Assessment of loliolide extracted from Biserula pelecinus, present during in vitro oocyte maturation, on fertilisation and embryo development in sheep

Experimental design

In Experiment 1, methanol–chloroform extracts of B. pelecinus were fractionated using rapid silica filtration (RSF) and solvents of increasing polarity. Each final RSF fraction (about 10%) was tested in the IVEP-system, with each of the seven fractions compared at three concentrations (0, 100 and 200 μg mL⁻¹). Fractions were added to the medium (final concentrations: 0, 100 or 200 μg mL⁻¹) used for in vitro maturation (IVM) of abattoir-derived ovine cumulus–oocyte complexes (COC). After IVM (24 h), mature oocytes underwent in vitro fertilisation (IVF) and subsequent IVC procedures. Cleavage rate, blastocyst rate, hatching rate, blastocyst efficiency and total blastocyst cell number (TCN) were assessed. The most active fraction was selected for Experiment 2.

In Experiment 2, the fraction with the greatest effect was further fractionated using semi-preparative high-performance liquid chromatography (HPLC). The most abundant compound was identified then added as a supplement to IVM medium at different concentrations (0, 2.5, 5, 10 and 25 μg mL⁻¹) to test for effects on cleavage rate, blastocyst rate, hatching rate, blastocyst efficiency and TCN.

In both experiments, dimethyl sulfoxide (DMSO) was used to dilute (1 in 500) the fractions/compound, and was therefore used as the control treatment (+DMSO). A negative control was also included (–DMSO).

Collection of ovaries and oocytes

Ovaries from adult ewes were collected from an abattoir and transported to the laboratory in warm (33°C) phosphate-buffered saline (PBS). COCs were aspirated from follicles ≥2 mm in diameter by using an 18 g needle and a vacuum pump (Cook Australia, Queensland, Australia) with a pressure equivalent to 25 mm Hg. The COCs were collected into 2 mL aspiration medium that comprised Hepes-buffered TCM199 supplemented with 2% (v/v) heat-inactivated ovine oestrous serum (SS), 100 IU mL⁻¹ heparin (Pharmacia and Upjohn; Bentley, Western Australia, Australia), 100 μg mL⁻¹ streptomycin sulfate (CSL, Parkville, Victoria, Australia), and 100 IU mL⁻¹ penicillin G (CSL). Unexpanded COCs were recovered using a stereo-microscope (×40 magnification) (Olympus, Tokyo, Japan). Oocytes that were atretic or showed signs of an expanded cumulus were discarded.

In vitro maturation (IVM)

The procedures used were similar to those outlined by Walker et al. (1996) and Kelly et al. (2007). In brief, COCs were rinsed in IVM maturation medium containing sodium bicarbonate-buffered TCM199 supplemented with 20% (v/v) SS, 5 μg/mL follicle-stimulting hormone (Folltropin, Bioniche, Inc.), 0.1 IU mL⁻¹ hCG (Chorulon, Intervet, USA) and 1 μg mL⁻¹ oestradiol-17β. COCs were matured in four-well culture dishes (Nunc Inc., Naperville, IL, USA) containing 500 μL maturation medium covered with 300 μL mineral oil for approximately 24 h at 38.8°C in a humidified atmosphere of 5% CO₂ in air. The COCs were allocated equally among treatments.

Sperm preparation and in vitro fertilisation (IVF)

After maturation, COCs were gently stripped of excess cumulus cells in 400 IU mL⁻¹ hyaluronidase by using a fine-bore pipette, followed by three washes in IVF medium containing synthetic oviduct fluid (SOF) supplemented with 2% (v/v) SS. Up to 25 COCs were placed into four-well culture dishes containing 450 μL IVF medium overlaid with 300 μL mineral oil. Motile sperm were obtained using the ‘swim-up’ procedure in which 200 μL of frozen-thawed semen, pooled from two rams of proven fertility, were layered for 30 min under 1 mL of IVF medium in a 14 mL tube (Falcon, Becton Dickinson, Melbourne, Victoria, Australia). Approximately 0.5 × 10⁶ sperm were placed in each well and co-incubated with the COCs for 24 h at 38.8°C in a humidified atmosphere of 5% CO₂ in air.

In vitro culture (IVC)

After approximately 24 h, remnant cumulus cells were removed by gentle pipetting with a fine bore pipette and the presumptive zygotes were washed three times in IVC medium, which included SOF containing 8 mg mL⁻¹ bovine serum albumin (Fraction V; Invitrogen Corporation, Auckland, New Zealand) and amino acids at concentrations typical of oviduct fluid [22–23]. Presumptive zygotes (n = 20–25 per well) were cultured in 600 μL IVC medium under 300 μL of mineral oil in a humidified atmosphere of 5% CO₂:5% O₂:90% N₂ at 38.8°C. Oocytes that failed to divide (‘single cell’) were removed after 24 h, allowing the cleavage (fertilisation) rate to be determined.

Embryo assessment

Embryo development was assessed on Day 7 (Day 0 = day of IVF). Blastocysts, expanded blastocysts, hatching blastocysts and totally hatched blastocysts were collected, and stained so that the cells could be counted. Embryos were mounted on a microscope slide in a drop of glycerol containing Hoechst 33342 (1 mg mL⁻¹) and covered with a coverslip. The total cell count was recorded using a fluorescent microscope (Olympus Optical Co., Tokyo, Japan). Blastocyst rate was defined as the total number of blastocysts expressed as a percentage of the number of cleaved oocytes; hatching rate was defined as the total number of hatching and totally hatched blastocysts as a percentage of the total number of blastocysts; blastocyst efficiency was defined as the total number of blastocysts produced, expressed as a percentage of the original number of COCs.

Extraction and fractionation of B. pelecinus

For HPLC, we used an Agilent 1200 system equipped with a photodiode array detector (PDA) and fraction collector. Analytical separations were conducted using an Apollo C18 column (250 mm × 4.6 mm i.d., 5 μm, Grace-Davison, Deerfield, USA). Absorbance of UV was routinely measured at wavelengths of 220, 254 and 280 nm, and full photodiode spectra were collected between 200 and 600 nm. Semi-preparative HPLC was conducted using an Apollo C18 column (250 mm × 10.0 mm i.d., 5 μm, Grace-Davison). For high-resolution mass spectrometry (HR-MS), we used a Waters Alliance e2695 HPLC interfaced to a Waters LCT Premier XE time-of-flight (TOF) mass spectrometer fitted with an atmospheric pressure chemical-ionisation source (APCI) scanning at a mass range of 80–1000 amu. The mobile phase was 50% water (0.1% formic acid) and 50% acetonitrile at a flow rate of 0.3 mL min⁻¹ operating at ambient temperature. Nitrogen (20 psi) was used as the nebulising gas, vaporiser temperature was 350°C, drying gas flow was 10 mL min⁻¹, and capillary voltage was set to 2000 V. To obtain nuclear magnetic resonance (NMR) spectra, we used a Bruker Avance 600, with the 1 H NMR spectra being recorded at 600 MHz. Chemical shifts were expressed as δ values in parts per million (ppm) and coupling constants (J) in Hertz (Hz). For gas chromatography-mass spectrometry (GC-MS), we used a Hewlett Packard 6890 gas chromatograph connected to a HP 5973 mass spectral detector. The GC was equipped with a BPX5 column [(5% phenyl polysilphenylene-siloxane), 30 m × 0.25 mm × 0.25 μm film thickness, SGE Australia], by using helium as a carrier gas. All solvents used were of technical grade and distilled before use. All solvents used for HPLC were of HPLC grade.

Experiment 1

Samples of B. pelecinus in the vegetative stage were collected from UWA Farm Ridgefield, Western Australia (32°29′S, 116°58′E). The plant material (15.1 g dry weight) was extracted by stirring overnight with 1:1 (v/v) methanol:chloroform (MeOH:CHCl₃, 2 × 150 mL). The solvent phase was filtered (Whatman No. 1, 32.0 cm) and evaporated to dryness to provide 1.82 g of crude extract. Analysis of the crude extract by HPLC showed that a complex mixture of compounds was present (Fig. 1a), indicating the need for further purification steps.

Fig. 1.

(a) HPLC chromatogram of a crude methanolic extract of B. pelecinus and (b) a fraction of BP-6, showing the presence of only two major compounds. Loliolide accounted for more than 80% of the sample.

To separate the crude extract into fractions, it was dissolved in a minimal volume of MeOH/hexanes and added to a RSF column (4.5 cm × 12 cm) containing silica gel (10 g, Grace-Davisil, 40–63 μm) equilibrated with hexanes. The column was first eluted with hexanes (200 mL), followed by different solvent mixtures of increasing polarity from 20% to 100% (v/v) ethyl acetate (EtOAc) in hexanes and finally mixtures of 20–100% MeOH in EtOAc (all 200 mL volumes). A sample of each of the 10 fractions (Supplementary Table S1) was analysed by HPLC. Fractions with similar chromatograms (BP.1 and BP.2; BP.6 and BP.7; BP.9 and BP.10) were pooled, giving a total of seven fractions to be tested during IVM (Table S1). The fractions were concentrated under reduced pressure and dried under a gentle flow of nitrogen gas at room temperature.

Each final RSF fraction (about 10%) was tested in the IVEP system, with each of seven fractions compared at three concentrations of 0, 100 and 200 μg mL⁻¹. DMSO was used to dilute (1 in 500) the fractions and was therefore used as one of the control treatments. The most active fraction was selected for Experiment 2.

Experiment 2

Fraction BP-6 was selected for Experiment 2 because it decreased blastocyst rate when tested at 100 μg mL⁻¹. Fraction BP-6 from the RSF separation (16.1 mg) was subjected to semi-preparative HPLC. The sample was dissolved in a minimal volume of methanol and a volume of 500 μL was injected. The column was eluted with a gradient mobile phase, beginning with 1% acetonitrile in water (+0.1% trifluoroacetic acid) and increasing to 100% acetonitrile over 35 min at a flow rate of 4 mL min⁻¹. Fractions were collected every 1 min. Allowing for solvent delay and column cleaning, 20 fractions were collected and, on the basis of similarities in the HPLC chromatogram, they were combined to give a total of six fractions for testing.

One of the subfractions of BP-6 was shown to be pure compared to the unfractionated extract and contained two compounds, one of which accounted for >80% of the fraction (Fig. 1b). High-resolution mass spectrometry (HR-MS) of the main compound gave a protonated molecular ion [M + H] + at m/z 197.1186, corresponding to a molecular formula of C₁₁H₁₆O₃ (C₁₁H₁₇O₃ requires 197.1178). Careful analysis and comparison of the HR-MS data and NMR spectroscopic properties (Supplementary Fig. S1, Table S2) with a commercial sample confirmed the isolated compound to be the monoterpene, (–)-loliolide.

Loliolide was purchased from Toronto Research Chemicals (Canada). The effect of pure loliolide was assessed by adding it to the IVM medium at five concentrations (0, 2.5, 5, 10 and 25 μg mL⁻¹). We used DMSO (1 in 200) to dilute the loliolide; so, DMSO was used as one of the control treatments (0 μg mL⁻¹) and we compared the two controls. The experiment was replicated four times.

For the minor compound in fraction BP-6, along with NMR, GC-MS and accurate mass measurements were also conducted. HR-MS returned a protonated molecular ion [M + H] + of m/z 225.1498 corresponding to a molecular formula of C₁₃H₂₀O₃ (C₁₃H₂₁O₃ requires 225.1491). Comparison of the NMR spectroscopic data and electron ionization mass spectrum with literature values (Table S3) confirmed the compound to be 3-hydroxy-5,6-epoxy-β-ionone. A commercial sample of this compound was not available for testing.

Statistical analyses

For both experiments, treatment effects were initially analysed by comparing the two controls (‒DMSO, +DMSO). The controls did not differ significantly for any of the variables; so, the +DMSO control was used as the zero concentration for both experiments. The seven fractions of B. pelecinus (Experiment 1), tested at three concentrations (0, 100 and 200 μg mL⁻¹) and replicated five times, were compared within fraction. Loliolide (Experiment 2) was tested at five concentrations (0, 2.5, 5, 10 and 25 ng mL⁻¹) with three replications. Data for embryonic development (cleavage rate, blastocyst rate, hatching rate, blastocyst efficiency) were analysed using the procedure CATMOD in SAS (Statistical Analysis System, Cary, NC, USA). Concentration (fraction or loliolide) and replicate were included in the model. Total cell numbers (Experiment 2) were analysed using the GLM procedure in SAS, with concentration, embryo stage (early blastocyst, blastocyst, hatched) and replicate being included in the model. Probability values of <0.05 were considered significant.

Ethics approval

The experimental protocol was approved by the Animal Ethics Committee of the University of Western Australia (RA/3/500/70 and RA/3/500/71) according to the recommendations of the Australian National Health and Medical Research Council.

Experiment 1

There were no significant differences between the two controls (+DMSO and –DMSO) for any of the measures of oocyte and embryo development (Table 1). For fractions BP-1, BP-4, and BP-5, there were no consistent effects on any measure of embryo development. Compared with the DMSO control, no fraction affected cleavage rate.

At 100 μg mL⁻¹, blastocyst development rate was about 22% lower for BP-6, and about 18% lower for BP-9, than for the DMSO control (Table 1). At 200 μg mL⁻¹, blastocyst rate was about 14% lower for BP-3 and 17% lower for BP-9 than for the DMSO control.

For BP-9, the effect of lower blastocyst development rate at 100 and 200 μg mL⁻¹ was reflected in lower blastocyst efficiency for both concentrations. BP-6 blastocyst development rate was significantly lower at 100 but not 200 μg mL⁻¹, and the lower blastocyst rate at 100 μg mL⁻¹ was reflected in a lower blastocyst efficiency (Table 1). For BP-6 and BP-8 at 200 μg mL⁻¹, blastocyst efficiency was not affected. Although hatching rate for BP-6 was not significantly affected, more blastocysts were produced, leading to a higher hatching rate than the control values (Table 1).

Experiment 2

As expected, TCN increased as the embryo developed. Least-square means (±s.e.m.) for TCN were 66.1 ± 3.2 at the blastocyst stage, 109.1 ± 2.4 at the expanded blastocyst stage, and 168.9 ± 3.3 at the hatching stage.

There were no significant differences between the two controls (+DMSO, –DMSO) for any of the measures of oocyte and embryo performance, including TCN (Table 2). No effects of loliolide were observed on any of the measures of embryonic development, over the range of loliolide concentrations tested (Table 2). For TCN, there were no significant interactions between loliolide concentration and stage of embryo development.