Identification of compatibility between ooplasmic factor and sperm gene in the intersubspecific crosses involving DDK and PWK mice strains

The DDK strain （Mus musculus domesticus） of inbred mouse has a unique peculiarity known as DDK syndrome. The DDK females are mostly infertile when crossed with males of other inbred strains, while DDK males exhibit normal fertility in the reciprocal crosses, as intrastrain matings. This DDK syndrome has been demonstrated to be caused by an incompatibility system between DDK ooplasmic factor and the sperm gene of other strains owing to the ovum mutant （Ore） locus on mouse Chromosome 11. Recently, it was reported that DDK females are fully fertile when crossed to males of MOM （M. m. molossinus） and CASP （M. m. castaneus） strains, indicating that no incompatibilities exist between DDK ooplasmic factor and sperm gene of MOM or CASP males. In the present study, DDK females were found to be also fully fertile when crossed to the males of PWK wild-derived inbred strain （originated from Czech Republic wild mice, M. m. musculus）. The crosses of DDK females × F1 （DDK9 × PWK♀） males also resulted in normal fertility. Furthermore, the transmission ratios of Om alleles from these Fl males to their backcross N2 offspring are 50%：50% as genotyped by microsatellite markers closely linked to Om locus. Moreover, it was demonstrated that PWK females are also fully fertile when crossed to DDK males. All above results indicated that no incompatibility exists between ooplasmic factor and sperm gene in the intersubspecific crosses with DDK and PWK strains. PWK strain would also be useful for further investigations on the DDK syndrome, and DDK strain can be used more widely for various studies in the mouse.

Age-dependent expression of the cystatin-related epididymal spermatogenic （Cres） gene in mouse testis and epididymis

Aim： To investigate the spatial and temporal expression of the cystatin-related epididymal spermatogenic （Cres） gene in mouse testis and epididymis during postnatal development. Methods： The QuantiGene assay and indirect immunofluorescence technique were used to examine the Cres mRNA and Cres protein level in mouse testis and epididymis on postnatal days 14, 20, 22, 28, 35, 49, 70 and 420. Results： （1） In both the testis and epididymis, Cres mRNA was fast detected on day 20, then it increased gradually from day 20 to day 70, and the high expression level maintained till day 420. （2） In the testis, the Cres protein was exclusively localized to the elongating spermatids and was first detected on day 22. The number of Cres-positive spermatids increased progressively till day 49. From day 49 to day 420, the number of Cres-positive cells was almost stable. （3） The Cres protein was first detected on day 20 in the proximal caput epididymal epithelium. By day 35, the expression level of the Cres protein increased dramatically and the high level was maintained till day 420. Moreover, the luminal fluid of the midcaput epididymis was also stained Cres-positive from day 35 on. No Cres-positive staining was observed in distal caput, corpus and cauda epididymis throughout. Conclusion： The Cres gene displays a specific age-dependent expression pattern in mouse testis and epididymis on both the mRNA and protein level.

Fluorescence Tracking of Exogenous DNA in Genetic Transformation of the Chinese Oak Silkmoth Antheraea Pernyi via Sperm-Mediated Gene Transfer

Exogenous DNA expressing green fluorescent protein( GFP) and labeled with fluorescein isothiocyanate( FITC) was used to transform the Chinese oak silkmoth Antheraea pernyi( A. pernyi)via sperm-mediated gene transfer( SMGT). Sperms entry into the female reproductive system and eggs were observed using fluorescence microscopy. The ability of A. pernyi sperms to uptake exogenous DNA was confirmed,and transfer of the exogenous DNA was shown by GFP expression in the transgenic eggs. Our result suggested that SMGT could also be used to directly generate transgenic A. pernyi expressing functional genes of interest.

体内干扰Zfy基因对驴精子发育相关基因表达的影响

【目的】研究体内干扰Zfy基因对驴睾丸、附睾组织中Zfy蛋白表达以及精子发育相关基因表达的影响。【方法】选取健康种公驴,将一侧睾丸注射空载体作为对照组,另一侧睾丸注射Zfy干扰载体作为干扰组。采集睾丸、附睾组织,分别制作冰冻切片、固定液中固定及液氮中保存。使用冰冻切片观察载体绿色荧光蛋白在组织中的表达情况。使用免疫组化和HE染色观察干扰组和对照组Zfy蛋白表达情况。通过RT-qPCR检测干扰Zfy基因对睾丸、附睾中精子生成相关基因SYCP3、STRA8、TNP2、FAS表达水平的影响。【结果】绿色荧光蛋白主要在驴曲细精管中的圆形精子细胞和部分长形精子细胞中表达,干扰载体成功转入;干扰组驴睾丸Zfy蛋白表达量显著下调;干扰Zfy基因使驴睾丸中SYC P3基因表达量显著下调、附睾中TNP 2和FAS基因表达量显著下调、而睾丸和附睾中STR A8基因表达量没有明显差异。【结论】驴Zfy基因主要在驴圆形精子中表达,干扰Zfy基因可显著抑制驴睾丸中Zfy蛋白表达、SYC P3基因表达和附睾中TNP 2、FAS基因表达。

中国荷斯坦公牛不同耐冻性精子的蛋白质组学分析

在以冷冻精液和人工授精为主导技术的奶牛繁育体系中,公牛精液的冻后活力备受关注。精子的耐冻性是影响精液冻后活力的关键。已有研究发现解冻后的精子活力在不同公牛之间存在显著差异。因此,迫切需要从遗传水平挖掘影响精子耐冻性的关键基因和分子标记,为提高我国种公牛的精子耐冻性提供理论依据。本研究筛选了精子高耐冻组公牛9头(鲜精活力>0.65,冻后活力>0.40)和精子低耐冻组公牛6头(鲜精活力>0.65,冻后活力≤0.27),使用Label-free蛋白质组学技术对15头公牛的精子细胞进行定量蛋白质组学分析和生物信息学分析。结果显示,在高、低耐冻组间共检测到432个差异表达蛋白,主要富集在“代谢途径”和“氧化磷酸化”等与精子能量供应密切相关的生物学过程。差异表达蛋白互作网络分析显示与能量代谢相关的蛋白之间具有强相互作用。与QTL数据库比对,发现9个差异表达蛋白与已定位到的冻后活精子百分比性状关联基因重叠。最终筛选到HSPA1A、BSP3、ACSL4等重要候选蛋白可作为公牛精子耐冻性的潜在生物标志物。本研究揭示了具有不同耐冻性公牛精子的蛋白质组特征,这些发现为我国种公牛冻后精液品质的分子选育提供了重要信息。

性别控制在畜牧生产中的应用研究进展

动物的性别控制一直是动物遗传育种与繁殖领域研究的热点。性别控制技术是一项能显著提高家畜繁殖效率的技术,该技术对快速种群扩繁、提高优良生产性能、推动育种进程和保护地方优良与濒危品种等具有非常重要的意义。本文主要综述了常见性别控制技术在畜牧生产中最新研究进展,以期为性别控制研究和应用提供参考和帮助。

维生素E对雄性畜禽繁殖性能的影响及其候选基因

维生素E可促进畜禽的睾丸发育和精子发生,目前大多数维生素E的相关研究倾向于从抗氧化的角度探究其对繁殖性能的影响,但也有部分研究发现了维生素E潜在的非抗氧化功能。为进一步揭示维生素E调控动物繁殖性能的机制,文章综述了维生素E对雄性家畜繁殖性能的影响,总结了介导维生素E促进睾丸发育及精子发生的细胞增殖、细胞凋亡、精子发生、激素受体等相关候选基因,以期为维生素E在畜禽繁殖方面的应用提供参考。

常染色体显性遗传2型多囊肾病基因参与精子尾部组装的作用研究

目的研究常染色体显性遗传2型多囊肾病基因(PKD2)在精子尾部组装过程中的作用。方法回顾分析1例携带PKD2基因突变的非梗阻性无精症(NOA)患者的临床资料,Sanger测序验证该基因突变,对睾丸组织进行HE和免疫荧光染色;通过对正常人精子涂片进行免疫荧光染色观察PKD2在精子的定位,利用人视网膜色素上皮(hRPE)细胞检测PKD2在纤毛发育过程中的定位。结果该NOA患者PKD2基因存在c.1571T>A(P.I524N)杂合突变,其精子细胞尾部发育缺陷;PKD2主要定位于精子鞭毛起始部,与参与精子尾部组装及发育的重要蛋白Ac-Tubulin及γ-Tubulin存在共定位,在hRPE细胞纤毛发育过程中PKD2主要定位于纤毛的基体。结论本研究报道了1例全新的PKD2杂合突变的NOA患者病例,结果提示PKD2可能参与精子尾部的起始组装,为进一步研究精子鞭毛组装的机制提供了基础,PKD2基因c.1571T>A(P.I524N)杂合突变可能是无精症诊疗的新靶点。

Progress in gene transfer by germ cells in mammals

Use of germ cells as vectors for transgenesis in mammals has been well developed and offers exciting prospects for experimental and applied biology, agricultural and medical sciences. Such approach is referred to as either male germ cell mediated gene transfer （MGCMGT） or female germ cell mediated gene transfer （FGCMGT） technique. Sperm-mediated gene transfer （SMGT）, including its alternative method, testis-mediated gene transfer （TMGT）, becomes an established and reliable method for transgenesis. They have been extensively used for producing transgenic animals. The newly developed approach of FGCMGT, ovary-mediated gene transfer （OMGT） is also a novel and useful tool for efficient transgenesis. This review highlights an overview of the recent progress in germ cell mediated gene transfer techniques, methods developed and mechanisms of nucleic acid uptake by germ cells.

Inhibition of mouse acrosome reaction and sperm-zona pellucida binding by anti-human sperm membrane protein 1 antibody

Aim： To investigate the possible functions of human sperm membrane protein （hSMP-1） in the process of fertilization. Methods： A 576-bp cDNA fragment of HSD-1 gene coding for the extracellular domain of hSMP-1 was cloned and expressed. The localization of this protein on human and mouse sperm was determined by indirect immunofluorescent staining by using anti-recombinant hSMP-1 （anti-rhSMP-1） antibodies. Sperm acrosome reaction and spermzona pellucida （ZP） binding assay were carried out in 10-week-old BALB/c mice. Results： Recombinant hSMP-1 was successfully cloned and expressed. The expression of the native protein was limited on the acrosome of human and mouse sperm. Treatment of anti-rhSMP-1 antibodies significantly decreased the average number of sperms bound to each egg. Meanwhile, the percentage of acrosome reaction was decreased in comparison to pre-immune control after treatment with anti-rhSMP-1 （P 〈 0.05）. Conclusion： The results suggest that anti-rhSMP-1 antibody inhibited mouse acrosome reaction and sperm-ZP binding.

The mechanism of sperm-egg interaction and the involvement of IZUMO1 in fusion

An average human ejaculate contains over 100 million sperm, but only a few succeed in accomplishing the journey to an egg by migration through the female reproductive tract. Among these few sperm, only one participates in fertilization. There might be an ingenious molecular mechanism to ensure that the very best sperm fertilize an egg. However, recent gene disruption experiments in mice have revealed that many factors previously described as important for fertilization are largely dispensable. One could argue that the fertilization mechanism is made robust against gene disruptions. However, this is not likely, as there are already six different gene-disrupted mouse lines （Calmegin, Adam Ia, Adam2, Adam3, Ace and Pgapl）, all of which result in male sterility. The sperm from these animals are known to have defective zona-binding ability and at the same time lose oviduct-migrating ability. Concerning spermzona binding, the widely accepted involvement of sugar moiety on zona pellucida 3 （ZP3） is indicated to be dispensable by gene disruption experiments. Thus, the landscape of the mechanism of fertilization is revolving considerably. In the sperm-egg fusion process, CD9 on egg and IZUMO1 on sperm have emerged as essential factors. This review focuses on the mechanism of fertilization elucidated by gene-manipulated animals.

Effects of Diabetes Mellitus upon Sperm Quality Insight into Molecular Level

Diabetes Mellitus is a chronic disease that affects important body organs in a very serious manner. The consequences of this disease turn out to be a significant problem for the patient, who tries to cope with the new condition his organism has been placed in. The most common effect of the disease, hyperglycaemia, leads over time to serious damage to various body systems, such as nerves and blood vessels. What is not widely known among the population is that diabetes may have harmful effects on the reproductive system of the men suffering from diabetes type 1 and 2 and that such a parameter could lead to or might be the reason for infertility problems for couples, for example, miscarriage or embryonic failure. AGEs is a number of products which are believed to play an important role, because their presence has been detected in increased level in diabetic men. This implies that those glycation products might play a key role in diabetic complications. Their receptor, RAGE, member of the immunoglobulin superfamily has been detected in the reproductive tract of diabetic men. Reactive oxygen species (ROS), a possible product of AGEs appear in high levels in seminal plasma and are believed to be the cause of DNA fragmentation. The objective of this review was to gather all the available material i.e. studies on diabetes mellitus in one article, to study the research which has already been conducted and the conclusions that have been drawn, in order to offer, if possible, new pathways and perspectives to the scientists, who focus on fertility problems, sometimes intractable.

Genetic Variation in the Testis-Specific HASPIN Gene Encoding a Serine/Threonine Protein Kinase in Infertile Japanese Males

HASPIN is a serine/threonine protein kinase predominantly expressed during spermatogenesis and localized in the nucleus. The HASPIN gene is conserved from yeast to mammals and plants. To investigate any possible associations between HASPIN polymorphisms and impaired spermatogenesis in Japanese males, we screened for mutations in the HASPIN coding sequence (CDS) using DNA from 282 sterile male patients and 262 fertile male volunteers. Polymorphisms were found at 10 positions within the HASPIN CDS. Among these 10 polymorphisms, 5 were found only in the infertile group, 3 of which were nonsynonymous. These polymorphisms found only in the infertile patients may be a cause of male infertility and thus valuable candidates for further study of this condition.

Alteration of ERβ gene Rsal polymorphism may contribute to reduced fertilization rate and embryonic developmental competence

This paper aims to determine the possible role of estrogen receptor-β （ERβ） gene Rsal polymorphism on sperm fertility and early embryonic development in humans. Three groups of Chinese men were recruited： in vitro fertilization （IVF） group, including 374 couples who underwent conventional IVF; intracytoplasmic sperm injection （ICSI） group, including 294 couples who underwent an ICSI procedure using ejaculated sperm; and azoospermic group, consisting of 197 couples who underwent ICSI using either testis or epididymis sperm. Rsal polymorphism in the ERβ gene was detected by polymerase chain reaction （PCR）-restriction fragment length polymorphism technique; fertilization and high-quality embryo rates were evaluated for each group. In each group, no significant differences were found in the overall rates of fertilization and high-quality embryos among GG, AG and AA genotypes. However, the proportion of cycles possessing a satisfactory high-quality embryo rate with the AA genotype was significantly lower than that in the wild-type GG genotype from each group. These results demonstrated that sperm possessing the ERβ RsalA genotype may have reduced fertilization ability and decreased early embryonic developmental potential, which could directly or indirectly contribute to the low fertilization rate and early embryonic developmental arrest in some cases.

基于高通量转录组测序的牦牛和犏牛附睾尾部差异表达基因分析

【目的】探明牦牛和犏牛的附睾尾部差异表达基因(DEGs),筛选出与精子成熟和贮存密切相关的功能基因,为揭示犏牛精子发生及其成熟过程的分子机制打下理论基础。【方法】以牦牛和犏牛的附睾尾部为研究对象,通过Illumina 127-HiSeq 2000平台完成高通量转录组测序,经过滤、质量控制及拼接组装后,依据FDR<0.05且|log_(2)Fold Change|>1的筛选标准,通过EBSeq筛选出DEGs,然后进行GO功能注释分析和KEGG信号通路富集分析,并以实时荧光定量PCR验证高通量转录组测序数据的准确性。【结果】在牦牛和犏牛的附睾尾部共筛选获得76个DEGs,其中43个DEGs上调、33个DEGs下调;76个DEGs在COG、GO、KEGG、KOG、Nr、Pfam、Swiss-Prot和eggNOG等数据库中均有注释信息,尤其在COG和Pfam数据库中的注释率最高(达88.16%)。GO功能注释分析结果显示,DEGs被注释到生物过程(Biological process)、细胞组分(Cellular component)和分子功能(Molecular function)三大功能类别上;KEGG信号通路富集分析发现76个DEGs主要显著富集在3条信号通路上,分别是胆汁分泌通路(ko04976:Bile secretion)、ABC转运器(ko02010:ABC transporters)和cAMP信号通路(ko04024:cAMP signaling pathway)。随机选择8个DEGs(SERPINA1、MMP7、ATP2C1、ABCC1、NMT1、NAT1、CFTR和PRX)进行实时荧光定量PCR检测验证,结果显示这8个DEGs的表达模式与高通量转录组测序的结果基本一致,表明转录组数据准确可靠。【结论】在牦牛和犏牛的附睾尾部存在76个DEGs(43个DEGs上调,33个DEGs下调),显著富集在胆汁分泌通路、ABC转运器及c AMP信号通路上,与精子获能相关的DEGs有MMP7、IGFBP2和ABCC4基因,且这3个基因在犏牛附睾尾部呈下调表达,即精子获能失败可能是导致犏牛雄性不育的主要原因。

A Qualitative Model of the Interaction of Sexual Behavior and Hormone Gene Transcription in Male Blue Gourami during Reproduction

In the present study, a model is suggested to describe hormone control in male blue gourami (Trichogaster trichopterus) along the gonadotropic brain-pituitary- gonad axis (BPG axis) and the hypothalamic-pituitary-somatotropic axis (HPS axis). This model is based on the cloning and transcription of genes encoding hormones of the two axes involved in spermatogenesis during blue gourami reproduction. Gene transcription is affected by environmental, biological, and behavioral factors. Mature males were examined in two different stages—nonreproductive in high-density habitats and reproductive in low-density habitats. Based on gene transcription, gonadotropin-releasing hormone 1 (GnRH1) was involved in controlling spermatogenesis (spermatogonia to spermatids) via the BPG axis in nonreproductive and reproductive stages by controlling follicle-stimulating hormone (FSH), 11-ketotestosterone (11KT) and 17β-estradiol (E2). However, GnRH3 had a larger effect during the reproductive stage via the BPG axis (spermatids to sperm) on luteinizing hormone (LH), 11KT, and 17α-hydroxyprogesterone (17P). At the same time, the HPS axis was involved in spermatogenesis via pituitary adenylate cyclase-activating polypeptide (PACAP) and its related peptide PRP (formerly known as GHRH-like peptide) in the brain, and growth hormone (GH) in the pituitary affected synthesis of insulin-like growth factor 1 (IGF1) in the liver.

畜禽精子活力调控基因的研究进展

精液品质决定着畜禽受精率和繁殖效率,是影响畜禽生产效益的重要因素。精子活力指精液中直线向前运动精子的百分率,是畜禽精液品质最重要的评价指标之一。提高精子活力是改善精液品质的关键。精子活力是受多基因多通路共同调控的复杂性状,为中高遗传力性状,因此遗传改良成为提高精子活力的有效途径。相比传统的遗传改良手段,基因编辑和分子标记辅助选择等育种新技术具有效率高、目的性强、周期短的特点,为畜禽育种提供了全新机遇。目前,已知影响畜禽精子活力的基因数量有限,限制了育种新技术在精子活力遗传改良工作中的应用。因此,鉴定调控畜禽精子活力的关键基因是育种工作的重要环节。基因主要从精子结构和功能两方面调控精子活力,包括对精子结构缺陷、能量代谢、离子通道、DNA完整性、精液成分等方面的调控。文章介绍了在精子结构和功能两方面调控精子活力的功能基因,概述了其作用机制和研究进展,旨在为畜禽精子活力相关的分子育种工作提供参考。

利用WGCNA挖掘种公鸡睾丸和附睾中影响精子活力的核心基因

种公鸡的精子活力对养禽业的可持续发展至关重要,通过加权基因共表达网络(WGCNA)分析法挖掘种公鸡睾丸、附睾中调控精子活力的基因共表达模块和核心基因,并构建与种公鸡精子活力相关的调控网络。基于团队前期对不同精子活力种公鸡睾丸、附睾组织转录组测序数据的分析,用WGCNA方法构建基因共表达网络,识别与表型性状显著相关的基因模块,并对关键模块基因进行GO功能注释、KEGG通路富集分析。用Cytoscape软件筛选每个关键模块的核心基因并构建可视化共表达网络。结果表明,14227个基因聚类到11个模块,以决定系数(R2)≥0.6、P<0.05为标准挖掘出青绿色(Turquoise)模块、黄色(Yellow)模块、红色(Red)模块与表型显著相关。对3个关键模块的基因进行功能分析,发现这些基因显著富集在核苷酸切除修复、同源重组、细胞色素P450对异类物质代谢、MAPK信号通路和细胞凋亡等通路上。选出的IFT家族基因与HMOX2、CYP4B1、ANG、ITGB2基因是与种公鸡精子活力相关的核心基因,可作为提高精子活力的潜在基因。

热应激下公兔睾丸组织形态和精液转录组分析

旨在深入了解公兔在热应激环境中睾丸组织形态改变及相应基因谱,对揭示热应激下公兔精液质量下降的机理具有重要意义。本研究选取了6只具有相同饲养水平,体况和体重相近的8月龄新西兰种公兔,通过建立热应激模型得到热应激组(8月份,HS)和未热应激组(5月份,NHS)各3只,分别采集2组公兔的睾丸和精液。通过HE染色,比较不同条件下睾丸组织结构的差异,并利用Illumina Hiseq高通量测序技术对精子进行转录组测序分析,通过生物信息学方法对2组样本间的差异表达基因进行GO、KEGG富集分析,并通过RT-qPCR方法验证转录组测序数据。公兔睾丸组织切片结果显示,与NHS组相比,HS组的睾丸中央出现明显的空泡化和撕裂,生精上皮较薄,受损情况显著,精子细胞数量下降。进一步以log_(2) FoldChange>1和P<0.05为阈值,共筛选出与精子热应激响应相关的差异表达基因1676个,包括ATP 5MC1、MDH2、ALDH 7A1、GAPDHS、PRPS 1等,其中上调基因894个,下调基因782个。GO分析结果显示,差异表达基因富集在应激反应、蛋白质代谢过程、催化活性等相关条目上,KEGG结果显示,差异表达基因富集到MAPK、PI3K-AkT、Toll-like receptor、Ras等信号通路上。RT-qPCR验证了ATP 5MC1、MDH2、PRPS1、ALDH 7A1和GAPDHS等5个差异表达基因的表达水平,与转录组结果趋势一致。综上,热应激能够调控精子发生相关基因的表达,影响相关信号通路及生物学功能。本研究结果为精子发生的分子调控机制提供线索,并为高温环境下家兔的选育提供理论基础。

附睾中非编码小RNA(sncRNA)的研究进展

非编码小RNA(sncRNA)主要通过抑制性调控靶基因发挥作用。近年的研究发现sncRNA在哺乳动物附睾的各个部位均有表达,并且在调控附睾不同节段的基因表达方面起着非常关键的作用。附睾上皮细胞中sncRNA的多样性及其表达水平的时空差异会影响很多潜在的靶基因,从而调节附睾的生理功能,另外,这些sncRNA还可以通过附睾小体传递给精子,对精子产生一系列的调控作用。综述了附睾中sncRNA表达、sncRNA对附睾和附睾中精子功能调节方面的研究进展,以期为探究sncRNA对雄性生殖细胞调控作用的分子机制提供参考。