Ontogeny of gonadal sex development relative to growth in fathead minnow

Ovarian differentiation of fathead minnow Pimephales promelas occurred at between 10 and 25 days post‐hatch (dph)(8–11 mm fork length, L _F, and 7–12 mg), and was characterized by the presence of meiotic cells in the centre of the gonad, location of the somatic cells at the periphery of the gonad and the formation of an ovarian cavity. In contrast with the developing ovary, in the presumptive testis somatic cells were scattered throughout the gonads and this was evident from 25 dph (fish >10 mm and >11 mg). In males, at 60 dph (15–26 mm and 39–220 mg) the efferent ducts (sperm ducts) were apparent and the testis lobules started to form, but germ cells (spermatogonia) did not enter meiosis until between 90 and 120 dph. Fish of both sexes reached full sexual maturity at between 120 and 150 dph (males: 33–59 mm and 400–2895 mg; females: 24–48 mm and 160–1464 mg). Differences in body size ( L _F and mass) between males and females were only apparent when the fish were approaching full sexual maturity (120 dph).     

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.     

Meiotic germ cells antagonize mesonephric cell migration and testis cord formation in mouse gonads

The developmental fate of primordial germ cells in the mammalian gonad depends on their environment. In the XY gonad, Sry induces a cascade of molecular and cellular events leading to the organization of testis cords. Germ cells are sequestered inside testis cords by 12.5 dpc where they arrest in mitosis. If the testis pathway is not initiated, germ cells spontaneously enter meiosis by 13.5 dpc, and the gonad follows the ovarian fate. We have previously shown that some testis-specific events, such as mesonephric cell migration, can be experimentally induced into XX gonads prior to 12.5 dpc. However, after that time, XX gonads are resistant to the induction of cell migration. In current experiments, we provide evidence that this effect is dependent on XX germ cells rather than on XX somatic cells. We show that, although mesonephric cell migration cannot be induced into normal XX gonads at 14.5 dpc, it can be induced into XX gonads depleted of germ cells. We also show that when 14.5 dpc XX somatic cells are recombined with XY somatic cells, testis cord structures form normally; however, when XX germ cells are recombined with XY somatic cells, cord structures are disrupted. Sandwich culture experiments suggest that the inhibitory effect of XX germ cells is mediated through short-range interactions rather than through a long-range diffusible factor. The developmental stage at which XX germ cells show a disruptive effect on the male pathway is the stage at which meiosis is normally initiated, based on the immunodetection of meiotic markers. We suggest that at the stage when germ cells commit to meiosis, they reinforce ovarian fate by antagonizing the testis pathway.     

RSPO1/β-catenin signaling pathway regulates oogonia differentiation and entry into meiosis in the mouse fetal ovary

Differentiation of germ cells into male gonocytes or female oocytes is a central event in sexual reproduction. Proliferation and differentiation of fetal germ cells depend on the sex of the embryo. In male mouse embryos, germ cell proliferation is regulated by the RNA helicase Mouse Vasa homolog gene and factors synthesized by the somatic Sertoli cells promote gonocyte differentiation. In the female, ovarian differentiation requires activation of the WNT/β-catenin signaling pathway in the somatic cells by the secreted protein RSPO1. Using mouse models, we now show that Rspo1 also activates the WNT/β-catenin signaling pathway in germ cells. In XX Rspo1(-/-) gonads, germ cell proliferation, expression of the early meiotic marker Stra8, and entry into meiosis are all impaired. In these gonads, impaired entry into meiosis and germ cell sex reversal occur prior to detectable Sertoli cell differentiation, suggesting that β-catenin signaling acts within the germ cells to promote oogonial differentiation and entry into meiosis. Our results demonstrate that RSPO1/β-catenin signaling is involved in meiosis in fetal germ cells and contributes to the cellular decision of germ cells to differentiate into oocyte or sperm.     

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.     

Gonad Differentiation in the Rabbit: Evidence of Species-Specific Features

The rabbit is an attractive species for the study of gonad differentiation because of its 31-day long gestation, the timing of female meiosis around birth and the 15-day delay between gonadal switch and the onset of meiosis in the female. The expression of a series of genes was thus determined by qPCR during foetal life until adulthood, completed by a histological analysis and whenever possible by an immunohistological one. Interesting gene expression profiles were recorded. Firstly, the peak of SRY gene expression that is observed in early differentiated XY gonads in numerous mammals was also seen in the rabbit, but this expression was maintained at a high level until the end of puberty. Secondly, a peak of aromatase gene expression was observed at two-thirds of the gestation in XX gonads as in many other species except in the mouse. Thirdly, the expression of STRA8 and DMC1 genes (which are known to be specifically expressed in germ cells during meiosis) was enhanced in XX gonads around birth but also slightly and significantly in XY gonads at the same time, even though no meiosis occurs in XY gonad at this stage. This was probably a consequence of the synchronous strong NANOS2 gene expression in XY gonad. In conclusion, our data highlighted some rabbit-specific findings with respect to the gonad differentiation process.     

Impaired spermatogenesis in the Japanese eel, Anguilla japonica: Possibility of the existence of factors that regulate entry of germ cells into meiosis

In the cultivated male Japanese eel, spermatogonia are the only germ cells present in the testis. Weekly injections of human chorionic gonadotropin (HCG) can induce complete spermatogenesis from proliferation of spermatogonia to spermiogenesis. In some cases, however, HCG injection fails to induce complete spermatogenesis. Testicular morphological observations revealed that HCG-injected eels could be classified into three types based on their testicular conditions. Type 1 eels had a well-developed testis and the milt could be acquired by hand-stripping. In type 2 eels, spermatogenesis was also induced by HCG injection, but testicular size was remarkably smaller than that of type 1 eels, and the milt could not be hand-stripped. At the end of the experiment, type 2 fish had only spermatogonia and a small amount of spermatozoa, but no spermatocytes or spermatids, in their testis. Type 3 eels had thready testis, which did not develop any germ cells during the experimental period. These results suggest that, despite elevations of plasma 11–ketotestosterone levels, HCG injections were not successful in inducing the completion of spermatogenesis in type 2 and type 3 eels. In most spermatogonia of type 2 eels, meiosis was not induced by HCG injections. Furthermore, only few mitotic divisions had occurred as evidenced by the presence of 2³ to 2⁶ late type B spermatogonia in most cysts. This suggests that spermatogonial stem cells undergo four or five, and occasionally six, mitotic divisions before the interruption of spermatogenesis in type 2 eels. It is proposed that those numbers of mitotic divisions are related to a mediator that regulates entry of spermatogonia of the Japanese eel into 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.     

A histological description of gonadal development and sex differentiation in the coho salmon (Oncorhynchus kisutch) for both untreated and oestradiol immersed fry

Gonadal development is described in detail for coho salmon ( Oncorhynchus kisutch ) between hatching and 1000 degree-days, post-hatch, to aid sex reversal research. Germ cell morphology and sequence, vascular and reproductive duct development, and gross characteristics of the gonads are presented. Sex differentiation occurs by 380 degree-days, post-hatch (800 degree-days, post-fertilization) and is direct to male and female. Oocytes proliferate rapidly after differentiation while the testes enter a period of quiescence. Fry immersed for short durations in oestradiol (200 μg ⁻¹) are also examined. Hormone immersion advanced sex differentiation by 70 degree-days. The immersions were applied early, at 20 and 90 degree-days, post-hatch, yet still altered the sex ratio and timing of differentiation. Definitive germ cells, which are abundant during this period, may be the type most receptive to steroid treatment.     

New testicular mechanisms involved in the prevention of fetal meiotic initiation in mice

In mammals, early fetal germ cells are unique in their ability to initiate the spermatogenesis or oogenesis programs dependent of their somatic environment. In mice, female germ cells enter into meiosis at 13.5 dpc whereas in the male, germ cells undergo mitotic arrest. Recent findings indicate that Cyp26b1, a RA-degrading enzyme, is a key factor preventing initiation of meiosis in the fetal testis. Here, we report evidence for additional testicular pathways involved in the prevention of fetal meiosis. Using a co-culture model in which an undifferentiated XX gonad is cultured with a fetal or neonatal testis, we demonstrated that the testis prevented the initiation of meiosis and induced male germ cell differentiation in the XX gonad. This testicular effect disappeared when male meiosis starts in the neonatal testis and was not directly due to Cyp26b1 expression. Moreover, neither RA nor ketoconazole, an inhibitor of Cyp26b1, completely prevented testicular inhibition of meiosis in co-cultured ovary. We found that secreted factor(s), with molecular weight greater than 10 kDa contained in conditioned media from cultured fetal testes, inhibited meiosis in the XX gonad. Lastly, although both Sertoli and interstitial cells inhibited meiosis in XX germ cells, only interstitial cells induced mitotic arrest in germ cell. In conclusion, our results demonstrate that male germ cell determination is supported by additional non-retinoid secreted factors inhibiting both meiosis and mitosis and produced by the testicular somatic cells during fetal and neonatal life.     

Interaction of gdt1 and protein kinase A (PKA) in the growth-differentiation-transition in Dictyostelium

Abstract The gdt1 gene is a negative regulator of the growth-differentiation-transition (GDT) in Dictyostelium . gdt1 ⁻ cells express the GDT marker discoidin earlier and at higher levels and prematurely enter the differentiation pathway. Protein kinase A is a positive regulator of the GDT and is required for multicellular development. Disruption of the PKA catalytic subunit or overexpression of a constitutively active mutant of the regulatory subunit results in cells which do not form multicellular aggregates and which show strongly reduced levels of discoidin. We have created PKA ⁻/gdt1⁻ double mutants and show that these display high levels of discoidin expression but no aggregation, suggesting that gdt1 may be a downstream target of PKA in a branched signalling cascade initiating differentiation. Data obtained with the PKA inhibitor H89 support these result: in wild type cells H89 inhibits discoidin expression while in gdt1 ⁻ mutants there is no obvious effect. However, since PKA⁻/gdt1⁻ cells display less discoidin expression than the single gdt1 mutant, we propose that PKA and gdt1 are in two parallel interacting pathways.
To get insight into the mechanism how PKA may block gdt1, we have tested two putative PKA phosphorylation sites in the protein and found that one of them is efficiently phosphorylated by PKA in vitro. A model for the interplay between PKA and gdt1 during the growth-differentiation-transition is discussed.     

Cadmium toxicity to the freshwater amphipod Gammarus pulex (L.) during the moult cycle

SUMMARY. 1. The toxicity of cadmium to mature Gammarus pulex at different stages in the moult cycle is described.
2. Immediate post-moult animals are significantly more sensitive than intermoult specimens at cadmium concentrations between 1.0 and 0.1 mg 1⁻¹ but not at 0.03 or 0.01 mg Cd 1⁻¹.
3. At a calcium concentration of 40mgl⁻¹, post-moult animals undergo recalcification within 7 days and thereafter there is little variation in their response to cadmium.
4. External calcium concentrations of 40 and 115mg 1⁻¹ do not affect cadmium toxicity but at 180 mg Ca1⁻¹ the sensitivity of immediate post-moult specimens is significantly reduced.
5. The results are discussed with regard to the protection of G. pulex by present water quality standards.     

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.     

Efferent duct differentiation during female-to-male sex change in honeycomb grouper Epinephelus merra

Efferent duct (ED) differentiation was examined histologically during female-to-male sex change in the honeycomb grouper Epinephelus merra . During natural sex change, ED differentiation began with the appearance of slit-like structures between the stromal tissue and the tunica ovary and small oval-shaped spaces within the wall of the ovarian cavity, accompanied by oocyte degeneration and the initiation of spermatogenesis, i.e. the early transitional phase. In the late transitional phase, ED structure formation expanded and further ED differentiation occurred, including the rapid multiplication of spermatogonial germ cells. In sex-changed males, the slit-like structures increased in size, fused with each other and finally formed a well-developed ED. The oval-shaped spaces also increased in size and fused to form an ED. In contrast, during artificial sex change, induced by aromatase inhibitor (AI, 1 mg kg⁻¹), ED differentiation in E. merra was first observed as the appearance of slit-like structures and small oval-shaped spaces in the restructuring gonads in the third week after AI treatment. These were accompanied by oocyte degeneration and the proliferation of gonial germ cells into spermatogonia. In the fifth week, the rapid multiplication of spermatogonial germ cells, increases in 11-ketotestosterone (11-KT), and further differentiation of EDs were observed. Sex-changed males had testes containing sperm in the completely differentiated EDs; the significantly highest levels of 11-KT were observed in the sixth week. Simultaneous increases in 11-KT and initiation of ED differentiation were observed, suggesting a role of 11-KT in ED differentiation during sex change. There were no basic differences in the mechanisms of natural and artificially induced ED differentiation. Two types of structure led to the formation of EDs in two different areas of the newly formed testis during sex change.     

Knock-down of DMY initiates female pathway in the genetic male medaka, Oryzias latipes

DMY is the second vertebrate sex-determining gene identified from the fish, Oryzias latipes. In this study, we used two different ways of sex reversal, DMY knock-down and estradiol-17beta (E2) treatment, to determine the possible function of DMY during early gonadal sex differentiation in XY medaka. Our findings revealed that the mitotic and meiotic activities of the germ cells in the 0 day after hatching (dah) DMY knock-down XY larvae were identical to those of the normal XX larvae, suggesting the microenvironment of these XY gonads to be similar to that of the normal XX gonad, where DMY is naturally absent. Conversely, E2 treatment failed to initiate mitosis in the XY gonad, possibly due to an active DMY, even though it could initiate meiosis. Present study is the first to prove that the germ cells in the XY gonad can resume the mitotic activity, if DMY was knocked down.     

Differential control of yolk protein gene expression in fat bodies and gonads by the sex-determining gene tra-2 of Drosophila.

We studied the regulation of the yolk protein (YP) genes in the somatic cells of the gonads, using temperature sensitive mutations (tra-2ts) of transformer-2, a gene required for female sexual differentiation. XX;tra-2ts mutant animals were raised at the permissive temperature so that they developed as females and were then shifted to the restrictive male-determining temperature either 1-2 days before or 0-2 h after eclosion. These animals formed vitellogenic ovaries. Likewise, mutant gonads transplanted into either normal female hosts or normal male hosts, kept at the restrictive temperature, underwent vitellogenesis. Thus, the ovarian follicle cells can mature and express their YP genes in the absence of a functional product of the tra-2 gene. Although the gonadal somatic cells of ovary and testis may derive from the same progenitor cells, the testicular cells of XX;tra-2ts pseudomales did not express their YP genes nor take up YP from the haemolymph at the permissive female-determining temperature. We conclude that in the somatic cells of the gonad, the YP genes are no longer under direct control of the sex-determining genes, but instead are regulated by tissue specific factors present in the follicle cells. It is the formation of follicle cells which requires the activity of tra-2.     

Intraperitoneal Germ Cell Transplantation in the Nile Tilapia Oreochromis niloticus

Germ cell transplantation offers promising applications in finfish aquaculture and the preservation of endangered species. Here, we describe an intraperitoneal spermatogonia transplantation procedure in the Nile tilapia Oreochromis niloticus. Through histological analysis of early gonad development, we first determined the best suitable stage at which exogenous germ cells should be transplanted into the recipients. For the transplantation procedure, donor testes from a transgenic Nile tilapia strain carrying the medaka β-actin/enhanced green fluorescent protein (EGFP) gene were subjected to enzymatic dissociation. These testicular cells were then stained with PKH26 and microinjected into the peritoneal cavity of the recipient fish. To confirm colonization of the donor-derived germ cells, the recipient gonads were examined by fluorescent and confocal microscopy. PKH26-labeled cells exhibiting typical spermatogonial morphology were incorporated into the recipient gonads and were not rejected within 22 days posttransplantation. Long-term survival of transgenic donor-derived germ cells was then verified in the gonads of 5-month-old recipients and in the milt and vitelogenic oocytes of 1-year-old recipients, by means of PCR using EGFP-specific primers. EGFP-positive milt from adult male recipients was used to fertilize non-transgenic oocytes and produced transgenic offspring expressing the donor-derived phenotype. These results imply that long-term survival, proliferation, and differentiation of the donor-derived spermatogonia into vitelogenic oocytes and functional spermatozoa are all possible. Upon further improvements in the transplantation efficiency, this intraperitoneal transplantation system could become a valuable tool in the conservation of genetic resources for cichlid species.     

Vessel differentiation along different tissue polarities

Organized vessel differentiation in an isolated system was studied on a quantitative basis. An auxin source was oriented on isolated pieces of turnip storage root ( Brassica campestris cv. Rapifera) to allow diffusion a) in the direction of, b) at right angle to, and c) opposite to the original polarity. New vessel members differentiated within 44 h, and a minimum average auxin (IAA) concentration of 3.10⁻⁶ M was required to induce initial vessel differentiation. The differentiation rates in three experimental orientations were 167, 60 and 43 cells h⁻¹ at 10⁻³ M IAA, and 1445, 1346 and 838 cells (log IAA concentration)⁻¹ after 96 h, respectively. The difference between the differentiation rates in the original polarity orientation (a) and in the orientation at right angle (b) is interpreted as reflecting reorientation itself, which requires a minimum time.     

Effect of Platelet-Activating Factor on the Process of Cellular Differentiation of Herpetomonas muscarum muscarum

ABSTRACT. The effects of platelet-activating factor (PAF), at doses ranging from 10⁻⁶ M to 10⁻¹⁰ M, on cell growth and on cell differentiation of Herpetomonas muscarum muscarum were investigated. Cell differentiation was evaluated by both light and electron microscopy. At the concentrations used, PAF did not interfere with the protozoan growth. However, parasites grown in the presence of PAF (10⁻⁶ M) were significantly more differentiated than those grown in the absence of PAF, since the first day of culture. On the first two days of culture, PAF doses ranging from 10⁻¹⁰ M to 10⁻⁷ M, did not significantly interfere with the differentiation of these parasites, although after the third day of culture, all PAF doses used significantly increased the protozoan differentiation. Specific PAF receptor antagonists totally abrogated (WEB 2086 and WEB 2170)or significantly decreased (BN 52021) PAF effect on cell differentiation. These findings indicate PAF triggers the process of cell differentiation in Herpetomonas muscarum muscarum and suggest these parasites have receptors for PAF.