Differential expression of key subunits of SWI/SNF chromatin remodeling complexes in porcine embryos derived in vitro or in vivo

In vitro embryo production is an established method for both humans and animals, but is fraught with inferior development and health issues in offspring born after in vitro fertilization procedures. Analysis of epigenetic changes caused by exposure to in vitro conditions should shed light on potential sources of these phenotypes. Using immunocytochemistry, we investigated the localization and relative abundance of components associated with the SWI/SNF (Switch/Sucrose non‐fermentable) chromatin‐remodeling complex—including BAF155, BAF170, BAF180, BAF53A, BAF57, BAF60A, BAF45D, ARID1A, ARID1B, ARID2, SNF5, and BRD7—in oocytes and in in vitro‐produced and in vivo‐derived porcine embryos. Differences in the localization of BAF155, BAF170, BAF60A, and ARID1B among these sources indicate that improper timing of chromatin remodeling and cellular differentiation might occur in early preimplantation embryos produced and cultured in vitro.

nucleosomes and alter the accessibility of transcription factors to chromatin. Common to all mammalian SWI/SNF complexes is a catalytic subunit that functions as an ATPase (either Brahma or BRG1). In addition, SWI/SNF complexes contain a core group of modulating subunits (BAF150/BAF170 and SNF5) and a multitude of accessory subunits (Euskirchen, Auerbach, & Snyder, 2012;Ryme, Asp, Böhm, Cavellán, & Farrants, 2009); these subunits are encoded by at least 29 genes belonging to 15 gene families (Kadoch & Crabtree, 2015).
Previous studies characterized multiple SWI/SNF complexes in various tissues and cell types (Kadoch et al., 2013). Some of these complexes were found to be involved in tumor suppression, whereby mutations and/or the loss of function of different SWI/SNF subunits are linked to a number of different cancers (Kadoch & Crabtree, 2015; Marquez-Vilendrer, Rai, Gramling, Lu, & Reisman, 2016;Reisman, Glaros, & Thompson, 2009). BRG1 is expressed in preimplantation embryos, while BRM appears to be expressed later during differentiation (Ryme et al., 2009). Knockout studies in mice identified requirements for these two SWI/SNF subunits that are consistent with their expression during embryo development: Brg1-null mice do not survive beyond early embryonic stages whereas Brm-null mice survive to adulthood and display only a slight overgrowth phenotype. Knockdown of BRG1 resulted in aberrant Pou5f1 (also known as Oct4) and Nanog expression in blastocyst-stage mouse embryos (Kidder, Plamer, & Knott, 2009). This phenotype is likely due to the BRG1 occupancy of the promoters of Pou5f1, Sox2, Nanog, and other significant pluripotency related genes, which supports a key role of this factor in the regulation of pluripotency and self-renewal in embryonic stem cells.
Additional genetic evidence revealed roles of other SWI/SNF factors during development. Baf155 +/− mice develop to term and are fertile, but brain defects (exencephaly) in adults were observed. In contrast, Baf155 −/− mice develop to the blastocyst stage, but the inner cell mass subsequently degenerates and fail to develop egg cylinders (Kim et al., 2001). Consistent with these null-phenotypes, BAF155 knockdown in mice results in aberrant expression of pluripotency genes while overexpression of BAF155 arrested development at the blastocyst stage (Panamarova et al., 2016). Furthermore, gene inactivation of Snf5 or Baf155 causes peri-implantation lethality; Baf180-null mice are lethal at embryonic Days 12.2-15.5, and show cardiac and placenta abnormalities; and murine embryos lacking ARID1A (AT-rich interaction domain protein 1A) arrest their development around embryonic Day 6.5, with failed development of a mesodermal layer (reviewed by de la Serna, Ohkawa, & Imbalzano, 2006;Gao et al., 2008;Xu, Flowers, & Moran, 2012).
Data regarding SWI/SNF complexes are limited for preimplantation embryos. Results obtained in cell culture and embryonic stem cells, however, provide a baseline understanding of the distinct SWI/SNF complexes present during critical stages of development and differentiation. We hypothesized that SWI/SNF-complexes in the early embryo should function similarly to SWI/SNF complexes found in pluripotent embryonic stem cells, which are derived from the inner cell mass in mice. We therefore analyzed the localization and relative abundance of a multitude of SWI/SNF subunits during early porcine embryo development, and compared their localization between embryos produced in vitro and those derived from insemination in vivo. We expected to identify changes in the localization and abundance of critical subunits around the time of zygotic gene activation, which occurs at the 4-cell stage in the pig, and/or morphological differentiation, such as blastocyst formation.

| RESULTS
The following results represent data from three independent replicates. Western blot analysis was performed twice to validate the antibodies used in this study (see Supplementary Figure S1).
Controls incubated with only secondary antibody exhibited no detectable staining above background levels in any of the oocytes or embryos analyzed. The data presented herein depict representative images of porcine oocytes and embryos probed with antibodies for SWI/SNF subunits, and counterstained with Hoechst to identify the nuclei (Figures 1-12). A summarized comparison of localization | 1239 patterns of the 12 SWI/SNF subunits between in vitro-produced and in vivo-derived embryos during the course of the first week of development is presented in Table 1. The descriptive localization of each subunit as well as the frequency of each pattern follows.

| BAF170
The majority of germinal vesicle-stage oocytes presented clear staining for BAF170 in the nucleus and perinuclear area (26/33), while 7 of 33 germinal vesicle-stage oocytes showed no detectable staining.

| BRD7
The distribution of BRD7 was variable in germinal vesicle-stage oocytes. While half of the germinal vesicle-stage oocytes appeared to In SWI/SNF complexes, the scaffolding subunits BAF155 and BAF170 are present as heterodimers or homodimers (Chen & Archer, 2005;Wang et al., 1996). Wang et al. (2016) found that BAF155 and BAF170 are present within the same remodeling complex in human The quantity of BAF155 and BAF170 in human cells determines the abundance of BAF57 (Chen & Archer, 2005). In murine embryonic stem cells, knockdown of BAF155 attenuated BAF57 and vice versa (Schaniel et al., 2009). BAF57 is found only in higher eukaryotes, and is a key subunit that facilitates interactions between SWI/SNF complexes and transcription factors (Lomelí & Castillo-Robles, 2016). With the exception of some pronuclear embryos, BAF57 was detectable in most oocytes and embryos throughout preimplantation developmentwhich is in accordance with reports that BAF57 is omnipresent in all mammalian assemblies (Lomelí & Castillo-Robles, 2016). Similarly, although knockout of Baf60a was tolerated in murine differentiate, pointing to its essential function during stem cell differentiation (Alajem et al., 2015). We found that BAF60A is present in only some in vitro-produced embryos, and is at low abundance in pronuclear and blastocyst-stage embryos. In vivo-derived embryos showed clear nuclear staining at the 4-cell stage, whereas localization and intensity of the signal was highly varied at the blastocyst stage.
Two additional subunits highlighted for their contribution to cell survival during the process of embryonic stem cell differentiation are SNF5 and BAF53A. SNF5, a core subunit of SWI/SNF chromatin remodeling complexes, is critical for cell survival during the transition from pluripotency to differentiation in murine embryonic stem cells because it controls POU5F1 levels (You et al., 2013). Consistent with this function, we detected SNF5 in the nuclei of the majority of porcine embryos analyzed. In germinal vesicle-stage oocytes, however, the localization of SNF5 was evenly distributed between the cytoplasm and nucleus. BAF53A is present in both human and murine embryonic stem cells (Lu et al., 2015;Zhang et al., 2014). Additionally in mice, BAF53A repressed differentiation into primitive endoderm (Lu et al., 2015). We detected the presence of BAF53A in a vast majority of porcine oocytes and embryos at all stages analyzed.
Three mutually exclusive members of ARIDs are found within SWI/SNF complexes: ARID1A, ARID1B, and ARID2. In mice, ARID1A is abundant in embryonic stem cells as well as early embryos. The ablation of ARID1A in mice caused developmental arrest around embryonic Day 6.5, and failure to develop a mesodermal layer (Gao et al., 2008). ARID1A was ubiquitously expressed in all regions and stages of early embryos, whereas ARID1B was barely detectable in early embryos, with first detection at the 8-cell stage in mice (Flores-Alcantar, Gonzalez-Sandoval, Escalante-Alcalde, & Lomelí, 2011).

Although murine embryonic stem cells could be established from
Arid1b −/− blastocyst-stage embryos, these cells possessed slower proliferation and an abnormal cell cycle, as well as a lower expression of pluripotency markers and accelerated differentiation in Arid1b −/− versus Arid1b +/+ embryonic stem cells (Yan et al., 2008). Similar to data in mice, we found porcine ARID1A in all stages of preimplantation embryos from both in vitro-and in vivo-derived conditions.
Conversely, ARID1B was absent in most oocytes and pronuclear and 4-cell embryos, but was subsequently detected in most in vitroproduced, blastocyst-stage embryos-yet, only one of seven in vivoderived blastocyst-stage embryo possessed detectable ARID1B. Such discrepancy in the abundance of ARID1B indicates a potentially altered epigenetic state and abnormal timing of differentiation between blastocyst-stage embryos produced in vitro versus in vivo.
ARID2, together with BAF180 and BRD7, represent signature subunits for the biochemically defined polybromo BAF complex, a subset of mammalian SWI/SNF chromatin remodeling complexes. This complex is critical in mouse embryo development; indeed Baf180-null mice displayed embryonic lethality (Xu et al., 2012). Our data reveal distinct localizations and abundances of BAF180, ARID2, and BRD7.
Very weak or no signal was observed for BAF180, indicating that it is present in low abundance in early porcine embryos or that the BAF180 antigens recognized by this antibody may be masked in whole-mount immunocytochemical staining due to higher-order chromatin structure or associations made between BAF180 and other SWI/SNF subunits; an alternative primary antibody for BAF180 could help obtain clearer, brighter images. Additionally-and in contrast to our prediction that members of the polybromo BAF complex would show similar staining patterns-we detected differences between ARID2, which was predominantly nuclear in all analyzed stages of embryo development, and BRD7, which was clearly cytoplasmic at 4-cell and blastocyst stages of both in vitro-produced and in vivo-derived embryos. We interpret these findings to indicate that the classic murine polybromo BAF complexes do not exist in early porcine embryos. Future immunoprecipitation analyses might reveal new combinations of subunits of SWI/SNF complexes in early embryos in the pig. For example, ARID1A/ARID1B and BAF180 can also co-exist in a subset of SWI/SNF complexes, as revealed in HeLa cells (Ryme et al., 2009).
The majority of the work presented here involved the interpretation of indirect immunofluorescence assays. While the commercially  Figure S1); the porcine protein extracts sometimes revealed much clearer banding than protein isolated from HeLa cells (e.g., ARID1A and BAF170). Yet, other antibodies (e.g. against BAF53A, BAF57, and BAF180) exhibited weak immunoreactivity, such that very faint or no bands were detected in porcine tissues. Although this may indicate that the antibody does not recognize the respective porcine orthologs, the absence of non-specific bands suggested that the equal-sized band detected is the correct porcine ortholog and that non-specific binding by immunocytochemical analysis is unlikely. Instead, the porcine tissues examined by immunoblot may not express these particular subunits, or these tissues may possess subunits at a quantity that is below the detection threshold of our assay. One particular example to highlight is BRD7, which was detected as an immunoreactive band of ∼37 kDa in HeLa cells whereas the same antibody detected a single band of ∼70 kDa in porcine tissue. These differing sizes may reflect changes in post-translational modifications, or degradation of endogenous BRD7 (the predicted mass is 74 kDa). In any case, the absence of non-specific staining strongly suggests one dominant antigen is recognized by the antibody.
Together, our data indicate that: (i) distinct differences between mouse and pig embryos, as well as differences between embryonic stem cells and early embryos, exist in regards to SWI/SNF subunit use and expression and (ii) the timing of differentiation during in vitro culturing of embryos might not be appropriately synchronized with the expression of certain SWI/SNF subunits, which is reflected by the observed range of SWI/SNF complex subunits present at particular stages of development. Such asynchrony could perturb the epigenetic state of the embryo, potentially resulting in long-term effects that impact both survival and health of the embryos and subsequent offspring. The variance in localization patterns was more prominent for the in vitro-produced embryos, suggesting that they are more likely to exhibit asynchronous epigenetic development. Of note, the blastocyst-stage embryos (both in vitro-produced and in vivo-derived) analyzed did not show distinct staining patterns between the inner cell mass and trophectoderm, although we did not explicitly examine the differential intracellular localization of SWI/SNF subunits in these two lineages. Future experiments will focus on SWI/SNF complex distribution in peri-implantation embryos to determine if embryos are capable of correcting differences in localization and abundance of the various SWI/SNF subunits as well as if these observed differences are carried on during the course of development.

| Collection of in vivo-derived embryos
Gilts from the Animal Sciences Research and Education Center at Purdue University were inseminated and slaughtered 2-5 days after the onset of estrus to obtain 4-cell and blastocyst-stage embryos (Purdue Animal Care and Use Committee protocol 31311000982).
Reproductive tracts were removed, and the oviducts/uteri were flushed with Hepes-buffered medium. Developmental stages and numbers of embryos were recorded, and embryos from each animal were considered as one biological replicate.

| Immunocytochemistry
All primary antibodies were obtained from Abcam (Cambridge, MA).