Reprod Dom Anim doi: 10.1111/rda.12344 ISSN 0936–6768

Ghrelin Accelerates In Vitro Maturation of Bovine Oocytes E Dovolou1,2, IE Messinis2, E Periquesta3, K Dafopoulos2, A Gutierrez-Adan3 and GS Amiridis1 1 Department of Obstetrics & Reproduction, Veterinary Faculty, University of Thessaly, Karditsa, Greece; 2Department of Obstetrics & Gynecology, Faculty of Medicine, University of Thessaly, Larissa, Greece; 3Departmento de Reproducciόn Animals y Conservaciόn de Recursos Zoogeneticos, INIA, Madrid, Spain

Contents Ghrelin, apart from its metabolic role, is nowadays considered as a basic regulator of reproductive functions of mammals, acting at central and gonadal levels. Here, we investigated for possible direct actions of ghrelin on in vitro maturation of bovine oocytes and for its effects on blastocyst yield and quality. In experiment 1, cumulus oocyte complexes (COCs) were matured in the presence of four different concentrations of ghrelin (0, 200, 800 and 2000 pg/ml). In vitro fertilization and embryo culture were carried out in the absence of ghrelin, and blastocyst formation rates were examined on days 7, 8 and 9. In experiment 2, only the 800 pg/ml dose of ghrelin was used. Four groups of COCs were matured for 18 or 24 h (C18, Ghr18, C24 and Ghr24), and subsequently, they were examined for oocyte nuclear maturation and cumulus layer expansion; blastocysts were produced as in experiment 1. The relative mRNA abundance of various genes related to metabolism, oxidation, developmental competence and apoptosis was examined in snap-frozen cumulus cells, oocytes and day-7 blastocysts. In experiment 1, ghrelin significantly suppressed blastocyst formation rates. In experiment 2, more ghrelintreated oocytes matured for 18 h reached MII compared with controls, while no difference was observed when maturation lasted for 24 h. At 18 and 24 h, the cumulus layer was more expanded in ghrelin-treated COCs than in the controls. The blastocyst formation rate was higher in Ghr18 (27.7  2.4%) compared with Ghr24 (17.5  2.4%). Differences were detected in various genes’ expression, indicating that in the presence of ghrelin, incubation of COCs for 24 h caused overmaturation (induced ageing) of oocytes, but formed blastocysts had a higher hatching rate compared with the controls. We infer that ghrelin exerts a specific and direct role on the oocyte, accelerating its maturational process.

Introduction Metabolic status is an important regulator of fertility in most mammalian species. In humans, pathological conditions, such as anorexia nervosa or periods of extreme energy outflow, such as intensive physical exercise, or starvation, are associated with reduced fertility (Tena-Sempere 2007). High milk yield in dairy cows causes a long-lasting negative energy balance (NEB) that is a serious determinant for impaired fertility (Wathes et al. 2007). Under those conditions elevated, ghrelin concentrations have been detected in both species (Jurimae et al. 2007; Bradford and Allen 2008; Usdan et al. 2008). Bovine in vitro embryo production can be used as model for human IVF providing, in general, more accurate information than the common laboratory animals (Menezo and Herubel 2002).

Place where the work was carried out: Karditsa – Greece. © 2014 Blackwell Verlag GmbH

In in vitro bovine embryo production, the main source of oocytes is the slaughterhouse ovaries; this inevitably leads to lower blastocyst formation rates and to inferior quality of produced embryos when compared to in vivo derived ones (Rizos et al. 2002). Although in vitro embryo culture conditions seriously affect embryo quality and viability, the same stands true for the in vitro oocyte maturation conditions, and consequently, the latter should not be overlooked. In cattle, early embryonic development – up to the 8 to 16 cell stage – is supported by the oocyte proteins and mRNA; after this stage, the embryonic genome is activated and takes over control of the development (Memili et al. 1998). Oocyte maturation, which to a great degree depends on the undisturbed oocyte–cumulus cell communication (Tanghe et al. 2003), includes both nuclear maturation – progression to metaphase II – and cytoplasmic maturation that is characterized by molecular and structural changes including the accumulation of mRNAs and various nutrients and substrates that are required to sustain fertilization and early embryonic development (Watson 2007). In 1999, the peptide ghrelin was discovered by Kojima et al. (1999) and was initially characterized as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In recent years, a body of evidence suggests that ghrelin does not possess a purely metabolic role, as the hormone and its receptor have been detected in many tissues (Dupont et al. 2010). A great number of published works provide empirical evidence that ghrelin has a pivotal role in regulating reproductive functions in many species. At central level, many studies report an inhibitory role of ghrelin on gonadotrophin – mainly LH – pulsatility, due to inhibition of hypothalamic GnRH neurons and/or due to direct action at pituitary level (Fernandez-Fernandez et al. 2004; Forbes et al. 2009; Dovolou et al. 2013). In comparison with a plethora of reports related to the effects of ghrelin on gonadotrophin secretion (reviewed by Tena-Sempere 2007), those related to its effects on oocyte maturation and on embryo development are scarce. The expression of ghrelin mRNA and GHSR-1a mRNA has been shown in in vitro matured oocytes and in vitro cultured embryos, with the expression level influenced by the maturational stage of the oocyte and the developmental stage of the embryo (Du et al. 2010; Deaver et al. 2013). The addition of ghrelin in the culture of human mid-luteal cell culture suppressed cell proliferation and reduced the ability of the cells to secrete estradiol and progesterone (Tropea et al. 2007). We have recently shown that addition of ghrelin

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E Dovolou, IE Messinis, E Periquesta, K Dafopoulos, A Gutierrez-Adan and GS Amiridis

in bovine embryo culture medium dramatically reduces blastocyst formation rate (Dovolou et al. 2014). The objectives of this study were to test an overall hypothesis that supernormal ghrelin concentrations can modulate in vitro bovine oocyte maturation. To this end, we tried to imitate ghrelin concentrations, which are typical for the preprandial period and fasting (WetrzLutz et al. 2006), in an in vitro maturation model that would allow the testing of the effects of ghrelin on oocyte developmental competence, and to assess the quality of the in vitro produced blastocysts through the relative abundance of various genes.

Materials and Methods In vitro embryo production Unless differently stated, all chemicals were purchased from the Sigma Chemical Company (Poole, UK). Bovine acylated ghrelin was purchased from Anaspec – Germany. The techniques for in vitro embryo production have been previously described (Dovolou et al. 2014). Immature cumulus oocyte complexes (COCs) were collected from abattoir material, by aspirating all follicles with a 3–8 mm diameter. The COCs were matured in TCM-199 supplemented with 10% (v/v) foetal calf serum (FCS), and 10 ng/ml epidermal growth factor, at 39°C under an atmosphere of 5% CO2 in air, and with maximum humidity. Depending on the experiment, the maturation medium was modified with the addition of 0, 200, 800 and 2000 pg/ml of acylated ghrelin. For IVF, matured COCs were inseminated with frozen–thawed, swim-up separated, bull sperm at a final concentration of 1 9 106 spermatozoa/ml. Gametes were co-incubated at 39°C under an atmosphere of 5% CO2 in air, with maximum humidity. At approximately 20 h post-insemination (hpi), presumptive zygotes were denuded and cultured at 39°C in groups of 25 in 25-ll droplets, under mineral oil, in an atmosphere of 5% CO2, 5% O2 and 90% N2. The basal medium for all embryo cultures consisted of synthetic oviduct fluid (SOF) supplemented with 5% FCS. Cleavage and blastocysts formation rates were recorded at 48 hpi and on days 7, 8 and 9pi, respectively. Pools of in vitro matured oocytes, cumulus cells and day-7 blastocysts were snap-frozen in PBS in liquid nitrogen and stored at 80°C for mRNA extraction and qRT-PCR. Oocytes were mechanically denuded by sequential passages of the COCs through a fine glass pipette; the cumulus cells were subsequently collected after centrifugation and washed three times in PBS. RNA extraction, reverse-transcription and quantification of mRNA transcript abundance Pools of 10 blastocysts, 10 denuded oocytes and cumulus cells from each experimental group were used to extract poly (A) RNA using the Dynabeads mRNA Direct Extraction Kit (Dynal Biotech, Oslo, Norway) according to manufacturer’s instructions after minor modifications. For the cumulus cells, the extracted RNA was a product of cumulus cells pooled from 10 COCs; moreover, RNA concentration of each sample was determined by spectrophotometry, and its quality was

evaluated by agarose gel electrophoresis. The reversetranscription (RT) reaction was carried out after extraction, following manufacturer’s instructions (Bioline, Ecogen, Madrid, Spain), using poly(T) primer, random primers and MMLV reverse transcriptase enzyme in a total volume of 40 ll to prime the RT reaction and to produce cDNA. cDNA preparation from cumulus cells was performed using 20 ng of total mRNA, and cDNA preparation from embryos and oocytes was performed using the total mRNA purified en each pool. Tubes were heated to 70°C for 5 min to denature the secondary RNA structure, and then, the RT mix was completed with the addition of 100 units of reverse transcriptase. Subsequent incubation at 42°C for 60 min allowed the reverse-transcription of RNA, followed by 70°C for 10 min for the denaturation of the RT enzyme. Before RT, the RNA amount from cumulus cells was quantified by a NanoDrop ND-1000 spectrophotometer (EuroClone S.p.A., Madrid, Spain). After quantification, RNA was incubated with DNAase. The amount of RNA extracted from each cumulus ranged from 0.4 to 0.7 lg. The same amount of RNA (0.4 lg) was used for reverse-transcription into cDNA. Real-time quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) was employed for the quantification of all mRNA transcripts. For qRT-PCR, three groups of cDNA per experimental group were used with two repetitions for all genes of interest. Experiments were conducted to contrast relative levels of each transcript and both beta actin (ACTB) and histone H2a.z (H2A.Z) in each sample. PCR was performed by adding a 2-ll aliquot of each sample to the PCR mix containing the specific primers to amplify histone H2Az (H2A.z), beta actin (ACTB), Bcl-2-associated X protein (BAX), prostaglandin G/H synthase-2 (PTGS2, also known as COX2), DNA (cytosine-5-) methyltransferase 3 alpha (DNMT3A), insulin-like growth factor receptor 2 (IGFR2), solute carrier family 2 (facilitated glucose transporter) member 1 (SLC2A1, also known as GLUT1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucose-6-phosphate dehydrogenase (H6PD), lactate dehydrogenase (LDHA), Mphase-promoting factor subunit Cyclin B1 (CCNB1), superoxide dismutase (SOD2), Gremlin 1 (GREM1) and glutathione peroxidase (GPX1). Primer sequences and the approximate sizes of the amplified fragments of all transcripts are detailed in Table S1. For quantification, real-time PCR was performed as previously described (Bermejo-Alvarez et al. 2010). PCR conditions were optimized to achieve efficiencies close to 1, and then, the comparative cycle threshold (CT) method was used to quantify expression levels. Quantification was normalized to the endogenous controls, H2A.z and beta actin. Fluorescence was acquired in each cycle to determine the threshold cycle or the cycle during the log-linear phase of the reaction at which fluorescence increased above background for each sample. Within this region of the amplification curve, a difference of one cycle is equivalent to the doubling of the amplified PCR product. According to the comparative CT method, the DCT value was determined by subtracting the housekeeping genes CT value for each sample from each gene CT value of the sample. The calculation of DDCT involved using the © 2014 Blackwell Verlag GmbH

Ghrelin and Bovine IVM

highest sample DCT value (i.e. the sample with the lowest target expression) as an arbitrary constant to subtract from all other DCT sample values. Fold changes in the relative gene expression of the target were determined using the formula 2-DDCT. Assessment of cumulus layer expansion, and stage of nuclear maturation All COCs used had at least 4 compact cumulus layers. After 18 or 24 h in IVM, the COCs were examined under stereomicroscopic visualization and the degree of expansion was expressed in a 4-grade scale, after modification of the classification described by Hunter and Moor (1987). Floating COCs with a ‘fluffy’ appearance and a complete expansion of all cellular layers of the cumulus mass were characterized as grade 4, while COCs with a complete expansion of all cumulus layers were characterized as grade 3. COCs with partial and moderate expansion of the cumulus layer were characterized as grade 2, and finally, COCs with the cumulus layer tightly adhered to the zona pelucida were characterized as grade 1. Next, the evaluation of cumulus expansion nuclear maturation was examined. Oocytes were completely denuded by vortexing, and they were placed on a glass slide overlaid with a coverslip that was supported by four droplets of a vaseline/paraffin mixture (40:1). After slight compression, oocytes were fixed for 48 h in acetic acid/methanol fixative (1:3), subsequently, they were stained with aceto-orcein (1% orcein in 45% acetic acid), and they were finally examined under phasecontrast microscopy. An oocyte was considered mature if it displayed a chromatin configuration corresponding to telophase I, or metaphase II. Experimental design Experiment 1 A total of 1994 COCs were divided into 4 groups: Control, n = 484; Ghr2000: n = 497, supplemented with 2000 pg/ml ghrelin; Ghr800 n = 524, supplemented with 800 pg/ml ghrelin; and Ghr200 n = 489, supplemented with 200 pg/ml ghrelin. A total of 10 replicates were carried out, and in every replicate, the number of available COCs was evenly distributed into each one of the aforementioned groups. Experiment 2 The aim of this experiment was to examine how ghrelin could influence the process of in vitro maturation of bovine oocytes. After having analysed the results of experiment 1, we decided to use only the 800 pg/ml concentration of ghrelin, as it was the one that had the most profound effect on the blastocyst formation rate. Oocytes were matured for 18 or 24 h in the presence of 0 (control C) or 800 pg/ml (Ghr) of ghrelin. Evaluation of in vitro oocyte maturation To access the degree of cumulus expansion and stage of nuclear maturation, we used 387 COCs divided in © 2014 Blackwell Verlag GmbH

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groups as described above; five replicates were carried out. After a macroscopic evaluation of the maturational stage, oocytes were completely denuded for an assessment of the stage of nuclear maturation; 12 oocytes with grade 3 cumulus expansion were examined from each group and replicate. Denuded oocytes (pools of 10) and cumulus cells separated from the suspension by centrifugation at 302 g for 10 min were snap-frozen in liquid nitrogen for mRNA extraction. Quantitative RT-PCR was used to quantify transcripts for COX2, GPX1, SOD2, CCNB1, LDHA, GREM1, GADH, and COX2, GPX1, SOD2, LDHA, CCNB1, G6PD, GLUT1, GADPH, GREM1 in oocytes and cumulus cells, respectively. In vitro embryo production For embryo production, a total of 1151 oocytes were used in five replicates divided in four groups (C18, n = 210; Ghr18, n = 481; C24, n = 243; Ghr 24, n = 217). For mRNA extraction, day-7 blastocysts from each group were snap-frozen in liquid nitrogen in groups of 10. The quantification of transcripts for IGF2R, SOD2, SLC2A1, H6PD, GPX1, DNMT3A and BAX was carried out by real-time qRT-PCR. Statistical analyses Data were analysed using the SIGMASTAT (Jandel Scientific, San Rafael, CA, USA) software package. Differences in cumulus expansion and nuclear maturation were examined by chi-square test. Differences in embryo development (cleavage and blastocysts formation rates) were analysed by one-way repeated measures ANOVA, with arcsine transformation. A two-factor analysis of variance (ANOVA) was applied to test for possible interactions between time of maturation and doses of ghrelin on the relative mRNA abundance. Because no interactions were detected, the differences on relative mRNA abundance among groups were finally analysed by one-way ANOVA with multiple pair-wise comparisons using Student–Newman–Kelus method post hoc. In all cases, significance was set at the 0.05 level.

Results Experiment 1 No difference was detected between groups in the cleavage rate. In all concentrations, ghrelin significantly reduced the embryo formation rate. No difference was detected between groups Ghr2000, Ghr800 and Ghr200 in the hatching rate; however, the latter was significantly increased in groups Ghr800 and Ghr200 in comparison with group C. All details related to embryo formation are presented in Table 1. Experiment 2 Assessment of cumulus expansion and oocyte maturation At 18 and 24 h, the proportion of grade 4 COCs was higher in Ghr800 (16.5% and 46.2%) compared with respective controls (4.5 and 16.6%), p < 0.001.

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E Dovolou, IE Messinis, E Periquesta, K Dafopoulos, A Gutierrez-Adan and GS Amiridis

Table 1. Effects of modification of maturation medium with 4 different concentrations of ghrelin, (0, 2000, 800 and 200 pg/ml, groups C, Ghr2000, Ghr800 and Ghr200, respectively) on cleavage, blastocysts formation and hatching rates. Values are expressed as means  standard error of the mean (SEM) of 10 replicates. Maturation lasted for 24 h Group

COCs in IVM (n)

C

Cleavage (%)

484

Ghr2000

497

Ghr800

524

Ghr200

489

366 75.6 347 69.8 353 67.3 335 68.5

Day-7 blast. (%) 109 22.5 68 13.7 64 12.2 78 15.9

 3.3  2.7  2.8  1.8

Day-8 blast. (%) 123 25.4 88 17.1 93 17.7 91 18.6

 1.2a  1.9b  1.24b  1.8b

 1.3a  2.0b  1.5b  2.6b

Day-9 blast. (%) 127 26.2 91 18.3 95 18.1 104 21.3

Hatched Day-9 blast. (%) 22 17.3 21 23.1 33 34.7 32 30.8

 1.3a  2.6b  2.3b  2.8ab

 6.2a  7.3ab  6.7b  4.6b

Different superscripts within columns denote significant difference (p < 0.05). Cleavage and blastocyst formation rates are expressed as proportion of COCs. Hatching rate is proportion of day-9 blastocysts. For all comparisons between C and Ghr800, p < 0.005.

At 18 and 24 h, no difference was detected in nuclear maturation rates among ghrelin-treated groups (86.7% and 90.0%, respectively). Conversely, more matured oocytes were found in C24 (85.0%) compared with C18 (71.7%), p < 0.05. At 18 h, more matured oocytes were found in group Ghr800 compared to those in group C18 (86.7% vs 71.7%, p < 0.05). Gene expression in oocytes At 18 h of maturation, ghrelin induced an over-expression of COX2, GPX1, SOD2, CCNB1 and GREM1 in comparison with respective controls. At 24 h, similar differences were detected for COX2, SOD2, CCNB1, LDHA, GAPDH. Data on oocyte gene expression are presented in Fig. 1. Gene expression in cumulus cells At 18 h of maturation, ghrelin induced up-regulation of COX2 and SLC2A1 in cumulus cells compared with respective controls. At 24 h, ghrelin caused downregulation of LDHA, SLC2A1, GAPDH, while CCNB1 was up-regulated. Data on gene expression in cumulus cells are detailed in Fig. 2.

Gene expression in blastocysts In blastocysts produced after 18 h and 24 h of oocyte maturation in the presence of ghrelin, SOD2 and G6PD were up-regulated compared with controls, respectively. In general, time and the presence of ghrelin affected the mode of gene expression as depicted in Fig. 3.

Discussion In this study, we have shown for the first time that the presence of high ghrelin concentrations in IVM medium accelerates in vitro bovine oocyte maturation. This observation could have serious implications both in bovine and human IVP, when oocyte donors are individuals with inherent hyperghrelinaemia.

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Ghrelin accelerates in vitro maturation of bovine oocytes.

Ghrelin, apart from its metabolic role, is nowadays considered as a basic regulator of reproductive functions of mammals, acting at central and gonada...
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