The murine seminiferous epithelial cycle is pre-figured in the Sertoli cells of the embryonic testis

The seminiferous epithelial cycle and spermatogenic wave are conserved features of vertebrate spermatogenic organisation that reflect the need for the rigorous maintenance of sperm production. Although the cycle and the wave of the adult seminiferous epithelium have been well characterised, particularly in rodent species, their developmental origins are unknown. We show that the Sertoli cells of the pre-pubertal mouse, including those of the germ cell-deficient XXSxra mutant, exhibit coordinated, cyclical patterns of gene expression, presaging the situation in the adult testis, where Sertoli cell function is coupled to the spermatogenic cycle. In the case of the galectin 1 gene (Lgals1), localised differential expression in the Sertoli cells can be traced back to neonatal and embryonic stages, making this the earliest known molecular marker of functional heterogeneity in mammalian testis cords. In addition, the timing of germ cell apoptosis in normal pre-pubertal testes is linked to the temporal cycle of the Sertoli cells. These data show that the cycle and wave of the murine seminiferous epithelium originate at a much earlier stage in development than was previously known, and that their maintenance in the early postnatal cords depends exclusively on the somatic cell lineages.     

Effects of diethylstilbestrol on the cytogenesis of prolactin cells in the pars distalis of the pituitary gland of the mouse

This study was carried out to examine the developmental stage when prolactin cells differentiate in mice and to examine the effects of diethylstilbestrol on the development of prolactin cells in the fetal and neonatal pituitary glands. A small number of immunoreactive prolactin cells appeared first on embryonic day 15 in control (injected with oil) pituitary glands, whereas they did not increase in number until postnatal day 2. In diethylstilbestrol-treated mice (5 mg/kg body weight, 24 h before killing), a small number of immunoreactive prolactin cells were detectable as early as embryonic day 14, but not on day 13. They increased in number on embryonic days 15 and 16, and decreased markedly on days 17 and 18, followed by a rapid increase after birth. This transient reduction in the response to diethylstilbestrol was partially restored by treatment with metyrapone, a specific inhibitor of corticosteroid production. These results suggest that in the mouse: (1) differentiation of prolactin cells occurs between embryonic days 13 and 14, (2) prolactin gene expression is suppressed in the nascent prolactin cells presumably due to the presence of high levels of estrogen-binding protein, alpha-fetoprotein, and (3) prolactin gene expression is also suppressed by elevation of circulating glucocorticoids during the perinatal period. The present results suggest that, in the mouse, at least a proportion of prolactin cells are not derived from growth hormone cells, because the diethylstilbestrol-induced prolactin cells appear earlier than growth hormone gene expression.     

Pituitary astrocytes from the neural lobe of rats

Summary Tissue culture preparations of adult and neonatal rat pituitary neural lobes were examined by use of cell-type specific immunohistochemical markers. Cultures obtained from explanted or dissociated adult tissue or explanted neonatal tissue produced cells immunoreactive for endothelial and fibroblast markers. In contrast, dissociated neonatal tissue produced, in addition, two distinct forms of astrocytic glial cells immunoreactive for glial fibrillary acidic protein, one of which was also immunoreactive for the ganglioside GD3.     

Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development

To demonstrate the cellular and subcellular localization of mouse vasa homologue protein during germ cell development, specific antibody was raised against the full-length MVH protein. The immunohistochemical analyses demonstrated that MVH protein was exclusively expressed in primordial germ cells just after their colonization of embryonic gonads and in germ cells undergoing gametogenic processes until the post-meiotic stage in both males and females. The co-culture of EG cells with gonadal somatic cells indicated inductive MVH expression caused by an intercellular interaction with gonadal somatic cells. In adult testis, MVH protein was localized in the cytoplasm of spermatogenic cells, including chromatoid bodies in spermatids, known to be a perinuclear nuage structure which includes polar granules that contain VASA protein in Drosophila.     

Immunohistochemical study of protein 4.1B in the normal and W/W(v) mouse seminiferous epithelium.

Cell-cell adhesion is crucial not only for mechanical adhesion but also for tissue morphogenesis. Protein 4.1B, a member of the protein 4.1 family named from an erythrocyte membrane protein, is a potential organizer of an adherens system. In adult mouse seminiferous tubules, protein 4.1B localized in the basal compartment, especially in the attaching region of spermatogonia and Sertoli cells. Protein 4.1B localization and appearance were not different in each spermatogenic stage. Developmentally, protein 4.1B was not detected at postnatal day 3 (P3), was diffusely localized at P15, and was found in the basal compartment during the third week. By double staining for protein 4.1B and F-actin, their localizations were shown to be different, indicating that protein 4.1B was localized in a region lower than the basal ectoplasmic specialization that formed the Sertoli-Sertoli junction. By electron microscopy, immunoreactive products were seen mainly on the membranes of Sertoli cells. In the W/W(v) mutant mouse, the seminiferous epithelium had few germ cells. Protein 4.1B and beta-catenin were not detected, although the basal ectoplasmic specialization was retained. These results indicate that protein 4.1B may be related to the adhesion between Sertoli cells and germ cells, especially the spermatogonium.     

Change of cyclin D2 mRNA expression during murine testis development detected by fragmented cDNA subtraction method

Using subtractive hybridization and polymerase chain reaction, we developed a differential cloning system, the fragmented cDNA subtraction method, that requires only small amounts of materials. The cloning system was used to isolate several cDNA fragments expressed more abundantly in the premeiotic day 3 post-natal mouse testis than in the adult mouse testis. The isolated cDNA fragments included cDNA encoding the murine cyclin D2. Northern blot and in situ hybridization analyses revealed that, during testis development, cyclin D2 expression was most abundant in the neonatal proliferating Sertoli cells. Those type A spermatogonia that were thought to divide mitotically also expressed cyclin D2 mRNA. Other spermatogenic cells, such as mitotically arrested gonocytes in neonatal testis and meiotically dividing germ cells in adult testis as well as adult Sertoli cells, were negative for the cyclin D2 signal. Adult W/W ^v mutant mice lacking germ cells expressed cyclin D2 mRNA in terminally differentiated Sertoli cells. Elimination of germ cells other than the undifferentiated type A spermatogonia by treating wild-type mice with an anti-c- kit monoclonal antibody did not result in the expression of cyclin D2 in Sertoli cells. These results demonstrate that there are lineage- and developmental-specific expression patterns of cyclin D2 mRNA during mouse testis development. At the same time, it is suggested that primitive type A spermatogonia affect the cyclin D2 expression of Sertoli cells.     

The presence of hepatocyte growth factor in the developing rat.

Hepatocyte growth factor (HGF), a heparin-binding polypeptide mitogen, stimulates DNA synthesis in adult rat and human hepatocytes and in several other cells of epithelial origin. Recently, it was determined that scatter factor (SF), a protein that has been shown to cause the dispersion and migration of epithelial cells in culture, is identical to HGF. Moreover, the receptor for HGF was identified as the product of the proto-oncogene, c-MET, a tyrosine kinase-containing transmembrane protein. c-MET expression has been reported in a variety of adult and embryonic mouse tissues. Similarly, we and others have demonstrated that HGF is expressed in various adult rat and human tissues. In the present study, the tissue distribution of HGF during rat development was determined by immunohistochemistry using an HGF-specific polyclonal antiserum. Between day 12 and day 19, immunoreactivity for HGF was present in various locations such as hematopoietic cells, somites, squamous epithelium of the esophagus and skin, periventricular germinal matrix of the brain, bronchial epithelium, renal collecting tubules and chondrocytes. After day 19, HGF immunoreactivity was also present in the pancreas, submaxillary glands and neural tissues. In addition to immunolocalizing HGF in tissue sections, bioreactive and immunoreactive HGF was extracted and purified from rat fetuses. Other studies demonstrated the presence of HGF and c-MET mRNA in total fetal rat, and in fetal and neonatal rat liver. Addition of purified HGF to fetal and neonatal rat liver cultures enriched for hepatocytes stimulated DNA synthesis up to six-fold over controls. These findings strongly suggest a pivotal role for this potent regulator of growth and development.     

Possible activation by the green tea amino acid theanine of mammalian target of rapamycin signaling in undifferentiated neural progenitor cells in vitro

We have shown marked promotion of both proliferation and neuronal differentiation in pluripotent P19 cells exposed to the green tea amino acid theanine, which is a good substrate for SLC38A1 responsible for glutamine transport. In this study, we evaluated the activity of the mammalian target of rapamycin (mTOR) kinase pathway, which participates in protein translation, cell growth and autophagy in a manner relevant to intracellular glutamine levels, in murine neural progenitor cells exposed to theanine. Exposure to theanine promoted the phosphorylation of mTOR and downstream proteins in neurospheres from embryonic mouse neocortex. Although stable overexpression of SLC38A1 similarly facilitated phosphorylation of mTOR-relevant proteins in undifferentiated P19 cells, theanine failed to additionally accelerate the increased phosphorylation in these stable transfectants. Theanine accelerated the formation of neurospheres from murine embryonic neocortex and adult hippocampus, along with facilitation of both 5-bromo-2’-deoxyuridine incorporation and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction in embryonic neurospheres. In embryonic neurospheres previously exposed to theanine, a significant increase was seen in the number of cells immunoreactive for a neuronal marker protein after spontaneous differentiation. These results suggest that theanine activates the mTOR signaling pathway for proliferation together with accelerated neurogenesis in murine undifferentiated neural progenitor cells.     

Immunocytochemical detection of orexin A in endocrine cells of the developing mouse gut.

Orexins are novel neuropeptides that were originally localized in neurons of the hypothalamus and neuronal fibers of the brain. Recently orexin A and its receptor have also been reported in neurons and endocrine cells of the gastrointestinal tract. Because no studies have been done at the embryonic period, we studied the appearance and distribution of orexin A during the development of mouse gastrointestinal tract using immunocytochemical methods. Immunoreactivity to orexin A was detected in neuroendocrine cells of the pyloric region of the stomach at gestational Day 14 and 1 day after in the small intestine. The numbers of immunoreactive cells progressively increased through development until the adult pattern was reached. Staining of reverse-face sections demonstrated that orexin A and serotonin co-localized in some endocrine cells of the mouse stomach and small intestine. These findings suggest that orexin A may be relevant in the growth and maturation of the gastrointestinal tract during intrauterine life.     

Differential distribution of immunoreactive angiotensinogen in the hind-brain and spinal cord of neonatal and adult rats

The present communication deals with the cytochemical localization of angiotensinogen (ATG) immunoactivity in the hind-brain and spinal cord of neonatal (1-day-old) and adult (3-month-old pregnant) female rats. In the neonatal hind-brain, the immunoreactive cells were more numerous than in that of adult rats. In the adult rat hind-brain, the number of ATG-positive cells was quite limited in each nucleus. Further, in some nuclei, only neurons or neuroglia were positive, while in others the immunoactivity was observed in both the components. Spinal cords of neonatal rats showed a few undifferentiated ATG-positive cells in the grey matter. Contrary to this, the spinal cord of adult animals contained numerous immunoreactive glial cells in the grey matter, fasciculus cuneatus and fasciculus gracilis. Immunoactivity in the neurons was localized in the Nissl bodies.     

Isoelectric forms of alpha-L-fucosidase in mouse teratocarcinoma-derived cell lines

The alpha-L-fucosidase isoenzyme pattern of mouse teratocarcinoma-derived cell lines was analyzed by isoelectric focusing and compared with the pattern of a mammary carcinoma as an example of a malignant somatic cell line. In addition, these isoenzyme patterns were compared with those of normal fetal and adult mouse tissues from an earlier study. In the normal early fetal and placental tissues as well as in embryonal carcinoma and yolk sac carcinoma cells the alpha-L-fucosidase activity is predominantly associated with basic forms of the enzyme. This embryonic pattern of alpha-L-fucosidase is characterized by one to three isoelectric forms of the enzyme with pI values ranging from 7 to 9.5 accounting for more than two-thirds of the total activity. In contrast, the mammary carcinoma pattern resembles adult somatic tissues and primarily expresses acidic enzymatic forms (which comprise approximately 80% of total activity). The somatic cell malignancies arising in retransplantable teratocarcinomas show varying isoenzyme patterns. Thus, a malignant fibrous histiocytoma expresses predominantly basic forms of the enzyme, whereas a leiomyosarcoma expresses approximately equal amounts of acidic and basic forms of the enzyme resembling in this respect late fetal or immature neonatal tissues. These findings show that the embryonal carcinoma and yolk sac carcinoma cells of the mouse express the embryonic isoenzyme pattern of alpha-L-fucosidase in contrast to malignant cells originating in somatic tissue, like mammary carcinoma, which express the adult pattern. Malignancies arising in somatic tissues of teratocarcinomas may retain the embryonic alpha-L-fucosidase phenotype or show a phenotype corresponding to late fetal or neonatal tissues in normal ontogeny.     

Myosin heavy chain composition of single cells from avian slow skeletal muscle is strongly correlated with velocity of shortening during development

We have determined the myosin heavy chain (MHC) composition (using a sensitive sodium dodecyl sulfate-polyacrylamide gel electrophoresis system) and the maximal velocity of shortening (Vmax) of single cells from neonatal and adult chicken anterior latissimus dorsi (ALD) muscles. In addition, the MHC, myosin light chain, and regulatory protein (i.e., troponin and tropomyosin subunits) compositions of bundles of ALD fibers were determined at late embryonic, neonatal, and adult ages. At young ages, there are two MHCs in ALD muscle, SM1 and SM2, with SM1 decreasing in relative amount with increasing age, as shown previously by others. The mean Vmax of single fibers also decreases from neonatal to adult ages. A strong quantitative correlation is demonstrated between the specific MHC composition and Vmax among individual cells of the ALD muscle at several ages. Since virtually no changes occur in the regulatory protein and myosin light chain compositions of the ALD muscle between late embryonic and adult ages, it appears that the MHC composition of an individual cell in this muscle is the primary determinant of the maximal shortening velocity. These results are the first to illustrate the functional significance of the developmental transition in myosin heavy chain composition of an avian slow skeletal muscle, consistent with our previous findings on mammalian muscle.     

Nestin expression is lost in ventricular fibroblasts during postnatal development of the rat heart and re-expressed in scar myofibroblasts

Studies have reported that the intermediate filament protein nestin was expressed in various non-stem/progenitor cells during development, downregulated during postnatal growth and re-expressed following injury. The present study tested the hypothesis that an analogous paradigm was prevalent for ventricular fibroblasts. In the neonatal rat heart, nestin protein levels were significantly higher than the adult heart and the isolation of cardiac cells revealed a selective expression in ventricular fibroblasts. In adult ventricular fibroblasts, nestin protein expression was markedly lower compared to neonatal ventricular fibroblasts. Following ischemic damage to the rat heart, nestin staining was detected in a subpopulation of scar myofibroblasts (37%) and the percentage of immunoreactive cells was greater than adult ventricular fibroblasts (7%) but significantly lower than neonatal ventricular fibroblasts (86%). Moreover, dissimilar rates of (3)H-thymidine uptake were observed among the fibroblast populations and may be related in part to the disparate percentage of nestin(+) cells. To assess the role of nestin in DNA synthesis, neonatal ventricular fibroblasts were infected with a lentivirus containing a shRNAmir directed against the intermediate filament protein. The partial depletion of nestin expression in neonatal ventricular fibroblasts significantly reduced basal DNA synthesis, in the absence of an apoptotic response. Thus, postnatal development of the rat heart was associated with a selective loss of nestin expression in ventricular fibroblasts and subsequent induction in a subpopulation of myofibroblasts following ischemic injury. The re-expression of nestin in scar myofibroblasts may represent an adaptive response to enhance their proliferative rate and accelerate the healing process.     

Characterization of an endothelial protein in the developing rabbit kidney

A new protein (EnPo 1 antigen) abundant on endothelial cells and glomerular podocytes has been characterized by means of the mouse monoclonal antibody EnPo 1. Following electrophoretical separation of rabbit kidney homogenates EnPo 1 recognized a protein with a molecular weight of 110 kDa and an isoelectric point of 5.9 in Western blots. Using immunohistological techniques, the EnPo 1 antigen has been localized in high concentrations on glomerular podocytes of different developmental stages. Furthermore, the EnPo 1 antigen was expressed on endothelial cells of all adult rabbit organs tested so far. Detailed analysis of neonatal rabbit kidney revealed the abundance of EnPo 1 antigen on both differentiated vessels as well as on immature endothelial cells and endothelium of the microvasculature. Thus, for the first time a marker for in situ investigations of angiogenic processes within the mammalian kidney is available. Analysis of kidney cryosections by confocal laser scan microscopy revealed a direct connection between mature and differentiating vessels in the outer kidney cortex. Furthermore, two differentially organized cell populations discriminated by their EnPo 1 binding pattern were localized in the embryonic renal cortex. Morphologically, these cells were not distinguishable from other mesenchymal cells.