Impaired Fertility in Transgenic Mice Overexpressing Betacellulin.

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.