Effects of exogenous estradiol-17β on early growth and gonadal development of diploid and triploid female rainbow trout (Oncorhyncus mykiss)

Triploidy is a viable condition in teleosts. However, in many salmonids, the triploid condition in the female results in sterility as gametogenesis appears to be disrupted. Although the underlying mechanisms regulating the gonadal development of teleosts have not been clearly elucidated, the reversal of phenotypic sex by the administration of the appropriate exogenous steroid during early development supports the argument that gonadal steroids play a pivotal role in sexual differentiation and subsequent gonad development in these fish. To determine whether the failure of normal ovarian development in triploid female rainbow trout (Oncorhynchus mykiss) is due to an absence or reduction of endogenous sex steroids, ovarian morphology was compared between diploid and triploid juvenile rainbow trout treated with exogenous estradiol-17β (E₂). The ovaries of both untreated and E₂ treated diploid fish, at 145 days post-fertilization, contained synchronously developing oocytes in the perinucleolar stage, whereas ovaries from untreated and estradiol-treated triploid fish of the same age were considerably smaller and devoid of developing oocytes. No differences in the ovaries of triploid untreated fish and triploid fish treated with E₂ were observed. It is reported that exposure to exogenous E₂ during the period of gonadal differentiation is not sufficient to induce oocyte development in triploid rainbow trout. © 1996 Wiley-Liss, Inc.     

PRIMORDIAL GERM CELL-DERIVED MOUSE EMBRYONIC GERM (EG) CELLSIN VITRORESEMBLE UNDIFFERENTIATED STEM CELLS WITH RESPECT TO DIFFERENTIATION CAPACITY AND CELL CYCLE DISTRIBUTION

Embryonic germ (EG) cells of line EG-1 derived from mouse primordial germ cells were investigated for theirin vitrodifferentiation capacity. By cultivation as embryo-like aggregates EG-1 cells differentiated into cardiac, skeletal muscle and neuronal cells accompanied by the expression of tissue-specific genes and proteins as shown by RT-PCR analysis and indirect immunofluorescence. In comparison to embryonic stem (ES) cells of line D3 the efficiency of differentiation into cardiac and muscle cells was comparatively low, whereas spontaneous neuronal differentiation was more efficient than in D3 cells. Furthermore, the distribution of cell cycle phases as a parameter for the differentiation state was analysed in undifferentiated EG cells and ES cells and compared to data obtained for embryonic carcinoma (EC) cells of line P19 and differentiated, epithelioid EPI-7 cells. Flow cytometric analysis revealed similar cell cycle phase distributions in EG, EC and ES cells. In contrast, the somatic differentiated EPI-7 cells showed a longer G₁-phase and shorter S- and G₂/M-phases. Together, our results demonstrate that the differentiation state and capacity of EG cellsin vitroresemble that of totipotent ES cells.     

Cause of the decreased number of PGC in albino Xenopus: analysis of the number and position of pPGC in albino embryos during and after cleavage

In order to understand the cause for the decreased number of primordial germ cells (PGC) in Xenopus albino (a(p)/a(p)) tadpoles, the number of presumptive PGC (pPGC) in the albino and wild-type embryos at Nieuwkoop and Faber's stages 6-37/38 were examined using the antibody specific to germ plasm. The positions of pPGC in the endodermal cell mass in embryos of both types at stages 28 and 33/34 were also observed to learn the migratory behavior of pPGC. The number of pPGC in the albino increased up to stage 28 with development, but decreased thereafter. In contrast, the number in the wild-type increased to stage 33/34 as development proceeded, and the number of pPGC in stage 33/34 embryos reached nearly that of PGC of the feeding tadpoles in the same batches. Judging from the positions of pPGC, the migration of pPGC from the median part through the lateral to the dorsal part of the endodermal cell mass in the albino was suspected to be somewhat later than that in the wild-type. These results, together with the results in previous studies, suggest that the decreased number of PGC in the albino would be closely related to the sudden decrease in number of pPGC at stage 33/34, as well as to the ectopic position of pPGC in endodermal cell mass, the latter of which had already been demonstrated to be responsible for the differentiation into PGC.     

Polarized endocytosis and transcytosis in the hypothalamic tanycytes of the rat

Four types of tanycytes can be distinguished in the rat hypothalamus: ₁ and ₂ tanycytes establish an anatomical link between the ventricular cerebrospinal fluid (CSF) and the arcuate nucleus, whereas ₁ and ₂ tanycytes establish a link between CSF and portal blood. Endocytosis and transcytosis in these cells have been investigated by (1) immunocytochemistry with antibodies against molecular markers of the endocytotic and transcytotic pathways; (2) the administration of wheat germ agglutinin (WGA) into the ventricular or subarachnoidal CSF and following its internalisation by and its routing through tanycytes. The four populations of tanycytes show marked differences concerning the expression and subcellular location of proteins involved in endocytosis and transcytosis, such as clathrin, caveolin-1, Rab4 and ARF6. Thus, _1,2 tanycytes express caveolin-1 at the ventricular cell pole and at their terminals contacting the portal capillaries, whereas _1,2 tanycytes do not, suggesting that caveolae-dependant endocytosis does not occur in the latter and that, in _1,2 tanycytes, it may occur at both cell poles. In _1,2 tanycytes, clathrin is only expressed at the ventricular cell pole indicating that clathrin-dependant endocytosis operates for compounds present in the ventricular CSF and not for those exposed to the terminals. This agrees with the property of _1,2 tanycytes of internalising WGA through the ventricular cell pole but not through the terminals. The subcellular distribution in _1,2 tanycytes of WGA and of the proteins clathrin and Rab4 indicates that part of the internalised WGA follows the degradative pathway and part is sorted to a transcytotic pathway and that the transcytotic and the secretory pathways might intersect. Financial support was provided by grants 01/1050, from FIS, Spain (to J.L.B.) and 1030265, from FONDECYT, Chile (to E.M.R.)     

Developmental and ultrastructual characteristics of mouse oocytes grown <Emphasis Type="Italic">in Vitro</Emphasis> from primordial germ cells

The aim of this study was to clarify the developmental and ultrastructual characteristics of oocytes grown in vitro from primordial germ cells. The female genital ridges at 12.5 days post coitus were cultured for 18 days on an insert membrane in Waymouth's MB752/1 medium, supplemented with 15% fetal bovine serum and 1 mM sodium pyruvate; subsequently, the follicles isolated from the tissue were cultured for eight days in Waymouth's medium supplemented with 5 microg/ml insulin, 5 microg/ml transferrin, 5 ng/ml selenium, 10 mIU/ml follicle stimulating hormone, and 100 ng/ml stem cell factor. The primordial germ cells developed in vitro into oocytes of more than 60 microm in diameter. The transmission electron microscopic analysis indicated that the oocytes, which developed in vitro, showed no obvious abnormality in their ultrastructure and had organelles appropriate for the oocyte size. However, a delay in the progressive changes of morphology in some of the organelles during oocyte growth was often found when comparing them to oocytes grown in vivo.     

Loss of PGC-specific expression of the orphan nuclear receptor ERR-beta results in reduction of germ cell number in mouse embryos

Estrogen related receptor beta (ERR-beta) is an orphan nuclear receptor specifically expressed in a subset of extra-embryonic ectoderm of post-implantation embryos. ERR-beta is essential for placental development since the ERR-beta null mutants die at 10.5dpc due to the placenta abnormality. Here, we show that the ERR-beta is specifically expressed in primordial germ cells (PGC), obviously another important cell type for reproduction. Expression of the ERR-beta mRNA in embryonic germ cells started at E11.5 as soon as PGC reached genital ridges, and persisted until E15-E16 in both sexes. Immunostaining with anti-ERR-beta antibody revealed that the ERR-beta protein is exclusively expressed in germ cells in both male and female gonads from E11.5 to E16. 5. To study function of the ERR-beta in PGC, we complemented placental defects of the ERR-beta null mutants with wild-type tetraploid embryos, and analyzed germ cell development in the rescued embryos. It was found that development of gonad and PGC was not apparently affected, but number of germ cells was significantly reduced in male and female gonads, suggesting that the ERR-beta appears to be involved in proliferation of gonadal germ cells. The rescued embryos could develop to term and grow up to adulthood. The rescued ERR-beta null male were found to be fertile, but both male and female null mutants exhibited behavioural abnormalities, implying that the ERR-beta plays important roles in wider biological processes than previously thought.     

Avian pluripotent stem cells

Pluripotent embryonic stem cells are undifferentiated cells capable of proliferation and self-renewal and have the capacity to differentiate into all somatic cell types and the germ line. They provide an in vitro model of early embryonic differentiation and are a useful means for targeted manipulation of the genome. Pluripotent stem cells in the chick have been derived from stage X blastoderms and 5.5 day gonadal primordial germ cells (PGCs). Blastoderm-derived embryonic stem cells (ESCs) have the capacity for in vitro differentiation into embryoid bodies and derivatives of the three primary germ layers. When grafted onto the chorioallantoic membrane, the ESCs formed a variety of differentiated cell types and attempted to organize into complex structures. In addition, when injected into the unincubated stage X blastoderm, the ESCs can be found in numerous somatic tissues and the germ line. The potential give rise to somatic and germ line chimeras is highly dependent upon the culture conditions and decreases with passage. Likewise, PGC-derived embryonic germ cells (EGCs) can give rise to simple embryoid bodies and can undergo some differentiation in vitro. Interestingly, chicken EG cells contribute to somatic lineages when injected into the stage X blastoderm, but only germ line chimeras have resulted from EGCs injected into the vasculature of the stage 16 embryo. To date, no lines of transgenic chickens have been generated using ESCs or EGCs. Nevertheless, progress towards the culture of avian pluripotent stem cells has been significant. In the future, the answers to fundamental questions regarding segregation of the avian germ line and the molecular basis of pluripotency should foster the full use of avian pluripotent stem cells.     

Prospects for transgenesis in the chick

Research to develop a useful method for genetic modification of the chick has been on-going since the first demonstrations in the mouse in the 1980s that genetic modification is an invaluable tool for the study of gene function. Manipulation of the chick zygote is possible but inefficient. Considerable progress has been made in developing potentially pluripotent embryo stem cells and their contribution to somatic chimeric birds well-established. Germ line transmission of gametes derived from genetically modified embryo cells has not been described. Transfer of primordial germ cells from a donor embryo to a recipient and production of functional gametes from the donor-derived cells is possible. Genetic modification of primordial germ cells before transfer and their recovery through the germ line has not been achieved. The first transgenic birds described were generated using retroviral vectors. The use of lentiviral vectors may make this approach a feasible method for transgenic production, although there are limitations to the applications of these vectors. It is likely that a method will be developed in the next few years that will enable the use of transgenesis as a tool in the study of development in the chick and for many other applications in basic research and biotechnology.