Progesterone-dependent and -independent modulation of luminal epithelial transcription to support pregnancy in cattle

In cattle, starting 4-5 days after estrus, pre-implantation embryonic development occurs in the confinement of the uterine lumen. Cells in the endometrial epithelial layer control the molecular traffic to and from the lumen and, thereby determine luminal composition. Starting early post-estrus, endometrial function is regulated by sex-steroids, but the effects of progesterone on luminal cells transcription have not been measured in vivo. First objective was to determine the extent to which progesterone controls transcription in luminal epithelial cells 4 d (D4) after estrus. Second objective was to discover luminal transcripts that predict pregnancy outcomes, when the effect of progesterone is controlled. Endometrial luminal epithelial cells were collected from embryo transfer recipients on D4 using a cytological brush and their transcriptome determined by RNASeq. Pregnancy by embryo transfer was measured on D30 (25 pregnant and 18 non-pregnant). Progesterone concentration on D4 was associated positively (n= 182) and negatively (n= 58) with gene expression. Progesterone-modulated transcription indicated an increase in oxidative phosphorylation, biosynthetic activity and proliferation of epithelial cells. When these effects of progesterone were controlled, different genes affected positively (n= 22) and negatively (n= 292) odds of pregnancy. These set of genes indicated that a receptive uterine environment was characterized by the inhibition of phosphoinositide signaling and innate immune system responses. A panel of 25 genes predicted the pregnancy outcome with sensitivity and specificity ranging from 64-96% and 44-83%, respectively. In conclusion, in the early diestrus, both progesterone-dependent and -independent mechanisms regulate luminal epithelial transcription associated with pregnancy outcomes in cattle.

Cooperation of membrane-translocated syntaxin4 and basement membrane for dynamic mammary epithelial morphogenesis

Mammary epithelia undergo dramatic morphogenesis after puberty. During pregnancy, luminal epithelial cells in ductal trees are arranged to form well-polarized cystic structures surrounded by a myoepithelial cell layer, an active supplier of the basement membrane (BM). Here, we identified a novel regulatory mechanism in this process by using a reconstituted BM-based three-dimensional culture and aggregates of a model cell line EpH4, which had been manipulated for inducible expression of a t-SNARE protein syntaxin4, either in an intact or signal peptide-connected form, and those genetically deficient in syntaxin4. We found that cells extruded syntaxin4 upon stimulation with the lactogenic hormone, prolactin, which in turn accelerated the turnover of E-cadherin. In response to extracellular expression of syntaxin4, cell populations that were less affected by BM actively migrated and integrated into the BM-faced cell layer. Concurrently, the BM-faced cells, which were simultaneously stimulated with syntaxin4 and BM, acquired unique epithelial characteristics to undergo dramatic cellular arrangement for cyst formation. These results highlight the importance of the concerted action of extracellular syntaxin4 extruded by the lactogenic hormone and BM components in epithelial morphogenesis.

Evidence for accelerated aging in mammary epithelia of women carrying germline BRCA1 or BRCA2 mutations

During aging in the human mammary gland, luminal epithelial cells lose lineage fidelity by expressing markers normally expressed in myoepithelial cells. We hypothesize that loss of lineage fidelity is a general manifestation of epithelia that are susceptible to cancer initiation. In the present study, we show that histologically normal breast tissue from younger women who are susceptible to breast cancer, as a result of harboring a germline mutation in BRCA1, BRCA2 or PALB2 genes, exhibits hallmarks of accelerated aging. These include proportionately increased luminal epithelial cells that acquired myoepithelial markers, decreased proportions of myoepithelial cells and a basal differentiation bias or failure of differentiation of cKit+ progenitors. High-risk luminal and myoepithelial cells are transcriptionally enriched for genes of the opposite lineage, inflammatory- and cancer-related pathways. We have identified breast-aging hallmarks that reflect a convergent biology of cancer susceptibility, regardless of the specific underlying genetic or age-dependent risk or the associated breast cancer subtype.

Highly metastatic claudin-low mammary cancers can originate from luminal epithelial cells

Claudin-low breast cancer represents an aggressive molecular subtype that is comprised of mostly triple-negative mammary tumor cells that possess stem cell-like and mesenchymal features. Little is known about the cellular origin and oncogenic drivers that promote claudin-low breast cancer. In this study, we show that persistent oncogenic RAS signaling causes highly metastatic triple-negative mammary tumors in mice. More importantly, the activation of endogenous mutant KRAS and expression of exogenous KRAS specifically in luminal epithelial cells in a continuous and differentiation stage-independent manner induces preneoplastic lesions that evolve into basal-like and claudin-low mammary cancers. Further investigations demonstrate that the continuous signaling of oncogenic RAS, as well as regulators of EMT, play a crucial role in the cellular plasticity and maintenance of the mesenchymal and stem cell characteristics of claudin-low mammary cancer cells.

Analysis of the transcriptome of bovine endometrial cells isolated by laser micro-dissection (2): impacts of post-partum negative energy balance on stromal, glandular and luminal epithelial cells

Background In post-partum dairy cows, the energy needs to satisfy high milk production induces a status of more or less pronounced Negative Energy Balance (NEB). NEB associated with fat mobilization impairs reproductive function. In a companion paper, we described constitutive gene expression in the three main endometrial cell types (stromal, glandular and luminal epithelial cells) isolated by laser capture micro-dissection (LCM) showing the specificities of their transcriptomic profiles. This study investigates the specific impact of NEB on gene expression in these cells around 80 days after parturition at day 15 of the oestrus cycle and describes their specific response to NEB. Results Following the description of their constitutive expression, the transcriptome profiles obtained by RNA sequencing of the three cells types revealed that differences related to the severity of NEB altered mainly specific patterns of expression related to individual cell types. Number of differentially expressed genes between severe NEB (SNEB) and mild NEB (MNEB) cows was higher in ST than in LE and GE, respectively. SNEB was associated with differential expression of genes coding for proteins involved in metabolic processes and embryo-maternal interactions in ST. Under-expression of genes encoding proteins with functions related to cell structure was found in GE whereas genes encoding proteins participating in pro-inflammatory pathways were over-expressed. Genes associated to adaptive immunity were under-expressed in LE. Conclusion The severity of NEB after calving is associated with changes in gene expression around 80 days after parturition corresponding to the time of breeding. Specific alterations in GEs are associated with activation of pro-inflammatory mechanisms. Concomitantly, changes in the expression of genes encoding proteins involved in cell interactions and maternal recognition of pregnancy takes place in ST. The combination of these effects possibly altering the uterine environment and embryo maternal interactions may negatively influence the establishment of pregnancy.

Involvement of Nestin in the Progression of Canine Mammary Carcinoma

Nestin, a class VI intermediate filament protein, is known to be expressed in various types of human neoplasms, including breast cancer, and is associated with their progression. However, its expression and role in canine mammary tumors remain unknown. We analyzed nestin expression in canine mammary tumors using in situ hybridization and immunohistochemistry. We also investigated its role in a canine mammary carcinoma cell line using RNA interference. Nestin expression was not observed in luminal epithelial cells of any of the 62 cases of benign mammary lesions examined, although myoepithelial cells showed its expression in most cases. In 16/50 (32%) primary mammary carcinomas and 6/15 (40%) metastases of mammary carcinomas, cytoplasmic nestin expression was detected in luminal epithelial cells. In luminal cells of primary mammary carcinomas, its expression was positively related to several pathological parameters that indicate high-grade malignancy, including histological grading ( P < .01), vascular/lymphatic invasion ( P < .01), Ki-67 index ( P < .01), and metastasis ( P < .05). Immunohistochemistry revealed that nestin expression was related to vimentin expression in mammary carcinomas ( P < .01). This relationship was confirmed using reverse transcription-quantitative polymerase chain reaction using 9 cell lines derived from canine mammary carcinoma ( P < .01). Finally, nestin knockdown in canine mammary carcinoma cells using small interfering RNA inhibited cell proliferation and migration based on WST-8, Boyden chamber, and cell-tracking assays. These findings suggest that nestin may at least partially mediate these behaviors of canine mammary carcinoma cells.

Sperm migration, selection, survival and fertilizing ability in the mammalian oviduct

In vitro fertilization (IVF) gives rise to embryos in a number of mammalian species and is currently widely used for assisted reproduction in humans and for genetic purposes in cattle. However, the rate of polyspermy is generally higher in vitro than in vivo and IVF remains ineffective in some domestic species like pigs and horses, highlighting the importance of the female reproductive tract for gamete quality and fertilization. In this review, the way the female environment modulates sperm selective migration, survival and acquisition of fertilizing ability in the oviduct is being considered under six aspects: (1) the utero-tubal junction which selects a sperm sub-population entering the oviduct; (2) the presence of sperm binding sites on luminal epithelial cells in the oviduct, which prolong sperm viability and plays a role in limiting polyspermic fertilization; (3) the contractions of the oviduct, which promote sperm migration toward the site of fertilization in the ampulla; (4) the regions of the oviduct, which play different roles in regulating sperm physiology and interactions with oviduct epithelial cells; (5) the time of ovulation and (6) the steroid hormonal environment which regulates sperm release from the luminal epithelial cells and facilitates capacitation in a finely orchestrated manner.