Estrogen Receptor Null Mice - Endocrine Reviews

June, 1999 ESTROGEN RECEPTOR NULL MICE 387
16 wk of age (315). Furthermore, the epididymal sperm collected
from the �ERKO males are characterized by significantly
decreased levels of motility and increased incidence of
sperm heads separated from the flagellum (315). Even those
sperm that possessed normal structure and motility were
unable to fertilize wild-type oocytes in an in vitro fertilization
assay (315). Therefore, despite levels of circulating gonadotropins
and androgen within the normal range, disruption of
the ER� gene has resulted in severe impairments in both
spermatogenesis and sperm function.
As shown in Fig. 7, histological analysis of testes from
sexually mature �ERKO males indicated significant atrophy
of the seminiferous epithelium and severe dilation of the
tubule lumen. At 10–20 days of age, no morphological difference
in the testis was apparent when comparing the
�ERKO with wild-type. However, a distinct morphological
phenotype becomes obvious by 40 days of age and
progresses to produce a completely atrophied testis by 150
days in the �ERKO male (315). Accordingly, sperm counts
decrease as the testicular phenotype worsens, although immunohistochemical
detection of Hsp70–2, a germ cell-specific
protein, was possible even in the most severely disrupted
tubules (315).
Further characterization of testes from mature �ERKO
males indicated a prominent rete testis that is dilated and
protrudes into the interior of the organ as well as severely
dilated efferent ductules (Fig. 7) (315, 327). The rete testes are
composed of a network of intercommunicating channels located
in the posterior-cranial portion of the testes and serve
as a pathway by which suspended spermatozoa can pass
from the testis to the epididymis. Connecting the rete testis
to the epididymis are the efferent ducts, a series of multiple
channels thought to play a significant role in reabsorption of
much of the testicular fluid, and therefore act to also concentrate
the sperm (303). Steroid autoradiography has indicated
that the efferent ducts of the mouse possess the highest
concentration of ER compared with any other region of the
excurrent duct system (309). This expression pattern is evident
in the mouse as early as neonatal day 3 (310). Immunohistochemical
and RNA analyses have shown that ER� is
the predominant form of ER in the efferent ducts and the
cranial portion of the epididymis (311, 328), although ER�
mRNA is also detectable (121, 311).
Previous studies employing surgical ligation of the efferent
ductules reported a severe dilation of the seminiferous
tubules similar to that observed in the �ERKO male (329,
330). Based on the similarity of phenotypes, it became clear
that the testicular anomaly observed in the mature �ERKO
male may be the result of a severe imbalance in the fluid
equilibrium. However, was it due to hypersecretion of fluid
from the testis or insufficient reabsorption of fluid by the
epithelial cells lining the efferent ducts, or possibly a combination
of both? Using surgical techniques to inhibit fluid
transport at different points in the excurrent duct system,
Hess et al. (327) demonstrated that the reabsorption abilities
of the efferent ductules in the �ERKO male were lacking and,
in fact, the secretory activity is actually reduced in the
�ERKO testis. Further characterization indicated a reduction
or often a complete lack of endocytotic vesicles and organelles
common to fluid uptake in the epithelial cells lining
the �ERKO efferent ducts (327). This study was the first
report of a direct ER�-mediated estrogen function in the male
reproductive tract. Interestingly, however, was the inability
of the pure antiestrogen, ICI-182,780, to produce a similar
phenotype in wild-type ductal fragments in in vitro experiments
(327). Although the antagonist was able to cause some
loss of fluid absorption in the wild-type ductal fragment, the
resulting phenotype was not nearly as extreme as that observed
in the �ERKO tissue fragments (327). The authors
proposed that perhaps ER� was possibly mediating an agonistic
effect of the ICI-182,780 and thereby may explain the
lack of full corroboration with the in vivo �ERKO phenotype
(327). This hypothesis was based on the work of Paech et al.
(91), which demonstrated that estrogen antagonists, including
ICI-164,384, may function as an agonist when interacting
with AP-1 complexes in vitro. We, as well as others, have
since shown that ER� mRNA expression in the �ERKO male
reproductive tract is not altered, although its function remains
unclear (93, 121). However, the preservation of ER�
expression in the �ERKO strongly indicates that the reabsorption
functions of the efferent ducts are indeed dependent
on the presence of functional ER�. This view is strengthened
by the lack of a similar testicular phenotype in �ERKO male
mice observed at ages as old as 14 months (47).
Interestingly, the luminal swelling, loss of germinal epithelium,
and atrophy in the seminiferous tubules of the
�ERKO testes appeared to commence at the caudal portion
of the organ and progress toward the cranial region as the
animal aged (Fig. 7) (315). This is thought to be due to a
gradual increases in testicular pressure leading to restricted
blood flow as the phenotype in the rete testis worsens and
fluid accumulates within the encapsulated organ (315). The
result of such decreased circulation is likely to become initially
manifested in the less vascularized caudal region of the
testis and eventually advance to affect the whole gonad.
Despite the severe testicular phenotype that occurs in the
�ERKO male with age, younger males do produce viable
sperm. However, the motility and fertilization abilities of
epididymal sperm collected from �ERKO males are severely
compromised. ER (326) as well as P450 arom (331) have been
reported in Sertoli cells and germ cells of the testis, respectively.
Therefore, a loss of ER�-mediated estrogen action in
the Sertoli cell may alter sperm function. It is also known that
spermatozoa entering the epididymis are unable to fertilize,
and undergo a critically active maturation process as they
pass through the epididymal cords (303). Estradiol treatment
of adult male mice has been reported to increase the rate at
which spermatozoa pass through the epididymis (332). Furthermore,
expression of ER� in the mouse epididymis appears
to be highest in the caput epididymis, where sperm
first enter after exiting the testis (309). Reports of ER� expression
in the epididymis indicate an opposite distribution,
i.e. highest levels are found in the cauda epididymis, in both
the rat (311) and mouse (121). This pattern of epididymal ER�
expression is preserved in the �ERKO male (121). Nonetheless,
normal fertility in the �ERKO male indicates that any
actions of estrogen required for sperm maturation and fertilization
capacity appear dependent on the presence of ER�.
The varied phenotypes leading to infertility in the �ERKO
male have provided great insight into the role that ER� plays