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.