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PURPOSE The prospective Neoadjuvant Breast Registry Symphony Trial compared the 80-gene molecular subtyping signature with clinical assessment by immunohistochemistry and/or fluorescence in situ hybridization in predicting pathologic complete response (pCR) and 5-year outcomes in patients with early-stage breast cancer. METHODS Standard-of-care neoadjuvant chemotherapy combined with trastuzumab or trastuzumab plus pertuzumab was given to patients with human epidermal growth factor receptor 2 (HER2)–positive tumors (n = 295). pCR was the primary end point, with secondary end points of distant metastasis-free survival and overall survival at 5 years. RESULTS Among clinically defined HER2-positive (cHER2) tumors, the 80-gene assay identified 29.5% (87 of 295) as Luminal-Type (cHER2/gLuminal), 14.9% (44 of 295) as Basal-Type (cHER2/gBasal), and 55.6% (164 of 295) as HER2-Type (cHER2/genomically classified as HER2 [gHER2]). Patients with cHER2/gHER2 tumors had a higher pCR rate (61.6%) compared with non-gHER2 tumors (26.7%; P < .001). Dual targeting for cHER2/gHER2 tumors yielded a higher pCR rate (75%) compared with those treated with single HER2-targeted therapy (54%; P = .006). For cHER2/gBasal tumors, the 42.9% pCR rate observed with dual targeting was not different from that with trastuzumab alone (46.4%; P = .830). Among those with cHER2/gBasal tumors, 5-year distant metastasis-free survival (68.6%; 95% CI, 49.1 to 81.9) was significantly worse than in patients with cHER2/gLuminal tumors (88.9%; 95% CI, 78.0 to 94.6) and cHER2/gHER2 tumors (87.4%; 95% CI, 80.2 to 92.2; P = .010), with similar corresponding overall survival differences. CONCLUSION The 80-gene assay identified meaningful genomic diversity in patients with cHER2 disease. Patients with cHER2/gHER2 tumors, who benefitted most from dual HER2-targeted therapy, accounted for approximately half of the cHER2 cohort. Genomically Luminal tumors had low pCR rates but good 5-year outcomes. cHER2/gBasal tumors derived no benefit from dual therapy and had significantly worse 5-year prognosis; these patients merit special consideration in future trials.

As more patients with early-stage breast cancer receive neoadjuvant endocrine therapy (NET), there is a need for reliable biomarkers that can identify patients with HR+ HER2- tumors who are likely to benefit from NET. NBRST (NCT01479101) compared the prognostic value of the 70-gene risk classification and 80-gene molecular subtyping signatures with conventional pathological classification methods in response to neoadjuvant therapy. We evaluated the association of these signatures with clinical response and 5-year outcome of patients treated with NET.1091 patients with early-stage breast cancer scheduled to receive neoadjuvant therapy were prospectively enrolled into NBRST, and a sub-analysis of 67 patients treated with NET was performed. Patients received standard of care genomic testing using the 70-gene and 80-gene signatures and were treated with NET, per physician's discretion. The primary endpoint was pathologic partial response (pPR) and secondary endpoints were distant metastasis-free survival (DMFS) and overall survival (OS). Clinical benefit was defined as having a pPR or stable disease (SD) with NET.Overall, 94.4% of patients with genomically (g) Luminal A-Type (50.0% pPR and 44.4% SD) and 95.0% with Luminal B-Type tumors (55.0% pPR and 40.0% SD) exhibited clinical benefit. At 5 years, patients with gLuminal B tumors had significantly worse DMFS (75.6%, 95% CI 50.8-89.1) than patients with gLuminal A (91.1%; 95% CI 74.8-97.1; p = 0.047), with a similar trend for OS, albeit not significant (81.0%, 95% CI 56.9-92.4 and 91.1%, 95% CI 74.8-97.1, respectively; p = 0.13).Genomic assays offer a broader understanding of the underlying tumor biology, which adds precision to pathology as a preoperative risk classifier. Patients with 70-gene signature Low Risk, gLuminal A tumors treated with endocrine therapy alone have excellent 5-year outcomes. Most patients with genomically-defined Luminal A- and B-Type tumors respond well to NET, suggesting these patients may be safely treated with NET, while those with gLuminal B tumors will also require post-operative chemotherapy or CDK4/6 inhibitors to improve long-term outcomes. Overall, these findings demonstrate that genomic classification, defined by the combined 70- and 80-gene signatures, is associated with tumor response and prognostic of long-term outcomes.

PURPOSE The 80-gene molecular subtyping signature (80-GS) reclassifies a proportion of immunohistochemistry (IHC)-defined luminal breast cancers (estrogen receptor–positive [ER+], human epidermal growth factor receptor 2–negative [HER2–]) as Basal-Type. We report the association of 80-GS reclassification with neoadjuvant treatment response and 5-year outcome in patients with breast cancer. METHODS Neoadjuvant Breast Registry Symphony Trial (NBRST; NCT01479101 ) is an observational, prospective study that included 1,069 patients with early-stage breast cancer age 18-90 years who received neoadjuvant therapy. Pathologic complete response (pCR) and 5-year distant metastasis-free survival (DMFS) and overall survival (OS) were assessed in 477 patients with IHC-defined ER+, HER2– tumors and in a reference group of 229 patients with IHC-defined triple-negative breast cancer (TNBC). RESULTS 80-GS reclassified 15% of ER+, HER2– tumors (n = 73) as Basal-Type (ER+/Basal), which had similar pCR compared with TNBC/Basal tumors (34% v 38%; P = .52), and significantly higher pCR than ER+/Luminal A (2%; P < .001) and ER+/Luminal B (6%; P < .001) tumors. The 5-year DMFS (%, [95% CI]) was significantly lower for patients with ER+/Basal tumors (66% [52.6 to 77.3]), compared with those with ER+/Luminal A tumors (92.3% [85.2 to 96.1]) and ER+/Luminal B tumors (73.5% [44.5 to 79.3]). Importantly, patients with ER+/Basal or TNBC/Basal tumors that had a pCR exhibited significantly improved DMFS and OS compared with those with residual disease. By contrast, patients with ER+/Luminal B tumors had comparable 5-year DMFS and OS whether or not they achieved pCR. CONCLUSION Significant differences in chemosensitivity and 5-year outcome suggest patients with ER+/Basal molecular subtype may benefit from neoadjuvant regimens optimized for patients with TNBC/Basal tumors compared with patients with ER+/Luminal subtype. These data highlight the importance of identifying this subset of patients to improve treatment planning and long-term survival.

The Neoadjuvant Breast Symphony Trial (NBRST) demonstrated the 70-gene risk of distant recurrence signature, MammaPrint, and the 80-gene molecular subtyping signature, BluePrint, precisely determined preoperative pathological complete response (pCR) in breast cancer patients. We report 5-year follow-up results in addition to an exploratory analysis by age and menopausal status.The observational, prospective NBRST (NCT01479101) included 954 early-stage breast cancer patients aged 18-90 years who received neoadjuvant chemotherapy and had clinical and genomic data available. Chemosensitivity and 5-year distant metastasis-free survival (DMFS) and overall survival (OS) were assessed. In a post hoc subanalysis, results were stratified by age (≤ 50 vs. 50 years) and menopausal status in patients with hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-) tumors.MammaPrint and BluePrint further classified 23% of tumors to a different subtype compared with immunohistochemistry, with more precise correspondence to pCR rates. Five-year DMFS and OS were highest in MammaPrint Low Risk, Luminal A-type and HER2-type tumors, and lowest in MammaPrint High Risk, Luminal B-type and Basal-type tumors. There was no significant difference in chemosensitivity between younger and older patients with Low-Risk (2.2% vs. 3.8%; p = 0.64) or High-Risk tumors (14.5% vs. 11.5%; p = 0.42), or within each BluePrint subtype; this was similar when stratifying by menopausal status. The 5-year outcomes were comparable by age or menopausal status for each molecular subtype.Intrinsic preoperative chemosensitivity and long-term outcomes were precisely determined by BluePrint and MammaPrint regardless of patient age, supporting the utility of these assays to inform treatment and surgical decisions in early-stage breast cancer.

Warfarin is used in paediatric populations, but dosing algorithms incorporating pharmacogenetic data have not been developed for children. Previous studies have produced estimates of the effect of polymorphisms in Cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) on stable warfarin dosing, but data on time in therapeutic range, initial dosing and adverse effects are limited. Participants (n=97) were recruited, and routine clinical data and salivary DNA samples were collected from all participants and analysed for CYP2C9*2, *3 and VKORC1-1639 polymorphisms.VKORC1 -1639 was associated with a greater proportion of the first 6 months' treatment time spent within the target International Normalised Ratio (INR) range, accounting for an additional 9.5% of the variance in the proportion of time. CYP2C9*2 was associated with a greater likelihood of INR values exceeding the target range during the initiation of treatment (odds ratio (OR; per additional copy) 4.18, 95% confidence interval (CI) 1.42, 12.34). CYP2C9*2 and VKORC1-1639 were associated with a lower dose requirement, and accounted for almost 12% of the variance in stable dose. VKORC1-1639 was associated with an increased likelihood of mild bleeding complications (OR (heterozygotes vs homozygotes) 4.53, 95% CI 1.59, 12.93). These data show novel associations between VKORC1-1639 and CYP2C9*2 and INR values in children taking warfarin, as well as replicating previous findings with regard to stable dose requirements. The development of pharmacogenomic dosing algorithms for children using warfarin has the potential to improve clinical care in this population.

The nonsteroidal estrogenic compound bisphenol A (BPA) is a monomer used in the manufacture of polycarbonate plastics and resins. BPA may be ingested by humans as it reportedly leaches from the lining of tin cans into foods, from dental sealants into saliva, and from polycarbonate bottles into their contents. Because BPA is weakly estrogenic--approximately 10,000-fold less potent than 17beta-estradiol--current environmental exposure levels have been considered orders of magnitude below the dose required for adverse effects on health. Herein we demonstrate measurable effects on the offspring of Sprague-Dawley female rats that were exposed, via their drinking water, to approximately 0.1 mg BPA/kg body weight (bw)/day (low dose) or 1.2 mg BPA/kg bw/day (high dose) from day 6 of pregnancy through the period of lactation. Offspring exposed to BPA exhibited an increase in body weight that was apparent soon after birth and continued into adulthood. In addition, female offspring exposed perinatally to the high dose of BPA exhibited altered patterns of estrous cyclicity and decreased levels of plasma luteinizing hormone (LH) in adulthood. Administration of neither the doses of BPA that caused effects during perinatal exposure nor a 10-fold higher dose was able to evoke a uterotropic response in ovariectomized postpubertal females. These data indicate an increased sensitivity to BPA during the perinatal period and suggest the need for careful evaluation of the current levels of exposure to this compound.

Background The objective of the present study was to define the morphological changes that occur in the epithelium of the isthmus oviduct of the sheep during the first few weeks of pregnancy. Materials: Isthmus oviducts were obtained from ovariectomized, estrous (day O) and pregnant (day 2, 3, 4, 6, and 16) ewes and analyzed using light and electron microscopy. Results The epithelium was low cuboidal in the isthmus from ovariectomized sheep, significantly increased in height at estrus, underwent an additional increase at day 3, and was significantly reduced by day 4 of pregnancy. Ciliated and nonciliated epithelial cells were present at all reproductive stages. Ciliated cells were always the predominant cell type. The percentage of ciliated and nonciliated cells did not significantly change at any stage examined. In addition, a third cell type, located adjacent to the basement membrane, was present. These “basal” cells were rounded in appearance and more frequently observed in the epithelial lining of ovariectomized ewes and after day 3 of pregnancy. The secretory organelles of nonciliated epithelial cells obtained from ovariectomized ewes were poorly developed. In estrous ewes, the Golgi apparatus in nonciliated cells consisted of stacked cisternae that atrophied later in pregnancy. The most striking alterations in the secretory apparatus occurred in ciliated cells. Two populations of ciliated cells were observed at estrus-one with abundant, membrane bound supranuclear granules and one without granules. The granules in ciliated cells were rare after day 3 of pregnancy and no evidence of secretion was ever observed. Large lipid droplets were present in the cytoplasm of ciliated cells at estrus. Lysosomes were abundant in nonciliated cells by day 16 of pregnancy. Regularly arranged microvilli were present in nonciliated cells obtained from ovariectomized, estrous, and pregnant ewes. No appreciable change in nuclear shape or chromatin content was observed at any reproductive stage examined. The ultrastructural characteristics and relative abundance of other cytoplasmic organelles such as the RER, SER, mitochondria, and glycogen varied slightly during pregnancy in ciliated and nonciliated cells. Conclusions These data show that the epithelial lining of the isthmus oviduct in the sheep undergoes subtle, yet distinctive, structural changes during the first few weeks of pregnancy. Anat. Rec. 247:368–378, 1997. © 1997 Wiley-Liss, Inc.

The deduced amino acid sequence of an estrogen-dependent sheep oviductal glycoprotein (M(r) 90,000-116,000) revealed the presence of several potential sites for glycan substitution on a protein backbone of M(r) approximately 66,500, and identity with chitinases. In order to further define the nature of the secreted glycoprotein, the objectives of the present study were 1) to devise a method to significantly enrich for the glycoprotein from oviductal secretions, 2) to biochemically characterize the glycoprotein by use of lectin blotting and enzymatic and chemical digestion, and 3) to determine whether unfractionated and enriched fractions containing the glycoprotein have chitinase activity. Oviducts were obtained from ovariectomized ewes treated with estradiol for 6 days and explant-cultured for 24 h. The oviductal glycoprotein was enriched approximately 80-85% from explant culture media by Maackia amurensis agglutinin (MAA) lectin affinity chromatography. Enriched fractions containing the oviductal protein were separated on SDS gels, transferred to polyvinyl difluoride, and probed with digoxigenin-labeled lectins. Lectin blotting revealed that the glycoprotein contained the carbohydrate moieties N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose, and sialic acid both in alpha(2,3) and alpha(2,6) linkages, typical of sialomucins. Enzymatic digestion with neuraminidase and N-glycanase indicated that approximately 20% and approximately 6% of the molecular weight of the oviductal glycoprotein can be accounted for by sialic acid and N-linked glycans, respectively. The oviductal glycoprotein was resistant to digestion with O-glycanase alone and chondroitinase ABC, with the latter indicating that it was not a proteoglycan. Treatment with trifluoromethanesulfonic acid resulted in a deglycosylated product of M(r) approximately 66,000 immunoreactive with antibodies to the oviductal glycoprotein. No chitinase activity could be detected for unfractionated culture medium proteins or enriched fractions containing the M(r) 90,000-116,000 oviductal glycoprotein when the substrate methylumbelliferyl chitotriose was used. These data show that 1) MAA lectin chromatography can significantly enrich for the M(r) 90,000-116,000 glycoprotein from oviductal secretions, 2) the secreted glycoprotein contains saccharide residues typical of sialomucins, and 3) despite primary amino acid sequence identity, the oviductal glycoprotein does not share an enzymatic relationship with chitinases.

Data from our laboratory have shown that an estrogen (E2)-dependent M(r) 90,000-92,000 protein and its mRNA are synthesized and expressed in abundant amounts at estrus from the fimbria and ampulla, not isthmus, oviduct of the sheep. Immunocytochemical studies have shown that the M(r) 90,000-92,000 protein is contained in apical secretory granules of oviduct epithelial cells. The objective of this study was to determine whether the mRNA for the E2-dependent oviduct protein was localized and compartmentalized in similar manner. Fimbria, ampulla, and isthmus oviducts obtained from estrous ewes were flash frozen in liquid nitrogen, cryosectioned, fixed in 4% paraformaldehyde, hybridized with digoxigenin (DIG)-labeled oviduct-specific riboprobes, incubated in anti-DIG antibodies conjugated with alkaline phosphatase, and developed in color substrate. Oviduct protein-specific transcripts were localized to basal perinuclear compartments and, surprisingly, at sites distant from the nucleus in the apical cytoplasm of epithelial cells in the fimbria and ampulla. No specific reaction product was observed in the underlying mucosa or smooth muscle layers. Oviduct protein mRNA was contained predominantly in the apical cytoplasm of epithelial cells at the free margins of mucosal folds and in the basal regions of cells located at the crypts of longitudinal folds. No reaction product was present when sections of the fimbria and ampulla oviduct of estrous ewes were incubated in sense riboprobe to the oviduct protein. In addition, when sections of the isthmus oviduct obtained from estrous ewes or fimbira and ampulla oviducts from long-term ovariectomized ewes were hybridized with antisense riboprobes no specific reaction product was detected. Electron microscopy of oviduct protein mRNA containing areas revealed the presence of secretory granules, rough endoplasmic reticulum (RER) and Golgi in the apical cytoplasm, and RER in the basal regions of epithelial cells. These data show that the mRNA encoding an E2-dependent oviduct-specific protein is distributed in epithelial cells at perinuclear foci and at sites distant from the nucleus, which are also the sites of protein localization and protein synthesizing organelles, implying translation at unique cytoplasmic foci.

The biosynthetic activity of the sheep ampulla oviduct changes during the first few days of pregnancy. An estrogen (E2)-dependent glycoprotein (M(r) 90,000-92,000) is expressed, synthesized, and released when fertilization and embryo development occur in the oviduct. The objective of the present study was to determine whether the secretory epithelial lining of the ampulla, the cellular source of the E2-dependent protein, undergoes morphological alterations during early pregnancy is a contemporaneous manner. Ampulla oviducts obtained from ovariectomized (ovx), estrous (Day 0), and pregnant (Days 1.5, 2, 3, 4, 6, and 16) ewes were analyzed with light and electron microscopy. The mean cell height of epithelial cells from estrus and from Days 1.5, 2, and 3 of pregnancy was significantly (p < 0.05) higher than in ovx animals. A significant reduction in cell height was observed at Day 4 of pregnancy, and this value remained unchanged at Day 6 and Day 16. Ciliated and nonciliated cells were present in the ampulla at all stages examined, with ciliated cells constituting the major cell type. Consistent with previous results, the nonciliated cells obtained from ovx ewes were synthetically inactive and at estrus were characterized by well-developed secretory organelles, including abundant amounts of secretory granules containing the E2-dependent glycoprotein. Extensive microvilli and cytoplasmic blebs protruding into the oviduct lumen distinguished the apical surface of secretory epithelial cells until Day 3. At Day 1.5 the secretory cells contained dilated cisternae of rough endoplasmic reticulum and enlarged Golgi, which were progressively diminished with increasing stages of pregnancy. Exocytosis, fusion of secretory granules, and a reduction in their electron density were seen until Days 3-4. By Day 4 and Day 6, fewer granules were observed in the ampulla cytoplasm and few, if any, were present by Day 16. Increases in the amount of heterochromatin were seen in nuclei at Day 6 and Day 16. Extrusion of epithelial cells into the oviduct-lumen was evident at Day 16 of pregnancy. These data show that 1) the secretory epithelium of the sheep ampulla oviduct undergoes morphological alterations in protein-synthesizing organelles and apical specializations that vary with the stage of pregnancy, 2) the secretory product contained in lamellar granules is released by the process of exocytosis until Days 3-4, and 3) cell death appears to occur at Day 16 by shedding of epithelial cells into the oviduct lumen.

This study was undertaken (1) to devise a method of inducing multiple follicular development and subsequent ovulation in the Djungarian or Siberian hamster (Phodopus sungorus) and (2) to assess the quality of ovulated oocytes collected from PMSG/hCG treated animals in comparison to naturally ovulating animals. Hamsters (4–5 weeks; n = 70) received 5 IU PMSG followed 50–52 hr later by 10 IU hCG. Ovulated oocytes were collected 14–20 hr after hCG injection. Ovulated oocytes were flushed from oviducts of cycling animals (7–12 weeks; n = 30) exhibiting two consecutive estrous cycles. Oocytes were fixed and subjected to triple fluorescence immunostaining using anti-tubulin antibodies, fluorescein phalloidin, and Hoechst 33258. The mean number of ovulated oocytes collected from cycling animals was 4.8 ± 0.4 (range 1–7). Ovulation occurred in 73% of the PMSG/hCG-stimulated animals. The mean number of oocytes ovulated from stimulated animals was 9.2 ± 0.8 (range 0–22). The ovaries of animals that did not ovulate or that ovulated few oocytes did respond to PMSG, as indicated by the presence of multiple follicular development and pre-ovulatory stigmata. There was no evidence of a polar body in ovulated oocytes collected from PMSG/hCG-treated or cycling animals, indicating that oocytes were arrested in meiosis I. In the majority (80%) of ovulated oocytes from PMSG/hCG-treated and cycling animals, cortically placed chromosomes were aligned on a metaphase plate equidistant from a bipolar spindle. Sparse f-actin staining was observed in the region of the ooplasm surrounding the chromosomes. As the interval between hCG injection and the time of collection increased, chromosomes lost their proper alignment and migrated away from the cortex of the oocyte concomitant with a disruption of spindle integrity. This collapse of proper chromosome alignment and disruption of spindle architecture also characterized aging oocytes collected from cycling animals. These data show that in the Djungarian or Siberian hamster (Phodopus sungorus), (1) there is individual animal variation in responsiveness to hCG following PMSG treatment, (2) there are no cytological differences in the quality of oocytes collected from hormonally treated animals when compared to cycling animals, and (3) oocytes are ovulated in meiosis I. © 1995 Wiley-Liss, Inc.

The luminal environment of the estrogen (E2)-dominated mammalian oviduct generates and sustains the environment in which the first embryonic cleavages take place. The objective of this study was to determine, by use of an antiserum against an E2-dependent sheep oviduct secretory glycoprotein (M(r) 90,000-92,000), whether the E2-dominated and pregnant oviduct of the Djungarian hamster (Phodopus sungorus) releases an antigenically related protein. If the protein was present, a secondary objective was to define its fate and association with filamentous-actin (f-actin) and chromatin patterns in early cleavage-stage embryos. Oviduct flushings containing embryos (1-cell fertilized, 2-, 4-, and 8-cells), and uterine flushings (> 16 cell embryos) were obtained from pregnant hamsters. Embryos were removed from flushings, and oviduct secretions were analyzed by Western blotting. The zona pellucida was removed with acid Tyrode's solution from approximately half of the 2-, 4-, and 8-cell embryos. Zona-intact and zona-free embryos were then fixed and subjected to triple immunofluorescence staining with an antiserum to the sheep oviduct protein, rhodamine phalloidin, and Hoechst 33258. An antigenically related protein M(r) 200,000) was detected in oviduct secretions of E2-treated, ovariectomized, and pregnant hamsters, and not in secretions from ovariectomized controls. In the zona pellucida of 1- and 2-cell embryos, the oviduct protein displayed an intertwining, reticular organization that was replaced by a diffuse and more intense accumulation in 4-, 8-, and > 16-cell embryos. In 2-cell embryos, punctate foci of the oviduct protein were distributed unevenly over the apical blastomere plasma membrane, forming patches in regions of f-actin exclusion, which were absent at later development stages. At the 4- and 8-cell stage of development, as blastomeres lost their spherical form by minimizing intercellular spaces, the oviduct protein took on a polarized arrangement and was intensely concentrated on membrane areas involved in cell-cell contact that were also the focus of f-actin. These data show that in early cleavage-stage hamster embryos, the intensity and pattern of staining for an E2-dependent oviduct protein (M(r) 200,000) that is released into the oviduct lumen during embryo transport can be distinguished on the basis of membrane f-actin display and blastomere number and shape. These events may mediate cellular processes related to blastomere cleavage, shaping, and/or adhesion that occur in the oviduct.

Administration of estradiol-17j7 (E) to ovariectomized (ovx) sheep results in the synthesis and release of an M, 90 00092 000 glycoprotein into the oviductal lumen and into culture medium of ampullar explants (Biol Reprod 1992; 47:889-902). The objective of this study was to determine when and from what region of the oviduct the Mr 90 000-92 000 glycoprotein is synthesized and released during early pregnancy. Estrous ewes were bred to intact rams of known fertility, and oviducts were obtained at estrus (Day 0) and at Days 1.5, 2, 3, 4, 6, and 16 of pregnancy. Pregnancy was verified by the presence of a fertilized egg or developing conceptus and a functional corpus luteum. Oviductal secretions were collected by flushing oviducts with saline and by explant culture. The oviductal fimbria, ampulla, and isthmus were individually cultured (24 h) in the presence of 5 H-leucine (3H-leu) or H-glucosamine (H-glcN). The presence of the Mr 90 000-92 000 glycoprotein in oviductal flushings and culture medium was determined by fluorography and Western blotting. The Mr 90 000-92 000 protein was present in SDS gels and blots of oviductal flushings from animals through Days 4-6 of pregnancy, but not in flushings from Day 16 pregnant animals or from ovx, untreated animals. This protein was present in H-leu- and 'H-glcN-labeled culture medium of the oviductal ampulla (Days 0, 1.5, 2, 3, 4, 6, and 16) and fimbria (Days 0, 1.5, 2, 3, and 4) during early pregnancy. The M, 90 000-92 000 protein was not detected in oviductal ampulla or fimbria culture medium obtained from ovx animals, nor was it present in culture medium of the isthmus oviduct obtained from estrous and pregnant animals. These data show that the E-dependent Mr 90 000-92 000 glycoprotein is synthesized and released by the oviduct in a temporally and regionally specific manner during early pregnancy as the gametes and fertilized egg are traversing the tube and prior to a systemic rise in progesterone.

This study was undertaken to determine the effects of 17 beta-estradiol (E) and progesterone (P) on polypeptide synthesis and release from the uterus of the sheep. Uterine flushings (UF) and endometrium were obtained from ovariectomized untreated animals, ovariectomized animals treated with E (approximately 5-10 pg/ml) for 6 days (6E) and ovariectomized animals primed with E for 6 days then treated with P (approximately 1.5-3 ng/ml), in the continued presence of E, for an additional 6 days (6EP). Endometrium was cultured (24 h) in the presence of 3H-leucine (3H-leu) or 3H-glucosamine (3H-glcN), and newly synthesized and released proteins were detected in culture media by fluorography of 10% SDS gels. The quantity of proteins in UF and radiolabeled proteins in explant culture media did not change between treatment groups (p 200,000 was present in 3H-leu-labeled culture media of endometrium obtained from 6E and 6EP animals. An M(r) 44,000 protein was present only in UF from 6EP animals but could not be detected in endometrial culture media from animals undergoing this steroid treatment. These data show that the endometrium of the ovariectomized sheep undergoes alterations in secretory protein patterns which depend on the presence of E and P.

Published reports have shown that an M(r) 90,000-92,000 protein is released into the oviductal lumen of the sheep, during estrus at a time corresponding to ovulation and fertilization, where it associates with the embryo. The objectives of this study were (a) to determine whether estradiol-17 beta (E) alone or in combination with progesterone (P) induces the synthesis of the M(r) 90,000-92,000 protein from the ampulla and/or isthmus oviduct; (b) to monitor structural alterations in oviductal epithelial cells associated with the synthesis of this protein; and (c) to generate a polyclonal antiserum to the protein and use the antiserum to verify its cellular location and tissue specificity. Oviductal flushings and explant culture media were obtained from ovariectomized animals treated with E alone or with E plus P. The M(r) 90,000-92,000 protein was present in 3H-leucine- and 3H-glucosamine-labeled culture media of the ampulla (not isthmus) oviduct in animals treated with E alone or with E plus P. The glycoprotein was detected in gels of oviductal flushings obtained from animals treated only with E. A specific polyclonal antiserum to the protein was made and cross-reacted on Western blots of oviductal flushings from E-treated animals and ampulla (not isthmus) oviduct culture media from animals treated with E alone or with E plus P. The secretory apparatus of the epithelial cells of the ampulla oviduct matured and differentiated in response to E. Light and electron microscopic immunocytochemistry localized the M(r) 90,000-92,000 glycoprotein to secretory granules in the nonciliated cells of the ampulla oviduct. Immunoperoxidase reaction product was absent in tissue sections and Western blots of other reproductive and nonreproductive tract tissues obtained from steroid-treated animals. Therefore, the secretory cells of the ampulla oviduct of the sheep synthesize and release an E-induced, oviduct-derived M(r) 90,000-92,000 glycoprotein.

The development of epithelial cells of the uterine glands of ovariectomized sheep in response to estradiol-17 beta (E) and progesterone (P) was studied using light and electron microscopy. Animals that had been ovariectomized for six weeks were placed in one of three experimental treatment groups. Group I animals (untreated controls) received no steroid treatment. Group II animals (E alone) received one 4-cm E implant (E approximately 5-10 pg/ml) and their uteri were removed after 2, 4, or 6 days. Group III animals (E-primed, P-treated) received an E implant (E approximately 5-10 pg/ml) for 6 days and then were treated with six 13-cm P implants (P approximately 1.5-3 ng/ml), in the continued presence of E, for 2, 4 or 6 days. Six weeks after ovariectomy the epithelial cells of the uterine glands were low cuboidal and morphologically appeared to be synthetically inactive. Following 2 days of E treatment the epithelial cells had significantly increased in cell height, and protein-synthesizing organelles were well developed. Maturation of the secretory apparatus continued throughout E treatment. The Golgi complex and rough endoplasmic reticulum (RER) were abundant. Lysosomal-like granules and granules of varying electron density were present in the cytoplasm. The chronic administration of P to E-primed animals did not result in any further increase in cell height. Elongated mitochondria, a cup-shaped Golgi apparatus, extensive apical microvilli, and irregularly shaped membranous profiles in the supranuclear cytoplasm characterized these uterine epithelial cells. Lysosomal-like granules, small vesicles, and scattered patches of glycogen were seen in the cytoplasm. These data show that the uterine epithelial cells of the ovariectomized sheep undergo morphological alterations in protein-synthesizing organelles and apical specializations that depend on the presence of E and P.

The cyclic fluctuations in circulating levels of 17 beta-estradiol and progesterone that occur during the menstrual or estrous cycle are responsible for dramatic, cyclic changes in the epithelial lining and secretory status of the mammalian oviduct. The timely transition in the synthesis and release of oviduct proteins, due to the ovarian steroids, and their interactions with oocytes, sperm, and the fertilized ovum underscore key biological events during gamete interactions and early embryonic cleavage. The regulation of these secretory alterations during the first few days of pregnancy is discussed with respect to the influence of the ovarian steroids, their interactions with the embryo microenvironment, and the possible ways in which they may mediate the critical reproductive events of fertilization and embryo development.

An estrogen (E2)-dependent 90,000-92,000 M(r) protein is synthesized and released by the sheep oviduct in a temporally and regionally specific manner during the first few days of pregnancy, where it associates with the embryo. The present study was undertaken to further define the nature of this protein and its regulation at a time when fertilization and early embryonic cleavage occur in the oviduct. The complete complementary DNA (cDNA) sequence for the 90,000-92,000 M(r) protein was determined, and steady state messenger RNA (mRNA) levels were measured during early pregnancy (estrus and days 1, 2, 3, 4, 6, and 16). The composite cDNA possessed an open reading frame of 1633 bases with a single potential N-glycosylation site. The inferred amino acid (aa) sequence predicted a prepolypeptide of 539 aa (69,151 M(r)) and a mature polypeptide of 518 aa (66,477 M(r)). Nucleotide and deduced aa sequence shared identity with translated E2-dependent cow, human, and partially sequenced baboon oviduct protein cDNAs; a human articular cartilage protein, gp 39; and chitinases. Northern blot hybridization revealed a single RNA species (2.2 kilobases) in the fimbria and ampulla, which was not detected in the isthmus or other reproductive and nonreproductive tract tissue RNAs. Steady state levels of mRNA encoding the 90,000-92,000 M(r) were highest in the fimbria and ampulla at estrus and on day 1 of pregnancy, when gamete transport and fertilization occur in the E2-dominated fallopian tube. Oviduct protein mRNA levels declined significantly (P < 0.05) on day 2 and underwent a further significant reduction on day 3 of pregnancy coincident with transport of the embryo from the oviduct to the uterus, a reproductive stage associated with rising progesterone levels. By day 16 of pregnancy, the mRNA encoding the E2-dependent oviduct protein was virtually undetectable. The temporally and regionally specific regulation of gene expression for the 90,000-92,000 M(r) E2-dependent protein correlates with protein synthesis data and emphasizes the precise regulation of its synthesis at a time when fertilization and embryonic cleavage are taking place in the oviduct. Identity with a growing class of steroid-regulated oviduct secretory proteins and chitinase enzymes suggests a possible structural and/or functional relationship, which may be important in mediating these latter reproductive events.

We previously reported the purification of an estrogen-dependent cat uterine protein (CUPED) and the preparation of a specific anti-CUPED serum in rabbits. Here, we describe a specific radioimmunoassay for CUPED using the purified CUPED and anti-C UPED serum that was utilized to quantify CUPED in daily uterine flushings obtained from steroid-treated ovariectomized cats. The radioimmunoassay was sufficiently sensitive to measure 0.1-100 ng C UPED. C UPED levels were low in un treated ovariecto mized cats, in creased within one day after the onset of treatment with estradiol, and remained elevated as long as estradiol was unopposed by progesterone. The levels of CUPED decreased when progesterone was added to the treatment regimen either 7, 14, or 28 days after the initiation of estradiol treatment. The data indicate 1) that the presence of CUPED in the uterine flushings is dependent on the presence of estradiol and the absence of progesterone, 2) that CUPED appears in the uterine lumen within one day after the onset of treatment with estradiol, and 3) that the levels of CUPED are sharply reduced within one day of administration of progesterone and become nondetectable after three days.

An estrogen-dependent polypeptide (CUPED), which was purified from uterine flushings of estrogen-treated cats, was localized in endometrial epithelial cells of cats using the peroxidase-antiperoxidase immunocytochemical staining procedure. Epithelial cells from animals treated with estradiol for 4, 7, or 14 days and estrogen-primed animals treated with progesterone for 2 days showed positive immunostaining. Staining was absent in untreated ovariectomized animals and in estrogen-primed animals treated with progesterone for 4 days. Specific cytoplasmic staining was confined to apical secretory granules in nonciliated cells of deep uterine glands. Staining was also commonly observed in the lumen of deep glands. Immunostaining was absent in the cells of the surface epithelium, stroma, and myometrium. In addition, other organs such as the oviduct, kidney, liver, pancreas, and lung showed no evidence of specific immunocytochemical staining. Therefore, the estrogen-dependent polypeptide obtained from uterine flushings of estrogen-treated ovariectomized cats is a uterine-specific secretory product that is packaged in apical cytoplasmic granules of uterine epithelial gland cells before being released into the uterine lumen.

Oviducts and uteri were obtained from cats at estrus and up to 14 days after coitus. Progesterone binding sites were estimated by Scatchard analysis, serum levels of progesterone were determined by radioimmunoassay, and key morphological parameters of the lining of the epithelium of the oviduct and the deep uterine glands were quantitated. Progesterone cytosol binding was hightest at estrus, significantly depleted by day 4, and continued a gradual decline out to day 14 in both tissues. Nuclear binding was markedly elevated at day 4; with time the binding decreased until by day 14 it was equal to or lower than detected at estrus. The initial depletion of cytosol binding and rise in nuclear binding coincided with the initial rise in serum progesterone levels. Within the oviduct, the increase in nuclear binding coincided with the onsdet of regressive changes within the epithelium. These changes included atrophy, deciliation, and apotosis. Within the uterine glands, the increase in nuclear binding coincided with the onset of the progestational response. This response included cell hypertrophy, glycogen synthesis and deposition, and with longer exposure to elevated levels of progesterone, the disappearance of these glycogen deposits. These results clearly illustrate the immediate effect of low levels of progesterone on the subcellular compartmentalization of the progesterone receptor system, and the down regulation of the total receptor system when target tissues are exposed to a continuous elevation of serum progesterone. These result also illustrate that similar changes in receptor compartmentalization induce different morphological responses within target tissues from the same animal.

cytosol and nuclear progesterone receptors in the cat uterus were measured by Scatchard analysis to determine the relationships between dose of progesterone administered and the time following administration with the content of receptor in these two cellular compartments. Cats were ovariectomized, treated for 7 days with estradiol and then injected via the saphenous vein with progesterone. One uterine horn was removed prior to, and the other uterine horn after the injection of progesterone. The amount of cytosol receptor translocated was found to be dosedependent over the range of 0-200 pg of progesterone. A maximum of 40% of the cytosol receptor was depleted even when the amount of progesterone injected was increased. In non-estradiolprimed animals it was also found that approximately 40% of the cytosol receptor was depleted following a progesterone injection. Within 3 h of the injection of 300 pg of progesterone, the cytosol and nuclear receptor levels had returned to preinjection values. A second administration of progesterone at 1 or 3 h after the first injection of progesterone caused a partial depletion of the cytosol receptor and an increase in nuclear progesterone receptor concentration. These data suggest that the translocation of cytosol receptor and the appearance of nuclear receptor is dosedependent until approximately 40% of the cytosol receptor is depleted following a single injection of progesterone, that the retention of nuclear receptor after an acute injection of progesterone is of short duration (

An estrogen-dependent secretory protein (CUPED) was detected and purified from uterine flushings of ovariectomized cats treated with 17 beta-estradiol. The protein was not detected in uterine flushings obtained from untreated ovariectomized animals or estrogen-primed animals treated with progesterone for 4 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of uterine flushings showed the presence of 1 or 2 protein bands with relative mobility values less than reduced and denatured thyroglobulin (Mr = 330,000). The protein was purified by differential centrifugation and gel filtration chromatography. Antiserum was raised against this purified protein in rabbits. The specificity of the antiserum to uterine fluid proteins was assessed by immunoblotting of electrophoretically transferred proteins. The antiserum cross-reacted with electrophoretically separated CUPED protein bands in uterine flushings. This protein may represent the content of the estradiol-induced secretory granules present in endometrial epithelial cells.

Media from cultured cat endometrial explants were analyzed for the presence of a previously characterized high molecular weight estrogen-dependent secretory protein (CUPED) by polyacrylamide gel electrophoresis and radioimmunoassay (RIA). Both L-[(3)H]-leucine and D-[(3)H]-glucosamine were incorporated into newly synthesized CUPED during the culture of endometrial explants obtained from estradiol-treated ovariectomized cats, but not during the culture of tissue obtained from untreated ovariectomized animals or estradiol-primed ovariectomized animals treated with progesterone. The addition of tunicamycin to the culture medium inhibited the synthesis and release of the glycosylated form of CUPED from endometrial explants obtained from estradiol-treated cats. These data demonstrate that CUPED is synthesized and released in vitro from endometrial explants obtained from estradiol-treated cats as a glycoprotein possessing N-linked oligosaccharide chains.