(373) Transcriptional Regulation of Prepubertal Male Germline by Core-Binding Factor Subunit-β (CBFβ) and Runt-Related Transcription Factor (RUNX) Family

Physiological and Metabolomic Differences in Canine Testes with or without Spermatogenesis

 Yuki Yamamoto¹; Ryosuke Shimizu¹; Mai Okazawa¹; Kazumi Shimada²; Ryo Tanaka³; Lina Hamabe⁴; Kentaro Nagaoka¹

1.  Laboratory of Veterinary Physiology, Cooperative Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo, Japan.

2.     Laboratory of Veterinary Surgery, Cooperative Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo, Japan.

3.     Animal Medical Center, Tokyo University of Agriculture and Technology, Tokyo, Japan.

4.    Laboratory of Veterinary Clinical Oncology, Cooperative Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo, Japan.

 


Abstract Text: Recent studies demonstrated in vitro spermatogenesis in the immature testis of rodents. This method would contribute to assisted reproductive techniques for human, livestock and endangered species, however, no success case has been reported with other mammals. In addition, in vitro spermatogenesis in adult testes has not been established yet. This suggests that optimal environment for spermatogenesis is different between immature and adult testes. To clarify the physiological and metabolomic characters in immature and mature testis of non-rodent animals, we investigated the germ cell (GC) and Sertoli cell (SC) states before and after organ culture, local testosterone levels and metabolomic profiles in canine testis tissues. The canine testes (4 m.o.-5 y.o.) were collected after clinical castration at Animal Medical Center in Tokyo University of Agriculture and Technology after informed consent from the owners. Tissues were fixed by 4% paraformaldehyde for histological analysis or kept at -80℃ until hormonal and metabolomic analyses. In addition, testicular fragments (1-2 mm³) were cultured for 2-6 weeks on half-soaked agarose gels with a culture medium followed by immunohistochemistry to examine the GC and SC states. GC and SC states were analyzed by immunohistochemistry for DDX4 (GC marker), c-KIT (differentiating spermatogonia marker), SOX9 and vimentin (SC marker), aldehyde dehydrogenase 1/2 (ALDH, retinoic acid production enzyme), and androgen receptor (AR, SC maturation marker). Testosterone levels in testis was analyzed by ELISA. Non-target metabolomics was performed using gas-chromatography mass spectrometry and the data were analyzed by MetaboAnalyst (https://www.metaboanalyst.ca). Firstly, testis tissues were classified as immature (no spermatogenesis, 4-10.5 m.o., n=6), matured (with spermatogenesis, n=6, 9.5-5 y.o.) by histological observation. Testes from matured unilateral cryptorchidism (n=2, no spermatogenesis in only cryptorchidism side) were also collected. Results of immunohistochemistry showed many differentiating spermatogonia, clear expression of ALDH and AR in SCs in matured testis. On the other hand, only undifferentiated GCs and faint expression of ALDH and AR in SCs were observed in immature testis. In cryptorchidism testis, few GCs, faint immunostaining of ALDH but clear expression of AR was observed in SCs. Testosterone levels of testis tissues were not significantly different between non-spermatogenic (immature and cryptorchidism) and spermatogenic (matured) testis. These results suggest that ALDH expression in SCs is more directly involved in spermatogenesis than local testosterone levels. In cultured fragments of immature testis (n=3), the expression of ALDH increased after culture, however, spermatogenesis was not observed. Although GCs and SCs remained on basal membrane of seminiferous tubules by 4-week of culture, AR expression was not observed in SCs and most of GCs were lost at 6-week of culture. Cultured matured testis (n=3) showed loss of GCs and SCs after 2-week, indicating seminiferous epithelium degraded immediately after start of culture. It is suggested that this culture condition could induce the ALDH expression but not AR expression and spermatogenesis. Furthermore, nutrient might be insufficient for cell survival especially in matured testes. To investigate the optimal nutrient environment for spermatogenesis, non-target metabolomics were compared between non-spermatogenic group and spermatogenic group. The results demonstrated 30 metabolites were significantly higher and 29 metabolites were significantly lower in spermatogenic group (p < 0.05), suggesting that these are involved in the spermatogenesis. The culture medium will be modified based on the metabolomics results to establish the organ culture methods suitable for spermatogenesis from canine immature and matured testis.