Supportive techniques to investigate in vitro culture and cryopreservation efficiencies of equine ovarian tissue: A review
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
References (197)
- et al.
Ovarian follicle development in vitro and oocyte competence: advances and challenges for farm animals
Domest Anim Endocrinol
(2016) - et al.
Overnight shipping of equine oocytes from remote locations to an ART laboratory enables access to the flexibility of ovum pick up-ICSI and embryo cryopreservation technologies
J Equine Vet Sci
(2016) - et al.
Mechanisms maintaining the dormancy and survival of mammalian primordial follicles
Trends Endocrinol Metabol
(2010) - et al.
Development of morulae from the oocytes of cultured sheep preantral follicles
Theriogenology
(2010) - et al.
In vitro production of a caprine embryo from a preantral follicle cultured in media supplemented with growth hormone
Theriogenology
(2011) - et al.
Equine preantral follicles obtained via the Biopsy Pick-Up method: histological evaluation and validation of a mechanical isolation technique
Theriogenology
(2013) - et al.
Quantification, morphology, and viability of equine preantral follicles obtained via the Biopsy Pick-Up method
Theriogenology
(2013) - et al.
Preservation of bovine preantral follicle viability and ultra-structure after cooling and freezing of ovarian tissue
Anim Reprod Sci
(2008) - et al.
Morphologic, viability and ultrastructural analysis of vitrified sheep preantral follicles enclosed in ovarian tissue
Small Rumin Res
(2012) - et al.
Effects of conditioned media from murine granulosa cell lines on the growth of isolated bovine preantral follicles
Theriogenology
(1997)
Isolation and characterization of primordial follicles from fresh and cryopreserved human ovarian tissue
Fertil Steril
Coculture of monkey ovarian tissue increases survival after vitrification and slow-rate freezing
Fertil Steril
Ultrastructure of follicles after vitrification of mouse ovarian tissue
Fertil Steril
Low temperature storage and grafting of human ovarian tissue
Mol Cell Endocrinol
Cryopreservation of caprine ovarian tissue using dimethylsulphoxide and propanediol
Anim Reprod Sci
Oocyte growth, capacitation and final maturation in cattle
Theriogenology
FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles
Theriogenology
In vitro culture and in vitro maturation of mouse preantral follicles with recombinant gonadotropins
Fertil Steril
Insulin improves in vitro survival of equine preantral follicles enclosed in ovarian tissue and reduces reactive oxygen species production after culture
Theriogenology
FSH supplementation to culture medium is beneficial for activation and survival of preantral follicles enclosed in equine ovarian tissue
Theriogenology
Role of EGF on in situ culture of equine preantral follicles and metabolomics profile
Res Vet Sci
Effects of FSH addition to an enriched medium containing insulin and EGF after long-term culture on functionality of equine ovarian biopsy tissue
Theriogenology
The challenges of using fluorescent probes to detect and quantify specific reactive oxygen species in living cells
Biochim Biophys Acta
Contribution of culture media to oxidative stress and its effect on human oocytes
Reprod Biomed Online
Effect of heat stress on the survival and development of in vitro cultured bovine preantral follicles and on in vitro maturation of cumulus-oocyte complex
Theriogenology
Anethole improves the in vitro development of isolated caprine secondary follicles
Theriogenology
Polymerase chain reaction
J Invest Dermatol
Detection of rare mRNAs via quantitative RT-PCR
Trends Genet
A novel growth differentiation factor-9 (GDF-9) related factor is co-expressed with GDF-9 in mouse oocytes during folliculogenesis
Mech Dev
Bone morphogenetic protein 15 expression in human ovaries from fetuses, girls, and women
Fertil Steril
Expression of growth-differentiating factor 9 and its type 1 receptor in human ovaries
Reprod Biomed Online
Glucocorticoid metabolism in equine follicles and oocytes
Domest Anim Endocrinol
Determination of follicle numbers in human ovarian biopsies—a method for estimation of outcome of ovarian cryopreservation?
Fertil Steril
The murine winged-helix transcription factor Foxl2 is required for granulosa cell differentiation and ovary maintenance
Development
Control of primordial follicle recruitment by anti-Mullerian hormone in the mouse ovary
Endocrinology
Cryopreservation and in vitro culture of caprine preantral follicles
Reprod Fertil Dev
A revised protocol for in vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence
Biol Reprod
Live offspring from cryopreserved embryos following in vitro growth, maturation and fertilization of oocytes derived from preantral follicles in mice
J Reprod Dev
Development of in vitro-matured oocytes from porcine preantral follicles following intracytoplasmic sperm injection
Biol Reprod
Dynamic medium produces caprine embryo from preantral follicles grown in vitro
Reprod Sci
Production of buffalo embryos using oocytes from in vitro grown preantral follicles
Zygote
Harvesting, processing, and evaluation of in vitro-manipulated equine preantral follicles: A review
Theriogenology
Factors that may influence the willingness of cancer patients to consent for biobanking
Biopreserv Biobank
Human ovarian tissue vitrification versus conventional freezing: morphological, endocrinological, and molecular biological evaluation
Reproduction
Isoform 165 of vascular endothelial growth factor in collagen matrix improves ovine cryopreserved ovarian tissue revascularisation after xenotransplantation in mice
Reprod Biol Endocrinol
Monitoring preantral follicle survival and growth in bovine ovarian biopsies by repeated use of neutral red and cultured in vitro under low and high oxygen tension
Theriogenology
Ultrastructure and viability of isolated bovine preantral follicles
Hum Reprod Update
Trypan blue exclusion test of cell viability
Curr Protoc Immunol
In vitro development of buffalo preantral follicles in co-culture with cumulus or granulosa cells
Vet Arh
Long-term in vitro culture of ovarian cortical tissue in goats: effects of FSH and IGF-I on preantral follicular development and FSH and IGF-I receptor mRNA expression
Cell Tissue Res
Cited by (14)
Towards clinical translation of the cell sheet engineering: Technological aspects
2023, Smart Materials in MedicineCitation Excerpt :The morphological structure/viability can remain at high levels upon thawing; moreover, all the studies cited in this review report the preservation of CS properties comparable to the initial ones. However, DNA fragmentation can be different with different methods and CPAs [154], which means that assessing CS viability, morphology, and essential marker expression upon thawing is probably not enough for clinical translation. More rigorous and standard characterization protocols should be developed.
Ovarian tissue features assessed in bovine fetuses after vitrification and xenotransplantation procedures
2021, Reproductive BiologyCitation Excerpt :Despite that ovarian tissue cryopreservation followed by autotransplantation has been successfully applied to women [3] and mice [4], there has been a correlated decrease in follicular reserve and low response to ovarian stimulation [5]. Cryopreservation and/or transplantation can compromise several ovarian tissue features, such as follicular viability [6], ovarian cells (stromal cell [7]; granulosa cell, [8]), vascularization [9], and DNA integrity [10], that has important rules on follicular growth, steroids production, and meiotic resumption [11]. Therefore, xenotransplantation can be accepted as an experimental approach to evaluate the functionality of cryopreserved ovarian tissue [12].
Heterotopic autotransplantation of equine ovarian tissue using intramuscular versus subvulvar grafting sites: Preliminary results
2021, TheriogenologyCitation Excerpt :Additionally, in young mares, at 3 and 7 days post-OTT, the SV transplantation site had a higher stromal cell density compared with the IM transplantation site. The importance of factors (e.g., estradiol, epidermal growth factor) produced via the interactions between stromal cells and follicles for the regulation of follicular growth has been well described (reviewed in [38,39]). Therefore, the utilization of a more vascularized transplantation site seems to be critical in order to maintain a greater density of stromal cells and may contribute to a favorable follicular environment.
Harvesting, processing, and evaluation of in vitro-manipulated equine preantral follicles: A review
2020, TheriogenologyCitation Excerpt :In this context, recent studies determined the spatial distribution of equine preantral follicles in different regions and portions of the whole ovary [61,92]. The results from the two abovementioned studies have been discussed in detail recently [for review, see 98]. In summary, the use of the whole equine ovary has recently helped to advance knowledge to support the concept that the ovary is a dynamic organ that passes through large plasticity of the internal structures (i.e., follicles and corpora lutea) within the stromal tissue.