DOI: 10.34810/data1762
title: Replication data for: "Impact of adding oocyte-secreted factors BMP15 and GDF9 to the IVM medium of prepubertal goat oocytes on cumulus-oocyte communication, EGF receptor and embryo developmental competence"

description: Raw data of research article "Impact of adding oocyte-secreted factors BMP15 and GDF9 to the IVM medium of prepubertal goat oocytes on cumulus-oocyte communication, EGF receptor and embryo developmental competence". The aim of this study was to assess the effect of adding two of the main OSFs, the BMP15 and GDF9, to IVM medium of prepubertal goat oocytes. Cumulus-oocyte complexes (COCs) were in vitro matured in absence (control group) or presence of 100 ng/mL of BMP15, GDF9, or both. To determine cumulus-oocyte communication, transzonal projections (TZP) density at 0h, 6h, 12h and 24h of IVM were evaluated. 
After IVM, the epidermal growth factor receptor (EGFR) expression in oocytes and their cumulus cells, cumulus expansion, mitochondrial activity and intracellular ROS and glutathione (GSH) levels were assessed. Finally, cleavage and blastocyst rates after parthenogenetic activation (PA) and IVF, and blastocyst quality by cell count were assessed.
description: METHODOLOGICAL INFORMATION
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1. Description of methods used for collection-generation of data:

Cumulus-oocyte complexes (COCs) were in vitro matured in absence (control group) or presence of 100 ng/mL of BMP15 (BMP15 group), 100 ng/mL of GDF9 (GDF9 group), or with 100 ng/mL of GDF9 plus 100 ng/mL of BMP15 (GDF9+BMP15 group) depending on the experiment.

Transzonal projections (TZPs) density was evaluated at several timepoints during IVM (0h, 6h, 12h and 24h) by staining actin filaments with 5 μg/mL phalloidin-FITC. TZPs were observed as thin actin filaments going through the zona pellucida from the cumulus cells to the oocyte. One image was taken per oocyte in the equatorial plane and TZPs density was quantified as phalloidin-FITC average fluorescence intensity in the whole zona pellucida area, delimited by polygon selection tool using ImageJ software.

After IVM:
- EGFR protein was quantified both in oocytes and their respective cumulus cells separately.
 · EGFR quantification in oocytes was obtained by photographing EGFR-immunostained oocytes with a microscope camera and average fluorescence intensity was quantified using ImageJ v1.51h software.
 · EGFR quantification in cumulus cells was obtained by western blotting and band optical density was quantified by Image Studio Digits v5.2 software. EGFR expression was calculated as the ratio between the optical density of the EGFR band and that of vinculin in the same lane.


- Cumulus expansion of each COC was scored on a 0 to 4 scale where 0: no expansion, 1: only the outer cell layers are expanded, 2: expansion extending inwards to several cell layers, 3: all cell layers except the corona radiata are expanded, 4: all cell layers are expanded. Cumulus expansion index was calculated as the mean value scored per replica.

- Mitochondrial activity was quantified in oocytes by photographing oocytes stained with 200 nM MitoTracker Orange CMTMRos. A total of 35 cuts in the region of major intensity for a total image thickness of 50 µm were taken. Fluorescence intensity was quantified with Imaris 9.2 imaging software (Oxford Instruments Group; UK) to express average intensity divided by the area of the object.

- Reactive oxigen species (ROS) and gluthatione (GSH) were quantified in oocytes by photographing oocytes stained with 10 µM dichlorodihydrofluorescein diacetate (H2DCF-DA) or 12.5 µM monochlorobimane (MCB), respectively, with a microscope camera and average fluorescence intensity was quantified using ImageJ v1.51h software.

- Oocytes were in vitro fertilized with frozen-thawed sperm or parthenogenetically activated with ionomycin and DMAP, and presumptive zygotes or activated oocytes were cultured for 8 days for blastocyst production assessment. Blastocyst quality was assessed by differential staining and cell count of the inner cell mass (ICM) and the trophectoderm (TE) cells


2. Methods for processing the data: 
Data are presented raw.
description: MS Excel, null