# Glutamine Synthetase 1 Functions in Spermatogenesis in the Silkworm, Bombyx mori

## TL;DR

This study explores the role of glutamine synthetase 1 in silkworm reproduction and identifies potential inhibitors that could reduce fertility.

## Contribution

The study identifies BmGS1 as a testis-specific enzyme in silkworms and discovers small-molecule inhibitors that may be used for pest sterility.

## Key findings

- BmGS1 is specifically expressed in silkworm testes and is regulated by the transcription factor Bmdsx.
- Two small-molecule inhibitors, ET and MSX, were found to bind to and inhibit BmGS1 activity.
- Inhibitor-treated silkworms showed reduced fertilization rates, suggesting BmGS1 as a sterility target.

## Abstract

While glutamine synthetase (GS) has been documented in mammalian spermatogenesis, its functional role in insect male reproductive systems remains largely unexplored. In this study, we investigated Bombyx mori GS1 (BmGS1) and demonstrated its testis-specific expression pattern, with peak expression observed during the moth stage. Subcellular localization analysis revealed its dual distribution in both mitochondria and cytoplasm. Investigation into its upstream regulatory factors demonstrated that the expression of BmGS1 is positively regulated by the reproduction-associated transcription factor Bmdsx. Through virtual screening and molecular dynamics simulation, two small-molecule inhibitors, ET and MSX, which can stably bind to the BmGS1 protein were identified. Enzymatic activity assays and fluorescence binding experiments verified the specific binding and inhibitory effects of the two small molecules on BmGS1. Experiments showed that injection of the inhibitors could reduce the fertilization rate of silkworms. This study provides a valuable clue and potential inhibitors for pest sterility technology.

Glutamine synthetase, a critical enzyme catalyzing the conversion of glutamate and ammonia into glutamine, has been shown to influence sperm development in mammals. Here, we carried out functional analysis of Bombyx mori homolog of glutamine synthetase 1 (BmGS1) and screened its small-molecule inhibitor. RT-PCR and qPCR showed that BmGS1 was specifically expressed in the testis of the silkworm, with the highest expression in the moth stage. Subcellular localization revealed that the BmGS1 protein was localized in mitochondria and cytoplasm. Identification of upstream regulatory factors revealed that the expression of BmGS1 is positively regulated by the sex-related transcription factor Bmdsx. Virtual screening, molecular docking and MD simulations showed that the small molecule Ethylhexyl triazone (ET), as well as the known GS inhibitor L-Methionine -DL-sulfoximine (MSX), could be stably bound to BmGS1. Subsequently, site-specific mutation and fluorescence binding assays revealed that the putative key sites of ET binding to the protein were E79 and R265, and the putative key sites of MSX binding to the protein were E81, R245, and R286. Both in vitro and in vivo experiments demonstrated that inhibitor treatment significantly attenuated BmGS1 enzymatic activity. Inhibitor-injected silkworms showed reduced fertilization rates compared to control groups. Our findings raise BmGS1 as a potential target for silkworm sterility.