Calcium and cyclic nucleotides have crucial roles in mammalian fertilization, but the molecules comprising the Ca2+-permeation pathway in sperm motility are poorly understood. Here we describe a putative sperm cation channel, CatSper, whose amino-acid sequence most closely resembles a single, six-transmembrane-spanning repeat of the voltage-dependent Ca2+-channel four-repeat structure. CatSper is located specifically in the principal piece of the sperm tail. Targeted disruption of the gene results in male sterility in otherwise normal mice. Sperm motility is decreased markedly in CatSper-/- mice, and CatSper-/- sperm are unable to fertilize intact eggs. In addition, the cyclic-AMP-induced Ca2+ influx is abolished in the sperm of mutant mice. CatSper is thus vital to cAMP-mediated Ca2+ influx in sperm, sperm motility and fertilization. CatSper represents an excellent target for non-hormonal contraceptives for both men and women.

Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a FACS-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically-active cells that exhibit a gene expression profile consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and spontaneously generate 35–50 µm oocytes, as determined by morphology, gene expression and attainment of haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1–2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, like adult mice, possess rare mitotically-active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo.

A basic doctrine of reproductive biology is that most mammalian females lose the capacity for germ-cell renewal during fetal life, such that a fixed reserve of germ cells (oocytes) enclosed within follicles is endowed at birth. Here we show that juvenile and adult mouse ovaries possess mitotically active germ cells that, based on rates of oocyte degeneration (atresia) and clearance, are needed to continuously replenish the follicle pool. Consistent with this, treatment of prepubertal female mice with the mitotic germ-cell toxicant busulphan eliminates the primordial follicle reserve by early adulthood without inducing atresia. Furthermore, we demonstrate cells expressing the meiotic entry marker synaptonemal complex protein 3 in juvenile and adult mouse ovaries. Wild-type ovaries grafted into transgenic female mice with ubiquitous expression of green fluorescent protein (GFP) become infiltrated with GFP-positive germ cells that form follicles. Collectively, these data establish the existence of proliferative germ cells that sustain oocyte and follicle production in the postnatal mammalian ovary.