During early porcine development a rapid morphological transformation of the conceptus from a spherical (9–10 mm) to filamentous (> 150 mm) morphology is required to establish adequate placenta to uterine contact necessary for survival . Characterization of the specific subset of genes regulating peri-implantation conceptus development and trophoblastic elongation in the pig provides valuable information concerning key developmental events essential to embryonic survival after trophoblastic elongation .
Previous information regarding genes critical to development in early pig pregnancy is limited. Using semi-quantitative RT-PCR, Yelich et al.  evaluated peri-implantation gene expression profiles for 17α-hydroxylase, aromatase, brachyury and leukemia inhibitory factor receptor, all of which are more greatly expressed in filamentous conceptuses. Messenger RNA for retinoic acid receptor α, retinal binding protein and transforming growth factor β-3 is also increased in filamentous conceptuses . Using a ribonuclease protection assay, Wilson et al.  indicated enhanced cyclooxygenase-2 expression was specific to filamentous conceptuses. An autocrine effect of conceptus estrogen synthesis has been suggested as filamentous conceptuses have greater gene expression for estrogen receptor β compared to its spherical and tubular counterparts .
Suppression subtractive hybridization allowed isolation of candidate genes expected to be differentially expressed during rapid trophoblastic elongation. Differentially expressed genes identified using SSH striking particular interest were interleukin-1β (IL-1β) and metallopanstimulin-1 (MPS-1), both of which are suspected to be up-regulated in the elongated conceptus. MPS-1 expression is notable in proliferating cells and has been identified as a nuclear protein that binds to DNA . Further analysis indicates cells stimulated with transforming growth factor β1 (TGF-β1) exhibit a 8-fold increase in MPS-1 gene expression . TGF-β1 and its receptors are localized in the trophectoderm of porcine conceptuses between days 10–14 of gestation . MPS-1 may have roles in transcriptional-mediation of the embryonic response to TGF-β1 having effects on continued intra-uterine elongation. However, MPS-1 gene expression needs to be quantitatively analyzed across the stages of conceptus development. IL-1β was the most predominant clone sequenced during SSH and was also the most abundant clone represented when mapping expressed sequence tags derived from a porcine early embryonic cDNA library in a study by Smith et al. . We have more thoroughly examined and confirmed IL-1β gene and ligand expression in developing conceptuses as well as the endometrium. Changes in conceptus IL-1β gene expression and ligand release in the uterine horn dramatically increase during the process of elongation (Ross and Geisert, unpublished results). Increase in IL-1β gene expression was specific to the conceptus as endometrial expression was unchanged between cyclic and pregnant gilts. Following completion of conceptus elongation, IL-1β gene expression and secretion rapidly decline and expression levels are more than 2000-fold lower in day 15 compared to day 12 filamentous conceptuses. It is possible IL-1β plays an important role in triggering conceptus elongation and initiating uterine-conceptus "cross-talk" in the pig.
In the present study, three transcripts; OSU-T1-50, SAHH and HSC70 were confirmed to be differentially expressed analogous to the pattern predicted using SSH. Although no confirmed identity was found in GenBank, the novel gene, OSU-T1-50 displayed a very dynamic enhancement of gene expression during trophoblastic elongation. When compared to spherical conceptuses there was almost a 12-fold increase in mRNA expression for OSU-T1-50 in tubular conceptuses while expression in filamentous conceptuses increased over 100-fold. Given the substantial increase in gene expression and temporal relationship to conceptus development, OSU-T1-50 may play an important role in trophoblastic elongation. Future studies will attempt to identify the gene and its translated protein to determine the biological function during conceptus development.
Gene expression of SAHH was similar between spherical, ovoid and tubular conceptuses, but there was nearly a 7-fold increase in gene expression in filamentous conceptuses. SAHH may have significant impact on the conceptuses ability to use folates during this transitional stage of development. Folates have long been known to be an essential requirement for developing embryos, predominately during neurulation . Vallet and coworkers  have previously shown the increase in maternal folate binding protein activity occurs in the uterine lumen of cyclic and pregnant gilts between days 10–12 post-estrus. The increase of folate in the uterine lumen is temporally associated with the increase in conceptus SAHH gene expression.
S-adenosylhomocysteine (SAH) is the resultant product following the release of a methyl group from s-adenosylmethionine (SAM), a universal methyl donor . Methyl donation from SAM has crucial developmental impacts governing DNA methylation  as well as methylation of amino acids, proteins, carbohydrates and polysaccharides . S-adenosylhomocysteine hydrolase is the only known enzyme capable of SAH hydrolysis. The breakdown of SAH, which is reversible, results in the release of free homocysteine which is converted to methionine while being used as a substrate for the reduction of 5-methylenetetrahydrofolate (5-MTHF) to tetrahydrofolate (THF). Elevated levels of methionine can then be used in the synthesis of SAM while THF has downstream effects on uracil to thymidine conversions involved with DNA repair and synthesis .
SAHH functions as a protective enzyme for adenosine toxicity by preventing nuclear accumulation of SAH  and may explain the nuclear localization of SAHH in cells that are transcriptionally active in Xenopus embryos . Vanaerts et al.  demonstrated that high concentrations of homocysteine are associated with embryotoxicity during early gestation in rats. These authors suggest the toxicity may be associated with the reverse hydrolysis of homocysteine to SAH resulting in the dramatic reduction of the SAM/SAH ratio to a point where methylation reactions are inhibited. Miller and co-workers  have revealed that the lethal nonagouti (ax) mutation in mice is characterized by deletion of the SAHH gene resulting in embryonic death prior to implantation. Addition of an inhibitor to SAHH, 3-deazaaristeromycin, inhibits inner cell mass proliferation and differentiation during in vitro development of non-mutant embryos .
As in other species, SAHH is likely a biological regulator of the SAM/SAH ratio controlling the occurrence of transmethylation reactions to the degree of which they are necessary for successful porcine conceptus development. Establishment of the maternal-fetal interface is extremely competitive among littermates in early swine gestation. Advanced conceptuses, those that elongate first, have a much greater advantage with regards to acquiring ample placental:uterine contact and also limit the available uterine capacity for those conceptuses lagging in development . While gene expression for SAHH was present at all stages evaluated, an approximate 7-fold increase in relative expression for this transcript over a 2–3 hour time period is noteworthy. It is possible that increased expression of SAHH by filamentous conceptuses serves as a protective function at the level of the nucleus by reducing SAH to homocysteine thereby maintaining the SAM/SAH ratio at appropriate levels for SAM-mediated transmethylation reactions to occur. Increased SAHH expression by advanced conceptuses suggests there would also be increased homocysteine released into the uterine lumen that may have an embryo-toxic effect on neighboring conceptuses lagging in development.
Gene expression for HSC70 was similar in spherical, ovoid and tubular conceptuses followed by a 10-fold increase in filamentous conceptuses. Gene expression changes for HSC70 during early development have previously been associated with neurulation in Xenopus  and chick  embryos. Negative mutations of HSC70 in the nervous system of Drosophila larvae resulted in both developmental defects and lethality . The increased HSC70 gene expression during trophoblastic elongation is temporally associated with neural tube development in pig conceptuses.
Traditionally, heat shock proteins are known for their function during cellular stress as molecular chaperones responsible for the folding, re-folding and transport of newly synthesized proteins. HSC70 is a constituitively expressed member of the 70 kDa heat-shock protein (HSP70) family. Members of the HSP70 family have also been proposed to be involved with HSP90 chaperones regulating signal transduction pathways . Unlike HSP70, HSC70 does not exhibit increased gene expression when exposed to heat stress or other agents such as sodium arsenate in the gastrula and neurula Xenopus embryo . Tsang  suggested because of their ability to bind and transport folded and un-folded cellular proteins, HSP70 family members may function as a cross-linker to couple cellular proteins to the cytoskeletal matrix. Association of HSP70 with the cytoskeleton suggests that conceptus produced HSC70 may be directly involved with the complex process of conceptus remodeling during trophoblastic elongation.
Through the method of SSH, we have detected several genes that may serve a vital role in differentiation, neurulation, as well as attachment and maintainence of pregnancy in the pig. Detection and confirmation of IL-1β, SAHH, HSC70 and OSU-T1-50 as being differentially expressed during the period of rapid trophoblastic elongation contributes important information towards understanding the mechanisms involved with this essential biological event in the pig.