Differentiation-related changes in the distribution of glycoconjugates in rat testis

Summary The distribution of glycoconjugates in differentiating rat testis was investigated by fluorescein labeled lectins during embryogenesis and postnatal development. Double immunofluorescence with rhodamine coupled laminin antibodies was used to delineate testicular cords from the interstitium in embryonic testes. Rat testis was found to be rich in various glycoconjugates, with distinct differentiation-related changes in their distribution. All types of germ cells contained carbohydrate rich compounds in their cytoplasm. Glycosylation in the embryonic testis was different from that in the adult rat. At an early stage of testicular differentiation, the labeling of germ cells and other testicular cells was almost identical. The lectin binding patterns of embryonic germ cells and somatic cells were related to the developmental age of the animal, with a graded disappearance of galactose containing glycoconjugates in embryonal spermatogonia. Spermatogenic cell differentiation was characterized by striking changes in lectin binding patterns of germ cells, particularly in the acrosomes of developing spermatids, in relation to their functional activation and the emergence of adult type of glycosylation during the postnatal maturation of the testis. As the knowledge of regular glycosylation throughout tissue differentiation is of significance for the analysis of aberrant glycosylations occurring in pathologic disorders, our findings suggest the usefulness of lectin histochemistry for the studies on germ cell differentiation.     

Stage-specific expression of three cell surface carbohydrate antigens during murine spermatogenesis detected with monoclonal antibodies

We have identified three germ cell surface carbohydrate antigens that exhibit a common, stage-specific pattern of expression during spermatogenesis in the mouse. IgM-class monoclonal antibodies designated "J1," "C6," and "A5" were absorbed by adult testis, but not by any adult somatic tissue tested. In indirect immunofluorescence assays using collagenase-dissociated prepuberal and adult testicular cells, these antibodies labeled the surfaces of early and late pachytene spermatocytes and round spermatids. Gonocytes from fetal and neonatal testes were not labeled. In paraffin sections of prepuberal and adult testes, sialidase treatment exposed antigens recognized by antibodies C6 and A5 on preleptotene, leptotene, and zygotene spermatocytes located near the perimeter of seminiferous tubules. The determinants recognized by antibodies J1, C6, and A5 were characterized partially using a sugar hapten inhibition assay. The binding of J1 to adult testicular cells was inhibited specifically by N-acetylglucosamine and the binding of both C6 and A5 was inhibited by N-acetyllactosamine. The glycoconjugates recognized by J1, C6, and A5 eluted from gel filtration columns with an apparent molecular weight greater than 1 X 10(6) and were sensitive to endo-beta-galactosidase (keratanase) treatment. The apparent high molecular weight of these glycoconjugates was confirmed by immunolabeling Western blots of testis extracts separated by SDS-polyacrylamide gel electrophoresis. The results suggest that polylactosamine (keratan) glycoconjugates of high molecular weight are associated with the plasma membranes of meiotic and haploid male germ cells. The effects of sialidase on antibody labeling patterns suggest that changes in cell surface sialylation accompany the transition of early meiotic germ cells to pachytene spermatocytes during spermatogenesis.     

Distribution of terminal sugar residues in the testis of the spotted ray Torpedo marmorata

Lectins represent a class of proteins/glycoproteins binding specifically to terminal sugar residues. The present investigation aims to identify lectin-binding sites in testis of Torpedo marmorata. Using a panel of lectins coupled with fluoresceine isothiocyanate, we demonstrated that germ and somatic cells present in Torpedo testis contain glycoconjugates, whose distribution at the level of the surface, the cytoplasm and the nucleus changes during germ cell differentiation. Moreover our observations demonstrate that the germ cells undergoing apoptosis (Prisco et al., 2003a: Mol Reprod Dev 64:341-348) overexpress a residual sugar recognised by WFA lectin that can be considered a specific marker for apoptotic germ cells. Finally, our results indicate that there is a progressive increase in glycosilation during spermatogenesis, especially at the level of the acrosome in the spermatocyte-spermatid step, and that Leydig cells are differently stained in relation to the spermatogenetic cycle.     

Changes in lectin binding pattern of gonads of developing mice

Changes in lectin binding of developing fetal mouse testes and ovaries were examined by light and electron microscopy, with much attention paid particularly to those in carbohydrates of germ cells. Characteristic binding patterns were observed with three lectins (BPA, GS-I, and GS-II) in the germ cells and the somatic cells during the process of testicular and ovarian development. GS-I and BPA, which showed similar binding patterns, preferentially bound to the plasma membrane and small dense bodies (SDB) of germ cells in both testes and ovaries during the 12th to 14th day post coitum (p.c.). In the fetal testes on day 16 p.c., the reaction with both GS-I and BPA completely disappeared. While, in the ovaries, a weak reaction with these lectins was retained as it was in germ cells until the 16th day p.c. The reaction with GS-II was restricted to Sertoli cells in the fetal testes during the 12th to 14th day p.c., and thereafter disappeared on day 16 p.c. The distribution of GS-II binding sites was in agreement with that of the glycogen granules. No positive staining with GS-II was seen in the ovaries throughout their development. These results indicate that certain glycoconjugates containing D-galactose and N-acetyl-D-galactosamine residues are expressed on the cell surface and in the SDB of germ cells during the period of the 12th to 14th day p.c., and that striking changes in function as well as in structure may take place in both germ cells and somatic cells during the 14th to 16th day p.c. in association with testicular and ovarian development.     

Differential expression of N-Myc downstream regulated gene 2 (NDRG2) in the rat testis during postnatal development

N-Myc downstream regulated gene 2 (NDRG2) is expressed in the testis of adult animals and is involved in cell differentiation and development. However, little is known about the expression pattern of NDRG2 in the testis during postnatal development. Here, we show that NDRG2 is consistently expressed in Leydig cells in the rat testis during postnatal development. However, its expression has also been detected at a high frequency in spermatogenic cells of the seminiferous tubules in young rats but at a much lower frequency in adult rats. Furthermore, high levels of NDRG2 expression have been found in methoxyacetic-acid-induced apoptotic germ cells, particularly at stages X–XIII of the seminiferous epithelium cycle of adult rats. Interestingly, high levels of NDRG2 expression have also been observed in spontaneously apoptotic germ cells in the seminiferous tubules of young and adult rats. Thus, the expression of NDRG2 in germ cells seems to alter during spermatogenesis. These findings suggest that NDRG2 regulates testicular development and spermatogenesis in rats and is involved in the physiological and pathological apoptosis of germ cells. Wu-Gang Hou, Yong Zhao, and Lan Shen contributed equally to this study. This study was supported by the Natural Science Foundation of China (2006: no. 30600340; 2007: no. 30771138; 2008: no. 30871309).     

Changes in lectin binding pattern of gonalds of developing mice

Summary Changes in lectin binding of developing fetal mouse testes and ovaries were examined by light and electron microscopy, with much attention paid particularly to those in carbohydrates of germ cells. Characteristic binding patterns were observed with three lectins (BPA, GS-I, and GS-II) in the germ cells and the somatic cells during the process of testicular and ovarian development. GS-I and BPA, which showed similar binding patterns, preferentially bound to the plasma membrane and small dense bodies (SDB) of germ cells in both testes and ovaries during the 12th to 14th day post coitum (p.c.). In the fetal testes on day 16 p.c., the reaction with both GS-I and BPA completely disappeared. While, in the ovaries, a weak reaction with these lectins was retained as it was in germ cells until the 16th day p.c. The reaction with GS-II was restricted to Sertoli cells in the fetal testes during the 12th to 14th day p.c., and thereafter disappeared on day 16 p.c. The distribution of GS-II binding sites was in agreement with that of the glycogen granules. No positive staining with GS-II was seen in the ovaries throughout their development. These results indicate that certain glycoconjugates containing d-galactose and N-acetyl-d-galactosamine residues are expressed on the cell surface and in the SDB of germ cells during the period of the 12th to 14th day p.c., and that striking changes in function as well as in structure may take place in both germ cells and somatic cells during the 14th to 16th day p.c. in association with testicular and ovarjan development.     

The Capacity of Testicular Cells of the Postnatal Rat to Reorganize into Histotypic Structures

Cell reorganization experiments in vitro were performed with dissociated rat testes at different ages of postnatal development namely, newborn, 8–10, 18–25, 35–40, and 90 days. Only newborn and juvenile rat testicular cells reassociated into testicular-like organization in rotation culture. Puberal and adult rat testicular cells show morphogenetic organization when they were deprived of germ cells by busulphan pretreatment. A factor present in testicular tissue of puberal and adult rats inhibits reorganization. The inhibitor is confined to the spermatic cell fraction in the testis.     

Immunolocalization of albumin and transferrin in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis

The localization of albumin and transferrin was examined immunohistochemically in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis. These proteins appeared as early as the 13th day of gestation in migrating primordial germ cells before Sertoli cell differentiation. In the fetal testis, strong immunoreactivity was only detected in the gonocytes. In the prepubertal testis, spermatogonia, primary spermatocytes, and some Sertoli cells accumulate albumin and transferrin. At puberty, different patterns of immunostaining of the germ cells were observed at the various stages of the cycle of the seminiferous epithelium. Diplotene spermatocytes at stage XIII, spermatocytes in division at stage XIV, and round spermatids at stages IV–VIII showed maximal staining. Labeling was evident in the cytoplasm of adult Sertoli cells. Albumin and transferrin staining patterns paralleled each other during ontogenesis.     

Spermatogonial depletion in adult Pin1-deficient mice

Spermatogonia in the mouse testis arise from early postnatal gonocytes that are derived from primordial germ cells (PGCs) during embryonic development. The proliferation, self-renewal, and differentiation of spermatogonial stem cells provide the basis for the continuing integrity of spermatogenesis. We previously reported that Pin1-deficient embryos had a profoundly reduced number of PGCs and that Pin1 was critical to ensure appropriate proliferation of PGCs. The current investigation aimed to elucidate the function of Pin1 in postnatal germ cell development by analyzing spermatogenesis in adult Pin1-/- mice. Although Pin1 was ubiquitously expressed in the adult testis, we found it to be most highly expressed in spermatogonia and Sertoli cells. Correspondingly, we show here that Pin1 plays an essential role in maintaining spermatogonia in the adult testis. Germ cells in postnatal Pin1-/- testis were able to initiate and complete spermatogenesis, culminated by production of mature spermatozoa. However, there was a progressive and age-dependent degeneration of the spermatogenic cells in Pin1-/- testis that led to complete germ cell loss by 14 mo of age. This depletion of germ cells was not due to increased cell apoptosis. Rather, detailed analysis of the seminiferous tubules using a germ cell-specific marker revealed that depletion of spermatogonia was the first step in the degenerative process and led to disruption of spermatogenesis, which resulted in eventual tubule degeneration. These results reveal that the presence of Pin1 is required to regulate proliferation and/or cell fate of undifferentiated spermatogonia in the adult mouse testis.     

Lectin receptors in the salivary glands of the rat during postnatal ontogenesis

Distribution of lectin-binding sites in rat submandibular and sublingual salivary glands during postnatal development has been investigated. Lectin preparations include con A, lentin lectin, castor beans agglutinin, peanut, soybean and Sophora japonica agglutinins, wheat germ agglutinin and lectin from the bark of Laburnum anagyroides. The direct and indirect peroxidase techniques are used. According to the similarities of histochemical patterns, all lectins are divided into four groups. Besides the general patterns of lectin binding sites, some details are noted. Lectins of peanut and Sophora japonica possess an extremely high affinity to mast cells, con A, lens lectin, castor beans and wheat germ agglutinins--to serous demilunes cells. Laburnum lectin--to salivary ducts epithelia in adult rat salivary glands. Lentin lectin, con A and Laburnum lectin preferentially stain cells with specific granularity in granular ducts at early stages of postnatal development. Considering the character of staining, we propose for further histochemical investigations of the salivary glands lentin lectin, peanut agglutinin, wheat germ agglutinin and Laburum anagyroides lectin.     

Testis cell-specific proteins in testosterone-treated rat ovaries

Abstract. Two-dimensional gel electrophoresis of rat gonads in various developmental stages (embryonic days 16, 17, 21 postnatal day 5) reveals an increasing number of polypeptide spots; 0.6%-2% of the polypeptides are gonad-specific and increase from embryonic day 16 onwards. Exposure of newborn rat ovaries to testosterone for 5 days results in the appearance of eight polypeptides. These polypeptides are absent in control ovaries but present in the testis from embryonic day 16 or 17 onwards. Three do not appear in the ovary at any developmental stage. These findings indicate that testosterone plays a physiologic role in normal testicular differentiation. After long-term testosterone treatment, ovaries are depleted of germ cells. This might explain the degeneration of oocytes in the abnormal environment of a testis (e.g., in XX true hermaphrodites).     

Changes in the testis interstitium of Sprague Dawley rats from birth to sexual maturity

Changes in the rat testis interstitium from birth to adulthood were studied using Sprague Dawley rats of 1, 7, 14, 21, 28, 40, 60, and 90 days of age. Our objectives were 1) to understand the fate of the fetal Leydig cells (FLC) in the postnatal rat testis, 2) to determine the volume changes in testicular interstitial components and testicular steroidogenic capacity in vitro with age, 3) to differentially quantify FLC, adult Leydig cells (ALC), and different connective tissue cell types by number and average volume, and 4) to investigate the relationship between mesenchymal and ALC numbers during testicular development. FLC were present in rat testes from birth to 90 days, and they were the only steroidogenic cells in the testis interstitium at Days 1 and 7. Except for FLC, all other interstitial cell numbers and volumes increased from birth to 90 days. The average volume of an FLC and the absolute volume of FLC per testis were similar at all ages except at Day 21, when lower values were observed for both parameters. FLC number per testis remained constant from birth through 90 days. The observations suggested that the significance of FLC in the neonatal-prepubertal rat testis is to produce testosterone to activate the hypothalamo-hypophyseal-testicular axis for the continued development of the male reproductive system. ALC were the abundant Leydig cell type by number and absolute volume per testis from Day 14 onwards. The absolute numbers of ALC and mesenchymal cells per testis increased linearly from birth to 90 days, with a slope ratio of 2:1, respectively, indicating that the rate of production of Leydig cells is 2-fold greater than that of mesenchymal cells in the postnatal rat testis through 90 days. In addition, this study showed that the mesenchymal cells are an active cell population during testis development and that their numbers do not decrease but increase with Leydig cell differentiation and testicular growth up to sexual maturity (90 days).     

Cell type-autonomous and non-autonomous requirements for Dmrt1 in postnatal testis differentiation

Genes containing the DM domain, a conserved DNA binding motif first found in Doublesex of Drosophila and mab-3 of Caenorhabditis elegans, regulate sexual differentiation in multiple phyla. The DM domain gene Dmrt1 is essential for testicular differentiation in vertebrates. In the mouse, Dmrt1 is expressed in pre-meiotic germ cells and in Sertoli cells, which provide essential support for spermatogenesis. Dmrt1 null mutant mice have severely dysgenic testes in which Sertoli cells and germ cells both fail to differentiate properly after birth. Here we use conditional gene targeting to identify the functions of Dmrt1 in each cell type. We find that Dmrt1 is required in Sertoli cells for their postnatal differentiation, and for germ line maintenance and for meiotic progression. Dmrt1 is required in germ cells for their radial migration to the periphery of the seminiferous tubule where the spermatogenic niche will form, for mitotic reactivation and for survival beyond the first postnatal week. Thus Dmrt1 activity is required autonomously in the Sertoli and germ cell lineages, and Dmrt1 activity in Sertoli cells is also required non-autonomously to maintain the germ line. These results demonstrate that Dmrt1 plays multiple roles in controlling the remodeling and differentiation of the juvenile testis.     

Carbohydrate determinants of organs of the reproductive tract of the mouse according to the results of using lectins with different specificities

Histotopography of lectin receptor sites in adult mice ovary, oviduct, uterus, testis and epididymis has been investigated on light-optic level by means of lectin-peroxidase technique. Paraffin sections are treated with peanut agglutinin (PNA), soybean agglutinin (SBA), wheat germ agglutinin (WGA) and Laburnum anagyroides lectin (LAL), conjugated with horseradish peroxidase. Concanavalin A (Con A) receptor sites are visualized by indirect method. The usefulness of lectins for selective histochemic evaluation of definite organ structures is demonstrated. Zona pellucida, luteocytes, oviductal and uterine epitheliocytes are rich in receptor sites for all lectin used in the investigation. The most intense binding to zonae pellucidae glycoconjugates possess WGA and LAL, to luteocytes--PNA, SBA and LAL, to oviductal and uterine epitheliocytes--Con A and LAL. The preferential SBA binding to the acrosomal system and plasma membranes of spermatogenic cells is demonstrated. Changes in lectin-binding patterns during the maturation of intraovarian oocytes and spermatogenic cells are also studied. The perspectives of practical application of the results obtained, as well as trends in further lectin histochemistry investigations of the reproductive system are discussed.     

Differing lectin binding profiles among human embryonic stem cells and derivatives aid in the isolation of neural progenitor cells

Human embryonic stem cells (hESCs) and their differentiated progeny allow for investigation of important changes/events during normal embryonic development. Currently most of the research is focused on proteinacous changes occurring as a result of differentiation of stem cells and little is known about changes in cell surface glycosylation patterns. Identification of cell lineage specific glycans can help in understanding their role in maintenance, proliferation and differentiation. Furthermore, these glycans can serve as markers for isolation of homogenous populations of cells. Using a panel of eight biotinylated lectins, the glycan expression of hESCs, hESCs-derived human neural progenitors (hNP) cells, and hESCs-derived mesenchymal progenitor (hMP) cells was investigated. Our goal was to identify glycans that are unique for hNP cells and use the corresponding lectins for cell isolation. Flow cytometry and immunocytochemistry were used to determine expression and localization of glycans, respectively, in each cell type. These results show that the glycan expression changes upon differentiation of hESCs and is different for neural and mesenchymal lineage. For example, binding of PHA-L lectin is low in hESCs (14±4.4%) but significantly higher in differentiated hNP cells (99±0.4%) and hMP cells (90±3%). Three lectins: VVA, DBA and LTL have low binding in hESCs and hMP cells, but significantly higher binding in hNP cells. Finally, VVA lectin binding was used to isolate hNP cells from a mixed population of hESCs, hNP cells and hMP cells. This is the first report that compares glycan expression across these human stem cell lineages and identifies significant differences. Also, this is the first study that uses VVA lectin for isolation for human neural progenitor cells.     

Commitment of fetal male germ cells to spermatogonial stem cells during mouse embryonic development

The continuous production of mammalian sperm is maintained by the proliferation and differentiation of spermatogonial stem cells that originate from primordial germ cells (PGCs) in the early embryo. Although spermatogonial stem cells arise from PGCs, it is not clear whether fetal male germ cells function as spermatogonial stem cells able to produce functional sperm. In the present study, we examined the timing and mechanisms of the commitment of fetal germ cells to differentiate into spermatogonial stem cells by transplantation techniques. Transplantation of fetal germ cells into the seminiferous tubules of adult testis showed that donor germ cells, at 14.5 days postcoitum (dpc), were able to initiate spermatogenesis in the adult recipient seminiferous tubules, whereas no germ cell differentiation was observed in the transplantation of 12.5-dpc germ cells. These results indicate that the commitment of fetal germ cells to differentiate into spermatogonial stem cells initiates between embryonic days 12.5 and 14.5. Furthermore, the results suggest the importance of the interaction between germ cells and somatic cells in the determination of fetal germ cell differentiation into spermatogonial stem cells, as normal spermatogenesis was observed when a 12.5-dpc whole gonad was transplanted into adult recipient testis. In addition, sperm obtained from the 12.5- dpc male gonadal explant had the ability to develop normally if injected into the cytoplasm of oocytes, indicating that normal development of fetal germ cells in fetal gonadal explant occurred in the adult testicular environment.     

RNA binding protein Musashi1 is expressed in sertoli cells in the rat testis from fetal life to adulthood.

The Musashi1 (Msi1) gene identified in mouse is a member of a subfamily of RNA binding proteins that are highly conserved across species. Msi1 expression is highly enriched in proliferative cells within the developing central nervous system. Within the testis, proliferation and differentiation of germ cells takes place within the seminiferous epithelium, where these cells are supported physically and functionally by Sertoli cells that do not themselves proliferate following the onset of puberty. RNA binding proteins expressed in testicular germ cells are essential for normal fertility. Preliminary data suggested the mRNA for Msi1 was present in ovary; therefore, we used an Msi1-specific cRNA and monoclonal antibody to investigate whether Msi1 was expressed in the testis. Msi1 mRNA was expressed in rat testis from birth until adulthood; in situ hybridization revealed silver grains within the seminiferous epithelium. Immunohistochemical studies demonstrated that at all ages examined (from Fetal Day 14.5 until adulthood) Msi1 protein was expressed in Sertoli cells. In fetal and adult rat ovaries, Msi1 was detected in granulosa cells and their precursors. In Sertoli cells, protein was detected in both cytoplasmic and nuclear compartments; in adult testes, the immunointensity of the nuclear staining was stage dependent, with highest levels of expression in Sertoli cells at stages I-VI. In rat gonads, the RNA binding protein Msi1 is expressed in both proliferating and nonproliferating Sertoli and granulosa cells.