Sexual dimorphic expression and release of transcription factors in bovine embryos exposed to oxidative stress

Sexually dimorphic differences in genome activity, which is orchestrated by transcription factors (TFs), could explain the differential response of male and female embryos to environmental stressors. To proof this hypothesis, the expression of cellular and extracellular TFs was investigated in male and female bovine embryos in vitro cultured either under low (5%) or high (20%) oxygen levels. The intracellular reactive oxygen species (ROS), total cell number, expression of nuclear factor (erythroid‐derived 2) factor 2 (NFE2L2), Krüppel‐like factor 4 (KLF4), notch receptor 1 (NOTCH1), E2F transcription factor 1 (E2F1), and SREBF2 along with extracellular vesicles (EVs) biogenesis genes were assessed at the blastocyst stage and their released EVs. Low blastocyst rate in both sexes due to oxidative stress (OS) was accompanied by increased ROS accumulation and reduced cell number in female embryos. The messenger RNA and protein levels of NFE2L2, as well as KLF4 expression, were higher in male embryos exposed to OS compared with female embryos. However, the expression of NOTCH1 and E2F1 was higher in female embryos cultured in high oxygen level. Male embryos exposed to OS released more EVs enriched with NFE2L2, superoxide dismutase 1, and NOTCH1 accompanied by elevated expression of EVs biogenesis genes. Accordingly, differential expression of TFs and their release into spent media could partially explain the sexual dimorphic response of bovine embryos to environmental stresses.

The sex ratio is believed to be affected by numerous factors either in vivo or in vitro including ovarian factors, maternal diets, and culture conditions (Gutiérrez-Adán, Granados, Pintado, & La Fuente, 2001;Hylan et al., 2009;Iwata, 2012;Rosenfeld & Roberts, 2004).  (Gómez et al., 2018), which may be associated with their differential response to environmental insults. So far, this sexual dimorphic response to stress between male and female embryos is controversial and not fully understood. Some argue that female embryos are more tolerant to heat stress than the male embryos (Pérez-Crespo et al., 2005). This could be attributed to the transcriptional dimorphism derived from unaccomplished X-chromosome inactivation during early embryo development (Bermejo-Alvarez et al., 2010). This phenomenon is not only correlated with the upregulation of several genes linked to sex chromosome but also to autosomal chromosomes (Pérez-Cerezales et al., 2018) like glucose-6-phosphate dehydrogenase (G6PD), which is an X-linked gene related to oxygen-free radicals. In bovine, the G6PD has been found to be upregulated in female morula than male counterparts exposed to OS accompanied by enhanced developmental components (Iwata et al., 2002). Contrastingly, others reported that the bovine female embryos were more susceptible to apoptosis than male counterparts (Ghys et al., 2016;Oliveira et al., 2016) following fertilization either with sex-sorted or unsorted semen and cultured under two different culture conditions (Ghys et al., 2016).  (Lawson et al., 2016), follicular fluid (Hung, Hong, Christenson, & McGinnis, 2015) and uterine aspirates (Campoy et al., 2016). On the basis of their size, EVs can be subdivided into apoptotic bodies (1,000-5,000 nm), microvesicles (100-1,000 nm), and exosomes (30-200 nm;Lawson et al., 2016;Sun et al., 2010;de la Torre Gomez, Goreham, Bech Serra, Nann & Kussmann, 2018). They carry a cargo of bioactive molecules such as DNA, RNAs, proteins to be transferred into the extracellular environment to maintain cellular homeostasis (Takahashi et al., 2017) and/or transfer genetic material to neighbor or distant recipient cells (Saeed-Zidane et al., 2017). In addition, its cargo content varies depending on the cell origin (de la Torre Gomez et al., 2018), physiological status (Hung et al., 2017;Navakanitworakul et al., 2016), inflammation (Jolly, Thompson, & Winchester, 1975) and stress status (Beninson et al., 2014;Bewicke-Copley et al., 2017) of the donor cells.
However, the effect and regulation of EVs biogenesis and secretion in bovine embryos during OS condition is not yet fully understood. Hence, the current study was designed to understand the cellular and extracellular defense mechanisms of male and female bovine embryos exposed to OS under in vitro culture condition.

| Effect of OS on cleavage and blastocyst rate of male and female embryos
The culture of male and female embryos under 5 or 20% oxygen levels revealed no significant difference in cleavage rate ( Figure 1a). However, blastocyst rates at Days 7 and 8 were significantly reduced under 20% compared with 5% oxygen level in both male and female embryos (Figure 1b,c). Interestingly, Day 8 blastocyst rate of male embryos was significantly higher compared to female counterparts at both oxygen concentrations. The sex of embryos produced using sexed semen was validated using a combination of primers and results are indicated in Figure S1.

| Higher atmospheric oxygen-induced ROS accumulation in both male and female embryos
Embryos cultured under 20% oxygen level showed significantly elevated intracellular ROS accumulation irrespective of the sex of embryo ( Figure 2). However, male embryos exhibited a significantly higher accumulation of ROS when cultured under 5% oxygen level compared with female counterparts. Nonetheless, there were no significant differences in ROS accumulation between male and female embryos cultured under 20% oxygen level.
2.3 | OS resulted in reduced total cell number OS due to high oxygen level reduced the blastocyst cell number in both male and female embryos ( Figure 3). Moreover, the impact of higher oxygen level was pronounced in female blastocysts, as evidenced by a significant reduction in total cell number compared with male embryos cultured under 20% oxygen level. The blastocyst cell number did not differ between male and female embryos cultured under 5% oxygen level.

| Oxygen level altered the expression of TFs in male and female embryos
Analysis on the differential response of male and female bovine embryos in terms of expression of TFs related to OS response (NFE2L2), differentiation and apoptosis (Krüppel-like factor 4 [KLF4], NOTCH1), cell cycle (E2F transcription factor 1 [E2F1]), and cholesterol biosynthesis (SREBF2) was performed at the blastocyst stage. Results showed that the expression pattern of NFE2L2 was significantly higher in male blastocysts cultured under 20% oxygen level compared with those cultured under 5% oxygen level ( Figure 4). Similarly, NFE2L2 tended to be higher in female embryos cultured under 20% compared with 5% oxygen level.
Likewise, the transcript abundance of KLF4 was significantly higher in male embryos exposed to higher oxygen tension compared to the low oxygen level. However, no significant difference was observed in the expression level of NOTCH1 in F I G U R E 1 The cleavage (a) and blastocyst rates at Day 7 (b) and Day 8 (c) of male and female preimplantation embryos cultured under 5% (white bar) or 20% (black bar) oxygen levels. Data are represented as mean ± SEM of four independent biological replicates F I G U R E 2 Intracellular ROS accumulation (a) and fluorescence intensity of ROS signal (b) of male and female preimplantation embryos derived from 5% (white bar) and 20% (black bar) oxygen levels. Scale bar = 50 µm, values of fluorescence intensity are represented as means ± SEM (*p ≤ .05, **p ≤ .01, ***p ≤ .001). ROS, reactive oxygen species

| OS-induced expression of NFE2L2 and KLF4 proteins
The protein expression of selected TFs in male and female blastocysts cultured under 5 or 20% oxygen levels revealed that NFE2L2 protein was significantly higher and proportionally localized in the nucleus of both male and female embryos cultured under 20% oxygen level than those cultured under 5% oxygen level. As opposed to the messenger RNA (mRNA) level, the protein level of NFE2L2 was higher in male embryos cultured under 20% oxygen than their female counterpart ( Figure 5), and no significant difference was noticed between male and female embryos cultured under 5% oxygen level. Even though the KLF4 protein level tended to be higher in embryos cultured under 20% compared with 5% counterparts, male and female embryo did not show a statistically significant difference in the KLF4 protein abundance irrespective of the oxygen level ( Figure 6).

| Dysregulation of antioxidant and differentiation genes after induced OS
Exposure of embryos to higher oxygen level significantly increased the expression of catalase (CAT) and superoxide dismutase 1 (SOD1) in male embryos ( Figure 7). However, only female embryos showed a significantly higher abundance of CAT in response to high oxygen level, as opposed to SOD1, which was indifferent in female embryos cultured under higher or lower oxygen levels. Similarly, the expression of differentiation-related gene POU class 5 homeobox 1 (POU5F1) was significantly higher in male and female embryos cultured under 20% oxygen level compared with the 5% counterparts. However, no F I G U R E 3 Total cell number of male and female blastocysts derived from 5% (white bar) and 20% (black bar). Data are represented as means ± SEM (**p ≤ .01, ***p ≤ .001) F I G U R E 4 The messenger RNA expression pattern of transcription factors (NFE2L2, KLF4, NOTCH1, E2F1, and SREBF2) in male and female blastocysts cultured in 5 or 20% oxygen level. Expression was compared between 5% (white bar) and 20% oxygen level within and between sexes. Data are represented as means ± SEM (*p ≤ .05, **p ≤ .01, ***p ≤ .001). The arbitrary units were multiplied by 100. E2F1, E2F transcription factor 1; KLF4, Krüppel-like factor 4; NFE2L2, nuclear factor (erythroid-derived 2) factor 2; NOTCH1, notch receptor 1 significant differences in transcript abundance of CAT, SOD1, and POU5F1 were observed between male and female bovine embryos cultured under the same oxygen level. The expression of GATA binding protein 4 (GATA4) was not different within the sex of embryo cultured under either 5 or 20% oxygen level. However, under both oxygen levels, male embryos exhibited elevated expression of GATA4 compared with female embryos.

| Characterization of EVs derived from male and female embryos spent media
The presence of EVs in the spent culture media of male and female embryos was confirmed using EV marker protein namely CD63 and their purity or absence cellular contamination was checked by the absence of cytochrome c (CYCS) in the isolated EVs ( Figure 8a).
Subsequently, the concentration and size of particles were determined using nanoparticle tracking analysis (NTA) and the morphology of the isolated EVs was assessed using transmission electron microscopy ( Figure 8b,c). The size distribution of EVs was ranging from 117 ± 2.1 to 145.2 ± 1.1 nm irrespective of the embryo sex and oxygen level. The concentration of EVs was higher in male embryos cultured under 20% oxygen level compared with those under 5% oxygen level. Interestingly, the average size of EVs was tended to be higher in male embryos derived from 20% oxygen level than those derived from 5% oxygen level. Interestingly, low concentration of EVs was found in female embryos derived from 20% compared with those derived from 5% oxygen level. However, the EVs released into cultured media of male embryos exposed to OS had a higher concentration and bigger average size than EVs released from female embryos.

| Expression of EV biogenesis and secretionrelated genes under OS condition
To evaluate whether the difference in the concentration of EVs is attributed to the impact of OS, the expression levels of genes related to EVs biogenesis (ALIX, vacuolar protein sorting 4 homolog B [VPS4B], and STEAP3 metalloreductase [TSAP6]) and secretion (RAB11 family interacting protein 2 [RAB11FIP1], RAB35, and RAB27A) were investigated. Results revealed that the expression levels of ALIX, VPS4B, RAB11FIP1, and RAB27A were significantly higher in male embryos cultured under 20% oxygen level compared with the 5% counterparts ( Figure 9). However, only the expression of ALIX was higher in female embryos cultured under 20% oxygen level.
Exposure of embryos to higher oxygen level led to elevated expression of EV biogenesis related-gene VPS4B and secretionrelated genes RAB11FIP1 and RAB27A in male embryos compared with female embryos cultured under the same oxygen level.
However, the expression of TSAP6 and RAB35 was not affected by both the sex of embryo and oxygen level.
F I G U R E 5 Detection and immunolocalization of NFE2L2 protein in male and female blastocyst-stage embryos derived from 5 or 20% oxygen levels. Red color reveals the localization of the NRF2 protein, while the blue color, is nuclear staining (DAPI). Scale bar = 50 µm. The bars represent the means ± SEM (*p ≤ .05, **p ≤ .01, ***p ≤ .001) of fluorescence signals as quantified by the ImageJ software. NFE2L2, nuclear factor (erythroid-derived 2) factor 2 TAQI ET AL.

| EV-coupled transcript releases due to OS
In an attempt to determine whether the changes occurred in the embryos due to OS could affect the communication between embryos via the release of stress indicators loaded in EVs, the mRNA content of released EVs was isolated and quantified for mRNA level of stress-related candidate TFs. Results revealed that the NFE2L2 ( Figure 10) carried by EVs released from male embryos cultured under 20% oxygen level was higher compared to EVs F I G U R E 6 Detection and immunolocalization of KLF4 protein in male and female blastocyst-stage embryos derived from 5 or 20% oxygen levels. Red color reveals the localization of the KLF4 protein, while the blue color, is nuclear staining (DAPI). Scale bar = 50 µm. The bars represent the means ± SEM of fluorescence intensity of the protein as quantified by the ImageJ software. KLF4, Krüppel-like factor 4 F I G U R E 7 The messenger RNA expression pattern of genes related to the antioxidant system (CAT and SOD1) and differentiation (POU5F1 and GATA4) in male and female blastocysts cultured under 5 or 20% oxygen levels. Expression was compared between 5% (white bar) and 20% oxygen level under the same sex and between sexes. Data are represented as means ± SEM (*p ≤ .05, **p ≤ .01, ***p ≤ .001). The arbitrary units were multiplied by 100. CAT, catalase; GATA4, GATA binding protein 4; POU5F1, POU class 5 homeobox 1; SOD1, superoxide dismutase 1 released from male embryos cultured under 5% oxygen level and female embryos cultured under 20% oxygen. Similarly, SOD1 was significantly higher in EVs released from male embryos exposed to OS compared with those cultured under normal conditions. The transcript level of CAT, KLF4, and E2F1 did not show significant differences among all investigated groups. It is noteworthy that the EVs released from male embryos exposed to OS showed a significantly higher amount of NOTCH1 transcript than those cultured under 5% oxygen level and the EVs released from female embryos exposed to OS. EVs released from female embryos under 5 and 20% oxygen levels did not show significant differences in terms of NOTCH1 transcript level.

| DISCUSSION
The suboptimal culture conditions of the in vitro production system are partly responsible for the reduction in number and quality of blastocysts (Farin & Farin, 1995;Gad et al., 2012). In addition to the culture media, the higher oxygen level in in vitro system is responsible for contributing to the suboptimal environment (Amin et al., 2014;Kelley & Gardner, 2016;Leite et al., 2017). However, previous evidence indicated that there was no significant difference observed in terms of embryo development between maturation followed by fertilization at high oxygen tension and maturation at low oxygen followed by fertilization at high oxygen level, but a dramatic effect was observed in case of fertilization at low oxygen level (Bermejo-Alvarez, Lonergan, Rizos, & Gutiérrez-Adan, 2010). Accordingly, the maturation and fertilization were done at high oxygen tension in the current study. Furthermore, here we showed that irrespective of the sex of embryo, those embryos cultured under 5% oxygen level showed higher development rate of blastocyst than those under 20% oxygen level, as it has been reported before (Amin et al., 2014;Leite et al., 2017). Differences in metabolism (Gómez et al., 2018) and genetics (Bermejo-Alvarez et al., 2010) between male and female bovine embryo could contribute to skewness in sex ratio of the resulted embryos (Oliveira et al., 2016;Xu, Yadav, King, & Betteridge, 1992).
Similarly, our results showed that the blastocyst rate at Days 7 and 8 ( Figure 1) were skewed toward the male embryos when cultured under either 5 or 20% oxygen levels. As it was been reported previously  (Figure 2). However, male embryos exhibited higher ROS accumulation than female embryos at lower oxygen level, which may be attributed to higher metabolism rate of male embryo compared with female counterparts (Tiffin, Rieger, Betteridge, Yadav, & King, 1991). Consistently, metabolite analysis of the spent culture media of male and female bovine embryos cultured under low oxygen tension revealed extensive metabolite exchange of male embryos than the female counterpart (Gómez et al., 2018). However, this difference was faded out under high oxygen tension, which may be associated with the potential role of glucose in ROS scavenging process (Andrisse et al., 2014).

F I G U R E 9
The messenger RNA expression pattern of extracellular vesicles biogenesis (ALIX, VPS4B, and TSAP6) and secretion (RAB11FIP1, RAB35, and RAB27A) genes in male and female blastocysts cultured under 5 or 20% oxygen levels. Expression was compared between 5% (white bar) and 20% oxygen levels under the same sex and between sexes. Data are represented as means ± SEM (*p ≤ .05, **p ≤ .01, ***p ≤ .001). The arbitrary units were multiplied by 100. RAB11FIP1, RAB11 family interacting protein 2; TSAP6, STEAP3 metalloreductase; VPS4B, vacuolar protein sorting 4 homolog B F I G U R E 1 0 The messenger RNA (mRNA) expression pattern of extracellular vesicles-coupled transcription factors (NFE2L2, KLF4, NOTCH1, and E2F1) and antioxidant genes (CAT and SOD1) released into culture media of male and female embryos cultured under 5 or 20% oxygen levels. Expression was compared between 5% (white bar) and 20% (black bar) oxygen levels within and between sexes. The mRNA level was measured by quantitative reverse transcription-polymerase chain reaction and normalized by the geometric means of three housekeeping genes (ACTB, GAPDH, and 18S). Data are represented as means ± SEM (*p ≤ .05, ***p ≤ .001). The arbitrary units were multiplied by 100. CAT, catalase; E2F1, E2F transcription factor 1; KLF4, Krüppel-like factor 4; NFE2L2, nuclear factor (erythroid-derived 2) factor 2; NOTCH1, notch receptor 1; SOD1, superoxide dismutase 1 The higher oxygen tension is also reported to compromise the cell proliferation, as evidenced by subsequent reduction in total cell number of embryos (Amin et al., 2014;Leite et al., 2017;van Soom et al., 2002;Yoon et al., 2014). Likewise, we found that embryos exposed to high oxygen level had a reduced total cell number ( Figure   3) independent of the sex of embryo, which could be associated with dysregulation of development and apoptosis-related genes (Leite et al., 2017). The negative effect of high oxygen level in terms of cell number was more pronounced in female embryos than male counterparts. This fact is in agreement with Dallemagne et al. (2018), who showed that exposing bovine embryos to OS from Days 5 to 7 in presence of FCS but not bovine serum albumin supplemented with insulin, transferrin and selenium (BSA-ITS) resulted in a reduction of total cell number and increasing apoptosis in female embryos compared with male counterparts. Moreover, higher cell number was observed in male embryos than female cultured under 5% oxygen level in presence FCS or BSA-ITS, which was not in agreement with the current study (Dallemagne et al., 2018). Supporting to our results, no significant difference in total cell numbers was observed between male and female bovine embryos cultured under low oxygen tension (Siqueira & Hansen, 2016), which indicates the contribution of culture media in the variation between male and female embryos under either normal or stress conditions.
In fact, one of the cellular mechanisms in response to suboptimal conditions is activation/suppression of transcription to maintain cellular homeostasis. This has been elucidated by the fact that higher intracellular ROS level induces the expression of NFE2L2 in bovine granulosa cell (Saeed-Zidane et al., 2017) and preimplantation embryos (Amin et al., 2014). NFE2L2 is a TF found to be sequestrated in the cytoplasm via Kelch-like ECH-associated protein 1 (KEAP1).
During stressor-induced NFE2L2 activation, the NFE2L2 disengages from KEAP1 and translocated in the nucleus to bind with a specific DNA motif called antioxidant response element to induce the antioxidant machinery (Bryan, Olayanju, Goldring, & Park, 2013;Zhang, 2006). In consistent with this, we found that the mRNA expression ( Figure 4) 2007). Interestingly, the expression of SREBF2 was significantly higher in male compared with female counterparts either cultured in 5 or 20% oxygen levels, which may be attributed to developmental dimorphism in favor of male embryos (Xu et al., 1992). Recent studies also evidenced that bovine male embryos release higher lipids and lipid-like molecules into culture media than female counterparts (Gómez et al., 2018).
NOTCH1 is one of NOTCH signaling pathway receptors, which is branching in various cell function including proliferation, differentiation, and apoptosis. Various combinations of NOTCH signaling pathway receptors and ligands might lead to different cellular response (E. R. Andersson, Sandberg, & Lendahl, 2011). In the present study, the expression of NOTCH1 was higher in those embryos exposed to OS coupled with elevated NFE2L2 transcript, as previously has been shown (Zhao et al., 2016). The upregulation of NOTCH1 under high oxygen level is attributed to cell apoptosis prevention via suppression of apoptosis signal-regulating kinase 1 (ASK1), and then preventing the p38 mitogenactivated protein kinases signaling pathway (Mo et al., 2013). This was more pronounced in embryos produced in vitro (Heras et al., 2016).
Consistent with this finding, in the current study higher expression of NOTCH1 was found in female embryos exposed to OS than male counterparts ( Figure 4). This phenomenon is believed to be deemed a reaction to the high level of apoptosis induced by the expression of E2F1. KLF4 belongs to KLFs TF family that are involved in the regulation of proliferation, apoptosis, and differentiation of various cell types (Miao, Wu, & Shi, 2017) as well as stem cell fate in coordination with NANOG and POU5F1 (Chan et al., 2009). In the current study, we found that the higher expression of KLF4 was accompanied by differential expression of KLF4 target genes under high oxygen tension namely: POU5F1 and GATA4 (Figure 7). This could explain a disturbed embryonic stemness pattern at higher oxygen level (Jang et al., 2014), which was remarkable in male embryos. Irrespective of the sex of the bovine embryo, transcript analysis of blastocysts showed upregulation of pluripotency-related genes (NANOG and SOX2), but not POU5F1 in response to OS (Leite et al., 2017). However, the mRNA level of GATA4 was upregulated in male embryos cultured either in 5 or 20% oxygen level compared with female counterparts.
The EVs are supposed to be involved in several biological functions including embryo-maternal communication (Saadeldin, Oh, & Lee, 2015;Szekeres-Bartho, Šućurović, & Mulac-Jeričević, 2018). Their size, as well as concentration, could vary according to cellular physiological status. Accordingly, our results indicated that the higher number and bigger size EV (Figure 8c) were found in spent media of male embryos cultured under high oxygen level. Similar results were obtained in our previous study in granulosa cells exposed to OS (Saeed-Zidane et al., 2017). In TAQI ET AL.

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contrast, the female embryos exhibited a lower number and bigger size EVs upon exposure to OS. A possible explanation for this sexual dimorphic phenotype can be differences in the expression of genes involved in biogenesis and release of EVs (Hessvik & Llorente, 2018;Kowal, Tkach, & Théry, 2014). Accordingly, the mRNA expression pattern of those genes revealed that ALIX and VPS4B were significantly increased in male embryos exposed to OS condition (Figure 9). However, female embryos showed higher expression of ALIX transcript only. In addition, the mRNA expression level of RAB11FIP1 and RAB27A, responsible for EVs secretion (Kowal et al., 2014), showed an upregulation in male embryos exposed to OS compared with 5% oxygen level and/or female counterparts. This may indicate the sexual dimorphic physiological need in biogenesis and secretion of EVs.
It is not only the number of EVs released into extracellular space but also the molecular cargo of those EVs, which was differed between male and female exposed to OS. As shown in Figure 10, EVs from male embryos at higher oxygen level contained higher level of NFE2L2 and SOD1 transcripts compared to those cultured under low oxygen level, which might be beneficial of development of embryos (Pavani et al., 2018). The role of EVs is not only in cell-cell communication (Saeed-Zidane et al., 2017) but also in the removal of undesirable cellular molecules (Takahashi et al., 2017). Contrary to cellular expression, the NOTCH1 was increased in EVs derived from male embryos exposed to OS compared with female counterparts, which may indicate the selectivity of cargo molecules to be exported by EVs (Bhome et al., 2018;Hinger et al., 2018) to facilitate cell to cell communication or maintain of cellular homeostasis. Herein, we can summarize that exposing preimplantation bovine embryos to OS compromised the transformation of blastocysts irrespective of embryo sex, due to increasing intracellular ROS accumulation, which in turn leads to increase apoptosis and delay of differentiation in response to dysregulating of the TFs related to stress response, development, differentiation, and apoptosis. Interestingly, the reduction in blastocyst formation and total cell count was more pronounced in female embryos exposed to OS compared with male counterparts, which was accompanied with alteration in cellular expression and subsequent release of TFs through EVs. Taken together, exposure either male or female embryos to OS altered their TFs expression pattern coupled with a reduction in development and delaying in differentiation. However, the male embryos are more tolerant to OS than female counterparts via activation of NFE2L2 and their downstream antioxidant genes at the cellular and extracellular levels, which partially regulate other TFs involved in apoptosis, differentiation, and development leading to skewed sex ratio toward male embryos.

| Oocyte collection and maturation
Ovaries were obtained from a nearby slaughterhouse and transported to the laboratory in 0.9% physiological saline solution (NaCl) within 2-3 hr. Upon arrival, the ovaries were rinsed with prewarmed (37°C) 70% ethanol followed by two to three times washing in fresh phosphate buffer saline (PBS), and cumulus-oocyte complexes (COCs) were then aspirated from small follicles (2-8 mm), using sterilized syringe attached to 18G needle. Oocytes with homogenous cytoplasm and surrounded by multiple layers of compacted cumulus cells were then selected for in vitro maturation. Groups of 50 COCs were transferred to TCM 199 modified culture media (Sigma-Aldrich, Munich, Germany) supplemented with 4.4 mM HEPES, 33.9 mM NaCHO 3 , 2 mM pyruvate, 2.9 mM calcium lactate, 55 mg/ml gentamicin and 12% (vol/vol) heat-inactivated estrus cow serum (Schellander, Fuhrer, Brackett, Korb, & Schleger, 1990) and then cultured for 22-24 hr at 39°C under 5% CO 2 in air with maximum humidity.

| Intracellular ROS detection
To investigate the effect of OS (20% O 2 ) on intracellular ROS accumulation in male and female bovine embryos, the ROS level in blastocyst was detected using the 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescent probe (Life Technologies, Darmstadt, Germany). Fifteen blastocysts from each group were incubated with 400 µl of 5 µM H2DCFDA for 20 min in the dark at 37°C. Thereafter, the embryos were washed twice in PBS-PVA and the images were immediately acquired under an inverted microscope (Leica DM IRB, Germany), using a green fluorescence filter. The fluorescent intensity was analyzed as a single embryo for each group using ImageJ 1.48v software (National Institutes of Health, http://imagej.nih.gov).

| Total cell count
Total blastocyst cell number of male and female bovine embryos cultured under low (5%) or high (20%) oxygen levels were quantified using nuclear fluorescence staining with the glycerol-based Hoechst 33342 (Sigma-Aldrich) according to the manufacturer's procedure. For that, 10 blastocysts from each group were fixed for 5 min in a solution containing 2% formalin and 0.25% glutaraldehyde. The fixed blastocysts were mounted and stained for 10 min with glycerol-based Hoechst 33342 (12.5 mg/ml) solution on clean glass slides. The stained nuclei were visualized using a fluorescent microscope (Olympus, Tokyo, Japan) with a blue filter (excitation: 330-385 nm; emission: 420 nm; dichromatic: 400 nm). The cell number was recorded for individual blastocysts from each cultured group using Zen 2.3V (blue edition; https://www.zeiss.com/ microscopy/int/products/microscope-software/zen-lite.html).

| RNA extraction and complementary DNA synthesis
Four biological replicates (pool of 10 blastocysts/each) embryos from each group were used for RNA extraction. Total RNA was extracted using a PicoPure RNA isolation kit (Arcturus, Munich, Germany) according to the manufacturer's instruction. On-column DNA digestion was performed using RNase-free DNase enzyme (Qiagen GmbH, Hilden, Germany). After two washes with washing buffer, the RNA was eluted in 12 µl elution buffer and stored at −80°C until further use. In addition, the total RNA from EVs of four biological replicates was isolated using Norgen's exosomal RNA isolation kit (Norgen Biotek, Canada) following the manufacturer's procedure. Briefly, a combination of lysis buffer was added to certain volume of PBS-CMF (calcium magnesium-free) containing EVs followed by vortex and incubation for 10 min at room temperature, respectively. After incubation, 500 µl of absolute ethanol was added to the mixture and mixed well by vertexing. Appropriate volume from mixture was transferred to a mini spin column and then centrifuged at 3,300g. After the withdrawal of the flow through, the mini spin column was washed twice with washing buffer each time followed by centrifugation at 3,300g for 30 s. Afterward, the mini spin column was dried with centrifugation at 13,000g for 1 min at room temperature.
Finally, the RNA was eluted in appropriate amount of elution buffer and then stored at −80°C till further application.
RNA concentration was measured (Tables S1 and S2) using 8000 NanoDrop ™ Spectrophotometer (Thermo Fisher Scientific, Germany) and equal quantities of RNA input was used for complementary DNA (cDNA) synthesis. The cDNA synthesis was performed using the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Briefly, an equal amount of RNA was incubated with 0.5 µl of oligo-dT and 0.5 µl random primer, followed by incubation at 65°C for 5 min. Besides this, a master mix containing 4 µl of 5X reaction buffer, 1 µl of RiboLock RNase inhibitor, 2 µl of 10 mM dNTP mix, and 2 µl of M-MuLV reverse transcripts were prepared and gently mixed by pipetting. At the end of incubation, a total of 9 µl was added to each specimen, and then incubated at 25°C for 5 min, then 37°C for 60 min followed by 70°C for 5 min to terminate the reaction. After the incubation time, the cDNA was kept at −20°C for gene expression analysis.

| Quantitative real-time PCR
The transcript level of developmental and stress-related TFs namely,  Table 1). The master mix containing cDNA, optimized amount of forward and reverse primers and SYBR green master mix was run in a thermal cycler program of 95°C for 3 min initial denaturation, followed by 40 cycles of 15 s at 95°C and 60 s at 60°C.
Melting curve was generated to verify the amplicon specificity. The expression level of ACTB and GAPDH was used for normalization for embryonic transcripts, while 18S was used as a third housekeeping gene for EV-coupled transcript profiling. The relative abundance of gene transcript was expressed as arbitrary unit calculated by t t a r g e t g e n e t housekeeping gene (Livak & Schmittgen, 2001).

| Immunohistochemistry
To quantify and localize the NFE2L2 and KLF4 proteins in male and female bovine embryos cultured under 5 and 20% oxygen levels, immunohistochemistry assay was conducted. For this, 10 embryos from each group were fixed using 4% paraformaldehyde and kept at 4°C overnight. Thereafter, the fixed embryos were washed four times for 5 min each with glycine in PBS, followed by permeabilization step using 0.5% (vol/vol) Triton X-100 (Sigma-Aldrich) in PBS for 3 hr at room temperature. After the incubation time, the samples were incubated with 4% donkey serum for 1 hr at room temperature, followed by incubation at 4°C overnight with primary rabbit polyclonal antibody for NFE2L2 ( procedure. Finally, the isolated EVs were suspended in PBS-CMF and kept at −80°C for further investigation.
The purity of the isolated EVs was examined using immunoblotting for the presence of EV marker CD63 protein and for the absence of mitochondrial cellular marker, CYCS. Briefly, an equal amount of EVs solution from each group was incubated with 2X sodium dodecyl sulfate (SDS) loading buffer at 95°C for 5 min and then loaded onto 12% SDS-PAGE gel. Following electrophoresis, the proteins were transferred to nitrocellulose membranes (Protran ® , Schleicher & Schuell Bioscience) using a semi-dry blotting system (Bio-Rad Laboratories GmbH). Subsequently, the membrane was blocked using 1X blocking solution (Carl Roth GmbH) for 1 hr at room temperature followed by incubation overnight at 4°C with the primary rabbit polyclonal antibody against CD63 (1:1,000; System BioSciences) and goat polyclonal against CYCS (1:350; Santa Cruz Biotechnology Inc, Germany). After the incubation time, the membrane was washed three times with diluted Tris-buffer saline with Tween 20 (TBST) followed by incubation for 1 hr at room temperature with goat anti-rabbit (1:5,000) (System BioSciences) and donkey anti-goat (1:5,000; Santa Cruz Biotechnology Inc) secondary antibodies. Afterward, the membrane was washed with diluted TBST followed by incubation with Clarity ECL Substrate (Bio-Rad Laboratories GmbH). Finally, the bands were visualized and the images were picked up using the ChemiDoc ™ XRS+ system (Bio-Rad Laboratories GmbH).
The concentration and size of EVs were evaluated using Nano-Sight NS300 following manufacturer protocols (Malvern Instruments, Malvern, UK). Briefly, a total of 50 µl from isolated EVs were diluted in 1 ml PBS-CMF, and then five consecutive videos were recorded.
The recorded videos for each sample were analyzed using NTA software to obtain mean, mode of the particles size and concentration per ml of volume.

| Electron microscopy
A total of 30 µl from isolated EVs were used for visualization under electron microscopy. Briefly, the sample was dropped on parafilm and coincubated with Formvar/carbon-coated grids for 5 min at room temperature. Afterward, the Formvar/carbon-coated gird was washed with PBS and subsequently stained with 2% uranyl acetate followed by washing with PBS. Finally, the grids were dried and the images were acquired using electron microscopy.

| Statistical analysis
The data were statistically analyzed using two-way analysis of variance (ANOVA) followed by multiple pair-wise comparisons using the Tukey post hoc test (GraphPad Prism Version 7). The data are presented as mean ± SEM of biological quadruplicate. Statistical significance was determined at p ≤ .05.

ACKNOWLEDGMENTS
We are grateful to Dr Reinhard Bauer at Life and Medical Sciences