Estrogen Receptor Null Mice - Endocrine Reviews

June, 1999 ESTROGEN RECEPTOR NULL MICE 365
et al. (88) have demonstrated colocalization of ER� and ER�
to select regions of the rat forebrain.
C. Generation of the ER null mice
The field of estrogen action has been and continues to be
a dynamic one with broadening scope. Although estradiol is
often called the female sex steroid, the list of tissues and
organs in which estrogen actions appear to be critical to
physiological function continues to expand, in both sexes.
The crucial actions of estradiol in the female reproductive
tract, breast, and hypothalamic-pituitary axis have been well
described. However, we now know of significant functions
fulfilled by estrogens in male reproduction. Furthermore, the
effects of estrogens in the physiology, maintenance, and
overall health of the cardiovascular, central nervous, bone,
immune, skin, and adipogenesis systems have been an area
of continued research. And finally, the role that estrogen and
normal and variant ERs may play in carcinogenesis, especially
in the breast and female reproductive tract, continues
to receive great attention.
The rationale for generating mice that possess no functional
ER is multifaceted, but in its most simple terms, was
founded on the classical ablation experiments of the early
part of this century. In 1900, Knauer (105) described the
ability of ovarian grafts to prevent uterine atrophy in the
castrated guinea pig. Five years later, Marshall and Jolly (106)
described the capacity of ovarian extracts to induce estrus
when administered to ovariectomized dogs. Similar protocols
were later elegantly employed by Jost (107) to substantiate
the endocrine function of the testis and the importance
of testosterone in sex determination. These studies were critical
to establishing the following basic criteria required to
verify an endocrine role for a particular organ or tissue: 1)
removal or destruction of the synthesizing organ should
result in predictable symptoms presumed to be related to the
absence of the hormone; 2) administration of material prepared
from the removed organ should relieve these symptoms;
and 3) the hormone should be present in and extractable
from both the organ and blood (108). In the spirit of these
earlier studies, the later part of this century has witnessed the
marriage of two relatively new methodologies to introduce
a modern version of the ablation experiment. The combination
of in vitro culture of mouse embryonic stem cells and
targeted homologous recombination has generated a tool
that allows for the precise disruption or knockout of a particular
gene of study and the passage of this mutation to
offspring. Although one can debate whether this new technique
is more or less invasive than the previous surgical
methods, it is obvious that the classic “ablation” experiment
has been elevated to a molecular level. The function of a
specific hormone can now be studied rather than the function
of a whole endocrine organ that may produce multiple secretions.
Furthermore, this new technology allows for the
study of a particular cellular component, such as a receptor,
that is intrinsic to one or more tissues. Such studies were
previously impossible or relied on the use of chemical antagonists,
which introduced their own inherent limitations.
Additionally, gene targeting provides for in vivo methods to
study the roles of a particular receptor throughout the life of
the animal, including the early development stages. The current
tools that allow for the generation of transgenic mice
have already made significant contributions to our knowledge
of particular genes, especially those involved in development
and reproduction (reviewed in Refs. 109 and 110).
At the time the ERKO mouse was envisioned, the ER� was
the only form of ER known to exist. Furthermore, there were
no reports of ER mutations in normal tissue that resulted in
estrogen insensitivity in humans or laboratory animals. This
was in contrast to the descriptions of syndromes of receptorbased
insensitivity to androgens (37, 38) and thyroid hormones
(40). Therefore, investigators were inclined to conclude
that mutations that resulted in insufficient estrogen
synthesis or resistance to the hormone at the level of the
target organ were lethal at the earliest developmental stages
(111, 112). This view was strengthened by reports of the
detection of ER� mRNA in both human (113) and mouse
(114) oocytes as well as in mouse blastocysts (115). Accordingly,
the concept of generating a mouse devoid of ER� was
initially met with skepticism but was pursued as a collaborative
effort between our laboratory and that of Dr. Oliver
Smithies of the University of North Carolina at Chapel Hill.
If disruption of the mouse ER� gene did prove lethal, a model
to study the precise time and locations of critical ER-mediated
actions during early development would be available.
However, providing the animal was viable, an in vivo model
of estrogen insensitivity would now be accessible for continued
study. Six years after the initial description of the
�ERKO mouse, we now take for granted that disruption of
the ER� gene proved not to be lethal, but rather the animal
develops normally and exhibits a life span comparable to its
wild-type litter mates (46). However, as will be elaborated in
detail in this review, the adult �ERKO mice exhibit several
abnormalities and deficiencies, most notable of which are the
phenotypic syndromes that result in infertility in both sexes.
Since this time, additional discoveries have been made to
enhance the utility of the �ERKO model. Soon after the
generation of the �ERKO mouse, Smith et al. (116) reported
the first and only known case of clinical estrogen insensitivity
due to an inactivating mutation of the ER� gene in a human
patient. Later came multiple descriptions of aromatase and
subsequent estradiol deficiency in humans (117–120). And
finally, a second collaborative effort resulted in the successful
generation of the �ERKO mice, which also survive to adulthood
and exhibit phenotypes unique from those of the
�ERKO (47). However, splicing variants of the disrupted
genes that may encode for receptors with decreased functional
activity have been detected in small amounts in each
of the respective ERKO models (as discussed below) and
therefore complicate the interpretation of the nonlethality of
the gene targeting. Nonetheless, we now know that a loss of
full function of any one of the two ERs is neither lethal nor
detrimental to embryonic and fetal development in both
mice and humans. Perhaps the survival of the ERKO mice,
the aromatase-deficient humans, and the ER�-deficient male
will prompt a renewed effort among clinicians to suspect and
investigate the possibility of estrogen insufficiency or resistance
in patients not responding to conventional therapies.
The ERKO mice provide broad and multiple advantages
to the research efforts toward understanding the function