Sex differentiation and pubertal development of gonads in the viviparous mosquitofish, Gambusia affinis

The ontogenetic development of gonads from embryo to adult was observed histologically in the viviparous teleost, Gambusia affinis. Primordial germ cells (PGCs) appeared in the subendodermal space of the embryo 14 days before birth, and then transferred to the dorsal mesentery to form paired genital ridges 12 days before birth. The PGCs proliferated in the genital ridge, forming gonadal primordia 10 days before birth. All gonadal primordia differentiated to the ovary containing oocytes 2 days before birth, but then redifferentiated to the ovary and testis just after birth. This indicates that the mosquitofish is a juvenile hermaphroditic species. The characteristics of gonadal sex differentiation just after birth were enlargement of the oocytes in females, and invasion of somatic cells from the hilar region to an inner portion of the gonad in males. The paired ovary fused at the basal area 5 days after birth, then on the ventral and dorsal portions, developing into a single ovary 10 days after birth. During this time a single ovarian cavity was formed on the dorsal portion of the ovary. The paired testes fused only at the basal area and became a single testis having two main lobes 10 days after birth. The oocytes gradually developed and began vitellogenesis 100 days after birth, but did not reach maturation until 110 days after birth. Spermatogenic cells formed cysts at 20 days, began meiosis at 70 days, and matured to form sperm balls 90 days after birth. The male fish sexually matured earlier than the female.     

A light- and electron-microscopic study on the migration of primordial germ cells in the teleost,Oryzias latipes

Summary The primordial germ cells (PGCs) of Oryzias latipes in migration to the gonadal anlage have been investigated by light and electron microscopy. The ultrastructure of the PGCs, which occur in the subendodermal space on the syncytial periblast, differ conspicuously from that of the surrounding endodermal cells. After the PGCs move to the cavity between lateral plate and ectoderm, they are taken into the somatomesodermal layer and transferred to the dorsal mesentery where they form gonadal anlage with mesodermal cells. During their translocation to the dorsal mesentery through the somatic mesoderm, apparently without formation of pseudopods, the PGCs are completely surrounded by mesodermal cells. Since these conditions seem unfavorable to the active translocation of the PGCs to the dorsal mesentery, it is more likely that the PGCs are transferred passively by the morphogenic activity of the lateral-plate mesoderm.Counts of the number of the PGCs revealed that they are mitotically dormant during the migratory period. After the completion of the migration, they regain their proliferative activity. The PGCs in the female proliferate more actively than those in the male, which provides the first morphological indication of sex differentiation in this species of fish.     

Estrogen-induced gonadal sex reversal in the tammar wallaby

Estrogens have a feminizing effect on gonadal differentiation in fish, amphibians, reptiles, and birds. However, the role of estrogen during gonadal differentiation in mammals is less clear. We investigated the effect of estrogen on gonadal differentiation of male tammar wallabies. Male pouch young were treated orally with estradiol benzoate or oil from the day of birth, before seminiferous cords develop, to Day 25 postpartum and were killed at Day 50 postpartum. In all estrogen-treated neonates, a decrease in gonadal volume, volume of the seminiferous cords, thickness of the tunica albuginea, and number of germ cells was found. The stage of treatment affected the magnitude of the response. Two of three male young born prematurely after 25 days of gestation and treated subsequently with estradiol had ovary-like gonads, with well-developed cortical and medullary regions and primordial follicle formation. Furthermore, at Day 50 postpartum, many (21%) of the germ cells in these sex-reversed ovaries were in the leptotene and zygotene stages of meiosis, similar to female germ cells at the same stage of development. In the other males born on Day 26 of gestation or later, estradiol treatment from the day of birth caused development of dysgenetic testes, with abnormal Sertoli cells, atrophy of the seminiferous tubules and tunica albuginea, and absence of meiotic germ cells. In this marsupial, therefore, estradiol can induce either partial or complete transformation of the male gonads into an ovary with meiotic germ cells. These results confirm that estrogen can inhibit early testicular development, and that testis determination occurs during a narrow window of time.     

Germ cells, gonads and sex reversal in marsupials.

The formation of the testis or ovary is a critical step in development. Alterations in gonadal development during fetal or postnatal life can lead to intersexuality or infertility. Several model systems have been particularly useful in studying gonadal differentiation, the eutherian mammal and amphibia, fish, and birds. However, marsupials provide a unique opportunity to investigate gonadal development and the interactions of genes and hormones in gonadal differentiation and germ cell development in all mammals. On the one hand the genetic mechanisms appear to be identical to those in eutherian mammals, including the testis-determining SRY gene. On the other hand, marsupials retain in part the plasticity of the amphibian gonad to hormonal manipulation. It is possible to induce female to male and also male to female gonadal sex reversal in marsupials by hormonal manipulation, and oestradiol can induce male germ cells to enter meiosis at the time the oogonia do. In addition, in marsupials the development of the scrotum and mammary glands are independent of testicular androgens and instead are controlled by a gene or genes on the X-chromosome. Thus marsupials provide a number of opportunities for manipulating the sexual differentiation of the gonads that are not possible in eutherian mammals and so provide a unique perspective for understanding the common mechanisms controlling sexual development.     

Germ cells are not the primary factor for sexual fate determination in goldfish

The presence of germ cells in the early gonad is important for sexual fate determination and gonadal development in vertebrates. Recent studies in zebrafish and medaka have shown that a lack of germ cells in the early gonad induces sex reversal in favor of a male phenotype. However, it is uncertain whether the gonadal somatic cells or the germ cells are predominant in determining gonadal fate in other vertebrate. Here, we investigated the role of germ cells in gonadal differentiation in goldfish, a gonochoristic species that possesses an XX-XY genetic sex determination system. The primordial germ cells (PGCs) of the fish were eliminated during embryogenesis by injection of a morpholino oligonucleotide against the dead end gene. Fish without germ cells showed two types of gonadal morphology: one with an ovarian cavity; the other with seminiferous tubules. Next, we tested whether function could be restored to these empty gonads by transplantation of a single PGC into each embryo, and also determined the gonadal sex of the resulting germline chimeras. Transplantation of a single GFP-labeled PGC successfully produced a germline chimera in 42.7% of the embryos. Some of the adult germline chimeras had a developed gonad on one side that contained donor derived germ cells, while the contralateral gonad lacked any early germ cell stages. Female germline chimeras possessed a normal ovary and a germ-cell free ovary-like structure on the contralateral side; this structure was similar to those seen in female morphants. Male germline chimeras possessed a testis and a contralateral empty testis that contained some sperm in the tubular lumens. Analysis of aromatase, foxl2 and amh expression in gonads of morphants and germline chimeras suggested that somatic transdifferentiation did not occur. The offspring of fertile germline chimeras all had the donor-derived phenotype, indicating that germline replacement had occurred and that the transplanted PGC had rescued both female and male gonadal function. These findings suggest that the absence of germ cells did not affect the pathway for ovary or testis development and that phenotypic sex in goldfish is determined by somatic cells under genetic sex control rather than an interaction between the germ cells and somatic cells.     

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.     

Morphological development of the gonads in zebrafish

Gonadogenesis of zebrafish Danio rerio was investigated by means of light microscopy to test the suitability of gonad histology as an endpoint in hazard assessment of endocrine‐active compounds. At age 2 weeks post‐fertilization (pf), primordial germ cells were found in a dorsocaudal position in the body cavity. At 4 weeks pf, the majority of the fish (86%) possessed paired gonads with meiotic germ cells; these gonads represented presumptive ovaries. At week 5 pf, 87% of the fish examined had ovaries with perinucleolar oocytes. Further development of the gonads in female zebrafish up to week 11 pf was characterized by an increase in gonad size as well as in the number and size of perinucleolar oocytes. Starting with week 5, some fish showed alterations of gonad morphology, including a decrease in the number and size of the oocytes, an enhanced basophilia and irregular shape of the oocytes, and finally their degeneration into residual bodies. With the decline in oocyte number, stromal cells became more numerous and they infiltrated the gonadal matrix. In several 7 week‐old zebrafish with altered gonadal morphology, enhanced numbers of gonial cells arranged in cyst‐like groups appeared. These gonads were interpreted as presumptive testes. In one fish out of 32 individuals examined, spermatocytes were detected, in addition to the gonial cells. During the subsequent weeks, the percentage of fish showing early testes with spermatogonia, spermatocytes and spermatids increased and reached 40% at 11 weeks pf. The sequence of gonadal alterations taking place in some of the individuals from week 5 pf onwards was interpreted to reflect the transition of protogynic ovaries into testes. The developmental pattern described identifies zebrafish to be a juvenile hermaphrodite. The results of this study are of relevance for the use of gonadal histopathology as endpoint in endocrine disruption testing, particularly in order to avoid false diagnoses of ‘intersex gonads’ in zebrafish.     

Gonadal morphogenesis and sex differentiation in the viviparous fish Chapalichthys encaustus (Teleostei, Cyprinodontiformes, Goodeidae)

This study describes the structural and ultrastructural characteristics of gonadal sex differentiation and expression of Vasa, a germline marker, in different developmental stages of embryos and newborn fry of the barred splitfin Chapalichthys encaustus, a viviparous freshwater teleost endemic to Mexico. In stage 2 embryos, the gonadal crest was established; gonadal primordia were located on the coelomic epithelium, formed by scarce germ and somatic cells. At stage 3, the undifferentiated gonad appeared suspended from the mesentery of the developing swimbladder and contained a larger number of germ and somatic cells. At stages 4 and 5, the gonads had groups of meiotic and non-meiotic germ cells surrounded by somatic cells; meiosis was evident from the presence of synaptonemal complexes. These stages constituted a transition towards differentiation. At stage 6 and at birth, the gonad was morphologically differentiated into an ovary or a testis. Ovarian differentiation was revealed by the presence of follicles containing meiotic oocytes, and testicular differentiation by the development of testicular lobules containing spermatogonia in mitotic arrest, surrounded by Sertoli cells. Nuage, electron-dense material associated with mitochondria, was observed in germ cells at all gonadal stages. The Vasa protein was detected in all of the previously described stages within the germ-cell cytoplasm. This is the first report on morphological characteristics and expression of the Vasa gene during sexual differentiation in viviparous species of the Goodeidae family. Chapalichthys encaustus may serve as a model to study processes of sexual differentiation in viviparous fishes and teleosts.     

PITX2 controls asymmetric gonadal development in both sexes of the chick and can rescue the degeneration of the right ovary

The gonads arise on the ventromedial surface of each mesonephros. In most birds, female gonadal development is unusual in that only the left ovary becomes functional, whereas that on the right degenerates during embryogenesis. Males develop a pair of equally functional testes. We show that the chick gonads already have distinct morphological and molecular left-right (L-R) characteristics in both sexes at indifferent (genital ridge) stages and that these persist, becoming more elaborate during sex determination and differentiation, but have no consequences for testis differentiation. We find that these L-R differences depend on the L-R asymmetry pathway that controls the situs of organs such as the heart and gut. Moreover, a key determinant of this, Pitx2, is expressed asymmetrically, such that it is found only in the left gonad in both sexes from the start of their development. Misexpression of Pitx2 on the right side before and during gonadogenesis is sufficient to transform the right gonad into a left-like gonad. In ZW embryos, this transformation rescues the degenerative fate of the right ovary, allowing for the differentiation of left-like cortex containing meiotic germ cells. There is therefore a mechanism in females that actively promotes the underlying L-R asymmetry initiated by Pitx2 and the degeneration of the right gonad, and a mechanism in males that allows it to be ignored or overridden.     

Presumptive Primordial Germ Cells (pPGCs) and PGCs in Tadpoles from UV-irradiated embryos of Xenopus

In order to determine whether or not tadpoles that once lacked primordial germ cells (PGCs) in the genital ridges and dorsal mesentery as a result of ultraviolet (UV) irradiation subsequently contained germ cells at more advanced stages of larval development, the numbers of presumptive PGCs or PGCs were carefully examined in Xenopus tadpoles at Nieuwkoop and Faber's stage 35/36–52 that developed normally from UV-irradiated eggs.
No late-appearing germ cells were observed in almost all the UV-irradiated tadpoles examined at stages 49–52. This same population had completely lacked PGCs at about stage 46. Moreover, presumptive PGCs (pPGCs) or cells with granular cytoplasm that reacted with a monoclonal antibody specific for the germ plasm of cleaving Xenopus eggs stayed in the central part of the endoderm cell mass in the irradiated tadpoles at stage 35/36, when the majority of those cells were located in the dorsal part of the endoderm in unirradiated controls. Furthermore, in the irradiated embryos pPGCs were demonstrated to decrease in number with development and eventually to disappear in tadpoles at about stage 40. The results strongly suggest that UV irradiation under the conditions used here totally eliminated germline cells from the irradiated animals.     

Characterization of stage-specific embryonic antigen-1 (SSEA-1) expression during early development of the turkey embryo.

SSEA-1 is a carbohydrate epitope associated with cell adhesion, migration and differentiation. In the present study, SSEA-1 expression was characterized during turkey embryogenesis with an emphasis on its role in primordial germ cell development. During hypoblast formation, SSEA-1 positive cells were identified in the blastocoel and hypoblast and later in the germinal crescent. Based on location and morphology, these cells were identified, as PGCs. Germ cells circulating through embryonic blood vessels were also SSEA-1 positive. During the active phase of migration, PGCs in the dorsal mesentery and gonad could no longer be identified using the SSEA-1 antibody. The presence of PGCs at corresponding stages was verified using periodic acid Schiff stain. Pretreatment of PGCs with trypsin, alpha-galactosidase and neuraminidase did not restore immunoreactivity to SSEA-1. In general, expression was not limited to the germ cell lineage. SSEA-1 was also detected on the ectoderm, yolk sac endoderm, gut and mesonephric tubules. During neural tube closure, SSEA-1 was expressed by the neural epithelium of the fusing neural folds. Later SSEA-1 was detected in regions of the developing spinal cord. Enzyme pretreatment unmasked the epitope on some neural crest cells and cells in the sympathetic ganglion. The temporal and spatial distribution of SSEA-1 in the turkey embryo suggests a role in early germ cell and neural cell development. The absence of SSEA-1 on turkey gonadal germ cells was different from that observed for the chick. Therefore, while features of avian germ cell development appear to be conserved, expression of SSEA-1 can vary with the species.     

Sexual differentiation of germ cell deficient gonads in the medaka, Oryzias latipes

To determine whether germ cells perform any function in gonadal sexual differentiation, development of gonads in the medaka, Oryzias latipes, after exposure to busulfan was investigated. Busulfan suppressed proliferation of early germ cells, thus significantly reducing the number of germ cells and generating regions without germ cells in the developing gonads. Globular structures were observed in the parenchyma in these regions. The structure was male specific, developed at the same time as acinus (seminiferous tubule precursor), surrounded by the basal lamina, and contained characteristic desmosomes. These results strongly suggest that these globular structures are the precursors of seminiferous tubules devoid of germ cells. In the ovary, no follicles were observed but a well-developed ovarian cavity was evident. From these results we conclude that differentiation of gonadal parenchyma cells, except for follicular ones, is not germ cell dependent, though morphological differentiation of the somatic cells seems to follow the differentiation of germ cells.     

Gonadal development and sex differentiation in the cichlid fish Cichlasoma dimerus (Teleostei, Perciformes): a light- and electron-microscopic study

Although the overall pattern and timing of gonadal sex differentiation have been established in a considerable number of teleosts, the ultrastructure of early stages of gonadal development is not well documented. In this study, gonads from larval and juvenile stages of laboratory-reared Cichlasoma dimerus were examined at the light-microscopic and ultrastructural levels. This freshwater species adapts easily to captivity and spawns with high frequency during 8 months of the year, providing an appropriate model for developmental studies. Larvae and juveniles were kept at a water temperature of 26.5 +/- 1 degrees C and a 12:12 hour photoperiod. Gonadal development was documented from 14-100 days postfertilization, covering the period of histologically discernible sex differentiation. Gonadal tissue was processed according to standard techniques for light and electron microscopy. C. dimerus, a perciform teleost, is classified as a differentiated gonochorist, in which an indifferent gonad develops directly into a testis or ovary. On day 14, the gonadal primordium consists of a few germ cells surrounded by enveloping somatic cells. Ovarian differentiation precedes testicular differentiation, as usual in teleost fishes. The earliest signs of differentiation, detected from day 42 onward, include the onset of meiotic activity in newly formed oocytes, which is soon accompanied by increased oogonial mitotic proliferation and the somatic reorganization of the presumptive ovary. The ovarian cavity is completely formed by day 65. Numerous follicles containing perinucleolar oocytes are observed by day 100. In contrast, signs of morphological differentiation in the presumptive testis are not observed until day 72. By day 100, the unrestricted lobular organization of the testis is evident. The latest stage of spermatogenesis observed by this time of testicular development is spermatocyte II.     

Transforming growth factor beta inhibits proliferation of somatic cells without influencing germ cell number in the chicken embryonic ovary

The gonadal development of chicken embryo is regulated by hormones and growth factors. Transforming growth factor beta (TGF-β) isoforms may play a critical role in the regulation of growth in chicken gonads. We have investigated the effect of the TGF-β isoforms on the number of germ and somatic cells in the ovary of the chicken embryo. Ovaries were obtained from chicken embryos at 9 days of incubation. They were organ-cultured for 72 h in groups treated with TGF-β1, TGF-β2, soluble betaglycan, TGF-β1 plus soluble betaglycan, or TGF-β2 plus soluble betaglycan, and untreated (control). TGF-β1 and TGF-β2 diminished the somatic cell number in the ovary of the chicken embryo at this age by inhibiting the proliferation of the somatic cells without increasing apoptosis. On the other hand, TGF-β1 and TGF-β2 did not affect the number of germ cells in the cultured ovary. The capacity of TGF-β1 and TGF-β2 to diminish the number of somatic cells in the ovary was blocked with soluble betaglycan, a natural TGF-β antagonist. However, changes in the location of germ cells within the ovary suggested that TGF-β promoted the migration of the germ cells from the ovarian cortex to the medulla. Thus, TGF-β affects germ and somatic cells in the ovary of the 9-day-old chicken embryo and inhibits the proliferation of somatic cells.This work was supported by DGAPA-UNAM (IN214403) and CONACYT (45030).     

Determination of c-Abl tyrosine kinase and mTERT catalytic subunit of telomerase expression level during prenatal-postnatal mouse ovary-testis development

Expression levels of genes involved in the development of germ cells vary throughout the process from bipotential gonadal period to adult gonadal formation. In mice, developments of female and male reproductive system are regulated by germ cell-specific factors and hormones, and determinative days in this regulation are very important. c-Abl is a non-receptor tyrosine kinase with cellular functions including cell proliferation, growth and development. mTERT is involved in maintaining telomerase activity and proliferation of surviving cells. We suggested that c-Abl and mTERT might be important for the healthy development of prenatal and postnatal mouse ovary and testis. We aim to demonstrate localization and expressions of c-Abl and mTERT in crucial days of ovary and testis development in prenatal and postnatal period in mouse by immunofluorescence staining and qRT-PCR, respectively. The importance of c-Abl and mTERT expressions during the healthy gonadal development is indicated in the prenatal and postnatal gonadal development. Also, protein expression levels were detected by Western Blot in only postnatal ovary and testis. Determining the functions of the c-Abl and mTERT throughout the process will be important in terms of understanding the infertility cases in the female and male with future studies.     

萍乡肉红鲫的性腺发育研究

萍乡肉红鲫(Pingxiang red-transparent crucian carp,Carassius auratus L.)是在江西省萍乡地区分布的天然三倍体鲫突变体经人工选育后获得的遗传性状基本稳定的后代,具有两性生殖和雌核生殖两种生殖方式.研究以F5代萍乡肉红鲫为材料,自孵化后每满1个月开始取性腺,观察了其卵巢1周年性成熟和精巢的发育过程,结果表明萍乡肉红鲫的性腺为1年成熟类型.卵巢发育进程町以分为6个时期,卵母细胞发育相应可分为6个时相.统计了卵巢成熟系数周年变化,体重为95 g左右的雌性萍乡肉红鲫,其成熟卵巢的成熟系数约为(11.73±2.8)%,成熟的卵母细胞内充满卵黄,相对怀卵量为(3018±310)粒/g.萍乡肉红鲫精巢属于小叶型,在精小叶中可观察到不同发育阶段的生殖细胞.由精原细胞分裂而来的仞级精母细胞经分裂增殖,产生次级精母细胞并最终发育成为精子.萍乡肉红鲫的精巢发育程序与普通鲫鱼和鲤鱼相似,卵巢和精巢的发育过程基本同步,孵化后50日龄内性腺分化不明显,到70日龄左右开始出现雌雄分化,3月龄发育为第1期,4-5月龄发育为第2期,6-7月龄发育至第3期,7-10月龄可见第4期卵巢,1年即可成熟产卵,精巢可排出精液.结果表明,该鲫鱼突变体的性腺发育与普通二倍体鲤(鲫)鱼的性腺发育方式类似.