家畜性腺分化和配子形成研究进展

哺乳动物胎儿发育早期，原始外胚层特定细胞分化为原始生殖细胞，即配子的祖细胞。原始生殖细胞随后迁移到未分化的性腺中，与体细胞共同形成卵巢或睾丸。原始生殖细胞经过有丝分裂增殖形成卵原细胞或精原细胞，随后进行减数分裂分化为配子。性别决定、性腺分化和配子发生过程涉及多种细胞的增殖分化和相互调控，但受到取样和检测技术的限制，以往的研究系统性较差。近十年，单细胞水平检测技术的快速发展为全面分析人和动物生殖能力形成的机制提供了重要机遇。本文综述了不同动物原始生殖细胞的分化、迁移与增殖、性腺分化、性别决定及雌雄配子形成过程的研究进展，并介绍了单细胞测序技术在这些过程中的应用现状和前景，为揭示动物性别和生殖能力形成的机制以及提高哺乳动物的繁殖效率提供理论参考。     

表观遗传学在动物繁育上的应用研究

动物生产是现代农业发展领域的关键产业,是改善并提高配子利用率的新型繁殖技术,利用体外生产胚胎实现批量生产动物包括转基因动物,以及培育优良品种等都将是推动动物产业化进程的手段。但由于卵泡发育、配子发生和胚胎发育的机制尚未阐明,尤其是表观遗传学理论的欠缺,已成为制约上述技术发展的瓶颈之一。本文拟对表观遗传学在配子发生和胚胎发育过程中的研究现状进行概述,旨在阐明这些领域的表观遗传机制问题和发展趋势,从而促进动物繁育技术的发展,加快动物产业化的进程。     

TET蛋白在配子形成和早期胚胎中的作用研究进展

在哺乳动物配子形成和胚胎发育过程中，DNA甲基化水平一直处于动态变化，DNA甲基化水平的规律变化决定了配子的生成、分裂和成熟，尤其是在早期胚胎发育过程中DNA去甲基化与DNA甲基化共同决定了1枚胚胎能否发育为完整的个体。TET蛋白家族的各成员可以通过不同作用机制发挥去甲基化作用，并且不同种类TET蛋白的缺失会对胚胎发育产生不同程度的阻碍。大量研究表明TET蛋白在配子形成和早期胚胎发育不同阶段动态调控甲基化水平，目前关于TET蛋白生物功能还存在很大的研究空间。本文综述了TET蛋白家族成员的结构、作用机制以及在配子形成和胚胎发育过程中的生物功能，为深入研究TET蛋白功能提供参考。     

毁灭泰泽球虫内生发育的超微结构研究 Ⅲ.小配子生殖与小配子

毁灭泰泽球虫的小配子生殖发生在小肠上皮细胞内由单层膜围成的带虫空泡中。其细胞核的分裂过程与裂殖生殖时期的相似,但小配子体的细胞核缺少核仁,染色质在核的浅层聚集成致密的斑块。在小配子的形成过程中,核附近的中心粒转变为基粒,其中央微管消失。随后,两根鞭毛从基粒长出,突入带虫空泡。细胞核的致密部分逐渐地进入小配子体的表面突起,并最终发育为小配子的细胞核;而核的疏浅部分则留在小配子体的细胞质中,成为残体的成份。成熟的小配子拥有两根纤长的鞭毛和香蕉形的身体,体内含有长形的细胞核及在其尖端嵌入的线粒体,一组微管(4 1 1)自基粒附近向体后延伸,但仅约3根微管抵达身体的末端。     

热休克蛋白70在配子发生和胚胎发育中的表达

热休克蛋白(hsp)是机体在应激状态下迅速合成的一组蛋白质。热休克蛋白70(hsp70)是hsp中成员最多、最重要的一族,具有普遍性、高度保守性以及应激性。hsp70作为蛋白成熟过程中的分子伴侣,参与细胞周期调节、DNA损伤修复及细胞凋亡,在配子发生及胚胎发育、机体衰老过程中具有重要的生理作用。本文主要就hsp70在配子形成和胚胎发育中的作用进行了简述。     

毛乌素沙地南缘柠条固沙恢复区植物多样性及生物量对水氮添加的响应

为研究大气氮沉降和降水变化对毛乌素沙地南缘柠条(Caragana korshinskii)固沙恢复区草本植物多样性及地上生物量的影响，采用随机区组设计，设置不增水(W₀)、增水量分别为年均降水量的 33%(W₁)、66%(W₂)和 100%(W₃)4 个水分添加处理，及 4 个氮添加处理，分别为添加纯氮含量为 0 (N₀)、5(N₅)、10(N₁₀)、20(N₂₀) g/(m² ·a)的(NH₄)₂SO₄，共 16 个处理组合，计 4 个区组，进行水氮添加试验。结果表明：1)水氮添加对植物群落组成、重要值产生显著影响(P＜0. 05)，一、二年生植物比多年生植物对增水和施氮的反应更敏感，但半灌木、小半灌木所受影响最小；2)水分处理对植物群落物种丰富度和多样性指数有显著影响(P＜0. 05)，二者都随水分添加呈先增加后减小的趋势，在添加量为 W1处理时达到水分阙值；3)氮素处理显著影响物种丰富度、优势度指数和多样性指数(P＜0. 01)，研究发现增水在一定程度上会抵消氮沉降增加对植物物种多样性的负面影响，进而改变植物群落组成与结构；4)水氮耦合处理对植物群落盖度和群落地上生物量有显著影响(P＜0. 05)；5)植物群落地上生物量与 Shannon-Wienner 多样性指数呈正相关，与物种丰富度、Simpson 优势度指数和 Pielou 均匀度指数呈负相关关系。主成分分析表明，W₃-N₁₀水氮添加方案对柠条固沙恢复区的草本植被生产力恢复效果最好，而 W₁-N₅水氮添加方案对该区草本植物群落多样性恢复效果最好。     

15.犬科动物的生殖生物学与辅助生殖

动物的生殖生物学技术,包括从对自然交配的控制到成年体细胞克隆等多种形式,主要包括人工授精(artificial insemination,AI)、卵母细胞的体外成熟(in vitro maturation,IVM)与体外受精(in vitrofertilization,IVF)、胚胎体外发育(in vitro production,IVP)、胚胎移植(embryo transfer,ET)和配子冷冻等,又称之为人工辅助生殖技术(assisted reproductive technology,ART).     

鸡住白细胞虫病的防治

正(一)病原在我国,寄生于鸡体的白细胞虫主要有两种:卡氏白细胞虫和沙氏白细胞虫。白细胞虫的发育过程需要两个宿主。卡氏住白细胞虫的传播媒介是库蠓;沙氏住白细胞虫的传播媒介是蚋。卡氏住白细胞虫的发育包括裂殖生殖、配子生殖、孢子生殖三个阶段。裂殖生殖和配子生殖的大部分在鸡体内完成,而配子生殖的一部分及孢子生殖则在库蠓体内完成。(二)流行病学     

谷胱甘肽和活性氧对哺乳动物配子作用的研究进展

文章综述了近年来关于谷胱甘肽对哺乳动物精子、卵母细胞和胚胎早期发育的作用及活性氧对精子功能的影响。在雄性和雌性配子中,谷胱甘肽起着保护精子和卵母细胞免受氧化损害的作用。在卵母细胞减数分裂中谷胱甘肽具有维持纺锤体形态的作用,受精后,巯基在精核的形成中起着积极的作用,并促进早期胚胎发育到囊胚阶段。在卵母细胞成熟过程中,谷胱甘肽的浓度发生着变化。它的合成受促性腺激素的调节,在精子的发生过程中随着精子的成熟,谷胱甘肽的浓度逐渐降低,但活性氧对精子功能也有多方面的作用。文章对卵丘细胞中小分子巯基化合物在谷胱甘肽合成中的重要作用也进行了综述。     

外泌体miRNA在配子发育和受精中的调控作用

外泌体(Exosomes,EXs)是一类膜状结构的纳米级细胞外囊泡,携带大量的生物活性调节分子进行细胞间信息转导。EXs携带的miRNA在配子发生、发育和受精过程中发挥重要的调控作用。本文主要对EXs中的miRNA在卵母细胞和精子成熟以及受精过程中的调控作用做一综述。     

Perspectives of germ cell development in vitro in mammals

Pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are able to differentiate into all cell lineages of the embryo proper, including germ cells. This pluripotent property has a huge impact on the fields of regenerative medicine, developmental biology and reproductive engineering. Establishing the germ cell lineage from ESCs/iPSCs is the key biological subject, since it would contribute not only to dissection of the biological processes of germ cell development but also to production of unlimited numbers of functional gametes in vitro. Toward this goal, we recently established a culture system that induces functional mouse primordial germ cells (PGCs), precursors of all germ cells, from mouse ESCs/iPSCs. The successful in vitro production of PGCs arose from the study of pluripotent cell state, the signals inducing PGCs and the technology of transplantation. However, there are many obstacles to be overcome for the robust generation of mature gametes or for application of the culture system to other species, including humans and livestock. In this review, we discuss the requirements for a culture system to generate the germ cell lineage from ESCs/iPSCs.     

Development of avian embryo manipulation techniques and their application to germ cell manipulation

The development of chicken embryo culture techniques, from single‐cell stage to hatching, makes it possible to manipulate developing embryos at any developmental stage. Production of germline chimeric chickens by the transfer of stage X blastodermal cells or primordial germ cells enables the manipulation of germline cells in vitro. Production of transgenic chickens has been attempted by the retroviral vector method, microinjection of DNA into a fertilized ovum at the single‐cell stage, use of chimeric intermediates produced by the transfer of stage X blastodermal cells or primordial germ cells, manipulation of spermatozoa, and in vivo manipulation of gonads. So far, the only non‐viral method that has successfully produced transgenic chickens is microinjection of DNA into a fertilized ovum. Manipulation of primordial germ cells could become an efficient system for producing transgenic chickens by combining it with the highly efficient transfection method or the in vitro culture system for primordial germ cells. Preservation of avian genetic resources has now become possible by cryopreservation of stage X blastodermal cells or primordial germ cells as well as spermatozoa. The development of nuclear transfer techniques for avian species is necessary.     

胚胎干细胞向生殖细胞分化的研究进展

生殖细胞来源于原始生殖细胞,其在体内的分化机制已基本清楚。随着生殖细胞研究的不断深入,通过体外诱导途径获得生殖细胞已成为现实。将胚胎干细胞体外分化为上胚层样细胞,进而分化为原始生殖样或原始生殖细胞。将它们迁移入胎儿生殖腺,具有减数分裂及产生精子能力,与卵子结合移入缺生精管的生殖腺的新生鼠可以产生健康后代。为此,体外诱导生殖细胞的成功,进一步阐明了胚胎干细胞向生殖细胞分化的机制,为更有效利用干细胞造福人类奠定了基础。     

Biological characteristics of fish germ cells and their application to developmental biotechnology

We have revealed several unique characteristics of germ cell development using rainbow trout, including the fact that spermatogonia transplanted into the peritoneal cavity of newly hatched embryos migrate toward recipient gonads, that spermatogonia transplanted into female recipients start oogenesis and produce functional eggs and that diploid germ cells transplanted into triploid trout can complete gametogenesis. By combining these unique features of fish germ cells, we established allogeneic and xenogeneic transplantation systems for spermatogonia in several fish species. Spermatogonia isolated from the mature testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout were transplanted into the peritoneal cavity of triploid masu salmon newly hatched embryos. These spermatogonia migrated toward recipient salmon genital ridges with extending pseudopodia and were subsequently incorporated into them. We further confirmed that the donor-derived spermatogonia resumed gametogenesis and produced sperm and eggs in male and female salmon recipients, respectively. By inseminating the resulting eggs and sperm, we obtained only rainbow trout offspring in the F1 generation, suggesting that the triploid salmon recipients produced functional gametes derived only from donor trout. We further confirmed that this intra-peritoneal transplantation of germ cells is applicable to several marine fishes, which could be of benefit in the production of bluefin tuna that has a large broodstock (>100 kg) and is difficult to maintain in captivity. Gamete production of bluefin tuna could be more easily achieved by generating a surrogate species, such as mackerel, that can produce tuna gametes.     

Donor primordial germ cell-derived offspring from recipient germline chimaeric chickens: Absence of long term immune rejection and effects on sex ratios

1. Germline chimaeric chickens were produced by the transfer of primordial germ cells, and the generation of donor-derived offspring was examined for a maximum of 146 weeks. 2. The frequencies of donor-derived offspring from the chimaeras were 47% to 97%, and no apparent changes in frequency were observed with increasing age during the test period. 3. Differentiation of donor primordial germ cells into functional gametes appeared to be restricted to a degree at some developmental stage in the gonads of chimaeric chickens of the opposite sex.     

Isolation and culture of rabbit primordial germ cells

Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they can also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin on inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.     

Bovine ovarian stem cells differentiate into germ cells and oocyte‐like structures after culture in vitro

Stem cells have been isolated from ovaries, and their ability to differentiate into oocytes in vitro has been demonstrated for mice and human, but not for bovine species. The aims of this study were to isolate germline stem cells from bovine ovaries and to evaluate the effects of bone morphogenetic proteins (BMPs) 2 and 4, and follicular fluid on the differentiation of these stem cells into oocyte‐like structures. The ovarian stem cells were isolated and cultured in α‐MEM⁺ supplemented with BMP2, BMP4 or follicular fluid. On days 0 and 14, cells were evaluated for their morphological appearance, viability, expression of alkaline phosphatase and for markers of germ cell formation (VASA and DAZL) and oocyte development (GDF9, ZPA and SCP3) by qPCR. Levels of mRNA were analysed using ANOVA and Bonferroni test (p < .05). The results showed that at day 0, ovarian stem cells expressed specific markers of pluripotency (OCT4, SOX). In addition, these cells were positive for alkaline phosphatase, which is a marker commonly used to identify primordial germ cells (PGCs). After the period of differentiation, cells had morphological features that resemble PGCs and oocyte‐like cells (OLCs). An increase, ranging from five to 14 times, in the expression of VASA was observed in cells cultured in medium supplemented with BMPs and follicular fluid, while the increase in DAZL expression ranged from four to six times. In addition, OLCs had an increase in expression of mRNAs for GDF9, ZPA and SCP3 that ranged from two to eight times. In conclusion, OLCs can be differentiated in vitro from ovarian stem cells and BMPs and follicular fluid are effective in stimulating the expression of mRNAs for germ cell and oocyte markers.     

Migration of primordial germ cells isolated from embryonic blood into the gonads after transfer to stage X blastoderms and detection of germline chimaerism by PCR

1. The present study was carried out to determine whether primordial germ cells isolated from embryonic blood can enter the bloodstream and successfully migrate to the germinal ridges of recipient embryos after transfer to stage X blastoderms, and also whether they can differentiate into blood cells, as is suggested in mice. 2. Primordial germ cells were transfected in vitro by lipofection and then transferred to stage X blastoderms. The introduced GFP gene was efficiently expressed in the gonads of 6-d incubated embryos. 3. Freshly collected primordial germ cells were transferred to stage X blastoderms. The fate of the transferred primordial germ cells was traced by detecting the single nucleotide polymorphism in the D-loop region of the mitochondrial DNA in White Leghorn and Barred Plymouth Rock chickens used in this study. The transferred donor primordial germ cell-derived cells were detected in the gonads, but not in the blood cells, of 17-d incubated embryos by PCR. 4. This procedure for primordial germ cell manipulation could provide a novel method of producing germline chimaeric chickens. 5. In conclusion, our findings indicate that primordial germ cells isolated from embryonic blood can migrate to the germinal ridges of recipient embryos after being transferred to stage X blastoderms. Although these transferred primordial germ cells differentiated into germ cells, no differentiation into blood cells was observed.     

In vitro differentiation of bovine bone marrow‐derived mesenchymal stem cells into male germ cells by exposure to exogenous bioactive factors

Mesenchymal stem cells (MSC) are multipotent progenitor cells defined by their ability to self‐renew and give rise to differentiated progeny. Previous studies have reported that MSC may be induced in vitro to develop into different types of specialized cells including male gametes. In vitro gamete derivation technology has potential applications as an alternative method for dissemination of elite animal genetics, production of transgenic animals and conservation of endangered species. This study aimed at investigating the in vitro effect of BMP4, TGFβ1 and RA on the potential for germ cell (GC) differentiation of bovine foetal MSC (bfMSC) derived from bone marrow (BM). The effect of BMP4, TGFβ1 and RA was analysed on the expression of pluripotent, GC and male GC markers on bfMSC during a 21‐day culture period. bfMSC cultured under in vitro conditions expressed OCT4, NANOG and DAZL, but lacked expression of mRNA of VASA, STELLA, FRAGILIS, STRA8 and PIWIL2. Treatment with exogenous BMP4 and TGFβ1 induced a transient increase (p < .05) in DAZL and NANOG mRNA levels, respectively. However, exposure to RA was more effective in increasing (p < .05) expression of DAZL and regulating expression of OCT4 and mRNA levels of NANOG. These data suggest that bfMSC may possess potential for early GC differentiation, where OCT4, NANOG and specially DAZL may play significant roles in controlling progression along the GC lineage.