Identification of motility-associated progesterone-responsive differentially phosphorylated proteins.

Progesterone is one of the regulators of sperm motility and hyperactivation. In human spermatozoa, the effects of progesterone are thought to be mediated by protein phosphorylation. In the present study, we identified 22 proteins that are differentially phosphorylated (12 phosphorylated and 10 dephosphorylated) by progesterone in human spermatozoa. Functionally, the differentially phosphorylated proteins are predicted to have cytoskeletal localisation and to be associated with sperm motility. 5µM of progesterone to capacitated increased the phosphorylation of tyrosine residues in the principal piece and protein tyrosine kinase activity increased by almost 3.5-fold. For the first time, we demonstrate that tyrosine phosphatases are also activated in response to progesterone and that inhibition of tyrosine phosphatases attenuates dephosphorylation of flagellar proteins. We propose that progesterone activates both kinase and phosphatase pathways, leading to changes in the phosphorylation of many proteins in sperm flagella to increase motility.

Progesterone activates Janus Kinase 1/2 and activators of transcription 1 (JAK1-2/STAT1) pathway in human spermatozoa.

Ejaculated spermatozoa undergo capacitation and acrosome reaction by responding to extrinsic clues and activate signalling cascades to induce protein tyrosine phosphorylation. In the present study, we investigated the existence, the Janus kinase (JAK) and activator of transcription (STAT) pathway and determined its physiological relevance. JAK1 and STAT1 are localised on the equatorial region and the midpiece of their human spermatozoa, JAK2 is detected on the sperm tail. Capacitation leads to phosphorylation of JAK2 but not JAK1 and STAT1. In the uncapacitated sperm, phosphorylated JAK2 (pJAK2) is localised mainly in the tail region; in response to capacitation, the JAK2 is phosphorylated in the midpiece and the head region along with the tail. Progesterone (5 µm) leads to phosphorylation of JAK1, JAK2 and STAT1 in a time-dependent manner. In progesterone-treated spermatozoa, the JAK2 in the tail is hyperphosphorylated, the JAK2 in the head and the midpiece is dephosphorylated. We conclude that in human spermatozoa, the JAK1/2 pathway is activated upon capacitation and is further modulated by progesterone; the biological processes controlled by this pathway in sperm need to be elucidated.

Differential concentration and time dependent effects of progesterone on kinase activity, hyperactivation and acrosome reaction in human spermatozoa.

Progesterone has been identified to be one of the physiological regulators of sperm hyperactivation and acrosome reaction. However, the high sensitivity of human spermatozoa to progesterone implies that many may undergo premature hyperactivation and acrosome reaction thereby compromising their ability to fertilize. We hypothesized that if a spermatozoon has to preclude the occurrence of these events prematurely, there should be differential dose- and time-dependent effects on motility and acrosome reaction. We observed that low concentrations of progesterone (10 and 100 nm) induce sperm motility and activate tyrosine kinase; higher concentrations (1-10 µm) are required to induce extracellular signal regulated kinases 1/2 (Erk1/2), p90 ribosomal S6 kinase (p90RSK), p38 mitogen-activated protein kinase (p38MAPK), c-Jun N-terminal kinase (JNK1) and AKT phosphorylation, hyperactivation and acrosome reaction. The induction of acrosome reaction and tyrosine phosphorylation in response to higher concentration of progesterone is not absolutely dependent on activation of T-type voltage-gated Ca(2+) channel or CatSper as Mibefradil did not completely abrogate progesterone-mediated effects. These results imply that although the spermatozoa are sensitive to low concentrations of progesterone, they only activate motility and tyrosine kinase activation; higher concentrations are required to induce hyperactivation and acrosome reaction probably by activating multiple kinase pathways including the MAPK and AKT.