Establishment of human sperm-specific voltage-dependent anion channel 3 recombinant vector for the production of a male contraceptive vaccine.

Background: The aim of this study was to construct a recombinant vector of human sperm specific VDAC3 gene for production of VDAC3 antibody, which is potential as male contraception vaccine. Methods: Target fragment sequence of VDAC3 gene was obtained through amplification of human sperm VDAC3 cDNA with primers covering exon 5 to exon 8. Its PCR product in size of 435 bp was cloned to the pET101/D-TOPO expression vector (5753 bp). E. coli bacteria were transformed with this vector. Cloning of VDAC3 fragment gene to the vector was confirmed by the using of XbaI restriction enzyme and PCR colony method with primers covering exons 5-8 of the human VDAC3 gene. Results: Alignment analysis of amplified fragment covering exon 5 to exon 8 of VDAC3 gene showed 94% homology to human VDAC3 gene from databank. After cloning to the expression vector and transformation to E. coli competent cells, twelve colonies could grow in culture media. Gel electrophoresis of sliced VDAC3 recombinant vector showed a single band in the size of 6181 bp in 8 colonies. After application of PCR colony and amplicon sequencing, the result showed a single band in the size of 435 bp and fragment sequence with 94% identity to human VDAC3 gene. Conclusion: The construction of human sperm specific VDAC3 gene recombinant vector was established in this study. In the future, this recombinant vector will be used to produce VDAC3 antibody for the development of a male contraception vaccine.

Continuous exposure of three successive generations of mice to electromagnetic fields: implication on double minute frequency.

Background: Epidemiological studies indicate increased risk of leukemia, lymphoma, and brain tumor among electrical workers exposed to electromagnetic field (EMF). Other investigator reported that continuous exposure of four successive generations of mice to EMF in doses of 1 kV to 5 kV caused tumor formation in offspring. The objective of this study was to evaluate the effect of continuous exposure of three successive generations of mice (Mus musculus L) to EMF of 3 kV, 4 kV, and 5 kV and its implication of chromosomal breakage, as detected by double minute formation. Methods: Four couples of mice of Swiss Webster strain, 3-4 months of age, and 7-40 gram of body weight were exposed to EMF at the doses of 3 kV, 4 kV, and 5 kV, and one couple served as control. Double minute formation was examined in all offspring, except one couple of each group to be exposed with the same doses of EMF to get the F2 generation, and so forth until F3 generation. Twenty metaphases of chromosomes were examined and frequencies of double minute were calculated in the three generations of all group. Results: Frequencies of double minute in F1, F2, and F3 of mice exposed to EMF of 3 kV were respectively 0.78 ± 0.08; 0.83 ± 0.09; and 0.80 ± 0.05. In the 4 kV group were 0.083 ± 0.11; 0.73 ± 0.03; and 0.96 ± 0.15, and in the 5 kV group were 0.96 ± 0.25; 0.75 ± 0.05; and 0.99 ± 0.33, whereas no double minute chromosomes were noted in control group. Frequencies of the double minute in mice exposed to EMF were significantly higher than control group. Conclusions: Continuous exposure of mice during three successive generations to EMF at doses of 3 kV, 4 kV, and 5 kV causes increased chromosomal breakage as detected as double minute chromosome formation.

Polyclonal VDAC3 antibody decreases human sperm motility: a novel approach to male contraception.

Background: Voltage dependent anion channels (VDAC) mediate transport of anions, cations and ATP which play an important role in sperm motility. This study was aimed to examine the effect of polyclonal VDAC3 antiserum to human sperm motility. Methods: Polyclonal VDAC3 antiserum used in this study was produced in rabbits by immunization of VDAC3-specific synthetic peptides. Preimmunserum was collected before immunization and used for control experiment. Recognition of VDAC3 antiserum to antigen in human sperm was performed by western blot. Thirty sperm samples obtained from fertile men which had high quality of sperm motility were washed and collected by Percoll gradient. Sperm motility was assessed by means of evaluation of sperm velocity (seconds per 0.1 mm distance) and the number of unmoved sperm (million per ml) which were observed 0 minute, 30 minutes and 60 minutes after addition of VDAC3 antiserum and preimmunserum as a control. Both data were analyzed by SPSS 13.0 software. Results: VDAC3 antiserum recognized VDAC3 protein in human sperm. Statistical analysis demonstrated that there were increasing numbers of unmoved spermatozoa after addition of anti-VDAC3 antiserum in vitro for 60 minutes observation compared with preimmunserum (control). We found also that sperm velocity decreased signifi cantly after giving anti-VDAC3 antiserum in vitro for 0 minute, 30 minutes, and 60 minutes compared with pre-immunee serum (control). Conclusion: VDAC3 antiserum can decrease motility of human sperm. and may provide a novel principle of male contraception in the future.

Testosterone undecanoate and depo medroxyprogesterone acetate induced azoospermia through increased expression of spermatogenic cell caspase 3.

The administration of a combination of testosterone undecanoate (TU, a long-acting androgen) and depo-medroxyprogesterone acetate (DMPA) were investigated in term of suppression of rat sperm concentration in vivo to azoospermia through increasing activity of spermatogenic cell caspase 3. Adult Sprague Dawley rats received TU and DMPA of 2.5 mg and 1.25 mg, respectively, a regimen known to rapidly reduce intra testicular testosterone and to produce azoospermia within 12 weeks. Caspase 3 positive sperm cells increased compared with control levels during 6 weeks post-injection and increased further through 60 weeks. Immunohistochemistry for caspase 3 revealed that spermatocytes represented the predominant caspase 3 positive germ cells. Modest immunoreactivity for caspase-3 was localized to nuclear region of the germ cells of control and treated testes. Immunohistochemistry study revealed significantly increased caspase-3 expression in nuclei of germ cells during administration of TU+DMPA to rats. Additionally, the caspase 3 content was significantly increased in germ cells during rats were administered TU+DMPA (453.90±84.88 cells/200 seminiferous tubules) and caspase 3 significant increase in immunoreactivity was localized to the nuclei of spermatogonia, spermatocytes, and spermatids. Taken together, these results indicated that azoospermia due to reduced intratesticular testosterone concentration was caspase-3 activation dependent and suggested that the increase in active caspase-3 in the nucleus may be involved in the induction of decreased sperm production.