Functional analysis of porcine conceptus estrogens and prostaglandins in the maintainence of pregnancy in the pig : the two essential conceptus maternal recognition signals for preventing luteolysis
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Bazer and Thatcher's endocrine-exocrine model was the long accepted theory on achievement of maternal recognition of pregnancy in the pig. The endocrine exocrine theory states that, in the presence of conceptus estrogen at day 12 of gestation, PGF2a is sequestered into the uterine lumen rather than being secreted into the uterine vasculature to prevent its action on the ovary (Bazer and Thatcher, 1982). After the publication of the endocrine-exocrine theory, conceptus estrogen was considered the sole maternal recognition signal for nearly 40 years. There are three aromatase paralogs that contribute to conceptus estrogen production up to day 30 in the pig: CYP19A1, CYP19A2, and CYP19A3 (Choi et al., 1997; Conley et al., 1997). A loss-of-function study targeting conceptus aromatase genes CYP19A1 and CYP19A2, surprisingly proved that conceptuses could not only signal for maternal recognition of pregnancy without estrogen but could maintain pregnancy to day 25 of gestation (Meyer et al., 2019; Lucas et al., 2023). Although Meyer's study was able to successfully show the effect of simultaneously ablating early aromatase isoforms on pregnancy, their individual roles during conceptus development are still unknown. Study one focuses on a loss-of-function model for CYP19A2, known as placental aromatase (Conley et al., 1997), to better understand the role of this individual isoform during early pregnancy in the pig. Because CYP19A2 is the primary isoform expressed from days 21 to 54 of gestation in the pig (Lucas et al., 2023), it was hypothesized that CYP19A2-/- (A2KO) conceptuses would not produce estrogen after day 21 and cause loss of pregnancy before day 30 of gestation. A2KO pregnancies were generated using CRISPR Cas-9 gene editing technology, SCNT, and embryo transfer. A2KO pregnancies were maintained to day 15 of gestation and appeared morphologically normal. Eight recipients were allowed to continue pregnancy to day 32. Only two A2KO recipients maintained pregnancy to this stage while the six other recipients extended luteal function past day 21 but did not maintain pregnancy. Surprisingly, the allantoic fluid of the d32 A2KO fetuses contained significantly higher levels of estradiol than their WT counterparts. Measurement of CYP19A1, CYP19A2, and CYP19A3 mRNA expression revealed that, unlike WT pregnancies that only express CYP19A2 at day 32, A2KO placenta expressed both CYP19A1 and CYP19A3. This suggests that CYP19A2 may play a potential role in downregulating the other aromatase isoforms, and/or the expression of CYP19A1 and CYP19A3 may be a compensatory mechanism to ensure estrogen production and maintenance of pregnancy. When it became apparent that estrogen was not essential to maternal recognition of pregnancy in the pig, a second loss-of-function study targeting PTGS2 (Pfeiffer et al., 2020) attempted to better understand the role of conceptus prostaglandin production during MRP. Conceptus PTGS2 was successfully ablated, but, once again, pregnancies were maintained up to day 30 of gestation. It was concluded that neither conceptus estrogens nor prostaglandins had a significant effect on maintenance of pregnancy alone, however, the current study two investigates the hypothesis that conceptus estrogens and prostaglandins can compensate for once another to ensure proper PGE production and successful maternal recognition in the pig. PGE, a known luteotrophic factor, can be produced through two synthesis pathways: 1) conceptus PTGS2 converts arachidonic acid into PGE precursors and 2) conceptus estrogens stimulate endometrial PGE production. To better understand the relationship between conceptus estrogens and prostaglandins and investigate their effects on maternal recognition in the pig, study two focuses on a triple loss-of function model generated for conceptus genes CYP19A1, CYP19A2 and PTGS2. CYP19A1-/- /CYP19A2-/- / PTGS2-/- (TKO) embryos were once again generated using CRISPR Cas-9 gene editing and SCNT. TKO embryos were transferred to recipient gilts, and the first collections happened at day 14 of gestation before the period of cyclic regression in the pig. TKO embryos were present in the uterine lumen at day 14 and appeared to be morphologically normal. Both conceptus estradiol and prostaglandin production were significantly lower as expected, but conceptus IFNG and ILIB2 production was not inhibited. Five recipient gilts were allowed to continue pregnancy to day 16 of gestation. Upon day 16 collections, all recipient gilts presented regressing corpora lutea and experienced a significant decrease in progesterone concentrations from days 14 to 16. Concentrations of PGF2a in the uterine lumen were significantly decreased compared to A2KO and WT pregnancies, indicating that PGF2a was not being sequestered into the uterine lumen but was rather being secreted into the uterine vasculature to initiate luteolysis exemplifying the endocrine model. It can be concluded from study two that, rather than estrogen alone, conceptus estrogen and prostaglandins ensure proper production of PGE, prevent luteolysis at day 15 of gestation, and serve as the true maternal recognition signals in the pig.
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