[Correlation between ERp57 and pregnancy of intrauterine insemination].

OBJECTIVE: To investigate the association between the pregnancy rate of intrauterine insemination (IUI) and the ERp57 expression level in donor sperm. METHODS: Forty-two sperm samples were divided into 3 groups according to the pregnancy rate: Group A (n = 16, pregnancy rate = 0), B (n = 13, pregnancy rate 10%-20%) and C (n = 13, pregnancy rate > or = 20%). The optical density (OD) was normalized to the beta-tubulin band for the evaluation of the ERp57 level. RESULTS: OD (ERp57/Tubulin) was 0.95 +/- 0.24 in Group A, 1.33 +/- 0.43 in Group B and 1.33 +/- 0.39 in Group C. The ERp57 expression level was significantly lower in Group A than in B and C (P < 0.05), with no significant differences between the latter two groups. CONCLUSION: The ERp57 expression level in donor sperm could be used as an index to predict the pregnancy rate of IUI and to avoid IUI failure by removing low-level donor sperm.

Preparation and immunogenicity of tag-free recombinant human eppin.

Human epididymal protease inhibitor (eppin) may be effective as a male contraceptive vaccine. In a number of studies, eppin with an engineered His(6)-tag has been produced using prokaryotic expression systems. For production of pharmaceutical-grade proteins for human use, however, the His(6)-tag must be removed. This study describes a method for producing recombinant human eppin without a His(6)-tag. We constructed plasmid pET28a (+)-His(6)-tobacco etch virus (TEV)-eppin for expression in Escherichia coli. After purification and refolding, the fusion protein His(6)-TEV-eppin was digested with TEV protease to remove the His(6)-tag and was further purified by NTA-Ni(2+) affinity chromatography. Using this procedure, 2 mg of eppin without a His(6)-tag was isolated from 1 l of culture with a purity of >95%. The immunogenicity of the eppin was characterized using male Balb/c mice.

Localization of epididymal protease inhibitor in adult rat and its transcription profile in testis during postnatal development.

To investigate the expression pattern of rat Eppin (epididymal protease inhibitor; official symbol Spinlw1), we detected mRNA transcripts and subsequent protein translation of Eppin in several sorts of tissues by RT-PCR and western blotting. Then immunohistochemistry was performed for more detailed observation. The testicular transcription level was monitored by real-time PCR throughout postnatal development. We found that rat Eppin was specifically expressed in the testis and epididymis. The testicular transcription was slight in neonatal (1-day) and infantile stages (5-, 7- and 10-day). It increased sharply thereafter, with maximum expression level (about 38-fold compared with that of 1-day old rat) detected in prepubertal stage (15-day). Then a slightly declined but stable level (about 20-fold compared with that of 1-day old rat) was kept in pubertal-early adult (30-day) and adult (60-day) stages of postnatal maturation. In the adult rat, EPPIN protein was mainly localized in the elongated spermatids and epididymal epithelial cells. Sperm in the epididymal duct were all covered with EPPIN and its level kept constant during incubation under conditions used to achieve capacitation. Its stage-specific expression in the testis suggests that EPPIN may be important during spermatogenesis especially for the spermatid elongation. The abundant production of epididymal EPPIN indicated indirectly that it might play a role in the function of the epididymis.