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Gratao, Ana Angelica (2007): Impaired Fertility in Transgenic Mice Overexpressing Betacellulin.. Dissertation, LMU München: Faculty of Veterinary Medicine
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Abstract

Impaired fertility in transgenic mice overexpressing Betacellulin Peptide growth factors regulate many cellular functions by autocrine, paracrine, juxtacrine or endocrine mechanisms. The epidermal growth factor (EGF)-like peptides are emerging as major players in regulating different aspects of animal and human physiology and pathology. The EGF family elicits essential actions in reproduction. For instance, different Egfr ligands have been shown to be involved in oocyte maturation and ovulation, preimplantational embryonic development, and implantation. Btc, a member of the Egf family, was initially isolated from a mouse insulinoma cell line, and it is expressed in a wide range of tissues in the mouse, with particularly high levels in the heart, lung, liver, kidney, pancreas, small intestine, colon, testis, ovary and uterus. Btc was identified as one of the Egfr ligands expressed in the mouse uterus exclusively at the time of implantation and can also participate as a mediator of luteinizing hormone (LH), prostaglandins (PGs) and progesterone receptor (PGR). Mice lacking Btc are viable, fertile and show no overt phenotype, but transgenic mice overexpressing Btc exhibit a whole array of phenotypical alterations. In the present study, Btc transgenic mice were employed to study the effects of increased growth factor levels in female and male reproduction. The observation of relatively ineffective matings involving transgenic females (non-productive matings and reduced litter size) during routine breeding led us to functionally evaluate the different stages of the reproductive process. The reduced fertility of Btc transgenic females could be attributed to one or more reproductive dysfunctions, such as a decreased ovulation, fertilization or implantation. Therefore, we have studied different aspects of Btc transgenic female’s and male’s reproduction, including puberty initiation, ovulation, in vivo and in vitro oocyte maturation, sperm parameters, in vivo and in vitro fertilization, and implantation in order to uncover the reason for their reduced fertility. Successive matings of Btc transgenic males and females mice with wild-type mice revealed a decrease in litter size as compared with litters produced by control matings. However, the interval to the first litter was not significantly different between groups. Litter size development showed a significant difference between Btc transgenic females and controls females. The onset of puberty occurred essentially at the same age in transgenic and non-transgenic females. The implantation of the Btc transgenic mice was delayed, but this was not the reason for the litter size reduction, because the mean number of total embryos either attached or recovered from the uterus of transgenic females was already markedly reduced when compared to the number of embryos present in the uterus of control females. For this reason, the explanation must be found in processes taking place before implantation (ovulation or fertilization). We evaluated the number of ovulated oocytes and observed that this parameter did not differ between the two genotypes, however, we observed a statistically significant reduction in the percentage of fertilized oocytes in transgenic as compared to control females, identifying the reason for the reduction in the litter size. Next, we carried out in vitro maturation of oocytes. The timing of nuclear maturation differed between transgenic and control oocytes. Therefore, we decided to evaluate the in vitro fertilization rate, which turned out to be impaired in the transgenic group. The expression pattern at the cellular level, studied by immunohistochemistry, revealed a high expression of Btc in the transgenic cumulus cells, which could be an explanation for the altered in vivo and in vitro fertilization. Although the fertility of Btc transgenic males appears to be impaired, these animals do not display evident alterations in sperm production. This study provides evidence that Btc overexpression does not negatively affect spermatogenesis, sperm motility, progression and concentration values. Future studies are needed to clarify whether the altered fertilization is in fact caused by the high expression of Btc in the transgenic cumulus cells. Furthermore, experiments involving the overexpression of a non-sheddable form of Btc are underway and will help to clarify the actions of precursor (membrane-bound) versus mature Egfr ligands during oocyte maturation and fertilization.