Recognition Of Pregnancy In Pigs Research Articles

New perspective on conceptus estrogens in maternal recognition and pregnancy establishment in the pig†.

The proposed signal for maternal recognition of pregnancy in pigs is estrogen (E2), produced by the elongating conceptuses between days 11 to 12 of pregnancy with a more sustained increase during conceptus attachment and placental development on days 15 to 30. To understand the role of E2 in porcine conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing aromatase (CYP19A1) using CRISPR/Cas9 technology. Wild-type (CYP19A1+/+) and (CYP19A1-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer, which were transferred into recipient gilts. Elongated and attaching conceptuses were recovered from gilts containing CYP19A1+/+ or CYP19A1-/- embryos on day 14 and 17 of pregnancy. Total E2 in the uterine flushings of gilts with CYP19A1-/- embryos was lower than recipients containing CYP19A1+/+ embryos with no difference in testosterone, PGF2α, or PGE2 on either day 14 or 17. Despite the loss of conceptus E2 production, CYP19A1-/- conceptuses were capable of maintaining the corpora lutea. However, gilts gestating CYP19A1-/- embryos aborted between days 27 and 31 of gestation. Attempts to rescue the pregnancy of CYP19A1-/- gestating gilts with exogenous E2 failed to maintain pregnancy. However, CYP19A1-/- embryos could be rescued when co-transferred with embryos derived by in vitro fertilization. Endometrial transcriptome analysis revealed that ablation of conceptus E2 resulted in disruption of a number biological pathways. Results demonstrate that intrinsic E2 conceptus production is not essential for pre-implantation development, conceptus elongation, and early CL maintenance, but is essential for maintenance of pregnancy beyond 30 days .

The whole blood transcriptome at the time of maternal recognition of pregnancy in pigs reflects certain alterations in gene expression within the endometrium and the myometrium

The whole blood transcriptome at the time of maternal recognition of pregnancy in pigs reflects certain alterations in gene expression within the endometrium and the myometrium

The alterations in endometrial and myometrial transcriptome at the time of maternal recognition of pregnancy in pigs

The maternal recognition of pregnancy in pigs occurs around day 12–13 of gestation. During this period embryo-derived signals act on the endometrium and the myometrium to trigger tissue-specific responses that lead to proper implantation and maintenance of pregnancy. The present study utilized gene expression microarrays to examine embryo-induced alterations in porcine endometrial and myometrial transcriptome on days 12–13 of pregnancy. It has been confirmed that the endometrium and the myometrium, in response to embryonic signals, express unique set of genes. However, a key discovery of this study is the fact that genes that encode crucial factors involved in maternal recognition of pregnancy and preparation of uterus for implantation are regulated similarly in both tissues. Genes responsible for PGs synthesis (PTGES2, PTGR2), uterine development and remodeling (HOXA10, GRB10, MTOR) and innate immune response and immunomodulation (NFKBIA, TRAFD1) were regulated similarly in the endometrium and the myometrium. This suggests that in pigs these tissues act in sync to support embryos during peri-implantation.

Proteomic analysis of porcine endometrial tissue during peri-implantation period reveals altered protein abundance

In mammals, successful pregnancy depends upon the readiness of uterus for implantation, followed by correct communication between the endometrium and the developing conceptus. The objective of this study was to elucidate changes in protein abundance associated with progression of estrous cycle and pregnancy from Day 9 to Day 12. We analyzed porcine endometrial tissue lysates by 2D-DIGE. Abundance of several proteins was altered depending upon the pregnancy status of animals. MALDI-TOF/TOF was used to identify a number of these proteins. Endometrial proteins that increased from Day 9 to Day 12 of cycle included annexin A4, beta-actin, apolipoprotein, ceruloplasmin and afamin. Changes in protein abundances associated with conceptus secreted factors, including haptoglobin, prolyl-4-hydroxylase, aldose-reductase and transthyretin, were also observed. Functional analysis revealed that endometrial proteins with altered abundance on Day 12 irrespective of the reproductive status were related to growth and remodeling, acute phase response and free radical scavenging, whereas transport and small molecule biochemistry were the functions activated in the pregnant endometrium as compared to the cyclic endometrium. These data provide information on dynamic physiological processes associated with uterine endometrial function of the cyclic and pregnant endometrium during period of maternal recognition of pregnancy in pigs and may potentially demonstrate a protein profile associated with successful pregnancy. Biological significanceIn pigs, the fertility rates are generally very high but the early embryonic loss that occurs during the second and third weeks of gestation critically affects the potential litter size. Temporal changes that take place in the uterine environment during the period of early pregnancy in pigs and a cross-talk between the uterus and the embryo play an important role in embryonic survival and successful pregnancy. A better understanding of the molecular changes associated with these processes will pave way for understanding of endometrial functions and help towards increasing embryo survival. In this study, we present a 2D-DIGE based analysis of changes in porcine endometrial proteome that are associated with progression of cycle and progression of pregnancy. The network analysis of the results clearly revealed the pathways that are involved in rendering the endometrium receptive to the presence of embryo and also the changes that are result of molecular communication between the endometrium and the conceptuses. This comprehensive identification of proteomic changes in the porcine endometrium could be a foundation for targeted studies of proteins and pathways potentially involved in abnormal endometrial receptivity, placentation and embryo loss.

Expression of VEGF-receptor system in conceptus during peri-implantation period and endometrial and luteal expression of soluble VEGFR-1 in the pig

In view of the importance of vascular events observed during gestation, it was hypothesized that the VEGF-receptor system plays a critical role during early pregnancy and maternal recognition of pregnancy in pigs. This hypothesis was tested by examining the expression of the VEGF-receptor system in the porcine conceptus. Additionally, the endometrium, corpus luteum (CL) and embryos were studied for the expression of soluble VEGF receptor 1 (sVEGFR-1), the strong endogenous antagonist of VEGF. The expression patterns show that VEGF164 mRNA levels increase gradually in line with conceptus development, whereas VEGF120 and VEGFR-2 remain unchanged during the peri-implantation period. Interestingly, elevated VEGFR-1 expression was observed in conceptuses on days 15–16 of gestation ( P < 0.05). Comparison of the endometrial sVEGFR-1 mRNA expression revealed up-regulation on days 12 and 15–16 of pregnancy ( P < 0.01 and P < 0.05, respectively). Furthermore, increased sVEGFR-1 levels were observed on day 12 of the estrous cycle in the CL ( P < 0.05). Concluding, it seems that conceptus-derived VEGF164 plays crucial role in peri-implantation vascular events in pigs. These results support a potential role of VEGFR-1 in the proper growth and development of porcine conceptus during pregnancy. Moreover, expression patterns of sVEGFR-1 in the endometrium of pregnant pigs suggest that it may participate in vascular remodeling important for successful implantation. Finally, luteal sVEGFR-1 may be involved in the maintenance of CL function whenever pregnancy occurs in pigs.

Identification of differentially expressed genes in the uterine endometrium on day 12 of the estrous cycle and pregnancy in pigs

Maternal recognition of pregnancy in pigs occurs approximately on Day (D) 12 of pregnancy and is critical for embryo implantation. The presence of the conceptus in the uterine lumen during this period changes uterine endometrial function to prepare for attachment of the conceptus to the endometrial epithelial cells and maintain luteal function in the ovary. Although much is known about endometrial gene expression, the genes expressed in the uterine endometria and the cellular and molecular mechanisms of those gene products during the period of implantation and maternal recognition of pregnancy in pigs are still not completely defined. To better understand the interactions between the maternal uterus and conceptus during the implantation process, we searched genes differentially expressed in the endometria on D12 of pregnancy compared to those on D12 of the estrous cycle. A new reverse transcription-polymerase chain reaction (RT-PCR)-based method that involves annealing control primers (ACPs) was employed. Using 120 ACPs, we sequenced 12 differentially expressed genes (DEGs) and identified those genes using the Basic Local Alignment Search Tool (BLAST). Northern blot hybridization analysis confirmed the differential expression of those DEGs in the uterine endometrium. In situ hybridization analysis determined the cell-type specific expression of the DEGs in the uterine endometrium. Further analysis of the DEGs found in this study will provide insights into the cellular and molecular basis of maternal and fetal interactions during the period of maternal recognition of pregnancy in the pig. Mol. Reprod. Dev. 76: 75-84, 2009. (c) 2008 Wiley-Liss, Inc.

Expression of prostaglandin synthesis pathway enzymes in the porcine corpus luteum during the oestrous cycle and early pregnancy

Prostaglandins (PGs) of luteal origin may have paracrine and/or autocrine actions on the functions of the corpus luteum (CL). Previously, we have shown that enzymes of PG synthesis pathway such as prostaglandin E synthase (mPGES-1), prostaglandin F synthase (PGFS) and prostaglandin 9-ketoreductase (CBR1) are important in regulation of PG production in the conceptuses and endometrium of cyclic and pregnant pigs. Therefore, localization and expression patterns of these enzymes were determinated in porcine CL. The PGFS protein content was lower in metestrus and higher around luteolysis, and then decreased in late regressing CL. PGFS protein levels were lower on days 5-8 of pregnancy and did not differ between days 10 and 25. Elevated expression of mPGES-1 mRNA was found in early luteal phase. The mPGES-1 protein content, similarly to PGFS, was higher during luteolysis. mPGES-1 mRNA and protein levels were constant between days 5 and 25 of pregnancy. PGFS and mPGES-1 expression was down-regulated on days 16-17 of the oestrous cycle when compared to the corresponding days of pregnancy. Enhanced mPGES-1/PGFS ratio occurred during early luteal phase and days 5-8 of pregnancy. Expression of CBR1 mRNA and protein was constant during the cycle and pregnancy. Our studies revealed higher mPGES-1/PGFS ratios in the CL during early luteal phase and corresponding days of pregnancy that could favor PGE(2) synthesis and may be important in the control of luteal development. However, PG synthesis in the endometrium/conceptus rather than in the CL could be involved in luteolysis and maternal recognition of pregnancy in pigs.

Secretion of estradiol-17β by porcine endometrium and myometrium during early pregnancy and luteolysis

Past studies of the source of estrogens secreted during maternal recognition of pregnancy in pigs have focused on embryonic rather than uterine origin of these steroids. The present study documents: (1) the expression of the gene CYP 17, encoding cytochrome P450 17alpha-hydroxylase/C(17-20) lyase and (2) the synthesis and secretion of estradiol-17 beta (E(2)) in endometrial and myometrial tissues in gilts. The expression of CYP 17 gene was shown in porcine endometrium and myometrium. Basal endometrial secretion of E(2) was higher in pregnant gilts than in cyclic gilts (days 14-16). The myometrium secreted more E(2) during the expected time of luteolysis compared to early pregnancy. Basal secretion of E(2) during pregnancy was higher from the endometrium than from the myometrium. Conversely, during luteolysis E(2) secretion was higher from the myometrium and lower from the endometrium. In pregnant and cyclic gilts (days 14-16), progesterone (P(4), 10(-5)M) in vitro significantly increased E(2) secretion regardless of reproductive status. Oxytocin (OT, 10(-7)M) had no influence on E(2) secretion and did not change the stimulatory effect of P(4) in both tissues examined. In conclusions: (1) the CYP 17 gene transcript is present in porcine endometrium and myometrium; (2) porcine endometrium and myometrium release E(2) in vitro; (3) the endometrium releases more E(2) than the myometrium during early pregnancy; (4) the myometrium releases E(2) mainly during luteolysis; (5) the endometrium and myometrium can increase E(2) release in vitro if substrate (P(4)) is provided during early pregnancy and luteolysis. These data suggest active estrogen production by the myometrium and endometrium as an alternative source for this signal for recognition of pregnancy in the pig.

Old, new and the newest concepts of inhibition of luteolysis during early pregnancy in pig

In 1977 Bazer and Thatcher proposed that maternal recognition of pregnancy in the pig involves the secretion of PGF 2α towards the uterine lumen (exocrine) rather than towards the uterine venous drainage (endocrine) as occurs in the non-pregnant pig during the mid to late stages of the estrous cycle. The retrograde transfer of PGF 2α from the venous blood and uterine lymph into the uterus and the ability of the uterine vein and artery wall to accumulate PGF 2α could constitute a part of putative mechanism of corpus luteum protection during early pregnancy. A luteotropic/anti-luteolytic effect of PGE 2 in the pig also has been frequently demonstrated and it seems that the most effective agent in changing PGE 2:PGF 2α secretion is estradiol. The role for oxytocin during luteolysis and early pregnancy is controversial. It appears, however, that the main function of this hormone is autocrine and/or paracrine stimulation of PGF 2α secretion. Pig trophoblastic interferons, unlike those of ruminants, do not themselves exert an anti-luteolytic effect in pigs. It is likely, that cytokines and angiogenic growth factors are involved in the initiation of luteolysis and/or maintenance of corpora lutea (CL). A discovery of functional LH receptors in porcine endometrium opened a new possibility for this hormone in luteolysis and perhaps in recognition of pregnancy in pigs. The endogenous LH pulses can provoke prostaglandin secretion from endometrium in pigs. On the other hand prolongation of up-regulation of LH receptors in endometrium of early pregnant gilts can additionally increase angiogenic factor production before the process of implantation is completed. Finally new integrated concepts of luteolysis and inhibition of luteolysis in pigs based on selectively reviewed information are presented.

Keratinocyte growth factor is up-regulated by estrogen in the porcine uterine endometrium and functions in trophectoderm cell proliferation and differentiation.

Keratinocyte growth factor (KGF) is expressed by uterine endometrial epithelial cells during the estrous cycle and during pregnancy in pigs, whereas KGF receptor is expressed in conceptus trophectoderm and endometrial epithelia. In particular, KGF expression in the endometrium is highest on day 12 of pregnancy. This corresponds to the period of maternal recognition of pregnancy in pigs, which is signaled by large amounts of estrogen secreted by conceptus trophectoderm acting on the endometrium. Our hypothesis is that estrogens of conceptus origin stimulate endometrial epithelial KGF expression, and, in turn, secreted KGF stimulates proliferation and differentiation of conceptus trophectoderm. To determine the factors affecting KGF expression in the uterus, endometrial explants from gilts on day 9 of the estrous cycle were cultured in the presence of 17beta-estradiol, catechol estrogens, or progesterone. 17beta-Estradiol stimulated the expression of KGF (P < 0.05), whereas catechol estrogens had no effect (P > 0.05). Between days 9 and 15 of pregnancy, proliferating cell nuclear antigen was abundant in conceptuses, but was barely detectable in uterine endometrial epithelia. To determine the effects of KGF on conceptus trophectoderm, porcine trophectoderm (pTr) cells were treated with recombinant rat KGF (rKGF). rKGF increased the proliferation of pTr cells (P < 0.01) as measured by [(3)H]thymidine incorporation. rKGF elicited phosphorylation of KGF receptor and activated the mitogen-activated protein kinase (ERK1/2) cascade in pTr cells. pTr cell differentiation was affected by rKGF, because it increased expression of urokinase-type plasminogen activator, a marker for differentiation in pTr cells. Collectively, these results indicate that estrogen, the pregnancy recognition signal from the conceptus in pigs, increases uterine epithelial KGF expression, and, in turn, KGF stimulates the proliferation and differentiation of conceptus trophectoderm.

Maternal Recognition of Pregnancy in Pigs

Pig is a polytocous animal and average number of newborn piglets is around ten. In pig, it is a interesting point that how many number of littermate embryos (conceptuses) in the early pregnant uterus is needed for the establishment of pregnancy. Frontier research showed that four embryos are necessary for establishment of pregnancy in pig. On the contrary, embryo transfer study provided that one embryo can not maintain a pregnancy, however two embryos establish it, and more than two embryos in the uterus improve a pregnancy rate. These facts support the evidence that plural growing embryos in the reproductive tracts of early pregnant pig synthesize some signal substances above the threshold level for the recognition of pregnancy to their maternal system. In pig, the most important substance from conceptus for the recognition of pregnancy is estradiol-17 β (E2). When conceptus starts an elongation in the uterus at during week 2 of pregnancy, concomitant E2 synthesized and secreted from trophoblast is a first signal to their mother for a recognition of pregnancy. This signal from conceptus is necessary for maintenance of corpora lutea during week 2 of pregnancy, and conceptus has to spread over both uterine lumens to play an effective role for maintenance of corpora lutea. In pig, as same as other domestic animals, prostaglandin (PG) F2α causes a luteolysis at the estrous cycle. An exocrine direction of synthesized and secreted PGF2α in the endometrium is critical at the time of recognition of pregnancy. Conversely, PGE2 probably plays a luteotropic role for corpora lutea. The recognition and establishment of pregnancy is the results of many successive reactions between sow and conceptuses in the maternal system. What kinds of substances and mechanisms are related to the recognition of pregnancy in pig, is not completely resolved. The relationship between conceptus and their maternal system, and signals for maintenance of corpora lutea may have still unknown parts. Therefore, in this article, following subjects are discussed, 1) E2 synthesis by conceptus, 2) Retinal binding protein synthesis by conceptus and uterine endometrium, 3) Secretion and its direction of PGE2 and PGF2α from endometrium, 4) proteins synthesized by conceptus.

Expression of c-fos in Porcine Endometrium during the Estrous Cycle and Early Pregnancy1

This study determined the pattern of expression of the c-fos oncogene in endometrium from pregnant and cyclic gilts. A transcript of the expected 2.2-kb size for the c-fos mRNA was detected in porcine endometrium from cyclic and pregnant gilts. Expression of endometrial c-fos mRNA was not different for Days 0, 5, 10, 15, or 18 cyclic or pregnant gilts. However, expression of c-fos mRNA was greater on Day 12 of pregnancy compared to Day 12 of the estrous cycle. Furthermore, expression of c-fos mRNA was related to developmental stages when pig conceptuses secrete estradiol, the primary determinant for maternal recognition of pregnancy. Throughout the estrous cycle and early pregnancy, immunoreactive fos protein was localized to the glandular and surface epithelial cells as well as myometrial cells. Results of this study indicate that endometrial expression of c-fos mRNA increases on Day 12 of pregnancy, coinciding with the establishment of maternal recognition of pregnancy in pigs.