Retention of Structure and Function of the Cat Germinal Vesicle after Air-Drying and Storage at Suprazero Temperature

ABSTRACT

The study explored a novel approach for preserving the maternal genome without the entire oocyte by air-drying the cat germinal vesicle (GV) in the presence of the disaccharide trehalose. Specifically, we examined GV structure and function after desiccation, storage at 4°C (up to 32 wk), and rehydration including the ability to resume meiosis after injection into a fresh, conspecific cytoplast. In experiment 1, DNA integrity was similar to fresh controls after 1 and 4 wk storage in the presence of trehalose, but was more fragmented at later time points (especially after 32 wk). Nuclear envelope integrity was sustained in >90% of oocytes stored for 0, 4, or 16 wk regardless of protective treatment. In experiment 2, compacted, air-dried GVs were stored for 2 or 4 wk, rehydrated, and injected into fresh cytoplasts. After culture for 24 h in vitro, up to 73% of oocytes reconstructed with desiccated GVs preserved in trehalose resumed meiosis compared to 30% of those dried in the absence of the disaccharide. At each storage time point, trehalose presence during air-drying was advantageous for resumption of meiosis, with >20% of oocytes completing nuclear maturation to metaphase II. This demonstrates a potential for preserving the female genome using the GV alone and for multiple weeks after desiccation. Trehalose enhanced the process by retaining the ability of a dried and rehydrated GV to resume communication with the surrounding cytoplasm of the recipient oocyte to permit reaching metaphase II and likely sustain subsequent embryo development.

Trehalose protects germinal vesicle DNA integrity and its ability to resume meiosis during air-drying and storage at cold temperature.

INTRODUCTION

It is known that the functionality of the germinal vesicle (GV) is regulated and enhanced by the quality and quantity of cytoplasm in the growing, immature cat oocyte [1]. The GV of these oocytes also appears biologically competent at the early antral follicular stage [1]. Particularly interesting is that a GV can be rescued from an incompetent or subpar oocyte to produce a good-quality, fertilizable counterpart after transfer to a conspecific cytoplast. When removed from the oocyte by enucleation, the cat GV can also withstand disruptions from nonphysiological conditions, including artificial compaction or vitrification [1]. Therefore, the cat model has been insightful for learning more about oocyte structure and function during folliculogenesis, including the role and resilience of the GV as an essential organelle interacting with the surrounding cytoplasm.

We have also considered the potential of the GV as an attractive target for fertility preservation [2, 3]. The question is: would it be possible to preserve the maternal genome by storing the GV alone, later transferring this organelle to a fresh cytoplast? If this approach is biologically possible, then it could also permit circumventing the well-established cell injuries and high costs commonly associated with freeze-storing biomaterials in liquid nitrogen [4, 5]. Thus, dehydration by air, evaporative, and vacuum drying is an appealing option for preserving the genome because desiccation is similar to natural approaches used by certain small organisms to suspend their life cycle. For instance, tardigrades are protostomal animals well known for their capability of surviving extreme conditions by undergoing anhydrobiosis at ambient temperature for extended periods [6]. This phenomenon is possible because of an innate ability to accumulate natural sugars (including the disaccharide trehalose) intracellularly to preserve membrane lipid bilayers and proteins [6, 7]. Trehalose has also been demonstrated to protect against oxidative stress to allow organisms to survive extremely hot or cold temperatures [8].

The idea of preserving gametes by desiccation is not new. Studies have shown the ability to freeze-dry mouse [9–12] and rabbit [13] spermatozoa. Although this process renders the spermatozoon immotile, the highly compacted DNA remains intact and functional. For example, mouse spermatozoa loaded with trehalose, then evaporatively dried and injected into oocytes after rehydration, can produce blastocysts and live-born offspring after embryo transfer [14, 15]. There has been a similar study in the cat demonstrating that sperm DNA can survive desiccation and result in early embryo formation postinjection into an oocyte [16]. Additionally, nuclei from somatic cells have been freeze-dried, stored at 4°C, then rehydrated and transferred into enucleated oocytes to consistently produce embryos in the mouse [17], sheep [18], and pig [19].

Collectively, these successes are incentive for examining the impact of desiccation on the GV. It is logical that the latter could be a highly vulnerable target. This organelle is relatively large in the cat (∼40 μm diameter) and contains decondensed and fragile DNA [1, 2]. Nonetheless, we have determined that this DNA can be at least partially safeguarded through reversible compaction of the GV using resveratrol exposure [1, 20]. Additional protection might be afforded by process supplementation with trehalose. Our assumption here is based on the investigation by Eroglu et al. [21], who have demonstrated that mouse oocytes injected with this disaccharide survive cryopreservation and retain the ability to fertilize and develop into blastocysts and healthy young.

Thus, our hypothesis here was that the cat GV retains significant structure and function after desiccation and cool (4°C) storage, especially when exposed to trehalose. Our objectives were to examine the potential of 1) preventing or mitigating DNA/chromatin damages, 2) ensuring the presence of the nuclear envelope, and 3) retaining an ability to resume meiosis and reach the metaphase II. Irreparable DNA strand breaks are known to commonly occur during exposures of oocytes to freezing, irradiation, and oxidation and can be lethal to gamete viability [22]. In addition, a stable nuclear envelope is essential for ensuring subsequent nucleo-cytoplasmic exchanges [23]. Lastly, maintaining the capacity to achieve metaphase II during maturation is the hallmark to subsequent ability of the oocyte to fertilize. Nuclear maturation is best evaluated by transferring the manipulated GV to a recipient, conspecific cytoplasm as has been demonstrated in the rabbit [24], mouse [25], and cow [26–28]. Successful GV transfer has also been demonstrated in the cat model [1], including determining that injection of a compacted GV into a cytoplast can circumvent the heteroplasmy encountered after classical karyoplast-oocyte fusions [3].

MATERIALS AND METHODS

Collection of Oocytes

Ovaries were collected from local veterinary clinics that were routinely performing ovario-hysterectomies on adult domestic cats. Freshly excised ovaries were placed immediately in phosphate-buffered saline (PBS; at 4°C) supplemented with 100 IU/ml penicillin and 100 μg/ml streptomycin (Sigma Chemical Co., St. Louis, MO) and transported within 6 h to the laboratory. Oocytes were recovered immediately by repeatedly slicing the ovaries while immersed in Hepes-buffered minimum essential medium (HMEM; Gibco Laboratories, Grand Island, NY) supplemented with 1.0 mM pyruvate, 1.0 mM glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 4 mg/ml bovine serum albumin (Sigma). Grade 1 and 2 oocytes, categorized according to the specific criteria described by Wood and Wildt [29] were selected and denuded of cumulus cells by exposure to 0.2% hyaluronidase for ∼5 min (38°C) followed by gentle pipetting and rinsing in HMEM.

Air-Drying and Rehydration

Denuded oocytes were exposed to 10 μg/ml hemolysin for 15 min (38°C) to permeabilize the cytoplasmic membrane and then rinsed in HMEM. Batches of 10 oocytes each were air-dried on a glass slide or were exposed to 1.5 M trehalose (Sigma) in deionized water for 10 min (38°C) prior to deposition on a different glass slide. Excess medium surrounding the oocytes was aspirated, and dehydration allowed to commence passively via air-drying at ambient (20–23°C) temperature and 40%–60% relative humidity. In the absence of trehalose, each oocyte acquired a dried, almost crystalline appearance with a white “halo” effect around the surface edge of the cell within 6 min of deposit (monitored by stereomicroscopy; Fig. 1A). By contrast, the presence of trehalose gave the oocyte a smoother, less encrusted appearance (Fig. 1B), with the batch deposit site having a glassy-like appearance. All slides were stored in airtight containers at 4°C in a laboratory refrigerator for multiple days (see Experimental Design). Rehydration simply involved pipetting 100 μl of HMEM (at ambient temperature) onto the oocyte deposition site.

FIG. 1.

Micrograph of germinal vesicle oocytes (white arrow) air-dried on a glass slide after permeabilization with hemolysin and exposure to 0.0 M (A) or 1.5 M (B) trehalose. Bar = 100 μm.

Assessment of DNA Integrity

The comet assay (Trevigen, Gaithersburg, MD) was used to assess incidence of DNA fragmentation within each GV. The manufacturer's recommendations for this assay were optimized for our experimental conditions. In brief, this involved exposing each oocyte (with a minimal amount of associated medium) to 75 μl of melted agarose on comet assay slides. After being held in the dark for 30 min at 4°C, each slide was exposed to the kit lysis solution for 1 h followed by the kit alkaline solution for 30 min. The process continued with two 5-min rinses with Tris-borate-EDTA solution and then electrophoresis for 10 min at 22 V and <500 A in Tris-borate-EDTA solution. Slides were rinsed in MilliQ water twice for 5 min, placed in ethanol for 5 min, and allowed to dry for 30 min. Then 50 μl of SYBR Green I staining solution was added to the slide, and each oocyte observed using an epifluorescence microscope (Olympus BX 41; Olympus Corporation, Melville, NY). The length of each oocyte's comet tail was measured using SPOT Advanced Software (Diagnostic Instrument Inc., Sterling Heights, MI; Fig. 2, A and B). To allow distinguishing normal versus abnormal comet tail length, we used measures from the fresh control group to calculate a confidence interval (CI). Any tail length less than or equal to the upper CI limit was considered normal; any length greater than the upper CI limit was classified as abnormal.

FIG. 2.

Air-dried oocytes' GVs assessed for DNA integrity (using comet assay [A, B] and TUNEL assay [C, D]), and presence/absence of a nuclear envelope (E, F). A) Intact GV with short comet tail. B) Damaged GV with long comet tail. C) Intact GV without TUNEL labeling. D) Damaged GV with positive TUNEL signal. E) Undamaged GV with intact nuclear envelope. F) Damaged GV with loss of nuclear envelope. Bar = 50 μm.

The TUNEL assay (in situ cell death detection kit, fluorescein; Roche Applied Science, Indianapolis, IN) was used to detect DNA strand breaks in GVs. Manufacturer's assay recommendations were slightly modified as follows. Each oocyte was fixed in 4% paraformaldehyde for 1 h, rinsed three times in PBS containing 1 mg/ml polyvinylpyrrolidone (PVP; Sigma), and then placed in 0.5% Triton X-100 solution for 30 min (both steps at room temperature). After three rinses in PBS-PVP, each oocyte was exposed to the TUNEL solution for 1 h at 38°C. Oocytes then were rinsed three times in PBS-PVP before the chromatin was stained with Hoechst 33342 (10 μg/ml; Sigma) and propidium iodide (400 μg/ml; Sigma). Each trial included a positive control (exposed to DNAse for 1 h prior to TUNEL staining) and a negative control (incubation in staining rather than TUNEL solution). All oocytes were mounted on slides with Vectashield (Vector Labs Inc., Burlingame, CA) and then examined using epifluorescence microscopy. Observation of green fluorescence in a GV was indicative of DNA damage (Fig. 2, C and D).

Assessment of Nuclear Envelope Presence

Procedures were performed at 38°C. Batches of oocytes were fixed in 4% paraformaldehyde for 30 min and then placed in a blocking solution (20% fetal calf serum, 0.5% Triton X-100 in PBS) for 30 min. Oocytes were incubated in anti-lamin A/C antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) at 4°C overnight in a working solution (2% fetal calf serum, 0.5% Triton X-100 in PBS). Each trial included a negative control (primary antibody was omitted). After being rinsed for 45 min in working solution, oocytes were incubated in anti-mouse IgG labeled with fluorescein isothiocyanate (Sigma) for 1 h. This was followed by a 30-min wash in working solution, and then the chromatin was counterstained with Hoechst 33342 (10 μg/ml) and propidium iodide (400 μg/ml) for 5 min. All oocytes were mounted on slides with Vectashield and then evaluated for presence/absence of the nuclear envelope using epifluorescence microscopy (Fig. 2, E and F).

Assessment of GV Meiotic Competence

The GV of each denuded oocyte was compacted by exposure to 1 mM resveratrol for 30 min [20]. All oocytes then were permeabilized and incubated in 0 or 1.5 M trehalose before air-drying (as described above). After storage for 2 or 4 wk (experimental design below), each GV was rehydrated and then injected using a 15-μm-diameter Piezo-actuated pipette (Origio-Humagen, Charlottesville, VA) into a conspecific, fresh cytoplast. The latter was prepared by earlier described cumulus removal and GV enucleation [1]. The control group included fresh, enucleated oocytes (collected simultaneously), each of which was injected with a fresh, compacted GV that was unexposed to trehalose or storage. To promote development in vitro [30], all reconstructed oocytes were cultured for 24 h with intact cumulus-oocyte complexes (ratio 1:1) in minimal essential medium (Sigma) supplemented with 4 mg/ml bovine serum albumin, 1 mM pyruvate, 1 mM glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, 1 μg/ml follicle-stimulating hormone, 1 μg/ml luteinizing hormone, and 1 μg/ml estradiol. Nonmicromanipulated cumulus-oocyte complexes served as controls and were denuded after culture. All oocyte groups were fixed with 95% ethanol on glass slides and stained with Hoechst and propidium iodide (as above) to assess GV meiotic resumption (Fig. 3, C–E).

FIG. 3.

Nuclear status of oocytes after reconstruction with an air-dried GV and in vitro maturation (chromatin stained with propidium iodide). A) Compacted GV. B) Normal, re-expanded GV. C) GV breakdown. D) Metaphase I. E) Metaphase II. Bar = 50 μm.

Experimental Design

Two experiments were conducted. The first examined the influence of trehalose on GV structure during air-drying and storage. In experiment 1a, batches of air-dried oocytes (with or without trehalose) were rehydrated immediately (time 0) or after storage at 4°C for 1, 2, 4, or 8 wk. On a given day, oocytes from the same batch were equally allocated to the different treatment groups. The controls were fresh oocytes recovered similarly from ovaries and directly placed in HMEM without any drying. Fresh controls and rehydrated oocytes were evaluated in parallel using the comet assay, TUNEL assay, or nuclear envelope presence at each time point (total of 1743 oocytes in five replicates). Experiment 1b explored the same metrics following a storage time extended to 16 or 32 wk (total of 619 oocytes in five replicates). For each condition of experiment 1, chi-square testing was used to compare the pooled percentages of oocytes among all groups. Based on experiment 1 results, experiment 2 focused on examining GV functionality and specifically ability to reach the metaphase II stage after 2 versus 4 wk of storage. In this case, all GVs were rehydrated and then each injected into a fresh cytoplast, cultured, and evaluated for stage of nuclear maturation (total of 500 oocytes in three replicates). Pooled percentages of nuclear stages were compared using chi-square testing.

RESULTS

Experiment 1: Ability of the GV to Withstand Air-Drying and Multi-Week Storage at 4°C with or Without Trehalose Exposure

Forty-two percent to 67% of GVs retained normal DNA integrity according to the comet assay compared to 18%–83% from TUNEL testing; only 5%–13% of all desiccated GVs were missing a nuclear envelope (Table 1; Fig. 2). Comparative values for these same metrics in control oocytes were 73%, 88%, and 0%, respectively. In some cases, there was a trehalose supplementation (P < 0.05), but no storage interval (P > 0.05) effect on DNA integrity (Table 1).

TABLE 1.

Proportion (%) of GVs (n) with intact DNA structure and a nuclear envelope after air-drying in the absence/presence of trehalose and after 0, 1, 2, 4, or 8 wk of storage (4°C).

^a–d Within each column, values with different superscript letters differ (P < 0.05). N/A, not applicable.

With the exception of the 8-wk storage time point in experiment 1a, presence of trehalose during drying resulted in a similar (P > 0.05) or higher (P < 0.05) incidence of intact DNA (based on both the comet and TUNEL assays) compared to desiccation in the absence of the disaccharide (Table 1). Both assays also revealed that the proportion of air-dried GVs supplemented with trehalose with intact DNA structure after 1 and 4 wk of storage was similar (P > 0.05) to fresh controls. The two DNA integrity testing methods appeared to differ in sensitivity at any given time point. For example, the TUNEL evaluation indicated a twofold (or higher; P < 0.05) advantage for supplemental trehalose at times 0, 1, and 4 wk, whereas comet testing revealed no significant (P > 0.05) benefit from the disaccharide at 0, 2, and 4 wk (Table 1). At time 2 wk, both assays indicated a lower incidence (P < 0.05) of DNA integrity with or without trehalose compared to fresh controls. From 0 through 8 wk, despite the variations between time points, there was no increase (P > 0.05) in DNA damage over storage time with or without trehalose (Table 1).

The integrity of the nuclear envelope generally was resistant to both desiccation and trehalose supplementation (Table 1). In the presence of the disaccharide, there were more missing (P < 0.05) envelopes at 0 and 4 wk compared to other time points, but only ∼10% less compared to controls. Full (100%) nuclear envelope integrity was measured in 7 of the 10 treatment groups where GVs were stored with or without trehalose for 1–8 wk. Incidence of nuclear envelope presence appeared unrelated to variations in DNA integrity (Table 1).

In the more protracted storage protocol associated with experiment 1b, the comet assay indicated that DNA structure was maintained (P > 0.05) at both the 16 and 32 wk time points (with or without trehalose) compared to controls (Table 2). By contrast, the TUNEL assay revealed extensive DNA damage (P < 0.05) in >85% of GVs at both storage intervals and no protective effect of the disaccharide. Again, most desiccated/rehydrated GVs retained an intact nuclear envelope (Table 2).

TABLE 2.

Proportion (%) of GVs (n) with intact DNA structure and a nuclear envelope after air-drying in the absence/presence of trehalose and after 16 or 32 wk of storage (4°C).

^a,bWithin each column, values with different superscript letters differ (P < 0.05). N/A, not applicable.

Experiment 2: Influence of Air-Drying and Storage with or Without Trehalose Exposure on GV Capacity to Resume Meiosis after Oocyte Reconstruction

For the two control groups, 100% of nonmanipulated whole oocytes and 99% of fresh GVs (introduced into conspecific cytoplasts) resumed meiosis, of which 85% and 42%, respectively, achieved metaphase II (Figs. 3E and 4). Therefore, although there was no influence of oocyte reconstruction on the ability to resume nuclear maturation (P > 0.05), transferring a fresh GV to an enucleated counterpart reduced (P < 0.05) the overall ability to achieve metaphase II by ∼50% (Fig. 4).

FIG. 4.

Proportions of oocytes that contained a compacted GV (gray bars) or resumed meiosis (white bars) after reconstruction (with fresh or dried GVs) and in vitro maturation for 24 h. The proportion of oocytes at the metaphase II stage (black bars) is relative to the number of oocytes that resumed meiosis. Columns with different superscript letters (a–c, a′–c′, a″–d″) are different (P < 0.05) across time points. GVBD, GV breakdown; M I, metaphase I; M II, metaphase II.

Desiccation, storage, and transfer into a fresh cytoplast further reduced meiotic ability compared to control values (Fig. 4). Nonetheless, the average proportions of all oocytes reconstructed with non-trehalose-dried GVs that resumed meiosis and then reached full maturation were 37% and 9%, respectively, after 2 wk and 26% and 2% after 4 wk storage (Fig. 4). Presence of trehalose was beneficial at both storage time points, reducing (P < 0.05) the proportions of GVs trapped in compaction and increasing (P < 0.05) the incidence of meiotic resumption (to as high as 73% at 2 wk and 70% at 4 wk) and capacity to achieve the metaphase II stage (to as high as 23% at 2 wk and 21% at 4 wk) (Fig. 4). Within storage periods, supplementation with the disaccharide enhanced (P < 0.05) the ability of reconstructed oocytes to reach metaphase II at 4 wk (Fig. 4). However, there was no difference (P > 0.05) at 2 wk in proportion of oocytes reaching metaphase II with or without trehalose. Overall, there were no differences (P > 0.05) in residual compaction, meiotic resumption, or metaphase II values for dried GVs exposed to trehalose and stored for 2 versus 4 wk.

DISCUSSION

This is the first evidence that the mammalian GV (the storehouse for the maternal genome) can retain critical structural and functional components after being removed from the host oocyte, desiccated, stored at a cool temperature, and then rehydrated weeks later. There were some induced injuries from these manipulations, including modest-to-severe DNA fragmentation, but a negligible impact on nuclear envelope integrity. Under the most favorable conditions, ∼75% of air-dried and stored GVs that were rehydrated and placed into enucleated cytoplasts resumed meiosis, with >20% completing nuclear maturation after in vitro culture. These capacities, including partial mitigation of DNA disruptions, generally were enhanced by the presence of the natural sugar trehalose.

We consider this the first step in assessing the feasibility of preserving the maternal genome by storing only the GV rather than the whole oocyte in a dehydrated state. As such, it was essential initially to ensure that this organelle could retain both fundamental structure and function after these significant manipulations. For these reasons, we focused considerable attention on characterizing and comparing DNA integrity using two different methods. The comet assay usually detects single-stranded and double-stranded DNA breaks and relies on observing a “comet tail” indicative of unwinding of the macromolecule; by contrast, the TUNEL assay detects DNA strand fractures in situ by labeling the free 3′OH end [22]. Until now, there have been no studies of DNA integrity in freshly collected cat GV-stage oocytes, and our finding of ∼80% intact status was similar to that recently reported for fresh dog GV oocytes [31]. Although both assays detected some damage from GV manipulations, the disruptions were more modest as revealed by comet compared to TUNEL testing. It is noteworthy that some of the minor DNA damages detected in human oocytes by the latter assay can naturally repair [32].

Because of 1) the significant differences between the fresh control GVs and those assessed at 0 wk, and 2) the lack of differences from 0 through 8 wk of storage (experiment 1a), clearly a significant proportion of DNA breaks occurred during the air-drying/dehydration process. Our laboratory currently is optimizing the dehydration techniques using microwave-assisted desiccation [33, 34], which considerably reduces the DNA damage. However, experiment 1b revealed major losses in DNA integrity after 16 and 32 wk storage, but largely based only on the TUNEL assessment. Unlike the nil (or negligible) changes that whole gametes undergo at constant, low-temperature storage in liquid nitrogen [4], desiccated and cooled GVs probably were experiencing some dynamic changes during storage. For example, although we discovered that trehalose supplementation generally was beneficial to sustaining DNA integrity at most time points, it appeared detrimental at 8, 16, and 32 wk of storage (from the comet assay results). Microscopic evaluation revealed that these “glassy,” encapsulated GVs also had crystal formations, which may have indicated continued drying over time while stored at 4°C. For instance, Hochi et al. [35] have described compromised structure and function over time in freeze-dried mammal spermatozoa. These investigators discovered reduced DNA quality and ability to fertilize an oocyte postinjection after the sperm were stored for >6 mo.

The key to effective storage at a suprazero temperature is achieving a desirable level of dehydration and then ensuring low moisture stability over time [4, 36]. Previous studies have reported that stable storage of somatic cells at suprazero temperatures requires a drying process that achieves ∼0.1 g water/g of dried sample (which represents a >60% loss in mass of the original sample weight [33, 37]). In the case of our cat model, we conducted a preliminary study where we monitored weight loss in a set of glass slides containing GVs in the presence or absence of trehalose (using methods described by Chakraborty et al. [33] and a precision scale to determine weight every 30 sec for 6 min). Our samples lost <55% of their original weight with or without disaccharide supplementation. In neither case were we achieving the dehydration target [33, 37]. Therefore, we speculate that the moisture content in samples from the present study still remained too high for storage at 4°C, thereby contributing to the compromised as well as inconsistent DNA integrity between time periods. The lack of controlled humidity in our storage containers (despite their being airtight) might also have been an issue [36]. This assertion probably also explained why it was possible to repeatedly observe more DNA damage at 2 compared to 4 wk storage. Simply put, the GVs were not in a totally inanimate state, and perhaps the improvement in DNA integrity observed over time resulted from some gradual change(s) that were occurring in this storage format. For example, the high incidence of DNA damage as well as the absent nuclear envelopes at Time 0 might have been due to the immediate rehydration after air-drying without enough time for trehalose to bind and protect structures as observed after weeks of storage [38]. It now is well known that the full benefit of trehalose supplementation is observed only after allowing sufficient time to ensure penetration, binding, and protection of DNA ultrastructure [39].

Unlike the significant variation in DNA integrity with treatment and time, there generally was negligible impact on the presence/absence of the nuclear envelope, including through 32 wk of storage. An intact membrane is important functionally for molecular trafficking between the nucleoplasm and the cytoplasm [23]. From an applied perspective, the envelope must also be present during micromanipulation to effectively transfer the GV to a recipient cytoplast while avoiding loss of nuclear proteins and factors critical for subsequent development. Our finding that the envelope itself was mostly retained after desiccation was similar to observations for cryopreserved and thawed oocytes [4, 40]. Although we confirmed here that envelope presence appeared resistant to air-drying and suprazero temperature storage, we currently are examining its functional integrity, including pore status and ability at molecular trafficking [41].

The storage time points chosen for experiment 2 (to assess functionality) were based on the results of the structural assays (no apparent benefit of trehalose at 2 wk vs. benefit of trehalose at 4 wk). Our microinjection protocol was highly effective, with only a small proportion of reconstructed oocytes (2%–7%) failing to contain a GV after fixation. The decrease in meiotic resumption ability between intact and micromanipulated control oocytes (∼50%) was similar to previous reports on GV transfer using electro-fusion in the mouse [42], the cow [26–28], and, most recently, the domestic cat [1]. For the latter species, we have discovered that microinjecting the GV into the cytoplast significantly enhances ability to progress with nuclear maturation while simultaneously eliminating the potential of heteroplasmy [3]. In the present study, 26%–73% of oocytes reconstructed using desiccated GVs retained the capacity to resume meiosis, of which 2%–23% were able to advance in vitro to metaphase II. The ability of the maternal genomic package to withstand drying, cool storage, and then transfer into a conspecific, enucleated oocyte was influenced by processing/storage conditions. For this study, we were particularly interested in examining the impact of trehalose that is known to be protective of 1) membranes, enzymes, and proteins in lower life forms that survive extreme natural temperatures [6, 7] and 2) DNA of mouse spermatozoa [39]. In the presence of trehalose exposure, more GVs re-expanded after compaction (thereby revealing their retained viability) and more reconstructed oocytes resumed meiosis and completed nuclear maturation. There was also some evidence that this disaccharide exerted similar time effects on both structural and functional metrics. For example, although there was no benefit at 2 wk storage for either retaining DNA integrity or promoting nuclear maturation, both were significantly enhanced at 4 wk. It was also noteworthy that, even in the absence of trehalose, ∼30% of desiccated GVs retained an ability to resume meiosis, with up to 8% reaching metaphase II. It is likely that some of the handling medium compounds are able to form a stable, protective structure during air-drying and storage. Collectively, these results suggested an inherent capacity of the GV for resilience and, likely, a strong potential for these reconstructed oocytes to have fertilizing potential (studies in progress).

In summary, critical structural and functional components of the oocyte's GV were preserved after air-drying and storage for multiple weeks at suprazero temperatures, with survival metrics enhanced by the presence of trehalose. A significant proportion of these GVs, once hydrated and incorporated into a recipient cytoplast, reanimated and resumed meiosis up to the metaphase II stage. A sequential assessment protocol over time was useful for demonstrating that these desiccated GVs were not entirely inanimate. Clearly, to minimize DNA damage and to stabilize structure and function over time, there is a need to reach a minimum water content that also ensures viability. For this study, we used simple air-drying, and even so produced promising results. We now are exploring a dehydration process using microwave-assisted desiccation [33, 34] that may well facilitate reaching a more homogeneous, low moisture content at ambient temperatures. Other priorities include detailed assessments of ploidy, developmental competence, and gene expression of reconstructed oocytes. Nonetheless, results to date demonstrate what appears to be a strong tolerance and extraordinary plasticity for the mammalian GV to compaction, drying, and rehydration, perhaps offering alternatives to whole oocyte/embryo storage as a means for fertility preservation.