Molecular markers for oocyte competence in bovine cumulus cells

Theriogenology xxx (2016) 1–10

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Molecular markers for oocyte competence in bovine cumulus cells N.R. Kussano a, L.O. Leme b, A.L.S. Guimarães b, M.M. Franco a, c, M.A.N. Dode b, c, * a b c

School of Veterinary Medicine, Federal University of Uberlândia, Uberlândia, Minas Gerais, Brazil School of Agriculture and Veterinary Medicine, University of Brasilia, Brasília, Distrito Federal, Brazil Laboratory of Animal Reproduction, Embrapa-Genetic Resources and Biotechnology, Brasília, Distrito Federal, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 August 2015 Received in revised form 28 November 2015 Accepted 30 November 2015

This study aimed to quantify the expression of candidate genes in cumulus cells (CCs) from cumulus-oocyte complexes (COCs) with high and low potential for in vitro development up to the blastocyst stage. First, the effects of individual culture and biopsy on embryo development were evaluated. Individuals cultured using the well of the well system were compared with individuals cultured in 20 mL droplets (microdroplets) and those cultured in groups (control). Blastocyst rates were lower for the individual culture systems (P < 0.05; well of the well ¼ 17.9%, n ¼ 95; microdrop ¼ 26.3%, n ¼ 95) than for the control group (45.0%, n ¼ 209). Second, the effects of biopsy on embryo production were compared between the control and microdroplet cultures, and no effects (P > 0.05) were observed for either group. Finally, the expression profiles of glypican 4 (GPC4), IGF4-binding protein, follicle-stimulating hormone receptor, growth hormone receptor, epidermal growth factor receptor, fibroblast growth factor 11, solute carrier family 2 member 1, solute carrier family 2 member 3, sprouty homolog 1, versican, and keratin protein 8 in CCs obtained by biopsy were quantified by real-time polymerase chain reaction. Cumulus cells were categorized on the basis of the fates of the COCs: expanded blastocyst, cleaved and arrested, and uncleaved. The GPC4 gene was overexpressed (P ¼ 0.007) in CCs from oocytes that formed embryos compared with those that produced cleaved and arrested embryos. We concluded that individual culture reduced blastocyst production; however, biopsy did not affect embryo development. The profile of GPC4 expression can be used as a marker to distinguish COCs with potential for embryo development from those with limited developmental potential. Ó 2016 Elsevier Inc. All rights reserved.

Keywords: Biopsy Oocyte competence Individual culture Molecular marker

1. Introduction Among assisted reproduction techniques, in vitro embryo production (IVP) is the most widespread and studied in cattle. This technique has been used to accelerate the multiplication of valuable donors, and its commercial application has continuously increased in the past decade. * Corresponding author. Tel.: þ55 61 34484659; fax: þ55 61 3340 3658.. E-mail address: [email protected] (M.A.N. Dode). 0093-691X/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.11.033

Despite all the progress that has been made in IVP techniques, the percentage of embryos that are able to demonstrate normal development has remained stable, with blastocyst rate of approximately 40% [1–3]. One of the primary reasons for the reduced rate of in vitro blastocyst production is the poor quality or lack of competence of the selected oocytes, as only competent oocytes can progress through normal embryonic development [4,5]. Oocyte selection based solely on morphologic criteria has been routinely used for IVP; however, morphologic evaluation is insufficient to differentiate oocytes that are

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more or less competent [5–7]. Therefore, it is essential to develop an objective and accurate test to assess oocyte quality to improve IVP outcome. Several studies have been performed to identify differences between competent and incompetent oocytes, and have evaluated various parameters such as the amount and distribution of cytoplasm organelles [8], oocyte diameter [9–11], and gene expression analysis [12–16]; however, these methods are invasive procedures that preclude further use of those oocytes. An alternative, noninvasive technique for selecting the most competent oocytes when maintaining oocyte integrity is based on the identification of markers for competence in cumulus cells (CCs). The maintenance of functional two-way communication between the CCs and the oocyte [17–19] indicates that these cells play an important role in maturation, fertilization, and early embryonic development [10,20]. Consequently, it is thought that CCs could be a mirror that reflects oocyte competence. Indeed, previous studies have shown a correlation between CC mRNA expression profiles and oocyte and embryo quality [21–23]. Several candidate genes for oocyte competence have been identified in CCs [15,23–28]; however, these markers have not yet been used in standard IVF programs, even for humans. The barriers blocking implementation of this technology include the absence of a ready-to-use device that produces consistent results. It seems that the problem primarily results from the absence of a strong correlation between oocyte competence and the expression profiles of identified genes that would make them useful markers. Gene expression quantification in CCs from cumulusoocyte complexes (COCs) that have been proven to either produce or not produce embryos is the best method for validating candidate genes as molecular markers for oocyte competence; however, in cattle, only a few studies have followed embryonic development to confirm the association of CC candidate gene expression profiles with oocyte quality [27,28]. This information is imperative to establish a pattern of expression patterns of a number of selected CC genes that could be used to develop a more accurate method for oocyte selection for use in assisted reproduction techniques. 2. Materials and methods Unless otherwise indicated, the reagents used in this research and any chemicals used in the preparation of the maturation, fertilization, and in vitro culture media were purchased from Sigma (St. Louis, MO, USA). 2.1. Oocyte recovery Ovaries from crossbred females (Bos indicus  Bos taurus) were collected from local abattoirs immediately after slaughter and transported in saline solution (0.9% NaCl) supplemented with antibiotics (100 mg/mL of streptomycin and 100 IU/mL of penicillin G) at 35 C to 36  C. Cumulus-oocyte complexes were aspirated from 3- to 8mm diameter follicles using a 10 mL syringe with a 40  12 needle and then pooled in a 15 mL conical tube (TPP, Trasadingen, Switzerland). After sedimentation, COCs

were recovered and selected using a stereomicroscope. Only those oocytes with a homogenous cytoplasm and at least four layers of CCs were used for subsequent work. 2.2. Cumulus cell biopsies Cumulus cell biopsies were performed using an ophthalmic blade (15 Straight; ACCUTOME, Malvern, PA, USA). This ophthalmic blade was chosen as it was found to be the easiest and most comfortable to handle; one blade was used for each COC. Immediately after selection, immature COCs were individually placed in 50 mL drops of follicular fluid (previously centrifuged at 700  g for 5 minutes). A small fragment of CCs was removed using a blade, and COCs were placed in maturation media and taken to the incubator after micromanipulation. Individual biopsies were washed in two 50 mL drops of PBS, identified, and stored in microtubes containing RNAlater (Ambion Life Technologies, Carlsbad, CA, USA) at 20  C until used. 2.3. In vitro maturation Immediately after selection, COCs were transferred to basic maturation media comprising tissue culture media199 supplemented with 10% foetal calf serum, 0.01 IU/mL porcine FSH (pFSH), 12 IU/mL LH, 0.1 mg/mL L-glutamine, 0.075 mg/mL amikacin, and 0.1 mM cysteamine. Cumulusoocyte complexes were then matured for 22 hours at 38.5  C with 5% CO2. The oocytes were then matured either in groups (control), or individually in a 20-mL microdrop or in the well of the well (WOW) system [29]. In the control group, COCs (20–25 per replicate) were washed in IVM media, transferred into a 200-mL drop of IVM media covered with mineral oil, and then cultured for 22 hours at 38.5  C in 5% CO2. For the WOW system, we used a four-well culture dish, in which two of the wells contained 36 microwells (WOW plate 2004; INGÁMED, Maringá, Brazil). After selection, each COC (16 per replicate) was individually washed in IVM media and placed in a microwell that was immediately identified. The microwells were covered with 200 mL of IVM media, overlaid with mineral oil, and then cultured for 22 hours at 38.5  C with 5% CO2. The microdrop system consisted of sixteen 20 mL microdrops in a 60-mm diameter Petri dish covered with mineral oil. Cumulus-oocyte complexes were individually washed and transferred to properly identified droplets and matured for 22 hours at 38.5  C in 5% CO2. Nuclear maturation was not determined because in our system, it is already established that 85% to 90% of the oocytes obtained from 3- to 8-mm follicles reach metaphase II stage at 22 hours of maturation. 2.4. In vitro fertilization After maturation, the control group COCs were transferred to a 200 mL of fertilization media consisting of TALP [30] supplemented with penicillamine (2 mM), hypotaurine (1 mM), epinephrine (250 mM), and heparin (10 mg/mL). Frozen semen from a bull of proven fertility was used for all treatments and replicates; this bull has been

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used for several years as the reference bull in our laboratory. Motile spermatozoa were obtained using the Percoll (GE Healthcare, Piscataway, NJ, USA) gradient method in microtubes [31] and then added to the fertilization droplet at a final concentration of 1  106 spermatozoa/mL. Spermatozoa and oocytes were coincubated for 18 hours at 39  C with 5% CO2. Day 0 was considered to be the day of in vitro insemination. For the WOW system, maturation media was aspirated from the dish of matured oocytes and replaced with fertilization media. For the microdrop system, sperm (1  106/mL) was added to 350 mL of fertilization media and used to prepare 16 microdrops (20 mL) in a dish, and the matured oocytes were individually transferred into the drops of fertilization media containing the sperm. 2.5. Embryo culture (IVC) Eighteen hours after insemination, the presumptive zygotes from the control group were washed and transferred to 200 mL drops of synthetic oviduct fluid media with amino acids, citrate, and inositol (SOFaaci [32]) supplemented with 5% foetal calf serum and incubated at 38.5  C with 5% CO2 for 8 days. Embryos were evaluated on Day 2 for cleavage and on Days 6–8 to determine the blastocyst rates. For the WOW system, microwells were covered with 200 mL of SOFaaci and overlaid with mineral oil. Presumptive zygotes were individually removed from the IVF dish microwells, washed in SOFaaci media, and transferred to the new culture drops, maintaining the positions they occupied in IVM and IVF.

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For the microdrop system culture, the presumptive zygotes were removed from the IVF media and transferred to a new dish containing 16 microdrops (20 mL) of SOF, maintaining the positions they occupied on the fertilization dish. 2.6. RNA extraction and complementary DNA (cDNA) synthesis Total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions with minor modifications. The total RNA was used for cDNA synthesis using the FirstStrand cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Reactions were incubated at 65  C for 5 minutes, 50  C for 50 minutes, and 85  C for 5 minutes. Real-time quantitative polymerase chain reactions (RT-qPCRs) were performed using the Fast SYBR Green Master Mix kit (Applied Biosystems). Each sample was analyzed in triplicate and PCR specificities were determined by examining the melting curves and amplicon sizes on an agarose gel. Reactions were performed in a final volume of 25 mL using template cDNA equivalent to 0.175 of a CCs biopsy. The PCR conditions were 95  C for 5 minutes followed by 50 cycles of denaturation at 95  C for 10 seconds and then annealing and extension at 60  C for 30 seconds. The gene names, primer sequences, amplicon sizes, and annealing temperatures are listed in Table 1. The amplification profiles for three housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), b-actin, and peptidylprolyl isomerase A were analyzed using the GeNorm program [34]. This analysis

Table 1 Primer sequences, annealing temperatures, amplicon sizes in base pairs (bp), and reference GenBank accession numbers. Genes GAPDH EGFR GHR IGFBP4 GPC4 SLC2A1 SLC2A3 FGF11 SPRY1 FSHR VCAN KRT8

Primer sequence 0

0

F 5 -GGCGTGAACCACGAGAAGTATAA-3 R 50 -CCCTCCACGATGCCAAAGT-30 F 50 -AAAGTTTGCCAAGGGACAAG-30 R 50 -AAAGCACATTTCCTCGGATG-30 F 50 -AGAGATTCATGCCGACATCC-30 R 50 -CGTTGTCTGGTTCTCACACG-30 F 50 -TGTGTGCGTGTGTGTTAATGAGCC-30 R 50 -TTGGAAACATACCAGGGCTCTCCT-30 F 50 -TGGTGAATCCCACAACCCAGTGTA-30 R-50 TCTCAGCCACCATCAGCATAGCAT-30 F 50 -CAGGAGATGAAGGAGGAGAGC-30 R 50 -CACAAATAGCGACACGACAGT-30 F 50 -ACTCTTCACCTGATTGGCCTTGGA-30 R-50 GGCCAATTTCAAAGAAGGCCACGA-30 F 50 -TTCACCCACTTCAACCTGATCCCT-30 R 50 -AGACGCACTCCTTAAAGCGACACT-30 F 50 -CATGTGCTTGGTCAAGGGCATCTT-30 R 50 -TGTGACTGTGAACAGGAGCAAGGA-30 F 50 -GGATGCCATCATCGACTCTG-30 R 50 -TGACTCGAAGCTTGGTGAGAAC-30 F 50 -TCATAGCCACCCCAGAGC-30 R 50 -TTCCTTCCCCATCATGTCTC-30 F 50 -TGTGAAGAAGATTGAGACCCGCGA-30 R 50 -AAACCTCAGGTCTCCTGTGCAGAT-30

Annealing temperature ( C)

Fragment size (bp)

GenBank accession/reference

60

119

NM_001034034.2

53

253

XM_002696890.3

54

210

JQ711177.1

60

108

NM_174557.4

60

192

NM_001205784.1

59

258

BT029806

62

145

NM_174603.3

60

148

XM_005220239.2

60

93

XM_005217601.2

60

133

NM_174061

60

143

NM_181035

60

160

X12877/ [33]

Abbreviations: EGFR, epidermal growth factor receptor; FGF11, fibroblast growth factor 11; FSHR, follicle-stimulating hormone receptor; GAPDH, glyceraldehyde3-phosphate dehydrogenase; GHR, growth hormone receptor; GPC4, glypican 4; IGFBP4, IGF4-binding protein; KRT8, keratin protein 8; RT-qPCR, real-time quantitative polymerase chain reaction; SLC2A, solute carrier family 2 member 1; SLC2A3, solute carrier family 2 member 3; SPRY1, sprouty homolog 1; VCAN, versican.

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indicated that GAPDH was the most stable gene and was thus used as a reference for data normalization. The relative expression of each gene was calculated using the DDCt method with efficiency correction [35]. 2.7. Experimental Design 2.7.1. Experiment 1: effect of CC biopsy and individual culture systems on IVP We initially tested several types of ophthalmic blades, e.g., Micro Feather (Osaka, Japan) no. 715 15 ; SHARPOINT Slit 2.75 mm; Stab EAGLE 15 ; Slit Angled ACCUTOME 2.75 mm; and Straight ACCUTOME 15 . We performed 50 biopsies from COCs for each blade type to evaluate which one would be the most suitable for our manipulations. We then determined which individual culture system would be the most suitable to our conditions. Cumulusoocyte complexes (399) were distributed into three groups for IVM, IVF, and IVC: control (C), in which the COCs were cultured in groups (209 COCs); WOW, in which the COCs were cultured individually in the WOW system (95 COCs); and microdrop, in which the COCs were cultured in 20 mL microdrops (95 COCs). After fertilization, we determined the cleavage rates on Day 2, blastocyst rates on Day 7, and hatching rates on Day 8. After choosing the 20-mL microdrop culture system, we evaluated the effects of biopsy on blastocyst production. Cumulus-oocyte complexes (478) were divided into four groups and used for IVM, IVF, and IVC: a control group in which COCs were cultured in groups (115); a control group with biopsy (121), which was similar to the first group but including a biopsy of the COC; COCs that were cultured individually in 20 mL microdrops without biopsies (119); and COCs that were cultured individually in 20 mL microdrops with biopsies (123). Embryos were evaluated for cleavage on Day 2 and for blastocyst formation and hatching on Days 7 and 8, respectively. Biopsies were washed in PBS and stored individually with RNAlater in identified microtubes at 20  C.

expanded blastocysts on Day 7; (2) those that were derived from COCs that cleaved after IVF but that did not reach the blastocyst stage; and (3) those that were derived from COCs that did not cleave after IVF. Five pools of seven biopsies were formed per treatment group and 11 genes were selected for RT-qPCR expression profiling: glypican 4 (GPC4), IGF4-binding protein (IGFBP4), follicle-stimulating hormone receptor (FSHR), growth hormone receptor (GHR), epidermal growth factor receptor (EGFR), fibroblast growth factor 11 (FGF11), solute carrier family 2 member 1 (SLC2A1), solute carrier family 2 member 3 (SLC2A3), sprouty homolog 1 (SPRY1), versican (VCAN), and keratin protein 8 (KRT8). Each sample was analyzed in triplicate using GAPDH as a constitutively expressed gene. Of the 11 genes evaluated in this study, nine (GHR, FSHR, EGFR, GPC4, FGF11, IGFBP4, SPRY1, SLC2A1, and SLC2A3) were selected from our results using the follicle size model as an indicator of competence [11,15,36]. The remaining genes were chosen for their functions and/or have been identified in other studies as candidate markers (VCAN and KRT8) [27,28,37–42].

2.8. Statistical analysis The results of maturation and embryo development were analyzed using the chi-square test (P < 0.05). The standardized gene expression quantification data were evaluated for normality. Those genes that were normally distributed were analyzed by ANOVA and Tukey’s test, whereas those that were not normally distributed were evaluated using the Kruskal–Wallis and Mann–Whitney tests. Differences were considered statistically significant at the 95% confidence level (P < 0.05). All analyses were performed using Prophet Statistics software, version 5.0 (BBN Systems and Technologies, Cambridge, MA, USA; 1996). 3. Results

2.7.2. Experiment 2: candidate gene expression profiles in CCs of oocytes that were or were not capable of producing embryos in vitro This experiment aimed to evaluate the expression profiles of candidate genes in relation to oocyte competence in bovine CCs. Before maturation, biopsies were removed from compact CCs and individually stored for gene expression analysis. The COCs were then matured, fertilized, and cultured individually up to Day 7. Biopsies were then classified into three groups based on embryo production: (1) those that were derived from COCs that formed

There was no difference (P > 0.05) between the WOW and microdrop individual culture systems; however, both had reduced rates of embryo production compared with the control group. The embryo production data are shown in Table 2. When developmental speed was evaluated, development at Day 7 was similar for all groups; however, on Day 8, the individually cultured embryos were delayed, as they presented reduced rates of hatched embryos compared with the control group (Table 3).

Table 2 In vitro bovine embryo production from matured, fertilized, and cultured cumulus-oocyte complexes from control and individual culture systems well of the well (WOW) and 20-mL drop (microdrop). Group

Number of oocytes

Cleavage, Day 2 (%)

Blastocyst, Day 7 (%)

Blastocyst, Day 8 (%)

Hatched blastocyst, Day 8 (%)

Control group WOW Microdrop 20 mL

209 95 95

174 (83.3)a 62 (65.3)b 61 (64.2)b

94 (45)a 17 (17.9)b 25 (26.3)b

101 (48.3)a 17 (17.9)b 26 (27.4)b

29 (28.7)a 1 (5)b 1 (3.8)b

a,b

Different letters in the same column indicate significant difference by c2 (P < 0.05).

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Table 3 Developmental stages on culture Days 7 and 8 of bovine embryos produced in vitro from matured, fertilized, and cultured cumulus-oocyte complexes from control and individual culture systems well of the well (WOW) and 20-mL drop (microdrop). Group

Blastocyst (Day 7)

Blastocyst (Day 8)

Bi (%)

Bl (%)

Bx (%)

Bn (%)

Be (%)

Total

Bl (%)

Bx (%)

Bn (%)

Be (%)

Total

Control group WOW Microdrop 20 mL

9 (9.5)a 2 (11.7)a 4 (16)a

24 (25.5)a 5 (29.4)a 9 (36)a

47 (50)a 10 (58.8)a 12 (48)a

11 (11.7)a 0 (0)a 0 (0)a

3 (3.1)a 0 (0)a 0 (0)a

94 17 25

7 (6.9)a 3 (17.6)a 5 (19.2)a

53 (52.4)a 12 (70.5)a 16 (61.5)a

12 (11.8)a 1 (5.8)a 4 (15.3)a

29 (28.7)a 1 (5.8)b 1 (3.8)b

101 17 26

a,b Different letters in the same column indicate significant difference by c2 (P < 0.05). Abbreviations: Be, hatched blastocyst; Bi, early blastocyst; Bl, blastocyst; Bn, hatching blastocyst; Bx, expanded blastocyst.

The biopsy did not affect embryo production of any group (Table 4). Embryo production and developmental speeds were reduced for the individually cultured groups compared with the control group (Table 5). From the genes selected only one was differentially expressed among the different groups (Fig. 1). Cumulus cells from oocytes that reached the blastocyst stage exhibited greater GPC4 expression than CCs of oocytes that cleaved but did not continue to develop (P ¼ 0.007). However, the relative abundances of GHR (P ¼ 0.09) and VCAN (P ¼ 0.06) transcripts tended to be higher in the CCs of oocytes that reached the blastocyst stage compared with those that did not cleave (Fig. 1). No significant change in transcript level between the groups was seen for the FSHR, EGFR, FGF11, IGFBP4, SPRY1, SLC2A1, SLC2A3, and KRT8 genes (Fig. 2). 4. Discussion It is well established that CCs play an important role in paracrine signaling to the oocyte and in the acquisition of oocyte development potential [43–45]. Therefore, identifying markers for oocyte competence in CCs is an option for the development of a noninvasive method to select the highest quality oocytes. Many studies have focused on the identification of biomarkers in CCs that are related to oocyte developmental potential, especially in humans. In the present study, we determined the expression profiles of selected candidate genes in CCs from COCs for which the exact embryonic developmental outcome was known. Studies using biopsies to collect a small sample of bovine CCs from immature COCs are scarce and have not clearly described the procedure or its possible effects on oocyte viability [27,28]. Moreover, a negative effect of CC biopsy on embryo development has been reported when immature COCs were used [27]. Therefore, we initially established the procedure for collecting CC samples from immature COCs by testing ophthalmic blades with different slopes. We choose the 15 Straight ACCUTOME blade that we considered to be the easiest to handle to remove biopsies from COCs. With regards to the effects of biopsy,

our results showed no impact on embryo development for any culture method. Matoba et al. [27] observed a significant drop in blastocyst development when they biopsied immature oocytes. The cause of the difference in results is not known; however, some factors, such as the method of biopsy removal and the number of cells recovered can certainly affect subsequent development. To evaluate CC molecular markers for oocyte quality, their expression profiles must be associated with COC capacity to produce embryos or even to establish pregnancy. This procedure has been used for humans by several research groups [41,46–50]. Most studies in cattle have assessed gene expression in CCs using different models for oocyte competence [11,14,15,22,51–55] and were performed using grouped culture conditions in which the exact embryonic development of each biopsied COC was unknown. In the present study, it was essential to monitor each COC to achieve our goals. Furthermore, as individual culture systems have reported variable results using bovine oocytes [27,29,56–60], we tested two different systems: the “WOW” system developed by Vajta et al. [29] and the 20-mL microdrop system [61–66]. Our results showed that embryo production was similar for the WOW and microdrop systems; however, both presented reduced rates (P < 0.05) compared with the control group (Table 2). According to Gardner and Lane [67], embryos secrete paracrine factors that have beneficial effects on the neighboring developing embryos, which could justify the reduced rate of development in individual culture. Notably, the WOW group should enjoy that benefit as all embryos are in the same culture drop; however, we observed that this culture method was also associated with reduced embryo development. Although our group had previously shown that embryo production using the WOW system was similar to the group culture [63], the individual culture was only used during embryo culture. In the present study, individual culture was instead used during IVM, IVF, and IVC, which increased the length of time each oocyte and embryo was handled, affecting embryo development. Because of its easy

Table 4 In vitro bovine embryo production from matured, fertilized, and cultured cumulus-oocyte complexes from the control and 20-mL drop (microdrop) individual culture systems, with or without cumulus cell biopsy. Group

Number of oocytes

Cleavage, Day 2 (%)

Control without Control with Microdrop without Microdrop with

115 121 119 123

98 109 81 78

(85.2)a (90.1)a (68.1)b (63.4)b

Different letters in the same column indicate significant difference by c2 (P < 0.05).

a,b

Blastocyst, Day 7 (%) 55 52 24 31

(47.8)a (43)a (20.2)b (25.2)b

Blastocyst, Day 8 (%) 57 58 25 31

(49.6)a (47.9)a (21)b (25.2)b

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Table 5 Developmental stage on culture Days 7 and 8 of bovine embryos produced in vitro from matured, fertilized, and cultured cumulus-oocyte complexes from the control and 20-mL drop (microdrop) individual culture systems, with or without cumulus cell biopsy. Group

Blastocyst (Day 7) Bi (%)

Bl (%)

Control without Control with Microdrop without Microdrop with

2 1 2 3

10 14 11 6

(3.6) (1.9) (8.3) (9.6)

(18.1)b (26.9)a,b (45.8)a (19.3)b

Blastocyst (Day 8) Bx (%) 38 31 11 21

(69.0)a (59.6)a,b (45.8)b (67.7)a,b

Bn (%)

Be (%)

Total

%

Bl (%)

Bx (%)

2 (3.6) 5 (9.6) 0 (0) 1 (3.2)

3 (5.4) 1 (1.9) 0 (0) 0 (0)

55 52 24 31

47.8 43.0 20.2 25.2

1 (1.7)a,b 0 (0)a 1 (4)a,b 2 (6.4)b

30 33 20 17

(52.6)a (56.8)a (80)b (54.8)a

Bn (%) 11 5 1 8

(19.2)a,b,c (8.6)a,c (4)a,c (25.8)b

Be (%) 15 20 3 4

(26.3)a,b (34.4)a (12)b (12.9)b

Total

%

57 58 25 31

49.6 47.9 21.0 25.2

a,b,c Different letters in the same column indicate significant difference by c2 (P < 0.05). Abbreviations: Be, hatched blastocyst; Bi, early blastocyst; Bl, blastocyst; Bn, hatching blastocyst; Bx, expanded blastocyst.

Fig. 1. Growth hormone receptor (GHR), VCAN, and GPC4 transcript levels analyzed by RT-qPCR using cumulus cells of bovine cumulus-oocyte complexes (COCs) from three groups: COCs that developed to the blastocyst stage (Embryo), COCs that cleaved but did not develop to the blastocyst stage (Cleaved), and COCs that were not cleaved (Not Cleaved). Each group had five pools of samples and was evaluated in triplicate. Data are presented as the mean  standard error. GPC4, glypican 4; RT-qPCR, real-time quantitative polymerase chain reaction.

handling, the 20-mL microdrop system was chosen for use in subsequent experiments. Of the genes evaluated, only one was differentially expressed among the groups. Glypican 4 was highly expressed in CC from COCs that developed into expanded blastocysts by Day 7 compared with CCs from COCs that arrested as two-cell embryos (Fig. 1). Although not statically different, the transcripts levels for VCAN (P ¼ 0.06) and GHR (P ¼ 0.09) genes showed a tendency to be more abundant in the in CCs derived from oocytes that formed expanded blastocysts by Day 7 than from those that remained uncleaved (Fig. 1). Therefore, we cannot discard the importance of these gene transcripts for oocyte competence. Versican is a proteoglycan that binds to hyaluronic acid in the expanded cumulus matrix. It acts to stabilize hyaluronic acid in the pericellular matrix and plays a central role in tissue maintenance and morphogenesis through its roles in proliferation, cell migration, and adhesion [46,68]. Several studies reported increased expression of this gene in human CCs from COCs that developed into embryos and/or had a successful pregnancy [41,42,69,70]. Matoba et al. [27] in his work did not observe differential expression of this gene in CCs from COCs that formed embryos; however, they used CCs from matured COCs. The differences in the expression at different stages of meiosis were reported by Wathlet et al. [41], who observed reduced expression of VCAN in CCs from matured COCs (metaphase II) compared with immature COCs (germinal vesicle); it is possible that this transcript is less abundant in matured oocytes as a consequence of its translation, which is required for maturation processes and CC expansion. Supplementation of the IVM media with GH accelerates nuclear maturation, induces CC expansion, and improves oocyte developmental competence [71–74]. The beneficial effects of GH on maturation and in vitro embryonic development occur only in the presence of CCs, indicating that it acts by binding its receptor to these cells. Here, we observed a tendency of GHR transcript levels to be more increased in CCs from COCs that gave rise to blastocysts compared with CCs from COCs that did not cleave. The association between GHR expression and oocyte competence was previously suggested in studies performed in our laboratory [15]. It is possible that binding of GH to its receptor can generate a signal that is transferred to the oocytes, stimulating mechanisms involved in competence acquisition. This signaling may play an important role during the final stage of follicular development.

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Fig. 2. IGF4-binding protein (IGFBP4), SPRY1, FSHR, SLC2A, KRT8, FGF11, EGFR, and SLC2A3 transcript levels analyzed by RT-qPCR in cumulus cells of bovine cumulus-oocyte complexes (COCs) from three groups: COCs that developed to the blastocyst stage (Embryo), COCs that cleaved but did not develop to the blastocyst stage (Cleaved), and COCs that were not cleaved (Not Cleaved). Each group had five pools of samples and was evaluated in triplicate. Data are presented as the mean  standard error. EGFR, epidermal growth factor receptor; FGF11, fibroblast growth factor 11; FSHR, follicle-stimulating hormone receptor; KRT8, keratin protein 8; RT-qPCR, real-time quantitative polymerase chain reaction; SLC2A, solute carrier family 2 member 1; SLC2A3, solute carrier family 2 member 3; SPRY1, sprouty homolog 1.

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A member of the glycan family, GPC4 is a heparan sulfate proteoglycan that is associated with the cell surface and is involved in several biological processes, including the regulation of growth factors, adhesion, signaling, proliferation, and differentiation [75,76]. These activities result from the structural variety of heparan sulfate, which facilitates their connections and interactions with a wide range of proteins, e.g., growth factors, chemokines, morphogenic substances, extracellular matrix components, enzymes, and so forth. [75]. More recently, growth differentiation factor-9 function in the induction of CC expansion was reported to also require heparan sulfate proteoglycan, which sequesters and facilitates the interaction of growth differentiation factor-9 with its receptor in COC [77]. In the present study, GPC4 transcript levels in CCs from COCs that formed expanded blastocysts were greater than in CCs from COCs that cleaved but did not form embryos by Day 7. Similar results were reported by Van Montfoort et al. [47], who evaluated GPC4 expression in human CCs and observed reduced expression in CCs from embryos that exhibited delayed cleavage compared with embryos that cleaved at the expected time. These data indicate that expression of this gene may indicate embryo viability. Among the evaluated genes, GPC4 was the only one that exhibited differential expression in COCs that cleaved but did not develop, and those that developed to blastocyst. Consequently, GPC4 appears to be the most appropriate for COC selection, which is consistent with previous findings from our laboratory. This information is critical to the identification of a CC gene expression pattern that can be used to identify the most competent oocytes. This capacity to noninvasively predict oocyte quality will greatly benefit IVP, bringing cost savings and increased pregnancy rates. According to the results of this study, immature oocyte CC biopsy does not affect embryo production. In combination with individual culture, this approach can be used to obtain RNA for molecular marker validation studies despite the reduced production resulting from the blastocyst culture system. Glypican 4 was differentially expressed in oocyte CCs that gave rise to expanded blastocysts by Day 7; therefore, it was the best indicator of the degree of competence and quality of the oocyte, and this gene could be used to noninvasively select competent oocytes. Acknowledgments The authors thank EMBRAPA (Grant: 03.13.06.001.00) and CAPES for their financial support, and the Qualimax (Luziania-GO) slaughterhouse for providing the necessary biological materials for this experiment. References [1] Pontes JHF, Melo-Sterza FA, Basso AC, Ferreira CR, Sanches BV, Rubnic KCP, et al. Ovum pick up, in vitro embryo production, and pregnancy rates from a large-scale commercial program using Nelore cattle (Bos indicus) donors. Theriogenology 2011;75:1640–6. [2] Guimarães ALS, Pereira AS, Leme LO, Dode MAN. Evaluation of the simulated physiological oocyte maturation system for improving bovine in vitro embryo production. Theriogenology 2015;83:52–7.

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