Progesterone activates the cyclic AMP-protein kinase A signalling pathway by upregulating ABHD2 in fertile men

Abstract

Objective: This was a prospective study to investigate whether progesterone affects sperm activity by regulating the cyclic AMP-protein kinase A (cAMP-PKA) signalling pathway via α/β hydrolase domain-containing protein 2 (ABHD2).

Methods: Spermatozoa were collected from healthy and infertile men (with oligoasthenospermia or abnormal acrosome; n = 30/group). The expression of and mutations in ABHD2 were detected by quantitative PCR, western blot, and gene sequencing. The expression of ABHD2 in the presence of progesterone was detected in all groups, and cAMP and PKA levels were detected by ELISA in fertile men after treatment with ABHD2 antibody and PKA inhibitor H-89, respectively.

Results: Expression of ABHD2 mRNA and protein were reduced in spermatozoa from infertile compared with fertile men. Four gene mutation sites were detected in spermatozoa from the infertile groups. Progesterone increased mRNA and protein levels of ABHD2 in healthy spermatozoa but not in spermatozoa from infertile men. The levels of cAMP and PKA were increased by progesterone in healthy spermatozoa, and the progesterone-increased cAMP and PKA were decreased by ABHD2 antibody and H-89, respectively.

Conclusion: Progesterone regulates the ABHD2-mediated cAMP-PKA signalling pathway in healthy spermatozoa, which provides a new target for clinical diagnosis and treatment of infertility.

Keywords: Sperm; abhydrolase domain containing 2; acylglycerol lipase; cyclic AMP-protein kinase A signalling pathway; gene mutation; oligoasthenospermia; progesterone.

Figure 1.

Expression of ABHD2 in spermatozoa from men in the fertile control, oligoasthenospermia, and abnormal acrosome groups. (a) Quantitative reverse transcription-PCR detection of ABHD2 mRNA; (b) western blot detection and quantitation of ABHD2. n = 30 per group; **p < 0.01; error bars represent standard deviations. ABHD2, abhydrolase domain containing 2, acylglycerol lipase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 2.

Mutation sites of ABHD2 gene in spermatozoa from men in the fertile control, oligoasthenospermia, and abnormal acrosome groups, including a C to G at position 480 in the oligoasthenospermia group, and a G deletion at 477 and a CC deletion at bases 804 and 805 in the abnormal acrosome group. No mutations in ABHD2 were detected in the healthy control group. ABHD2, abhydrolase domain containing 2, acylglycerol lipase.

Figure 3.

Effect of progesterone on expression of ABHD2 in spermatozoa from men in the fertile control and oligoasthenospermia groups. Spermatozoa were treated with progesterone for 2 hours. (A) Western blot; (b) quantitative data. n = 30 per group; **p < 0.01; error bars represent standard deviations. ABHD2, abhydrolase domain containing 2, acylglycerol lipase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 4.

Levels of PKA and cAMP in spermatozoa in the presence of progesterone. Spermatozoa were treated with progesterone with or without ABHD2 antibody or PKA inhibitor H89 for 2 hours. (A) ELISA detection of cAMP, (B) cell PKA activity. n = 10 per group, *p < 0.05; **p < 0.01; error bars represent standard deviations. PKA, protein kinase A; cAMP, cyclic adenosine monophosphate; ABHD2, abhydrolase domain containing 2, acylglycerol lipase; NADH, nicotinamide adenine dinucleotide.

Figure 5.

Sperm acrosome reaction was detected by FITC-PSA staining. Spermatozoa from fertile controls were treated with progesterone with or without ABHD2 antibody or the PKA inhibitor H89 for 2 hours. (A) Confocal images (arrows indicate the differences between spermatozoa); and (B) quantification of intensity. **p < 0.01; error bars represent standard deviations. FITC, fluorescein isothiocyanate; PSA, Pisum sativum agglutinin; ABHD2, abhydrolase domain containing 2, acylglycerol lipase; PKA, protein kinase A.