Stem cell and niche development in the postnatal rat testis

Adult tissue stem cells self-renew and differentiate in a way that exactly meets the biological demand of the dependent tissue. We evaluated spermatogonial stem cell (SSC) activity in the developing rat testis and the quality and accessibility of the stem cell niche in wild type, and two busulfan-treated models of rat pup recipient testes using an SSC transplantation technique as a functional assay. While our results revealed a 69-fold increase in stem cell activity during rat testis development from neonate to adult, only moderate changes in SSC concentration were observed, and stem cells from neonate, pup, and adult donor testes produce spermatogenic colonies of similar size. Analysis of the stem cell niche in recipient rat testes demonstrated that pup testes support high levels of donor stem cell engraftment when endogenous germ cells are removed or compromised by busulfan treatment. Fertility was established when rat pup donor testis cells were transplanted into fetal- or pup-busulfan-treated recipient rat pup testes, and the donor genotype was transmitted to subsequent generations. These results provide insight into stem cell/niche interactions in the rat testis and demonstrate that techniques originally developed in mice can be extended to other species for regenerative medicine and germline modification.     

Morphological changes in the rat sertoli cell induced by the microtubule poison carbendazim

Early morphological changes in the rat Sertoli cell induced by the fungicide carbendazim (methyl-2-benzimidazole carbamate; MBC), a metabolite of benomyl, were examined. Adult rats were treated with single doses of MBC (400mg/kg) or vehicle and examined by light and electron microscopy at 3 hr post-treatment. Sloughing of elongating spermatid clusters was observed in all stages of spermatogenesis, except for Stages III–V. Cleavage occurred near the apical region of the seminiferous epithelium where cytoplasmic processes of the Sertoli cell surround the heads of elongating spermatids. The cleaved cytoplasm remained attached to the sloughed spermatids and ectoplasmic specializations remained undamaged. Intact microtubules were observed in the apical Sertoli cell cytoplasm (including sloughed tissues) but were decreased in the body region, where aggregates of mitochondria were found. Cytoplasm near the cleavage site exhibited rarefaction, which was associated with swollen cisternae of endoplasmic reticulum. It appears that the mechanism of germ cell sloughing induced by MBC treatment involves the disruption of microtubules in the body region of the Sertoli cell, the retraction of cytoplasmic organelles and the swelling of endoplasmic reticulum.     

Trout sertoli and leydig cells: Isolation,separation, and culture

Trout testes at various stages of maturation were dissociated by perfusion at 12°C with collagenase plus pronase and then with collagenase alone, followed by slight shaking overnight in 1% bovine albumin. This step provided a suspension of isolated somatic and germ cells, clusters of interstitial cells, and either intact spermatogenetic cysts (meiotic testes) or clusters of Sertoli cells (other testes). Most of the spermatozoa were removed from the testis cell suspension by centrifugation in Percoll (density 1.065 g/ml). Sertoli and Leydig cells were prepared by a two-step separation method: (1) the testis cell suspension was separated by sedimentation at unit gravity into “isolated cell” and “cell cluster” populations; (2) these populations were fractionated by isopyknic centrifugation in Percoll gradients. In terms of somatic cell composition, a nearly pure Sertoli cell (clusters) population was obtained between 1.017 and 1.033 g/ml and a Leydig cell (clusters) enriched population of between 1.033 and 1.048 g/ml (testes resuming spermatogenesis) or 1.048 and 1.062 g/ml (other testes). These various cell populations were cultured in modified Leibovitz L15 medium for 10–15 days. When seeded, the Sertoli cells had a normal ultrastructure that remained unchanged for at least 10 days, and the steroidogenic activity of Leydig cells could be stimulated by salmon gonadotropin. Leydig cells remained 3β-HSD positive and produced progesterone and 17α, 20β-OH progesterone for at least 11 days. This study points out that viable and differentiated trout somatic testicular cells can be prepared and cultured for several days.     

β-hexosaminidase immunolocalization and α- and β-subunit gene expression in the rat testis and epididymis

β-hexosaminidase is an essential lysosomal enzyme whose absence in man results in a group of disorders, the G_M2 gangliosidoses. β-hexosaminidase activity is many times higher in the epididymis than in other tissues, is present in sperm, and is postulated to be required for mammalian fertilization. To better understand which cells are responsible for β-hexosaminidase expression and how it is regulated in the male reproductive system, we quantitated the mRNA expression of the α- and β-subunits of β-hexosaminidase and carried out immunocytochemical localization studies of the enzyme in the rat testis and epididymis. β-hexosaminidase α-subunit mRNA was abundant and differentially expressed in the adult rat testis and epididymis, at 13- and 2-fold brain levels, respectively. In contrast, β-subunit mRNA levels in the testis and epididymis were 0.3- and 5-fold brain levels. During testis development from 7–91 postnatal days of age, testis levels of α-subunit mRNA increased 10-fold and coincided with the appearance of spermatocytes and spermatids in the epithelium; in contrast, β-subunit mRNA was expressed at low levels throughout testis development. In isolated male germ cells, β-hexosaminidase α-subunit expression was most abundant in haploid round spermatids, whereas the β-subunit mRNA was not detected in germ cells. Within the epididymis both α- and β-subunit mRNA concentrations were highest in the corpus, with 1.5-fold and 9-fold initial segment values, respectively. Light microscopic immunocytochemistry revealed that β-hexosaminidase was localized to Sertoli cells and interstitial macrophages in the testis. In the epididymis, β-hexosaminidase staining was most intense in narrow cells in the initial segment, principal cells in the caput, and proximal corpus, and clear cells throughout the duct. Electron microscopic immunocytochemistry revealed that β-hexosaminidase was predominantly present in lysosomes in Sertoli and epididymal cells. The cellular and regional specificity of β-hexosaminidase immunolocalization suggest an important role for the enzyme in testicular and epididymal functions. Mol. Reprod. Dev. 46:227–242, 1997. © 1997 Wiley-Liss, Inc.     

Glycogen content during the postnatal differentiation of the Leydig cell in the mouse testis

Summary The concentration and distribution of glycogen in relation to postnatal differentiation of the mouse Leydig cell are studied by biochemical and ultrastructural methods. Glycogen decreases to less than one third in the first twelve days after birth. This decrease is accompanied by modifications of its distribution in the cytoplasm. In the newborn it is abundant and arranged in clusters of beta particles; in the mature Leydig cell, glycogen is found scattered in extremely low concentration interspersed among elements of the endoplasmic reticulum.The role of glycogen during Leydig cell differentiation can be interpreted as a source of energy and/or as a source of building material in the biogenesis of membranous components.This work was supported by Grant M 63,121 from the Population Council, U.S.A.Fellow Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina.     

Expression of a novel recombinant dual human stem cell factor in insect cells

Stem cell factor (SCF) is a hematopoietic cytokine that promotes the survival, proliferation, and differentiation of hematopoietic cells. A dual human stem cell factor (dhSCF) cDNA was constructed, which consisted of a full-length human stem cell factor cDNA plus a truncated hSCF cDNA (1-145aa), linked by a peptide (GGGGSGGGGSGG) coding region. The dhSCF gene was cloned into baculovirus transfer vector pAcSecG2T under the control of polyhedrin promoter. The Sf9 cells infected with the recombinant virus expressed rdhSCF up to 6000 U/10(6) cell in flask and 8300 U/10(6) cell in spinner flask. The rdhSCF was purified by two-step chromatography. The molecular mass of rdhSCF was examined by western blotting and HPLC analysis. The specific activity of rdhSCF was up to 3.1x10(6) U/mg, about 8.7 times as high as that of monomer rhSCF from Escherichia coli.     

小鼠睾丸支持细胞体外培养特性

支持细胞是睾丸的重要组成部分,其主要功能是为生精细胞提供适宜的生长环境。从幼鼠睾丸中分离得到支持细胞,并通过苏木精-伊红和Fas-L免疫组化染色对分离得到的细胞进行了鉴定。通过对原代小鼠睾丸支持细胞的贴壁、生长和培养液中葡萄糖、谷氨酰胺、氨基酸等营养底物及其副产物乳酸、铵根离子等的代谢以及培养液渗透压和pH的研究,发现支持细胞的贴壁时间主要集中在接种后2～4h;当培养液中氨根离子浓度高于2.3mmol/L,渗透压高于326mosm/kg,pH≤6.8时支持细胞生长进入衰亡期;在氨基酸代谢方面,发现培养过程中丙氨酸和谷氨酸浓度迅速增加,缬氨酸、亮氨酸和异亮氨酸浓度略有降低,丝氨酸、精氨酸和甘氨酸浓度基本保持不变。因此培养液中铵根离子浓度的过量积累、渗透压和pH的异常和贴壁面积不足是限制支持细胞静态生长的主要因素。研究结果为支持细胞大规模培养及工艺优化奠定了基础。     

Apoptosis and cell removal in the cryptorchid rat testis

In order to determine that apoptosis is responsible for large-scale germ cell elimination, we analyzed cells from cryptorchid testes both in histological sections and among those isolated in vitro. Apoptotic testicular cells during 3 to 7 days were only 8 to 30%, reaching a maximum of 80% by the end of 15 days of cryptorchidism. A similar trend was also observed with the number of dead cells. The process of large-scale germ cell removal in the initial stages was facilitated by the formation of multinucleated giant cells, which stained negative for apoptosis. Increase in oxidative stress and decrease in intratesticular testosterone was also observed. The above findings indicate that large-scale germ cell removal, at least during initial stages of cryptorchidism is not solely as a result of apoptosis. Declined intra testicular testosterone, elevated temperature and high oxidative stress following cryptorchidism probably affect cell viability and trigger a fast pace cell removal through giant cell formation.     

Spermatogenesis in the golden hamster: the role of c-kit.

c-kit is related to the family of transmembrane tyrosine kinase receptors. Mutations in genes for either c-kit or its ligand, Steel factor, result in infertility, but the role of c-kit/SCF system in spermatogenesis is not well understood. In this study Western blot analysis together with confocal microscopy were used to follow c-kit expression in hamsters during the first spermatogenic wave in mature animals and in old age. Three antibodies raised against different domains of c-kit were tested on Western Blot. Confocal microscopy was performed after incubation of fixed seminiferous tubules with tested antibodies followed by binding of FITC-labeled secondary antibody. Longitudinal sections of seminiferous tubule were observed by confocal microscopy to determine in which stages of spermatogenesis and in which cell types c-kit was found. C-kit bands of 80,140, and 150 kDa were observed on Western blot, indicating that c-kit is a name related to several proteins sharing some common domains. Only the band of 150 kDa correlated with positive staining of c-kit in tubules using confocal microscopy. We term this protein c-kit150T (150 kDa, testis). We demonstrated that c-kit150T appeared in differentiating hamster spermatogonia at stages VII-VIII of adult spermatogenesis and at day 13-14 during the first spermatogenic wave. It remained attached to the cell until late pachytene. This suggests that c-kit may play a role in preparing the germinal cells to enter meiosis. In order to evaluate the effect of aging on the number of germ cells, B2 spermatogonia/Sertoli cell ratio was calculated in the group of young animals (5-7 months) compared to this ratio in older ones (20-26 months). A significant decrease (P < 0.01) in the number of B2 spermatogonia in the group of old hamsters as compared to young ones was seen. The calculated value for the B2 spermatogonia/Sertoli cell ratio was 5.6 +/- 0.7 in young animals and 3.8 +/- 1.2 in the 20-26 months ones. In addition, decrease in the intensity of staining for c-kit was detected in the old hamsters. These may be the reasons for subfertility in old age and in other cases of testicular disorders.     

Quantitative analysis of male germline stem cell differentiation reveals a role for the p53-mTORC1 pathway in spermatogonial maintenance

p53 protects cells from DNA damage by inducing cell-cycle arrest upon encountering genomic stress. Among other pathways, p53 elicits such an effect by inhibiting mammalian target of rapamycin complex 1 (mTORC1), the master regulator of cell proliferation and growth. Although recent studies have indicated roles for both p53 and mTORC1 in stem cell maintenance, it remains unclear whether the p53-mTORC1 pathway is conserved to mediate this process under normal physiological conditions. Spermatogenesis is a classic stem cell-dependent process in which undifferentiated spermatogonia undergo self-renewal and differentiation to maintain the lifelong production of spermatozoa. To better understand this process, we have developed a novel flow cytometry (FACS)-based approach that isolates spermatogonia at consecutive differentiation stages. By using this as a tool, we show that genetic loss of p53 augments mTORC1 activity during early spermatogonial differentiation. Functionally, loss of p53 drives spermatogonia out of the undifferentiated state and causes a consistent expansion of early differentiating spermatogonia until the stage of preleptotene (premeiotic) spermatocyte. The frequency of early meiotic spermatocytes is, however, dramatically decreased. Thus, these data suggest that p53-mTORC1 pathway plays a critical role in maintaining the homeostasis of early spermatogonial differentiation. Moreover, our FACS approach could be a valuable tool in understanding spermatogonial differentiation.     

Immunolocalization of proliferating cell nuclear antigen (PCNA) in cynomolgus monkey (Macaca fascicularis) testes during postnatal development

The age-related distribution of proliferating cell nuclear antigen (PCNA) in the testes of cynomolgus monkeys (Macaca fascicularis) during postnatal development was detected using light-microscopic immunohistochemistry. In neonatal testes, some PCNA-positive spermatogonia, Sertoli cells, peritubular cells, and Leydig cells were detected. In early infantile testes, only a few of these cell types were positive. In late infantile testes, the numbers of positive cells were greater than in the earlier developmental stages. In pubertal testes, the numbers of positive spermatogonia, spermatocytes, Sertoli cells, peritubular cells, and Leydig cells were considerably higher. In adult testes, a larger percentage of spermatogonia and spermatocytes was positive, and peritubular cells and Leydig cells were occasionally positive; secondary spermatocytes, spermatids, and Sertoli cells were not positive. We concluded that immunolocalization of PCNA can serve as a tool for studying proliferation status in developing testes of cynomolgus monkeys. A relatively low proliferative activity in early infantile testes and a remarkable increase of proliferative activity in pubertal testes correlate with the fluctuations of steroidogenic functions during postnatal development in cynomolgus monkeys.     

Expression of a novel recombinant stem cell factor/macrophage colony-stimulating factor fusion protein in baculovirus-infected insect cells

A novel human stem cell factor (SCF)/macrophage colony-stimulating factor (M-CSF) fusion protein gene was constructed, in which the coding regions of human SCF cDNA (1-165aa) and the truncated M-CSF cDNA (1-149aa) were connected by a linker sequence encoding a short peptide GGGGSGGGGSGG. The SCF/M-CSF gene was cloned into baculovirus transfer vector pVL1392 under the control of polyhedrin promoter and expressed in the Sf9 cells (Spodoptera frugiperda). SDS-PAGE and Western blot analysis showed that the purified fusion protein was a homodimer with a molecular weight about 84kDa under non-reducing conditions or a monomer about 42kDa under reducing conditions. The specific activity of rhSCF/M-CSF was 17 times as high as that of monomeric rhSCF to stimulate the proliferation of TF-1 cell. The results of macrophages colony-forming (CFU-M) assay performed with human bone marrow mononuclear cells demonstrated that rhSCF/M-CSF was more potent in promoting CFU-M than the equimolar of SCF, M-CSF or that of two cytokines mixture.     

Single-cell analysis of the developing human testis reveals somatic niche cell specification and fetal germline stem cell establishment

1. Download : Download high-res image (168KB)
2. Download : Download full-size image

     

Differentiation of the adult Leydig cell population in the postnatal testis

Five main cell types are present in the Leydig cell lineage, namely the mesenchymal precursor cells, progenitor cells, newly formed adult Leydig cells, immature Leydig cells, and mature Leydig cells. Peritubular mesenchymal cells are the precursors to Leydig cells at the onset of Leydig cell differentiation in the prepubertal rat as well as in the adult rat during repopulation of the testis interstitium after ethane dimethane sulfonate (EDS) treatment. Leydig cell differentiation cannot be viewed as a simple process with two distinct phases as previously reported, simply because precursor cell differentiation and Leydig cell mitosis occur concurrently. During development, mesenchymal and Leydig cell numbers increase linearly with an approximate ratio of 1:2, respectively. The onset of precursor cell differentiation into progenitor cells is independent of LH; however, LH is essential for the later stages in the Leydig cell lineage to induce cell proliferation, hypertrophy, and establish the full organelle complement required for the steroidogenic function. Testosterone and estrogen are inhibitory to the onset of precursor cell differentiation, and these hormones produced by the mature Leydig cells may be of importance to inhibit further differentiation of precursor cells to Leydig cells in the adult testis to maintain a constant number of Leydig cells. Once the progenitor cells are formed, androgens are essential for the progenitor cells to differentiate into mature adult Leydig cells. Although early studies have suggested that FSH is required for the differentiation of Leydig cells, more recent studies have shown that FSH is not required in this process. Anti-Müllerian hormone has been suggested as a negative regulator in Leydig cell differentiation, and this concept needs to be further explored to confirm its validity. Insulin-like growth factor I (IGF-I) induces proliferation of immature Leydig cells and is associated with the promotion of the maturation of the immature Leydig cells into mature adult Leydig cells. Transforming growth factor alpha (TGFalpha) is a mitogen for mesenchymal precursor cells. Moreover, both TGFalpha and TGFbeta (to a lesser extent than TGFalpha) stimulate mitosis in Leydig cells in the presence of LH (or hCG). Platelet-derived growth factor-A is an essential factor for the differentiation of adult Leydig cells; however, details of its participation are still not known. Some cytokines secreted by the testicular macrophages are mitogenic to Leydig cells. Moreover, retarded or absence of Leydig cell development has been observed in experimental models with impaired macrophage function. Thyroid hormone is critical to trigger the onset of mesenchymal precursor cell differentiation into Leydig progenitor cells, proliferation of mesenchymal precursors, acceleration of the differentiation of mesenchymal cells into Leydig cell progenitors, and enhance the proliferation of newly formed Leydig cells in the neonatal and EDS-treated adult rat testes.     

Effect of 17-alpha-ethynylestradiol on germ cell proliferation in organ and primary culture of medaka (Oryzias latipes) testis

Organ cultures and primary cell cultures of medaka (Oryzias latipes) testis were compared with respect to cell viability and cell proliferation. The analysis by fluorescence microscopy and flow cytometry showed that in both cultures, the cells remained viable for at least 1 day and cell proliferation could be analyzed reliably by BrdU incorporation. The proliferating cells were mostly spermatogonia located at the periphery of the testis in tissue sections. Both culture systems were used to study the effect of 17-alpha-ethynylestradiol on cell proliferation. The results obtained with organ and primary cultures were consistent: low concentrations (0.01 and 1 nm) of synthetic estrogen stimulated cell proliferation slightly, while a higher concentration (100 nm) had an inhibitory effect. Both culture methods are suitable for the analysis of substances that might interfere with germ cell proliferation or other functions in spermatogenesis.