Out of millions of ejaculated sperm, a few reach the fertilization site in mammals. Flagellar Ca²⁺signaling nanodomains, organized by multi-subunit CatSper calcium channel complexes, are pivotal for sperm migration in the female tract, implicating CatSper-dependent mechanisms in sperm selection. Here using biochemical and pharmacological studies, we demonstrate that CatSper1 is an O-linked glycosylated protein, undergoing capacitation-induced processing dependent on Ca²⁺and phosphorylation cascades. CatSper1 processing correlates with protein tyrosine phosphorylation (pY) development in sperm cells capacitated in vitro and in vivo. Using 3D in situ molecular imaging and ANN-based automatic detection of sperm distributed along the cleared female tract, we demonstrate that spermatozoa past the utero-tubal junction possess the intact CatSper1 signals. Together, we reveal that fertilizing mouse spermatozoa in situ are characterized by intact CatSper channel, lack of pY, and reacted acrosomes. These findings provide molecular insight into sperm selection for successful fertilization in the female reproductive tract.

In mammalian sperm cells, regulation of spatiotemporal Ca²⁺ signaling relies on the quadrilinear Ca²⁺ signaling nanodomains in the flagellar membrane. The sperm-specific, multi-subunit CatSper Ca²⁺ channel, which is crucial for sperm hyperactivated motility and male fertility, organizes the nanodomains. Here, we report CatSperτ, the C2cd6-encoded membrane-associating C2 domain protein, can independently migrate to the flagella and serve as a major targeting component of the CatSper channel complex. CatSperτ loss of function in mice demonstrates that it is essential for sperm hyperactivated motility and male fertility. CatSpertτ targets the CatSper channel into the quadrilinear nanodomains in the flagella of developing spermatids, whereas it is dispensable for functional channel assembly. CatSperτ interacts with ciliary trafficking machinery in a C2-dependent manner. These findings provide insights into the CatSper channel trafficking to the Ca²⁺ signaling nanodomains and the shared molecular mechanisms of ciliary and flagellar membrane targeting.

The flagellar-specific Ca²⁺channel CatSper is the predominant Ca²⁺entry site in mammalian sperm. CatSper-mediated Ca²⁺signaling affects nearly every event that regulates sperm to acquire fertilizing capability. In this review, we summarize some of the main findings from 20 years of CatSper research and highlight recent progress and prospects.

Males have evolved species-specifical sperm morphology and swimming patterns to adapt to different fertilization environments. In eutherians, only a small fraction of the sperm overcome the diverse obstacles in the female reproductive tract and successfully migrate to the fertilizing site. Sperm arriving at the fertilizing site show hyperactivated motility, a unique motility pattern displaying asymmetric beating of sperm flagella with increased amplitude. This motility change is triggered by Ca²⁺ influx through the sperm-specific ion channel, CatSper. However, the current understanding of the CatSper function and its molecular regulation is limited in eutherians. Here, we report molecular evolution and conservation of the CatSper channel in the genome throughout eutherians and marsupials. Sequence analyses reveal that CatSper proteins are slowly evolved in marsupials. Using an American marsupial, gray short-tailed opossum (Monodelphis domestica), we demonstrate the expression of CatSper in testes and its function in hyperactivation and unpairing of sperm. We demonstrate that a conserved IQ-like motif in CatSperζ is required for CatSperζ interaction with the pH-tuned Ca²⁺ sensor, EFCAB9, for regulating CatSper activity. Recombinant opossum EFCAB9 can interact with mouse CatSperζ despite high sequence divergence of CatSperζ among CatSper subunits in therians. Our finding suggests that molecular characteristics and functions of CatSper are evolutionarily conserved in gray short-tailed opossum, unraveling the significance of sperm hyperactivation and fertilization in marsupials for the first time.

Ca²⁺ is a key secondary messenger that determines sperm motility patterns. Mammalian sperm undergo capacitation, a process to acquire fertilizing ability, in the female reproductive tract. Capacitated sperm change their flagellar waveform to develop hyperactivated motility, which is crucial for successful sperm navigation to the eggs and fertilization. The sperm-specific channel, CATSPER, and an ATPase transporter, PMCA4, serve as major paths for Ca²⁺ influx and efflux, respectively, in sperm. The ionic paths coordinate Ca²⁺ homeostasis in the sperm, and their loss-of-function impairs sperm motility, to cause male infertility. In this review, we summarize the physiological significance of these two Ca²⁺ gates and suggest their potential applications in novel male contraceptives.

Journal of Animal Science and Technology (JAST) is a peer-reviewed, open access journal publishing original research, review articles and notes in all fields of animal science. Topics covered by the journal include: genetics and breeding, physiology, nutrition of monogastric animals, nutrition of ruminants, animal products (milk, meat, eggs and their by-products) and their processing, grasslands and roughages, livestock environment, animal biotechnology, animal behavior and welfare.