BmPMFBP1 regulates the development of eupyrene sperm in the silkworm, Bombyx mori

Abstract

Sperm deliver the male complement of DNA to the ovum, and thus play a key role in sexual reproduction. Accordingly, spermatogenesis has outstanding significance in fields as disparate as infertility treatments and pest-control, making it a broadly interesting and important focus for molecular genetics research in a wide range of species. Here we investigate spermatogenesis in the model lepidopteran insect Bombyx mori (silkworm moth), with particular focus on the gene PMFBP1 (polyamine modulated factor 1 binding protein 1). In humans and mouse, PMFBP1 is essential for spermatogenesis, and mutations of this gene are associated with acephalic spermatozoa, which cause infertility. We identified a B. mori gene labeled as “PMFBP1” in GenBank’s RefSeq database and sought to assess its role in spermatogenesis. Like in mammals, the silkworm version of this gene (BmPMFBP1) is specifically expressed in testes. We subsequently generated BmPMFBP1 mutants using a transgenic CRISPR/Cas9 system. Mutant males were sterile while the fertility of mutant females was comparable to wildtype females. In B. mori, spermatogenesis yields two types of sperm, the nucleated fertile eupyrene sperm, and anucleated unfertile apyrene sperm. Mutant males produced abnormal eupyrene sperm bundles but normal apyrene sperm bundles. For eupyrene sperm, nuclei were mislocated and disordered inside the bundles. We also found the BmPMFBP1 deficiency blocked the release of eupyrene sperm bundles from testes to ejaculatory seminalis. We found no obvious abnormalities in the production of apyrene sperm in mutant males, and double-matings with apyrene-deficient sex-lethal mutants rescued the ΔBmPMFBP1 infertility phenotype. These results indicate BmPMFBP1 functions only in eupyrene spermatogenesis, and highlight that distinct genes underlie the development of the two sperm morphs commonly found in Lepidoptera. Bioinformatic analyses suggest PMFBP1 may have evolved independently in lepidoptera and mammals, and that despite the shared name, are likely not homologous genes.

The Prmt5-Vasa module is essential for spermatogenesis in Bombyx mori

“…As mutation of BmPrmt5 does not affect the production or stability of BmVasa, we speculate that BmPrmt5-mediated sDMA modification of BmVasa activates BmVasa allowing it to regulate spermatogenesis. We therefore propose Previous studies have demonstrated that morphology defects in the bundles of either eupyrene or apyrene sperm compromise their physiological function [10,11,14,19,20]. In BmPrmt5 and BmVasa mutants, there are severe defects in both sperm morphs.…”

“…Previous studies have demonstrated that morphology defects in the bundles of either eupyrene or apyrene sperm compromise their physiological function [ 10 , 11 , 14 , 19 , 20 ]. In BmPrmt5 and BmVasa mutants, there are severe defects in both sperm morphs.…”

“…To date, the studies on dichotomous spermatogenesis in B. mori and other lepidopteran species have been limited mostly to aspects related to cytology and developmental timing carried out by microscopic observations. Only few genes such as poly(A)-specific ribonuclease-like domaincontaining 1 (BmPnldc1), Hua enhancer 1 (BmHen1), Sex-lethal (BmSxl), Maelstrom (BmMael), and Polyamine modulated factor 1 binding protein (BmPMFBP1) have been experimentally linked to this process in B. mori [10,11,[14][15][16][17][18][19][20]. Therefore, the molecular and genetic framework that controls spermatogenesis remains largely unknown, and it is important to continue investigating additional genes and molecular mechanisms that may be governing dichotomous spermatogenesis.…”

The RNase III enzyme Dicer1 is essential for larval development in Bombyx mori

“…The resultant F 1 progeny expressed both Cas9 and sgRNAs, causing DNA lesions in BmDicer1, resulting in independent de novo mutations in each of the cross's offspring. Thus, we referred to the mosaic mutagenesis of the mutant as "somatic" (Zhang et al, 2017;Yang et al, 2022). Genomic DNA from day 2 of the 3rd instar larvae was used as a template for PCR-based analysis.…”

Differential gene expression underpinning production of distinct sperm morphs in the wax mothGalleria mellonella

“…The final product of this deliberate and orchestrated process is two morphs with different morphologies made with different proteomes, comprising some shared proteins and some proteins unique to one or other morph (9, 10). A small number of genes have been demonstrated via CRISPR-Cas9/RNAi experiments to be important for Lepidopteran sperm heteromorphism, including Sex-lethal (Sxl) (Table S1) (5, 6, 11, 12). However, there has not been a systematic, unbiased identification of differentially expressed genes that ensure normal differentiation of spermatocytes towards eupyrene and apyrene sperm fates.…”