RAB10 Interacts with the Male Germ Cell-Specific GTPase-Activating Protein during Mammalian Spermiogenesis

Abstract

According to recent estimates, 2%–15% of couples are sterile, and approximately half of the infertility cases are attributed to male reproductive factors. However, the reasons remain undefined in approximately 25% of male infertility cases, and most infertility cases exhibit spermatogenic defects. Numerous genes involved in spermatogenesis still remain unknown. We previously identified Male Germ Cells Rab GTPase-Activating Proteins (MGCRABGAPs) through cDNA microarray analysis of human testicular tissues with spermatogenic defects. MGCRABGAP contains a conserved RABGAP catalytic domain, TBC (Tre2/Bub2/Cdc16). RABGAP family proteins regulate cellular function (e.g., cytoskeletal remodeling, vesicular trafficking, and cell migration) by inactivating RAB proteins. MGCRABGAP is a male germ cell-specific protein expressed in elongating and elongated spermatids during mammalian spermiogenesis. The purpose of this study was to identify proteins that interact with MGCRABGAP during mammalian spermiogenesis using a proteomic approach. We found that MGCRABGAP exhibited GTPase-activating bioability, and several MGCRABGAP interactors, possible substrates (e.g., RAB10, RAB5C, and RAP1), were identified using co-immunoprecipitation (co-IP) and nano liquid chromatography-mass spectrometry/mass spectrometry (nano LC-MS/MS). We confirmed the binding ability between RAB10 and MGCRABGAP via co-IP. Additionally, MGCRABGAP–RAB10 complexes were specifically colocalized in the manchette structure, a critical structure for the formation of spermatid heads, and were slightly expressed at the midpiece of mature spermatozoa. Based on these results, we propose that MGCRABGAP is involved in mammalian spermiogenesis by modulating RAB10.

Deficiency of the Tbc1d21 gene causes male infertility with morphological abnormalities of the sperm mitochondria and flagellum in mice

“…In our previous study, we found that TBC1D21 are localized to the peri-acrosomal and manchette regions in elongated spermatids and to the tail in mature sperms [31,32]. However, sperm-head defects have not been detected in Tbc1d21 -/sperm; moreover, the morphology of the sperm revealed the severe defects in the mitochondria and the axoneme (Figs 3 and 4).…”

“…Grey lines indicate the possible interaction between two interactors via the STRING software. Red lines present the interactions demonstrated through co-IP in our previous studies[31,32,47].…”

“…In our previous studies, we identified interactors of TBC1D21, a RabGAP, through co-IP and nano-LC/MS [32,47]. One group of interactors consists of small GTP-binding proteins, including RAB10, RAB5C, RAB1A, and RAP1A.…”

“…One group of interactors consists of small GTP-binding proteins, including RAB10, RAB5C, RAB1A, and RAP1A. One of these, RAB10, showed similar localization to TBC1D21 at the manchette structure of the elongating spermatids and midpiece region of mature sperm, during murine spermiogenesis [32]. In this study, we filtered out the interactors as; (1) mitochondria-, (2) microtubule-, and (3) actin-related proteins, based on the phenotypes of the Tbc1d21 knockout mice (Fig 5).…”

Sperm Differentiation: The Role of Trafficking of Proteins

“…RAB3 and RAB27 are present in the acrosomal membrane and are essential for acrosomal exocytosis [97]. RAB27b was detected in the manchette of human and monkey spermatids [98], while RAB10 localizes to the manchette of mouse spermatids [99]. Moreover, RABL2 is essential for sperm tail function and male fertility and RAB8B is known to assist in junction dynamics of the testis [100].…”