Molecular mechanism for human sperm chemotaxis mediated by progesterone

Abstract

Sperm chemotaxis is a chemical guiding mechanism that may orient spermatozoa to the egg surface. A picomolar concentration gradient of Progesterone (P), the main steroidal component secreted by the cumulus cells that surround the egg, attracts human spermatozoa. In order to elucidate the molecular mechanism of sperm chemotaxis mediated by P, we combine the application of different strategies: pharmacological inhibition of signaling molecules, measurements of the concentrations of second messengers and activation of the chemotactic signaling. Our data implicate a number of classic signal transduction pathways in the response and provide a model for the sequence of events, where the tmAC-cAMP-PKA pathway is activated first, followed by protein tyrosine phosphorylation (equatorial band and flagellum) and calcium mobilization (through IP(3)R and SOC channels), whereas the sGC-cGMP-PKG cascade, is activated later. These events lead to sperm orientation towards the source of the chemoattractant. The finding proposes a molecular mechanism which contributes to the understanding of the signal transduction pathway that takes place in a physiological process as chemotaxis.

Figure 1. AC-cAMP-PKA pathway during sperm chemotaxis mediated by P.

A, B and E: Percentage of oriented spermatozoa (OS) towards 10 pM P gradient after treating the cells with ddAdo (15 min), KH7 (15 min) and KT5720 (5 min), respectively. C: cAMP intracellular concentration in the absence or presence of 10 pM P. D: Percentage of oriented spermatozoa towards db-cAMP. HAM was assayed as a negative control. Each bar represents the mean ± SEM. ^a Significant differences vs. without inhibitor (p<0.05). ^b Significant differences vs. without P (p<0.05). ^c Significant differences vs. HAM (p<0.05).

Figure 2. GC-cGMP-PKG pathway during sperm chemotaxis mediated by P.

A, B and E: Percentage of oriented spermatozoa (OS) towards 10 pM P gradient after treating the cells with ODQ (15 min), LY-83.583 (15 min) and KT5823 (5 min), respectively. C: cGMP intracellular concentration in the absence or presence of 10 pM P. D: Percentage of oriented spermatozoa towards db-cGMP. HAM was assayed as a negative control. Each bar represents the mean ± SEM. ^a Significant differences vs. without inhibitor (p<0.05). ^b Significant differences vs. without P (p<0.05). ^c Significant differences vs. HAM (p<0.05).

Figure 3. Calcium as a second messenger for sperm chemotaxis.

A, B, D, E, F, G and H: Percentage of oriented spermatozoa (OS) towards 10 pM P gradient after treating the cells with BAPTA-AM (1 min), sEBSS culture medium with and without extracellular calcium, Nifedipine (1 min), SKF96365 (1 min), TMB-8 (1 min), 2-APB (15 min) and Tetracaine (15 min), respectively. C: Ca²⁺ signal (change in Oregon green 1 BAPTA florescence) in immobilized human spermatozoa, black line shows the mean of cells that responded to a step application of pM P and gray line shows the mean of those cells in which no response was detectable. Black arrow marks time of arrival of P in the imaging chamber. HAM was assayed as a negative control. Each bar shows mean ± SEM. ^a Significant differences vs. without inhibitor (p<0.05).

Figure 4. Sperm protein tyrosine phosphorylation.

A: Immunocytochemistry for protein Tyr-phosphorylation in the absence or presence of a 10 pM P gradient. ^A flagellum; ^B principal and end piece; ^C neck, principal and end piece; ^D punctuate staining of the acrosome region and flagellum; ^E equatorial band; ^F no label; ^G punctuate staining of the whole spermatozoon; ^H equatorial band and flagellum; ^I midpiece. Percentage of each identified pattern of Tyr-phosphorylation after treating the cells with (dark) or without (white) 10 pM P gradient in: (B) spermatozoa incubated under capacitating or (C), non-capacitating conditions. Data are expressed as the mean ± SEM. ^a Significant differences vs. without P (p<0.05).

Figure 5. Sequence of chemotactic signaling events.

A and B: Percentage of oriented spermatozoa (OS) towards: (A) 10⁻⁹ M db-cAMP or (B) 10⁻¹⁰ M db-cGMP gradient in the presence of effective doses of the following inhibitors ddAdo (300 µM), ODQ (30 µM), KT5720 (100 µM), SKF96365 (50 µM), sEBSS medium without calcium, TMB-8 (10 µM); 2-APB (50 µM) and KT5823 (10 µM). C and D: Percentage of spermatozoa showing protein Tyr-phosphorylation pattern “H” after exposure to 10 pM P gradient and pre-treated with (C) ddAdo (300 µM) or KT5720 (100 µM) inhibitors and (D) ODQ (30 µM) or KT5823 (10 µM) inhibitors. E: Percentage of cells showing pattern “H” after exposure to 10 pM P, 10⁻⁹ M db-cAMP or 10⁻¹⁰ M db-cGMP gradient. HAM was assayed as a negative control. Data are expressed as the mean ± SEM. ^a Significant differences vs. without inhibitor (p<0.05). ^b Significant differences vs. HAM (p<0.05).

Figure 6. Molecular mechanisms involved in the sperm chemotaxis mediated by progesterone.

1, P-P_R interaction; 2, tmAC activation and cAMP synthesis; 3, PKA activation; 4, protein phosphorylation; 5, stored Ca²⁺ released through IP₃R channel; 6, first Ca²⁺ influx through SOC channel; 7, activation of sGC with an intracellular cGMP increase; 8, PKG activation and a second Ca²⁺ influx probably through other calcium channels; 9, change in sperm swimming direction. Data from this study are shown with black arrows while unknown connecting steps are marked with red arrows. See text for further explanation of the model.