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Structural integrity and developmental potential of spermatozoa following microwave-assisted drying in the domestic cat model
Theriogenology 103 (2017) 36e43
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Structural integrity and developmental potential of spermatozoa following microwave-assisted drying in the domestic cat model Jennifer L. Patrick a, b, Gloria D. Elliott a, Pierre Comizzoli b, * a b
Department of Mechanical Engineering and Engineering Science, University of North Carolina at Charlotte, Charlotte, NC, USA Smithsonian Conservation Biology Institute, National Zoological Park, Washington DC, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 June 2017 Received in revised form 25 July 2017 Accepted 25 July 2017 Available online 26 July 2017
Characterizing the resilience of mammalian cells to non-physiological conditions is necessary to develop preservation and long-term storage strategies at low or ambient temperatures. Using the domestic cat model, the objective of the study was to characterize structural integrity (morphology and DNA damage) as well as functional properties (sperm aster formation and embryo formation after sperm injection) of spermatozoa after microwave-assisted drying to a moisture content compatible with storage in a glassy state at supra-zero temperatures. In Experiment 1, cat epididymal spermatozoa were porated with hemolysin and dried (using a commercial microwave oven set to 20% power) in the presence of trehalose for up to 50 min in a low humidity environment (11%) before measuring moisture content and sample temperature. In Experiment 2, morphology and DNA integrity were evaluated in sperm dried for up to 30 min (using the same method as above) versus fresh spermatozoa. In Experiment 3, the functionality of sperm dried for 30 min versus fresh sperm cells was evaluated after injection into oocytes based on sperm aster formation (5 h post-injection) and embryo development in vitro over 7 days. Moisture contents compatible with dry state storage were reached after 30 min of microwave-assisted drying. After rehydration, sperm morphology was not affected and the percentages of cells with damaged DNA (~6.5%) was similar to the fresh controls. Sperm aster diameters appeared to be generally smaller for dried-rehydrated cells compared to the fresh controls. This observation was consistent with a lower proportion of blastocyst formation after injection with dried spermatozoa (6.5%) compared to fresh spermatozoa (15%). However, the blastocyst quality based on the total blastomere number was not affected by the sperm treatment. This is the first and encouraging report in any species so far demonstrating that spermatozoa can be dried using microwaves without causing irreversible damage to the cellular structure and function. Published by Elsevier Inc.
Keywords: Spermatozoa Drying Domestic cat Sperm injection Sperm aster Embryo development
1. Introduction Cryopreservation is still considered as the reference method to preserve and store spermatozoa for the long-term in livestock species, human, and laboratory animals [1]. However, frozen biomaterials are stored using classical approaches e in electrical, subzero freezers or liquid nitrogen containers that require complex maintenance, alarms and specialized rooms with back-up power and heating, ventilation and air conditioning systems. Electricity and liquid nitrogen are expensive and not always readily available.
* Corresponding author. Smithsonian Conservation Biology Institute, Smithsonian National Zoological Park, 3001 Connecticut Ave., NW, Washington, DC 20008, USA. E-mail address: [email protected] (P. Comizzoli). http://dx.doi.org/10.1016/j.theriogenology.2017.07.037 0093-691X/Published by Elsevier Inc.
Contemporary storage systems also are fraught with failure opportunities e from human error to equipment breakdown [2]. Rather than relying on subzero-temperature to suspend cellular activities, it might be possible to preserve biomaterials by mimicking a natural phenomenon called ‘anhydrobiosis’. This mechanism is exploited by certain, nematodes, tardigrades, insects and brine shrimps to survive extreme cellular water loss [3]. In response to water stress, these organisms synthesize and accumulate disaccharides (e.g., trehalose) in their cells. As dehydration proceeds sugars replace water within the cells and eventually the intracellular and extracellular solutions convert to a glass e a high viscosity liquid state that immobilizes enzymes and prevents chemical activities e at ambient temperature. Dehydration of mammalian somatic cells in the presence of trehalose already has provided encouraging results in terms of preservation of structural
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and functional properties [4]. We also have demonstrated that desiccating the cat oocyte nucleus (germinal vesicle; GV) in the presence of trehalose circumvents the many disadvantages associated with relying on liquid nitrogen for maternal genome preservation [5,6]. Specifically, microwave-assisted dehydration of the GV allows reaching an equilibrium moisture content that is nonlethal. More than two-thirds of GVs retain intact DNA regardless of additional moisture loss over 8 wk of storage at supra-zero temperatures [6]. These GVs also have been used to reconstruct viable oocytes after incorporation into healthy cytoplasts, with the resulting reconstituted oocytes capable of resuming meiosis [5], being fertilized in vitro and forming transferable embryos [7]. Interestingly, the domestic cat shares important traits with humans in anatomy, physiology and pathology [8]. There also now is an impressive database on cat reproductive physiology and biotechnologies [7], much of which has emerged from our laboratory, including that this species is an excellent model for fertility preservation in human and rare species [9]. Sperm DNA damage is positively correlated with lower percentages of fertilization rates and embryo development in reproductive technologies [10,11] even though oocytes and early embryos can help repair paternal DNA damage [12]. Fertilization of an oocyte by a spermatozoa damaged by extensive double stranded DNA fragmentation can be incompatible with complete fertilization and embryo development. Activation checkpoints during embryogenesis slow down cell cycle progression until DNA damage is resolved, and if it remains unrepaired, cellular senescence and apoptosis are initiated [13]. It therefore is critical to determine the level of sperm DNA after exposure to non-physiological conditions. Importantly, there is a significant difference between sperm function in rodent and non-rodent mammalian species with respect to centrosomal inheritance, with rodent mammalian species inheriting the centrosome from the oocyte, and non-rodent mammalian species like the domestic cat inheriting the centrosome from the spermatozoa [14]. So far, most of the studies on sperm dry preservation have been conducted in non-rodent mammalian species using freeze-drying, with examples in rabbit, horse, bull, pig, and primate [14]. Recent studies on lyophilized cat spermatozoa have only led to limited embryo development [15]. In the domestic cat, decreases in the size of the sperm aster formed post fertilization have been correlated with delayed first cleavage divisions, slower developmental rates, and reduced morulae and blastocyst formations rates [16]. Centrosomal functions therefore are good indicators in addition to the sperm DNA integrity to assess developmental potential after exposure to non-physiological conditions. The objective of the study was to (1) optimize the microwave-assisted desiccation method and (2) characterize structural integrity and the functional properties of dried versus fresh spermatozoa. 2. Materials and methods Cat ovaries and testes were obtained (after owner consents) from a local local spay and neuter clinic as by-products of routine castrations and ovario-hysterectomies (UNC Charlotte IACUC protocol # 14-501). 2.1. Sperm preparation, drying, and rehydration Testes from domestic cats were harvested and transported at 4 C in PBS to the laboratory within 4 h post routine orchiectomy. Testes were dissected to remove the cauda epididymidis in F10 Hams Hepes medium (Irvine Scientific, Santa Ana, Ca) supplemented with 1.0 mM pyruvate, 1.0 mM glutamate, 100 IU/mL penicillin, 100 mg/mL streptomycin, and 5% fetal calf serum. The
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caudae epididymides were sliced through several times and after 15 min of incubation at room temperature in supplemented Hams medium, sperm suspension were centrifuged at 300g for 8 min and the pellet was re-suspended in 100 mL of Hams medium. Sperm suspension was analyzed for concentration, motility, and forward progression according to standard criteria [17]. Only viable good quality samples (>75% sperm motility) were retained for further experiments. Before drying, the membranes of epididymal spermatozoa were porated by exposure to 0.5 mg/mL a-hemolysin (Sigma) for 15 min at 38.5 C. After centrifugation at 300 g for 8 min, the pellet was resuspended in 100 mL of 0.3 M trehalose (in Tris-EDTA buffer [6]) for 30 min at room temperature (including a control without poration). A volume of 40 mL of sperm preparation was deposited on 13 mm coverslips (Thermanox) and dried using a commercial microwave oven (CEM SAM 225, Matthews, NC) set to 20% power in a stable relative humidity environment (11.0± 0.7%) for up to 50 min as previously reported [18]. After each drying time point (0, 5, 10, 15, 20 25, 30, 35, 40, 45, 50 min), dried sperm samples were rehydrated in 1 mL Hams Hepes media at 38.5 C for 1 h with mild shaking. Percentage of sperm recovery was expressed as the total number spermatozoa post-drying/total number of spermatozoa pre-drying) x 100. Sperm morphology (head, midpiece, tail) also was recorded (Fig. 1 A). 2.2. Assessment of end moisture content and temperature of microwaved samples End moisture content was determined as previously reported [6]. Briefly, a drying curve was established for the 0.3 M trehalose buffer solution at 20% microwave power, with the end moisture content determined for every 5 min of exposure up to 50 min. Drying curves were established in controlled humidity of 11.0± 0.7%. Integral to the development of this curve was the use of Karl Fisher titration (V20, Mettler-Toledo, Columbus, OH), a technique that determines water content of samples accurately to levels as low as 100 ppm. The moisture content was expressed as g H2O per g dried weight (gH2O/gDW). At each time point, the temperature of the titrated sample was measured immediately post drying using a hand held infra-red thermometer (IRT207, General Tools, Secaucus, NJ). 2.3. TUNEL assay Levels of sperm nuclear DNA fragmentation damage were assessed in both fresh samples and dehydrated samples using the TUNEL assay (Roche Diagnostics, Indianapolis, IN). A volume of 10 mL of sperm suspension was smeared onto microscope slides (positive control, negative control, and experimental samples) and fixed in 4% paraformaldehyde for 30 min at room temperature. Slides were washed in methanol-free ethanol for 5 min at room temperature, followed by 3 5 min washes at room temperature in a 1 mg/mL PVP-PBS solution. Cells were permeabilized with 0.5% Triton-X-100 in PBS for 5 min at RT, followed by 3 5 min washes at room temperature in 1 mg/mL PVP-PBS. Positive controls were incubated with 100 mL RQ1 DNAse 10 Reaction Buffer for 5 min at room temperature. Following reaction buffer incubation, 6 mL of RQ1 RNase-Free DNase was added to the mixture and incubated for an additional 30 min at room temperature. Positive control samples were washed 3 times with 1 mL of deionized water for 5 min. All positive controls, negative controls and experimental samples were incubated in 100 mL Equilibration Buffer for 5 min at room temperature. Positive (Equilibration Buffer, nucleotides, and rTDT enzyme), experimental (Equilibration Buffer, nucleotides, and rTDT enzyme), and negative control (Equilibration Buffer with 5% fetal calf serum and nucleotides) samples were covered with a cover slip
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and incubated for 1 h at 37 C in the dark. After 1 h, 1 mL of Saline Sodium Citrate solution was added to each sample and followed by incubation for 15 min at room temperature. All samples were washed in 1 mL of 0.2% Triton-X-100 for 5 min at room temperature, followed by 3 5 min washes at room temperature in 1 mL of deionized water, and then by 3 5 min washes at room temperature in 1 mL of 1 mg/mL PVP in PBS. Excess liquid was removed and 30 mL of Vectashield/DAPI Fluoromount-G (Vector Laboratories, Burlingame, CA) was added to each slide. Samples were then covered with a coverslip and allowed to dry for 15 min at room temperature before observation with a microscope equipped with epifluorescence (Olympus IX73; Olympus Corporation, Melville, NY) using SPOT software 5.0 (Diagnostic Instruments, Inc., Sterling Heights, MI). A minimum of 400 spermatozoon were analyzed per sample to determine the level of nuclear DNA fragmentation
damage. The quantification of DNA fragmentation damage was based on the intensity of fluorescence (measured by analog to digital units, ADU) within each sperm head relative to the background fluorescence [6]. TUNEL positive/negative sperm heads were classified based on the presence/absence of green fluorescence in sperm heads (Fig. 1BeG). 2.4. Oocyte collection and in vitro maturation Ovaries from domestic cats were harvested from a local spay and neuter clinic and transported at 4 C in PBS to the laboratory within 4 h post routine ovario-hysterectomy. The ovaries were sliced in H-MEM (Hepes-buffered Minimal Essential Medium, supplemented with 0.4 mM cysteine, 0.4 mM glutamate, 4.0 mM pyruvate, 100 mg/mL penicillin, 100 mg/mL streptomycin) to recover
Fig. 1. Representative morphology of cat spermatozoa after microwave-assisted drying (phase contrast; A), TUNEL assay (B, D, F) and counterstaining with Hoechst (C, E, G). Negative control (B, C), positive control (D, E), and normal rehydrated sperm sample (F, G). Bar ¼ 15 mm.
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the immature oocytes. Grade I immature oocytes, characterized by dark, homogenous cytoplasm surrounded by several layers of compacted cumulous cells, were selected for in vitro maturation, using methodology as previously described [19]. Oocytes then were cultured in vitro in Protein Plus Blastocyst Embryo Culture Medium (Sage, Trumbull, CT) supplemented with 1 mg/mL FSH (Sigma), 1 mg/ mL LH (Sigma), and mg/mL estradiol (Sigma) for 24e28 h at 38.5 C, 6.0% CO2 and 5.0% O2 in groups of 5 oocytes per 50-mL drops. Oocytes then were denuded by gentle pipetting in 0.2% cumulase (Origio, Trumbull, CT). Only oocytes with a first polar body were selected for Intracytoplasmic sperm injection (ICSI). 2.5. Intracytoplasmic Sperm Injection (ICSI) and embryo culture A volume of 2 mL of sperm suspension was added to 10 mL of 10% polyvinylpyrrolidone (PVP, Irvine Scientific) in the center drop of a 50 9 mm ICSI dish (NUNC) surrounded by 8 10 mL drops of complete Hams Hepes medium and covered with oil. Due to the poor mixing of the trehalose in PVP, for dehydrated sperm suspensions, spermatozoa were first added to a drop of Hams Hepes medium in the ICSI dish, collected, and moved to the middle PVP drop for immobilization, then aspirated into the injection pipette, and injected into a mature oocyte. In vitro matured oocytes were added to the Hams Hepes drops of the ICSI dish. The dish was maintained on a heated stage (38.5 C) of an inverted microscope (Olympus IX73) equipped with holding pipette and micromanipulators (Narishige, Sterling, VA). A single morphologically normal spermatozoon was selected, immobilized by drawing the injection pipette across the midpiece, aspirated into the injection pipette, and injected into a mature oocyte (polar body at 12 or 6 o-clock). Motile fresh spermatozoa as well as immotile dehydrated spermatozoa were immobilized across the midpiece prior to injection. Injected mature oocytes then were cultured in groups of 5 oocytes in 50 mL drops in Protein Plus Blastocyst Medium (Sage, Trumbull, CT) at 38.5 C 6% CO2 and 5% O2 for 7 days. Additional 10 mature oocytes were sham injected (no sperm) for each replicate to serve as parthenogenetic controls. 2.6. Sperm aster evaluation Presumptive zygotes were fixed at 5 h post insemination (hpi) in 2.5% paraformaldehyde for 30 min at 38.5 C, followed by 3 5 min washes in 1 PBS at room temperature. Nonspecific antigenic sites were saturated with 0.5% Triton-X-100 and 20% fetal calf serum in PBS for 30 min at 38.5 C. Presumptive zygotes were incubated overnight with anti-a-tubulin (Sigma) and anti-b-tubulin (Sigma) monoclonal antibodies (1/1000 in PBS with 0.5% Triton-X-100 and 2% fetal calf serum) at 4 C. Embryos were washed in PBS (3 15 min at room temperature), and incubated with FITC-labeled anti-mouse IgG (Sigma), diluted 1/150 in PBS, and held for 1 h at 38.5 C. Chromatin was counterstained with 10 mg/mL Hoechst 33342 in PBS for 5 min at 38.5 C, followed by mounting on microscope slides with a 30 mL volume of Vectashield and covered with a coverslip. Presumptive zygotes were observed with a confocal microscope (Olympus Fluoview Laser Scanning Microscope) to assess the size of the male and female pronuclei and the sperm aster formation according to our standard criteria (Fig. 2) [16]. Unfertilized oocytes were categorized by the absence of decondensed sperm heard or pronuclei in oocytes remained at the metaphase II stage. 2.7. Embryo assessment Embryos were assessed on Day 3 and Day 7 post fertilization (Day 0 being ICSI day) for embryo cell number and embryonic stage.
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On Day 3, embryos were observed with an inverted microscope (Olympus IX 73) with a heated stage (38.5 C), and the cell number was recorded for each embryo within 4 categories (1-cell; 2-4 cell; 5-8 cell; 9-16 cell). Following embryo assessment, embryos were returned to the incubator for further culture. On Day 7, embryos were observed and fixed on microscope slides using 2.5% paraformaldehyde incubation for 30 min at 38.5 C followed by chromatin staining with 10 mg/mL Hoechst 33342 in PBS for 5 min at 38.5 C. Embryos were mounted with Vectashield covered with a coverslip prior to imaging with epifluorescence microscopy. Embryonic cell number or stage was determined for each embryo within 6 categories (1-cell; 2-4 cell; 5-8 cell; 9-16 cell; Morula; Blastocyst). 2.8. Experimental design and statistical analysis In Experiment 1, epididymides from 6 testes per replicate (n ¼ 6 replicates) were exposed to microwave-assisted drying. In each replicate, aliquotes were analyzed at each time point (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 min) for moisture content/temperature. In Experiment 2, epididymides from 6 testes per replicate (n ¼ 4 replicates) were dried (with or without prior membrane poration). In each replicate, aliquotes were analyzed at each time point (0, 10, 20, 30 min) for morphology/DNA integrity. In Experiment 3a, epididymides from 3 testes (n ¼ 6 replicates) were exposed to 30 min of microwave-assisted drying before sperm injection into oocytes (n ¼ 350 including for sham injections) and sperm aster assessment. In Experiment 3b, epididymides from 3 testes (n ¼ 4 replicates) were exposed to 30 min of drying before injection into oocytes (n ¼ 450 including for sham injections) and assessment of embryo development. In all experiments, values were expressed as mean ± standard deviation (SD) of the multiple replicates. Percentage data were transformed using arcsine transformation before statistical analysis. Comparisons between treatments and among replicates were analyzed by analysis of variance (ANOVA), Tukey's multiple test for mean comparison, and Bartlett test for homogeneity of the variances. Data not normally distributed were analyzed by Kruskal-Wallis ANOVA on ranks and Dunn method for all pairwise comparisons. Differences were considered significant at P < 0.05 (GraphPad Software Inc.). 3. Results The average moisture content of the sperm suspension in trehalose decreased rapidly to reach 0.16 gH2O/gDW after 30 min of drying with no further decrease beyond that time point (Fig. 3A and inset). At the successive drying time points, the sample temperature progressed from 27.9 C after 5 min to a maximum of 37.3 C after 50 min (Fig. 3B). After rehydration, the percentages of sperm recovery from the coverslips (ranging from 83.5 to 89.7%) were not different across all drying time points. Additionally, gross morphology of the sperm cells was unchanged with no abnormalities on the sperm head, midpiece, and flagellum (Fig. 1A) although no sperm motility was reported after any of the drying time points. After only 10 min of dehydration, the percentage of sperm cells with damaged DNA ranged from 35.1 to 42.4% in the non-porated group exposed to trehalose which was higher (P < 0.05) than in the membrane porated counterparts (ranging from 5.2 to 7.9%). However, following membrane poration and trehalose exposure, the percentages of sperm cells with DNA damage were not different after 10, 20, or 30 min of desiccation (ranging from 4.5 to 8.2%) compared to the fresh controls (6.6± 0.5%). In TUNEL positive cells, the degree of DNA damage (ranging from 395 to 407 ADU) was not influenced by the microwave-assisted drying across time points
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Fig. 2. Confocal images of oocytes 5 h after injection with fresh or dried cat spermatozoa. Immunostaining of a-tubulin and b-tubulin (with FITC; green) and counterstaining of chromatin (with Hoechst; light blue). Oocyte with large sperm aster (A), small sperm aster (B), no sperm aster (C), non-fertilized oocytes (D). Bar ¼ 10 mm; pb: polar body, mPN: male pronucleus; fPN: female pronucleus; MII: metaphase II. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
compared to the positive sperm cells in the fresh controls (385 ± 11 ADU). Percentages of oocyte reaching the metaphase II stage ranged between 60 and 66% in all replicates. Percentages of fertilization were higher (P < 0.05) after injection of fresh spermatozoa than with dried sperm cells (Table 1). Five hours after the sperm injection, the majority of sperm asters had a small diameter regardless of the sperm treatment (Table 1). However, more large sperm asters (P < 0.05) were observed after injection with fresh than dried spermatozoa (Table 1). At that same time point, diameters of male pronuclei were similar after fresh or dried sperm injection (range, 4.9e6.3 mm) confirming that zygotes were all at a comparable stage of the first cell cycle when analyzed. None of the sham injected oocytes (parthenogenetic controls) were activated. Percentages of oocytes reaching the metaphase II stage ranged between 62 and 65% in all replicates. Percentages of fertilization were higher (P < 0.05) after injection of fresh spermatozoa than with dried sperm cells (Table 2). After 3 days of in vitro culture (Day 0 being the day of ICSI), more embryos were at the 9-16 cell stage after injection with fresh sperm cells (P < 0.05) than with dried spermatozoa while more embryos were observed at the 2-4 cell stage (P < 0.05) after the injection of the latter (Table 2). Regardless of the sperm treatment, blastomeres had equal sizes in those early embryo stages. After 7 days of in vitro culture, more embryos reached the morula or blastocyst stage (P < 0.05) after injection with fresh spermatozoa than with dried sperm cells (Table 3). With the latter, more embryos arrested their development before the 58 cell stage (P < 0.05; Table 3). However, blastomere numbers
within the blastocysts were not different between the fresh (92 ± 15) and dried (87 ± 11) sperm groups (Fig. 4). None of the sham injected oocytes (parthenogenetic controls) were activated or cleaved after 3 or 7 days of culture. 4. Discussion The moisture content reached by sperm samples after microwave-assisted drying was minimally damaging and could be compatible with storage at non-freezing temperatures. Sperm morphology and DNA integrity were not affected by membrane poration, trehalose exposure, and drying. However, the centrosomal functions of some dried sperm cells appeared to be impaired which likely led to more arrests of the embryo development before the blastocyst formation. Most of the sperm drying studies in non-rodent mammalian species have been conducted through freeze-drying and reported successful embryo development after sperm injection in cattle [20,21] and primates [22] or livebirths after transfer in rabbits [23] and horses [24]. However, the present study is the first report on sperm microwave-assisted drying in any species. Drying for 30 min at 20% power produced the lowest end moisture content with the highest estimated storage temperature. The estimated glass transition temperature (Tg) for that moisture content was approximately 7 C, based on reported values for a binary solution of trehalose and water [25]. Importantly, the sample temperature during the microwaving remained within physiological temperature range for cat spermatozoa. Although sperm motility was lost
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Fig. 3. Moisture content (average ± SD; n ¼ 6 replicates) with insert for values between 30 and 50 min drying (A) and sample temperature (average ± SD; n ¼ 6 replicates; B) of 0.3 M Trehalose in TE Buffer (40 mL) during microwaving (20% power) on coverslips in 11% Relative Humidity.
Table 1 Fertilization success and proportions of sperm aster types 5 h after injection with fresh or dried epididymal spermatozoa from domestic cats. Values are expressed mean ± SD (n ¼ 6 replicates).
Total fertilized oocytes (%) Fertilized oocyte with: Large sperm aster (%) Small sperm aster (%) No sperm aster (%)
Fresh sperm injection (n ¼ 137 injected oocytes)
Dried sperm injection (n ¼ 144 injected oocytes)
93.5 ± 1.0a
84.0 ± 2.5b
14.6 ± 3.0a 62.1 ± 4.5a 16.8 ± 1.7a
5.6 ± 1.8b 58.3 ± 2.5a 20.1 ± 4.1a
Within rows, values with different superscripts differ significantly (P < 0.05).
during the treatment, recovery and morphology of sperm cells were not affected during the drying process. This confirms what has already been observed in other cell types treated the same way
[6,26]. Inversely, freeze-dried felid cat spermatozoa have displayed evidence of surface damage, broken membranes, and decapitation [15] that were not observed here.
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Table 2 Fertilization success and proportions of different embryo stages obtained after oocyte injection with fresh or dried spermatozoa from domestic cats followed by 3 days of in vitro culture. Values are expressed mean ± SD (n ¼ 4 replicates). Total number of injected oocytes
Total fertilized oocytes (%)
Proportion of embryo stages (%) 2-4 cells
Fresh spermatozoa Dried spermatozoa
192 203
a
5-8 cells a
24.0 ± 5.1 32.0 ± 3.4b
88.6 ± 1.2 77.9 ± 7.9b
37.5 ± 10.2 29.1 ± 6.3a
9-16 cells a
27.1 ± 4.9a 16.8 ± 4.3b
Within columns, values with different superscripts differ significantly (P < 0.05).
Table 3 Proportions of different embryo stages obtained after oocyte injection with fresh or dried spermatozoa from domestic cats followed by 7 days of in vitro culture. Values are expressed mean ± SD (n ¼ 4 replicates).
Fresh spermatozoa Dried spermatozoa
Total number of injected oocytes
Proportion of embryo stages (%) 2-4 cells
5-8 cells
9-16 cells
Morulae
Blastocysts
192 203
8.9 ± 3.2a 23.2 ± 5.6b
18.2 ± 3.5a 17.2 ± 6.4a
20.3 ± 2.8a 18.7 ± 8.0a
26.6 ± 3.3a 12.3 ± 5.7b
15.1 ± 3.3a 6.4 ± 3.7b
Within columns, values with different superscripts differ significantly (P < 0.05).
The proportions of sperm cells with damaged DNA (as well as the degree of damage) were not affected by the drying for up to 30 min resulting in a low moisture content of 0.16 gH2O/gDW. Epididymal spermatozoa already have highly compacted chromatin [13] which contributes to the resilience to non-physiological conditions like cryopreservation [27] or drying like in the present study. Former studies in frozen-dried human sperm also demonstrated the resilience to DNA damage [28]. However, drying without prior membrane poration with a-hemolysin showed a significant increase in damage after only 10 min of drying. Without poration of the plasma and nuclear membranes, trehalose was unable to enter the cell and surround the nuclear chromatin, associated proteins, and centrosome, to create the protective milieu. These data confirm that trehalose is required to be present on both sides of the membrane to confer protection like in the mouse sperm cells [29]. This protective effect also is similar to previous a report on the microwave-assisted drying of germinal vesicles [6]. The fertilization success after dried sperm injection was slightly lower than with fresh spermatozoa and could not be explained by a loss of DNA integrity. One or multiple sperm components involved
in the oocyte activation may have been impaired and will have to be investigated further [30]. At 5 h post-injection, the diameter of male pronuclei (or decondensed sperm chromatin) was in the same range after fresh and dried sperm injections. Zygotes therefore were all observed at a comparable stage of the first cell cycle when analyzed [16]. The majority of smaller sperm asters observed with dried sperm cells likely revealed damaged centrosomal functions that then led to early arrest of the embryo development [16,31]. This particular detrimental effect of the desiccation has already been reported in bovine spermatozoa [32]. Strategies to better protect the centrosome or to supplement the sperm with critical proteins such as centrin [33] might be a good strategy to overcome the issue. Importantly, there likely was no embryo toxic effect of remnant trehalose as demonstrated in the mouse [34]. Embryo development in vitro obtained in the present study was in the same range as previous reports from our laboratory [16]. Previous studies form other groups on lyophilized cat spermatozoa have only been taken out to the 8-day/blastocyst stage [15]. When oocytes were activated with ethanol to over-ride deficiencies in the ability of freeze-dried spermatozoa to induce the required intracellular calcium spikes, nearly 28% of fertilized oocytes formed
Fig. 4. Micrographs or morulae (A-B, E-F) and blastocysts (C-D, G-H) after 7 days of in vitro culture, produced after fresh (A, B, C, D) or dried cat sperm (E, F, G, H) injection. Before (A, C, E, F) and after fixation and Hoechst staining (B, D, F, H). Bar ¼ 25 mm.
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blastocysts was achieved compared to 54% in fresh controls. Same results may be expected with ejaculated spermatozoa. Feline embryo development using epididymal sperm was previously reported as 25% blastocyst development [35] and 26.8% in ejaculated feline sperm, which has been noted as equivalent to epididymal sperm in terms of embryo development potential [16]. Further studies to investigate embryonic genome activation and fetal development after embryo transfer with dried spermatozoa are warranted. This is the first in-depth assessment of structure and function in sperm cells processed by microwave-assisted drying. Although the levels of drying achieved with the current processing conditions would suggest that storage below 7 C may be necessary to maintain the sample in the glassy state, it is possible that storage slightly above Tg would be adequate for multi-year shelf-life. Storage studies at ambient and moderate refrigeration temperatures are ongoing and will further advance the fields of biobanking and fertility preservation. Acknowledgments The authors are indebted to Dr. Alida Kinney and staff at the Cabarrus Spay and Neuter Clinic for generously facilitating the transfer of resources from consenting pet-owners to UNC Charlotte. References [1] Comizzoli P, Wildt DE. Mammalian fertility preservation through cryobiology: value of classical comparative studies and the need for new preservation options. Reprod Fertil Dev 2013;26:91e8. http://dx.doi.org/10.1071/RD13259. [2] Baker M. Biorepositories: building better biobanks. Nature 2012;486:141e6. http://dx.doi.org/10.1038/486141a. [3] Crowe JH. Introduction: stabilization of dry biological materials. Biopreserv Biobank 2012;10. http://dx.doi.org/10.1089/bio.2012.1043. 375e375. [4] Loi P, Iuso D, Czernik M, Zacchini F, Ptak G. Towards storage of cells and gametes in dry form. Trends Biotechnol 2013;31:688e95. http://dx.doi.org/ 10.1016/j.tibtech.2013.09.004. [5] Graves-Herring JE, Wildt DE, Comizzoli P. Retention of structure and function of the cat germinal vesicle after air-drying and storage at suprazero temperature. Biol Reprod 2013;88:139. http://dx.doi.org/10.1095/ biolreprod.113.108472. [6] Elliott GD, Lee P-C, Paramore E, Van Vorst M, Comizzoli P. Resilience of oocyte germinal vesicles to microwave-assisted drying in the domestic cat model. Biopreserv Biobank 2015;13:164e71. http://dx.doi.org/10.1089/ bio.2014.0078. [7] Comizzoli P, Wildt DE. On the horizon for fertility preservation in domestic and wild carnivores. Reprod Domest Anim 2012;47(Suppl 6):261e5. http:// dx.doi.org/10.1111/rda.12010. [8] Wildt DE, Comizzoli P, Pukazhenthi B, Songsasen N. Lessons from biodiversitythe value of nontraditional species to advance reproductive science, conservation, and human health. Mol Reprod Dev 2010;77:397e409. http:// dx.doi.org/10.1002/mrd.21137. [9] Comizzoli P, Songsasen N, Wildt DE. Protecting and extending fertility for females of wild and endangered mammals. Cancer Treat Res 2010;156: 87e100. http://dx.doi.org/10.1007/978-1-4419-6518-9_7. [10] Larson KL, DeJonge CJ, Barnes AM, Jost LK, Evenson DP. Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques. Hum Reprod 2000;15:1717e22. ndez-Gonzalez R, Moreira PN, Pe rez-Crespo M, Sa nchez-Martín M, [11] Ferna Ramirez MA, Pericuesta E, et al. Long-term effects of mouse intracytoplasmic sperm injection with DNA-fragmented sperm on health and behavior of adult offspring. Biol Reprod 2008;78:761e72. http://dx.doi.org/10.1095/ biolreprod.107.065623. [12] Aitken RJ, Krausz C. Oxidative stress, DNA damage and the Y chromosome. Reproduction 2001;122:497e506. lez-Marín C, Gosa lvez J, Roy R. Types, causes, detection and repair of [13] Gonza DNA fragmentation in animal and human sperm cells. Int J Mol Sci 2012;13:
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14026e52. http://dx.doi.org/10.3390/ijms131114026. [14] Patrick J, Comizzoli P, Elliott G. Dry preservation of spermatozoa: considerations for different species. Biopreserv Biobank 2017;15:158e68. http:// dx.doi.org/10.1089/bio.2016.0087. [15] Ringleb J, Waurich R, Wibbelt G, Streich WJ, Jewgenow K. Prolonged storage of epididymal spermatozoa does not affect their capacity to fertilise in vitromatured domestic cat (Felis catus) oocytes when using ICSI. Reprod Fertil Dev 2013;23:818. http://dx.doi.org/10.1071/RD10192. [16] Comizzoli P, Wildt DE, Pukazhenthi BS. Poor centrosomal function of cat testicular spermatozoa impairs embryo development in vitro after intracytoplasmic sperm injection. Biol Reprod 2006;75:252e60. http://dx.doi.org/ 10.1095/biolreprod.106.051342. [17] Hribal R, Jewgenow K, Braun BC, Comizzoli P. Influence of culture medium composition on relative mrna abundances in domestic cat embryos. Reprod Domest Anim 2013:48. http://dx.doi.org/10.1111/j.1439-0531.2012.02139.x. [18] Cellemme SL, Van Vorst M, Paramore E, Elliott GD. Advancing microwave technology for dehydration processing of biologics. Biopreserv Biobank 2013;11:278e84. http://dx.doi.org/10.1089/bio.2013.0024. [19] Comizzoli P, Wildt DE, Pukazhenthi BS. In vitro development of domestic cat embryos following intra-cytoplasmic sperm injection with testicular spermatozoa. Theriogenology 2006;66:1659e63. http://dx.doi.org/10.1016/ j.theriogenology.2006.01.038. [20] Keskintepe L, Pacholczyk G, Machnicka A, Norris K, Curuk MA, Khan I, et al. Bovine blastocyst development from oocytes injected with freeze-dried spermatozoa. Biol Reprod 2002;67:409e15. [21] Bhowmick S. Desiccation Tolerance of Spermatozoa Dried at Ambient Temperature: Production of Fetal Mice, 1786; 2003. p. 1779e86. http://dx.doi.org/ 10.1095/biolreprod.102.009407. [22] S anchez-Partida LG, Simerly CR, Ramalho-Santos J. Freeze-dried primate sperm retains early reproductive potential after intracytoplasmic sperm injection. Fertil Steril 2008;89:742e5. http://dx.doi.org/10.1016/ j.fertnstert.2007.02.066. [23] Liu J-L, Kusakabe H, Chang C-C, Suzuki H, Schmidt DW, Julian M, et al. Freezedried sperm fertilization leads to full-term development in rabbits. Biol Reprod 2004;70:1776e81. http://dx.doi.org/10.1095/biolreprod.103.025957. [24] Choi YH, Varner DD, Love CC, Hartman DL, Hinrichs K. Production of live foals via intracytoplasmic injection of lyophilized sperm and sperm extract in the horse. Reproduction 2011;142:529e38. http://dx.doi.org/10.1530/REP-110145. [25] Chen T, Fowler A, Toner M. Literature review: supplemented phase diagram of the trehalose-water binary mixture. Cryobiology 2000;40:277e82. http:// dx.doi.org/10.1006/cryo.2000.2244. [26] Chakraborty N, Biswas D, Elliott GD. A simple mechanistic way to increase the survival of Mammalian cells during processing for dry storage. Biopreserv Biobank 2010;8:107e14. http://dx.doi.org/10.1089/bio.2010.0010. _ n ski W, Partyka A. Comparative analysis of in vitro char[27] Prochowska S, Niza acteristics of fresh and frozen-thawed urethral and epididymal spermatozoa from cats (Felis domesticus). Theriogenology 2016;86:2063e72. http:// dx.doi.org/10.1016/j.theriogenology.2016.07.002. [28] Gianaroli L, Magli MC, Stanghellini I, Crippa A, Crivello AM, Pescatori ES, et al. DNA integrity is maintained after freeze-drying of human spermatozoa. Fertil Steril 2012;97:1067e73. http://dx.doi.org/10.1016/j.fertnstert.2012.02.014. e1. [29] McGinnis LK. Mouse sperm desiccated and stored in trehalose medium without freezing. Biol Reprod 2005;73:627e33. http://dx.doi.org/10.1095/ biolreprod.105.042291. [30] Anifandis G, Messini CI, Dafopoulos K, Daponte A, Messinis IE. Sperm contributions to oocyte activation: more that meets the eye. J Assist Reprod Genet 2016;33:313e6. http://dx.doi.org/10.1007/s10815-016-0653-0. [31] Comizzoli P, Wildt DE. Centrosomal functions and dysfunctions in cat spermatozoa. 2012. http://dx.doi.org/10.1007/978-1-62703-035-9_3. [32] Hara H, Abdalla H, Morita H, Kuwayama M, Hirabayashi M, Hochi S. Procedure for bovine ICSI, not sperm freeze-drying, impairs the function of the microtubule-organizing center. J Reprod Dev 2011;57:428e32. [33] Rowlison T, Ottinger MA, Comizzoli P. Deciphering the mechanisms involving cenexin, ninein and centriolin in sperm maturation during epididymal transit in the domestic cat. Reprod Domest Anim 2017;52(Suppl 2):193e6. http:// dx.doi.org/10.1111/rda.12831. [34] Eroglu A, Elliott G, Wright DL, Toner M, Toth TL. Progressive elimination of microinjected trehalose during mouse embryonic development. Reprod Biomed Online 2005;10:503e10. [35] Waurich R, Ringleb J, Braun BC, Jewgenow K. Embryonic gene activation in in vitro produced embryos of the domestic cat (Felis catus). Reproduction 2010;140:531e40. http://dx.doi.org/10.1530/REP-10-0298.
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Structural integrity and developmental potential of spermatozoa following microwave-assisted drying in the domestic cat model Jennifer L. Patrick a, b, Gloria D. Elliott a, Pierre Comizzoli b, * a b
Department of Mechanical Engineering and Engineering Science, University of North Carolina at Charlotte, Charlotte, NC, USA Smithsonian Conservation Biology Institute, National Zoological Park, Washington DC, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 June 2017 Received in revised form 25 July 2017 Accepted 25 July 2017 Available online 26 July 2017
Characterizing the resilience of mammalian cells to non-physiological conditions is necessary to develop preservation and long-term storage strategies at low or ambient temperatures. Using the domestic cat model, the objective of the study was to characterize structural integrity (morphology and DNA damage) as well as functional properties (sperm aster formation and embryo formation after sperm injection) of spermatozoa after microwave-assisted drying to a moisture content compatible with storage in a glassy state at supra-zero temperatures. In Experiment 1, cat epididymal spermatozoa were porated with hemolysin and dried (using a commercial microwave oven set to 20% power) in the presence of trehalose for up to 50 min in a low humidity environment (11%) before measuring moisture content and sample temperature. In Experiment 2, morphology and DNA integrity were evaluated in sperm dried for up to 30 min (using the same method as above) versus fresh spermatozoa. In Experiment 3, the functionality of sperm dried for 30 min versus fresh sperm cells was evaluated after injection into oocytes based on sperm aster formation (5 h post-injection) and embryo development in vitro over 7 days. Moisture contents compatible with dry state storage were reached after 30 min of microwave-assisted drying. After rehydration, sperm morphology was not affected and the percentages of cells with damaged DNA (~6.5%) was similar to the fresh controls. Sperm aster diameters appeared to be generally smaller for dried-rehydrated cells compared to the fresh controls. This observation was consistent with a lower proportion of blastocyst formation after injection with dried spermatozoa (6.5%) compared to fresh spermatozoa (15%). However, the blastocyst quality based on the total blastomere number was not affected by the sperm treatment. This is the first and encouraging report in any species so far demonstrating that spermatozoa can be dried using microwaves without causing irreversible damage to the cellular structure and function. Published by Elsevier Inc.
Keywords: Spermatozoa Drying Domestic cat Sperm injection Sperm aster Embryo development
1. Introduction Cryopreservation is still considered as the reference method to preserve and store spermatozoa for the long-term in livestock species, human, and laboratory animals [1]. However, frozen biomaterials are stored using classical approaches e in electrical, subzero freezers or liquid nitrogen containers that require complex maintenance, alarms and specialized rooms with back-up power and heating, ventilation and air conditioning systems. Electricity and liquid nitrogen are expensive and not always readily available.
* Corresponding author. Smithsonian Conservation Biology Institute, Smithsonian National Zoological Park, 3001 Connecticut Ave., NW, Washington, DC 20008, USA. E-mail address: [email protected] (P. Comizzoli). http://dx.doi.org/10.1016/j.theriogenology.2017.07.037 0093-691X/Published by Elsevier Inc.
Contemporary storage systems also are fraught with failure opportunities e from human error to equipment breakdown [2]. Rather than relying on subzero-temperature to suspend cellular activities, it might be possible to preserve biomaterials by mimicking a natural phenomenon called ‘anhydrobiosis’. This mechanism is exploited by certain, nematodes, tardigrades, insects and brine shrimps to survive extreme cellular water loss [3]. In response to water stress, these organisms synthesize and accumulate disaccharides (e.g., trehalose) in their cells. As dehydration proceeds sugars replace water within the cells and eventually the intracellular and extracellular solutions convert to a glass e a high viscosity liquid state that immobilizes enzymes and prevents chemical activities e at ambient temperature. Dehydration of mammalian somatic cells in the presence of trehalose already has provided encouraging results in terms of preservation of structural
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and functional properties [4]. We also have demonstrated that desiccating the cat oocyte nucleus (germinal vesicle; GV) in the presence of trehalose circumvents the many disadvantages associated with relying on liquid nitrogen for maternal genome preservation [5,6]. Specifically, microwave-assisted dehydration of the GV allows reaching an equilibrium moisture content that is nonlethal. More than two-thirds of GVs retain intact DNA regardless of additional moisture loss over 8 wk of storage at supra-zero temperatures [6]. These GVs also have been used to reconstruct viable oocytes after incorporation into healthy cytoplasts, with the resulting reconstituted oocytes capable of resuming meiosis [5], being fertilized in vitro and forming transferable embryos [7]. Interestingly, the domestic cat shares important traits with humans in anatomy, physiology and pathology [8]. There also now is an impressive database on cat reproductive physiology and biotechnologies [7], much of which has emerged from our laboratory, including that this species is an excellent model for fertility preservation in human and rare species [9]. Sperm DNA damage is positively correlated with lower percentages of fertilization rates and embryo development in reproductive technologies [10,11] even though oocytes and early embryos can help repair paternal DNA damage [12]. Fertilization of an oocyte by a spermatozoa damaged by extensive double stranded DNA fragmentation can be incompatible with complete fertilization and embryo development. Activation checkpoints during embryogenesis slow down cell cycle progression until DNA damage is resolved, and if it remains unrepaired, cellular senescence and apoptosis are initiated [13]. It therefore is critical to determine the level of sperm DNA after exposure to non-physiological conditions. Importantly, there is a significant difference between sperm function in rodent and non-rodent mammalian species with respect to centrosomal inheritance, with rodent mammalian species inheriting the centrosome from the oocyte, and non-rodent mammalian species like the domestic cat inheriting the centrosome from the spermatozoa [14]. So far, most of the studies on sperm dry preservation have been conducted in non-rodent mammalian species using freeze-drying, with examples in rabbit, horse, bull, pig, and primate [14]. Recent studies on lyophilized cat spermatozoa have only led to limited embryo development [15]. In the domestic cat, decreases in the size of the sperm aster formed post fertilization have been correlated with delayed first cleavage divisions, slower developmental rates, and reduced morulae and blastocyst formations rates [16]. Centrosomal functions therefore are good indicators in addition to the sperm DNA integrity to assess developmental potential after exposure to non-physiological conditions. The objective of the study was to (1) optimize the microwave-assisted desiccation method and (2) characterize structural integrity and the functional properties of dried versus fresh spermatozoa. 2. Materials and methods Cat ovaries and testes were obtained (after owner consents) from a local local spay and neuter clinic as by-products of routine castrations and ovario-hysterectomies (UNC Charlotte IACUC protocol # 14-501). 2.1. Sperm preparation, drying, and rehydration Testes from domestic cats were harvested and transported at 4 C in PBS to the laboratory within 4 h post routine orchiectomy. Testes were dissected to remove the cauda epididymidis in F10 Hams Hepes medium (Irvine Scientific, Santa Ana, Ca) supplemented with 1.0 mM pyruvate, 1.0 mM glutamate, 100 IU/mL penicillin, 100 mg/mL streptomycin, and 5% fetal calf serum. The
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caudae epididymides were sliced through several times and after 15 min of incubation at room temperature in supplemented Hams medium, sperm suspension were centrifuged at 300g for 8 min and the pellet was re-suspended in 100 mL of Hams medium. Sperm suspension was analyzed for concentration, motility, and forward progression according to standard criteria [17]. Only viable good quality samples (>75% sperm motility) were retained for further experiments. Before drying, the membranes of epididymal spermatozoa were porated by exposure to 0.5 mg/mL a-hemolysin (Sigma) for 15 min at 38.5 C. After centrifugation at 300 g for 8 min, the pellet was resuspended in 100 mL of 0.3 M trehalose (in Tris-EDTA buffer [6]) for 30 min at room temperature (including a control without poration). A volume of 40 mL of sperm preparation was deposited on 13 mm coverslips (Thermanox) and dried using a commercial microwave oven (CEM SAM 225, Matthews, NC) set to 20% power in a stable relative humidity environment (11.0± 0.7%) for up to 50 min as previously reported [18]. After each drying time point (0, 5, 10, 15, 20 25, 30, 35, 40, 45, 50 min), dried sperm samples were rehydrated in 1 mL Hams Hepes media at 38.5 C for 1 h with mild shaking. Percentage of sperm recovery was expressed as the total number spermatozoa post-drying/total number of spermatozoa pre-drying) x 100. Sperm morphology (head, midpiece, tail) also was recorded (Fig. 1 A). 2.2. Assessment of end moisture content and temperature of microwaved samples End moisture content was determined as previously reported [6]. Briefly, a drying curve was established for the 0.3 M trehalose buffer solution at 20% microwave power, with the end moisture content determined for every 5 min of exposure up to 50 min. Drying curves were established in controlled humidity of 11.0± 0.7%. Integral to the development of this curve was the use of Karl Fisher titration (V20, Mettler-Toledo, Columbus, OH), a technique that determines water content of samples accurately to levels as low as 100 ppm. The moisture content was expressed as g H2O per g dried weight (gH2O/gDW). At each time point, the temperature of the titrated sample was measured immediately post drying using a hand held infra-red thermometer (IRT207, General Tools, Secaucus, NJ). 2.3. TUNEL assay Levels of sperm nuclear DNA fragmentation damage were assessed in both fresh samples and dehydrated samples using the TUNEL assay (Roche Diagnostics, Indianapolis, IN). A volume of 10 mL of sperm suspension was smeared onto microscope slides (positive control, negative control, and experimental samples) and fixed in 4% paraformaldehyde for 30 min at room temperature. Slides were washed in methanol-free ethanol for 5 min at room temperature, followed by 3 5 min washes at room temperature in a 1 mg/mL PVP-PBS solution. Cells were permeabilized with 0.5% Triton-X-100 in PBS for 5 min at RT, followed by 3 5 min washes at room temperature in 1 mg/mL PVP-PBS. Positive controls were incubated with 100 mL RQ1 DNAse 10 Reaction Buffer for 5 min at room temperature. Following reaction buffer incubation, 6 mL of RQ1 RNase-Free DNase was added to the mixture and incubated for an additional 30 min at room temperature. Positive control samples were washed 3 times with 1 mL of deionized water for 5 min. All positive controls, negative controls and experimental samples were incubated in 100 mL Equilibration Buffer for 5 min at room temperature. Positive (Equilibration Buffer, nucleotides, and rTDT enzyme), experimental (Equilibration Buffer, nucleotides, and rTDT enzyme), and negative control (Equilibration Buffer with 5% fetal calf serum and nucleotides) samples were covered with a cover slip
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and incubated for 1 h at 37 C in the dark. After 1 h, 1 mL of Saline Sodium Citrate solution was added to each sample and followed by incubation for 15 min at room temperature. All samples were washed in 1 mL of 0.2% Triton-X-100 for 5 min at room temperature, followed by 3 5 min washes at room temperature in 1 mL of deionized water, and then by 3 5 min washes at room temperature in 1 mL of 1 mg/mL PVP in PBS. Excess liquid was removed and 30 mL of Vectashield/DAPI Fluoromount-G (Vector Laboratories, Burlingame, CA) was added to each slide. Samples were then covered with a coverslip and allowed to dry for 15 min at room temperature before observation with a microscope equipped with epifluorescence (Olympus IX73; Olympus Corporation, Melville, NY) using SPOT software 5.0 (Diagnostic Instruments, Inc., Sterling Heights, MI). A minimum of 400 spermatozoon were analyzed per sample to determine the level of nuclear DNA fragmentation
damage. The quantification of DNA fragmentation damage was based on the intensity of fluorescence (measured by analog to digital units, ADU) within each sperm head relative to the background fluorescence [6]. TUNEL positive/negative sperm heads were classified based on the presence/absence of green fluorescence in sperm heads (Fig. 1BeG). 2.4. Oocyte collection and in vitro maturation Ovaries from domestic cats were harvested from a local spay and neuter clinic and transported at 4 C in PBS to the laboratory within 4 h post routine ovario-hysterectomy. The ovaries were sliced in H-MEM (Hepes-buffered Minimal Essential Medium, supplemented with 0.4 mM cysteine, 0.4 mM glutamate, 4.0 mM pyruvate, 100 mg/mL penicillin, 100 mg/mL streptomycin) to recover
Fig. 1. Representative morphology of cat spermatozoa after microwave-assisted drying (phase contrast; A), TUNEL assay (B, D, F) and counterstaining with Hoechst (C, E, G). Negative control (B, C), positive control (D, E), and normal rehydrated sperm sample (F, G). Bar ¼ 15 mm.
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the immature oocytes. Grade I immature oocytes, characterized by dark, homogenous cytoplasm surrounded by several layers of compacted cumulous cells, were selected for in vitro maturation, using methodology as previously described [19]. Oocytes then were cultured in vitro in Protein Plus Blastocyst Embryo Culture Medium (Sage, Trumbull, CT) supplemented with 1 mg/mL FSH (Sigma), 1 mg/ mL LH (Sigma), and mg/mL estradiol (Sigma) for 24e28 h at 38.5 C, 6.0% CO2 and 5.0% O2 in groups of 5 oocytes per 50-mL drops. Oocytes then were denuded by gentle pipetting in 0.2% cumulase (Origio, Trumbull, CT). Only oocytes with a first polar body were selected for Intracytoplasmic sperm injection (ICSI). 2.5. Intracytoplasmic Sperm Injection (ICSI) and embryo culture A volume of 2 mL of sperm suspension was added to 10 mL of 10% polyvinylpyrrolidone (PVP, Irvine Scientific) in the center drop of a 50 9 mm ICSI dish (NUNC) surrounded by 8 10 mL drops of complete Hams Hepes medium and covered with oil. Due to the poor mixing of the trehalose in PVP, for dehydrated sperm suspensions, spermatozoa were first added to a drop of Hams Hepes medium in the ICSI dish, collected, and moved to the middle PVP drop for immobilization, then aspirated into the injection pipette, and injected into a mature oocyte. In vitro matured oocytes were added to the Hams Hepes drops of the ICSI dish. The dish was maintained on a heated stage (38.5 C) of an inverted microscope (Olympus IX73) equipped with holding pipette and micromanipulators (Narishige, Sterling, VA). A single morphologically normal spermatozoon was selected, immobilized by drawing the injection pipette across the midpiece, aspirated into the injection pipette, and injected into a mature oocyte (polar body at 12 or 6 o-clock). Motile fresh spermatozoa as well as immotile dehydrated spermatozoa were immobilized across the midpiece prior to injection. Injected mature oocytes then were cultured in groups of 5 oocytes in 50 mL drops in Protein Plus Blastocyst Medium (Sage, Trumbull, CT) at 38.5 C 6% CO2 and 5% O2 for 7 days. Additional 10 mature oocytes were sham injected (no sperm) for each replicate to serve as parthenogenetic controls. 2.6. Sperm aster evaluation Presumptive zygotes were fixed at 5 h post insemination (hpi) in 2.5% paraformaldehyde for 30 min at 38.5 C, followed by 3 5 min washes in 1 PBS at room temperature. Nonspecific antigenic sites were saturated with 0.5% Triton-X-100 and 20% fetal calf serum in PBS for 30 min at 38.5 C. Presumptive zygotes were incubated overnight with anti-a-tubulin (Sigma) and anti-b-tubulin (Sigma) monoclonal antibodies (1/1000 in PBS with 0.5% Triton-X-100 and 2% fetal calf serum) at 4 C. Embryos were washed in PBS (3 15 min at room temperature), and incubated with FITC-labeled anti-mouse IgG (Sigma), diluted 1/150 in PBS, and held for 1 h at 38.5 C. Chromatin was counterstained with 10 mg/mL Hoechst 33342 in PBS for 5 min at 38.5 C, followed by mounting on microscope slides with a 30 mL volume of Vectashield and covered with a coverslip. Presumptive zygotes were observed with a confocal microscope (Olympus Fluoview Laser Scanning Microscope) to assess the size of the male and female pronuclei and the sperm aster formation according to our standard criteria (Fig. 2) [16]. Unfertilized oocytes were categorized by the absence of decondensed sperm heard or pronuclei in oocytes remained at the metaphase II stage. 2.7. Embryo assessment Embryos were assessed on Day 3 and Day 7 post fertilization (Day 0 being ICSI day) for embryo cell number and embryonic stage.
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On Day 3, embryos were observed with an inverted microscope (Olympus IX 73) with a heated stage (38.5 C), and the cell number was recorded for each embryo within 4 categories (1-cell; 2-4 cell; 5-8 cell; 9-16 cell). Following embryo assessment, embryos were returned to the incubator for further culture. On Day 7, embryos were observed and fixed on microscope slides using 2.5% paraformaldehyde incubation for 30 min at 38.5 C followed by chromatin staining with 10 mg/mL Hoechst 33342 in PBS for 5 min at 38.5 C. Embryos were mounted with Vectashield covered with a coverslip prior to imaging with epifluorescence microscopy. Embryonic cell number or stage was determined for each embryo within 6 categories (1-cell; 2-4 cell; 5-8 cell; 9-16 cell; Morula; Blastocyst). 2.8. Experimental design and statistical analysis In Experiment 1, epididymides from 6 testes per replicate (n ¼ 6 replicates) were exposed to microwave-assisted drying. In each replicate, aliquotes were analyzed at each time point (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 min) for moisture content/temperature. In Experiment 2, epididymides from 6 testes per replicate (n ¼ 4 replicates) were dried (with or without prior membrane poration). In each replicate, aliquotes were analyzed at each time point (0, 10, 20, 30 min) for morphology/DNA integrity. In Experiment 3a, epididymides from 3 testes (n ¼ 6 replicates) were exposed to 30 min of microwave-assisted drying before sperm injection into oocytes (n ¼ 350 including for sham injections) and sperm aster assessment. In Experiment 3b, epididymides from 3 testes (n ¼ 4 replicates) were exposed to 30 min of drying before injection into oocytes (n ¼ 450 including for sham injections) and assessment of embryo development. In all experiments, values were expressed as mean ± standard deviation (SD) of the multiple replicates. Percentage data were transformed using arcsine transformation before statistical analysis. Comparisons between treatments and among replicates were analyzed by analysis of variance (ANOVA), Tukey's multiple test for mean comparison, and Bartlett test for homogeneity of the variances. Data not normally distributed were analyzed by Kruskal-Wallis ANOVA on ranks and Dunn method for all pairwise comparisons. Differences were considered significant at P < 0.05 (GraphPad Software Inc.). 3. Results The average moisture content of the sperm suspension in trehalose decreased rapidly to reach 0.16 gH2O/gDW after 30 min of drying with no further decrease beyond that time point (Fig. 3A and inset). At the successive drying time points, the sample temperature progressed from 27.9 C after 5 min to a maximum of 37.3 C after 50 min (Fig. 3B). After rehydration, the percentages of sperm recovery from the coverslips (ranging from 83.5 to 89.7%) were not different across all drying time points. Additionally, gross morphology of the sperm cells was unchanged with no abnormalities on the sperm head, midpiece, and flagellum (Fig. 1A) although no sperm motility was reported after any of the drying time points. After only 10 min of dehydration, the percentage of sperm cells with damaged DNA ranged from 35.1 to 42.4% in the non-porated group exposed to trehalose which was higher (P < 0.05) than in the membrane porated counterparts (ranging from 5.2 to 7.9%). However, following membrane poration and trehalose exposure, the percentages of sperm cells with DNA damage were not different after 10, 20, or 30 min of desiccation (ranging from 4.5 to 8.2%) compared to the fresh controls (6.6± 0.5%). In TUNEL positive cells, the degree of DNA damage (ranging from 395 to 407 ADU) was not influenced by the microwave-assisted drying across time points
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Fig. 2. Confocal images of oocytes 5 h after injection with fresh or dried cat spermatozoa. Immunostaining of a-tubulin and b-tubulin (with FITC; green) and counterstaining of chromatin (with Hoechst; light blue). Oocyte with large sperm aster (A), small sperm aster (B), no sperm aster (C), non-fertilized oocytes (D). Bar ¼ 10 mm; pb: polar body, mPN: male pronucleus; fPN: female pronucleus; MII: metaphase II. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
compared to the positive sperm cells in the fresh controls (385 ± 11 ADU). Percentages of oocyte reaching the metaphase II stage ranged between 60 and 66% in all replicates. Percentages of fertilization were higher (P < 0.05) after injection of fresh spermatozoa than with dried sperm cells (Table 1). Five hours after the sperm injection, the majority of sperm asters had a small diameter regardless of the sperm treatment (Table 1). However, more large sperm asters (P < 0.05) were observed after injection with fresh than dried spermatozoa (Table 1). At that same time point, diameters of male pronuclei were similar after fresh or dried sperm injection (range, 4.9e6.3 mm) confirming that zygotes were all at a comparable stage of the first cell cycle when analyzed. None of the sham injected oocytes (parthenogenetic controls) were activated. Percentages of oocytes reaching the metaphase II stage ranged between 62 and 65% in all replicates. Percentages of fertilization were higher (P < 0.05) after injection of fresh spermatozoa than with dried sperm cells (Table 2). After 3 days of in vitro culture (Day 0 being the day of ICSI), more embryos were at the 9-16 cell stage after injection with fresh sperm cells (P < 0.05) than with dried spermatozoa while more embryos were observed at the 2-4 cell stage (P < 0.05) after the injection of the latter (Table 2). Regardless of the sperm treatment, blastomeres had equal sizes in those early embryo stages. After 7 days of in vitro culture, more embryos reached the morula or blastocyst stage (P < 0.05) after injection with fresh spermatozoa than with dried sperm cells (Table 3). With the latter, more embryos arrested their development before the 58 cell stage (P < 0.05; Table 3). However, blastomere numbers
within the blastocysts were not different between the fresh (92 ± 15) and dried (87 ± 11) sperm groups (Fig. 4). None of the sham injected oocytes (parthenogenetic controls) were activated or cleaved after 3 or 7 days of culture. 4. Discussion The moisture content reached by sperm samples after microwave-assisted drying was minimally damaging and could be compatible with storage at non-freezing temperatures. Sperm morphology and DNA integrity were not affected by membrane poration, trehalose exposure, and drying. However, the centrosomal functions of some dried sperm cells appeared to be impaired which likely led to more arrests of the embryo development before the blastocyst formation. Most of the sperm drying studies in non-rodent mammalian species have been conducted through freeze-drying and reported successful embryo development after sperm injection in cattle [20,21] and primates [22] or livebirths after transfer in rabbits [23] and horses [24]. However, the present study is the first report on sperm microwave-assisted drying in any species. Drying for 30 min at 20% power produced the lowest end moisture content with the highest estimated storage temperature. The estimated glass transition temperature (Tg) for that moisture content was approximately 7 C, based on reported values for a binary solution of trehalose and water [25]. Importantly, the sample temperature during the microwaving remained within physiological temperature range for cat spermatozoa. Although sperm motility was lost
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Fig. 3. Moisture content (average ± SD; n ¼ 6 replicates) with insert for values between 30 and 50 min drying (A) and sample temperature (average ± SD; n ¼ 6 replicates; B) of 0.3 M Trehalose in TE Buffer (40 mL) during microwaving (20% power) on coverslips in 11% Relative Humidity.
Table 1 Fertilization success and proportions of sperm aster types 5 h after injection with fresh or dried epididymal spermatozoa from domestic cats. Values are expressed mean ± SD (n ¼ 6 replicates).
Total fertilized oocytes (%) Fertilized oocyte with: Large sperm aster (%) Small sperm aster (%) No sperm aster (%)
Fresh sperm injection (n ¼ 137 injected oocytes)
Dried sperm injection (n ¼ 144 injected oocytes)
93.5 ± 1.0a
84.0 ± 2.5b
14.6 ± 3.0a 62.1 ± 4.5a 16.8 ± 1.7a
5.6 ± 1.8b 58.3 ± 2.5a 20.1 ± 4.1a
Within rows, values with different superscripts differ significantly (P < 0.05).
during the treatment, recovery and morphology of sperm cells were not affected during the drying process. This confirms what has already been observed in other cell types treated the same way
[6,26]. Inversely, freeze-dried felid cat spermatozoa have displayed evidence of surface damage, broken membranes, and decapitation [15] that were not observed here.
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Table 2 Fertilization success and proportions of different embryo stages obtained after oocyte injection with fresh or dried spermatozoa from domestic cats followed by 3 days of in vitro culture. Values are expressed mean ± SD (n ¼ 4 replicates). Total number of injected oocytes
Total fertilized oocytes (%)
Proportion of embryo stages (%) 2-4 cells
Fresh spermatozoa Dried spermatozoa
192 203
a
5-8 cells a
24.0 ± 5.1 32.0 ± 3.4b
88.6 ± 1.2 77.9 ± 7.9b
37.5 ± 10.2 29.1 ± 6.3a
9-16 cells a
27.1 ± 4.9a 16.8 ± 4.3b
Within columns, values with different superscripts differ significantly (P < 0.05).
Table 3 Proportions of different embryo stages obtained after oocyte injection with fresh or dried spermatozoa from domestic cats followed by 7 days of in vitro culture. Values are expressed mean ± SD (n ¼ 4 replicates).
Fresh spermatozoa Dried spermatozoa
Total number of injected oocytes
Proportion of embryo stages (%) 2-4 cells
5-8 cells
9-16 cells
Morulae
Blastocysts
192 203
8.9 ± 3.2a 23.2 ± 5.6b
18.2 ± 3.5a 17.2 ± 6.4a
20.3 ± 2.8a 18.7 ± 8.0a
26.6 ± 3.3a 12.3 ± 5.7b
15.1 ± 3.3a 6.4 ± 3.7b
Within columns, values with different superscripts differ significantly (P < 0.05).
The proportions of sperm cells with damaged DNA (as well as the degree of damage) were not affected by the drying for up to 30 min resulting in a low moisture content of 0.16 gH2O/gDW. Epididymal spermatozoa already have highly compacted chromatin [13] which contributes to the resilience to non-physiological conditions like cryopreservation [27] or drying like in the present study. Former studies in frozen-dried human sperm also demonstrated the resilience to DNA damage [28]. However, drying without prior membrane poration with a-hemolysin showed a significant increase in damage after only 10 min of drying. Without poration of the plasma and nuclear membranes, trehalose was unable to enter the cell and surround the nuclear chromatin, associated proteins, and centrosome, to create the protective milieu. These data confirm that trehalose is required to be present on both sides of the membrane to confer protection like in the mouse sperm cells [29]. This protective effect also is similar to previous a report on the microwave-assisted drying of germinal vesicles [6]. The fertilization success after dried sperm injection was slightly lower than with fresh spermatozoa and could not be explained by a loss of DNA integrity. One or multiple sperm components involved
in the oocyte activation may have been impaired and will have to be investigated further [30]. At 5 h post-injection, the diameter of male pronuclei (or decondensed sperm chromatin) was in the same range after fresh and dried sperm injections. Zygotes therefore were all observed at a comparable stage of the first cell cycle when analyzed [16]. The majority of smaller sperm asters observed with dried sperm cells likely revealed damaged centrosomal functions that then led to early arrest of the embryo development [16,31]. This particular detrimental effect of the desiccation has already been reported in bovine spermatozoa [32]. Strategies to better protect the centrosome or to supplement the sperm with critical proteins such as centrin [33] might be a good strategy to overcome the issue. Importantly, there likely was no embryo toxic effect of remnant trehalose as demonstrated in the mouse [34]. Embryo development in vitro obtained in the present study was in the same range as previous reports from our laboratory [16]. Previous studies form other groups on lyophilized cat spermatozoa have only been taken out to the 8-day/blastocyst stage [15]. When oocytes were activated with ethanol to over-ride deficiencies in the ability of freeze-dried spermatozoa to induce the required intracellular calcium spikes, nearly 28% of fertilized oocytes formed
Fig. 4. Micrographs or morulae (A-B, E-F) and blastocysts (C-D, G-H) after 7 days of in vitro culture, produced after fresh (A, B, C, D) or dried cat sperm (E, F, G, H) injection. Before (A, C, E, F) and after fixation and Hoechst staining (B, D, F, H). Bar ¼ 25 mm.
J.L. Patrick et al. / Theriogenology 103 (2017) 36e43
blastocysts was achieved compared to 54% in fresh controls. Same results may be expected with ejaculated spermatozoa. Feline embryo development using epididymal sperm was previously reported as 25% blastocyst development [35] and 26.8% in ejaculated feline sperm, which has been noted as equivalent to epididymal sperm in terms of embryo development potential [16]. Further studies to investigate embryonic genome activation and fetal development after embryo transfer with dried spermatozoa are warranted. This is the first in-depth assessment of structure and function in sperm cells processed by microwave-assisted drying. Although the levels of drying achieved with the current processing conditions would suggest that storage below 7 C may be necessary to maintain the sample in the glassy state, it is possible that storage slightly above Tg would be adequate for multi-year shelf-life. Storage studies at ambient and moderate refrigeration temperatures are ongoing and will further advance the fields of biobanking and fertility preservation. Acknowledgments The authors are indebted to Dr. Alida Kinney and staff at the Cabarrus Spay and Neuter Clinic for generously facilitating the transfer of resources from consenting pet-owners to UNC Charlotte. References [1] Comizzoli P, Wildt DE. Mammalian fertility preservation through cryobiology: value of classical comparative studies and the need for new preservation options. Reprod Fertil Dev 2013;26:91e8. http://dx.doi.org/10.1071/RD13259. [2] Baker M. Biorepositories: building better biobanks. Nature 2012;486:141e6. http://dx.doi.org/10.1038/486141a. [3] Crowe JH. Introduction: stabilization of dry biological materials. Biopreserv Biobank 2012;10. http://dx.doi.org/10.1089/bio.2012.1043. 375e375. [4] Loi P, Iuso D, Czernik M, Zacchini F, Ptak G. Towards storage of cells and gametes in dry form. Trends Biotechnol 2013;31:688e95. http://dx.doi.org/ 10.1016/j.tibtech.2013.09.004. [5] Graves-Herring JE, Wildt DE, Comizzoli P. Retention of structure and function of the cat germinal vesicle after air-drying and storage at suprazero temperature. Biol Reprod 2013;88:139. http://dx.doi.org/10.1095/ biolreprod.113.108472. [6] Elliott GD, Lee P-C, Paramore E, Van Vorst M, Comizzoli P. Resilience of oocyte germinal vesicles to microwave-assisted drying in the domestic cat model. Biopreserv Biobank 2015;13:164e71. http://dx.doi.org/10.1089/ bio.2014.0078. [7] Comizzoli P, Wildt DE. On the horizon for fertility preservation in domestic and wild carnivores. Reprod Domest Anim 2012;47(Suppl 6):261e5. http:// dx.doi.org/10.1111/rda.12010. [8] Wildt DE, Comizzoli P, Pukazhenthi B, Songsasen N. Lessons from biodiversitythe value of nontraditional species to advance reproductive science, conservation, and human health. Mol Reprod Dev 2010;77:397e409. http:// dx.doi.org/10.1002/mrd.21137. [9] Comizzoli P, Songsasen N, Wildt DE. Protecting and extending fertility for females of wild and endangered mammals. Cancer Treat Res 2010;156: 87e100. http://dx.doi.org/10.1007/978-1-4419-6518-9_7. [10] Larson KL, DeJonge CJ, Barnes AM, Jost LK, Evenson DP. Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques. Hum Reprod 2000;15:1717e22. ndez-Gonzalez R, Moreira PN, Pe rez-Crespo M, Sa nchez-Martín M, [11] Ferna Ramirez MA, Pericuesta E, et al. Long-term effects of mouse intracytoplasmic sperm injection with DNA-fragmented sperm on health and behavior of adult offspring. Biol Reprod 2008;78:761e72. http://dx.doi.org/10.1095/ biolreprod.107.065623. [12] Aitken RJ, Krausz C. Oxidative stress, DNA damage and the Y chromosome. Reproduction 2001;122:497e506. lez-Marín C, Gosa lvez J, Roy R. Types, causes, detection and repair of [13] Gonza DNA fragmentation in animal and human sperm cells. Int J Mol Sci 2012;13:
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