TRANSCRIPTOMIC ANALYSIS OF BOVINE ENDOMETRIUM WITH SHORT UTERINE TRACT LEADING TO INFERTILITY

Abstract

Background: Beef is one of the most highly consumed meat worldwide. With the increasing human population, the demand for beef will be increased by 43% in 2050. However, the production of beef cattle has declined in recent years. Infertility/subfertility is one of the main reasons causing a decline in beef production. Infertility is the diminished or incapability of females to produce viable offspring, causing a substantial economic loss. Since 1980, infertility has been increasing among the cattle population worldwide. Infertility is triggered by several factors, including developmental defects of the reproductive tract. Recently, Hawaii beef producers reported that heifers selected for breeding had good body condition scores and were bred several times with fertile bulls but failed to conceive. After slaughtering at a local abbatoir, these heifers had small uterine tract with normal ovaries. The uterus provides the biological environment for the embryo’s growth and development. Bovine endometrium consists of caruncular (CAR) and Intercaruncular (ICAR) regions. The caruncular region serves as the implantation and placentation site, whereas the intercaruncular region contains numerous glands for preparing the biological environment for the embryo's growth and development. Studies have suggested that secretions from the ICAR are necessary for the conceptus growth, development, and survival. So far, there is no report on infertility induced by smaller reproductive tract in bovines. Therefore, we hypothesized that the heifers with short uterine tract might have abnormal uterine transcriptomic profiles and developmental defects leading to infertility. The objectives of this study are to 1) analyze the morphological difference between the short and normal uterine heifers, and 2) determine the genes and biological pathways in the intercaruncular endometrium of the heifers with short uterine tract compared to normal heifers.Methods: We examined the reproductive tracts of over 171 cattle from local slaughterhouses; among them, 51 heifers had a smaller uterine tract. Uterine tissues were collected from a subset of infertile heifers (n=6, designated as the short uterine tract). Uterine tissues were also collected from cyclic heifers having normal uterine tract (n=6, cyclic heifers, designated as the normal uterine tract). Samples were selected, matching the heifers' corpus luteum stage (II). For the histological study, uterine tissues were collected and fixed in 10% Neutral buffered formalin. The fixed tissues were sectioned and stained with Hematoxyline and Eosin. For the transcriptomic study, intercaruncular tissues were collected in RNAlater solution. Total RNAs were isolated and were subjected to high-throughput RNA-sequencing (n=5/group). The differentially expressed genes (DEGs) were determined between the short uterine tract compared to normal. To delineate the DEGs and associated biological pathways, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Ingenuity Pathway Analysis were done. The most highly upregulated and downregulated genes expression were validated using real-time quantitative PCR (qPCR). Results and conclusion: Total uterine layer length, endometrial layer length, and gland numbers were significantly lower in the short uterine tract than normal. A total of 521 genes were differentially expressed in the short uterine tract compared to normal. The top 20 downregulated genes in the short bovine endometrium were NLRP12, ENSBTAG00000052892, ENSBTAG00000055111, ENSBTAG00000036102, LOC112444733, LOC100847874, OTOG, CLCNKA, NPPC, CYP26A1, LOC788915, VNN2, PRKCG, LOC782061, MYOT, PLA2G3, ANKS1B, GPR183, ST8SIA6, and TMIE. Metabolic pathways, carbon metabolism, and Wnt signaling pathways were the top three enriched pathways by the downregulated DEGs. The qPCR result confirmed the expression of the DEGs in short uteri compared to normal. In conclusion, heifers with short uterine tract had significantly decreased endometrial layers, uterine glands, and altered transcriptomic profiles. The decrease in uterine glands probably resulted in lower uterine secretions necessary to support embryo growth and development. As a result, heifers with short uteri were infertile even when they were bred by fertile bulls.