Sperm Malformation Research Articles

Seminal plasma metabolomics and sperm lipidomics profiles of bull semen with different total progressive motile sperm count.

Total progressive motile sperm count (TPMSC) is a reliable index of fecundity evaluation of bull semen. It is an important determinant frozen semen yield and conception rate of females artificially inseminated. Seminal plasma metabolites and sperm lipids are closely related to sperm survival and motility, but their relationship with TPMSC is not well known. In the present study, Simmental bulls with higher (H, n = 6) or lower (L, n = 6) TPMSC (P < 0.01) were selected from a cohort of 100 animals aged 2 to 5 years based on semen quality. Analysis of semen quality and biochemical markers of seminal plasma revealed that H bulls had greater ejaculate volume (P < 0.05), sperm motility, plasma membrane integrity rate (P < 0.01), seminal plasma neutral α-glucosidase (P < 0.05), alkaline phosphatase, acid phosphatase, cortisol and phosphatidylcholine (P < 0.01), and lower sperm malformation rate (P < 0.05) and reactive oxygen species (ROS) (P < 0.01). Semen metabolites and sperm liposome profiles of H and L groups were compared using LC-MS/MS analysis. A total of 120 differentially abundant metabolites (VIP > 1; P < 0.05) and 59 differentially abundant lipids (VIP > 1; P < 0.05) were identified between H and L groups. Oxidative stress, sperm motility and sperm plasma membrane integrity were among the enriched biological pathways. Cyclic ADP-ribose (cADPR), up-regulated in H bulls, is associated with energy for sperm motility and maintenance of membrane stability. Thymidineglycol (Tg), levanbiose, thymidine (Thd), and CE (3M5) were down-regulated in H bulls, and may have negatively affected sperm motility. Correlation analyses revealed that TPMSC and sperm motility were significantly positively correlated with cADPR, while Tg, Levanbiose, Thd and CE (3M5) were significantly negatively correlated with TPMSC and sperm motility. Thus, we speculate that these molecules may be exploited as potential biomarkers for non-invasive evaluation of TPMSC in bull semen.

Elevated Lipid Concentrations in Seminal Plasma Can Reduce Sperm Motility in Simmental Bulls.

Sperm motility is a key factor influencing male fertility and is associated with metabolic and lipid profiles across species. The aim of this study was to investigate the relationship between sperm motility and the seminal plasma lipid profile in Simmental bulls, and to identify key lipids potentially influencing sperm motility. Semen samples were collected from 26 healthy Simmental bulls with an average age of 4.9 years. Sperm quality was evaluated using computer-assisted sperm analysis (CASA). Based on motility, the samples were divided into two groups: high sperm motility (HSM > 65%) and low sperm motility (LSM < 65%). Compared to the LSM group, the HSM group exhibited significantly higher sperm viability, motility, straight-line velocity, beat-cross frequency, and sperm acrosome integrity, while the sperm malformation rate was lower (p < 0.05). Lipid profiles were determined using LC-MS/MS, and 40 differential lipids were identified by multivariate statistical analysis. Among them, 39 lipids were upregulated in the LSM group compared to the HSM group. They were primarily triglycerides and carnitines, mainly involved in four metabolic pathways related to glycerophospholipid and linoleic acid metabolism. Notably, PC (16:0/20:4; 14:0/18:3), LPC (22:4/0:0; 22:6/0:0), and PE (14:0/18:1; 18:1/20:3) were diagnosed with great accuracy (AUC > 0.7), which means they may serve as potential biomarkers for sperm motility.

Lanthanum nitrate demonstrated no genotoxicity in the conducted tests

Given the widespread applications in industrial and agricultural production, the health effects of rare earth elements (REEs) have garnered public attention, and the genotoxicity of REEs remains unclear. In this study, we evaluated the genetic effects of lanthanum nitrate, a typical representative of REEs, with guideline-compliant in vivo and in vitro methods. Genotoxicity assays, including the Ames test, comet assay, mice bone marrow erythrocyte micronucleus test, spermatogonial chromosomal aberration test, and sperm malformation assay were conducted to assess mutagenicity, chromosomal damage, DNA damage, and sperm malformation. In the Ames test, no statistically significant increase in bacterial reverse mutation frequencies was found as compared with the negative control. Mice exposed to lanthanum nitrate did not exhibit a statistically significant increase in bone marrow erythrocyte micronucleus frequencies, spermatogonial chromosomal aberration frequencies, or sperm malformation frequencies compared to the negative control (P > 0.05). Additionally, after a 24-h treatment with lanthanum nitrate at concentrations of 1.25, 5, and 20 μg/ml, no cytotoxicity was observed in CHL cells. Furthermore, the comet assay results indicate no significant DNA damage was observed even after exposure to high doses of lanthanum nitrate (20 μg/ml). In conclusion, our findings suggest that lanthanum nitrate does not exhibit genotoxicity.

Diquat causes mouse testis injury through inducing heme oxygenase-1-mediated ferroptosis in spermatogonia

Diquat dibromide (DQ) is a globally used herbicide in agriculture, and its overuse poses an important public health issue, including male reproductive toxicity in mammals. However, the effects and molecular mechanisms of DQ on testes are limited. In vivo experiments, mice were intraperitoneally injected with 8 or 10 mg/kg/ day of DQ for 28 days. It has been found that heme oxygenase-1 (HO-1) mediates DQ-induced ferroptosis in mouse spermatogonia, thereby damaging testicular development and spermatogenesis. Histopathologically, we found that DQ exposure caused seminiferous tubule disorders, reduced germ cells, and increased sperm malformation, in mice. Reactive oxygen species (ROS) staining of frozen section and transmission electron microscopy (TEM) displayed DQ promoted ROS generation and mitochondrial morphology alterations in mouse testes, suggesting that DQ treatment induced testicular oxidative stress. Subsequent RNA-sequencing further showed that DQ treatment might trigger ferroptosis pathway, attributed to disturbed glutathione metabolism and iron homeostasis in spermatogonia cells in vitro. Consistently, results of western blotting, measurements of MDA and ferrous iron, and ROS staining confirmed that DQ increased oxidative stress and lipid peroxidation, and accelerated ferrous iron accumulation both in vitro and in vivo. Moreover, inhibition of ferroptosis by deferoxamine (DFO) markedly ameliorated DQ-induced cell death and dysfunction. By RNA-sequencing, we found that the expression of HO-1 was significantly upregulated in DQ-treated spermatogonia, while ZnPP (a specific inhibitor of HO-1) blocked spermatogonia ferroptosis by balancing intracellular iron homeostasis. In mice, administration of the ferroptosis inhibitor ferrostatin-1 effectively restored the increase of HO-1 levels in the spermatogonia, prevented spermatogonia death, and alleviated the spermatogenesis disorders induced by DQ. Overall, these findings suggest that HO-1 mediates DQ-induced spermatogonia ferroptosis in mouse testes, and targeting HO-1 may be an effective protective strategy against male reproductive disorders induced by pesticides in agriculture.

High Concentration of Iron Ions Contributes to Ferroptosis-Mediated Testis Injury.

In order to explore the effect of excessive iron supplementation on ferroptosis in mouse testes, Kunming mice received injections of varying concentrations of iron. The organ weight, sperm density, and malformation rate were measured. Observations of pathological and ultrastructural alterations in spermatogenic tubules were conducted using haematoxylin eosin (HE) staining and transmission electron microscopy(TEM). Transcript levels of related genes and serum biochemical indicators were measured in mouse testicular tissue. The results showed that higher iron concentration inhibited the growth of mice; reduced the organ coefficients of the testis, heart, and liver; and increased the rate of sperm malformation and mortality. Supplementation with high levels of iron ions can adversely affect the male reproductive system by reducing sperm count, damaging the structure of the seminiferous tubules and causing sperm cell abnormalities. In addition, the iron levels also affected the immune response and blood coagulation ability by affecting the red blood cells, white blood cells and platelets. The results showed that iron ions can affect mouse testicular tissue and induce ferroptosis by altering the expression of ferroptosis-related genes. However, the degree of effect was different for the different concentrations of iron ions. The study also revealed the potential role of deferoxamine in inhibiting the occurrence of ferroptosis. Nevertheless, the damage caused to the testis by deferoxamine supplementation suggests the need for further research in this direction. This study provides reference for reproductive toxicity induced by environmental iron exposure and clarifies the mechanism of reproductive toxicity caused by iron overload and the important role of iron in the male reproductive system.

Safety and efficacy evaluation of halicin as an effective drug for inhibiting intestinal infections.

Halicin, the first antibacterial agent discovered by artificial intelligence, exerts broad-spectrum antibacterial effects and has a unique structure. Our study found that halicin had a good inhibitory effect on clinical isolates of drug-resistant strains and Clostridium perfringens (C. perfringens). The safety of halicin was evaluated by acute oral toxicity, genotoxicity and subchronic toxicity studies. The results of acute toxicity test indicated that halicin, as a low-toxicity compound, had an LD50 of 2018.3mg/kg. The results of sperm malformation, bone marrow chromosome aberration and cell micronucleus tests showed that halicin had no obvious genotoxicity. However, the results of the 90-day subchronic toxicity test indicated that the test rats exhibited weight loss and slight renal inflammation at a high dose of 201.8mg/kg. Teratogenicity of zebrafish embryos showed that halicin had no significant teratogenicity. Analysis of intestinal microbiota showed that halicin had a significant effect on the intestinal microbial composition, but caused a faster recovery. Furthermore, drug metabolism experiments showed that halicin was poorly absorbed and quickly eliminated in vivo. Our study found that halicin had a good therapeutic effect on intestinal infection model of C. perfringens. These results show the feasibility of developing oral halicin as a clinical candidate drug for treating intestinal infections.

Ameliorative effects of zinc and vitamin E against phthalates-induced reproductive toxicity in male rats.

Phthalates (PEs) could cause reproductive harm to males. A mixture of three widely used PEs (MPEs) was used to investigate the ameliorative effects of zinc (Zn) and vitamin E (VE) against male reproductive toxicity. Fifty male SD rats were randomly divided into five groups (n = 10). Rats in MPEs group were orally treated with 160 mg/kg/d MPEs, while rats in MPEs combined Zn and/or VE groups were treated with 160 mg/kg/d MPEs plus 25 mg/kg/d Zn and/or 25 mg/kg/d VE. After intervention for 70 days, it's was measured of male reproductive organs' weight, histopathological observation of sperms and testes, serum hormones, PIWI proteins and steroidogenic proteins. Compared with control, anogenital distance, testes weight, epididymides weight, and sex hormones were significantly decreased, while the sperm malformation rate was markedly increased in MPEs group (p < .05); the testicular tissues were injured in MPEs group with disordered and decreased spermatids, and arrested spermatogenesis. PIWIL1, PIWIL2, StAR, CYP11A1 and CYP19A1 were down-regulated in MPEs group (p < .05). However, the alterations of these parameters were restored in MPEs combined Zn and/or VE groups (p < .05). Zn and/or VE improved steroid hormone metabolism, and inhibited MPEs' male reproductive toxicity.

Acute toxicity and genotoxicity evaluation of irradiated ophiopogonis radix at 25 kGy with electron beam

As concerned about the potential risks of irradiated Ophiopogonis Radix (IOR) at a high radiation level (25 kGy) by high-energy electron beam irradiation, this study evaluated oral acute toxicity and genotoxicity of IOR. Moisture, total ash, water-soluble extracts, and ruscogenin content in IOR meet the requirement of Ophiopogonis Radix described in the Chinese Pharmacopoeia. When the mice received by oral 21.5 g/kg BW administration of IOR in the acute toxicity test, neither mortality nor any other abnormal general clinical signs were found within 14 days, except that 10% of mice suffered from diarrhea on day 1, which disappeared and returned to normal on day 4. The acute toxicity test indicated the LD50 of IOR was higher than 21.5 g/kg BW. Ames test showed no dose-dependent increase in TA97a, TA98, TA100, TA102, and TA1535 strains with or without rat liver microsomal enzyme mixture (S9) metabolic activation, at a quantity range of 50–5000 μg IOR. Mouse micronucleus test of bone marrow cells demonstrated no significant increase in the frequencies of micronucleated polychromatic erythrocytes (MN PCEs) among each IOR dose. Mouse sperm abnormality test showed that there was no evidence of the potential to induce sperm malformation with oral administration at doses of 2.5, 5.0, and 10.0 g/kg BW. Overall, the acute toxicity or genotoxicity of IOR were not observed, indicating that IOR at 25 kGy with a high-energy electron beam is toxicologically safe.

Deficiency of nucleosome-destabilizing factor GLYR1 dampens spermatogenesis in mice

Aberrant sperm morphology hinders sperm motility and causes male subfertility. Spermatogenesis, a complex process in male germ cell development, necessitates precise regulation of numerous developmental genes. However, the regulatory pathways involved in this process remain partially understood. We have observed the widespread expression of Glyr1, the gene encoding a nucleosome-destabilizing factor, in mouse testicular cells. Our study demonstrates that mice experiencing Glyr1 depletion in spermatogenic cells exhibit subfertility characterized by a diminished count and motility of spermatozoa. Furthermore, the rate of sperm malformation significantly increases in the absence of Glyr1, with a predominant occurrence of head and neck malformation in spermatozoa within the cauda epididymis. Additionally, a reduction in spermatocyte numbers across different meiotic stages is observed, accompanied by diminished histone acetylation in spermatogenic cells upon Glyr1 depletion. Our findings underscore the crucial roles of Glyr1 in mouse spermiogenesis and unveil novel insights into the etiology of male reproductive diseases.

Rreb1 is a key transcription factor in Sertoli cell maturation and function and spermatogenesis in mouse.

Spermatogenesis is a developmental process driven by interactions between germ cells and Sertoli cells. This process depends on appropriate gene expression, which might be regulated by transcription factors. This study focused on Rreb1, a zinc finger transcription factor, and explored its function and molecular mechanisms in spermatogenesis in a mouse model. Our results showed that RREB1 was predominantly expressed in the Sertoli cells of the testis. The decreased expression of RREB1 following injection of siRNA caused impaired Sertoli cell development, which was characterized using a defective blood-testis barrier structure and decreased expression of Sertoli cell functional maturity markers; its essential trigger might be SMAD3 destabilization. The decreased expression of RREB1 in mature Sertoli cells influenced the cell structure and function, which resulted in abnormal spermatogenesis, manifested as oligoasthenoteratozoospermia, and we believe RREB1 plays this role by regulating the transcription of Fshr and Wt1. RREB1 has been reported to activate Fshr transcription, and we demonstrated that the knockdown of Rreb1 caused a reduction in follicle-stimulating hormone receptor (FSHR) in the testis, which could be the cause of the increased sperm malformation. Furthermore, we confirmed that RREB1 directly activates Wt1 promoter activity, and RREB1 downregulation induced the decreased expression of Wt1 and its downstream polarity-associated genes Par6b and E-cadherin, which caused increased germ-cell death and reduced sperm number and motility. In conclusion, RREB1 is a key transcription factor essential for Sertoli cell development and function and is required for normal spermatogenesis.

Flaxseed oil attenuates PFOS-induced testicular damage by regulating RNA alternative splicing.

Background: Perfluorooctane sulfonate (PFOS) is a persistent, widely present environmental pollutant, and its toxicity to male reproduction has gradually attracted attention. Flaxseed oil (FO) is a dietary oil abundant in α-linolenic acid and has been demonstrated to possess multiple health benefits. However, whether FO protects against PFOS-induced testicular injury and its mechanism remain unclear. Methods: C57/BL6 mice were gavaged with different concentrations of FO or PFOS (10 mg kg-1) for 28 days. Blood and testicular tissues were collected for histopathology, proteomics, and biochemical and molecular analyses. Results: Our results showed that FO supplementation significantly attenuated PFOS-induced testicular injury, as indicated by histopathological changes, decreased oxidative stress level, increased sperm count, decreased rate of sperm malformation, and improved functional markers of spermatogenesis. Proteomic analysis showed that differentially expressed proteins were notably enriched in spliceosome pathways. Machine learning algorithms were used to screen the hub gene, and PRPF3 and PUF60 proteins were found to be important for FO to exert protective benefits to testicular injury. Western blot results confirmed that FO supplementation could increase the protein expression of PRPF3 and decrease the protein expression of PUF60 in PFOS-exposed mice. Conclusions: This study revealed that FO can alleviate PFOS-induced testicular dysfunction by regulating RNA alternative splicing. The spliceosome-related proteins PRPF3 and PUF60 may be the potential targets for FO to alleviate PFOS-induced testicular injury. FO supplementation may be an effective dietary intervention to prevent adverse effects of PFOS on testes.

T-2 toxin-induced testicular impairment by triggering oxidative stress and ferroptosis

T-2 toxin is a trichothecene mycotoxin of significant danger to humans and animals. Its impact on reproductive toxicity is attributed to oxidative stress, which ultimately leads to cell death. Ferroptosis is a programmed cell death that characterized by lipid peroxidation. This study aimed to investigate the toxic effects of T-2 toxin on mouse testis and the potential mechanism of T-2 toxin-induced ferroptosis. T-2 toxin significantly altered the morphology of the testis and decreased testosterone level, sperm concentration, and increased sperm malformation rate, as well as induced oxidative damage with reactive oxygen species and malondialdehyde accumulated, and activity of superoxide dismutase, glutathione peroxidase decreased. Additionally, T-2 toxin induced ferroptosis by accumulating iron ions, increasing prostaglandin endoperoxide synthase 2, downregulating glutathione peroxidase 4 and ferritin heavy chain 1, as well as manifesting ferroptotic morphological alterations, ultimately leading to testicular impairment. Administration of ferroptosis inhibitor liproxstatin-1 or antioxidant resveratrol effectively mitigated the T-2 toxin-induced ferroptosis and testicular injury. These findings provided novel insights into the fundamental mechanism of T-2 toxin-induced cell death and furnished further proof of the potential therapeutic effect in addressing T-2 toxin-induced testicular impairment.

Early-life long-term ibuprofen exposure reduces reproductive capacity involved in spermatogenesis impairment and associated with the transcription factor DAF-5 in Caenorhabditis elegans

Pharmaceuticals and personal care products (PPCPs) are emerging environmental contaminants and have raised significant concern due to their potential adverse impact on the environment. Ibuprofen is one of the most extensively used non-steroidal anti-inflammatory drugs (NSAIDs) and is also considered an environmental contaminant. The negative impact of ibuprofen on non-target organisms has been documented; however, the molecular mechanisms behind its reproductive toxicity remain unclear. We investigated the impact of early-life long-term ibuprofen exposure on reproductive capacity and its involvement of spermiogenesis in the non-target model organism Caenorhabditis elegans. Hermaphrodites were exposed to various ibuprofen concentrations (0.1, 1, 10, and 100 mg/L), resulting in a dose-dependent inhibition of reproduction. In addition, the lowest observed adverse effect concentration (LOAEC) for ibuprofen exposure on the total brood size of C. elegans was 0.1 mg/L, a concentration that falls within the environmentally relevant range for ibuprofen. Outcross progeny assays revealed a significant 47% reduction in total brood size for larval males (him-5) exposed to ibuprofen, while females (fog-2) exhibited only a minor effect. We found that early-life long-term ibuprofen exposure impairs spermatogenesis. The number of mitotic cells significantly reduced by 31%. The rate of sperm malformation in exposed males was 63%, much higher than in unexposed males (11%). Additionally, the percentage of sperm activation decreased from 89% to 39% in ibuprofen-exposed worms. Mechanistic insights indicated that ibuprofen downregulated mRNA levels of genes related to spermatogenesis and DAF-7/TGF-β signaling. RNAi assays provided evidence for the crucial role of the transcription factor DAF-5 in mediating the spermatogenesis impairment by ibuprofen. Our study provides insight into the environmental impacts of pharmaceutical contaminants, such as ibuprofen, on both male and female reproductive systems to safeguard environmental health.

Shortwave radiation-induced reproductive organ damage in male rats by enhanced expression of molecules associated with the calpain/Cdk5 pathway and oxidative stress

ABSTRACT Shortwave radiation has been reported to have harmful effects on several organs in humans and animals. However, the biological effects of 27 MHz shortwave on the reproductive system are not clear. In this study, we investigated the effects of shortwave whole-body exposure at a frequency of 27 MHz on structural and functional changes in the testis. Male Wistar rats were exposed to 27 MHz continuous shortwaves at average power densities of 0, 5, 10, or 30 mW/cm2 for 6 min. The levels of insulin-like factor 3 (INSL3) and anti-sperm antibodies (AsAb) in the peripheral serum, sperm motility, sperm malformation rate, and testicular tissue structure of rats were analyzed. Furthermore, the activity of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) content, calpain, and Cdk5 expression were analyzed at 1, 7, 14, and 28 days after exposure. We observed that the rats after radiation had decreased serum INSL3 levels (p < 0.01), increased AsAb levels (p < 0.05), decreased percentage of class A+B sperm (p < 0.01 or p < 0.05), increased sperm malformation (p < 0.01 or p < 0.05), injured testicular tissue structure, decreased SOD and CAT activities (p < 0.01 or p < 0.05), increased MDA content (p < 0.01), and testicular tissue expressions of calpain1, calpain2, and Cdk5 were increased (p < 0.01 or p < 0.05). In conclusion, Shortwave radiation caused functional and structural damage to the reproductive organs of male rats. Furthermore, oxidative stress and key molecules in the calpain/Cdk5 pathway are likely involved in this process.

Melatonin or vitamin C attenuates lead acetate-induced testicular oxidative and inflammatory damage in mice by inhibiting oxidative stress mediated NF-κB signaling

Lead (Pb) acts as an environmental endocrine disruptor and has negative effects in animals; excessive accumulation of lead causes reproductive dysfunction in male animals. Oxidative stress plays a vital role in Pb-induced injury. However, the mechanisms underlying chronic testicular toxicity of Pb remain unclear. In this study, we aimed to determine the effects of lead acetate on reproductive function in male mice, identify the underlying mechanisms, and test counter measures to alleviate the toxic effects. Male mice were dosed with lead acetate (500 mg/L) in free drinking water for 12 weeks, and administered melatonin (5 mg/kg) or vitamin C (500 mg/kg) by intraperitoneal injection. Blood from the eyeball, testicles, and sperm from the caudal epididymis were collected after 12 weeks and analyzed. Pb exposure reduced sperm count and motility, increased sperm malformation (P < 0.01), disrupted testicular morphology and structure, and decreased the expression of steroid hormone synthesis-related enzymes and serum testosterone concentration (P < 0.01). Pb also increased the number of inflammatory cells and the levels of the pro-inflammatory cytokines TNF-α and IL-6 (P < 0.01), and activated NF-κB signaling. Furthermore, the ROS yield and oxidation indicators LPO and MDA were significantly increased (P < 0.01), and the antioxidant indicators T-AOC, SOD, and GSH were significantly reduced (P < 0.01). Treatment with melatonin or vitamin C reversed the effects of lead acetate; vitamin C was more effective in restoring SOD activity (P < 0.01) and enhancing ZO-1 protein levels (P < 0.01). Thus, long-term exposure to lead acetate at low concentrations could adversely affect sperm quality and induce inflammatory damage by oxidative stress mediated NF-κB signaling. Vitamin C could act as a protective agent and improve reproductive dysfunction in male animals after lead accumulation.

Arsenic exposure caused male infertility indicated by testis and sperm metabolic dysfunction in SD rats

Arsenic containment is one of the most severe environmental problems. It has been reported that arsenic exposure could cause male reproductive damage. However, the evidence chain from sodium arsenite (NaAsO2) exposure to adverse male fertility outcomes has not been completed by molecular events. In this study, adult male rats were exposed to NaAsO2 for eight weeks via drinking water for verifying their reproductive capacity by checking the phenotypes of testis damage, sperm quality, and female pregnancy rate. H&E staining indicated testicular cells had atrophied, and necrosis was observed under transmission electron microscopy. Sperm viability tended to decrease, and sperm malformation increased. Notably, metabolites in the testes and sperm showed substantial disruption, especially sperm metabolites. The pregnancy rate tests showed that arsenic decreased male rats' reproduction, with some adverse outcomes of the increased numbers of unpregnant females. However, the fetal crown-rump length remained unaltered, indicating that the pregnancy rate was impacted by arsenic exposure but not fetal growth. On arsenic toxicometabolomics analysis, docosahexaenoic acid (DHA) in sperm was the clearest metabolic sign to correlate with the unpregnant rate. In summary, arsenic exposure can cause male infertility via the injured sperm, which results in decreased female pregnancy. The DHA information may imply the dietary intervention for improving sperm quality. Although the fetal growth of the successful pregnancy has not been affected, the changes in epigenetic phenotypes carried by sperms still need to be verified.

Alpha-tocopherol enhances spermatogonial stem cell proliferation and restores mouse spermatogenesis by up-regulating BMI1.

Spermatogonial stem cells (SSCs) are essential for maintaining reproductive function in males. B-lymphoma Mo-MLV insertion region 1 (BMI1) is a vital transcription repressor that regulates cell proliferation and differentiation. However, little is known about the role of BMI1 in mediating the fate of mammalian SSCs and in male reproduction. This study investigated whether BMI1 is essential for male reproduction and the role of alpha-tocopherol (α-tocopherol), a protective agent for male fertility, as a modulator of BMI1 both in vitro and in vivo. Methyl thiazolyl tetrazolium (MTT) and 5-ethynyl-2'-deoxyuridine (EDU) assays were used to assess the effect of BMI1 on the proliferative ability of the mouse SSC line C18-4. Real-time polymerase chain reaction (PCR), western blotting, and immunofluorescence were applied to investigate changes in the mRNA and protein expression levels of BMI1. Male mice were used to investigate the effect of α-tocopherol and a BMI1 inhibitor on reproduction-associated functionality in vivo. Analysis revealed that BMI1 was expressed at high levels in testicular tissues and spermatogonia in mice. The silencing of BMI1 inhibited the proliferation of SSCs and DNA synthesis and enhanced the levels of γ-H2AX. α-tocopherol enhanced the proliferation and DNA synthesis of C18-4 cells, and increased the levels of BMI1. Notably, α-tocopherol rescued the inhibition of cell proliferation and DNA damage in C18-4 cells caused by the silencing of BMI1. Furthermore, α-tocopherol restored sperm count (Ctrl vs. PTC-209, p = 0.0034; Ctrl vs. PTC-209 + α-tocopherol, p = 0.7293) and normalized sperm malformation such as broken heads, irregular heads, lost and curled tails in vivo, as demonstrated by its antagonism with the BMI1 inhibitor PTC-209. Analysis demonstrated that α-tocopherol is a potent in vitro and in vivo modulator of BMI1, a transcription factor that plays an important role in in SSC proliferation and spermatogenesis. Our findings identify a new target and strategy for treating male infertility that deserves further pre-clinical investigation.

Semen quality and seminal plasma metabolites in male rabbits (Oryctolagus cuniculus) under heat stress.

Heat stress causes infertility in male rabbits in summer. This study was conducted to determine the effects of heat stress on semen quality and seminal plasma metabolites of male rabbits. To achieve these objectives, the temperature and humidity index (THI) was used to determine the stress state of male rabbits during different months, thereby the rabbits were divided into heat stress and no heat stress groups. The quality of the semen and the biochemical indices of seminal plasma were then analyzed. Next the plasma metabolites of rabbits in both groups were evaluated using the ultra-high performance liquid chromatography-mass spectroscopy (UPLC-MS)/MS technique. Our results showed that the THI value of the rabbit housing in May was 20.94 (no heat stress). The THI value of the housing in August was 29.10 (heat stress group, n = 10). Compared with the non-heat stress group, the sperm motility, density, and pH in the heat stress group (n = 10) were significantly decreased (P < 0.01); the semen volume decreased significantly (P < 0.05); and the sperm malformation rate increased significantly (P < 0.01). The number of grade A sperm significantly decreased, while the numbers of B and C grade sperm significantly increased (P < 0.01). The total sperm output (TSO), total motile sperm (TMS), and total functional sperm fraction (TFSF) decreased significantly (P < 0.01). Heat stress protein 70 (HSP70) and acid phosphatase (ACP) in the seminal plasma of rabbits in the heat stress group (n = 20) were significantly increased (P < 0.01). Seminal plasma testosterone (T), α-glucosidase (α-Glu), and fructose decreased significantly (P < 0.01). The concentrations of Mg2+ (P < 0.05), Na+ (P < 0.01), and K+ (P < 0.01) in metal ions were significantly decreased. These findings indicated that heat stress severely affected the quality of the male rabbit semen. Furthermore, UPLC-MS/MS technology was used to analyze the seminal plasma samples of rabbits in the heat stress group and non-heat stress group (n = 9 for each group). In total, 346 metabolites were identified, with variable importance in project (VIP) > 1.0, fold change (FC) > 1.5 or < 0.667, and P < 0.05 as the threshold. A total of 71 differential metabolites were matched, including stearic acid, betaine, arachidonic acid, L-malic acid, and indole. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differential metabolites revealed 51 metabolic pathways, including synthesis and degradation of ketones, serine and threonine metabolism, tryptophan metabolism, and the citric acid cycle. Our study has shown that the sperm motility, sperm pH value, and sperm density of male rabbits decreased significantly under heat stress, and the sperm malformation rate increased significantly. Furthermore, the quality of semen was shown to deteriorate and the energy metabolism pathway was disturbed. These findings provide a theoretical reference for alleviating the adaptive heat stress in male rabbits.

Difenoconazole Exposure Induces Retinoic Acid Signaling Dysregulation and Testicular Injury in Mice Testes.

Difenoconazole (DFZ) is a broad-spectrum triazole fungicide that is widely utilized in agriculture. Although DFZ has been demonstrated to induce reproductive toxicity in aquatic species, its toxic effects on the mammalian reproductive system have yet to be fully elucidated. In vivo, male mice were administered 0, 20 or 40 mg/kg/d of DFZ via oral gavage for 35 days. Consequently, DFZ significantly decreased testicular organ coefficient, sperm count and testosterone levels, augmented sperm malformation rates, and elicited histopathological alterations in testes. TUNEL assay showed increased apoptosis in testis. Western blotting results suggested abnormally high expression of the sperm meiosis-associated proteins STRA8 and SCP3. The concentrations of retinoic acid (RA), retinaldehyde (RE), and retinol (ROL) were increased in the testicular tissues of DFZ-treated groups. The mRNA expression level of genes implicated in RA synthesis significantly increased while genes involved in RA catabolism significantly decreased. In vitro, DFZ reduced cell viability and increased RA, RE, and ROL levels in GC-2 cells. Transcriptome analysis revealed a significant enrichment of numerous terms associated with the RA pathway and apoptosis. The qPCR experiment verified the transcriptome results. In conclusion, our results indicate that DFZ exposure can disrupt RA signaling pathway homeostasis, and induce testicular injury in mice testes.

Correlation analysis of sperm DNA fragmentation index with semen parameters and the effect of sperm DFI on outcomes of ART

Routine semen analysis provides limited information about a man’s male reproductive potential and can not always fully explain male infertility. Many male infertilities are caused by sperm DNA defects that routine semen quality analyses fail to detect. In this study, we analyzed the association of sperm DNA fragmentation index (DFI) with the semen routine, sperm morphology, in vitro fertilization embryo transfer (IVF-ET)/intracytoplasmic sperm injection (ICSI). Further, we explored the predictive value of sperm DFI in evaluating male fertility and the outcome of IVF-ET/ICSI. Data on sperm DFI, sperm routine, and sperm morphology were collected from 1462 males with infertility. According to DFI levels, there were 468 cases in group I (DFI ≤ 15%), 518 cases in group II (15% < DFI < 30%), and 476 cases in group III (DFI ≥ 30%). The correlations of sperm DFI with semen routine and malformation rate were analyzed. Seminal plasma malondialdehyde (MDA), and total antioxidant capacity (TAC) were assessed. Sperm DFI, semen routine, and sperm morphology were detected in male patients of 101 pairs of IVF-ET/ICSI infertile couples and subdivided into IVF-I group (DFI ≤ 15%), IVF-II group (15% < DFI < 30%), IVF-III group (DFI ≥ 30%), ICSI-I group (DFI ≤ 15%), ICSI-II group (15% < DFI < 30%) and ICSI-III group (DFI ≥ 30%) according to DFI value. The effect of sperm DFI on the outcome of IVF-ET/ICSI was analyzed. There were significant differences in sperm survival rate, sperm concentration, and PR% between groupIII and group II (P < 0.01). There were significant differences in sperm survival rate, sperm concentration and PR% between group III and group I (P < 0.01). There was no significant difference in semen volume, age, abstinence days, or percentage of normal sperm between the three groups (P > 0.05). DFI was positively correlated with MDA content ( P < 0.01) and negatively correlated with TAC (P < 0.01). Sperm DFI was negatively correlated with sperm survival rate, sperm concentration, and PR% (P < 0.01). There was no correlation with age, abstinence days, semen volume, or percentage of normal-form sperm (r = 0.16, 0.05, 0.04, -0.18, p > 0.05). Compared with IVF-I group, the sperm concentration and PR were decreased in IVF-III group. The sperm malformation rate was higher in IVF-III group than that in IVF-II group. Comparatively, the PR was decreased in ICSI-III group. The sperm malformation rate was higher in ICSI-III group than that of the ICSI-I group (P < 0.05). There were no statistically significant differences in fertilization rate, cleavage rate, embryo rate, and clinical pregnancy rate between IVF group or ICSI group, and between all subgroups (P > 0.05). Sperm DFI is negatively associated with sperm survival rate, sperm concentration, and PR%. Antioxidants can decrease the rate of DNA fragmentation. Sperm DFI has proven to be very valuable in the male fertility evaluation, but its significance as a predictor of pregnancy outcomes following assisted reproductive technology. (ART) requires further investigation.