The Influence of Retinoic Acid on Cell Proliferation and Differentiation in Lamb Testis Tissue

Abstract

The majority of wild ungulate species are threatened or endangered by extinction. Populations managed in zoos and breeding centers serve as ‘insurance’ for species sustainability and future reintroductions. However, 10-15% of animals born in ex situ collections die before reaching puberty and therefore fail to contribute to conservation breeding. Understanding the in vitro culture requirements for producing gametes from gonadal tissues could facilitate the rescue of germplasm from genetically valuable individuals. Most studies on this topic have focused on the laboratory mouse, with limited information on larger animal models. Retinoic acid (RA) is the biologically active form of vitamin A and is involved in the early steps of spermatogenesis by promoting meiosis to occur in progenitor cells. Furthermore, RA is also involved in Sertoli cell and gonocyte proliferation and/or differentiation in various species. It was hypothesized that RA would promote Sertoli cell and gonocyte proliferation and/or differentiation in testicular explants that were cryopreserved and thawed before in vitro culture. Testicular pieces (1-2 mm3) from 6-7 week-old lambs (n = 6) were cryopreserved using the slow cool method. Tissues were thawed at room temperature (1 min), then in water (25°C; 1 min), followed by three washes (5 min each) in MEM containing 20% FBS, 25 mM HEPES, and antibiotics). Thawed explants (5 pieces/treatment/week/lamb) were cultured for 5 weeks in the absence (0 μM, control) or presence (1 μM, 2 μM, and 5 μM) of RA (RA1, RA2, and RA5, respectively). Tissues were cultured on agarose blocks in MEM supplemented with 10% (v/v) FBS, sphingosine-1-phosphate (2 μM; for the first 2 weeks), insulin (2 μg/ml), transferrin (1.1 μg/ml), selenium (1 μg/ml), pyruvate (0.1 mM), glutamine (2 mM) and antibiotics. Tissues were harvested weekly to be assessed histologically and for gene expression studies. Twenty tubules of uniform size per piece were evaluated for the number of gonocytes and Sertoli cells. Tissues were analyzed for the expression of PCNA (cell proliferation), c-Kit (cell differentiation), Stra8 (synthesis of RA-responsive protein), and HSD3-β (testosterone synthesis). Analyses were performed with a linear mixed model followed by a Tukey’s test for post-hoc comparisons. Gonocyte counts changed continuously over 5 weeks of culture, and responded the most favorably to RA1 (p < 0.05). Under all treatment groups, Sertoli cell counts were maintained for the first three weeks of culture (p > 0.05), before sharply declining by week 4 (p < 0.05). In addition, Sertoli cells did not respond differently to RA1 or RA2 compared to the control (p > 0.05), but declined when treated with RA5 (p < 0.05). Both c-Kit and PCNA expression changed over 5 weeks, but PCNA was negatively impacted by RA5 (p < 0.05) while c-Kit responded positively to either RA2 or RA5 (p <0.05). Stra8 experienced dramatic changes over the culture period, and responded positively to RA1 (p < 0.05) compared to the other treatments. HSD3-β had the highest expression at week 4 before declining by week 5 (p < 0.05), but there was no effect of RA. This study demonstrates for the first time that lamb testicular explants can be 1) cultured in vitro for up to five weeks and 2) retinoic acid stimulates pathways involved in germ cell differentiation while promoting cell proliferation and steroidogenesis. Although advanced stages of spermatogenesis were not achieved in this study, results addressed critical knowledge gaps pertaining to long term culture of testicular tissue and the role of retinoic acid in lamb spermatogenesis in vitro.