Date Published: June 21, 2019
Publisher: Public Library of Science
Author(s): Swati Sharma, Stefan Schlatt, Ans Van Pelt, Nina Neuhaus, Shree Ram Singh.
From a biological and clinical perspective, it is imperative to establish primate spermatogonial cultures. Due to limited availability of human testicular tissues, the macaque (Macaca fascicularis) was employed as non-human primate model. The aim of this study was to characterize the expression of somatic as well as germ cell markers in testicular tissues and to establish macaque testicular primary cell cultures.
Characterization of macaque testicular cell population was performed by immunohistochemical analyses for somatic cell markers (SOX9, VIM, SMA) as well as for germ cell markers (UTF1, MAGEA4, VASA). Testicular cells from adult macaque testes (n = 4) were isolated and cultured for 21 days using three stem cell culture media (SSC, PS and SM). An extended marker gene panel (SOX9, VIM, ACTA2; UTF1, FGFR3, MAGEA4, BOLL, DDX4) was then employed to assess the changes in gene expression levels and throughout the in vitro culture period. Dynamics of the spermatogonial population was further investigated by quantitative analysis of immunofluorescence-labeled MAGEA4-positive cells (n = 3).
RNA expression analyses of cell cultures revealed that parallel to decreasing SOX9-expressing Sertoli cells, maintenance of VIM and ACTA2-expressing somatic cells was observed. Expression levels of germ cell marker genes UTF1, FGFR3 and MAGEA4 were maintained until day 14 in SSC and SM media. Findings from MAGEA4 immunofluorescence staining corroborate mRNA expression profiling and substantiate the overall maintenance of MAGEA4-positive pre- and early meiotic germ cells until day 14.
Our findings demonstrate maintenance of macaque germ cell subpopulations in vitro. This study provides novel perspective and proof that macaques could be used as a research model for establishing in vitro germ cell-somatic cell cultures, to identify ideal culture conditions for long-term maintenance of primate germ cell subpopulation in vitro.
Spermatogonia present at the basement membrane of the seminiferous tubules are the least differentiated germ cells in adult primate testes [1,2,3]. Previous experimental studies postulate the role of testicular niche factors and Sertoli cells in regulating germ cell dynamics and maintaining testicular homeostasis [4,5,6]. However, the specific mechanisms regulating self-renewal and differentiation of spermatogonia in primates are not yet completely understood [7,8,9,10,11]. For understanding spermatogonial regulation in primates, establishing primate-specific in vitro approaches for co-culturing testicular germ cells and somatic cells isolated from adult testes are considered highly valuable.
Here, for the first time we characterized adult macaque testicular sub-populations, and thereafter isolated and cultured testicular cells from adult macaque testes (Macaca fascicularis). Effect of three distinct culture media was analyzed by assessing the change in germ cell-somatic cell expression levels in vitro during culture. Differences in depletion and enrichment patterns of specific germ cell and somatic cell sub-populations during culture were detected. Expression analysis using eight characterized marker genes indicates that in accordance with the selective depletion of Sertoli cells amongst the somatic cell sub-populations, a relative depletion of germ cell (undifferentiated, pre- and meiotic and differentiating) sub-populations was observed overtime. The changing germ cell dynamics during culture further confirms the maintenance of pre- and early meiotic germ cells until day 14 in culture.