In vitro reduction of bovine oocyte ATP production with oligomycin affects embryo epigenome.

In brief: Epigenetic programming is a crucial process during early embryo development that can have a significant impact on the results of assisted reproductive technology (ART) and offspring health. Here we show evidence using a bovine in vitro experiment that embryo epigenetic programing is dependent on oocyte mitochondrial bioenergetic activity during maturation. Abstract: This study investigated if oocyte and early embryo epigenetic programming are dependent on oocyte mitochondrial ATP production. A bovine in vitro experiment was performed in which oocyte mitochondrial ATP production was reduced using 5 nmol/L oligomycin A (OM; ATP synthase inhibitor) during in vitro maturation (IVM) compared to control (CONT). OM exposure significantly reduced mitochondrial ATP production rate in MII oocytes (34.6% reduction, P = 0.018) and significantly decreased embryo cleavage rate at 48 h post insemination (7.6% reduction, P = 0.031). Compared to CONT, global DNA methylation (5mC) levels were decreased in OM-exposed MII oocytes (9.8% reduction, P = 0.019) while global histone methylation (H3K9me2) was increased (9.4% increase, P = 0.024). In zygotes, OM exposure during IVM increased 5mC (22.3% increase, P < 0.001) and histone acetylation (H3K9ac, 17.3% increase, P = 0.023) levels, while H3K9me2 levels were not affected. In morulae, 5mC levels were increased (10.3% increase, P = 0.041) after OM exposure compared to CONT, while there was no significant difference in H3K9ac and H3K9me2 levels. These epigenetic alterations were not associated with any persistent effects on embryo mitochondrial ATP production rate or mitochondrial membrane potential (assessed at the four-cell stage). Also, epigenetic regulatory genes were not differentially expressed in OM-exposed zygotes or morulae. Finally, apoptotic cell index in blastocysts was increased after OM exposure during oocyte maturation (41.1% increase, P < 0.001). We conclude that oocyte and early embryo epigenetic programming are dependent on mitochondrial ATP production during IVM.

Risks of using mitoquinone during in vitro maturation and its potential protective effects against lipotoxicity-induced oocyte mitochondrial stress.

PURPOSE: Oxidative stress and mitochondrial dysfunction play central roles in reduced oocyte quality and infertility in obese patients. Mitochondria-targeted treatments containing co-enzyme Q10 such as mitoquinone (MitoQ) can increase mitochondrial antioxidative capacity; however, their safety and efficiency when supplemented to oocytes under lipotoxic conditions have not been described. METHODS: We tested the effect of different concentrations of MitoQ or its cationic carrier (TPP) (0, 0.1, 0.5, 1.0 µM each) during bovine oocyte IVM. Then, we tested the protective capacity of MitoQ (0.1 µM) against palmitic acid (PA)-induced lipotoxicity and mitochondrial dysfunction in oocytes. RESULTS: Exposure to MitoQ, or TPP only, at 1 µM significantly (P<0.05) reduced oocyte mitochondrial inner membrane potential (JC-1 staining) and resulted in reduced cleavage and blastocyst rates compared with solvent control. Lower concentrations of MitoQ or TPP had no effects on embryo development under control (PA-free) conditions. As expected, PA increased the levels of MMP and ROS in oocytes (CellROX staining) and reduced cleavage and blastocyst rates compared with the controls (P<0.05). These negative effects were ameliorated by 0.1 µM MitoQ. In contrast, 0.1 µM TPP alone had no protective effects. MitoQ also normalized the expression of HSP10 and TFAM, and partially normalized HSP60 in the produced blastocysts, indicating at least a partial alleviation of PA-induced mitochondrial stress. CONCLUSION: Oocyte exposure to MitoQ may disturb mitochondrial bioenergetic functions and developmental capacity due to a TPP-induced cationic overload. A fine-tuned concentration of MitoQ can protect against lipotoxicity-induced mitochondrial stress during IVM and restore developmental competence and embryo quality.

Preconception Diet Interventions in Obese Outbred Mice and the Impact on Female Offspring Metabolic Health and Oocyte Quality.

Obese individuals often suffer from metabolic health disorders and reduced oocyte quality. Preconception diet interventions in obese outbred mice restore metabolic health and oocyte quality and mitochondrial ultrastructure. Also, studies in inbred mice have shown that maternal obesity induces metabolic alterations and reduces oocyte quality in offspring (F1). Until now, the effect of maternal high-fat diet on F1 metabolic health and oocyte quality and the potential beneficial effects of preconception dietary interventions have not been studied together in outbred mice. Therefore, we fed female mice a high-fat/high-sugar (HF/HS) diet for 7 weeks and switched them to a control (CONT) or caloric-restriction (CR) diet or maintained them on the HF/HS diet for 4 weeks before mating, resulting in three treatment groups: diet normalization (DN), CR, and HF/HS. In the fourth group, mice were fed CONT diet for 11 weeks (CONT). HF/HS mice were fed an HF/HS diet from conception until weaning, while all other groups were then fed a CONT diet. After weaning, offspring were kept on chow diet and sacrificed at 11 weeks. We observed significantly elevated serum insulin concentrations in female HF/HS offspring and a slightly increased percentage of mitochondrial ultrastructural abnormalities, mitochondrial size, and mitochondrial mean gray intensity in HF/HS F1 oocytes. Also, global DNA methylation was increased and cellular stress-related proteins were downregulated in HF/HS F1 oocytes. Mostly, these alterations were prevented in the DN group, while, in CR, this was only the case for a few parameters. In conclusion, this research has demonstrated for the first time that a maternal high-fat diet in outbred mice has a moderate impact on female F1 metabolic health and oocyte quality and that preconception DN is a better strategy to alleviate this compared to CR.

Genesis of clinical and subclinical endometritis in dairy cows.

In brief: Clinical and subclinical endometritis are different manifestations of reproductive tract inflammatory disease in dairy cows. This review addresses the genesis of clinical and subclinical endometritis considering metabolic stress, innate immune dysfunction, and shifts in the composition of the uterine microbiota in the postpartum period. Abstract: Up to half of dairy cows may develop one or more types of reproductive tract inflammatory disease within 5 weeks after calving. Clinical endometritis (CE) results from uterine bacterial dysbiosis with increased relative abundance of pathogenic bacteria associated with luminal epithelial damage. These bacteria cause endometrial stromal cell lysis, followed by massive polymorphonuclear neutrophil (PMN) migration, and pyogenesis. CE is defined as endometrial inflammation accompanied by purulent discharge. Purulent discharge is not always accompanied by uterine inflammation (being (rarely) vaginitis or (commonly) cervicitis), hence referred to as purulent vaginal discharge (PVD). Subclinical endometritis (SCE) is an asymptomatic uterine disease defined by a threshold of PMN on cytology that is associated with worse reproductive performance; it has not been linked with bacterial dysbiosis. Current evidence suggests that SCE is a result of metabolic and inflammatory dysfunction that impairs innate immune function and the ability of endometrial PMN to undergo apoptosis, necrosis, and ultimately achieve resolution of inflammation. CE and SCE are diagnosed between 3 and 5 weeks postpartum and commonly overlap, but they are considered distinct manifestations of reproductive tract inflammatory disease. This review addresses the genesis of CE and SCE in postpartum dairy cows considering metabolic stress, innate immune dysfunction, and shifts in the composition of the uterine microbiota.

Viability and function dynamics of circulating versus endometrial polymorphonuclear leukocytes in postpartum dairy cows with subclinical or clinical endometritis.

We aimed to compare the viability of circulating polymorphonuclear leukocyte (cPMN) and endometrial PMN (ePMN) and their function dynamics in postpartum dairy cows with subclinical (SCE) or clinical endometritis (CE). To do so, blood samples from 38 Holstein cows were collected at -7, 9, 21, and 36 d relative to calving, and endometrial cytology samples from 32 Holstein cows were harvested at 9, 21, and 36 d postpartum. Uterine health status was assessed at 36 d postpartum, and cows were classified as healthy (absence of abnormal vaginal discharge and ≤5% ePMN), SCE (absence of abnormal vaginal discharge and >5% ePMN), or CE (mucopurulent or purulent vaginal discharge and >5% ePMN). Viability (viable, apoptotic, and necrotic) and function parameters phagocytosis (PC), oxidative burst, and intracellular proteolytic degradation were evaluated for cPMN via flow cytometry. For ePMN, only viability and PC were evaluated. The association of cPMN and ePMN viability and functional parameters with reproductive tract health classification were fitted in mixed linear regression models, accounting for repeated measures, sampling day, and interactions of reproductive tract status and day. Cows with CE had a lower proportion of cPMN viability (84.5 ± 2.1%; least squares means ± standard error) and a higher proportion of apoptosis (14.4 ± 2.0%) than healthy (92.4 ± 1.3 and 6.7 ± 1.3%, respectively) or SCE (95.3 ± 2.4 and 3.8 ± 2.3%, respectively) at 9 d postpartum. Interestingly, cPMN intracellular proteolytic degradation was lower [6.2 ± 0.1 median fluorescence intensity (MFI)] in SCE compared with healthy (6.7 ± 0.08 MFI) or CE (6.8 ± 0.1 MFI) at d 9 postpartum. No other differences in cPMN function were found among experimental groups. The proportion of necrotic ePMN was higher for healthy (49.6 ± 5.1%) than SCE (27.4 ± 7.3%) and CE (27.7 ± 7.3%) cows at 36 d postpartum. Also, at 36 d postpartum, the proportion of ePMN performing PC was higher in CE (47.0 ± 8.6%) than in healthy (18.4 ± 7.6%) cows, but did not differ from SCE cows (25.9 ± 8.7%). Results of the present study suggest that cPMN viability and function at 9 d postpartum are associated with the development of uterine disease. Furthermore, ePMN at 36 d postpartum are mostly necrotic in healthy cows but viable and functional in cows with CE, probably due to active uterine inflammation. Remarkably, ePMN in cows with SCE at 36 d postpartum are also mostly viable but seem to display a numerically lower proportion of PC compared with ePMN in CE cows.

Cellular Stress Responses in Oocytes: Molecular Changes and Clinical Implications.

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.

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.

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 ß-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.

Maladaptation to the transition period and consequences on fertility of dairy cows.

After parturition, dairy cows undergo a plethora of metabolic, inflammatory, and immunologic changes to adapt to the onset of lactation. These changes are mainly due to the homeorhetic shift to support milk production when nutrient demand exceeds dietary intake, resulting in a state of negative energy balance. Negative energy balance in postpartum dairy cows is characterized by upregulated adipose tissue modelling, insulin resistance, and systemic inflammation. However, half of the postpartum cows fail to adapt to these changes and develop one or more types of clinical and subclinical disease within 5 weeks after calving, and this is escorted by impaired reproductive performance in the same lactation. Maladaptation to the transition period exerts molecular and structural changes in the follicular and reproductive tract fluids, the microenvironment in which oocyte maturation, fertilization, and embryo development occur. Although the negative effects of transition diseases on fertility are well-known, the involved pathways are only partially understood. This review reconstructs the mechanism of maladaptation to lactation in the transition period, explores their key (patho)physiological effects on reproductive organs, and briefly describes potential carryover effects on fertility in the same lactation.

Diet normalization or caloric restriction as a preconception care strategy to improve metabolic health and oocyte quality in obese outbred mice.

BACKGROUND: Maternal metabolic disorders are linked to reduced metabolic health and oocyte quality. Obese women are advised to lose weight before conception to increase pregnancy chances. However, as human studies show no univocal guidelines, more research is necessary to provide fundamental insights in the consequences of dietary weight loss on oocyte quality. Therefore, we investigated the impact of diet normalization or calorie restricted diet for two, four or six weeks, as preconception care intervention (PCCI), in obese mice on metabolic health and oocyte quality. METHODS: Outbred female mice were fed a control (CTRL) or high-fat (HF) diet for 7 weeks (7w). Afterwards, HF-mice were put on different PCCIs, resulting in four treatment groups: 1) control diet up to 13w, 2) HF diet up to 13w (HF_HF), switch from a HF (7w) to 3) an ad libitum control diet (HF_CTRL) or 4) 30% calorie restricted control diet (HF_CR) for two, four or six weeks. Body weight, metabolic health, oocyte quality and overall fertility results were assessed. RESULTS: Negative effects of HF diet on metabolic health, oocyte quality and pregnancy rates were confirmed. HF_CTRL mice progressively improved insulin sensitivity, glucose tolerance, serum insulin and cholesterol from PCCI w2 to w4. No further improvements in metabolic health were present at PCCI w6. However, PCCI w6 showed best oocyte quality improvements. Mature oocytes still showed elevated lipid droplet volume and mitochondrial activity but a significant reduction in ROS levels and ROS: active mitochondria ratio compared with HF_HF mice. HF_CR mice restored overall insulin sensitivity and glucose tolerance by PCCI w4. However, serum insulin, cholesterol and ALT remained abnormal. At PCCI w6, glucose tolerance was again reduced. However, only at PCCI w6, oocytes displayed reduced ROS levels and restored mitochondrial activity compared with HF_HF mice. In addition, at PCCI w6, both PCCI groups showed decreased mitochondrial ultrastructural abnormalities compared with the HF_HF group and restored pregnancy rates. CONCLUSIONS: Diet normalization for 4 weeks showed to be the shortest, most promising intervention to improve metabolic health. Most promising improvements in oocyte quality were seen after 6 weeks of intervention in both PCCI groups. This research provides fundamental insights to be considered in developing substantiated preconception guidelines for obese women planning for pregnancy.

Preservation of connexin 43 and transzonal projections in isolated bovine pre-antral follicles before and following vitrification.

PURPOSE: Gap junctions and transzonal projections play a crucial role in intercellular communication between different follicular components and are necessary for follicle development. We aimed to demonstrate gap junction protein connexin 43 (Cx43) and transzonal projections (TZPs) in viable, category 1, isolated bovine pre-antral follicles (PAFs) during short-term culture and after vitrification and warming. METHODS: This study involved four experimental groups: fresh control, 2-day culture, 4-day culture, and vitrified secondary PAFs. Isolated PAFs were vitrified using a simple and efficient cryopreservation method by means of mini cell strainers. RESULTS: Cx43 and TZPs were detected in pre-antral follicles of all stages, as well as in every experimental group. The group fresh follicles showed a higher percentage of follicles that were positive for Cx43 (91.7%) than the follicles that were vitrified (77.4%). All follicles that were cultured for 2 days were Cx43-positive (100%). Follicles cultured for 4 days (65.8%) (P = 0.002) showed the lowest percentage of follicles that were Cx43-positive. The percentages of the presence or (partial) absence of the TZP network were shown to be very heterogeneous between follicles in different treatment groups. CONCLUSIONS: These results suggest the maintenance of communication between the oocyte and the somatic companion cells after vitrification and warming. The varying percentages of the expression of the TZP network within groups suggests that it will be of interest to investigate whether this is truly due to variability in TZP integrity and follicle quality or due to methodological limitations.

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.

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-τ secretion; IFN-τ). 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.

Molecular interactions at the bovine embryo-endometrial epithelium interface.

In cattle, pre-implantation embryo development occurs within the confinement of the uterine lumen. Current understanding of the bi-lateral molecular interactions between embryo and endometrium that are required for a successful pregnancy is limited. We hypothesized that the nature and intensity of reciprocal embryo-endometrium interactions depend on the extent of their physical proximity. Bovine endometrial epithelial cells (bEECs) and morulae were co-cultured in juxtacrine (Contact+) or non-juxtacrine (Contact-) apposition. Co-culture with bEECs improved blastocyst rates on day 7.5, regardless of juxtaposition. Contact+ regulated transcription of 1797 endometrial genes vs only 230 in the Contact- group compared to their control (no embryos) counterparts. A subset of 50 overlapping differentially expressed genes (DEGs) defined embryo-induced effects on bEEC transcriptome irrespective of juxtaposition. Functional analysis revealed pathways associated with interferon signaling and prostanoid biosynthesis. A total of 175 genes displayed a graded expression level depending on Contact+ or Contact-. These genes were involved in interferon-related and antigen presentation pathways. Biological processes enriched exclusively in Contact+ included regulation of cell cycle and sex-steroid biosynthesis. We speculate that, in vivo, embryonic signals fine-tune the function of surrounding cells to ultimately maximize pregnancy success.

Rescue Potential of Supportive Embryo Culture Conditions on Bovine Embryos Derived from Metabolically Compromised Oocytes.

Elevated non-esterified fatty acid (NEFA), predominantly palmitic acid (PA), concentrations in blood and follicular fluid are a common feature in maternal metabolic disorders such as obesity. This has a direct negative impact on oocyte developmental competence and the resulting blastocyst quality. We use NEFA-exposure during bovine oocyte in vitro maturation (IVM) as a model to mimic oocyte maturation under maternal metabolic stress conditions. However, the impact of supportive embryo culture conditions on these metabolically compromised zygotes are not known yet. We investigated if the addition of anti-apoptotic, antioxidative and mitogenic factors (namely, Insulin-Transferrin-Selenium (ITS) or serum) to embryo culture media would rescue development and important embryo quality parameters (cell proliferation, apoptosis, cellular metabolism and gene expression patterns) of bovine embryos derived from high PA- or high NEFA-exposed oocytes when compared to controls (exposed to basal NEFA concentrations). ITS supplementation during in vitro culture of PA-exposed oocytes supported the development of lower quality embryos during earlier development. However, surviving blastocysts were of inferior quality. In contrast, addition of serum to the culture medium did not improve developmental competence of PA-exposed oocytes. Furthermore, surviving embryos displayed higher apoptotic cell indices and an aberrant cellular metabolism. We conclude that some supportive embryo culture supplements like ITS and serum may increase IVF success rates of metabolically compromised oocytes but this may increase the risk of reduced embryo quality and may thus have other long-term consequences.

Action mechanisms of n-3 polyunsaturated fatty acids on the oocyte maturation and developmental competence: Potential advantages and disadvantages.

Infertility is a growing problem worldwide. Currently, in vitro fertilization (IVF) is widely performed to treat infertility. However, a high percentage of IVF cycles fails, due to the poor developmental potential of the retrieved oocyte to generate viable embryos. Fatty acid content of the follicular microenvironment can affect oocyte maturation and the subsequent developmental competence. Saturated and monounsaturated fatty acids are mainly used by follicle components as primary energy sources whereas polyunsaturated fatty acids (PUFAs) play a wide range of roles. A large body of evidence supports the beneficial effects of n-3 PUFAs in prevention, treatment, and amelioration of some pathophysiological conditions including heart diseases, cancer, diabetes, and psychological disorders. Nevertheless, current findings regarding the effects of n-3 PUFAs on reproductive outcomes in general and on oocyte quality more specifically are inconsistent. This review attempts to provide a comprehensive overview of potential molecular mechanisms by which n-3 PUFAs affect oocyte maturation and developmental competence, particularly in the setting of IVF and thereby aims to elucidate the reasons behind current discrepancies around this topic.

Mitochondria-targeted therapy rescues development and quality of embryos derived from oocytes matured under oxidative stress conditions: a bovine in vitro model.

STUDY QUESTION: Can we use a mitochondrial-targeted antioxidant (Mitoquinone) during in vitro embryo culture to rescue developmental competence of oocytes matured under lipotoxic conditions, exhibiting mitochondrial dysfunction and oxidative stress? SUMMARY ANSWER: Supplementation of embryo culture media with Mitoquinone reduced oxidative stress and prevented mitochondrial uncoupling in embryos derived from metabolically compromised oocytes in vitro, leading to higher blastocyst rates and lower blastomeric apoptosis. WHAT IS KNOWN ALREADY: Maternal metabolic disorders, such as obesity and type-II diabetes are associated with hyperlipidemia and elevated free fatty acid (FFA) concentrations in the ovarian follicular fluid (FF). Oocyte maturation under these lipotoxic conditions results in increased oxidative stress levels, mitochondrial dysfunction, reduced developmental competence and disappointing IVF results. STUDY DESIGN, SIZE, DURATION: A well-described bovine oocyte IVM model was used, where a pathophysiologically relevant elevated FF concentrations of palmitic acid (PA; 150 µM or 300 µM) were added to induce oxidative stress. After fertilization (Day 0, D0), zygotes were in vitro cultured (IVC, from D1 to D8) in standard fatty acid-free media in the presence or absence of Mitoquinone or its carrier triphenyl-phosphonium. PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryo cleavage and fragmentation (D2) and blastocyst rates (D8) were recorded. Mitochondrial activity and oxidative stress in cleaved embryos at D2 were determined using specific fluorogenic probes and confocal microscopy. D8 blastocysts were used to (i) examine the expression of marker genes related to mitochondrial unfolded protein responses (UPRmt; HSPD1 and HSPE1), mitochondrial biogenesis (TFAM), endoplasmic reticulum (ER) UPR (ATF4, ATF6 and BiP) and oxidative stress (CAT, GPX1 and SOD2) using real time RT-PCR; (ii) determine cell differentiation and apoptosis using CDX-2 and cleaved caspase-3 immunostaining; and (iii) measure mtDNA copy numbers. This was tested in a series of experiments with at least three independent replicates for each, using a total of 2525 oocytes. Differences were considered significant if a P value was <0.05 after Bonferroni correction. MAIN RESULTS AND THE ROLE OF CHANCE: Exposure to PA during IVM followed by culture under control conditions resulted in a significant increase in oxidative stress in embryos at D2. This was associated with a significant reduction in mitochondrial inner membrane potential (uncoupling) compared with solvent control (P < 0.05). The magnitude of these effects was PA-concentration dependent. Consequently, development to the blastocysts stage was significantly hampered. Surviving blastocysts exhibited high apoptotic cell indices and upregulated mRNA expression indicating persistent oxidative stress, mitochondrial and ER UPRs. In contrast, supplementation of PA-derived zygotes with Mitoquinone during IVC (i) prevented mitochondrial uncoupling and alleviated oxidative stress at D2; and (ii) rescued blastocyst quality; normalized oxidative stress and UPR related genes and apoptotic cell indices (P > 0.01 compared with solvent control). Mitoquinone also improved blastocyst rate in PA-exposed groups, an effect that was dependent on PA concentration. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This is a fundamental study performed using a bovine in vitro model using PA-induced lipotoxicity during oocyte maturation. PA is the most predominant FFA in the FF that is known to induce lipotoxicity; however, in vivo maturation in patients suffering from maternal metabolic disorders involve more factors that cannot be represented in one model. Nevertheless, focusing on the carryover oxidative stress as a known key factor affecting developmental competence, and considering the novel beneficial rescuing effects of Mitoquinone shown here, we believe this model is of high biological relevance. WIDER IMPLICATIONS OF THE FINDINGS: Human oocytes collected for IVF treatments from patients with maternal metabolic disorders are vulnerable to lipotoxicity and oxidative stress during in vivo maturation. The results shown here suggest that mitochondrial targeted therapy, such as using Mitoquinone, during IVC may rescue the developmental competence and quality of these compromised oocytes. After further clinical trials, this may be a valuable approach to increase IVF success rates for infertile patients experiencing metabolic disorders. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by a BOF/KP grant number 34399, from the University of Antwerp, Belgium. W.F.A.M. was supported by a postdoctoral fellowship from the Research Foundation-Flanders (FWO), grant number 12I1417N, Antwerp, Belgium. The Leica SP 8 confocal microscope used in this study was funded by the Hercules Foundation of the Flemish Government (Hercules grant AUHA.15.12). All authors have no financial or non-financial competing interests to declare.

Oleic acid in the modulation of oocyte and preimplantation embryo development.

Potential reproductive effects are considered as the major aspect of biomolecules functionality in an organism. The recent identification of differential patterns of fatty acids across ovarian follicles and their association with levels of sexual maturity highlights the importance of these biomolecules. It is well known that fatty acids are highly diverse in terms of their functional properties. Oleic acid is chemically classified as an unsaturated omega-9 fatty acid. Besides serving as an important energy source, oleic acid is involved in metabolic and structural roles. Free and esterified oleic acids are compartmentalized into discrete extracellular fluids, cell organelles and found within the cytosol. This review summarizes the current knowledge on the contribution of oleic acid in regulating female fertility, particularly its involvement in female germ cell growth and development. Oleic acid has been identified as a blastomeric and post-cryopreservation survival biomarker in bovine. Several related studies have shown the critical role of oleic acid in counteracting the detrimental effects of saturated fatty acids and in paracrine support of oocyte development. Although available data are not ideally detailed, most data suggest that oleic acid can contribute to normal oocyte and preimplantation embryo development via mechanisms involving metabolic partitioning of fatty acids, change in the membrane structural organization, attenuation of oxidative stress and regulation of intracellular signalling. Thus, oleic acid may play a significant role in oocyte and early embryo development, suggesting that future studies should explore in more detail its potential effects on the physiopathology of female reproduction.

Effects of vitrification on the viability of alginate encapsulated isolated bovine pre-antral follicles.

PURPOSE: Individual follicle cryopreservation techniques, without hydrogel support, are labor-intensive and a substantial proportion of isolated follicles are lost during handling and after warming. Therefore, the viability and morphology of isolated bovine (as a model for human) pre-antral follicles after vitrification and warming, when encapsulated in alginate beads, were investigated. METHODS: Bovine pre-antral follicles were mechanically isolated and divided into four different groups: (1) culture in 2% alginate beads (3D system) and vitrification in beads using mesh cups (3DVIT), (2) culture in 2% alginate beads (3DCUL), (3) culture in 96-well plates (2D system) and vitrification using High Security Vitrification straws® (2DVIT), (4) culture in a 2D system (2DCUL). The same vitrification and warming protocols were used for embedded (3DVIT) and non-embedded follicles (2DVIT). RESULTS: No differences were observed in follicle viability between group 2DCUL and 3DCUL. Group 3DVIT showed the lowest viability (45.9%) according to calcein and neutral red staining among all groups. Group 2DVIT displayed the highest viability (87.5%) and largest percentage of follicles with a well-preserved morphology. CONCLUSIONS: Our results show that, using a vitification protocol optimized for non-embedded follicles, 2D culture is more effective in vitrifying isolated follicles. However, embedding in alginate allow to handle follicles more efficiently, i.e., without excessive manipulation and thus less labor-intensive in combination with a reduced loss of follicles during the procedure. Based on the increased work efficiency, but lower viability and higher proportion of follicles showing impaired morphology, we consider it advantageous to optimize the protocol for the vitrification of embedded follicles to increase survival and maintain morphology after vitrification.

Alpha-linolenic acid protects the developmental capacity of bovine cumulus-oocyte complexes matured under lipotoxic conditions in vitro.

Elevated concentrations of free fatty acids (FFAs), predominantly palmitic, stearic, and oleic acids (PSO), exert detrimental effects on oocyte developmental competence. This study examined the effects of omega-3 alpha-linolenic acid (ALA) during in vitro oocyte maturation (IVM) in the presence of PSO on subsequent embryo development and quality, and the cellular mechanisms that might be involved. Bovine cumulus-oocyte complexes (COCs) were supplemented during IVM with ALA (50 µM), PSO (425 µM), or PSO+ALA. Compared with FFA-free controls (P < 0.05), PSO increased embryo fragmentation and decreased good quality embryos on day 2 postfertilization. Day 7 blastocyst rate was also reduced. Day 8 blastocysts had lower cell counts and higher apoptosis but normal metabolic profile. In the PSO group, cumulus cell (CC) expansion was inhibited with an increased CC apoptosis while COC metabolism was not affected. Mitochondrial inner membrane potential (MMP; JC-1 staining) was reduced in the CCs and oocytes. Heat shock protein 70 (HSP70) but not glucose-regulated protein 78 kDa (GRP78, known as BiP; an endoplasmic reticulum stress marker) was upregulated in the CCs. Higher reactive oxygen species levels (DCHFDA staining) were detected in the oocytes. In contrast, adding ALA in the presence of PSO normalized embryo fragmentation, cleavage, blastocyst rates, and blastocyst quality compared to controls (P > 0.05). Combined treatment with ALA also reduced CC apoptosis, partially recovered CC expansion, abrogated the reduction in MMP in the CCs but not in the oocytes, and reduced BiP and HSP70 expression in CCs, compared with PSO only (P < 0.05). In conclusion, ALA supplementation protected oocyte developmental capacity under lipotoxic conditions mainly by protecting cumulus cell viability.

Targeted deletion of the Kv6.4 subunit causes male sterility due to disturbed spermiogenesis.

Electrically silent voltage-gated potassium (KvS) channel subunits (i.e. Kv5-Kv6 and Kv8-Kv9) do not form functional homotetrameric Kv channels, but co-assemble with Kv2 subunits, generating functional heterotetrameric Kv2--KvS channel complexes in which the KvS subunits modulate the Kv2 channel properties. Several KvS subunits are expressed in testis tissue but knowledge about their contribution to testis physiology is lacking. Here, we report that the targeted deletion of Kv6.4 in a transgenic mouse model (Kcng4-/-) causes male sterility as offspring from homozygous females were only obtained after mating with wild-type (WT) or heterozygous males. Semen quality analysis revealed that the sterility of the homozygous males was caused by a severe reduction in total sperm-cell count and the absence of motile spermatozoa in the semen. Furthermore, spermatozoa of homozygous mice showed an abnormal morphology characterised by a smaller head and a shorter tail compared with WT spermatozoa. Comparison of WT and Kcng4-/- testicular tissue indicated that this inability to produce (normal) spermatozoa was due to disturbed spermiogenesis. These results suggest that Kv6.4 subunits are involved in the regulation of the late stages of spermatogenesis, which makes them a potentially interesting pharmacological target for the development of non-hormonal male contraceptives.

Effect of nutritionally induced hyperlipidaemia on in vitro bovine embryo quality depends on the type of major fatty acid in the diet.

The present study examined whether the effects of dietary-induced hyperlipidaemia on preimplantation embryo development depend on the predominant fatty acid (FA) type in the diet. In a combined in vivo-in vitro bovine model, two groups of cows (n=3 in each group) were fed with three diets consecutively (4 weeks feeding for each): (1) a maintenance control diet (CONT); (2) a high-starch diet rich in saturated fat (SAT); and (3) a high-starch diet rich in omega-3 unsaturated fat (UNSAT). Two feeding sequences were used to test for carry-over effects: Group A was fed CONT, SAT1 and then UNSAT2, whereas Group B was fed CONT, UNSAT1 and then SAT2. Serum was collected after each dietary period, analysed and tested in bovine in vitro embryo culture. Introducing SAT and UNSAT diets induced hyperlipidaemia (specifically hypercholesterolaemia and elevated free FAs) and reduced insulin sensitivity. Carry-over effects in serum metabolites and FA profile were dependent on the diet and feeding sequence. SAT1 and SAT2 serum decreased blastocyst rates and altered blastocyst mRNA expression related to apoptosis and oxidative stress. UNSAT1 and UNSAT2 serum resulted in normal embryo development and quality. Other in vitro effects depended on the sequence of feeding. In conclusion, substitution of saturated fat with omega-3 fat in a high-caloric diet induced hyperlipidaemia with an FA profile yielding similar rates and quality of blastocysts compared with normolipidaemic controls.