Successful pregnancy establishment in all mammalian species is considerably influenced by P4. In cows, low concentrations of circulating P4 are associated with altered endometrial gene expression and a reduced ability of the uterus to support embryo development [35, 36]. In the current study, we used early pregnant gilts with PMSG/hCG-induced estrus accompanied by impaired luteal P4 synthesis, which were previously described [12, 21], to test the hypothesis that insufficient P4 concentrations alter both endometrial and myometrial receptivity; in particular, the expression levels of steroid, PG, cytokine, and oxytocin receptors were examined. Our results clearly showed that the low concentrations of P4 observed in the luteal tissue and blood serum of gilts with gonadotropin-induced estrus [12] had no effect on PGR expression in the uterus but negatively affected the endometrial expression of genes encoding estrogen, androgen, PGE2, PGF2α, IL6, TNFα, and oxytocin receptors, which was primarily visible on day 12 of gestation. The response of the myometrial tissue was more differentiated because both inhibitory and stimulatory effects were detected. These results are the first demonstrating that estrus induction with PMSG/hCG, accompanied by reduced P4 synthesis during the peri-implantation period, contributes to changes in the transcriptome in the myometrium of farm animals.
The uterus is the main target of luteal P4, and P4 receptors are expressed in the porcine uterus during both the estrous cycle and pregnancy [37]. Interestingly, the loss of PGR protein from the luminal epithelium of the endometrium is a prerequisite for pregnancy establishment in various species [13, 16], and this phenomenon is observed on days 10 to 12 after ovulation in pigs [38]. As we reported here, the concentrations of PGR mRNA in the endometrial tissue did not change between days 10 and 15 of pregnancy in either group of animals. Such an observation is in agreement with previous data showing relatively stable PGR transcript abundance in the porcine endometrium on days 9 to 25 of pregnancy [37]. However, PGR mRNA expression did not differ between gilts with natural and gonadotropin-induced estrus (current results), which seems to contradict the theory regarding the negative correlation between concentrations of circulating P4 and the abundance of PGR in the endometrium of the pig [16, 38]. Similarly, no differences in PGR mRNA expression in the endometrium were detected on day 13 in cyclic beef heifers with normal and low concentrations of circulating P4 [36]. The lack of differences in endometrial PGR expression in gilts with normal and reduced luteal P4 synthesis observed in the present study suggests that concentrations of circulating P4 in gonadotropin-treated gilts were sufficient to induce PGR down-regulation. Moreover, the mechanisms controlling the decline in PGR expression in pigs are activated before day 6 of pregnancy because the blocking of P4 action with P4 antagonist on days 3 to 5 but not on days 6 and 7 of pregnancy elevates PGR mRNA levels in the endometrium and prevent PGR protein down-regulation [39]. In the current study, CLs and blood samples for P4 analyses were collected after day 9 of pregnancy [12]; therefore, we can only speculate that the amount of P4 secreted before day 6 by CLs of PMSG/hCG-treated and control gilts were similar.
As previously demonstrated, the highest PGR mRNA expression in the myometrium was detected at estrus, followed by much lower levels on days 11 and 19 of pregnancy in mature sows [40]. In fact, PGR protein is localized within in the myometrium on days 9 to 85 in pregnant gilts [37]. In the present study, the concentrations of PGR transcripts in the myometrial tissue were similar to those detected in the endometrium and were not affected by either the day of pregnancy or PMSG/hCG treatment. This indicates that PGR mRNA expression in the myometrium is not directly controlled by luteal P4. To support this statement, PGR protein was localized in the myometrial cells of ovariectomized gilts injected with both P4 and corn oil vehicle [37].
In addition to P4, ovarian estrogens influence morphological and physiological changes in the uterus [20, 41, 42]. In pigs, conceptus-derived estradiol-17β (E2) is the main pregnancy recognition signal, and it significantly affects uterine functions during early pregnancy [14, 16]. Both endometrial and myometrial cells of the porcine uterus express the estrogen receptor proteins ERα and ERβ, with ERα being the dominant form [43]. Our present results also showed that ESR1 mRNA is much more abundantly expressed than ESR2 in both tissues. Furthermore, ESR1 showed similar expression profiles in the endometrium and myometrium, which decreased gradually during the examined pregnancy period. Such results are consistent with previously published profiles of ESR1 mRNA and ERα protein expression in pregnant sows [40, 43]. In contrast to ESR1, ESR2 mRNA expression in the endometrium and myometrium increased during the studied days of early pregnancy. In the endometrium of naturally ovulating gilts, concentrations of ESR2 transcripts increased substantially from day 12, while in PMSG/hCG-treated gilts, the up-regulation of ESR2 mRNA expression was delayed and observed on day 15. Such delay in ESR2 mRNA expression and lower concentrations of ESR1 transcripts in the endometrium of gilts with gonadotropin-induced estrus indicate that sustained P4 synthesis is required for the development of the proper endometrial receptivity to E2 action.
In contrast to the endometrium, ESR2 mRNA expression in the myometrium was not affected by estrus induction with gonadotropins. Although a similar expression of ERβ on days 10–11 and 18–19 in the endometrium and myometrium of pregnant sows was reported [40, 43], the transient up-regulation of ESR2 mRNA may occur on days 12 and 15 (current data) in response to conceptus-derived E2, followed by a decrease on day 19 to a level comparable to that observed on day 11 [40]. The potential regulation of ERβ expression by E2 was suggested for porcine peri-implantation conceptuses [44].
Compared with E2 and P4, there is little data concerning regulation of AR expression in the porcine uterus. Both endometrial and myometrial cells of the uteri of cyclic and pregnant gilts express AR [45]. Further studies have demonstrated the marked up-regulation of AR mRNA expression in the porcine endometrium on day 12 of pregnancy and a potent stimulatory effect of E2 on AR transcript abundance in vitro [46]. In the present study, AR mRNA expression in the endometrium and myometrium did not change during the studied period of pregnancy in gilts with natural estrus, but lower levels of these receptors were found on day 12 in PMSG/hCG-induced animals. Because gonadotropin-treated gilts showed decreased luteal P4 synthesis [12], AR expression in the porcine uterus seems to be regulated not only by E2 from conceptuses [46] but also by P4 from the CL (present data). It has been demonstrated that the androgens present in the pig uterus may modulate the biological effect of E2 in the endometrium [46]. Therefore, lower levels of AR mRNA in PMSG/hCG-treated gilts may influence E2-dependent gene expression and uterine growth, which are required for successful implantation.
The PGs are important regulators of the CL lifespan, and PGF2α is responsible for luteal regression in non-pregnant animals [47], while PGE2 functions as a luteotropic and antiluteolytic factor promoting CL maintenance during pregnancy [48]. Additionally, PGs act locally to modulate uterine functions [49, 50]. In pregnant gilts, PGE2 and PGF2α activate their membrane receptors expressed in the endometrium to regulate vascular permeability, angiogenesis, immune response, and implantation [15, 51]. As we previously demonstrated, the treatment of gilts with PMSG/hCG to induce estrus resulted in the decreased expression of enzymes involved in PGE2 synthesis in the endometrium on day 12 of pregnancy and lower levels of PGE2 in the uterine lumen observed on day 15 [12, 22]. Moreover, much lower concentrations of PGF2α in endometrial tissue and blood plasma were detected in pregnant gilts with gonadotropin-induced estrus [22]. In the present study, insufficient P4 synthesis in gilts with precocious estrus induction negatively affected the expression of PTGER2, PTGER4, and PTGFR mRNA in the endometrium and/or myometrium. It indicates that sustained P4 synthesis during the peri-implantation period in pigs is required not only for the proper synthesis of PGE2 and PGF2α but also for the expression of PG receptor system. Considering the important role of PGs during early pregnancy, the decreased expression of PG receptors in the endometrium may influence embryo-maternal interactions and conceptus implantation in the pig.
To our knowledge, this is the first report demonstrating the profiles of PGE2 and PGF2α receptor expression in the myometrium of early pregnant pigs. In gilts with natural estrus, PTGFR mRNA levels did not vary during the examined period of gestation, while PTGER2 mRNA expression increased markedly after day 10. Moreover, levels of both types of receptors were lower at the maternal recognition of pregnancy in gonadotropin-treated than in naturally ovulating gilts. In the porcine myometrium, PTGER2 has been described as a relaxant, and PTGFR has been reported as a contractile receptor [52]. However, both PGE2 and PGF2α increase the tension of the contractions of the porcine myometrium during early pregnancy [24]. Therefore, the decreased expression of PG receptors in gilts with impaired luteal P4 may result in abnormal myometrial contractility during conceptus elongation and initial implantation.
PGI2 has been primarily described as a potent vasodilator and inhibitor of platelet aggregation in the vascular system [53]. PGI2 is also involved in pregnancy establishment and implantation in various species [17, 54–56]. Specifically, PGI2, acting via its membrane receptor, PTGIR, stimulates the expression of pro-angiogenic factors in the porcine endometrium [57]. In the present study, endometrial PTGIR expression was not affected by PMSG/hCG treatment. However, concentrations of PTGIR transcripts in the myometrium were greater in gilts with induced estrus, characterized by lower P4 synthesis, than in the control group. PGI2 is a smooth muscle relaxant in the uteri of non-pregnant women and pigs [58, 59]. P4, in turn, promotes uterine quiescence during pregnancy [60]. Moreover, the supplementation of early pregnant gilts with P4 stimulates PTGIR mRNA expression in the CL [61]. Therefore, the up-regulation of PTGIR mRNA in the myometrium of PMSG/hCG-induced gilts is surprising, but it may point to the activation of some local mechanisms in the uterus to compensate for reduced P4 synthesis and prevent contractions. This suggestion, however, requires more detailed research.
The period of the maternal recognition of pregnancy and early implantation is accompanied by the presence of cytokines in the porcine uterus [62, 63]. Among these, IL6 and TNFα contribute to local inflammatory reactions related to conceptus implantation [64, 65]. Both the endometrium and myometrium of the pig uterus express IL6R and TNFR mRNA during pregnancy [27, 66, 67]. In the present study, concentrations of IL6R, TNFRSF1A, and TNFRSF1B transcripts in the endometrium increased after day 10 of gestation, which is partly consistent with previously published data showing a stimulatory effect of conceptus estrogens on the expression of TNFα receptors [66] but not on IL6R [67]. Furthermore, we observed a negative effect of estrus induction on the expression of all three types of receptors. IL6 and TNFα are important regulators of cell proliferation, differentiation, and apoptosis [64, 68, 69] and also substantially modulate PGE2 and/or PGFα synthesis in the porcine endometrium [70–72]. Therefore, the reduced expression of IL6R, TNFRSF1A, and TNFRSF1B during early pregnancy may be detrimental to endometrial preparation for implantation.
In the myometrium, greater IL6R and TNFRSF1B mRNA expression was observed on day 15 of pregnancy. This implies a possible role of IL6 and TNFα in this tissue during the period of early implantation. Notably, the incubation of myometrial explants collected on day 15–16 of pregnancy, but not the estrous cycle, with IL6 and TNFα resulted in the elevated secretion of E2, supplementing, in this way, the amount of estrogens synthesized by the endometrium and conceptuses [27]. Estrus induction with PMSG/hCG did not affect IL6 and TNFα receptor expression in the myometrium, which points to other than P4-dependent mechanisms controlling IL6R and TNFRSF1B abundance in this tissue.
A number of cytokines released by developing porcine conceptuses activate inducible transcription factors, including NFkB, which are thought to be involved in pro-inflammatory and immune responses in the maternal uterus [73]. The results of our current research showed an increase in NFKB1 mRNA expression in the endometrium between days 10 and 12 of pregnancy in both groups of animals. Such up-regulation of NFKB1 is in line with the role of this factor as a receptivity marker in the porcine endometrium [74] and coincides with the elevated secretion of IL1β by elongating pig conceptuses, which has been shown to activate NFkB in the endometrium [63]. Estrus induction with gonadotropins had no effect on NFKB1 mRNA expression in the endometrium, which is consistent with the previously described lack of correlation between P4 action and NFkB activation in this tissue [39]. By contrast, the myometrial expression of NFKB1 mRNA was lower on day 12 in PMSG/hCG-treated gilts as compared with the control animals, indicating a transient inhibition of inflammatory response in the myometrium of gonadotropin-stimulated gilts.
Oxytocin is involved in luteal regression in both ruminants and pigs [47], and its stimulatory effect on luteolytic PGF2α secretion from the porcine endometrium has been documented [75]. Interestingly, OXTR was found in the endometrium and myometrium of pigs not only during the estrous cycle but also during early pregnancy [76]. In the current study, greater OXTR mRNA expression in the endometrium was detected around the time of conceptus implantation (day 15). This was consistent with elevated levels of oxytocin detected on days 10 and 14 in the uterine lumen of pregnant gilts [77]. Moreover, oxytocin stimulates the synthesis of PGE2 in porcine endometrial epithelial cells collected on days 11–12 of pregnancy but not on the corresponding days of the estrous cycle [70]. Therefore, the lower concentrations of OXTR transcripts detected in the endometrium of gilts with PMSG/hCG-induced estrus (present data) may participate in the disturbed PGE2 synthesis reported previously for these animals [22].
Although oxytocin plays an important role during parturition, much less is known about the role of this hormone in the myometrium of early pregnant pigs. Its involvement, however, in the regulation of PGF2α secretion from the myometrial slices collected on days 15–16 of pregnancy has been demonstrated previously [78]. Nevertheless, the results of our present study showed that concentrations of OXTR transcripts in the porcine myometrium were not affected by gonadotropin treatment to induce estrus.
The PPARγ isoform belongs to the nuclear receptor family of transcription regulators and its activation in the reproductive tract has been observed during steroidogenesis, angiogenesis, apoptosis, and tissue remodeling [79, 80]. The presence and activation of PPARγ in the porcine endometrium has been previously documented [33, 81]. No effect of estrus induction on PPARG mRNA was observed in the endometrium indicating no correlation between luteal P4 and PPARG transcript abundance in this tissue. Similarly, no difference in endometrial PPARG expression was found in beef heifers with normal and low concentrations of circulating P4 [36]. To our knowledge, this is the first study demonstrating PPARG mRNA expression in the myometrium of early pregnant gilts. Relatively stable PPARG mRNA expression was detected during the peri-implantation period in gilts with natural estrus. Interestingly, greater PPARG mRNA expression in gonadotropin-treated as compared to control gilts was found in this tissue on day 15. Because PPARγ is primarily involved in glucose homeostasis, such up-regulation may be attributed to the higher energy demands of the myometrium in gonadotropin-treated gilts.
The key element involved in the regulation of embryo-maternal interactions and myometrial activity is cell-to-cell communication mediated via gap junctions. Among the gap junction proteins, connexin 43 was localized in both the endometrium and myometrium of cyclic and pregnant gilts [82]. Greater expression of GJA1 mRNA was found in the porcine endometrium on day 12 of pregnancy as compared with day 12 of the estrous cycle [83]. The results of our present study showed that GJA1 mRNA expression increased in the endometrium and decreased in the myometrium between days 10 and 12 of pregnancy in gilts with natural estrus. This indicates a distinct response of both tissues to conceptus signals. Although GJA1 is more expressed in the myometrium than in the endometrium, both tissues responded with greater concentrations of GJA1 transcripts in PMSG/hCG-induced gilts on day 15. Such results are consistent with a negative correlation between the number of connexin 43 gap junctions in the myometrium and P4 concentrations in this tissue, as demonstrated in non-pregnant gilts [84]. However, the consequences of the elevated expression of GJA1 during early pregnancy remain to be elucidated.