Asymmetrical Development of the Gonads in the Embryos and Fry of the Fish, Oryzias celebensis

The development of the gonad in Oryzias celebensis was studied by light and electron microscopy. The path of PGC-migration, the time of the sex-differentiation, and the pattern of germ cell proliferation were almost identical to those in O. latipes. The most conspicuous difference was in the distribution of germ cells after migration. The gonadal anlage in O. celebensis developed only on the right side of the dorsal mesentery, although PGCs were stituated on the both sides of the embryos before migration. The testis retained its unilateral condition throughout development and acquired a unilobed shape. In the female, the right presumptive ovary developed over the mesentery, and the ovary became bilobed. Thereafter, right and left parts of the presumptive ovary fused to develop into a single ovary in the adult fish. The situation appears to be comparable to the gonadal asymmetry in birds, but the present observations suggest that the developmental processes of the asymmetrical gonads in this fish are quite different from those in birds.     

Differentiation of mouse primordial germ cells into female or male germ cells.

Mouse primordial germ cells (PGCs) migrate from the base of the allantois to the genital ridge. They proliferate both during migration and after their arrival, until initiation of the sex-differentiation of fetal gonads. Then, PGCs enter into the prophase of the first meiotic division in the ovary to become oocytes, while those in the testis become mitotically arrested to become prospermatogonia. Growth regulation of mouse PGCs has been studied by culturing them on feeder cells. They show a limited period of proliferation in vitro and go into growth arrest, which is in good correlation with their developmental changes in vivo. However, in the presence of multiple growth signals, PGCs can restart rapid proliferation and transform into pluripotent embryonic germ (EG) cells. Observation of ectopic germ cells and studies of reaggregate cultures suggested that both male and female PGCs show cell-autonomous entry into meiosis and differentiation into oocytes if they were set apart from the male gonadal environments. Recently, we developed a two-dimensional dispersed culture system in which we can examine transition from the mitotic PGCs into the leptotene stage of the first meiotic division. Such entry into meiosis seems to be programmed in PGCs before reaching the genital ridges and unless it is inhibited by putative signals from the testicular somatic cells.     

Sexually dimorphic development of mouse primordial germ cells: switching from oogenesis to spermatogenesis

During embryogenesis, primordial germ cells (PGCs) have the potential to enter either spermatogenesis or oogenesis. In a female genital ridge, or in a non-gonadal environment, PGCs develop as meiotic oocytes. However, male gonadal somatic cells inhibit PGCs from entering meiosis and direct them to a spermatogenic fate. We have examined the ability of PGCs from male and female embryos to respond to the masculinising environment of the male genital ridge, defining a temporal window during which PGCs retain a bipotential fate. To help understand how PGCs respond to the male gonadal environment, we have identified molecular differences between male PGCs that are committed to spermatogenesis and bipotential female PGCs. Our results suggest that one way in which PGCs respond to this masculinising environment is to synthesise prostaglandin D(2). We show that this signalling molecule can partially masculinise female embryonic gonads in culture, probably by inducing female supporting cells to differentiate into Sertoli cells. In the developing testis, prostaglandin D(2) may act as a paracrine factor to induce Sertoli cell differentiation. Thus part of the response of PGCs to the male gonadal environment is to generate a masculinising feedback loop to ensure male differentiation of the surrounding gonadal somatic cells.     

革胡子鲇卵巢在第一次性周期内分化与发育的研究

用光镜和电镜研究了革胡子鲇（Ｃｌａｒｉａｓｌａｚｅｒａ）原生殖细胞的起源迁移,卵巢在第１次性周期内的分化与发育以及各发育时期卵母细胞的超微结构。原生殖细胞起源于内胚层,有固定的迁移路线,进入生殖嵴后,生殖嵴进一步分化,出现卵巢腔分化成卵巢,卵母细胞在第１次性周期内的发育可分成６个时相,描述了各时相卵母细胞的显微结构与超微结构。同时,叙述了卵母细胞中卵黄发生的形态形成。     

Expression of low density lipoprotein receptor-related protein 4 (Lrp4) gene in the mouse germ cells

The low density lipoprotein receptor-related protein 4 gene (Lrp4) was identified by subtractive screening of cDNAs of the migratory primordial germ cells (PGCs) of E8.5-9.5 embryo and E3.5 blastocysts. Lrp4 is expressed in PGCs in the hindgut and the dorsal mesentery of E9.5 embryos, and in germ cells in the genital ridges of male and female E10.5-13.5 embryos. Lrp4 is also expressed in spermatogonia of the neonatal and adult testes and in the immature oocytes and follicular cells of the adult ovary. The absence of Lrp4 expression in the blastocyst, embryonic stem cells and embryonic germ cells suggests the Lrp4 is a molecular marker that distinguishes the germ cells from embryo-derived pluripotent stem cells.     

Gonadal morphology and reproductive traits of the Amazonian fish Arapaima gigas (Schinz, 1822)

An account of gonadal morphology and reproductive traits of the Amazonian arapaimatid, Arapaima gigas (Schinz 1822), is given. The only functional ovary (left) was foliaceous in shape having the ventral surface without an ovarian capsule. The absence of the ovarian capsule ventral portion, absence of oviduct and funnel‐shaped coelomic cavity are features that imply that oocytes are freed in the coelomic cavity before being shed to the aquatic environment through a genital papilla. The only functional testis (left) was cord‐like with a maximum diameter of 1–1.5 cm and was connected to the genital papilla through a spermatic duct. Size at sexual maturity was 145–154 cm and 115–124 cm, total length class, for females and males, respectively.     

Stage- and sex-dependent expressions of Usp9x, an X-linked mouse ortholog of Drosophila Fat facets, during gonadal development and oogenesis in mice

During the Drosophila oogenic processes, Fat facets (Faf), an ubiquitin-specific protease essential for normal development of oocyte and eye, becomes localized at the posterior pole and is incorporated into the pole cells. This is dependent on Oskar, a key factor for pole cell determination, and suggests a role for Faf in germ cell differentiation and development. Here we show that Usp9x, an X-linked ortholog of Faf, is predominantly expressed in both germ cell and supporting cell lineages during mouse gonadal development in stage- and sex-dependent manners. Usp9x was first detected in PGCs at 10.5 days post coitum (dpc), and thereafter its expression both at mRNA and protein levels was enhanced in PGCs of both sexes at 11.5-13.5 dpc. In testis, Usp9x expression rapidly decreased to an undetectable level by 15.5 dpc and after birth to adult, no expression was found in any spermatogenic cells, except for weak expression in Sertoli cells. In the ovary, Usp9x expression in embryonic oocytes was also reduced at the newborn stage, its expression reappeared in oocytes at secondary follicle stage, and its products were highly accumulated in the cytoplasm of Graaffian follicles in adults. Although follicular epithelial cells also expressed Usp9x at a moderate level during postnatal development, its expression was downregulated from early secondary follicle stage. Thus, the present study is not only the first to demonstrate a conserved expression of fat facets in PGCs between mouse and fly, but also sex- and stage-dependent changes of a specific component of the deubiquitylation system during mammalian gonadal development.     

Gonadal morphogenesis and sex differentiation in cultured Ussuri catfish Tachysurus ussuriensis

The objective of this study was to investigate the optimal developmental time to perform sex reversal in Ussuri catfish Tachysurus ussuriensis, to develop monosex breeding in aquaculture. Systematic observations of gonadal sex differentiation of P. ussiriensis were conducted. The genital ridge formed at 9 days post fertilization (dpf) and germ cells begin to proliferate at 17 dpf. The ovarian cavity began forming on 21 dpf and completed by 25 dpf while presumptive testis remained quiescent. The primary oocytes were at the chromatin nucleolus stage by 30 dpf, the peri‐nucleolus stage by 44 dpf and the cortical alveoli stage by 64 dpf. The germinal vesicle migrated towards the animal pole (polarization) at 120 dpf. In presumptive testis, germ cells entered into mitosis and blood vessels appeared in the proximal gonad on 30 dpf. The efferent duct anlage appeared on 36 dpf and formation of seminal lobules with spermatogonia and lobules interstitium occurred at 120 dpf. Therefore, gonadal sex differentiation occurred earlier in females than in males, with the histological differentiation preceding cytologic differentiation in T. ussuriensis. This indicates that undifferentiated gonads directly differentiate into ovary or testis between 17 and 21 dpf and artificial induction of sexual reversal by oral steroid administration must be conducted before 17 dpf.     

Stage- and sex-dependent expressions of Usp9x,an X-linked mouse ortholog of Drosophila Fat facets,during gonadal development and oogenesis in mice

During the Drosophila oogenic processes, Fat facets (Faf), an ubiquitin-specific protease essential for normal development of oocyte and eye, becomes localized at the posterior pole and is incorporated into the pole cells. This is dependent on Oskar, a key factor for pole cell determination, and suggests a role for Faf in germ cell differentiation and development. Here we show that Usp9x, an X-linked ortholog of Faf, is predominantly expressed in both germ cell and supporting cell lineages during mouse gonadal development in stage- and sex-dependent manners. Usp9x was first detected in PGCs at 10.5 days post coitum (dpc), and thereafter its expression both at mRNA and protein levels was enhanced in PGCs of both sexes at 11.5-13.5 dpc. In testis, Usp9x expression rapidly decreased to an undetectable level by 15.5 dpc and after birth to adult, no expression was found in any spermatogenic cells, except for weak expression in Sertoli cells. In the ovary, Usp9x expression in embryonic oocytes was also reduced at the newborn stage, its expression reappeared in oocytes at secondary follicle stage, and its products were highly accumulated in the cytoplasm of Graaffian follicles in adults. Although follicular epithelial cells also expressed Usp9x at a moderate level during postnatal development, its expression was downregulated from early secondary follicle stage. Thus, the present study is not only the first to demonstrate a conserved expression of fat facets in PGCs between mouse and fly, but also sex- and stage-dependent changes of a specific component of the deubiquitylation system during mammalian gonadal development.     

Gonadal morphogenesis and sex differentiation in intraovarian embryos of the viviparous fish Zoarces viviparus (Teleostei, Perciformes, Zoarcidae): a histological and ultrastructural study

It is essential to know the timing and process of normal gonadal differentiation and development in the specific species being investigated in order to evaluate the effect of exposure to endocrine-disrupting chemicals on these processes. In the present study gonadal sex differentiation and development were investigated in embryos of a viviparous species of marine fish, the eelpout, Zoarces viviparus, during their intraovarian development (early September to January) using light and electron microscopy. In both sexes of the embryos at the time of hatching (September 20) the initially undifferentiated paired bilobed gonad contains primordial germ cells. In the female embryos, ovarian differentiation, initiated 14 days posthatch (dph), is characterized by the initial formation of the endoovarian cavity of the single ovary as well as by the presence of some early meiotic oocytes in a chromatin-nucleolus stage. By 30 dph, the endoovarian cavity has formed. By 44 dph and onward, the ovary and the oocytes grow in size and at 134 dph, just prior to birth, the majority of the oocytes are at the perinucleolar stage of primary growth and definitive follicles have formed. In the presumptive bilobed testis of the male embryos, the germ cells (spermatogonia), in contrast to the germ cells of the ovary, remain quiescent and do not enter meiosis during intraovarian development. However, other structural (somatic) changes, such as the initial formation of the sperm duct (30 dph), the presence of blood vessels in the stromal areas of the testis (30 dph), and the appearance of developing testicular lobules (102 dph), indicate testicular differentiation. Ultrastructually, the features of the primordial germ cells, oogonia, and spermatogonia are similar, including nuage, mitochondria, endoplasmic reticulum, and Golgi complexes.     

Genital ridges exert long-range effects on mouse primordial germ cell numbers and direction of migration in culture

The functional gametes of all vertebrates first arise in the early embryo as a migratory population of cells, the primordial germ cells (PGCs). These migrate to, and colonise, the genital ridges (GR) during the early organogenesis period, giving rise to the complete differentiating gonad. PGCs first become visible by alkaline phosphatase staining in the root of the developing allantois at 8.5 days post coitum (dpc). At 9.5 dpc they are found in the wall of the hind-gut and, during the following three days, they migrate along the hind-gut mesentery to the dorsal body wall, and then to the genital ridges. By 12.5 dpc, the great majority of PGCs have colonised the genital ridges. During this period the number of PGCs increases from less than 100 to approximately 4000. In a previous paper (Donovan et al. 1986), we showed that 10.5 dpc PGCs can be explanted from the hind-gut mesentery, and will spread and migrate on feeder cell layers. We showed also that the intrinsic ability of PGCs to spread and migrate changes as they colonise the genital ridges. In this paper, we examine extrinsic factors that control PGC behaviour in vitro. Using PGCs taken from 8.5 dpc embryos, at the beginning of their migratory phase, we show that culture medium conditioned by 10.5 dpc genital ridges causes an increase in the number of PGCs in these cultures. We also show that PGCs migrate towards 10.5 dpc genital ridges in preference to other explanted organs. These experiments show that genital ridges exert long-range effects on the migrating population of PGCs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trichinella spiralis: Morphological characteristics of male and female intestine-infecting larvae

The sex of encysted and excysted intestine-infecting T. spiralis larvae can be distinguished by the following morphological characteristics: the male larva has a long (approx 50 μm) rectum, and the anterior part of the testis is curved posteriorly. The female larva has a shorter rectum (approx 25 μm), a telogonic ovary, coiled uterine and seminal receptacle primordia, and a vaginal primordium. In paraffin sections males can be recognized by the spermatocytes which are of the same size. The oocytes vary in size: the smallest are located in the ventral portion, the largest on the dorsal portion of the ovary. Sex of the larvae can be differentiated by the length of the rectum as early as the tenth day, by the curvature of the anterior part of the testis and by the uterine primordium by the eleventh day, and by the presence of the vaginal primordium by the thirteenth day of intramuscular development. Farre's Organ is believed to be the primordium of the seminal receptacle.     

Two isoforms of vasa homologs in a teleost fish: their differential expression during germ cell differentiation.

Two isoforms of vasa mRNA and protein are present in a teleost fish, tilapia. One (vas-s) lacks a part of the N-terminal region found in the other isoform (vas). Both isoforms are expressed in oocytes through the embryonic stage when primordial germ cells (PGCs) localize in the lateral plate mesoderm. After PGC localization in the gonadal anlagen, vas-s expression increased and vas expression became undetectable. Expression of both isoforms was observed again after morphological gonadal sex differentiation, irrespective of genotypic sex. In ovary, compared with vas expression vas-s expression predominated throughout oogenesis. In testis, vas expression was predominant compared with vas-s during spermatogenesis. These results indicate that relative expression of two vasa isoforms is dependent upon germ cell differentiation and sex.     

Ultrastructural Morphology of the Male and Female Genital Tracts of <Emphasis Type="Italic">Psoroptes</Emphasis> spp. (Acari: Astigmata: Psoroptidae)

The structure of the male and female genital systems of the astigmatid mite Psoroptes ovis (Hering) is described. The male genital system is composed of a paired testis, fused at its proximal part, two vasa deferentia, an ejaculatory duct, into which a single accessory gland opens, and a copulatory organ. The testis is characterized by a peripheric syncytial cell surrounding spermatogonia, spermatocytes, spermatids and spermatozoa which are distributed regularly in the gonad according to the sequence of spermatogenesis. The female genital system consists of a copulatory pore (the bursa copulatrix), a seminal receptacle, paired ovaries and oviducts, a glandular uterus and an ovipositor which leads to the oviporus. Ovaries are composed of somatic cells, germ cells and a central cell, with a multilobular nucleus, connected to oocytes by a stalk. Similarities with other astigmatic mites belonging to Psoroptidia and Acaridia are also discussed.     

Production of quail (Coturnix japonica) germline chimeras derived from in vitro-cultured gonadal primordial germ cells

A previous report from our laboratory documented successful production of quail (Coturnix japonica) germline chimeras by transfer of gonadal primordial germ cells (gPGCs). Subsequently, this study was designed to evaluate whether gPGCs can be maintained in vitro for extended period, and furthermore, these cultured PGCs can induce germline transmission after transfer into recipient embryos. In experiment 1, gonadal cells from the two strains (wild-type plumage (WP) and black (D) quail) were cultured in vitro for 10 days. Using antibody QCR1, we detected a continuous, significant (P = 0.0002) increase in the number of WP, but not D, PGCs. QCR1-positive WP colonies began to form after 7 days in culture. On Day 10 of culture, 803 WP PGCs were present as a result of a continuous increase, whereas no D PGC colonies could be detected and the D gonadal stroma cells were rolled up. Differences in the PGCs or the gonadal stroma cells of the two different strains might account for these differences. In experiment 2, WP PGC colonies were maintained in vitro up to Day 20 of culture, and 10- or 20-day-cultured PGCs were microinjected into dorsal aortas of 181 recipient D embryos. Thirty-five (19.3%) of the transplanted embryos hatched after incubation, and 25 (71.4%) of the hatchlings reached sexual maturity. Testcrossing of the sexually mature hatchlings resulted in three (10 days, 33.3%) and eight (20 days, 50.0%) germline chimeras respectively. This report is the first to describe successful production of germline chimera by transfer of in vitro-cultured gPGCs in quail.     

Commitment of fetal male germ cells to spermatogonial stem cells during mouse embryonic development

The continuous production of mammalian sperm is maintained by the proliferation and differentiation of spermatogonial stem cells that originate from primordial germ cells (PGCs) in the early embryo. Although spermatogonial stem cells arise from PGCs, it is not clear whether fetal male germ cells function as spermatogonial stem cells able to produce functional sperm. In the present study, we examined the timing and mechanisms of the commitment of fetal germ cells to differentiate into spermatogonial stem cells by transplantation techniques. Transplantation of fetal germ cells into the seminiferous tubules of adult testis showed that donor germ cells, at 14.5 days postcoitum (dpc), were able to initiate spermatogenesis in the adult recipient seminiferous tubules, whereas no germ cell differentiation was observed in the transplantation of 12.5-dpc germ cells. These results indicate that the commitment of fetal germ cells to differentiate into spermatogonial stem cells initiates between embryonic days 12.5 and 14.5. Furthermore, the results suggest the importance of the interaction between germ cells and somatic cells in the determination of fetal germ cell differentiation into spermatogonial stem cells, as normal spermatogenesis was observed when a 12.5-dpc whole gonad was transplanted into adult recipient testis. In addition, sperm obtained from the 12.5- dpc male gonadal explant had the ability to develop normally if injected into the cytoplasm of oocytes, indicating that normal development of fetal germ cells in fetal gonadal explant occurred in the adult testicular environment.     

Transfer of blood containing primordial germ cells between chicken eggs development of embryonic reproductive tract

The present study was carried out to investigate development of recipient chicken embryonic reproductive tracts which are transferred chicken primordial germ cells (PGCs). It is thought that differentiation of PGCs is affected by the gonadal somatic cells. When female PGCs are transferred to male embryos, it is possible that they differentiate to W-spermatogonia. However, the relationship development between PGCs and gonads has not been investigated. At stage 12–15 of incubation of fertilized eggs, donor PGCs, which were taken from the blood vessels of donor embryos, were injected into the blood vessels of recipient embryos. The gonads were removed from embryos that died after 16 days of incubation and from newly hatched chickens and organs were examined for morphological and histological features. The survival rate of the treated embryos was 13.6% for homo-sexual transfer of PGCs (male PGCs to male embryo or female PGCs to female embryo) and 28.9% for hetero-sexual transfer PGCs (male PGCs to female embryo or female PGCs to male embryo) when determined at 15 days of incubation. The gonads of embryos arising from homo-sexual transfer appeared to develop normally. In contrast, embryos derived from hetero-sexual transfer of PGCs had abnormal gonads as assessed by histological observation. These results suggest that hetero-sexual transfer of PGCs may influence gonadal development early-stage embryos.     

Erasure of methylation imprinting of Igf2r during mouse primordial germ-cell development

During germ cell differentiation in mice, the genome undergoes specific epigenetic modifications. These include demethylation of imprinted genes and subsequent establishment of parental allele-specific methylation. The mouse Igf2r gene is an imprinted gene that shows maternal-specific expression. Maternal-specific methylation of differentially methylated region 2 (DMR2) of this gene may be necessary for its maternal-specific expression. Before the allele-specific methylation is established, DMR2 is demethylated in both male and female primordial germ cells (PGCs) by 13.5 days post coitum (dpc), indicating that the demethylation of this region occurs earlier in PGC development. The timing of the demethylation has been, however, unknown. In this study, we attempted to determine the timing of methylation erasure of Igf2r DMR2 in developing PGCs, using transgenic mice expressing green fluorescent protein specifically in the germ line. We purified migrating PGCs from the transgenic mice and examined the methylation status of DMR2. The results show that some CpG sites within DMR2 start demethylation at 9.5 dpc in some migrating PGCs, before the cells colonize genital ridges, and the progression of demethylation is rapid after colonization of the genital ridges. To examine whether the gonadal environment is involved in demethylation, we analyzed the methylation of DMR2 after culturing migrating PGCs in the absence of a gonadal environment. These culture experiments support the idea that a gonadal environment is not required for demethylation of the region in at least a fraction of PGCs.