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.     

哺乳动物原始卵泡形成与发育的研究进展

在大多数雌性哺乳动物中,早期原始卵泡的形成主要包括3个过程：原始生殖细胞的迁移、生殖细胞减数分裂和生殖细胞巢破裂。原始卵泡形成与发育过程涉及到诸多因子和信号通路的调节作用,且原始卵泡库的大小及储备能力将决定雌性动物终生生殖能力。本文就参与原始卵泡形成和发育过程中的因子和信号通路作一综述,旨在深入了解参与原始卵泡形成与发育的细胞和分子机制,为维持原始卵泡库及促进原始卵泡激活提供研究思路。     

p27(Kip1) negatively regulates the activation of murine primordial oocytes

In mice, small oocytes (primordial oocytes) are enclosed within flattened granulosa cells to form primordial follicles around birth. A small number of primordial oocytes enter the growth phase, whereas others are quiescent. The mechanism regulating this selection of primordial oocytes is not well understood. The objective of the present study was to understand the role of p27(Kip1), which regulates cell cycle progression in somatic cells, in the growth initiation of primordial oocytes in neonatal mice. We studied the localization of p27(Kip1) in 0-, 3-, 5-, 7- and 21-day-old mouse ovaries by immunohistochemistry. Ovaries from 3-day-old mice were treated with p27(Kip1) siRNAs (small interfering RNAs), and knockdown of p27(Kip1) was determined by immunohistochemistry and Western blotting. Ovaries treated with siRNAs were organ-cultured for 6 days, and oocyte growth was estimated histologically. Expression of p27(Kip1) was undetectable in the primordial oocytes of newborn mice. In the 3-day-old ovaries (n=3), p27(Kip1) was demonstrated in the nucleus of 36 ± 6% primordial oocytes. The percentage of p27(Kip1)-positive primordial oocytes increased to 72 ± 8 (n=3), 85 ± 7 (n=3) and 93 ± 5 (n=3) in the 5-, 7- and 21-day-old mouse ovaries, respectively. After knockdown of the p27(Kip1) protein by siRNAs, a higher proportion of oocytes entered the growth phase in cultured ovaries than those in the control. These results suggest that p27(Kip1) negatively regulates primordial oocyte growth and that knockdown of p27(Kip1) leads primordial oocytes to enter the growth phase in vitro.     

哺乳动物PGCs用于胚胎干细胞培养的研究

本文综述了哺乳动物原始生殖细胞（PGCs）的研究进展；归纳了影响用PGCs分离胚胎干培养的因素。阐述了在PGCs用于胚胎干细胞培养研究中存在的问题及应用前景。     

Morphological Studies on the Development of the Bronchial Epithelium of the Fetal Camel Lung

Light microscopic observations revealed that in camel foetuses of 25 mm crown‐to‐rump length (CRL) the primordial tubular system of the prospective lung was formed of several tubules lined by undifferentiated columnar epithelium. Intra‐epithelial neuroendocrine cells were the first elements to be differentiated in the lining epithelium of the primordial tubular system of the prospective lung as early as 25 mm CRL. On reaching 50–67 mm CRL, the primordial tubular system started to differentiate into two systems of primordial tubules, the prospective bronchial or light tubules and the future respiratory or dark tubules. The lining epithelium of the prospective bronchial tubules revealed a clear evidence of ciliogenesis as early as 80 mm CRL. From 800 mm CRL onwards, the bronchial epithelium demonstrated ciliated and non‐ciliated secretory cells. The non‐ciliated secretory cells of the bronchial epithelium of fetal camel lung showed moderate reaction to AB/PAS technique, for the first time, in fetuses reaching 600 mm CRL.     

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.     

On the Development of the Feline Skeleton

Light microscopic observations revealed that in camel foetuses of 25 mm crown-to-rump length (CRL) the primordial tubular system of the prospective lung was formed of several tubules lined by undifferentiated columnar epithelium. Intra-epithelial neuroendocrine cells were the first elements to be differentiated in the lining epithelium of the primordial tubular system of the prospective lung as early as 25 mm CRL. On reaching 50–67 mm CRL, the primordial tubular system started to differentiate into two systems of primordial tubules, the prospective bronchial or light tubules and the future respiratory or dark tubules. The lining epithelium of the prospective bronchial tubules revealed a clear evidence of ciliogenesis as early as 80 mm CRL. From 800 mm CRL onwards, the bronchial epithelium demonstrated ciliated and non-ciliated secretory cells. The non-ciliated secretory cells of the bronchial epithelium of fetal camel lung showed moderate reaction to AB/PAS technique, for the first time, in fetuses reaching 600 mm CRL.     

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。     

A morphological and immunohistochemical study of healthy and atretic follicles in the ovary of the sexually immature ostrich (Struthio camelus)

The morphology of healthy and atretic follicles in the ovary of the sexually immature ostrich was described in the present study. In addition, the distribution of the intermediate filaments desmin, vimentin and smooth muscle actin, in these ovarian follicles, was demonstrated. Healthy and atretic primordial, pre-vitellogenic and vitellogenic follicles were present in the ovaries of the sexually immature ostrich. Atresia occurred during all stages of follicular development. Atretic primordial and pre-vitellogenic follicles were characterized by the presence of a shrunken oocyte surrounded by a multilayered granulosa cell layer. Two forms of atresia (types 1 and 2) were identified in vitellogenic follicles. In the advanced stages of type 1 atresia the follicle was dominated by a hyalinized mass. In contrast, in type 2 atresia the granulosa and theca interna cells differentiated into interstitial gland cells. Positive immunostaining for desmin was observed in the granulosa cells of only healthy primordial and pre-vitellogenic follicles. Atretic primordial and pre-vitellogenic follicles were immunonegative for desmin. Vimentin immunoreactivity was demonstrated in the granulosa cells of all follicles except the vitellogenic atretic follicles. The results of the present study indicate that ovarian follicles in the sexually immature ostrich undergo a cycle of growth and regression, which is similar to that reported in other avian species. Furthermore, based on the results of the immunohistochemical study, it would appear that the distribution and immunostaining of intermediate filaments changes during follicular development and atresia.     

Expression Pattern of Vascular Endothelial Growth Factor in Canine Folliculogenesis and its Effect on the Growth and Development of Follicles after Ovarian Organ Culture

In this study, the expressions of VEGF in dog follicles were detected by immunohistochemistry and the effects of VEGF treatment on the primordial to primary follicle transition and on subsequent follicle progression were examined using a dog ovary organ culture system. The frozen‐thawed canine ovarian follicles within slices of ovarian cortical tissue were cultured for 7 and 14 days in presence or absence of VEGF. After culture, the ovaries were fixed, sectioned, stained and counted for morphologic analysis. The results showed that VEGF was expressed in the theca cells of antral follicles and in the granulosa cells nearest the oocyte in preantral follicle but not in granulosa cells of primordial and primary follicles; however, the VEGF protein was expressed in CL. After in vitro culture, VEGF caused a decrease in the number of primordial follicles and concomitant increase in the number of primary follicles that showed growth initiation and reached the secondary and preantral stages of development after 7 and 14 days. Follicular viability was also improved in the presence of VEGF after 7 and 14 days in culture. In conclusion, treatment with VEGF was found to promote the activation of primordial follicle development that could provide an alternative approach to stimulate early follicle development in dogs.     

An immunohistochemical study of ovarian follicle histogenesis in the early post-hatch Japanese quail (Coturnix coturnix japonica)

The early post-hatch development of immunoreactivity to vimentin, desmin, smooth muscle actin (SMA) and laminin, in relation to follicle histogenesis, was described in this study. Ovigerous cords in day old quails contained pre-granulosa cells and oocytes. Pre-granulosa cells at the cortico-medullary junction were vimentin immunopositive. A laminin immunopositive basement membrane and desmin immunopositive mesenchymal cells lined the ovigerous cords. Ovigerous cords in 3-day-old quails contained developing primordial follicles, the vimentin immunopositive pre-granulosa cells of which were partially encircled by a basement membrane and desmin immunopositive mesenchymal cells. In 5- to 7-day-old quails, ovigerous cords formed an outer cortical region, while primordial follicles formed the inner cortical region. Early pre-vitellogenic follicles were present in 9- to 13-day-old quails. Underlying the granulosa cells of these follicles was a laminin immunopositive basement membrane and a layer of desmin immunopositive thecal cells. Early and late pre-vitellogenic follicles dominated the ovary in 15- to 17-day-old quails. The thecal layer in these follicles was desmin immunopositive, but SMA immunonegative. The results of the study have shown that the process of primordial follicle development in the Japanese quail is similar to that reported in mammals. The study suggests that in the quail pre-granulosa cells originate predominantly from the medulla. The study has shown that, in the Japanese quail, thecal cells are derived from desmin immunopositive mesenchymal cells lining the ovigerous cords.     

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.     

四氯联苯对鸡胚性腺原始生殖细胞的毒性影响

采用四氯联苯处理发育至Ⅹ期的白来航鸡种蛋,孵化5.5天后取出胚胎进行全固定,用石蜡切片和PAS染色(高碘酸希夫试剂)研究四氯联苯对鸡胚性腺原始生殖细胞(PGCs)的影响.结果表明,四氯联苯对发育至5.5日胚龄的鸡胚性腺PGCs数量和结构有明显影响,但雌二醇处理的5.5日胚龄鸡胚PGCs数量变化不显著,其结构无明显变化.表明四氯联苯对5.5日胚龄的鸡胚性腺PGCs的影响主要来自于其毒性作用.     

IFN-γ在胚泡植入期大鼠卵巢的分布

为研究IFN-γ对妊娠的免疫调控机制,本文采用免疫组化SP法,对大鼠胚泡植入期卵巢组织内IFN-γ的分布进行了研究。结果表明,IFN-γ在不同发育阶段卵泡的颗粒细胞中均有分布,原始卵泡、初级卵泡、次级卵泡和成熟卵泡的卵母细胞呈中等阳性反应,在原始卵泡、初级卵泡及次级卵泡的卵泡内膜细胞呈IFN-γ强阳性反应,成熟卵泡内膜细胞则呈弱阳性反应;在卵巢组织基质细胞、血管内皮细胞、粒性黄体细胞、生殖上皮也出现强阳性反应,卵泡液呈阳性着色。结果表明,妊娠有机体生理剂量的IFN-γ通过影响卵巢功能参与了胚泡的植入,与妊娠的建立和维持密切相关。     

An immunohistochemical study of the distribution of intermediate filaments in the ovary of the emu (Dromaius novaehollandiae)

The immunohistochemical localization of the intermediate filaments desmin, vimentin and smooth muscle actin (SMA) in the ovary of the emu was described in the present study. The cortical region of the ovary contained developing and atretic primordial, pre-vitellogenic and vitellogenic follicles. Vimentin immunostaining was demonstrated in the granulosa cell layer of primordial, pre-vitellogenic and vitellogenic developing and atretic follicles. An interesting finding of the present study was the localization of SMA in fibroblasts located in the theca externa of late vitellogenic follicles. The presence of SMA in these fibroblasts suggests that they possess characteristics of smooth muscle cells.     

Early organogenesis of the caprine stomach

Early organogenesis of the caprine stomach was studied in a series of 11 embryos ranging from 6.5 mm neck-rump length (NRL) to 13.3 mm crown-rump length (CRL). In embryos with 6.5-6.7 mm NRL, a part of the primordial proper esophagus extended to the dorsal side of the primordial stomach. The primordial proper esophagus and its extension were lined with a simple epithelium and stained dark brown with Con A III, while the primordial stomach was weakly stained. In embryo with 7.3 mm NRL, the esophageal extension was separated from the proper esophagus by constriction, and became a primordial forestomach situated in an area outside the omental sac. In embryos with 8.3 mm NRL-10.7 mm CRL, primordial forestomach and primordial stomach were united and formed a spindle shaped primordial ruminant stomach with foregut rotation. The primordial ruminant stomach was similar to the primordial simple stomach except that it was more flattened laterally with the convex at the area of 'lesser curvature'. Primordial rumen, omasum and abomasum appeared from the spindle shaped primordial ruminant stomach in an embryo with 12.9 mm CRL. In an embryo with 13.3 mm CRL, primordial reticulum originated from an area between the primordial rumen and omasum.     

Oocyte Morphology from Primordial to Early Tertiary Follicles of Yak

The objective of this study was to investigate the developmental morphology of yak oocytes from the primordial follicle to the tertiary follicle. Yak oocytes from resting primordial (n = 6), activated primordial (n = 12), primary (n = 9), secondary (n = 7) and early tertiary (n = 5) follicles were processed and analysed by light and transmission electron microscopy. The resting primordial follicular oocyte was characterized by relatively smooth surface on the oolemma, the accumulation of free and organelle‐related smooth (SER) and rough endoplasmic reticulum (RER), round or oval mitochondria, and polyribosomes on the surface of the RER and throughout the ooplasm. The activated primordial follicular oocyte was dominated by numerous coated pits and coated vesicles on the oolemma, and round mitochondria. Up to the secondary follicular stage the oocyte displayed an increase in the number of microvilli, polyribosome, Golgi complexes and mitochondria with distinct cristae. During the secondary follicular stage, formation of the zona pellucida, development of a desmosome‐like connection between the oocyte and the granulosa cells, formation of the cortical granules in the oocyte and elongated mitochondria in nearly all oocytes were seen. In the early tertiary follicular oocyte, the perivitelline space was present and a decrease in free SER and RER in the ooplasm occurred; finally, the nucleus migrated from an eccentric to a peripheral location. In conclusion, the growth of the yak oocyte is associated with the relocation and modulation of a number of cytoplasmic organelles as well as the development of oocyte‐specific structures such as the zona pellucida, desmosome‐like connection and cortical granules.     

哺乳动物原始生殖细胞与EG细胞研究进展

本文综述了哺乳动物原始生殖细胞的来源、增殖、迁移及生物学特点及近年来国内外用原始生殖细胞分离EG细胞的研究进展，探讨了EG细胞作为另一种多能性干细胞的优越性。     

Caspase-9在肉用绵羊主要生殖器官中的分布研究

通过试验研究凋亡因子Caspase-9在肉用绵羊主要生殖器官中的表达,并探讨其生理意义.利用免疫组织化学的方法对Caspase-9在肉用绵羊主要生殖器官中的表达进行研究.研究结果显示,Caspase-9只在细胞质中表达,其在卵巢中主要分布于原始卵泡、初级卵泡及颗粒性黄体细胞,而卵泡膜性黄体细胞中未见表达;黄体期,主要表达于子宫的浅层腺体腺上皮细胞、子宫内膜上皮细胞、子宫颈黏膜固有层的淋巴小结、输卵管黏膜上皮分泌细胞和纤毛细胞、峡部的浆液性腺的腺上皮细胞;卵泡期,子宫和输卵管各个部位阳性反应不明显.在正常生理情况下,Caspase-9参与了肉用绵羊主要生殖器官周期性变化的调控和子宫黏膜免疫,对生殖器官功能的稳定发挥起着重要作用.