Development and early differentiation of gonad in the european eel (Anguilla anguilla [L.], Anguilliformes,Teleostei): A cytological and ultrastructural study

The structure of the gonad of the European eel (Anguilla anguilla [L.]), an “undifferentiated” gonochoristic teleost, was investigated by transmission electron microscopy from 6–8 cm elvers to 22 cm yellow eels with juvenile hermaphroditic gonads. The pear-shaped gonads of 6–8 cm elvers assume, in 12–15 cm eels, a lamellar shape and enlarge by migration of germ cells, which we refer to as primary primordial germ cells. In the gonads of ∼ 16 cm eels, the primary primordial germ cells multiply, giving rise to clusters of germ cells that have ultrastructural characteristics of the primary primordial germ cells but show giant mitochondria, enlarged Golgi complexes, and round bodies not limited by membranes. We refer to these as secondary primordial germ cells. In 16–18 cm eels, syncytial clones of oogonia interconnected by cytoplasmic bridges are also observed. In 18–22-cm-long eels, the gonads contain primordial germ cells, oogonial clones, early oocyte cysts, single oocytes in early growth stages, and primary spermatogonia. Such germ cells are present in the same cross section where they are either intermingled or are in areas of predominantly female germ cells close to areas with predominantly male germ cells. These gonads are juvenile hermaphroditic and should be considered ambisexual because in larger eels they differentiate either into an ovary or into a testis. Somatic cells always envelop the germ cells following their migration into the gonad. These somatic cells first show similar ultrastructural features and then differentiate either into early Sertoli cells investing spermatogonia, or into early follicular (granulosa) cells investing the early previtellogenic oocytes. In eels ∼ 14 cm long, primitive steroid-producing cells also migrate into the gonad. In the ambisexual gonad they differentiate either into immature Leydig cells in the male areas, or into early special cells of the theca in the female areas. Nerve fibers are joined to the steroid-producing cells. Gonad development and differentiation are also associated with structural changes of the connective tissue characterized by the progressive appearance and deposition of collagen fibrils first in the mesogonadium, then in the gonad vascular region, and then in the germinal region. The collagen-rich areas are massive in the male areas and reduced in the female ones. J. Morphol. 231:195–216, 1997. © 1997 Wiley-Liss, Inc.     

Immunolocalization of steroidogenic enzymes in gonads of European eel, Anguilla anguilla (L.)

The localization of cytochrome P450 cholesterol side-chain cleavage (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and aromatase (P450arom) was investigated using polyclonal antibodies during gonad development in wild European eels, Anguilla anguilla (L.), from the River Po Delta (Ferrara, Italy). The first steroidogenic cells, observed in undifferentiated gonads of 14–16 cm yellow eels, showed no P450scc, 3β-HSD or P450arom activity, but positive regions appeared in head kidney insulae from this stage until the silver eel stage. In undifferentiated gonads of 16–20 cm yellow eels the steroidogenic cells were positive to all enzymes. Pre-Leydig steroidogenic cells, identified in Syrski organs of yellow eels of 22–26 cm evolving into testes, were positive to 3β-HSD and P450scc, but negative to P450arom. However, steroidogenic cells in Syrski organs evolving towards ovaries and in small but fully differentiated ovaries were positive to all enzymes. Immature testes of yellow and silver eels had Leydig cells positive to P450scc and 3β-HSD; the same reactions were also observed in some Sertoli cells of silver eel testes containing meiotic cells. Sex differentiation in A. anguilla apparently occurs through an initial female stage controlled by P450arom activity. Leydig and Sertoli cells appear involved in different steps of hormonal control of spermatogenesis: Leydig cells begin their steroidogenic activity before meiosis, while Sertoli cells begin their activity during meiosis.     

Sex differentiation in the European eel: histological analysis of the effects of sex steroids on the gonad

The effects of sex steroids on sex differentiation in the European eel were studied. The steroids, 17α-methyltestosterone (MT) and 17α-ethynylestradiol (EE), were given in the diet to 6–8 cm elvers and to 15–18 cm and 22–25 cm yellow eels. In our rearing conditions a very large percentage of the untreated eels developed as males. No masculinizing effect of MT could be demonstrated. The EE, administered at a dose of 10 mg kg^-1 of diet to 6–8 cm elvers and 15–18 cm eels, induced ovarian differentiation in about 90 and 65% of eels respectively, while in the control <5% of females was recorded. In 22–25 cm yellow eels a moderated feminizing effect was observed.
Histological analysis of the gonads of treated eels showed that sex steroids affect the gonadal structure. The androgen stimulates hypertrophy of compact connective tissue, early differentiation of Leydig cells, Sertoli cells and early formation of the spermatic duct. Oestrogen inhibits the differentiation of these structural components and stimulates the differentiation of follicular cells and an ovarian structure.
The involvement of gonadal structural components is discussed in relation to the effect of steroid treatment and to the peculiarities of sex differentiation in the European eel.     

Effects of diet supplementation with carp ovary on gonad differentiation and growth of the European eel

Treating elvers of European eel Anguilla anguilla with mature carp ovary for 3–6 months during early growth induced female differentiation in 51·6–66·7% of treated animals compared with c . 5% in controls. The treatment also induced differentiation of ovaries in eels <13 cm L _T and a higher number of Syrski organs with ambisexual characters, and was most effective when administered at an early growth stage. The results could be attributed to the natural steroid content of the carp ovary. The total weight of treated animals at the end of the farm experiment was 84·7% higher than controls. The specific growth rate for weight was significantly higher in female yellow eels than in males, for both control and treated groups. The enhanced growth was related to induced feminization. A diet supplementation with mature carp ovary could be a good approach to control of sex differentiation and growth in eels.     

On the sex and distribution of the freshwater eel (Anguilla anguilla)

This paper reports the result of an examination of about 8000 eels mainly from various parts of several Welsh rivers made to try and find out whether the distribution of eels was determined by their sex. Contrary to the popular belief, males and females were found living together in freshwater and in brackish water. The organ of Syrski, which was supposed to be diagnostic of the male sex has been found ambiguous in nature and not always a precursor of a male gonad. The consequent difficulties of the identification of the sex of moderately sized eels is discussed and it is suggested that the abnormal sex ratio, as reported by some investigators, is related to the obscure structure of the organ of Syrski and the fact that gonad differentiations may not occur until the fish is quite large. The possibility that other factors such as selective fishing and a sex differential in growthrates and duration of freshwater life may also be contributory factors isdiscussed. It is suggested that the environment plays little part in sex determination.     

Gonad differentiation and body growth in Anguilla anguilla L.

Using histological sections, the gonads of samples of yellow and silver eels of two populations were examined. The populations were previously analysed for growth and sex ratio. The histological structures observed are similar to those described in previous publications for the European eel, Anguilla anguilla and to those indicated for the Pacific eel, A. japonica . Well differentiated gonads are present in the silver eels. In the yellow eels, ranging in age from 0 + to 2 + years and from a length of 20 cm to that at which they become silver, undif-ferentiated and both sex gonads are found. Histological evidence is presented which suggests that the ovary, found even in young and small eels, is completely differentiated at a very early stage. The testis-like gonad of the yellow eel is a more primitive, and possibly reversible, gonad which differentiates completely at the beginning of sexual maturation and the change to the silver phase.     

Determination of the sensitive stages for gonadal sex-reversal in Xenopus laevis tadpoles

The response of developing gonads of the clawed toad Xenopus laevis tadpoles to estradiol benzoate (EB) was studied between stages 44 and 67 using high resolution techniques. In presumptive genetic males the following results were obtained: 1) 100% sex reversal was induced when EB was administered before translocation of primordial germ cells (PGCs) from the gonadal epithelium into the medullary region (stages 44-50). 2) Ambiguous gonads were formed when EB treatment was initiated at stages 51-54, when PGCs were migrating into the medullary region. 3) Finally, normal testes differentiated when EB treatment began after the primordial germ cells had completed their translocation into the medulla (stages 55-56). These results suggest that EB might induce sex-reversal in genetic males by disruption of early somatic-germ cell interactions in the medullary region of the gonad. Consequently, later morphogenetic events might be deranged, preventing differentiation of testis. We propose a hypothesis in which precocious production of estradiol (E2) by genotypic females is the mechanism for primary sex differentiation.     

Freemartin in the amphibian Pleurodeles waltl: parabiosis between individuals from opposite sex triggers both germ and somatic cells alterations during female gonad development

Wild type embryos of the newt Pleurodeles waltl were used to realize parabiosis, a useful model to study the effect of endogenous circulating hormones on gonad development. The genotypic sex of each parabiont (ZZ male or ZW female) was determined early from the analysis of the sex chromosome borne marker peptidase-1. In ZZ/ZZ and ZW/ZW associations, gonads develop according to genetic sex. In ZZ/ZW associations, the ZZ gonads differentiate as normal testes while ZW gonads development shows numerous alterations. At the beginning of sex differentiation, these ZW gonads possess a reduced number of germ cells and a reduced expression of steroidogenic factor 1 and P450-aromatase mRNAs when compared to gonads from ZW/ZW associations. During gonad differentiation, conversely to the control situation, these germ cells do not enter meiosis as corroborated by chromatin status and absence of the meiosis entry marker DMC1; the activity of the estradiol-producing enzyme P450-aromatase is as low as in ZZ gonads. At adulthood, no germ cells are observed on histological sections, consistently with the absence of VASA expression. At this stage, the testis-specific marker DMRT1 is expressed only in ZZ gonads, suggesting that the somatic compartment of the ZW gonad is not masculinized. So, when exposed to ZZ hormones, ZW gonads reach the undifferentiated status but the ovary differentiation does not occur. This gonad is inhibited by a process affecting both somatic and germ cells. Additionally, the ZW gonad inhibition does not occur in the case of an exogenous estradiol treatment of larvae.     

Cross talk between germ cells and gonadal somatic cells is critical for sex differentiation of the gonads in the teleost fish, medaka (Oryzias latipes)

To evaluate the possible role of germ cells on sex differentiation of the gonads in vertebrates, the teleost fish, medaka ( Oryzias latipes ), was used to generate a gonad without germ cells. The germ cell-deficient medaka reveals multiple effects of germ cells on the process of sex differentiation. The previously isolated mutant medaka, hotei , with the excessive number of germ cells may support the contention that the proliferation of germ cells is related to feminization of the gonad. Futhermore, we show that two modes of proliferation for either maintenance of germ cells or commitment to gametogenesis are important components of the sex differentiation of medaka developing gonads. An intimate cross talk between germ cells and gonadal somatic cells during the sex differentiation will be discussed.     

Immunohistochemical localization of the carbohydrate antigen 4C9 in the mouse embryo: a reliable marker of mouse primordial germ cells

Expression of 4C9, a Lex[Gal beta 1----4(Fuc alpha 1----3)GlcNAc] antigen, during mouse embryogenesis was studied by immunohistochemical methods. Distribution of 4C9 was similar to, but not identical with that of SSEA-1 (stage-specific embryonic antigen-1). Notably, 4C9 was detected in some of the inner cell mass cells of late blastocysts, ectoderm cells migrating from the primitive streak to the mesoderm space and primordial germ cells just formed from the migrating cells. Thus, 4C9 was considered to be continuously expressed in the cell lineage starting at the totipotent 8 cell stage and leading to primordial germ cells. While 4C9 gradually decreased from the surface of primordial germ cells after they have settled in the gonad, the antigen remained in cytoplasmic granules for some period in a sex determined manner. In male gonads, cytoplasmic granules positive for 4C9 tended to be polarized to one side of cytoplasm. The 4C9 reactive material completely disappeared from male germ cells by day 16 of gestation. In female gonads, granules scattered throughout the cytoplasm and cell surface were positive for 4C9. On day 16 of gestation the cell surface antigenicity was lost, but some cytoplasmic antigenicity still remained. As above, 4C9 is a reliable marker to study the origin, migration and differentiation of primordial germ cells, and to distinguish male and female germ cells. By immunoelectron microscopy, 4C9 was detected at the plasma membrane, the Golgi apparatus, and dense-cored vesicles in primordial germ cells on 10-11 days of gestation.     

Sexual differentiation in Salaria (=Blennius) pavo

The sexual differentiation of Salaria (= Blennius ) pavo is described from the stage of hatching to a body length of 35 mm. At hatching, the primordial germ cells (PGCs) can be recognized clearly. At a standard body length of 5 mm, they begin to protrude into the peritoneal cavity and at 14 mm they transform to oogonia. At 17 mm length, the first oocytes can be observed. In males at a standard length of 16–17 mm, the first signs of a differentiation into a testis can be recognized. Shortly after the differentiation of the male sex, the division of the male gonad into a testis and a testicular gland can be seen. The fine structural characteristics of the PGCs and of differentiation stages are presented.     

Sex Differentiation of Avian Gonads In Vitro

The analysis of avian sex differentiation in vitro has beenlimited to the following problems: morphological sex differentiationof gonads cultured in vitro; analysis of the chemical natureof the hormonal secretion; differentiation of germ cells inrelation to their somatic environment. Morphological sex differentiationof avian gonads occurs in vitro. Differentiated gonads of thechick embryo carry out biosynthesis of sex hormones from severalradioactive precursors. Female gonads in particular synthesizeestrogens while male gonads synthesize testosterone. Some experimentshave given evidence of estrogen synthesis by undifferentiatedfemale gonads. Embryonic gonads of quail, like those of chick,are able to synthesize sex steroids from radioactive precursors.However, in the quail and mainly in the testes, a delayed appearanceand a lower activity of the enzyme system 3ß-HSDHs-4-isomerase was found. Histoenzymological results corroboratethe biochemical ones. Combination of culture and grafting experimentshave shown that male germ cells when they are forced into femaledifferentiation by early colonization of a female gonad degenerateafter entering the premeiotic stage. The reasons for this delayedfailure of sex differentiation of "male oocytes" have certainlyto be searched for at the level of perturbation in the mechanismsof meiosis.     

Ultrastructural aspects of gonadal morphogenesis in Bufo bufo (Amphibia Anura) 1. Sex differentiation

The morphogenesis of gonads in Bufo bufo tadpoles was studied, and ultrastructural differences between sexes were identified. All specimens analyzed initially developed gonads made up of a peripheral fertile layer (cortex) surrounding a small primary cavity. Subsequently a central layer of somatic cells (medulla) developed. Both layers were separated by two uninterrupted basal laminae between which a vestige of the primary cavity persisted. During female differentiation, the peripheral layer continued to be the fertile layer. In males, the central layer blended into the peripheral layer and the basal laminae disappeared. The somatic cells of the central layer came into direct contact with the germ cells; this did not occur in females. Testicular differentiation continued with the migration of germ cells towards the center of the gonad. The somatic elements surrounding the germ cells appeared to play an active role in their transfer to the center of the gonad. The peripheral layer shrank and became sterile. Two basal laminae then re-formed to separate the fertile central layer from the peripheral sterile one. Germ cells have always been thought to perform a passive role in sex differentiation in amphibians. Following the generally accepted "symmetric model", the mechanism of gonad development is symmetrical, with cortical somatic cells determining ovarian differentiation and medullary somatic cells determining testicular differentiation. In contrast, we found that sex differentiation follows an "asymmetric" pattern in which germ cells tend primarily toward a female differentiation and male differentiation depends on a secondary interaction between germ cells and medullary somatic cells.     

Sexual dimorphism and gonadal development of the Australian longfinned river eel

The sex and stage of gonadal development of longfinned river eels Anguilla reinhardtii , captured from nine river catchments in New South Wales, Australia, between 1999 and 2001, were determined macroscopically. Sex was verified by histology. Histology was also necessary, however, to accurately define stages of gonadal development, particularly in individuals <600 mm in total body length. Anguilla reinhardtii displayed asynchronous gamete development. The most advanced cells present in migrating male and female A. reinhardtii were spermatocytes and pre-vitellogenic oocytes, respectively. Gonadal development stages were positively correlated with body size in both sexes. Females, however, were significantly larger than males and their gonads matured over a broader size range. Size at sexual differentiation (42–60 cm for males and 50–76 cm for females) was much larger than for most other anguillids that have been studied, with the exception of the New Zealand longfinned eel Anguilla dieffenbachii . Corresponding with its large range in size at sexual differentiation was a relatively large range in size at migration for both males (44–62 cm) and females (74–142 cm).     

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.     

Inhibition of primordial germ cell proliferation by the medaka male determining gene Dmrt1bY

Background  

Dmrt1 is a highly conserved gene involved in the determination and early differentiation phase of the primordial gonad in vertebrates. In the fish medaka dmrt1bY, a functional duplicate of the autosomal dmrt1a gene on the Y-chromosome, has been shown to be the master regulator of male gonadal development, comparable to Sry in mammals. In males mRNA and protein expression was observed before morphological sex differentiation in the somatic cells surrounding primordial germ cells (PGCs) of the gonadal anlage and later on exclusively in Sertoli cells. This suggested a role for dmrt1bY during male gonad and germ cell development.     

Sexual phenotype of avian chimeric gonads with germinal and stromal cells of opposite genetic sexes

The respective roles of germinal and stromal cells in determining the sexual phenotype of the gonad were analyzed in chimeric gonads obtained by surgical recombination between young avian blastodiscs in ovo. Equivalent territories were exchanged between two blastodisc, in order that the germinal crescent and the gonad territory had a different origin (fig. 3). Embryos used for these experiments carried a sex linked pigment mutation, that made it possible to diagnose the genetic sexes of germ cells and stroma at the time when the gonad was retrieved for examination. On the basis of species, three types of combination were performed: chick germ cells in chick or quail stroma, quail germ cells in chick stroma. In each chimera, the genetic sexes of the two gonadal cell populations could be identical or opposite. However it appeared that the germ cell population was not always homogeneous. In some grafting schemes, ectopic germ cells, located outside the germinal crescent, contributed to the colonization of the experimental gonad. These germ cells were from the same territory as the stroma element of the gonad, i.e., they were of the same species and the same genetic sex. Whatever the case, in 87 chimeras that were studied, the sex phenotype of the gonads always corresponded to the genetic sex of the stroma. Thus the genetic sex of germ cells has no role in the sexual differentiation of the gonadal rudiments.     

Female‐specific increase in primordial germ cells marks sex differentiation in threespine stickleback (Gasterosteus aculeatus)

Gonadal sex differentiation is increasingly recognized as a remarkably plastic process driven by species‐specific genetic or environmental determinants. Among aquatic vertebrates, gonadal sex differentiation is a frequent endpoint in studies of endocrine disruption with little appreciation of underlying developmental mechanisms. Work in model organisms has highlighted the diversity of master sex‐determining genes rather than uncovering any broad similarities prompting the highly conserved developmental decision of testes versus ovaries. Here we use molecular genetic markers of chromosomal sex combined with traditional histology to examine the transition of the bipotential gonads to ovaries or testes in threespine stickleback (Gasterosteus aculeatus). Serially‐sectioned threespine stickleback fry were analyzed for qualitative and quantitative indications of sexual differentiation, including changes in gonadal morphology, number of germ cells and the incidence of gonadal apoptosis. We show that threespine stickleback sampled from anadromous and lacustrine populations are differentiated gonochorists. The earliest sex‐specific event is a premeiotic increase in primordial germ cell number followed by a female‐specific spike in apoptosis in the undifferentiated gonad of genetic females. The data suggest that an increase in PGC number may direct the undifferentiated gonad toward ovarian differentiation. J. Morphol., 2008. © 2007 Wiley‐Liss, Inc.     

FGF9 promotes survival of germ cells in the fetal testis

In addition to its role in somatic cell development in the testis, our data have revealed a role for Fgf9 in XY germ cell survival. In Fgf9-null mice, germ cells in the XY gonad decline in numbers after 11.5 days post coitum (dpc), while germ cell numbers in XX gonads are unaffected. We present evidence that germ cells resident in the XY gonad become dependent on FGF9 signaling between 10.5 dpc and 11.5 dpc, and that FGF9 directly promotes XY gonocyte survival after 11.5 dpc, independently from Sertoli cell differentiation. Furthermore, XY Fgf9-null gonads undergo true male-to-female sex reversal as they initiate but fail to maintain the male pathway and subsequently express markers of ovarian differentiation (Fst and Bmp2). By 14.5 dpc, these gonads contain germ cells that enter meiosis synchronously with ovarian gonocytes. FGF9 is necessary for 11.5 dpc XY gonocyte survival and is the earliest reported factor with a sex-specific role in regulating germ cell survival.