Recent progress of interferon-tau research and potential direction beyond pregnancy recognition

Abstract

Since the discovery of interferon-tau (IFNT) over 30 years ago as the trophectodermal cytokine responsible for the maintenance of the maternal corpus luteum (CL) in ruminants, exhaustive studies have been conducted to identify genes and gene products related to CL maintenance. Recent studies have provided evidence that although CL maintenance, with the up- and down-regulation of IFNT, is important, its regulatory role in the endometrial expression of interferon-stimulated genes (ISGs) is far more important for conditioning the uterine environment for successful conceptus implantation and thereafter. This review initially describes the mammalian implantation process, briefly but focuses on recent findings, as there appears to be a common phenomenon during early to mid-pregnancy among mammalian species.

In mammals, implantation is a critical step for a successful pregnancy and the completion of sequential events, such as maternal uterine modification, conceptus (embryo plus extra-embryonic membrane) development, and attachment/invasion to the endometrium, followed by placental formation, must be tightly controlled temporally and spatially. In fact, conceptus and uterine adaptation must be well synchronized and coordinated through physical, cellular, and biochemical communication. From blastocyst development to placental formation, detailed physiological events and their time courses as well as placental structures, differ among mammalian species; however, the basic processes of implantation and the requirement for placental formation are similar among eutherians [1,2,3,4,5] (Fig. 1).

Fig. 1.

Pre-implantation periods in mice and cows. Implantation in mice—an important laboratory animal model—and in cattle, domestic animal, are illustrated. Although the basic processes of implantation and placentation are similar among mammalian species, the details of these events, their time courses, and placental structures are species-specific. Notably, soon after hatching, the blastocyst implants into the maternal endometrium in mice, while the bovine conceptus elongates prior to its attachment to the uterine epithelium. In both cases, trophoblast cells cover the entire surface of the uterine lumen and this is followed by placentation.

In mice, fertilized eggs reach the uterine lumen on day 3.5 as floating blastocysts. Blastocysts undergo rapid implantation with apposition and attachment to the uterine epithelium, followed by invagination of the uterine endometrium, forming the implantation chamber—a characteristic of eccentric implantation [1]. As blastocyst invagination progresses, trophectodermal cells begin to cover the surface of the uterine lumen. In ruminants, such as bovine, ovine, and caprine species, blastocysts are formed several days after fertilization, as in other mammals, but they do not attach to the uterine epithelium for several days. Trophoblast elongation is one of the unique features of ruminant conceptus development. The blastocyst remains spherical in shape and unattached to the uterine epithelium for several days before its elongation, which begins around day 12–13 (day 0 = day of estrus). Around day 14, the conceptus becomes an ovoid structure and then begins to elongate rapidly. Around day 20, when conceptus elongation slows down, bovine conceptus starts to attach to the endometrial epithelium [6, 7]. At this point, trophoblast cells start to cover the entire uterine lumen, including uterine caruncles, from which placental structures unique to ruminants develop [8] (Fig. 1). Thus, sequential events during conceptus implantation in the maternal endometrium differ in rodents and ruminants, while the events in which the uterine lumen is entirely covered with trophectodermal cells are similar, if not the same, in these mammals.

In mammals, maintenance of corpus luteum (CL) function, from which the continued secretion of progesterone (P4) is ensured, is required for the establishment and maintenance of pregnancy. P4 is involved in several uterine functions; however, the mechanisms by which CL is maintained for P4 production differ among mammalian species. In humans, luteolysis is prevented by chorionic gonadotropin (CG), a hormone produced following the implantation of trophoblast cells [9]. CG binds to the luteinizing hormone/CG receptor, modulating the uterine environment and ensuring that the CL continuously secretes P4 [10]. In rodents, CL is maintained through the release of copulation-induced pituitary prolactin (PRL) surges [11]. PRL surges are believed to convert CL to the CL of pregnancy and lead to the secretion of sufficient P4 to support pregnancy [12]. In ruminants, interferon-tau (IFNT) is produced by trophoblast cells of the elongating conceptus during the peri-implantation period [13, 14]. IFNT prevents luteolysis by attenuating prostaglandin F2α (PGF2α) secretion from the uterine endometrium, resulting in continued secretion of P4. The attenuation of PGF2α results from the down-regulation of estrogen receptor and estrogen-induced oxytocin receptor expression [15, 16]. IFNT is now well known as the key factor for maternal recognition of pregnancy in ruminants [17,18,19,20,21].

It was reported that homogenates of day 14–16 conceptuses, but not those of days 21–23, extended CL life span, suggesting that these conceptuses actively produced the anti-luteolytic factor trophoblastin [22]. Using two-dimensional polyacrylamide gel electrophoresis, Godin et al. (1982) identified a group of proteins responsible for CL maintenance and named the protein as ovine trophoblast protein-1 (oTP-1) [17]. A similar protein was also identified in bovines [13]. In 1987, through the analysis of cDNA and amino acid sequences, Imakawa et al. identified that the substance was an interferon [18], and later named it interferon τ (tau) because it was derived from the embryonic trophectoderm [21]. More than 30 years after its discovery, IFNT remains a key player in the mechanism of pregnancy establishment in ruminants and has the potential to act beyond pregnancy recognition.

Trophectodermal IFNT is a Product of a Gene Family

Interferons (IFN) are cytokines with antiviral activity induced by a viral infection or double-stranded RNAs. They are divided into three groups: type I, II, and III IFNs [23], and type I IFNs are further divided into the following subfamilies: IFN-alpha (IFNA), beta (IFNB), delta (IFND), epsilon (IFNE), kappa (IFNK), tau (IFNT), omega (IFNW), and zeta (IFNZ) [24, 25]. While IFNW is conserved in most mammals, IFNT is found only in ruminants and is a relatively new IFN that is thought to have derived from IFNW approximately 36 million years ago [26, 27]. The amino acid sequence of IFNT shows high similarity (~70%) with IFNW, moderate similarity (~50%) with IFNA, and low similarity (~25%) with IFNB [28, 29].

All IFN-type genes are located on chromosome 8 in domestic cattle [25]. At least 13 IFNA, 6 IFNB, and 24 IFNW genes have been found in the bovine genome, and although the exact number of IFNT genes has not been determined, as many as 40 IFNT sequences have been suggested to exist [25]. However, a comprehensive analysis of transcripts using peri-implantation bovine conceptuses revealed that among numerous IFNT genes, only two, IFNT1 and IFNTc1, are predominantly expressed in utero during the peri-implantation period [30, 31]. In sheep, multiple IFNT genes are present as well, even though only a single gene (IFNT-o10) accounts for up to 75% of the total mRNA expressed during the equivalent period of early pregnancy [32].

It remains unclear why many duplicated genes have arisen in the IFNT gene family. Although the regulatory mechanisms of these genes have not been definitively elucidated [30], no clear differences in transcriptional regulation, function, or downstream gene targets have been found [33]. Similarly, the exact reason for the presence of multiple IFNT genes and how specific each IFNT variant functions, is still unknown, it could be a molecular mechanism that ensures the maximum level of IFNT production during early pregnancy. Alternatively, the presence of multiple genes ensures IFNT production even when major IFNT genes could be dysfunctional. The presence of multiple IFNT genes suggests that their expression in ruminants must be critical and needs to be ensured during early pregnancy. The evolution of IFNs, including IFNT, has been well described in more detail elsewhere [23, 29].

Transcriptional Regulation of IFNT Gene

a) Up-regulation

IFNT exhibits structural and functional similarities to type I IFNs [34]. Type I IFNs, induced by a viral infection, include several members such as IFNA, IFNW, and IFNB, which are produced by leukocytes and fibroblasts, respectively. IFN gamma (IFNG), a type II IFN, is produced by T cells and Natural Killer (NK) cells following mitogen treatment. IFNT, which has a high degree of structural similarity to IFNW, consists of four cysteine residues that are well conserved across the type I IFN gene family. However, IFNT is not secreted by blood cells, strongly suggesting that IFNT is regulated differently from other type I IFNs.

One of the differences between IFNT and other type I IFNs is that presence and expression of IFNT are unique to ruminant trophoblast cells. The coding sequences of IFNT genes are similar to those of IFNW, but the 5′ UTR and 3′ UTR regions differ. The ancestral IFNT gene is predicted to have arisen by the insertion of a novel chorionic membrane-specific 5′ UTR (promoter/enhancer) and 3′ UTR into the gene regulatory region of IFNW [23]. The first 400 bases upstream of the IFNT gene transcription start site are unique to the IFNT gene, and no apparent virus-inducible transcriptional elements are found in this region [28, 35].

Numerous transcription factor binding sites exist in the regulatory region of the IFNT gene, and several transcription factors have been found to regulate its transcription [36]. IFNT is intrinsically produced by the ruminant trophectoderm (TE) and no other cell types that produce IFNT have been identified to date. However, in non-trophoblastic cells, overexpression of the transcription factor CDX2 can induce endogenous IFNT gene expression in Mardin-Darby bovine kidney (MDBK) cells, suggesting that CDX2 is responsible for trophoblast cell-specific regulation of IFNT expression [37]. Together with CDX2 regulation, IFNT gene expression is epigenetically regulated [37, 38]. The binding sites of the transcription factors ETS2 and DLX3 are well conserved in several IFNT genes, and they work together to promote IFNT expression, making them common regulators of IFNT genes [39, 40]. In addition to these main transcription factors, the coactivators CREB binding protein (CREBBP) [41] and p300 [42] have also been reported to be involved in the regulation of IFNT transcription.

b) Down-regulation

Coinciding with conceptus elongation, IFNT expression increases dramatically for several days, and then rapidly declines as the TE attaches to the maternal uterine epithelium [43, 44]. The exact reason and mechanism for this down-regulation have not been definitively elucidated, but it is presumed that IFNT expression needs to decline soon after pregnancy recognition and the initiation of conceptus attachment to the uterine epithelium.

Transcription factors, such as Octamer-binding transcription factor 4 (OCT4) [45] and eomesodermin (EOMES) [46], have been reported to suppress IFNT gene expression. These factors are known to play an important role in embryonic development in mice. Oct4^–/– embryos die at the time of implantation due to failure to form the inner cell mass (ICM) [47]. Eomes-deficient mouse embryos are arrested at the blastocyst stage, suggesting that EOMES may be required for trophoblast stem cell development [48]. The downregulation of IFNT transcription by EOMES has been demonstrated through the use of in vitro co-culture systems of trophoblasts and endometrial epithelial cells [46]. The transcription cofactor Yes-associated protein (YAP) and the TEA domain family transcription factor (TEAD) are major components of the Hippo signaling pathway and play an essential role in cell fate specification of TE and ICM [49, 50, 52, 53]. It has also been shown that the transcription cofactor YAP inhibits IFNT expression by altering the localization of TEAD2 and TEAD4 from the nucleus to the cytoplasm [51].

Epigenetic regulation, such as histone modification and DNA methylation, also regulates IFNT gene expression by altering chromatin conformation. Indeed, chromatin immunoprecipitation (ChIP) assay using ovine conceptuses (days 14–20) revealed that histone proteins wrapped in the upstream region of the IFNT gene are highly acetylated on days 14 and 16, followed by a decrease in acetylation after conceptus attachment to the uterus with a simultaneous increase in histone methylation [38]. In addition, Nojima et al. examined the methylation status of the upstream region of the IFNT gene using non-IFNT-producing cells (endometrium, leukocytes) and IFNT-producing day 14 (high IFNT production) and day 20 (low IFNT production) conceptuses. They reported that changes in the degree of methylation may be one of the major mechanisms leading to the down-regulation or silencing of ovine IFNT transcription [52]. These epigenetic modifications probably ensure trophoblast specificity of IFNT expression. Indeed, the upstream regions of ovine IFNT genes in non-trophoblastic tissues, such as the uterine endometrium and peripheral blood mononuclear cells (PBMCs), were highly methylated [52]. Thus, in addition to transcription factor expression, epigenetic modifications are required for trophoblast-specific IFNT expression temporally and spatially.

Together, these findings indicate that IFNT genes use multiple transcriptional mechanisms to up-regulate and later down-regulate their transcription. The reason why ruminants acquired the IFNT gene has not yet been elucidated, nor has its gene duplication made it possible for massive IFNT production during this limited pre-attachment period. It is generally accepted that the expression of the IFNT gene is finely regulated by not only various transcription factors but also by epigenetic mechanisms. For more information on the regulation of IFNT expression, please refer to the excellent review [36].

Common Transcription Factors are Involved in Both Trophectodermal Cell Development and IFNT Gene Transcription

Several transcription factors play essential roles in the development and maintenance of mammalian trophoblast cells, and in particular, CDX2 has been extensively studied during early embryonic development in mice. In fact, CDX2 is required for correct cell fate specification and TE differentiation in mouse blastocysts. Cdx2 homozygous mutant embryos die around the time of implantation [53]. In cattle, CDX2 mRNA is detected from the germinal vesicle stage to the expanded blastocyst stage, and CDX2 protein is restricted to the TE in blastocysts [2]. ETS2 expression is restricted to trophoblasts in the early post-implantation stages and is also essential for trophoblast development in mice [54, 55]. In bovines, ETS2 expression begins in trophoblasts at the spherical stage [56].

GATA2 and GATA3 also play key roles in the development and function of mouse trophoblast cells [57, 58]. Although the GATA binding site is found only in the upstream region of IFNTc1, one of the two bovine IFNT genes expressed by the pre-implantation conceptus in utero, they can also induce the transcriptional activity of IFNT genes in non-trophoblast cells and regulate the expression of IFNT as well as other TE factors [59,60,61]. In the human choriocarcinoma JEG3 cell line, GATA2 failed to exert its effect, probably because JEG3 cells express abundant endogenous GATA2 and GATA3. In addition, single-cell analysis revealed that there are four subpopulations of TEs and that the genes strongly expressed in all of them include IFNT, CDX2, GATA2, and GATA3 [62]. In addition to CDX2, GATA expression in trophoblasts can provide an adequate microenvironment that supports IFNT expression [59]. Evidence has accumulated that the transcription factors required for trophectodermal lineage specification are also associated with ruminant-specific IFNT gene transcription.

These findings suggest that although the time course of implantation processes, mechanisms of pregnancy recognition/CL maintenance, and placental formation differ among mammalian species, a group of transcription factors that function in TE is generally conserved (Fig. 2). Therefore, understanding the TE-specific factors associated with the regulatory mechanism of IFNT gene expression may help us identify common mechanisms required for pregnancy establishment in ruminant ungulates as well as in other mammals. In other words, the identification of molecular and cellular mechanisms associated with the temporal and spatial expression of IFNT may provide an alternative method to identify what promotes TE cell development and the regulation of their associated gene expression.

Fig. 2.

Transcriptional regulation of IFNT gene. Although IFNT is ruminant-specific, most factors important for trophoblast development are conserved in mammals, and these factors, such as CDX2, ETS2, and GATA are also involved in the regulation of IFNT gene expression. Mammals had existed long before the appearance of the first ruminants with IFNT. As such, ruminants did not acquire new genes to control IFNT genes but were able to use genes that were already functioning in trophoblasts.

Use of IFNT to Improve Pregnancy Success

Since the discovery of IFNT, attempts have been made to utilize IFNT to improve pregnancy rates. At the time of its discovery, a recombinant protein was not available, and thus, recombinant IFNA, another type I IFN, was initially tested for its efficacy in extending inter-estrous intervals or improving pregnancy rates. IFNA administration inhibits oxytocin-induced PGF2α induction and prolongs luteal lifespan [63,64,65]. However, it has not been definitively shown to date whether IFN administration compensates for inadequate conceptus production of IFNT in utero. Recombinant IFNT administration was reported to inhibit luteal regression and extend the luteal phase for only a few days [66, 67]. Following a series of experiments involving the administration of recombinant IFNT proteins with limited success, a new methodology was developed to administer IFNT; the use of trophoblastic vesicles which produce intrinsic IFNT. Trophoblastic vesicles were obtained by cutting elongated conceptuses into small pieces and culturing them. These vesicles produce IFNT and are intended to compensate for IFNT. It has been reported that the co-transfer of trophoblastic vesicles obtained from 14-day blastocysts improves the conception rate in cattle [68]. In addition, it has also been reported that co-transfer of trophoblastic vesicles from pregnant day 14 bovine and day 11–13 sheep embryos maintained the CL [69], and that administration of trophoblastic vesicles from pregnant day 7–8 bovine embryos into the uterine horn prolonged the estrous cycle in approximately half of the examined cows [70].

Co-transfer of trophoblastic vesicles derived from IVF embryos has also been reported to improve pregnancy rates in bovines [71, 72]. Although this treatment was very effective in relatively early pregnancies (days 26–43), there was no significant difference in mid-pregnancy (days 38–73) or final delivery outcomes (days 280–299) [71]. In other words, these findings suggest that IFNT supplementation may improve conception rates from pregnancy recognition to the early stage of gestation, the first 4–5 weeks, but factors other than IFNT are involved in the maintenance of pregnancy after 5 weeks of pregnancy.

Embryo transfer (ET) following artificial insemination (AI) is used to improve the conception rate of repeat-breeder cows [73, 74]. In such cows, improvement in conception rates was observed in about half of the cases [73, 75]. The combination of AI and ET doubled the chance of pregnancy resulting from the effectiveness of IFNT supplementation [75]. However, there is concern that twin pregnancies would increase since embryos could develop from both AI and ET. It was found that IFNT is produced by parthenogenetic embryos as well as by in vivo or in vitro produced embryos and trophoblastic vesicles [76], and their use was expected to increase due to their ease of preparation and cost. Thus, the use of parthenogenetic embryos, which do not become embryos but produce IFNT, is also under investigation. The co-transfer of parthenogenetic embryos enhances maternal recognition of pregnancy and promotes the viability of poor-quality embryos until day 40 of gestation [77]. In addition, it has also been reported that AI plus parthenogenic embryos may be beneficial for improving maternal recognition of pregnancy in repeat-breeder cattle while avoiding twin generation [78]. These findings further emphasize that IFNT is important for the recognition and establishment of pregnancy, especially in the early stages of gestation, and further testing is needed for practical applications to improve pregnancy rates in the future.

Common Maternal Responses Exist during the Early Mid-pregnancy in Mammals

IFNT not only prevents CL regression but also modulates endometrial gene expression as the pregnancy to proceed. For example, IFNT induces the expression of several chemokines in uterine endometrial tissues such as chemokine ligand 10 (CXCL10) and 9 (CXCL9) [79, 80]. CXCL10 attracts immune cells, especially NK cells, to the implantation site by acting through the CXCL10 receptor, CXCR3, which regulates TE cell migration and integrin expression [81, 82].

In addition to these chemokines, IFNT induces the expression of numerous interferon-stimulated genes (ISGs) in the maternal uterus [83,84,85]. Although the detection of ISGs expression in the uterus is well documented [85,86,87], elevated ISGs expression has been reported in various tissues other than the uterus, such as blood cells, luteal tissue, and liver during pregnancy [88,89,90,91,92,93,94]. Since the expression of type I IFN and ISGs during the implantation period has also been found in mice and humans [95, 96], it is likely that these factors play important roles in the maternal uterine environment necessary for conceptus implantation processes and/or the consequence of conceptus implantation into the uterine endometrium. In fact, the immune function of type I IFN and ISGs during pregnancy has been recognized in the human placenta [97].

When compared to non-pregnant ewes, expression of another ISG protein, MX, in pregnant ewes was higher in PBMCs up to 30 days after AI [98]. In addition, it was also reported that although ISG15 mRNA decreased after day 25 of pregnancy, its protein remained in the endometrium until day 40 of pregnancy in sheep [99]. In cows, ISG15 expression is up-regulated in the CL on day 16 of gestation and is still detectable on day 60 [90]. In mouse uteri, ISG15 expression is detectable on embryonic day 3.5 (E3.5) and increases by day 9.5 (E9.5) [95]. In the human placenta, ISG15 is expressed from the first to the second trimester of pregnancy, suggesting that ISG15 is associated with placental development, fetal growth, and/or potential defense mechanisms against infections [100]. These results suggest that ISGs are important not only for the pre-implantation period but also thereafter until mid-pregnancy. These observations are consistent with the fact that ISG expression persists long after IFNT expression subsides. It has been suggested that another type I IFN, such as IFNA or IFNW, could be present, which also up-regulates ISG expression. However, the presence and effects of type I IFN have not been definitively elucidated. Thus, further studies are needed to elucidate the molecular mechanisms associated with ISG regulation toward the mid-gestation.

The presence of extracellular vesicles (EVs) in the uterine lumen has been previously reported [101,102,103,104]. In addition to the direct release of IFNT into the uterine lumen, IFNT has been detected in EVs produced by bovine conceptus [105]. The IFNT produced is confined to the uterine lumen [15, 106], although a few studies have reported that IFNT exists in the uterine vein [107]. The presence of IFNT in uterine EVs suggests that such EVs can circulate, and the effect of IFNT may be more widespread. The release of EVs from various tissues is a common phenomenon, and EVs have been detected in the uteri of non-ruminant animals [108,109,110,111]. These results provide evidence that although EVs contain IFNT in ruminants, they may also contain other substances that play a common role in pregnancy establishment in other or all mammals. In cattle, the effects of EVs due to other factors other than IFNT have been reported [112, 113].

Although the role of ISGs during the period following IFNT expression has not yet been well characterized, their expression could be used as a marker for early pregnancy detection in ruminants. Recently, we identified ISGs expression in bovine tissues, such as the cervical and vaginal mucosa, from which a less invasive pregnancy diagnosis was developed. [114, 115]. It appears that the expression of ISGs is seen in humans, mice, and cows, and future validation is needed to understand the species-specific types as well as those common across mammalian species. ISGs induced by type I IFNs are diverse, comprising several hundred types, and are potentially involved in a variety of phenomena during the implantation-placentation processes. In humans, IFN plays no role in pregnancy recognition; however, type I IFN signaling is required for normal pregnancy progression [116, 117]. Although the substances and mechanisms of pregnancy recognition may differ among mammalian species, the existence of type I IFN and ISGs during pregnancy across various mammalian species suggests that the expression of various ISGs and possibly EV release may be a common feature necessary for the establishment and/or maintenance of pregnancy (Fig. 3).

Fig. 3.

Ruminant-specific pregnancy recognition and potential factors required for pregnancy establishment in ruminants and beyond. CL maintenance by IFNT is specific to ruminants (dotted line box). However, the expression of type I IFN from the embryo and responses by the uterus, such as interferon-stimulated genes (ISGs) expression, are similar among mammals, and ISGs and possibly EVs play a common role in successful pregnancy in these animals (solid-line box).

Conclusion

Although the expression of IFNT is required for the maintenance of CL function in ruminants, the expression of ISGs in the uteri, as well as other tissues during early to mid-pregnancy, appears to be common in mammals. Therefore, elucidation of the molecular mechanisms associated with ISGs expression and understanding of ISGs and even EV’s functions after the first weeks of the pregnancy recognition period is crucial, if pregnancy rates are to be improved in mammalian species.

Conflict of interests

The author declares that there are no conflicts of interest that could be perceived as prejudicing the impartiality of this review.