Moorkens, K., J. L. M. R. Leroy, J. Quanico, G. Baggerman, and W. F. A. Marei, "How the Oviduct Lipidomic Profile Changes over Time after the Start of an Obesogenic Diet in an Outbred Mouse Model.", Biology, vol. 12, issue 7, 2023. Abstract

We investigated whether a high-fat/high-sugar (HF/HS) diet alters the lipidomic profile of the oviductal epithelium (OE) and studied the patterns of these changes over time. Female outbred Swiss mice were fed either a control (10% fat) or HF/HS (60% fat, 20% fructose) diet. Mice ( = 3 per treatment per time point) were sacrificed and oviducts were collected at 3 days and 1, 4, 8, 12 and 16 weeks on the diet. Lipids in the OE were imaged using matrix-assisted laser desorption ionisation mass spectrometry imaging. Discriminative / values and differentially regulated lipids were determined in the HF/HS versus control OEs at each time point. Feeding the obesogenic diet resulted in acute changes in the lipid profile in the OE already after 3 days, and thus even before the development of an obese phenotype. The changes in the lipid profile of the OE progressively increased and became more persistent after long-term HF/HS diet feeding. Functional annotation revealed a differential abundance of phospholipids, sphingomyelins and lysophospholipids in particular. These alterations appear to be not only caused by the direct accumulation of the excess circulating dietary fat but also a reduction in the de novo synthesis of several lipid classes, due to oxidative stress and endoplasmic reticulum dysfunction. The described diet-induced lipidomic changes suggest alterations in the OE functions and the oviductal microenvironment which may impact crucial reproductive events that take place in the oviduct, such as fertilization and early embryo development.

Xhonneux, I., W. F. A. Marei, B. Meulders, S. Andries, and J. L. M. R. Leroy, "The impact of a maternal and offspring obesogenic diet on daughter's oocyte mitochondrial ultrastructure and bioenergetic responses. Insights from an outbred mouse model.", Frontiers in physiology, vol. 14, pp. 1288472, 2023. Abstract

Obesity affects oocyte mitochondrial functions and reduces oocyte quality and fertility. Obesity may also increase the risk of metabolic disorders in the offspring. Children are likely to follow their parents lifestyle and diet, which also contributes to the increased prevelance of obesity across generations. We hypothesise that the impact of obesogenic (OB) diet and obesity on oocyte mitochondrial functions is different in offspring born to obese mothers compared to those born to healthy mothers. To test this hypothesis, we fed a control (C, 10% fat, 7% sugar) or an OB diet (60% fat, 20% sugar) to female mice (for 7 weeks (w)) and then to their female offspring (for 7w after weaning) in a 2 × 2 factorial design (C » C, n = 35, C » OB, n = 35, OB » C n = 49 and OB » OB, n = 50). Unlike many other studies, we used an outbred Swiss mouse model to increase the human pathophysiological relevance. Offspring were sacrificed at 10w and their oocytes were collected. Offspring OB diet increased oocyte lipid droplet content, mitochondrial activity and reactive oxygen species (ROS) levels, altered mitochondrial ultrastructure and reduced oocyte pyruvate consumption. Mitochondrial DNA copy numbers and lactate production remained unaffected. Mitochondrial ultrastructure was the only factor where a significant interaction between maternal and offspring diet effect was detected. The maternal OB background resulted in a small but significant increase in offspring's oocyte mitochondrial ultrastructural abnormalities without altering mitochondrial inner membrane potential, active mitochondrial distribution, mitochondrial DNA copy numbers, or ROS production. This was associated with reduced mitochondrial complex III and V expression and reduced pyruvate consumption which may be compensatory mechanisms to control mitochondrial inner membrane potential and ROS levels. Therefore, in this Swiss outbred model, while offspring OB diet had the largest functional impact on oocyte mitochondrial features, the mitochondrial changes due to the maternal background appear to be adaptive and compensatory rather than dysfunctional.

Meulders, B., W. F. A. Marei, I. Xhonneux, P. E. J. Bols, and J. L. M. R. Leroy, "Effect of lipotoxicity on mitochondrial function and epigenetic programming during bovine in vitro embryo production.", Scientific reports, vol. 13, issue 1, pp. 21664, 2023. Abstract

Maternal metabolic disorders may cause lipotoxic effects on the developing oocyte. Understanding the timing at which this might disrupt embryo epigenetic programming and how this is linked with mitochondrial dysfunction is crucial for improving assisted reproductive treatments, but has not been investigated before. Therefore, we used a bovine in vitro model to investigate if pathophysiological palmitic acid (PA) concentrations during in vitro oocyte maturation and in vitro embryo culture alter embryo epigenetic patterns (DNA methylation (5mC) and histone acetylation/methylation (H3K9ac/H3K9me2)) compared to control (CONT) and solvent control (SCONT), at the zygote and morula stage. Secondly, we investigated if these epigenetic alterations are associated with mitochondrial dysfunction and changes in ATP production rate, or altered expression of epigenetic regulatory genes. Compared to SCONT, H3K9ac and H3K9me2 levels were increased in PA-derived zygotes. Also, 5mC and H3K9me2 levels were increased in PA-exposed morulae compared to SCONT. This was associated with complete inhibition of glycolytic ATP production in oocytes, increased mitochondrial membrane potential and complete inhibition of glycolytic ATP production in 4-cell embryos and reduced SOD2 expression in PA-exposed zygotes and morulae. For the first time, epigenetic alterations in metabolically compromised zygotes and morulae have been observed in parallel with mitochondrial dysfunction in the same study.

Marei, W. F. A., and J. L. M. R. Leroy, "Cellular Stress Responses in Oocytes: Molecular Changes and Clinical Implications.", Advances in experimental medicine and biology, vol. 1387, pp. 171-189, 2022. Abstract

The oocyte may be exposed to several sources of stress during its growth and maturation, which may lead to reduced fertility. Unfolded protein responses (UPRs) play a central role to maintain cell survival and repair. Transcription of heat shock proteins (HSPs) is a key element to facilitate reestablishment of cellular homeostasis. Unlike somatic cells, cellular mechanisms by which oocytes can sense and respond to stress are not well described. In here, we provide an overview about the impact of cellular stress, particularly due to lipotoxicity, oxidative stress, and heat stress on oocyte developmental competence. Next, we focus on the expression of HSPs in oocytes and their potential role in UPRs in oocytes and embryos. This is based on a comprehensive shotgun proteomic analysis of mature bovine oocytes performed in our laboratory, as well as a literature review. The topic is discussed in light of our understanding of similar mechanisms in other cell types and the limited transcriptional activity in oocytes. More fundamental research is needed both at the transcriptomic and proteomic levels to further understand cell stress response mechanisms in oocytes and early developing embryos, their critical interactions, and their long-term effects. Strategies to provide targeted external support to prevent or reduce cell stress levels during oocyte maturation or early embryo development under maternal metabolic stress conditions should be developed to maximize the odds of producing good quality embryos and guarantee optimal viability.

Marei, W. F. A., J. De Bie, I. Xhonneux, S. Andries, J. H. Britt, and J. L. M. R. Leroy, "Metabolic and antioxidant status during transition is associated with changes in the granulosa cell transcriptome in the preovulatory follicle in high-producing dairy cows at the time of breeding.", Journal of dairy science, vol. 105, issue 8, pp. 6956-6972, 2022. Abstract

In this study, we hypothesized that early postpartum (pp) metabolic and oxidative stress conditions in dairy cows (particularly those with severe negative energy balance, NEB) are associated with long-term changes in granulosa cell (GC) functions in the preovulatory follicle at the time of breeding. Blood samples were collected at wk 2 and wk 8 pp from 47 healthy multiparous cows. Follicular fluid (FF) and GC were collected from the preovulatory follicle after estrous synchronization at wk 8. Several metabolic and antioxidant parameters were measured in blood and FF, and their correlations were studied. Subsequently, 27 representative GC samples were selected for RNA sequencing analysis. The GC gene expression data of LH-responsive genes and the estradiol:progesterone ratio in FF were used to identify pre- and post-LH surge cohorts. We compared the transcriptomic profile of subgroups of cows within the highest and lowest quartiles (Q4 vs. Q1) of each parameter, focusing on the pre-LH surge cohort (n = 16, at least 3 in each subgroup). Differentially expressed genes (DEG: adjusted P-value < 0.05, 5% false discovery rate) were determined using DESeq2 analysis and were functionally annotated. Blood and FF β-carotene and vitamin E concentrations at wk 2, but not at wk 8, were associated with the most pronounced transcriptomic differences in the GC, with up to 341 DEG indicative for lower catabolism, increased oxidoreductase activity and signaling cascades that are known to enhance oocyte developmental competence, increased responsiveness to LH, and a higher steroidogenic activity. In contrast, elevated blood NEFA concentrations at wk 2 (and not at wk 8) were associated with a long-term carryover effect detectable in the GC transcriptome at wk 8 (64 DEG). These genes are related to response to lipids and ketones, oxidative stress, and immune responses, which suggests persistent cellular stress and oxidative damage. This effect was more pronounced in cows with antioxidant deficiencies at wk 8 (up to 148 DEG), with more genes involved in oxidative stress-dependent responses, apoptosis, autophagy and catabolic processes, and mitochondrial damage. Interestingly, within the severe NEB cows (high blood NEFA at wk 2), blood antioxidant concentrations (high vs. low) at wk 8 were associated with up to 194 DEG involved in activation of meiosis and other signaling pathways, indicating a better oocyte supportive capacity. This suggests that the cow antioxidant profile at the time of breeding might alleviate, at least in part, the effect of NEB on GC functions. In conclusion, these results provide further evidence that the metabolic and oxidative stress in dairy cows early postpartum can have long-term effects on GC functions in preovulatory follicles at the time of breeding. The interplay between the effects of antioxidants and NEFA illustrated here might be useful to develop intervention strategies to minimize the effect of severe NEB on fertility.

Leroy, J., B. Meulders, K. Moorkens, I. Xhonneux, J. Slootmans, L. De Keersmaeker, A. Smits, O. Bogado Pascottini, and W. F. A. Marei, "Maternal metabolic health and fertility: we should not only care about but also for the oocyte!", Reproduction, fertility, and development, vol. 35, issue 2, pp. 1-18, 2022. Abstract

Metabolic disorders due to obesity and unhealthy lifestyle directly alter the oocyte's microenvironment and impact oocyte quality. Oxidative stress and mitochondrial dysfunction play key roles in the pathogenesis. Acute effects on the fully grown oocytes are evident, but early follicular stages are also sensitive to metabolic stress leading to a long-term impact on follicular cells and oocytes. Improving the preconception health is therefore of capital importance but research in animal models has demonstrated that oocyte quality is not fully recovered. In the in vitro fertilisation clinic, maternal metabolic disorders are linked with disappointing assisted reproductive technology results. Embryos derived from metabolically compromised oocytes exhibit persistently high intracellular stress levels due to weak cellular homeostatic mechanisms. The assisted reproductive technology procedures themselves form an extra burden for these defective embryos. Minimising cellular stress during culture using mitochondrial-targeted therapy could rescue compromised embryos in a bovine model. However, translating such applications to human in vitro fertilisation clinics is not simple. It is crucial to consider the sensitive epigenetic programming during early development. Research in humans and relevant animal models should result in preconception care interventions and in vitro strategies not only aiming at improving fertility but also safeguarding offspring health.

Moorkens, K., J. L. M. R. Leroy, S. Verheyen, and W. F. A. Marei, "Effects of an obesogenic diet on the oviduct depend on the duration of feeding.", PloS one, vol. 17, issue 9, pp. e0275379, 2022. Abstract

RESEARCH QUESTION: How long does it take for an obesogenic (high-fat/high-sugar, HF/HS) diet to influence the oviductal microenvironment? What are the affected cellular pathways and are they dependent on the genetic background of the mouse model?

DESIGN: Female Swiss (outbred) and C57BL/6N (B6, inbred) mice were fed either a control (10% fat) or HF/HS (60% fat, 20% fructose) diet. Body weight was measured weekly. Mice were sacrificed at 3 days (3d), 1 week (1w), 4w, 8w, 12w and 16w on the diet (n = 5 per treatment per time point). Total cholesterol concentrations and inflammatory cytokines were measured in serum. Oviductal epithelial cells (OECs) were used to study the expression of genes involved in (mitochondrial) oxidative stress (OS), endoplasmic reticulum (ER) stress and inflammation using qPCR.

RESULTS: Body weight and blood cholesterol increased significantly in the HF/HS mice in both strains compared to controls. In Swiss mice, HF/HS diet acutely increased ER-stress and OS-related genes in the OECs already after 3d. Subsequently, mitochondrial and cytoplasmic antioxidants were upregulated and ER-stress was alleviated at 1w. After 4-8w (mid-phase), the expression of ER-stress and OS-related genes was increased again and persisted throughout the late-phase (12-16w). Serum inflammatory cytokines and inflammatory marker-gene expression in the OECs were increased only in the late-phase. Some of the OEC stress responses were stronger or earlier in the B6.

CONCLUSIONS: OECs are sensitive to an obesogenic diet and may exhibit acute stress responses already after a few days of feeding. This may impact the oviductal microenvironment and contribute to diet-induced subfertility.

Smits, A., W. F. A. Marei, K. Moorkens, P. E. J. Bols, D. De Neubourg, and J. Leroy, "Obese outbred mice only partially benefit from diet normalization or calorie restriction as preconception care interventions to improve metabolic health and oocyte quality.", Human reproduction (Oxford, England), vol. 37, issue 12, pp. 2867-2884, 2022. Abstract

STUDY QUESTION: Can diet normalization or a calorie-restricted diet for 2 or 4 weeks be used as a preconception care intervention (PCCI) in Western-type diet-induced obese Swiss mice to restore metabolic health and oocyte quality?

SUMMARY ANSWER: Metabolic health and oocyte developmental competence was already significantly improved in the calorie-restricted group after 2 weeks, while obese mice that underwent diet normalization showed improved metabolic health after 2 weeks and improved oocyte quality after 4 weeks.

WHAT IS KNOWN ALREADY: Maternal obesity is linked with reduced metabolic health and oocyte quality; therefore, infertile obese women are advised to lose weight before conception to increase pregnancy chances. However, as there are no univocal guidelines and the specific impact on oocyte quality is not known, strategically designed studies are needed to provide fundamental insights in the importance of the type and duration of the dietary weight loss strategy for preconception metabolic health and oocyte quality.

STUDY DESIGN, SIZE, DURATION: Outbred female Swiss mice were fed a control (CTRL) or high-fat/high-sugar (HF/HS) diet. After 7 weeks, some of the HF mice were put on two different PCCIs, resulting in four treatment groups: (i) only control diet for up to 11 weeks (CTRL_CTRL), (ii) only HF diet for up to 11 weeks (HF_HF), (iii) switch at 7 weeks from an HF to an ad libitum control diet (HF_CTRL) and (iv) switch at 7 weeks from an HF to a 30% calorie-restricted control diet (HF_CR) for 2 or 4 weeks. Metabolic health and oocyte quality were assessed at 2 and 4 weeks after the start of the intervention (n = 8 mice/treatment/time point).

PARTICIPANTS/MATERIALS, SETTING, METHODS: Changes in body weight were recorded. To study the impact on metabolic health, serum insulin, glucose, triglycerides, total cholesterol and alanine aminotransferase concentrations were measured, and glucose tolerance and insulin sensitivity were analyzed at PCCI Weeks 2 and 4. The quality of in vivo matured oocytes was evaluated by assessing intracellular lipid droplet content, mitochondrial activity and localization of active mitochondria, mitochondrial ultrastructure, cumulus cell targeted gene expression and oocyte in vitro developmental competence.

MAIN RESULTS AND THE ROLE OF CHANCE: Significant negative effects of an HF/HS diet on metabolic health and oocyte quality were confirmed (P < 0.05). HF_CTRL mice already showed restored body weight, serum lipid profile and glucose tolerance, similar to the CTRL_CTRL group after only 2 weeks of PCCI (P < 0.05 compared with HF_HF) while insulin sensitivity was not improved. Oocyte lipid droplet volume was reduced at PCCI Week 2 (P < 0.05 compared with HF_HF), while mitochondrial localization and activity were still aberrant. At PCCI Week 4, oocytes from HF_CTRL mice displayed significantly fewer mitochondrial ultrastructural abnormalities and improved mitochondrial activity (P < 0.05), while lipid content was again elevated. The in vitro developmental capacity of the oocytes was improved but did not reach the levels of the CTRL_CTRL mice. HF_CR mice completely restored cholesterol concentrations and insulin sensitivity already after 2 weeks. Other metabolic health parameters were only restored after 4 weeks of intervention with clear signs of fasting hypoglycemia. Although all mitochondrial parameters in HF_CR oocytes stayed aberrant, oocyte developmental competence in vitro was completely restored already after 2 weeks of intervention.

LARGE SCALE DATA: N/A.

LIMITATIONS, REASONS FOR CAUTION: In this study, we applied a relevant HF/HS Western-type diet to induce obesity in an outbred mouse model. Nevertheless, physiological differences should be considered when translating these results to the human setting. However, the in-depth study and follow-up of the metabolic health changes together with the strategic implementation of specific PCCI intervals (2 and 4 weeks) related to the duration of the mouse folliculogenesis (3 weeks), should aid in the extrapolation of our findings to the human setting.

WIDER IMPLICATIONS OF THE FINDINGS: Our study results with a specific focus on oocyte quality provide important fundamental insights to be considered when developing preconception care guidelines for obese metabolically compromised women wishing to become pregnant.

STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Flemish Research Fund (FWO-SB grant 1S25020N and FWO project G038619N). The authors declare there are no conflicts of interest.

Marei, W. F. A., and J. L. M. R. Leroy, "Cellular Stress Responses in Oocytes: Molecular Changes and Clinical Implications.", Advances in experimental medicine and biology, vol. 1387, pp. 171-189, 2022. Abstract2021_hsps_in_oocyte_development_book_chapter_wmarei_.pdf
application/pdf icon2021_hsps_in_oocyte_development_book_chapter_wmarei_.pdf

The oocyte may be exposed to several sources of stress during its growth and maturation, which may lead to reduced fertility. Unfolded protein responses (UPRs) play a central role to maintain cell survival and repair. Transcription of heat shock proteins (HSPs) is a key element to facilitate reestablishment of cellular homeostasis. Unlike somatic cells, cellular mechanisms by which oocytes can sense and respond to stress are not well described. In here, we provide an overview about the impact of cellular stress, particularly due to lipotoxicity, oxidative stress, and heat stress on oocyte developmental competence. Next, we focus on the expression of HSPs in oocytes and their potential role in UPRs in oocytes and embryos. This is based on a comprehensive shotgun proteomic analysis of mature bovine oocytes performed in our laboratory, as well as a literature review. The topic is discussed in light of our understanding of similar mechanisms in other cell types and the limited transcriptional activity in oocytes. More fundamental research is needed both at the transcriptomic and proteomic levels to further understand cell stress response mechanisms in oocytes and early developing embryos, their critical interactions, and their long-term effects. Strategies to provide targeted external support to prevent or reduce cell stress levels during oocyte maturation or early embryo development under maternal metabolic stress conditions should be developed to maximize the odds of producing good quality embryos and guarantee optimal viability.

Desmet, K. L. J., W. F. A. Marei, C. Richard, K. Sprangers, G. T. S. Beemster, P. Meysman, K. Laukens, K. Declerck, W. Vanden Berghe, P. E. J. Bols, et al., "Oocyte maturation under lipotoxic conditions induces carryover transcriptomic and functional alterations during post-hatching development of good-quality blastocysts: novel insights from a bovine embryo-transfer model", Hum Reprod, 2020/03/01, vol. 35, no. 2, pp. 293-307, Feb 29, 2020. AbstractWebsite

STUDY QUESTION: Does oocyte maturation under lipolytic conditions have detrimental carry-over effects on post-hatching embryo development of good-quality blastocysts after transfer? SUMMARY ANSWER: Surviving, morphologically normal blastocysts derived from bovine oocytes that matured under lipotoxic conditions exhibit long-lasting cellular dysfunction at the transcriptomic and metabolic levels, which coincides with retarded post-hatching embryo development. WHAT IS KNOWN ALREADY: There is increasing evidence showing that following maturation in pathophysiologically relevant lipotoxic conditions (as in obesity or metabolic syndrome), surviving blastocysts of good (transferable) morphological quality have persistent transcriptomic and epigenetic alteration even when in vitro embryo culture takes place under standard conditions. However, very little is known about subsequent development in the uterus after transfer. STUDY DESIGN, SIZE, DURATION: Bovine oocytes were matured in vitro in the presence of pathophysiologically relevant, high non-esterified fatty acid (NEFA) concentrations (HIGH PA), or in basal NEFA concentrations (BASAL) as a physiological control. Eight healthy multiparous non-lactating Holstein cows were used for embryo transfers. Good-quality blastocysts (pools of eight) were transferred per cow, and cows were crossed over for treatments in the next replicate. Embryos were recovered 7 days later and assessed for post-hatching development, phenotypic features and gene expression profile. Blastocysts from solvent-free and NEFA-free maturation (CONTROL) were also tested for comparison. PARTICIPANTS/MATERIALS, SETTING, METHODS: Recovered Day 14 embryos were morphologically assessed and dissected into embryonic disk (ED) and extraembryonic tissue (EXT). Samples of EXT were cultured for 24 h to assess cellular metabolic activity (glucose and pyruvate consumption and lactate production) and embryos' ability to signal for maternal recognition of pregnancy (interferon-tau secretion; IFN-tau). ED and EXT samples were subjected to RNA sequencing to evaluate the genome-wide transcriptome patterns. MAIN RESULTS AND THE ROLE OF CHANCE: The embryo recovery rate at Day 14 p.i. was not significantly different among treatment groups (P > 0.1). However, higher proportions of HIGH PA embryos were retarded in growth (in spherical stage) compared to the more elongated tubular stage embryos in the BASAL group (P < 0.05). Focusing on the normally developed tubular embryos in both groups, HIGH PA exposure resulted in altered cellular metabolism and altered transcriptome profile particularly in pathways related to redox-regulating mechanisms, apoptosis, cellular growth, interaction and differentiation, energy metabolism and epigenetic mechanisms, compared to BASAL embryos. Maturation under BASAL conditions did not have any significant effects on post-hatching development and cellular functions compared to CONTROL. LARGE-SCALE DATA: The datasets of RNA sequencing analysis are available in the NCBI's Gene Expression Omnibus (GEO) repository, series accession number GSE127889 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE127889). Datasets of differentially expressed genes and their gene ontology functions are available in the Mendeley datasets at http://dx.doi.org/10.17632/my2z7dvk9j.2. LIMITATIONS, REASONS FOR CAUTION: The bovine model was used here to allow non-invasive embryo transfer and post-hatching recovery on Day 14. There are physiological differences in some characteristics of post-hatching embryo development between human and cows, such as embryo elongation and trophoblastic invasion. However, the main carry-over effects of oocyte maturation under lipolytic conditions described here are evident at the cellular level and therefore may also occur during post-hatching development in other species including humans. In addition, post-hatching development was studied here under a healthy uterine environment to focus on carry-over effects originating from the oocyte, whereas additional detrimental effects may be induced by maternal metabolic disorders due to adverse changes in the uterine microenvironment. RNA sequencing results were not verified by qPCR, and no solvent control was included. WIDER IMPLICATIONS OF THE FINDINGS: Our observations may increase the awareness of the importance of maternal metabolic stress at the level of the preovulatory oocyte in relation to carry-over effects that may persist in the transferrable embryos. It should further stimulate new research about preventive and protective strategies to optimize maternal metabolic health around conception to maximize embryo viability and thus fertility outcome. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Flemish Research Fund (FWO grant 11L8716N and FWO project 42/FAO10300/6541). The authors declare there are no conflicts of interest.

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