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Abstract: Vascular endothelial growth factor (VEGF) is known to influence the testis function. To establish the role of VEGF in the testis of a variety of species, we analyzed the expression of VEGF transcript using human gene-specific primers by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis in the testes of 18 vertebrates, including a few endangered species. An amplicon of 566 bp representing VEGF165 was identified in testis of all species in this study. Sequence analysis of these amplicons revealed 84 to 96% homology to available human VEGF sequence and to the VEGF sequences of other species in GenBank. Immunohistochemical analysis revealed expression of VEGF protein, primarily in Sertoli and Leydig cells and occasionally in the germ cells of the testis sections. It can be concluded from this study that expression of VEGF transcript is conserved in the testis of several vertebrates and may have a role in the process of spermatogenesis. [Copyright &y& Elsevier]

Growth and development of immature testis xenograft from various domestic mammals has been shown in mouse recipients; however, buffalo testis xenografts have not been reported to date. In this study, small fragments of testis tissue from 8-week-old buffalo calves were implanted subcutaneously onto the back of immunodeficient male mouse recipients, which were either castrated or left intact (non-castrated). The xenografts were retrieved and analyzed 12 and 24 weeks later. The grafted tissue survived and grew in both types of recipient with a significant increase in weight and seminiferous tubule diameter. Recovery of grafts from intact recipients 24 weeks post-grafting was significantly lower than that from the castrated recipients. Seminal vesicle indices and serum testosterone levels were lower in castrated recipients at both collection time points in comparison to the intact recipients and non-grafted intact mouse controls. Pachytene spermatocytes were the most advanced germ cells observed in grafts recovered from castrated recipients 24 weeks post-grafting. Complete spermatogenesis, as indicated by the presence of elongated spermatids, was present only in grafts from intact recipients collected 24 weeks post-grafting. However, significant number of germ cells with DNA damage was also detected in these grafts as indicated by TUNEL assay. The complete germ cell differentiation in xenografts from intact recipients may be attributed to efficient Sertoli cell maturation. These results suggest that germ cell differentiation in buffalo testis xenograft can be completed by altering the recipient gonadal status. [ABSTRACT FROM AUTHOR]

Spermatogonia are germ cells that initiate spermatogenesis in mammalian testis and they are the only cells in adult body capable of dividing both mitotically and meiotically. Therefore, isolation and preservation of spermatogonia can provide an alternative method for preservation of genetic pool of endangered animals. To achieve this objective, it is essential to identify markers that can specifically distinguish spermatogonia from other cells in the testis. In the present study, anti-ubiquitin C-terminal hydroxylase-1 (UCHL-1/PGP 9.5) antibody specifically recognized spermatogonia in the testis of wild and domestic bovid. The size of the UCHL-1 protein in various species of bovid testes was identical, and similar to that in mice. Furthermore, UCHL-1 staining could be utilized for the identification of spermatogonia in isolated testicular cells from wild bovids suggesting that UCHL-1 protein expression could be used as a specific marker for spermatogonia in bovid family. [ABSTRACT FROM AUTHOR]

Abstract: Buffalo is an economically important livestock species in Asia. Little is known about male germ line technology owing to lack of sufficient understanding regarding expression of germ- and somatic-cell specific-proteins in the testis. In this study, we identified UCHL-1 (PGP 9.5) and lectin- Dolichos biflorus agglutinin (DBA) as specific markers for spermatogonia in buffalo testis. Expression of germ-cell and pluripotency-specific proteins such as DDX4 (VASA) and POU5F1 (OCT3/4) were also present in spermatogonia. Interestingly, the expression of somatic cell-specific proteins such as VIMENTIN and GATA4 were also detected in germ cells. Using two-step enzymatic digestion followed by differential plating and Percoll density-gradient centrifugation, an approximately 55% spermatogonia-enriched cell population could be obtained from the prepubertal buffalo testis. Isolated spermatogonia could survive and proliferate in vitro in DMEM/F12 medium containing 10% fetal bovine serum in the absence of any specific growth factors for a week. Cultured spermatogonia showed DBA affinity and expressed DDX4 and POU5F1. These results may help to establish a long-term culture system for buffalo spermatogonia. [Copyright &y& Elsevier]