Depletion of Gtsf1L impairs development of eupyrene sperm and ovary in Gtsf1L.

Sperm delivers genetic information from the male to the ovum, playing vital roles in sexual reproduction. Like other Lepidoptera, Bombyx mori exhibits dimorphic spermatogenesis, generating coexisting nucleated eupyrene sperm and anucleated apyrene sperm. The mechanism of dimorphic spermatogenesis is still to be clarified. In a previous study, we demonstrated that Gametocyte-specific factor1 (Gtsf1) is essential for female sex determination and PIWI-interacting RNA (piRNA) mediated transposon silencing in B. mori. Here, we performed functional analysis of the Gtsf1 paralog BmGtsf1L using a binary transgenic CRISPR/Cas9 system. BmGtsf1L is dispensable for sex determination but critical for fertility in both males and females. We separated different types of sperm and found that BmGtsf1L is highly expressed in both types of sperm. BmGtsf1L deficiency (△BmGtsf1L) impaired the formation and migration of eupyrene sperm, whereas the development and movement of apyrene sperm were normal. Furthermore, through a sperm culture experiment, we confirmed that eupyrene spermatogenesis defects appeared before the elongation stage. Double copulations of a female with △BmGtsf1L and Sex-lethal mutant males can rescue infertility phenotypes, revealing that the apyrene sperm of BmGtsf1L mutants is functional. We also found that the depletion of BmGtsf1L impacted proper oogenesis. This study provided the first functional analysis of Gtsf1 paralogs on physiology, demonstrating the critical role of BmGtsf1L in the development of eupyrene sperm and the ovary.