Ethylene Glycol and Dimethyl Sulfoxide Combination

BIOPRESERVATION AND BIOBANKING Volume 15, Number 6, 2017 ª Mary Ann Liebert, Inc. DOI: 10.1089/bio.2017.0078

Ethylene Glycol and Dimethyl Sulfoxide Combination Reduces Cryoinjuries and Apoptotic Gene Expression in Vitrified Laying Hen Ovary

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Reihane Nateghi,1 AliReza Alizadeh,2 Yousef Jafari Ahangari,1 Rouhollah Fathi,2 and Amir Akhlaghi 3

Successful cryopreservation of avian gonads is important not only for avian breeding but is also crucial for preservation of species, especially of endangered birds. The aim of this study was to evaluate the effect of vitrification by several cryoprotectants on the ovarian tissues of laying hens. Ovarian tissues were randomly divided into six groups: control (nonvitrified: C), dehydrated using ethylene glycol (EG), dehydrated with propylene glycol (PROH), dehydrated using dimethyl sulfoxide (DMSO), and two combined groups, EG+DMSO and EG+PROH. The composition of vitrification solutions was as follows: EG group: V1 = 7.5% EG and V2 = 15% EG +0.5 M sucrose, DMSO group: V1 = 7.5% DMSO and V2 = 15% DMSO +0.5 M sucrose, PROH group: V1 = 7.5% PROH and V2 = 15% PROH +0.5 M sucrose, EG+DMSO group: V1 = 7.5% EG +7.5% DMSO and V2 = 15% EG +15% DMSO +0.5 M sucrose and EG+PROH group: V1 = 7.5% EG +7.5% PROH and V2 = 15% EG +15% PROH +0.5 M sucrose. Ovarian tissues of each group were dehydrated for 10 minutes with V1 solution and 2 minutes with V2. Among the vitrified groups, intact primordial and primary follicles showed significant increase in EG+DMSO, but follicular attrition had the highest rate in the PROH group ( p < 0.05). Immunohistochemical analysis showed that the percentage of active caspase 3-positive cells was lower ( p < 0.05) when using EG+DMSO versus PROH. Further gene expression of caspase 3, 8, and 9 was highest in the PROH group ( p < 0.05). Vitrification of ovaries of laying hens using EG+DMSO can afford effective protection of primordial and primary follicles during preservation and may therefore be successfully used for storing avian gonadal tissues. Keywords: vitrification, cryoprotectant, ovary, laying hen

Introduction

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varian tissues and oocyte cryopreservation comprise an approach for preservation of species that are extinct or endangered.1 One issue that worries the poultry breeding industry is the loss of genetic variability resource and an established method for preserving premier livestock breeds, and the conservation of those animal species that are endangered is cryoconservation of reproductive cells or gonads.2 Deansely3 was the first to report the successful cryopreservation of ovarian tissue in immature rat. Several attempts have been made to cryopreserve whole or partial ovaries of various species such as mice,4 rat,5 rabbit,6 cattle,7,8 sheep,9 goat,10 humans,11 and recently in quail.12 The use of freezing methods helps facilitate this process by allowing access to the genome. Thus, recent years have

seen a rise in research involving the use of freezing techniques to cryopreserve primordial germ cells. An important issue in cryopreservation is identifying the best kind of cryoprotectant that leads to minimal damage to the tissue for each animal species.13 Thus, numerous studies compare various cryoprotectants in human,14 mouse,15 and rat16 ovaries. Among several cryoprotectants, propylene glycol (PROH), dimethyl sulfoxide (DMSO), and ethylene glycol (EG) are viable options and researchers have commonly used them due to their efficiency of cryopreserving reproductive organs.17 While DMSO (C2H6OS) has a molecular weight and density of 78.13 g/mol and 1.1004 g/cm3, respectively, EG (C2H6O2) has the least molecular weight and is the most dense (1.1132 g/cm3). PROH (1,2-propanediol, C3H8O2), which has a molecular weight of 76.06 g/mol and density of 1.0597 g/ cm3, is less penetrable than EG. EG is more penetrable than

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Faculty of Animal Science, Gorgan University of Agricultural Science and Natural Resource, Gorgan, Iran. Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. 3 Department of Animal Science, College of Agriculture, Shiraz University, Shiraz, Iran. 2

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other cryoprotectants,18 but the size and imbalance density of cells specifically in the cells of ovarian tissue change the effect of this penetration.19 The effects of cryoprotectants vary according to the type of substance and animal species.19 For instance, DMSO is a secure cryoprotectant for several species such as mouse oocytes,20 bovine oocytes,21 and human germinal vesicle oocytes,22 but in some species such as zebra fish it is poisonous.23 Using the adult laying hen as a model, it may be that results can be extended to cryopreservation of ovarian tissues of other avian species. The single ovary of the chicken is irregularly shaped. This organ is attached to the dorsal surface of the left side of the abdominal cavity wall.24 Follicles of the ovary have different sizes, and usually an ovary contains 5–10 follicles greater than 1 cm in diameter. Primary oocytes are enclosed by the vitelline membrane. These oocytes become organized into a primordial follicle (up to *80 mm in diameter). The average diameter of primary follicles is 0.8 to 1 mm and ovulation occurs in the largest follicle. The accumulation of lipoprotein-rich, white yolk and differentiation of the theca into internal and external layers occur and further growth leads to the prehierarchical follicle stage (1–8 mm).25 As cryoprotectants are cytotoxic,26 the concentration of exposure to cryoprotectants should be selected carefully, because it is a key factor to minimize the toxic effects. Meanwhile, there are a few reports about quantitative assessment of follicles at different categories immediately after vitrification warming of laying hen ovarian tissue. For this purpose, in the present study, we used adult hen ovaries, rather than the small, juvenile ovaries used by the other studies. Some previous studies have illustrated that ovarian tissue cryopreservation of 1-week-old Japanese quail has successfully been done.12 More studies on avian gonadal transplantation27 have an important role in improving the functional recovery of cryopreserved gonadal tissue. Although the vitrification of avian ovarian tissue seems to be well established,28 a few studies report differences in the sensibility of types of follicles (primordial, primary, and prehierarchical) to a variety of cryoprotectants and gene expression mechanisms regarding vitrification of the laying hen ovary. Furthermore, numerous studies have confirmed that vitrification could result in the emergence of apoptosis in vitrified ovaries and embryos and change the epiphany of apoptosis-related genes.29,30 The objective of the current investigation was to assess the efficiency of vitrification solution, which contains different cryoprotectants (EG, DMSO, and PROH), for cryopreservation of mature laying hen ovaries, and quantitative and qualitative assessment of different follicular categories after vitrification. Furthermore, gene expression (Bak, Bcl-2, Bak/Bcl-2, Cas3, Cas8, and Cas9) of ovarian tissue from adult hen ovaries after vitrification was compared to noncryopreserved control tissue.

Materials and Methods Birds, tissue preparation, and study design Seven White Leghorn hens (6 months old) were handled in compliance with existing guidelines and subject to the approval of Royan Institute Ethics Committee (IR.ACECR. ROYAN.REC.1394.30). Laying hens were euthanized by

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cervical dislocation and ovarian tissues were removed from the body, immediately transferred to the laboratory in phosphate-buffered saline (PBS), and washed three times in fresh PBS. Follicles larger than 3 mm were separated from the ovarian tissue. Then, the ovarian tissues were randomly distributed into six experimental groups: nonvitrified control (C), dehydrated by using EG, dehydrated with PROH, dehydrated with DMSO, and two combined groups: EG+DMSO and EG+PROH. The size of each piece of ovarian tissue was *3 · 3 · 2 mm. The experiments were followed by morphological evaluations, follicular counts, and gene expression studies (Bak, Bcl-2, Cas3, Cas8, and Cas9) (Fig. 1).

Vitrification procedure (dehydration and plunging of samples into liquid nitrogen) The vitrification procedure was performed according to Liu et al.,12 with a minor modification. Base medium (BM) in equilibrium (V1) and vitrification (V2) solutions were composed of Dulbecco’s modified Eagle’s medium (Gibco, Invitrogen, Carlsbad, CA) and 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY) (Table 1). First, all the ovarian tissues from vitrified groups were dehydrated through immersing in V1 and V2 solutions for 10 and 2 minutes, respectively, at room temperature (RT), and the tissue samples were picked up with the needle of an insulin syringe (cryopin)31 and immersed in liquid nitrogen. Dehydrated ovarian tissues were placed in cryovials and stored in a nitrogen tank for more than 3 weeks.

Warming procedure (rehydration and reviving the vitrified samples) The warming procedure was carried out as described by Liu et al.,12 with some modifications from our pretests. In this study, there were no acceptable results regarding warming for 5 minutes according to Liu’s study. Therefore, 1 and 3 minutes of warming were also examined and histological evaluations showed that the optimum time for warming is 1 minute. The BM in warming solutions was composed of Dulbecco’s modified Eagle’s medium and 10% FBS. During the warming process, first the vitrified ovaries were transferred from the liquid nitrogen tank to W1 solution (BM+sucrose 1 M) and kept for 1 minute. Then, the tissues were transferred from W1 solution to W2 solution (BM+sucrose 0.5 M) and then to W3 solution (BM+sucrose 0.25 M) and kept in each solution for 1 minute. The entire washing process was performed at RT. Eventually, to complete the warming process, the washed ovaries were transferred to W4 solution (BM) and incubated in a 37C, 5% CO2 humidified incubator (New Brunswick Scientific Co., Inc., Edison, NJ) for 30 minutes until histological assessments were made.

Morphology and follicle structure The ovary samples were fixed at RT in Bouin’s fluid and formalin for 24 and 72 hours, respectively. Then, they were dehydrated in alcohol and embedded in paraffin wax. Thick sections (5 mm) were cut and stained with hematoxylin and eosin (H&E) for histopathological studies.

CRYOPROTECTANTS IN VITRIFIED AVIAN OVARIAN

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FIG. 1. Outline of the experimental procedure.

In normal follicles, the layer of granulosa cells is attached to the spherical oocyte surrounding it. Furthermore, the homogenous ooplasm (cytoplasm of the oocyte) contains a tiny granulated nucleus. In contrast to normal follicles, in atretic follicles, aggregation and shrinkage of nuclear chromatin and wrinkling of the nuclear membrane were regarded as signs of atresia. To avoid double counting, only the follicles with a visible nucleus were counted. Determination of the different types of follicles was done according to a study by Johnson and Woods25 (Fig. 2). Accordingly, follicles with a maximum size of 80 mm were counted as primordial follicles and those 0.08–1 mm were considered primary follicles. Also, follicles with a size greater than 1 mm were considered prehierarchical follicles. The best preserved group (EG+DMSO) and the worst group (PROH) as determined by morphological

evaluations were selected for immunohistochemical and gene expression assays.

Caspase 3-positive follicles detected by immunohistochemistry For immunohistochemistry, the section of each tissue was randomly selected and stained with active caspase 3 antibody. To suppress endogenous peroxidase activity, tissues were treated with 3% H2O2 in methanol for 30 minutes.32 Then, the sections were soaked in citrate buffer (pH = 6.0) and warmed up to 98C for 1 hour. After that, tissue sections were blocked for 1 hour with protein block and incubated at 4C overnight in 1:200 dilution of active caspase 3 antibody (ab4051). They were washed well and incubated with a

Table 1. Cryoprotectant Concentrations in Vitrification Groups Groups EG DMSO PROH EG+DMSO EG+PROH

Equilibrium solution (V1) 7.5% 7.5% 7.5% 7.5% 7.5%

EG +10% FBS DMSO +10% FBS PROH +10% FBS EG +7.5% DMSO +10% FBS EG +7.5% PROH +10% FBS

Vitrification solution (V2) 15% 15% 15% 15% 15%

EG +0.5 M sucrose +10% FBS DMSO +0.5 M sucrose +10% FBS PROH +0.5 M sucrose +10% FBS EG +15% DMSO +0.5 M sucrose +10% FBS EG +15% PROH +0.5 M sucrose +10% FBS

DMSO, dimethyl sulfoxide; EG, ethylene glycol; FBS, fetal bovine serum; PROH, propanediol.


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FIG. 2. Vitrified warmed laying hen ovarian tissues. Atretic primordial follicles (white arrows) and intact primordial follicles (dash arrows). Atretic primary follicles (arrowhead) and intact primary follicles (black arrows). Intact prehierarchical follicles (star). Control (nonvitrified) (A), EG group (B), DMSO group (C), PROH group (D), EG+DMSO group (E), and EG+PROH group (F). Hematoxylin and eosin staining (scale bar = 100 mm). Follicles with a maximum size of 80 mm were counted as primordial follicles and those 0.08–1 mm were considered primary follicles. Also, follicles with a size greater than 1 mm were considered prehierarchical follicles. DMSO, dimethyl sulfoxide; EG, ethylene glycol; PROH, propanediol.

secondary antibody (ab97051) for 1 hour at RT. Eventually, the blocks were washed and the attached antibody was visible after the addition of 3, 3-diaminobenzidine tetrachloride (DAB) solution. For accurate cell counting by light microscopy, counterstaining of slides with hematoxylin was carried out.32 In this study, follicles in which oocytes or more than 50% of granulosa cells showed positive reaction for caspase 3 were considered apoptotic follicles (Fig. 3).31

DNA (Table 2). Gene expression levels were measured using Applied Biosystems (ABI 7500 thermocycler) Step One and Step One Plus real-time PCR systems. Expression levels of Bak, Bcl-2, Cas3, Cas8, and Cas9 were evaluated. The housekeeping gene GAPDH was used for normalization. Design of specific primers was done using the software Allele ID (Premier Biosoft, Palo Alto, CA) (Table 2).

Statistical analysis Quantitative real-time polymerase chain reaction for apoptotic genes Soon after warming, fragments from each group were stored in RNAlater reagent (Ambion) and preserved at -80C until RNA extraction. Total RNA was extracted from the samples through a manual method using TRIzol (Sigma-Aldrich) as follows: small ovarian fragments were added to TRIzol and homogenized. The fragments were incubated on ice for 5 minutes and centrifuged (12,000 rpm, 15 minutes, 4C). Then, chloroform (Merck, Germany) was added followed by cold isopropanol (Merck, Germany). The mixture was incubated for 60 minutes at -20C and then centrifuged (12,000 rpm, 15 minutes, 4C). Finally, the RNA pellets were washed with 70% ethanol, air-dried, and dissolved in diethyl pyrocarbonatetreated water. Evaluations of concentration and purity of the total RNA were done using a spectrophotometer at 260/280 nm. A cDNA Synthesis Kit (Fermentas, Leon-Rot, Germany) and random hexamers were used to synthesize complementary

Statistical analysis was carried out using SPSS 22.0 (IBM Crop., Armonk, NY). Continuous variables were expressed as mean – standard error of mean. The Kolmogorov–Smirnov test was used to evaluate the normality of the data. A one-way ANOVA, followed by the Duncan posthoc test, was used to compare the groups. All statistical tests were two sided and a p-value