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

Out of millions of ejaculated sperm, a few reach the fertilization site in mammals. Flagellar Ca 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 Ca and phosphorylation cascades.

CatSperζ regulates the structural continuity of sperm Ca signaling domains and is required for normal fertility.

We report that the () and () genes encode novel subunits of a 9-subunit CatSper ion channel complex. Targeted disruption of reduces CatSper current and sperm rheotactic efficiency in mice, resulting in severe male subfertility. Normally distributed in linear quadrilateral nanodomains along the flagellum, the complex lacking CatSperζ is disrupted at ~0.

Rheotaxis guides mammalian sperm.

Background: In sea urchins, spermatozoan motility is altered by chemotactic peptides, giving rise to the assumption that mammalian eggs also emit chemotactic agents that guide spermatozoa through the female reproductive tract to the mature oocyte. Mammalian spermatozoa indeed undergo complex adaptations within the female (the process of capacitation) that are initiated by agents ranging from pH to progesterone, but these factors are not necessarily taxic. Currently, chemotaxis, thermotaxis, and rheotaxis have not been definitively established in mammals.

ATP-activated P2X2 current in mouse spermatozoa.

Sperm cells acquire hyperactivated motility as they ascend the female reproductive tract, which enables them to overcome barriers and penetrate the cumulus and zona pellucida surrounding the egg. This enhanced motility requires Ca(2+) entry via cation channel of sperm (CatSper) Ca(2+)-selective ion channels in the sperm tail. Ca(2+) entry via CatSper is enhanced by the membrane hyperpolarization mediated by Slo3, a K(+) channel also present in the sperm tail.

Cleavage of disulfide bonds in mouse spermatogenic cell-specific type 1 hexokinase isozyme is associated with increased hexokinase activity and initiation of sperm motility.

During epididymal transit, sperm acquire the ability to initiate rapid forward progressive motility on release into the female reproductive tract or physiological media. Glycolysis is the primary source of the ATP necessary for this motility in the mouse, and several novel glycolytic enzymes have been identified that are localized to the principal piece region of the flagellum. One of these is the spermatogenic cell-specific type 1 hexokinase isozyme (HK1S), the only member of the hexokinase enzyme family detected in sperm.

Bicarbonate-induced phosphorylation of p270 protein in mouse sperm by cAMP-dependent protein kinase.

Signaling by cAMP-dependent protein kinase (PKA) plays an important role in the regulation of mammalian sperm motility. However, it has not been determined how PKA signaling leads to changes in motility, and specific proteins responsible for these changes have not yet been identified as PKA substrates. Anti-phospho-(Ser/Thr) PKA substrate antibodies detected a sperm protein with a relative molecular weight of 270,000 (p270), which was phosphorylated within 1 min after incubation in a medium supporting capacitation.

Energy metabolism and sperm function.

Energy metabolism is a key factor supporting sperm function. Sustaining sperm motility and active protein modifications such as phosphorylation could be the reason why sperm require exceptionally more ATP than other cells. Many methods have been used to understand the relationship between energy metabolism and sperm function.

Multiple glycolytic enzymes are tightly bound to the fibrous sheath of mouse spermatozoa.

The fibrous sheath is a cytoskeletal structure located in the principal piece of mammalian sperm flagella. Previous studies showed that glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS), a germ cell-specific glycolytic isozyme that is required for sperm motility, is tightly bound to the fibrous sheath. To determine if other glycolytic enzymes are also bound to this cytoskeletal structure, we isolated highly purified fibrous sheath preparations from mouse epididymal sperm using a sequential extraction procedure.

Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility.

Although glycolysis is highly conserved, it is remarkable that several unique isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like its human ortholog (GAPD2), is the sole GAPDH isozyme in sperm. It is tightly bound to the fibrous sheath, a cytoskeletal structure that extends most of the length of the sperm flagellum.

A-kinase anchoring protein 4 binding proteins in the fibrous sheath of the sperm flagellum.

The fibrous sheath is a unique cytoskeletal structure located in the principal piece of the sperm flagellum and is constructed of two longitudinal columns connected by closely spaced circumferential ribs. Cyclic AMP-dependent protein kinases are secured within specific cytoplasmic domains by A-kinase anchoring proteins (AKAPs), and the most abundant protein in the fibrous sheath is AKAP4. Several other fibrous sheath proteins have been identified, but how the fibrous sheath assembles is not understood.

Mouse testis brain RNA-binding protein/translin selectively binds to the messenger RNA of the fibrous sheath protein glyceraldehyde 3-phosphate dehydrogenase-S and suppresses its translation in vitro.

The testis brain RNA-binding protein (TB-RBP/translin) is a DNA- and RNA-binding protein with multiple functions. As an RNA-binding protein, TB-RBP binds to conserved sequence elements often present in the 3' untranslated regions (UTRs) of specific mRNAs modulating their translation and transport. To identify additional mRNA targets of TB-RBP, immunoprecipitation and reverse transcription-polymerase chain reaction (RT-PCR) assays were carried out using an affinity-purified antibody to TB-RBP with testicular extracts.

Targeted disruption of the Akap4 gene causes defects in sperm flagellum and motility.

A-kinase anchoring proteins (AKAPs) tether cyclic AMP-dependent protein kinases and thereby localize phosphorylation of target proteins and initiation of signal-transduction processes triggered by cyclic AMP. AKAPs can also be scaffolds for kinases and phosphatases and form macromolecular complexes with other proteins involved in signal transduction. Akap4 is transcribed only in the postmeiotic phase of spermatogenesis and encodes the most abundant protein in the fibrous sheath, a novel cytoskeletal structure present in the principal piece of the sperm flagellum.

Tumor necrosis factor receptor 1 is an ATPase regulated by silencer of death domain.

Self-aggregation of tumor necrosis factor receptor type 1 (TNFR1) induces spontaneous downstream signaling and results in cell death. It has been suggested that silencer of death domain (SODD) binds TNFR1 monomers to prevent self-aggregation. We found that SODD binds through its BAG domain to the ATPase domain of Hsp70.