From January 2004 until December 2005 patients were included in this prospective study at the department of Histology and Biology of Reproduction at Tenon Hospital.
Patient selection
Twenty-four men participated to this study. All of them were in good physical and mental health. All the men (cases and controls) have a primary infertility and are members of couples undergoing their first in vitro fertilization cycle. Patients with genital tract infection, congenital disease or treated with ACE inhibitors were excluded from this study.
The case subjects (n = 10) have normal semen parameters (number, motility, morphology) according to the WHO guidelines [18] but a total in vitro fertilization failure occurring during an IVF cycle. The causes of infertility were tubal infertility (4 cases) and unexplained infertility (6 cases).
The control subjects (n = 14) are men members of infertile couples undergoing an in vitro fertilization cycle during the same week as the case-subjects and having normal semen parameters and a fertilization rate ≥ 65%. The causes of infertility in this group were: tubal infertility (6 cases), polycystic ovary syndrome (1 case), endometriosis (5 cases) and unexplained infertility (2 cases).
All the female partners of both the case and the control subjects must have at least 3 mature oocytes inseminated with husband’s fresh ejaculated spermatozoa. These patients underwent pituitary down-regulation using a GnRH analog (Decapeptyl 0.1 mg subcutaneously per day, Ipsen, France) starting on the 21st day of the previous cycle and continuing until the day of hCG administration. When E2 levels fell below 40 pg/ml, patients underwent stimulation with FSH (Gonal F, Merck Serono, France). Follicular development was monitored by E2 levels and transvaginal ultrasound measurements daily, beginning 5 to 6 days of stimulation. An injection of hCG (Ovitrelle, Merck Serono, France) was given when at least 3 leading follicles reached a mean diameter ≥17 mm and the serum E2 level exceeded 250 pg/ml per follicle. Transvaginal follicular aspiration was performed under ultrasound guidance 36 hours after the administration of hCG.
Samples
Informed consent was obtained from all subjects for using their semen and blood samples in this study. The study was approved by the local Ethical CPP (Comité de Protection des Personnes).
Samples were collected in the IVF laboratory of Tenon Hospital. Semen samples were obtained at day 1 after sperm insemination (when assessing oocytes’fertilization) or later by calling the patients for a new semen sample.
At day 1 after sperm insemination, for each patient, the entire remained semen sample and the pellet obtained after density gradient are pooled so that we collect the maximum of spermatozoa to have a sufficient protein extract later. Then, spermatozoa were separated from seminal plasma by centrifugation (10 min, 12000 rpm). The supernatant is removed and 1 ml PBS (Phosphate Buffer Solution) is added to the pellet. A second centrifugation is carried, and just after, the supernatant is removed and the final pellet is frozen at −80°C.
If a semen sample is not obtained at day one after IVF, the patient is asked for a new semen sample after a sexual abstinence of 3 to 5 days. After a spermatozoa count, the semen is centrifuged, the supernatant removed and the pellet frozen at −80°C.
Centrifugation and supernatant removal allow us to eliminate sACE normally present in the seminal plasma.
Protein extraction
Frozen pellets of spermatozoa were thawed, resuspended in cold PBS and centrifuged twice (5 min, 12000 rpm). The supernatant was removed and spermatozoa were extracted by mixing the last pellet with 500 μl RIPA1X buffer and grinding them. After a last centrifugation, the supernatant was removed and stored at −80°C. The supernatants containing ACE were quantified by Biorad Protein Assay (Biorad, France).
Gel electrophoresis and protein blotting
Ten to 20 μg of total proteins were subjected to 7.5% SDS-PAGE (Biorad, France), and blotted onto polyvinylidene difluoride membrane (Biorad, France). A prestained protein ladder (BenMark Pre-stained Protein Ladder, Invitrogen, France) was used to determine the molecular weight of the protein. So together with anti-ACE antibody specificity and molecular weight (100 kDa) we are sure that it is the testicular isoform, the somatic one is 170 kDa and is absent or too weak in the protein extracts. After blocking with 5% dried milk in PBS-T (0.05% Tween 20 in PBS), the membrane was incubated with a diluted (1/100th) goat anti ACE polyclonal antibody (ACE C20 peptide, Sc-12187, TEBU-BIO SA, France) at 4°C overnight. This antibody have been previously tested for its cross reactivity by using the 293 T whole cell lysate as a positive control (Santa Cruz Biotechnology, France) and the sc-12187 Blocker Peptide (Santa Cruz Biotechnology) as a negative one. The membrane was washed three times (10 min for each) in PBS-T, and incubated with rabbit anti-goat (1/10000th) (Rabbit anti-goat IgG HRP conjugate, Sc-2768, TEBU-BIO SA, France) for 1 hour at room temperature. After washing 3 times in PBS-T, the immunoreactive signal was detected by using ECL™ Western Blotting Detection Reagents (Amersham, France). These membranes were then incubated in a tris-SDS-β-mercaptoethanol (30 min in a wet chamber at 50°C). After blocking as mentioned above, the membrane was incubated with a diluted goat (1/500th) anti Pyruvate Kinase (standard protein) polyclonal antibody (Biogenesis, France) overnight at 4°C. Incubation with the secondary antibody (the same as the one used for ACE) and detection of the immunoreactive signal were done as mentioned above.
DNA extraction and PCR
A total of 10 case-subjects and 14 control-subjects were screened for PCR of the tACE gene. Genomic DNA was extracted from peripheral blood using QIAamp Blood Kit (Qiagen, France). Specific primers were designed to amplify each of the 14 exons of the tACE. The primer sequences are available upon request.
The PCR amplification was carried out in a total volume of 30 μl reaction mixture containing MgCl2 (1.5 mM), 0.1 mM of each dNTP, 100 ng of genomic DNA, 0.11 μm of each primer (primers are previously diluted at a 3.3 μM concentration) and 1 IU of gold Taq DNA polymerase. We used a touch-down PCR. The cycling profile consisted of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds except for the first cycle where denaturation was extended to 9 minutes.
Direct DNA sequencing
Purified PCR products were submitted to direct sequencing. Sequencing reaction were performed in forward and reverse orientations using the ABI BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems, France).
Statistical analysis was performed using chi-squared test as a test of significance. A p ≤ 0.05 was considered as significative.