Germinal stage vitrification is superior to MII stage vitrification in prepubertal mouse oocytes

Journal Pre-proof Germinal stage vitrification is superior to MII stage vitrification in prepubertal mouse oocytes Akshatha Daddangadi, Shubhashree Uppangala, Guruprasad Kalthur, Riccardo Talevi, Satish Kumar Adiga PII:

S0011-2240(19)30315-3

DOI:

https://doi.org/10.1016/j.cryobiol.2020.02.012

Reference:

YCRYO 4193

To appear in:

Cryobiology

Received Date: 21 October 2019 Revised Date:

24 February 2020

Accepted Date: 25 February 2020

Please cite this article as: A. Daddangadi, S. Uppangala, G. Kalthur, R. Talevi, S.K. Adiga, Germinal stage vitrification is superior to MII stage vitrification in prepubertal mouse oocytes, Cryobiology (2020), doi: https://doi.org/10.1016/j.cryobiol.2020.02.012. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Inc.

1

Title: Germinal stage vitrification is superior to MII stage vitrification in

2

prepubertal mouse oocytes.

3

4

Author affiliation: Akshatha Daddangadi1 Shubhashree Uppangala1, Guruprasad Kalthur1,

5

Riccardo Talevi2, Satish Kumar Adiga1

6 7

1

8

Higher Education, Manipal-576 104, India.

9

2

10

Department of Clinical Embryology, Kasturba Medical College, Manipal, Manipal Academy of

Dipartimento di Biologia, Università di Napoli "Federico II", Complesso Universitario di Monte

S Angelo, Napoli, Italy.

11 12

Corresponding authors:

13

Satish Kumar Adiga, PhD

14

Department of Clinical Embryology, Kasturba Medical College, Manipal, Manipal Academy of

15

Higher Education, Manipal-576 104, India Tel: 91-820-29-22320,

16

E mail: [email protected]

17 18 19

Short title: Vitrification of prepubertal oocytes

20

Key words: prepubertal oocyte, fertility preservation, oocyte vitrification, in vitro maturation

21

Word count: 4035

1

22

Abstract

23

This study investigated if in vitro maturation (IVM) before or after vitrification would be more

24

successful for prepubertal oocytes. To mimic prepubertal conditions in an experimental setup,

25

oocytes were collected from healthy 14, 21 and 28day old Swiss albino mice. The germinal

26

vesicle (GV) stage oocytes and in vitro matured MII oocytes were subjected to vitrification-

27

warming. Both structural (meiotic spindle morphology, mitochondrial integrity, cortical

28

granules) and functional (sperm zona binding, fertilization) characteristics were assessed in

29

oocytes after warming. This study demonstrated that IVM was more detrimental to prepubertal

30

oocytes than to young adults. Further, vitrification of the IVM oocytes resulted in an increase in

31

the number of abnormal meiotic spindles, a change in the cortical distribution pattern, a

32

reduction in sperm zona binding and the fertilization rate. Importantly, oocyte integrity was

33

better when prepubertal oocytes were vitrified before, rather than after, IVM. The above

34

observations support GV stage vitrification for prepubertal oocytes requiring fertility

35

preservation. Understanding the mechanisms behind the differing outcomes for oocytes from

36

immature females will help in refining current protocol, thereby retaining the oocytes’ maximum

37

structural and functional integrity Further investigation is necessary to determine whether human

38

prepubertal oocytes also behave in a similar way. It is to be noted here, with great emphasis, that

39

a major limitation of this study is that the oocytes’ abilities were tested only until fertilisation, as

40

a consequence of which the study cannot reveal the developmental potentials of the embryos

41

beyond fertilisation.

42

2

43

1. Introduction

44

Chemotherapy and radiation used in the treatment of cancer can have a significant impact on

45

ovarian function of young women and girls [7]. Hence, ovarian tissue cryopreservation and re

46

implantation of cryopreserved tissue is the most preferred option available to preserve fertility

47

before gonadotoxic therapy [30]. However, malignant cells present in the ovary may pose the

48

risk of reintroducing cancer post transplantation [8]. Therefore, the ability of an immature oocyte

49

to mature in vitro would be of relevance to fertility preservation for overcoming this problem.

50

Tissue harvested from prepubertal females contains only immature oocytes within the follicles,

51

which would need to be matured in vitro either within the follicle [2] or extracted and subjected

52

to in vitro maturation (IVM). Matured oocytes will then be fertilized by assisted reproductive

53

technology (ART) in hope of establishing successful pregnancy in the survivors [18]. Though,

54

IVM of germinal vesicle (GV) oocytes to metaphase II (MII) stage oocytes has been the subject

55

of research for almost four decades [24], experimental data suggest that immature oocytes

56

obtained from a prepubertal ovary have lower ability to form viable offspring after IVM of

57

immature oocytes from the adult ovary [25,26]. However, the underlying mechanisms are poorly

58

understood.

59

Due to the increasing interest in fertility preservation to help cancer affected prepubertal girls

60

[14], it is important for oncologists and fertility experts to be aware of the success rates and

61

limiting factors of oocyte vitrification in order to provide patients with proper counseling. GV

62

oocytes retrieved from the ovarian tissues of prepubertal girls who need an urgent intervention of

63

chemo- or radiotherapy may be of great value in identifying appropriate fertility preservation

64

techniques. In such cases, the question is whether GV oocytes should be vitrified before or after

65

IVM in order to maintain the highest developmental competence [3,4]. 3

66

Oocyte maturation is a complex process that involves both nuclear and cytoplasmic maturation

67

[35], which if disturbed can alter spatial and temporal dynamics of the oocyte and affect their

68

functional competence[31]. One specific major problem associated with cryopreservation of

69

metaphase II oocytes is the sensitivity of the microtubular spindle to cryoprotectants and low

70

temperatures [27]. To circumvent this problem, vitrification of whole follicles or immature

71

oocytes at the GV stage can be used although this requires in vitro maturation after warming. We

72

hypothesized that prepubertal GV stage oocytes have limited functional ability than oocytes from

73

adults and that subjecting them to in vitro maturation after vitrification will further reduce their

74

structural and functional integrity. Hence, using mouse model, this study investigated whether

75

IVM is more successful before or after vitrification in maintaining the structural and functional

76

integrity of prepubertal oocytes.

77

78

2. Materials and methods

79

2.1 Animals and oocyte collection

80

Female Swiss albino mice, housed and maintained at controlled conditions of 24±2ºC

81

temperature, 50-60 % humidity, 12:12h light-dark cycle fed with water ad libitum were used in

82

the study. The animal handling and all the experiments were implemented in accordance with the

83

institutional guidelines for animal experimentation after obtaining prior approval from the

84

Institutional Animal Ethics Committee (IAEC/KMC/07/2018). To mimic prepubertal effects in

85

an experimental setup, healthy female siblings were sacrificed on postnatal day 14, postnatal day

86

21 and postnatal day 28; (hereafter referred to as D14, D21 and D28 respectively) to collect

87

oocytes. The ovaries were gently teased using fine needles and germinal vesicle (GV) oocytes

4

88

were released from the follicles to HEPES based, prewarmed M2 medium. Oocyte imaging was

89

done using an inverted phase contrast microscope (40X, IX 73, Olympus, Japan). Ooplasmic

90

diameter was measured using Q-Capture software (Media Cybernetics Inc., USA).

91

2.2 Oocyte vitrification and warming

92

Oocytes retrieved from D14, D21 and D28 groups were divided into non-vitrified control, GV

93

vitrified in vitro matured (GV-V) and GV in vitro matured vitrified (MII-V) groups.

94

Accordingly, vitrification was done as per the manufacturer’s protocol (Cat. No. VT601 &

95

VT602 Kitazato Corporation, Japan). The entire process was performed at room temperature as

96

per the protocol of [28], with minor modifications. Two droplets each of 100 µl for the basic

97

solution (BS), equilibrated solution (ES), and the vitrification solution (VS) was used. About 10

98

oocytes were placed in a BS droplet, mixed with an ES droplet and left for 3 min. Oocytes were

99

then transferred to a new ES droplet and left for 9 min. Finally, each group of oocytes was placed

100

in VS for 1 min, loaded into the Cryolock® (Cat. No. CL-R-CT Biotech, Inc., USA) using a

101

small volume of media (about 2µl) and plunged into the liquid nitrogen and stored in the

102

immersion phase.

103

The process of thawing was performed at room temperature except for the first step. There were

104

three warming media: thawing solution (TS), dilution solution (DS) and washing solution (WS).

105

The Cryolock® was removed under liquid nitrogen and dipped into a 400 µl of pre warmed TS

106

for one minute on a petridish. Then the oocytes were placed in 100 µl of DS for 3 min, 100 µl of

107

WS for 5 min before cultured in M16 medium (Cat. No. M7292, Sigma Aldrich, USA) for 3 h

108

prior to survival check. Only those oocytes having homogenous cytoplasm, intact plasma

109

membrane and zona pellucida were considered as survived and used for further analysis. Control

110

oocytes were subjected to identical conditions except vitrification – warming. 5

111

112

2.3 In vitro maturation (IVM)

113

The GV oocytes devoid of cumulus cells were subjected to in vitro maturation (IVM) as

114

described previously [33]. D14 and D21 oocytes did not have cumulus cells attached to them

115

whereas only cumulus free oocytes were considered in case of D28, Briefly, oocytes were

116

cultured in 20µL Dulbecco’s Modified Eagle’s Medium (DMEM, Cat. No. D5648 Sigma

117

Aldrich, USA) supplemented with non-essential α-amino acids (1 %, Cat. No. M7145 Sigma

118

Aldrich, USA), Insulin-Transferrin-Selenium (1 %, Cat. No. 51500-056 Gibco, India), pyruvate

119

(10 µM, Cat. No. P3662 Sigma Aldrich, USA), and bovine serum albumin (0.3 %, Cat. No.

120

MB083 Sigma Aldrich, USA) overlaid with oil. IVM medium did not contain any hormones.

121

Oocytes were incubated at 37°C in 5 % CO2 for 24 h and then assessed for nuclear maturity,

122

extrusion of polar bodies, mitochondrial potential, spindle morphology and cortical granule

123

distribution. Oocytes were also inseminated to evaluate fertilization rates and sperm zona

124

binding.

125

2.4 Analysis of spindle morphology

126

Metaphase II oocytes were rinsed in phosphate buffered saline supplemented with 0.1 % BSA

127

and permeabilized at 37°C for 1h using extraction buffer (50mM Potassium chloride, Cat. No.

128

P5405 Sigma Aldrich, USA; 5mM ethylenediaminetetraacetic acid disodium salt, Cat. No. 54960

129

Sisco Research Laboratories, India; 0.5 mM Magnesium chloride, Cat. No. 1349130 Sisco

130

Research Laboratories, India; 25 mM HEPES, Cat. No. H-3375 Sigma Aldrich, USA; 25 %

131

Glycerol, Cat. No. G9012 Sigma Aldrich, USA; 2 % Triton X-100, Cat. No. 1.8603.1000,

132

Merck; 20 µM phenylmethane sulphonyl fluoride, Cat. No. 1592 Himedia, India) adjusted to pH

133

6.75. Then, oocytes were fixed using ice-cold methanol for 15 min at -20°C, and then blocked 6

134

using 5 % knock out serum (Cat. No. 10828-010 Gibco, India) and 0.25 % Triton-X for 1 h at

135

37°C. The oocytes were incubated overnight at 4°C in 1:150 diluted primary anti-α-tubulin

136

antibody (Cat. No. T9026 Sigma Aldrich, USA) followed by treatment with 1:500 FITC tagged

137

goat anti-mouse IgG antibody (Cat. No. D0408 Santa Cruz Biotechnology, India) for 1h at 37°C.

138

The chromosomes were stained with 4 µg/mL DAPI (4',6-Diamidino-2-phenylindole Cat. No.

139

D9542 Sigma Aldrich, USA), and observed under the fluorescent microscope (Imager-A1, Zeiss,

140

Gottingen, Germany). The spindle images were captured under 40X objective using Q-Capture

141

software (Media Cybernetics Inc., USA).

142

2.5 Oocyte mitochondrial potential

143

Mitochondria in oocytes provides energy for fertilization and subsequent preimplantation

144

development. Here we used JC1, a mitochondrial specific probe to determine the mitochondrial

145

potential in relation to the age of the animals. JC1 fluoresces red in the presence of active

146

mitochondria by forming aggregates. In the presence of inactive mitochondria as the probe

147

remains in monomeric state, the probe fluoresces green. The mitochondrial potential was

148

measured as previously described [33] with minor modification. Oocytes were briefly washed

149

and

150

tetraethylbenzimidazolylcarbocyanine iodide; Cat. No. T3168, Molecular Probes, Life

151

Technologies, USA) in M199 media (Cat. No. M5017 Sigma Aldrich, USA) supplemented with

152

0.1 % BSA at 37°C, and 5 % CO2. After 30 min, excess stain was washed with M199 medium

153

containing 0.1 % BSA and mounted on a glass slide using mounting media (Cat. No. S3023

154

DAKO, US). Labelled oocytes were observed under the fluorescent microscope. The ratio of JC-

155

1 aggregates (orange fluorescence) to JC-1 monomer (green fluorescence) was analyzed using

156

ImageJ software (National Institute of Health, Bethesda, Maryland, USA).

then

treated

with

pre-warmed

1

µg/mL

7

JC-1(5,5’,6,6’-Tetrachloro-1,1’,3,3’-

157

2.6 Staining of cortical granules (CGs) and confocal microscopy

158

Cortical granules change their location during oocyte maturation leading to cortical granule free

159

domain (CGFD) in metaphase II mouse oocytes which is an indicator of cytoplasmic maturation.

160

Cortical granule staining and analysis was done as described earlier [23] with few modifications.

161

Briefly, the oocyte zona pellucida was removed by exposing oocytes to acid tyrode (Cat. No.

162

T1788 Sigma Aldrich, USA) for < 1 min. Zona free oocytes were then fixed using 4 %

163

paraformaldehyde (Cat. No. 8.18715.1000 Merck, India) at room temperature for 1h followed by

164

blocking in 1 % BSA, 0.01 M glycine and 0.05 % TritonX-100 in DPBS (Dulbecco’s phosphate-

165

buffered saline Cat. No. 56064C SAFC) for one hour. The cortical granules were stained using 5

166

µg/mL rhodamine tagged lens culinaris agglutinin (Cat. No. RL-1042 Vector Laboratories, Inc.,

167

CA, USA) for 30 min in dark. Oocytes were washed in DPBS and counterstained using DAPI

168

(4',6-Diamidino-2-phenylindole Cat. No. D9542 Sigma Aldrich, USA). Cortical granule

169

distribution was visualized, and images were acquired using the confocal microscope (LSM 510

170

META-Confocal Zeiss, Germany) and the percentage of oocytes displaying CGFD was

171

determined.

172

2.7 Evaluation of sperm zona binding and fertilization

173

Caudal spermatozoa extracted from 6-week-old male mice were used to evaluate the sperm-zona

174

binding. Spermatozoa were allowed to capacitate in M199 medium supplemented with 10 %

175

BSA for 2 h at 37°C in 5 % CO2 [17]. Sperm concentration was adjusted to 1X105 sperm/mL

176

prior to insemination. Metaphase II oocytes were incubated in 80 µL sperm suspension for 30

177

min at 37°C and 5 % CO2 thereafter washed gently to remove unbound sperm and fixed in 4 %

178

PFA. Oocytes were stained using DAPI (4',6-Diamidino-2-phenylindole Cat. No. D9542, Sigma

179

Aldrich, USA) and the number of sperm nuclei bound to the zona was assessed under the

8

180

fluorescent microscope. Fertilization ability was tested in separate set of experiments after 13 h

181

of insemination. The presence of two pronuclei and two polar bodies indicated successful normal

182

fertilization.

183

2.8 Statistical analysis

184

Data represented either as Mean ± Standard Error of Mean (SEM) or in percentage (%). Chi

185

square test was applied for percentage data whereas one-way analysis of variance (ANOVA) was

186

used (if data had followed normality) otherwise Kruskal Wallis test was used to test the

187

difference between the study groups. P < 0.05 was considered as significant. The statistical tests

188

were done using GraphPAD Instat software (Graphpad Inc., La Jolla, CA, USA) and the

189

graphical representation of the data was prepared using Microcal Origin 6.0 software (Origin Lab

190

Corporation, Northampton, MA, USA).

191 192

3. Results

193

194

3.1 Age, in vitro maturation potential and vitrification-warming survival

195

The diameter of the GV oocytes soon after collection was assessed from the different age groups.

196

The mean ooplasmic diameter in D14, D21 and D28 was 60.39 ± 0.61, 70.86 ± 0.64 and 77.44 ±

197

0.31 uM respectively. Oocyte size increased significantly with the age of the animals (P < 0.001).

198

Age dependent changes in the in vitro maturation potential was observed when GV oocytes

199

isolated from D14, D21 and D28 mice were subjected to in vitro maturation (Fig 1a). At the end

200

of 24 h, the maturation rate of D28 and D21 mice was similar (79 % and 78 %), whereas D14

201

oocytes showed a significant reduction in the maturation potential (63 %) in comparison to D28

202

(P < 0.05) and D21 (P < 0.001). 9

203

In order to determine whether IVM is suitable before or after vitrification-warming for

204

prepubertal oocytes, we assessed the survival rate and maturation potential post vitrification-

205

warming of oocytes from various cohorts. No significant association was observed between the

206

prepubertal age and vitrification-warming survival. Vitrification warming did not affect the IVM

207

potential of GV oocytes (Fig 1a). Similarly, maturation status of the oocytes (viz GV or MII) did

208

not affect the survival post warming (Fig 1b).

209

3.2 Effect of IVM prior to vitrification-warming (MII-V) on spindle morphology, mitochondrial

210

potential and cortical granule redistribution.

211

Initially, to understand whether poor maturation potential of D14 oocytes has affected the

212

nuclear maturation, we assessed the meiotic spindle integrity in IVM oocytes (Fig 2a). About 26

213

% of the oocytes in D28 animals had an abnormal spindle pattern. Approximately 32 % of D14

214

oocytes had meiotic abnormalities post IVM which was significantly higher than D21 (P <

215

0.01). Interestingly, abnormal spindle structure was observed in D21 was about 14 %. However,

216

no statistical power was demonstrated with other groups.

217

To determine the appropriate oocyte stage that can protect the structural integrity post

218

vitrification-warming, both GV stage oocytes and IVM oocytes were assessed for the spindle

219

morphology (Fig 2b). Interestingly, it was found that IVM post vitrification-warming of GV

220

stage (GV-V) oocytes preserved the spindle morphology irrespective of the age of the animals.

221

In contrast, significant alteration in spindle structure was observed in MII-V group (Fig 2a).

222

Importantly, almost two-fold increase in the number of oocytes with a spindle abnormality was

223

found in D14 and D21 oocytes in comparison to GV-V (P < 0.001 and 0.01 respectively). On the

224

other hand, differences were not significantly different in D28 group.

10

225

JC1 ratio (red to green) was comparable across the age groups (Fig 3a). Images showing various

226

levels of mitochondrial potential of IVM oocytes from prepubertal ovary are shown in the Fig

227

3b. Further, skewed JC-1 ratio was observed in MII-V oocytes irrespective to the age of the

228

animals (P < 0.001). However, significant differences were also observed between GV-V and

229

MII-V groups except in D14 (Fig 3a).

230

When percentage of oocytes presenting CGFD were determined post IVM, D28 group displayed

231

about 84 % CGFD whereas it was about 89 % in D21 oocytes. On the other hand, D14 oocytes

232

showed a significant decline in CGFD compared to D21 group (76 %; P < 0.05; Fig 4a). Figure

233

4b depicts patterns of cortical granule redistribution in oocytes. The number of oocytes with

234

CGFD was significantly lower (P < 0.01) only in MII-V, D21 group though the trend was similar

235

in other groups (Fig 4a).

236

3.3 Effect of vitrification on sperm zona binding ability and fertilization potential

237

The changes found in cortical granule redistribution in prepubertal oocytes could affect the

238

binding ability of sperm to the zona pellucida. The analysis revealed an age dependent, but non-

239

significant increase in the number of capacitated spermatozoa bound to the zona pellucida (46.96

240

± 6.18, 58.33 ± 7.03, 69.55 ± 5.65 in D14, D21 and D28 groups) (Fig 5a). Importantly,

241

fertilization ability in D14 group was found to be significantly lower in comparison to D28 group

242

(Fig 5b). These results suggest that IVM oocytes from early prepubertal ovary have reduced

243

competence to undergo successful fertilization than young adults.

244

Further, significant impairment in sperm-zona binding was observed only in D21 group of GV-V

245

oocytes (P < 0.01). On the other hand, MII-V oocytes had significantly reduced sperm-zona

246

binding irrespective of the age of the animals (P < 0.001). The number of sperm bound to zona

11

247

was almost 2-3 fold lower compared to other groups (Fig 5a). Though, fertilization rate was low

248

in both vitrification groups, lowest fertilization rate was evident in MII-V oocytes (P < 0.01 -

249

0.001) (Fig 5b).

250

4 Discussion

251

The initial observations made in this study suggested that prepubertal oocytes are susceptible to

252

IVM when compared to that of young adults. Further, subjecting the prepubertal IVM oocytes to

253

vitrification-warming resulted in an increase in the number of abnormal meiotic spindles and

254

cortical distribution pattern in oocytes, reduced sperm zona binding and fertilization rate.

255

In vitro maturation (IVM) is clinically used to derive metaphase II oocytes that are competent to

256

be fertilized and capable of producing viable embryos [1, 34]; hence, considered as a potential

257

alternative to ovarian tissue transplantation in fertility preservation programs. However, one of

258

the major concerns in metaphase II oocyte vitrification is the cryo-sensitivity of microtubular

259

spindle organization and subsequent impact on its integrity [27]. To circumvent this problem,

260

GV stage vitrification is proposed where in vitro maturation is carried out post-warming. Based

261

on our initial observation, we hypothesized that as prepubertal GV stage oocytes have limited

262

functional ability in comparison to young adults, hence subjecting in vitro matured prepubertal

263

oocytes to vitrification-warming may further deteriorate the structural and functional integrity.

264

Our results clearly suggested that IVM is more successful after vitrification-warming of

265

prepubertal GV oocytes.

266

267

Though it was possible to fertilize in vitro matured prepubertal oocytes to produce viable

268

embryos in animals, the fertilization rate, blastocyst development and reproductive outcome 12

269

were found inferior to adults [26]. This could be attributed to the maternal sexual maturity or

270

difference in the follicular micro-environment or oocyte ultrastructure [10]. Oocytes of young

271

mice (especially the D14) have not completed their in vivo growth phase and exhibit poor

272

maturation rates. The average ooplasmic diameter in our study was significantly smaller in D14.

273

Earlier studies have used oocytes “greater than 70 µM” with a specified cumulus status [5].

274

However, oocytes from D14 used in this study had a mean ooplasmic diameter of 60.39±0.61

275

µM which could be a reason for poor functional competence. To have uniformity in our

276

experiments, we have mechanically removed the cumulus cells from D28 oocytes before

277

vitrification or IVM. Oocytes isolated from 12-day-old mice were totally incompetent to undergo

278

GVB in vitro, whereas the GVB rate increased progressively with mouse age and oocyte

279

diameter [20]. Few studies have also reported in vitro follicle culture from D14 mice [22,29] or

280

superovulating D21 mice before oocyte collection [15,36], however, this was not attempted in

281

our study as it was not mimicking the human clinical situation.

282

283

IVM of oocytes from the vitrified warmed ovarian tissue is an ideal option where ovarian tissue

284

transplantation is contraindicated due to the risk of reintroduction of the malignant cells.

285

However, important question is that whether the maturation ability of prepubertal oocytes is

286

similar to that of adult oocytes. Interestingly, IVM ability of vitrified warmed oocytes in this

287

study was comparable between prepubertal and young adults which was in contrary to the

288

observation made earlier where a significant reduction in the maturation rates of human GV

289

oocytes after vitrification when compared to IVM oocytes [4]. This observation made us to look

290

into cryo-susceptibility of GV and IVM derived MII oocytes. Surprisingly, survival was not

13

291

significantly different in relation to the age of the animals and stage of maturity prior to

292

vitrification (Fig 1).

293 294

Vitrification technique made a revolution in oocyte cryopreservation, in terms of oocyte survival

295

and IVF outcomes [6]. However, whether vitrification affects the physiology of oocytes

296

especially when used from prepubertal age group remains to be determined. Oocyte vitrification

297

has shown to damage organelles such as endoplasmic reticulum lysosomes and mitochondria

298

[13]. Mitochondria play a pivotal role in oocyte competence for production of the energy

299

required to perform all the cellular processes coordinated, such as programmed cell death or

300

spindle formation [9] and it has been shown that vitrification can alter mitochondrial distribution

301

and activity in mouse oocytes [21]. We investigated the mitochondrial potential between GV-V

302

and MII-V groups with respect to the age of the animal. Interestingly, mitochondrial potential

303

was significantly lower in MII-V oocytes from both prepubertal and young adult groups which

304

clearly suggested that mitochondria of in vitro matured MII oocytes have higher tendency to get

305

damaged. In addition, significantly higher incidence of abnormal spindle morphology was

306

observed in MII-V oocytes especially in D14 group. Though, oocyte vitrification at MII stage

307

alters spindle integrity [32], the extent of vitrification-warming induced abnormalities in D14

308

group was almost two-fold higher in prepubertal group whereas no significant difference was

309

observed in young adult oocytes.

310 311

Earlier studies have shown that both vitrification and IVM can impact intracellular calcium level

312

and thereby affect cortical granule redistribution [12]. Although, the current study demonstrated

14

313

moderate decrease in CGFD in both MII-V and GV-V group oocytes, significant reduction was

314

observed only in MII-V oocytes in D21 group.

315 316

A transient increase in intracellular calcium during oocyte vitrification can cause zona hardening

317

and eventually reduce sperm binding and fertilization [16, 19]. Though, GV-V oocytes showed

318

reduced zona binding and fertilization rate irrespective of the age of the animal, the extent of

319

reduction in the functional parameters of MII-V oocytes was significantly lower than GV-V

320

oocytes. At present we do not have any supporting mechanistic evidence to address this

321

interesting observation. Exploring the integrity of zona pellucida receptors between two groups

322

may provide valuable information. In contrast to our findings, IVM procedure is more efficient

323

when it is performed before oocyte vitrification in human adult oocytes [11]. However, we argue

324

that due to structural and functional uniqueness of prepubertal oocytes and lack of exposure to

325

endocrine milieu within the follicular environment, their ability to retain the functional

326

competence, post IVM is impaired. Two distinct categories of primordial follicles i.e first wave

327

and the adult primordial follicles seen in rodent and primate ovaries exhibit distinct

328

developmental dynamics and contribute differently to ovarian physiology and functionality [37,

329

38]. Hence, it is possible that pre-pubertal oocytes with less developmental capacity are more

330

susceptible to interventions such as IVM or vitrification. On the other hand, post-pubertal ovaries

331

have a new wave of primordial follicles with higher developmental capacity post IVM and

332

vitrification. It is important to note that this is the first report, where functional and

333

developmental competence of prepubertal oocytes were found diminished when IVM was done

334

before vitrification. However, the limitations of our study are i) functional ability of the oocytes

335

beyond fertilization was not tested ii) the oldest group used in this study D28 which is ‘young

15

336

adults’ or ‘sub adult’ and oocytes from animals beyond D28 was not assessed for ‘adult status’

337

and iii) in vitro growth (IVG) of oocytes within the follicles was not performed for the D14

338

group to overcome their very immature status.

339

340

Though these observations are in favor of GV stage oocyte vitrification, understanding the

341

mechanisms behind poor outcome in MII-V oocytes will help in modifying the current protocol

342

thereby retain their maximum structural and functional integrity to apply in fertility preservation

343

practice. It is to be noted here, with great emphasis, that a major limitation of this study is that

344

the oocytes’ abilities were tested only until fertilisation, as a consequence of which the study

345

cannot reveal the developmental potentials of the embryos beyond fertilisation.

346

Conflict of interest:

347

Authors declare that there is no conflict of interest.

348

Author contribution statement:

349

Conceived and designed the experiments: SKA. Performed the experiments: AD. Analyzed the

350

data: AD, SU. Prepared/reviewed the manuscript: SKA, SU, GK, RT. AD is the guarantor of this

351

work and as such, had full access to all the data and takes responsibility for the integrity of the

352

data and the accuracy of the data analysis.

353

Acknowledgement

354

The authors thank IMPRINT (grant#4511) for providing project assistanceship to AD. Technical

355

support from Mrs. Shobha Uday Sonawane, Senior technical officer and Mrs. Reshma Santosh

356

Gaonkar, Technical officer, Confocal facility, NIRRH (ICMR), Mumbai is thankfully

357

acknowledged.

16

358 359

References

360 361 362

1. D.F. Albertini, A. Sanfins, C.M.H. Combelles, Origins and manifestations of oocyte maturation competencies, Reprod Biomed Online 6 (2003) 410-415.

363

2. R.A. Anderson, M. McLaughlin, W.H.B. Wallace, D.F. Albertini, E.E. Telfer, The immature

364

human ovary shows loss of abnormal follicles and increasing follicle developmental

365

competence through childhood and adolescence, Hum Reprod. 29 (2014) 97–106.

366 367 368 369

3. A. Borini, V. Bianchi, Cryopreservation of mature and immature oocytes, Clin Obstet Gynecol. 53 (2010) 763–774. 4. Y.X. Cao, R.C. Chian, Fertility preservation with immature and in vitro matured oocytes, Semin Reprod Med. 27 (2009) 456–64.

370

5. F. Chen, J. Lin, X. Sun, B. Xiao, S.-F. Ning, S. Zhu, H.-L. Wang, J.-H. Tan, Mechanisms by

371

which in vitro meiotic arrest and sexual maturity improve developmental potential of mouse

372

oocytes, Sci. Rep. 7 (2017) 15763.

373 374 375 376

6. A. Cobo, C. Diaz, Clinical application of oocyte vitrification: A systematic review and metaanalysis of randomized controlled trials, Fertil Steril. 96 (2011) 277–285. 7. C.M. Cosgrove, R. Salani, Ovarian effects of radiation and cytotoxic chemotherapy damage, Best Pract Res Clin Obstet Gynaecol. 55 (2019) 37–48.

377

8. M.M. Dolmans, V. Luyckx , J. Donnez , C.Y. Andersen, T.Greve, Risk of transferring

378

malignant cells with transplanted frozen-thawed ovarian tissue, Fertil Steril. 99 (2013) 1514–

379

1522.

380

9. R. Dumollard, M. Duchen, J. Carroll, The Role of Mitochondrial Function in the Oocyte and

17

381

Embryo, Curr Top Dev Biol. 77 (2007) 21–49.

382

10. G. Fasano, J. Dechène, R. Antonacci, J. Biramane, A.S. Vannin, et al., Outcomes of

383

immature oocytes collected from ovarian tissue for cryopreservation in adult and prepubertal

384

patients, Reprod Biomed Online 34 (2017) 575–582.

385 386 387 388 389 390

11. G. Fasano, I. Demeestere, Y. Englert, In-vitro maturation of human oocytes: Before or after vitrification?, J Assist Reprod Genet. 29 (2012) 507–512. 12. Y. Ghetler, E. Skutelsky, I. Ben Nun, L. Ben Dor, D. Amihai, R. Shalgi, Human oocyte cryopreservation and the fate of cortical granules, Fertil Steril. 86 (2006) 210–216. 13. D.A. Gook, D.H. Edgar, Human oocyte cryopreservation, Hum Reprod Update 13 (2007) 591–605.

391

14. S. Knight, A. Lorenzo, A.M. Maloney, A. Srikanthan, R. Donen, E. Greenblatt, et al., An

392

approach to fertility preservation in prepubertal and postpubertal females: A critical review

393

of current literature, Pediatr Blood Cancer 62 (2015) 935–939.

394

15. T. Kolbe, S. Sheety, I. Walter, R. Palme, T. Rülicke, Impact of superovulation and mating on

395

the wellbeing of juvenile and adult C57BL/6N mice, Reprod. Fertil. Dev. 28 (2016) 969–973.

396

16. N. Koutlaki, B. Schoepper, G. Maroulis, K. Diedrich, S. Al-Hasani, Human oocyte

397

cryopreservation: Past, present and future, Reprod Biomed Online 13 (2006) 427–436.

398

17. D. Kumar, D. Upadhya, S. Uppangala, S.R. Salian, G. Kalthur, S.K. Adiga, Nuclear DNA

399

fragmentation negatively affects zona binding competence of y bearing mouse spermatozoa,

400

J. Assist. Reprod. Genet. 30 (2013) 1611–1615.

401 402 403

18. G.M. Lantinga, A.M.H. Simons, W.A. Kamps, A. Postma, Imminent ovarian failure in childhood cancer survivors, Eur J Cancer. 42 (2006) 1415–1420. 19. M.G.

Larman,

C.B.

Sheehan,

D.K.

Gardner, Calcium-free vitrification

18

reduces

404

cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse

405

oocytes, Reproduction 131 (2006) 53–61.

406

20. B. Lefèvre, E. Nagyova, A. Pesty, J. Testart, Acquisition of meiotic competence is related to

407

the functionality of the phosphoinositide/calcium signaling pathway in the mouse oocyte,

408

Exp. Cell Res. 236 (1997) 193–200.

409

21. T. Lei, N. Guo, M.H. Tan, Y.F. Li, Effect of mouse oocyte vitrification on mitochondrial

410

membrane potential and distribution, J Huazhong Univ Sci Technol Med Sci. 34 (2014) 99–

411

102.

412

22. J. Liu, J. Van Der Elst, R. Van Den Broecke, F. Dumortier, M. Dhont, Maturation of mouse

413

primordial follicles by combination of grafting and in vitro culture, Biol. Reprod. 62 (2000)

414

1218–1223.

415

23. R. Ma, J. Zhang, X. Liu, L. Li, H. Liu, R. Rui, et al., Involvement of Rab6a in organelle

416

rearrangement and cytoskeletal organization during mouse oocyte maturation, Sci Rep. 6

417

(2016) 23560.

418 419 420 421

24. D. Nogueira, J.C. Sadeu, J. Montagut, In vitro oocyte maturation: Current status, Semin Reprod Med. 30 (2012) 199–213. 25. J.K. O’Brien, Developmental capacity, energy metabolism and ultrastructure of mature oocytes from prepubertal and adult sheep, Reprod Fertil Dev. 8 (1996) 1029–37.

422

26. G. Ptak, K. Matsukawa, C. Palmieri C, L.D. Salda, P.A. Scapolo, P. Loi, Developmental and

423

functional evidence of nuclear immaturity in prepubertal oocytes, Hum Reprod. 21 (2006)

424

2228–2237.

425

27. L. Rienzi, F. Martinez, F. Ubaldi, M.G. Minasi, M. Iacobelli, J. Tesarik, et al., Polscope

426

analysis of meiotic spindle changes in living metaphase II human oocytes during the freezing

19

427

and thawing procedures, Hum Reprod. 19 (2004) 655–659.

428

28. L. Rienzi, S. Romano, L. Albricci, R. Maggiulli, A. Capalbo, E. Baroni, S. Colamaria, F.

429

Sapienza, F. Ubaldi, Embryo development of fresh “versus” vitrified metaphase II oocytes

430

after ICSI: A prospective randomized sibling-oocyte study, Hum. Reprod. 25 (2010) 66–73.

431

29. I. Segers, T. Adriaenssens, E. Ozturk, J. Smitz, Acquisition and loss of oocyte meiotic and

432

developmental competence during in vitro antral follicle growth in mouse, Fertil. Steril. 93

433

(2010) 2695–2700.

434 435 436 437

30. S. Silber, Ovarian tissue cryopreservation and transplantation: scientific implications, J Assist Reprod Genet. 33 (2016) 1595–603. 31. M.A. Sirard, F. Richard, P. Blondin, C. Robert, Contribution of the oocyte to embryo quality, Theriogenology 65 (2006) 126–136.

438

32. A.N. Tamura, T.T. Huang, Y. Marikawa, Impact of Vitrification on the Meiotic Spindle and

439

Components of the Microtubule-Organizing Center in Mouse Mature Oocytes, Biol Reprod.

440

89 (2013) 1–10.

441

33. S. Uppangala, S. Dhiman, S.R. Salian, V.J. Singh, G. Kalthur, S.K. Adiga, In vitro matured

442

oocytes are more susceptible than in vivo matured oocytes to mock ICSI induced functional

443

and genetic changes, PLoS One 10 (2015) e0119735.

444 445 446 447 448 449

34. P.M. Wassarman, L. Jovine, H. Qi, Z. Williams, C. Darie, E.S. Litscher, Recent aspects of mammalian fertilization research, Mol Cell Endocrinol. 234 (2005) 95–103. 35. A.J. Watson, Oocyte cytoplasmic maturation: a key mediator of oocyte and embryo developmental competence, J Anim Sci. 85 (2007) E1-E3. 36. M.X. Zarrow, E.D. Wilson, The influence of age on superovulation in the immature rat and mouse., Endocrinology. 69 (1961) 851–855.

20

450

37. W. Zheng, H. Zhang, N. Gorre, S. Risal, Y. Shen, K. Liu, Two classes of ovarian primordial

451

follicles exhibit distinct developmental dynamics and physiological functions, Hum Mol

452

Genet. 23 (2014) 920-928.

453

38. W. Zheng, H. Zhang, K. Liu, The two classes of primordial follicles in the mouse ovary: their

454

development, physiological functions and implications for future research, Mol Hum Reprod.

455

20 (2014) 286-292.

456 457 458

Figure legends

459 460

Figure 1 Age, in vitro maturation potential and vitrification-warming survival. (a) Oocyte

461

maturation potential of non-vitrified (teal bar) (N=468, 274, 218; D14, D21, D28 respectively)

462

and vitrified germinal vesicle (orange bar) (N=374, 273, 221; D14, D21, D28 respectively) to

463

MII in relation to the age. (b) Survival rate of GV stage vitrification-warming (teal bar) (N=351,

464

264, 205; D14, D21, D28 respectively) and in vitro matured MII oocytes vitrification-warming

465

(orange bar) (N=212, 209, 150; D14, D21, D28 respectively).

466

Figure 2 IVM prior to vitrification-warming alters spindle morphology (a) Meiotic spindle

467

abnormalities (D14: N=55,41,43; D21: N=50,33,46; D28: N=30,35,32 Control, GV-V and MII-

468

V respectively) Control (teal bar); GV-V (orange bar); MII-V (green bar). (b) Schematic

469

representation of spindle abnormalities in MII oocytes (40X). Please note that i-iv represents

470

normal spindle structure and v-xii represent abnormal forms. Scale bar=20um.

471

Figure 3 Mitochondrial potential in GV-V and MII-V oocytes (a) Mitochondrial potential

472

(JC-1 ratio) (D14: N=125,85,87; D21: N=62,75,93; D28: N=54,44,40 Control, GV-V and MII-V 21

473

respectively) Control (teal bar); GV-V (orange bar); MII-V (green bar). (b) Fluorescent

474

microscopic images (40X) of oocytes stained by JC-1 in reducing order of mitochondrial

475

potential: Scale bar=20um.

476

Figure 4 Cortical granule redistribution in vitrified prepubertal oocytes (a) Cortical granule

477

free domain (CGFD) (D14: N=33,25,26; D21: N=28,26,27; D28: N=29,26,31 Control, GV-V

478

and MII-V respectively) Control (teal bar); GV-V (orange bar); MII-V (green bar). (b) Confocal

479

microscopic images (40X) of oocytes stained by LCA showing CGFD: Scale bar=20um.

480

Figure 5 MII-V oocytes had decreased sperm-zona binding and fertilizing abilities (a)

481

Sperm zona binding ability (D14: N=25,20,24; D21: N=30,27,22; D28: N=20,26,22; Control,

482

GV-V and MII-V respectively) and (b) fertilization rate (D14 N=56,50,46; D21 N=45,44,37;

483

D28 N=40,37,42; Control, GV-V and MII-V respectively). Control (teal bar); GV-V (orange

484

bar); MII-V (green bar).

485

22