Tsagakis, I. | bioRxiv Science

bioRxiv2023

Differences in binding preferences for XIST partners are observed in mammals with different early pregnancy morphologies

In comparison to protein-coding genes, long non-coding RNAs (lncRNAs) are poorly conserved between species at the sequence level yet they often perform conserved and essential regulatory roles. The lncRNA XIST mediates X chromosome inactivation (XCI) through the interaction of numerous proteins, most of which bind to its repeat regions. Whilst XIST is present across placental mammals, its sequence is divergent. In addition, the timing and mechanistic details of the process of XCI also vary across placental mammals. Here, we sought to determine whether XIST interactor proteins previously identified in a model mammalian species (mouse) also bind in other mammals which exhibit divergent timing of XCI (human and bovine) and differing pre-implantation embryo morphology. We show that XIST and its putative protein partners are coordinately expressed in the endometria of mammals with different pre-implantation embryo morphologies. RNA immunoprecipitations revealed that human WTAP, SPEN, hnRNPK and CIZ1 proteins bind human XIST. CIZ1-XIST binding was also detected in bovine. In both human and cow, we found CIZ1 binds to the repeat E element of XIST. However, human CIZ1 protein is not able to bind bovine repeat E, indicating a species-specific binding interaction. Together these data indicate that several of the key RNA-protein interactions with XIST are common across placental mammals, even though we observe divergence in both the RNA sequence of XIST, and early embryo morphologies. This work sheds light upon the evolution of RNA-protein binding interactions, revealing that the binding events can be conserved even when the precise mechanism of binding has changed. Statements and DeclarationsAuthors have no financial or non-financial interests that are directly or indirectly related to the work submitted for publication.

developmental biology↗

bioRxiv2022

Sex-bias in utero alters ovarian reserve but not uterine capacity in female offspring

Environmental stressors to which a foetus is exposed, affect a range of physiological functions in post-natal offspring. Such stressors include disproportionate steroid hormone concentrations in the uterine environment. We aimed to determine the in-utero effect of steroid hormones on reproductive potential of female offspring using a porcine model. Hypothesising that an in-utero sex bias will influence ovarian reserve and endometrial morphology in the breeding gilt. Reproductive tracts of pigs from female-biased litters (>65% female, n=15), non-biased litters (45-54.9% female, n=15), and male-biased litters (<35% females, n=9) were collected at slaughter (95-115 kg). Ovaries and uterine horns were processed for histological approaches and stained using H&E or IHC techniques. All measurements were conducted in QuPath (Bankhead et al, 2017). Variability of data within groups was analysed with a Levenes test, whilst data was analysed using linear models in R. In the ovarian reserve, there was a significant interaction between the birth weight and the sex ratio of a litter from which a pig originated (p=.015), with low-birth-weight pigs from male-biased litters having a higher number of primordial follicles and the opposite trend seen in pigs from female-biased litters. This was not reflected in recruited, nor atretic follicles. In the uterine horn sex bias held no effect on development as seen in this study. Birth weight held more effects on the gilts. A lower BW decreased the proportion of glands found in the endometrium (p=.045). BW was found to be far more variable in both male-biased and female-biased litters (p=.026). The variability of primordial follicles from male-biased litters was greater than non-and female-biased litters (p=.014). Similarly, endometrial stromal nuclei had a greater range in male- and female-biased litters than non-biased litters (p=.028). There was a greater effect on both ovarian reserve and uterine development of piglet BW than the litter bias. There seems a benefit of being androgenised on ovarian reserve whilst no effects were found for the morphology or endometrial gland proliferation of the uterine horns. However, a crucial finding was in the variability of the data. Both primordial follicles in the male-biased ovary, and stromal nuclei in the male- and female-biased uterine horns had a wider spread in numbers than non-biased litters. This could be inflating the variability of reproductive success seen in animals form male-biased litters by two means. Firstly, by a higher likelihood of insufficient primordial pools. Secondly, through a potential impact on stromal-derived growth factors or insufficient support of the underlying implantation structures, leading to an increased variability in uterine implantation capabilities, and thus survival of the embryo.

developmental biology↗