Steroid hormones acutely regulate expression of a Nudix protein-encoding gene in the endometrial epithelium of sheep

Steroid hormones regulate endometrial gene expression to meet the needs of developing embryos. Our hypothesis is that steroid hormones transiently induce expression of genes in the endometrial epithelium to make the uterine environment different between the earliest days of pregnancy. We identified one such gene product using differential display-polymerase chain reactions. The gene product that was strongly induced in ewes between day 3 and 6 of the estrous cycle was cloned and sequenced to identify it as encoding a member of the Nudix family of hydrolase enzymes. Northern blot analyses indicated that NUDT16 mRNA concentrations were elevated 10-fold in the endometrium of sheep from day 5 to 9 of the estrous cycle and returned to basal levels by day 11. In assays of RNA samples from 15 different tissues from an adult ewe, the concentrations of NUDT16 mRNA were greatest in endometrium. In situ hybridization localized NUDT16 mRNA exclusively to the endometrial epithelial cells of the glands and uterine lumen. In ovariectomized ewes, NUDT16 mRNA was induced by a regimen of alternating estrogen and progesterone therapy designed to mimic the hormonal experiences of a ewe at day 6 of the estrous cycle. The final estrogen treatment in the regimen was critical to the expression of NUDT16 as well as progesterone receptor and estrogen receptor-beta genes. Characterization of the NUDT16 gene identified putative steroid hormone response elements, which can now be investigated to understand its unique pattern of regulation in the earliest days of pregnancy.     

Ovine uterine gland knock-out model: effects of gland ablation on the estrous cycle

Ovine endometrial gland development is a postnatal event that can be inhibited epigenetically by chronic exposure of ewe lambs to a synthetic progestin from birth to puberty. As adults, these neonatally progestin-treated ewes lack endometrial glands and display a uterine gland knockout (UGKO) phenotype that is useful as a model for study of endometrial function. Here, objectives were to determine: 1) length of progestin exposure necessary from birth to produce the UGKO phenotype in ewes; 2) if UGKO ewes display normal estrous cycles; and 3) if UGKO ewes could establish and/or maintain pregnancy. Ewe lambs (n = 22) received a Norgestomet (Nor) implant at birth and every two weeks thereafter for 8 (Group I), 16 (Group II), or 32 (Groups III and IV) weeks. Control ewe lambs (n = 13) received no Nor treatment (Groups V and VI). Ewes in Groups I, II, III, and VI were hemihysterectomized (Hhx) at 16 weeks of age. After puberty, the remaining uterine horn in Hhx ewes was removed on either Day 9 or 15 of the estrous cycle (Day 0 = estrus). Histological analyses of uteri indicated that progestin exposure for 8, 16, or 32 weeks prevented endometrial adenogenesis and produced the UGKO phenotype in adult ewes. Three endometrial phenotypes were consistently observed in Nor-treated ewes: 1) no glands, 2) slight glandular invaginations into the stroma, and 3) limited numbers of cyst- or gland-like structures in the stroma. Overall patterns of uterine progesterone, estrogen, and oxytocin receptor expression were not different in uteri from adult cyclic control and UGKO ewes. However, receptor expression was variegated in the ruffled luminal epithelium of uteri from UGKO ewes. Intact UGKO ewes displayed altered estrous cycles with interestrous intervals of 17 to 43 days, and they responded to exogenous prostaglandin F(2 approximately ) (PGF) with luteolysis and behavioral estrus. During the estrous cycle, plasma concentrations of progesterone in intact control and UGKO ewes were not different during metestrus and diestrus, but levels did not decline in many UGKO ewes during late diestrus. Peak peripheral plasma concentrations of PGF metabolite, in response to an oxytocin challenge on Day 15, were threefold lower in UGKO compared to control ewes. Intact UGKO ewes bred repeatedly to intact rams did not display evidence of pregnancy based on results of ultrasound. Collectively, results indicate that 1) transient, progestin-induced disruption of ovine uterine development from birth alters both structural and functional integrity of the adult endometrium; 2) normal adult endometrial integrity, including uterine glands, is required to insure a luteolytic pattern of PGF production; and 3) the UGKO phenotype, characterized by the absence of endometrial glands and a compact, disorganized endometrial stroma, limits or inhibits the capacity of uterine tissues to support the establishment and/or maintenance of pregnancy.     

蛋白激酶H11基因在小鼠子宫中的表达及其性激素调节

为研究蛋白激酶H11基因在生殖系统中的作用,我们采用半定量RT-PCR和原位杂交方法,研究了蛋白激酶H11基因在小鼠中的组织特异性表达,在妊娠初始期胚胎植入位点、妊娠期子宫和胎盘以及正常动情周期子宫中的表达及其受性激素的调节。结果发现:蛋白激酶H11基因在小鼠多种组织中都有表达,在卵巢及子宫等一些生殖相关的组织中表达水平较高;妊娠初始期,蛋白激酶H11基因在小鼠子宫内膜植入位点处有明显的高表达,其mRNA定位于腔上皮细胞和基质细胞中。在动情周期中,蛋白激酶H11基因在动情前期子宫中表达水平较低;卵巢切除模型显示雌激素和孕激素均可显著上调蛋白激酶H11基因的表达。以上结果提示蛋白激酶H11可能参与了胚胎植入过程中腔上皮细胞凋亡和基质细胞增殖与蜕膜化以及动情周期小鼠子宫内膜细胞的功能调节[动物学报51(3):462-468,2005]。     

Estrogen receptor mRNA in uteri of normal estrous cycling and ovariectomized rats by in situ hybridization.

Biological effects of estrogen are mediated via its binding to the estrogen receptor (ER), the contents of its protein and mRNA varying during the estrous cycle. In the present study, the ERalpha mRNA expression in different cell components of the uterus was investigated in normal estrous cycling rats using nonisotopic in situ hybridization. Additionally, ovariectomized (OVX) rats treated with 17beta-estradiol (E2: 5 microg/kg, sc injection daily) were also investigated to clarify the effects of exogenous E2. At proestrus and diestrus, and especially the former, the luminal and glandular epithelial (LE and GE) cells were strongly positive, along with stromal cells beneath the luminal epithelium. At estrus, the expression was slightly diminished in LE cells, but almost completely lacking in GE cells. At metestrus, positive signals appeared again in GE cells. In the myometrium, ER mRNA was demonstrated to be constantly positive in all estrous cycle stages. OVX rat uteri underwent marked atrophy, but ER mRNA still remained in all cell types. After 2 consecutive days of E2 treatment, markedly increased intensity was observed, especially in LE and GE cells. The uteri of OVX rats treated with E2 for 14 days, however, showed slightly diminished expression, whereas the serum concentration of E2 was comparable to that in rats after 2 days. These results provide evidence that cell-type specific patterns of ER mRNA expression characterize the uteri of both normal estrous cycling rats and OVX rats after estrogen treatment.     

Lectin binding patterns in normal canine endometrium and in bitches with pyometra and cystic endometrial hyperplasia

Cystic endometrial hyperplasia (CEH) and pyometra in the bitch are dioestral syndromes, supposed to be caused by hormonal disturbances and changes in endometrial steroid hormone receptor levels. Histologically, the endometria show cystic dilated glands and, if bacteria succeed in invading the uterus, pyometra may develop in the following metoestrus. In this study, lectin histochemistry was performed on paraffin sections to compare carbohydrate expression of uterine glands and surface epithelium in healthy dogs and in dogs with CEH and pyometra. Lectin binding is a useful tool to identify glycoconjugates, especially of the glycocalyx, which has essential functions in the endometrium during reproduction. Uterine tissue was obtained from 18 healthy bitches in metoestrus or anoestrus and 18 bitches with a clinical diagnosis of CEH or pyometra. Normal endometria showed cycle-dependent changes in SBA, PNA, HPA and UEA binding during metoestrus and anoestrus. LCA did not show cycle-dependent changes and WGA bound to Golgi regions in the apical parts of surface epithelial cells only in metoestrous. Endometria with inflammatory alterations lost cycle-specific lectin binding patterns and, with increasing severity of pathological changes, showed a marked decrease in binding intensity to the glandular and surface epithelial glycocalyx and secretions. In dogs with CEH, unaltered glands with generally strong lectin binding to the glycocoalyx and Golgi regions were found adjacent to altered glands. The decrease of lectin binding in pyometra cases is supposed to be a result of glandular exhaustion after cystic hyperplasia. In addition, bacterial adhesion to sugar residues on the uterine surface epithelium might impede lectin binding.     

Contact of dog spermatozoa with homologous uterine tube epithelium prolongs flagellar activity in relation to the stage of the estrous cycle

There is scant information about the storage of spermatozoa within the reproductive tract of the bitch. In several species the uterine tube plays a significant role in sperm storage. The present study was performed to investigate the interaction between spermatozoa and the epithelium of the uterine tube, in particular how this interaction might influence the flagellar activity of spermatozoa in relation to the stage of the estrous cycle. Epithelium was harvested from uterine tubes of 24 bitches at various stages of the estrous cycle (estrus, luteal phase or anestrus), and cultured with pooled spermatozoa collected from 6 dogs. Spermatozoa rapidly bound to the epithelial surface by their heads and the majority of attached spermatozoa were motile. The intimate association between spermatozoa and the uterine tube epithelium maintained motility in a manner that was related to the stage of the estrous cycle. Flagellar activity was significantly greater for spermatozoa bound to estrous epithelium than epithelium from the luteal phase or anestrus. On average, approximately 10% of spermatozoa that were attached to the uterine tube epithelium of estrous bitches retained their flagellar activity for 48 h after innoculation. There was no apparent influence of the region of the uterine tube on this effect. These findings suggest that the uterine tube may form a functional spermatozoal reservoir in the bitch.     

Uterine gland development begins postnatally and is accompanied by estrogen and progesterone receptor expression in the dog

During neonatal and juvenile life, mammalian uteri undergo extensive structural and functional changes, including uterine gland differentiation and development. In sheep and mice, inhibition of neonatal uterine gland development induced by progestin treatment led to a permanent aglandular uterine phenotype and adult infertility, suggesting that this strategy might be useful for sterilizing dogs and other companion animals. The goal of this study was to define temporal patterns of adenogenesis (gland development), cell proliferation, and progesterone and estrogen receptor expression in uteri of neonatal and juvenile dogs as a first step toward determining whether neonatal progestin treatments might be a feasible contraceptive approach in this species. Uteri obtained from puppies at postnatal wk 1, 2, 4, 6, or 8 were evaluated histologically and immunostained for MKI67, a marker of cell proliferation, estrogen receptor-1, and progesterone receptor. Adenogenesis was under way at 1 wk of age, as indicated by the presence of nascent glands beginning to bud from the luminal epithelium, and rapid proliferation of both luminal epithelial and stromal cells. By Week 2, glands were clearly identifiable and proliferation of luminal, glandular, and stromal cells was pronounced. At Week 4, increased numbers of endometrial glands were evident penetrating uterine stroma, even as proliferative activity decreased in all cell compartments as compared with Week 2. Whereas gland development was most advanced at Weeks 6 to 8, luminal, glandular, and stromal proliferation was minimal, indicating that the uterus was nearly mitotically quiescent at this age. Both estrogen receptor-1 and progesterone receptor were expressed consistently in uterine stromal and epithelial cells at all ages examined. In summary, canine uterine adenogenesis was underway by 1 wk of age and prepubertal glandular proliferation was essentially complete by Week 6. These results provided information necessary to facilitate development of canine sterilization strategies based on neonatal progestin treatments designed to permanently inhibit uterine gland development and adult fertility.     

Epidermal growth factor, transforming growth factor-α, and epidermal growth factor receptor expression and localization in the canine endometrium during the estrous cycle and in bitches with pyometra

Gene expression and immunohistochemical localization of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), and epidermal growth factor receptor (EGF-R) were compared between the endometrium of bitches (Canis familiaris) with pyometra accompanied by cystic endometrial hyperplasia (CEH) and that of healthy bitches at similar stages of the estrous cycle. In normal bitches, endometrial TGF-α mRNA levels were highest at proestrus and gradually decreased as the cycle progressed to anestrus. Epidermal growth factor receptor mRNA levels were not significantly affected by the stage of the estrous cycle. Epidermal growth factor mRNA levels were higher at Day 35 of diestrus than at other stages of the estrous cycle (P < 0.05). In bitches with pyometra, endometrial TGF-α and EGF-R mRNA levels did not differ significantly from those at diestrus in normal bitches, but EGF mRNA levels were lower than those at Day 35 of diestrus in normal bitches (P < 0.05). In normal bitches, positive immunohistochemical staining for TGF-α, EGF, and EGF-R was mainly present in the glandular and luminal epithelial cells of the endometrium. In contrast, in bitches with pyometra, immunoreactivity for EGF was clearly present in endometrial stromal cells. Inflammatory cells that had infiltrated the endometrial stroma stained strongly for TGF-α and EGF-R. Luminal and glandular epithelial cells also stained positive for EGF-R. In conclusion, expression of TGF-α by inflammatory cells and a low level of expression and differential localization of EGF may be involved in aberrant growth of endometrial glands and development of CEH.     

富含脯氨酸小蛋白-2在小鼠子宫中的表达及调节

富含脯氨酸小蛋白(Sprrs)参与构建复层扁平上皮的角化细胞壳（CE）,它们在子宫单层上皮中的作用还不清楚.采用RNA印迹和半定量RT-PCR方法,研究了Sprr2在小鼠动情周期和妊娠子宫中的表达及其激素调控.实验结果发现:Sprr2在动情前期和动情期表达上调,而动情后期和间情期表达下调.在妊娠初期表达迅速下调,直至临产期表达重新受到诱导并在产后达到高峰.鉴于其在不同生殖阶段子宫中独特的表达模式和在复层上皮中保护性的功能,推测Sprr2与子宫对交配和分娩所产生的应激反应有关.     

Localization and correlation between NADPH-diaphorase and nitric oxide synthase isoforms in the porcine uterus during the estrous cycle

Nitric oxide (NO), a highly reactive free radical is involved in vasodilation, neurotransmission, hormone secretion, and reproduction. Since all known nitric oxide synthase (NOS) isoforms possess NADPH-diaphorase (NADPH-d) activity, NADPH-d histochemistry was used as a commonly accepted procedure for NOS identification. The aim of our study was to determine the cellular localization of NADPH-d, eNOS, and iNOS in the porcine uterus and the correlation between NADPH-d and NOS activity in the early, middle, late luteal, and follicular phase of the estrous cycle. Light-microscopic observations of the sections revealed the differential expression of the NADPH-d in the analyzed stages of the estrous cycle. The most intense staining was observed in the luminal epithelium in the late luteal phase and in some groups of the endometrial glands in all studied stages. Positive reaction was also found in the endothelial cells of blood vessels and in the myometrium itself. Immunostaining for eNOS was observed in the luminal and glandular epithelium in all studied stages, but no clear fluctuations were observed. The endothelium of both endometrial and myometrial blood vessels displayed pronounced eNOS immunostaining. Strong iNOS staining was observed in the luminal epithelium in the late luteal and follicular phase and in selected groups of endometrial glands. Thus, only NADPH-d and iNOS undergo cyclic changes in the studied stages of the estrous cycle. The differential expression of NADPH-d/NOS in the porcine uterine horn during the estrous cycle suggests a role for NO in modulating uterine function.     

A morphological study of uterine alterations in mice due to exposure to cadmium

We investigated the morphologic and molecular effects of exposure to cadmium (Cd) for 30 and 60 days on the uteri of mice. We assessed uterine morphometric measurements, eosinophilia, mast cell numbers, endometrial apoptosis, proliferation and estrogen receptor alpha (ERα) immunoreactivity. We examined vaginal smears that reflected the hormonal alterations in the female reproductive tract. Because the female reproductive tract exhibits different morphology at each stage of the estrous cycle, we sacrificed all animals at estrus to make appropriate comparisons. Female BALB/c mice were exposed to 200 ppm Cd in their drinking water for either 30 or 60 days. Cd exposure caused significant decreases in endometrial thickness and number of glands in estrus phase uteri. The endometrial eosinophilia in the groups exposed to Cd also decreased compared to controls. Cd exposure increased the number of mast cells. Luminal and glandular epithelia were examined using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and by immunostaining proliferating cell nuclear antigen (PCNA) and estrogen receptor α (ERα). Compared to controls, the apoptotic index increased with time in both Cd exposed groups, while the proliferation index decreased. ERα immunoreactivity was decreased in both Cd exposed groups compared to controls; the decrease was most apparent in the 30 day Cd group. We found that 60 day Cd exposure increased apoptosis in the endometrium, which may affect the receptivity of the uterus for implantation.     

Differential expression of Toll-like receptor 4 (TLR4) in healthy and infected canine endometrium

This study provides the first report into immunohistochemical localization of Toll-like receptor (TLR) in the canine reproductive tract. TLR4 was investigated in endometrium during the estrous cycle and in pyometra. Pyometra is the most important pathological condition of the uterus due to bacterial infection in dogs. To protect against invading pathogens, the female reproductive tract has evolved immune mechanisms. TLRs are the cellular components of the afferent arm of the innate immune system. The expression of TLR4 was significantly higher in the endometrial stroma compared to the endometrial surface epithelium and glandular epithelium in proestrus. The glandular epithelium and stroma at the diestrous stage expressed TLR4 significantly higher than surface epithelium. Furthermore, when compared to other healthy groups, the glandular epithelium at diestrus also higher expressed TLR4 than other stages. The expression of TLR4 in the surface epithelium was higher in dogs with pyometra compared with all other groups. And, the surface epithelium of dogs suffering from pyometra also expressed TLR4 more intensely than the glandular epithelium. The innate immunity of infected canine endometrium response to bacterial infection is intensely extremely increased by the expression of TLR4. Furthermore, the different levels of TLR4 expression seems related to physiological changes in distinct cell types of endometrium, leukocytes populations, cytokines and sex hormones.     

G0101C03-3和L0254H10-3 基因在小鼠动情周期子宫中的差异表达

正常动情周期的维持是小鼠子宫多功能的必要条件 ,但对于其分子基础至今尚不十分明了。我们曾通过基因芯片技术分析了小鼠动情期与间情期子宫的基因表达谱 ,发现了许多差异表达的基因或表达序列标签(ESTs)。本实验选取了G0 1 0 1C0 3 3及L0 2 5 4H1 0 3两个差异表达的EST ,通过Northern印迹与原位杂交方法分析了它们在小鼠动情周期子宫中的时空表达模式。结果表明 :这两个基因的表达水平都发生周期性变化 ,在动情期表达量较少 ,而在间情期表达量较高 ;G0 1 0 1C0 3 3在动情期的表达量是间情期的 2 7% ,L0 2 5 4H1 0 3在动情期检测不到有表达 ,提示它们的表达受卵巢类固醇激素的调控 ;G0 1 0 1C0 3 3基因主要在子宫的腔上皮与腺上皮中表达 ,可能与子宫细胞的程序性死亡有关 ;而L0 2 5 4H1 0 3基因则主要位于基质细胞中     

Hysterographic appearance and uterine histology at different stages of the reproductive cycle and after progestagen treatment in the domestic cat

The aims of this study were to characterize the hysterographic and histological features of the uteri and to perform immunohistochemistry with proliferating cell nuclear antigen (PCNA) in the cat endometrium at various stages of the reproductive cycle and after treatment with exogenous progestagen. Seventy-four female domestic cats submitted for routine ovariohysterectomy were categorized into six groups: inactive (n=20), follicular (n=9), luteal (n=18), and postpartum (n=12) stages of the reproductive cycle; cats given medroxyprogesterone acetate for estrus prevention (MPA group) (n=12); and cats with uterine pathological lesions (n=3). Hysterography was performed and the relation of the uterine and luminal shape in the hysterogram with the stage of the reproductive cycle as well as with any pathological conditions of the uterus was evaluated. The uteri and ovaries were thereafter surgically removed and sectioned for histological examination. The PCNA was used to demonstrate the expression of endometrial epithelial cell growth. The hysterographic appearance was found to differ between the six groups of cats. A straight uterine cavity was characteristic for cats in the inactive stage, whereas a wavy uterine cavity was characteristic for cats in the follicular stage. In the luteal stage, the luminal cavity of the uteri differed in shape with increasing progesterone concentration from straight to irregular wavy or coiled. The coil shaped uterine lumen seen in the MPA treated and pathological groups was considered also to be an expression of a progestagenic effect. Waviness and coiling of the uterine lumen was related to a proliferation of the endometrial glands, whereas irregular filling defects were indicative of endometrial cystic changes. This study is the first to demonstrate the expression of PCNA in the cat endometrium although no differences were found between the six groups of cats. The hysterographic appearance was found to differ according to stage of the reproductive cycle and pathological conditions. Thus, a normative hysterogram is now available for diagnosing the reproductive stage and uterine changes in cats developing endometrial hyperplasia with and without cystic changes.     

Apoptosis in the canine endometrium during the estrous cycle

Apoptotic cell death in the endometria of 58 female dogs in different stages of the estrous cycle was assessed (in formalin-fixed, paraffin-embedded sections) with both the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay and immunohistochemical detection of caspase-3 activity. For both techniques, the apoptotic index was determined in the surface epithelium, stroma, crypts, and basal glands by counting the percentage of stained cells in a total of 500 cells in each category. In the surface epithelium and stroma, TUNEL- and caspase-3-positive cells were rare (apoptotic index<1) throughout the estrous cycle. However, caspase-3 detection showed a significant increase in the apoptotic index in the stroma during anestrus as well as an increase in the index in both the stroma and surface epithelium in late metestrus. The apoptotic index increased during late metestrus and anestrus in the crypts and basal glands; in the crypts, this increase was significant only when caspase-3 detection was used, whereas in basal glands, significant differences were found for both techniques. In conclusion, apoptosis was present in canine endometrial cells during the estrous cycle, but caspase-3 detection showed more significant differences than the TUNEL assay. Furthermore, a high apoptotic index (suggestive of endometrial desquamation) was not detected in the surface epithelium and there was no significant correlation between the apoptotic index in any cell group and serum progesterone concentrations.     

Possible role for insulin-like growth factor-I in the pathogenesis of cystic endometrial hyperplasia pyometra complex in the bitch

Cystic endometrial hyperplasia (CEH) is an important pathologic condition in the canine uterus and recognized as a common cause of illness and death in this species. The underlying cause and pathogenic mechanism responsible for this condition remains incompletely understood. Aberrant sex steroid hormone receptor expression in the uterus of dogs with CEH has been documented but not explained. In the dog there is an exceptionally high, progestin induced production of insulin-like growth factor-I (IGF-I) which is now generally accepted to be one of the most important growth factors with a high mitogenic effect on the uterus. Therefore, in this study the immunohistochemical staining intensity for IGF-I was compared among the uteri of 25 adult female dogs that had developed CEH and 14 healthy dogs in comparable stages of the estrus cycle. Specific staining for IGF-I was found in the cytoplasm epithelial cells and in smooth muscle cells of endometrium and myometrium. A marked increase in specific staining intensity for IGF-I was found in the surface epithelium, glandular epithelium and in the stroma of the uteri of dogs with CEH. The increase in IGF-I specific staining intensity was most prominent in the superficial endometrial stroma. Based on the known role of IGF-I in endometrial proliferation, it was concluded from the present study that high concentrations of IGF-I located in and around the epithelial cells of the endometrium in dogs with CEH, could play an important role in the development of CEH.     

The rat uterine oestrogen receptor in relation to intra-uterine devices and the oestrous cycle [proceedings

There are changes in the nuclear content of the estrogen receptor in the rat uterus during the estrous cycle that are associated with changes in its physiology. The changes correlate with the concentrations of circulating estradiol. It appears that uterotrophic response to estradiol is a function of the nuclear receptor. The insertion of an IUD leads to changes in the treated uterine horn which appear to be the result of an increased responsitivity to circulating estradiol. The presence of an IUD did not alter the estrous cycle, gonadotropin, or corpus luteum function. The intracellular distribution of the estrogen receptor was investigated in normal uterine horns and in the horns with devices throughout the estrous cycle. Groups of 30 Wistar rats had a silk suture fitted in the lumen of 1 uterine horn. After 14 days the progress of these estrous cycles was determined. Rats were grouped according to the stage of the cycle on the 4th day. Rats were then killed and the uteri removed. Cytosol receptors were measured. The capacity of the cytosol estrogen receptor to bind to oligo(dT)-cellulose was determined. Cytosol protein, nuclear protein, and DNA were measured. At all stages of the estrous cycle, the wet weight and cytosol receptor of the treated horns were greater than the control horns. A slight increase in the capacity of cytosol receptor to bind to oligo(dT)-cellulose was noted at proestrus. The response elicited by the IUD was not considered to be due to an estrogenic response since the changes observed were not accompanied by a corresponding increase in the content of nuclear receptor.