Arrest Of Spermatogenesis Research Articles

Heterozygous mutation of eEF1A1b resulted in spermatogenesis arrest and infertility in male tilapia, Oreochromis niloticus

Eukaryotic elongation factor 1 alpha (eEF1A) is an essential component of the translational apparatus. In the present study, eEF1A1b was isolated from the Nile tilapia. Real-time PCR and Western blot revealed that eEF1A1b was expressed highly in the testis from 90 dah (days after hatching) onwards. In situ hybridization and immunohistochemistry analyses showed that eEF1A1b was highly expressed in the spermatogonia of the testis. CRISPR/Cas9 mediated mutation of eEF1A1b resulted in spermatogenesis arrest and infertility in the F0 XY fish. Consistently, heterozygous mutation of eEF1A1b (eEF1A1b+/−) resulted in an absence of spermatocytes at 90 dah, very few spermatocytes, spermatids and spermatozoa at 180 dah, and decreased Cyp11b2 and serum 11-ketotestosterone level at both stages. Further examination of the fertilization capacity of the sperm indicated that the eEF1A1b+/− XY fish were infertile due to abnormal spermiogenesis. Transcriptomic analyses of the eEF1A1b+/− testis from 180 dah XY fish revealed that key elements involved in spermatogenesis, steroidogenesis and sperm motility were significantly down-regulated compared with the control XY. Transgenic overexpression of eEF1A1b rescued the spermatogenesis arrest phenotype of the eEF1A1b+/− testis. Taken together, our data suggested that eEF1A1b is crucial for spermatogenesis and male fertility in the Nile tilapia.

QUANTITATIVE KARYOLOGICAL ANALYSIS OF IMMATURE GERM CELLS FROM EJACULATE AS PART OF EXAMINATION OF PATIENTS WITH INFERTILITY IN MARRIAGE

Male factor contributes significantly to the problem of infertile marriages. Alterations of morphological and/or functional characteristics of germ cells can result in infertility. We analyzed 122 semen samples – 55 samples with normal (≥ 15 million/ml) sperm concentration (group I) and 67 samples with cryptozoospermia or severe oligozoospermia (concentration < 1 million/ml, group II). Standard semen analysis and quantitative karyological analysis of immature germ cells from ejaculate (QKA IGC) (patent L.F. Kurilo № 2328736, 2007) were performed for all samples. The results of QKA IGC are significantly different in the two groups analyzed. The proportion of IGC at stages of preleptotene-zygotene and pachytene is verified to be higher (p < 0.01) in the sperm samples with normal concentration (group 1), indicating spermatogenesis arrest at prophase I of meiosis. In the semen samples of group 2 (patients with cryptozoospermia or with severe oligozoospermia, sperm concentration < 1 million/ml) proportion of spermatocytes II and spermatids is significantly higher than in the group with normal sperm concentration, that indicates the spermatogenesis arrest at the stage of sperm formation (spermiogenesis). IGC index in 58 % of the samples from the group I is higher than normal IGC index. For the majority of the samples from the group II the IGC index was not estimated because of lack of spermatozoa. In all sperm samples with suspected azoospermia examined through QKA IGC spermatozoa were identified (44 samples from group II). Thus, if azoospermia is suspected, QKA IGC can be recommended to patients as an alternative to diagnostic testicular or epididimal biopsy. QKA IGC is unique in the assessment of the of spermatogenesis dynamics, as it is precise, safe, non-invasive and time-saving method that allows multiple repeated evaluation of IGC at different stages of spermatogenesis.

Experimental pathology of arsenic toxicity and its alleviation by Moringa oleifera in Japanese quails

Arsenic occurs naturally in water and soil and can be source of intoxication. The present experiment was carried out to explore the medicinal property of Moringa oleifera in restoration of toxic effects of arsenic in Japanese quails. Group T1 birds were fed with commercial diet and served as control while group T2 was treated with arsenic trioxide @ 300 ppm in the feed. The birds in group T3 and T4 were fed with Moringa oleifera seed powder at 5% and 10% level, respectively. The T5 and T6 group birds were fed with arsenic trioxide @ 300 ppm with Moringa oleifera powder at 5% and 10% level, respectively. Lowest carcass and organ weight was recorded in T2 group. Grossly, testes and ovary of T2 and T5 group birds were smaller in size compared to other groups. Microscopically, hydropic degeneration and fatty changes of liver, reduced numbers of glomeruli in kidneys, increased Virchow Robin space and neuronophagia in brain, severe destruction of mucosal villi of duodenum, arrest of spermatogenesis in testesand irregular sized ova in ovaries were observed in T2 group birds. The birds of T3 and T4 group and those fed arsenic with Moringa oleifera seeds powder (group T5 and T6) showed normal histological architecture of various organs and active spermatogenesis. Arsenic residual level in plasma as well as in the liver, kidneys, muscles and tibia bone was significantly higher in T2 group compared to the control group.

Short-term Effects of Date Palm Extract (Phoenix dactylifera) on Ischemia/Reperfusion Injury Induced by Testicular Torsion/Detorsion in Rats.

Background:Antioxidants are potent scavengers of free radicals and have beneficial effects on human health.Objective:The aim of this study was to investigate the potential protective antioxidant activity of the edible portion of date fruit extract in an experimental testicular torsion/detorsion (T/D) model in rats.Materials and Methods:To investigate the potential protective effects of date palm (DP), 30 male Spraque-Dawley rats were divided into three groups: sham-operated, T/D, and T/D + DP-treated (500 mg/kg, PO) groups. Testicular ischemia was induced via keeping the left testis under 720° clockwise torsion for 2 h (h), afterward, detorsion was performed. All rats were sacrificed 4 h after detorsion. Serum malondialdehyde (MDA) concentration, total oxidative status (TOS), total antioxidant status (TAS), oxidative stress index (OSI), and histopathological damage score were evaluated.Results:Serum MDA, TOS, and OSI levels rose significantly in the T/D group. These values were lower in the T/D + DP group. TAS values decreased significantly in T/D group and rose in T/D + DP group. Severe injury was seen in the twisted testes of T/D group. In contrast, ipsilateral-twisted testicular tissue in the DP-treated group showed moderate-to-mild changes. Contralateral testicular tissue in the T/D group had a mild-to-moderate tissue injury; meanwhile, treated group revealed normal-to-mild changes. Spermatogenesis was significantly improved in DP-treated group when compared with the T/D group.Conclusion:The findings suggest a possible protective effect of DP against testicular oxidative damage induced by T/D; however, more detailed studies are warranted.SUMMARYGiven the presence of several phenolic compounds possessing high antioxidant activity in DP, it could potentially be used to reduce testis ischemia/reperfusion-induced damage.Abbreviations Used: TAS: Total antioxidant status,TOS: Total oxidative status; OSI: Oxidative stress index; MDA: Malondialdehyde; C: Congestion; H: Hemorrhage, E: Edema; SG: Sloughed germinal cells; SA: Spermatogenesis arrest; STD: Seminiferous tubules disorganization; STA: Seminiferous tubules atrophy; G: Giant cells; T/D: Torsion/detorsion; DP: Date palm

Azoospermia and Testicular Hypoplasia in a Boar Carrier of a Novel Y-Autosome Translocation

Few sex-autosome chromosome abnormalities have been documented in domestic animal species. In humans, Y-autosome chromosome abnormalities may occur at a rate of 1/2,000 live births, whereas in the domestic pig only 2 Y-autosome reciprocal translocations have been previously described. During a routine cytogenetic screening of young boars, we identified a new Y-autosome translocation carrier, which after puberty showed semen devoid of sperm and testicular hypoplasia with spermatogenesis arrest. Whole chromosome painting by FISH analysis corroborated the reciprocal nature of the chromosomal exchanges between the Y chromosome and SSC13. The possible causes for the observed meiotic arrest of the carrier are reviewed.

TESTICULO-PROTECTIVE EFFECT OF MORINGAOLEIFERA SEED EXTRACT ON COPPER SULPHATE INDUCED INJURY IN WISTAR RATS

The aim of this study was to observe the testiculo-protective effect of aqueous extract of Moringaoleifera seed also known as Moringa seed on copper sulphate induced injury in Wistar rats. Twenty adult male Wistar rats (200-300g) were randomly selected into four groups (5 rats per group). Group A served as control group and were given 2ml/kg of distilled water. Group B was administered aqueous extract of Moringa seed only (200mg/kg body weight). Group C was administered aqueous extract of Moringa seed (200mg/kg body weight) and copper sulphate (100mg/kg body weight) concurrently. Group D was administered copper sulphate only (100mg/ kg body weight). All administrations lasted for three weeks. Results showed that in group D, there was proliferation of interstitial cells, degeneration of the seminiferous tubules as evidenced by hyalinization and blockage of the tubular lumen by immature spermatogenic series due to an arrest of spermatogenesis. There was also a significant decrease in testosterone level anda significant increase in lactate dehydrogenase level when compared with the control at p< 0.05. In group C rats, the results show mild interstitial connective tissue loosening and normal sequential maturation of spermatogenic series and normal levels of testosterone and lactate dehydrogenase. Conclusively, administration of copper sulphate causes testicular damage as evidenced by testicular hyalinization and arrest of spermatogenesis. However concurrent treatment of the rats with Moringa oleifera seed extract and copper sulphate have no effect on the testes, establishing the fact that Moringa oleifera seed extract offers a protective effect to the testes. Keywords: Testosterone; Moringa; copper sulphate; wistar rat; Moringa oleifera

Y-chromosomal genes affecting male fertility: A review.

The mammalian sex-chromosomes (X and Y) have evolved from autosomes and are involved in sex determination and reproductive traits. The Y-chromosome is the smallest chromosome that consists of 2-3% of the haploid genome and may contain between 70 and 200 genes. The Y-chromosome plays major role in male fertility and is suitable to study the evolutionary relics, speciation, and male infertility and/or subfertility due to its unique features such as long non-recombining region, abundance of repetitive sequences, and holandric inheritance pattern. During evolution, many holandric genes were deleted. The current review discusses the mammalian holandric genes and their functions. The commonly encountered infertility and/or subfertility problems due to point or gross mutation (deletion) of the Y-chromosomal genes have also been discussed. For example, loss or microdeletion of sex-determining region, Y-linked gene results in XY males that exhibit female characteristics, deletion of RNA binding motif, Y-encoded in azoospermic factor b region results in the arrest of spermatogenesis at meiosis. The holandric genes have been covered for associating the mutations with male factor infertility.

Transcription Factor RFX2 Is a Key Regulator of Mouse Spermiogenesis.

The regulatory factor X (RFX) family of transcription factors is crucial for ciliogenesis throughout evolution. In mice, Rfx1-4 are highly expressed in the testis where flagellated sperm are produced, but the functions of these factors in spermatogenesis remain unknown. Here, we report the production and characterization of the Rfx2 knockout mice. The male knockout mice were sterile due to the arrest of spermatogenesis at an early round spermatid step. The Rfx2-null round spermatids detached from the seminiferous tubules, forming large multinucleated giant cells that underwent apoptosis. In the mutants, formation of the flagellum was inhibited at its earliest stage. RNA-seq analysis identified a large number of cilia-related genes and testis-specific genes that were regulated by RFX2. Many of these genes were direct targets of RFX2, as revealed by chromatin immunoprecipitation-PCR assays. These findings indicate that RFX2 is a key regulator of the post-meiotic development of mouse spermatogenic cells.

Knockout of BRD7 results in impaired spermatogenesis and male infertility.

BRD7 was originally identified as a novel bromodomain gene and a potential transcriptional factor. BRD7 was found to be extensively expressed in multiple mouse tissues but was highly expressed in the testis. Furthermore, BRD7 was located in germ cells during multiple stages of spermatogenesis, ranging from the pachytene to the round spermatid stage. Homozygous knockout of BRD7 (BRD7−/−) resulted in complete male infertility and spermatogenesis defects, including deformed acrosomal formation, degenerative elongating spermatids and irregular head morphology in postmeiotic germ cells in the seminiferous epithelium, which led to the complete arrest of spermatogenesis at step 13. Moreover, a high ratio of apoptosis was determined by TUNEL analysis, which was supported by high levels of the apoptosis markers annexin V and p53 in knockout testes. Increased expression of the DNA damage maker λH2AX was also found in BRD7−/− mice, whereas DNA damage repair genes were down−regulated. Furthermore, no or lower expression of BRD7 was detected in the testes of azoospermia patients exhibiting spermatogenesis arrest than that in control group. These data demonstrate that BRD7 is involved in male infertility and spermatogenesis in mice, and BRD7 defect might be associated with the occurrence and development of human azoospermia.

Blood-testis barrier and Sertoli cell function: lessons from SCCx43KO mice.

The gap junction protein connexin43 (CX43) plays a vital role in mammalian spermatogenesis by allowing for direct cytoplasmic communication between neighbouring testicular cells. In addition, different publications suggest that CX43 in Sertoli cells (SC) might be important for blood-testis barrier (BTB) formation and BTB homeostasis. Thus, through the use of the Cre-LoxP recombination system, a transgenic mouse line was developed in which only SC are deficient of the gap junction protein, alpha 1 (Gja1) gene. Gja1 codes for the protein CX43. This transgenic mouse line has been commonly defined as the SC specific CX43 knockout (SCCx43KO) mouse line. Within the seminiferous tubule, SC aid in spermatogenesis by nurturing germ cells and help them to proliferate and mature. Owing to the absence of CX43 within the SC, homozygous KO mice are infertile, have reduced testis size, and mainly exhibit spermatogenesis arrest at the level of spermatogonia, seminiferous tubules containing only SC (SC-only syndrome) and intratubular SC-clusters. Although the SC specific KO of CX43 does not seem to have an adverse effect on BTB integrity, CX43 influences BTB composition as the expression pattern of different BTB proteins (like OCCLUDIN, β-CATENIN, N-CADHERIN, and CLAUDIN11) is altered in mutant males. The supposed roles of CX43 in dynamic BTB regulation, BTB assembly and/or disassembly and its possible interaction with other junctional proteins composing this unique barrier are discussed. Data collectively indicate that CX43 might represent an important regulator of dynamic BTB formation, composition and function.

Alleviating effect of Mallotus roxburghianus in heat-induced testicular dysfunction in Wistar rats

Context: Hyperthermia causes detrimental effects on the testes leading to fertility problems. Mallotus roxbhurghianus Muell. Arg. (Euphorbiaceae) is used in traditional medicine and possesses antioxidant property. However, the mechanisms remain unknown in the context of alleviative action of M. roxburghianus against heat stress.Objective: The objective of this study was to demonstrate the alleviating activity of M. roxburghianus and its mechanism in scrotal hyperthermia.Materials and methods: Scrotal hyperthermia experiments were performed in three groups (n = 7 per group) consisting of (i) the control group (C) maintained at 22 °C for 30 min, (ii) the heat stress-induced group (HS), and (iii) the heat stress-induced M. roxburghianus-treated group (HSM – 400 mg/kg each) in a thermostatically controlled water bath at 43 °C for 30 min. Subsequent to the heat treatment HS group, rats were treated with saline p.o and methanol extract of M. roxburghianus was administered to the rats of HSM group along with their standard food for 14 d. Scrotal hyperthermic effects were evaluated.Results: Scrotal hyperthermia significantly (p < 0.0001) elevated malondialdehyde levels while decreasing the body and testes weights, serum testosterone, and antioxidant enzyme levels due to oxidative stress. Disorganisation of seminiferous tubules and arrest of spermatogenesis were observed in the HS group. The administration of methanol extract of M. roxburghianus (400 mg/kg) for 14 d after heat treatment significantly suppressed the lipid peroxidation, restored the antioxidant enzyme and testosterone levels, revived the spermatogenesis, and increased the cell proliferation activity in the HSM group.Discussion and conclusion: The methanol extract of M. roxburghianus accelerates testicular recovery from the damaging influence of hyperthermia.

Semen parameters in polyzoospermic men

Polyzoospermia is extremely high (above 250 mln/ml) sperm concentration with normal volume of ejaculate. In the laboratory of genetic disorders of reproduction of Research Centre for Medical Genetics in 2007–2012 years we analyzed more than 14 000 semen samples from infertile men and men with reproductive disorders in order to study the distribution and characteristics of spermatogenesis in polyzoospermic men. Only polyzoospermic men semen analysis results were interpretered in present article. Polyzoospermia (sperm count over 250 mln/ml in volume over 1,5 ml) was detected in 191 semen samples (1.3 %). At the same time 15 % of the samples with polyzoospermia were normozoospermic. Among 85 % of the sperm pathology samples asthenozoospermia occured most frequently (77 %), astenoteratozoospermia was detected in 8 % of cases with polyzoospermia. The average proportion of vitality was 90.09 ± 10.02 %, normal morphology – 14.93 ± 8.51 %, and progressive motility – 17.72 ± 11.81 %. The average concentration of spermatozoa in the ejaculate in the examined samples was 313.29 ± 64.78 mln/ml. More than half of the tested samples with polyzoospermia had a concentration 250–300 mln/ml. Concentration of 450 mln/ml and more detected in 3 % of samples. The maximum total number of spermatozoa in the ejaculate in our study was more than 2 billion sperm cells and was observed in two polyzoospermic men. One of these patients had the maximum concentration (615 mln/ml) also. We found a high correlation (r = 0.89; p &lt; 0.01) between the volume of ejaculate and the total number of spermatozoa. Immature germ cells from ejaculate were estimated in 7 patients. There was a partial spermatogenesis arrest at meiosis I prophase in 5 out 7 of the examined semen samples.

Effects of psychological stress on male fertility.

Psychological stress can be defined as any uncomfortable 'emotional experience' accompanied by predictable biochemical, physiological and behavioural changes or responses. Many clinical studies looking at the effects of psychological stress on male fertility have shown that stress is associated with reduced paternity and abnormal semen parameters. Enough scientific evidence exists to suggest that psychological stress could severely affect spermatogenesis, mainly as a result of varying testosterone secretion. The hypothalamic-pituitary-adrenal axis has a direct inhibitory action on the hypothalamic-pituitary-gonadal (HPG) axis and Leydig cells in the testes. The newly discovered hormone, gonadotropin-inhibitory hormone (GnIH), also has an inhibitory effect on the HPG axis. Inhibition of the HPG axis results in a fall in testosterone levels, which causes changes in Sertoli cells and the blood-testis barrier, leading to the arrest of spermatogenesis. Germ cells also become vulnerable to gonadotoxins and oxidation. However, the extent and severity of the effects of psychological stress on human testes is difficult to study and data mostly come from animal models. Despite this limitation, stress as a causative factor in male infertility cannot be ignored and patients should be made aware of its effects on testicular function and fertility and helped to manage them.

Boule gene expression underpins the meiotic arrest in spermatogenesis in male rainbow trout (Oncorhynchus mykiss) exposed to DEHP and butachlor

Boule, the ancestor of the DAZ (Deleted in AZoospermia) gene family, in most organisms is mainly involved in male meiosis. The present study investigates the effects of the plasticizer DEHP (50mg/kg body weight) and herbicide butachlor (0.39mg/L) on male rainbow trout (Oncorhynchus mykiss) for a 10-day period in two independent experiments. The results showed that plasma testosterone (T) concentrations were significantly lower in fish exposed to either DEHP or butachlor compared to the control fish (P<0.05). Fish showed a significantly elevated hepatosomatic index (HSI) in the butachlor treatment (P<0.05). However, no significant difference was observed in HSI values in the DEHP treatment (P>0.05). In addition, no significant differences were found in the gonadosomatic index (GSI) in both DEHP and butachlor treatments (P>0.05). Histologically, testes of male trout in the control groups were well differentiated and filled with large numbers of cystic structures containing spermatozoa. In contrast, the testes of male trout contained mostly spermatocytes with few spermatozoa in both treated group, suggesting that DEHP and butachlor may inhibit the progression of meiosis. Also, boule gene expression was significantly lower in the testes of male trout affected by DEHP and butachlor in comparison with their control groups (P<0.05), which confirmed the meiotic arrest in affected trout. Based on the results, the present study demonstrated that DEHP and butachlor can inhibit the progression of spermatogenesis in male trout, potentially by causing an arrest of meiosis, maybe due to down-regulation of boule gene expression through T and/or IGF1 via ERK1/2 signaling in T-independent pathways. In addition, these results confirmed that boule can be considered as a predictive marker to assess meiotic efficiency.

Complementary expression and phosphorylation of Cx46 and Cx50 during development and following gene deletion in mouse and in normal and orchitic mink testes.

Gap junction-mediated communication helps synchronize interconnected Sertoli cell activities. Besides, coordination of germ cell and Sertoli cell activities depends on gap junction-mediated Sertoli cell-germ cell communication. This report assesses mechanisms underlying the regulation of connexin 46 (Cx46) and Cx50 in mouse testis and those accompanying a "natural" seasonal and a pathological arrest of spermatogenesis, resulting from autoimmune orchitis (AIO) in mink. Furthermore, the impact of deleting Cx46 or Cx50 on the expression, phosphorylation of junction proteins, and spermatogenesis is evaluated. Cx46 mRNA and protein expression increased, whereas Cx50 decreased with adulthood in normal mice and mink. Cx46 mRNA and protein expression increased, whereas Cx50 decreased with adulthood in normal mice and mink. During the mink active spermatogenic phase, Cx50 became phosphorylated and localized to the site of the blood-testis barrier. By contrast, Cx46 was dephosphorylated and associated with annular junctions, suggesting phosphorylation/dephosphorylation of Cx46 and Cx50 involvement in the barrier dynamics. Cx46-positive annular junctions in contact with lipid droplets were found. Cx46 and Cx50 expression and localization were altered in mink with AIO. The deletion of Cx46 or Cx50 impacted on other connexin expression and phosphorylation and differently affected tight and adhering junction protein expression. The level of apoptosis, determined by ELISA, and a number of Apostain-labeled spermatocytes and spermatids/tubules were higher in mice lacking Cx46 (Cx46-/-) than wild-type and Cx50-/- mice, arguing for life-sustaining Cx46 gap junction-mediated exchanges in late-stage germ cells secluded from the blood by the barrier. The data show that expression and phosphorylation of Cx46 and Cx50 are complementary in seminiferous tubules.

Cytological evaluation of spermatogenesis: a novel and simple diagnostic method to assess spermatogenesis in non-obstructive azoospermia using testicular sperm extraction specimens.

Most of the non-obstructive azoospermia (NOA)-patients have only focal spermatogenesis which results in insufficient numbers of spermatozoa to reach the ejaculate. In ≈50% of these NOA-patients testicular sperm extraction (TESE) is successful and intracytoplasmic sperm injection (ICSI) is pursued. We studied whether (i) spermatogenesis can be evaluated by defining the ratios between Sertoli cells, pachytene spermatocytes and spermatozoa in a testicular cell suspension, and (ii) these ratios are associated with the outcome of fertility treatment. A retrospective cohort study was conducted between June 2007 and August 2012. In this period, 441 consecutive ICSI-TESE cycles were performed in 212 couples. For each TESE biopsy, the ratios between Sertoli cells, pachytene spermatocytes and spermatozoa were calculated. A control population of 32 vasectomized men was used to define cut-off values for complete spermatogenesis. Based on the pachytene to sperm ratio (P/Sp) and number of spermatozoa per 100 Sertoli cells (#Sp/100SC) groups were defined as complete spermatogenesis, hypospermatogenesis and partial maturation arrest (MA). Validation of the cytological diagnoses was performed by comparing the results of cytology to the histological evaluation of spermatogenesis in 40 cases. In 92.5%, a perfect match was observed and in the three remaining cases cytology corresponded well with the results of TESE. Couples with complete spermatogenesis have a higher ongoing pregnancy rate after the first treatment cycle compared to couples with hypospermatogenesis (34 vs. 16%; p=0.02) and partial MA (34 vs. 19%; p=0.11). In conclusion, pachytene spermatocytes, spermatozoa and Sertoli cells can be easily identified and counted in a cell suspension and their ratios can be successfully used to diagnose the level of spermatogenic impairment. This pilot study indicates that once successful spermatozoa retrieval is achieved, treatment outcome declines when spermatogenesis is impaired in NOA. The predictive value of cytological evaluation of spermatogenesis has to be established in a future prospective trial.

An ENU-induced mutation in the mouse Rnf212 gene is associated with male meiotic failure and infertility

The ENU-induced repro57 mutation was identified in an unbiased screen for the discovery of novel genes for fertility. Male repro57 homozygous mice are infertile and exhibit significantly reduced testis weight compared with WT mice. Histological examination of mutant testes revealed that spermatocytes degenerated during late prophase, and no mature spermatozoa were found in the seminiferous epithelium, suggesting that infertility is caused by the arrest of spermatogenesis at late meiotic prophase. Consistent with this hypothesis, the number of foci with MLH1, a protein essential for crossing over, is greatly reduced in repro57 mutant spermatocytes, which also lack chiasmata between homologs and exhibit premature dissociation of XY chromosomes. In repro57 mutant mice, we identified a mutation in the Rnf212 gene, encoding Ring finger protein 212. The overall phenotype of repro57 mice is consistent with the recently reported phenotype of the Rnf212 knockout mice; slight differences may be due to genetic background effects. Thus, the repro57 nonsense mutation provides a new allele of the mouse Rnf212 gene.

High‐energy diets: a threat for male fertility?

Male fertility is declining in developed countries, as well as in developing countries. External factors linked to lifestyle, such as eating disorders, negatively affect spermatogenesis, both at central and gonadal levels. The overconsumption of high-energy diets (HED) alters the functioning of the male reproductive axis and consequently affects the testicular physiology, disrupting its metabolism and bioenergetic capacity. Testicular metabolism presents unique characteristics, partly because of its cellular heterogeneity and to the specific functions that each cell type plays within the testicular environment. Disruption of the tightly regulated metabolic pathways leads to adverse reproductive outcomes, such as inefficient energy supply to germ cells, sperm defects or spermatogenesis arrest. Testicular metabolic alterations induced by HED intake may also lead to mitochondrial dysfunction, which is closely associated to reactive oxygen species (ROS) overproduction and oxidative stress. ROS easily target spermatozoa DNA and lipids, contributing to decreased sperm quality. Thus, understanding the detrimental effects of HED overconsumption on the pathways underlying testicular metabolism and sperm production is imperative; otherwise, one may favour a transgenerational amplification of subfertility. Herein, we present an up-to-date overview of the effects of HED on testicular metabolism, sperm parameters and the subsequent consequences for male fertility.

AB193. Isolation and long-term culture of human spermatogonia stem cell

ObjectiveIn vitro culture of spermatogonia stem cell would be benefit to the research on spermatogenesis mechanism. For those who suffer from azoospermia with a spermatogenesis arrest, there would be a hope to gain functional gamete via inducing their own spermatogonia stem cells if we could achieve long-term culture of these cells in vitro. However, in spite of that the rodent spermatogonia stem cells had been cultured in vitro over months successfully, there was few report on the long-term culture of human spermatogonia stem cells, which was mainly due to the fragile of human spermatogonia cells and the critical culture condition essential for them. This work aimed to explore a gentle way to isolate human spermatogonia stem cells and achieve a long-term in vitro culture of them.DesignExploratory research.Materials and methodsHuman testis tissues of obstructive azoospermia patients were obtained via surgery and digested into cell suspension via two-step enzymatic digestion. Matrix selection was applied to isolate the human spermatogonia stem cells. SG medium was used as the condition medium for human spermatogonia stem cell culture. Immunofluorescence was carried out to characterize the isolated cells.ResultsThe isolated cells survived over 3 weeks in vitro with a good morphology that was identical with human spermatogonia stem cells and some of them former colonies. Immunofluorescence showed that over 90% isolated cells expressed the spermatogonia stem cell markers such as GPR125, UCHL1, PLZF and GFRα-1.ConclusionsMatrix selection would be a good way to isolated human spermatogonia stem cells and SG medium was suitable for the long-term culture of these cells. Improvements should be made for a longer period of culture and a higher purity.SupportThis work was supported by China National Key Project (2010CB945200 to ZL), a key grant from National Nature Science Foundation of China (31230048 to ZH), and grants from National Science Foundation of China (31171422 to ZH)

Isolation and long-term culture of human spermatogonia stem cell

In vitro culture of spermatogonia stem cell would be benefit to the research on spermatogenesis mechanism. For those who suffer from azoospermia with a spermatogenesis arrest, there would be a hope to gain functional gamete via inducing their own spermatogonia stem cells if we could achieve long-term culture of these cells in vitro. However, in spite of that the rodent spermatogonia stem cells had been cultured in vitro over months successfully, there was few report on the long-term culture of human spermatogonia stem cells, which was mainly due to the fragile of human spermatogonia cells and the critical culture condition essential for them. This work aimed to explore a gentle way to isolate human spermatogonia stem cells and achieve a long-term in vitro culture of them. Exploratory research. Human testis tissues of obstructive azoospermia patients were obtained via surgery and digested into cell suspension via two-step enzymatic digestion. Matrix selection was applied to isolate the human spermatogonia stem cells. SG medium was used as the condition medium for human spermatogonia stem cell culture. Immonuflorescence was carried out to characterize the isolated cells. The isolated cells survived over 4 weeks in vitro with a good morphology that was identical with human spermatogonia stem cells and some of them formed colonies. Immonuflorescence showed that over 90% isolated cells expressed the spermatogonia stem cell markers such as GPR125, UCHL1, PLZF and GFRα-1. Matrix selection would be a good way to isolated human spermatogonia stem cells and SG medium was suitable for the long-term culture of these cells. Improvements should be made for a longer period of culture and a higher purity.