Nutrition has a strong influence on female bovine reproductive
performance. In recent years, there has been a
growing interest in investigating the relationship between
nutrition and reproduction. In dairy cows, high milk yield
leads to negative energy balance (NEB) which has adverse
effects for fertility (
Previous studies have indicated that ghrelin regulates
several reproductive functions (
To perform this experimental research, bovine ovaries
were obtained from an abattoir and transported to the
laboratory in sterile NaCl solution (9 g/L) including the
antibiotics streptomycin (100 mg/L) and penicillin (59
mg/L) at 37°C within 3 hours after slaughter. Ovaries
were pooled, regardless of the estrous cycle stage of the
donor. The COCs were aspirated from 3 to 8 mm follicles,
using an 18-G needle connected to a sterile syringe.
COCs with evenly granulated cytoplasms were selected under a low power (20-30 X) stereomicroscope (Nikon, Japan), and washed twice in TCM-199 buffered with 15 mM HEPES and IVM medium. Groups of 10 COCs were transferred into 50 μL of IVM medium under mineral oil (Squibb, USA). Incubation was performed at 39°C in an atmosphere of 5% CO2 in air with saturated humidity for 24 hours. COCs were matured in IVM medium supplemented with 0, 20, 40, and 60 pM acylated ghrelin. The total number of maturated COC was 1152. This total was divided on 200 COC for polymerase chain reaction (PCR) analysis, 480 for viability assay and 472 for pronuclear formation rates after
After IVM, COCs were pipetted several times with a narrow-bore pipette in TCM-199 buffered with HEPES, and washed three times in calcium- and magnesium-free phosphate buffer solution (PBS) containing 1 mg/mL polyvinylpyrrolidone (PVP). Total RNA was isolated from CCs and oocytes with TRIzol (Invitrogen, CA) according to the manufacturer’s instructions. Samples were then treated with a RNase-Free DNase kit (Qiagen, Germany). The RNA content of each sample was calculated through 260 nm absorbance. RNA quality was evaluated by the ratio of absorbance at 260 and 280 nm with a NanoVue spectrophotometer (NanoVue™-NV-General Electrics Healthcare Limited, UK). Complementary DNA (cDNA) was synthesized using a reaction mixture containing 1.5 μg of total RNA, random hexamers and the M-MLV reverse transcriptase (Invitrogen-Life Technologies, USA), following the procedure suggested by the manufacturer. Polymerase chain reaction (PCR) was subsecquently performed on the cDNA from oocytes and CCs. The reaction were performed at a final volume of 25 μL containing 4 μL cDNA, 0.85 pmol/ mL of each primer, 0.2 mmol/L of each deoxynucleoside triphosphate, PCR buffer 1X (50 mmol/L KCl and 10 mmol/L TriseHCl, pH=8.3) and 0.1% Triton X-100, 1.2 mmol/L MgCl2, and 1.5 units of Taq DNA polymerase (Invitrogen, CA). The cDNA amplification reactions for (
At the end of IVM, oocyte and CC viability were evaluated as follows. Oocytes were stripped of surrounding CCs by repeated pipetting in PBS containing 1 mg/mL PVP. Oocytes and CCs were incubated separately in the dark in 2.5 μg/L fluorescein diacetate fluorochrome and 2.5 g/L trypan blue in PBS medium for 10 minutes at 37°C. Then, they were washed three times in PBS. The CCs were centrifuge at 200 x g for 5 minutes. The pellet was resuspended in 50 μL of PBS. Oocytes and CC samples were transfered onto slides, which were immediately covered with cover slips and observed under a fluorescent microscope Olympus BX40 (Olympus, Japan) equipped with a 330-490 nm excitation filter and 420-520 nm emission filter. Live cells were visible with green fluorescence, whereas dead ones showed a characteristic blue staining under white light (
Oocyte and cumulus cells viability evaluated through fluorescein diacetate/trypan blue assay. The cell population was classified using the combined microscopic images obtained through light and fluorescence microscopy images. A. Alive cumulus cell (green) in the fluorescent field (×1,000 magnification), B. Dead cumulus cells (*) show a characteristic blue staining under white light (×1,000 magnification), and C. Alive (green) and dead (blue,*) bovine oocytes in a combined light and fluorescence field (×40 magnification).
The effect of different concentrations of ghrelin in the IVM medium on pronuclear formation was assessed after IVF (
Sequences of the primers for ghrelin (GHRL), growth hormone secretagogue receptor 1A (GHS-R1A) and the sizes of the reverse transcription polymerase chain reaction (RT-PCR) products
|Gene||Primer sequence (5´-3´)||Temperature annealing (°C)||Amplicon size (pb)|
|F: CTGAAGAAA CCCTGGCTAAC||57||107|
Fertilization status of putative zygotes produced in vitro with various ghrelin concentrations in IVM medium
|Treatments||Number of oocytes||n (%) 1 PN||n (%) 2 PN||n (%)>2 PN||n (%) penetrated|
|0 pM ghrelin||115||33a (28.6)||69a (60.0)||1a (0.8)||103a (89.5)|
|20 pM ghrelin||116||59b (50.8)||48b (41.3)||0a (0)||107a (92.2)|
|40 pM ghrelin||122||64b (52.4)||45b (36.8)||1a (0.8)||110a (90.1)|
|60 pM ghrelin||119||69b (57.9)||46b (38.6)||2a (1.6)||109a (91.5)|
IVM; In vitro maturation, PN; Pronucleus, a, b; Within a column, values without a common superscript are significantly different (P<0.05), and COCs; Cumulus oocyte complex. Pronuclear rate was recorded 18 hours after insemination (472 COCs were matured and fertilized in three replicates). The presumptive zygotes were incubated in Hoechst 33342 and then examined under a fluorescent microscope at ×200 and ×400 magnification.
We used completely randomized block designs. Statistical models included the fixed effect of treatment (0 vs. 20 vs. 40 vs. 60 pM ghrelin) and the random effects of block (day of COCs collection, n=3). Oocyte and CC viability and rate of pronuclei presence were analyzed with logistic regression using the GENMOD procedure (SAS Institute, NC). Data for oocyte and CC viability and rate of pronuclei presence were expressed as a percentage. The level of significance was P≤0.05.
Using total RNA prepared from bovine oocytes and CCs and the specific primers for
Oocyte viability was not significantly different (P=0.77) among COCs treated with 0, 20, 40, or 60 pM of ghrelin during IVM (89.0, 87.1, 88.0 and 89.1%, respectively). However, CC viability was significantly lower (P=0.04) in COCs matured with ghrelin (72.10, 66.32 and 46.86% for 20, 40, and 60 pM of ghrelin, respectively) than in COCs matured with 0 pM of ghrelin (77.65%). No differences were found between 20 and 40 pM of ghrelin. The lowest CC viability rate was observed with 60 pM of ghrelin (P=0.04).
The incidence of polyspermy (>2 pronuclei) and the percentage of mature oocytes penetrated by spermatozoa did not differ among treatments (P=0.96). However, the chance of two pronuclei forming (normal fertilization) were higher when ghrelin was not added to IVM medium (P= 0.03,
Agarose gel (2%) electrophoresis of polymerase chain reaction (PCR) products of
To our knowledge, this is the first study to report the expression of ghrelin and its receptor
Cumulus cells play a key role in the acquisition of nuclear and cytoplasmic oocyte maturation (