[Show abstract][Hide abstract] ABSTRACT: Reduced oocyte quality has been associated with poor fertility of high performance dairy cows during peak lactation, due to negative energy balance. We examined the role of non-esterified fatty acids (NEFA), known to accumulate within follicular fluid during under- and over-nutrition scenarios, in causing endoplasmic reticulum (ER) stress of in vitro maturated (IVM) cattle cumulus-oocyte complexes (COCs). NEFA concentrations were: palmitic acid (150 μM), oleic acid (200 μM) and steric acid (75 μM). Abattoir-derived COCs were randomly matured for 24h in the presence of NEFAs and/or an ER stress inhibitor, salubrinal. Total and hatched blastocyst yields were negatively impacted by NEFA treatment compared with controls, but this was reversed by salubrinal. ER stress markers, activating transcription factor 4 (Atf4) and heat shock protein 5 (Hspa5), but not Atf6, were significantly up-regulated by NEFA treatment within whole COCs, but reversed by co-incubation with salubrinal. Likewise, glucose uptake and lactate production, measured in spent medium samples, showed a similar pattern, suggesting that cumulus cell metabolism is sensitive to NEFAs via an ER-stress mediated process. In contrast, while mitochondrial DNA copy number was recovered in NEFA treated oocytes, oocyte autofluorescence of the respiratory chain co-factor, FAD, was lower following NEFA treatment of COCs and this was not reversed by salubrinal, suggesting the negative impact was via reduced mitochondrial function. These results reveal the significance of NEFA-induced ER stress on bovine COC developmental competence, revealing a potential therapeutic target for improving oocyte quality during peak lactation.
Full-text · Article · Dec 2015 · Biology of Reproduction
[Show abstract][Hide abstract] ABSTRACT: The c-kit/kit ligand (KITL) signalling axis is an essential component of ovarian folliculogenesis in mammals, but little is known about expression and localisation of its key components in the ovaries of reproductive age women. This study aimed to characterise mRNA expression of c-kit and KITL isoforms and the localisation of c-kit and KITL proteins in adult human premenopausal ovaries.
This study utilised granulosa cells obtained from the preovulatory follicles of women undergoing assisted reproduction, pieces of ovarian tissue obtained from premenopausal women undergoing gynaecological surgeries and archival paraffin-embedded premenopausal ovarian tissues. Methodology included PCR for gene expression and Western blot or immunohistochemistry for protein expression.
Both c-kit mRNA isoforms, known as GNNK+ and GNNK-, were detected in human ovarian cortex, while KITL protein isoforms (KITL1 and KITL2) were present in ovarian cortex and human granulosa cells. Immunohistochemistry showed expression of KITL and c-kit protein in multiple cell types within follicles throughout development, from primordial follicles to large antral follicles, in addition to atretic follicles. Oocytes of all follicle stages expressed c-kit protein exclusively. Interestingly, unlike animal models, expression of both proteins displayed a less cell-type specific distribution with immunostaining present in granulosa, theca and stromal cells, suggesting that autocrine signalling occurs within the human ovary.
The results of this study indicate that c-kit/KITL signalling also occurs in the human ovary, as established in various animal models, and may involve previously unknown autocrine signalling.
Full-text · Article · May 2015 · Journal of Ovarian Research
[Show abstract][Hide abstract] ABSTRACT: To investigate the impacts that a paternal high fat diet (HFD) has on embryology, ovarian/cumulus cell gene expression and COC metabolism from female offspring, using a mouse model.
Founder male mice were either fed a control diet (CD) or a HFD for 12 weeks. The HFD induced obesity but not diabetes, and founder males were then mated to normal weight CD fed female mice. Female offspring were maintained on a CD, super-ovulated, mated and the resultant zygotes were cultured to the blastocyst stage for embryo morphology, blastocyst cell number and apoptosis assessment. Ovaries and cumulus cells from offspring were collected for gene expression analysis of selected genes that maintain chromatin remodeling and endoplasmic reticulum (ER), metabolic and inflammatory homeostasis. Cumulus/oocyte complexes were also investigated for glucose uptake and lipid accumulation.
Female offspring sired by obese fathers produced embryos with delayed development and impaired quality, displayed increases in ovarian expression of Glut1, Glut3 and Glut4, and an increase in cumulus cell expression of Glut4. Interestingly their COCs did take up more glucose, but did accumulate more lipid.
A paternal HFD is associated with subfertility in female offspring despite the offspring being fed a CD and this subfertility is concomitant with ovarian/cumulus cell molecular alterations and increased lipid accumulation.
No preview · Article · Apr 2015 · Journal of Assisted Reproduction and Genetics
[Show abstract][Hide abstract] ABSTRACT: In vitro maturation of oocytes is suboptimal to in vivo maturation with altered gene expression and compromised oocyte quality. The large proteoglycan versican is abundant in mouse cumulus oocyte complexes (COCs) matured in vivo but absent in cultured COCs. Versican is also positively correlated with human oocyte quality. Versican contains an EGF motif and based on epidermal growth factor (EGF)-like activities in other systems we hypothesized that versican acts as an EGF-like signaling factor during COC maturation. Here we purified recombinant versican and compared its function to EGF during in vitro maturation (IVM). Versican significantly increased cumulus expansion and induced cumulus-specific genes Ptgs2, Tnfaip6 and Has2, which was blocked by EGF receptor antagonist. Microarray analysis revealed that versican has overlapping function with EGF however a subset of genes were uniquely altered following 6 h IVM with either treatment. Following 6 h of IVM both Areg and Ereg were significantly increased by both treatments while Egln3, Nr4a1, Nr4a2, Nr4a3, and Adamts5 were significantly higher following versican treatment compared to EGF. In contrast Sprr1a and Aqp3 were increased after 6h EGF and not versican treatment. To determine whether there were temporal differences, COCs were cultured with EGF or versican for 0-12 h. Versican-induced expression occurred later, but remained elevated for longer compared to EGF for Ptgs2, Ereg and Nr4a3. The unique expression profiles of Aqp3 and Nr4a3 during IVM were similarly regulated in vivo. These data indicate that versican has EGF-like effects on COC gene expression, but with distinct temporal characteristics.
Copyright 2015 by The Society for the Study of Reproduction.
No preview · Article · Mar 2015 · Biology of Reproduction
[Show abstract][Hide abstract] ABSTRACT: Obesity is associated with decreased pregnancy rates due, in part, to compromised oocyte quality. The aim of the present cross-sectional study of 84 women undergoing oocyte aspiration was to: (1) compare insulin, lipids and glucose in follicular fluid with serum; (2) determine whether increased body mass index (BMI) and waist circumference, hyperinsulinaemia, dyslipidaemia or metabolic syndrome altered follicular fluid metabolites; and (3) determine relative lipid content in oocytes to reveal any influence of these parameters on oocyte quality and IVF outcomes. Insulin, glucose, triglyceride and free fatty acids were lower in follicular fluid than blood and not strictly correlated between compartments. Insulin, glucose and triglyceride positively correlated with increasing BMI and waist circumference in blood and follicular fluid. Insulin increased in follicular fluid in association with metabolic syndrome. Free fatty acid composition analysis showed saturated fatty acids, particularly palmitic and stearic acid, to be more prevalent in follicular fluid than blood. There were no associations between follicular fluid metabolites or oocyte lipid content and clinical outcomes; however, oocyte immaturity correlated with follicular fluid glucose and fatty acid levels, as well as metabolic syndrome. The present study confirms that the human ovarian follicular environment surrounding the oocyte exhibits a unique metabolite profile compared with blood, with distinct localisation of lipids within follicular fluid and oocytes.
No preview · Article · Mar 2015 · Reproduction Fertility and Development
[Show abstract][Hide abstract] ABSTRACT: Maternal diabetes and obesity are characterised by elevated blood glucose, insulin and lipids, resulting in upregulation of specific fuel-sensing and stress signalling pathways. Previously, we demonstrated that, separately, upregulation of the hexosamine biosynthetic pathway (HBP; under hyperglycaemic conditions) and endoplasmic reticulum (ER) stress (due to hyperlipidaemia) pathways reduce blastocyst development and alter oocyte metabolism. In order to begin to understand how both glucose and lipid metabolic disruptions influence oocyte developmental competence, in the present study we exposed mouse cumulus-oocyte complexes to hyperglycaemia (30mM) and/or lipid (40?M) and examined the effects on embryo development. The presence of glucosamine (GlcN; a hyperglycaemic mimetic) or increased lipid during in vitro maturation severely perturbed blastocyst development (PO-linked glycosylation (O-GlcNAcylation) of proteins (PGfpt2) and O-linked ?-N-acetylglucosaminyltransferase (Ogt) was repressed following lipid treatment (PO-GlcNAcylation and ER stress as likely contributors to compromised fertility of obese women.
No preview · Article · Feb 2015 · Reproduction Fertility and Development
[Show abstract][Hide abstract] ABSTRACT: Insulin resistance is a key defect associated with obesity, type 2 diabetes and other metabolic diseases. While a number of factors have been suggested to cause defects in insulin action, there is a very strong association between inappropriate lipid deposition in insulin target tissues and the development of insulin resistance. In recent times, a large number of studies have reported changes in markers of mitochondrial metabolism in insulin-resistant individuals, leading to the theory that defects in mitochondrial substrate oxidation are responsible for the buildup of lipid intermediates and the development of insulin resistance. The primary support for the mitochondrial theory of insulin resistance comes from studies in skeletal muscle; however, there is recent evidence in murine models that mitochondrial dysfunction in oocytes may also play a role. Oocytes from obese or insulin-resistant mice have been shown to exhibit abnormalities in many different mitochondrial parameters, including mitochondrial morphology and membrane potential. Here we review the findings regarding the link between mitochondrial dysfunction and insulin resistance, and propose that abnormalities in mitochondrial metabolism in oocytes may predispose to the development of obesity and insulin resistance and thus contribute to the inter-generational programming of metabolic disease.
No preview · Article · Jan 2015 · Molecular Human Reproduction
[Show abstract][Hide abstract] ABSTRACT: Individuals conceived by in vitro fertilization (IVF) may be at increased risk of cardio-metabolic disorders. We recently reported that IVF conceived male mice displayed impaired glucose metabolism at normal and high body weights. In this study, we examined glucose metabolism in mature female C57BL/6J mice that were conceived by natural conception (NC), by ovarian stimulation (OS) or by IVF following chow or high-fat diet (HFD) for 8 weeks. By design, litter size was comparable between groups, but interestingly the birth weight of IVF and OS females was lower than NC females (p≤0.001). Mature IVF female mice displayed increased fasting glucose as compared to NC and OS mice, irrespective of diet. Mature IVF and OS mice were also more susceptible to the metabolic consequences of high fat diet as compared with NC females, with impaired glucose tolerance (p≤0.01), whereas peripheral insulin resistance and increased hepatic expression of gluconeogenic genes Ppargc1α, Pck1 and G6pc was observed in IVF mice only (p<0.05). This study suggests that ovarian stimulation alone and IVF program distinct metabolic effects in females, but that high fat diet may be required to unmask these effects. This study adds to the growing body of literature that assisted reproduction procedures may increase the risk of developing type 2 diabetes in an obesity prone environment.
[Show abstract][Hide abstract] ABSTRACT: An increasing number of non-erythroid tissues are found to express hemoglobin mRNA and protein. Hemoglobin is a well described gas transport molecule, especially for O2 but also for NO, CO O2 and CO, and also acts as a reactive oxygen species scavenger. We previously found Hba-a1 and Hbb mRNA and protein at high levels within mouse peri-ovulatory cumulus cells, but not in cumulus following in vitro maturation. This led us to investigate the temporal and spatial regulation in follicular cells during the peri-ovualtiry period. Cumulus oocyte complexes (COCs) were collected from eCG/hCG-treated peri-pubertal SV129 female mice and collected and analyzed for gene expression and protein localization at a variety of time points over the periovulatory period. A further cohort matured in vitro with different forms of hemoglobin (ferro- and ferri-hemoglobin) under different O O2 atmospheres conditions (2, 5 and 20% O O2) were subsequently fertilized in vitro and cultured to the blastocyst stage. Murine mRNA transcripts for hemoglobin were regulated by stimulation of the ovulatory cascade, in both granulosa and cumulus cells, and expression of HBA1 and HBB was highly significant in human granulosa and cumulus, but erythrocyte cell marker genes were not. Several other genes involved in hemoglobin function were similarly LH-regulated, including genes for heme biosynthesis. Immunohistochemistry revealed a changing localization pattern of HBA-A1 protein in murine cumulus cells and oocytes following the ovulatory signal. Significantly, no positive staining for HBA-A1 protein was observed within in vitro matured oocytes, but if co-incubated with ferro- or ferri-hemoglobin, cytoplasmic HBA-A1 was observed, similar to in vivo derived oocytes. Addition of ferro-, but not ferri-hemoglobin had a small, positive effect on blastocyst yield but only under either 2 or 20% O2 gas atmosphere. The identification of hemoglobin within granulosa and cumulus cells poses many questions as to its function in these cells. There are several possible roles, the most likely of which is either an O2 or NO sequestering molecule; perhaps both roles are engaged. The strong endocrine regulation during the peri-ovulatory period suggests to us that one potential function of hemoglobin is to provide a short-lived hypoxic environment by binding very tightly any available O2. This in turn facilitates the differentiation of the follicle towards corpus luteum formation, by enabling the stabilization of a key transcription factor known to initiate such differentiation; hypoxia inducible factor.
Full-text · Article · Nov 2014 · Biology of Reproduction
[Show abstract][Hide abstract] ABSTRACT: At fertilization, the gametes endow the embryo with a genomic blueprint, the integrity of which is affected by the age and
environmental exposures of both parents. Recent studies reveal that parental history and experiences also exert effects through
epigenomic information not contained in the DNA sequence, including variations in sperm and oocyte cytosine methylation and
chromatin patterning, noncoding RNAs, and mitochondria. Transgenerational epigenetic effects interact with conditions at conception
to program the developmental trajectory of the embryo and fetus, ultimately affecting the lifetime health of the child. These
insights compel us to revise generally held notions to accommodate the prospect that biological parenting commences well before
birth, even prior to conception.
[Show abstract][Hide abstract] ABSTRACT: Kit ligand (KITL) is an important granulosa cell-derived growth factor in ovarian folliculogenesis, but its expression and function in human granulosa cells are currently poorly understood. Based on studies performed in animal models, it was hypothesised that KITL gene expression in human granulosa cells is regulated by androgens and/or growth differentiation factor 9 (GDF9). We utilised two models of human granulosa cells, the KGN granulosa tumour cell line and cumulus granulosa cells obtained from preovulatory follicles of women undergoing assisted reproduction. Cells were treated with combinations of 5α-dihydrotestosterone (DHT), recombinant mouse GDF9, and the ALK4/5/7 inhibitor SB431542. KITL mRNA levels were measured by quantitative real-time PCR. No change in KITL mRNA expression was observed after DHT treatment under any experimental conditions, but GDF9 treatment resulted in a significant decrease in KITL mRNA levels in both KGN and cumulus cells. The effect of GDF9 was abolished by the addition of SB431542. These results indicate that KITL is not directly regulated by androgen signalling in human granulosa cells. Moreover, this study provides the first evidence that GDF9 negatively regulates KITL gene expression in human granulosa cells providing new information on the regulation of these important growth factors in the human ovary.
[Show abstract][Hide abstract] ABSTRACT: The fertility of high performance (high milk yield) dairy breeds such as the Holstein within the Australian dairy herd has been on the decline for the past two decades. The 12-month calving interval for pasture-based farming practises results in oocyte maturation coinciding with peak lactation, periods of negative energy balance and energy partitioning for lactation, causing energy deficiency in some organ systems, including the reproductive system. Oocyte developmental competence (the ability to undergo successful fertilisation, embryo development and establishment of pregnancy) is intrinsically linked with the composition of follicular fluid (FF). The aim of this study was to determine if there was a relationship between the fat and carbohydrate levels in plasma and FF and the ability to support in vitro oocyte maturation (IVM). Plasma and FF were collected in vivo from eight Holstein cows between 52-151 days post-partum. Plasma glucose trended (P = 0.072) higher and triglyceride levels were significantly higher than in FF (P < 0.05) but there were no relationships between FF and plasma composition. Glucose FF concentration was negatively related to follicular lactate and NEFA levels and days post-partum. Conversely, FF triglyceride concentrations were positively related to FF NEFA levels and negatively related to milk fat and protein composition. Abattoir-derived cumulus oocyte complexes (COCs) were cultured in either 50% FF (FF-IVM) or 50% plasma (plasma-IVM), with on time embryo development then assessed. While there were no differences between animals, blastocyst rates following FF-IVM were negatively related to plasma glucose and days post-partum and positively related to body condition score (BCS) and plasma NEFA levels. In comparison to previous studies, total NEFA levels in FF were not related to animal parameters and did not influence oocyte developmental competence in vitro. Results from this study suggest that days post-partum and BCS influence carbohydrate metabolism within the follicular environment and this may be attributed to the pasture-based feed system applied in the Australian dairy industry.
[Show abstract][Hide abstract] ABSTRACT: The STAT3 transcription factor is a pleiotropic transducer of signalling by hormones, growth factors and cytokines that has been identified in the female reproductive tract from oocytes and granulosa cells of the ovary to uterine epithelial and stromal cells. In the present study we used transgenic models to investigate the importance of STAT3 for reproductive performance in these different tissues. The Cre-LoxP system was used to delete STAT3 in oocytes by crossing Stat3fl/fl with Zp3-cre+ mice, or in ovarian granulosa cells and uterine stroma by crossing with Amhr2-Cre+ mice. Surprisingly, deletion of STAT3 in oocytes had no effect on fertility indicating that the abundance of STAT3 protein in maturing oocytes and fertilized zygotes is not essential to these developmental stages. In Stat3fl/fl;Amhr2-cre+ females impaired fertility was observed through significantly fewer litters and smaller litter size. Ovulation rate, oocyte fertilization and development to blastocyst were unaffected in this line; however, poor recombination efficiency in granulosa cells had yielded no net change in STAT3 protein abundance. In contrast, uteri from these mice showed STAT3 protein depletion selectively from the stomal compartment. A significant reduction in number of viable fetuses on gestational day 18, increased fetal resorptions and disrupted placental morphology were evident causes of the reduced fertility. In conclusion, this study defines an important role for STAT3 in uterine stromal cells during embryo implantation and the development of a functional placenta.
[Show abstract][Hide abstract] ABSTRACT: Oviducts play a critical role in gamete and embryo transport, as well as supporting early embryo development. Progesterone receptor (PGR) is a transcription factor highly expressed in oviductal cells, while its activating ligand, progesterone, surges to peak levels as ovulation approaches. Progesterone is known to regulate oviduct cilia beating and muscular contractions in vitro, but how PGR may mediate this in vivo is poorly understood. We used PGR null mice to identify genes potentially regulated by PGR in the oviducts during the periovulatory period. Histologically, oviducts from PGR null mice showed no gross structural or morphological defects compared to normal, littermates. However, microarray analysis of oviducts at 8 h post-hCG revealed over 1000 PGR-dependent genes. Ten genes were selected for validation using reverse-transcription polymerase chain reaction (RT-PCR) based on their potential roles in oocyte/embryo transport and support. Eight genes were confirmed to be down-regulated (Adamts1, Itga8, Edn3, Prlr, Ptgfr, Des, Myocd and Actg2) and one up-regulated (Agtr2) in PGR null oviducts. Expression of these genes was also assessed in oviducts of naturally cycling mice during ovulation, day 1 and day 4 of pregnancy. Adamts1, Itga8, Edn3, Prlr and Ptgfr were significantly up-regulated in oviducts at ovulation/mating. However, most genes showed basal levels of expression at other times. The exceptions were Prlr and Ptgfr which showed pulsatile increases on day 1 and/or day 4 of pregnancy. This is the first, comprehensive study to elucidate putative PGR-regulated genes in the oviduct and reveals key downstream targets potentially mediating oocyte and embryo transport.
Full-text · Article · Jun 2014 · Physiological Genomics
[Show abstract][Hide abstract] ABSTRACT: In-vitro fertilization (IVF) may influence the metabolic health of children. However, in humans, it is difficult to separate out the relative contributions of genetics, environment, or the process of IVF, which includes ovarian stimulation and embryo culture. Therefore, we examined glucose metabolism in young adult humans and in adult male C57BL/6J mice conceived by IVF versus naturally, under energy balanced and high-fat overfeeding conditions. In humans, peripheral insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamp (80mU/m(2)/min), was lower in IVF (n=14) versus controls (n=20) after 3 days of an energy-balanced diet (30% fat). In response to 3-days of overfeeding (+1250 kcal/day, 45% fat), there was a greater increase in systolic blood pressure in IVF versus controls (P=0.02). Mice conceived following either ovarian stimulation alone or IVF weighed significantly less at birth versus controls (P<0.01). However, only mice conceived by IVF displayed increased fasting glucose, impaired glucose tolerance and reduced insulin-stimulated Akt phosphorylation in liver following 8 weeks of either chow or high-fat diet (60% fat). Thus, ovarian stimulation impaired fetal growth in mouse, but only embryo culture resulted in changes in glucose metabolism that may increase the risk of developing metabolic diseases later in life, and in both mouse and humans.
[Show abstract][Hide abstract] ABSTRACT: Metabolism and ATP levels within the oocyte and adjacent cumulus cells are associated with oocyte quality and optimal development of a healthy embryo. Lipid metabolism provides a potent source of energy and its importance during oocyte maturation is being increasingly recognised. The triglyceride and fatty acid composition of ovarian follicular fluid has been characterised for many species and is influenced by nutritional status (i.e. dietary fat, fasting, obesity and season) as well as lactation in cows. Lipid in oocytes is primarily triglyceride of specific fatty acids which differ by species, stored in distinct droplet organelles that re-localise during oocyte maturation. The presence of lipids, particularly saturated versus unsaturated fatty acids, in in vitro maturation (IVM) systems affects oocyte lipid content as well as developmental competence. Triglycerides are metabolised by lipases which have been localised to cumulus cells as well as oocytes. Fatty acids generated by lipolysis are further metabolised byβ-oxidation in mitochondria for the production of ATP.β-oxidation is induced in cumulus-oocyte complexes by the LH surge and pharmacological inhibition ofβ-oxidation impairs oocyte maturation and embryo development. Promotingβ-oxidation with L-carnitine improves embryo development many species. Thus fatty acid metabolism in the mammalian cumulus-oocyte complex is regulated by maternal physiological and in vitro environmental conditions; and is important for oocyte developmental competence.