Expression of progesterone receptor(s) during capacitation and incidence of acrosome reaction induced by progesterone and zona proteins in boar spermatozoa.
Sperm acrosome reaction (AR) is a prerequisite step for in vivo fertilization. In the vicinity of the oocyte, zona protein(s) (ZP) and progesterone (P4), a component of follicular fluid, are proven to be responsible for physiological AR induction. In the present study, a thorough analysis of the role of the progesterone receptor (PR) in this processing including in vitro physiological studies and biochemical isolation and characterization of the receptor protein was conducted. Following capacitation for 0, 2, 4 and 6h, pooled fertile boar semen samples (n=6) with >70% sperm motility were labeled with P4-BSA-FITC (100 microg/ml) to detect the activation of PR. Parallel sperm samples were treated with P4 (10 microg/ml) for 20 min to test AR inducing efficiency at different time points. To compare the ability of ZP and P4 to induce AR, spermatozoa capacitated in a modified medium supplemented with 1mg/ml heparin for 4h, were then treated with heat solubilized ZP (150 microg/ml), P4 (10 microg/ml) or ZP+P4 for 20 min. FITC-peanut agglutinin staining was applied to observe the disrupt acrosomal morphology. A purification protocol for crude boar sperm membrane proteins was developed based on ligand-receptor affinity chromatography procedures. The PR proteins were then identified by using mAb C262 raised against intracellular PR, combined with second antibody (SDS-PAGE, Western blotting). Their N-terminal amino acid sequence was determined. The amount of PR-activated spermatozoa was enhanced with time (onset: 27+/-5%, 2h: 41+/-4%, 4h: 49+/-3% and 6h: 52+/-4%, mean+/-S.E., n=6) as evidenced by increasing percentage of spermatozoa with completed cap fluorescent staining. In parallel sperm samples, percentages of AR induced by P4 were 9+/-2, 14+/-2, 18+/-2, and 24+/-2%, respectively. In solvent control at all time points, less than 10% spermatozoa had undergone AR. Capacitation for 4h or greater time periods resulted in optimal percentage of PR-activated and acrosome-reacted spermatozoa. After sperm incubation in heparin-medium, ZP+P4 treatment induced greater amounts of AR than either P4 or ZP alone (13+/-1% compared with 8+/-1 and 10+/-1%, P<0.01). Inducing capacity of P4 was comparable to that of ZP. The molecule weights of two apparent PR molecular masses were detected to be at Mr 74 kDa and Mr 63 kDa. N-terminal amino acid sequence of 74 kDa protein was XPXNIVLIFADXLXY, which had 78% homology to arylsulfatase A and 88% homology to 72 kDa protein from boar spermatozoa. The activation of PR is associated with the capacitating process and that appears to be required for P4-induced AR. P4 and ZP appear to be equally capable of independently inducing the AR but lack synergetic or additive effects in this induction process. Both might represent alternative pathways thus resulting in alternative systems for induction of the prerequisite acrosomal exocytosis (supported by NSC 90-2313-B-005-114; 91-2313-B-005-131).
Wu
Jui-Te
JT
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, 250 Ko-Koung Road 402 Taichung City, Taiwan.
Chiang
Kai-Chieh
KC
Cheng
Feng-Pang
FP
D020101:Acrosome Reaction / Q000187:drug effects* / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D008297:Male; D008970:Molecular Weight; D011374:Progesterone / Q000494:pharmacology*; D011980:Receptors, Progesterone / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013075:Sperm Capacitation / Q000502:physiology*; D013081:Sperm Motility; D013552:Swine / Q000502:physiology*
D011980:Receptors, Progesterone; D011374:Progesterone