Beta-defensin 126 on the surface of macaque sperm mediates attachment of sperm to oviductal epithelia

Beta-defensin 126 (DEFB126) coats the entire surface of macaque sperm until sperm become capacitated, and the removal of DEFB126 from over the head of sperm is required for sperm-zona recognition. Viable sperm collected from cervix and the uterine lumen of mated female macaques had DEFB126 coating the entire surface, suggesting that DEFB126 is retained on sperm en route to the oviduct. DEFB126 plays a major role in attachment of sperm to oviductal epithelial cells (OECs). Following treatment to either remove or alter DEFB126, sperm were coincubated with explants of OECs, which were assessed for sperm binding following rinsing to remove superficially attached sperm. Sperm treated with either 1 mM caffeine + 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) (induces capacitation and complete release of DEFB126 from sperm), 2 mM caffeine (removes DEFB126 from over the head and midpiece but does not induce capacitation), anti-DEFB126 immunoglobulin, or neuraminidase (cleaves sialic acid from terminal positions on glycosylation sites of DEFB126) resulted in similar and significant levels of inhibition of sperm-OEC binding. Preincubation of OECs with soluble DEFB126 also resulted in significantly reduced sperm-OEC binding. Furthermore, reduced OEC binding capability of sperm lacking DEFB126 could be restored by addition of soluble DEFB126 to the sperm surface prior to incubation with OECs. Finally, purified DEFB126, infused into oviducts in situ, associated primarily with the apical membranes of secretory-type epithelial cells. In summary, treatments of macaque sperm that result in either removal, masking, or alteration of DEFB126 result in loss of sperm-OEC binding that is independent of changes in sperm motility. DEFB126 may be directly involved in the formation of a reservoir of sperm in the oviduct of macaques.     

ENU‐induced mutant allele of Dnah1, ferf1, causes abnormal sperm behavior and fertilization failure in mice

Given attention to both contraception and treatment of infertility, there is a need to identify genes and sequence variants required for mammalian fertility. Recent unbiased mutagenesis strategies have expanded horizons of genetic control of reproduction. Here we show that male mice homozygous for the ethyl‐nitroso‐urea‐induced ferf1 (fertilization failure 1) mutation are infertile, producing apparently normal sperm that does not fertilize oocytes in standard fertilization in vitro fertilization assays. The ferf1 mutation is a single‐base change in the Dnah1 gene, encoding an axoneme‐associated dynein heavy chain, and previously associated with male infertility in both mice and humans. This missense mutation causes a single‐amino‐acid change in the DNAH1 protein in ferf1 mutant mice that leads to abnormal sperm clumping, aberrant sperm motility, and the inability of sperm to penetrate the oocyte's zona pellucida; however, the ferf1 mutant sperm is competent to fertilize zona‐free oocytes. Taken together, the various mutations affecting the DNAH1 protein in both mouse and human produce a diversity of phenotypes with both subtle and considerable differences. Thus, future identification of the interacting partners of DNAH1 might lead to understanding its unique function among the sperm dyneins.     

A novel mutation in PCD-associated gene DNAAF3 causes male infertility due to asthenozoospermia

Primary ciliary dyskinesia (PCD) is a rare autosomal-recessive disease manifested with recurrent infections of respiratory tract and infertility. DNAAF3 is identified as a novel gene associated with PCD and different mutations in DNAAF3 results in different clinical features of PCD patients, such as situs inversus, sinusitis and bronchiectasis. However, the sperm phenotypic characteristics of PCD males are generally poorly investigated. Our reproductive medicine centre received a case of PCD patient with infertility, who presented with sinusitis, recurrent infections of the lower airway and severe asthenozoospermia; However, no situs inversus was found in the patient. A novel homozygous mutation in DNAAF3(c.551T>A; p.V184E) was identified in the PCD patient by whole-exome sequencing. Subsequent Sanger sequencing further confirmed that the DNAAF3 had a homozygous missense variant in the fifth exon. Transmission electron microscopy and immunostaining analysis of the sperms from the patient showed a complete absence of outer dynein arms and partial absence of inner dynein arms, which resulted in the reduction in sperm motility. However, this infertility was overcome by intracytoplasmic sperm injections, as his wife achieved successful pregnancy. These findings showed that the PCD-associated pathogenic mutation within DNAAF3 also causes severe asthenozoospermia and male infertility ultimately due to sperm flagella axoneme defect in humans. Our study not only contributes to understand the sperm phenotypic characteristics of patients with DNAAF3 mutations but also expands the spectrum of DNAAF3 mutations and may contribute to the genetic diagnosis and therapy for infertile patient with PCD.     

Deficiency of TPPP2, a factor linked to oligoasthenozoospermia,causes subfertility in male mice

Oligoasthenozoospermia is a major cause of male infertility; however, its etiology and pathogenesis are unclear and may be associated with specific gene abnormalities. This study focused on Tppp2 (tubulin polymerization promoting protein family member 2), whose encoded protein localizes in elongating spermatids at stages IV‐VIII of the seminiferous epithelial cycle in testis and in mature sperm in the epididymis. In human and mouse sperm, in vitro inhibition of TPPP2 caused significantly decreased motility and ATP content. Studies on Tppp2 knockout (KO) mice demonstrated that deletion of TPPP2 resulted in male subfertility with a significantly decreased sperm count and motility. In Tppp2^?/? mice, increased irregular mitochondria lacking lamellar cristae, abnormal expression of electron transfer chain molecules, lower ATP levels, decreased mitochondrial membrane potential and increased apoptotic index were observed in sperm, which could be the potential causes for its oligoasthenozoospermia phenotype. Moreover, we identified a potential TPPP2‐interactive protein, eEf1b (eukaryotic translation elongation factor 1 beta), which plays an important role in protein translation extension. Thus, TPPP2 is probably a potential pathogenic factor in oligoasthenozoospermia. Deficiency of TPPP2 might affect the translation of specific proteins, altering the structure and function of sperm mitochondria, and resulting in decreased sperm count, motility and fertility.     

Choline dehydrogenase polymorphism rs12676 is a functional variation and is associated with changes in human sperm cell function

Approximately 15% of couples are affected by infertility and up to half of these cases arise from male factor infertility. Unidentified genetic aberrations such as chromosomal deletions, translocations and single nucleotide polymorphisms (SNPs) may be the underlying cause of many cases of idiopathic male infertility. Deletion of the choline dehydrogenase (Chdh) gene in mice results in decreased male fertility due to diminished sperm motility; sperm from Chdh(-/-) males have decreased ATP concentrations likely stemming from abnormal sperm mitochondrial morphology and function in these cells. Several SNPs have been identified in the human CHDH gene that may result in altered CHDH enzymatic activity. rs12676 (G233T), a non-synonymous SNP located in the CHDH coding region, is associated with increased susceptibility to dietary choline deficiency and risk of breast cancer. We now report evidence that this SNP is also associated with altered sperm motility patterns and dysmorphic mitochondrial structure in sperm. Sperm produced by men who are GT or TT for rs12676 have 40% and 73% lower ATP concentrations, respectively, in their sperm. rs12676 is associated with decreased CHDH protein in sperm and hepatocytes. A second SNP located in the coding region of IL17BR, rs1025689, is linked to altered sperm motility characteristics and changes in choline metabolite concentrations in sperm.     

Hereditary haemochromatosis gene (HFE) H63D mutation shows an association with abnormal sperm motility

The aim of this study was to screen infertile men for HFE H63D mutation in correlation with clinical characteristics of infertile men (sperm concentration, sperm motility, morphology, testicular volume, Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH) and total Testosterone levels) and find out if the HFE H63D mutation has an effect on male infertility. After excluding hormonal treatment, any scrotal pathology, having any systemic diseases such as diabetes mellitus, sickle cell anemia and microdeletions of the Y chromosome, a total of 148 infertile men with age range between 17 and 52-years-old (average age 29.6 ± 7.2) were enrolled into the study. Our analysis indicates that the mean FSH levels are significantly higher (6.3 ± 4.6 mIU/ml, P = 0.03), whereas sperm motility is significantly lower (36.6 ± 28.1%, P = 0.01) in the infertile men with the HFE H63D mutation compared with subjects lacking this mutation. Comparison of allele frequencies of the infertile men with Ts < 50% versus the infertile men with Ts > 50% revealed a significant difference as expected (P = 0.001, OR = 0.14, %95 CI = 0.04–0.44). Comparison of allele frequencies of infertile men with abnormal sperm motility versus infertile men with normal sperm motility revealed a highly significant difference (P = 0.005, OR = 3.11, %95 CI = 1.41–6.86). Thus, the HFE H63D mutation seems to be an important risk factor for impaired sperm motility and is clinically associated with male infertility.     

精子端粒长度与特发性男性不育相关

端粒是真核生物染色体末端的多功能特异性DNA-蛋白结构,覆盖在染色体末端,保护基因组的稳定性。端粒在减数分裂过程中起到了十分重要的作用,协助染色体配对、联会、同源重组和分离。精子中的端粒可能在精子的受精能力和胚胎发育中起到重要作用。近年来,端粒与生殖的相关性研究成为一个新的热点,但精子端粒与男性不育间的相关性并不明确。本文采用实时荧光定量PCR方法检测中国特发性男性不育人群(126例)和正常可育男性人群(138例)的精子相对端粒长度,结果发现,特发性男性不育病例的精子平均相对端粒长度(2.894±0.115)低于正常对照组(4.016±0.603),差异具有统计学意义(P=5.097×10^-5);并且精子相对端粒长度与精子密度、精子总数和精子活力都有显著的相关性：精子数量较多和/或精子活力较高,精子相对端粒长度较长。研究结果提示,在中国人群中,精子端粒长度与特发性男性不育具有相关性,精子的端粒长度可能影响精子发生和精子的功能,精子端粒的缩短导致精子数目及活力的降低从而导致男性不育。     

The Carbohydrate Structure of DEFB126, the Major Component of the Cynomolgus Macaque Sperm Plasma Membrane Glycocalyx

Based on the amino-acid sequence of the macaque epididymal secretory protein, ESP 13.2 (Q9BEE3/AJ236909), it has now been classified as β-defensin DEFB126. DEFB126 is one of the five β-defensins with genes that are clustered along chromosome 20pl3, and all five proteins have an extended carboxy terminus that continues beyond the 6-cysteine β-defensin core region. This 60-amino acid carboxyl tail extension of the DEFB126 molecule is extraordinarily rich in threonine and serine (40%), many of which appear to be likely candidates for having O-glycosylation. DEFB126 has been shown to coat the entire surface of cynomolgus macaque sperm as they move through the corpus/caudal region of the epididymis. It is a major glycocalyx barrier to the external environment and is retained until the completion of capacitation. Sperm exposed to fluorescein-conjugated poly-L-lysine or Alexa488-histone showed a very uniform fluorescent labeling pattern over the entire sperm surface, almost identical to that observed with anti-DEFB126 Ig label. Sperm surface components that were released following treatment with caffeine/cAMP (in vitro capacitation) were blotted and probed with three different lectins which are known to recognize terminal sialic acid residues, and all three recognized the 35 kDa DEFB126 band. Neuraminidase treatment of sperm shifted the molecular weight of DEFB126 from 34–36 kDa to approximately 38–40 kDa and removed or greatly inhibited sialic acid-specific lectin recognition. O-Glycanase treatment alone was ineffective at removal of the oligosaccharides, but prior treatment with neuraminidase was sufficient to enable the O-glycanase treatment to effectively change the apparent molecular weight to 10 kDa, confirming that a major portion of the molecular mass is associated with the carbohydrate portion. Western blots of neuraminidase-treated DEFB126 showed strong recognition with a number of lectins that identify β-galactose and also lectins that recognize the N-acetylgalactosamine-serine/threonine, the proposed connection site of O-glycosylation. All of the lectins that recognized DEFB126 on Western blots were used to fluorescently probe sperm. The fluorescent patterns that were observed with poly-L-lysine, Alexa488-histone, sialic acid-specific lectins, and galactose-specific lectins showed even distributions over the entire sperm surface and the patterns were identical to sperm labeled with anti-DEFB126 Ig, and all but the antibody did not recognize neuraminidase-treated sperm.     

Purification and characterization of a motility initiating protein from caprine epididymal plasma

Numerous reports have appeared on the occurrence of undefined protein factors in male reproductive fluids that promote motility of mature sperm and initiate forward motility in the immature (immotile) caput‐epididymal sperm. This study reports for the first time purification to apparent homogeneity of a motility initiating protein (MIP) from epididymal plasma and its characterization using the caprine sperm model. It is a 125 kDa (approximately) dimeric protein made up of two subunits: 70 and 54 kDa. MIP is an acidic protein with an isoelectric point of 4.75. The motility protein at 30 µg/ml (240 nM) level showed nearly maximal motility‐promoting activity. MIP is heat stable and it is maximally active at pH 8. It is a glycoprotein that binds with high affinity to concanavalin A and it contains mannose, galactose, and N‐acetyl glucosamine approximately in the ratios of 6:1:6. It is sensitive to the actions of α‐mannosidase and β‐N‐acetylglucoseaminidase thereby demonstrating that the sugar side chain of the glycoprotein is essential for its biological activity. Epididymal plasma is its richest source. It is also capable of enhancing forward motility of mature cauda‐sperm. Its antibody markedly inhibits sperm motility. MIP antibody is highly immunospecific and it recognizes both the subunits. MIP causes significant increase of the intrasperm level of cyclic AMP. MIP: the physiological motility‐activating protein has potential for use as a contraceptive vaccine and for solving some of the problems of human infertility and animal breeding. J. Cell. Physiol. 222:254–263, 2010. © 2009 Wiley‐Liss, Inc.     

Oxidation of glyceraldehyde-3-phosphate dehydrogenase decreases sperm motility

The relation between the activity of the sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) and the motility of sperms was investigated. It was found that the mean value of GAPDS activity in sperm samples with low motility is 2.5–3-fold lower than that in samples with high motility. Sperm motility was shown to diminish in the presence of superoxide anion, hydroxyl radical, and hydrogen peroxide. The decrease in sperm motility in the presence of hydrogen peroxide was proportional to the concentration of the oxidant and correlated with the decrease in GAPDS activity (r = 0.96). Based on the literature data on the importance of GAPDS for the motility of sperms together with the presented observations, it was concluded that the decrease in the sperm motility in the presence of reactive oxygen species is due to the oxidation of GAPDS and inhibition of glycolysis.     

Deficiency in fertilization by morphologically abnormal sperm produced by Azh mutant mice

Male mice homozygous for the azh mutation produce spermatozoa with abnormal head shapes and have significantly reduced fecundity, to between 5% and 10% that of wild-type or heterozygous mice. Several possible causes of this infertility were investigated. No gross endocrine disorders in azh/azh male mice were observed, and they exhibited apparently normal mating behavior. In addition, their sperm were motile, were capable of hyperactivated motility, and did not show premature acrosome reactions. However, quantitative analysis revealed slight but significant reductions in several motility parameters. Analysis of embryos following mating of azh/azh males with superovulated females indicated a reduction in the number of fertilized eggs compared to control matings. In vitro, spermatozoa from azh/azh mice failed to fertilize cumulus-intact/zona-intact and cumulus-free/zona-intact ova, although they successfully fertilized zonafree ova. These results indicate that the primary defect in fertility of azh/azh male mice is a result of sperm quality, likely, in sperm morphology, and is manifest at the level of interaction with the zona pellucida. © 1994 Wiley-Liss, Inc.     

Normal fertility in male mice with deletion of β‐catenin gene in germ cells

As a dual function protein, β‐catenin affects both cell adhesion and mediates canonical Wnt/β‐catenin cell signaling. β‐Catenin is prominently expressed in somatic Sertoli cells in the testis and postmeiotic germ cells, suggesting an additional role in spermatogenesis. It was reported previously that Cre/loxP‐mediated conditional inactivation of the β‐catenin gene (Ctnnb1) in male gonads using a protamine promoter‐driven Cre transgene (Prm‐cre) resulted in partial infertility, reduced sperm count, and abnormal spermatogenesis. In this report, we demonstrated that the conditional deletion of Ctnnb1 using a germ cell specific Cre transgene (Stra8‐icre) had no effect on male fertility. We have shown that the Stra8‐icre transgene was highly efficient in generating deletion in early pre‐meiotic and post‐meiotic cells. No differences in anatomical or histological presentation were found in the mutant testis, the production of viable sperm was similar, and no abnormalities in DNA sperm content were detected. We concluded that β‐catenin is fully dispensable in germ cells for spermatogenesis. The conflicting results from the earlier study may have been due to off‐target expression of Prm‐cre in testicular somatic cells. In future studies, the analysis of conditional mutants using several Cre‐transgenes should be encouraged to reduce potential errors. genesis 52:328–332, 2014. © 2014 Wiley Periodicals, Inc.     

Fertilization and male fertility in the rotifer Brachionus plicatilis

The role of males in fertilization in the rotifer Brachionus plicatilis was examined. Neonate mictic females sometimes escaped fertilization even when sperm were present in their pseudocoeloms. Males made a major contribution to this fertilization failure through temporary infertility. As males aged, sperm numbers and motility decreased. Maternal diet was found to play a primary role in determining the fertilizing capacity of the F₁ males. Females from log phase populations produced males with higher sperm counts and motility than females from stationary phase populations. A conditioning factor in the medium may increase sperm number and fertilization rate.     

Large and persistent effect of a female steroid pheromone on ejaculate size in goldfish Carassius auratus

This study determined if ejaculate size in male goldfish Carassius auratus is increased by the female preovulatory steroid pheromone 4‐pregnen‐17,20β‐diol‐3‐one (17,20βP), which previously has been shown to affect male behaviour and to increase sperm motility and stripped sperm number, and also to increase paternity in competitive spawning and competitive in vitro fertilization. Experimental males were exposed overnight to 17,20βP whereas control males were not. The morning following exposure, each male was placed with a reproductively active female and, after one to 20 spawning acts, aquarium water was sampled to quantify released sperm. Although exposure to 17,20βP induced a five‐fold difference in the number of sperm that could be stripped, the median number of sperm in first ejaculates of pheromone‐exposed males was >60 sixty times that of control males, a pheromonal effect on ejaculate size that persisted for at least 20 spawning acts. The magnitude of the pheromone effect on ejaculate size indicates that it is a critical component of C. auratus sperm allocation, and that examining this effect in concert with other factors (e.g. presence of competitors, male and female size and frequency of spawning) will reveal the contribution of the preovulatory pheromone to male fitness in this promiscuous species.     

Plasma membrane calcium ATPase 4 (PMCA4) co‐ordinates calcium and nitric oxide signaling in regulating murine sperm functional activity

Reduced sperm motility (asthenospermia) and resulting infertility arise from deletion of the Plasma Membrane Ca²⁺‐ATPase 4 (Pmca4) gene which encodes the highly conserved Ca²⁺ efflux pump, PMCA4. This is the major Ca²⁺ clearance protein in murine sperm. Since the mechanism underlying asthenospermia in PMCA4's absence or reduced activity is unknown, we investigated if sperm PMCA4 negatively regulates nitric oxide synthases (NOSs) and when absent NO, peroxynitrite, and oxidative stress levels are increased. Using co‐immunoprecipitation (Co‐IP) and Fluorescence Resonance Energy Transfer (FRET), we show an association of PMCA4 with the NOSs in elevated cytosolic [Ca²⁺] in capacitated and Ca²⁺ ionophore‐treated sperm and with neuronal (nNOS) at basal [Ca²⁺] (ucapacitated sperm). FRET efficiencies for PMCA4‐eNOS were 35% and 23% in capacitated and uncapacitated sperm, significantly (p < 0.01) different, with the molecules being <10 nm apart. For PMCA4‐nNOS, this interaction was seen only for capacitated sperm where FRET efficiency was 24%, significantly (p < 0.05) higher than in uncapacitated sperm (6%). PMCA4 and the NOSs were identified as interacting partners in a quaternary complex that includes Caveolin1, which co‐immunoprecipitated with eNOS in a Ca²⁺‐dependent manner. In Pmca4^?/? sperm NOS activity was elevated twofold in capacitated/uncapacitated sperm (vs. wild‐type), accompanied by a twofold increase in peroxynitrite levels and significantly (p < 0.001) increased numbers of apoptotic germ cells. The data support a quaternary complex model in which PMCA4 co‐ordinates Ca²⁺ and NO signaling to maintain motility, with increased NO levels resulting in asthenospermia in Pmca4^?/? males. They suggest the involvement of PMCA4 mutations in human asthenospermia, with diagnostic relevance.     

The testis‐specific gene 1700102P08Rik is essential for male fertility

Male infertility is a rising problem around the world. Often the cause of male infertility is unclear, and this hampers diagnosis and treatment. Spermatogenesis is a complex process under sophisticated regulation by many testis‐specific genes. Here, we report the testis‐specific gene 1700102P08Rik is conserved in both the human and mouse and highly expressed in spermatocytes. To investigate the role of 1700102P08Rik in male fertility, knockout mice were generated by CRISPR‐Cas9. 1700102P08Rik knockout male mice were infertile with smaller testis and epididymis, but female knockout mice retained normal fertility. Spermatogenesis in the 1700102P08Rik knockout male mouse was arrested at the spermatocyte stage, and no sperm were found in the epididymis. The deletion of 1700102P08Rik causes apoptosis in the testis but did not affect the serum concentration of testosterone, luteinizing hormone, and follicle‐stimulating hormone or the synapsis and recombination of homologous chromosomes. We also found that 1700102P08Rik is downregulated in spermatocyte arrest in men. Together, these results indicate that the 1700102P08Rik gene is essential for spermatogenesis and its dysfunction leads to male infertility.     

Release of DEFB126 from macaque sperm and completion of capacitation are triggered by conditions that simulate periovulatory oviductal fluid

Capacitation of macaque sperm in vitro has been achieved efficiently only with the addition of both cyclic nucleotides and methylxanthines. The use of these exogenous sperm activators clouds an understanding of the normal mechanisms underlying capacitation and may slow early embryo development following in vitro fertilization (IVF). We demonstrate that culture medium which simulates periovulatory oviductal fluid with respect to bicarbonate (HCO(3)(-)) and glucose concentration induces capacitation in a high percentage of macaque sperm as determined by the ability of sperm to undergo both the release of coating protein DEFB126 and the zona pellucida-induced acrosome reaction (AR). Few sperm were able to undergo the AR following 6 hr incubation in medium containing either 35 mM HCO(3)(-) (approximately 7.2 pH) or 90 mM HCO(3)(-) (approximately pH 7.8) with 5 mM glucose. When glucose concentration was lowered to 0.5 mM to match levels reported for women at midcycle, the AR rate increased significantly in sperm incubated in both levels of HCO(3)(-), indicating that glucose interferes with sperm responsiveness to increasing HCO(3)(-) concentration observed in the primate oviduct during ovulation. Even greater synchronization of capacitation could be achieved with nonphysiologic extremes of alkalinity or energy substrate deprivation. In the latter case, sperm achieved high rates of IVF. A shift in pH from 7.2 to 7.8 in a HEPES-buffered medium was sufficient to remove DEFB126 from the surface of most sperm after only 3 hr. The loss of DEFB126 from sperm under periovulaory fluid conditions has implications for the timing of release of sperm from the oviductal reservoir.     

Impact of Cu and Fe concentrations on oxidative damage in male infertility

Oxidative stress in the reproductive system is thought to have an effect on the fertilizing ability of sperm. The purpose of this study was to assess the interaction of iron (Fe) and copper (Cu) ions in suspected subfertile and fertile male groups and to find out the relationships of the semen parameters (sperm count, motility, and abnormal morphology), glutathione, malondialdehyde, and reactive oxygen species with these variables. Semen and blood obtained from 60 subfertile men and from 40 fertile volunteers were examined. The sperm count and motility in the subfertile male group were found lower than those in fertile male group (p<0.001). Cu levels in serum and seminal plasma in the subfertile male group were significantly higher than those in the fertile male group (p<0.001 and p<0.05, respectively). There was also a significant increase in the Fe level of seminal plasma in the subfertile male group (p<0.001). However, there was no significant difference in the Fe level of serum in the subfertile male group. In conclusion, these findings suggest that Cu and Fe might be mediators of the effects of oxidative damage and play an essential role in spermatogenesis and male infertility; the determination of Fe and Cu levels in serum and seminal plasma during infertility investigation is recommended.     

Dead end1 is an essential partner of NANOS2 for selective binding of target RNAs in male germ cell development

RNA‐binding proteins (RBPs) play important roles for generating various cell types in many developmental processes, including eggs and sperms. Nanos is widely known as an evolutionarily conserved RNA‐binding protein implicated in germ cell development. Mouse NANOS2 interacts directly with the CCR4‐NOT (CNOT) deadenylase complex, resulting in the suppression of specific RNAs. However, the mechanisms involved in target specificity remain elusive. We show that another RBP, Dead end1 (DND1), directly interacts with NANOS2 to load unique RNAs into the CNOT complex. This interaction is mediated by the zinc finger domain of NANOS2, which is essential for its association with target RNAs. In addition, the conditional deletion of DND1 causes the disruption of male germ cell differentiation similar to that observed in Nanos2‐KO mice. Thus, DND1 is an essential partner for NANOS2 that leads to the degradation of specific RNAs. We also present the first evidence that the zinc finger domain of Nanos acts as a protein‐interacting domain for another RBP, providing a novel insight into Nanos‐mediated germ cell development.     

Epigenetic heterogeneity of developmentally important genes in human sperm: Implications for assisted reproduction outcome

The molecular basis of male infertility is poorly understood, the majority of cases remaining unsolved. The association of aberrant sperm DNA methylation patterns and compromised semen parameters suggests that disturbances in male germline epigenetic reprogramming contribute to this problem. So far there are only few data on the epigenetic heterogeneity of sperm within a given sample and how to select the best sperm for successful infertility treatment. Limiting dilution bisulfite sequencing of small pools of sperm from fertile donors did not reveal significant differences in the occurrence of abnormal methylation imprints between sperm with and without morphological abnormalities. Intracytoplasmic morphologically selected sperm injection was not associated with an improved epigenetic quality, compared to standard intracytoplasmatic sperm injection. Deep bisulfite sequencing (DBS) of 2 imprinted and 2 pluripotency genes in sperm from men attending a fertility center showed that in both samples with normozoospermia and oligoasthenoteratozoospermia (OAT) the vast majority of sperm alleles was normally (de)methylated and the percentage of epimutations (allele methylation errors) was generally low (<1%). However, DBS allowed one to identify and quantify these rare epimutations with high accuracy. Sperm samples not leading to a pregnancy, in particular in the OAT group, had significantly more epimutations in the paternally methylated GTL2 gene than samples leading to a live birth. All 13 normozoospermic and 13 OAT samples leading to a child had <1% GTL2 epimutations, whereas one (7%) of 14 normozoospermic and 7 (50%) of 14 OAT samples without pregnancy displayed 1–14% GTL2 epimutations.