High and low performing sires differ in their contributions to early embryonic stress in the bovine
Lindsey Fallon A C , Kelsey N. Lockhart A , Thomas E. Spencer A and M. Sofia Ortega
B *
A
B
C Present address:
Abstract
Sires differ in their ability to produce viable blastocysts, yet our understanding of the cellular mechanisms regulated by the sire during early embryo development is limited.
The first aim was to characterise autophagy and reactive oxygen species (ROS) in embryos produced by high and low performing sires under normal and stress culture conditions. The second aim was to evaluate DNA damage and lipid peroxidation as mechanisms that may be impacted by increased cellular stress, specifically oxidative stress.
Embryos were produced using four high and four low performing sires based on their ability to produce embryos. Autophagy and ROS were measured throughout development. To evaluate oxidative stress response, autophagy, and ROS were measured in 2–6 cell embryos exposed to heat stress. To understand how cellular stress impacts development, DNA damage and lipid peroxidation were assessed.
Under normal conditions, embryos from low performing sires had increased ROS and autophagy. Under heat stress, embryos from low performing sires had increased ROS, yet those from high performing sires had increased autophagy. There was no difference in DNA damage or lipid peroxidation.
Results suggest that embryos from low performing sires may begin development under increased cellular stress, and autophagy potentially increases to mitigate the impacts of stress.
There is potential for improving embryonic competence through selection of sires with lower stress-related markers.
Keywords: autophagy, cellular stress, embryo development, reactive oxygen species, sire fertility.
Introduction
Pregnancy loss is one of the primary contributors to economic failure in the dairy cattle industry (Maurer and Chenault 1983; Diskin and Morris 2008; Cerri et al. 2009; Wiltbank et al. 2016). Pregnancy loss can be classified under two predominant categories: early pregnancy loss, which occurs between days 1 and 27; and late pregnancy loss, which occurs from day 28 throughout gestation (Wiltbank et al. 2016). As gestation continues, the probability of pregnancy loss decreases. Specifically, the first week following fertilisation is a critical time for embryo development and survival. Events such as the first cellular divisions (cleavage), embryonic genome activation, clearance of paternal mitochondria, cell polarisation and compaction, and cell lineage specifications are all crucial steps for embryo survival (Fig. 1) (van Soom et al. 1997; Memili and First 1999; Graf et al. 2014). If an embryo is unable to complete any of these steps, it will arrest development.
There is evidence that the sire influences pregnancy success during the events of fertilisation, early embryo development, and conceptus elongation (Ortega et al. 2018; Lockhart et al. 2023a), yet our understanding of the malfunction of cellular mechanisms regulated by sire during early embryo development is still lacking. Previous studies in our laboratory have identified sires with a decreased ability to produce blastocysts (classified as low performing), and have found them to produce a greater proportion of less viable spermatozoa, indicated by pre-mature membrane alteration and capacitation as well as a significant population of spermatozoa containing protein aggresomes in their heads which may potentially be incorporated into a zygote upon fertilisation (Fallon et al. 2023). Additionally, these low performing sires produce embryos with an increased expression of genes related to DNA damage, apoptosis, oxidative stress, and increased autophagic activity, and have resulted in a higher proportion of embryos undergoing arrested development at the 4–6 cell stage (Lockhart et al. 2023b).
Autophagy is a cell recycling mechanism that degrades targeted proteins and organelles and allows for the subsequent byproducts to be re-used (Mizushima 2007; Song et al. 2012). Autophagic activity is also known to be upregulated during times of cellular stress such as the response to misfolded or aggregated proteins and cell starvation (Song et al. 2012). Another mechanism upregulated during times of stress is the production of reactive oxygen species (ROS), which are molecules derived from molecular oxygen that have an increased ability to oxidise other molecules that they encounter (Bayr 2005; Sies and Jones 2020), and are primarily produced in the mitochondria during oxidative phosphorylation (Kuznetsov et al. 2011). High levels of intracellular ROS can damage the cell and lead to DNA damage, lipid peroxidation, and endoplasmic reticulum (ER) stress which causes the misfolded protein response (Guerin 2001; Bayr 2005; Kuznetsov et al. 2011).
Utilising sires that are known to produce less viable spermatozoa which contain aggregated protein defects, and are known to produce embryos with increased gene expression related to oxidative stress, is an ideal model to further investigate the cellular stress mechanisms impacted by sire during early embryo development. This leads to the hypothesis of the current study, that embryos produced by low performing sires have increased autophagic activity in response to an increase in cellular stress and ROS production, compared to embryos produced by high performing sires, therefore resulting in impaired early development. The first aim of this study was to characterise the autophagic activity and ROS production of embryos produced by high and low performing sires under both normal culture conditions and induced environmental stress conditions. The second aim was to evaluate mechanisms that may be impacted downstream from cellular stress such as DNA damage and lipid peroxidation in preimplantation bovine embryos.
Materials and methods
Embryo production
All media for oocyte collection and maturation, as well as in vitro embryo production, was prepared in-house, as previously described (Ortega et al. 2017, 2018). Briefly, cumulus–oocyte complexes (COCs) were collected from abattoir-derived ovaries and washed with a medium consisting of Tissue Culture Medium-199 with Hanks salts plus 25 mM HEPES. COCs with at least three layers of compact cumulus cells and homogeneous cytoplasm were selected and placed in an oocyte maturation medium warmed to 38.5°C and equilibrated with air containing 5% (v/v) CO2. COCs, in groups of 50, were incubated for approximately 22 h to mature prior to fertilisation. The recipe of maturation medium was as follows: Tissue Culture Medium-199 with Earle salts (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, 0.2 mM sodium pyruvate, 2 mM L-glutamine, 50 ng/mL recombinant human epidermal growth factor (Invitrogen, Waltham, MA, USA), and 5.0 μg/mL of follicle-stimulating hormone (FSH; Folltropin; Bioniche Animal Health, Athens, GA, USA). Following maturation, COCs were washed three times in HEPES-TALP and placed in a culture dish containing 1.7 mL of IVF-TALP medium. Semen straws used in all experiments were gifted by Select Sires Inc. (Great Plains, OH, USA) and were processed in the same commercial house. Semen was collected from Holstein sires at approximately 28 months of age on average. Up to four high performing sires and four low performing sires were selected for the experiments of this study based on previous classification as having high or low capacity to produce embryos (Clark and Ortega 2021; Fallon et al. 2023; Lockhart et al. 2023b). Spermatozoa were prepared for fertilisation using an isolate gradient, as previously described by Fallon et al. (2023). Specifically, semen straws were thawed in a water bath at 37°C for 40–45 s. Concomitantly, 600 μL of 50% density upper layer gradient was gently pipetted onto 600 μL of 90% density lower layer gradient (Isolate™, Irvine Scientific, Santa Ana, CA, USA), in a 1.5 mL microcentrifuge tube. Semen was extruded from the straw into the gradient tube and centrifuged at 700g for 5 min. The sperm pellet (100 μL) was removed from the bottom of the tube, placed into an Eppendorf tube containing 1 mL of warm Hepes-TALP medium, and centrifuged again at 700g for 3 min. This wash step was then repeated once more, for a total of two washes in Hepes-TALP per sample. Sperm were then diluted in fertilisation medium (IVF-TALP) to a final concentration of 1 × 106/mL in the fertilisation plate. Mature oocytes and sperm were incubated together for 18–20 h at 38.5°C with air containing 5% (v/v) CO2, following which cumulus cells were removed, and putative zygotes were placed in synthetic oviductal fluid (SOF-BEII) culture medium in a controlled environment (38.5°C with a humidified atmosphere of 5% (v/v) CO2, 5% (v/v) O2, 90% (v/v) N2). Cleavage rates were assessed on the third day of embryo culture (66–72 h post insemination, HPI) and blastocyst rates were assessed in the morning on the eighth day of embryo culture (186–192 HPI) (Fallon et al. 2023). Throughout the experiments of Fallon et al. (2023) and the experiments reported in this manuscript, cleavage and blastocyst rates were recorded for a similar number of embryos produced by all four low performing sires and four high performing sires.
Fertilisation rate
A total of 117–130 putative zygotes per sire classification (four high and four low performing sires) were collected at 18–20 HPI, throughout four replicates. Putative zygotes were stripped of cumulus cells both with mechanical motion, as well as treatment with pronase (Thermo-Fisher Scientific, Carlsbad, CA, USA) for 60 s. They were then washed three times in phosphate-buffered saline with polyvinylpyrrolidone (PBS-PVP: 1 mg PVP per 1 mL of 1× PBS), fixed in 4% paraformaldehyde for 15 min at room temperature, and washed three more times in PBS-PVP. Putative zygotes were then incubated with Hoechst 33342 nuclear stain (Thermo-Fisher Scientific) at a concentration of 1 μg/mL, diluted in PBS-PVP, for 15 min before being mounted on glass slides with 6 μL of slow fade gold antifade solution (Thermo-Fisher Scientific). Slides were imaged on a Leica DM5500 B fluorescent microscope with an attached Leica DFC450 C camera at the pro-nuclear stage to assess the fertilisation rate. Zygotes containing two pronuclei were considered successfully fertilised.
Autophagy and ROS during development
A total of 60–80 embryos per stage, per fertility classification (four high and four low performing sires), were collected at four stages of development, namely 2–6 cell (30–55 HPI), 8–16 cell (60–75 HPI), morula (120–144 HPI), and blastocyst (168–192 HPI), over the course of three replicates and co-stained to assess both autophagy and ROS production in each live embryo. Autophagosomes were tagged using the Cyto-ID Autophagy Detection Kit 2.0 (Enzo Life Sciences) as previously described (Chan et al. 2012), and ROS were tagged using CellROX Deep Red Reagent (Thermo-Fisher Scientific). Briefly, 1 μL of Cyto-ID Green Autophagy Stain was added to 500 μL of the provided assay buffer, and 0.5 μL of that mixture was added to 250 μL of SOF-PVP (1 mg PVP per 1 mL SOF-BEII) warmed to 38.5°C. CellROX Deep Red Reagent was also diluted in warm SOF-PVP to a 10 μM concentration. Then, 80 μL of each stain was added to a 96-well plate and mixed thoroughly. Embryos were washed three times in warm SOF-PVP, then placed into stain mixture and incubated for 25 min at 38.5°C with a humidified atmosphere of 5% (v/v) CO2, 5% (v/v) O2, and 90% (v/v) N2. The efficacy of this staining protocol was validated during method development using the positive control of rapamycin treatment which was recommended by the manufacturer. Two to six cell embryos were treated with 500 nM rapamycin prior to receiving the stain mixture. Positive controls were not continuously run throughout the experiment though. Following incubation, embryos were immediately washed again in warm SOF-PVP and mounted on glass slides in 6 μL of antifade solution (Thermo-Fisher Scientific). All embryos were imaged under identical lighting conditions on a Leica DM5500 B fluorescent microscope with an attached Leica DFC450 C camera and mean fluorescent intensity (MFI) of each image was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA). MFI was calculated by subtracting the mean background intensity from the mean embryo intensity, on each channel individually. ROS was measured at 590 nm wavelength with red fluorescence and autophagy was measured at 510 nm wavelength with green fluorescence.
Autophagy and ROS under normal vs heat stress conditions
Following the removal of cumulus cells via mechanical motion during in vitro embryo production, zygotes were randomly assigned to the treatment group or control group. Treatment embryos were exposed to heat stress at 41°C for 4 h before being placed in normal culture conditions (38.5°C with a humidified atmosphere of 5% (v/v) CO2, 5% (v/v) O2, 90% (v/v) N2). Heat stress temperature was chosen based on previous literature (Al-Katanani and Hansen 2002). Control embryos were placed directly into normal culture conditions. A total of 60–80 embryos per stage, per fertility classification (two high and two low performing sires, determined by the previous experiment) were collected at the 2–6 cell stage of development (30–55 HPI) over the course of four replicates and co-stained to assess both autophagy and ROS production. Staining, imaging, and data analysis for autophagic activity and ROS production were performed as in the previous experiment. Developmental data for this experiment were analysed including cleavage rates recorded from IVF replicates that produced embryos to be collected at the 2–6 cell stage for staining, and therefore do not have corresponding blastocyst rates.
DNA damage
A total of 108–135 embryos per fertility classification (four high and four low performing sires) were collected at the 2–6 cell stage (30–55 HPI) over the course of three replicates and stained using the In Situ Cell Death Detection kit (Roche Diagnostics, Indianapolis, IN, USA) to assess the presence of DNA fragmentation in each embryo. Embryos were washed three times in PBS-PVP, fixed in 4% paraformaldehyde for 15 min at room temperature, washed three times again, and permeabilised in permeabilisation solution (0.005% Triton X-100 diluted in PBS) for 40 min at room temperature. TUNEL solution was prepared by adding 15 μL of the provided enzyme to 135 μL of the label solution. For each replicate, three to five embryos were prepared to be positive controls, and three to five were set aside for negative controls. Positive control embryos were incubated in 8 μL of DNAse for 10 min before adding 25 μL of the TUNEL solution. At that time, embryos were placed in either a negative control group (into 25 μL the label solution only) or placed into 25 μL of prepared TUNEL solution and both were incubated for 1 h at 38.5°C in a humidified atmosphere of 5% (v/v) CO2, 5% (v/v) O2, and 90% (v/v) N2. Following incubation, all embryos were washed three times in PBS-PVP and placed into Hoechst 33342 nuclear stain (Thermo-Fisher Scientific) at a concentration of 1 μg/mL, diluted in PBS-PVP, for 15 min. Embryos were then mounted on glass slides with antifade solution (Thermo-Fisher Scientific) and imaged under identical lighting conditions on a Leica DM5500 B fluorescent microscope with an attached Leica DFC450 C camera. The presence or absence of DNA fragmentation, indicated by fluorescent labelling when measured at 510 nm wavelength with green fluorescence, was recorded, and then the percentage of TUNEL-positive cells per embryo was estimated. If an embryo contained at least one TUNEL-positive cell it was considered positive in the analysis.
Lipid peroxidation
A total of 108–130 embryos per fertility classification (four high and four low performing sires) were collected at the 2–6 cell stage (30–55 HPI) over the course of six replicates and stained with the Image-iT™ Lipid Peroxidation Kit (Thermo-Fisher Scientific) to quantify the percentage of oxidised lipids in each live embryo. Lipid peroxidation stain was used according to manufacturer recommendations. Embryos were washed three times in warmed (38.5°C) SOF-PVP before three to five were set aside to be positive controls. To prepare the positive control, 1 μL of the provided cumene hydroperoxide was diluted in 54 μL of pure ethanol, and then 0.5 μL of this stock solution was added to 500 μL of 1× PBS for a final concentration of 100 mM. Embryos assigned to the positive control group were added to 100 μL of this solution for 10 min at 38.5°C. The lipid peroxidation stain was diluted to a 10 μM concentration in warm 1× PBS-PVP. At this time, all embryos were placed into the stain and incubated for 30 min at 38.5°C with a humidified atmosphere of 5% (v/v) CO2, 5% (v/v) O2, and 90% (v/v) N2. Following incubation, embryos were immediately washed again three times in warm SOF-PVP, mounted on glass slides in 6 μL of antifade solution (Thermo-Fisher Scientific), and imaged on a Leica DM5500 B fluorescent microscope with an attached Leica DFC450 C camera. To quantify the percentage of lipids oxidised, the MFI of each embryo was measured using ImageJ software. All lipids were measured at 590 nm wavelength and oxidised lipids were measured at 510 nm wavelength, with the background fluorescence subtracted from the embryo fluorescence, before dividing the oxidised lipid value by the total lipid value for a final percentage.
Statistical analysis
Analyses were performed using SAS (ver. 9.4), and significance was considered when P < 0.05. All variables were tested for normality and log-transformed as needed using the link function in the GLIMMIX procedure. Fertilisation was analysed with a binomial logistic regression model using the GLIMMIX procedure; the model included a fixed effect of performance and a random replicate, and differences in means between sire performance groups were determined using the pdiff option of LSMEANS with the Tukey adjustment. To evaluate autophagy and ROS during development, a generalised linear mixed model was used using the GLIMMIX procedure; the model included a fixed effect of fertility classification (performance), developmental stage, and their interaction, and replicate as a random effect. Differences in means between sire performance groups were determined using the pdiff option of LSMEANS with the Tukey adjustment. Autophagy and ROS under normal or heat stress conditions were analysed in a 2 × 2 factorial design considering the effects of two factors, performance and treatment (each with two levels), and their interaction. Differences in means between sire performance groups were determined using the pdiff option of LSMEANS with the Tukey adjustment. DNA damage and lipid peroxidation were with a binomial logistic regression model, using the GLIMMIX procedure in SAS. The model included a fixed effect of performance and a replicate as random. Differences in means between sire performance groups were determined using the pdiff option of LSMEANS with the Tukey adjustment.
Results
Fertilisation rate and embryo development
Putative zygotes were determined to be successfully fertilised by the presence of two pronuclei at 18–20 HPI (Fig. 2a). The fertilisation rates of each fertility classification (n = 117–131) are shown in Fig. 2b. There was no difference observed between high (77.86 ± 3.62%) and low (74.78 ± 4.05%) performing sires (P = 0.571). Cleavage and blastocyst rates are shown in Fig. 2c, d. Cleavage rates from embryos produced by high performing sires (76.73 ± 2.57%) were significantly higher (P = 0.029) than cleavage rates from low performing sires (68.25 ± 2.74%) (Fallon et al. 2023). Similarly, blastocyst rates from embryos produced by high performing sires (29.04 ± 1.78%) were significantly higher (P = 0.001) than blastocyst rates from low performing sires (19.94 ± 1.90%) (Fallon et al. 2023).
Fertilisation rate and embryonic development. (a) Representative image of a zygote containing both male and female pronuclei just prior to syngamy. (b) No difference was observed (P = 0.571) between high performing and low performing sires’ ability to successfully fertilise oocytes (n = 117–131). (c, d) Embryos produced by high performing sires had significantly higher cleavage rates (*, P = 0.029) and blastocyst rates (***, P = 0.001) than embryos produced by low performing sires (Fallon et al. 2023). All data are shown as LS means ± s.e.m.
Autophagy and ROS during development
When comparing the ROS production and autophagic activity of embryos produced from either high or low performing sires, under normal culture conditions, the most noticeable differences were observed at the 2–6 cell stage of development (Fig. 3b, c). Embryos at the 2–6 cell stage, produced by low performing sires exhibited an increase in both ROS production (21.89 ± 0.79) and autophagic activity (13.28 ± 0.47) compared to embryos from high performing sires (16.18 ± 0.94, P < 0.0001; 11.28 ± 0.56, P = 0.007), respectively. Embryos at the morula stage, produced by low performing sires, also exhibited an increase (P = 0.009) in ROS production (22.45 ± 1.23) compared to embryos from high performing sires (18.16 ± 1.09), but no corresponding increase in autophagy was observed. There were no significant differences observed between the groups at the 8–16 cell or blastocyst stages of development (P > 0.05).
Autophagy and reactive oxygen species (ROS) production during development. (a) Representative images of the 2–6 cell stage in embryos from high performing sires (left) and low performing sires (right). Fluorescence emitted from ROS activity is shown in magenta and fluorescence emitted from autophagic activity is shown in green. Mean fluorescent intensity (MFI) for each process is listed below the corresponding image. (b) Differences in ROS production between high a low performing sires (n = 60–80 embryos per stage, per classification). Low performing sires had increased ROS at the 2–6 cell stage (****, P < 0.001), and the morula stage (**, P = 0.009). (c) Two to six cell embryos produced by low performing sires had significantly increased (**, P = 0.007) autophagic activity compared to embryos produced from high performing sires (n = 60–80 embryos per stage, per sire classification). All data are shown as LS means ± s.e.m.
Autophagy and ROS under normal vs heat stress conditions
To assess the impact of induced stress on embryos from each group, ROS production and autophagic activity were compared at the 2–6 cell stage of development, and cleavage and blastocyst rates were recorded (Fig. 4). ROS production was affected by sire performance (P < 0.0001) and by heat stress (P = 0.0004); no main effect of interaction was found (P = 0.1544). Embryos at the 2–6 cell stage produced by low performing sires under heat stress had increased ROS production (25.52 ± 0.62) when compared to embryos produced by high performing sires under heat stress (21.68 ± 0.82; P = 0.0002), embryos from low performing sires under normal conditions (22.31 ± 0.52; P < 0.0001), and embryos from high performing sires under normal conditions (20.29 ± 0.56; P < 0.0001). Additionally, the ROS production from embryos produced by low performing sires under normal conditions was significantly increased when compared to embryos produced by high performing sires under normal conditions (P = 0.0086). In contrast, in this experiment, autophagic activity was affected by heat stress (P = 0.004), but no main effect of performance (P = 0.3508) or interaction was found (P = 0.129). However, 2–6 cell embryos produced by high performing sires under heat stress had increased autophagic activity (25.53 ± 3.41) when compared to embryos from low performing sires under normal conditions (15.30 ± 2.16; P = 0.0117) and embryos from high performing sires under normal conditions (13.74 ± 2.35; P = 0.0046). Cleavage rates from embryos produced by high performing sires cultured under normal conditions (70.74 ± 2.71%) were significantly higher (P = 0.015) than cleavage rates from embryos produced by low performing sires cultured under heat stress (57.95 ± 4.37%). There was no difference in blastocyst rate observed between embryos of any group (P > 0.05).
Autophagy and reactive oxygen species (ROS) production under induced environmental stress compared to normal culture conditions. (a) Representative images of 2–6 cell, heat-stressed embryos from high performing (left) and low performing sires (right). Fluorescence emitted from ROS activity is shown in magenta and fluorescence emitted from autophagic activity is shown in green. Mean fluorescent intensity (MFI) for each process is listed below the corresponding image. (b) Embryos from low perfoming sires had increased ROS production compared to high performing sires under heat stress (***, P = 0.0002), low performing sires under normal conditions (****, P < 0.0001), and high performing sires under normal conditions (****, P < 0.0001). Embryos produced by low sires under normal conditions had increased ROS compared with embryos produced by high sires under normal conditions (**, P = 0.0086) (n = 60–80 embryos per sire classification). (c) Embryos produced by high performing sires under heat stress had increased autophagic activity when compared to embryos from either low (*, P = 0.0117) or high performing sires (**, P = 0.0046) under normal conditions (n = 60–80 embryos per sire classification); each point is an embryo. (d) Embryos produced by high performing sires under normal conditions had significantly higher cleavage rates (*, P = 0.015) than embryos produced by low performing sires under heat stress conditions. (e) There was no difference in blastocyst rate observed between embryos of any group (P > 0.05); each point is an in vitro production run. All data are shown as LS means ± s.e.m. Triangles represent embryos subjected to heat stress, and circles represent embryos cultured under normal conditions.
DNA damage
The DNA damage present in embryos produced from either high or low performing sires is shown in Fig. 5a. There was no difference (P = 0.334) in the percent of TUNEL-positive cells in embryos produced from high performing sires (6.72 ± 2.16%) when compared to embryos produced from low performing sires (10.19 ± 2.91%).
DNA damage and percent of lipid peroxidation present in 2–6 embryos from high and low performing sires. (a) No difference was observed (P = 0.334) in the percent of TUNEL-positive cells in embryos produced by high and low performing sires; a positive control picture is shown. (b) No difference was observed (P = 0.079) in the percent of peroxidised lipids in embryos produced by high and low performing sires. (c) Representative images of 2–6 cell embryos produced by high performing sires (left) and low performing sires (right), stained for the quantification of peroxidised lipids. Each point is an in vitro production run (replicate). All data are shown as LS means ± s.e.m.
Lipid peroxidation
The percentage of lipids oxidised in embryos produced from either high or low performing sires is shown in Fig. 5b. There was no difference (P = 0.079) in the percent of lipids that had been oxidised in embryos produced from high performing sires (24.44 ± 0.71%) when compared to embryos produced from low performing sires (22.57 ± 0.79%).
Discussion
This study confirmed that sire influences early embryo development and has direct effects on embryonic stress and the ability to respond to stress. There was no difference found in the fertilisation rate between groups of sires, which shows that sires with varying fertility status in this study do produce a population of spermatozoa that have an equal ability to penetrate the zona pellucida of an oocyte. However, differences in cleavage and blastocyst rates indicate that sires do vary in their ability to produce a viable embryo that will develop to the blastocyst stage (Fallon et al. 2023). Following penetration of the zona pellucida, the spermatozoon causes meiosis resumption and begins undergoing changes in the oocyte cytoplasm that are crucial for subsequent embryo success. First, the reduction of disulfide bonds between protamine proteins that were incorporated into the sperm’s nucleosomes during maturation in the epididymis (Collas and Poccia 1998; Senger 2003) and the replacement of protamine proteins by oocyte-derived histones, initially by the linker H1FOO histone (Gao et al. 2004). This parallels the release of the proximal centriole from the spermatozoon, which will recruit maternal pericentriolar material to form a sperm aster. The sperm aster is responsible for bringing the male and female pronuclei together to allow for pronuclear apposition, which is critical for the formation of the mitotic spindle and the first zygotic cleavage (Avidor-Reiss et al. 2020). Alterations to any of these processes would prevent the first cleavage division and result in arrest. This could explain, in part, the sire’s influence during early embryo development.
In this study, embryos produced from low performing sires appear to begin development with an increased level of cellular stress under normal culture conditions, specifically oxidative stress, demonstrated by an increase in ROS production and autophagic activity as early as the 2–6 cell stage of development. Previous studies have demonstrated that high levels of intracellular ROS lead to cellular damage, and in embryos, may lead to impaired developmental competence (Yang et al. 1998; Sakatani 2017). In murine embryos, it was shown that there is an increase in ROS production around the 2 cell stage, which coincided with embryo arrest in that study (Nasr-Esfahani and Johnson 1991). In the bovine, a reduction in oocytes that developed to the blastocyst stage has been correlated with increased ROS and decreased glutathione levels (Hashimoto et al. 2000).
Under induced stress conditions in the form of a heat shock, 2–6 cell embryos produced by low performing sires continued to have increased ROS production compared to all other embryos. Alternatively, 2–6 cell heat-stressed embryos produced by high performing sires had the highest autophagic activity compared to control embryos produced by either group. Interestingly, there was no difference observed in the autophagic activity in embryos produced by high and low performing sires assigned to control conditions. Although the same sires were used in both experiments, and a similar number of embryos were assessed in both experiments, higher variation was observed in this analysis. This difference in variation is one limitation of the present study. It is hypothesised that it could potentially be due to an oocyte effect on early embryo development, and the accumulation of appropriate mRNA transcripts to produce proteins necessary for autophagic machinery. These data show that higher cellular stress leads to an increase in both autophagy and ROS accumulation, with autophagy potentially increasing to mitigate the deleterious impacts of stress. Embryos from low performing sires appear to be unable to upregulate their autophagic pathway to the same capacity that embryos from high sires do under induced environmental stress, indicating that embryos produced by low performing sires have a less robust mechanism for moderating stress via this pathway, or that this pathway becomes overwhelmed and begins leading to apoptosis. Previous literature has shown that the autophagic pathway is initiated to promote cell survival, such as during cell starvation in vitro (Mizushima 2007; Song et al. 2012), and has been correlated with embryo development to the blastocyst stage in vitro (Song et al. 2012; Balboula et al. 2020).
Low performing sires used in this study have previously been identified as having spermatozoa with an increased incidence of aggresome defects in their heads (Fallon et al. 2023). Aggresomes form when misfolded proteins are not efficiently degraded, which then provides an opportunity for them to interact with other proteins, leading to continuously larger protein aggregates (Garcia-Mata et al. 2002). Aggresomes must be cleared via autophagic degradation to prevent further enlargement, and may result in cell damage and apoptosis if not cleared quickly (Garcia-Mata et al. 2002; Driscoll and Chowdhury 2012). If aggresomes are brought into the zygote by the spermatozoon upon fertilisation, they would elicit an upregulation in autophagic activity in early embryos and potentially lead to increased ROS production related to cellular stress.
Although we observed increased ROS in embryos produced from low performing sires, and it is well documented that ROS may lead to DNA fragmentation (Guerin 2001; Bayr 2005), there was no difference found in the DNA damage in embryos from low performing sires in this study. Previous experiments in our group did identify increased expression of genes related to DNA damage in 4–6 cell embryos produced by low performing sires such as cyclin-dependent kinases (CDKs), histone PARylation factor (HPF1), DNA damage-inducible transcript 3 (DDIT3), and growth arrest and DNA damage gamma (GADD45G) (Lockhart et al. 2023b), but we did not see this translate to detectable levels of DNA damage in the embryos of the current experiment. Similarly, there was also no difference found in the peroxidation of lipids in embryos from low sires. This leaves open questions about which pathways are being negatively impacted by increased levels of ROS in early embryos and contributing to lower developmental rates (Fallon et al. 2023; Lockhart et al. 2023b).
Bayr, as well as others, have also shown that ROS can lead to ER stress and the misfolded protein response in embryonic cells (Bayr 2005; Yoon et al. 2014). Additionally, it has been demonstrated that the induction of autophagy can alleviate impacts of ER stress in embryos, and inversely, the defects found in autophagy-inhibited embryos can also be alleviated if treated with an ER stress inhibitor as well (Song et al. 2012). Given this data, one hypothesis is that embryos produced by low performing sires may be experiencing ER stress due to their increased ROS production and already overwhelmed autophagic pathways, therefore preventing timely clearance of sperm-derived aggresomes, causing cellular damage. In conclusion, sire contributions do regulate early embryo success via cellular stress pathways and may negatively impact development by bringing aggresomes into the zygote that lead to upregulated autophagy and ROS production. Still, further investigation is needed to clarify which downstream mechanisms are impacted in embryos that begin development under high stress conditions and how those mechanisms result in lower developmental competence.
Acknowledgements
The authors would like to thank Missouri Prime Beef Packers for providing ovaries for this study, and Katy Stoecklein for her help in oocyte collection. Additionally, the authors would like to thank Select Sires for their generous contributions to this project.
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