Hot topic: Pregnancy-induced expression of interferon-stimulated genes in the cervical and vaginal mucosal membranes

Abstract

In ruminants, IFN-tau (IFNT) is a pregnancy recognition signal secreted by the embryonic trophectoderm before implantation, and it induces the expression of IFN-stimulated genes (ISG) in the uterine endometrium and blood leukocytes. The expression of ISG in blood leukocytes could indicate the presence of a viable conceptus before return of the next estrus; however, expression levels have high variation for confirming pregnancy. We hypothesized that the secreted IFNT in the uterus would affect ISG expression in cervical and vaginal tissues because they are directly adjacent to the uterus. To prove the hypothesis, we investigated the expression of 3 ISG (ISG15, MX1, and MX2) in cervical and vaginal mucosal membranes collected from pregnant (n = 12) and nonpregnant (n = 11) lactating Holstein cows at 17 to 18 d after artificial insemination. Mucosal membrane samples of the cervical canal near the external os (cervix) and deep vaginal wall surrounding the external os (vagina) were collected separately by simply scraping with a curette on d 17 or 18 of pregnancy (d 1 = ovulation), at which time IFNT secretion into the maternal uterus is maximal. After pregnancy diagnosis on d 30 and 60, separately collected samples confirmed as pregnant and nonpregnant were used for evaluation of the expression of IFN-stimulated protein 15 kDa (ISG15) and myxovirus-resistance protein 1 and 2 (MX1, MX2) with quantitative real-time PCR. The collected mucosal membrane samples from cervix contained mostly cell clots showing membrane structure and a low content of blood cells. The expression levels of all 3 genes were significantly increased in pregnant cows compared with nonpregnant cows in both cervical and vaginal samples. These results suggest that increased expression of ISG in the cervix and vagina is a pregnancy-associated phenomenon and is highly affected by IFNT secreted from the conceptus through the uterus.

New approaches to diagnose and target reproductive failure in cattle

“…Maternal recognition of pregnancy in cattle occurs during the period of embryo elongation when IFNT is secreted by trophectoderm cells of the developing conceptus (Bazer et al, 1997;Gray et al, 2002) to prevent the release of PGF 2α by the endometrium, thus maintaining the CL function and P4 production (Bazer et al, 2015). In addition to its role in the process of maternal recognition of pregnancy, IFNT is also able to induce the expression of several ISG by the endometrium (Mirando et al, 1991) and reaches the peripheral circulation through the uterine vein (Oliveira et al, 2008;Bott et al, 2010), leading to the expression of ISG in extra-uterine sites such as the CL (Oliveira et al, 2008), liver (Ruhmann et al, 2017) vaginal and cervical mucosa (Kunii et al, 2018) and peripheral blood leukocytes (Yankey et al, 2001;Han et al, 2006;Kizaki et al, 2013).…”

Changes in expression of interferon-stimulated genes and ubiquitin activating enzyme E1-like in ovine thymus during early pregnancy

“…Mx1 gene is expressed in maternal liver during late periimplantation period, which is induced by embryonic signal IFNT in the bovine (Meyerholz et al, 2016). Mx1 mRNA is upregulated in the cervix, vagina and blood neutrophils during early pregnancy in cows (Kunii et al, 2018;Sheikh et al, 2018). Mx1 interacts with tubulin beta in the ovine glandular epithelial cells during interphase and mitosis, and is implicated in intracellular trafficking and secretion in ovine uterus during early pregnancy (Racicot et al, 2012).…”

Feasibility and accuracy of using different methods to detect pregnancy by conceptus-stimulated genes in dairy cattle

“…Many studies have investigated ISG mRNA abundance us-ing peripheral blood mononuclear cells (PBMC). Recently, other studies have used total immune cells from whole blood (Yoshino et al, 2018) or milk (Schanzenbach et al, 2017) or cervical and vaginal mucosa cells (Kunii et al, 2018) for accessing gene expression and predicting pregnancy status. In addition, novel potential pregnancy markers were recently reported by our group in beef heifers (Rocha et al, 2020) and need to be tested for early pregnancy diagnosis in dairy cattle.…”

“…Therefore, although the use of whole blood for extraction of mRNA in circulating immune cells is simple and faster for applied use, this method results in reduced accuracy for pregnancy prediction and is still not feasible to replace the use of PBMC or granulocytes. Kunii et al (2018) reported that ISG15 expression in cervical and vaginal mucosal membranes of pregnant cows is significantly greater than that in nonpregnant cows. These authors also observed a greater fold change of ISG15 expression in CC of pregnant animals, indicating that the closer location of the cervix in relation to the embryo may result in greater IFN-τ stimuli for ISG transcription.…”

“…After the time of maternal recognition of pregnancy, the conceptus begins the processes of implantation that involve attachment and adhesion of the trophectoderm to the endometrial luminal epithelium (Spencer and Bazer, 2004). Its increased expression in this cell type is interesting because the involvement of LGALS3BP in the adhesion process of the conceptus is limited to the luminal epithelium of the uterus (Bauersachs et al, 2006), and, in addition to the different function of cervical and endometrial cells, they are not exposed to the same stimuli during pregnancy (Kunii et al, 2018). However, Baba et al (2019) also observed that LGALS3BP modulates the maternal immune response during early pregnancy in buffaloes, suggesting that its higher expression at this period has an important role in maternal recognition of pregnancy and implantation.…”