Expression of p63 in the mouse primordial germ cells

Proteins encoded by p63 gene a have structural similarity with tumor suppressor p53, and were thought to induce cell cycle arrest and apoptosis during development. The p63 proteins are also expressed in the basal cells of many epithelial tissues in the adult, and supposed to play important roles in maintaining the epidermal stem cells. Previously, we reported the p63 expression in the testis of mouse embryos, suggesting their involvement in the growth arrest and apoptosis of testicular germ cells (Nakamuta and Kobayashi, J. Vet. Med. Sci. 65:853-856). In this study, we investigated the timing of this p63 expression in the germ cells during migration and colonization to the gonads. Immunohistochemical analysis of mice from embryonic day (E) 7.5 to E12.5 demonstrated that p63 positive reactivity was seen as early as E8.5 when the founder cells of germ cells, primordial germ cells (PGCs), were located in the hind gut epithelium, but PGCs were negative for p63 at E7.5 when they first appeared. p63 is expressed as six isoforms, resulting from alternative splicing at C-terminus and by the use of two promoters that generate variations at N-terminal end. RT-PCR analyses suggested that different types of p63 mRNAs were likely to be expressed in PGCs during development. These results imply that p63 may be involved in the regulation of PGC development by controlling the gene expression required for their migration and colonization to the gonads.     

N,N'-Bis(2-chloroethyl)-N-nitrosourea (BCNU)-induced Apoptosis of Neural Progenitor Cells in the Developing Fetal Rat Brain

N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) is one of the major drugs used in chemotherapy against malignant gliomas due to its effects, such as induction of bifunctional alkylation of DNA and formation of interstrand DNA cross-linkages, and induces cortical malformations in the fetal and neonatal rat brain. In this study, pregnant rats were treated with 7.5 mg/kg of BCNU on gestational day 13 (GD 13), and their fetuses were collected from 12 to 72 hours after BCNU treatment in order to examine the timecourses of morphological and immunohistochemical changes in neural progenitor cells in the developing brain. The number of pyknotic cells in the telencephalon peaked at 24 h and then gradually decreased until 72 h. The majority of these pyknotic cells were positive for cleaved caspase-3, a key executioner of apoptosis. The pyknotic cells showed the ultrastructural characteristics of apoptosis. The number of p53-positive cells began to increase prior to the appearance of apoptotic cells and p21-positive cells. The number of phosphorylated-histone H3-positive cells (mitotic cells) decreased from 24 to 36 h. The number of Iba1-positive cells (microglial cells) in the telencephalon increased from 12 to 48 h. These results suggest that BCNU induces p53-dependent apoptosis and reduces proliferative activity, resulting in reduction of the weight of the telencephalon and the thickness of the telencephalic wall in the fetal brain. This study will help to clarify the mechanisms of BCNU-induced fetal brain toxicity.     

Reduced mitotic activity and increased apoptosis of fetal sertoli cells in rat hypogonadic (hgn/hgn) testes

Sterility in male hypogonadic (hgn/hgn) rats results from congenital testicular dysplasia caused by a single recessive gene hgn on rat chromosome 10. We recently identified an insertion mutation in the Spag5/astrin gene of hgn/hgn rats that may cause defective proliferation of immature Sertoli cells in the postnatal hgn/hgn testis. Since the pathological alterations were present in the testes at birth, we examined the involvement of defective mitosis and apoptotic cell death in embryonic development of hgn/hgn testes. Testicular hypoplasia was apparent at embryonic day (ED) 18.5. Immunostaining of hgn/hgn testes at ED 21.5 with antibody to GATA-4, which is specific for fetal Sertoli cells in the seminiferous cords, showed that the significant decrease in the number of fetal Sertoli cells was accompanied by a two fold increase in their mitotic index and abnormal mitosis and apoptosis. Prior to this, we observed a decrease in the number of BrdU-labeled cells, an increase in the number of TUNEL-positive apoptotic cells, and presence of MIS-positive apoptotic cells in hgn/hgn testes on ED 17.5 and 18.5. These results suggest that the Spag5 mutation may cause a reduction in mitotic activity and an increase in apoptosis of fetal Sertoli cells in hgn/hgn testes.     

绵羊羊水、胎儿肾脏组织中表达Oct-4细胞的分离培养及其分子学特性研究简

试验旨在建立从绵羊羊水、胎儿肾脏组织中分离培养表达Oct-4细胞的新方法,同时,通过实时荧光定量PCR（Real-time PCR）初步探索二者在分子生物学特性方面的联系及差异。采用0.01%胰酶培养液,从同一只受孕中期绵羊的羊水和胎儿肾脏组织中各分离1株细胞。Real-time PCR分析结果表明,从绵羊羊水、胎儿肾脏组织中分离的细胞均表达胚胎干细胞相关标志基因Oct-4、胚胎生殖细胞标志基因SSEA-1、主要组织相容性抗原MHC-Ⅱ、细胞凋亡基因Bax及抑制细胞凋亡基因Bcl-2,不表达MHC-Ⅰ、Nanog及TERT,因此,将羊水中表达Oct-4的细胞暂命名为羊水来源多潜能干细胞(amniotic fluid stem cells,AFSC),胎儿肾脏组织中表达Oct-4的细胞暂命名为肾脏组织干细胞(renal tissue stem cells,RTSC)。相对定量分析结果显示,二者的Oct-4、Bax及Bcl-2基因的相对表达量存在明显差异。绵羊羊水中表达Oct-4的细胞（AFSC）和胎儿肾脏组织中表达Oct-4的细胞（RTSC）体外悬浮培养7 d均能形成类胚体。试验成功建立了从绵羊羊水、胎儿肾脏组织中分离培养表达Oct-4细胞的新方法,实时荧光定量PCR分析初步显示绵羊羊水中表达Oct-4的细胞和胎儿肾脏组织中表达Oct-4的细胞具有相同的起源,并在体内处于一种动态的变化之中。     

Distribution of apoptotic cells and apoptosis-related molecules in the developing murine palatine rugae

Distribution of apoptotic cells and expression of the apoptosis-related factors p53, bcl-2 and bad during morphogenesis of the murine palatine rugae (PR) were examined histochemically using the terminal deoxynucleotidyl transferase-mediated UTP nick end-labeling (TUNEL) technique and specific antibodies against apoptosis and cell cycle-related molecules. Formation of the PR rudiment was controlled by cell proliferation and apoptosis in the palatal epithelium. TUNEL-positive cells were detected only at the epithelial placode area at 12.5-13.5 days post coitus (dpc), but only a few cells were positive at the protruding PR area at 14.5-16.5 dpc. Bcl-2 protein was expressed mainly in the areas outside of those containing TUNEL-positive cells at 15.5 -6.5 dpc. P53 protein was not detected throughout gestation. Bad was detected in the epithelial layer at 13.5 and 15.5 dpc and overlapping the apoptotic area at 13.5-15.5 dpc. Apoptosis of palatal epithelial cells might therefore involve spatiotemporally regulated expression of bad during murine PR development.     

雄性关中奶山羊胎儿生殖细胞增殖分化的研究

旨在阐明山羊胚性腺的发生规律,这对于山羊的繁殖育种、丰富胚胎学有关研究内容都具有重要意义.该研究对22枚从39日胎龄胎儿至初生奶山羊性腺进行石蜡组织切片,用组织学和免疫组化、免疫荧光方法对山羊胎儿性腺发育的组织学特点,山羊胎儿性腺发育过程中VASA及细胞增殖核抗原(PCNA)的表达规律进行了系统研究.结果表明:奶山羊性腺在39～47日胎龄时已出现精索和白膜,已发生性别分化,并可分辨出生殖细胞、支持细胞及间质细胞,细胞增殖率较高.在大约50日胎龄前生殖细胞可称作性原细胞,细胞为球形、核质比高.在51～59日胎龄,生殖细胞发生剧烈变化:体积增大、核质比降低.此后逐渐由性原细胞发育为前精原细胞,并且在此期间表达VASA的生殖细胞增多.50～90日胎龄,生殖细胞增殖率最高,生殖细胞所占比例也达到峰值.在90日胎龄后生殖细胞增殖率下降,比例下降,间质细胞大量退化.结果表明,山羊胎儿的生殖细胞在50日胎龄后由性原细胞发育为前精原细胞,生殖细胞在50～90日胎龄的增殖率最高,VASA可作为雄性山羊胎儿前精原细胞的标记.     

The Effects of GABA on embryonic gonadotropin-releasing hormone neurons in rat hypothalamic primary culture

Gonadotropin-releasing hormone (GnRH) neurons arise in the olfactory placode, migrate into the preoptic area (POA), and then extend axons to the median eminence during embryogenesis. Little information is available concerning the properties of GnRH neurons during the late gestational period when GnRH neurons reach the POA and form neuronal networks, although many studies have examined such properties during earlier developmental stages or the postnatal period. The present study was performed to elucidate the involvement of gamma-aminobutyric acid (GABA), one of the major neurotransmitters modifying GnRH neural activity, in regulation of GnRH gene expression on embryonic day 18.5 (E18.5) using transgenic rats expressing enhanced green fluorescence protein (EGFP) under the control of GnRH promoter. First, using RT-PCR, the mRNA of two isoforms of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), GAD65 and GAD67 was detected in E18.5 embryonic POA-containing tissues. GAD67-positive cells were also demonstrated in close vicinity to GnRH-positive cells by immunohistochemistry, and immunoreactivity for both the GABA-A and GABA-B receptor subunits was detected in GnRH neurons. Next, primary cultures derived from anterior hypothalamic tissue of E18.5 embryos were prepared, and the effects of GABA and its agonists on GnRH promoter activity were evaluated using EGFP expression as a marker. GABA and the GABA-A receptor agonist muscimol, but not the GABA-B receptor agonist baclofen, significantly increased the EGFP-positive/GnRH-positive cell ratio. These results suggest that GABA plays a role in stimulating GnRH gene expression through GABA-A receptors in embryonic GnRH neurons in late gestational stages.     

Labelling of rat endothelial cells with antibodies to vWF, RECA-1, PECAM-1, ICAM-1, OX-43 and ZO-1

Labelling with endothelium specific monoclonal antibodies, von Willebrand Factor (vWF), rat endothelial cell antigen-1 (RECA-1), platelet-endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), OX-43 and zonula occludentes-1 (ZO-1), was investigated in cryostat sections of vessels from rats of different ages using a confocal microscope. The results showed that labelling of the vWF was positive in endothelial cells from adult, fetal and different ages of embryonic rat. Labelling with RECA-1 was weakly positive in adult rat aorta and lung endothelial cells but not in embryonic yolk sac endothelial cells. Labelling using PECAM-1, ICAM-1 and OX-43 was negative in both adult and embryonic endothelial cells. ZO-1 showed positive but very weak reactivity in embryonic yolk sac endothelial cells. The expression of vWF on vessels from adult and 19.5-day fetal tissues was strongly positive. However, the expression of vWF in embryonic endothelial cells was dependent on the gestational age. While the 11.5-day yolk sac vessels stained weakly, staining gradually increased in 13.5-, 15.5- and 17.5-day-old yolk sac vessels. The results suggest that vWF is a reliable endothelial cell marker in rat vascular endothelial cells, including both fetal and embryonic stages.     

Full-term development of offspring using round spermatids produced ectopically from fetal male germ cells

The continuous production of mammalian sperm is maintained by the proliferation and differentiation of spermatogonial stem cells, which originate from primordial germ cells in the early embryo. Previously, we reported that the transplantation of fetal male gonadal tissue into the recipient testis was effective obtaining functional sperm. This transplantation technique is a promising new approach for the preservation of testicular function in a mutant animal with embryonic lethality. In the present study, we examined whether spermatogenesis from fetal male germ cells is induced under ectopic conditions in male and female recipients. Nine to 10 weeks after the transplantation of male gonads prepared from embryos at 12.5 or 16.5 days post gestation, male germ cell differentiation occurred under the skin of male and female recipient nude mice. Histological analyses revealed that grafted gonads contained haploid germ cells such as round or elongated spermatids. Furthermore, we succeeded in obtaining normal progeny by injecting the ectopically produced round spermatids into the cytoplasm of oocytes, even when the male germ cells had been generated in female recipients. These results indicate that the transplantation of fetal male gonads under the skin of recipient mice is a useful technique for obtaining functional male gametes.     

Time-course changes in 5-fluorouracil-induced neural progenitor cell damages in the developing rat brain

5-Fluorouracil (5-Fu) is a DNA-damaging agent and teratogenic in rodents. This study aimed to investigate its influence on neural progenitor cells (NPCs) in the developing fetal rat brain. Dams were intraperitoneally injected with 5-Fu (50 mg/kg b.w.) on gestation day 13 and its effects on fetal NPCs were observed from 3 to 72 hours after treatment (HAT), via periodic examination at six intervals. In NPCs of the fetal brain, the p53-labeling index (LI%) was markedly elevated at 3 HAT. Pyknosis and cleaved caspase-3-LI% also increased at 3 HAT, reaching peak values at 9 and 12 HAT. These parallel changes suggested the induction of apoptosis through a p53-mediated pathway. Pyknotic NPCs were distributed across the ventricular zone (VZ) of the telencephalic wall until 12 HAT, and became localized in the medial and dorsal layers at 12 and 48 HAT. Significant decreases in the numbers of mitotic NPCs and BrdU-LI% were noted from 3 HAT and 24 HAT, respectively. BrdU-positive NPCs were located in the ventral and middle layer at 24 and 48 HAT. p21-positive cells were detected at 12 and 24 HAT. The present results demonstrated that p53-mediated apoptosis was induced in all phases of the cell cycle of the NPCs in the early stage after 5-FU treatment. Furthermore, apoptosis of NPCs and suppression of cell proliferative activity are the events that take place in parallel leading to prominent reduction in the width of the telencephalic wall.     

Immunohistochemical identification and quantitative analysis of cytoplasmic Cu/Zn superoxide dismutase in mouse organogenesis

Cytoplasmic Cu/Zn superoxide dismutase (SOD1) is an antioxidant enzyme that converts superoxide to hydrogen peroxide in cells. Its spatial distribution matches that of superoxide production, allowing it to protect cells from oxidative stress. SOD1 deficiencies result in embryonic lethality and a wide range of pathologies in mice, but little is known about normal SOD1 protein expression in developing embryos. In this study, the expression pattern of SOD1 was investigated in post-implantation mouse embryos and extraembryonic tissues, including placenta, using Western blotting and immunohistochemical analyses. SOD1 was detected in embryos and extraembryonic tissues from embryonic day (ED) 8.5 to 18.5. The signal in embryos was observed at the lowest level on ED 9.5-11.5, and the highest level on ED 17.5-18.5, while levels remained constant in the surrounding extraembryonic tissues during all developmental stages examined. Immunohistochemical analysis of SOD1 expression on ED 13.5-18.5 revealed its ubiquitous distribution throughout developing organs. In particular, high levels of SOD1 expression were observed in the ependymal epithelium of the choroid plexus, ganglia, sensory cells of the olfactory and vestibulocochlear epithelia, blood cells and vessels, hepatocytes and hematopoietic cells of the liver, lymph nodes, osteogenic tissues, and skin. Thus, SOD1 is highly expressed at late stages of embryonic development in a cell- and tissue-specific manner, and can function as an important antioxidant enzyme during organogenesis in mouse embryos.     

Changes in intracellular and cell surface localization of Le(x) epitope during germ cell differentiation in fetal mice.

Changes in the intracellular and cell surface localization of Lewis x (Le(x)) determinants in germ cells during fetal development in mice were examined by light and electron microscopy. Light microscopically, in undifferentiated gonads on day 12 post coitum (p.c.), the anti-Le(x) monoclonal antibody (MAb) was specifically bound to the plasma membranes and to the cytoplasmic granule-like structures of germ cells. In the testes on day 13 p.c., most of the germ cells were enclosed within the testicular cords and showed an MAb-positive reaction which was restricted mainly to the cytoplasmic granule-like structures. The reaction on the plasma membranes almost disappeared. On the other hand, the ectopic germ cells still showed a positive reaction on their plasma membranes. In the ovaries on day 13 p.c., the germ cells also exhibited positive reactions both on the plasma membranes and on the granule-like structures. Immunoelectron microscopic observations agreed well with these light microscopic observations in such a way that both the plasma membranes and the "small dense bodies" (SDB) were positive in undifferentiated gonads on day 12 p.c. In the germ cells organized into the testicular cords, the reaction to anti-Le(x) MAb became restricted to the SDB. These results may indicate that such intracellular changes in Le(x) determinants during germ cell differentiation are associated with the enclosure of germ cells within the testicular cords.     

From Zygote to Implantation: Morphological and Molecular Dynamics during Embryo Development in the Pig

The increasing focus on the pig as a biomedical model calls for studies which investigate morphological and molecular mechanisms during initial embryonic development in this species. In the pig, the paternal genome is actively demethylated in the zygote, whereas the maternal genome remains methylated. The major genome activation occurs at the four-cell stage, when prominent ribosome-synthesizing nucleoli develop in the blastomeres, allowing for trophectoderm and inner cell mass (ICM) differentiation. Unlike in mice, the pluripotency gene OCT4 is initially expressed in both compartments. The ICM differentiates into epiblast and hypoblast approximately at the time of hatching from the zona pellucida, and subsequently the loss of the Rauber's layer results in an uncovered epiblast establishing the embryonic disc again in contrast to mice. This particular and protracted ICM/epiblast biology may contribute to the lack of success in culturing porcine embryonic stem cells. The embryonic disc subsequently becomes polarized by a posterior thickening, which includes ingression of the first extra-embryonic mesoderm. Thereafter, the primitive streak forms and gastrulation results in formation of the somatic germ layers and germline, i.e. the primordial germ cells. The latter remain pluripotent for a period and may be isolated and cultured as embryonic germ cells in vitro .     

Synergistic role of Igf2 and Dlk1 in fetal liver development and hematopoiesis in bi-maternal mice

Mouse bi-maternal embryos (BMEs) that contain two haploid sets of genomes from non-growing (ng) and fully-grown (fg) oocytes develop to embryonic day (E) 13.5. However, the ng/fg BMEs never develop beyond E13.5 because of repression of the paternally expressed imprinted genes, Igf2 and Dlk1. The present study was conducted to address the issue of whether fetal hematopoietic disorder is involved in the restricted development of BMEs. FACS analysis revealed that the livers of ng(wt)/fg BMEs contained increased numbers of immature c-kit(+)/ter119(-) hematopoietic cells, were while the numbers of mature c-kit(-)/ter119(+) hematopoietic cells were decreased. This finding was supported by histological observations. Quantitative gene expression analysis revealed that Igf2 and Dlk1 expression was repressed in the liver. To understand the role of paternally-methylated imprinted genes on chromosomes 7 and 12, particularly Igf2 and Dlk1, in fetal liver hematopoiesis, we constructed ng(Deltach7)/fg, ng(Deltach12)/fg and ng(DeltaDouble)/fg BMEs using ng oocytes harboring deletion of differentially methylated regions at distal chromosomes 7 and/or 12. The ng(Deltach7)/fg, ng(Deltach12)/fg and ng(DeltaDouble)/fg BMEs, respectively, express Igf2, Dlk1 and both, and these embryos developed to term with specific phenotypes; the ng(Deltach7)/fg and ng(Deltach12)/fg BMEs develop to term with severe growth retardation, and the ng(DeltaDouble)/fg BMEs can survive to become normal female adults. By inducing Igf2 and Dlk1 expression, the proportions of mature and immature hematopoietic cells in the livers of the ng(Deltach7)/fg, ng(Deltach12)/fg and ng(DeltaDouble)/fg BMEs were considerably restored, and particularly in the ng(DeltaDouble)/fg BMEs, hematopoiesis occurred normally with appropriate expressions of the related genes. These data suggest that inappropriate expression of Igf2 and Dlk1 is involved in impaired fetal hematopoiesis.