96 Alert or absent: how attentive is the oviduct?

The oviduct provides an optimal and adaptive microenvironment for the establishment of pregnancy. The oviduct comprises four main regions: infundibulum (oocyte pick-up), ampulla (fertilisation), isthmus (sperm capacitation and reservoir, preimplantation embryonic development), and uterotubal junction (sperm and embryo transport). The rise in assisted reproductive technologies (ARTs) has spurred increased interest in the oviduct’s influence on gametes and the embryo, as a basis for improved culture in vitro. There is mounting evidence in livestock and rodents that gametes alter gene expression in secretory and ciliated epithelial cells of the oviduct during fertilisation. To elucidate whether adaptive interactions between the oviduct and the preimplantation embryo exists, we performed bulk RNA-sequencing on murine C57BL/6J oviducal tissues at various developmental stages (0.5, 1.5, 2.5, and 3.5 days post-coitus; dpc). In addition to samples taken after natural fertilisation, samples were collected during oestrus and during Days 0.5, 1.5, 2.5, and 3.5 of pseudopregnancy. Pseudopregnancy was induced by mating females with vasectomised males. Tissues were dissected into infundibulum-ampulla (IA) and isthmus-uterotubal junction (IU) regions. Tissue samples were collected and flushed with L15 (Leibovitz’s) medium and promptly flash frozen in liquid nitrogen and stored at −80°C. Total RNA was extracted utilising the RNeasy Micro Kit (Qiagen). Samples were sequenced with a depth of 25M reads/sample (n = 3/region/time-point). Our findings from oestrus recapitulated published work by Roberson et al. (2021 Dev. Biol. 476, 240-248), in that we observed strong region-specific expression of large clusters of genes. We also reproduced the expression of Foxa2 as IA-specific and Pax2 as IU-specific, nominating these genes as potential markers for future knockout model systems in the mouse. At 0.5 dpc, large sets of differentially expressed genes were observed in pregnancy compared to pseudopregnancy in both IA and IU regions. Genes involved in anti-inflammatory responses were detected in both IU and IA regions. We also found that multiple Dusp genes were up-regulated, likely suppressing proinflammatory MAPK activity. Pregnant and pseudopregnant IA samples showed similar differential expression indicating ovulation as the major proximal influencer. Similarly, Ano2 was significantly up-regulated (up to 8.7 log₂FC) in the IU region at 1.5, 2.5, and 3.5 dpc (absent at oestrus or 0.5 dpc) in pregnant and pseudopregnant samples. ANO family proteins are calcium-activated chloride transmembrane channels shown to increase the intensity of both uterine and oviducal muscle contractions by increasing intracellular chloride levels. In conclusion, we have sequenced and are exploring the transcriptomic landscape of the oviduct at four timepoints for both natural pregnancy and pseudopregnancy. Our findings reveal unique transcriptomic profiles of different oviducal regions that may be specialised to influence sperm migration, fertilisation, embryo transport, and development.