to utilities for human fertility preservation, improving animal agriculture, and wildlife conservation are actively being pursued. Here, we provide overviews of these aspects covering both historical and current states of understanding., (© 2024 The Author(s). Molecular Reproduction and Development published by Wiley Periodicals LLC.)

Purpose: Adropin is an emerging metabolic hormone that has a role in regulating energy homeostasis. The present study aimed to explore the impact of adropin on redox homeostasis and its possible role in testicular functions in adult mouse testis., Methods: Western blot, flow-cytometry, and TUNEL assay were performed to explore the impact of intra-testicular treatment of adropin (0.5 μg/testis) on testicular functions of adult mice. Hormonal assay was done by ELISA. Further, antioxidant enzyme activities were measured., Results: Adropin treatment significantly increased the sperm count and testicular testosterone by increasing the expression of GPR19 and steroidogenic proteins. Also, adropin treatment reduced the oxidative/nitrosative stress by facilitating the translocation of NRF2 and inhibiting NF-κB into the nucleus of germ cells. Enhanced nuclear translocation of NRF2 leads to elevated biosynthesis of antioxidant enzymes, evident by increased HO-1, SOD, and catalase activity that ultimately resulted into declined LPO levels in adropin-treated mice testes. Furthermore, adropin decreased nuclear translocation of NF-κB in germ cells, that resulted into decreased NO production leading to decreased nitrosative stress. Adropin/GPR19 signaling significantly increased its differentiation, proliferation, and survival of germ cells by elevating the expression of PCNA and declining caspase 3, cleaved caspase 3 expression, Bax/Bcl2 ratio, and TUNEL-positive cells. FACS analysis revealed that adropin treatment enhances overall turnover of testicular cells leading to rise in production of advanced germ cells, notably spermatids., Conclusion: The present study indicated that adropin improves testicular steroidogenesis, spermatogenesis via modulating redox potential and could be a promising target for treating testicular dysfunctions., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Male ageing is always accompanied by decreased fertility. The forkhead O (FOXO) transcription factor FOXO4 is reported to be highly expressed in senescent cells. Upon activation, it binds p53 in the nucleus, preventing senescent cell apoptosis and maintaining senescent cells in situ. Leydig cells play key roles in assisting spermatogenesis. Leydig cell senescence leads to deterioration of the microenvironment of the testes and impairs spermatogenesis. In this study, we observed that FOXO4-DRI, a specific FOXO4- p53 binding blocker, induced apoptosis in senescent Leydig cells, reduced the secretion of certain Senescence-Associated Secretory Phenotype and improved the proliferation of cocultured GC-1 SPG cells. In naturally aged mice, FOXO4-DRI-treated aged mice exhibited increased sperm quality and improved spermatogenesis., Competing Interests: Declaration of competing interest All of the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

HSP47, a chaperone whose main function is the maturation of collagen molecules, is considered a marker of fibrotic diseases. Increased collagen synthesis in the testis has been associated with various pathologies leading to seminiferous tubule regression. Our aim was to study whether HSP47 is expressed in hamster Sertoli cells both in the adult and in two physiological situations of seminiferous tubule atrophy: irreversible testicular ageing and testicular regression due to short photoperiod (reversible). Eighteen animals were divided as follows: a group of 6 young animals aged 6 months, a group of 6 animals aged 24 months, which were exposed to a long photoperiod, and a final group of 6 young animals subjected to a short photoperiod. Testicular samples were fixed in methacarn and an immunohistochemical technique was used to detect HSP47. A semiquantitative study of of this protein expresion was performed between tubular sections of aged animals with complete spermatogenesis and arrested spermatogenesis and tubular sections with arrest spermatogenesis of photoinhibited testes. Sertoli cells were positive for HSP47, the intensity being greater in tubular sections with arrested spermatogenesis in both aged and photoinhibited animals. Semiquantitative analysis corroborated this observation in the sense that the expression of this protein differed according to the functional state of the seminiferous tubules. Thus, the radio of immunoreactivity was significantly higher in tubular sections with arrested spermatogenesis in aged animals compared with regressed animals, and in the latter compared with those whose tubular sections showed complete spermatogenesis. In conclusion, HSP47 expression in Sertoli cells was found for the first time in mammals. Moreover, increased expression seemed to be related to the degree of atrophy of the seminiferous epithelium and to the reversible or non-reversible physiological state of the seminiferous epithelium., (©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

In this study, we have explored the role of the KATNB1 gene, a microtubule-severing protein, in the seminiferous epithelium of the rat testis. Our data have shown that KATNB1 expressed in rat brain, testes, and Sertoli cells. KATNB1 was found to co-localize with α-tubulin showing a unique stage-specific distribution across the seminiferous epithelium. Knockdown of KATNB1 by RNAi led to significant disruption of the tight junction (TJ) permeability barrier function in primary Sertoli cells cultured in vitro with an established functional TJ-barrier, as well as perturbations in the microtubule and actin cytoskeleton organization. The disruption in these cytoskeletal structures, in turn, led to improper distribution of TJ and basal ES proteins essential for maintaining the Sertoli TJ function. More importantly, overexpression of KATNB1 in the testis in vivo was found to block cadmium-induced blood-testis barrier (BTB) disruption and testis injury. KATNB1 exerted its promoting effects on BTB and spermatogenesis through corrective spatiotemporal expression of actin- and microtubule-based regulatory proteins by maintaining the proper organization of cytoskeletons in the testis, illustrating its plausible therapeutic implication. In summary, Katanin regulatory subunit B1 (KATNB1) plays a crucial role in BTB and spermatogenesis through its effects on the actin- and microtubule-based cytoskeletons in Sertoli cells and testis, providing important insights into male reproductive biology., (© 2024 Federation of American Societies for Experimental Biology.)

Zebrafish serve as a valuable model organism for studying germ cell biology and reproductive processes. The AB strain of zebrafish is proposed to exhibit a polygenic sex determination system, where most males initially develop juvenile ovaries before committing to male fate. In species with chromosomal sex determination, gonadal somatic cells are recognized as key determinants of germ cell fate. Notably, the loss of germ cells in zebrafish leads to masculinization, implying that germ cells harbor an intrinsic feminization signal. However, the specific signal triggering oogenesis in zebrafish remains unclear. In the present study, we identified foxl2l as an oocyte progenitor-specific gene essential for initiating oogenesis in germ cells. Results showed that foxl2l -knockout zebrafish bypassed the juvenile ovary stage and exclusively developed into fertile males. Further analysis revealed that loss of foxl2l hindered the initiation of oocyte-specific meiosis and prevented entry into oogenesis, leading to premature spermatogenesis during early gonadal development. Furthermore, while mutation of the pro-male gene dmrt1 led to fertile female differentiation, simultaneous disruption of foxl2l in dmrt1 mutants completely blocked oogenesis, with a large proportion of germ cells arrested as germline stem cells, highlighting the crucial role of foxl2l in oogenesis. Overall, this study highlights the unique function of foxl2l as a germ cell-intrinsic gatekeeper of oogenesis in zebrafish.

Serine protease 50 (PRSS50/TSP50) is highly expressed in spermatocytes. Our study investigated its role in testicular development and spermatogenesis. Initially, PRSS50 knockdown was observed to impair DNA synthesis in spermatocytes. To further explore this, we generated PRSS50 knockout ( Prss50 -/- ) mice ( Mus musculus ), which exhibited abnormal spermatid nuclear compression and reduced male fertility. Furthermore, dysplastic seminiferous tubules and decreased sex hormones were observed in 4-week-old Prss50 -/- mice, accompanied by meiotic progression defects and increased apoptosis of spermatogenic cells. Mechanistic analysis indicated that PRSS50 deletion resulted in increased phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and elevated levels of MAP kinase phosphatase 3 (MKP3), a specific ERK antagonist, potentially accounting for testicular dysplasia in adolescent Prss50 -/- mice. Taken together, these findings suggest that PRSS50 plays an important role in testicular development and spermatogenesis, with the MKP3/ERK signaling pathway playing a significant role in this process.

sion="1.0" encoding="utf-8"?> RD Reproduction, Fertility and Development Reprod. Fertil. Dev. 1031-3613 1448-5990 CSIRO Publishing 36 Gardiner Road Clayton 3168 Victoria Australia RD24134 10.1071/RD24134 Foreword Dedication to Jim Cummins Spermatology and testis biology - honouring the contributions of James M. Cummins M. Yeste and G. B. Martin https://orcid.org/0000-0002-2209-340X Yeste Marc A * https://orcid.org/0000-0002-1905-7934 Martin Graeme B. B Department of Biology, Faculty of Sciences, University of Girona , Girona , Spain . Faculty of Agriculture and Natural Sciences, University of Western Australia , Stirling Highway , Crawley , WA , Australia . * Correspondence to: Marc Yeste Department of Biology, Faculty of Sciences, University of Girona, Girona, Spain Email: marc.yeste@udg.edu 12 September 2024 36 15 Collection: Dedication to Jim Cummins RD24134 17 August 2024 Received: 17 August 2024 20 August 2024 Accepted: 20 August 2024 12 September 2024 Published: 12 September 2024 © 2024 The Author(s) (or their employer(s)). Published by CSIRO Publishing 2024 The Author(s) (or their employer(s)). This collection celebrates the important contributions James (Jim) Michael Cummins made to the field of reproductive biology. It covers a variety of topics but with a clear focus on spermatology where Jim's impact, as a scientist and teacher, was most profound. As a core member of the Editorial Board of Reproduction, Fertility and Development , for a series of Editors-in-Chief, he was a font of wisdom for all aspects of the field, including reproductive technology in humans, and acted as a guardian of editorial principles and ethics. This article belongs to the Collection Dedication to Jim Cummins.

This study explores the effects of Trib3 gene knockout on adult male rat spermatogenesis. Using CRISPR/Cas9, we knocked out the Trib3 gene in Wistar rats. Results indicate altered expression of PLZF, ID4, and c-KIT in knockout rats, suggesting impaired spermatogonial stem cell proliferation and differentiation. Histological analysis reveals reduced seminiferous tubule area and decreased spermatocyte numbers. Mating experiments demonstrate reduced offspring rates after the second self-mating in knockout rats. SYCP3, a meiosis marker, shows elevated expression in knockout rat testes at 14 days postpartum, suggesting an impact on reproductive processes. ELISA results indicate decreased testosterone, FSH, and FGF9 levels in knockout rat testicular tissues. In conclusion, Trib3 gene deletion may impede spermatogonial self-renewal and promote differentiation through the FSH-FGF9- c-KIT interaction and p38MAPK pathway, affecting reproductive capacity. These findings contribute to understanding the molecular mechanisms regulating spermatogenesis., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2024 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Proper connection between the sperm head and tail is critical for sperm motility and fertilization. Head-tail linkage is mediated by the head-tail coupling apparatus (HTCA), which secures the axoneme (tail) to the nucleus (head). However, the molecular architecture of the HTCA is poorly understood. Here, we use Drosophila to investigate formation and remodeling of the HTCA throughout spermiogenesis by visualizing key components of this complex. Using structured illumination microscopy, we demonstrate that key HTCA proteins Spag4 and Yuri form a 'centriole cap' that surrounds the centriole (or basal body) as it invaginates into the surface of the nucleus. As development progresses, the centriole is laterally displaced to the side of the nucleus while the HTCA expands under the nucleus, forming what we term the 'nuclear shelf'. We next show that the proximal centriole-like (PCL) structure is positioned under the nuclear shelf, functioning as a crucial stabilizer of centriole-nucleus attachment. Together, our data indicate that the HTCA is a complex, multi-point attachment site that simultaneously engages the PCL, the centriole and the nucleus to ensure proper head-tail connection during late-stage spermiogenesis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

This study investigated the effects of transplanted testicular stromal stem cells (tSSCs) on surgically damaged testis tissue. Ten-week-old male Wistar albino rats were divided into three groups: control (n = 6), damage (DG) (n = 6) and testicular stromal stem cell (TSSC) (n = 6) groups. Surgically induced damage was inflicted on the left testes of both the DG and TSSC groups, with no intervention on the right testes. In the TSSC group, damaged testes were treated with transplanted tSSCs, followed by orchiectomy after 15 days. Testes tissues were stained with haematoxylin-eosin (H&E), and recovery rates of functional structures were assessed by modified Johnsen scoring. The effects of tSSCs on testicular tissue were demonstrated by immunohistochemistry using BAX, BCL-2 and caspase 3. Serum testosterone levels were analysed using the enzyme-linked immunosorbent assay (ELISA) method. Surgical damage caused germ cell degeneration in some seminiferous tubules and a decrease in interstitial areas. With tSSC treatment, improvements in testicular architecture were identified through spermatogenesis in the seminiferous tubules and normal histological structures in the interstitial areas. Correspondingly, in the modified Johnsen score, the DG group showed a significant difference compared to the other groups (p = 0.001). High expressions of BAX, BCL-2 and caspase-3 in the DG group revealed prominent features of apoptosis. With the injection of tSSCs, these expressions significantly normalized according to H score analysis (all p = 0.004). Although serum testosterone levels in the tSSC group were higher compared to the control and DG groups, this difference was not statistically significant (p = 0.119). This study suggests transplanting tSSCs could accelerate tissue healing after testicular sperm extraction (TESE) surgery for azoospermia patients, potentially paving the way for a new and important clinical treatment., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Currently, there is limited histological data for spermatid morphologies within the testes of squamates. There are only 10 species of lizard that have complete ultrastructural data across the entire process of spermiogenesis, including several species of Sceloporus. These studies have shown that differences can be seen between spermatids of saurians within the same family or genus. Thus, the present study continues to test the hypothesis that differences exist in spermatid morphology between species within the same family. We collected five Phrynosoma cornutum males from Arizona. Their testes were extracted and processed with standard TEM techniques. Many of the characteristics of spermiogenesis within P. cornutum are conserved and similar in morphology to other phrynosomatid lizards. These similarities include the development of the acrosome, perforatorium, subacrosomal cone, nuclear rostrum, and epinuclear lucent zone. However, there were also differences observed in P. cornutum spermatids that are distinct compared to other phyrnosomatids. For example, P. cornutum spermatids include a wider and more robust perforatorium and less spiraling of the chromatin during condensation than that of other phrynosomatid lizards. The present results corroborate previous studies and indicate that even with morphological conservation within saurian spermatids, character differences between species can be recognized. Further studies on spermiogenesis are required to judge the relevance of these ontogenetic changes in terms of using them in amniotic or squamate spermatid/spermatozoa phylogenic analysis., (© 2024 Wiley Periodicals LLC.)

The ricefield eel (Monopterus albus) is inherently timid and highly sensitive to stress. Our previous studies have shown that low-temperature weather could significantly affect the sperm vitality of ricefield eels. This study aims to investigate the regulatory mechanism of low-temperature effects on testicular function and sperm vitality in ricefield eels. The ricefield eels were initially reared at low (10 °C) and normal (25 °C) temperatures for 24 h. Low temperatures were found to induce the expression of pituitary pro-opiomelanocortin (POMC) and testes insulin-like growth factor-binding protein 1 (IGFBP1) mRNA expression, suggesting that the reduction in sperm vitality could be attributed to the activation of the stress axis. Moreover, the results indicated a significant decrease in sperm occupancy and count in the testes, along with a reduced percentage of motile sperm. Subsequent transcriptome analysis showed substantial inhibition of reproductive hormone genes (gnrh1, lh, and fsh) in the brain and pituitary, and downregulation of meiosis-related genes (dmc1, rec8, and sycp3) in the testes. These findings suggest that low temperatures might disrupt testicular development and spermatogenesis by inhibiting the reproductive axis. Metabolomics analysis then demonstrated a significant reduction in the levels of metabolites related to glycolysis, fatty acid metabolism, and the tricarboxylic acid (TCA) cycle in the testes after low-temperature treatment. Interestingly, the expression of zona pellucida sperm-binding proteins 3 and 4 (ZP3 and ZP4), which may affect sperm vitality and spermatogenesis, was significantly induced by low temperatures in the testes. In conclusion, these findings suggested that low temperatures might affect testicular function and sperm vitality by simultaneously activating the stress axis and inhibiting the reproductive axis and energy metabolism in the testes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Testicular development and spermatogenesis are critical for male reproduction, with histone (de)acetylation playing a key role in chromatin remodeling within germ cells. Sirt1, a key histone deacetylase, is implicated in chromatin remodeling, but its expression pattern and specific role in testicular development and spermatogenesis need further study. This study comprehensively analyzed Sirt1 expression in adult and juvenile mouse testicular tissues and across various male germ cells, utilizing RT-qPCR, Western blot, immunofluorescence, and cell transfection. GO and KEGG enrichment analyses were performed to elucidate the biological functions and pathways associated with Sirt1 and its related genes. Multiple miRNA databases were utilized to predict miRNAs targeting Sirt1, and their expression levels were validated using RT-qPCR. Lentiviral transfection was used to knockdown candidate miRNAs to assess their functional roles. The results revealed a significant downregulation of Sirt1 expression in adult mouse testicular tissues compared to juvenile tissues, with pronounced variation across diverse male germ cells. Sirt1 was highly expressed in spermatogonia and mature sperm, but comparatively lower in spermatocytes and spermatids. GO and KEGG enrichment analyses highlighted Sirt1's role in key biological processes, including chromatin organization, regulation of cell proliferation, and energy homeostasis, as well as its association with signaling pathways like cellular senescence, the FoxO signaling pathway, and the AMPK signaling pathway. Bioinformatic analysis and subsequent RT-qPCR validation identified miR-9-5p as a miRNA targeting Sirt1. The expression of miR-9-5p was significantly higher in adult mouse testicular tissues compared to juvenile tissues, inversely correlating with Sirt1 levels. Moreover, the knockdown of miR-9-5p led to a notable increase in Sirt1 mRNA and protein expression. In conclusion, Sirt1 is a key player in mouse testicular development and spermatogenesis. The discovery that miR-9-5p negatively regulates Sirt1 suggests a critical regulatory axis that may govern these processes, providing novel insights into male fertility and potential targets for therapeutic intervention., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interest The authors have no conflict of interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The ubiquitination is crucial for controlling cellular homeostasis and protein modification, in which ubiquitin-conjugating enzyme (E2) acts as the central player in the ubiquitination system. Ubiquitin-conjugating enzymes, which have special domains that catalyse substrates, have sequence discrepancies and modulate various pathophysiological processes in different cells of multiple organisms. E2s take part in the mitosis of primordial germ cells, meiosis of spermatocytes and the formation of mature haploid spermatids to maintain normal male fertility. In this review, we summarize the various types of E2s and their functions during distinct stages of spermatogenesis., (© 2024. The Author(s).)

Reproduction is paramount to animals. For it to be successful, a coordination of social behavior, physiology, and gamete production is necessary. How are social cues perceived and how do they affect physiology and gametogenesis? While females, ranging from insects to mammals, have provided multiple insights about this coordination, its existence remains largely unknown in males. Here, by using the Drosophila male as a model, we describe a phenomenon by which the availability of potential mating partners triggers an activation state on the stem cell populations of the testis, boosting spermatogenesis. We reveal its reliance on pheromonal communication, even in the absence of mating or other interactions with females. Finally, we identify the interorgan communication signaling network responsible-muscle-secreted tumor necrosis factor alpha (TNF-α)/Eiger and neuronally secreted octopamine trigger, respectively, the Jun N-terminal kinase (JNK) pathway and a change in calcium dynamics in the cyst stem cells. As a consequence, germ line stem cells increase their proliferation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The horned octopod Eledone cirrhosa, a medium-sized species found in Arabian Gulf off Saudi Arabia, was collected monthly from the Arabian Gulf off Dammam city during January to December 2022. Samples were dissected and prepared for examination using transmission electron microscopy. During genital maturation, seminiferous tubules are established in the testis, with active spermatogonia dividing. Spermatocytes 1 are observed in the tubule, followed by an increase in spermatogonia and spermatocytes in August. Spermiogenesis begins, with spherical spermatids evolving into elongated spermatids. In September, active spermatogonia, meiotic divisions, and increased spermiogenesis continue. Spermatozoa appear in Needham's pouch, indicating sexual maturity. The ovary undergoes various stages of development, with oocytes at stage I in June and July, followed by stage II in October and November. In stage III, follicular cords invade the oocyte's cytoplasm, forming numerous lipid inclusions and protein granules. The cytoplasm contains cisternae of endoplasmic reticulum and a poorly developed Golgi apparatus. Stage IV occurs in November, characterized by the maximum development of follicular cords and the beginning of vitellogenesis. The ooplasm contains numerous lipid inclusions, a syncytium, and secretory cells. From December, stage V oocytes are mainly present, indicating the activity phase of maximum secretion. Yolk platelets accumulate in the oocyte ooplasm, and chorion forms at the zona pellucida. In January, the first smooth eggs are found in some octopuses' ovary, with their proportion increasing steadily. This study aimed to investigate the mitogenic action of gonadotropin and identify the periods of intense cell multiplication during the sexual cycle using cytological methods.

Heat stress (HS) poses a substantial challenge to livestock. Studies have demonstrated that HS reduces fertility and leads to gut microbiota dysbiosis in bulls. However, the impact of the gut microbiota on fertility in bulls during HS is still unclear. Our research revealed that HS exposure decreased semen quality in bulls, and fecal microbiota transplantation (FMT) from heat-stressed bulls to recipient mice resulted in a significant decrease in number of testicular germ cells and epididymal sperm. Untargeted metabolomics methodology and 16S rDNA sequencing conjoint analysis revealed that Akkermansia muciniphila (A. muciniphila) seemed to be a key bacterial regulator of spermatogenesis after HS exposure. Moreover, the research indicated that A. muciniphila regulated secondary bile acid metabolism by promoting the colonization of bile salt hydrolase (BSH)-metabolizing bacteria, leading to increase of retinol absorption in the host gut and subsequently elevation of testicular retinoic acid level, thereby improving spermatogenesis. This study sheds light on the relationship between HS-induced microbiota dysbiosis and spermatogenesis, offering a potential therapeutic approach for addressing bull spermatogenic dysfunction triggered by HS exposure., Competing Interests: Declaration of competing interest The author has declared that no competing interest exists., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Diets leading to caloric overload are linked to metabolic disorders and reproductive function impairment. Metabolic and hormonal abnormalities stand out as defining features of metabolic disorders, and substantially affect the functionality of the testis. Metabolic disorders induce testicular metabolic dysfunction, chronic inflammation and oxidative stress. The disruption of gastrointestinal, pancreatic, adipose tissue and testicular hormonal regulation induced by metabolic disorders can also contribute to a state of compromised fertility. In this Review, we will delve into the effects of high-fat diets and metabolic disorders on testicular metabolism and spermatogenesis, which are crucial elements for male reproductive function. Moreover, metabolic disorders have been shown to influence the epigenome of male gametes and might have a potential role in transmitting phenotype traits across generations. However, the existing evidence strongly underscores the unmet need to understand the mechanisms responsible for transgenerational paternal inheritance of male reproductive function impairment related to metabolic disorders. This knowledge could be useful for developing targeted interventions to prevent, counteract, and most of all break the perpetuation chain of male reproductive dysfunction associated with metabolic disorders across generations., (© 2024. Springer Nature Limited.)

Spermatogonial stem cells (SSCs) are essential for the maintenance of male fertility and survival of species. Environmental conditions, notably heat stress, have been identified as important causes of male infertility and have a negative impact on SSCs. Animals with cryptorchid testes (CT) are optimal models for the study of long-term heat stress-related changes in germ cells. The effect of heat stress on germ cells differs depending on the spermatogenesis stage. Thus, verifying whether the specific phase of spermatogenesis is dependent or independent of heat stress in stallions is important. We evaluated the heat stress-related response of SSCs by comparing the relative abundance of mRNA transcripts and expression patterns of the undifferentiated embryonic cell transcription factor 1 (UTF-1) and deleted in azoospermia-like (DAZL) in the seminiferous tubules of CT and normal testes (NT) of stallions using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, and western blotting. We also analyzed the relative abundance of mRNA of different proliferative markers, including minichromosome maintenance 2 (MCM2), marker of proliferation Ki-67 (MKI-67), and proliferating cell nuclear antigen (PCNA). Testicular tissues from four Thoroughbred unilateral cryptorchid postpubertal stallions were used in this study during the breeding season. The relative abundance of the mRNA transcripts of UTF-1 and MCM2 was significantly upregulated in the CT group than that of those in the NT group. In contrast, the relative abundance of the mRNA transcripts of DAZL was significantly downregulated in the CT group than that of those in the NT group. Western blot quantification showed that the relative intensity of UTF-1 protein bands was significantly higher, while that of DAZL protein bands was significantly lower in the CT group than in the NT group. Immunofluorescence studies showed that the number of germ cells immunostained with UTF-1 was significantly higher while immunostained with DAZL was significantly lower in the CT group than that in the NT group. The higher expression level of UTF-1 in the CT group shows that undifferentiated SSCs are not affected by long-term exposure to heat stress. These results also indicate that germ cells after differentiation phase are directly affected by heat-stress conditions, such as cryptorchidism, in stallions., Competing Interests: Declaration of competing interest No conflicts of interest have been disclosed by the authors., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Drosophila melanogaster is an ideal model organism for investigating spermatogenesis due to its powerful genetics, conserved genes and visible morphology of germ cells during sperm production. Our previous work revealed that ocnus (ocn) knockdown resulted in male sterility, and CG9920 was identified as a significantly downregulated protein in fly abdomen after ocn knockdown, suggesting a role of CG9920 in male reproduction. In this study, we found that CG9920 was highly expressed in fly testes. CG9920 knockdown in fly testes caused male infertility with no mature sperms in seminal vesicles. Immunofluorescence staining showed that depletion of CG9920 resulted in scattered spermatid nuclear bundles, fewer elongation cones that did not migrate to the anterior region of the testis, and almost no individualization complexes. Transmission electron microscopy revealed that CG9920 knockdown severely disrupted mitochondrial morphogenesis during spermatogenesis. Notably, we found that CG9920 might not directly interact with Ocn, but rather was inhibited by STAT92E, which itself was indirectly affected by Ocn. We propose a possible novel pathway essential for spermatogenesis in D. melanogaster, whereby Ocn indirectly induces CG9920 expression, potentially counteracting its inhibition by the JAK-STAT signaling pathway., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Males of the bat Eptesicus furinalis show at least one process of testicular regression, in which the testes regress and temporarily interrupt the production of sperm, during its annual reproductive cycle. As the process of spermatogenesis is under hormonal control, mainly of pituitary and androgen hormones, our aim was to analyze the morphological variations and the hormonal control of the testes of E. furinalis during the four phases of its reproductive cycle. Testes of 18 adult males, divided into four sample groups (active, regressing, regressed, and recrudescence phases), were submitted to morphological, morphometric, and immunohistochemical analyzes. The results demonstrate that the processes of testicular regression and recrudescence of E. furinalis are under the control of pituitary, androgen and estrogen hormones. The regulation is exerted mainly through the activation and cross signaling of AR and FSHR in Sertoli cells and of LHR in Leydig cells. The testicular regression appears to be activated by an inhibition/reduction of AR expression in Sertoli cells, which inhibits the proliferation and differentiation of new spermatogonia and causes the deactivation of spermatogenesis. Conversely, the testicular recrudescence occurs by the increasing of the expression of LHR in Leydig cells, and AR and FSHR in Sertoli cells, which reactivates the testicular production of androgens and estrogens, the proliferation of spermatogonia and restarts the spermatogenesis., (© 2023 American Association for Anatomy.)

Background: Spermatogenesis is a temperature-sensitive process, and elevation in temperature hampers this process quickly and significantly. We studied the molecular effects of testicular heating on piRNAs and gene expression in rat testicular germ cells., Methods: We generated a cryptorchid rat model by displacing the testis from the scrotal sac (34 °C) to the abdominal area (37 °C) and sacrificed animals after 1 day, 3 days, and 5 days. Pachytene spermatocytes and round spermatids were purified using elutriation centrifugation and percoll gradient methods. We performed transcriptome sequencing in pachytene spermatocytes and round spermatids to identify differentially expressed piRNAs and their probable targets, i.e., TE transcripts and mRNAs., Results: As a result of heat stress, we observed significant upregulation of piRNAs and TE transcripts in testicular germ cells. In addition to this, piRNA biogenesis machinery and heat shock proteins (Hsp70 and Hsp90 family members) were upregulated. mRNAs have also been proposed as targets for piRNAs; therefore, we shortlisted certain piRNA-mRNA pairs with an inverse relationship of expression. We observed that in testicular heat stress, the heat shock proteins go hand-in-hand with the upregulation of piRNA biogenesis machinery. The dysregulation of piRNAs in heat-stressed germ cells, increased ping-pong activity, and disturbed expression of piRNA target transcripts suggest a connection between piRNAs, mRNAs, and TE transcripts., Conclusions: In heat stress, piRNAs, piRNA machinery, and heat shock proteins are activated to deal with low levels of stress, which is followed by a rescue approach in prolonged stressaccompained by high TE activity to allow genetic mutations, perhaps for survival and adaptability., (© 2024. The Author(s).)

The specialized cell cycle of meiosis transforms diploid germ cells into haploid gametes. In mammals, diploid spermatogenic cells acquire the competence to initiate meiosis in response to retinoic acid. Previous mouse studies revealed that MEIOC interacts with RNA-binding proteins YTHDC2 and RBM46 to repress mitotic genes and to promote robust meiotic gene expression in spermatogenic cells that have initiated meiosis. Here, we have used the enhanced resolution of scRNA-seq and bulk RNA-seq of developmentally synchronized spermatogenesis to define how MEIOC molecularly supports early meiosis in spermatogenic cells. We demonstrate that MEIOC mediates transcriptomic changes before meiotic initiation, earlier than previously appreciated. MEIOC, acting with YTHDC2 and RBM46, destabilizes its mRNA targets, including the transcriptional repressors E2f6 and Mga, in mitotic spermatogonia. MEIOC thereby derepresses E2F6- and MGA-repressed genes, including Meiosin and other meiosis-associated genes. This confers on spermatogenic cells the molecular competence to, in response to retinoic acid, fully activate the transcriptional regulator STRA8-MEIOSIN, which is required for the meiotic G1/S phase transition and for meiotic gene expression. We conclude that, in mice, mRNA decay mediated by MEIOC-YTHDC2-RBM46 enhances the competence of spermatogenic cells to initiate meiosis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Infertility is a global health problem affecting one in six couples, with 50% of cases attributed to male infertility. Spermatozoa are male gametes, specialized cells that can be divided into two parts: the head and the flagellum. The head contains a vesicle called the acrosome that undergoes exocytosis and the flagellum is a motility apparatus that propels the spermatozoa forward and can be divided into two components, axonemes and accessory structures. For spermatozoa to fertilize oocytes, the acrosome and flagellum must be formed correctly. In this Review, we describe comprehensively how functional spermatozoa develop in mammals during spermiogenesis, including the formation of acrosomes, axonemes and accessory structures by focusing on analyses of mouse models., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Sertoli cells act as highly polarized testicular cells that nutritionally support multiple stages of germ cell development. However, the gene regulation network in Sertoli cells for modulating germ cell development has yet to be fully understood. In this study, we report that heterogeneous nuclear ribonucleoproteins C in Sertoli cells are essential for germ cell development and male fertility. Conditional knockout of heterogeneous nuclear ribonucleoprotein C in mouse Sertoli cells leads to aberrant Sertoli cells proliferation, disrupted cytoskeleton of Sertoli cells, and compromised blood-testis barrier function, resulting in loss of supportive cell function and, ultimately, defective spermiogenesis in mice. Further ribonucleic acid-sequencing analyses revealed these phenotypes are likely caused by the dysregulated genes in heterogeneous nuclear ribonucleoprotein C-deficient Sertoli cells related to cell adhesion, cell proliferation, and apoptotic process. In conclusion, this study demonstrates that heterogeneous nuclear ribonucleoprotein C plays a critical role in Sertoli cells for maintaining the function of Sertoli cells and sustaining steady-state spermatogenesis in mice., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Heat stress induces testicular oxidative stress, impairs spermatogenesis, and increases the risk of male infertility. Recent studies have highlighted the antioxidative properties of the Sestrins family in reducing cellular oxidative damage. However, the role of Sestrins (Sestrin1, 2, and 3) in the testicular response to heat stress remains unclear. Here, we found that Sestrin2 and 3 were highly expressed in the testis relative to Sestrin1. Then, the Sestrin2-/- and Sestrin3-/- mice were generated by CRISPR/Cas9 to investigate the role of them on spermatogenesis after heat stress. Our data showed that Sestrin2-/- and Sestrin3-/- mice testes exhibited more severe damage manifested by exacerbated loss of germ cells and higher levels of oxidative stress as compared to wild-type counterparts after heat stress. Notably, Sestrin2-/- and Sestrin3-/- mice underwent a remarkable increase in heat-induced spermatocyte apoptosis than that of controls. Furthermore, the transcriptome landscape of spermatocytes and chromosome spreading showed that loss of Sestrin2 and Sestrin3 exacerbated meiotic failure by compromising DNA double-strand breaks repair after heat stress. Taken together, our work demonstrated a critical protective function of Sestrin2 and Sestrin3 in mitigating the impairments of spermatogenesis against heat stress., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

RNA-binding proteins (RBPs), as key regulators of mRNA fate, are abundantly expressed in the testis. However, RBPs associated with human male infertility remain largely unknown. Through bioinformatic analyses, we identified 62 such RBPs, including an evolutionarily conserved RBP, DEAD-box helicase 20 (DDX20). Male germ-cell-specific inactivation of Ddx20 at E15.5 caused T1-propsermatogonia (T1-ProSG) to fail to reenter cell cycle during the first week of testicular development in mice. Consequently, neither the foundational spermatogonial stem cell (SSC) pool nor progenitor spermatogonia were ever formed in the knockout testes. Mechanistically, DDX20 functions to control the translation of its target mRNAs, many of which encode cell-cycle-related regulators, by interacting with key components of the translational machinery in prospermatogonia. Our data demonstrate a previously unreported function of DDX20 as a translational regulator of critical cell-cycle-related genes, which is essential for cell-cycle reentry of T1-ProSG and formation of the SSC pool., Competing Interests: Declaration of interests A patent for a screening method for male infertility genes has been issued to W.S., D.Z., L.S., and K.L. (patent number: ZL 2023 1 1149986.4; issue number: CN 116884488 B)., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The present study aims to identify spermatogenesis in testicular seminiferous tubules (ST) and testicular tissue of adult normal and busulfan-treated mice utilizing PCA and Raman spectroscopy. Raman measurements were conducted on single tubules and testes samples from adult and immature mice, comparing them with those from busulfan-treated adult mice, with validation through histological examination. The analysis revealed a higher signal variability (30 %-40 % at the peaks), prompting scrutiny of individual Raman spectra as a means of spermatogenesis measurement. However, principal component analysis (PCA) demonstrated significant cluster separation between the ST of mature and immature mice. Similar investigations were performed to compare ST from normal mature mice and those from busulfan-treated (BS-treated) mature mice, revealing substantial separation along PC1 and PC2 for all comparison sets. Additionally, comparing testicular samples from mature and immature mice revealed distinct separation in PCA. The study concludes that the combined approach of PCA and Raman spectroscopy proves to be a noninvasive and potentially valuable method for identifying spermatogenesis in seminiferous tubules and testicular samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Context A population of sperm progenitor cells, known as Asingle spermatogonia, has been described in mammalian testes. During division cycles in spermatogenesis, some cells will form part of the Asingle spermatogonia group, while others form primary spermatocytes. Thus, during spermatogenesis, spermatogonia are the progenitor cells of spermatozoa. Aims In this study, we characterise the spermatogonial stem cells (SSCs) in the testicles of Artibeus jamaicensis and Sturnira lilium bats. The knowledge generated from this will contribute to the understanding of the biology of germ cells and the mechanisms of spermatogenesis in mammals, generating information on wildlife species that are important for biodiversity. Methods Testes were analysed by light and electron microscopy. Likewise, the expression of specific factors of stem cells (Oct4 and C-kit), germ cells (Vasa), cell proliferation (pH3 and SCP1) and testicular somatic cells (MIS, 3βHSD and Sox9) was characterised by immunofluorescence and western blot. Key results The histological analysis enabled the location of type Asingle, Apaired and Aaligned spermatogonia in the periphery of the seminiferous tubules adjacent to Sertoli cells. The expression of genes of stem and germ cells made it possible to corroborate the distribution of the SSCs. Conclusions Results indicate that type Asingle spermatogonia were not randomly distributed, since proliferative activity was detected in groups of cells adjacent to the seminiferous tubules membrane, suggesting the localisation of spermatogonial niches in a specific region of testes. Implications This study provides evidence for the existence of SSCs in the testis of chiropterans that contribute to the renewal of germline progenitor cells to maintain the reproduction of the organisms.

Although the order Rodentia does not present a high risk of extinction compared to mammals as a whole, several families demonstrate high levels of threat and/or data deficiency, therefore highlighting the need for targeted research and the application of ecological and reproductive data to the development of conservation actions. The order Rodentia, the largest among mammals, includes 9 families, and the family Cricetidae is the most diverse of the Brazilian rodents. In Brazil, 12 of the 16 genera of Oecomys are found. Oecomys bicolor is known in Brazil as the 'arboreal rat' and is, found in dry, deciduous and tropical forests. The mean body weight of Oecomys bicolor was 35.8 g and the gonadal, tubular and epithelial somatic indexes were, 0.53%, 0.47% and 0.37%, respectively. Seminiferous tubules volume density was 89.72% and the mitotic and meiotic indexes corresponded to 8.59 and 2.45 cells, respectively, and the yield of spermatogenesis was 23.83 cells. The intertubular compartment represented 10.28% of the testis parenchyma and around 5% of the interstitial space was occupied by Leydig cells, whose number per gram of testis was 11.10 × 10 7 cells. By evaluating the biometric and histomorphometric characteristics of the testis, there is evidence that this species has a high investment in reproduction. Due to the high contribution of the seminiferous epithelium and the intertubular compartment in this species, compared to the others of the same family, it is possible to infer that the species Oecomys bicolor has a promiscuous reproductive behaviour., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Osteoglossomorpha, the bony tongue fishes, show great variation in morphology, behavioural strategies, reproductive biology and gamete ultrastructure. The order Osteoglossiformes is the only vertebrate taxon, in which four types of sperm (monoflagellate, biflagellate and aflagellate aquasperm and the complex introsperm) have been described. It is also the only vertebrate lineage in which aflagellate spermatozoa exist. The aim of this study was to analyse the structure of the testis and the process of spermiogenesis in the mormyrid Campylomormyrus compressirostris during the breeding season using light and electron microscopy (transmission and scanning). Males of this species have a single testis of the anastomosing tubular type. The tubules of the anterior part of the testis contain cysts with developing germ cells, and this region is much wider than the posterior part, which consists of efferent ducts filled with sperm cells. The cysts are filled with single or mitotic spermatogonia, primary and secondary spermatocytes and early spermatids. At the stage of spermatids with fine granular chromatin, the cysts rupture and successive stages of spermatid differentiation take place in the testicular lumen; we therefore characterise this process as 'extracystic spermiogenesis'. Sperm development in C. compressirostris is extremely simple and involves chromatin condensation in the central region of the nucleus, a slight decrease in nuclear volume, the appearance of numerous vesicles in the cytoplasm that form a tubular-vesicular system at the base of the nucleus. Both centrioles and mitochondria are translocated to the peripheral region of the midpiece, which forms the opposite pole to the nucleus. There are many differences between the types of spermiogenesis described so far in teleosts and that found in C. compressirostris, including the loss of flagellum formation. This unique type of spermiogenesis is restricted to species of the families Mormyridae and Gymnarchidae, all of which possess aflagellate spermatozoa. Our data demonstrate that the spermatid differentiation and existence of the aflagellate spermatozoon are a unique phenomena not only among teleosts but also in the whole vertebrate lineage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier GmbH.)

The present study investigates the changes in M1/M2 macrophage polarization resulting from unilateral testicular torsion in the bilateral testis. The study sample included 63 male Sprague-Dawley rats, which were randomly divided into nine groups (n = 7): Control, Sham (4 h (4 h), 24 h, 7 days (7d), 14d), and Torsion/Detorsion (T/D) (4 h, 24 h, 7d, 14d). Histopathological evaluations revealed no changes in the Sham groups, while T/D was noted to cause edema, vascular occlusion, disruption of seminiferous tubule epithelial organization, germ cell abnormalities and structural anomalies in the experimental rats, the severity and extent of which increased from 4 h to 14d after T/D. The Cosentino scores used to determine the degree of histological damage were consistent with the histopathological findings in all groups, while the Johnsen scores, as a marker of spermatogenesis, were lower in the T/D groups. Seminiferous tubule diameters and germinal epithelial thickness decreased significantly in parallel with increased tubule damage in the ipsilateral testicles. Testicular torsion significantly affected sperm motility, with significant reductions observed in the T/D 7d and T/D 14d groups. A hormone profile analysis revealed decreased testosterone levels in both the Sham and T/D groups when compared to the Controls. CD68 and CD163 immunoreactivities, as M1 and M2 macrophage surface markers, were determined in the testicular tissue using the avidin-biotin-peroxidase complex method. T/D interventions caused M1/M2 macrophage polarization changes and increased M1 macrophages, particularly in contralateral testicular tissue. The increase in M1 macrophages in contralateral testicular tissue following T/D in the present study suggests that cell processes, including macrophages, may play an important role in contralateral testicular injury., (© 2024. The Author(s).)

Germ cells are highly conserved in the gonads, nurtured to either develop into a gamete or self-renew into a stem cell reserve. Preserving the germ cell pool and protecting the reproductive organs is essential for maintaining an individual's fertility. Several factors, including a sedentary lifestyle, pollutants, hormonal disruption, drugs, and a disease condition, have been shown to impair normal reproductive function. Irisin has recently been identified as an adipomyokine involved in modulating physiological functions based on the body's metabolic status. It is being studied for its role in various functions, including fertility. Findings show the localization of irisin in various parts of the reproductive axis, with the highest levels observed during puberty and pregnancy. This raises questions about its role and function in reproduction. Studies support irisin's role in protecting against disease-induced reproductive abnormalities and infertility. Therefore, the current review focuses on how irisin influences spermatogenesis and ovarian follicular development and plays a significant role in indirectly preserving the germ cell pool by protecting the gonads against oxidative stress and inflammation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Gravity in space can have a negative impact on the reproductive system. Given that the reproductive system is one of vitamin D's objectives, this study will use a simulated microgravity model to evaluate its impact on the rat reproductive system.Twenty-two male Wistar rats were allocated into four groups at random. Under microgravity circumstances, the rats were housed in both special and standard cages. Each group was then separated into two subgroups, one of which received vitamin D3 and the other did not. Blood was drawn twice to determine blood levels of vitamin D3, LH, FSH, and testosterone. Rat testes were isolated for histological analysis, as well as a piece of epididymis for sperm count and morphological examination.Microgravity had a detrimental effect on testicular tissue, resulting in lower serum levels of LH and testosterone (p-value < 0.001). Spermatogenesis was largely inhibited under microgravity. During microgravity conditions, however, vitamin D3 had a good effect on testicular structure, and the total number of sperm. Simulated microgravity affects the male reproductive system, compromising testicular morphology, sperm parameters, and hormonal balance. However, this study shows that vitamin D3 supplementation can act as a preventative strategy, minimizing the negative consequences of microgravity. The beneficial effect of vitamin D3 on testicular health and sperm quality implies that it may be useful in protecting male reproductive function in space-related situations., (© 2024. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Notch is a conserved cell-signaling pathway involved in spermatogenesis regulation. This study firstly evaluated the presence, localization patterns, acquisition origin and relation to acrosome reaction of Notch proteins in bull sperm. Western Blot analysis detected all Notch proteins in ejaculated bull sperm, and immunostaining described their specific sperm localization. Recovery of sperm from different segments showed that Notch proteins have testicular origin (NOTCH1, NOTCH2, DLL4), are sequentially acquired during sperm maturation along epididymal transit (NOTCH3, DLL3, JAGGED1-2), or post-ejaculation (DLL1, NOTCH4). Testis NOTCH2 is ubiquitously expressed in all germ-cell lines, whereas DLL4 is expressed in round and elongated spermatids during the Golgi, Cap, Acrosome and Maturation phases. In vitro spontaneous and induced sperm acrosome reaction induce consistent sperm regional relocation of NOTCH2, DLL4 and JAGGED1, and these relocation patterns are significantly associated to sperm acrosome status. NOTCH2 and JAGGED1 are relocated from the head apical to the post-equatorial regions, whereas DLL4 is lost along with the acrosome, evidencing that sperm spatial redistribution of NOTCH2 and JAGGED1 is linked to acrosome reaction onset, whereas DLL4 loss is linked to AR completion. Overall, results prompt for a relevant Notch role in bull sperm acrosome testicular development, epididymal maturation and acrosome reaction., (© 2024. The Author(s).)

Spermatogonial stem cells (SSCs) undergo self-renewal division to sustain spermatogenesis. Although it is possible to derive SSC cultures in most mouse strains, SSCs from a 129 background never proliferate under the same culture conditions, suggesting they have distinct self-renewal requirements. Here, we established long-term culture conditions for SSCs from mice of the 129 background (129 mice). An analysis of 129 testes showed significant reduction of GDNF and CXCL12, whereas FGF2, INHBA and INHBB were higher than in testes of C57BL/6 mice. An analysis of undifferentiated spermatogonia in 129 mice showed higher expression of Chrna4, which encodes an acetylcholine (Ach) receptor component. By supplementing medium with INHBA and Ach, SSC cultures were derived from 129 mice. Following lentivirus transduction for marking donor cells, transplanted cells re-initiated spermatogenesis in infertile mouse testes and produced transgenic offspring. These results suggest that the requirements of SSC self-renewal in mice are diverse, which has important implications for understanding self-renewal mechanisms in various animal species., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

pH homeostasis is crucial for spermatogenesis, sperm maturation, sperm physiological function, and fertilization in mammals. HCO 3 - and H + are the most significant factors involved in regulating pH homeostasis in the male reproductive system. Multiple pH-regulating transporters and ion channels localize in the testis, epididymis, and spermatozoa, such as HCO 3 - transporters (solute carrier family 4 and solute carrier family 26 transporters), carbonic anhydrases, and H + -transport channels and enzymes (e.g., Na + -H + exchangers, monocarboxylate transporters, H + -ATPases, and voltage-gated proton channels). Hormone-mediated signals impose an influence on the production of some HCO 3 - or H + transporters, such as NBCe1, SLC4A2, MCT4, etc. Additionally, ion channels including sperm-specific cationic channels for Ca 2+ (CatSper) and K + (SLO3) are directly or indirectly regulated by pH, exerting specific actions on spermatozoa. The slightly alkaline testicular pH is conducive to spermatogenesis, whereas the epididymis's low HCO 3 - concentration and acidic lumen are favorable for sperm maturation and storage. Spermatozoa pH increases substantially after being fused with seminal fluid to enhance motility. In the female reproductive tract, sperm are subjected to increasing concentrations of HCO 3 - in the uterine and fallopian tube, causing a rise in the intracellular pH (pH i ) of spermatozoa, leading to hyperpolarization of sperm plasma membranes, capacitation, hyperactivation, acrosome reaction, and ultimately fertilization. The physiological regulation initiated by SLC26A3, SLC26A8, NHA1, sNHE, and CFTR localized in sperm is proven for certain to be involved in male fertility. This review intends to present the key factors and characteristics of pH i regulation in the testes, efferent duct, epididymis, seminal fluid, and female reproductive tract, as well as the associated mechanisms during the sperm journey to fertilization, proposing insights into outstanding subjects and future research trends.

Cystic spermatogenesis in the subadult, maturing and adult Greenland shark (Somniosus microcephalus) displays multiple novel features, characterized early on by an unorganized internal cellular environment of the spermatocysts (anatomically discrete follicle-like units containing a single germ cell stage and its complement of co-developing Sertoli cells). These typically show polar asymmetries due to asymmetrically distributed germ and Sertoli cells. These arise from several novel cellular rearrangements at the immature pole, including fusion of a cluster of somatic cells with newly formed cysts containing only one to three spermatogonia and that already display an excess of Sertoli cells. The subadult's germinative zone revealed an additional novelty, namely numerous previously formed somatic cell-lined rings into which spermatogonia were incorporated. A striking finding was the conspicuous rarity of the routinely discernible Sertoli mitotic figures in the hallmark cyst stage of diametric elasmobranch spermatogenesis that is known for the peak display of the latter. Scrutiny of sequentially unfolding phenomena in the linearly arranged spermatogonial generations revealed that the cellular developments at the most common type of cyst-duct transition area (comprising slender to spindle-like basophilic cells with pointed ends) were concurrent with the discreet appearance of a second dark Sertoli nucleus, a development that persisted in spermiated cysts. Spermatogenically active mature males displayed vigorous meiotic divisions. However, a scattering of their spermatid cysts also displayed shark-atypical asynchronous passage through spermiogenesis, phenomena which were exacerbated as arrested spermiogenesis in an archival collection of tissues from 13 maturing specimens. Subadult specimens revealed meiotic arrest, and foci of infiltration of leukocytes that originate from a mass of eosinophilic, granule-laden immune cells dorsally under the testis capsule. This tissue was identical to the testis-affixed bone marrow equivalent in other shark species. This tissue is likely developmentally regulated in the Greenland shark as it is absent in adults., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 McClusky et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)