Regulation of hyperactivation of hamster spermatozoa by progesterone

Abstract

Aim: Although it is accepted that progesterone (P) induces acrosome reaction through non-genomic regulation, it is not well known if P also affects hyperactivation of sperm. Methods: Hamster spermatozoa were hyperactivated by incubation for 4 h on modified Tyrode's albumin lactate pyruvate medium and recorded on a DVD via a charge-coupled device camera attached to a microscope with phase-contrast illumination and a small CO₂ incubator. Phosphorylation of proteins was detected by western blotting using antiphosphotyrosine antibodies. Results: Sperm hyperactivation was significantly increased and accelerated by a non-genomic signal of P. Although acceleration of motility of hyperactivated sperm occurred with 10, 20 and 40 ng/mL P, the most effective concentration was 20 ng/mL. Progesterone also significantly increased 80-kDa tyrosine phosphorylation of sperm proteins. Both extracellular Ca²⁺ and albumin were essential for sperm hyperactivation, and the former was also essential for maintaining sperm flagellar movement. Moreover, phospholipase C (PLC) was associated with the regulation of hyperactivation by P. Conclusion: It is likely that P regulates sperm hyperactivation by a non-genomic signal in relation to tyrosine phosphorylation and PLC. (Reprod Med Biol 2008; 7: 63-74).

Keywords: capacitation; hyperactivation; non‐genomic regulation; progesterone; spermatozoa.

Figure 1

Effects of progesterone (P) on hamster sperm hyperactivation. (a) Significant increase with 20 ng/mL P. Values are means ± standard deviation. Modified Tyrode's albumin lactate pyruvate (mTALP) + 0.1% ethanol (EtOH) (vehicle, ), mTALP + 20 ng/mL P + 0.1% EtOH (P; ) and mTALP + 20 ng/mL P + 23.4 µmol/L RU486 + 0.1% EtOH (P and RU486, ). ^aSignificantly different from vehicle (P < 0.01); ^bsignificantly different from P and RU486 (P < 0.01). (b) Sperm hyperactivation significantly increased by P in a concentration‐dependent manner. Values are means ± standard deviation. Vehicle (), 10 ng/mL P (), 20 ng/mL P () and 40 ng/mL P (). ^aSignificantly different from vehicle (P < 0.01); ^bsignificantly different from 10 ng/mL P (P < 0.01); ^csignificantly different from 40 ng/mL P (P < 0.01); ^dsignificantly different from vehicle (P < 0.05). (c) Sperm hyperactivation increased by non‐genomic signals of P. Values are means ± standard deviation. Vehicle (), 20 ng/mL P (), mTALP + 7 nmol/L fluorescein isothiocyanate and bovine serum albumin conjugated progesterone + 0.1% EtOH (FITC/BSA‐P, ) and mTALP + 7 nmol/L FITC/BSA‐P + 23.4 µmol/L RU486 + 0.1% EtOH (FITC/BSA‐P and RU486, ). ^aSignificantly different from vehicle (P < 0.01); ^bsignificantly different from FITC/BSA‐P and RU486 (P < 0.01); ^csignificantly different from FITC/BSA‐P and RU486 (P < 0.05). Experiments were carried out four times using four hamsters.

Figure 2

Binding of progesterone (P) to sperm heads. (a,d) Hamster spermatozoa incubated in modified Tyrode's albumin lactate pyruvate (mTALP) with 3.5 nmol/L fluorescein isothiocyanate and bovine serum albumin conjugated progesterone (FITC/BSA‐P), which converted into approximately 10 ng/mL P, and 0.1% ethanol (EtOH). (b,e) Hamster spermatozoa incubated in mTALP with 7 nmol/L FITC/BSA‐P and 0.1% EtOH. (c,f) Hamster spermatozoa incubated in mTALP with 14 nmol/L FITC/BSA‐P, which converted into approximately 40 ng/mL P, and 0.1% EtOH. (g,i) Hamster spermatozoa incubated in mTALP with 7 nmol/L FITC/BSA‐P, 23.4 µmol/L RU486 and 0.1% EtOH. (h,j) Hamster spermatozoa incubated in vehicle. (a–c,g,h) Observed under a light field; (d–f,i,j) observed under a fluorescent field. Fluorescence of the mitochondria sheath in the flagellum was autofluorescence. Bar represents 100 µm.

Figure 3

Acceleration and increasing of tyrosine phosphorylation of 80‐kDa sperm proteins by progesterone (P). (a) Typical Coomassie Brilliant Blue‐stained membrane after blotting. (b) Western blotting against proteins obtained from spermatozoa that were incubated in vehicle. (c) Western blotting against proteins obtained from spermatozoa that were incubated in 20 ng/mL P (P). (d) Western blotting against proteins obtained from spermatozoa that were incubated in 20 ng/mL P and RU486 (P and RU486). Arrows show tyrosine phosphorylation of 80‐kDa sperm proteins. Numbers on the left‐hand side indicate the molecular weight standard of 80 kDa. (e) Density of upper bands detected on (b–d). (F) Density of lower bands detected on (b–d). ^aSignificantly different than vehicle (P < 0.01); ^bsignificantly different than vehicle (P < 0.05); ^csignificantly different than P and RU486 (P < 0.01). Lanes a–f and g–l illustrate the results from urea extract and urea–thiourea extract, respectively. Lanes a and g were incubated for 0 h after supplying P. Lanes b and h were incubated for 0.5 h, lanes c and i were incubated for 1 h, lanes d and j were incubated for 2 h, lanes e and k were incubated for 3 h, and lanes f and l were incubated for 4 h.

Figure 4

Effects of Ca²⁺ on sperm motility and hyperactivation. Rates of motile spermatozoa (a) and hyperactivated spermatozoa (b). Values are means ± standard deviation. Vehicle (), 20 ng/mL P (P, ), modified Tyrode's albumin lactate pyruvate (mTALP) without Ca²⁺ and with added 1 mmol/L ethyleneglycol bis(2‐aminoethyl ether)tetraacetic acid (EGTA) + 0.1% EtOH (Ca²⁺(–), ), mTALP without Ca²⁺ and with added 20 ng/mL P + 1 mmol/L EGTA + 0.1% EtOH (Ca²⁺(–) and P, ). ^aSignificantly different from vehicle (P < 0.01); ^bsignificantly different from P (P < 0.01); ^csignificantly different from vehicle (P < 0.05); ^dsignificantly different from P (P < 0.05). Experiments were carried out four times using four hamsters.

Figure 5

Effects of albumin on sperm hyperactivation. Values are means ± standard deviation. Vehicle (), 20 ng/mL P (P, ), modified Tyrode's albumin lactate pyruvate (mTALP) without bovine serum albumin (BSA) and with added 0.1% ethanol (EtOH) (BSA(–), ) and mTALP without BSA and with added 20 ng/mL P + 0.1% EtOH (BSA(–) and P, ). ^aSignificantly different from vehicle (P < 0.05); ^bsignificantly different from P (P < 0.05); ^csignificantly different from vehicle (P < 0.01); ^dsignificantly different from P (P < 0.01). Experiments were carried out four times using four hamsters.

Figure 6

Effects of phospholipase C (PLC) inhibitors on sperm hyperactivation. (a) U73122, which is an inhibitor of PLCδ, and U73343, which is a negative control of U73122, were added to the modified Tyrode's albumin lactate pyruvate (mTALP) medium. Values are means ± standard deviation. Vehicle (), 1 µmol/L U73122 (U73122, ), 1 µmol/L U73343 (U73343, ), 20 ng/mL P (P, ), 20 ng/mL P + 1 µmol/L U73122 (P and U73122, ) and 20 ng/mL P + 1 µmol/L U73343 (P and U73343, ). (b) D609, which is an inhibitor of phosphatidylcholine‐PLC (PC‐PLC), was added to the mTALP medium. Values are means ± standard deviation. Vehicle (), 10 µmol/L D609 (D609, ), 20 ng/mL P (P, ), 20 ng/mL P + 10 µmol/L D609 (P and D609, ). (c) ET‐18‐OCH3, which is an inhibitor of phosphatidylinositol‐PLC (PI‐PLC), was added to the mTALP medium. Values are means ± standard deviation. Vehicle (), 15 µmol/L ET‐18‐OCH3 (ET‐18‐OCH3, ), 20 ng/mL P (P, ) and 20 ng/mL P + 15 µmol/L ET‐18‐OCH3 (P and ET‐18‐OCH3, ). (d) Neomycine, which is a non‐specific inhibitor of PLC, was added to the mTALP medium. Values are means ± standard deviation. Vehicle (), 65 µmol/L neomycine (Neomycine, ), 20 ng/mL P (P, ) and 20 ng/mL P + 65 µmol/L neomycine (P and Neomycine, ). (e) Spermine, which is an inhibitor of PLCα and activator of PLCδ, was added to the mTALP medium. Values are means ± standard deviation. Vehicle (), 1 mmol/L spermine (Spermine, ), 20 ng/mL P (P, ) and 20 ng/mL P + 1 mmol/L spermine (P and Spermine, ). ^aSignificantly different from vehicle and inhibitor (P < 0.01); ^bsignificantly different from P and inhibitor (P < 0.01); ^csignificantly different from vehicle and inhibitor (P < 0.05); ^dsignificantly different from P and inhibitor (P < 0.05). Experiments were carried out four times using four hamsters.

Figure 7

Regulatory mechanism of the acrosome reaction (AR) and hyperactivation induced by progesterone (P). Dotted arrows indicate the regulatory signals of the AR induced by P. Arrows indicate the regulatory signals of hyperactivation induced by P. A high concentration of P stimulates Phospholipase Cδ (PLCδ) and induces the AR. A low concentration of P stimulates PLC and induces hyperactivation. AC, adenylate cyclase; CAMK, calmodulin‐dependent protein kinase; cAMP, cyclic adenosine monophosphate; IP3R, inositol 1,4,5‐trisphosphate receptor; PC, phosphatidylcholine; PI, phosphatidylinositol; PKA, protein kinase A; PLC, phospholopase C; PR, progesterone receptor; PTK, protein tyrosine kinase.