Recent strategies to mitigate reproductive aging in male broiler breeders: A review.

The continued improvement of genetics, nutrition, and management has resulted in rapid growth, better feed efficiency, and higher meat yield with competitive prices in the broiler industry. Nowadays, however, it is well-documented that productive traits and fertility are negatively correlated, and male broiler breeders are exposed to a fertility decline after 45 wk of age. Considering a low male-to-female ratio in breeder flocks, roosters have a prominent impact on flock fertility. Consequently, strategies to maintain the fertility of male broiler breeders could guarantee the reproductive performance of commercial herds. Understanding reproductive aging demands deep insights into its molecular and physiological mechanisms. Over-weighting, Sertoli and Leydig cell dysfunctions, compromised antioxidant capacity, imbalance in sexual hormones, and epididymal lithiasis are among candidate culprits associated with reproductive aging in roosters. Nutritional and managing strategies have been successfully applied to modulate body weight, improve sperm fatty acid profile and antioxidant status, and boost spermatogenic and steroidogenic pathways. The current review characterizes the physiology and biochemistry of reproductive aging in male broiler breeders and then highlights strategies and their underlying mechanisms to mitigate this failure. In summary, applying one or more of the abovementioned strategies might result in consistent post-peak reproduction and benefit producers in the poultry industry.

In Vitro Generation of Haploid Germ Cells from Human XY and XXY Immature Testes in a 3D Organoid System.

Increasing survival rates of children following cancer treatment have resulted in a significant population of adult survivors with the common side effect of infertility. Additionally, the availability of genetic testing has identified Klinefelter syndrome (classic 47,XXY) as the cause of future male infertility for a significant number of prepubertal patients. This study explores new spermatogonia stem cell (SSC)-based fertility therapies to meet the needs of these patients. Testicular cells were isolated from cryopreserved human testes tissue stored from XY and XXY prepubertal patients and propagated in a two-dimensional culture. Cells were then incorporated into a 3D human testicular organoid (HTO) system. During a 3-week culture period, HTOs maintained their structure, viability, and metabolic activity. Cell-specific PCR and flow cytometry markers identified undifferentiated spermatogonia, Sertoli, Leydig, and peritubular cells within the HTOs. Testosterone was produced by the HTOs both with and without hCG stimulation. Upregulation of postmeiotic germ cell markers was detected after 23 days in culture. Fluorescence in situ hybridization (FISH) of chromosomes X, Y, and 18 identified haploid cells in the in vitro differentiated HTOs. Thus, 3D HTOs were successfully generated from isolated immature human testicular cells from both euploid (XY) and Klinefelter (XXY) patients, supporting androgen production and germ cell differentiation in vitro.

Radiotherapy and Testicular Function: A Comprehensive Review of the Radiation-Induced Effects with an Emphasis on Spermatogenesis.

This comprehensive review explores the existing literature on the effects of radiotherapy on testicular function, focusing mainly on spermatogenic effects, but also with a brief report on endocrine abnormalities. Data from animal experiments as well as results on humans either from clinical studies or from accidental radiation exposure are included to demonstrate a complete perspective on the level of vulnerability of the testes and their various cellular components to irradiation. Even relatively low doses of radiation, produced either from direct testicular irradiation or more commonly from scattered doses, may often lead to detrimental effects on sperm count and quality. Leydig cells are more radioresistant; however, they can still be influenced by the doses used in clinical practice. The potential resultant fertility complications of cancer radiotherapy should be always discussed with the patient before treatment initiation, and all available and appropriate fertility preservation measures should be taken to ensure the future reproductive potential of the patient. The topic of potential hereditary effects of germ cell irradiation remains a controversial field with ethical implications, requiring future research.

Elucidating the Transcriptional States of Spermatogenesis-Joint Analysis of Germline and Supporting Cell, Mice and Human, Normal and Perturbed, Bulk and Single-Cell RNA-Seq.

In studying the molecular underpinning of spermatogenesis, we expect to understand the fundamental biological processes better and potentially identify genes that may lead to novel diagnostic and therapeutic strategies toward precision medicine in male infertility. In this review, we emphasized our perspective that the path forward necessitates integrative studies that rely on complementary approaches and types of data. To comprehensively analyze spermatogenesis, this review proposes four axes of integration. First, spanning the analysis of spermatogenesis in the healthy state alongside pathologies. Second, the experimental analysis of model systems (in which we can deploy treatments and perturbations) alongside human data. Third, the phenotype is measured alongside its underlying molecular profiles using known markers augmented with unbiased profiles. Finally, the testicular cells are studied as ecosystems, analyzing the germ cells alongside the states observed in the supporting somatic cells. Recently, the study of spermatogenesis has been advancing using single-cell RNA sequencing, where scientists have uncovered the unique stages of germ cell development in mice, revealing new regulators of spermatogenesis and previously unknown cell subtypes in the testis. An in-depth analysis of meiotic and postmeiotic stages led to the discovery of marker genes for spermatogonia, Sertoli and Leydig cells and further elucidated all the other germline and somatic cells in the testis microenvironment in normal and pathogenic conditions. The outcome of an integrative analysis of spermatogenesis using advanced molecular profiling technologies such as scRNA-seq has already propelled our biological understanding, with additional studies expected to have clinical implications for the study of male fertility. By uncovering new genes and pathways involved in abnormal spermatogenesis, we may gain insights into subfertility or sterility.

ZMYND12 serves as an IDAd subunit that is essential for sperm motility in mice.

Inner dynein arms (IDAs) are formed from a protein complex that is essential for appropriate flagellar bending and beating. IDA defects have previously been linked to the incidence of asthenozoospermia (AZS) and male infertility. The testes-enriched ZMYND12 protein is homologous with an IDA component identified in Chlamydomonas. ZMYND12 deficiency has previously been tied to infertility in males, yet the underlying mechanism remains uncertain. Here, a CRISPR/Cas9 approach was employed to generate Zmynd12 knockout (Zmynd12-/-) mice. These Zmynd12-/- mice exhibited significant male subfertility, reduced sperm motile velocity, and impaired capacitation. Through a combination of co-immunoprecipitation and mass spectrometry, ZMYND12 was found to interact with TTC29 and PRKACA. Decreases in the levels of PRKACA were evident in the sperm of these Zmynd12-/- mice, suggesting that this change may account for the observed drop in male fertility. Moreover, in a cohort of patients with AZS, one patient carrying a ZMYND12 variant was identified, expanding the known AZS-related variant spectrum. Together, these findings demonstrate that ZMYND12 is essential for flagellar beating, capacitation, and male fertility.

Functional Analysis of Forkhead Transcription Factor Fd59a in the Spermatogenesis of Drosophila melanogaster.

Spermatogenesis is critical for insect reproduction and is regulated by many different genes. In this study, we found that Forkhead transcription factor Fd59a functions as a key factor in the spermatogenesis of Drosophila melanogaster. Fd59a contains a conversed Forkhead domain, and it is clustered to the FoxD subfamily with other FoxD members from some insect and vertebrate species. Mutations in Fd59a caused swelling in the apical region of the testis. More importantly, fewer mature sperm were present in the seminal vesicle of Fd59a mutant flies compared to the control flies, and the fertility of Fd59a2/2 mutant males was significantly lower than that of the control flies. Immunofluorescence staining showed that the homeostasis of the testis stem cell niche in Fd59a2/2 mutant and Fd59a RNAi flies was disrupted and the apoptosis of sperm bundles was increased. Furthermore, results from RNA sequencing and qRT-PCR suggested that Fd59a can regulate the expression of genes related to reproductive process and cell death. Taken together, our results indicated that Fd59a plays a key role in the spermatogenesis of Drosophila.

Ultrastructure of the Spermiogenesis in Halyomorpha halys (Hemiptera: Pentatomidae): X-Irradiation and New Insights on the Centriolar Region Organization.

Halyomorpha halys (Heteroptera: Pentatomidae) is an insect pest native to Asia that has spread over the last two decades to most of the North America, parts of South America, Europe and North Africa. Its impact is significant as it can feed on more than 300 host plants, rendering affected fruits and vegetable crops unsellable or of lower quality. Various chemical and biological methods have been used to control this pest, with varying degrees of success. The sterile insect technique (SIT) is a pest control method involving the sterilization of insects via ionizing radiation and their subsequent mass release into the field. In the present contribution, the spermiogenesis of H. halys was studied from an ultrastructural point of view in both irradiated and non-irradiated adult males. In both cases, we observed ultrastructural characteristics typical of hemipteran sperm cells: bridges connecting the mitochondrial derivatives and the axonemal microtubules, the absence of accessory bodies, and the presence of two or three crystalline inclusions within the mitochondrial derivatives, an acrosome composed of tightly packed tubules, and an atypical, plaque-shaped microtubular organizing center (MTOC) in the centriolar region. Moreover, in the same region, we seldom observed the presence of two centrioles in the spermatids, one of which disappeared at a later stage of maturation. This feature is a novelty for insect spermiogenesis. The cysts of irradiated adults were not all uniformly affected by the radiation. However, irradiated cysts sometimes exhibited a general disorganization of sperm arrangement, incomplete divisions of sperm cells resulting in multiple copies of the same organelle within the same cell, failure to reabsorb the cytoplasm, and the lack of axonemes. Finally, rod-shaped viruses or virus-like particles were observed in vasa deferentia independently of irradiation.

Whole genome sequencing identifies a homozygous splicing variant in TDRKH segregating with non-obstructive azoospermia in an Iranian family.

Non-obstructive azoospermia (NOA) resulting from primary spermatogenic failure represents one of the most severe forms of male infertility, largely because therapeutic options are very limited. Beyond their diagnostic value, genetic tests for NOA also hold prognostic potential. Specifically, genetic diagnosis enables the establishment of genotype-testicular phenotype correlations, which, in some cases, provide a negative predictive value for testicular sperm extraction (TESE), thereby preventing unnecessary surgical procedures. In this study, we employed whole-genome sequencing (WGS) to investigate two generations of an Iranian family with NOA and identified a homozygous splicing variant in TDRKH (NM_001083965.2: c.562-2A>T). TDRKH encodes a conserved mitochondrial membrane-anchored factor essential for piRNA biogenesis in germ cells. In Tdrkh knockout mice, de-repression of retrotransposons in germ cells leads to spermatogenic arrest and male infertility. Previously, our team reported TDRKH involvement in human NOA cases through the investigation of a North African cohort. This current study marks the second report of TDRKH's role in NOA and human male infertility, underscoring the significance of the piRNA pathway in spermatogenesis. Furthermore, across both studies, we demonstrated that men carrying TDRKH variants, similar to knockout mice, exhibit complete spermatogenic arrest, correlating with failed testicular sperm retrieval.

[The Roles of N6-Methyladenosine Modification and Its Regulators in Male Reproduction]. / N6-ç”²åŸºè ºå˜Œå‘¤ä¿®é¥°åŠå ¶è°ƒæŽ§å› å­åœ¨ç”·æ€§ç”Ÿæ®–ä¸­çš„ä½œç”¨.

Infertility affects an estimated 10 to 15 percent of couples worldwide, with approximately half of the cases attributed to male-related issues. Most men diagnosed with infertility exhibit symptoms such as oligospermia, asthenospermia, azoospermia, and compromised sperm quality. Spermatogenesis is a complex and tightly coordinated process of germ cell differentiation, precisely regulated at transcriptional, posttranscriptional, and translational levels to ensure stage-specific gene expression during the development of spermatogenic cells and normal spermiogenesis. N6-methyladenosine (m6A) stands out as the most prevalent modification on eukaryotic mRNA, playing pivotal roles in various biological processes, including mRNA splicing, transportation, and translation. RNA methylation modification is a dynamic and reversible process primarily mediated by "writers", removed by "erasers", and recognized by "readers". In mammals, the aberrant methylation modification of m6A on mRNA is associated with a variety of diseases, including male infertility. However, the precise involvement of disrupted m6A modification in the pathogenesis of human male infertility remains unresolved. Intriguingly, a significant correlation has been found between the expression levels of m6A regulators in the testis and the severity of sperm concentration, motility, and morphology. Aberrant expression patterns of m6A regulatory proteins have been detected in anomalous human semen samples, including those of oligospermia, asthenozoospermia, and azoospermia. Furthermore, the examination of both sperm samples and testicular tissues revealed abnormal mRNA m6A modification, leading to reduced sperm motility and concentration in infertile men. Consequently, it is hypothesized that dysregulation of m6A modification might serve as an integral link in the mechanism of male infertility. This paper presents a comprehensive review of the recent discoveries regarding the spatial and temporal expression dynamics of m6A regulators in testicular tissues and the correlation between deregulated m6A regulators and human male infertility. Previous studies predominantly utilized constitutive or conditional knockout animal models for testicular phenotypic investigations. However, gene suppression in additional tissues could potentially influence the testis in constitutive knockout models. Furthermore, considering the compromised spermatogenesis observed in constitutive animals, distinguishing between the indirect effects of gene depletion on testicular development and its direct impact on the spermatogenic process is challenging, due to their intricate relationship. Such confounding factors might compromise the validity of the findings. To address this challenge, an inducible and conditional gene knockout model may serve as a superior approach. To date, nearly all reported studies have concentrated solely on the level changes of m6A and its regulators in germs cells, while the understanding of the function of m6A modification in testicular somatic cells remains limited. Testicular somatic cells, including peritubular myoid cells, Sertoli cells, and Leydig cells, play indispensable roles during spermatogenesis. Hence, comprehensive exploration of m6A modification within these cells as an additional crucial regulatory mechanism is warranted. In addition, exploration into the presence of unique methylation mechanisms or m6A regulatory factors within the testes is warranted. To elucidate the role of m6A modification in germ cells and testicular somatic cells, detailed experimental strategies need to be implemented. Among them, manipulation of the levels of key enzymes involved in m6A methylation and demethylation might be the most effective approach. Moreover, comprehensive analysis of the gene expression profiles involved in various signaling pathways, such as Wnt/ß-catenin, Ras/MAPK, and Hippo, in m6A-modified germ cells and testicular somatic cells can provide more insight into its regulatory role in the spermatogenesis process. Further research in this area could provide valuable insights for developing innovative strategies to treat male infertility. Finally, considering the mitigation impact of m6A imbalance regulation on disease, investigation concerning whether restoring the equilibrium of m6A modification regulation can restore normal spermatogenesis function is essential, potentially elucidating the pivotal clinical significance of m6A modulation in male infertility.

Motor proteins, spermatogenesis and testis function.

The role of motor proteins in supporting intracellular transports of vesicles and organelles in mammalian cells has been known for decades. On the other hand, the function of motor proteins that support spermatogenesis is also well established since the deletion of motor protein genes leads to subfertility and/or infertility. Furthermore, mutations and genetic variations of motor protein genes affect fertility in men, but also a wide range of developmental defects in humans including multiple organs besides the testis. In this review, we seek to provide a summary of microtubule and actin-dependent motor proteins based on earlier and recent findings in the field. Since these two cytoskeletons are polarized structures, different motor proteins are being used to transport cargoes to different ends of these cytoskeletons. However, their involvement in germ cell transport across the blood-testis barrier (BTB) and the epithelium of the seminiferous tubules remains relatively unknown. It is based on recent findings in the field, we have provided a hypothetical model by which motor proteins are being used to support germ cell transport across the BTB and the seminiferous epithelium during the epithelial cycle of spermatogenesis. In our discussion, we have highlighted the areas of research that deserve attention to bridge the gap of research in relating the function of motor proteins to spermatogenesis.

The proliferation and differentiation of spermatogonial stem cells in the frist wave of spermatogenesis in rats with Trib3 gene knockout.

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.

Triptolide induced spermatogenesis dysfunction via ferroptosis activation by promoting K63-linked GPX4 polyubiquitination in spermatocytes.

Triptolide (TP) is a major bioactive compound derived from Tripterygium wilfordii Hook. F. (TwHF) known for its medicinal properties, but it also exhibits potential toxic effects. It has been demonstrated to induce severe male reproductive toxicity, yet the precise mechanism behind this remains unclear, which limits its broad clinical application. This study aimed to investigate the mechanisms underlying testicular damage and spermatogenesis dysfunction induced by TP in mice, using both mouse models and the spermatocyte-derived cell line GC-2spd. In the present study, it was found that TP displayed significant testicular microstructure damaged and spermatogenesis defects including lower concentration and abnormal morphology by promoting ROS formation, MDA production and restraining GSH level, glutathione peroxidase 4 (GPX4) expression in vivo. Furthermore, Ferrostatin-1 (FER-1), a ferroptosis inhibitor, was found to significantly reduce the accumulation of lipid peroxidation, alleviate testicular microstructural damage, and enhance spermatogenic function in mice. Besides, notably decreased cell viability, collapsed mitochondrial membrane potential, and elevated DNA damage were observed in vitro. The above-mentioned phenomenon could be reversed by pre-treatment of FER-1, indicating that ferroptosis participated in the TP-mediated spermatogenesis dysfunction. Mechanistically, TP could enhance GPX4 ubiquitin degradation via triggering K63-linked polyubiquitination of GPX4, thereby stimulating ferroptosis in spermatocytes. Functionally, GPX4 deletion intensified ferroptosis and exacerbated DNA damage in GC-2 cells, while GPX4 overexpression mitigated ferroptosis induced by TP. Overall, these findings for the first time indicated a vital role of ferroptosis in TP induced-testicular injury and spermatogenic dysfunction through promoting GPX4 K63-linked polyubiquitination, which hopefully offers a potential therapeutic avenue for TP-related male reproductive damage. In addition, this study also provides a theoretical foundation for the improved clinical application of TP or TwHF in the future.

Effects of chronic CuNPs treatment followed by termination for two spermatogenic cycles in the testicular functions of mice.

The present study investigated the possible effects of copper nanoparticles (CuNPs) after discontinuing treatment on testicular activity in a mouse model. The male mice were given continuous CuNPs treatment for 70 days and left untreated for 70 days. The results show that even after the discontinuation of CuNPs treatment, the testicular impairment was persistent till 140 days at a higher dose (200 mg/kg group). The spermatogenesis, sperm parameters, proliferation and antioxidant status were suppressed in the higher dose groups. However, these effects were also observed at moderate levels in the other CuNPs treated groups, such as at 10 mg/kg and 100 mg/kg. The apoptosis was stimulated at a higher dose compared to the other groups. The testosterone, LH levels and AR expression were suppressed in all the CuNPs treated groups, along with slight elevation in the estrogen levels and up-regulated ERß expression. The fertility data also showed a decline in all CuNPs treated groups with the lowest litter size in the 200 mg/kg treated group. Despite testis, epididymis and accessory sex organs like prostate, seminal vesicle, and vas deferens, histoarchitecture also showed impairment. This is the first report on how CuNPs affect the male reproductive system in mice even after treatment was terminated. The current study also demonstrated possible negative effects on male reproductive function that might last for longer at higher dosages of chronic CuNPs exposure even after termination.

ERK/CREB and p38 MAPK/MMP14 Signaling Pathway Influences Spermatogenesis through Regulating the Expression of Junctional Proteins in Eriocheir sinensis Testis.

The hemolymph-testis barrier (HTB) is a reproduction barrier in Crustacea, guaranteeing the safe and smooth process of spermatogenesis, which is similar to the blood-testis barrier (BTB) in mammals. The MAPK signaling pathway plays an essential role in spermatogenesis and maintenance of the BTB. However, only a few studies have focused on the influence of MAPK on crustacean reproduction. In the present study, we knocked down and inhibited MAPK in Eriocheir sinensis. Increased defects in spermatogenesis were observed, concurrently with a damaged HTB. Further research revealed that es-MMP14 functions downstream of ERK and p38 MAPK and degrades junctional proteins (Pinin and ZO-1); es-CREB functions in the ERK cascade as a transcription factor of ZO-1. In addition, when es-MMP14 and es-CREB were deleted, the defects in HTB and spermatogenesis aligned with abnormalities in the MAPK. However, JNK impacts the integrity of the HTB by changing the distribution of intercellular junctions. In summary, the MAPK signaling pathway maintains HTB integrity and spermatogenesis through es-MMP14 and es-CREB, which provides insights into the evolution of gene function during barrier evolution.

Differential Impairment Mechanism of Sperm Production via Induction of miR-34c-Activated Apoptosis and Spermatogenesis Pathway in Diet-Induced Obesity and Resistant Mice and GC-1 Spg Cells.

Male reproductive dysfunction is a clinical disease, with a large number of cases being idiopathic. Reproductive disorders have been found in obese (diet-induced obesity and diet-induced obesity-resistant) mice, but the mechanism behind the male reproductive dysfunction between them may be different. The purpose of this study was to explore the possible role and mechanism of miR-34c on sperm production in high-fat-diet-induced obesity-resistant (DIO-R) mice and GC-1 spg cells, which may differ from those in high-fat-diet-induced obesity (DIO) mice. In vivo and in vitro experiments were performed. C57BL/6J mice were fed a high-fat diet for 10 weeks to establish the DIO and DIO-R mouse model. GC-1 spg cells were used to verify the mechanism of miR-34c on sperm production. During in vivo experiments, sperm production damage was found in both DIO and DIO-R male mice. Compared to the control mice, significantly decreased levels of testosterone, LH, activities of acrosome enzyme (ACE), HAse, and activating transcription factor 1 (ATF1) were found in both DIO and DIO-R male mice (p < 0.05). Compared with the control group, the ratio of B-cell lymphoma-2 (Bcl-2)/bcl-2-associated X protein (Bax) in the DIO group was significantly decreased, and the expression level of cleaved caspase-3 was significantly increased (p < 0.05). Compared with the control group, the Bcl-2 protein expression level in the testes of the DIO-R group significantly decreased (p < 0.05). However, the Bax expression level increased. Thus, the Bcl-2/Bax ratio significantly decreased (p < 0.01); however, the factor-related apoptosis (Fas), Fas ligand (FasLG), cleaved caspase-8, caspase-8, cleaved caspase-3, and caspase-3 protein expression levels significantly increased (p < 0.05). Compared with the DIO group, in DIO-R mice, the activities of ACE, ATF1, Bcl-2, and Bcl-2/Bax's spermatogenesis protein expression decreased, while the apoptosis-promoting protein expression significantly increased (p < 0.05). During the in vitro experiment, the late and early apoptotic ratio in the miR-34c over-expression group increased. MiR-34c over-expression enhanced the expression of apoptosis-related proteins Fas/FasLG and Bax/Bcl-2 while inhibiting the expression of ATF1 and the sperm-associated protein in GC-1 spg cells. DIO and DIO-R could harm sperm production. DIO-R could impair sperm production by inducing the miR-34c-activated apoptosis and spermatogenesis pathway, which may be different from that of DIO.

Deficiency of ValRS-m Causes Male Infertility in Drosophila melanogaster.

Drosophila spermatogenesis involves the renewal of germline stem cells, meiosis of spermatocytes, and morphological transformation of spermatids into mature sperm. We previously demonstrated that Ocnus (ocn) plays an essential role in spermatogenesis. The ValRS-m (Valyl-tRNA synthetase, mitochondrial) gene was down-regulated in ocn RNAi testes. Here, we found that ValRS-m-knockdown induced complete sterility in male flies. The depletion of ValRS-m blocked mitochondrial behavior and ATP synthesis, thus inhibiting the transition from spermatogonia to spermatocytes, and eventually, inducing the accumulation of spermatogonia during spermatogenesis. To understand the intrinsic reason for this, we further conducted transcriptome-sequencing analysis for control and ValRS-m-knockdown testes. The differentially expressed genes (DEGs) between these two groups were selected with a fold change of ≥2 or ≤1/2. Compared with the control group, 4725 genes were down-regulated (dDEGs) and 2985 genes were up-regulated (uDEGs) in the ValRS-m RNAi group. The dDEGs were mainly concentrated in the glycolytic pathway and pyruvate metabolic pathway, and the uDEGs were primarily related to ribosomal biogenesis. A total of 28 DEGs associated with mitochondria and 6 meiosis-related genes were verified to be suppressed when ValRS-m was deficient. Overall, these results suggest that ValRS-m plays a wide and vital role in mitochondrial behavior and spermatogonia differentiation in Drosophila.

Dietary supplementation with ginseng extract enhances testicular function, semen preservation, and fertility rate of mature and aging Thai native roosters.

The decrease in fertility in aging roosters is related to the reduced quality of ejaculated sperm. This study aimed to investigate the effect of dietary supplementation with ginseng extract at various concentrations (0-150 mg/kg) on testicular function, semen preservation, and fertility at different stages of sexual maturity (mature and aging roosters) in Thai native roosters. Pradu Hang Dum roosters at 32 (mature; n = 24) and 75 (aging; n = 24) weeks of age were fed diets with non-supplemented or supplemented ginseng extracts (50, 100, and 150 mg/kg) until the end of the experiment. In experiment 1, fresh semen samples were examined for the quality parameters of semen volume, sperm concentration, sperm motility, sperm viability, lipid peroxidation, and enzymatic activities. In experiment 2, semen was preserved at 5 °C for up to 48 h, and the semen quality and fertility potential were determined. In experiment 3, testicular function and testosterone concentrations were evaluated. The results showed that ginseng extract supplementation in the diets of both mature and aging roosters at 50 and 100 mg/kg improved fresh semen quality (P < 0.05). A decrease in malondialdehyde levels in fresh semen was observed with increasing enzyme activities. In mature roosters, the progressive motility of cold-stored semen and fertility rates were higher in the G50 and G100 groups compared to the control and G150 groups after 24 h of storage (P < 0.05). In aging roosters, the highest significant differences in progressive motility, viability, and fertility rates were observed in the G50 and G100 groups at all storage times (P < 0.01). These improvements might be attributed to good testicular function in spermatogenesis, as revealed by the results of histological examination and testosterone concentrations. However, higher doses of ginseng extract supplementation negatively affected sperm quality. In summary, the recommended dose of ginseng extract supplementation in diets is 50 mg/kg. Fertility results indicated that insemination with semen preserved for 24 h was satisfactory in both mature and aging roosters.

Dietary restriction promote sperm remodeling in aged roosters based on transcriptome analysis.

BACKGROUND: The breeder rooster has played a pivotal role in poultry production by providing high-quality semen. Typically, fertility peaks between 30 and 40 weeks of age and then declines rapidly from 45 to 55 weeks of age. Research into improving fertility in aging roosters is essential to extend their productive life. While progress has been made, enhancing fertility in aging roosters remains a significant challenge. METHODS: To identify the genes related to promoting sperm remodeling in aged Houdan roosters, we combined changes in testis and semen quality with transcriptome sequencing (RNA-seq) to analyze the synchrony of semen quality and testis development. In this study, 350-day-old Houdan breeder roosters were selected for RNA-seq analysis in testis tissues from induced molting roosters (D group) and non-induced molting roosters (47DG group). All analyses of differentially expressed genes (DEGs) and functional enrichment were performed. Finally, we selected six DEGs to verify the accuracy of the sequencing by qPCR. RESULTS: Compared with the 47DG group, sperm motility (P < 0.05), sperm density (P < 0.01), and testis weight (P < 0.05) were significantly increased in roosters in the D group. Further RNA-seq analysis of the testis between the D group and 47DG group identified 61 DEGs, with 21 up-regulated and 40 down-regulated. Functional enrichment analysis showed that the DEGs were primarily enriched in the cytokine-cytokine receptor interaction, Wnt signaling pathway, MAPK signaling pathway, TGF-ß signaling pathway, and focal adhesion pathway. The qRT-PCR results showed that the expression trend of these genes was consistent with the sequencing results. WNT5A, FGFR3, AGTR2, TGFß2, ROMO1, and SLC26A7 may play a role in testis development and spermatogenesis. This study provides fundamental data to enhance the reproductive value of aging roosters.

The role of immune cell death in spermatogenesis and male fertility.

The male reproductive system provides a distinctive shield to the immune system, safeguarding germ cells (GCs) from autoimmune harm. The testis in mammals creates a unique immunological setting due to its exceptional immune privilege and potent local innate immunity. which can result from a number of different circumstances, including disorders of the pituitary gland, GC aplasia, and immunological elements. Apoptosis, or programmed cell death (PCD), is essential for mammalian spermatogenesis to maintain and ensure an appropriate number of GCs that correspond with the supporting capability of the Sertoli cells. Apoptosis is substantial in controlling the number of GCs in the testis throughout spermatogenesis, and any dysregulation of this process has been linked to male infertility. There is a number of evidence about the potential of PCD in designing novel therapeutic approaches in the treatment of infertility. A detailed understanding of PCD and the processes that underlie immunological infertility can contribute to the progress in designing strategies to prevent and treat male infertility. This review will provide a summary of the role of immune cell death in male reproduction and infertility and describe the therapeutic strategies and agents for treatment based on immune cell death.

Spermatogenesis in mouse testicular organoids with testis-specific architecture, improved germ cell survival and testosterone production.

This study presents a biphasic approach to overcome the limitations of current testicular organoid (TO) cultures, including histological heterogeneity, germ cell loss and absence of spermatogenesis. Agarose microwells were utilized to create TOs from prepubertal C57BL/6J testicular cells. First emphasis was on improving germ cell survival during the initial 2-week reorganization phase by comparing α-MEM + 10% KSR medium, known to support TO generation in mice, to three optimized media (1-3). Cell densities and culture dynamics were also tested to recreate histological resemblance to testes. After optimizing germ cell survival and cell organization, the effect of growth factors and immunomodulation through CD45+ immune cell depletion or dexamethasone (DEX) supplementation were assessed for enhancing spermatogenesis during the subsequent differentiation phase. Testicular cells self-reorganized into organoids resembling the testicular anatomical unit, characterized by one tubule-like structure surrounded by interstitium. Media 1 3 proved superior for organoid growth during the reorganization phase, with TOs in medium 3 exhibiting germ cell numbers (7.4 ± 4.8%) comparable to controls (9.3 ± 5.3%). Additionally, 37 ± 30% demonstrated organized histology from 32 × 103 cells under static conditions. Switching to α-MEM + 10% KSR during the differentiation phase increased formation efficiency to 85 ± 7%, along with elevated germ cell numbers, testosterone production (3.1 ± 0.9 ng/mL) and generation of γH2AX+ spermatid-like cells (steps 8-11, 1.2 ± 2.2% of the total). Adding differentiation factors to the α-MEM increased spermatid-like cell numbers to 2.9 ± 5.9%, confirmed through positive staining for CREM, TP1, and PNA. Although, these remained diploid with irregular nuclear maturation. DEX supplementation had no additional effect, and immune cell depletion adversely impacted TO formation. The manipulability of TOs offers advantages in studying male infertility and exploring therapies, with scalability enabling high-throughput chemical screening and reducing animal usage in reproductive toxicity and drug discovery studies.