Application of testicular organ culture system for the evaluation of spermatogenesis impairment.

Recently, it was reported that a testicular organ culture system (TOCS) using polydimethylsiloxane (PDMS) chips with excellent oxygen permeability and biocompatibility, called the PDMS-chip ceiling (PC) method, enables improved spermatogenesis efficiency. We investigated whether this PC method is useful for detecting impaired spermatogenesis caused by busulfan (Bu), a typical testicular toxicant. In this study, testicular tissue fragments from Acro3-EGFP mice, which express the green fluorescent protein (GFP) and reflect the progression of spermatogenesis, were subjected to the PC method. When treated with Bu, cultured tissues shrank in volume, and their GFP-expressing area decreased or disappeared. Histological examination confirmed the regression of spermatogenesis. In addition, immunohistochemical examination revealed that spermatogonia, including spermatogonial stem cells (SSCs), were the primary targets of Bu toxicity. Time-course analysis demonstrated that the recovery of spermatogenesis, dependent on Bu concentration, correlated closely with the severity of damage to these target cells. These results suggest that the PC method is a useful approach for detecting spermatogenesis impairment accurately through faithful recapitulation of spermatogenesis in vivo.

Organophosphate pesticide chlorpyrifos and its metabolite 3,5,6-trichloropyridinol downregulate the expression of genes essential for spermatogenesis in caprine testes.

Organophosphate pesticides have potent endocrine disrupting effects, hence banned in many countries. However, many organophosphates like chlorpyrifos, malathion et cetera continue to be used in some countries (Wolejko et al., 2022; Wolejko et al., 2022)including India. Fodder mediated ingestion of these substances may be harmful for livestock fertility. We have investigated the effect of the widely used organophosphate pesticide chlorpyrifos (CPF) and its metabolite, 3,5,6-trichloropyridinol (TCPy) on the expression of genes essential for spermatogenesis in goat testicular tissue. The testicular Sertoli cells (Sc) regulate germ cell division and differentiation under the influence of follicle stimulating hormone (FSH) and testosterone (T). Impaired FSH and T mediated signalling in Sc can compromise spermatogenesis leading to sub-fertility/infertility. As Sc express receptors (R) for FSH and T, they are highly susceptible to the endocrine disrupting effects of pesticides affecting fertility by dysregulating the functioning of Sc. Our results indicated that exposure to different concentrations of CPF and TCPy can compromise Sc function by downregulating the expression of FSHR and AR which was associated with a concomitant decline in the expression of genes essential for germ cell division and differentiation, like KITLG, INHBB, CLDN11 and GJA1. CPF also induced a significant reduction in the activity of acetylcholinesterase in the testes and increased the total testicular antioxidant capacity. Our results suggested that CPF and its metabolite TCPy may induce reproductive toxicity by dysregulating the expression of Sc specific genes essential for spermatogenesis.

[Development of a Novel Male Reproductive Toxicity Assessment Method to Predict Male-mediated Effects on the Next Generation].

Less than 10% of the candidate drug compounds are associated with male reproductive toxicity. Genetic and/or epigenetic information on sperm may be crucial for fetal development. Therefore, developmental toxicity, such as paternally transmitted birth defects, is possible if genetic abnormalities in the male germ line persist and accumulate in the sperm during spermatogenesis. First, this study provides an overview of chemical and male reproductive toxicity, which may lead to developmental toxicity from the perspective of male reproduction. Second, we demonstrate methods for evaluating male reproductive toxicity to anticipate male-mediated developmental toxicity. We developed a novel staining technique for evaluating sperm quality, as well as a noninvasive imaging analysis of male reproductive toxicity. The former is a mammalian male germ cell-specific staining method using reactive blue 2 dye (RB2), as previously confirmed in human sperm, and a method for detecting the early-stage DNA fragmentation in a single nucleus from mouse spermatozoa using single-cell pulsed-field gel electrophoresis. The latter is a new, ready-to-use, and compact magnetic resonance imaging (MRI) platform utilizing a high-field permanent magnet to evaluate male reproductive toxicity. The histopathological analysis supported the suitability of the MRI platform. The present study, for the first time, revealed a rapid, noninvasive evaluation of male reproductive toxicity in vivo using compact MRI. These novel toxicity assessments can help predict male-mediated developmental toxicity, contributing to accelerated drug discovery and drug repositioning.

[Intervention of traditional Chinese medicine in spermatogenic epithelium injury by regulating the ferroptosis pathway: Advances in studies].

The incidence of male infertility has been increasing year by year, and one of the major causes is testicular spermatogenic epithelial injury, which affects the spermatogenic function of the testis. Ferroptosis is a novel mode of cell death and plays an important role in testicular cell injury. Some traditional Chinese medicines can intervene in the progression of testicular injury by regulating the ferroptosis pathway in testicular spermatogenic epithelia. This paper focuses on the effect of traditional Chinese drugs in regulating the ferroptosis pathway in testicular cells, and summarizes the advances in the studies of traditional Chinese medicines in the treatment of testicular spermatogenic epithelial injury, aiming to provide a theoretical basis for the selection of relevant medicines and their clinical application.

Icariin alleviates the injury of Sertoli cell junction function by upregulating PKR pathway via ERα/c-fos signaling in aged mice.

ETHNOPHARMACOLOGICAL RELEVENACE: Sertoli cells are vital to maintain spermatogenesis and their function decline during aging. Epimedium has the effects of tonifying kidney-yang, strengthening bones and muscles, and expelling wind and dampness, and is commonly used in the treatment of kidney-yang deficiency, impotence and spermatorrhea. Icariin is the main active ingredients from Epimedium exhibiting delaying aging effects and improving male reproductive dysfunction. Whereas, it remains poorly understood how icariin alleviates age-associated decline in testicular function by protecting against the damage of junction function of Sertoli cells. AIM OF THE STUDY: This study aimed to evaluate the improvement effect of icariin on Sertoli cell junction function damage and explore the underlying mechanisms. MATERIALS AND METHODS: Male C57BL/6 mice and mouse Sertoli cell line TM4 cells were utilized to assess the improvement effect of icariin on aging-associated Sertoli cell junction function injury. H&E staining, transmission electron microscopy, qPCR, Western blot, molecular docking, siRNA transfection, and immunofluorescence were performed in this study. RESULTS: Dietary administration of icariin remarkly attenuated age-associated deterioration in spermatogenic function as evidenced by elevated testicular weight and index, sperm concentration and sperm viability. In addition, icariin protected Sertoli cell junction function from age-associated damage as proven by increased Sertoli cell numbers, improved tight junction ultrastructure, and upregulated junction-related proteins (ZO-1, Occludin and ß-Catenin). Moreover, icariin significantly upregulated ERα/c-fos signaling and PKR pathway in testicular Sertoli cells. Similarly, in vitro studies revealed that deletion of ERα, c-fos or PKR abolished the improvement effects of icariin on Sertoli cell junction function damage. CONCLUSIONS: Icariin effectively mitigates age-associated decline in testicular function by diminished Sertoli cell junction function damage through upregulating PKR pathway via ERα/c-fos signaling. Therefore, attenuating Sertoli cell junction function injury by the upregulation of PKR pathway via ERα/c-fos signaling probably indicates an effective target for the prevention and treatment of testicular spermatogenic function with aging.

The protective effect of glucose selenol on cadmium-induced testicular toxicity in male rat.

This study aimed to investigate the protective effects of glucose selenol on cadmium (Cd)-induced testicular toxicity. Twenty-four male Sprague-Dawley (SD) rats were randomly divided into four groups. Cd was administered orally at a dose of 40 mg/L or in combination with orally administered glucose selenol at doses of 0.15 mg/L and 0.4 mg/L for 30 days. The results showed that sperm quality decreased and testicular tissue was damaged in the Cd group; Glucose selenol significantly attenuated the negative effects by improving sperm quality and reducing testicular damage. Transcriptome sequencing analysis showed that Cd stress affected spermatogenesis, sperm motility, oxidative stress, blood-testis barrier and protein metabolism. Four clusters were obtained using the R Mfuzz package, which clustered highly expressed genes under different administrations, and 36 items were enriched. Notably, protein phosphorylation was enriched in the Cd group and is considered to play a key role in the response to Cd stress. We identified fifty-six target selenium (Se) and Cd co-conversion differentially expressed genes (DEGs), including three genes relating to spermatogenesis (Dnah8, Spata31d1b, Spata31d1c). In addition, the obtained DEGs were used to construct a protein-protein interaction network, co-processed with Se and Cd, and 5 modules were constructed. Overall, the analyses of rat testicular physiology and gene expression levels offer new insights into the reproductive toxicity of Cd in rats, and provide potential application prospects for glucose selenol in alleviating the impact of Cd-induced testicular damage.

Efficacy of propolis and royal jelly in attenuating cadmium-induced spermatogenesis and steroidogenesis dysregulation, causing infertility in rats.

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

Multiple omics integration analysis reveals the regulatory effect of chitosan oligosaccharide on testicular development.

Infertility is a global health problem affecting millions of people of reproductive age worldwide, with approximately half caused by males. Chitosan oligosaccharide (COS) has strong antioxidant capacity, but its impact on the male reproductive system has not been effectively evaluated. To address this, we integrated RNA-seq, serum metabolomics and intestinal 16 S rDNA analysis to conduct a comprehensive investigation on the male reproductive system. The results showed that COS has potential targets for the treatment of oligospermia, which can promote the expression of meiotic proteins DDX4, DAZL and SYCP1, benefit germ cell proliferation and testicular development, enhance antioxidant capacity, and increase the expression of testicular steroid proteins STAR and CYP11A1. At the same time, COS can activate PI3K-Akt signaling pathway in testis and TM3 cells. Microbiome and metabolomics analysis suggested that COS alters gut microbial community composition and cooperates with serum metabolites to regulate spermatogenesis. Therefore, COS promotes male reproduction by regulating intestinal microorganisms and serum metabolism, activating PI3K-Akt signaling pathway, improving testicular antioxidant capacity and steroid regulation.

Exosomes-mediated retinoic acid disruption: A link between gut microbiota depletion and impaired spermatogenesis.

Gut microbiota symbiosis faces enormous challenge with increasing exposure to drugs such as environmental poisons and antibiotics. The gut microbiota is an important component of the host microbiota and has been proven to be involved in regulating spermatogenesis, but the molecular mechanism is still unclear. A male mouse model with gut microbiota depletion/dysbiosis was constructed by adding combined antibiotics to free drinking water, and reproductive parameters such as epididymal sperm count, testicular weight and paraffin sections were measured. Testicular transcriptomic and serum metabolomic analyses were performed to reveal the molecular mechanism of reproductive dysfunction induced by gut microbiota dysbiosis in male mice.This study confirms that antibiotic induced depletion of gut microbiota reduces sperm count in the epididymis and reduces germ cells in the seminiferous tubules in male mice. Further study showed that exosomes isolated from microbiota-depleted mice led to abnormally high levels of retinoic acid and decrease in the number of germ cells in the seminiferous tubules and sperm in the epididymis. Finally, abnormally high levels of retinoic acid was confirmed to disrupted meiotic processes, resulting in spermatogenesis disorders. This study proposed the concept of the gut microbiota-exosome-retinoic acid-testicular axis and demonstrated that depletion of the gut microbiota caused changes in the function of exosomes, which led to abnormal retinoic acid metabolism in the testis, thereby impairing meiosis and spermatogenesis processes.

Protective effect of Cordycepin on blood-testis barrier against pre-puberty polystyrene nanoplastics exposure in male rats.

Plastic pollution is an emerging environmental issue, with microplastics and nanoplastics raising health concerns due to bioaccumulation. This work explored the impact of polystyrene nanoparticle (PS-NPs) exposure during prepuberty on male reproductive function post maturation in rats. Rats were gavaged with PS-NPs (80 nm) at 0, 3, 6, 12 mg/kg/day from postnatal day 21 to 95. PS-NPs accumulated in the testes and reduced sperm quality, serum reproductive hormones, and testicular coefficients. HE staining showed impaired spermatogenesis. PS-NPs disrupted the blood-testis barrier (BTB) by decreasing junction proteins, inducing inflammation and apoptosis. Transcriptomics identified differentially expressed genes related to metabolism, lysosome, apoptosis, and TLR4 signaling. Molecular docking revealed Cordycepin could compete with polystyrene for binding to TLR4. Cordycepin alleviated oxidative stress and improved barrier function in PS-NPs treated Sertoli cells. In conclusion, prepubertal PS-NPs exposure induces long-term reproductive toxicity in male rats, likely by disrupting spermatogenesis through oxidative stress and BTB damage. Cordycepin could potentially antagonize this effect by targeting TLR4 and warrants further study as a protective agent. This study elucidates the mechanisms underlying reproductive toxicity of PS-NPs and explores therapeutic strategies.

Activated dormant stem cells recover spermatogenesis in chemoradiotherapy-induced infertility.

Male infertility is a recognized side effect of chemoradiotherapy. Extant spermatogonial stem cells (SSCs) may act as originators for any subsequent recovery. However, which type of SSCs, the mechanism by which they survive and resist toxicity, and how they act to restart spermatogenesis remain largely unknown. Here, we identify a small population of Set domain-containing protein 4 (Setd4)-expressing SSCs that occur in a relatively dormant state in the mouse seminiferous tubule. Extant beyond high-dose chemoradiotherapy, these cells then activate to recover spermatogenesis. Recovery fails when Setd4+ SSCs are deleted. Confirmed to be of fetal origin, these Setd4+ SSCs are shown to facilitate early testicular development and also contribute to steady-state spermatogenesis in adulthood. Upon activation, chromatin remodeling increases their genome-wide accessibility, enabling Notch1 and Aurora activation with corresponding silencing of p21 and p53. Here, Setd4+ SSCs are presented as the originators of both testicular development and spermatogenesis recovery in chemoradiotherapy-induced infertility.

Use of a slow-release GnRH implant in an adult billy goat.

A 9.4 mg deslorelin slow-release implant was inserted into an adult, healthy billy goat to achieve temporary infertility and a reduction in sexual behavior. The implant was inserted in late autumn. No significant change in testis size was observed over the following 6 weeks. The endocrine function of the testis, which was examined by stimulation with human chorionic gonadotropin, was also unchanged after 6 weeks compared to the initial examination. Histological examination revealed a preserved spermatogenesis.In conclusion, the application of a GnRH analogue implant in the adult male goat has no influence on the investigated parameters - and thus probably also on its fertility.

[Improving effect of selenium on spermatogenesis in mice with cyclophosphamide-induced spermatogenic impairment and its underlying mechanism].

OBJECTIVE: To investigate the effect of selenium on cyclophosphamide (CTX)-induced spermatogenic impairment (SI) in mice and its underlying mechanism. METHODS: We equally randomized 36 male KM mice into 3 SI model and 3 control groups, the first 3 treated by intraperitoneal injection of CTX at 100 mg/kg (the SI model control group), CTX plus SI model control group, selenium deficient model group (－Se SI), selenium supplemented model group (+Se SI), while latter 3 by intraperitoneal injection of normal saline (the normal control), selenium deficiency control group (－Se control), selenium addition control group (+Se control), respectively, all once a week for 6 successive weeks. Then we observed the histopathological changes in the testes of all the mice by HE staining, obtained the sperm count in the epididymides, determined the expressions of glutathione peroxidase 4 (GPx4) and SLC7A11 proteins by Western blot and ferroptosis-related genes by RT-qPCR, and examined the changes in the expressions of ferroptosis-related proteins and genes in the GC2-spd cells treated with ferroptosis inhibitors and inducers in combination with different concentrations of inorganic sodium selenite (SeS) and organic selenomethionine (SeM). RESULTS: Compared with the normal controls, the SI model mice showed significantly decreased testicular and prostatic organ coefficients, reduced spermatogenic layers, increased voids, decreased serum ferritin concentration (P<0.05), and elevated transferrin concentration (P<0.05). The organ coefficients were significantly higher in the +Se SI and +Se control than in the －Se SI and －Se control groups (P<0.05, P<0.01), with evident pathological improvement of the testis tissue in the +Se controls. The expressions of the GPx4 and solute carrier family 7 members 11（SLC7A11） genes in the testis were dramatically down-regulated in the SI model controls (P<0.01), but up-regulated in the +Se SI and +Se control compared with those in the －Se SI and －Se control group (P<0.01 and P<0.05), but there were no statistically significant differences between their protein expressions. The results of in vitro GC2 spd cell experiments indicated that the GPx4 gene and GPx4 protein levels in the - Se group were significantly lower than those in the normal control group (P<0.05), while the SLC7A11 gene level decreased (P<0.01). Different doses of SeS and SeM significantly increased the GPx4 protein expression compared to the average Se group. Low doses of SeM promoted a significant increase in GPx4 gene levels, while high doses of SeS increased the expression levels of SLC7A11 gene and SLC7A11 protein (P<0.05, P<0.01). The Se group showed a significant decrease in the levels of acsl4 and ptgs2 genes compared to the normal control group. SeM promoted the expression of acsl4, while SeS promoted the expression of ptgs2 and fth1 (P<0.01, P<0.05). The intervention results of GC2 spd showed that the Erastin group had a decrease in ptgs2 compared to the normal control group, while the SeS+Erastin and SeM+Erastin groups had an increase in ptgs2 gene expression compared to the Erastin group. However, the ptgs2 expression of Fer-1 was lower than that of the normal control group, and the ptgs2 gene level of SeS+Fer-1 and SeM+Fer-1 groups was lower than that of Fer-1 group (P<0.05); The gene quantity of GPx4 in the SeM+Erastin and SeM+Fer-1 groups increased compared to the Erastin and Fer-1 groups (P<0.01, P<0.05); SeM+Erastin and SeS+Erastin showed a decrease in SLC7A11 compared to the Erastin group, as well as SeM+Fer-1 and SeS+Fer-1 groups compared to the Fer-1 group, accompanied by an increase in acsl4 and fth1 (P<0.01). CONCLUSION: Selenium deficiency causes the reduction of the SLC7A11 and GPx4 gene levels, disorder of ferroptosis-related genes and down-regulation of the GPx4 protein expression in the mouse testis and spermatocytes. Selenium can promote the expression of GPx4, up-regulate the level of SLC7A11, and improve spermatogenesis in the testis of the mouse with SI. There are differences between organic SeM and inorganic SeS in regulating the ferroptosis pathway-related genes.

[Effect and safety of pulsatile GnRH therapy for male congenital hypogonadotropic hypogonadism].

OBJECTIVE: To investigate the efficiency and safety of the pulsatile GnRH therapy in the treatment of male congenital hypogonadotropic hypogonadism (CHH). METHODS: We retrospectively analyzed the clinical data on 45 CHH males treated by pulsatile GnRH therapy in our hospital from January 2013 to March 2023. We treated the patients with gonadorelin at 7－15 µg, one pulse/90 min, and followed them up every month in the first 3 months and then every 3 to 6 months after treatment, for an average of 19.1±4.3 months, during which we recorded the height, body weight, penile length, testis volume, Tanner stages, levels of FSH, LH and T, semen parameters and adverse reactions of the patients, followed by comparison of the data obtained with the baseline. RESULTS: The levels of FSH, LH and T of the patients were dramatically elevated after treatment (P < 0.01). The T level of the 6 cases of cryptorchidism, however, failed to reach the normal value within 18.2 ± 8.6 months of follow-up. Significant improvement was seen in the external genitalia and secondary sexual characteristics of all the patients, and spermatogenesis was observed in the semen in 33 cases (73.3%), with a mean sperm concentration of (18.2 ± 6.2) 106/ml, sperm progressive motility of (19.7 ± 6.5) %, and semen volume of (1.8 ± 0.6) ml. Eight of the cases achieved natural fertility, and another 3 achieved childbirth by assisted reproductive technology. As for adverse events, gynecomastia was observed in 8, subcutaneous induration in 6, and allergic reaction to therapeutic agent in 3 cases. CONCLUSION: Pulsatile GnRH therapy is an effective and safe strategy for male CHH. However, clinicians should choose appropriate approaches to different individual cases.

Icariin protects testicular damage in streptozotocin-induced diabetic rats through regulation of glycolysis pathway.

OBJECTIVE: This study aims to investigate potential beneficial actions of icariin (ICA) on testicular spermatogenic function in male rats with streptozotocin (STZ)-induced diabetes and to explore the underlying mechanisms. Background: ICA was found to reduce blood glucose, regulate the endocrine function of the reproductive system, and improve testicular spermatogenic function. METHODS: Adult rats were intraperitoneally injected with STZ (65 mg/kg) to induce type 1 diabetes mellitus (T1DM). Diabetic rats were randomly classified intoT1DM (n = 6) and T1DM + ICA (n = 6) groups. Rats without STZ and ICA treatment were assigned as control group (n = 6). The morphology of testicular tissues was examined by histological staining. The mRNA and protein expression levels were determined by quantitative real-time PCR, Western blot and immunostaining, respectively. RESULTS: Rats from T1DM group showed a reduction in epididymis and testis weight, and a decrease in sperm count when compared to control group (p < 0.01), which was attenuated by ICA treatment (p < 0.05) Diabetic rats from T1DM group also exhibited reduced diameter and area of seminiferous tubules, along with decreased spermatogonia and primary spermatocytes number when compared to control group (p < 0.01), which was partially reversed by ICA treatment (p < 0.05) Rats from T1DM group exhibited down-regulation of PCNA mRNA and protein in the testis when compared to control group (p < 0.01); while ICA treatment up-regulated PCNA expression in the testis of diabetic rats compared to T1DM group (p < 0.05). Rats from T1DM group showed up-regulation of Bax and capase-3 and down-regulation of Bcl-2, PKM2, HK2 and lactate dehydrogenase A in the testes when compared to control group (p < 0.05), which was reversed by ICA treatment (p < 0.05). CONCLUSION: These findings suggest that ICA may exert its protective effects on testicular damage in diabetic rats through modulation of glycolysis pathway and suppression of apoptosis.

Oral administration of GnRH via a cricket vehicle stimulates spermiation in tiger salamanders (Ambystoma tigrinum).

More than 50% of caudates are threatened with extinction and are in need of ex-situ breeding programs to support conservation efforts and species recovery. Unfortunately, many salamander populations under human care can experience reproductive failure, primarily due to missing environmental cues necessary for breeding. Assisted reproductive technologies (ARTs) are a useful suite of techniques for overcoming or bypassing these missing environmental cues to promote breeding. Exogenous hormones are used to stimulate natural breeding behaviors or gamete expression for in-vitro fertilization or biobanking and are typically administered intramuscularly in caudates. While effective, intramuscular injection is risky to perform in smaller-bodied animals, resulting in health and welfare risks. This research investigated the spermiation response to hormone administration through a non-invasive oral bioencapsulation route using the tiger salamander (Ambystoma tigrinum) as a model species. Male salamanders were randomly rotated six weeks apart through four treatments (n = 11 males/treatment) in which animals received a resolving dose of gonadotropin-releasing hormone (GnRH) as follows: (1) Prime-Only (0.0 µg/g); (2) Low (0.25 µg/g); (3) Medium (1.0 µg/g); and (4) High (2.0 µg/g). All males were given a GnRH priming dose (0.25 µg/g) 24 hours prior to the resolving dose. Exogenous hormone was delivered inside of a cricket (Gryllodes sigillatus) that was presented as a food item by tweezers. Sperm samples were collected at 1, 3, 6, 9, 12, and 24 hours after the resolving dose and analyzed for quantity and quality. For all treatments, sperm concentration was produced in an episodic pattern over time. The Prime-Only treatment had a lower (p < 0.05) percent of sperm exhibiting normal morphology compared to treatments utilizing a resolving dose of GnRH. Overall, oral administration of GnRH is a feasible route of inducing spermiation in salamanders, yielding sperm of sufficient quantity and quality for in-vitro fertilization and biobanking efforts.

The influence of ghrelin agonist ipamorelin acetate on the hypothalamic-pituitary-testicular axis in a cichlid fish, Oreochromis mossambicus.

Ghrelin, a peptide found in the brain and gut, is predicted to play a significant role in the control of various physiological systems in fish. The objective of this study was to examine the impact of ipamorelin acetate (IPA), a ghrelin agonist, on the reproductive axis of the tilapia Oreochromis mossambicus. The administration of either 5 or 30 µg of IPA for 21 days led to a significant and dose-dependent rise in food intake concomitant with a significant increase in the numbers of primary spermatocytes, secondary spermatocytes, and early spermatids compared to the control group. There was a significant rise in the number of late spermatids, as well as the areas of the lobule and lumen, in fish treated with 30 µg of IPA, compared to the control group. Moreover, there was no significant difference in the percentage of gonadotropin-releasing hormone (GnRH)-immunoreactive fibres in the hypothalamus and anterior pituitary gland across different groups. However, a significant elevation in the expression of androgen receptor protein was observed in fish treated with 30 µg of IPA. Furthermore, the concentrations of luteinizing hormone (LH) and 11-ketotestosterone (11-KT) in the serum of fish treated with either 5 or 30 µg of IPA were significantly elevated in comparison to the control group. Collectively, these findings suggest that the administration of ghrelin enhances the development of germ cells during the meiosis-I phase and that this effect might be mediated via the stimulation of 11-KT and androgen receptors at the testicular level and LH at the pituitary level in the tilapia.

FOXO4-DRI improves spermatogenesis in aged mice through reducing senescence-associated secretory phenotype secretion from Leydig cells.

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.

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.

Effect of Humanin and MOTS-c on ameliorating reproductive damage induced by prepubertal cyclophosphamide chemotherapy in male mice.

Male patients who undergo prepubertal chemotherapy face the dual problems of fertility preservation in adulthood, including low testosterone, hypersexual function, and infertility. Humanin, as a small polypeptide coded within the mitochondrial DNA, with the mitochondrial short open reading frame named MOTS-c, both was believed to regulate mitochondrial homeostasis, be anti-inflammatory, improve metabolism, anti-apoptosis, and multiple pharmacological effects. However, there exists little evidence that reported Humanin and MOTS-c 's effects on moderating male spermatogenic function of patients after prepubertal chemotherapy. Here, we found that in vivo, mitochondrial polypeptides Humanin analog (HNG) and MOTS-c efficaciously protected the testicular spermatogenic function from reproductive injury. Moreover, transcriptomic sequencing analysis was performed to verify the differentially expressed genes such as Piwil2, AGT (angiotensinogen), and PTGDS (glycoprotein prostaglandin D2 synthase), which are related to the regulation of male reproductive function of male mice induced by prepubertal chemotherapy. Collectively, our data revealed that both Humanin analogs HNG and MOTS-c are the feasible approaches attached to the protective effect on the male reproductive function damaged by prepubertal chemotherapy.