Expression of recombinant human ZP3 protein using the baculovirus expression system / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the methods and solve the technical bottlenecks in the preparation of recombinant human protein hZP3 using the baculovirus expression system and pave the technical ground for the production and application of recombinant hZP3.</p><p><b>METHODS</b>The recombinant vector pFASTBAC HTa-hZP3 was constructed and transferred to competent E. coli cells carrying bacmid to produce recombinant bacmid by homologous recombination. Sf9 cells were transfected with the recombinant bacmid to produce recombinant baculovirus. Full-length recombinant hZP3 (amino acids 1-424) and truncated recombinant hZP3 (amino acids 23-348) were expressed in the sf9 cells by infection with the recombinant baculovirus. The expression time of hZP3 was determined by Western blot and its purification was explored.</p><p><b>RESULTS</b>The recombinant bacmid and baculovirus were successfully constructed for expressing both the full-length and truncated hZP3. The maximal expression of recombinant hZP3 in the sf9 cells was achieved at 72-96 hours after baculovirus infection. Some of the recombinant hZP3 with His-tag could bind affinity matrix and got purified but most of the solubilized hZP3 passed through and the reasons remained unknown. Purified recombinant hZP3 labeled with Dylight Dye488 was able to bind human sperm.</p><p><b>CONCLUSION</b>It is feasible to express recombinant hZP3 in insect cells using the baculovirus system though the yield of hZP3 needs to be optimized. The methods for efficient enrichment and purification of recombinant hZP3 require further exploration.</p>

Reference values of semen parameters for normal fertile men in Shanghai / 中华男科学杂志

<p><b>OBJECTIVE</b>To analyze the distribution characteristics of the main semen parameters of healthy semen donors and normal fertile men in Shanghai, compare the semen quality between the two groups, and investigate the normal reference values of the semen parameters of the fertile population in Shanghai.</p><p><b>METHODS</b>We obtained semen samples from 100 healthy donors and 41 fertile men, performed semen analyses according to the WHO (2010) guidelines, and determined the semen volume, sperm concentration, sperm progressive motility, total sperm count and total progressively motile sperm count. We analyzed the distribution of the semen parameters of the normal fertile men, and obtained the lower limits of their normal reference values.</p><p><b>RESULTS</b>There were no statistically significant differences in the main semen parameters between the healthy donors and normal fertile men (P < 0.05). The lower reference limits for the semen parameters of normal fertile men in Shanghai (P < 0.05) were as follows: sperm concentration > or = 27.3 x 10(6)/ml, sperm progressive motility > or = 8.1%, semen volume > or = 0.82 ml, total sperm count > or = 44.73 x 10(6) per ejaculate, and total progressively motile sperm count > or = 24.68 x 10(6) per ejaculate.</p><p><b>CONCLUSION</b>For the evaluation of male fecundity, total sperm count and total progressively motile sperm count may be two better predictors than others.</p>

Direct fumigation for freeze-thawing of human sperm: an experimental study / 中华男科学杂志

<p><b>OBJECTIVE</b>To study the effect of direct fumigation on the post-thaw recovery rate of cryopreserved spermatozoa, and to search for a best method for human sperm cryopreservation.</p><p><b>METHODS</b>We collected semen samples from 100 donors conforming to the normal reference values in WHO Laboratory Manual for the Examination and Processing of Human Semen (5th Ed), divided them into two groups, and subjected them to cryopreservation by programmable freezing (Group A) and direct fumigation (Group B), respectively. We detected the progressive motility of pre-freezing and post-thaw sperm with a computer-assisted semen analyzer, and compared the effects of the two methods on the functional integrity of sperm membrane and the rate of abnormal sperm using the percentage of hypo-osmotic swelling sperm and modified Papanicolaou staining.</p><p><b>RESULTS</b>Statistically significant differences were found in post-thaw sperm progressive motility between the Groups A and B ([34.0 +/- 18.4]% vs [43.0 +/- 19.5]%, P<0.05), both remarkably decreased as compared with pre-freezing ([57.0 +/- 16.7]%, P<0.05). Such differences were also found in the post-thaw recovery rate of progressively motile sperm between the two groups ([52.2 +/- 20.6]% vs [67.1 +/- 20.0]%, P<0.05). The post-thaw percentage of hypo-osmotic swelling sperm was obviously decreased in both Groups A and B ([67.1 +/- 11.1]% and [70.6 +/- 10.0]%) in comparison with pre-freezing ([84.5 +/- 7.5]%, P<0.05), with significant differences between A and B (P<0.05). However, the rate of sperm abnormality was evidently increased in Groups A and B ([85.0 +/- 8.7% and [85.7 +/- 9.1]%), significantly higher than pre-freezing ([77.8 +/- 9.6]%, P<0.05), but with no significant differences between A and B (P>0.05).</p><p><b>CONCLUSION</b>Direct fumigation is superior to programmable freezing for its easier operation, wider application, and higher sperm recovery rate.</p>