Ethylene Glycol and Egg Yolk as Cryoprotectant

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AIP Conference Proceedings 2168, 020097 (2019); https://doi.org/10.1063/1.5132524 2168, 020097

The profile of ovarian tissue of female

Sprague-Dawley rats (Rattus norvegicus L.) after vitrification using ethylene glycol and egg yolk as cryoprotectant

Cite as: AIP Conference Proceedings 2168, 020097 (2019); https://doi.org/10.1063/1.5132524 Published Online: 04 November 2019

A. Awanis, N. Anita, A. Abinawanto, et al.

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The Profile of Ovarian Tissue of Female Sprague-Dawley Rats (Rattus norvegicus L.) After Vitrification Using

Ethylene Glycol and Egg Yolk as Cryoprotectant

A. Awanis

¹

, N. Anita

^{1, a)}

, A. Abinawanto

¹

, A. A. Jusuf

²

, I. Muhiardi

¹

, M. A. Rosvita

¹

and Y. Q. A’yun

¹

1Department of Biology, Faculty of Mathematics and Natural Sciences (FMIPA), Universitas Indonesia, Depok 16424, Indonesia

2Department of Histology, Faculty of Medicine Universitas Indonesia, Jakarta 10430, Indonesia

a)Corresponding author: [email protected]

Abstract. Vitrification has been widely used to preserve ovarian tissue. However, an efficient method with minimal damaging effect towards the tissue has not been established, yet. Accordingly, the combination method among intra- and extracellular cryoprotectant was conducted in this study. The aim of the research is to evaluate combination of ethylene glycol (EG) as intracellular cryoprotectant with egg yolk as extracellular cryoprotectant in reducing coldshock effect on ovarian tissue vitrification after 48 hours. Twenty one samples of twelve week-old female Sprague-Dawley rats are used in this study. Ovarian samples (n = 21) were washed in saline to clear excessive blood and were weighed. Samples for control groups (n = 3) were immediately fixated. Control treatment groups (n = 9) plunged into cryotube filled with 3.75

%, 7.5 % and 15 % of EG solution, respectively, while treatment groups (n = 9) were 3.75 %, 7.5 % and 15 % of egg yolk + EG solution, respectively. All samples were then frozen in liquid nitrogen (- 196 °C). After 48 hours, the samples were thawed and weighed. The result shown that based on Anova test there were no significant different (p < 0.05) in weight of ovarian tissue before and after vitrification. In conclusion, vitrification using a combination of ethylene glycol (EG) and egg yolk may have significant impact in preventing coldshock in the ovary.

Keywords: Egg yolk, ethylene glycol, female rats, ovarian tissue, vitrification

INTRODUCTION

Technology in tissue preservation recently has been widely developed in order to find the right method that can support the success of assisted reproductive technology (ART). One of the main purposes of ART was to maintain reproductive functions [1]. Reproductive tissue preservations such as ovary can be used for prior female gamete culture and subsequently utilized in in-vitro embryo formation [2]. Ovarian preservation in animals can be applied for conserving either an endangered species or live stocks which has high economic value [3]. Besides, this method can also be used in humans to save and restore women reproductive function, particularly in women who will be undergoing chemotheraphy [4].

Vitrification is a method of preservation of cells or intact tissue at low temperatures (-196 °C) over a certain period of time with a minimum risk of damage [5]. Vitrification can be applied to preserve organs including the ovary [6]. Ovarian vitrification is more recommended because it is more efficient and has lower technical difficulty and also shorter processing time [7, 8].

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One of the challenges in vitrification of the ovary is the risk of cryoinjury or damage due to the formation of ice crystals in the process of freezing and thawing. The fluctuation within extreme temperatures in both processes can trigger the formation of either intracellular ice crystals or extracellular ice crystals. Ice crystals in the intracellular cytoplasm can damage the organelles and the cell cytoskeleton, whereas the extracellular ice crystals can reduce the membrane fluidity, especially during rehydration (thawing). Low membrane fluidity can cause damage of cell membranes, which could directly affected the diffusion process and eventually lead to shrinkage of ovarian tissue [9, 10].

Ovarian protection from the formation of ice crystals during the vitrification process was supported by giving cryoprotectants to the vitrification medium. Cryoprotectants will increase the viscosity of either intracellular or extracellular fluids, so that ovarian tissue solidifies without crystallization during cooling [11]. Previous studies using vitrification on whole ovaries [12] and oocytes [13] in mice showed that intracellular cryoprotectants effectively protect the intracellular components of the ovary from the formation of ice crystals. However, the extracellular membrane cannot be protected by intracellular cryoprotectants. Therefore, to maximize intracellular and extracellular membrane integrity, an addition of extracellular cryoprotectants is a must.

Combination of ethylene glycol (EG) as intracellular cryoprotectants and egg yolk as extracellular cryoprotectants has been successfully carried out by Isachenko and Nayudu [14] in vitrification of mouse oocytes.

The results of this study indicate that egg yolks can maintain the integrity of post-vitrification oocyte morphology as much as 46–83 %. Fathi et al. [13] showed that the optimum concentration ratio of two types of cryoprotectant was 1:1, with concentrations of 7.5 % and 15 % respectively. Concentration of 3.75 % was added in order to determine the minimum concentration that possibly has an optimum effect on the ovary. The use of natural extracellular cryoprotectants in vitrification methods of ovarian tissue has never been done before. Accordingly, early research using rats as a model animal was conducted to determine the effect of egg yolk as natural extracellular cryoprotectants on ovarian weight after vitrification.

MATERIALS AND METHOD Animal Model and Treatment Groups

Twenty one female Sprague−Dawley rats (Rattus norvegicus L.) were used as test animal. Each were twelve week-old, weighed 180 to 200 grams and were in proestrus phase when performed ovarian isolation. The animal was obtained from Health Research and Development Agency of Ministry of Health and habituated in new cages for two weeks. Every day the animals fed by mineral water and pellet in ad libitum and weighed to achieve a favorable body weight. Test animals were grouped in five, each group kept in 43 cm x 37 cm x 15 cm cages. The cages were cleaned and disinfected every two days. The storing room was maintained in constant temperature of 26–28 °C, air circulation were aided by exhaust fan and the lighting were set in 12 hours light and dark system. The research was carried using the Randomized Complete Design (CRD) method. Twenty-one test animals were divided into seven treatment groups as shown in Table 1, each with three repetitions. The treatment groups consist of NC (Normal Control, which are fresh samples without vitrification treatment); TC1 or Treatment Control 1 (vitrification with only ethylene glycol 3.75 %); TC2 or Treatment Control 2 (vitrification with only ethylene glycol 7.5 %); TC3 or Treatment Control 3 (vitrification with only ethylene glycol 15 %); T1 or Treatment 1 (vitrification with combination of ethylene glycol 3.75 % and egg yolk 3.75 %); T2 or Treatment 2 (vitrification with combination of ethylene glycol 7.5 % and egg yolk 7.5 %) and T3 or Treatment 3 (vitrification with combination of ethylene glycol 15 % and egg yolk 15 %). Test animal maintenance and treatment were approved by Ethical Clearance Committee of Faculty of Medicine Universitas Indonesia.

Determination of Proestrous Phase

Estrous cycle of tests animals were tested on day fifteen of acclimatization. The tests were performed according to Marcondes et al. [15] and Sjahfirdi et al. [16] by using vaginal smear methods. Vaginal fluid of each animal was collected as much as 10 µL using micropipette inserted approximately 1 cm into the rat vagina. The fluid was dropped onto glass slide, then fixated in 80% methanol and stained by 10 % Giemsa stain solution [15, 16]. The phase of estrous cycle were determined no longer than two hours after it was obtained on slide glass and performed

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TABLE 1. The composition of vitrification solution for each treatment group.

Treatment Group Ethylene Glycol (EG) (µ)

NaCl 0.9 % (µ)

Egg Yolk (µ)

NaCl 0.9 % (µ)

NC (without vitrification) - - - -

TC 1 (EG 3.75 %) 75 1925 - -

TC 2 (EG 7.5 %) 150 1850 - -

TC 3 (EG 15 %) 300 1700 - -

T1 (EG 3.75 % + egg yolk 3.75 %) 37.5 962.5 37.5 962.5

T2 (EG 7.5 % + egg yolk 7.5 %) 7.5 925 7.5 925

T3 (EG 15 % + egg yolk 15 %) 150 850 150 850

according to Paccola et al. [17] and Cora et al. [18]. The slides were observed under a Leica DM500 light microscope and image driving software LAS EZ v.2.0.0. Proestrous phase were indicated by a dominant population of small nucleated ephitellial cell (small NAC).The first results were used to predict excecution day for each animal.

The second test were performed on the predicted excecution day after test animal turned twelve week-old. The animal confirmed on proestrous phase were immediately sacrificed within an hour [17, 18].

Preparation of Vitrifying Solution

Preparation of vitrifying solution was performed according to Milenkovic [12], Fathi et al. [13] and Isachenko and Nayudu [14]. Ethylene glycol solution was made by dissolving ethylene glycol with NaCl solution 0.9 % to obtain a concentration of 3.75 %, 7.5 % and 15 %. NaCl 0.9 % solution was first put into a container then added with ethylene glycol. Fresh eggs for the egg yolk were obtained from a certified egg supplier UD Tirta Soeper Teloer, Jakarta. The yolk was separated from the egg white. Membrane of egg yolk was torn so that the yolk flew out. The egg yolk was then filtered using filter paper and collected in a container. Egg yolks solution was made by dissolving the yolk with NaCl 0.9 % solution to obtain a concentration of 3.75 %, 7.5 % and 15 %.

Ovary Tissue Preparation and Initial Data Sampling

Test animals were sacrificed by using ketamine with anasthetic dose through intraperitoneal injection (IP) and followed with cervical dislocation. Ovarian tissue preparation was performed according to Kittel et al. [19]. Test animals that have stopped breathing were immediately positioned with the dorsal attached to the paraffin tray. The animal skin was sprayed with 70 % alcohol, then cut from the top of the ureter to the lower part of the thorax. The digestive organs were removed so as not to block the view. The right and left ovaries were separated from the reproductive tract and cleaned from the attached fat. The process of surgery and ovarian isolation was carried out in less than 10 minutes. The ovary that was successfully isolated was immediately weighed with an analytical scale to obtain the initial weight. Ovary weighing must be done quickly and carefully. The ovaries were washed three times with NaCl 0.9 % solution. Fresh ovary samples were then immersed in formal saline 10 % solution, meanwhile ovary with vitrification treatment were dipped in vitrification medium.

Vitrification and Final Data Sampling

The vitrification method performed was a modification of the research that had been carried out by Milenkovic et al. [12], Mohamad et al. [20] and Rosadi et al. [21]. The ovarium of the vitrification treatment group was plunged into the cryotube which contained 2 mL of vitrified medium respectively. Cryotube that contain the ovary was then exposed to liquid nitrogen vapor for approximately 10 seconds to allow diffusion of cryoprotectant into the cell.

Cryotube is then drowned in -196 °C liquid nitrogen. After 48 hours, thawing is carried out by transferring the cryotube from the nitrogen tube into the waterbath at 37 °C for 2–3 minutes until it melts. The ovary was taken out and immediately weighed with an analytical scale to obtain final weight. Ovary samples were then immersed in formal saline 10 % solution.

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Statistical Analysis

Data obtained were tabulated descriptively in form of mean values and standard deviations using Microsoft Excel. The data were then tested using IBM SPSS Statistics 24 for normality by Saphiro-Wilk test and for homogeneity by Levene test. Data that was normally distributed and homogeneous were tested using one-way ANOVA test (P > 0.05) and Post Hoc LSD to analyze the difference on ovarian weight.

RESULTS AND DISCUSSION

The weight of twenty-one ovary samples observed before and after vitrification are shown in Table 2 and Fig. 1.

The average of initial weight of ovary ranged between 0.0478 ± 0.0050 gram to 0.0692 ± 0.0106 gram, with total mean of 0.0594 ± 0.070 gram (P = 0.0460). The sample was normally distributed and homogenic. ANOVA test (P >

0.05) showed no significant difference on the initial ovarian weight among treatment groups. Meanwhile, the average of final weight of ovary was ranged between 0.0469 ± 0.0039 gram to 0.0675 ± 0.0309 gram, with total mean of 0.0562 ± 0.0086 gram (P = 0.0647). The sample was also normally distributed and homogenic. ANOVA test (p > 0.05) shown no significant difference on the initial ovarian weight among treatment groups. Although, the average weight of ovary before and after vitrification are slightly decreased. ANOVA test of ovarian weight before and after treatment showed no significant difference, therefore LSD test was not performed.

A previous report carried out by Murasawa et al. [22] have shown that follicular density inside of ovary could have an impact on ovarian weight and that it reflected on the potential of ovary in producing mature follicles. The density of healthy ovarian follicle was closely related with the integrity of follicle. Its integrity can be affected by its lipid bilayers structure after treatment with vitrification. Therefore, the role of cryoprotectant was crucial. The use of a combination of cryoprotectant was expected to maintain the final weight of ovary as closely to NC groups and the initial weight.

Egg yolk as cryoprotectant has been previously used in the vitrification of mouse oocyte [14]. Egg yolks are a type of natural extracellular cryoprotectant which is known to have a membrane stabilizing effect which caused mainly by the components of phospholipids, LDL (lipoproteins) and their derivatives [23]. The phospholipid of the yolk, especially phosphatidylcholine (73 %), is known to provide protection to the cell membrane by coating and isolating the extracellular membrane from toxic trait of intracellular cryoprotectants, while maintaining the fluidity and flexibility of the membrane during cooling [14]. Low density lipoprotein (LDL) in egg yolks, namely lipovitelin and phosphitein, protect the plasma membrane from cold shock during the freezing process. Egg yolk lecithin content is known as membrane coating which also functions to envelop the cell membrane from the formation of ice crystals.

TABLE 2. Weight of ovarian tissue before and after treatment with or without egg yolk added.

Treatment Groups (gram) Total

Mean

NC TC1 TC2 TC3 T1 T2 T3

BT 0.0610

± 0.0223

0.0478

± 0.0050

0.0520

± 0.0183

0.0498

± 0.0082

0.0683

± 0.0162

0.0690

± 0.0254

0.0692

± 0.0106

0.0596

± 0.075

AT 0.0610

± 0.0223

0.0469

± 0.0039

0.0498

± 0.0160

0.0454

± 0.0107

0.0614

± 0.0148

0.0645

± 0.0257

0.0607

± 0.0121

0.0557

±0.0073 Percentage

of decrease 0 % 2 % 4 % 9 % 10 % 7 % 12 %

The values were mean and standard deviation of three sample repetitions. BT = Before Treatment, AT = After Treatment.

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FIGURE 1. Weight of ovarian tissue before and after vitrtification, except for NC group. The red graphics are for groups with vitrification treatment. The letter ‘a’ indicates no significant differences among treatment groups (P > 0.05, one-way ANOVA)

The cholesterol content of the yolk will insert between phospholipid bilayer and prevent shrinkage of the cell membrane due to the formation of bonds between the membrane-forming lipids during cooling which can cause cell membrane malformation [23]. In addition to lipid and protein components, chicken egg yolks also contain minerals such as sulfur, potassium, sodium, phosphorus, calcium, magnesium and iron which can act as ions to help maintain the extracellular fluid concentration gradient and accelerate the diffusion process during dehydration and rehydration [24]. The composition of egg yolk was expected to work sinergically in maintaining the lipid bilayer structure of ovary throughout vitrification, therefore the final weight of ovary should not much differ from initial weights and NC groups.

In this research, the results of ovarian weight before and after vitrification showed no significant differences between NC (normal control) groups and treatment groups; between three different concentration on TC (treatment control) group (TC1, TC2 and TC3); and between three different concentration on (T) treatment groups (T1, T2 and T3) (Fig. 1). The mean of the ovarian weight of NC groups or ovaries without vitrification treatment was 0.0610 ± 0.0223 gram. However, a decrease in ovarian weight after vitrification compared to before vitrification occured in almost all groups treated with vitrification (TC1, TC2, TC3, T1, T2 and T3), with the most significant decrease could be seen on treatment groups (T1, T2 and T3). The decrease of weight in TC (treatment control) groups were as much as 2 %, 4 % and 9 % for TC1, TC2 and TC3 respectively. From this result, the decrease of ovarian weight seem to be aligned with increase in concentration of ethelyne glycol. Although the cause of decrease is still unknown. Meanwhile, the percentage of decrease in ovarian weight of treatment groups were even bigger than TC groups, each were 10 %, 7 % and 12 % for T1, T2 and T3 respectively. Eventhough T2 group showed percentage of decrease less than 10 % and still in range with treatment control (TC) groups, the addition of egg yolk into ethylene glycol solution seem to show no effect on maintaining the final weight of ovary. However, the cause of weight decrease should not be wholly settled on the use of egg yolk.

CONCLUSION

This research has proved that the combination of ethylene glycol (EG) and egg yolk in ovary vitrification might not have any impact on the weight of rat ovary after vitrification. Nonetheless, an optimum methods has not yet to be concluded and further study is needed to evaluate the effect of the same combination of cryoprotectants on histological scale of the ovary.

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ACKNOWLEDGMENTS

The authors would like to send our gratitude to Universitas Indonesia and Directorate of Research and Public Service (DRPM) UI for providing financial assistance by Hibah Publikasi Internasional Terindeks untuk Tugas Akhir (PITTA) UI 2018. We would like to thank to researchers and staffs of Health Research and Development Agency of Ministry of Health and the Laboratory of Animal Physiology of Biology Department, Universitas Indonesia for their courtesy and assistance.

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