UTF1, a Putative Marker for Spermatogonial Stem Cells in Stallions

Abstract

Spermatogonial stem cells (SSCs) continuously undergo self-renewal and differentiation to sustain spermatogenesis throughout adulthood in males. In stallions, SSCs may be used for the production of progeny from geldings after cryopreservation and therapy for infertile and subfertile stallions. Undifferentiated cell transcription factor 1 (UTF1) is a putative marker for undifferentiated spermatogonia in humans and rats. The main purposes of this study are to determine the following: 1) changes in the expression pattern of UTF1 at various reproductive stages of stallions, 2) subpopulations of spermatogonia that express UTF1. Testicular samples were collected and categorized based on the age of the horses as follows: pre-pubertal (<1 yr), pubertal (1–1.5 yr), post-pubertal (2–3 yr), and adult (4–8 yr). Western blot analysis was utilized to determine the cross-activity of the UTF1 antibody to horse testes tissues. Immunohistochemistry was conducted to investigate the UTF1 expression pattern in germ cells at different reproductive stages. Whole mount staining was applied to determine the subpopulation of UTF1-positive spermatogonia. Immunohistological analysis showed that most germ cells in the pre-pubertal and pubertal stages were immunolabeled with UTF1, whereas only a few germ cells in the basal compartment of the seminiferous tubule cross-sections of post-pubertal and adult tissues were UTF1-positive. No staining was observed in the Sertoli or Leydig cells at any reproductive stages. Whole mount staining showed that As, Apr, and chains of 4, 8, 16 Aal spermatogonia were immunolabeled with UTF1 in the post-pubertal stallion tubule. Isolated single germ cells were also immunolabeled with UTF1. In conclusion, UTF1 is expressed in undifferentiated spermatogonia, and its antibody can be used as a putative marker for SSCs in stallions.

The Lin28 Expression in Stallion Testes

“…Based on the age and presence of lumina in the cross-sections of the seminiferous tubules of stallions, their reproductive stages were categorized as follows: pre-pubertal (< 1.5 yr, n = 3) and post-pubertal (2–4 yr, n = 3) [4, 11]. …”

“…Preparation of testicular tissue samples was performed as previously described [4] with slight modifications. Briefly, after castration, the testes were transported to the laboratory in an icedbox maintained at 4°C.…”

“…Western blot analysis was performed to verify the cross-reactivity of rabbit anti-human Lin28 antibody to Lin28 protein in the equine testis as previously described [4] with minor modification. Briefly, for protein sample preparation, thawed samples were homogenized using a Polytron PT 1200 CL (Kinematica AG, Littau-Lucerne, Switzerland) in a radioimmunoprecipitation assay buffer for approximately 5 min.…”

“…For immunocytochemistry, single germ cells were isolated from testes in pre-pubertal (n = 3) and post-pubertal (n = 3) stallions using a two-step enzyme protocol as previously reported [4], with slight modifications. Briefly, the sliced testicular tissues (1 cm 3 ) were incubated with collagenase type IV (1 mg/mL; Sigma) dissolved in Hank’s balanced salt solution (HBSS; Invitrogen) at 37°C for 15 min with vigorous shaking in a shaking incubator (Vision Scientific, Yuseong Gu, Daejeon, Korea).…”

“…However, the molecular markers specific for different stages of spermatogonia have not been identified because whole-mount staining is not feasible with these markers. Previously, we have reported that UTF1 is a molecular marker for undifferentiated type A spermatogonia [4]. However, the UTF1 protein was found to be expressed in A single (A s ) , A paired (A pr ), and chains of 4, 8, and 16 A aligned (A al ) types of spermatogonia.…”