Elucidation of the Cellular and Molecular Events Associated with Bovine Uterine Development and Disruptive effects of Tamoxifen

Abstract

Development of the bovine uterus begins prenatally and is completed postnatally with the formation of endometrial glands. Adenogenesis, uterine gland formation, is steroid hormone-sensitive and is required for proper uterine development and function. Uterine glands produce histotroph, secretions which are essential for conceptus development, recognition and maintenance of pregnancy, and placental and fetal growth. Exposure of developing uterine tissues to steroids, including estrogens and progestins, can alter patterns of uterine development such that uterine endometrial structure and function are compromised in the adult. Disruption of estrogen receptor alpha (ESR1) and progesterone receptor (PR) -mediated organizational events in ruminants can induce such effects. However, little is known about mechanisms regulating bovine endometrial development. Studies described here were designed to define cellular and molecular events associated with postnatal bovine endometrial histogenesis and cytodifferentiation. Multilabel immunohistochemistry (IHC), multispectral imaging (MSI) and digital image processing (DIP) were used to detect, capture and extract qualitative and quantitative data for multiple targeted signals associated with specific endometrial proteins, including transcription factors, and endometrial cell proliferation simultaneously in single uterine tissue sections. In study one, for purposes of technical validation, uterine samples from a single Holstein heifer obtained on postnatal day (PND) 40, treated with 5’bromo-2- deoxyuridine (BrdU) five days prior to euthanasia, were used to establish and validate imaging protocols. Single- and multilabel IHC protocols were evaluated in uterine cross-sections. Images were captured using MSI, and wavelength-specific data were extracted and analyzed using DIP tools. Resulting image data were compared qualitatively and quantitatively for transcription factors including ESR1 and PR forms-A, -B (PR), as well as for markers of cell proliferation Ki67 and BrdU. On PND 40 both endometrial luminal epithelium (LE) and glandular epithelium (GE) were observed. Nascent glands extended into the underlying stroma (ST) as coiled tubular structures. Both transcription factors (ESR1 and PR) and cell proliferation markers (Ki67 and BrdU) were identified in LE and GE and, to a lesser extent, in ST. Transcription factor (ESR1 and PR) expression was higher (P < 0.01) in epithelium than in stroma. Results obtained using protocols for multilabel and single-label IHC were comparable, particularly for targeted transcription factors. Thus, multilabel IHC, combined with MSI and DIP technologies, can be employed to identify, capture and analyze multiple targets of interest simultaneously in single bovine uterine tissue sections. In study two, bovine uterine histoarchitecture as well as temporospatial patterns of PR expression and cell proliferation (marked by BrdU labeling) were evaluated in Holstein heifers from birth (PND 0) to PND 42 using validated IHC, MSI and DIP technologies. Uteri were obtained from four heifers each day on PND 0, 7, 14, 21, 28, 35, and 42. Nascent GE was present on PND 0 as defined histologically by shallow epithelial invaginations or buds penetrating underlying ST. From PND 7 to 14 epithelial buds elongated into tubular structures and developed into coiled structures by PND 21, extending through the ST and approaching the stromal-myometrial junction by PND 42. Overall, endometrial PR expression was greater (P < 0.0001) in the epithelial than in the stromal compartment. Epithelial PR expression (LE and GE) was greatest on PND 0, waned thereafter to PND 21 and increased again to PND 42 (P < 0.0001). Patterns of endometrial cell proliferation, reflected by BrdU labeling, differed with age between epithelial and stromal compartments (Cell x Age, P < 0.0001). At birth, BrdU labeling index (LI = percent labeled cells) was higher in stroma than in epithelium. This relationship was also observed on PND 14 and 42. Otherwise, BrdU LI was higher in epithelium than in stroma. Epithelial BrdU LI increased from PND 0 to PND 14 and decreased gradually thereafter to PND 42. Results reflect histoarchitectural changes similar to those observed for postnatal endometrial histogenesis in other ungulate species. Study three examined the role of ER-mediated events in the developing prepubertal bovine uterus and endometrial adenogenesis as evaluated on PND 120. Here, tamoxifen (TAM), a mixed-function ER agonist/antagonist and selective estrogen receptor modulator (SERM), was used as a tool to alter normal ER signaling patterns in prepubertal uterine tissues. Holstein heifers were given either TAM (3 ml/kg BW/day, i.m.) or vehicle alone (N = 7-8 heifers/group) from PND 28 to PND 120 when tissues were collected. Compared to controls on PND 120, effects of TAM on endometrial histoarchitecture were varied and pronounced. Where control tissues contained regularly dispersed, coiled tubular endometrial glands throughout intercaruncular stroma and extending to the endometrial-myometrial interface. Uterine gland genesis in TAM-treated heifers was generally inhibited and irregular. While endometrial thickness was not affected, gland penetration depth was reduced (P < 0.05) in TAM-treated heifers. Moreover, effects were endometrial zone (superficial vs deep) – specific. Generally, uterine gland cross-sectional area was greater (P < 0.05) in TAM than in control groups. Cyst-like uterine glands were seen regularly in TAM-treated animals. Effects of TAM on endometrial cell proliferation as reflected by BrdU LI were pronounced in deep endometrium. Generally, BrdU LI was reduced (P < 0.05) in TAM-treated heifers, particularly in deep stromal tissues. Treatment with TAM altered patterns of ESR1 expression without marked effects on PR. On PND 120, expression of both ESR1 and PR was most pronounced in epithelium. Similar to patterns observed for BrdU LI, TAM treatment generally reduced (P < 0.05) ESR1 signal and LI in the endometrium. Overall, results: (1) provide evidence for the utility of IHC, in combination with MSI and DIP, for evaluation of bovine endometrial development; (2) indicate that, as observed in other ungulate species, endometrial histogenesis occurs, to a large extent, postnatally in the cow; (3) support the idea that neonatal bovine uterine tissues are likely to be progestin-sensitive from birth; and (4) show that organizational events associated with postnatal endometrial development involve ER-mediated signaling events. The latter observation is based on results obtained from evaluation of the effects of TAM on uterine development.