Estrogen Receptor Null Mice - Endocrine Reviews

380 COUSE AND KORACH Vol. 20, No. 3
(�5.7) oocytes per female in the wild-type and heterozygous
animals, respectively (Table 3) (47). Additionally, the cumulus
mass that surrounded the ovulated follicles from the �ERKO
females was consistently composed of a decreased number of
cells and a lessened integrity when compared with ova yielded
from wild-type controls. Most interesting was the histology of
the ovaries from the superovulated �ERKO females, which
indicated the presence of numerous preovulatory but unruptured
follicles (Fig. 5). It therefore appeared that the follicles of
the �ERKO ovary were able to respond to the proliferative
effects of PMSG in terms of increased size and antrum formation.
However, a severe deficit in the response to the gonadotropin
surge (hCG), required to induce luteinization and rupture
of the follicle, was obvious in the �ERKO. A small number
of the selected follicles were able to be expelled, as evidenced
by the presence of ova in the oviduct and of corpora lutea in the
corresponding �ERKO ovary. Therefore, a lack of ER� resulted
in a drastic reduction in ovulatory capacity, yet with incomplete
penetrance. Until �ERKO females are treated and tested in a
similar manner to the �ERKO studies described, it will be difficult
to determine the precise role for each ER in the ovary.
The observation of numerous unruptured Graafian follicles
in the ovaries of superovulated �ERKO females is strikingly
similar to the phenotype reported for mice possessing
a targeted disruption of the cyclin-D2 gene (246). Cyclin-D2
is a positive regulator of cell cycle progression that is highly
expressed in granulosa cells (reviewed in Ref. 258). Robker
and Richards (259) demonstrated strong up-regulation of the
cyclin-D2 gene by estradiol in rat granulosa cells and suggested
that this protein may be a downstream mediator of the
synergistic actions of FSH and estradiol that result in increased
granulosa cell numbers in the maturing follicle. The
dramatic increases in cyclin-D2 induced by estradiol are evident
in vivo at both the mRNA and protein levels in the rat.
Furthermore, assays on primary granulosa cell cultures from
the rat indicate that estradiol stimulation of the cyclin-D2
gene can be inhibited by the estrogen antagonist, ICI-164,384,
strongly suggesting that it is an ER-mediated process (259).
Therefore, given that ER� is the predominant form of ER in
the granulosa cells, disruption of the ER� gene may likely
result in significant deficits in cyclin-D2 expression in the
granulosa cells of the growing follicles in the �ERKO female.
However, FSH is also able to stimulate increases in cyclin-D2,
although the temporal pattern of regulation by FSH is distinct
from that elicited by estradiol (259). Nonetheless, it is
possible that FSH action in the follicles of the �ERKO ovary
have provided for some degree of cyclin-D2 expression and
thereby may explain the incomplete penetrance of the
�ERKO ovarian phenotype.
The �ERKO ovarian phenotype that becomes apparent
after superovulation is also similar to that reported for
knockout models of the genes for the PR (PRKO) (44) and
prostaglandin synthase-2 (189). The dramatic increases in
both PR (260) and prostaglandin synthase-2 (261) in the granulosa
cells of the ovulatory follicle shortly after the gonadotropin
surge have been well documented. Furthermore, the
lack of follicular rupture in the respective knockout models
supports a critical role for each of these components in ovulation
(reviewed in Ref. 262). Lydon et al. (44) reported infertility
in the PRKO and a consistent inability of super-
physiological doses of hCG to induce follicular rupture.
Although regulation of the PR gene is strongly influenced by
estradiol in the uterus (see Section III.A), sufficient evidence
exists to indicate that this may not be the case in the ovary.
For example, although the wild-type ovary possesses extremely
high intraovarian levels of estradiol and the presence
of ER�, levels of PR mRNA and protein remain at a modest
basal level in granulosa cells of the pre- and antral follicle
(260). However, within 4–6 h after the gonadotropin surge,
transcription of the PR gene has peaked at levels several fold
that before the surge, only to return to near-basal levels
within 20 h (260). The mechanism of this strong and transient
induction of the PR gene by LH is known to include significant
increases in intracellular cAMP, but may also involve
phosphorylation of ER� and/or a coactivator, which then
combine to act in a synergistic nature (262). Therefore, the
elevation in PR levels in the granulosa cells of the ovulatory
follicle that is critical to follicular rupture may be attenuated
in the �ERKO ovary.
Another possibility for a lack of spontaneous ovulation in
the �ERKO female may be not intaovarian in nature, but
rather due to altered gonadotropin synthesis and secretion
from the hypothalamic-pituitary axis. The sex steroids play
an important role as a positive regulator of the preovulatory
surge (reviewed in Refs. 263 and 264). Although the exact
mechanism of action by which estrogens may be involved is
not well defined, studies have shown that estradiol can induce
GnRH release from the hypothalamus as well as cause
increases in the level of GnRH receptors in the anterior pituitary
(263). The ER� may be the predominant form of ER
in the pituitary of the adult female mouse (93); however, both
ER� and ER� have been detected in various regions of the
hypothalamus (88, 97, 265). Preliminary data in the �ERKO
female indicate that tonic levels of serum LH are within the
normal range. However, a lack of hypothalamic ER� may
have reduced the potential for positive regulation by estradiol
in the hypothalamic-pituitary axis and thereby may
result in a reduction in the frequency and/or amplitude of
the preovulatory gonadotropin surge. Nonetheless, the results
of the superovulation studies described above, in which
an artificial bolus of gonadotropin is administered to induce
ovulation, indicate a severe phenotype that can be localized
to the ovary of the �ERKO female.
IV. Mammary Gland
In mammals, the mammary gland is essentially undeveloped
at birth and does not undergo full growth until the
completion of puberty and, in fact, remains undifferentiated
until pregnancy and lactation. Development of the mammary
gland may be divided into five distinct stages: embryonic
and fetal, prepubertal, pubertal, sexually mature adult,
and pregnancy/lactation (reviewed in Ref. 266). The influential
factors involved at each stage differ in both type and
magnitude. Although the developmental factors involved
during the embryonic and fetal stages of the female mammary
gland are poorly understood, estrogen action does not
appear to be essential (266). However, studies have shown
that the fetal and neonatal mammary gland of rodents is