The mechanisms of MEIG1 complex in mammalian spermiogenesis and fertilization - Natural fertilization and embryo development require heathy spermatozoa to carry male genetic material to fertilize the egg. During the final phase of spermatogenesis, the spermatids undergo dramatic changes including the formation of the flagella, condensation of chromatin and so on. A spermatid-specific structure, the manchette, is believed to play an essential role during spermiogenesis. Two functions for the manchette have been proposed: 1) transporting cargo proteins by intra-manchette transport (IMT) for sperm flagella assembly and 2) remodeling chromatin by replacing histones with germ cell-specific nuclear proteins. However, little is known about how protein complexes are assembled and transported in the manchette, or the manchette contributes to chromatin remodeling; and the IMT process has never been observed. The long-term objective of this research is to explore the mechanisms of meiosis-expressed gene 1 (MEIG1) complex in IMT for sperm flagella formation and in remodeling the chromatin for normal embryogenesis. The proposed studies are based our findings that MEIG1 plays an indispensable role in normal sperm flagella formation and chromatin remodeling. MEIG1 is present in the whole cell bodies in the spermatocytes and rounds spermatids, but it is recruited to the manchette by Parkin co-regulated gene (PACRG). MEIG1/PACRG localization in the manchette is dependent on a PACRG binding partner, the axonemal dynein light intermediate chain 1 (DNALI1). DNALI1 is a binding partner of cytoplasmic dynein heavy chain 1 (DHC1), which directly binds to microtubules for cargo transport. Both DNALI1 and DHC1 are localized to the manchette independent of PACRG and MEIG1. More recently, intracytoplasmic sperm injection (ICSI) using sperm from the PACRG mutant mice and a Meig1 KO mouse revealed failure of normal embryogenesis, indicating a functional defect of sperm chromatin in these KO/mutant mice. Based on these observations, we propose that MEIG1 complex plays important roles in transporting cargos through IMT for sperm tail formation and in the formation of male-germ cell specific chromatin essential for normal embryogenesis. To test these hypotheses, we propose the following studies.1: To dissect a motor-based complex in the manchette and study its role in sperm formation; 2: To establish an in vivo system to investigate the protein traffic through IMT; and 3: To examine the contribution and mechanisms of the MEIG1 complex for remodeling nuclear chromatin during spermiogenesis. We expect that DNALI1/DHC1 motors form a cargo transport system with MEIG1 complex in elongating spermatids for normal sperm formation; the dynamic traffic process of IMT can be visualized in live germ cells using knock-in mouse models to express fluorescence-tagged proteins; and MEIG1 complex plays an essential role in for chromatin remodeling by replacing histones with male germ cell-specific nuclear proteins. The proposed studies will dissect the macromolecular complexes in the manchette that are essential for spermiogenesis and establish an in vivo system to study IMT. The proposed studies will also reveal a novel mechanism in chromatin remodeling for normal embryogenesis.
