3D in situ imaging of the female reproductive tract reveals molecular signatures of fertilizing spermatozoa in mice.

Lukas Ded,Huanan F Shi,Jae Yeon Hwang,Kiyoshi Miki,Jean-Ju Chung

doi:10.7554/elife.62043

Abstract

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.

Highlights

In most mammals, millions or billions of spermatozoa are deposited into the cervix upon coitus.Yet less than 100 spermatozoa are found at the fertilization site, called ampulla, and only 10-12 spermatozoa are observed around an oocyte (Kolle, 2015; Suarez, 2002)
3D in situ molecular imaging and artificial neural network (ANN)-based automatic detection of sperm distributed along the cleared female tract, we demonstrate that all spermatozoa past the utero-tubal junction (UTJ) possess intact CatSper[1] signals
Recent ex vivo imaging studies combined with mouse genetics have shown that surface molecules on the sperm plasma membranes such as ADAM family proteins are essential for the sperm to pass through the utero-tubal junction (UTJ) (Fujihara et al, 2018)

Summary

Introduction

Millions or billions of spermatozoa are deposited into the cervix upon coitus.Yet less than 100 spermatozoa are found at the fertilization site, called ampulla, and only 10-12 spermatozoa are observed around an oocyte (Kolle, 2015; Suarez, 2002). Recent ex vivo imaging studies combined with mouse genetics have shown that surface molecules on the sperm plasma membranes such as ADAM family proteins are essential for the sperm to pass through the utero-tubal junction (UTJ) (Fujihara et al, 2018). By contrast, whether such selection and elimination within the oviduct requires specific molecular signatures and cellular signaling of spermatozoa is not fully understood. Previous in vitro studies that represent the population average at a given time may or may not have observed molecular details of a small number of the most fertilizing sperm cells

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