Inhibition of bovine sperm-oocyte fusion by a monoclonal antibody recognising the TEC-2 epitope on bovine oocytes.

The TEC-2 antigenic determinant is a carbohydrate epitope located on a glycoprotein carrier molecule. In the mouse, this epitope is expressed on the zona pellucida and plasma membrane of the oocyte and is associated with the ZP2 glycoprotein and involved in the secondary sperm receptor mechanism. On the bovine oocyte expression is confined to the plasma membrane. The aim of this study was to determine the role the TEC-2 epitope plays during fertilization in the bovine species using the monoclonal antibody TEC-02. Incubating oocytes with the TEC-02 antibody prior to fertilization inhibited cleavage in a dose-dependent manner-the cleavage rate decreased as the concentration of the antibody increased. Significantly more sperm were bound to oocytes exposed to TEC-02 (12 sperm/oocyte) compared to oocytes that were not incubated with the antibody (4 sperm/oocyte). Oocytes treated with the TEC-02 antibody had a 7.5 +/- 3.2% fusion rate and no cortical granule exocytosis compared with oocytes not exposed to the antibody, with 86.5 +/- 5.8% of sperm-oocyte fusions and release of cortical granules. The block to sperm-oocyte fertilization observed in the pretreated group was overcome using intracytoplasmic sperm injection as the method of fertilization that bypassed the fusion process. Although sperm were binding to the oolemma these results suggest that fusion was not occurring and this may be due to the antibody occupying TEC-2 epitope sites involved in the fusion process. In conclusion, the TEC-2 epitope seems to be involved in sperm-oocyte interaction in the bovine species and appears to be involved specifically during the fusion events of fertilization.

Inhibition of bovine sperm-oocyte fusion by the carbohydrate GalNAc.

The TEC-2 epitope is a carbohydrate located on the plasma membrane (oolemma) of the oocyte and appears to be involved in bovine sperm-oolemma fusion. The carbohydrates N-acetylgalactosamine (GalNAc) and galactose are part of the TEC-2 epitope and this study investigated the involvement of these carbohydrates during bovine fertilization. Gametes were exposed to the carbohydrates GalNAc, galactose, and fructose, and the lectins DBA and Con A to determine whether there was an effect on fertilization. The DBA lectin recognizes the carbohydrate GalNAc, whereas Con A recognizes the carbohydrates glucose and mannose. Oocytes pretreated with the DBA lectin prior to fertilization showed a reduction in cleavage corresponding to an increase in lectin concentrations. There was a significant increase in sperm-oolemma binding although fusion was inhibited. Oocytes exposed to GalNAc prior to sperm insemination had no effect on fertilization, however, sperm pretreatment with the carbohydrate caused inhibition of fertilization, with a reduction in cleavage rates as the GalNAc concentration increased. There was also a significant decrease in sperm-oolemma fusion and a significant increase in sperm-oolemma binding. When gametes were exposed to GalNAc at the time of fertilization a similar response to that seen with sperm pretreatment was observed. The carbohydrates galactose and fructose and the lectin Con A did not affect fertilization. In conclusion, the carbohydrate GalNAc, which is associated with the TEC-2 epitope, has a specific role during bovine sperm-oolemma fusion. This study also suggests that there is a carbohydrate-binding molecule on the sperm that binds GalNAc.

Linkage between male infertility and trinucleotide repeat expansion in the androgen-receptor gene.

BACKGROUND: Androgens acting via the androgen receptor bring about stimulation and maintenance of spermatogenesis. If mutations in the androgen-receptor gene interfere with the receptor's function, this effect may partly account for impaired spermatogenesis. We aimed to find out whether expansion of a trinucleotide repeat in the androgen-receptor gene is associated with male infertility. METHODS: We analysed 67 coded semen and blood samples from a predominantly white group of male infertility patients and controls. Clinical analyses included cause of infertility, sperm count, and reproductive hormone concentrations. Analysis of trinucleotide (CAG) repeat length and point mutations in the androgen-receptor gene was done by PCR, single-stranded conformational polymorphism, and DNA sequencing. FINDINGS: Screening and characterisation of the androgen-receptor gene in 35 patients and 32 controls showed no point mutations in the gene. 30 of the infertile patients had idiopathic azoospermia or oligozoospermia, and these men had significantly longer CAG repeat tracts than controls (mean 23.2 [SE 0.7] vs 20.5 [0.3], p=0.0001). The odds of having CAG repeat lengths of 20 were six-fold higher for fertile men than for men with a spermatogenic disorder. INTERPRETATION: Our results indicate a relation between CAG repeat length in the androgen-receptor gene and the risk of defective spermatogenesis. With the use of intracytoplasmic sperm injection, this mutation could be inherited, possibly leading to an increase in male infertility in future generations. Should further elongation of the CAG repeat occur in these future generations, there is an added risk of increased severity of male infertility, and potentially an increased incidence of neurodegenerative disease.

Comparison of the expression of specific cell surface epitopes on in vitro fertilized and parthenogenetic bovine embryos.

Parthenogenetic embryos, which are produced by the spontaneous or artificial stimulation of the oocyte, partially develop in the complete absence of the male gamete but fail to produce live young in many mammalian species. The identification of developmentally regulated molecules on the cell surface of embryos has implicated their possible role in cell interactions during embryogenesis and differentiation. In this study the expression patterns of four stage-specific cell surface antigenic determinants (TEC-1, -2, -3, and -4) were investigated in both parthenogenetic and in vitro fertilized bovine embryos. When compared to embryos produced using in vitro fertilization methods the parthenogenotes, although appearing morphologically normal, differed markedly in their TEC epitope pattern of presentation. TEC-1, -2, -3, and -4 epitope presentation on in vitro fertilized embryos occurred during specific stages of preimplantation development. TEC-1 and -2 presentation was detected on oocytes and blastocysts only, TEC-3 on morulae and blastocysts, and TEC-4 on oocytes through to 8-cell embryos, with all subsequent stages negative. Parthenogenetic embryos did not show TEC-1, -2, or -3 epitope presentation whereas the TEC-4 epitope was present throughout the developmental period examined. Enzymatic cleavage of sialic acid residues on in vitro fertilized and parthenogenetic embryos resulted in presentation of the TEC epitopes during all the embryonic stages. Western blot analysis of the embryos showed the TEC epitopes to be present on all the embryonic stages examined. This study suggests the mechanisms responsible for control and presentation of each of the TEC epitopes may not be functioning the same in parthenogenetic embryos that undergo changed glycosylation or deglycosylation resulting in altered patterns of sialylation. The study also shows TEC epitope presentation may prove to be a useful indicator of parthenogenetically activated bovine embryos. J. Exp. Zool. 284:392-400, 1999.

The stage-specific expression of TEC-1, -2, -3, and -4 antigens on bovine preimplantation embryos.

The preimplantation developmental period is associated with constant changes within the embryo, and some of these changes are apparent on the embryo cell surface. For example, during transition from maternal to embryonic genome control and the compaction and differentiation of embryonic cells, the cell surface undergoes morphologic alterations that reflect changes in gene control. In order to gain insight into the events occurring during embryonic development and cellular differentiation, monoclonal antibodies specific for cell surface antigens (TEC antigens) of embryonic cells have been generated previously and shown to recognise either the carbohydrate moiety of embryoglycan or a developmentally regulated protein epitope. The TEC antigens have been identified on mouse preimplantation embryos, and their expression is specific to particular developmental stages. To determine whether these antigens are conserved in higher mammals, we examined the expression of four TEC antigens (TEC-1 to TEC-4) on in vitro-derived bovine and murine embryos during the preimplantation stage of development. It was found that bovine oocytes and embryos derived from in vitro maturation (IVM) and in vitro fertilisation (IVF) showed stage-specific expression of each of the TEC antigens investigated, with the pattern of expression overlapping but not identical to that seen in the mouse. Immunoprecipitation together with Western blot analysis showed that the TEC monoclonal antibodies recognised a single glycoprotein band with an apparent molecular weight of 70 kDa. Confocal microscopy of immunofluorescence staining of the bovine cells showed this protein to be located on the cell surface. The apparent negative expression of these TEC antigens by immunohistochemistry and immunoprecipitation at particular stages of development appears to be due to the epitopes being inaccessible to the TEC antibodies, since Western blotting revealed the TEC antigens to be present at all stages of development examined. Antibodies identifying stage-specific antigens will provide useful markers to characterise early embryonic cells, monitor normal embryonic development in vitro, and identify cell surface structures having a function in cell-cell interactions during embryogenesis and differentiation.

Enucleation by centrifugation of in vitro-matured bovine oocytes for use in nuclear transfer.

Nuclear transfer has the potential to produce large numbers of identical progeny. Current limitations of the technique are associated with the use of micromanipulation for the demanding enucleation and reconstitution procedure. With the overcoming of this limitation, increased numbers of nuclear transfer embryos could be produced. Centrifugation of bovine oocytes at 15,000 x g for 2 min resulted in the stratification of organelles within the cytoplasm which positioned the metaphase II spindle for enucleation. After removal of the zona pellucida with Pronase, the oocytes were centrifuged in a Percoll density gradient so that the oocytes were stretched apart to form cytoplasts and the metaphase II spindle was separated from the majority of oocytes. Enucleation by centrifugation efficiently produced a consistent population of enucleated cytoplasts from bovine in vitro-matured oocytes. The population of enucleated cytoplasts was enriched by exclusion of the cytoplasts that exhibited an extrusion cone containing metaphase II chromosomes 6 h after centrifugation. The enucleated oocyte cytoplasts were aggregated with blastomeres isolated from in vivo-collected morulae. The aggregated embryonic cells were electrofused to obtain nuclear transfer embryos that were placed into a sodium alginate false zona and were capable of cleavage and development in vitro. The development of nuclear transfer embryos produced through use of centrifugation and aggregation techniques was comparable with that of nuclear transfer embryos produced by micromanipulation techniques.