Mouse models in male fertility research

Limited knowledge of the genetic causes of male infertility has resulted in few treatment and targeted therapeutic options. Although the ideal approach to identify infertility causing mutations is to conduct studies in the human population, this approach has progressed slowly due to the limitations described herein. Given the complexity of male fertility, the entire process cannot be modeled in vitro. As such, animal models, in particular mouse models, provide a valuable alternative for gene identification and experimentation. Since the introduction of molecular biology and recent advances in animal model production, there has been a substantial acceleration in the identification and characterization of genes associated with many diseases, including infertility. Three major types of mouse models are commonly used in biomedical research, including knockout/knockin/gene-trapped, transgenic and chemical-induced point mutant mice. Using these mouse models, over 400 genes essential for male fertility have been revealed. It has, however, been estimated that thousands of genes are involved in the regulation of the complex process of male fertility, as many such genes remain to be characterized. The current review is by no means a comprehensive list of these mouse models, rather it contains examples of how mouse models have advanced our knowledge of post-natal germ cell development and male fertility regulation.     

An epididymis-specific carboxyl esterase CES5A is required for sperm capacitation and male fertility in the rat

Despite the fact that the phenomenon of capacitation was discovered over half century ago and much progress has been made in identifying sperm events involved in capacitation, few specific molecules of epididymal origin have been identified as being directly involved in this process in vivo. Previously, our group cloned and characterized a carboxyl esterase gene Ces5a in the rat epididymis. The CES5A protein is mainly expressed in the corpus and cauda epididymidis and secreted into the corresponding lumens. Here, we report the function of CES5A in sperm maturation. By local injection of Lentivirus-mediated siRNA in the CES5A-expressing region of the rat epididymis, Ces5a-knockdown animal models were created. These animals exhibited an inhibited sperm capacitation and a reduction in male fertility. These results suggest that CES5A plays an important role in sperm maturation and male fertility.     

Evaluation of mid- and long-term impact of COVID-19 on male fertility through evaluating semen parameters

BackgroundThe coronavirus disease 2019 (COVID-19) has spread worldwide with alarming levels of spread and severity. The distribution of angiotensin converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) from bioinformatics evidence, the autopsy report for COVID-19 and the published study on sperm quality indicated COVID-19 could have a negative impact on male fertility. However, whether the negative impact of COVID-19 on male fertility is persistent remains unknown, which requires long-term follow-up investigation.MethodsSemen samples were collected from 36 male COVID-19 patients with a median recovery time of 177.5 days and 45 control subjects. Then, analysis of sperm quality and alterations of total sperm number with recovery time were performed.ResultsThere was no significant difference in semen parameters between male recovered patients and control subjects. And the comparisons of semen parameters between first follow-up and second follow-up revealed no significant difference. In addition, we explored the alterations of sperm count with recovery time. It showed that the group with recovery time of ≥120 and <150 days had a significantly lower total sperm number than controls while the other two groups with recovery time of ≥150 days displayed no significance with controls, and total sperm number showed a significant decline after a recovery time of 90 days and an improving trend after a recovery time of about 150 days.ConclusionsThe sperm quality of COVID-19 recovered patients improved after a recovery time of nearly half a year, while the total sperm number showed an improvement after a recovery time of about 150 days. COVID-19 patients should pay close attention to the quality of semen, and might be considered to be given medical interventions if necessary within about two months after recovery, in order to improve the fertility of male patients as soon as possible.     

Determination of sperm acrosin activity for evaluation of male fertility

Aim: To investigate a simple method for assaying acrosin activity for the evaluation of male fertility. Methods:The acrosin activity of 7.5 × 10~6 sperm without seminal plasma and acrosin activity inhibitors was assayed using N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and detergent (Triton X-100) as substrate. Results: The acrosin ac-tivity of 60 normal fertile men (35 ± 10 μIU/10~6 sperm ) was higher than that of 168 infertile men ( 16 ± 8 μIU/10~6sperm) (P <0.01). It was indicated that there was a significant positive correlation between the acrosin activity andthe sperm motility ( r ≥ 0.6534, P < 0.01) and a significant negative correlation between the sperm malformed rateand the WBC number ( r ≤ -0.5426, P < 0.01). The temperature and time of incubation and the sperm concentrationcould influence the assay results. Conclusion: Acrosin activity is an important index for the evaluation of male fer-tility. The approach developed by the authors is a simple method for the determination of acro     

Aqueous fruit extract of Mimusops elengi causes reversible suppression of spermatogenesis and fertility in male mice

Antifertility efficacy of oral administration of aqueous fruit extract of Mimusops elengi (200, 400 and 600 mg kg^?1 body weight/day for 35 days) was evaluated in Parkes strain male mice. Various reproductive end points such as histopathology, sperm parameters, testosterone level, haematology, serum biochemistry and fertility indices were assessed; activities of 3β‐ and 17β‐hydroxysteroid dehydrogenases, and immunoblot expressions of StAR and P450scc in the testis were also assessed. Histologically, testes in Mimusops‐treated mice showed nonuniform and diverse degenerative changes in the seminiferous tubules; both affected and normal tubules were observed in the same sections of testis. The treatment had adverse effects on testicular hydroxysteroid dehydrogenases and StAR and P450scc, serum level of testosterone and on motility, viability and number of spermatozoa in cauda epididymis. However, serum levels of alanine aminotransferase, aspartate aminotransferase and creatinine, and haematological parameters were not affected by the treatment. Also, libido was not affected in treated males, but their fertility was markedly suppressed. By 56 days of treatment withdrawal, the alterations caused in the above parameters recovered to control levels, suggesting that Mimusops treatment causes reversible suppression of spermatogenesis and fertility in Parkes mice. Further, there were no detectable signs of toxicity in treated males.     

Impact of the SARS-CoV-2 virus on male reproductive health

The coronavirus 2019 (COVID-19) pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to more than 160 million infections and 3.5 million deaths globally. Men are disproportionately affected by COVID-19, having more severe disease with higher mortality rates than women. Androgens have been implicated as the underlying cause for more severe disease, as the androgen receptor has been noted to upregulate the cell surface receptors that mediate viral cell entry and infection. Unfortunately, despite testosterone’s potential role in COVID-19 prognosis, androgen deprivation therapy is neither protective nor a treatment for COVID-19. Interestingly, the male reproductive organs have been found to be vulnerable in moderate to severe illness, leading to reports of erectile dysfunction and orchitis. COVID-19 viral particles have been identified in penile and testis tissue, both in live patients who recovered from COVID-19 and post mortem in men who succumbed to the disease. Although sexual transmission remains unlikely in recovered men, moderate to severe COVID-19 infection can lead to germ cell and Leydig cell depletion, leading to decreased spermatogenesis and male hypogonadism. The objective of this review is to describe the impact of SARS-CoV-2 on male reproductive health. There are still many unanswered questions as to the specific underlying mechanisms by which COVID-19 impacts male reproductive organs and the long-term sequelae of SARS-CoV-2 on male reproductive health.     

Contemporary and future insights into fertility preservation in male cancer patients

In recent years, survival rates of cancer patients have increased, resulting in a shift of focus from quantity to quality of life. A key aspect of quality of life is fertility potential; patients suffering from iatrogenic infertility often become depressed. Since many cancer therapies—chemotherapy, radiotherapy and/or surgery—and even cancer itself have detrimental effects on the male reproductive system, it is important to preserve fertility before any treatment commences. Currently, the only reliable method of male fertility preservation is sperm banking. For patients who are unable to provide semen samples by the conventional method of masturbation, there are other techniques such as electroejaculation, microsurgical epididymal sperm aspiration and testicular sperm extraction that can be employed. Unfortunately, it is presently impossible to preserve the fertility potential of pre-pubertal patients. Due to the increasing numbers of adolescent cancer patients surviving treatment, extensive research is being conducted into several possible methods such as testicular tissue cryopreservation, xenografting, in vitro gamete maturation and even the creation of artificial gametes. However, in spite of its ease, safety, convenience and many accompanying benefits, sperm banking remains underutilized in cancer patients. There are several barriers involved such as the lack of information and the urgency to begin treatment, but various measures can be put in place to overcome these barriers so that sperm banking can be more widely utilized.     

Effect of nordihydroguaiaretic acid on spermatogenesis and fertility in rats

Nordihydroguaiaretic acid (NDGA) is a naturally occurring lignan with potent antioxidant activity. Currently, it is in clinical trials as anticancer agent. As there is no earlier report on the effect of NDGA on spermatogenesis and fertility, this study was designed to investigate this aspect. Administration of NDGA to rats for 60 days produced degenerative changes in testis but had no effect on sperm DNA integrity test and androgen receptor expression. Ultrastructural studies revealed loss of integrity of cells in seminiferous tubules, vacuolation and presence of apoptotic bodies. Derangement of the outer dense fibres was noted in some sperm flagella. Acrosome formation appears to be normal. About 13.7% of epididymal spermatozoa had deformations like short tail or rounded head. This may explain the lower fertility index in NDGA‐treated group. No external deformations in newborns were noted. In conclusion, NDGA may have adverse effects on spermatogenesis.     

Biobanking efforts and new advances in male fertility preservation for rare and endangered species

Understanding and sustaining biodiversity is a multi-disciplinary science that benefits highly from the creation of organized and accessible collections of biomaterials (Genome Resource Banks). Large cryo-collections are invaluable tools for understanding, cataloging, and protecting the genetic diversity of the world''s unique animals and plants. Specifically, the systematic collection and preservation of semen from rare species has been developed significantly in recent decades with some biobanks now being actively used for endangered species management and propagation (including the introduction of species such as the black-footed ferret and the giant panda). Innovations emerging from the growing field of male fertility preservation for humans, livestock species, and laboratory animals are also becoming relevant to the protection and the propagation of valuable domestic and wild species. These new approaches extend beyond the “classical” methods associated with sperm freezing to include testicular tissue preservation combined with xenografting or in vitro culture, all of which have potential for rescuing vast amounts of unused germplasm. There also are other options under development that are predicted to have a high impact within the next decade (stem cell technologies, bio-stabilization of sperm cells at ambient temperatures, and the use of genomics tools). However, biobanking efforts and new fertility preservation strategies have to expand the way beyond mammalian species, which will offer knowledge and tools to better manage species that serve as valuable biomedical models or require assistance to reverse endangerment.     

Profertility effects of Shilajit on cadmium‐induced infertility in male mice

Shilajit is claimed as a Vajikarak (aphrodisiac) and used for the treatment of male infertility by traditional healers of the Indian subcontinent. Therefore, the present investigation was designed to assess the effectiveness of Shilajit for treatment of male infertility resulting from exposure to perilous chemicals. Effect of daily oral administration (p.o.) of Shilajit (50 mg, 100 mg and 200 mg/Kg BW) was investigated for a single spermatogenic cycle (35 days) in cadmium‐induced (2 mg/Kg BW, p.o. for 35 days) infertile adult (12–14 week) swiss male mice. Shilajit treatment increased weights of reproductive organs, testicular daily sperm production, activities of testicular Δ⁵ 3β‐HSD and 17 β‐HSD enzymes and serum level of testosterone. Histopathological evaluation of testis revealed that Shilajit restored spermatogenesis as reflected by a gradual augmentation in germ cell layers with increased doses of Shilajit compared to cadmium‐treated mice. Further, Shilajit treatment reverted back the adverse effects of cadmium on motility and concentration of spermatozoa. Secretory activities of the epididymis and seminal vesicle and libido, fertility and the number of litters per female were also improved by Shilajit in cadmium‐treated mice. Results thus suggest the potent androgenic nature of Shilajit and its role in fertility improvement against cadmium‐induced infertility.     

Pro-oxidative and anti-oxidative imbalance in human semen and its relation with male fertility

Oxidative stress is a common condition suffered by biological systems in aerobic conditions.Human semen also has its own molecular guard against the free radicals created by normal respiratory process or from immune reactions.The equilibrium of the creation and scavenging of free radicals is mandatory in the spermatozoa to fertilize and initiate a full-term pregnancy.The paper is a systematic review of publications that evaluate oxidative stress in semen.The Cochrane Library,Medline (1966-2003),Embase (1988-2003),SciSearch (1981-2003) and the conference papers were searched,When sperm samples from fertile and infertile males were analyzed,some of the mechanisms that determine the oxidative stress level were found to be impaired while others were unaltered.In conclusion,the literature as a whole provides contradictory findings and it is necessary to carry out more work to identify all the enzymatic and non-enzymatic systems involved in oxidative stress in the ejaculate,in order to develop new diagnostic systems and therapeutic strategies for combating detrimental free radical imbalance in the semen.(Asian J Androl 2004 Mar;6:59-65)     

育龄男性癌症患者生育力保护知识及需求调查

目的了解育龄男性癌症患者对于生育力保护的知识水平及生育力保护需求现况。方法采用自制的育龄男性癌症患者生育力保护知识水平及需求调查问卷对332例育龄男性癌症患者进行横断面调查。结果男性癌症患者生育力保护知识得分(3. 5±0. 7)分。77. 71%患者意识到癌症治疗会损害生育能力,63. 86%和80. 72%患者不知晓生育力保护的常规方法及场所。仅10. 54%患者在肿瘤治疗前选择精子库进行生育力保存,有68. 67%患者在治疗过程中希望进行生育力保存。多因素Logistic回归分析结果显示,家庭月收入、年龄是患者生育力保护知识水平的影响因素(均P 0. 05);年龄、有无子女是患者生育力保护需求的影响因素(均P 0. 05)。结论育龄男性癌症患者对生育力保护的知识水平不高,多数患者在治疗前并未选择生育力保护措施,但是在治疗期间有生育力保护的需求。年轻及家庭月收入≥8 000元的患者具备更多生育力保护知识。年轻及无子女的患者在治疗过程中有生育力保存的需求。医务人员需要在患者进行治疗前充分告知生育力保护的相关信息,全面评估患者对信息的掌握程度,为患者提供切实可行的生育力保护措施和指导。     

New perspectives on male fertility evaluation: Innovative approach for the qualitative analysis of spermatozoa

The identification of idiopathic infertility cases, actually, is impossible. Among new functional tests, developed to improve the male fertility diagnosis, the evaluation of spermatic myo-inositol (MI) level, known as Andrositol^® test (AT), is one of the most interesting, considering its weak economic burden and ease of use. The aim of this study was to evaluate the predictive power of AT and its potential use for a preliminary evaluation of semen samples. To evaluate the predictive power of AT, 87 sperm samples were analysed in comparison with spermiogram and sperm chromatin dispersion (SCD) Test, the gold standard analyses for male fertility evaluation. The application of AT resulted very useful for a preliminary sample evaluation, predicting the absence of DNA fragmentation in case of Low Responder samples precisely, and the presence of DNA fragmentation in case of medium or High Responder samples with abnormal morphology, predicting SCD results with a probability of 80% for Medium Responder sample and of 96.7% for High Responder sample. Considering the predictive power of this method, we could imagine, as preliminary qualitative analysis, its application before SCD test, deepening sperm analysis, improving the daily activities of laboratory operators and maintaining a good reliability of sperm evaluation.     

Mitochondria‐targeted antioxidant SkQ1 improves spermatogenesis in Immp2l mutant mice

Previous studies have confirmed that spermatogenesis in homozygous Immp2l mutant male mice was normal at the age of 6 months, but was significantly abnormal at the age of 13 months. Meanwhile, oxidative stress is reported to be involved in spermatogenic impairment in old mutant mice. However, it is unclear whether antioxidant treatment is a suitable intervention for improving spermatogenesis in old mutant mice. This study sought to investigate the effect of mitochondria‐targeted antioxidant SkQ1 on spermatogenesis in homozygous Immp2l mutant mice. Immp2l mutant mice were treated with the mitochondria‐targeted antioxidant SkQ1 from the age of 6 weeks until 13 months. SkQ1 treatment significantly improved spermatogenesis in old Immp2 l mutant mice. Moreover, SkQ1 treatment improved the morphology of testicular seminiferous tubules, significantly reduced the apoptosis of germ cells and increased the level of GPX4 expression in old Immp2 l mutant mice. In conclusion, our data suggest that the mitochondria‐targeted antioxidant SkQ1 is effective in improving spermatogenesis in Immp2 l mutant mice and might be used for the treatment of male infertility.     

MMP2, MMP9 and TIMP2 polymorphisms affect sperm parameters but not fertility in Polish males

Proper function of the blood–testis barrier is pivotal to spermatogenesis. Synchronised action of matrix metalloproteinases (MMP) and their inhibitors (TIMP) is mandatory to maintain dynamic balance of the barrier. Therefore, the association of functional genetic variants of MMP‐2, MMP‐9 and TIMP‐2 and male infertility was studied. A total of 416 infertile males and 421 healthy subjects were genotyped for 7 SNPs within MMP2, MMP9 and TIMP2 genes, along with the assessment of semen parameters (concentration, motility and morphology of spermatozoa). No association was observed between the studied genotypes and male infertility. However, higher sperm concentration was associated with TIMP2 rs8080623 C and rs2277698 T variants among infertile men, and with MMP9 rs17576 A minor allele in controls (p < .05). TIMP2 rs9900972 T and rs2277698 T allele were associated with higher percentage of morphologically normal spermatozoa among controls. MMP2 rs2285053 TT homozygous infertile patients presented higher percentage of spermatozoa displaying nonprogressive motility. Haplotype analysis revealed strong linkage disequilibrium between the studied loci (5 of 8 possible TIMP2 haplotypes, and 3 of 4 possible MMP2 and MMP9 were found). None of the haplotypes showed association with infertility. This study results suggest an association between MMP9 and TIMP2 SNPs with sperm parameters, but not infertility.     

E3 ubiquitin ligase ASB17 is required for spermiation in mice

BackgroundA major goal of spermiation is to degrade the apical ectoplasmic specialization (ES) junction between Sertoli cells and elongating spermatids in preparation for the eventual disengagement of spermatids into the lumen. E3 ubiquitin ligases mediate the process of ubiquitination and the subsequent proteasomal degradation, but their specific role during spermiation remains largely unexplored.MethodsAnkyrin repeat and SOCS box protein 17 (Asb17)-knockout mice were generated via a CRISPR/Cas9 approach. Epididymal sperm parameters were assessed by a computer-assisted sperm analysis (CASA) system, and morphological analysis of testicular tissues were performed based on histological and immunostaining staining, and transmission electron microscopy (TEM). The interactions between ASB17 and Espin (ESPN) were predicted by HawkDock server and validated through protein pull-down and immunoprecipitation assays.ResultsWe report that ASB17, an E3 ligase, is required for the completion of spermiation and that mice lacking Asb17 are oligozoospermic owing to spermiation failure. ASB17-deficient mice are fertile; however, spermatids exhibit a disorganized ES junction, resulting in retention within the seminiferous epithelium. Mechanistically, ASB17 deficiency leads to excess accumulation of ESPN, an actin-binding essential structural component of the ES. We determined that ASB17 regulates the removal of the ES through ubiquitin mediated protein degradation of ESPN.ConclusionsIn summary, our study describes a role for ASB17 in the regulation of cell-cell junctions between germ cells and somatic cells in the testis. These findings establish a novel mechanism for the regulatory role of E3 ligases during spermatogenesis.     

Potential mechanisms of SARS-CoV-2 action on male gonadal function and fertility: Current status and future prospects

The novel coronavirus was recognised in December 2019 and caught humanity off guard. The virus employs the angiotensin-converting enzyme 2 (ACE2) receptor for entry into human cells. ACE2 is expressed on different organs, which is raising concern as to whether these organs can be infected by the virus or not. The testis appears to be an organ enriched with levels of ACE2, while the possible mechanisms of involvement of the male reproductive system by SARS-CoV-2 are not fully elucidated. The major focus of the present studies is on the short-term complications of the coronavirus and gains importance on studying the long-term effects, including the possible effects of the virus on the male reproductive system. The aim of this review was to provide new insights into different possible mechanisms of involvement of male gonads with SARS-CoV-2 including investigating the ACE2 axis in testis, hormonal alterations in patients with COVID-19, possible formation of anti-sperm antibodies (ASA) and subsequently immunological infertility as a complication of SARS-CoV-2 infection. Finally, we suggest measuring the sperm DNA fragmentation index (DFI) as a determiner of male fertility impairment in patients with COVID-19 along with other options such as sex-related hormones and semen analysis. Invasion of SARS-CoV-2 to the spermatogonia, Leydig cells and Sertoli cells can lead to sex hormonal alteration and impaired gonadal function. Once infected, changes in ACE2 signalling pathways followed by oxidative stress and inflammation could cause spermatogenesis failure, abnormal sperm motility, DNA fragmentation and male infertility.     

Insulin-like growth factor 2 and its enterocyte receptor are not required for adaptation in response to massive small bowel resection

Purpose

Enhanced structural features of resection-induced intestinal adaptation have been demonstrated following the administration of multiple different growth factors and peptides. Among these, the insulin-like growth factor (IGF) system has been considered to be significant. In this study, we employ mutant mouse strains to directly test the contribution of IGF2 and its enterocyte receptor (IGF1R) toward the adaptation response to massive small bowel resection (SBR).

Methods

IGF2-knockout (IGF2-KO) (n = 8) and intestine specific IGF1R-knockout mice (IGF1R-IKO) (n = 9) and their wild type (WT) littermates (n = 5, n = 7, respectively) underwent 50% proximal SBR. At post-operative day 7, structural adaptation was measured as crypt depth and villus height. Rates of enterocyte proliferation and apoptosis were also recorded.

Results

The successful deletion of IGF2 and IGF1R expression in the enterocytes was confirmed by RT-PCR and Western blot, respectively. Normal adaptation occurred in both IGF2-KO and IGF1R-IKO mice after 50% SBR. Post-operative rates of proliferation and apoptosis in both IGF2-KO and IGF1R-IKO mice were no different than their respective controls.

Conclusion

IGF2 and functional IGF1R signaling in enterocytes are both dispensable for resection-induced adaptation responses. The mechanism for IGF-stimulation of intestinal adaptation may involve other ligands or cellular compartments within the intestine.