Studies on the role of specific molecules in the human fertilization process and additional assessments of potential applications for these proteins are hampered by the limited amount of available biological material. However, this drawback might be circumvented by the recent cloning of several gamete-specific genes, which opens possibilities for the production of recombinant proteins. By use of cDNA and genomic DNA fragments of the human ZP3 gene, which encodes a major constituent of the zona pellucida surrounding the oocyte, a 2.7-kb minigene was constructed containing the natural third and fourth introns of the gene and a truncated intron between exons 2 and 3. This ZP3 DNA was transfected to Chinese hamster ovary cells, and a single-cell clone producing the recombinant ZP3 protein (recZP3) was generated. Western blot analysis of culture medium from these cells showed that recZP3 has a molecular mass +/- 5 kDa smaller than that of natural ZP3. Under reducing conditions, it migrates at an apparent molecular mass of 55-60 kDa. RecZP3 induced the sperm acrosome reaction and promoted fusion of human spermatozoa with zona-free hamster oocytes, indicating that the recombinant protein is biologically active. RecZP3 provides an attractive tool for studying the initial stage of the human fertilization process. Furthermore, it might have clinical applications in the development of diagnostic tests for male infertility and serve as target antigen in the design of contraceptive vaccines.
Bovine follicular fluid (bFF) injected ip in mice during 2 days (65,000 U inhibin/day, 1 U inhibin the activity in 1 micrograms bFF protein) caused a significant decrease in the numbers of A4, intermediate (In), and B spermatogonia to 91%, 74%, and 67% of the control values, respectively. The numbers of undifferentiated spermatogonia remained unchanged. These injections suppressed peripheral FSH levels to 6% of the control values, suggesting that FSH might be the modulator of the effects on spermatogenesis. However, in the Chinese hamster, intratesticular injections of bFF during 4 days (6500 U inhibin/day into one testis) also caused a significant decrease in the numbers of A3. In, B1, and B2 spermatogonia to 86%, 61%, 55%, and 94% of the control values, respectively. Similarly, treatment with a partially purified inhibin preparation from rat Sertoli cell-conditioned medium (rSCCM) during 4 days (Mono Q fraction; 1512 U inhibin/day; 37.8 micrograms protein) caused a significant decrease in the numbers of A3, In, B1, and B2 spermatogonia to 90%, 87%, 66%, and 93% of the control values, respectively. Treatment with a highly purified inhibin preparation from rSCCM during 4 days (30K inhibin; 750 U inhibin/day; 100 ng protein) significantly decreased the numbers of In and B1 spermatogonia to, respectively, 87% and 91% of the control values. These effects were limited to the testis into which the material was injected; the contralateral testis or testes injected with control fluid always showed normal numbers of spermatogonia. This implies that the effects on the seminiferous epithelium are not FSH mediated. Intratesticular injections of bFF or pure inhibin did not affect the number of undifferentiated spermatogonia. However, the Mono Q fraction caused a significant increase in the numbers of undifferentiated spermatogonia in stages IV-VII of the cycle, suggesting the presence of a mitogenic factor for undifferentiated spermatogonia in rSCCM which is not present or is counteracted in bFF. The results suggest that inhibin may have a role in the regulation of spermatogonial development in the adult animal.
The development of a contraceptive vaccine based on a gamete-specific antigen requires knowledge of the ability of the antigen to elicit an immune response that inhibits fertilization. A well-defined immune response, as elicited by a synthetic peptide comprising a dominant B-cell epitope coupled to a common promiscuous T-cell epitope, might be preferable. In this study, the immunodominant B-cell epitope of sperm antigen Sp17 has been identified and synthesized as a chimeric peptide with the promiscuous T-cell epitope bovine RNase[94-104] at the N terminal. Immunization of female BALB/c mice with this peptide induced a dose-dependent reduction in fertility. Although antibodies to recombinant and native Sp17 were elicited in these mice, there was no strict correlation between the level of these antibodies and the reduction in fertility. Moreover, the induction of infertility was strain-specific since no effect on fertility could be induced in B6AF1 mice. To understand the mechanism behind this apparent strain-specific infertility induction, a more extended study on both the humoral and the cellular immune response to the chimeric peptide was performed. The antigen-specific T-cell response and the levels of antigen-specific cytokines are the major factors that affect fertility outcome.
Inhibin bioactivity and mRNA for inhibin subunits were measured in four dog Sertoli cell tumours and in the testes of five normal control dogs. The tumours contained increased levels of inhibin (P less than 0.05) and mRNA for the alpha and beta B subunits when compared with controls, whereas the mRNA for the beta A subunit was not detected in tumours or control testes. The inhibin bioactivity was associated with a 32 kDa molecule in both Sertoli cell tumours and normal dog testes; no higher molecular weight forms were found after sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Peripheral levels of immunoassayable inhibin in dogs with Sertoli cell tumours were higher than those in the controls (P = 0.01), indicating that it might be possible to use this parameter as a marker for Sertoli cell tumours. Other testicular tumours, however, might also secrete immunoactive inhibin. The increased inhibin concentrations are likely to be the cause of the suppressed peripheral levels of FSH (P less than 0.02). However, peripheral levels of LH (P less than 0.02) and testosterone (P less than 0.01) were also suppressed in the dogs with Sertoli cell tumours, whereas the concentrations of oestradiol in the peripheral plasma of both groups did not differ. Finally, i.v. injection of the LHRH agonist buserelin caused a significant increase in LH and testosterone in the control dogs, but not in the dogs with Sertoli cell tumours. It was concluded that secretory products from the Sertoli cell tumours suppressed pituitary gonadotrophin secretion. It is unlikely that testosterone or oestradiol play a role in this respect. FSH may be suppressed by the high levels of inhibin in tumour-bearing dogs, but it remains unclear whether inhibin or another Sertoli cell product is responsible for the unresponsiveness of the pituitary gland to LHRH and the suppression of LH.
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