Cellular and molecular characterization of the testicular development of GDF9-edited pigs.

Growth differentiation factor (GDF) 9 is a member of the transforming growth factor (TGF)-β superfamily, which plays an important role in mammalian ovarian follicular development and female fertility. However, its regulatory role in the male reproductive system remains largely unknown. This study aimed to investigate the regulatory mechanism of GDF9 in porcine testicular development. We established a GDF9-edited pig model through CRISPR-Cas9 technology and somatic cell nuclear transplantation. The testicular development of one edited piglet at birth and one at Day 14 were characterized via Western blotting, haematoxylin-eosin staining, and immunohistochemistry at cellular and molecular levels. Based on the preliminary analysis of one edited piglet at birth and one at Day 14, we did not observe changes to the gross morphological and anatomical structures of porcine testes at early developmental stages. However, the developmental process or cellular functions of Sertoli cells, Leydig cells and spermatogonia may be affected, as reflected by the altered expression patterns of SOX9, AR, DHRS9, DDX4 and ANXA2 in the testes. Noticeably, the altered expression of StAR may indicate an impact of GDF9 editing on the steroid hormone synthesis functions of porcine Leydig cells. These findings indicate that GDF9 may affect porcine testicular development at the early postnatal stage. These preliminary results provide a valuable foundation for future comprehensive studies toelucidate the regulatory mechanism of GDF9 in porcine testicular development.