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.     

Primordial germ cells in the embryos of Medaka fish.

In the second International Microgravity Laboratory (IML-2) mission in 1994, four small Japanese killifish (Medaka, Oryzias latipes) made a space travel of 15 days aboard a space shuttle. These four adult Medaka fish successfully mated in space for the first time among vertebrate animals. Moreover, the eggs they laid developed normally, at least in their external appearance, hatching as fry (baby fish) in space. Fish mated and laid eggs every day during the first week. Near the end of the mission most of the eggs had a well-developed body with two pigmented eyes. In total, 43 eggs were laid (detected), out of which 8 fry hatched in space, as truly 'space-originated' babies. A further 30 fry hatched within 3 days after landing. This is the normal hatching rate, compared with the ground-based data. Among the 8 space-originated fry, four were killed for histological sections, and germ cells at the gonadal region were counted for each fry. Their numbers were in the range of the germ cells of the normal control fry (ground-kept samples). Thus, as embryos developed normally in their external appearance, inside the embryos the formation of primordial germ cells took place normally in space, and their migration to the genital ridges was not hindered by microgravity. The two of the remaining space-originated fry have grown up and been creating their offspring in the laboratory. This proved that the primordial germ cells formed in space were also normal from a functional point of view. The four space-travelled adult fish re-started mating and laying eggs on the 7th day after landing and continued to do so every day afterward. Fertilization rate and hatchability of these eggs were as high as the eggs laid by the laboratory-kept fish. This fact implies that in gametogenesis of adult fish, there are no specific stages of germ cells extremely susceptible to microgravity.     

Dynamics of folliculogenesis in the mouse ovary after the intrauterine elimination of germ cells by administering cyclophosphamide

Injection of cytostatic--cyclophosphamide--to pregnant mice produces elimination of germ cells in their offspring ovaries. Independently on the elimination degree of the germ cells, the amount of growing follicles, as well as dynamics of their growth do not differ from those in the majority of control (intact) animals. In some cases the delay in the rate of the follicular growth is connected with regional outgrowth of the interstitial tissue. This, in its turn, depends on uneven distribution of the germ cells, preserved after cyclophosphamide effect. Unlike the control, for the experimental animals ovaries it is normal that the amount of the growing follicles predominates over the nongrowing ones. This results in exhaustion of the germ cells population and in further sterility. The results obtained contradict the universally recognized data that the process of initiation of the follicular growth depends on the number of the nongrowing follicles. The cause disturbing this dependence is, evidently, certain change in the balance of germ and somatic cells in the ovary development, produced by injection of cyclophosphamide.     

Effects of estradiol-17beta on germ cell proliferation and DMY expression during early sexual differentiation of the medaka Oryzias latipes

Sex reversal of XY male to functional females was induced by estrogen treatment during the embryonic period in the medaka Oryzias latipes. The present study aimed to examine whether exogenous estrogen (estradiol-17beta; E(2)) affects early sex differentiation, paying particular attention to DMY expression and proliferation activity of germ cells in estrogen treated XY individuals. Our results showed that germ cell number was not affected by E(2) treatment at hatching, and that DMY expression was not suppressed under conditions of sex reversal. Therefore, male differentiation of germ cells, which is triggered by the expression of DMY in the supporting cell lineage, proceeds even in E(2) treated XY individuals until hatching, and early sex differentiation is not altered by estrogen. However, sex reversal occurred after hatching probably because of estrogen remaining in the yolk. Interestingly, DMY expression was also detected in the large follicle layer of E(2 )treated XY ovary. These results suggested that DMY regulates male determination in early embryonic stage but does not suppress female follicle development.     

The planarian <Emphasis Type="Italic">nanos</Emphasis>-like gene Smednos is expressed in germline and eye precursor cells during development and regeneration

Planarians are highly regenerative organisms with the ability to remake all their cell types, including the germ cells. The germ cells have been suggested to arise from totipotent neoblasts through epigenetic mechanisms. Nanos is a zinc-finger protein with a widely conserved role in the maintenance of germ cell identity. In this work, we describe the expression of a planarian nanos-like gene Smednos in two kinds of precursor cells namely, primordial germ cells and eye precursor cells, during both development and regeneration of the planarian Schmidtea mediterranea. In sexual planarians, Smednos is expressed in presumptive male primordial germ cells of embryos from stage 8 of embryogenesis and throughout development of the male gonads and in the female primordial germ cells of the ovary. Thus, upon hatching, juvenile planarians do possess primordial germ cells. In the asexual strain, Smednos is expressed in presumptive male and female primordial germ cells. During regeneration, Smednos expression is maintained in the primordial germ cells, and new clusters of Smednos-positive cells appear in the regenerated tissue. Remarkably, during the final stages of development (stage 8 of embryogenesis) and during regeneration of the planarian eye, Smednos is expressed in cells surrounding the differentiating eye cells, possibly corresponding to eye precursor cells. Our results suggest that similar genetic mechanisms might be used to control the differentiation of precursor cells during development and regeneration in planarians. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sex reversal and aromatase in the European pond turtle: treatment with letrozole after the thermosensitive period for sex determination

In the European pond turtle (Emys orbicularis), gonadal sex differentiation is temperature-dependent. The temperature sensitive period (TSP) of gonadogenesis lies between stages 16 and 22 of embryonic development. Previous studies have shown that embryos incubated at 30 degrees C, a temperature yielding 100% phenotypic females, can be sex reversed by treatments with an aromatase inhibitor administered during TSP or even somewhat after TSP (as of stage 22+). The goal of the present study was to determine whether the ovary still retains male potential at later stages of embryonic development and whether the induced male characters persist after hatching. For this purpose, eggs of E. orbicularis were treated with letrozole, a nonsteroidal aromatase inhibitor, at or as of stages 23, 24 or 25, then gonadal aromatase activity in each individual and the related gonadal structure were studied at hatching (stage 26) and for one year after hatching. Two kinds of treatments were carried out: 1) repeated applications of 10 microg of letrozole in ethanolic solution onto the eggshell; and 2) a single injection of 10 microg of letrozole in olive oil. Similar results were obtained with either application or injection of the aromatase inhibitor. In treatments as of or at stage 23, individuals with gonadal aromatase activity lower than 20 fmoles/hour/gonad had ovotestes, i.e., 22% of the treated individuals. At hatching, the inner part of these ovotestes contained testicular cords and also mixed lacunae presenting various degrees of transdifferentiation of the epithelium into a Sertolian epithelium. The cortex was maintained, although some germ cells degenerated within it. These processes continued after hatching. However, at 12 months, gonads were still ovotestes displaying some follicles with a growing oocyte in the remaining parts of the cortex. In treatments as of or at stages 24 or 25, only a few individuals were masculinized. One had ovotestes; in others, the cortex was absent in some parts and when it was present oocytes were degenerating. These results show that in the European pond turtle, differentiation of ovotestes from ovaries can be induced by treatment with an aromatase inhibitor starting at late stages of embryonic development (between the end of TSP and hatching), although such differentiation is less frequent as embryonic development proceeds. Sex reversal persists for at least one year after hatching. J. Exp. Zool. 290:490-497, 2001.     

Effects of temperature experienced during embryonic development on biomass and C and N composition at hatching in Palaemon serratus (Pennant, 1777)

In decapod crustaceans, the conditions experienced during embryonic development trigger phenotypic plasticity of the larvae at hatching. The objective of this study was to test the effects of temperature during embryonic development of Palaemon serratus on the phenotypic plasticity of hatching larvae. We incubated egg-bearing females from eggs laying to hatching at four temperatures (10, 15, 18 and 20°C). Weight, carbon and nitrogen contents were measured on newly laid eggs and on freshly hatched larvae. The duration of embryonic development was negatively correlated with incubation temperature. At 20°C, all females abandoned their eggs during development. Incubation temperature had no effect on the weight and the percentage of N of the larvae at hatching, while it did affect their percentage of C and their C/N ratio. Embryos incubated at 10°C seemed to produce larvae with fewer lipid reserves than those incubated at 15 and 18°C. They probably overconsumed their lipid reserves to compensate for the metabolic losses due to the low temperature. These results provide information on the link between maternal investment per egg and larval development in P. serratus.     

On the control of germ cell development in Caenorhabditis elegans

After hatching, the germ line progenitor cells in C. elegans begin to divide mitotically; later, some of the germ line cells enter meiosis and differentiate into gametes. In the adult, mitotic germ cells, or stem cells, are found at one end (the distal end) and meiotic cells occupy the rest of the elongate gonad. Removal of two somatic gonadal cells, the distal tip cells, by laser microsurgery has a dramatic effect on germ cell development. In either sex, this operation leads to the arrest of mitosis and the initiation of meiosis in germ cells. The function of the distal tip cell in the intact animal appears to be the inhibition of meiosis (or stimulation of mitosis) in nearby germ cells. During development, this permits growth and, in the adult, it maintains the germ line stem cell population. A change in the position of the distal tip cell in the gonad at an early point in development is correlated with a change in the axial polarity of the germ line tissue. This suggests that the localization of the distal tip cell's inhibitory activity at the distal end of the gonad establishes the axial polarity of the germ line tissue in the intact animal.     

南方鲶性腺分化的组织学观察

用芳香化酶抑制剂(Fadrozole)、雌激素受体拮抗剂(Tamoxifen)对人工孵化的南方鲶(Silurus meridionalis)幼鱼进行雄性化诱导处理(口服),获得雄鱼。对孵化后第5—130d的南方鲶幼鱼性腺进行组织学观察,结果表明,在实验条件下,南方鲶性腺分化发生在孵化后7d左右,雌雄性分化过程差异明显。雌鱼卵巢腔在孵化后12d左右形成,生殖细胞在孵化后35d左右快速增殖,成熟分裂最早发生在孵化后55d左右;雄鱼生殖细胞在孵化后130d左右快速增殖,成熟分裂最早发生在孵化后130d左右。雌性性腺分化早于雄性。     

Theory of the continuity of germ plasma according to the findings of modern biology

The main principles on the theory of germ plasma by A. Weismann are briefly presented; a number of his genetic-embryological hypotheses proved to be prophetic. Modern notions on the germ plasma are critically discussed, as well as the resulting from them the conception on continuity of totipotent cells (the source of germ cells) in the line of generations, that is historically connected with M. Nussbaum--A. Weismann's notions on continuity of the embryonic pathway. The term totipotency is sometimes used inaccurately; it means ability to formation of a whole organism. In Metazoa zygota and isolated blastomeres, at a regulative type of development, and groups of somatic cells or fragments of the organism, at an asexual reproduction and somatic embryogenesis, possess this ability. In ontogenesis totipotency is lost both by the somatic and by the germ cells because of their specialization and is recreated with the beginning of every ontogenesis when zygota is formed. The germ cells are always a product of the organism--unicellular or multicellular, and their specialization in all its manifestations is the result of integrative influences of the organism as a whole of them. Certain reasons are presented for supporting ideas on germ cells as one of the lines of cell differentiation. The main, if not the only contradiction in the problem concerning relation of the germ and somatic cells is, at the present time, the thesis on continuity of totipotent cells in the line of generations.     

Dietary manipulation implicates lipid signaling in the regulation of germ cell maintenance in C. elegans

Reproduction in C. elegans relies on continuously proliferating germ cells which, during germline development, exit mitosis, undergo meiosis and differentiate into gametes. Supplementing the diet of C. elegans with dihommogamma-linolenic acid (20:3n-6, DGLA), a long chain omega-6 polyunsaturated fatty acid, results in sterile worms that lack germ cells. The effect is remarkably specific for DGLA, as eicosapentaenoic acid (20:5n-3, EPA) and other long-chain polyunsaturated fatty acids with similar physical properties have little or no effect on fertility. Germ cells undergoing mitosis during larval stages are especially sensitive to DGLA, but exposure to DGLA during adulthood also reduces germ cells and brood size, in part by inducing inappropriate apoptosis of meiotic germ cells. Mutant strains with defects in fatty acid desaturation and elongation display altered susceptibility to DGLA, indicating that the sterility effect of the dietary lipid depends on the amount of DGLA present in membranes as well as on the capacity to convert DGLA to other fatty acids. We propose that DGLA produces a signal that interacts with one or more pathways regulating germ cell survival. Our DGLA findings are the first report of a role for a specific fatty acid affecting the development and maintenance of germ cells in C. elegans.     

A cell surface differentiation antigen of Drosophila

A monoclonal antibody, generated by immunization with gastrula stage Drosophila melanogaster embryonic cells, recognizes a cell surface antigen which shows tissue and stage specificity. The antigen appears for the first time during cellularization of the blastoderm embryo and is present on all cells until around 12 hr of development. It becomes progressively restricted to specific tissues during the second half of embryogenesis. By the time of hatching, only the nervous system, germ cells, and imaginal cells are positive. During metamorphosis differentiating imaginal tissues become negative so that in the adult only the nervous system and undifferentiated germ cells are positive, with gonadal sheaths showing some staining. A third wave of antigen loss occurs during gametogenesis, resulting in negative staining on the mature sperm and oocyte. All positive tissues appear to contain the same 63-kDa cell surface antigen. The antigen behaves as a general differentiation marker lost by tissues as they approach their terminal differentiated state. The nervous system and possibly gonadal sheaths may be exceptions to this general behavior.     

Dynamic expression patterns of <Emphasis Type="Italic">vasa</Emphasis> during embryogenesis in the cricket <Emphasis Type="Italic">Gryllus bimaculatus</Emphasis>

The specification of germ cells during embryogenesis is an important issue in the development of metazoans. In insects, the mode of germ cell specification appears to be highly variable among species and molecular data are not sufficient to provide an evolutionary perspective to this issue. Expression of vasa can be used as a germ line marker. Here, we report the isolation of a vasa-like gene in a hemimetabolous insect, the cricket Gryllus bimaculatus (Gb'vas), and its expression patterns during oogenesis and embryogenesis. Gb'vas is preferentially expressed in the germarium and the expression of Gb'vas is detectable throughout vitellogenesis including mature eggs subjected to oviposition, suggesting that Gb'vas is maternally contributed to the cricket eggs. The zygotic expression of Gb'vas appears to start at the mid blastoderm stage in the posterior region of the egg, expanding in a developing germ anlage. In early germbands, an intense expression of Gb'vas is restricted to the posterior end. In later embryos, Gb'vas expression extends over the whole body and then distinctly localized to the embryonic gonad at the stage immediately before hatching. These results suggest that, in the cricket, germ cells are specified early in development at the posterior end of an early germband, as proposed by Heymons (1895) based on cytological criteria.     

Egg temperature and initial brood patch area determine hatching asynchrony in Magellanic penguin Spheniscus magellanicus

In birds, the adaptive significance of hatching asynchrony has been under debate for many years and the parental effects on hatching asynchrony have been largely assumed but not often tested. Some authors suggest that hatching asynchrony depends on the incubation onset and many factors have been shown to influence hatching asynchrony in different species. Our objective was to analyze the exact timing of the onset of incubation and if this affects hatching asynchrony; and, in addition, which other factors (brood patch development, incubation position, adult body condition, intra‐clutch egg dimorphism, laying date and year) affect hatching asynchrony in Magellanic penguins Spheniscus magellanicus. We first estimated the eggshell temperature at which embryo development starts, with a non‐destructive and novel method. We then recorded individual egg temperatures in 61 nests during incubation, and related them, and other breeding parameters, to hatching asynchrony. We also observed incubation positions in 307 nests. We found a significant positive relationship between hatching asynchrony and the temperature that the first‐laid egg experienced during egg laying and between hatching asynchrony and the initial brood patch area. We also found a negative relationship between hatching asynchrony and the difference in temperature between second and first‐laid eggs within a clutch, measured after the egg‐laying period was finished. We ruled out position of the eggs during incubation, adult body condition, egg volume, laying date, and study year as factors influencing hatching asynchrony. The egg temperature during laying and the difference in temperature between eggs of a clutch are determinants of hatching asynchrony in Magellanic penguins.     

Differential expression of vasa homologue gene in the germ cells during oogenesis and spermatogenesis in a teleost fish, tilapia, Oreochromis niloticus

Vas (a Drosophila vasa homologue) gene expression pattern in germ cells during oogenesis and spermatogenesis was examined using all genetic females and males of a teleost fish, tilapia. Primordial germ cells (PGC) reach the gonadal anlagen 3 days after hatching (7 days after fertilization), the time when the gonadal anlagen was first formed. Prior to meiosis, no differences in vas RNA are observed in male and female germ cells. In the ovary, vas is expressed strongly in oogonia to diplotene oocytes and becomes localized as patches in auxocytes and then strong signals are uniformly distributed in the cytoplasm of previtellogenic oocytes, followed by a decrease from vitellogenic to postvitellogenic oocytes. In the testis, vas signals are strong in spermatogonia and decrease in early primary spermatocytes. No vas RNA expression is evident in either diplotene primary spermatocytes, secondary spermatocytes, spermatids or spermatozoa. The observed differences in vas RNA expression suggest a differential function of vas in the regulation of meiotic progression of female and male germ cells.     

Embryogenesis in mouth-breeding cichlids (Osteichthyes,Teleostei) structure and fate of the enveloping layer

The eggs of African mouth-brooders are of unusual size and shape. Studying their development may help to more clearly understand epiboly, gastrulation, and the relation between enveloping layer (periderm) and epidermis. When epiboly has progressed over just one fifth of the yolk mass, the germ ring and embryonic shield are already well established. Behind the germ ring very few deep cells are present at this early stage of epiboly, except in the embryonic shield. When the blastodisc covers the animal half of the yolk mass, the future body is already well established with notochord, somites and developing neural keel. Apart from these structures, no deep cells can be detected between enveloping layer and yolk surface; not even a germ ring remains behind the advancing edge of the enveloping layer. Epiboly over the greater part of the yolk is achieved only by the enveloping layer and the yolk syncytial layer. As the margin of the enveloping layer begins to reduce its circumference when closing around the vegetal pole, groups of cells in the advancing edge become spindle-shaped, with a single cell in between of each of these groups broadening along the edge. The enveloping layer (called periderm after epiboly) remains intact until after hatching, when, together with the underlying ectoderm, it forms the double-layered skin of the larval fish. Thereafter, cells deriving from the subperipheral ectoderm gradually replace the decaying periderm cells to form the final epidermis. Thus, in the cichlids studied, the enveloping layer alone forms the yolk sac to begin with, and it covers the larval body until some days after hatching.     

Analysis of germ line development in the chick embryo using an anti-mouse EC cell antibody

We have found that EMA-1, a monoclonal antibody originally raised against mouse embryonal carcinoma (Nulli SCC1) cells (Hahnel & Eddy, 1982), also labels chick primordial germ cells (PGCs). We have used this antibody in immunohistological studies to follow the development of PGCs in the chick embryo from the time of their initial appearance beneath the epiblast, through their migratory phase and subsequent colonization of the germinal epithelium. During hypoblast formation, individual EMA-1-labelled cells appeared to separate from the basal surface of the epiblast and enter the blastocoel, coincident with the appearance of morphologically identifiable PGCs in this same area. EMA-1 continued to label germ cells until the initiation of gametogenesis in each sex; specifically, labelling was absent by 7-8 days of incubation in females and started to decrease at 11 days of incubation in males. There was a recurrence of the epitope on oogonia at 15 days of incubation, but not on spermatogonia during the remainder of development through hatching. These observations are consistent with an epiblast origin for the avian germ line, and are strikingly similar to those reported for the early mouse embryo using the same antibody (Hahnel & Eddy, 1986).     

Differentiation potential of germ line stem cells derived from the postnatal mouse ovary

General belief in reproductive biology is that in most mammals female germ line stem cells are differentiated to primary oocytes during fetal development and oogenesis starts from a pool of primordial follicles after birth. This idea has been challenged previously by using follicle kinetics studies and demonstration of mitotically active germ cells in the postnatal mouse ovary (Johnson et al., 2004; Kerr et al., 2006; Zhang et al., 2008). However, the existence of a population of self-renewing ovarian germ line stem cells in postnatal mammals is still controversial (Eggan et al., 2006; Telfer et al., 2005; Gosden, 2004). Recently, production of offspring from a germ line stem cell line derived from the neonatal mouse ovary was reported (Zou et al., 2009). This report strongly supports the existence of germ line stem cells and their ability to expand in vitro. Recently, using a transgenic mouse model in which GFP is expressed under a germ cell-specific Oct-4 promoter, we isolated and generated multipotent cell lines from male germ line stem cells (Izadyar et al., 2008). Using the same strategy we isolated and derived cell lines from postnatal mouse ovary. Interestingly, ovarian germ line stem cells expanded in the same culture conditions as the male suggesting that they have similar requirements for their self-renewal. After 1 year of culture and many passages, ovarian germ line stem cells maintained their characteristics and telomerase activity, expressed germ cell and stem cell markers and revealed normal karyotype. As standard protocol for differentiation induction, these cells were aggregated and their ability to form embryoid bodies (EBs) was investigated. EBs generated in the presence of growth factors showed classical morphology and expressed specific markers for three germ layers. However, in the absence of growth promoting factors EBs were smaller and large cells with the morphological and molecular characteristics of oocytes were formed. This study shows the existence of a population of germ line stem cell in postnatal mouse ovary with multipotent characteristics.     

Fine structural observations on the origin and associations of primordial germ cells of the mouse.

This study explores the origin of primordial germ cells (PGCs) of the mouse and examines their morphology and associations with other cells during early development. PGCs have been selectively stained by the alkaline phosphatase histochemical reaction and viewed by light and electron microscopy from the time they are first detectable in the yolk sac endoderm until they enter the gonadal ridges. There are conflicting reports as to whether the PGCs originate from endodermal cells or whether they originate elsewhere and subsequently enter the endoderm. The observations in the present study favor the premise that PGCs of the mouse do not originate in the endoderm. Furthermore, it was observed that PGCs undergo specific changes in morphology during the developmental period studied and this was interpreted to mean that, although PGCs are set aside early in development as a distinct cell line, they also continue to become more specialized within time. The germ cell line is rather unusual in that it does not exist as a discrete tissue but, instead, resides within various other tissues during its life history. This apparent dependence upon somatic cells is maintained even in adult animals and may be important in serving to maintain or modify the environment of the germ cells.