A novel mechanism of sperm midpiece epididymal maturation and the role of CCDC112 in sperm midpiece formation and establishing an optimal flagella waveform.

Abstract

Background: The mitochondrial sheath is a defining feature of mammalian sperm with proposed functions in structural support and energy production for flagella movement. Recently, coiled coil domain containing (CCDC) protein 112 (CCDC112) was suggested to play a role in the regulation of ciliogenesis. CCDC112 is a poorly characterised protein and there is virtually no knowledge of its in vivo function.

Methods: Here, we define CCDC112 as crucial for male fertility using a Ccdc112 loss-of-function mouse line. To characterize and analyze male fertility, and to identify a novel process of epididymal midpiece maturation, we utilized a range of assays including fertility testing, scanning electron microscopy, high-resolution sperm motility and power output analysis, in vitro fertilization, intracytoplasmic sperm injection, mitochondria stress test assays and glycolytic flux assays. Localization of CCDC112 in cilia was assessed via the transfection of IMCD-3 cells with a CCDC112-eGFP vector and subsequent immunofluorescent staining.

Results: Results reveal CCDC112 as a requirement for male fertility in the mouse with an essential role in mitochondrial sheath formation. Our data reveal the critical role of CCDC112 in mitochondrial morphogenesis during midpiece formation, with the lack of CCDC112 leading to significantly reduced respiration capacity, irregular flagellar waveforms, diminished progressive motility and ultimately male sterility. In the absence of CCDC112, sperm are unable to traverse the female reproductive tract to the site of fertilization and in vitro have a poor capacity to penetrate the zonae pellucidae of oocytes or fuse with the oocyte. We further unveil a previously unrecognized process of epididymal mitochondrial sheath maturation. We show the sperm midpiece is structurally immature upon exiting the testis and maturation continues during transit from the caput to the cauda epididymis. Finally, we identify CCDC112 as a component of the distal appendages of the mother centriole in IMCD-3 cells suggestive of a facilitative role for CCDC112 in protein entry into the ciliary compartment within germ cells.

Conclusion: Collectively, we establish CCDC112 as a key regulator of sperm midpiece assembly and function while further expanding our understanding on functional sperm production, energy generation and flagella kinematics.