Abstract Sperm cooperation has been observed in multiple species, yet its existence and benefit for reproductive success in mammals remains underexplored. Here, combining tissue-clearing with deep three-dimensional imaging, we demonstrate that postcopulatory mouse sperm congregate into unidirectional sperm cooperative clusters at the utero-tubal junction (UTJ), a key physical barrier for passage into the oviduct. Reducing sperm number in male mice by unilateral vasoligation or busulfan-treatment impairs sperm cluster formation and oviduct entry. Interestingly, sperm derived from Tex101 −/− mouse has normal number, motility and morphology, yet they cannot form sperm cluster and fail to pass through the UTJ, which is at least in part due to the altered tail beating pattern of the Tex101 −/− sperm. Moreover, Tex101 −/− sperm’s defect in oviduct entry cannot be rescued by the presence of wild-type (WT) sperm in the same uteri by sequential mating, suggesting sperm cooperative cluster as an essential behavior contributing to male fertility, which could be related to human infertility or subfertility.

Cooperation-based sperm clusters mediate sperm oviduct entry and fertilization