Elsevier

Theriogenology

Volume 114, 1 July 2018, Pages 244-251
Theriogenology

Positive effect of resveratrol against preantral follicles degeneration after ovarian tissue vitrification

https://doi.org/10.1016/j.theriogenology.2018.04.004Get rights and content

Highlights

  • The addition of resveratrol to the vitrification/thawing media improved the cryotolerance of preantral follicles.

  • After vitrification, a negative association between stage of follicular development and viability was observed.

  • The resveratrol had a positive effect against degeneration of preantral follicles enclosed in ovarian fragments.

Abstract

This study aimed to evaluate whether the addition of resveratrol to vitrification/thawing medium improves the cryotolerance of preantral follicles enclosed in bovine ovarian fragments. Ovarian fragments were obtained from bovine fetuses and distributed to the following groups: fresh ovarian fragments (control), vitrified (VIT), and vitrified with resveratrol (VIT + RESV). Overall, the mean percentage of normal follicles was greater (P < 0.05) in the VIT + RESV compared to the VIT group. Moreover, the probability of finding normal follicles was 2.5 greater (P < 0.05) in the VIT + RESV group. In class comparison, the primordial and transitional follicles have ∼3.0 times (P < 0.05) greater odds of being normal after vitrification compared to the secondary follicles. Additionally, a negative association (P < 0.05) was observed between the proportion of viable follicles and the stage of follicular development. ROS levels were similar (P > 0.05) between the VIT and VIT + RESV groups, and both were lower (P < 0.05) than the control group. The tissue viability in the VIT + RESV group was similar (P > 0.05) to the control group. In summary, the resveratrol provided greater ovarian tissue viability and has a positive effect against degeneration of preantral follicles enclosed in ovarian fragments.

Introduction

Cryopreservation of ovarian tissue followed by transplantation procedure has been used as a strategy to preserve the fertility potential of human and animals [1,2]. However, the cell functionality of remaining structures in cryopreserved ovarian tissue is essential for a potential clinical benefit [[3], [4], [5]].

The vitrification technique has become routinely applied for gamete preservation in different species (murine [6]; ovine [7]; caprine [8]; bovine [9]; swine [10]; primate [11]; and human [12]). However, during the vitrification procedure reactive oxygen species (ROS) may be generated by different mechanisms, such as increased oxidative metabolism, osmotic stress, or even by changes in cell defense mechanisms [13,14]. The ROS levels seem to increase, especially after cell warming, once oxygen is reintroduced in cryopreserved cells and consequently cause oxidation and reduction reactions that lead to its production [15].

Studies have been carried out in order to minimize the cell damage induced by cryopreservation such as apoptotic activation, accumulate of reactive oxygen species (ROS), efflux of glutathione, and progressive DNA and cell membrane damage [16,17]. In this way the resveratrol, a polyphenol synthesized by many plants, have several biological activities that can be applied in therapeutic purposes such as anti-carcinogenic, antioxidant, anti-inflammatory, protection of the cardiovascular system, anti-obesity, and reproductive disturbances (aging-related infertility and polycystic ovary syndrome) [[18], [19], [20], [21], [22]]. As an antioxidant, resveratrol protects the mitochondrial function by activation of sirtuin-1 gene (SIRT1), found in granulosa cells, cumulus cells, oocytes, and blastocysts [16,23]. Moreover, experimental studies showed that the resveratrol could protect against the reduction of fertility caused by oxidative stress [19] and ageing related changes [24].

Several species have been used animal models for studies with ovarian tissue (mare [25]; rat [19]; sheep [26]; and monkey [27]). Thus, these animals represent a comparative experimental model for genetic improvement within its specie and provide, at the same time, important information that may be applied to human reproductive clinical practice [28,29]. Ovaries of bovine fetuses over 180 days of gestation have preantral follicles in all developmental stages, enabling the assessment of vitrification effects on ovarian tissue. Therefore, based on biological activities of resveratrol, the present study evaluate whether the addition of resveratrol to the vitrification/thawing media improved the cryotolerance of preantral follicles enclosed in bovine ovarian fragments. The following end points were evaluated: ovarian tissue viability, ROS production, follicle and oocyte diameters, and preantral follicle morphology and class distribution.

Section snippets

Ovaries

Ovaries (n = 18) of bovine fetuses (180 days of gestation) were collected in a local slaughterhouse. The age of the fetuses was determined by the length (cm) from the atlanto-occipital joint to the origin of the tail [30]. After collection, ovaries were washed in alcohol 70% for 10 s, washed twice in PBS, and then transported to the laboratory in isothermal containers at 4 °C [31].

Experimental design

In the laboratory, the ovarian cortex was cut in 3 × 3 × 1 mm fragments and randomly distributed in the following

Results

A total of 53,547 follicles distributed in 880 histological sections were evaluated. Overall, the mean percentage of normal follicles was different among treatments (P < 0.05), and it was greater (P < 0.05) in the VIT + RESV compared to the VIT group (Table 1). Moreover, the proportion of normal follicles of VIT + RESV group was greater (P < 0.05) in primordial, transitional, and primary follicles compared with VIT group. Within the group vitrified with resveratrol, primordial follicles showed

Discussion

To our knowledge, this is the first study that evaluated the effects of ovarian fragments vitrification with resveratrol on tissue viability, ROS production, follicle and oocyte diameters, preantral follicle morphology, and classification.

The present study has shown that the overall percentage of morphologically normal follicles was 1.7-fold higher in the group of fragments vitrified with resveratrol than the group vitrified without the use of antioxidant. Previous studies reported the benefits

Acknowledgment

Research was supported by the Foundation for Research Support of the State of Minas Gerais (FAPEMIG, grant #APQ-01905-14), Brazil.

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