N-Oleoyldopamine promotes the differentiation of mouse trophoblast stem cells into parietal trophoblast giant cells.

The placenta plays various roles in a healthy pregnancy, and abnormalities in the placenta result in adverse outcomes. Adequate differentiation of trophoblast subtypes is necessary for placental function, but the molecular mechanisms that determine trophoblast cell fate remain unclear. Here, we screened small molecular compound (SMC) libraries (1904 SMCs) to identify particular SMCs which regulate trophoblast differentiation in mouse trophoblast stem cells (mTSCs) to understand the molecular mechanisms underlying cell fate decision in trophoblast cells. The two-step screening revealed a novel effect of N-oleoyldopamine (OLDA), an endogenic vanilloid, to promote differentiation into parietal trophoblast giant cells (P-TGCs) and repress them into spongiotrophoblast cells in mTSCs. Analyses by gene deletion and inhibitor treatments indicated that transient receptor potential cation channel subfamily V member 3 (Trpv3), one of the candidates for targeting by OLDA, was involved in maintaining stem status and P-TGC differentiation in mTSCs. Finally, transcriptome analysis revealed that Fosl1, a key regulatory factor in differentiation into P-TGCs, was upregulated by OLDA treatment, suggesting that OLDA promoted the differentiation of mTSCs into P-TGCs via regulation of Fosl1 expression.

CDX2 downregulation in mouse mural trophectoderm during peri-implantation is heteronomous, dependent on the YAP-TEAD pathway and controlled by estrogen-induced factors.

Purpose: To investigate the transition of CDX2 expression patterns in mouse trophectoderm (TE) and its regulatory mechanisms during implantation. Methods: Mouse E3.5-4.5 blastocysts were used to immunostain CDX2, YAP, TEAD4, and ESRRB. Endogenous estrogen signaling was perturbed by administrating estrogen receptor antagonist ICI 182,780 or ovariectomy followed by administration of progesterone and ß-estradiol to elucidate the relationship between the transition of CDX2 expression patterns and ovarian estrogen-dependent change in the uterine environment. Results: CDX2 expression was gradually downregulated in the mural TE from E4.0 in vivo, whereas CDX2 downregulation was not observed in blastocysts cultured in KSOM. Fetal bovine serum (FBS) supplementation in KSOM induced CDX2 downregulation independently of blastocyst attachment to dishes. CDX2 downregulation in the mural TE was repressed by administration of ICI 182,780 or by ovariectomy, and administration of ß-estradiol into ovariectomized mice retriggered CDX2 downregulation. Furthermore, Cdx2 expression in the mural TE might be controlled by the YAP-TEAD pathway. Conclusions: CDX2 downregulation was induced heteronomously in the mural TE from E4.0 by uterus-derived factors, the secretion of which was stimulated by ovarian estrogen.

Loop-mediated isothermal amplification (LAMP) and machine learning application for early pregnancy detection using bovine vaginal mucosal membrane.

An early and accurate pregnancy diagnosis method is required to improve the reproductive performance of cows. Here we developed an easy pregnancy detection method using vaginal mucosal membrane (VMM) with application of Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) and machine learning. Cows underwent artificial insemination (AI) on day 0, followed by VMM-collection on day 17-18, and pregnancy diagnosis by ultrasonography on day 30. By RNA sequencing of VMM samples, three candidate genes for pregnancy markers (ISG15 and IFIT1: up-regulated, MUC16: down-regulated) were selected. Using these genes, we performed RT-LAMP and calculated the rise-up time (RUT), the first-time absorbance exceeded 0.05 in the reaction. We next determined the cutoff value and calculated accuracy, sensitivity, specificity, positive prediction value (PPV), and negative prediction value (NPV) for each marker evaluation. The IFIT1 scored the best performance at 92.5% sensitivity, but specificity was 77.5%, suggesting that it is difficult to eliminate false positives. We then developed a machine learning model trained with RUT of each marker combination to predict pregnancy. The model created with the RUT of IFIT1 and MUC16 combination showed high specificity (86.7%) and sensitivity (93.3%), which were higher compared to IFIT1 alone. In conclusion, using VMM with RT-LAMP and machine learning algorithm can be used for early pregnancy detection before the return of first estrus.

Cell proliferation potency is independent of FGF4 signaling in trophoblast stem cells derived from androgenetic embryos.

We previously established trophoblast stem cells from mouse androgenetic embryos (AGTS cells). In this study, to further characterize AGTS cells, we compared cell proliferation activity between trophoblast stem (TS) cells and AGTS cells under fibroblast growth factor 4 (FGF4) signaling. TS cells continued to proliferate and maintained mitotic cell division in the presence of FGF4. After FGF4 deprivation, the cell proliferation stopped, the rate of M-phase cells decreased, and trophoblast giant cells formed. In contrast, some of AGTS cells continued to proliferate, and the rate of M-phase cells did not decrease after FGF4 deprivation, although the other cells differentiated into giant cells. RO3306, an ATP competitor that selectively inhibits CDK1, inhibited the cell proliferation of both TS and AGTS cells. Under RO3306 treatment, cell death was induced in AGTS cells but not in TS cells. These results indicate that RO3306 caused TS cells to shift mitotic cell division to endoreduplication but that some of AGTS cells did not shift to endoreduplication and induced cell death. In conclusion, the paternal genome facilitated the proliferation of trophoblast cells without FGF4 signaling.

Dual barrier system against xenomitochondrial contamination in mouse embryos.

Heteroplasmic mammalian embryos between genetically distant species fail to develop to term, preventing transmission of xenomitochondrial DNA to progeny. However, there is no direct evidence indicating the mechanisms by which species specificity of the mitochondrial genome is ensured during mammalian development. Here, we have uncovered a two-step strategy underlying the prevention of xenomitochondrial DNA transmission in mouse embryos harboring bovine mitochondria (mtB-M embryos). First, mtB-M embryos showed metabolic disorder by transient increase of reactive oxygen species at the 4-cell stage, resulting in repressed development. Second, trophoblasts of mtB-M embryos led to implantation failure. Therefore, we tested cell aggregation with tetraploid embryos to compensate for the placentation of mtB-M embryos. The 14 mtB-M embryos harboring bovine mtDNAs developed to term at embryonic day 19.5. Taken together, our results show that contamination of bovine mtDNA is prohibited by embryonic lethality due to metabolic disruption and failure of placentation, suggesting these represent xenomitochondrial elimination mechanisms in mammalian embryos.

A tripartite paternally methylated region within the Gpr1-Zdbf2 imprinted domain on mouse chromosome 1 identified by meDIP-on-chip.

The parent-of-origin specific expression of imprinted genes relies on DNA methylation of CpG-dinucleotides at differentially methylated regions (DMRs) during gametogenesis. To date, four paternally methylated DMRs have been identified in screens based on conventional approaches. These DMRs are linked to the imprinted genes H19, Gtl2 (IG-DMR), Rasgrf1 and, most recently, Zdbf2 which encodes zinc finger, DBF-type containing 2. In this study, we applied a novel methylated-DNA immunoprecipitation-on-chip (meDIP-on-chip) method to genomic DNA from mouse parthenogenetic- and androgenetic-derived stem cells and sperm and identified 458 putative DMRs. This included the majority of known DMRs. We further characterized the paternally methylated Zdbf2/ZDBF2 DMR. In mice, this extensive germ line DMR spanned 16 kb and possessed an unusual tripartite structure. Methylation was dependent on DNA methyltransferase 3a (Dnmt3a), similar to H19 DMR and IG-DMR. In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed. The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome. This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.

Dynamic Changes of Gene Expression in Mouse Mural Trophectoderm Regulated by Cdx2 During Implantation.

Implantation of the blastocyst into the uterus is a specific and essential process for mammalian embryonic development. In mice, implantation is initiated from the mural trophectoderm of the blastocyst and the mTE controls implantation progression by acquiring the ability to attach and invade into the endometrium while differentiating into primary trophoblast giant cells. Nevertheless, it remains largely unclear when and how the mTE differentiates and acquires this ability during implantation. Here, by RNA sequencing analysis with the pre- and peri-implantation mTE, we show that the mTE undergoes stage-specific and dynamic changes of gene expression during implantation. We also reveal that the mTE begins down-regulating Cdx2 and up-regulating differentiation marker genes during the peri-implantation stage. In addition, using trophectoderm (TE) -specific lentiviral vector-mediated gene transduction, we demonstrate that TE-specific Cdx2 overexpression represses differentiation of the mTE into the primary trophoblast giant cells. Moreover, we reveal that TE-specific Cdx2 overexpression also represses the up-regulation of cell adhesion- and migration-related genes, including Slc6a14, Slc16a3, Itga7, Itgav and Itgb3, which are known to regulate migration of trophectoderm cells. In particular, the expression of Itgb3, an integrin subunit gene, exhibits high inverse correlation with that of Cdx2 in the TE. Reflecting the down-regulation of the genes for TE migration, TE-specific Cdx2 overexpression causes suppression of the blastocyst outgrowth in vitro and abnormal progression of implantation in vivo. Thus, our results specify the time-course changes of global gene expression in the mTE during implantation and uncover the significance of Cdx2 down-regulation for implantation progression.

Deletion of Gtl2, imprinted non-coding RNA, with its differentially methylated region induces lethal parent-origin-dependent defects in mice.

The cluster of imprinted genes located in the Dlk1-Dio3 domain spanning 1 Mb plays an essential role in controlling pre- and postnatal growth and differentiation in mice and humans. The failure of parent-of-origin-dependent gene expression in this domain results in grave disorders, leading to death in some cases. However, little is known about the role of maternally expressed non-coding RNAs (ncRNAs) including many miRNAs and snoRNAs in this domain. In order to further understand the role of these ncRNAs, we created Gtl2-mutant mice harboring a 10 kb deletion in exons 1-5. The mutant mice exhibited a very unique inheritance mode: when the deletion was inherited from the mother (Mat-KO), the pups were born with normal phenotypes; however, all of them died within 4 weeks after birth, probably due to severely hypoplastic pulmonary alveoli and hepatocellular necrosis. Mice carrying the paternal deletion (Pat-KO) showed severe growth retardation and perinatal lethality. Interestingly, the homozygous mutants (Homo-KO) survived and developed into fertile adults. Our results show that these phenotypes occur due to altered expression of the Dlk1-Dio3 cluster genes including miRNAs and snoRNAs via the cis and trans effects.

Progesterone depletion results in Lamin B1 loss and induction of cell death in mouse trophoblast giant cells.

Trophoblast giant cells (TGCs), a mouse trophoblast subtype, have large amounts of cytoplasm and high ploidy levels via endocycles. The diverse functions and gene expression profiles of TGCs have been studied well, but their nuclear structures remain unknown. In this study, we focus on Lamin B1, a nuclear lamina, and clarify its expression dynamics, regulation and roles in TGC functions. TGCs that differentiated from trophoblast stem cells were used. From days 0 to 9 after differentiation, the number of TGCs gradually increased, but the amount of LMNB1 peaked at day 3 and then slightly decreased. An immunostaining experiment showed that LMNB1-depleted TGCs increased after day 6 of differentiation. These LMNB1-depleted TGCs diffused peripheral localization of the heterochromatin marker H3K9me2 in the nuclei. However, LMINB1-knock down was not affected TGCs specific gene expression. We found that the death of TGCs also increased after day 6 of differentiation. Moreover, Lamin B1 loss and the cell death in TGCs were protected by 10-6 M progesterone. Our results conclude that progesterone protects against Lamin B1 loss and prolongs the life and function of TGCs.

Birth of parthenogenetic mice that can develop to adulthood.

Only mammals have relinquished parthenogenesis, a means of producing descendants solely from maternal germ cells. Mouse parthenogenetic embryos die by day 10 of gestation. Bi-parental reproduction is necessary because of parent-specific epigenetic modification of the genome during gametogenesis. This leads to unequal expression of imprinted genes from the maternal and paternal alleles. However, there is no direct evidence that genomic imprinting is the only barrier to parthenogenetic development. Here we show the development of a viable parthenogenetic mouse individual from a reconstructed oocyte containing two haploid sets of maternal genome, derived from non-growing and fully grown oocytes. This development was made possible by the appropriate expression of the Igf2 and H19 genes with other imprinted genes, using mutant mice with a 13-kilobase deletion in the H19 gene as non-growing oocytes donors. This full-term development is associated with a marked reduction in aberrantly expressed genes. The parthenote developed to adulthood with the ability to reproduce offspring. These results suggest that paternal imprinting prevents parthenogenesis, ensuring that the paternal contribution is obligatory for the descendant.

The Differentiation Potency of Trophoblast Stem Cells from Mouse Androgenetic Embryos.

In mice, trophoblast stem (TS) cells are derived from the polar trophectoderm of blastocysts. TS cells cultured in the presence of fibroblast growth factor 4 (Fgf4) are in an undifferentiated state and express undifferentiated marker genes such as Cdx2. After removing Fgf4 from the culture medium, TS cells drastically reduce the expression of undifferentiated marker genes, stop cell proliferation, and differentiate into all trophoblast cell subtypes. To clarify the roles of the parental genomes in placentation, we previously established TS cells from androgenetic embryos (AGTS cells). AGTS cells are in the undifferentiated state when cultured with Fgf4 and express undifferentiated marker genes. After removing Fgf4, AGTS cells differentiate into trophoblast giant cells (TGCs), but not into spongiotrophoblast cells, and some of the AGTS cells continue to proliferate. In this study, we investigated the differentiation potency of AGTS cells by analyzing the expression of undifferentiated marker genes and all trophoblast cell subtype-specific genes. After removing Fgf4, some undifferentiated marker genes (Cdx2, Eomes and Elf5) continued to be expressed. Interestingly, TGCs differentiated from AGTS cells also expressed Cdx2, but not Prl3d1. Moreover, the expression of Gcm1 and Synb was induced after the differentiation, indicating that AGTS cells preferentially differentiated into labyrinth progenitor cells. Cdx2 knockdown resulted in increased Prl3d1 expression, suggesting that Fgf4-independent Cdx2 expression inhibited the functional TGCs. Moreover, Fgf4-independent Cdx2 expression was activated by Gab1, one of the paternally expressed imprinted genes via the mitogen-activated protein kinase kinase (MEK)-extracellular signal regulated protein kinase (ERK) pathway. These results suggested that the paternal genome activates the MEK-ERK pathway without the Fgf4 signal, accelerates the differentiation into labyrinth progenitor cells and controls the function of TGCs.

DNA methylation imprints on the IG-DMR of the Dlk1-Gtl2 domain in mouse male germline.

Mouse genomes show a large cluster of imprinted genes at the Dlk1-Gtl2 domain in the distal region of chromosome 12. An intergenic-differentially methylated region (IG-DMR) located between Dlk1 and Gtl2 is specifically methylated in the male germline; IG-DMR regulates the parental allele-specific expression of imprinted genes. Here, we show the resetting of IG-DMR methylation marks during male germ-cell differentiation. For parental allele-specific methylation analysis, polymorphisms were detected in a 2.6-kb IG-DMR in three mouse strains. Bisulfite methylation analysis showed erasure of the marks by E14 and re-establishment before birth. The IG-DMR methylation status was maintained in spermatogonia and spermatocytes of mature testes. The IG-DMR methylation status established before birth is thus maintained throughout the lifetime in the male germline.

Disruption of parental-specific expression of imprinted genes in uniparental fetuses.

In mammals, imprinted genes show parental origin-dependent expression based on epigenetic modifications called genomic imprinting (GI), which are established independently during spermatogenesis or oogenesis. Due to GI, uniparental fetuses never develop to term. To determine whether such expression of imprinted genes is maintained in uniparental mouse fetuses, we analyzed the expression of 20 paternally and 11 maternally expressed genes in androgenetic and parthenogenetic fetuses. Four genes of each type were expressed in both groups of fetuses. Furthermore, quantitative analysis showed that expression levels deviated from the presumed levels for some imprinted genes. These results suggest that mechanisms acting in trans between paternal and maternal alleles are involved in the appropriate expression of some imprinted genes.

Deficiency of genomic reprogramming in trophoblast stem cells following nuclear transfer.

To examine the genomic reprogrammability of trophoblast stem (TS) cells using a nuclear transfer technique, we produced TS cloned embryos using five TS cell lines from three strains of mice (ICR, B6D2F1, and B6CBF1) as donors and observed developmental ability during preimplantation development. The developmental rates of the TS cloned embryos that developed to the two-cell, four- to eight-cell, morula, and blastocyst stages were 58-83%, 0-38.6%, 0-21.3%, and 0-15.9%, respectively, indicating that more than 50% of TS cloned embryos arrested at the two-cell stage. These TS cloned two-cell embryos were expressed low level of Dappa3 (also known as PGC7/Stella), indicating that zygotic gene activation (ZGA) was disrupted in these embryos. However, a small portion of the TS cloned embryos (0-15.9%) reached the blastocyst stage. In these TS cloned blastocysts, the numbers of trophectoderm (TE) and inner cell mass (ICM) cells were 31.9 ± 4.6 and 12.1 ± 3.0, respectively, which were not significantly different from those in the fertilized embryos. In addition, the gene expression analysis showed that Oct3/4, and Cdx2, which are ICM- and TE-specific marker genes, respectively, and Dppa3, and Hdac1, which are zygotic gene activation-related genes, were expressed in TS cloned blastocysts at the same levels as in the fertilized blastocysts. These results indicate that although TS cloned embryos are able to differentiate into ICM cells, the genomic reprogrammability of TS cells is very low following nuclear transfer.

Epigenetic and transcriptional features of the novel human imprinted lncRNA GPR1AS suggest it is a functional ortholog to mouse Zdbf2linc.

Long non-coding RNAs (lncRNAs), transcribed from the intergenic regions of animal genomes, play important roles in key biological processes. In mice, Zdbf2linc was recently identified as an lncRNA isoform of the paternally expressed imprinted Zdbf2 gene. The functional role of Zdbf2linc remains undefined, but it may control parent-of-origin-specific expression of protein-coding neighbors through epigenetic modification in cis, similar to imprinted Nespas, Kcnq1ot1 and Airn lncRNAs. Here, we identified a novel imprinted long-range non-coding RNA, termed GPR1AS, in the human GPR1-ZDBF2 intergenic region. Although GPR1AS contains no human ZDBF2 exons, this lncRNA is transcribed in the antisense orientation from the GPR1 intron to a secondary, differentially methylated region upstream of the ZDBF2 gene (ZDBF2 DMR), similar to mouse Zdbf2linc. Interestingly, GPR1AS/Zdbf2linc is exclusively expressed in human/mouse placenta with paternal-allele-specific expression and maternal-allele-specific promoter methylation (GPR1/Gpr1 DMR). The paternal-allele specific methylation of the secondary ZDBF2 DMR was established in human placentas as well as somatic lineage. Meanwhile, the ZDBF2 gene showed stochastic paternal-allele-specific expression, possibly methylation-independent, in placental tissues. Overall, we demonstrated that epigenetic regulation mechanisms in the imprinted GPR1-GPR1AS-ZDBF2 region were well-conserved between human and mouse genomes without the high sequence conservation of the intergenic lncRNAs. Our findings also suggest that lncRNAs with highly conserved epigenetic and transcriptional regulation across species arose by divergent evolution from a common ancestor, if they do not have identical exon structures.

Identification of genes aberrantly expressed in mouse embryonic stem cell-cloned blastocysts.

During development, cloned embryos often undergo embryonic arrest at any stage of embryogenesis, leading to diverse morphological abnormalities. The long-term effects resulting from embryo cloning procedures would manifest after birth as early death, obesity, various functional disorders, and so forth. Despite extensive studies, the parameters affecting the developmental features of cloned embryos remain unclear. The present study carried out extensive gene expression analysis to screen a cluster of genes aberrantly expressed in embryonic stem cell-cloned blastocysts. Differential screening of cDNA subtraction libraries revealed 224 differentially expressed genes in the cloned blastocysts: eighty-five were identified by the BLAST search as known genes performing a wide range of functions. To confirm their differential expression, quantitative gene expression analyses were performed by real-time PCR using single blastocysts. The genes Skp1a, Canx, Ctsd, Timd2, and Psmc6 were significantly up-regulated, whereas Aqp3, Ak3l1, Rhot1, Sf3b3, Nid1, mt-Rnr2, mt-Nd1, mt-Cytb, and mt-Co2 were significantly down-regulated in the majority of embryonic stem cell-cloned embryos. Our results suggest that an extraordinarily high frequency of multiple functional disorders caused by the aberrant expression of various genes in the blastocyst stage is involved in developmental arrest and various other disorders in cloned embryos.

Apoptosis in the porcine uterine endometrium during the estrous cycle, early pregnancy and post partum.

The mammalian uterus changes dramatically during the estrous cycle, pregnancy, and involution post partum. Dynamic changes in the uterine endometrium are a type of homeostasis and proceed with proliferation and exclusion of cells. Homeostasis of the uterus is closely related to apoptosis involving various hormones and cytokines. The objective of the present study was to determine the morphological features and occurrence of apoptosis in the porcine endometrium during the estrous cycle, early pregnancy, and post partum. Cyclic changes in the morphology of the surface epithelium were observed during the estrous cycle. The heights of surface epithelia were significantly high on day 4 of the estrous cycle and the early pregnancy. The heights of the surface epithelium remained low from days 1 to 31 post partum. We then used terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling (TUNEL) of the 3'-terminal of fragmented DNA, which is effective for detection of apoptosis in various tissues. We found that apoptosis in the porcine endometrium contributed to homeostasis of the endometrium during the estrous cycle through control of cell proliferation and exclusion. Conversely, apoptosis on days 4 and 8 of gestation before the implantation window depended on the plasma estrogen and progesterone levels; however, suppressive homeostasis of apoptosis occurred at the time of implantation on days 15, 18 and 21 of gestation. Our study is the first to demonstrate apoptotic cell death in the porcine endometrium directly by TUNEL method. The results strongly suggest that uterine homeostasis is mainly controlled by apoptosis during the estrous cycle and early pregnancy.

Regulated expression of two sets of paternally imprinted genes is necessary for mouse parthenogenetic development to term.

Mouse parthenogenetic embryos (PEs) are developmentally arrested until embryo day (E) 9.5 because of genomic imprinting. However, we have shown that embryos containing genomes from non-growing (ng) and fully grown (fg) oocytes, i.e. ng(wt)/fg(wt) PE (wt, wild type), developed to E13.5. Moreover, parthenogenetic development could be extended to term by further regulation of Igf2 and H19 expression using mice with deletion of the H19 transcription unit (H19Delta13) together with its differentially unit (DMR). To gain an insight into the extended development of the parthenotes to term, we have here investigated the expression levels of paternally imprinted genes in ng(H19Delta13)/fg(wt) PE throughout their development. In ng(H19Delta13)/fg(wt) Pes that died soon after recovery, the expression of Igf2 and H19 was restored to the appropriate levels except for low Igf2 expression in the liver after E15.5. Further, the paternally expressed Dlk1 and Dio3 were repressed, while the expression levels of the maternal Gtl2 and Mirg were twice those of the controls. However, the above-mentioned four genes showed almost normal expression in the surviving ng(H19Delta13)/fg(wt) PEs. The methylation analysis revealed that the intragenic DMR of the Dlk1-Gtl2 domain was hypermethylated in the ng(H19Delta13)/fg(wt) PEs that survived, but not in the PEs that died soon after recovery. The present study suggests that two sets of co-ordinately regulated but oppositely expressed genes, Igf2-H19 and Dlk1-Gtl2, act as a critical barrier to parthenogenetic development in order to render a paternal contribution obligatory for descendants in mammals.

Trophinin is expressed in the porcine endometrium during the estrous cycle.

We investigated endometrial expression of trophinin mRNA and protein, homophilic cell adhesion molecules, during the estrous cycle of gilts. An immunopositive reaction for trophinin was observed in the luminal and glandular epithelia of the endometrium at all stages of the estrous cycle, but not in endometrial stromal cells or the myometrium. A partial coding sequence of porcine trophinin was similar to sequences in humans and mice, with homologies of 75% and 70%, respectively. As in humans and mice, the trophinin gene is expressed in the endometrium. Trophinin, however, is expressed in the endometrium of the pig throughout the estrous cycle, higher expression levels were observed at some points of the luteal phase, as in humans. These findings suggest that regulation of trophinin gene expression in the pig is different from that in mice, but similar to that in humans. Furthermore, the present results suggest that the pig might be a suitable model for studying the physiological importance of trophinin in early pregnancy in humans.