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Articles |
a Developmental Endocrinology Section,
b Reproductive Toxicology Group, Laboratory of Toxicology, Environmental Toxicology Program and Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, Environmental Diseases and Medicine Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ERß was mapped in the developing gonads and reproductive tracts of male and female mice from fetal day 14 to postnatal day 26 (PND 26). This study was designed to determine the pattern of expression of both ER subtypes in specific tissue compartments during development. In ovaries, ER mRNA was detected at all ages examined; ERß mRNA was seen as early as PND 1, and its expression increased with age. Immunolocalization showed ERß in differentiating granulosa cells of the ovary, whereas ER was predominantly seen in interstitial cells. The remainder of the female reproductive tract showed ER mRNA at all ages examined with little or no significant levels of ERß, except on PND 1 when a low level of message appeared. In males, ER and ERß mRNA were detected in the fetal testis; however, ERß gradually increased until PND 5 and subsequently diminished to undetectable levels by PND 26. Immunolocalization showed ER in the interstitial compartment of the testis, whereas ERß was seen predominantly in developing spermatogonia. The remainder of the male reproductive tract showed varying amounts of both receptors by RPA and immunostaining throughout development. These studies provide information useful in studying the role of both ER subtypes in normal differentiation, and they provide indications of differential tissue expression during development.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) [4–6]. The biological significance of two ER subtypes is unclear but may provide an explanation for the selective actions of estrogens in different target tissues. The fact that both ER and ERß are found in numerous mammalian species suggests that selective effects of estrogens may reside in promoters that are differentially responsive to ER and ERß.
The ontogeny of ER in the developing reproductive tract of a number of species has been documented for many years [7–10]. The presence of ERß has only recently been described in adult reproductive tract tissues of mice [4,11], rats [12–14], cows [15], and humans [3,16]. Developmental expression of ERß has been reported in the prostate [17] and testis [18,19] of the rat, and in the fetal reproductive tract of humans [5]. ERß has also been reported in several reproductive tract tissues of the mouse during embryogenesis [20]. Our particular interest has been in the expression of the two ER forms in the developing reproductive tract tissues of male and female mice. Since the differentiating mammalian reproductive tract has been shown to be uniquely sensitive to various estrogenic compounds, and exposure either prenatally or neonatally results in long-term abnormalities including neoplasia [21–23], the role of both ER and ERß in the induction of these lesions warrants further investigation. Synthetic and naturally occurring xenoestrogens have different relative in vitro affinities for ERß and ER receptors, and some ligands preferentially bind to ER, whereas others bind more efficiently to ERß [24–26]. Therefore, the presence and relative abundance of both ER and ERß in a specific tissue may be a determining factor in the relative effect of a particular compound on that tissue. The present study describes ER and ERß mRNA and protein expression in specific tissue compartments of reproductive tract tissues in male and female mice at various stages of normal development and differentiation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult CD-1 (Crl:CD-1 [ICR] BR) mice were obtained from Charles River Breeding Laboratories (Raleigh, NC) and bred to male mice of the same strain in the breeding facility at the National Institute of Environmental Health Sciences (NIEHS; Research Triangle Park, NC). Vaginal plug detection was considered day 0 of pregnancy. Pregnant mice were housed under controlled lighting (12L:12D) and temperature (21–22°C) conditions. Mice were provided with NIH 31 laboratory mouse chow (National Institutes of Health, Bethesda, MD, purchased from Zeigler Brothers, Inc., Gardners, PA) and fresh water ad libitum. All animal procedures complied with an approved NIEHS/NIH animal care protocol.
Pregnant mice were killed and fetuses were removed at 14 and 16 days postconception (dpc; plug day = 0 dpc). For the remaining time points, pregnant mice delivered their young on 19 dpc and were randomly standardized to 8 pups per litter. Mice were killed on days 1, 5, 12, 19, and 26 of life (day of birth = postnatal day [PND] 1).
Reproductive tract tissues were quickly removed and processed by one of two methods: 1) tissues collected for ribonuclease protection assay (RPA) were snap-frozen in liquid nitrogen and stored at -70°C until further processed; 2) tissues collected for immunohistochemical analysis were fixed in formalin for 6 h at 4°C, transferred to 70% ethyl alcohol at 4°C, and processed for histological evaluation. Tissues were embedded in paraffin and serially cut into 4-µm sections.
RNA Isolation and RPA
RNA isolation For fetal tissue collection, 6 pregnant females were killed at each time point (14 dpc and 16 dpc); the abdominal cavity was opened, and fetuses were quickly removed. Using a dissecting scope, the reproductive tract was collected en bloc into cold PBS, and the gonad was dissected away from the remainder of the reproductive tract. Reproductive tracts and gonads were frozen and pooled from approximately 30 female and 30 male mice for each age. At PNDs 1, 5, 12, 19, and 26, reproductive tracts and gonads were collected into cold PBS, dissected, and frozen. Approximately 8 mice per sex per time point were pooled on PNDs 1, 5, and 12, and 4 mice per sex per time point were pooled on PNDs 19 and 26. Tissues collected from the females included ovary/oviduct and uterus; tissues from the males were testis, epididymis, vas deferens, seminal vesicle, and coagulating gland complex, and the remainder of the reproductive tract including the prostate. For PND 1 males, seminal vesicles were not separated from reproductive tract tissues.
Tissues were pulverized and homogenized with a hand-held homogenizer in Trizol (Gibco-BRL, Grand Island, NY), and total RNA was isolated according to the manufacturer's instructions. A small aliquot of the final preparation was used to determine the amount of total RNA by A260, and the integrity of the RNA was checked on 1% agarose gels by viewing ribosomal RNA bands. RNA was frozen at -70°C until further use.
RPA
The probes used in this study have been previously described in detail [4,27]. Antisense riboprobes were generated from linearized templates using Maxiscript reagents (Ambion, Austin, TX), the appropriate RNA polymerase (T3 or T7), and the incorporation of [32P]CTP (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's protocol. The full-length mouse ER antisense riboprobe was 490 nucleotides (nt) and produced a specific protected fragment of 366 nt [27]. The mouse ERß antisense riboprobe was generated from the cloned cDNA fragment and was 318 nt in full length and generated a protected fragment of 262 nt [4]. An antisense riboprobe specific for mouse cyclophilin, used to equate loading among lanes, was generated from the template pTRI-cyclophilin (Ambion) at a full length of 165 nt and produced a protected fragment of 103 nt.
For all postnatal endpoints, 10 µg of total RNA was used, and for the fetal end points 2.5 µg was used. All RPA reactions consisted of 5 x 104 cpm of each probe, sample RNA, and yeast tRNA (for a final total RNA equal to 25 µg), which were mixed and ethanol-precipitated at -70°C for a minimum of 3 h to a maximum of overnight. The resulting pellets were then processed through the RPA using the Hybspeed RPA reagents (Ambion) according to the manufacturer's protocol. Final analysis of protected fragments was carried out by electrophoresis on a 1.5-mm-thick, 6% bis-acrylamide/8.3 M urea/single-strength Tris-borate-EDTA (TBE) gel (National Diagnostics, Atlanta, GA), which was then fixed, dried, and exposed to a Phosphorimager screen (Molecular Dynamics, Sunnyvale, CA) and then to x-ray film.
ER Immunohistochemistry
ER
Tissue sections from a minimum of three mice were immunostained for each age and sex. Tissue sections were deparaffinized in xylene, hydrated in a series of graded ethanols, and washed in single-strength automation buffer (AB; Fisher Scientific, Norcross, GA). Sections were then treated with 3% hydrogen peroxide to eliminate endogenous peroxidase. After washes in AB, sections were incubated with 0.015 mg/ml trypsin in AB for 4 min and with 0.025 mg/ml deoxyribonuclease I in AB for 3 min, and washed with AB. Sections were then incubated with normal goat serum (Vector Laboratories, Burlingame, CA) to block nonspecific binding for 20 min. The sections were then incubated with primary antibody, rat anti-human ER (H222; Abbott Laboratories, Chicago, IL) at a dilution of 1:3 overnight at room temperature. Negative controls were run on adjacent tissue sections with preimmune serum (Abbott Laboratories) or without the primary antibody (buffer only). Sections were then washed with AB and incubated with biotinylated goat anti-rat IgG (Vector Laboratories) for 1 h. After this incubation, sections were washed and incubated with ExtrAvidin peroxidase (Sigma Chemical Company, St. Louis, MO) at a dilution of 1:50 for 30 min. Visualization of the peroxidase was carried out by covering the sections with diaminobenzidine (DAB; Sigma Chemical Company) at 0.5 mg/ml in AB containing 0.01% H2O2 for 10 min. Sections were rinsed in distilled water and dehydrated in a graded series of ethanols and xylenes, and coverslipped for evaluation by light microscopy.
ERß
Adjacent tissue sections of the samples immunostained for ER were also immunostained for ERß. Sections were deparaffinized and rehydrated, and endogenous peroxidase was eliminated as described for ER above. After washes in AB, tissue sections were rinsed with water and microwaved for 5 min in dH2O for antigen retrieval. Sections were then incubated with 10% BSA in AB for 20 min, then incubated 2 h at room temperature using rabbit anti-mouse ERß at a dilution of 1:50 in AB pH 6.8 (310; Affinity Bioreagents, Golden, CO). Negative controls of adjacent tissue sections were incubated with anti-mouse ERß preabsorbed with ERß peptide (Affinity Bioreagents), or preimmune serum at the same dilution. Sections were then washed with AB and incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories) at a concentration of 1:500 in AB for 1 h. Sections were washed with AB and incubated with ExtrAvidin peroxidase for 30 min, and treated with DAB in AB containing 1% H2O2 for 10–20 min for color development. Sections were rinsed in distilled water, dehydrated, coverslipped, and evaluated using light microscopy.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two distinct bands differing in size by approximately 15 nt appeared in several tissues although a single protected fragment representative of ERß mRNA was expected. It is important to note that these bands appeared only in those tissues that also exhibited positive ERß immunoreactivity, and therefore both are probably representative of detectable ERß mRNA. As described previously [2], the ERß riboprobe was transcribed from a partial cDNA clone of the mouse ERß gene generated by PCR using primers specific to the rat ERß gene. Because the rat primers become incorporated into the clone during this process and are not 100% homologous to the corresponding mouse sequences, there exist some degenerate sequences in the termini of the clone and resulting riboprobe. This small difference in the riboprobe and protected mRNA sequence can be partially exploited during the ribonuclease (RNase) step of the assay, resulting in incomplete RNase digestion and the appearance of two bands.
The results of the RPA on the fetal tissues are shown in Figure 1; 16 dpc ovaries and uteri showed no detectable ERß mRNA, whereas 16 dpc testes did show the presence of ERß mRNA; the remainder of the male reproductive tract showed no evidence of ERß transcripts. ER transcripts were detected in all male and female 16 dpc tissues examined by RPA (data not shown). Total RNA from the 14 dpc testis was positive for ERß mRNA expression (data not shown).
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The results of the RPA on postnatal female tissues are shown in Figure 2. All time points showed the presence of both ER and ERß mRNA in the ovaries, with a peak in ERß mRNA transcripts at PND 19. The uteri showed ER at all ages examined; a low level of ERß was seen in the uterus on PND 1 but was not apparent in older tissues. Longer exposure time (2 days) did not reveal ERß mRNA in the uterus at later ages.
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Results of RPA analysis of ER and ERß mRNA in male tissues are seen in Figure 3. The neonatal testis showed the presence of ER that decreased with age starting on PND 19. An abundant amount of ERß mRNA was seen on PNDs 1 and 5. Although ERß mRNA was present at later time points, levels were relatively much lower. RPA analysis of the remainder of the reproductive tract showed detectable ER mRNA in the epididymis, vas deferens, seminal vesicle, reproductive tract (including the prostate), and bladder; varying levels of ERß were seen in the same tissues (Fig. 3). Although not readily apparent in Figure 3, longer exposure times (2 days) revealed faint ERß bands in the vas deferens and the epididymis (data not shown). The bladder also demonstrated the presence of ER and ERß mRNA early in development at varying levels for both transcripts.
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Immunohistochemistry
Immunohistochemical localization of ERß protein in the gonads during development is shown in Figure 4. The presence of ERß as early as PND 5 in the ovary (C) followed the differentiation of granulosa cells, with the most abundant protein expression seen at PND 26 (G). The protein was localized to the nucleus of the granulosa cells of primary, secondary, and tertiary follicles. No immunostaining of ERß was seen in the interstitial cells of the ovary. Adjacent tissue sections immunostained for the presence of ER exhibited specific staining in the interstitial cells of the ovary at all ages examined (data not shown). The presence of ERß protein in the testis was observed in the germ cells as early as 16 dpc (B). The most abundant ERß expression appeared at PND 12 in spermatocytes (F), after which the intensity decreased and was not apparent by PND 26 (H). Adjacent tissue sections immunostained for the presence of ER indicated immunoreactivity in the interstitial cells of the testis at all ages examined (data not shown).
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Immunostaining for both ER proteins was also determined in the female reproductive tract during development. ER immunoreactivity was observed in all time points tested as previously reported [7]. ER was detected in stromal cells of the uterus at all ages examined and in the epithelial cells as early as PND 5 and throughout the rest of development. There was no detectable expression of ERß protein in the uterus at any time point examined even though levels of ERß mRNA were seen at PND 1. An example of immunohistochemical localization of ER and ERß in the female reproductive tract can be seen in Figure 5. At PND 26, intense ER immunoreactivity can be seen in the oviduct (A) and the uterus (C) in both epithelial and stromal cells. In contrast, ERß immunoreactivity was not detectable in either the oviduct (B) or the uterus (D) in either cell type.
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A comparison of ER and ERß immunoreactivity in the male reproductive tract at 12 days of age is shown in Figure 6. The epididymis exhibited strong ER immunostaining in epithelial cells and light immunostaining in stromal cells (A); ERß immunostaining was easily detectable in stromal cells and epithelial cells (B). The presence of both receptor forms in vas deferens was not as intense as in the epididymis; ER was predominantly present in stromal cells (C), and ERß was predominantly seen in epithelial cells (D). Seminal vesicles exhibited no detectable ER immunoreactivity (E) although ER mRNA was present in the RPA. ERß immunoreactivity appeared in the epithelial cells of the seminal vesicle, but at a low level (F). The reproductive tract (seminal colliculus) exhibited immunostaining for ER in the stromal cells (G) and for ERß in the epithelial cells (H).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ERß was mapped in developing male and female mouse reproductive tracts. Fetal, neonatal, and immature female reproductive tract tissues exhibited an overall expression pattern of ER and ERß mRNAs that was similar to that reported in the adult [4]. In the ovary, ER immunoreactivity was present in the interstitial cells, whereas ERß immunoreactivity was seen in granulosa cells. ERß levels in the ovary appeared to increase with age and the onset of follicular maturation, which involved increases in the granulosa cell population. An increase in cellular ERß staining intensity indicated that the level of expression per cell was also increasing with age. The developing uterus showed an abundant amount of ER in both stromal cells and epithelial cells but little or no expression of ERß. Although a weak signal for ERß mRNA was detected in the uterus in the neonatal mouse, specific immunoreactivity could not be detected by the methods used in this study. The general lack of detection of ERß in the uterus is confirmed by the lack of function demonstrated by the uterine phenotypes described in ER [28] and ERß [29] knock-out mice.
Of particular significance was the presence of ERß in the testis of fetal and neonatal mice, which was in contrast to the lack of expression we previously reported in adult mice [4]. In the current study, the developing testis contained ERß immunoreactivity in the spermatocytes, but the immature testis showed no apparent ERß immunoreactivity. ER immunoreactivity was constitutively expressed in interstitial cells at all ages examined. By PND 26, the expression of ERß in the testis diminished to undetectable levels when assayed by both RPA and immunohistochemistry. Thus, ERß expression is apparently dependent on the stage of differentiation of the gonadocytes and disappears as the animal matures. Rosenfeld et al. [11] reported ERß in Leydig cells of adult mice, a finding not consistent with our findings by either RPA or protein localization; however, they used a C57Bl/6J/129 mouse while we used an outbred CD-1 strain. Strain differences and different ages examined may account for the differences.
The relationship of ERß message and protein mapped during development in the testis was interesting. The peak of ERß mRNA occurred at PND 5 while the peak of immunoreactivity occurred at PND 12. Similar delays in translation of mRNA into protein have been reported for other transcripts found during oogenesis [30] and spermatogenesis [31]. During spermatogenesis, cessation of transcription occurs within germ cells at certain stages of differentiation; apparently, it is necessary to synthesize and store mRNAs before their translation later during spermatogenesis [32]. For example, transcripts have been shown to be repressed during male germ cell differentiation by repressor proteins for several days before translation; protamine 1 mRNA was repressed by prm-1 RNA-binding protein [33]. Also, the developmental regulation of RNA binding proteins during spermatogenesis, like testis-brain RNA-binding protein, showed that mRNA translation could be regulated by proteins expressed during critical stages of development [34]. The mechanism of translational repression has been reviewed and is considered essential for normal spermatogenesis; premature translation of some of these mRNAs has been suggested to cause an arrest in spermatid differentiation resulting in male sterility [31]. The presence of ERß in the neonatal mouse testis during critical periods of development could play an important role in normal differentiation. Any delay in the initial transcription or premature translation of the mRNA into protein could result in permanent alterations in the differentiation of the testis. Studies are currently underway to examine the mRNA levels as well as the protein expression of ERß in the testis following neonatal exposure to exogenous estrogens.
The finding of ERß only in spermatocytes of the neonatal mouse testis is in contrast to reports in the developing rat testis by Saunders et al. [18] and van Pelt et al. [35] showing ERß in multiple cell types including Sertoli and peritubular cells. Both mice and rats, however, showed ERß in spermatocytes in developing testis. The differences in mRNA and other protein cell types in the testis may be due to species differences, or to the antibody and procedures used for detection of the protein. The conflicting data in ERß localization between the rat studies, specifically the differences seen in cell type distribution in the testis, are more striking than the differences between rat and mouse.
Tissues of the male reproductive tract exhibited expression of both receptor forms throughout development, similar to expression described in adults [4,9,10]. The developing male reproductive tract generally demonstrated the presence of both receptors in epithelial cells or stromal cells within a particular tissue. However, the seminal vesicle expressed ERß immunoreactivity in epithelial cells but no detectable ER immunoreactivity in either tissue compartment. This is in contrast to our RPA data that showed the presence of ER in the seminal vesicles. Since ER immunoreactivity was seen in the coagulating gland, ER transcripts were probably contributed by this tissue, which was collected with the seminal vesicles for RNA analysis. Additional studies will be necessary to verify this possibility. The epididymis appeared to be the only tissue that shows the presence of both receptors colocalized in the same cell type. A gradient of ER expression in the epithelial cells of the epididymis was apparent and agrees with that previously described at the RNA level [9,12,36]; highest expression was observed in the initial segment. ERß expression also appeared to be higher in some areas of the epididymis than others. A more comprehensive study of the expression pattern within different segments of the epididymis is warranted to determine the specific receptor content throughout the epididymis. This gradient of expression of both receptors within the epididymis is not understood, but differential effects of estrogens through each receptor may be studied using this tissue. Effects demonstrated in one section or the other of the epididymis may be a key in understanding the mechanism of action of various exogenous estrogens on one receptor or the other.
The differences in tissue and cellular expression, as well as the appearance and disappearance of the two receptors, may play an important role in development and differentiation. Further, these differences may help explain the varied effects of exposure to different environmental estrogens at different stages of development. Specifically, the affinity of a compound for one receptor or the other may direct the adverse effects of that estrogen on certain tissues within the reproductive tract and be unique for a particular stage of developmental exposure. For example, since genistein preferentially binds to ERß [25] and expression of ERß is high in the ovary, this compound may cause changes in the ovary at doses lower than another estrogen that binds more efficiently to ER. Studies are underway to test this hypothesis.
The presence of these two estrogen receptor subtypes in various tissues and their distribution in different cellular compartments adds further complexity to understanding of the role of estrogens in the developing animal. It is of interest to note that a recent publication [37] reported ERß to be the dominant receptor subtype in the adult rat ovary and further localized it in the granulosa cells, which is in agreement with our findings in the developing mouse ovary. These investigators demonstrated that the ERß protein was functional and was regulated by ovulatory doses of gonadotropins [37]. This suggests that the ERß subtype most likely is a mediator of estrogen action in rodent granulosa cells during normal follicular development. The subfertility and ovarian dysfunction described in the ERß knockout mice further suggests this hypothesis [29]. Therefore, the possibility now exists that ERß may be involved in effects of various exogenous estrogens, particularly those that preferentially bind to ERß. Experiments are underway in our laboratory to address this possibility. In summary, the data described in this report of normal developmental expression of the two receptor subtypes can be used as a map to further investigate abnormal differentiation of mice developmentally exposed to various environmental xenoestrogens.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Correspondence: Retha R. Newbold, Mail Drop E4-02, NIEHS, 111 Alexander Drive, South Campus, P.O. Box 12233, Research Triangle Park, NC 27709. FAX: 919 541 4634; newbold1{at}niehs.nih.gov 
Accepted: September 14, 1999.
Received: May 12, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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J. Lassurguere, G. Livera, R. Habert, and B. Jegou Time- and Dose-Related Effects of Estradiol and Diethylstilbestrol on the Morphology and Function of the Fetal Rat Testis in Culture Toxicol. Sci., May 1, 2003; 73(1): 160 - 169. [Abstract] [Full Text] [PDF]
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 S.A. Adeoya-Osiguwa, S. Markoulaki, V. Pocock, S.R. Milligan, and L.R. Fraser 17{beta}-Estradiol and environmental estrogens significantly affect mammalian sperm function Hum. Reprod., January 1, 2003; 18(1): 100 - 107. [Abstract] [Full Text] [PDF]
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A. Rivas, J. S. Fisher, C. McKinnell, N. Atanassova, and R. M. Sharpe Induction of Reproductive Tract Developmental Abnormalities in the Male Rat by Lowering Androgen Production or Action in Combination with a Low Dose of Diethylstilbestrol: Evidence for Importance of the Androgen-Estrogen Balance Endocrinology, December 1, 2002; 143(12): 4797 - 4808. [Abstract] [Full Text] [PDF]
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 Q. Zhou, R. Nie, G. S. Prins, P. T. K. Saunders, B. S. Katzenellenbogen, and R. A. Hess Localization of Androgen and Estrogen Receptors in Adult Male Mouse Reproductive Tract J Androl, November 1, 2002; 23(6): 870 - 881. [Abstract] [Full Text] [PDF]
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W. N. Jefferson, J. F. Couse, E. Padilla-Banks, K. S. Korach, and R. R. Newbold Neonatal Exposure to Genistein Induces Estrogen Receptor (ER){alpha} Expression and Multioocyte Follicles in the Maturing Mouse Ovary: Evidence for ER{beta}-Mediated and Nonestrogenic Actions Biol Reprod, October 1, 2002; 67(4): 1285 - 1296. [Abstract] [Full Text] [PDF]
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K. M. Robertson, L. O'Donnell, E. R. Simpson, and M. E. E. Jones The Phenotype of the Aromatase Knockout Mouse Reveals Dietary Phytoestrogens Impact Significantly on Testis Function Endocrinology, August 1, 2002; 143(8): 2913 - 2921. [Abstract] [Full Text] [PDF]
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M. R. Fielden, R. G. Halgren, C. J. Fong, C. Staub, L. Johnson, K. Chou, and T. R. Zacharewski Gestational and Lactational Exposure of Male Mice to Diethylstilbestrol Causes Long-Term Effects on the Testis, Sperm Fertilizing Ability in Vitro, and Testicular Gene Expression Endocrinology, August 1, 2002; 143(8): 3044 - 3059. [Abstract] [Full Text] [PDF]
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M. H. Tong and W.-C. Song Estrogen Sulfotransferase: Discrete and Androgen-Dependent Expression in the Male Reproductive Tract and Demonstration of an in Vivo Function in the Mouse Epididymis Endocrinology, August 1, 2002; 143(8): 3144 - 3151. [Abstract] [Full Text] [PDF]
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C. Borgeest, D. Symonds, L. P. Mayer, P. B. Hoyer, and J. A. Flaws Methoxychlor May Cause Ovarian Follicular Atresia and Proliferation of the Ovarian Epithelium in the Mouse Toxicol. Sci., August 1, 2002; 68(2): 473 - 478. [Abstract] [Full Text] [PDF]
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P. Yang, A. Kriatchko, and S. K. Roy Expression of ER-{alpha} and ER-{beta} in the Hamster Ovary: Differential Regulation by Gonadotropins and Ovarian Steroid Hormones Endocrinology, June 1, 2002; 143(6): 2385 - 2398. [Abstract] [Full Text] [PDF]
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D. Coveney, G. Shaw, and M. B. Renfree Estrogen-Induced Gonadal Sex Reversal in the Tammar Wallaby Biol Reprod, August 1, 2001; 65(2): 613 - 621. [Abstract] [Full Text] [PDF]
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 S. F. Palter, A. B. Tavares, A. Hourvitz, J. D. Veldhuis, and E. Y. Adashi Are Estrogens of Import to Primate/Human Ovarian Folliculogenesis? Endocr. Rev., June 1, 2001; 22(3): 389 - 424. [Abstract] [Full Text] [PDF]
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 G. LECCE, G. MEDURI, M. ANCELIN, C. BERGERON, and M. PERROT-APPLANAT Presence of Estrogen Receptor {beta} in the Human Endometrium through the Cycle: Expression in Glandular, Stromal, and Vascular Cells J. Clin. Endocrinol. Metab., March 1, 2001; 86(3): 1379 - 1386. [Abstract] [Full Text]
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 P. T.K. Saunders, R. M. Sharpe, K. Williams, S. Macpherson, H. Urquart, D.S. Irvine, and M. R. Millar Differential expression of oestrogen receptor {{alpha}} and {beta} proteins in the testes and male reproductive system of human and non-human primates Mol. Hum. Reprod., March 1, 2001; 7(3): 227 - 236. [Abstract] [Full Text] [PDF]
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H.O. Goyal, T.D. Braden, M. Mansour, C.S. Williams, A. Kamaleldin, and K.K. Srivastava Diethylstilbestrol-Treated Adult Rats with Altered Epididymal Sperm Numbers and Sperm Motility Parameters, but Without Alterations in Sperm Production and Sperm Morphology Biol Reprod, March 1, 2001; 64(3): 927 - 934. [Abstract] [Full Text]
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N. Atanassova, C. McKinnell, K. Williams, K. J. Turner, J. S. Fisher, P. T. K. Saunders, M. R. Millar, and R. M. Sharpe Age-, Cell- and Region-Specific Immunoexpression of Estrogen Receptor {{alpha}} (But Not Estrogen Receptor {beta}) during Postnatal Development of the Epididymis and Vas Deferens of the Rat and Disruption of This Pattern by Neonatal Treatment with Diethylstilbestrol Endocrinology, February 1, 2001; 142(2): 874 - 886. [Abstract] [Full Text] [PDF]
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H. Wang, H. Eriksson, and L. Sahlin Estrogen Receptors {alpha} and {beta} in the Female Reproductive Tract of the Rat During the Estrous Cycle Biol Reprod, November 1, 2000; 63(5): 1331 - 1340. [Abstract] [Full Text]
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