Insulin during in vitro oocyte maturation has an impact on development, mitochondria, and cytoskeleton in bovine day 8 blastocysts

Accepted Manuscript Insulin during in vitro oocyte maturation has an impact on development, mitochondria, and cytoskeleton in bovine day 8 blastocysts Denise Laskowski, Renée Båge, Patrice Humblot, Göran Andersson, Marc-André Sirard, Ylva Sjunnesson PII:

S0093-691X(17)30276-5

DOI:

10.1016/j.theriogenology.2017.06.002

Reference:

THE 14138

To appear in:

Theriogenology

Received Date: 18 February 2017 Revised Date:

30 May 2017

Accepted Date: 4 June 2017

Please cite this article as: Laskowski D, Båge René, Humblot P, Andersson Gö, Sirard MarcAndré, Sjunnesson Y, Insulin during in vitro oocyte maturation has an impact on development, mitochondria, and cytoskeleton in bovine day 8 blastocysts, Theriogenology (2017), doi: 10.1016/ j.theriogenology.2017.06.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

REVISED

ACCEPTED MANUSCRIPT 1

TITLE

2

Insulin during in vitro oocyte maturation has an impact on development, mitochondria, and

3

cytoskeleton in bovine Day 8 blastocysts

RI PT

4 AUTHOR NAMES AND AFFILIATIONS

6

Denise Laskowski1, 3,*, Renée Båge1,3, Patrice Humblot1,3, Göran Andersson2,3, Marc-André Sirard4,

7

Ylva Sjunnesson1,3

8

Departments of 1Clinical Sciences and 2 Animal Breeding and Genetics, Swedish University of

9

Agricultural Sciences, 1 P.O. Box 7054; 2 P.O. Box 7023; SE-750 07 Uppsala, Sweden

M AN U

SC

5

10

3Centre

for Reproductive Biology in Uppsala (CRU), Uppsala, Sweden

11

4Departement

12

Intergénérationnelle (CRDSI) Pavillon Des Services, local 2732 University Laval Québec G1V 0A6,

13

Canada

TE D

des Sciences Animales, Centre de Recherche en Développement Reproduction et Santé

EP

14

15

*CORRESPONDING AUTHOR

17

Denise Laskowski

18

Correspondence should be addressed to: Department of Clinical Sciences, Swedish University of

19

Agricultural Sciences, P.O. Box 7054, SE-750 07 Uppsala, Sweden.

20

[email protected]

AC C

16

21 22

ABSTRACT

1

ACCEPTED MANUSCRIPT Insulin is a key metabolic hormone that controls energy homeostasis in the body, including playing a

24

specific role in regulating reproductive functions. Conditions associated with hyperinsulinemia can

25

lower developmental rates in bovine in vitro embryo production and are linked to decreased fertility in

26

humans, as in cases of obesity or type 2 diabetes. Embryo quality is important for fertility outcome

27

and it can be assessed by choosing scoring standards for various characteristics, such as

28

developmental stage, quality grade, cell number, mitochondrial pattern or actin cytoskeleton structure.

29

Changes in the embryo’s gene expression can reflect environmental impacts during maturation and

30

may explain morphological differences. Together with morphological evaluation, this could enable

31

better assessment and possibly prediction of the developmental potential of the embryo. The aim of

32

this study was to use a bovine model to identify potential gene signatures of insulin-induced changes

33

in the embryo by combining gene expression data and confocal microscopy evaluation. Bovine

34

embryos were derived from oocytes matured in two different insulin concentrations (10 µg/ml and

35

0.1µg/ml), then stained to distinguish f-Actin, DNA and active mitochondria. The total cell number of

36

the embryo, quality of the actin cytoskeleton and mitochondrial distribution were assessed and

37

compared to an insulin-free control group. A microarray-based transcriptome analysis was used to

38

investigate key genes involved in cell structure, mitochondrial function and cell division. Our results

39

indicate that insulin supplementation during oocyte maturation leads to lower blastocyst rates and a

40

different phenotype, characterised by an increased cell number and different actin and mitochondrial

41

distribution patterns. These changes were reflected by an up-regulation of genes involved in cell

42

division (MAP2K2; DHCR7), cell structure (LMNA; VIM; TUBB2B; TUBB3; TUBB4B) and

43

mitochondrial activation (ATP5D; CYP11A1; NDUFB7; NDUFB10; NDUFS8). Taken together, we

44

hypothesise that the increased proliferation in the insulin-treated groups might impair the

45

developmental potential of the embryos by inducing metabolic stress on the molecular level, which

46

could be detrimental for the survival of the embryo.

47 48

AC C

EP

TE D

M AN U

SC

RI PT

23

ACCEPTED MANUSCRIPT 1. INTRODUCTION

50

1.1 Insulin and fertility

51

Insulin has important functions in regulating lipid and glucose metabolism in mammals [1] and

52

insulin-dependent signaling is mitogenic [2]. In the dairy cow, elevated insulin levels during oocyte

53

maturation have detrimental effects on oocyte quality [3] and early embryo development [4]. The

54

dairy cow can experience periods of metabolic imbalance, such as negative energy balance (NEB)

55

postpartum or increased body condition score (BCS) during the dry period or in repeat-breeding

56

heifers [5–7]. Both conditions have negative impacts on reproductive functions in the cow [3,8,9].

57

Decreased reproductive performance may lead to economic losses for farmers and also negatively

58

impacts animal health and welfare [10,11]. Human patients with hypo- or hyperinsulinemia are known

59

to be affected by decreased fertility, as in anorectic or obese women [12,13]. The prevalence of

60

obesity and metabolic syndrome complex is increasing rapidly in our modern society [14] and often,

61

women suffering from obesity or type 2 diabetes are sub- or infertile, with secondary diseases such as

62

polycystic ovary syndrome (PCOS) [15]. However, while euglycemia and insulin levels are well

63

monitored during pregnancy [16], less focus on these parameters is given in the periconceptional

64

period. Moreover, insulin or insulin-like growth factors can be added in high concentrations as a

65

growth factor during different steps of in vitro production (IVP) of both human and bovine embryos

66

[17,18]. Since early embryonic development is a very sensitive period, during which metabolic

67

programming is orchestrated, high insulin levels during oocyte maturation could have harmful effects

68

on embryo developmental competence and even have consequences for the offspring later in life [19].

69

The objective of this study was to obtain further insights into insulin-induced changes in bovine

70

embryos deriving from oocytes matured under elevated insulin conditions. This model could also be

71

suitable for studying potential embryo-related factors underlying impaired fertility in obese women.

72

1.2. Embryo morphology and gene expression

73

The morphological evaluation of embryos has been performed for many years in both human and

74

bovine IVP in order to choose the best quality embryos for embryo transfer [20–24]. For scientific

75

questions, different types of staining methods can be used to further investigate embryo structure,

AC C

EP

TE D

M AN U

SC

RI PT

49

ACCEPTED MANUSCRIPT including cell number, as well as cellular organelles and structures, e.g. mitochondria and actin

77

filaments [25]. This additional information enables the assessment of differences among individual

78

embryos. The total cell number of the Day 8 blastocyst (BC8) can be counted and used as an indicator

79

for embryo quality [26], as in vivo embryos show a different average cell number compared to in

80

vitro-derived embryos [27–30]. Actin filaments are important constituents of the cytoskeleton and are

81

involved in important steps during early development [31,32]. The actin structure’s quality in

82

blastocysts has been shown to be a valuable indicator for embryo quality in the porcine species [25].

83

Mitochondria are maternally-inherited organelles that convert energy in cells by oxidative

84

phosphorylation [33]. They provide the energy for many processes including mitosis, molecular

85

transports and key events during embryo development [34]. Changes in mitochondrial distribution in

86

the oocyte’s cytoplasm during maturation is important for further developmental steps and viability

87

during early cleavage [35]. Previous studies have investigated the mitochondrial distribution in bovine

88

blastocysts [36] and identified seemingly favorable mitochondrial patterns in embryos [37]. Their

89

important regulatory role in early embryo development [38] may explain why mitochondrial

90

distribution is a good predictor for embryo quality. A disproportional distribution in the blastomeres

91

leads to arrested development [39], as a marked aggregation can be seen in arrested embryos [40–42].

92

Many studies have been performed to investigate the effects of certain factors or substances on

93

embryonic gene expression [43,44] and they have proved that transcriptional activity of certain genes

94

can also be used as a predictor for embryo quality. Gene expression analysis can possibly explain why

95

oxidative stress or other induced metabolic pathways are observed after specific treatments, and can

96

therefore help to obtain a broad picture of ongoing processes in the early embryo.

97

In this study, we aimed to combine both morphological evaluations and transcriptome analysis to

98

investigate the insulin-induced changes in bovine blastocysts.

99

2. MATERIALS AND METHODS

100

AC C

EP

TE D

M AN U

SC

RI PT

76

2.1. Experimental design

ACCEPTED MANUSCRIPT Bovine insulin was added during in vitro oocyte maturation in two different concentrations

102

(INS10=10 µg/ml; INS0.1=0.1 µg/ml) and a parallel insulin-free control group (INS0) was run as a

103

control in each production series. Concentrations were chosen based on a literature review with

104

respect to insulin concentrations in serum, follicular fluid and in vitro embryo production (IVP)

105

systems [45]. Since insulin is considered to be unstable in in vitro culture [46], the aim was to cover

106

both a typical dose used for IVP and a dose closer to what can be measured in vivo. On Day 8 after

107

fertilisation (Day 0), bovine blastocysts (BC8) were morphologically assessed and either stained for

108

detailed morphological analysis (n=168) or frozen at -80 ºC for gene expression studies (n=193).

109

2.2. In vitro production of embryos (IVP)

110

IVP was performed according to standard procedures [47] as previously described in our related

111

studies [47,48]. Media components were purchased from Sigma-Aldrich (Stockholm, Sweden) unless

112

stated otherwise, and media were freshly produced in the laboratory (order numbers are given below).

113

Embryos were produced in vitro from slaughterhouse-derived oocytes (n=1144, producing BC8 for

114

detailed morphological evaluation; n=1102, producing embryos for gene expression studies) in serum-

115

free conditions.

SC

M AN U

TE D

116

RI PT

101

2.2.1. Oocyte collection and washing

118

Ovaries were collected at a local abattoir and were kept in 0.9% sodium chloride at a temperature of

119

approximately 35 ºC until arrival at our lab within 4 hours after collection. After arrival, ovaries were

120

briefly washed twice with sterile 0.9% sodium chloride solution at 35 ºC. Follicles with diameters of

121

3–8 mm were aspirated by using a 5-mL syringe with an 18-gauge hypodermic needle. Aspirates were

122

collected into TCM 199 (M7528)-based search medium, consisting of HEPES-buffered TCM 199

123

supplemented with 0.2% w/v bovine serum albumin (BSA), fraction V (A3311) and 50 µg mL-1

124

gentamicin (gentamicin sulfate; G1264). The COCs were washed and only COCs of excellent or good

125

quality (Grade 1 or 2; [47]) were selected and put into maturation.

AC C

EP

117

ACCEPTED MANUSCRIPT 2.2.2. Oocyte maturation

127

Oocytes were randomly divided into three groups. Groups of 30–45 cumulus-oocyte complexes

128

(COCs) were matured in separate wells containing 500 mL basic in vitro maturation (IVM) medium,

129

consisting of bicarbonate-buffered TCM199 (M2154) supplemented with 0.68 mM L-glutamine

130

(G8540), 0.5 µg mL-1 FSH and 0.1 µg mL-1 LH (Stimufol; PARTNAR Animal Health, Port Huron,

131

Canada), 50 µg mL-1 gentamicin and 0.4% w/v BSA. Bovine insulin (I5500) was added to the

132

medium during the IVM period, at either 0.1 µg mL-1 (INS0.1) or 10 µg mL -1 (INS10). As a control,

133

medium without insulin supplementation (INS0) was used. All COCs were incubated for 22 h (24 h

134

after the end of aspiration) at 38.5 ºC under a 5% O2, 5% CO2 atmosphere.

135

2.2.3. In vitro fertilisation

136

After maturation, COCs were washed twice with washing media consisting of modified Tyrode's

137

albumin lactate pyruvate (mTALP), supplemented with 0.3% w/v fraction V BSA and 50 µg mL-1

138

gentamicin. Cumulus cells were removed by pipetting until 3-5 layers of cumulus cells remained. The

139

COCs were transferred to four-well dishes containing 460 mL fertilisation medium, mTALP

140

containing 0.6% w/v fatty acid-free BSA, 50 µg mL-1 gentamicin, 3 µg mL-1 heparin (H3149) and

141

penicillamine, hypotaurine and adrenaline (PHE), giving a final concentration of 1.7 mM sodium

142

chloride (S5886), 10 mM hypotaurine (H1384), 20 mM penicillamine (P4875), 1.5 mM adrenaline

143

(E1635), 42 mM and sodium metabisulphite (S9000). Two sperm straws were thawed and prepared

144

using the swim-up procedure [49]. In brief, spermatozoa were put into four tubes, each containing

145

1mL capacitation medium, consisting of mTALP without CaCl2, containing 1.25 mg mL-1 glucose and

146

50 µg mL-1 gentamicin and 0.6% w/v BSA, and incubated for 45 min at 38.5 ºC in a 5% CO2

147

incubator. After swim-up, the spermatozoa were removed and pooled in a centrifugation tube.

148

Centrifugation (300 x g, 8 min, RT) produced a sperm pellet, which was washed and diluted in

149

fertilisation medium. Sperm suspension giving a final concentration of 1 x 106 spermatozoa per mL

150

was added to the pre-incubated COCs. Approximately 30–45 COCs per well and spermatozoa were

151

co-incubated at 38.5ºC in a maximal humidified atmosphere of 5% CO2, 5% O2 and 90% N2 for 22 h.

AC C

EP

TE D

M AN U

SC

RI PT

126

ACCEPTED MANUSCRIPT 2.2.4. Embryo culture

153

For embryo culture, modified synthetic oviducal fluid (mSOF) was used, consisting of 0.11 M sodium

154

chloride (S5886), 7 mM potassium chloride (P5405), 1.19 mM potassium phosphate monobasic

155

(P5655), 25 mM sodium bicarbonate (S5761), 0.33 mM pyruvic acid sodium salt (P4562), 1 mM

156

L-glutamine (G8540), 0.171 mM calcium chloride (C7902), 1.5 mM glucose (G6152), 110 mM

157

sodium lactate (L7900) and 0.49 mM magnesium chloride (M2393), with addition of minimum

158

essential medium (MEM) non-essential amino acids solution (100 X ; M7145), amino acids solution

159

(50 X ; B6766), 0.4% w/v fatty acid-free BSA and 50 µg mL -1 gentamicin. The presumed zygotes

160

were denuded 22 h after fertilisation by pipetting and incubated in a humidified atmosphere of 5%

161

CO2, 5% O2 and 90% N2 at 38.5 ºC in 500 mL mSOF medium per well (covered with oil: Ovoil;

162

Vitrolife AB, Gothenburg, Sweden) until Day 8.

163

2.3. Morphological classification of blastocysts

164

On Day 7 and 8 after fertilisation, all embryos were evaluated by means of light microscopy.

165

Evaluation of developmental stage (blastocyst, expanded blastocyst, hatching or hatched blastocyst),

166

quality grade (Grades 1–4 with 1=best grade) was performed according to the IETS guidelines [21].

167

2.4. Embryo staining and microscopy protocol

168

Unless otherwise indicated, phosphate-buffered saline and 0.1% polyvinyl alcohol (PVA, P8136,

169

Thermo Fisher Scientific, USA) (PBS+0.1% PVA) was used as dilution and washing buffer.

170

Blastocysts were stained according to a triple staining protocol using 200 nM Mitotracker Orange

171

(MTO, Invitrogen M7510, Sweden), 4.45 mM Hoechst 33342 (B2261), and 5 µl Alexa Fluor

172

Phalloidin (AFP, A12379; Thermo Fisher Scientific, USA) in 200 µl buffer [41]. Blastocysts were

173

first stained with 200 nM MTO dissolved in mSOF, then incubated for at least 1 hour before staining

174

in humidified air at 38.5 °C and 5% CO2. After 30 min of staining, blastocysts were washed three to

175

four times in mSOF and incubated overnight in fixing solution (2% paraformaldehyde). Actin staining

176

was performed the following day with AFP. After 10 min of permeabilisation using 0.1% Triton X

177

(CAS 9002931), blastocysts were stained for 1 hour with AFP (2.5% solution). Following four 15-

AC C

EP

TE D

M AN U

SC

RI PT

152

ACCEPTED MANUSCRIPT min washes, Hoechst staining was performed for a period of 20 min using 4.45 µM Hoechst dye.

179

Mounting in black well plates (ThermoFisher Scientific) in Vectashield (Vector Laboratories, USA)

180

was performed after three washes to clean the embryos of the remaining dye. Embryos were placed in

181

approximately 2 µl fluid in the center of the black well plate and 1 µl Vectashield was added around

182

the drop until the well was full. Images were taken immediately after staining or on the following day

183

using an epifluorescence microscope (LSM 510; Carl Zeiss, Germany) and a fluorescence microscope

184

camera (AxioCam MRm; Carl Zeiss). The Argon laser (488 nm) and Helium-neon (HeNe) laser (543

185

nm) were used with the filters BP (bans pass) 505-530 for AFP and P 560-615 for MTO. For image

186

analysis, ZEN Black Edition Software (Carl Zeiss; http://www.zeiss.com, accessed 13 January 2017)

187

was used and all BC8 images were taken according to a standard setting for magnification (x20) and

188

one camera epifluorescence image with the focus area in the central area of the blastocyst (CAM).

189

Stage and grade was used to identify each individual embryo in the well.

190

The number of nuclei was counted by two independent evaluators in a blinded study from the CAM

191

picture (blue Hoechst staining). The average of both counts was calculated and the count repeated if

192

sums differed by more than 10%. If the repeated counts still differed by over 10%, the count was

193

excluded from analysis.

194

2.5. Classification of actin staining

195

Actin cytoskeleton quality was assessed for categories I to III as described by [25,41], where ACTIN1

196

was the best category and indicated a sharp staining of cell borders and an abundant microfilament

197

complement in the cytoplasm in at least 75% of all blastomeres. Embryos were classified as ACTIN2

198

if gross maintenance of the cell outline was given but some clumped or scant microfilaments in the

199

cytoplasm were present. If cell integrity was lost and actin filaments were mainly visible as clumps or

200

aggregates, embryos were categorized as ACTIN3 (Figure 1 and Table 1). Twelve consecutive

201

Z-stack planes were merged to produce a three-dimensional image in order to assess actin

202

cytoskeleton grade in each embryo. All images were evaluated twice by the same person in a blinded

203

procedure.

AC C

EP

TE D

M AN U

SC

RI PT

178

ACCEPTED MANUSCRIPT 2.6. Classification of mitochondria staining

205

Two categories were used for evaluation of the spatial distribution pattern of active mitochondria. The

206

category MITO contained three grades as described previously [25,41]: MITO1 as the best category

207

with active mitochondria in almost all cells (>90%) and an even distribution pattern (in detail, the

208

embryo should neither contain outlier cells with extreme staining nor large accumulations or clusters

209

of dye); MITO3 as embryos lacking active mitochondria in some cells or areas, an uneven distribution

210

of staining or overstained cells, lack of distinct “points” in cells or blurry staining, and more than two

211

large accumulations or unclear cell borders; and MITO2 as an intermediate class if the embryo had

212

few of the characteristics of class MITO3 but still some of class MITO1.

213

Mitochondria were further categorised into three quality classes (MitoC, mitochondria centered)

214

depending on their spatial distribution in the cytoplasm. Embryos were denoted MitoC1 if there was a

215

clear, even mitochondria distribution pattern around the nucleus in all blastomeres. Embryos with this

216

pattern in approximately 50-75% of all cells were categorized as MitoC2 and all embryos with lower

217

presence of this characteristic were graded as MitoC3 (Figure 1 and Table 1).

SC

M AN U

TE D

218

RI PT

204

Twelve consecutive Z-stack planes were merged to produce a three-dimensional image in order to assess MITO and MitoC grade in each embryo. All images were evaluated twice by the

220

same person in a blinded procedure.

221

EP

219

2.7. Statistical analysis

223

The effect of the insulin treatment, stage, grade and corresponding second-order interactions on

224

numbers of nuclei per embryo were analysed by ANOVA (SAS Version 9.2, Proc GLM).

225

Non-significant main effects or interactions were removed and adjusted estimates from the final

226

model presented. The significance of individual differences among groups was tested following

227

Scheffe’s adjustment. The contrast option was used to compare treated groups taken together versus

228

controls. The effect of insulin treatment, stage, grade and corresponding second-order interactions on

229

development rates and on categorical morphological variables were analysed by logistic regression

AC C

222

ACCEPTED MANUSCRIPT models (SAS Version 9.2, Proc LOGISTIC). Morphological response variables were first analysed

231

while using the 3 initial categories. In order to obtain sufficient numbers of observations in each box

232

of the contingency tables data, morphological variables belonging to class 2 and 3 responses were

233

gathered and the variables were analysed as a 0/1 response. Percentages of observations belonging to

234

class 1 responses (best type of morphological appearance) corresponding to significant effects from

235

the model are presented. The numbers of embryos on which calculations of differences are based are

236

presented in the respective figure.

237

2.8. Microarray-based gene expression study

238

In total, 120 of the 193 BC8s were used for microarray-based transcriptome analysis at Embryogene®

239

Canada. The detailed description of the method and analysis pipeline is described in [49,50] and our

240

previous experiment for gene expression data [4]. Briefly, embryos were pooled in groups of ten (4

241

replicates with 10 embryos for 3 different treatments) and total RNA and genomic (g) DNA were

242

extracted in parallel using the AllPrepDNA/RNA micro kit (Qiagen, Germany). RNA was amplified

243

and antisense RNA labeled, the hybridisation step on the microarray was followed by scanning the

244

slides with a PowerScanner (Tecan, Switzerland) and feature extraction was performed using Array-

245

pro6.3 (Media Cybernetics, USA). Background subtraction and data normalisation of the raw data

246

was performed, after which the obtained data was analysed using Ingenuity Pathway Analysis (IPA®,

247

Qiagen) for further interpretation of gene expression patterns and constructing pathways of

248

significantly differentially expressed genes (DEGs) following insulin treatment. RT-qPCR validation

249

of candidate genes was performed to confirm microarray accuracy.

250

2.9. RT-qPCR validation of candidate genes

251

Validation of microarray results was performed by RT-qPCR on the RNA from the same pools used

252

for the microarray analysis as described in a previous study [4]. Candidate genes for morphological

253

changes were chosen to validate the results of the current morphology study: VIM, MAP2K2,

254

CYP11A1, DHCR7, NDUFA10. Detailed PCR protocol, primer sequences and annealing temperature

255

and accession numbers for the experiment are summarised in Table 2. Data analysis was performed

AC C

EP

TE D

M AN U

SC

RI PT

230

ACCEPTED MANUSCRIPT using LightCycler 480 Software 1.5.0 SP4 (version 1.5.0.39) with the second-derivative maximum

257

analysis method. Data normalisation used GeNORM normalisation factor (Biogazelle, Ghent,

258

Belgium) from expression values of three reference genes. According to GeNORM, the two most

259

stable housekeeping genes (B2M and ACTB) were used to transform the data. Differences in

260

expression between the INS0 and the INS0.1 groups, as well as between the INS0 and the INS10

261

groups, were compared by means of unpaired t-testing (Prism 5; GraphPad Software Inc., La Jolla,

262

CA, USA) following log transformation of data. Differences in expression were considered to be

263

significant at one-tailed P value <0.05.

SC

RI PT

256

264

M AN U

265 266 3. RESULTS

268

Evaluation of blastocyst rate and embryo stage and grade revealed that the insulin-treated groups

269

exhibited lower blastocyst development rates (based on immature oocytes) than the control group

270

(Table 3), while there was no observable significant influence of insulin on cleavage, embryo grade or

271

stage.

272

Total cell number differed significantly in the INS groups compared to control (Table 3), with the

273

highest cell number observed following the INS0.1 treatment.

274

Actin and mitochondria categories were distributed in the different treatment groups as indicated in

275

Table 4. The best assessment categories of actin and mitochondria (ACTIN1, MITO1, and MitoC1)

276

were significantly more frequent in the INS0.1 and INS10 groups than in the control group.

277

Evaluation of the actin cytoskeleton revealed significant higher frequency of ACTIN1 correlated to a

278

more advanced stage (Figure 2). Evaluation of the total mitochondrial distribution showed that more

279

advanced stages have a higher percentage of MITO1, independent of the insulin treatment (Figure 3).

280

Mitochondria with the best category of spatial distribution in the cytoplasm, MitoC1, were more

281

abundant in embryos deriving from insulin-treated oocytes (Table 4). Here, we observed higher

AC C

EP

TE D

267

ACCEPTED MANUSCRIPT incidences of MitoC1 in the best grade embryos: MitoC1 is more frequent in good embryo quality

283

(80% of MitoC1 were assessed as grade 1 (very good/good embryos), 22.2% of MitoC1 were

284

categorized as grade 2 (moderate embryos) and no MitoC1 were present if the embryo was assessed

285

as grade 3 (poor). Even when sorted according to stage, a difference in MitoC1 was observed, with

286

most progressed stages differing significantly from stage 1 and 2 (Figure 4). The transcriptome data

287

revealed changes in genes related to some of the observed morphological changes in the blastocyst

288

(Table 5). Genes related to both cell structure and proliferation pathways and mitochondrial functions

289

were identified, and these indicated an increased expression in the INS0.1 and INS10 groups. Five of

290

the 12 described genes were validated by RT-qPCR with good concordance (Figure 5).

SC

RI PT

282

M AN U

291 292 293

4. DISCUSSION

294

4.1 Impact of insulin on developmental competence, cell number, and proliferation

296

In a previous study [4], we showed that addition of insulin during oocyte maturation decreased

297

blastocyst rates but did not significantly affect cleavage, embryo stage or grade. However, the cell

298

number was increased and multiple changes on the gene expression level could be observed. A similar

299

developmental rate pattern was observed in the embryos in this study, and the nuclei number

300

following insulin treatment increased by approximately 20% compared to the controls. This increased

301

cell proliferation may be explained by the mitogenic effect of insulin as an important growth hormone

302

[52,53]. Often, large embryos are assessed as good quality embryos since e.g. in vivo embryos contain

303

more cells than in vitro embryos, which are known to have a generally lower viability [28,30]. Our

304

results indicate that the increased growth and proliferation of the insulin-treated embryos results in

305

changes in morphological characteristics, but that this treatment might be stressful for the embryo, as

306

fewer embryos survived after treatment with insulin during maturation. These findings are in

307

agreement with the theory proposed by Leese (“quiet embryo hypothesis”) in which a moderate

308

growth was observed to be beneficial for embryo quality and long-term survival [54,55]. On the gene

AC C

EP

TE D

295

ACCEPTED MANUSCRIPT 309

expression level, we observed significant differences in the fold-change of genes related to cell

310

proliferation. We cannot rule out the potential selection process resulting from insulin treatment, with

311

fewer embryos surviving, but those that do being more “fit” for in vitro culture, though not

312

necessarily healthier. Of all DEGs identified in this experiment, candidate genes possibly related to

RI PT

313

phenotype differences (actin, mitochondria and cell number) of insulin-derived embryos were

315

selected. MAP2K2 is directly involved in insulin receptor-signaling and plays a critical role in

316

mitogen growth factor signal transduction [56]. DHCR7 is responsible for the final step of cholesterol

317

production [57]. Cholesterol has important functions as the structural component of cell membranes, a

318

precursor for steroid hormone synthesis and, through involvement, in the control of early development

319

[58,59]. The increased DHCR7 expression observed in the insulin-treated groups could reflect an

320

increased need for cholesterol for different cellular functions and cell proliferation and, together with

321

the increased MAP2K2 transcription, may explain the increased nuclei number in the insulin-treated

322

groups.

323

4.2 Insulin impact on actin-, cytoskeleton-, and cell structure- related genes

324

The actin content of embryos and actin distribution play important function roles in cell division, and

325

impaired actin microfilament distribution could negatively influence normal cell division [60–62].

326

Consequently, one explanation for the better actin grades in INS0.1 and INS10 embryos could be that

327

the insulin stimuli have created a more ATP-rich environment. This could result in faster mitosis and

328

the embryos coping with this metabolic stress would have the advantage of progressing faster and

329

showing a better actin quality than non-insulin-treated control embryos. Nevertheless, we cannot

330

assess the ones that did not survive the increased growth stimuli, as they died before reaching the

331

blastocyst stage. The decreased blastocyst rates of insulin-derived embryos indicate that, even if found

332

to have exhibited a better actin quality, fewer embryos survived the insulin stimuli compared to

333

controls. The INS0 embryos grew more slowly and might therefore have been less sensitive if the

334

actin skeleton quality was poorer, since cell division frequency was lower and less susceptible to

335

disturbances. Interestingly, we could observe that the probability to be assigned the best actin quality

AC C

EP

TE D

M AN U

SC

314

ACCEPTED MANUSCRIPT category increased with embryo stage, which also confirms the hypothesis that more progressed

337

stages or more actively proliferating embryos are often found to have a better actin quality. Indeed,

338

the ACTIN1 category was more frequent in both insulin-exposed groups. It is difficult to exclude the

339

possibility that more progressed embryos may have a subjectively higher chance of receiving better

340

actin grades, but it is clear that the insulin treatment seems to induce a different actin phenotype than

341

observed in controls and that this phenotype might resemble embryos at more advanced stages.

RI PT

336

The cytoskeleton consists of intermediate filament, tubulin-based microtubules and

343

actin-based microfilaments [63]. The LMNA gene codes for the structural proteins Lamin A and C,

344

which belong to the family of intermediate filaments, are responsible for cell stability and strength

345

[64]. Vimentin functions as a cell shape stabiliser and is responsible for cytoplasm integrity and

346

stabilising cytoskeletal interactions. Interestingly, vimentin is also involved in cholesterol transport in

347

the cell by controlling the transport of low-density lipoprotein (LDL) [65]. TUBB2B, TUBB3 and

348

TUBB4B are genes that encode different subfamilies of tubulins. Thus, there was significant upward

349

regulation of all three components of the cytoskeleton in the insulin-treated groups, which leads to the

350

conclusion that observed morphological changes of these embryos compared to controls could be

351

explained and confirmed by the underlying changes in gene expression and expected protein level.

352

Whether these changes are beneficial or detrimental for the later development of the embryo can only

353

be the subject of speculation. Further investigations of later embryonic stages would be of interest in

354

order to follow the development and viability of these embryos with different phenotypes, as pertains

355

to their cytoskeleton character.

356

4.3 Impact of insulin on mitochondrial distribution and function

M AN U

TE D

EP

AC C

357

SC

342

Two classes of mitochondria quality were chosen in order to better understand if both findings are

358

correlated and if any observations could be made about a stage- or insulin-dependent active

359

mitochondria pattern. Since insulin is a key metabolic hormone regulating energy homeostasis in the

360

body and at cell level [1], and hyperinsulinemia as in obesity or type 2 diabetes is associated with

361

mitochondrial damage and oxidative stress [66–68], our results could help to better understand the

362

role of insulin in mitochondrial stress. Here, we have shown that classification as MITO1 is more

ACCEPTED MANUSCRIPT likely for embryos obtained from the INS groups and for more advanced embryo stages. This

364

confirms our hypothesis following the actin and cell number evaluations, which postulates that insulin

365

treatment during IVM leads to a phenotype of accelerated development. With regard to the new

366

classes defined as MitoC1, insulin-derived embryos were more frequently categorised as MitoC1 than

367

controls. Additionally, good quality embryos or hatching/hatched embryos were more likely to be

368

assessed as MitoC1. The location of the active mitochondria around the unstained region of the

369

nucleus could indicate a highly active cell, metabolising more ATP for mitosis in order to proliferate

370

and grow.

SC

RI PT

363

ATP5D, CYP11A1 and the various NDUFA subunits are genes related to mitochondrial

372

functions. ATP5D encodes a subunit of mitochondrial ATP synthase which catalyses ATP synthesis in

373

the inner mitochondrial membrane during oxidative phosphorylation [69]. The increased expression

374

of ATP5D in the INS groups could be a signature of increased metabolic activity and thus increased

375

oxidative stress for the embryos, a possible explanation for their decreased viability (past and future)

376

[70,71]. In another study focusing on the influence of insulin supplementation on oocyte competence

377

[72], the authors showed better blastocyst development rates if insulin is added during oocyte

378

maturation at a concentration of 0.1µg mL-1. The doses used were quite low compared to other insulin

379

concentrations in media for cell culture systems [18], which could explain why a negative effect on

380

development may have been avoided. In this study, gene expression studies of the oocytes after

381

maturation showed decreased abundance of transcripts protective against oxidative stress, which the

382

authors interpret as stimulation of translation and thus less abundance of these transcripts and thus less

383

abundance. Comparison of gene expression of oocytes and blastocysts is made difficult by metabolic

384

programming occurring during early embryonic development, and short-term effects on oocytes might

385

differ from the effect of insulin treatment during maturation on later embryo stages after major

386

genomic activation[73].

387

AC C

EP

TE D

M AN U

371

The mitochondrial enzyme cholesterol side-chain cleavage enzyme CYP11A1 catalyses

388

the first step of steroidogenesis, the conversion of cholesterol to pregnenolone [74]. Because

389

cholesterol is an important component of cell membranes [75] and is also present in mitochondria, the

ACCEPTED MANUSCRIPT increased expression level of CYP11A1 may affect the morphological phenotype by inducing higher

391

mitochondrial activity and/or changes in steroid and cholesterol metabolism. Conditions involving

392

increased levels of steroids, such as hyperandrogenism in women suffering from PCOS, are associated

393

with decreased fertility with little knowledge about the underlying causes [76]. At the same time,

394

steroids are discussed as a blastocyst factor inducing capillary permeability during early pregnancy

395

and important for implantation [77,78]. In the porcine species, the regulation of steroidogenic enzyme

396

gene expression in the peri-implantation conceptus is strongly related to IGF-1 [79], which shares

397

important signaling pathways and receptor mechanisms with insulin. Therefore, more knowledge

398

about the possible beneficial or detrimental effects of increased steroidogenesis in embryos could help

399

to better understand the function of different steroids during early embryonic development.

SC

M AN U

400

RI PT

390

NDUFB7, NDUFB10 and NDUFS8 are subunits of the NADH dehydrogenase (ubiquinone) complex, which is located in the mitochondrial inner membrane and involved in the

402

electron transport chain [80]. Activated expression of these genes in response to insulin-dependent

403

signaling could be explained by an increased need for NADH dehydrogenase activity and

404

oxidoreductase activity, as in situations of metabolic stress [81].

405

4.4 Conclusions

406

The combined results from morphological evaluation and gene expression data provide support for an

407

increased metabolic turnover in embryos derived from insulin-treated oocytes. Our findings showed

408

that insulin treatment during oocyte maturation leads to embryos with characteristics of a more

409

advanced phenotype. This, together with the fact that the INS embryos show signs of oxidative stress,

410

increased proliferation, and up-regulation of genes involved in mitochondrial functions, implies that

411

the increased energy turnover induced by insulin seems to change important structural and functional

412

characteristics of the embryo.

413

Our study contributes new insights into how a challenge with elevated insulin levels during oocyte

414

maturation has lasting effects on the phenotype and molecular pattern of the early embryo. It remains

415

to be investigated if these early changes will be transient or long-lasting, and whether the embryo can

AC C

EP

TE D

401

ACCEPTED MANUSCRIPT 416

cope with the increased metabolic activity and phenotype changes. This bovine model could be of

417

comparative value for finding explanations for reduced fertility on the embryo level in

418

hyperinsulinaemic obese women and also indicates the importance of surveillance of insulin levels in

419

the periconceptional period.

RI PT

420 421 ACKNOWLEDGEMENTS

423

The authors acknowledge Isabelle Dufort, Dominic Gagné and Eric Fournier at EmbryoGENE for

424

their support in the laboratory and bioinformatics work, as well as SCAN (Linköping, Sweden) for

425

providing the ovaries for the present study. This work was funded by FORMAS, The Swedish

426

Research Council for Environment, Agricultural Sciences and Spatial Planning (Grant no. 222–2010–

427

1132). Collaboration with EmbryoGENE was supported by The Natural Sciences and Engineering

428

Research Council of Canada (NSERC) and travel grants by KSLA (The Royal Swedish Academy of

429

Agriculture and Forestry).

431

REFERENCES

432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451

[1]

[3]

[4]

[5] [6]

[7]

EP

Saltiel AR, Kahn CR. Insulin signalling and the regulation of glucose and lipid metabolism. Nature 2001;414:799–806. doi:10.1038/414799a. Spicer L, Alpizar E, Echternkamp S. Effects of insulin, insulin-like growth factor I, and gonadotropins on bovine granulosa cell proliferation, progesterone production, estradiol production, and (or) insulin-like growth factor I production in vitro. J Anim Sci 1993;71:1232– 41. Adamiak SJ, Mackie K, Watt RG, Webb R, Sinclair KD. Impact of nutrition on oocyte quality: cumulative effects of body composition and diet leading to hyperinsulinemia in cattle. Biol Reprod 2005;73:918–26. doi:10.1095/biolreprod.105.041483. Laskowski D, Sjunnesson Y, Humblot P, Sirard M-A, Andersson G, Gustafsson H, et al. Insulin exposure during in vitro bovine oocyte maturation changes blastocyst gene expression and developmental potential. Reprod Fertil Dev 2016. Butler WR. Relationships of negative energy balance with fertility. Adv Dairy Tech 2005;17:35–46. Awasthi H, Saravia F, Rodríguez‐Martínez H, Båge R. Do Cytoplasmic Lipid Droplets Accumulate in Immature Oocytes from Over‐Conditioned Repeat Breeder Dairy Heifers? Reprod Domest Anim 2010;45:e194–8. Ingvartsen KL. Feeding-and management-related diseases in the transition cow: Physiological adaptations around calving and strategies to reduce feeding-related diseases. Anim Feed Sci Technol 2006;126:175–213.

AC C

[2]

TE D

430

M AN U

SC

422

ACCEPTED MANUSCRIPT

[13] [14] [15] [16] [17]

[18]

[19] [20] [21] [22] [23]

[24]

[25]

[26]

[27]

[28]

RI PT

[12]

SC

[11]

M AN U

[10]

TE D

[9]

Pryce J, Coffey M, Simm G. The relationship between body condition score and reproductive performance. J Dairy Sci 2001;84:1508–15. Rukkwamsuk T, Kruip TAM, Wensing T. Relationship between overfeeding and overconditioning in the dry period and the problems of high producing dairy cows during the postparturient period. Vet Q 1999;21:71–7. doi:10.1080/01652176.1999.9694997. Royal M, Darwash A, Flint A, Webb R, Woolliams J, Lamming G. Declining fertility in dairy cattle: changes in traditional and endocrine parameters of fertility. Anim Sci 2000;70:487–501. Inchaisri C, Jorritsma R, Vos P, Van der Weijden G, Hogeveen H. Economic consequences of reproductive performance in dairy cattle. Theriogenology 2010;74:835–46. Clark A, Ledger W, Galletly C, Tomlinson L, Blaney F, Wang X, et al. Weight loss results in significant improvement in pregnancy and ovulation rates in anovulatory obese women. Hum Reprod 1995;10:2705–12. Hart RJ. Physiological aspects of female fertility: role of the environment, modern lifestyle, and genetics. Physiol Rev 2016;96:873–909. Seidell JC. Obesity, insulin resistance and diabetes—a worldwide epidemic. Br J Nutr 2000;83:S5–8. Norman RJ, Masters S, Hague W. Hyperinsulinemia is common in family members of women with polycystic ovary syndrome. Fertil Steril 1996;66:942–7. Graves CR. Antepartum fetal surveillance and timing of delivery in the pregnancy complicated by diabetes mellitus. Clin Obstet Gynecol 2007;50:1007–13. Lighten AD, Moore GE, Winston R, Hardy K. Routine addition of human insulin-like growth factor-I ligand could benefit clinical in-vitro fertilization culture. Hum Reprod 1998;13:3144– 50. Laskowski D, Sjunnesson Y, Gustafsson H, Humblot P, Andersson G, Båge R. Insulin concentrations used in in vitro embryo production systems: a pilot study on insulin stability with an emphasis on concentrations measured in vivo. Acta Vet Scand 2016;58:66. Patel MS, Srinivasan M. Metabolic programming: causes and consequences. J Biol Chem 2002;277:1629–32. Lindner GM, Wright RW. Bovine embryo morphology and evaluation. Theriogenology 1983;20. doi:10.1016/0093-691X(83)90201-7. Stringfellow DA G MD. Manual of the International Embryo Transfer Society (IETS). 4th ed., Champaign, IL: IETS.; 2010. Kirkegaard K, Agerholm IE, Ingerslev HJ. Time-lapse monitoring as a tool for clinical embryo assessment. Hum Reprod 2012;27:1277–85. Desai NN, Goldstein J, Rowland DY, Goldfarb JM. Morphological evaluation of human embryos and derivation of an embryo quality scoring system specific for day 3 embryos: a preliminary study. Hum Reprod 2000;15:2190–6. Abe H, Yamashita S, Itoh T, Satoh T, Hoshi H. Ultrastructure of bovine embryos developed from in vitro–matured and–fertilized oocytes: Comparative morphological evaluation of embryos cultured either in serum‐free medium or in serum‐supplemented medium. Mol Reprod Dev 1999;53:325–35. Zijlstra C, Kidson A, Schoevers E, Daemen A, Tharasanit T, Kuijk E, et al. Blastocyst morphology, actin cytoskeleton quality and chromosome content are correlated with embryo quality in the pig. Theriogenology 2008;70:923–35. Watson AJ, De Sousa P, Caveney A, Barcroft LC, Natale D, Urquhart J, et al. Impact of Bovine Oocyte Maturation Media on Oocyte Transcript Levels, Blastocyst Development, Cell Number, and Apoptosis 1. Biol Reprod 2000;62:355–64. de la Fuente R, King WA. Use of chemically defined system for the direct comparison of inner cell mass and trophectoderm distribution in murine, porcine and bovine embryos. Zygote 1997;5:309–20. Iwasaki S, Yoshiba N, Ushijima H, Watanabe S, Nakahara T. Morphology and proportion of inner cell mass of bovine blastocysts fertilized in vitro and in vivo. J Reprod Fertil 1990;90:279–84.

EP

[8]

AC C

452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

[29] Shapiro BS, Harris DC, Richter KS. Predictive value of 72-hour blastomere cell number on blastocyst development and success of subsequent transfer based on the degree of blastocyst development. Fertil Steril 2000;73:582–6. [30] Lane M, Gardner DK. Effect of incubation volume and embryo density on the development and viability of mouse embryos in vitro. Hum Reprod 1992;7:558–62. [31] Ferreira E, Vireque A, Adona P, Meirelles F, Ferriani R, Navarro P. Cytoplasmic maturation of bovine oocytes: structural and biochemical modifications and acquisition of developmental competence. Theriogenology 2009;71:836–48. [32] Sun Q-Y, Schatten H. Regulation of dynamic events by microfilaments during oocyte maturation and fertilization. Reproduction 2006;131:193–205. [33] Bruce Alberts A, Lewis J, Raff M, Roberts K, Walter P. Molecular biology of the cell. 2002. Garland Sci N Y n.d.:263–399. [34] Barnett DK, Kimura J, Bavister BD. Translocation of active mitochondria during hamster preimplantation embryo development studied by confocal laser scanning microscopy. Cell 1996;40:44. [35] Båge R, Petyim S, Larsson B, Hallap T, Bergqvist A-S, Gustafsson H, et al. Oocyte competence in repeat-breeder heifers: effects of an optimized ovum pick-up schedule on expression of oestrus, follicular development and fertility. Reprod Fertil Dev 2003;15:115–23. [36] Tarazona A, Rodriguez J, Restrepo L, Olivera‐Angel M. Mitochondrial activity, distribution and segregation in bovine oocytes and in embryos produced in vitro. Reprod Domest Anim 2006;41:5–11. [37] Bavister BD, Squirrell JM. Mitochondrial distribution and function in oocytes and early embryos. Hum Reprod 2000;15:189–98. [38] Van Blerkom J. Mitochondria as regulatory forces in oocytes, preimplantation embryos and stem cells. Reprod Biomed Online 2008;16:553–69. [39] Van Blerkom J, Davis P, Alexander S. Differential mitochondrial distribution in human pronuclear embryos leads to disproportionate inheritance between blastomeres: relationship to microtubular organization, ATP content and competence. Hum Reprod 2000;15:2621–33. [40] Ahn HJ, Sohn IP, Kwon HC, Jo DH, Park YD, Min CK. Characteristics of the cell membrane fluidity, actin fibers, and mitochondrial dysfunctions of frozen‐thawed two‐cell mouse embryos. Mol Reprod Dev 2002;61:466–76. [41] González R, Sjunnesson YCB. Effect of blood plasma collected after adrenocorticotropic hormone administration during the preovulatory period in the sow on oocyte in vitro maturation. Theriogenology 2013;80:673–83. [42] González R, Gómez M, Pope C, Brandt Y. Characterization of Mitochondrial and Actin Patterns in Cat Oocytes and Blastocysts. Reprod Domest Anim 2012;47:118–20. [43] Cagnone GL, Dufort I, Vigneault C, Sirard M-A. Differential gene expression profile in bovine blastocysts resulting from hyperglycemia exposure during early cleavage stages. Biol Reprod 2012;86:50. [44] Van Hoeck V, Rizos D, Gutierrez-Adan A, Pintelon I, Jorssen E, Dufort I, et al. Interaction between differential gene expression profile and phenotype in bovine blastocysts originating from oocytes exposed to elevated non-esterified fatty acid concentrations. Reprod Fertil Dev 2015;27:372–84. [45] Laskowski D, Sjunnesson Y, Humblot P, Andersson G, Gustafsson H, Båge R. The functional role of insulin in fertility and embryonic development—What can we learn from the bovine model? Proc 18th ICAR 2016;86:457–64. doi:10.1016/j.theriogenology.2016.04.062. [46] Hayashi I, Larner J, Sato G. Hormonal growth control of cells in culture. In Vitro 1978;14. doi:10.1007/BF02618171. [47] Gordon I. Laboratory Production of Cattle Embryos. CAB International: Cambridge University Press; 1994. [48] Abraham MC, Ruete A, Brandt YCB. Breed influences outcome of in vitro production of embryos in cattle [abstract]. Reprod Fertil Dev 2009;22. doi:10.1071/RDv22n1Ab260. [49] Ng FLH, Liu DY, Baker HWG. Comparison of Percoll, mini-Percoll and swim-up methods for sperm preparation from abnormal semen samples. Hum Reprod 1992;7:261–6. doi:10.1093/oxfordjournals.humrep.a137628.

AC C

505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

[50] Shojaei Saadi HA, O’Doherty AM, Gagne D, Fournier E, Grant JR, Sirard M-A, et al. An integrated platform for bovine DNA methylome analysis suitable for small samples. BMC Genomics 2014;15:451. doi:10.1186/1471-2164-15-451. [51] Robert C, Nieminen J, Dufort I, Gagne D, Grant JR, Cagnone G, et al. Combining resources to obtain a comprehensive survey of the bovine embryo transcriptome through deep sequencing and microarrays. Mol Reprod Dev 2011;78:651–64. doi:10.1002/mrd.21364. [52] Augustin R, Pocar P, Wrenzycki C, Niemann H, Fischer B. Mitogenic and anti-apoptotic activity of insulin on bovine embryos produced in vitro. Reproduction 2003;126:91–9. doi:10.1530/rep.0.1260091. [53] Waters SB, Yamauchi K, Pessin JE. Functional expression of insulin receptor substrate-1 is required for insulin-stimulated mitogenic signaling. J Biol Chem 1993;268:22231–4. [54] Leese HJ, Sturmey RG, Baumann CG, McEvoy TG. Embryo viability and metabolism: obeying the quiet rules. Hum Reprod 2007;22:3047–50. [55] Baumann CG, Morris DG, Sreenan JM, Leese HJ. The quiet embryo hypothesis: molecular characteristics favoring viability. Mol Reprod Dev 2007;74:1345–53. [56] Sidarala V, Kowluru A. The Regulatory Roles of Mitogen-activated Protein Kinase (MAPK) Pathways in Health and Diabetes: Lessons Learned from the Pancreatic β-cell. Recent Pat Endocr Metab Immune Drug Discov 2016. [57] Singh P, Saxena R, Srinivas G, Pande G, Chattopadhyay A. Cholesterol biosynthesis and homeostasis in regulation of the cell cycle. PloS One 2013;8:e58833. [58] Farese Jr RV, Herz J. Cholesterol metabolism and embryogenesis. Trends Genet 1998;14:115– 20. [59] Roux C, Wolf C, Mulliez N, Gaoua W, Cormier V, Chevy F, et al. Role of cholesterol in embryonic development. Am J Clin Nutr 2000;71:1270s–1279s. [60] Wang W-H, Abeydeera LR, Han Y-M, Prather RS, Day BN. Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo. Biol Reprod 1999;60:1020–8. [61] Wang W-H, Abeydeera LR, Prather RS, Day BN. Polymerization of Nonfilamentous Actin into Microfilaments Is an Important Process for Porcine Oocyte Maturation and Early Embryo Development 1. Biol Reprod 2000;62:1177–83. [62] Matsumoto H, Shoji N, Umezu M, Sato E. Microtubule and microfilament dynamics in rat embryos during the two‐cell block in vitro. J Exp Zool Part Ecol Genet Physiol 1998;281:149– 53. [63] Wickstead B, Gull K. The evolution of the cytoskeleton. J Cell Biol 2011;194:513–25. [64] Dechat T, Pfleghaar K, Sengupta K, Shimi T, Shumaker DK, Solimando L, et al. Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin. Genes Dev 2008;22:832–53. [65] Cheng W, JeBailey L, Ridgway ND. Oxysterol-binding-protein (OSBP)-related protein 4 binds25-hydroxycholesterol and interacts with vimentin intermediate filaments. Biochem J 2002;361:461–72. [66] Facchini FS, Hua NW, Reaven GM, Stoohs RA. Hyperinsulinemia: the missing link among oxidative stress and age-related diseases? Free Radic Biol Med 2000;29:1302–6. [67] Urakawa H, Katsuki A, Sumida Y, Gabazza EC, Murashima S, Morioka K, et al. Oxidative stress is associated with adiposity and insulin resistance in men. J Clin Endocrinol Metab 2003;88:4673–6. [68] Morino K, Petersen KF, Shulman GI. Molecular mechanisms of insulin resistance in humans and their potential links with mitochondrial dysfunction 2006. [69] Yoshida M, Muneyuki E, Hisabori T. ATP synthase—a marvellous rotary engine of the cell. Nat Rev Mol Cell Biol 2001;2:669–77. [70] Guerin P, El Mouatassim S, Menezo Y. Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Hum Reprod Update 2001;7:175– 89. [71] Leese HJ. Quiet please, do not disturb: a hypothesis of embryo metabolism and viability. Bioessays 2002;24:845–9.

AC C

560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613

ACCEPTED MANUSCRIPT

SC

RI PT

[72] Mota GB, e Silva IO, de Souza DK, Tuany F, Pereira MM, de Almeida Camargo LS, et al. Insulin influences developmental competence of bovine oocytes cultured in α-MEM plus follicle-simulating hormone. Zygote 2015;23:563–72. [73] Martin-Gronert MS, Ozanne SE. Metabolic programming of insulin action and secretion. Diabetes Obes Metab 2012;14:29–39. doi:10.1111/j.1463-1326.2012.01653.x. [74] Auchus RJ, Adams CM. Cholesterol, Steroid and Isoprenoid Biosynthesis. eLS 2001. [75] Yeagle PL. Cholesterol and the cell membrane. Biochim Biophys Acta BBA-Rev Biomembr 1985;822:267–87. [76] Diamanti-Kandarakis E, Papavassiliou AG, Kandarakis SA, Chrousos GP. Pathophysiology and types of dyslipidemia in PCOS. Trends Endocrinol Metab 2007;18:280–5. [77] Dickmann Z, Dey SK. Steroidogenesis in the preimplantation rat embryo and its possible influence on morula-blastocyst transformation and implantation. J Reprod Fertil 1974;37:91–3. [78] Dickmann Z, Gupta J, Dey S. Does “blastocyst estrogen” initiate implantation? Science 1977;195:687. doi:10.1126/science.841306. [79] Green ML, Simmen RC, Simmen FA. Developmental regulation of steroidogenic enzyme gene expression in the periimplantation porcine conceptus: a paracrine role for insulin-like growth factor-I. Endocrinology 1995;136:3961–70. doi:10.1210/endo.136.9.7649105. [80] Weiss H, Friedrich T, Hofhaus G, Preis D. The respiratory‐chain NADH dehydrogenase (Complex I) of mitochondria. Eur J Biochem 1991;197:563–76. [81] Kowaltowski AJ, Vercesi AE. Mitochondrial damage induced by conditions of oxidative stress. Free Radic Biol Med 1999;26:463–71.

M AN U

614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635

AC C

EP

TE D

636

ACCEPTED MANUSCRIPT

SC

RI PT

Figure 1: Actin and mitochondria staining and categories Examples for actin categories in f-Actin (Alexa Fluor 488 Phalloidin) - stained Day 8 blastocysts: A1=ACTIN1; A2=ACTIN2 and A3=ACTIN3 (arrows indicating 1)aggregated microfilaments and 2) lost cell integrity). Examples for mitochondria category in active mitochondria (MitoTracker Orange)–stained Day 8 blastocysts: M1=MITO1; M2=MITO2, M3=MITO3 (arrows indicating 3)accumulations/clusters and 4) “empty” areas); MC1=MitoC1(arrows indicating 5)clear cell borders and 6)unstained nucleus area); MC2=MitoC2 (arrows indicating 7)cells with mitochondria distributed around nucleus and 8) areas with absence of cells with clear mitochondria distribution around nucleus); MC3=MitoC3 (arrows indicating 9)large areas without distribution pattern around nucleus and 10)areas with no clear cell borders). A high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM04069

M AN U

Figure 2: Actin quality in different developmental stages Percentage to be assessed as Actin quality 1 (ACTIN1) increases by embryo stage (a/b/c < 0.05). Numbers of embryos classified as ACTIN1 of total numbers of embryos belonging to the respective blastocyst stage are indicated above each bar.

TE D

Figure 3: Mitochondrial pattern MITO at different developmental stages MITO1 is more present in advanced stages (BC=blastocyst, Exp. BC=expanding/expanded blastocyst, Hatch. BC= hatching/hatched blastocyst), irrespective of the insulin treatment (a/b/c p< 0.05). Numbers of embryos classified as MITO1 of total numbers of embryos belonging to the respective blastocyst stage are indicated above each bar.

AC C

EP

Figure 4: MitoC1 percentage at different developmental stages. MitoC1 is more frequent in advanced stage embryos (BC=blastocyst, Exp. BC=expanding/expanded blastocyst, Hatch. BC= hatching/hatched blastocyst) irrespective of insulin treatment (a/b p< 0.05). Numbers of embryos classified as MitoC1 of total numbers of embryos belonging to the respective blastocyst stage are indicated above each bar.

Figure 5: 5 of the 12 genes related to morphological changes with significant fold-change validated by RT-qPCR (significant*) Expression levels from RT-qPCR of 5 selected genes, corrected with 2 housekeeping genes (relative quantities of expression after normalization of data). Significant differences in expression from RT-qPCR between INS0.1 and INS0 group or INS10 and INS0 group are indicated with *p < 0.05; **p < 0.01; ***p < 0.001. ¤ Set of genes for which a strong relationship is observed between results of the microarray and validation by RT-qPCR (DHCR7, CYP11A1, NDUFA10). ºSet of genes for which the same tendency is observed in microarray and RTqPCR.

ACCEPTED MANUSCRIPT

Category Definition

ACTIN

1

Sharp cell borders, abundant microfilament in cytoplasm

2

Gross maintenance of cell outline but some aggregated/clumped microfilaments

3

Aggregated clumps and lost cell integrity in main part of embryo

1

Evenly distributed, distinctly present in almost all cells, distinct points of staining, no or few small accumulations, no extreme stained cells (“cluster/blobs”)

2

1 or 2 parameters of MITO1 wrong but some fulfilled

Actin structure

MITO

RI PT

Abbreviation

SC

Parameter

3 MitoC

1

Mitochondria distribution

2 3

M AN U

Mitochondria

More than 3 serious deviations from class 1

Centred around nucleus in all cells Centred around nucleus in 50-75% of cells No centred pattern visible or in <25% of all cells

AC C

EP

TE D

Table 1: Summary of the different morphological categories and characteristics used to assess actin and mitochondria (1= best category for all parameters)

ACCEPTED MANUSCRIPT Experimental design Definition of experimental and control groups

Control group INS0: Blastocysts obtained in regular maturation IVF media. Insulin groups, INS0.1 and INS10: Blastocysts obtained in maturation IVF media containing 0.1 µg/ml and 10 µg/ml of insulin, respectively.

Number within each group

n=4 (pool of 10)

Description

RI PT

Samples

For each experiment, RNA extractions were performed with 4 independent experimental groups. Each RNA sample was obtained from an experimental group including 10 different Blastocysts. Real time RT-PCR was performed once for each examined gene, using 4 replicates for each cDNA.

SC

Nucleic acid extraction

RNA was extracted using an AllPrepDNA/RNA micro kit (Qiagen). The DNA was eluted in 30 µl of water and the RNA was eluted in 15 µl of elution buffer and both were kept at -80 °C

DNase treatment

No DNase treatment was performed, as recommended in the kit.

Contamination

Absence of genomic DNA contamination in the RNA samples was tested using the Bioanalyzer (Agilent)

Quantification

Bioanalyzer (Agilent)

Integrity

RNA integrity number : 8.5-9.3

M AN U

Procedure/kit

TE D

Reverse transcription

qScript™ Flex cDNA Kit (Quanta Biosciences) with oligo-dT (10uM)

Amount of RNA

Equivalent of 5 blastocysts of total RNA

Reaction volume

20 µl

65 °C for 5 minutes 42 °C for 1 hour 70 °C for 15 minutes

AC C

Temperature and time

EP

Procedure/kit

Storage condition of cDNA

-20 °C

RT-qPCR target information and oligonucleotide Gene symbol

Gene Name

Genebank

CYP11A1

cytochrome P450, family 11, subfamily A, polypeptide 1

NM_176644.2

DHCR7

7dehydrocholestero l reductase

NM_00101492 7.1

NDUFA10

NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 10, 42kDa

Primer sequence (5’-3’)

Fwr Rev Fwr Rev Fwr NM_176655.2

Rev

TAGCATCAAGGAGAC GCTGAGA TAGCTGGATTGGTGG AAAGGG CCCACAGGTATTCTT GACTTT CCTGCACTAACTCTG TTAGAC AGGTGGTCGAGGATA TTGAG CTCTGTGAACTCCTG GAAGA

Annealing Temperature

Length

Efficiency

57

469

1.86

57

208

1.89

57

198

1.90

ACCEPTED MANUSCRIPT MAP2K2

NM_00103807 1.2

Fwr Rev Fwr

vimentin

NM_173969.3

Real time RT-PCR instrument Data Analysis Statistical methods for results significance

Analysis of expression stability of endogenous reference genes

221

1.97

57

256

1.89

LightCycler® 480 SYBR Green I Master (Roche) Reaction volume: 20 µl Amount of cDNA: equivalent of 5 blastocysts Primer: 0.5 µM (final of each primer) Polymerase, nucleotides, MgCl2 and buffer are included in the LightCycler® 480 SYBR Green I Master (Roche) Hold: 95 °C for 10 minutes 50 cycles: 95 °C for 5 seconds Specific for each set of primers °C for 5seconds 72 °C for 20 seconds Light Cycler 480 (Roche)

M AN U

Complete thermocycling parameters

57

SC

RT-qPCR protocol Complete reaction conditions Reaction volume amount of cDNA/primers/polymerase/ buffer

Rev

CGAGGTGGAAGAAGT GGATTT GAAGCAGGATCTGAG AAGACAG GTCCAAGTTTGCTGA CCTCT GAGCCATCTCTTCCT TCATGTT

RI PT

VIM

mitogen-activated protein kinase kinase 2

Differences in expression between the INS0 group and the INS0.1 group and between INS0 group and the INS10 group were compared by means of unpaired t test (GraphPad Software ©, Prism 5) following log transformation of data. Differences in expression with p values <0.05 were considered significant To analyse gene expression stability, Ct values of 3 reference genes (ACTB, PPIA and B2M ) were evaluated using Genorm software (Biogazelle). Under our experimental conditions, the 2 most stable reference genes were ACTB and B2M and the constant of their geometrical mean was use to normalise the genes.

SE 0.01 0.01 0.01

BC8 15.97 a 16.29 a 20.29 b

EP

CLEAV 79.82 a 76.92 a 78.64 a

AC C

Treatment INS10 INS0.1 INS0

TE D

Table 2: PCR protocol, primer sequence and annealing temperature for the experiment.

SE 0.02 0.02 0.02

NUCLEI 101 a 104 a 86 b

SE 3.7 5.38 3.088

Table 3: Developmental rates (cleaved embryos/immature oocytes=CLEAV, blastocyst day 8/immature oocytes=BC8) and nuclei number in day 8 blastocysts (NUCLEI) in the different groups. a/b p < 0.05 for NUCLEI, a/b < 0.07 for BC8.

ACCEPTED MANUSCRIPT

Group

MITO 2 28.00

MITO 3 20.00

TN

INS10

MITO 1 52.00 a

50

MitoC 1 50.00 a

MitoC 2 26.00

MitoC 3 24.00

INS0.1

47.37 a

43.86

8.77

56

55.36 a

26.79

INS0

23.73 b

50.85

25.42

60

30.51 b

37.29

TN

ACTIN 2 44.90

ACTIN 3 22.45 a

TN

50

ACTIN 1 32.65 a

17.86

56

38.60 a

43.86

17.54 a

57

32.20

60

20.00 b

38.46

41.54 b

65

49

Gene symbol MAP2K2*

Fold change INS0.1 1.996

Fold change INS10 2.218

AC C

EP

TE D

M AN U

Gene name Mitogen-Activated Kinase Kinase 2 DHCR7* 7-Dehydrocholesterol Reductase LMNA Lamin A/C VIM* Vimentin TUBB2B Tubulin Beta 2B Class IIb TUBB3 Tubulin Beta 3 TUBB4B Tubulin Beta 4B Class IVb ATP5D ATP Synthase, H+ Transporting, Mitochondrial F1 Complex CYP11A1* Cholesterol side-chain cleavage enzyme NDUFB7(*) NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 7 NDUFB10(*) NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 NDUFS8(*) NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8

SC

RI PT

Table 4: Actin and mitochondria category percentages in insulin (INS0.1 and INS10) and control group (INS0), a/b p<0.05. TN=Total number of embryos used for evaluation of the respective categories.

Associated with Cell proliferation

1.933 2.196 1.920 2.000 1.880 1.840 2.163

2.325 2.578 1.924 1.800 2.200 2.100 2.457

Cell proliferation Cell structure Cell structure Cell structure Cell structure Cell structure Mitochondrial function Mitochondrial function Mitochondrial function

1.338

2.325

1.970

1.790

1.662

1.800

Mitochondrial function

1.720

1.700

Mitochondrial function

Table 5: Gene expression data related to the observed morphological changes in the blastocyst; Fold change differences are for the insulin groups INS0.1 and INS10 compared to the control group INS0. *Genes validated with significant concordance by RT-qPCR, (*) related gene NDUFA10 validated by RT-qPCR (see Figure 5)

ACCEPTED MANUSCRIPT

c 60

25/46

b

30

25/93

20

a

10 1/33 0 Exp. BC

Hatch. BC

M AN U

BC

RI PT

40

SC

% ACTIN1

50

Blastocyst Stage

AC C

EP

TE D

Figure 2: Actin quality in different developmental stages Percentage to be assessed as Actin quality 1 (ACTIN1) is increasing for embryo stage (a/b/c < 0.05). Numbers of embryos classified as ACTIN1 of total numbers of embryos belonging to the respective blastocyst stage are indicated above each bar.

ACCEPTED MANUSCRIPT

70.00

29/44

b

40.00

33/90

30.00

a

20.00

5/32

SC

50.00

M AN U

% of MITO1

60.00

RI PT

c

10.00 0.00 BC

Exp. BC

Hatch. BC

Blastocyst stage

AC C

EP

TE D

Figure 3: Mitochondrial pattern MITO at different developmental stages MITO1 is more present in advanced stages (BC=blastocyst, Exp. BC=expanding/expanded blastocyst, Hatch. BC= hatching/hatched blastocyst), irrespective of the insulin treatment (a/b/c p< 0.05). Numbers of embryos classified as MITO1 of total numbers of embryos belonging to the respective blastocyst stage are indicated above each bar.

b

80

33/43

60 50

a

a

32/90

40 30

9/32

M AN U

% of MitoC1

70

SC

90

RI PT

ACCEPTED MANUSCRIPT

20 10 0 BC

Exp.BC

Hatch.BC

Blastocyst stage

AC C

EP

TE D

Figure 4: MitoC1 percentage at different developmental stages. MitoC1 is more frequent in advanced stage embryos (BC=blastocyst, Exp. BC=expanding/expanded blastocyst, Hatch. BC= hatching/hatched blastocyst) irrespective of insulin treatment (a/b p< 0.05). Numbers of embryos classified as MitoC1 of total numbers of embryos belonging to the respective blastocyst stage are indicated above each bar.

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

7,00E-07 6,00E-07

4,00E-07 3,00E-07 2,00E-07

0,00E+00 INS0

INS0.1

INS10

1,6E-08 1,4E-08 1,2E-08 1E-08 8E-09

TE D

6E-09 4E-09 2E-09 0

INS0.1

INS10

EP

INS0

MAP2K2

AC C

1,40E-06

VIMº 1.00 1.92 1.92

M AN U

VIM

Microarray INS0 INS0.1 INS10

SC

1,00E-07

RI PT

5,00E-07

1,20E-06

**

Microarray INS0 INS0.1 INS10

MAP2K2º 1.00 2.00 2.22

Microarray INS0 INS0.1 INS10

CYP11A1¤ 1.00 1.34 2.33

**

1,00E-06 8,00E-07 6,00E-07 4,00E-07 2,00E-07 0,00E+00

INS0

INS0.1 CYP11A1

INS10

ACCEPTED MANUSCRIPT 3,00E-06

***

**

2,50E-06 2,00E-06 1,50E-06

0,00E+00 INS0

INS0.1

INS10

DHCR7

3,00E-07

DHCR7¤ 1.00 1.93 2.33

M AN U

***

Microarray INS0 INS0.1 INS10

SC

5,00E-07

RI PT

1,00E-06

2,50E-07 2,00E-07 1,50E-07

5,00E-08 0,00E+00 INS0

TE D

1,00E-07

INS0.1

AC C

EP

NDUFA10

INS10

Microarray INS0 INS0.1 INS10

NDUFA10¤ 1.00 1.66 1.89

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

Insulin during oocyte maturation changes the phenotype of bovine day 8 blastocysts These changes include cell number, actin cytoskeleton and mitochondria distribution Gene expression studies confirm the observed morphological changes

AC C

• • •