Global analysis of differential gene expression within the porcine conceptus transcriptome as it transitions through spherical, ovoid, and tubular morphologies during the initiation of elongation

Abstract This study aimed to identify transcriptome differences between distinct or transitional stage spherical, ovoid, and tubular porcine blastocysts throughout the initiation of elongation. We performed a global transcriptome analysis of differential gene expression using RNA‐Seq with high temporal resolution between spherical, ovoid, and tubular stage blastocysts at specific sequential stages of development from litters containing conceptus populations of distinct or transitional blastocysts. After RNA‐Seq analysis, significant differentially expressed genes (DEGs) and pathways were identified between distinct morphologies or sequential development stages. Overall, 1898 significant DEGs were identified between distinct spherical and ovoid morphologies, with 311 total DEGs between developmental stages throughout this first morphological transition, while 15 were identified between distinct ovoid and tubular, with eight total throughout these second morphological transition developmental stages. The high quantity of DEGs and pathways between conceptus stages throughout the spherical to ovoid transition suggests the importance of gene regulation during this first morphological transition for initiating elongation. Further, extensive DEG coverage of known elongation signaling pathways was illustrated from spherical to ovoid, and regulation of lipid signaling and membrane/ECM remodeling across these early conceptus stages were implicated as essential to this process, providing novel insights into potential mechanisms governing this rapid morphological change.


| INTRODUCTION
During the preimplantation period of porcine conceptus development, the conceptus undergoes a dramatic transformation from a spherical morphology (∼1-2 mm), through ovoid (∼3-9 mm), and tubular (>10 mm) morphologies, and finally to a long, thin filament (>100 mm) between Days 9 and 12 of gestation Geisert et al., 1982;Miles et al., 2008;Pope & First, 1985). Once initiated, this process of conceptus elongation is very rapid, with the remodeling of the trophoblast and changes in conceptus length occurring at a rate of 35-40 mm per hour on Days 11-12 (Bazer et al., 1988). During elongation, both the conceptus and maternal uterine endometrium undergo extensive changes in gene expression that are essential for the establishment of pregnancy, and for providing a favorable uterine environment for conceptus growth and development (Waclawik et al., 2017). These gene expression changes involve crosstalk between the maternal endometrium and the conceptus, as molecular factors produced by each influence gene expression and morphological changes of the conceptus via the stimulation of signaling cascades (Geisert et al., 2014). Successful conceptus elongation is critical for subsequent embryonic development and survival, as deficiencies in elongation contribute to an estimated 20% of embryonic loss (Pope, 1994) and directly influence within-litter birth weight variability and postnatal piglet survival (Vallet et al., 2009). However, there is currently a lack of understanding of the factors and mechanisms critical to successful elongation in the pig, particularly during the initiation of elongation. Therefore, a more complete understanding of the changes in porcine conceptus gene expression contributing to successful elongation is essential for increasing the overall efficiency of porcine reproduction.
However, to the best of our knowledge, no studies have been performed to characterize changes in global gene expression between the initial spherical, ovoid, and tubular porcine conceptus morphologies of elongation utilizing RNA sequencing (RNA-Seq) technology, which allows for the nontargeted identification of specific genes differentially regulated between early conceptus stages that potentially drive the drastic morphological changes occurring throughout later elongation stages. As such, the specific gene regulation and global expression patterns contributing to the critical period of rapid initiation of porcine conceptus elongation, specifically throughout the initial spherical, ovoid, and tubular morphological stages, or the mechanisms and pathways by which these genes operate, have not yet been fully elucidated.
Therefore, the current study aimed to further elucidate mechanisms of porcine conceptus elongation by examining differences in global gene expression between distinct spherical, ovoid, and tubular porcine conceptus morphologies as the conceptus progress through the initiation of elongation in vivo, as well as by utilizing heterogeneous conceptus morphological stage pregnancies (i.e., litters containing spherical/ovoid or ovoid/tubular blastocysts) to evaluate changes between transitional development stages at a higher temporal resolution amidst these distinct morphologies. Thus, the overall objectives of this study were to characterize porcine conceptus gene expression changes that occur throughout the initiation of elongation between distinct conceptus morphologies, derived from homogeneous morphological stage pregnancies, and between blastocysts at specific sequential stages of development, derived from homogeneous and heterogeneous morphological stage pregnancies. Specifically, we performed a global transcriptome analysis of differential gene expression between distinct spherical (S), ovoid (O), and tubular (T) conceptus morphologies, derived from litters containing homogeneous morphological stage blastocysts, using RNA-Seq to better understand the mechanisms governing the rapid morphological changes that occur during the initiation of porcine conceptus elongation (Figure 1). Additionally, we performed a global transcriptome analysis of differential gene expression with higher temporal resolution between blastocysts at specific sequential stages of development from litters containing homogeneous spherical (S), heterogeneous spherical (S T1 ) and ovoid (O T1 ), homogeneous ovoid (O), heterogeneous ovoid (O T2 ) and tubular (T T2 ), or homogeneous tubular (T) stage blastocysts using RNA-Seq to establish profiles of select significantly differentially expressed genes (DEGs) identified between distinct porcine conceptus morphologies with increased temporal precision, uncovering potential conceptus mechanisms of gene regulation driving the initiation of elongation (Figure 1).

| RESULTS
Blastocysts from homogeneous and heterogeneous pregnancies were sorted into seven conceptus morphological stage treatment groups based on conceptus morphology and length, as shown in Figure 1.
Global gene expression of individual blastocysts within each distinct and transitional conceptus treatment group was analyzed using RNA-Seq, and significant DEGs, pathways, biological processes (BPs), molecular functions (MFs), and cellular components (CCs) were determined between conceptus stages throughout the initiation of elongation ( Figure 1). Specifically, gene expression was compared between distinct conceptus morphologies: (1) at the first morpholo- across the overall morphological transition (OMT) from distinct S to distinct T blastocysts (T vs. S) (Figure 1c), to identify differences in gene expression between distinct conceptus morphologies throughout the initiation of elongation. Additionally, to characterize changes in gene expression with fine temporal resolution as the conceptus progresses through specific developmental stages during the initiation of elongation, gene expression was compared between F I G U R E 1 Comparisons among distinct and transitional conceptus morphological stages analyzed with RNA-Seq to examine porcine conceptus transcriptome differences throughout the initiation of elongation. Blastocysts were collected from pregnant gilts harvested on Days 9-11 of gestation and classified into spherical, ovoid, and tubular morphological stages according to conceptus morphology and length. Conceptus populations of individual litters were classified according to the uniformity of the most advanced conceptus morphological stage present within the respective litter. Individual blastocysts from these homogeneous and heterogeneous pregnancies were further selected and sorted into conceptus treatment groups based on conceptus morphological stage and conceptus morphological stage uniformities of corresponding litters: distinct spherical blastocysts (S, n = 8 blastocysts), from homogeneous spherical stage pregnancies (n = 8 gilts); transitional spherical blastocysts (S T1 , n = 8 blastocysts) or transitional ovoid blastocysts (O T1 , n = 8 blastocysts) during the first morphological transition, from the same heterogeneous spherical/ovoid stage pregnancies (n = 8 gilts); distinct ovoid blastocysts (O, n = 8 blastocysts), from homogeneous ovoid stage pregnancies (n = 8 gilts); transitional ovoid blastocysts (O T2 , n = 8 blastocysts) or transitional tubular blastocysts (T T2 , n = 8 blastocysts) during the second morphological transition, from the same heterogeneous ovoid/tubular stage pregnancies (n = 8 gilts); or distinct tubular blastocysts (T, n = 6 blastocysts), from homogeneous tubular stage pregnancies (n = 6 gilts). The average conceptus morphological stage uniformities for each conceptus treatment group were: S = 100%; S T1 = 34%; O T1 = 66%; O = 100%; O T2 = 43%; T T2 = 55%; and T = 90%. To identify differences in gene expression between distinct porcine conceptus morphologies throughout the initiation of elongation, global gene expression of individual blastocysts within each distinct conceptus morphological stage treatment group was analyzed using RNA-Seq, and significant DEGs, pathways, BPs, MFs, and CCs were determined between distinct conceptus morphologies (a) at the first morphological transition (1st MT) from S to O (1: O vs. S), (b) at the second morphological transition (2nd MT) from O to T (2: T vs. O), and (c) across the overall morphological transition (OMT) from S to T (3: T vs. S). To characterize changes in gene expression with fine temporal resolution as the porcine conceptus progresses through specific developmental stages during the initiation of elongation, global gene expression of individual blastocysts within each distinct and transitional conceptus morphological stage treatment group was analyzed using RNA-Seq, and significant DEGs, pathways, BPs, MFs, and CCs were determined between blastocysts at sequential stages of development throughout ( 2.2 | Abundant and similar changes in gene expression occur between distinct O versus S morphologies at the 1st MT and distinct T versus S morphologies across the OMT during the initiation of elongation Differential gene expression, pathway, and gene ontology (GO) analysis were performed between S and O blastocysts ( Figure 1a) and between S and T blastocysts ( Figure 1c) to determine significant DEGs, pathways, BPs, MFs, and CCs between distinct conceptus morphologies at the 1st MT and across the OMT during the initiation of elongation. A total of 1898 genes were significantly differentially expressed between O versus S after false discovery rate (FDR) adjustment (p ≤ 0.05) and fold change (FC) filtering (|log 2 FC| ≥ 1; i.e., FC ≥ 2) (Tables 1 and S2). Of these significant DEGs, 1384 genes were upregulated and 514 were downregulated (Tables 1 and S2) from S to O conceptus morphology ( Figure 2a). Between T versus S, 2291 total genes were significantly differentially expressed (Tables 1 and S2), with 1608 DEGs upregulated and 683 downregulated (Tables 1 and S2) from S to T conceptus morphology ( Figure 2b) (Tables 1 and   Table S3). The most significantly enriched BPs between O versus S and T versus S were mainly terms related to signaling, adhesion, and development (Tables 6 and Table S3), while many of the most significant MFs were terms involving signaling and transport (Tables 7   and S3).

| Limited changes in gene expression occur between distinct T versus O morphologies at the 2nd MT
Differential gene expression, pathway, and GO analysis were performed between O and T blastocysts ( Figure 1b)  which are related to cytokine and growth factor signaling and steroidogenesis (Table 13) (Figure 3d). Between T T2 versus O T2 blastocysts, only four genes were significantly differentially expressed (Tables 10 and 14), two of which were upregulated and two downregulated (Tables 10 and 14) from O T2 to T T2 blastocysts ( Figure 3e). Lastly, only two genes were significantly differentially expressed between T versus T T2 blastocysts (Tables 10   and 14), both of which were upregulated (Tables 10 and 14) from T T2 to T (Figure 3f).   (Table 10).

| Phospholipid membrane remodeling and lipid signaling
In the current study, numerous DEGs within multiple enriched pathways related to phospholipid metabolism indicate potential remodeling of the trophectoderm as the conceptus transitions from S T A B L E 6 (Continued)   to O morphology and S to T morphology ( Figure 5; Table S4).

Contrast
Phospholipase A2 (PLA2) group XIIB (PLA2G12B), PLA2 group VII (PLA2G7), and PLA2 group XVI (PLA2G16 or PLAAT3) were significantly upregulated from S to O and/or S to T conceptus morphologies, similar to a previously reported increase in porcine conceptus PLA2 expression and activity during elongation (Davis et al., 1983). During preimplantation development, the porcine conceptus undergoes remodeling of the lipid composition within the trophoblast phospholipid membrane, including PLA2-stimulated release of arachidonic acid (AA), to allow for trophectoderm cell motility that is essential to rapid conceptus elongation (Geisert et al., 2017). In addition to increasing fluidity of the trophoblast membrane, free AA can serve as precursors for bioactive signaling lipids and phospholipids during membrane remodeling (Geisert et al., 2014;Hanna & Hafez, 2018). In the current study, several genes involved in phospholipid and glycerophospholipid metabolism  (Cao et al., 2008;Shulga et al., 2011;Tafesse et al., 2007;Takahashi & Suzuki, 2012). Taken together, the results highlighted above identify specific PLA2 genes that potentially act as stimulators of the porcine conceptus trophectoderm membrane remodeling that is fundamental to the initiation of elongation, which may also involve changes in glycerophospholipid and sphingolipid composition.
The preimplantation period of porcine conceptus development is also associated with significant changes in various aspects of lipid metabolism that are involved in lipid molecular signaling within the uterine environment, including altered lipid uptake, activation, and modification, and biosynthesis of lipid signaling molecules (Blitek & Szymanska, 2017). In the current study, many of the significant DEGs and pathways between conceptus stages within the 1st MT and OMT reflect lipid metabolic changes involved in lipid signaling during the initiation of porcine conceptus elongation ( Figure 5;  Rokach, 2015), all of which play essential roles in elongation of the porcine conceptus. Another mechanism of lipid signaling within the uterine environment during porcine pregnancy is through glucocorticoids, including cortisol. In the current study, hydroxysteroid 11-beta dehydrogenase 2 (HSD11B2), encoding an enzyme that generates inactive cortisone from active cortisol (Simmons et al., 2010), was significantly upregulated from S to O conceptus morphologies, specifically from S to S T1 , and from S to T. Cortisol has been implicated as an important regulator of conceptus elongation in sheep and cattle via binding and activation of the glucocorticoid receptor (GR) in the maternal endometrium (Dorniak et al., 2013). GR (or NR3C1) is also expressed in the conceptus, as glucocorticoids play a role in a number of important processes during development (Brooks et al., 2015). It has been hypothesized that conceptus expression of HSD11B2 functions to protect the conceptus from the abundance of uterine luminal cortisol that regulates endometrial expression of genes essential to elongation (Brooks et al., 2015;   (Ribeiro et al., 2016). A major mechanism of lipid signaling is through activation of peroxisome proliferatoractivated receptors (PPARs) that have been implicated as essential regulators of conceptus elongation in pigs and ruminants (Blitek & Szymanska, 2017). The FATP gene SLC27A6 has been shown to increase during bovine conceptus elongation in conjunction with PPAR expression (Ribeiro et al., 2016), similar to the increased PPAR expression occurring in the elongating ovine and porcine conceptus (Blitek & Szymanska, 2017

| ECM remodeling and cellular adhesion
In the current study, multiple significant DEGs and pathways between conceptus stages throughout the 1st MT and across the OMT are related to ECM remodeling and cellular adhesion ( Figure 6; Table S4). During the peri-implantation period, the porcine conceptus trophectoderm undergoes reorganization of the ECM to facilitate adhesion between the conceptus and uterine epithelium, which is essential for the trophectoderm cell migration that occurs during rapid elongation (Erikson et al., 2009). It has been proposed that initial attachment of the conceptus to the luminal epithelium involves low-affinity contacts, such as carbohydrate ligand-lectin receptor binding, which are then replaced by more stable adhesions involving ECM molecules and integrins to promote conceptus elongation and implantation Erikson et al., 2009;Geisert et al., 2015). Within the current study, galactoside alpha-(1,2)fucosyltransferase 1 (FUT1), encoding an enzyme that catalyzes formation of the H type 1 antigen (J. K. Powell et al., 2000), was significantly downregulated from S to O, specifically from S to S T1 and O T1 to O, and from S to T. It has been suggested that the H type 1 antigen may function as a carbohydrate ligand in initial low-affinity contacts established between trophectoderm and endometrium at the initiation of the adhesion cascade (Bowen & Burghardt, 2000;Geisert et al., 2015). Therefore, downregulation of FUT1 throughout the initial stages of elongation may be due to the decreasing utilization of this low-affinity adhesion mechanism as the conceptus begins to more firmly attach to the endometrium through the actions of integrins and ECM molecules, such as fibronectin, laminin, secreted phosphoprotein 1 (SPP1), and hyaluronan (HA) (Ashworth et al., 2010;Ziecik et al., 2011), to promote morphological change.
Within the current study, HA synthase 2 (SHAS2) and HAS1, encoding trans-membrane enzymes that synthesize HA (Raheem, 2018), and cluster domain 44 (CD44), encoding the principal cell surface receptor for HA (Stojkovic et al., 2003), were significantly upregulated from S to O, with SHAS2 and CD44 specifically upregulated from O T1 to O and from S to T blastocysts. HA-CD44 binding has been hypothesized to act as a bridge between the conceptus and uterine epithelium (Berneau et al., 2019), and additional molecules present at the maternal-conceptus interface at the time of conceptus attachment may associate with this CD44-anchored HA (Ashworth et al., 2010)  migration-inducing hyaluronidase 1 (CEMIP), encoding an HA-binding protein with HA-degradation capability (Nagaoka et al., 2015), was significantly downregulated from S to O, specifically from O T1 to O, and from S to T in the current study, potentially indicating reduced degradation of HA and maintenance of ECM stability as the adhesion cascade proceeds and the abundance of low-affinity contacts continues to decrease throughout the initiation of porcine conceptus elongation.
Further indication of increasing stability of trophectodermendometrial attachments throughout the initiation of porcine conceptus elongation was the significant upregulation of genes forming fibronectin and laminin ECM proteins, as well as several integrin subunits, from S to O and S to T conceptus morphologies within the current study. This increased expression of integrin subunits is consistent with a recently reported upregulation of several of these integrin subunits by the porcine conceptus over the elongation period (Zeng et al., 2019). During porcine conceptus elongation, both the soluble ECM adhesion protein SPP1 and expression of integrin subunits for the SPP1 receptor are present at the maternal-conceptus interface (Erikson et al., 2009;Garlow et al., 2002). It is hypothesized that binding of the Arg-Gly-Asp (RGD) sequence of porcine SPP1 to integrins on both the uterine epithelium and conceptus trophectoderm facilitates trophectoderm attachment, spreading, and migration during conceptus elongation via adhesion at the maternalconceptus interface (Erikson et al., 2009;Garlow et al., 2002). Furthermore, covalent conjugation of the RGD peptide to an alginate matrix T A B L E 11 Ten most highly upregulated significant DEGs (p ≤ 0.05 and |log 2 FC| ≥ 1) between blastocysts at sequential stages of development (S T1 vs. S, O T1 vs. S T1 , and O vs. O T1 ) throughout the first morphological transition during the initiation of elongation  81.3% pregnancy rate), all blastocysts were recovered (n = 1267) and T A B L E 12 Ten most highly downregulated significant DEGs (p ≤ 0.05 and |log 2 FC| ≥ 1) between blastocysts at sequential stages of development  S) and across the OMT from S to T blastocysts (T vs. S) that indicate differential conceptus regulation of lipid metabolism throughout the initiation of elongation. Differential expression of (a) steroidogenesis-related genes within the conceptus may lead to the increase in synthesis and secretion of E2. PLA2s within the conceptus release AA from phospholipids composing the trophectoderm membrane, which can be utilized for PG synthesis by PTGS2 within the endometrium or for generating 12S-HETE by ALOX12 within the conceptus. Additionally, differential expression of genes involved in (b) phospholipid metabolism, (c) PC/PE metabolism, and (d) sphingolipid metabolism may indicate changes in conceptus phospholipid membrane composition. SLC27A6 facilitates uptake of FAs, including AA, by the conceptus, and PGs, 12S-HETE, AA, and FAs, as well as their derivatives, can bind and activate conceptus PPAR-γ. HSD11B1 generates cortisol in the endometrium, which can bind and activate GR, inducing PTGS2 expression and upregulation of PG synthesis. Both PGs and activated GR can also induce endometrial HSD11B1 expression. Endometrial cortisol produced by HSD11B1 as well as GCs can activate conceptus GR; however, conceptus HSD11B2 can covert active cortisol into inactive cortisone, which is unable to bind GR. All highlighted conceptus DEGs were significant between both O versus S and T versus S distinct morphologies unless specifically indicated by an asterisk: orange asterisk = significant between O versus S, while not significant between T versus S; and purple asterisk = significant between T versus S, while not significant between O versus S. Significant DEGs between blastocysts at sequential stages of development throughout the 1st MT are indicated by bubbles corresponding to specific developmental stage comparisons: 1a = S T1 versus S; 1b = O T1 versus S T1 ; and 1c = O versus O T1 . Green DEGs = upregulated; red DEGs = downregulated; 12S-HETE = 12S-hydroxyeicosatetraenoic acid; 17OH-preg = 17-hydroxy-pregnenolone; 17OH-prog = 17-hydroxyprogesterone; A4 = androstenedione; A5 = androstenediol; AA = arachidonic acid; C1P = ceramidE−1-phosphate; CE = cholesteryl ester; Cer = ceramide; ch = choline; CL = cardiolipin; CM = chylomicron; DAG = diacylglycerol; DHCer = dihydroceramide; DHEA = dehydroepiandrosterone; dLDLR = degraded LDLR; E1 = estrone; E2 = estradiol-17β; eth = ethanolamine; FA = fatty acid; FC = free cholesterol; Gal2Cer = galabiosylceramide; GalCer = galactosylceramide; GCs = glucocorticoids; Glbs = globo-series glycosphingolipids; GlcCer = glucosylceramide; GM4 = sialylgalactosylceramide; Gngs = ganglio-series glycosphingolipids; GR = glucocorticoid receptor; HDL = high-density lipoprotein; IDL = intermediatE−density lipoprotein; LacCer = lactosylceramide; Lcts = lacto-series glycosphingolipids; LDL = low-density lipoprotein; LPA = lysophosphatidic acid; MAG = monoacylglycerol; PAF = platelet-activating factor; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PG = prostaglandin; PGL = phosphatidylglycerol; PhA = phosphatidic acid; PI = phosphatidylinositol; PIPs = phosphoinositides; preg = pregnenolone; prog = progesterone; PS = phosphatidylserine; rCM = chylomicron remnant; S1P = sphingosinE−1phosphate; Sa = sphinganine; So = sphingosine; SPH = sphingomyelin; T = testosterone; VLDL = very low-density lipoprotein System (Illumina, Inc.). Corresponding sequence data are available in NCBI Sequence Read Archive under the accession number PRJNA723617.

| Processing and statistical analysis of RNA-Seq data
The quality of the raw paired-end sequence reads in individual fastq files was assessed using FastQC (Version 0.11.5; www. bioinformatics.babraham.ac.uk/projects/fastqc). Reads were trimmed to remove adapter sequences and low-quality bases (average base quality < 15 in a 4-bp window) using the Trimmomatic software (Version 0.35) (Bolger et al., 2014). The remaining reads were mapped to the Sscrofa 11.1 genome assembly (Genbank Accession GCA_000003025.6) using Hisat2 (Version 2.1.0) (D. Kim et al., 2015), and the NCBI annotation for Sscrofa 11.1 (Release 106) was used to guide the alignment. Stringtie (Pertea et al., 2015) was used to determine read counts for each of the 29,847 genes in the Sscrofa 11.1 reference annotation (Release 106). For each comparison, genes with low read counts, <15 reads in at least eight samples, were filtered out. DEGs were identified using DESeq. 2 (Love et al., 2014). An FDRcorrected p ≤ 0.05 and |log 2 fold change| ≥ 1 (i.e., fold change ≥ 2) between treatments was considered statistically significant. O T2 , and T vs. T T2 ) comparisons, and relevance of function in terms of plausible potential mechanisms contributing to elongation during this period of development.

| GO enrichment and pathway analysis
F I G U R E 6 Proposed mechanisms of interactions among highlighted significant DEGs throughout the 1st MT from S to O blastocysts (O vs. S) and across the OMT from S to T blastocysts (T vs. S) that indicate differential conceptus regulation of ECM remodeling throughout the initiation of elongation. Conceptus FUT1 may synthesize H-1 to serve as a carbohydrate ligand that binds to LCTNs on the endometrium; these low-affinity contacts may facilitate initial attachment of the conceptus to the uterus. Additionally, interactions between HA and CD44 may act as an initial bridge between conceptus and endometrium. As elongation initiates, conceptus CD44-anchored HA may interact with TSG-6 and IαIHs at the maternal-conceptus interface to stabilize the ECM and allow for more firm attachments between the conceptus and endometrium. TGF-β1 may contribute to this mechanism by inducing conceptus HAS1 and SHAS2 expression to up-regulate HA synthesis, as well as by inhibiting conceptus expression of CEMIP to reduce HA degradation. Lastly, attachment of SPP1, LN, and FN to ITGs on both the conceptus and endometrium may be essential mechanisms for forming stable adhesions to drive elongation. All highlighted conceptus DEGs were significant between both O versus S and T versus S distinct morphologies unless specifically indicated by an asterisk: orange asterisk = significant between O versus S, while not significant between T versus S; and purple asterisk = significant between T versus S, while not significant between O versus S. Significant DEGs between blastocysts at sequential stages of development throughout the 1st MT are indicated by bubbles corresponding to specific developmental stage comparisons: 1a = S T1 versus S; and 1c = O versus O T1 . Green DEGs = upregulated; red DEGs = downregulated; FN = fibronectin; H-1 = H type 1 antigen; HA = hyaluronan; ITGs = integrins; IαIHs = inter-alpha-trypsin inhibitor heavy chains; LCTNs = lectin receptors; LN = laminin; SPP1 = secreted phosphoprotein 1; TSG-6 = tumor necrosis factor-alpha-induced protein 6

| CONCLUSION
The results of this study highlight extensive differences in gene expression between distinct S, O, and T porcine conceptus morphologies, as well as changes in gene expression as the porcine conceptus transitions through specific developmental stages amidst these distinct morphologies, during the initiation of elongation. In particular, the high quantity and fold change of significant DEGs and pathways throughout the spherical to ovoid transition observed in this study, as opposed to minimal changes from ovoid to tubular, suggest that this 1st MT may be most critical to the initiation of elongation. Further, identification of significant DEGs between specific sequential stages of development throughout the 1st MT provided select gene expression profiles with higher temporal resolution, identifying specific DEGs with substantial and rapid changes in expression that may be crucial to the initiation of porcine conceptus elongation. Altogether, these results illustrate extensive DEG coverage of known elongation signaling pathways, implicate conceptus regulation of phospholipid membrane remodeling and lipid molecular signaling as potential key elongation mechanisms, and suggest that the adhesion cascade involving ECM remodeling that occurs throughout the periimplantation period may begin during the initiation of elongation.
Overall, the information gained from this study can be used to further elucidate mechanisms essential to the successful initiation of elongation as the porcine conceptus transitions between specific developmental stages and distinct morphologies, advancing knowledge of porcine conceptus elongation and development to improve swine reproductive outcomes.