Zebrafish germline chimeras produced by transplantation of ovarian germ cells into sterile host larvae

High frequency production of zebrafish germline chimeras was achieved by transplanting ovarian germ cells into sterile Danio hybrid recipients. Ovarian germ cells were obtained from 3-mo-old adult Tg(vasa:DsRed2-vasa);Tg(bactin:EGFP) double transgenic zebrafish by discontinuous Percoll gradient centrifugation. An average of 755 ± 108 DsRed-positive germ cells was recovered from each female. For transplantations, a total of approximately 620 ± 242 EGFP-positive cells of which 12 ± 4.7 were DsRed-positive germ cells were introduced into the abdominal cavity under the swim bladder of 2-wk-old sterile hybrid larvae. Six weeks after transplantation, a total of 10 recipients, obtained from 2 different transplantations, were examined, and 2 individuals (20%) were identified that possessed a large number of DsRed- and EGFP-positive cells in the gonadal region. The transplanted ovarian germ cells successfully colonized the gonads and differentiated into sperm in the male hybrid recipients. Of 67 adult recipients, 12 (18%) male chimeric fish reproduced and generated normal offspring when paired with wild-type zebrafish females. The fertilization efficiency ranged from 23% to 56%. Although the fertile male chimeras were generated by transplantation of ovarian germ cells, the F1 generation produced by the male chimeras contained both male and female progeny, indicating that male sex determination in zebrafish is not controlled by sex chromosome heterogamy. Our findings indicate that a population of ovarian germ cells that are present in the ovary of adult zebrafish can function as germline stem cells, able to proliferate and differentiate into testicular germ cells and functional sperm in male recipients. The high frequency of germline chimera formation achieved with the ovarian germ cells and the convenience of identifying the chimeras in the sterile host background should make this transplantation system useful for performing genetic manipulations in zebrafish.     

The Role of DNA Methylation Reprogramming During Sex Determination and Transition in Zebrafish

DNA methylation is a prevalent epigenetic modification in vertebrates, and it has been shown to be involved the regulation of gene expression and embryo development. However, it remains unclear how DNA methylation regulates sexual development, especially in species without sex chromosomes. To determine this, we utilized zebrafish to investigate DNA methylation reprogramming during juvenile germ cell development and adult female-to-male sex transition.We reveal that primordial germ cells(PGCs) undergo significant DNA methylation reprogramming during germ cell development, and the methylome of PGCs is reset to an oocyte/ovary-like pattern at 9 days post fertilization(9 dpf). When DNA methyltransferase(DNMT) activity in juveniles was blocked after 9 dpf, the zebrafish developed into females. We also show that Tet3 is involved in PGC development. Notably, we find that DNA methylome reprogramming during adult zebrafish sex transition is similar to the reprogramming during the sex differentiation from 9 dpf PGCs to sperm. Furthermore, inhibiting DNMT activity can prevent the female-to-male sex transition, suggesting that methylation reprogramming is required for zebrafish sex transition. In summary, DNA methylation plays important roles in zebrafish germ cell development and sexual plasticity.     

Generation of functional oocytes and spermatids from fetal primordial germ cells after ectopic transplantation in adult mice

Primordial germ cells (PGCs) are undifferentiated germ cells in developing fetuses. As these cells give rise to definitive oocytes and spermatozoa that contribute to new life in the next generation, their development must be under strict control, regarding genetic and epigenetic aspects. However, we do not know to what extent their development depends on the specific milieu. In this study, we transplanted mouse PGCs collected from male and female gonads at 12.5 days postcoitum, together with gonadal somatic cells, under kidney capsules of adult mice. The transplanted PGC and gonadal somatic cells constructed testis-like and ovary-like tissues, respectively, under the kidney capsules within 4 wk. Normal-appearing round spermatids and fully grown germinal vesicle (GV) oocytes developed within these tissues. Ectopic spermatogenesis continued thereafter, while oogenesis consisted of only a single wave. The injection of these round spermatids directly into mature in vivo-derived oocytes led to the birth at term of normal pups. PGC-derived GV oocytes were isolated, induced to mature in vitro, and injected with normal spermatozoa. The injected oocytes were successfully fertilized and developed into normal pups. Our findings demonstrate the remarkable flexibility of PGC development, which can proceed up to the functional gamete stage under spatially and temporally noninnate conditions. This transplantation system may provide a unique technical basis for induction of the development of early germ cells of exogenous origins, such as those from embryonic stem cells.     

A study of meiosis in chimeric mouse fetal gonads

The influence of somatic environment on the onset and progression of meiosis in fetal germ cells was studied in chimeric gonads produced in vitro by dissociation-reaggregation experiments. Germ cells isolated from testes or ovaries of 11.5-13.5 days post coitum (dpc) CD-1 mouse embryos were loaded with the fluorescent supravital dye 5-6 carboxyfluorescein diacetate succinimyl ester (CFSE) and mixed with a cell suspension obtained by trypsin-EDTA treatment of gonads of various ages and of the same or opposite sex. Whereas 11.5 dpc donor germ cells appeared unable to survive in the chimeric gonads obtained, about 76% of the CFSE-labeled female germ cells obtained from 12.5 dpc donor embryos (premeiotic germ cells) found viable within host ovarian tissues showed a meiotic nucleus. In contrast, a smaller number (about 19%) were in meiosis in chimeric testes. None or very few of donor male germ cells entered meiosis in testes or ovarian host tissues. Aggregation of meiotic 13.5 dpc female germ cells with testis tissues from 13.5 to 14.5 dpc embryos resulted in inhibition of meiotic progression and pyknosis in most donor germ cells. These results support the existence of a meiosis-preventing substance or a factor causing oocyte degeneration in the fetal mouse testis, but not of a meiosis-inducing substance in the fetal ovary.     

Sox9b/sox9a2-EGFP transgenic medaka reveals the morphological reorganization of the gonads and a common precursor of both the female and male supporting cells

We have established an enhanced green fluorescent protein (EGFP) transgenic medaka line that mimics the expression of sox9b/sox9a2 to analyze the morphological reorganization of the gonads and characterize the sox9b-expressing cells during gonadal formation in this fish. After the germ cells have migrated into the gonadal areas, a cluster of EGFP-expressing cells in the single gonadal primordium was found to be separated by the somatic cells along the rostrocaudal axis and form the bilateral lobes. We observed in these transgenic fish that EGFP expression persists only in the somatic cells directly surrounding the germ cells. As sex differentiation proceeds, dmrt1 and foxl2 begin to be expressed in the EGFP-expressing cells in the XY and the XX gonads, respectively. This indicates that the sox9b-expressing cells reorganize into two lobes of the gonad and then differentiate into Sertoli or granulosa cells, as common precursors of the supporting cells. Hence, our sox9b-EGFP medaka system will be useful in future studies of gonadal 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.     

Usefulness of double gene construct for rapid identification of transgenic mice exhibiting tissue-specific gene expression.

Identification of transgenics still requires PCR and genomic Southern blot hybridization of genomic DNA isolated from tail pieces. Furthermore, identification of transgene-expressing transgenics (hereafter called "expressor") requires mRNA analyses (RT-PCR and Northern blot hybridization) or protein analysis (Western blotting and immunohistochemical staining using specific antibodies). These approaches are often labor-intensive and time-consuming. We developed a technique that simplifies the process of screening expressor transgenics using enhanced green fluorescent protein (EGFP), a noninvasive reporter recently utilized in a variety of organisms, including mice, as a tag. We constructed a MNCE transgene consisting of two expression units, MBP-NCre (termed "MN") and CAG-EGFP (termed "CE"). MN consists of a myelin basic protein (MBP) promoter and NCre gene (Cre gene carrying a nuclear localization signal (NLS) sequence at its 5' end). CE consists of a promoter element, CAG composed of cytomegalovirus (CMV) enhancer and chicken beta-actin promoter, and EGFP cDNA. Of a total of 72 F0 mice obtained after pronuclear injection of MNCE at 1-cell egg stage, 15 were found to express EGFP when the tail, eye, and inner surface of the ear were inspected for EGFP fluorescence under UV illumination at weaning stage. These fluorescent mice were found to possess MNCE and to express NCre mRNA in a brain-specific manner. Mice exhibiting no fluorescence were transgenic or nontransgenic. Mice carrying MNCE, but exhibiting no fluorescence, never expressed NCre mRNA in any organs tested. These findings indicate that (i) direct inspection of the surface of mice for fluorescence under UV illumination enables identification of expressor transgenics without performances of the molecular biological analyses mentioned above, and (ii) systemic promoters such as CAG do not affect the tissue-specificity of a tissue-specific promoter such as MBP promoter, which is located upstream of CAG by approximately 2 kb.     

Identification of side population cells in mouse primordial germ cells and prenatal testis

In mammals, the stem cells of spermatogenesis are derived from an embryonic cell population called primordial germ cells (PGCs). Spermatogonial stem cells displaying the “side population” (SP) phenotype have been identified in the immature and adult mouse testis, but noting is known about the expression of the SP phenotype during prenatal development of germ cells. The SP phenotype, defined as the ability of cells to efflux fluorescent dyes such as Hoechst, is common to several stem/progenitor cell types. In the present study, we analyzed and characterized the Hoechst SP via cytofluorimetric analysis of disaggregated gonads at different time points during embryonic development in mice. To directly test the hypothesis that the SP phenotype is a feature of germ cell lineage, experiments were performed on transgenic animals expressing enhanced green fluorescent protein (EGFP) under the control of the Oct4 promoter, to identify early germ cells up to PGCs. We found that prenatal gonads contain a fraction of SP cells at each stage analyzed, and the percentage of cells in the SP fraction decreases as development proceeds. Surprisingly, more than 50% of the PGCs displayed the SP phenotype at 11.5 dpc (days post coitum). The percentage of germ cells with the SP phenotype decreased steadily with development, to less than 1% at 18.5 dpc. Cytofluorimetric analysis along with immunocytochemistry performed on sorted cells indicated that the SP fraction of prenatal gonads, as in the adult testis, was heterogeneous, being composed of both somatic and germ cells. Both cell types expressed the ABC transporters Abcg2, Abcb1a, Abcb1b and Abcc1. These findings provide evidence that the SP phenotype is a common feature of PGCs and identifies a subpopulation of fetal testis cells including prospermatogonia whose differentiation fate remains to be investigated.     

Fishing for function: zebrafish BAC transgenics for functional genomics

Transgenics using bacterial artificial chromosomes (BACs) offers a great opportunity to look at gene regulation in a developing embryo. The modified BAC containing a reporter inserted just before the translational start site of the gene of interest allows for the visualization of spatio-temporal gene expression. Though this method has been used in the mouse model extensively, its utility in zebrafish studies is relatively new. This review aims to look at the utility of making BAC transgenics in zebrafish and its applications in functional genomics. We look at the various methods to modify the BAC, some limitations and what the future holds.     

Establishment and characterization of CAG/EGFP transgenic rabbit line

Cell marking is a very important procedure for identifying donor cells after cell and/or organ transplantation in vivo. Transgenic animals expressing marker proteins such as enhanced green fluorescent protein (EGFP) in their tissues are a powerful tool for research in fields of tissue engineering and regenerative medicine. The purpose of this study was to establish transgenic rabbit lines that ubiquitously express EGFP under the control of the cytomegalovirus immediate early enhancer/beta-actin promoter (CAG) to provide a fluorescent transgenic animal as a bioresource. We microinjected the EGFP expression vector into 945 rabbit eggs and 4 independent transgenic candidate pups were obtained. Two of them died before sexual maturation and one was infertile. One transgenic male candidate founder rabbit was obtained and could be bred by artificial insemination. The rabbit transmitted the transgene in a Mendelian manner. Using fluorescence in situ hybridization analysis, we detected the transgene at 7q11 on chromosome 7 as a large centromeric region in two F1 offspring (one female and one male). Eventually, one transgenic line was established. Ubiquitous EGFP florescence was confirmed in all examined organs. There were no gender-related differences in fluorescence. The established CAG/EGFP transgenic rabbit will be an important bioresource and a useful tool for various studies in tissue engineering and regenerative medicine.     

Functional morphology of the male reproductive system in Callichirus major (Crustacea: Decapoda: Axiidea): Evidence of oocytes in the gonad

Callichirus major inhabits the intertidal region of marine ecosystems and it is frequently used as live bait for fishing. This study aimed to describe the functional anatomy of the male reproductive system by microscopic techniques. The animals were collected along the Corujão beach, Piuma—ES, Brazil, and, in laboratory, the males were classified into two phases: immature (IM) and developed (DE) based on the macroscopic characteristics of the gonads. The gonad and vas deferens were dissected for histological routine and histochemical tests. Histologically, it was noted that in both phases, the more distal region of gonads has ovarian characteristics, showing developing oocytes. Also, different male germ cells were identified: spermatogonium (SPG), spermatocytes I and II (SPTCI, SPTCII), initial and final spermatid (IS, FS) and sperm (SPZ). Accessory cells with spherical or pyramidal nuclei were also present inside the testicular lobules. According to the vas deferens structure, three regions can be characterized: proximal (PVD), middle (MVD) and distal (DVD). In the lumen of the vas deferens, a spermatophoric matrix highly reactive for histochemical tests was observed. The presence of female germ cells in males suggests the occurrence of intersexuality or hermaphroditism in this species.     

Differentiation of female chicken primordial germ cells into spermatozoa in male gonads

In avian species, the developmental fate of different-sex germ cells in the gonads is unclear. The present study attempted to confirm whether genetically female germ cells can differentiate into spermatozoa in male gonads using male germline chimeric chickens produced by the transfer of primordial germ cells (PGC), and employing molecular biological methods. As a result of Southern hybridization, specific sequences of the W chromosome (the female specific sex chromosome in birds) were detected in the genomic DNA extracted from one out of four male germline chimeric chickens. When two-color in situ hybridization was conducted on the spermatozoa of this germline chimera, 0.33% (average) of the nuclei of each semen sample showed the fluorescent signal indicating the presence of the W chromosome. The present study shows that female PGC can differentiate into spermatozoa in male gonads in the chicken. However, the ratio of produced W chromosome-bearing (W-bearing) spermatozoa fell substantially below expectations. It is therefore concluded that most of the W-bearing PGC could not differentiate into spermatozoa because of restricted spermatogenesis.     

A Cre gene directed by a human TSPY promoter is specific for germ cells and neurons

The testis-specific protein Y-encoded (TSPY) gene is a candidate for the gonadoblastoma locus on the Y chromosome and is expressed in normal testicular germ cells and gonadoblastoma cells of XY sex-reversed females. Although TSPY expression has been demonstrated in gonadoblastoma tissues, it is uncertain if such expression is involved in a causative or consequential event of the oncogenic process. We postulate that if TSPY is involved in gonadoblastoma development, its promoter should be functional in the female gonad before and/or at early stages of tumorigenesis. To test this hypothesis, we generated several lines of transgenic mice harboring a Cre-recombinase transgene directed by a 2.4-kb hTSPY promoter. These mice were crossed with the Z/EG reporter line that expresses EGFP only after a Cre-mediated recombination. Our results showed that hTSPY-Cre;Z/EG double transgenic mice expressed EGFP specifically in the germ cells of both male and female gonads. Further, neurons of the central and peripheral nervous systems also expressed EGFP as early as E12.5 embryonic stage. EGFP was particularly observed in the trigeminal nerve, trigeminal ganglion, dorsal root of the ganglia, and in postnatal and adult brains. These observations support the hypothesis that TSPY plays an active role in gonadoblastoma. The tissue-specific expression of the hTSPY-Cre transgene should also be useful in studies utilizing Cre-mediated gene activation/inactivation strategies in gamatogenesis and/or neurogenesis.     

Origin of Germ Cells and Early Differentiation of Gonads in the Starfish, Asterina pectinifera

The origin of the germ cells and the development of the genital system in the annually spawning starfish, Asterina pectinifera , were studied by light and electron microscopy. Characteristic germ cells were first characterized in gonads after spawning: the gonia are larger than somatic cells, have large nuclei (with electron-lucent nucleoplasm), and show mitochondrial aggregation associated with nuage (electron-dense bodies). In young starfish without gonads similar cells were detected in the haemal sinus, where they were termed primordial germ cells (PGCs). Brachiolariae and metamorphosed juveniles had a cellular cluster in the coelomic epithelium, near the hydroporic canal. The cluster was comprised of cells endowed with the above-mentioned characteristics of the germ cells. The germ cell counts indicated that PGCs migrate from the aboral haemal sinus near the hydroporic canal, through the haemal sinus to the gonads, where they settle, proliferate, and differentiate into gonia.     

Investigation of the fate of Sry-expressing cells using an in vivo Cre/loxP system

Sry (sex-determining region on Y chromosome) is expressed in the undifferentiated, bipotential genital ridges of mammalian XY fetuses. The expression of Sry initiates testis development, but the lineage of Sry-expressing cells is unclear. In this study, double-transgenic mice were analyzed using the Cre/loxP system. Cre under the control of the Sry promoter was expressed in the fetal gonads of transgenic mice similarly to endogenous Sry. The Sry/Cre-transgenic mice were crossed with CAG(cytomegalovirus immediate-early enhancer, chicken beta-actin promoter and fusion intron of chicken beta-actin and rabbit beta-globin)/loxP/CAT/loxP/LacZ-transgenic mice, in which the transgene expressed beta-galactosidase after a Cre-mediated recombination event. Sertoli cells, germ cells of testes and granulosa cells of ovaries of double-transgenic mice stained positive with X-gal. Cre expression was detected in germ cells and peritubular/Sertoli cells in adult testes. It is not clear whether beta-galactosidase expression in the Sertoli cells of the testes occurred as a result of Cre expression in the adult or in the fetal gonads. These analyses indicate that cells expressing Sry-inducing factors in female fetal gonads become granulosa cells.     

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.     

Ubiquitous transgene expression and Cre-based recombination driven by the ubiquitin promoter in zebrafish

Molecular genetics approaches in zebrafish research are hampered by the lack of a ubiquitous transgene driver element that is active at all developmental stages. Here, we report the isolation and characterization of the zebrafish ubiquitin (ubi) promoter, which drives constitutive transgene expression during all developmental stages and analyzed adult organs. Notably, ubi expresses in all blood cell lineages, and we demonstrate the application of ubi-driven fluorophore transgenics in hematopoietic transplantation experiments to assess true multilineage potential of engrafted cells. We further generated transgenic zebrafish that express ubiquitous 4-hydroxytamoxifen-controlled Cre recombinase activity from a ubi:cre(ERt2) transgene, as well as ubi:loxP-EGFP-loxP-mCherry (ubi:Switch) transgenics and show their use as a constitutive fluorescent lineage tracing reagent. The ubi promoter and the transgenic lines presented here thus provide a broad resource and important advancement for transgenic applications in zebrafish.