Use of synthetic polymers improves the quality of vitrified caprine preantral follicles in the ovarian tissue

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Use of synthetic polymers improves the quality of vitrified caprine preantral follicles in the ovarian tissue Diego Alberto Montano Vizcarraa, Yago Pinto Silvaa, Jamily Bezerra Brunoa, Danielle Cristina Calado Britoa, Deysi Dipaz Berrocala, Luciana Mascena Silvaa, Maria Luana Gaudencia dos Santos Moraisa, Benner Gerardo Alvesb, Kele Amaral Alvesb, Francielli Weber Santos Cibinc, José Ricardo Figueiredoa, Mary B. Zelinskid, Ana Paula Ribeiro Rodriguesa,* a

Faculty of Veterinary Medicine, Laboratory of Manipulation of Oocytes and Preantral Follicles (LAMOFOPA), State University of Ceará, Fortaleza, CE, Brazil Department of Animal Reproduction, Federal University of Uberlândia, MG, Brazil c Federal University of Pampa, Uruguaiana, RS, Brazil d Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, Beaverton, OR 97006, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: In vitro culture Vitrification SuperCool X-1000 SuperCool Z-1000 Polyvinylpyrrolidone

The aim of this study was to evaluate whether the addition of synthetic polymers to the vitrification solution affected follicular morphology and development and the expression of Ki-67, Aquaporin 3 (AQP3) and cleaved Caspase-3 proteins in ovarian tissue of the caprine species. Caprine ovaries were fragmented and two fragments were immediately fixed (Fresh Control) for morphological evaluation, while other two were in vitro cultured for 7 days (Cultured Control) and fixed as well. The remaining fragments were distributed in two different vitrification groups: Vitrified and Vitrified/Cultured. Each group was composed of 4 different treatments: 1) Sucrose (SUC); 2) SuperCool X-1000 0.2 % (X-1000); 3) SuperCool Z-1000 0.4 % (Z-1000) or 4) with polyvinylpyrrolidone K-12 0.2 % (PVP). Also, Fresh Control, Cultured Control, SUC and X-1000 were destined to immunohistochemical detection of Ki-67, AQP3 and cleaved Caspase-3 proteins. Morphologically, the treatment with X-1000 showed no significant difference with the Fresh Control group and was superior to the other treatments. After the cleaved caspase-3 analysis, X-1000 showed the lowest percentages of strong immunostaining while Cultured Control showed the highest. Also, a positive correlation was found between the percentages of degenerated follicles and the percentages of strong staining intensity follicles. Regarding the AQP3 analysis, the highest percentages of strong AQP3 staining intensity were found in X-1000. In conclusion, we have demonstrated that the addition of the synthetic polymer SuperCool X-1000 to the vitrification solution improved the current vitrification protocol of caprine ovarian tissue.

1. Introduction

excellent method for ovary cryopreservation due to its capacity of avoiding the formation of intracellular ice crystals, extremely dangerous to the cells and commonly observed in slow freezing procedures (Carvalho et al., 2011). During the vitrification procedure, the conversion of a system from a fluid to an amorphous vitreous state takes place by increasing the solution viscosity during cooling (Fahy et al., 1984, 2004). However, this process requires the use of ultra-rapid freezing rates that could lead to an osmotic stress in the ovarian tissue (Amorim et al., 2011). In addition, it demands the use of high concentrations of permeating cryoprotectant agents (CPAs). These substances enter the cell through water channel proteins, known as

The cryopreservation of ovarian tissue fragments is an excellent assisted reproduction technique with the potential to preserve the female fertility through the protection of the endocrine and exocrine functions of the ovary (Faustino et al., 2011). The current standard procedure for ovarian tissue cryopreservation is the slow freezing method, with over 80 reported live births, in contrast with the only 2 live births reports for the vitrification method (Jensen et al., 2017; Kawamura et al., 2013; Suzuki et al., 2015). Despite this lack of reports, many researchers have shown that the vitrification procedure is an

⁎ Corresponding author at: Programa de Pós-Graduação em Ciências Veterinárias (PPGCV), Laboratório de Manipulação de Oócitos e Folículos Pré-Antrais (LAMOFOPA), Universidade Estadual do Ceará (UECE), Av. Paranjana, 1700, Campus do Itaperi, Fortaleza, Ceará CEP: 60740 903, Brazil. E-mail address: [email protected] (A.P. Ribeiro Rodrigues).

https://doi.org/10.1016/j.acthis.2019.151484 Received 19 August 2019; Received in revised form 27 November 2019; Accepted 2 December 2019 0065-1281/ © 2019 Elsevier GmbH. All rights reserved.

Please cite this article as: Diego Alberto Montano Vizcarra, et al., Acta Histochemica, https://doi.org/10.1016/j.acthis.2019.151484

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AQP3 were evaluated.

aquaporins, such as the AQP3, and act by removing the intracellular water to prevent ice crystal formation (Sales et al., 2013). Despite the toxicity risk associated with the use of high concentrations of permeating CPAs remains as the greatest obstacle (Best, 2015), their use is extremely necessary in the cryopreservation procedures to minimize the injuries. Some non-permeating CPAs (sugars) are being used in numerous cryopreservation protocols in a variety of ovarian tissues across a wide range of species (Carvalho et al., 2011; Hovatta et al., 1996; Jimenez et al., 2016; Santos et al., 2006; Tanpradit et al., 2015; Zhang et al., 2009). The idea behind the use of these sugars (glucose, sucrose, trehalose, etc.) as additives in the cryopreservation protocols with the intention of reducing the cryoinjuries came from the fact that some of these substances are naturally used by various species of plants and animals as a mechanism of protection from extreme cold climates (Crowe and Crowe, 2000). It is important to note that several sugars provide different levels of protection during cryopreservation and this can vary depending on media composition and cooling conditions. These differences may depend, in part, on the differential effects of specific sugars on raising the glass transition temperature of vitrification solutions (Kuleshova et al., 1999). Caprine ovarian tissue has been previously vitrified using both permeating and non-permeating CPAs in the vitrification solution. Using this association of CPAs, the percentage of morphologically normal preantral follicles ranged from 55 % (Carvalho et al., 2014) to 58 % (Carvalho et al., 2013) immediately after vitrification/warming or was 36 % after vitrification/warming followed by in vitro culture. To our understanding, these results could be improved through a protocol adjustment. Adding macromolecules such as synthetic polymers in the vitrification solution, which have been included as additives, could be a viable alternative to achieve this goal. Previous studies have reported that the freezing resistance shown by some Antarctic fishes is explained by the presence of specific antifreeze proteins that exert their protective mechanism through the binding to heterogeneous ice nucleators to avoid the formation of new ice crystals (DeVries and Wohlschlag, 1969; Knight et al., 1984; Kristiansen and Zachariassen, 2005). Antifreeze proteins tend to increase the viscosity of the cryoprotectant solution and form interactions through hydrogen bonding with water thereby decreasing the propensity for ice crystal formation (Fuller, 2004). Since this discovery, many synthetic analogs like the copolymer of polyvinyl alcohol (SuperCool X-1000), polyglycerol polymer (SuperCool Z-1000) and polyvinylpyrrolidone (PVP K12) have been produced at lower costs and proposed as additives for the vitrification solutions (Wowk, 2005). They block ice crystal growth along the main axes in the nucleation sites, preventing effectively the formation of new ice crystals and improving the vitrification process (Marco-Jiménez et al., 2014; Wowk, 2005). The addition of synthetic polymers to the vitrification solutions facilitates the reduction in the concentration of permeating CPAs, hence attenuating the chemical toxicity (Wowk et al., 2000). Additionally, these synthetic polymers depress the freezing point through a process known as thermal hysteresis, to avoid the occurrence of ice recrystallization during the thawing process (Congdon et al., 2013; Gibson et al., 2009; Mitchell et al., 2015; Wowk et al., 2000). While these properties have led to some relevant results in the cryopreservation of ovarian tissue from primates (Hashimoto et al., 2010; Ting et al., 2011, 2013) and leporids (Marco-Jiménez et al., 2014), there are no reports of the use of synthetic polymers in the vitrification protocol of caprine ovarian tissue, which might represent an opening for the improvement of the vitrification protocol. Therefore, the aim of this study was to assess the effect of the addition of synthetic polymers (SuperCool X-1000, SuperCool Z-1000 and PVP K-12) in the vitrification protocol of caprine ovarian tissue. After vitrification, the ovarian tissue was in vitro cultured for 7 days to analyze the morphology and development of preantral follicles. Additionally, cell proliferation, apoptosis and immunolocalization of

2. Materials and methods The permeating (ethylene glycol and dimethyl sulfoxide) and nonpermeating CPAs (sucrose) were obtained from Dinâmica (Dinâmica Química Contemporânea, Diadema, SP, Brazil). The synthetic polymers (SuperCool X-1000, SuperCool Z-1000 and PVP K-12) were received as a donation from 21 st Century Medicine (21CM, Fontana, CA, USA) and the other chemicals were obtained from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). 2.1. Collection and transport of ovaries Ten ovaries were collected from five adult non-pregnant cross-bred goats at a local slaughterhouse. Immediately postmortem, the ovaries were washed once in 70 % ethanol for 10 s and then washed twice in HEPES-buffered minimum essential medium (MEM-HEPES) supplemented with 100 μg/mL penicillin and 100 μg/mL streptomycin. Then, the ovaries were transported to the laboratory in MEM-HEPES at 20 °C within 1 h after collection (Carvalho et al., 2013). 2.2. Experimental design and vitrification/warming procedures Ovaries were stripped of the surrounding fat and fibrous tissue and the cortex from each ovarian pair was cut into small fragments (n = 20; 1 × 1 × 0.5 mm) using a Tissue Slicer® (Thomas Scientific, Swedesboro, NY, USA) under sterile conditions. The ovarian fragments from each ovarian pair (each animal represented a replicate) were immediately evaluated under a stereoscope (selecting only those that contained preantral follicles) and randomly distributed among the treatments. Two fresh fragments were fixed in 4 % paraformaldehyde (PFA) for 2 h and named as Fresh Control (FCTR), while two others were in vitro cultured for 7 days and considered Cultured Control (CCTR). After the 7 days, the fragments from the CCTR group were fixed in the same conditions as with FCTR for morphology evaluation. The remaining fragments (n = 16) were distributed in two different groups: Vitrified group (Vitrified), when the fragments were immediately fixed after vitrification in the same conditions as with FCTR; and Vitrified/ Cultured group (Vitrified/Cultured), when they were vitrified and cultured in vitro for 7 days and then fixed in the same conditions as with FCTR. The fragments of the Vitrified and Vitrified/Cultured groups (n = 16) were distributed in four different vitrification treatments (2 fragments/treatment/group): 1) Vitrification with the addition of sucrose (SUC); 2) Vitrification with the addition of SuperCool X-1000 0.2 % (X-1000); 3) SuperCool Z-1000 0.4 % (Z-1000) or 4) with PVP K-12 0.2 % (PVP), totaling four experimental treatments. The concentrations chosen for the present experiment were according to previous reports in the literature (Ting et al., 2012, 2013). The whole vitrification procedure was performed using the Ovarian Tissue Cryosystem (OTC) device according to Carvalho et al. (2013). Briefly, the fragments were exposed to two vitrification solutions (vs). The VS1 consisted of MEM supplemented with 10 mg/mL bovine serum albumin (BSA), 20 IU/ml catalase, 10 % ethylene glycol (EG; v/v) and 10 % dimethyl sulfoxide (DMSO; v/v). The VS2 had a similar composition but with higher concentration of permeating CPAs (20 % EG and 20 % DMSO). Both solutions (VS1 and VS2) were supplemented with 0.25 M sucrose; 0.2 % [v/v] X-1000; 0.4 % [v/v] Z-1000 or 0.2 % [v/v] PVP. Initially, the ovarian fragments were exposed to VS1 for 4 min at 20 °C followed by an exposition to VS2 for 1 min at 20 °C. The vitrification solution was then removed and the OTC containing the ovarian tissue was closed and immediately immersed vertically into liquid nitrogen (-196 °C). After 1 week, the OTCs containing the vitrified ovarian fragments were warmed in air at room temperature (RT ∼25 °C) for 1 min, followed by immersion in a water bath (37 °C) for 30 s. After this, the CPAs 2

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antibody followed by incubation with the biotinylated goat anti-polyvalent secondary antibody. Next, the slides were washed, allowed to react with 3,3′-diaminobenzidine in chromogen solution (DAB) in substrate and, finally, the sections were counterstained with hematoxylin. Negative controls were performed by not using the primary antibody. Spleen and kidney tissue sections obtained from a healthy mouse were used as positive controls for Ki-67 and AQP3, respectively. Kidney tissue, used as positive control for cleaved Caspase-3, was previously exposed to toxic levels of snake venom. Follicles with at least one Ki-67stained granulosa cell were considered to have initiated proliferation and considered as Ki-67-positive (Amorim et al., 2012). Regarding the AQP3 immunohistochemical analysis, the intensity of each protein positive staining whether in the oocyte or granulosa cells of each preantral follicle was graded as + (weak), ++ (moderate) or strong (++ +) (Sales et al., 2016). Finally, the cleaved caspase-3 analysis was performed to detect the presence of the protein in the cytoplasm and nucleus of the follicles and classify them as a positive or negative cleaved-caspase 3 follicle (Amorim et al., 2012).

were removed using a three-step exposure to washing solutions (5 min each) at 20 °C. These washing solutions (WS) were composed of: WS1 (MEM +3 mg/mL BSA + 0.5 M sucrose); WS2 (MEM +3 mg/mL BSA + 0.25 M sucrose) and WS3 (MEM +3 mg/mL BSA). The three WS did not contain antioxidants (catalase) nor synthetic polymers. 2.3. In vitro culture Non-vitrified and vitrified/warmed fragments were cultured in 500 μl of culture medium, for 7 days, at 39 °C in a humidified incubator with 5 % CO2. The culture medium consisted of base medium α-MEM (pH 7.2–7.4), supplemented with ITS (10 μg/mL insulin, 5.5 μg/mL transferrin; 0.5 ng/mL selenium), glutamine (2 mM), hypoxanthine (2 mM) and BSA (1.25 mg/mL). In addition, following previous findings by Alves et al. (2013), growth differentiation factor-9 (GDF-9; 200 ng/ mL-1) and follicle stimulating hormone (FSH; 50 ng/mL) were added. Full medium replacements were performed every two days. 2.4. Evaluation of follicular morphology

2.6. Statistical analysis Ovarian fragments (Fresh Control, Cultured Control or vitrified/ warmed treatments) were fixed in 4 % PFA in phosphate-buffered saline (PBS) for 2 h at 37 °C, dehydrated in a graded series of ethanol, clarified with xylene, embedded in paraffin wax and cut in 7 μm thin slices using a conventional microtome. Slides with those slices were then stained with Periodic Acid Schiff (PAS)-hematoxylin. For morphological evaluation, the slides were coded and examined under a light microscope (Nikon, Japan) with a magnification of 400 × . Preantral follicles were classified as previously defined by Silva et al. (2004) as: primordial, with a single layer of granulosa cells flattened around the oocyte; intermediate or transition, with one layer of flattened with few cuboidal granulosa cells; primary, showing a full layer of cuboidal granulosa cells; and secondary, with two or more layers of cuboidal granulosa cells around the oocyte. These follicles were classified morphologically as normal or degenerated according to the absence or presence of the following characteristics: pyknotic bodies, cytoplasmic shrinkage and disorganization of granulosa cells, respectively (Rodgers and IrvingRodgers, 2010). Also, intermediate, primary and secondary follicles were classified as the developing preantral follicles population (Silva et al., 2004).

Statistical analyses were carried out using the Sigma Plot software version 11.0 (Systat Software Inc, San Jose, California, USA). Proportion variables were compared among treatments by Fisher's exact or chi-square tests. Mean levels of reactive oxygen species were analyzed by ANOVA and t-test. Pearson correlation test was used to assess the association between degenerated preantral follicles and apoptosis staining intensity (cleaved caspase-3). Data were presented as mean ( ± standard error of the mean) and percentage, unless otherwise indicated. Statistical significance was defined as p < 0.05 and probability values > 0.05 and ≤ 0.1 indicated that a difference approached significance. 3. Results 3.1. Evaluation of morphology, development and proliferation of preantral follicles Each fragment allowed for an average of 5 histological slides, each slide having an average of 40 histological sections, all of them were stained for morphological evaluation. In total, we obtained 1952 follicles from all 5 animals. Up to two-hundred (200) follicles from each of the following 10 different experimental treatments (Fresh Control (FCTR), Cultured Control (CCTR), Vitrified and Vitrified/Cultured groups (4 treatments each) (Sucrose (SUC), SuperCool X-1000 (X1000), SuperCool Z-1000 (Z-1000), PVP K-12 (PVP))). Preantral follicles were classified as previously defined by Silva et al. (2004) as: primordial, with a single layer of granulosa cells flattened around the oocyte; intermediate or transition, with one layer of flattened with few cuboidal granulosa cells; primary, showing a full layer of cuboidal granulosa cells; and secondary, with two or more layers of cuboidal granulosa cells around the oocyte. The morphologically normal preantral follicles found in this study had a centrally located oocyte showing a round shape and no signs of pyknosis degeneration and well organized surrounding granulosa cells. On the other hand, the degenerated follicles presented oocytes with cytoplasm retraction, nucleus pyknosis and disorganized granulosa cells (Fig. 1). Compared to Fresh Control, the percentage of morphologically normal preantral follicles decreased significantly (p < 0.05) in all treatments, except for the ovarian tissue vitrified with SuperCool X1000 followed by in vitro culture for 7 days (Table 1). It is important to note that the percentage of normal follicles in the vitrified ovarian tissue (in all treatments) followed by in vitro culture was superior (p < 0.05) than those observed in the Cultured Control. Regarding follicular development (Table 2), all vitrified and in vitro cultured fragments maintained the percentage of developing follicles

2.5. Immunohistochemical analysis For the immunohistochemical the following treatments were selected the that presented the best results in the morphological analysis: vitrified/cultured SuperCool X-1000 (X-1000), the current vitrification protocol used in our laboratory (SUC), Fresh Control (FCTR) and Cultured Control (CCTR). KI67, AQP3 and cleaved Caspase-3 assays were performed to determine granulosa cell proliferation, presence of water channels and apoptosis, respectively. Tissue samples were fixed with 4 % paraformaldehyde in PBS (pH 7.2) and subsequently dehydrated and embedded in paraffin wax. Tissue sections (5 μm) mounted on Superfrost Plus slides (Knittel Glass, Bielefeld, NW, Germany) were deparaffinized and rehydrated in a graded ethanol series. Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC kit (ab64264; Abcam Inc., Cambridge, MA, USA) was used according to manufacturer’s instructions. Antigen retrieval was performed by incubating tissue sections in 0.01 M sodium citrate buffer (pH 6.0) for 5 min at 96 °C. To block endogenous peroxidase the slides were incubated with the manufacturer’s solution for 10 min and then washed with PBS for 5 min. After this, the slides were incubated during 1 h for non-specific blocking (25 ml PBS +0.00125 g BSA + 75 μl Triton X). Then, the slides were incubated for 1 h either with anti-Ki67 rabbit polyclonal (1:1000 dilution; ab15580, Abcam Inc., Cambridge, MA, USA), anti-cleaved Caspase 3 rabbit polyclonal (1:1000 dilution; ab2302, Abcam Inc.) or anti-AQP3 rabbit polyclonal (1:1000 dilution; ab153694, Abcam Inc.) primary 3

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Fig. 1. Representative microscopy images of morphological evaluation of ovarian goat tissue after acid-periodic Schiff staining from Fresh Control (A), Cultured Control (B), and both vitrified and vitrified/cultured groups: Sucrose (C, D), SuperCool X-1000 (E, F), SuperCool Z-1000 (G, H) and PVP K-12 (I, J). Note in all images arrows represent morphologically normal follicles and arrowheads represent morphologically degenerated follicles. Scale bars =50 μm.

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immunostaining intensity when compared with the other treatments (Table 4).

Table 1 Percentages (%) of morphologically normal preantral follicles in fresh or vitrified ovarian fragments before and after in vitro culture for 7 days. Control

Sucrose SuperCool X-1000 SuperCool Z-1000 PVP K-12

Fresh Control: 84 (168/ 200)

Cultured Control: 44 (88/200)

Vitrified 47 (94/200) 48 (96/200) 43 (85/200) 43 (65/152)

Vitrified/Cultured 60 (119/200) * †Bb 77 (153/200) †Ab 61 (121/200) * †Bb 68 (135/200) * †Bb

*Aa *Aa *Aa *Aa

4. Discussion Although the use of non-permeating synthetic polymers has already been proven in the vitrification of ovarian tissue (Hashimoto et al., 2010; Ting et al., 2012, 2013), this is the first report in which these macromolecules (SuperCool X-1000, SuperCool Z-1000 and PVP K-12) are used to help prevent ice formation in the vitrification of caprine ovarian tissue. Previous vitrification protocols have included these iceblockers with success in animal models like bovine (Zhou et al., 2010), equine (Curcio et al., 2014), piscine (Cabrita et al., 2006), leporids (Marco-Jiménez et al., 2014), murine (Badrzadeh et al., 2010), porcine (Marco-Jiménez et al., 2012) and primate (Hashimoto et al., 2010; Ting et al., 2012, 2013). Regarding the follicular morphology results, the use of synthetic polymer SuperCool X-1000 (a copolymer of polyvinyl alcohol) has been proved worthy as a highly effective ice-inhibiting agent (Wowk et al., 2000). In previous reports, after the vitrification of ovarian tissue, the percentages of normal follicles were inferior when compared with the Fresh Control, without the addition of synthetic polymers (Carvalho et al., 2013, 2014; Ting et al., 2013;). Macromolecules such as SuperCool X-1000 were reported to block ice growth preventing the formation of ice crystals (Zachariassen and Kristiansen, 2000). This property is the same by which the natural antifreeze proteins effectively block rapid ice crystal growth (Olijve et al., 2016) and perhaps, could be the mechanism by which this synthetic polymer acted. Additionally, SuperCool X-1000 at low concentrations (0.001 %) can help to reduce the concentration of permeating cryoprotectants needed to vitrify, which can help to reduce the toxicity (Wowk et al., 2000). On the other hand, the use of SuperCool Z-1000 or PVP K-12 didn’t show satisfactory results regarding follicular morphology and follicle survival, suggesting that it might not be ideal to test them alone. Previous results indicate that we might obtain better results if we combine SuperCool Z-1000 with Super Cool X-1000 due to its combined action in the inhibition of heterogeneous ice nucleators (Wowk and Fahy, 2002). The PVP K-12 synthetic polymer has been shown to be most effective when is used in combination with ethylene glycol rather than DMSO (Hashimoto et al., 2010). Additionally, it has also been suggested that the exposure time to the synthetic polymers should be tested in future experiments, since, according to other authors, an exposure for 3 min gives the best results and avoids a possible toxicity risk (Ting et al., 2012) in comparison with the 5 min of exposure used in the current study. The vitrification process, regardless of the treatment used (sucrose, X-1000, Z-1000 and PVP K-12), affected the follicle morphology, perhaps due to the longer time exposure to the cryoprotectant agents. However, immediate analysis after vitrification/warming does not accurately represent the real status of the tissue because to restart the metabolism of the cells, an incubation period is needed (Hovatta et al., 1996). Additionally, based on previous investigations, ovarian tissue cells being subjected to stressful conditions like the vitrification procedure require a higher energy intake which could be provided by the in vitro culture medium (Castro et al., 2014; Gosden, 2000). Regarding follicular development, all vitrified/cultured treatments, except the SuperCool Z-1000 treatment which showed a significant decrease, maintained the percentages of developing follicles like the Fresh Control (Table 2). However, there was a significant reduction in the follicular development, except for the Sucrose treatment, between the vitrified and vitrified/cultured groups. On the other hand, previous reports showed that the follicular growth was promoted even after the vitrification of ovarian tissue followed by in vitro culture (Bandeira et al., 2015; Donfack et al., 2018; Ramezani et al., 2018). A possible explanation for our results would be that secondary follicles are more sensitive to degenerative events than primordial follicles (Silva et al., 2002), which suggests that the vitrification with synthetic polymers

A, B Indicates difference (p < 0.05) among treatments within the vitrified or vitrified/cultured groups; a,b Indicates difference (p < 0.05) between vitrified and vitrified/cultured groups. *Different from Fresh Control. † Different from Cultured Control. Table 2 Percentages (%) of developing preantral follicles in fresh and vitrified ovarian fragments before and after in vitro culture for 7 days. Control

Fresh Control: 38 (63/168)

Cultured Control: 45 (40/88)

Sucrose SuperCool X-1000 SuperCool Z-1000 PVP K-12

Vitrified 32 (30/94) 42 (40/96) 41 (35/85) 46 (30/65)

Vitrified/Cultured 31 (37/119) †a 29 (45/153) †b 24 (29/121) * †b 32 (43/135) †b

a a a a

a,b Indicates difference (p < 0.05) between vitrified and vitrified/cultured groups. *Different from Fresh Control. † Different from Cultured Control.

like the Fresh Control, except those vitrified with Z-1000 that showed a significant reduction (p < 0.05) in the follicular development percentages after 7 days of in vitro culture. Furthermore, it was observed that the percentage of developing follicles present in the vitrified tissue were significantly reduced (p < 0.05) after in vitro culture compared to Cultured Control. Only the Sucrose treatment showed no difference in percentages of developing follicles compared with the other vitrified/ cultured treatments (X-1000, Z-1000 and PVP K-12). After the immunohistochemical assay in search for Ki-67-positive granulosa cells, only the Fresh Control (Fig. 2) showed granulosa cells staining positive to that protein. Absence of Ki-67-positive granulosa cells was observed in the Cultured Control treatment, as well as in the Sucrose and SuperCool X-1000 treatments. 3.2. Immunohistochemical evaluation of apoptosis (cleaved caspase-3) and presence of water channels (AQP3) Immunohistochemical detection of cleaved caspase-3 in the cellular cytoplasm and nucleus indicative of apoptosis was observed in all treatments (Fig. 2). The cleaved caspase 3-positive preantral follicles were differentiated by their relative immunostaining intensity (Table 3). Super Cool X-1000 and Sucrose treatments showed similar percentages of weak (+) immunostaining intensity, when compared with the Fresh Control. Exceptionally, Cultured Control showed the highest percentages of strong (+++) immunostaining intensity when compared with the other groups (Table 3). The correlation test showed a positive correlation between the follicles showing strong (+++) immunostaining intensity and those that show signs of degeneration (Fig. 3). Regarding AQP3 detection (Fig. 2), Fresh Control showed higher percentages of follicles with weak (+) immunostaining intensity when compared with the other treatments. The sucrose treatment showed higher percentages of follicles with moderate (++) immunoreactivity intensity when compared with other treatments. SuperCool X-1000 was the treatment with the highest percentages of strong (+++) 5

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Fig. 2. Immunolocalization of aquaporin 3, cleaved caspase-3 and Ki-67 in preantral follicles from different treatments, Fresh Control (G–I); Cultured Control (J–L); Sucrose (M–O) and SuperCool X-1000 (P–R). Non-specific and specific bindings for these proteins are shown as Negative Controls (A–C) and Positive Controls (D–F). Kidney and spleen tissue sections obtained from a healthy mouse were used as positive controls for AQP3 (A) and Ki-67 (C), respectively; and kidney tissue, which was previously exposed to toxic levels of snake venom, was used as positive control for cleaved Caspase-3 (B). The immunostaining of KI67 in the nucleus of granulose cells is visible in panel I (see arrow). Scale bars =50 μm.

followed by in vitro culture for 7 days might have affected mainly the developing follicle population. The primordial follicles containing an immature oocyte represent almost all the ovarian follicle population (approximately 95 %). They appear to be more tolerant to the cryoinjuries inherent to the cryopreservation process because the contained oocyte has a low metabolism, as well as absence of the spindle in the metaphase, zona pellucida and cortical granules. The small size of the primordial follicle also greatly favors penetration of the cryoprotective agent (Oktay et al., 1997). Thus, it could be hypothesized that the synthetic polymers better preserved the pre-antral follicles at the early stages (primordial and transition) to secure their future development

Table 3 Percentages (%) of cleaved caspase 3-positive preantral follicles differentiated by their relative immunostaining intensity. Treatment Fresh Control Cultured Control Sucrose SuperCool X-1000

+

++ AB

18 (7/39) 6 (3/52) A 22 (13/60) 5 (2/41) A

B

46 42 52 73

(18/39) (22/52) (31/60) (30/41)

+++ A A A B

36 52 27 22

(14/39) AB (27/52) A (16/60) B (9/41) B

A, B within column indicates difference between treatments (p < 0.05).

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the apoptotic cell via the extrinsic (death receptors) and intrinsic pathways (mitochondrial pathway). Once the DNA fragmentation cascade is active, a positive feedback response ensures that the cell will inevitably enter apoptosis (Cohen, 1997). All treatments, no matter the group, stained positive to cleaved-caspase 3. Regardless of the presence of cryoprotectant agents and antioxidative additives, the results obtained are in accordance with previous studies in which the freezing and thawing induced certain levels of apoptosis in oocytes and ovarian stroma cells (Fauque et al., 2007). However, in this study the Cultured Control treatment showed the highest percentages of strong (+++) immunostaining intensity when compared with the other treatments (SUC and X-1000). In vitro culture promotes an acceleration in the metabolism of the cells, stimulating a rapid growth, activating mechanisms and sometimes these rapid changes could not be accompanied by the cells themselves. There is a recent study (Apolloni et al., 2016) that concludes that this accelerated follicle growth impacted negatively the morphology of caprine preantral follicles after in vitro culture. Therefore, we could hypothesize that the vitrification process could be helping to reduce this metabolism acceleration and help the follicle properly accompany the growth process, improving the percentages of morphologically normal follicles. In conclusion, we have demonstrated that the vitrification with the synthetic polymer SuperCool X-1000 followed by in vitro culture for 7 days maintained the follicular morphology and the percentages of strong (+++) cleaved caspase-3 immunostaining intensity like the observed in the Fresh Control treatment, implying a possible protective role by the synthetic polymers during the vitrification process. Furthermore, this treatment (SuperCool X-1000) showed higher percentages of follicles with strong (+++) AQP3 immunostaining intensity when compared with other treatments, highlighting the importance of these proteins in the vitrification process and suggesting a possible interaction between them and the synthetic polymers. Future experiments testing combinations of non-permeating polymers as well as optimal exposure times may further improve the survival and development of preantral follicles following vitrification.

Fig. 3. Correlation analysis between the percentages (%) of degenerated follicles and the percentages (%) of cleaved caspase-3 strong (+++) immunostaining intensity.

Table 4 Percentages (%) of AQP3-positive preantral follicles differentiated by their relative immunostaining intensity. Treatment Fresh Control Cultured Control Sucrose SuperCool X-1000

+ 100 (28/28) 11 (5/47) B 16 (3/19) B 6 (2/35) B

A

++

+++

– 43 (20/47) A 79 (15/19) B 14 (5/35) C

– 47 (22/47) 5 (1/19) B 80 (28/35)

A

C

A, B, C within column indicates difference between treatments (p < 0.05).

since they are more resistant to cryo-injury. Regarding the Ki-67 immunohistochemical results, compared to other proliferation markers such as Proliferating cell nuclear antigen (PCNA), Ki-67 is a superior indicator of true post thaw viability since it is labeled only if nuclear deoxyribonucleic acid (DNA) is functioning at the time the stain is incorporated (Nubani et al., 1998). Regardless of treatment, evidence of cell proliferation was absent in the early categories of preantral follicles and present in secondary follicles. Although Ki-67 is a nuclear antigen expressed in all phases of the cell cycle (Hadar et al., 2005), the cellular appearance and location of this protein throughout the cell cycle is not homogeneous (Urruticoechea et al., 2005). According to these authors, the levels of Ki-67 are low during G1 and early S-phase and progressively increase to reach a maximum during mitosis. Hence, it is the stage of cellular development of the cell in question that might compromise a Ki-67-positive result, getting a better chance of protein expression when it gets closer to the M-stage. Regarding the AQP3 labeling in the preantral follicles, the highest percentages of strong (+++) immunostaining intensity were observed in the SuperCool X-1000 treatment when compared with the other treatments. In comparison, the Sucrose treatment showed the highest percentages of moderate (++) immunostaining intensity in comparison with the other treatments, while the Fresh Control treatment only showed follicles with weak (+) immunostaining intensity. AQP3 water channels are known as aquaglyceroporins, this is an aquaporin with a bigger pore size that is permeable to water, urea and other solutes, playing an important role in transporting permeating ACPs such as ethylene glycol, propylene glycol, glycerol, and others (Sales et al., 2013). The presence of the strong AQP3 labeling in the earliest follicular categories in the SuperCool X-1000 treatment, in comparison with other treatments, could be correlated with the morphology results, which showed a higher follicular morphology preservation in comparison with other treatments. Thus, AQP3 could be playing an important role during the exposure and removal of CPAs in the vitrification and warming steps (Sales et al., 2016). Caspase-3 is part of the group of effector caspases and is activated in

Funding sources This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Scholarship, Mr. Diego Alberto Montano Vizcarra) and by National Counsel of Technological and Scientific Development (CNPq) (4572262013-7; Mrs. Ana Paula Ribeiro Rodrigues and Mr. José Ricardo de Figueiredo). Ana Paula is recipient of a grant from National Counsel of Technological and Scientific Development (CNPq). Animal rights declaration All procedures performed in studies involving animals were carried out in accordance with the ethical standards of the Ethics Committee for Animal Use of the State University of Ceará, registered as 2269277/ 2016. Also, they comply with the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, or the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). Authors' contributions “APRR and DAMV performed the experimental design. DAMV, YPS, BGA and KAA performed the in vitro culture of the preantral follicles. DAMV, DJDB, LMS and MLGSM performed the cryopreservation of ovarian tissue before and after in vitro culture. DAMV performed the histological examination of the ovarian sections. DAMV, YPS, JBB and DCCB performed the immunohistochemistry and analyzed the labeling of the proteins. BGA performed the statistical analysis. FWSC performed 7

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the reactive oxygen species analysis. JRF and MBZ were collaborators in the revision of the manuscript. APRR was the supervisor and the major contributor in writing the manuscript. All authors read and approved the final manuscript”.

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