Examination of basement membrane components associated with the bovine seminiferous tubule basal lamina.
ABSTRACT Immunohistology has been used to examine the distribution of certain components of the basement membrane (BM) associated with bovine spermatogonial germ cells that are located within the seminiferous tubules. Histology was performed on testis tissue from Brahman cattle (Bos indicus) of three different age groups: pre-pubescent (4-6 months), juvenile (8-10 months) and adult (18-24 months) animals. There were no major changes in the BM composition apparent between these three age groups, except for certain lectin staining. These data suggest that the predominant collagen type IV component may have an alpha3 and alpha4 composition, although other chains, including the alpha5 and alpha6 chains, were also present. Possibly the main laminin type present was laminin 121 (alpha1beta2gamma1), although other variants were also present. Both nidogen-1 and perlecan, which are normal BM components, were also found as part of the seminiferous tubule BM. Interstitial collagens, such as type I, III and VI collagens, were found in the peritubular space, but were not part of the BM itself, although type VI collagen was most visible in the peritubular zone adjacent to the tubules. Examination of the BM with a range of lectins gave strong staining for (glcNAc)(2) entities, weak positive staining for alpha-l-fuc, but little or no staining for alpha-galNAc and (glcNAc)(3) at all ages, whereas staining for alpha-gal, beta-gal(1-->3)galNAc and alpha-man showed developmental changes.
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ABSTRACT: The objective of the present study was to identify an efficient and practical enrichment method for bovine type A spermatogonia. Four different enrichment methods were compared: differential plating on laminin- or Datura stramonium agglutinin (DSA)-coated flasks, percoll-gradient isolation, magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). The isolated cells were characterised with Dolichos biflorus agglutinin (DBA) lectin staining for type A spermatogonia and vimentin-antibody staining for Sertoli cells. A 2 x 2 factorial design was used to investigate the enrichment efficiency on laminin and DSA. In the laminin-enrichment groups, 2 h incubation in plates coated with 20 microg mL(-1) laminin yielded a 3.3-fold increase in DBA-positive cells in the adherent fraction, while overnight incubation in flasks coated with 20 microg mL(-1) DSA produced a 3.6-fold increase in the non-adherent fraction. However, the greatest enrichment (5.3-fold) of DBA-positive cells was obtained after 2 h incubation in control flasks (coated with bovine serum albumin). Percoll-gradient centrifugation yielded a 3-fold increase in DBA-positive cells. MACS results showed a 3.5- to 5-fold enrichment while FACS produced a 4-fold increase in DBA-positive cells. It is concluded that differential plating is a better method of recovering large numbers of type A spermatogonia for germ cell transplantation, while MACS or FACS can provide highly enriched viable type A spermatogonia for in vitro culture. Further, the combination of differential plating and other enrichment techniques may increase the purification efficiency of type A spermatogonia.Reproduction Fertility and Development 02/2009; 21(3):393-9. · 2.58 Impact Factor
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ABSTRACT: Asymmetric division of germline stem cells in vertebrates was proposed a century ago; however, direct evidence for asymmetric division of mammalian spermatogonial stem cells (SSCs) has been scarce. Here, we report that ubiquitin carboxy-terminal hydrolase 1 (UCH-L1) is expressed in type A (A(s), A(pr), and A(al)) spermatogonia located at the basement membrane (BM) of seminiferous tubules at high and low levels, but not in differentiated germ cells distant from the BM. Asymmetric segregation of UCH-L1 was associated with self-renewal versus differentiation divisions of SSCs as defined by co-localization of UCH-L1(high) and PLZF, a known determinant of undifferentiated SSCs, versus co-localization of UCH-L1(low/-) with proteins expressed during SSC differentiation (DAZL, DDX4, c-KIT). In vitro, gonocytes/spermatogonia frequently underwent asymmetric divisions characterized by unequal segregation of UCH-L1 and PLZF. Importantly, we could also demonstrate asymmetric segregation of UCH-L1 and PLZF in situ in seminiferous tubules. Expression level of UCH-L1 in the immature testis where spermatogenesis was not complete was not affected by the location of germ cells relative to the BM, whereas UCH-L1-positive spermatogonia were exclusively located at the BM in the adult testis. Asymmetric division of SSCs appeared to be affected by interaction with supporting somatic cells and extracelluar matrix. These findings for the first time provide direct evidence for existence of asymmetric division during SSCs self-renewal and differentiation in mammalian spermatogenesis.Journal of Cellular Physiology 05/2009; 220(2):460-8. · 4.22 Impact Factor
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ABSTRACT: Odontogenic ameloblast-associated (ODAM) and amelotin (AMTN) are secreted by maturation stage ameloblasts and accumulate at the interface with enamel where an atypical basal lamina (BL) is present. This study aimed at determining and quantifying the ultrastructural distribution of ODAM and AMTN at the cell-tooth interface. Ultrathin sections of enamel organs from the early to mid- and late maturation stage of amelogenesis were processed for immunogold labeling with antibodies against ODAM, AMTN or with the lectins wheat germ agglutinin, Helix pomatia agglutinin (HPA) and Ricinus communis I agglutinin. Immunolabeling showed that both ODAM and AMTN localized to the BL. Quantitative analyses indicated that at the beginning of maturation there is a concentration of ODAM on the cell side of the BL while AMTN appears more concentrated on the enamel side. In the late maturation stage, such differential distribution is no longer apparent. All three lectins are bound to the BL. Competitive incubation with native lectins did not affect the binding efficiency of ODAM; however, AMTN binding was significantly reduced after incubation with HPA. In conclusion, ODAM and AMTN are bona fide components of the BL associated with maturation stage ameloblasts and they organize into different subdomains during the early maturation stage. The data also suggest that the BL is a dynamic structure that rearranges its organization as enamel maturation advances. Finally, the abrogation of AMTN antibody labeling by HPA supports the presence of O-linked sugars in the molecule and/or its close association with other O-glycosylated molecules.Histochemie 03/2012; 137(3):329-38. · 2.61 Impact Factor