Effects of palmitic acid on localization of embryo cell fate and blastocyst formation gene products

As obese and overweight patients commonly display hyperlipidemia and are increasingly accessing fertility clinics for their conception needs, our studies are directed at understanding the effects of hyperlipidemia on early pregnancy. We have focused on investigating palmitic acid (PA) and oleic acid (OA) treatment alone and in combination from the mouse two-cell stage embryos as a model for understanding their effects on the mammalian preimplantation embryo. We recently reported that PA exerts a negative effect on mouse two-cell progression to the blastocyst stage, whereas OA co-treatment reverses that negative effect. In the present study, we hypothesized that PA treatment of mouse embryos would disrupt proper localization of cell fate determining and blastocyst formation gene products and that co-treatment with OA would reverse these effects. Our results demonstrate that PA treatment significantly (P  < 0.05) reduces blastocyst development and cell number but did not prevent nuclear localization of YAP in outer cells. PA treatment significantly reduced the number of OCT4+ and CDX2+ nuclei. PA-treated embryos had lower expression of blastocyst formation proteins (E-cadherin, ZO-1 and Na/K-ATPase alpha1 subunit). Importantly, co-treatment of embryos with OA reversed PA-induced effects on blastocyst development and increased inner cell mass (ICM) and trophectoderm (TE) cell numbers and expression of blastocyst formation proteins. Our findings demonstrate that PA treatment does not impede cell fate gene localization but does disrupt proper blastocyst formation gene localization during mouse preimplantation development. OA treatment is protective and reverses PA’s detrimental effects. The results advance our understanding of the impact of FFA exposure on mammalian preimplantation development.


Introduction
The World Health Organization defines obesity as a BMI of 30 kg/m 2 or greater (WHO 2000) and obesity is reaching epidemic proportions. The incidence of diabetes, hypertension, heart disease and high serum cholesterol demonstrates a graded increase as BMI rises (Paeratakul et al. 2002, Knight et al. 2010. Obese females face many challenges including menstrual irregularity, endometrial pathologies, higher miscarriage rates and infertility (Broughton & Moley 2017). As BMI increases, women experience increased time-topregnancy and overall decreased fecundity (Wise et al. 2010). Furthermore, when obese women conceive, their pregnancies have increased incidence of preterm birth, high-and low-birth weight infants, increased risk of congenital anomalies (Knight et al. 2010, Schummers et al. 2015, Stang & Huffman 2016, pre-eclampsia and gestational diabetes (Stang & Huffman 2016). Assisted reproductive technologies (ARTs) are increasingly being required to achieve successful pregnancy in sub-fertile obese couples. Increasing BMI is positively correlated with pregnancy loss following ART treatment (Fedorcsák et al. 2004).
Obesity is accompanied by increased serum (Gaudet et al. 2012) and follicular fluid palmitic acid (PA) levels (Niu et al. 2014, Valckx et al. 2014. PA is a 16-carbon fully saturated fatty acid that increases the synthesis of harmful lipids and impedes normal organelle functioning (Yuzefovych et al. 2010, Palomer et al. 2018. It is the most abundantly saturated fatty acid in the human body (Carta et al. 2017). PA is present in many foods including meat, dairy and palm oil (Carta et al. 2017). The dysregulation of PA levels has detrimental longterm effects such as atherosclerosis, neurodegenerative disorders and cancer (Fatima et al. 2019).
Oleic acid (OA) is an 18-carbon fatty acid with a single double bond (Lopez et al. 2014). It is the most abundant  (Palomer et al. 2018). OA can be obtained in the human diet through olive, canola and peanut oils (Lopez et al. 2014) and is a primary component of the Mediterranean diet which lessens disease incidence such as diabetes (Martınez-Gonzalez et al. 2008). OA decreases PA-induced apoptosis and ROS (Yuzefovych et al. 2010), decreases PA-induced inflammation and relieves ER stress (Palomer et al. 2018). We have recently reported that PA treatment reduces mouse blastocyst development in a concentration-dependent manner and alters ER stress pathway transcript levels, while co-treatment of OA with PA reversed these negative effects (Yousif et al. 2020). Interestingly, we observed a small percentage of PA (100 µM) treated two-cell stage embryos that still cavitate, but for this study, we were especially interested in those that were unable to cavitate and become blastocysts.
Mammalian preimplantation development is characterized by a series of cleavage cell divisions from fertilized oocyte to multi-cellular blastocyst (Watson & Barcroft 2001). The initial cleavages occur without concomitant embryo growth (Hardy et al. 1993), and eventually the cells compact and form a morula (Watson & Barcroft 2001, Pfeffer 2018. At the mouse 32-cell stage, a cavity begins to form, leading to the blastocyst stage and the specification of two distinct cell lineages: the TE and ICM (Watson & Barcroft 2001, Niwa et al. 2005. OCT4 (POU5F1)is a pluripotency cell factor that gradually becomes restricted to the mouse ICM, acting to regulate transcription of target genes and maintain ICM pluripotency (Nichols et al. 1998). CDX2 is expressed in all cells at the 8-cell stage and gradually becomes restricted to the future trophectoderm in the blastocyst (Niwa et al. 2005). CDX2-deficient embryos have a poorly formed TE with deficiencies in maintaining tight junctions . OCT4 is downregulated when CDX2 reaches a specific threshold in mouse outer cells and collectively this asymmetry in cell lineage expression drives the formation of the ICM and TE (Niwa et al. 2005). CDX2 expression is induced by the transcription factor TEAD4 (Nishioka et al. 2009) and also requires Hippo pathway member YAP (Ota & Sasaki 2008). Failure to properly generate the first cell lineages can prevent the initiation of pregnancy (Piliszek et al. 2016).
Blastocyst development depends on the timely expression of critical gene families including: E-cadherincatenin cell adhesion genes, the tight junction genes and Na/K-ATPase subunits (reviewed in Watson & Barcroft 2001, Bell et al. 2008). Na/K-ATPase develops the ionic gradient which drives the movement of water through aquaporins across the epithelium to form the fluid-filled blastocyst cavity (Watson & Barcroft 2001). The cell-tocell adhesion provided by E-cadherin-catenin and tight junctional proteins such as ZO-1 inhibits the leakage of blastocyst fluid and maintains a polarized Na + /K + -ATPase distribution (Watson & Barcroft 2001). These events are interconnected, as interrupting expression or activity of Na/K-ATPase in embryos affects localization of ZO-1 (Violette et al. 2006, Madan et al. 2007. Because of the many fertility challenges obese women face and the importance of preimplantation development to successful pregnancy (Knight et al. 2010), we have pursued an understanding of how preimplantation development may be affected by PA and OA exposure. To date, research has not explored the effects of PA and OA on cell fate gene product and blastocyst formation gene product localization during preimplantation development. The primary objective was to determine how PA and OA treatment, alone and in combination, will affect YAP, OCT4, CDX2, Na/K ATPase α-1, E-cadherin and ZO-1 localization in mouse preimplantation embryos. We hypothesized that PA treatment of mouse preimplantation embryos would disrupt normal cell fate gene product and blastocyst formation gene product localization and that co-treatment with OA would reverse these effects. Our findings demonstrate that PA treatment does not impede cell fate gene product localization but reduces cell margin localization of blastocyst formation proteins. OA co-treatment reverses these effects of PA treatment. The results advance our understanding of the possible impact of obesity and FFA exposure on mammalian preimplantation development.

Animal source and ethics approval
CD1 mice were acquired from Charles River Canada (Saint-Constant, Quebec, Canada). The mice had free access to food and water while being housed in 12 h light: 12 h darkness cycles. All mice were handled according to the Canadian Council on Animal Care and Western University's Animal Care and Use Policies (protocol #: 2018-075 to Dr Andrew J. Watson).

Mouse superovulation and mating
Four-to six-week-old female CD-1 mice received a 5-7.5 international units (IU) intraperitoneal (i.p.) injection with pregnant mare's serum gonadotropin (Folligon, Merck Animal Health, Canada). This was followed by a 5-7.5 IU i.p. injection of human chorionic gonadotrophin (Chorulon, Merck) 46-48 h later. Following the second injection, each female was placed in a cage with a single male CD-1 mouse (3-8 months of age) for mating overnight. The following morning, female mice were checked for presence of a seminal plug. Forty-six hours post-injection of hCG, female mice were sacrificed by CO 2 asphyxiation (standard operating procedure). Their oviducts were collected and flushed with M2 flushing medium (Sigma-Aldrich). Flushed two-cell stage embryos were washed 3× in 50 μL drops of potassium simplex optimization medium with amino acids (KSOMaa Evolve, Zenith Biotech, Reproduction (2022) 163 133-143 Canada) for OCT4/CDX2 and cell counting experiments. Due to discontinuation of this product, YAP, Na/K ATPase α-1, E-cadherin and ZO-1experiments were conducted with KSOM+AA (IVL04, Caisson Laboratories, Smithfield, UT). After washing, embryos were distributed equally among experimental treatment groups and cultured in 20 μL drops under mineral oil (LiteOil LGOL-500, Cooper Surgical, Trumbull, CT) at a density of 1 embryo per μL for 46 h under a 5% CO 2, 5% O 2 and 90% N 2 culture atmosphere.

Free-fatty acid (FFA) preparation and embryo culture
A 20% FFA-free BSA (A6003, Sigma) was prepared in PBS, filter sterilized and used to conjugate with PA (P0500, Sigma) or OA (75090, Sigma) as outlined in Alsabeeh et al. (2018). Stock PA and OA solutions were prepared by solubilizing each FFA in RNAse-free water and NaOH at 70°C to create a 20 mM solution that was stored at −20°C. This stock was heated to 70°C and conjugated in a 1:3 (v/v) ratio with 20% BSA initially, followed by a dilution to 500 µM FFA solution in KSOM that was filtered and stored at 4°C. This results in a 2:1 molar ratio of PA or OA to BSA. The BSA control had a similar amount of added NaOH. All treatment groups thus contained 0.6% added fatty acid-free BSA final concentration.

PA and OA concentration response and treatment experiments
PA and OA treatment concentrations were derived from concentration responsive experiments conducted and reported in our recent study (Yousif et al. 2020); 100 µM PA had a negative effect on blastocyst development and 100 µM OA was able to overcome these deficits. PA is in the low physiological range, as 300-400 µM PA was measured in the regular chow-fed mouse oviduct (Yousif et al. 2020). Third trimester women with normal glucose tolerance had serum levels of 122 µM PA and 83 µM OA (Chen et al. 2010) which increased in obese women with gestational diabetes to 151 and 104 µM, respectively. For each experimental replicate, treatment groups consisted of 2-to 4-cell stage mouse embryos randomly allocated to the following treatments: (1) control KSOMaa medium (120 μL BSA control in a total volume of 300 μL balance KSOM); (2) KSOMaa medium +100 μM PA (60 μL + 60 μL BSA control in 300 μL); 3) KSOMaa medium + 100 µM OA (60 μL + 60 μL BSA control in 300 μL) and 4) KSOMaa + 100 µM PA (60 μL) and 100 µM OA (60 μL in 300 μL). Embryos were cultured under a 5% CO 2 , 5% O 2 and 90% N 2 atmosphere at 37°C for 46 h to assess progression to the blastocyst stage. Blastocysts were defined as embryos with a visible fluid-filled cavity of any size and the proportion was calculated based on number of two-cell cultured. Following this assessment, embryos were fixed for application of indirect immunofluorescence localization of OCT4, CDX2, YAP, Na/K-ATPase ∝ 1 subunit, ZO-1 or E-cadherin combined with 4,6-diamindino-2-phenylindole (DAPI) nuclear staining, actin cytoskeleton staining with rhodamine phalloidin and confocal microscopy.

Quantitation of Na + /K + ATPase alpha1 subunit, E-cadherin and ZO1 fluorescence intensity
Na + /K + ATPase alpha1 subunit, E-cadherin and ZO-1 fluorescence intensity were quantified using Zen and Ilastik (www.ilastik.org). From the embryo group Z-stacks czi files taken at 10×, rectangular boxes were drawn around individual embryos and the entire stack of green channel was converted to a TIFF file. In Ilastik, a single bright embryo slice was chosen in each replicate experiment, and the foreground and background fluorescence were delineated. All the other TIFF files were then imported and calibrated against this standard. For each embryo, all tissue slices were summed from 16 to Reproduction (2022) 163 133-143 18 5 µm Z-stack slices to give an individual embryo value. The average fluorescence of the negative control embryos was subtracted, embryos that had lower fluorescence than the negative were set to 0. For each antibody, there were at least two (OCT4 and CDX2), three (YAP, Na + /K + ATPase alpha1 subunit or E-cadherin) or four (ZO-1) different embryo collections subjected to separate staining and quantification protocols. There was a minimum of 22 to a maximum of 68 embryos evaluated per treatment.

Statistical analysis
GraphPad PRISM (https://www.graphpad.com/scientificsoftware/prism/) was employed to perform statistical analyses. For cell fate protein localization studies, each embryo examined represented a single n value within a treatment. However, for assessments of developmental stage, a biological replicate consisted of a single pool of embryos within a treatment group. Blastocyst development was analyzed using a one-way ANOVA test, and means were compared to one another by an ad hoc Tukey's multiple comparisons test. Data analysis for DAPI-, OCT4-, CDX2-and YAP-positive nuclei included applying Kruskal-Wallis non-parametric ANOVA (as values were not normally distributed) with Dunn's multiple comparisons tests. Data analysis of Na+/K+ ATPase, ZO-1 and E-cadherin fluorescence intensity was performed with non-parametric Kruskal-Wallis ANOVA with Dunn's multiple comparisons tests. For all tests, P values of less than or equal to 0.05 were considered significantly different from one another.

PA alone reduces blastocyst formation and total cell number while development is restored in combination with OA development
Preimplantation embryos from each treatment group were assessed using conventional light microscopy. Blastocysts were identified as having fluid-filled cavities (Fig. 1A) and were quantified as a percentage of all preimplantation embryos observed in each culture treatment group (Fig. 1C). A statistically significant decrease (P < 0.001) in the number of blastocysts was observed in the PA-treated group compared to all other groups (Fig. 1C), which is consistent with our previous study (Yousif et al. 2020). There were no significant differences in blastocyst developmental frequency between the control, OA alone and PA and OA combination (PA/OA) treatment groups (Fig. 1C).
We assessed the total number of DAPI-stained nuclei observed in each embryo composing each treatment group ( Fig. 1B and D). PA-treated embryos displayed a significant reduction (P < 0.001) in total number of nuclei compared to all other treatment groups and control group (Fig. 1D). PA/OA had lower cell number than control (P < 0.04, Fig. 1D). The majority of PA-treated embryos appeared as uncompacted cleavage stage embryos (Fig. 2Ab). The mean cell number of the PA-treated group was approximately 21 cells.

Effects of PA and OA treatment alone and in combination of YAP + nuclei
The number of cells with YAP localized to the nucleus following each treatment was counted ( Fig. 2A and C). The presence of binucleate cells in the PA-treated group was commonly observed ( Fig. 2Ab and Bb). Some YAP + and YAP − cells were binucleate; however, cells with YAPnuclei were properly localized to the inside. PA-treated embryos displayed a significantly (P < 0.0001) lower number of YAP + nuclei compared to all other treatment groups (Fig. 2C). Interestingly, the proportion of YAP + nuclei to total nuclei did not vary significantly (P > 0.05) between the control, PA and OA treatment groups (Fig.  2D). The proportion of YAP + nuclei to total cell nuclei in the PA and OA combination treatment was significantly (P < 0.05) higher than that observed for the control, OA and PA alone treatments (Fig. 2D).

PA reduced the number of OCT4 + nuclei, while OA in combination with PA restored OCT4 + number
PA-treated embryos displayed a significantly lower (P < 0.0001) number of OCT4 + nuclei compared to the vehicle-treated control group and other treatment groups  Fig. 3Ab and D). The mean proportion of OCT4 + nuclei in the control, OA and PA/OA embryos was significantly greater than the PA-treated embryos (P < 0.0001). The proportion of OCT4+ nuclei in OA-treated embryos (P < 0.005) and PA and OA combination-treated embryos (P < 0.004) was lower than control embryos (Fig. 3E). There was no significant difference in the proportion of OCT4 + nuclei between OA-and PA/OA-treated embryos (Fig. 3E).

PA reduced the number of CDX2 + nuclei, while OA in combination with PA restored CDX2+ cell number
The number of CDX2 + nuclei in the PA-treated embryos was significantly lower (P < 0.0001) compared to all other groups (Fig. 3Bb and F). The mean proportion of CDX2 + cells in PA-treated group was significantly lower (P < 0.0001) than the control and OA treatment groups and lower than the combined PA/OA group (P < 0.005, Fig. 3G). Control, OA and PA/OA treatments were not significantly different from each other.

PA reduced the amount of Na + /K + -ATPase α-1 subunit, E-cadherin and ZO-1 membrane localization
The level of Na + /K + -ATPase fluorescence intensity in PA-treated embryos was significantly lower (P < 0.005) compared to CON and P < 0.02 for both OA and PA/OA (Fig. 4A, B and C). The level of E-cadherin fluorescence intensity was significantly lower (P < 0.02) in PA-treated embryos compared to the OA group (Fig. 5A, B and C). The level of ZO-1 fluorescence intensity was also significantly lower (P < 0.004) in PA-treated embryos compared to the CON group (Fig. 6A, B and C).

Discussion
Treatment of mouse embryos with PA resulted in impaired development similar to previous studies (Jungheim et al. 2011a, Yousif et al. 2020. The concentrations of PA and OA used are like those of serum levels in normal pregnant women of about 100 µM (Chen et al. 2010), but PA can increase 30-60% in serum and follicular fluid in obese patients (Jungheim et al. 2011b, Valckx et al. 2014. Cleavage (O'Gorman et al. 2013) and pregnancy (Mirabi et al. 2017) in ART cycles are negatively correlated with follicular fluid PA concentrations. In the embryos treated with 100 µM PA, the cell number averaged 21 cells, many embryos arrested at the 8-16 cell stage and 22% developed to the blastocyst stage. Short-term exposure of mammalian female gametes to PA showed long-lasting effects, as development was delayed (Desmet et al. 2020) and fetuses and liveborn animals were smaller following embryo transfer (Jungheim et al. 2011a). As well, gene expression was altered in both embryo and extra-embryonic tissue (Desmet et al. 2020). ER stress pathways are activated in mammalian female gametes and granulosa cells subjected to PA treatment in vitro (Wu et al. 2012, Yousif et al. 2020. We observed that PA-treated embryos developed multi-nuclear cells. Multi-nuclear blastomeres in human embryos are quite common (Hardy et al. 1993), but they are rare in cultured CD1 mouse embryos. In human embryos, multi-nucleation is associated with aneuploidies (Staessen & Van Steirteghem 1998). However, binucleation can resolve if it occurs at the two-cell stage (Aguilar et al. 2016, Paim & FitzHarris 2020 but not later (Paim & FitzHarris 2019). Paim and FitzHarris (2020) suggest that binucleate cells in later stage mouse embryos arise due to a failure of cytokinesis, as was observed earlier in arrested human embryos (Hardy et al. 1993). Zhang et al. (2010) demonstrated that PA treatment resulted in the formation of binuclear HeLa cells due to disrupted cytokinesis. Difficulties in completing cytokinesis may underlie the reduced

Figure 4
Representative Na + /K + ATPase α-1 immunofluorescence images from each treatment group. Confocal microscopy images of preimplantation embryos imaged at 40× following processing for indirect immunofluorescence (A) Na + /K + ATPase α-1 subunit (green), F-actin (red), DAPI (blue), merged images on left panel and Na + /K + ATPase α-1 subunit alone on right panel. (a) Control embryo, (b) PA-treated embryo, (c) OA-treated embryo and (d) PA and OA combination-treated embryo. Scale bars = 20 µm. (B) Embryo group photo of Na + /K + ATPase α-1 (green), 10× magnification, (C) quantification of Na + /K + ATPase α-1 intensity in each treatment group. Data shown represent the values of the individual embryos and the mean Na + /K + ATPase α-1 fluorescence ± s.e.m. for each treatment group. Bars without letters in common are significantly different, P < 0.05. Control embryos (n = 3 experimental replicates, 13-16 embryos per replicate), PA-treated embryos (n = 3, 12-21 embryos per replicate), OA-treated embryos (n =3, 12-15 embryos per replicate) and PA and OA combination-treated embryos (n = 3, 14-15 embryos per replicate).  cell numbers of the PA treatment group in the present study. Further studies are required to determine whether this is the case and when binucleation is occurring in PA-treated 2-cell embryos.
While we have an excellent understanding of the basic mechanisms and molecular players involved in cell lineage specification in mammalian embryos, there is more to learn about governing mechanisms that coordinate the timing of cell fate events and their sequence (Piliszek et al. 2016). Early blastomeres transition from totipotency to obtaining positional information (outside or inside cells), triggering polarization, adherens junction cell to cell adhesion, and compaction leading to epithelialization and differentiation of TE cells (Piliszek et al. 2016). The role of the Hippo pathway in TE differentiation is to differentially regulate YAP phosphorylation in TE and ICM development (Nishioka et al. 2009, Anani et al. 2014, Sasaki 2017. Normally following compaction at the 8-cell stage, each blastomere acquires polarity (Johnson et al. 1986). At the 16-cell stage, nuclear YAP is expressed in the outer cells, controlled mainly by blastomeres inheriting apical membrane components (Anani et al. 2014, Hirate et al. 2015, Sasaki 2017). In the outer cells, i.e. TE progenitors, Hippo activity is inactivated thus allowing YAP to localize to the nucleus and interact with TEA domain transcription factor (TEAD) resulting in CDX2 expression and TE cell differentiation (Nishioka et al. 2009, Anani et al. 2014, Sasaki 2017. In contrast, the Hippo pathway is active in the ICM which results in phosphorylated YAP that remains cytoplasmic (Nishioka et al. 2009, Anani et al. 2014, Sasaki 2017. By the 32-cell stage, differential activation of Hippo signaling in the two-cell lineages is cemented by the formation of stable E-cadherin-mediated adherens junctions (Nishioka et al. 2009, Anani et al. 2014, Sasaki 2017. PA-treated embryos were predominantly 8-16-cells; still, YAP immunofluorescence localization appeared to be correct for regulating cell fate in outer cells, i.e. nuclear in outer cells. The proper localization of YAP may indicate that PA treatment did not alter membrane structure or domain inheritance. Since they are chronological at a normal developmental time point for these mechanisms to reveal themselves, we suggest that the PA-treated embryos were simply activating a normal cell fate developmental program according to the polar or apolar status of the blastomeres to the best of their ability. The number of YAP + nuclei was lower in the PA group in comparison to other groups; however, the proportion of YAP+ nuclei was unaffected. PA embryos including cleavage stage embryos displayed nuclear YAP immunofluorescence. Other studies have found that PA reduced the number of endothelial cells with nuclear YAP (Yuan et al. 2017); however, studies with β-cell lines have shown that PA induced nuclear localization of YAP (Deng et al. 2016). Inhibition of YAP resulted in increased PA-induced apoptosis, suggesting that nuclear YAP may have a protective role in β-cells (Deng et al. 2016). Since, the proportion of YAP + nuclei did not vary significantly between PA and control groups, we would propose that cell lineage specification mechanisms were activated, but for the majority of PA-treated embryos, this was not sufficient to enable cavitation and blastocyst formation.
We observed lower numbers and proportion of OCT4 + cells in PA-treated embryos, which is indicative of ICM. Similarly, the proportion of ICM cells was lower in embryos retrieved from high-fat diet fed mice (Minge et al. 2008, McPherson et al. 2015 and the number of ICM cells was lower in embryos from overweight women (Leary et al. 2015). CDX2 + cell numbers and proportion were also reduced in PA-treated embryos, which is indicative of reduced numbers of TE cells. Similarly, the number of TE cells is reduced in embryos from overweight women (Leary et al. 2015). Both decreased proliferation and increased apoptosis were observed in trophoblast stem cells cultured with PA (Jungheim et al. 2011a). The reduction in total cell number as well as both ICM and TE cells suggests that PA treatment promotes metabolic changes within the early embryo (McKeegan & Sturmey 2011). Leary et al. (2015) observed reductions in glucose uptake, alterations in amino acid turnover and increases triglyceride storage in embryos from overweight women.
We also observed that PA treatment reduced the fluorescent levels of Na + /K + ATPase, E-cadherin and ZO1 properly localized to blastomere cell margins. Exposure to high-fat diet reduced mRNA expression and reduced assembly of tight junctions and adherens junctions as well as Na + /K + ATPase expression and function in corneal cells (Bu et al. 2020). In addition, increased amounts of PA in the lipid bilayer reduced Na + /K + ATPase function (Gianfrancesco et al. 2019). Na + /K + ATPase activity is linked to membrane fluidity and negatively associated with levels of saturated fatty acids in erythrocytes (Rodrigo et al. 2014). Similarly, palmitate decreased integrity of tight and adherens junctions and increased permeability of the gut epithelium (Ghezzal et al. 2020). Failure to establish these junctions appropriately and produce Na/K-ATPase activity required for establishing a trans-trophectoderm ion gradient is likely the main factor underlying the reduced ability of PA-treated embryos to cavitate and form blastocysts , Betts et al. 1998, Watson & Barcroft 2001. Furthermore, blastocyst formation gene and cell fate promoting gene expression are interconnected, as CDX2-deficient embryos have deficiencies in forming and regulating trophectoderm apical tight junctions  and ZO-1 gene knockout decreased expression of OCT4 and CDX2 (Wang et al. 2008).
The total cell number in blastocysts and the proportion of ICM cells are important determinants Reproduction (2022) 163 133-143 of embryo viability following transfer (Papaionnou & Ebert 1995, Lane & Gardner 1997. However, a precise cell number is not required for blastocyst formation to occur. For example, it is possible to split embryos in half and still observe cavitation in embryos with half their normal cell number (Tarkowski 1959, Papaionnou & Ebert 1995. Since early mammalian embryos have this capacity to cavitate with significantly reduced cell number, we were compelled to determine if their reduced ability to cavitate was associated with an alteration in the expression of known blastocyst formation genes including E-cadherin, ZO-1 and Na/K-ATPase alpha 1 subunit genes. We have extensive experience with these gene products and have significantly helped with the understanding of their essential roles in driving cavitation and thus blastocyst formation , Betts et al. 1998, Watson & Barcroft 2001, Violette et al. 2006. The fluorescence signal of all three blastocyst formation genes was significantly reduced in 48-h PA-treated embryos. We thus conclude that overall, it is likely that PA treatment impedes proper blastocyst formation gene product expression, and while cell fate gene products are properly localized in PA-treated embryos, their reduced ability to cavitate could be due to a disruption in normal E-cadherin, ZO-1 and Na/K-ATPase alpha subunit expression. In conclusion, our study has reinforced the harmful effects of PA exposure on mouse embryo preimplantation development. Our observations are applicable of understanding the consequences that obesity may impose on human preimplantation embryo development as FFA within follicular fluid is positively correlated with BMI (Valckx et al. 2014) and negatively correlated with oocyte quality (Jungheim et al. 2011b). PA and other saturated fatty acids in the Western diet may hinder the growth of the embryo and lead to lower conception rates. In this study, treatment of twocell preimplantation mouse embryos with 100 µM PA significantly reduced total embryo cell number and the numbers of YAP + , OCT4 + , and CDX2 + expressing nuclei as well as decreasing cell adhesion, tight junction and Na + /K + ATPase proteins in the blastomere membranes. In contrast, OA co-treatment alleviates these effects of PA treatment. There are many other FFAs that should be investigated for their influence on preimplantation development, and as progress continues in this manner, eventual understanding of the full possible effects of FFA exposure to early development will unfold. It will be important to conduct further experiments that are directed at dissecting stage-specific and treatment time course variation effects of PA and OA treatment on preimplantation development. Our findings offer potential therapeutic interventions as supplementation of embryo culture medium with OA may improve development in obese patients with elevated PA serum levels.

Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.