Estrogen Receptor Null Mice - Endocrine Reviews

June, 1999 ESTROGEN RECEPTOR NULL MICE 377
cells of antral follicles, were apparently preserved in the
follicles of the �ERKO ovary. Follicular atresia in the ovary
is a hormonally controlled process that is critical to oocyte
selection. Although the factors that trigger atresia are not
well understood, it is characterized by apoptosis of the granulosa
cells of the follicle (reviewed in Refs. 223 and 224).
Estradiol has been shown to be one of the many factors
reported to protect the follicle from becoming atretic (250).
Furthermore, androgens reportedly accelerate the process,
and it may be an altered estrogen/androgen synthesis ratio
in the follicle that leads to atresia (250). However, despite
elevated androgen production, the presence of androgen
receptor (AR) mRNA, and a lack of ER� action in the mature
�ERKO ovary, an inordinate amount of apoptosis is not
observed in the follicles (142). Estradiol has also been shown
to facilitate the FSH induction of LH receptors in the granulosa
cells of the mature ovulatory follicle in both in vivo (225,
231, 252) and in vitro experiments (229, 230). Nonetheless, the
granulosa cells of the growing follicles, in addition to the
enlarged cysts in the �ERKO ovary, possess significant levels
of LH receptor mRNA when assayed by in situ hybridization
(142). The most plausible explanation for these observations
is that these estrogen actions are mediated by ER�, which has
been shown to be expressed in a normal pattern in the granulosa
cells of the �ERKO ovary, and concomitant with the
expression of LH receptor (93, 142).
Therefore, with data suggesting that disruption of the ER�
gene did not result in an ovary completely refractory to estrogen,
other aspects of ovarian physiology must be considered as
possible factors in the etiology of the �ERKO ovarian phenotype.
As previously discussed, ovarian function is tightly controlled
by pituitary gonadotropins (see Section III.D.1). In turn,
gonadotropin synthesis and secretion from the anterior pituitary
are at least partially regulated by gonadal steroids acting
via classical feedback mechanisms in the hypothalamic-pituitary
axis (reviewed in Ref. 251). Indeed, disruption of the ER�
gene has resulted in significant phenotypes in the hypothalamic-pituitary
axis of the �ERKO female (see Section VI.A). Most
notable is the increased and chronic secretion of LH in the
�ERKO female that results in levels that are 4–7 times that
found in wild-type females (Table 2) (252). As discussed above,
synchronized increases in serum FSH and LH levels are critical
to follicular maturation and ultimate ovulation in the ovary.
However, it has been proposed that the follicular requirements
for LH are finite, and the presence of abnormally high levels
may force maturing follicles to either prematurely luteinize or
become atretic (253).
Therefore, the �ERKO ovarian phenotype is likely caused
by the chronic exposure to abnormally high levels of LH.
Support for this hypothesis can be drawn from a number of
studies. Investigations involving prolonged treatment with
antiestrogens over a period of at least 28 days have produced
an ovarian phenotype in both mice (195, 196) and rats (197)
that is similar to that of the �ERKO. Of course, interpretation
of these studies is complicated by the ability of the antiestrogens
to inhibit both ERs as well as estrogen action in both
the ovary and hypothalamic-pituitary axis. However, these
studies reported that the “�ERKO” phenotype of enlarged
cystic follicles was produced only after prolonged treatments
with those antiestrogens that possessed the ability to cross
the blood-brain barrier and concurrently produce serum LH
levels that were several fold higher than controls. Therefore,
whereas the estrogen antagonists ZM-189,154 (197) and EM-
800 (195, 196) produced chronically elevated LH levels and
an ovarian phenotype strikingly similar to that of the
�ERKO, tamoxifen did neither (195–197). More definitively,
Risma et al. (254, 255) report that targeted transgenic overexpression
of the LH�-subunit in the mouse that results in a
15-fold increase in serum LH levels produces females that are
anovulatory and exhibit an ovarian phenotype almost indistinguishable
from that of the adult �ERKO female.
Therefore, the similarity in the ovarian phenotypes described
in the above studies, in which the models presumably
possessed normal levels of ovarian ER�, combined with our
observations in the �ERKO, strongly indicate that the ER� is
not directly involved in the development of ovarian cysts due
to hypergonadotropism. However, there are descriptions of
at least two models in which serum LH is chronically elevated,
yet do not manifest an ovarian phenotype similar to
the �ERKO or those induced by antiestrogens or transgenics
as described above. Female mice that are homozygous for a
targeted disruption of the FSH�-subunit gene exhibit an
approximate 5-fold increase in serum LH but do not show
indications of enlarged cystic follicles in the ovary (241),
indicating a role for FSH in this process as well. Bogovich
(256) has provided supporting evidence by demonstrating
that FSH is required along with prolonged exposure to LH
(in the form of human CG) to induce follicular cysts in the
rat. A likely role for FSH is the induction of LH receptor in
the granulosa cells of the maturing follicles, thereby rendering
the follicle responsive to the increased levels of LH.
Another contrasting knockout model is that of the P450 arom
gene (ArKO), in which the homozygous females possess
significantly elevated serum LH and FSH levels but lack the
capacity to synthesize estradiol (257). Although folliculogenesis
is arrested at an antral stage in the ArKO ovary, no
�ERKO-like cystic structures are reported (257). Therefore,
assuming that a lack of estradiol synthesis would disrupt
ligand-dependent activity of the ER� in the granulosa cells,
the lack of ovarian cysts in the ArKO indicate an intraovarian
role for estradiol in this phenotype. Therefore, although the
�ERKO phenoytpe may be triggered by hyperstimulation of
the follicles by LH, it is likely influenced by both FSH and
ER� actions in the granulosa cells as well. Current studies
utilizing prolonged treatment of �ERKO females with a
GnRH antagonist to reduce serum gonadotropins are being
carried out to further define the etiology of the ovarian
phenotype (J. F. Couse, D. O. Bunch, J. Lindzey, D. W.
Schomberg, and K. S. Korach, manuscript in preparation).
Since the �ERKO ovarian phenotype develops and worsens
only after sexual maturity, we have began studies to
characterize ovarian function in the immature �ERKO female
(J. F. Couse, D. O. Bunch, J. Lindzey, D. W. Schomberg,
and K. S. Korach, manuscript in preparation). Although superovulation
with exogenous gonadotropins was not successful
in eliciting ovulation in the older �ERKO females,
immature (�28 days) females do respond and produce viable
oocytes that can be collected from the oviduct. However, the
average number of oocytes collected from superovulated
�ERKO females is significantly less than that yielded from