Cryopreservation and in vitro maturation of germinal vesicle stage oocytes of animals for application in assisted reproductive technology

Abstract

Cryopreservation, in vitro maturation, fertilization and culture can be applied to various processes across a wide range of species, that is, for the breeding and reproduction of farm animals, preservation of genetic variants in laboratory animals, and the conservation of wild species. In particular, the storage of oocytes by cryopreservation and IVM following cryopreservation, might become effective alternative assisted reproduction treatments for infertile patients. For example, in a clinical context, these techniques might be important for patients who are at risk of losing their ovarian function because of extirpative therapy, chemotherapy or radiation. Thus, it is important for assisted reproductive technology to improve IVM and cryopreservation techniques. In the present review, we introduce our recent studies on vitrification and IVM of germinal vesicle stage oocytes in animals.

INTRODUCTION

CRYOPRESERVATION OF OOCYTES is an effective technology in assisted reproductive technology. The cryopreservation of female gametes is an important technology that is applied in both human and animal reproduction. It might be especially important for patients who are at risk of losing their ovarian function because of medical treatment. Women with cancer undergo potentially sterilizing chemotherapy or radiotherapy treatments. If ovarian tissue could be cryopreserved before such treatment, the patient's own oocytes could be conserved for future child bearing. Oocytes could be matured and fertilized in vitro before embryo transfer. It is possible that such preserved ovarian tissue might be autografted when cancer treatment is concluded. Once the problems of in vitro maturation (IVM) are solved, donated ovarian tissue might provide a rich source of donor oocytes for patients who experience premature ovarian failure or those with gonadal dysgenesis. Ultimately, IVM and the cryopreservation of oocytes might be useful for many infertile patients undergoing in vitro fertilization (IVF).

Furthermore, the complete maturation of oocytes is essential for the developmental competence of embryos. Immature oocyte collection and in vitro oocyte maturation have been successfully applied to laboratory animals, and attempts are now being made to use this treatment method for human infertility. The potential benefits of developing an effective IVM program as an alternative clinical strategy to conventional IVF are many; it would not only substantially reduce both the costs of drug treatment and waste of immature oocytes collected during standard IVF, but could also lessen the risks of hyperstimulation syndrome. Additionally, IVM might provide a valuable model for investigating the causes of meiotic aberrations and aneuploidies that are remarkably common in mature human oocytes. Finally, IVM might open the door to oocyte cryopreservation if the freeze storage of germinal vesicle (GV) stage oocytes avoids the problem of damaging the meiotic spindle of metaphase II (MII) stage oocytes. ¹ Therefore, IVM is an important technique for assisted reproductive technology and deserves rigorous experimental evaluation to determine whether it is practicable in reproductive medicine. In addition, it has been reported that GV transplantation might improve the quality of oocytes and contribute to elucidating the difference between nuclear and cytoplasmic maturation. ² , ³ , ⁴ For the GV transplantation, large numbers of recipient and/or donor GV oocytes are required. Therefore, the cryopreservation of GV oocytes might be an important technique in reproductive biology and the treatment of infertility.

In this review, we will introduce our recent study on the vitrification of GV stage oocytes and factors as indications of oocyte quality after IVM.

CRYOPRESERVATION OF MAMMALIAN OOCYTES

Cryopreservation of mammalian female gametes

THE STORAGE OF gametes by cryopreservetion might contribute to advances in infertility treatment and reproductive biology. Although oocytes remain one of the most difficult cell types to successfully cryopreserve because of their sensitivity to cooling and freezing, researchers have developed an efficient cryopreservation method for human and domestic animal oocytes. The first successful mammalian embryo cryopreservation was carried out using a slow‐cooling method where ice seeding was carried out in the mouse at a few degrees below the freezing point, followed by cooling the samples very slowly (0.3–0.4°C/min) to –80°C before storage in liquid nitrogen. ⁵ After this success, the slow‐cooling method was shown to be effective for embryos of several species, but has some disadvantages in that it requires as long as 2–3 h for cooling, and that special equipment and a large amount of liquid nitrogen are required to control the cooling rate. In 1985, Rall and Fahy devised an extremely rapid cooling method known as vitrification, by which 8‐cell mouse embryos were plunged directly into liquid nitrogen from a temperature above 0°C, thus taking only a few seconds to cool. ⁶ This method did not require expensive coolers or special skill and could be carried out very quickly. In other words, vitrification was simpler than the conventional method. Since then, vitrification has been widely used and is now regarded as an alternative to slow‐rate freezing.

In livestock, where large numbers of ovaries can be easily obtained from a slaughter house, many oocytes can be collected and incorporated into an in vitro embryo production (IVP) system when considered appropriate, thus diminishing seasonal variations or sanitary constraints. In the course of this system, the proper storage of oocytes is a prerequisite for use in the species whose gamete availability is restricted. For this purpose, because of the establishment of IVM of oocytes, many attempts have been carried out to cryopreserve matured oocytes in MII. Oocytes, however, are sensitive to low temperature and it is difficult to cryopreserve them compared with embryos. In fact, although successful procedures for cryopreservation of human MII oocytes have also been reported, ⁷ , ⁸ these results have been shown to be unsatisfactory and appear to be in need of an improvement to the methods. In MII oocytes, their microtubular spindles, to which the chromosomes are attached, are sensitive to temperature changes and chromatid disjunctions might occur during cooling, resulting in aneuploidy after fertilization. ⁹ , ¹⁰ , ¹¹ Because oocytes in the GV stage do not have any microtubular spindles, ¹² the cryopreservation of GV oocytes might be an alternative approach to the storage of female gametes.

For the successful freezing of cells, the most important issue is the total elimination of ice crystal injury inside the cytoplasm. When vitrification is applied to cryopreserved embryos, it is expected that ice crystal formation would be prevented by the use of high concentrations of cryoprotectants (CPA) that can draw water out of the cytoplasm through high rates of cooling and warming. From the time of the first trials until the present, many modifications in the process of vitrification have been tested, paying special attention to the fact that the solution of CPA might have toxic effects, resulting in irreversible damage to the cytoskeletal organization of the cells. ¹³ Thus, the main objective of the different approaches has been to minimize toxic, osmotic and other injuries to cells.

Container for vitrification of a large number of oocytes

The cooling rate is an important parameter for the success of vitrification, because cryopreservation strategies are based on CPA and on cooling or warming rates. The original vitrification method, which was developed to cryopreserve 8‐cell murine embryos, ⁶ used a standard straw for holding the embryos during cooling, storage and warming, although the use of the straw imposed limitations of the maximum cooling and warming rates to less than 2000°C/min. To increase the cooling and warming rates, different containers were applied to minimize the volume and to submerge the sample quickly in the liquid nitrogen (LN2). Recently, ultrarapid vitrification has been developed, which is characterized by more rapid cooling and warming rates than those in conventional vitrification. This ultrarapid method requires a small droplet of the vitrification solution for the handling of the oocytes, after which the oocytes are plunged directly into LN2 using cryodevices, such as an open pulled straw, ¹⁴ , ¹⁵ electron microscopy grid, ¹⁶ cryoloop, ¹⁷ nylon mesh ¹⁸ or plastic sheet, ¹⁹ which avoids ice crystal formation inside the oocyte. Thus, ultrarapid vitrification requires a small volume of CPA to achieve higher cooling and warming rates. Also, higher concentrations of CPA are needed to attain the glassy status, yet the procedure should not lead to chemical toxicity during equilibration. Recently, we have shown that a sheet of nylon mesh (pore size 60 µm) might be useful for vitrifing large quantities of bovine GV oocytes. Usually, bovine GV oocytes are collected as cumulus oocyte complexes (COC), so that their size is larger than the size of an oocyte only, and consequently open pulled straw or cryoloops are not applicable for their vitrification. Because EM grids can be used for larger numbers of embryos compared with other unsealed container systems, ¹⁶ we compared EM grids and nylon mesh. ¹⁸ Nylon mesh used as a physical support is a thin bar grid T400H mesh (pore size 55 µm). Bovine COC were exposed to a vitrification solution, EFS40, ²⁰ which consisted of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll‐70 and 0.3 mol/L sucrose in PB1, loaded either on a sheet of nylon mesh or on an EM grid, and then plunged directly into liquid N₂. The number of the COC loaded was 10–20 or 40–65 per nylon mesh, and 10–15 per grid. Over 90% of the oocytes vitrified on the nylon mesh were recovered, which was higher than the recovery of those on the EM grid (84.2%, P < 0.05). A higher recovery rate after vitrification on the nylon mesh was obtained for 40–65 COC (97.4%) than in the 10–20 COC (94.3%, P < 0.05). After the in vitro maturation of vitrified COC, the rate of germinal vesicle breakdown (GVBD) was higher in the group using the nylon mesh than in that using the EM grid (P < 0.05). The developmental rates beyond 2‐ and to 8‐cell stages were not significantly different between the oocytes vitrified on the nylon mesh and on the EM grid. Although only the maturation rate to MII in the 40–65 COC vitrified on the nylon mesh was lower than that in those vitrified on the EM grid (P < 0.05), neither cleavage nor the developmental rate were significantly different between them. Furthermore, we recently produced a viable calf after transfer of blastocysts that were derived from GV oocytes vitrified on the nylon mesh. ²¹ Considering that recent progress in assisted reproductive technology, cloning, stem cell biology and so on has been achieved, a large number of oocytes will be required. Therefore, when GV oocytes are obtained as COC, vitrification of GV oocytes using nylon mesh might be helpful in facilitating the application of these technologies.

Effect of stepwise exposure to CPA on subsequent development of oocytes

Vitrification is a physical process in which cryoprotectant solutions form a glassy solid, so the vitrification solution must be sufficiently concentrated to avoid crystallization when cooled, but must not produce chemical toxicity or osmotic injury during equilibration or dilution. ⁶ When this approach is applied to cryopreserve oocytes and embryos, their viability and developmental ability should be considered as the functional aim. In this regard, the stepwise addition of CPA might be considered to minimize damage that is a result of cryoprotactant toxicity. ¹³ In addition, during the removal of CPA, the water influx might cause extreme swelling and subsequent damage to the cellular membranes. For pre‐equilibrium before vitrification, mouse GV oocytes were treated as COC using four different methods of exposure to the vitrification solution in six experimental groups. ²² The COC were transferred to the vitrification solution, which was composed of 15% ethylene glycol, 15% dimethyl sulfoxide and 0.5 mol/L sucrose, either in a single step manner (non‐pre‐equilibrium) or in a stepwise manner (single‐, two‐ or 10‐step pre‐equilibrium). After an ultrarapid vitrification and storage in liquid nitrogen, the COC were warmed, washed by diluting the vitrification solution in five steps, and then subjected to in vitro maturation, fertilization and culture. Higher rates of survival and maturation to MII were obtained and followed by the efficient production of blastocysts when the oocytes were vitrified with an increased frequency of pre‐equilibration with the vitrification solution. These results suggest that the process of gradual equilibration with the vitrification solution seems to adjust the permeability of cellular membranes, so that the connection between the oocytes and cumulus cells in COC might be sufficiently maintained, and that rapid changes in osmotic pressure can be decreased or avoided.

Vitrification of germinal vesicle‐stage oocytes by stepwise equilibrium in one dish method

Germinal vesicle oocytes do not have microtubular spindles. Because of this, the cryopreservation of GV oocytes might be an alternative approach for the storage of female gametes. At present, successful cryopreservation of GV oocytes has been reported, ²³ , ²⁴ , ²⁵ but low rates of survival, fertilization and subsequent development have been observed. ²⁶ , ²⁷ , ²⁸ , ²⁹ Many studies on cryopreservation of immature (GV) and matured (MII) mouse oocytes have been reported using a slow‐cooling method, ⁹ , ²² , ²⁵ , ³⁰ but with limited success. These studies showed a lower embryo development rate in vitrified immature oocytes than in matured oocytes ²³ , ²⁶ and showed a loss of cumulus cells from vitrified immature oocytes, in which cumulus cells were no longer connected to the oocyte, ³⁰ , ³¹ suggesting that cumulus cells of COC might be disrupted physically because of ice formation in the slow‐cooling method.

The connection between cumulus cells and the oocyte is important for the completion of IVM in the case of COC. ³² Therefore, to maintain developmental potency after the cryopreservation of immature oocytes, the minimization of such disruption by ice formation must be ensured.

In this regard, vitrification seeks to circumvent the formation of intracellular ice that can potentially damage a cell, although toxic injury caused by higher concentrations of CPA can occur more frequently in vitrification than in the slow‐cooling method. In fact, cryopreservation of mouse immature oocytes by vitrification has been reported, ²⁸ , ²⁹ , ³³ but with unfortunately low resultant rates of fertilization and development. Although egg yolk, in the vitrification of immature oocytes, might provide beneficial effects on both oocyte and cumulus cell integrity, and also on survival and meiotic normality, ³⁴ its effects on subsequent fertilization and development have not been examined. Germinal vesicle oocytes can be cryopreserved by slow‐cooling or vitrification methods. Although slow‐cooling is the most widespread method for the majority of cells, cryopreservation by vitrification has shown greater results than that by slow‐cooling. ³⁵ , ³⁶ For this, we have attempted to pretreat GV oocytes in a series of high‐viscosity solutions before vitrification. In the previous report, we showed that the vitrification of mouse GV oocytes using a stepwise pre‐equilibrium method was advantageous for producing more blastocysts. ²² Furthermore, we showed that after the stepwise exposure to CPA, the degree of the entirety of cytoplasmic organelles in the bovine GV oocytes vitrified was similar to those in the control. ²¹ When examined by transmission electron microscopy, we found that fewer abnormalities were observed in the stepwise exposure; the ultrastructure was similar to the control (non‐vitrified oocytes), whereas in the single‐step exposure, most of the oocytes had cytoplasms with many vacuoles and membrane ruptured mitochondria. In the stepwise pre‐equilibrium applied, the oocytes were handled with a microcapillary pipette to be transferred and exposed to several solutions containing the CPA in droplets of the one dish or of several dishes. Thus, increasing the steps for pre‐equilibration required a concomitant increasing of the frequency in handling COC, suggesting that it might be a cause of the loss of cumulus cells from COC and/or oocytes during handling. Careful pipetting is important to allow the retention of the majority of cumulus cells. ³¹

In a recent study, we modified the pre‐equilibrium method by making it stepwise, showing that our serial stepwise method (stepwise equilibrium in one dish method [SWEID]) can enable us to increase the steps easily for pre‐equilibration to CPA without increasing the frequency of direct pipetting of COC, and at the same time with a decrease in the amount of frustrating labor. ³⁷ This method includes a substantial number of steps, the same as in the previous report, ¹⁸ but might resemble a single step because COC are handled just once during pre‐equilibrium. Therefore, if more steps must be added for pre‐equilibration, it might still be easy to carry out.

Our results are consistent with the report that the vitrification of bovine blastocysts after a 16‐step equilibration resulted in the minimization of ultrastructural damage to the plasma membrane. ³⁸ These results suggest that the process of gradual equilibrium conversion of CPA appears to adjust the permeability of the plasma membrane, which might contribute to maintain the connection between the oocyte and cumulus cells and/or might decrease rapid changes in osmotic pressure. In addition, the integrity of the normal characteristics of the plasma membrane, especially in the cryopreservation of GV oocytes, might be important for subsequent maturation and development abilities.

In conclusion, we achieved the production of offspring by the embryo transfer of two‐cell–stage embryos that were derived from vitrified GV oocytes. Ultrarapid vitrification accompanied by our serial stepwise equilibration, SWEID, in mouse GV oocytes showed improved results for the viability and production of blastocysts and offspring, suggesting that this method might be a useful strategy for the cryopreservation of immature oocytes. This model could be applied similarly to infertility treatment in humans.

IN VITRO MATURATION: INDICATORS OF CYTOPLASMIC MATURATION IN OOCYTES

Mitochondria activity

IN VITRO MATURATION is a very useful research tool and a commercially viable technique in domestic animal biotechnologies. Furthermore, IVM has the potential to become a viable alternative to ovarian stimulation, especially for the treatment of patients with fertility disorders who are at an increased risk of developing ovarian hyperstimulation syndrome when treated with exogenous hormones; for example, polycystic ovarian syndrome. In most species examined, however, oocytes matured in vitro are compromised in their development compared with oocytes matured in vivo. ³⁹ , ⁴⁰ , ⁴¹ As one of the causes of this, it is thought that cytoplasmic maturation might be insufficient under the present IVM conditions. Oocyte maturation has two aspects; nuclear maturation and cytoplasmic maturation. For IVM, both nuclear and cytoplasmic maturation of the oocyte are important to attain subsequent developmental competence. Early studies showed that media supporting high incidences of nuclear maturation were not necessarily able to support cytoplasmic maturation. ⁴² With recent improvements in methods for cytoplasmic maturation, as measured by the incidence and timing of male pronuclear development and the decreased frequency of polyspermy during IVF, IVM and IVF procedures are available that can consistently result in blastocyst formation in approximately 35% of immature porcine oocytes treated. ⁴³ However, full‐term development to produce piglets has been reported only within the past few years, suggesting that some problems remain to be overcome.

Reaching the full developmental potential requires synchronous nuclear maturation and cytoplasmic maturation. ⁴⁴ With regard to cytoplasmic maturation, many researchers have investigated the glutathione concentration, ⁴⁵ , ⁴⁶ cytosolic free Ca^{2+ 47} and cortical granules. ⁴⁸ However, there are few reports regarding the relationship between the dynamic state of mitochondria and IVM. Mitochondria have controlled energy production and metabolism that affect embryo development. ⁴⁹ In order to investigate the difference of in vivo and in vitro oocyte quality, therefore, we focused on the mitochondria. The shape and intercellular distribution of mitochondria are known to relate to the level of cell metabolism, proliferation and differentiation for the generation of the essential energy required in crucial periods of the cell cycle. ⁵⁰ Thouas et al. ⁵¹ reported that oocyte mitochondria can make a necessary physiological contribution to the cytoplasmic regulation of preimplantation embryo development. Thus, we observed the distribution of active mitochondria among the oocytes in vivo matured (ovulated) and in vitro matured (IVM) with two different culture media in mice. Then, we investigated whether there is the relationship between mitochondria distribution and the subsequent embryo development after IVF. Germinal vesicle oocytes were cultured in Waymouth's or HTF medium to mature to MII. The mitochondria distribution was analyzed with a mitochondria‐specific fluorescent probe, rhodamine 123, followed by confocal scanning laser microscopy. ⁵⁰ The mitochondria distribution was quite differently observed among the matured oocytes; in vivo matured oocytes had uniform localization with strong expression, whereas IVM MII oocytes in Waymouth's medium had a homogeneous distribution in the perinuclear area, resembling distributions of in vivo oocytes, but with weak expression. The IVM oocytes in HTF media showed spot‐like aggregation. Although the rates of MII oocytes in Waymouth's and HTF media were 72.9% and 88.1%, respectively, the corresponding cleaved rates after IVF were 82.2% and 16.1%. In other words, two kinds of culture media showed quite different results in the following development to blastocyst stage after IVF, although both media had an almost equal rate in in vitro maturation to metaphase II of mouse GV oocytes. By analyzing active mitochondria in mouse oocytes matured in vitro with two kinds of culture media, we showed that the localization of active mitochondria in oocytes at metaphase II is likely to be correlated with the subsequent developmental competence. Although the details are not known, IVM conditions might cause the incomplete movement of mitochondria to the inner cytoplasm, and thus affect the cytoplasmic maturation, which results in developmental incompetence. These results suggest that the status of mitochondria distribution might be an indication of the cytoplasmic maturation of GV oocytes. ⁵² Also, we examined bovine in vitro matured oocytes, showing that the pattern of active mitochondria was categorized by their distribution in the ooplasm and that oocytes having uniform distribution of active mitochondria were well developed (unpubl.). Further study will be needed to clarify the relationship between cytoplasmic maturation and mitochondria activity.

Maturation/M‐phase promoting factor activity

Mammalian follicular oocytes aspirated from follicles can be matured in vitro, and these oocytes are then arrested at MII, until such time as spermatozoa penetrate the oocytes. The nuclear status and morphology of the matured oocytes do not change during this meiotic arrest. However, cytoplasmic changes affecting oocyte quality (e.g. a decreased ability to be fertilized and to develop) occur when the arrest period is prolonged. ⁵³ , ⁵⁴ An increased tendency for spontaneous oocyte activation and subsequent fragmentation, an abnormal cleavage after activation characterized by unequal blastomeres, ⁵⁴ , ⁵⁵ has also been observed in porcine oocytes that were cultured for a long period. Furthermore, it has been reported that the time from meiotic progression of oocytes matured in vitro or in vivo influenced fertility and subsequent development. ⁵⁶ , ⁵⁷ , ⁵⁸ These findings suggest that the duration of IVM culture influences developmental ability in vitro after IVF. These quality changes occur in MII arrested oocytes during prolonged culture. Likewise, MPF activity also decreases during prolonged culture. ⁵⁹ MPF induces M‐phase in eukaryotic cells, including oocytes. ⁶⁰ MPF activity in porcine oocytes is assessed by the measurement of histone H1 kinase activity. It has been reported that porcine aged oocytes, showing enhanced activation ability and higher rates of fragmentation, have decreased MPF activities. ⁵⁵ MPF activation triggers a series of reactions ultimately leading to GVBD, chromosome condensation and spindle formation; events that are vital to supporting successful fertilization and early embryo development. High MPF activity can be detected in metaphase‐I and ‐II mammalian oocytes, ⁶¹ , ⁶² and its inactivation in matured oocytes is induced by fertilization ⁶¹ , ⁶³ or parthenogenetic activation. ⁵⁵ It was shown that the MPF activity of pig oocytes matured in vitro was influenced by the culture medium, ⁶⁴ and a correlation between the MPF activity in MII oocytes and their ability to develop pronuclei was reported by Naito et al. ⁶² Thus, it might be that the determination of MPF activity could be used as an indicator of the cytoplasmic maturation of pig oocytes and, therefore, of oocyte quality. One of the mechanisms that regulates MPF activity is a modification of the phosphorylation status of p34^cdc2 kinase by Myt1/Wee1 and cdc25. The addition of caffeine into the culture medium suppresses Myt1/Wee1 activity, resulting in the inhibition of the shift from active MPF to pre‐MPF, the inactive form, and an increase of MPF activity. ⁶⁵ It has been reported that caffeine supplementation inhibited the decrease of MPF activity in pig oocytes. ⁶⁵ , ⁶⁶ Caffeine treatment causes a significant decrease of aging phenomena in oocytes cultured for a long period; furthermore, this treatment is very simple to apply. In vitro matured oocytes are widely used in advanced reproductive technologies, such as in vitro fertilization and sperm injection. The control of oocyte aging might have many advantages for these procedures, because limitations on the manipulation time can be removed and oocyte quality could be refined. As a result, the establishment of methods for aging control might assist progress in these technologies.