Abstract
The molecular markers for specific germ cell stages can be utilized for identifying, monitoring, and separating a particular stage of germ cells. The RNA-binding protein Lin28 is expressed in gonocytes of human fetal testes. The Lin28 expression is restricted to a very small population of spermatogonial cells in human, mice, and monkey. The main objective of this study was to investigate the expression pattern of Lin28 in stallion testes at different reproductive stages. Based on the presence or absence of full spermatogenesis and lumina in seminiferous tubules, the testicular samples were categorized into two reproductive stages pre-pubertal and post-pubertal. We performed a reverse transcription polymerase chain reaction to confirm the presence of Lin28 mRNA in the testicular tissues and a western blot analysis to verify the cross-reactivity of rabbit Lin28 antibody with horse testicular tissue. For immunohistochemistry, Lin28 (rabbit anti-human), GATA4 (goat anti-human) or DAZL (goat anti-human) antibodies were used. The results of RT-PCR confirmed the expression of Lin28 mRNA in the stallion testes. The western blot analysis showed that the expression of 28 kDa Lin28 protein was localized in the cytoplasm of spermatogonia at both reproductive stages. The numbers of Lin28-positive germ cells per 1000 Sertoli cells in pre- and post-pubertal stages were 253 ± 8.66 and 29.67 ± 2.18, respectively. At both reproductive stages, all Lin28 positive cells showed no co-stained with GATA4 antibody, whereas only some of the Lin28-positive germ cells showed co-staining with DAZL antibody. The results from whole-mount staining showed that the Lin28 expression was limited to Asingle (As) and Apaired (Apr) spermatogonia. In conclusion, Lin28 might be utilized as a molecular marker for undifferentiated spermatogonial stem cells when used with DAZL antibody.
Highlights
Spermatogonial stem cells (SSCs) have the potential to undergo self-renewal and differentiation for continuous sperm production, and can be used as a resource to preserve the genetic value of stallions
The results suggest that Lin28 expression pattern in spermatogonia is conservative throughout the species
In the post-pubertal stallions, Lin28 positive germ cells are rarely observed and they were located adjacent to the basement membrane of seminiferous tubule
Summary
Spermatogonial stem cells (SSCs) have the potential to undergo self-renewal and differentiation for continuous sperm production, and can be used as a resource to preserve the genetic value of stallions. The utilization of putative molecular markers for undifferentiated. SSCs has been introduced as an alternative method to identify certain developmental stages of SSCs [2]. GFRα1, PLZF, and CSF1R have been identified as markers for undifferentiated spermatogonia [3]. The molecular markers specific for different stages of spermatogonia have not been identified because whole-mount staining is not feasible with these markers. We have reported that UTF1 is a molecular marker for undifferentiated type A spermatogonia [4]. We sought to identify another putative molecular marker for a stage of spermatogonia earlier than 16 Aal
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