Elsevier

Theriogenology

Volume 156, 15 October 2020, Pages 296-309
Theriogenology

Supportive techniques to investigate in vitro culture and cryopreservation efficiencies of equine ovarian tissue: A review

https://doi.org/10.1016/j.theriogenology.2020.06.043Get rights and content

Abstract

During the reproductive lifespan of a female, only a limited quantity of oocytes are naturally ovulated; therefore, the mammalian ovary possesses a substantial population of preantral follicles available to be handled and explored in vitro. Hence, the manipulation of preantral follicles enclosed in ovarian tissue aims to recover a considerable population of oocytes of high-value animals for potential application in profitable assisted reproductive technologies (ARTs). For this purpose, the technique of preantral follicle in vitro culture (IVC) has been the most common research tool, achieving extraordinary results with offspring production in the mouse model. Although promising outcomes have been generated in livestock animals after IVC of preantral follicles, the quantity and quality of embryo production with those oocytes are still poor. In recent years, the mare has become an additional model for IVC studies due to remarkable similarities with women and livestock animals regarding in vivo and in vitro ovarian folliculogenesis. For a successful IVC system, several factors should be carefully considered to provide an optimum culture environment able to support the viability and growth of preantral follicles enclosed in ovarian tissue. The cryopreservation of the ovarian tissue is another important in vitro manipulation technique that has been used to preserve the reproductive potential in humans and, in the future, may be used in highly valuable domestic animals or endangered species. Several improvements in cryopreservation protocols are necessary to support the utilization of ovarian tissue of different species in follow-up ARTs (e.g., ovarian fragment transplantation). This review aims to provide an update on the most current advances regarding supportive in vitro techniques used in equids to evaluate and manipulate preantral follicles and ovarian tissue, as well as methodological approaches used during IVC and cryopreservation techniques.

Introduction

Although the mammalian ovary inherits a massive potential source of fertilizable oocytes, only a limited number (0.01%) of follicles will ovulate. Therefore, the manipulation of oocytes enclosed in preantral follicles (MOEPF) aims to use those otherwise lost oocytes to maximize the reproductive potential of highly profitable animals and endangered species [1]. In horses, although the ovum pick-up (OPU) technique has been successful in enhancing the use of in vivo-grown oocytes to produce embryos through intracytoplasmic sperm injection (ICSI) technique [2], an enormous population of oocytes enclosed in preantral follicles remains to be explored. In this regard, studies on the preantral follicle population have been important to advance knowledge of the basic physiological mechanisms controlling early folliculogenesis. Under physiological conditions, the preantral follicle population experiences three potential fates: (i) remains in a dormant stage under the influence of inhibitory factors (e.g., Forkhead box L2 [3] and anti-Müllerian hormone (AMH) [4]; (ii) starts developing after follicle activation (transition from primordial to more advanced stages), or (iii) dies by the process of atresia [5]. Therefore, to mitigate follicular atresia, the improvement of strategies capable of promoting the survival and growth of preantral follicles until more advanced stages is an important goal during the in vitro manipulation of mammalian ovaries.

The IVC technique has been considered the ultimate strategy to provide information about preantral follicle dynamics [6]. The outstanding results achieved to this point in murine [7,8], with the production of offspring from preantral follicles cultured in vitro, unfortunately have not been repeatable yet in other species, including humans and livestock. In this regard, promising results with the production of embryos (e.g., morulas and blastocysts) from in vitro-grown preantral follicles have been reported in porcine [9], ovine [10], caprine [11,12], and buffalo [13]. However, since the quantity and quality of in vitro-produced embryos from oocytes enclosed in ovarian tissue have been limited, the MOEPF technique has the potential to improve the outstanding results previously reported in the murine model [7,8] to be translated to multiple species.

In the last two decades, the horse has emerged as an attractive and reliable animal model for in vivo and in vitro studies of folliculogenesis (for review, see [14]), also having the potential to be used for future improvement of MOEPF results in livestock. Improvements in in vitro manipulation of preantral follicles, in the near future, will potentially support a lucrative establishment of germ cell biobanks for high-value animals [15], the restoration of hormonal production in individuals [16], and the preservation of the gonadal reserve [17]. In this regard, efficient protocols for ovarian cryopreservation are also crucial in several species. Therefore, the optimization of techniques capable of cryopreserving equine ovarian tissue and preantral follicles appropriately is of great and rewarding interest in assisted reproductive technologies (ARTs).

The goal of this review, focusing on the equine model, is to provide an update on the most current advances regarding (1) the supportive techniques used to investigate preantral follicle quality (i.e., fluorescence, transmission electronic microscopy, hormonal analyses, reactive oxygen species (ROS) levels, metabolomics, gene expression, follicular density and spatial distribution, and stromal cell density), (2) IVC conditions for preantral follicles and ovarian tissue (i.e., base medium, hormones, and growth factors), and (3) cryopreservation techniques (i.e., cryoprotective agents – CPAs, and cryopreservation methods) of equine ovarian fragments.

Section snippets

Follicular viability evaluation using cell dyes and fluorescent probes

An important strategy for evaluating viability of preantral follicles consists of using cell dyes such as trypan blue [18] and neutral red [19], and viability fluorescent probes like calcein-AM, ethidium homodimer-1, and rhodamine 123 [20]. The trypan blue technique (Fig. 1A and B), an exclusion test used to determine the number of viable follicles after isolation [21], has been used efficiently in mares [18,22], cows [23], buffalo [24], sheep [25], goats [26], cats [27], and mice [28]. In

In vitro culture of equine preantral follicles

In general, the IVC of preantral follicles in any species has the primary goal of providing a feasible environment for the growth of immature follicles until more advanced stages of development, ultimately producing a large number of fully competent oocytes for follow-up manipulations. The applicability of the IVC process has been addressed in several review articles focusing on direct comprehension of folliculogenesis [132], effect of ovary transportation [133], IVC medium optimization [134],

Cryopreservation of equine ovarian tissue

Ovarian tissue cryopreservation has become a feasible technique to preserve and recover the fertility of humans who have been affected by diseases that may jeopardize fertility potential [187]. Although ovarian tissue cryopreservation in domestic animals and endangered species has been successfully achieved, few steps have been made toward the recovery of fertility [188]. Despite the undeniable advancements strengthening the protocols currently used for ovarian tissue cryopreservation [189],

Final considerations

The field of in vitro manipulation of equine ovarian tissue, especially IVC and cryopreservation, has evolved substantially in the last 8 years. The vast majority of the recent studies used preantral follicles enclosed in ovarian tissue and produced encouraging results concerning preantral follicle survival and primordial follicle activation after IVC. However, currently in horses, only low rates of follicle and oocyte growth, and no secondary follicles have been obtained post-IVC. To overcome

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