Human protamines and the developing spermatid: their structure, function, expression and relationshipwith male infertility

<abstract>During spermiogenesis, the protamine proteins play an integral role in spermatid chromatin compaction. Recent research has focused on many facets of protamine biology, including protamine gene and protein structure/ function relationships, mechanisms of protamine expression regulation and involvement of the protamines in male fertility. In this paper, we review our current understanding of the structure and function of the protamine-1 (P1) and protamine-2 (P2) proteins and genes, the expression and regulation of these genes and the relationship between the protamines and male fertility. In addition, we offer a brief outlook on future investigation into protamine proteins.

The paternal epigenome and embryogenesis： poising mechanisms for development

The scope of paternal contributions during early embryonic development has long been considered limited. Dramatic changes in chromatin structure throughout spermatogenesis have been thought to leave the sperm void of complex layers of epigenetic regulation over the DNA blueprint, thus leaving the balance of that regulation to the oocyte. However, recent work in the fields of epigenetics and male factor infertility has placed this long-held, and now controversial dogma, in a new light. Elegant studies investigating chromatin and epigenetic modifications in the developing sperm cell have provided new insights that may establish a more critical role for the paternal epigenome in the developing embryo. DNA methylation, histone tail modifications, targeted histone retention and protamine incorporation into the chromatin have great influence in the developing sperm cell. Perturbations in the establishment and/or maintenance of any of these epigenetic marks have been demonstrated to affect fertility status, ranging in severity from mild to catastrophic. Sperm require this myriad of chromatin structural changes not only to serve a protective role to DNA throughout spermatogenesis and future delivery to the egg, but also, it appears, to contribute to the developmental program of the future embryo. This review will focus on our current understanding of the epigenetics of sperm. We will discuss sperm-specific chromatin modifications that result in genes essential to development being poised for activation early in embryonic development, the disruption of which may result in reduced fecundity.

Epigenetic marks in zebrafish sperm： insights into chromatin compaction, maintenance of pluripotency, and the role of the paternal genome after fertilization

Human sperm chromatin, and the sperm of most mammals, undergoes extensive remodeling during spermiogenesis during which 85%-95% of the histones are removed and replaced with protamines. The replacement of most histones with protamines facilitates a tighter packaging of the chromatin that is necessary for normal sperm function, and may help protect sperm DNA from damage during transport.

特发性少精子症和无精子症与Pygo2基因蛋白编码区SNPs的相关性

男性不育常伴随精子数量减少。Pygo2基因在染色质重塑的伸长精细胞中表达,其功能受损会导致精子形成阻滞和精子生成减少而引发不育。文章旨在检测引起人特发性少精子症和无精子症的Pygo2基因突变。从77例正常生育力男性和195例特发性少精子症和无精子症患者静脉血提取DNA,采用聚合酶链式反应-测序方法对Pygo2基因3个蛋白质编码区进行测序对比,非同义单核苷酸多态性(Single nucleotide polymorphisms,SNPs)位点分别用SIFT、Polyphen-2和Mutation Taster软件进行诱发蛋白质结构和表型改变的检测和分析。结果表明,195例患者中,178例(30例轻度或中度少精子症,57例重度少精子症和91例无精子症)基因序列分析报告完好,无精子症中3例患者分别在2个位点(rs61758740,rs141722381)发生了非同义突变SNPs,重度少精子症中1例患者在位点rs61758741发生了非同义突变,3个突变位点在SNPs基因数据库都已有报道,轻度或中度少精子症患者以及正常生育力男性中不存在SNPs。rs61758740可使PYGO2蛋白第141位蛋氨酸(M)变为异亮氨酸(I),rs61758741使PYGO2蛋白第261位碱性赖氨酸(K)变为酸性谷氨酸(E),rs141722381使PYGO2蛋白第240位亲水侧链天冬酰胺(N)变为疏水侧链异亮氨酸(I)。软件分析表明,在所发现的3个SNP非同义突变位点中,rs141722381引起的单个氨基酸改变会导致PYGO2蛋白空间结构破坏和诱发相关疾病。因此,Pygo2基因蛋白质编码序列区SNPs可能是特发性少精子症和无精子症的诱发因素之一,导致男性不育。

Analysis of the methylation pattern of six gene promoters in sperm of men with abnormal protamination

It has recently been shown that alteration of the methylation pattern of imprinted genes is associated with different types of male infertility. The objective of our study was to investigate the methylation pattern of selected gene promoters in sperm of patients with abnormal protamine replacement. The promoters of OCT4, SOX2, NANOG, HOXC11, miR-17and CREMwere analyzed using bisulfite sequencing and the percentage of DNA methylation was compared between patients with an abnormal protamine l/protamine 2 （P1/ P2） ratio and normozoospermic controls. No significant quantitative differences were found between groups of patients with either an abnormally high or low P1/P2 ratio compared to normal controls. However, two individual samples from infertile subjects （2/20, 10%） showed an altered methylation pattern for the CREMgene promoter that was not found in control samples. These two samples had a significantly higher （P〈0.05） promoter methylation （5.58 and 4.23%, respectively） compared to the control group （0.46%）. In conclusion, in our pilot study, extreme methylations defects were not seen broadly in severely infertile men. However, two patients exhibited altered methylation of the CREMgene, which may be either causative or a result of abnormal protmaine replacement.

Novel UBE2B-associated polymorphisms in an azoospermic / oligozoospermic population

Aim： To assess whether abnormalities exist in the UBE2B gene in a population of infertile human males, and to establish biologic plausibility of any discovered mutations. Methods： We carried out polymerase chain reaction （PCR） amplification and sequence analysis of the 5＇-untranslated region and six exons of the UBE2B gene, including flanking intronic regions, in a group of fertile and infertile men. Following the identification of a putative promoter region that contained single or dual triplet deletions within a 10-CGG repeat island, we evaluated the binding affinity of these identified polymorphisms as compared to the wild-type sequence to transcription factor SP1 using a DNA-protein gel shift assay. Results： There was a novel exonic single nucleotide polymorphism （SNP） noted in exon 4 in 5% of infertile men. In silico 3D modeling of the altered protein showed an innocuous isoleucine for valine substitution. There were no mutations noted within any of the other exons. Three novel intronic SNPs were identified within the fertile group, and seven novel intronic SNPs identified in the infertile group. The DNA-protein gel shift assay noted that both single CGG deletion and double CGG deletion bands had approximately twice the binding affinity compared to the wild-type for SP1. The negative control confirmed no non-specific protein binding. Conclusion- By themselves, a single or double CGG deletion is unlikely to pose biologic significance. However, such deletions in this suspected promoter region are associated with increased binding affinity for SP1, and might represent one of several factors required for alteration of UBE2B gene expression.

卵母细胞体外成熟与后继的延迟受精可使非整倍体胚胎增加

Failed fertilization and the appearance of immature oocytes are common in IVF practice; rescue intracytoplasmic sperm injection can be used as a therapy. However, this study indicates that embryos created after in vitro maturation and delayed intracytoplasmic sperm injection contain an increase in aneuploidy (79.7%) over control embryos (60.5%). Therefore, patients should be informed of the possible risk when presented with delayed intracytoplasmic sperm injection.

Proton-pump inhibitor use does not affect semen luality in subfertile men

Proton-pump inhibitors （PPIs） are among the most widely used drugs worldwide. PPI use has recently been linked to adverse changes in semen quality in healthy men; however, the effects of PPI use on semen parameters remain largely unknown specifically in cases with male factor infertility. We examined whether PPI use was associated with detrimental effects on semen parameters in a large population of subfertile men. We retrospectively reviewed data from 12 257 subfertile men who had visited our fertility clinic from 2003 to 2013. Patients who reported using any PPIs for 〉3 months before semen sample collection were included; 7698 subfertile men taking no medication served as controls. Data were gathered on patient age, medication use, and conventional semen parameters; patients taking any known spermatotoxic medication were excluded. Linear mixed-effect regression models were used to test the effect of PPI use on semen parameters adjusting for age. A total of 248 patients （258 samples） used PPIs for at least 3 months before semen collection. In regression models, PPI use （either as the only medication or when used in combination with other nonspermatotoxic medications） was not associated with statistically significant changes in semen parameters. To our knowledge, this is the largest study to compare PPI use with semen parameters in subfertile men. Using PPIs was not associated with detrimental effects on semen quality in this retrospective study.