The reproductive system of male mice homozygous for a mutation in the estrogen receptor (ER) gene (ER knock-out; ERKO) appears normal at the anatomical level. However, these males are infertile, indicating an essential role for ER-mediated processes in the regulation of male reproduction. Adult ERKO male mice have significantly fewer epididymal sperm than heterozygous or wild-type males. Although spermatogenesis is occurring in some seminiferous tubules of 3- to 5-month-old ERKO males, other tubules either have a dilated lumen and a disorganized seminiferous epithelium with few spermatogenic cells or lack a lumen and contain mainly Sertoli cells. There are no obvious differences in seminiferous tubules at 10 days of age between wild-type and ERKO mice, but the lumen in ERKO males is dilated in all seminiferous tubules by 20 days. However, spermatogenesis progresses and similar numbers of sperm are present in the cauda epididymis of ERKO and wild-type males until 10 weeks of age. Disruption of spermatogenesis and degeneration of the seminiferous tubules become apparent after 10 weeks in the caudal pole of the testis and progresses in a wave to the cranial pole by 6 months. However, the seminal vesicles, coagulating glands, prostate, and epididymis do not appear to be altered morphologically in ERKO mice. Serum testosterone levels are somewhat elevated, but LH and FSH levels are not significantly different from those in wild-type males. Sperm from 8- to 16-week-old mice have reduced motility and are ineffective at fertilizing eggs in vitro. In addition, ERKO males housed overnight with hormone-primed wild-type females produce significantly fewer copulatory plugs than do heterozygous or wild-type males. These results suggest that estrogen action is required for fertility in male mice and that the mutation of the ER in ERKO males leads to reduced mating frequency, low sperm numbers, and defective sperm function.

Epidermal growth factor (EGF) reproduces many of the effects of estrogen on the murine female reproductive tract and may partially mediate estroged-induced growth and differentiation. This study was performed to investigate the mechanism by which EGF elicits estrogen-like actions in the whole animal. EGF was ad tered to adult ovariectomized mice by slow release pellets implanted under the kidney capsule. The induction ofuterine DNA synthesis and phosphatidylinositol lipid turnover by EGF or admintation of diethylstilbestrol (5 pg/kg), a potent estrogen, was attenuated by the estrogen receptor antagonist ICI 164,384. Furthermore, EGF mimicked the effects of estrogen on enhanced nuclear localization of the estrogen receptor and the formation of a unique form of the estrogen receptor found exclusively in the nucleus. These results suggest that EGF may induce effects similar to those of estrogen in the mouse uterus by an interaction between the EGF signaling pathway and the classical estrogen receptor. The demonstration of cross-talk between polypeptide growth factors and steroid hormone receptors may be of importance to our understanding of the regulation of normal growth and differentiation as well as the mechanisms of transmission of extracellular mitogen signals to the nucleus.It has been proposed that polypeptide growth factors may act as autocrine or paracrine mediators of estrogen-induced mitogenesis (1, 2). The observations that estrogen induces mRNA and protein for both epidermal growth factor (EGF) (3,4) and its receptor (5-7) in rodent uterus are consistent with this hypothesis and implicate a role for the EGF receptor signaling pathway in steroid hormone regulation of uterine tract growth. Furthermore, exogenous EGF administration to adult ovariectomized mice mimics the effects of estrogen on proliferation and differentiation in the murine female reproductive tract (8). EGF-induced mitogenesis in this model is not affected by adrenalectomy or hypophysectomy, which indicates that adrenal or pituitary hormones do not mediate the effects of EGF. In addition, the fact that an EGF-specific antibody administered prior to estradiol partially blocks estrogen-induced uterine epithelial cell proliferation (8) suggests that production of EGF may be necessary for estrogen-induced responses.Presently, the mechanism by which the actions of estrogens and EGF converge is unknown. This study addresses the intriguing possibility that some of the physiological actions of EGF, an extracellular ligand, may be mediated through a nuclear steroid hormone receptor, namely, the estrogen receptor (ER). Two recent reports by Power et al. (9,10) support such a hypothesis. These studies demonstrated that dopamine, an extracellular ligand, was able to stimulate transcriptional enhancement by three members of the steroid receptor superfamily [the progesterone receptor (PR), ER, and COUP (chicken ovalbumin upstream promoter) transcriptional enhancer], which were transfected into CV1 monkey kidney cells. Furthermore, in MCF-7 hu...

We employed homologous recombination in mouse embryonic stem cells to disrupt the estrogen receptor (ER) gene. Subsequently generated mice that are homozygous for the gene disruption, termed ERKO, possess no demonstrable wild-type ER by Western blot analysis. However, the presence of residual high affinity binding, as detected by [3H]estradiol binding assays and sucrose gradients in uterine extracts from ERKO females prompted further investigation of transcription and translation products from the disrupted ER gene. Analysis of ERKO uterine messenger RNA (mRNA) by reverse transcriptase-polymerase chain reaction demonstrated that although no full-length wild-type ER mRNA was present, two smaller transcripts, labeled E1 and E2, were identified and partially sequenced. Both ERKO transcripts are splicing variants that result in the disrupting NEO sequence being partially or completely removed from the mRNA. In the ERKO-E2 variant, this results in a frame shift and the creation of at least two stop codons downstream. In the ERKO-E1 variant, the ER reading frame is preserved and encodes for a smaller mutant ER that could be the source of the residual estradiol binding. When this mutant form is overexpressed and characterized in vitro, it results in a smaller protein of the predicted size that possesses significantly reduced estrogen-dependent transcriptional activity compared with that of the wild-type ER. Despite residual amounts of an impaired ER variant, estrogen insensitivity in the female ERKOs was confirmed by the failure of estrogen treatment to induce known uterine markers of estrogen action, such as increased DNA synthesis, and transcription of the progesterone receptor, lactoferrin, and glucose-6-phosphate dehydrogenase genes. Furthermore, serum levels of estradiol in the ERKO female are more than 10-fold higher than those in the wild type, consistent with a syndrome of hormone insensitivity.