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Effect of chilling on porcine germinal vesicle stage oocytes at the subcellular level
Cryobiology 59 (2009) 54–58
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Effect of chilling on porcine germinal vesicle stage oocytes at the subcellular level q Bou Gerelchimeg, Liu Li-Qing, Zheng Zhong, Tian Jiang-Tian, Kong Qing-Ran, Song Jun, Wang Xue-Dong, Liu Zhong-Hua * College of Life Science, Northeast Agricultural University, Harbin, Heilongjiang Province 150030, China
a r t i c l e
i n f o
Article history: Received 4 December 2008 Accepted 16 April 2009 Available online 24 April 2009 Keywords: Porcine germinal vesicle stage oocyte Chilling injury Transmission electron microscope
a b s t r a c t The potential subcellular consequence of chilling on porcine germinal vesicle (GV) stage oocytes was examined. Prior to in vitro maturation (IVM), Cumulus-oocyte complexes (COCs) freshly collected from antral follicles (3–6 mm in diameter) were evenly divided into four groups and immediately incubated in PVA-TL-HEPES medium at the temperature of 39 °C (control group), 23 °C (room temperature), 15 °C and 10 °C for 10 min, respectively. Following 42 h of IVM at 39 °C, the survival rates were examined. There was no significant difference between the survival rate of 23 °C chilled group and control group (77.92 and 91.89%), but the survival rate of 15 and 10 °C chilled group were significantly decreased (46.34 and 4.81%, P < 0.01). A further experiment on15 °C group showed that most oocytes died from 2 to 4 h of IVM. In order to investigate the effects of chilling on oocytes at the subcellular level, the control and 15 °C chilled group COCs fixed at different time points of the IVM cultures (2, 2.5, 3, 3.5 and 4 h of IVM) were prepared for transmission electron microscope (TEM) observation. As the result, compared with the control group, there were two significant changes in the ultrastructural morphology of 15 °C treatment group: (1) dramatic reduction of heterogeneous lipid, (2) disorganized mitochondria–endoplasmic reticulum–lipid vesicles (M–E–L) combination. These results indicate that 15 °C is a critical chilling temperature for porcine GV stage oocyte and the alteration of cellular chemical composition and the destruction of M–E–L combination maybe responsible for chilling injury of porcine oocyte at this stage. Ó 2009 Elsevier Inc. All rights reserved.
Reproductive cells exhibit species-specific sensitivity to chilling. The porcine oocytes are excessively intolerant of low temperature compared with other mammalian oocytes. In the 1990s, Didion reported that cumulus-intact pig oocytes at the germinal vesicle stage became irreversibly damaged following exposure to 15 °C, or lower [7]; but for bovine oocytes, the irreversible damage happens at 4 °C [5]. Some calf births have been reported after transfers of embryos derived from vitrified oocytes [11,23,34,35]. However, there is no report regarding the successful production of piglet from cryopreserved porcine immature or mature oocyte. Intracellular lipid has been previously speculated to be responsible for porcine oocyte and embryo hypothermic sensitivity. Nagashima et al. [21] provided the first landmark information that intracellular lipid was associated with cooling or cryo-sensitivity of porcine embryos, and his data indicated that early cleavage stage embryos could survive after cryopreservation following delipidation. It was clear that the hypothermic tolerance of porcine oocytes and embryos was increased when their lipid content was reduced, but the mechanism remains unclear. q This study was supported by the National Natural Science Foundation of China (No.30871431). * Corresponding author. Fax: +86 0451 55190413. E-mail address: [email protected] (L. Zhong-Hua).
0011-2240/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2009.04.005
Chilling injury, a type of cell damage associated with exposure to supra zero temperature has been described in several cell types [36]. Damages associated with chilling injury include disruption of membrane [4,39], cytoskeletal organization [2,6,27,31,37,38], intracellular organelles (mitochondria, endoplasmic reticulum) [29], protein synthesis and chromosome structure [3]. Chilling injury is considered as one of key factors contributing to low survival after cryopreservation of reproductive cells, especially the oocyte and embryo [26]. A few calf births have been reported after transfers of embryos derived from vitrified immature oocytes [34,35]; however, so far, no piglet were produced from vitrified immature porcine oocytes. Researchers only succeed in producing blastocysts: Fujihira et al. reported the production of blastocysts derived from porcine vitrified GV oocytes after ICSI [9], Gupta et al. described the production of blastocysts derived from porcine vitrified GV oocytes with IVF [10], and Nakagawa et al. successfully produced blastocysts using porcine vitrified GV oocytes matured in vitro in medium containing a mitochondrial permeability transition inhibitor- ruthenium red after IVF [22]. Thus, improving our understanding in the mechanisms underlying chilling injury will certainly help in developing an effective cryopreservation method for chilled sensitive cell types including the porcine oocyte. In this study, we examined the survival rate of porcine germinal vesicle stage (GV) stage oocytes after chilling at different temper-
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atures. And we also observed the temporal cytoplasmic events at the subcellular level by transmission electron microscopy (TEM), in order to research the intrinsic cause, which induced the lethality after chilling. Materials and methods All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise indicated. Oocyte collection Ovaries were collected from slaughtered prepubertal gilts at a local abattoir, and transported to laboratory in 0.9% NaCl containing 75 lg/ml penicillin G and 50 lg/ml streptomycin at 37 °C within 2 h. Cumulus-oocyte complexes (COCs) were aspirated into PVATL-HEPES [18] from antral follicles (3–6 mm in diameter) with a 12-gauge needle connected to a 10 ml disposable syringe. Chilling treatment COCs with homogeneous cytoplasm with several layers were selected and rinsed three times in PVA-TL-HEPES at 39 °C. Then, the COCs were randomly divided into four groups in PVA-TL-HEPES medium and exposed to 39 °C (control), 23 °C (room temperature), 15 °C or 10 °C for 10 min before culture for in vitro maturation. IVM After chilling, the COCs were rinsed and cultured in TCM199 (Gibco; Invitrogen Corp., Carlsbad, CA, USA) supplemented with 0.1% (w/v) PVA, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.5 lg/ml of luteinizing hormone, 0.5 lg/ml of follicle stimulating hormone, 10 ng/ml of epidermal growth factor, 10% porcine follicular fluid, 75 lg/ml of penicillin G and 50 lg/ml of streptomycin for 42 h in a humidified atmosphere of 5% CO2 and 95% air at 39 °C. Determination of oocytes survival rate In Experiment 1, the oocyte survival rates of the four parallel groups (39, 23, 15 and 10 °C) were detected at 42 h of IVM. Furthermore, in Experiment 2, for narrowing the crucial time window of the lysing of oocyte, the survival rate of COCs in control group and 15 °C treatment group were checked at 0, 2, 4, 6 and 12 h of IVM, respectively. In order to determine the survival rate, the cumulus cells were removed from the oocytes by vortexing in PVA-TL-HEPES supplemented with 1 mg/ml hyaluronidase for 3 min. Oocytes with one of the followed criteria were considered as the survived in Experiment 1: (1) with first polar body, distinguished perivitelline space and intact membrane; (2) without polar body, but with distinguished perivitelline space, intact membrane and normal cytoplasmic transparency. For Experiment 2, the criterion is a distinguished perivitelline space, intact membrane and normal cytoplasmic transparency.
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through graded ethanol/acetone, and embedded followed by polymerization at 40 °C for 17 h, 45 °C for 24 h and 60 °C for 17 h. Ultrathin (50–70 nm) sections were cut with ultramicrotome LKB-m (LKB, Sweden). Sections were stained with uranil acetate (30 min) and Reynold lead citrate (15 min). Stained sections were observed by JEM-1200EX transmission electron microscope (JEOL, Tokyo, Japan). For those 10 groups, at least 20 COCs were examined by TEM experiment in each group. Statistical analysis One-way ANOVA on SPSS 13.0 for MicroSoftTM Windows was used to assess the differences of survival rates between groups. Duncan method was employed for pairwise comparison and followed by bonferroni correction. A difference of P < 0.01 was considered to be statistically significant. Results Survival rate of porcine GV stage oocytes treated with different temperatures As shown in Fig. 1, when porcine GV stage oocytes were exposed to 15 °C for 10 min, the survival rate significantly decreased compared to the control group (46.34% vs. 91.89%; P < 0.01) after 42 h of culture. The survival rate of oocytes exposed to room temperature (23 °C) was not significantly different from that of the control (77.92% vs. 91.89%). Since 10 °C treatment results in an extremely low survival rate (4.81%), 15 °C was chosen as the chilling temperature for further investigation. Survival rate of porcine oocytes at different time points of IVM following exposure to 39 °C (control) and 15 °C As shown in Fig. 2, the survival rate of oocytes significantly decreased during 2–4 h post chilling treatment of 15 °C for 10 min (80.15% at 2 h vs. 43.19% at 4 h, P < 0.01). Beyond 4 h, the survival rate did not significantly change. Depending on these results, oocytes at the time points of 2, 2.5, 3, 3.5 and 4 h of IVM were used to carry on TEM specimen preparation and investigation. Micrographs of porcine oocytes exposed to 39 °C (control) and 15 °C followed by IVM for 2–4 h To investigate the cryogenic effects on oocyte ultrastructure, COCs fixed at 2, 2.5, 3, 3.5 and 4 h of IVM were processed for TEM. The ultra-structures of representative oocytes are shown in
Section preparation and transmission electron microscope (TEM) observation In Experiment 3, the COCs of the control and 15 °C treatment group taken at 2, 2.5, 3, 3.5, 4 h of IVM were fixed in 1 ml fixative containing 3% glutaraldehyde and 5% paraformaldehyde (pH = 7.2). After storage in fixative at 4 °C for 1–7 days, the COCs were treated in 1% OsO4 for 2 h at room temperature, then rapidly dehydrated
Fig. 1. Survival rate of porcine GV stage oocytes treated with different temperatures. The values were assessed following 42 h of IVM. Data are presented as mean percentages ± standard deviation (mean ± SD) of seven replicates. Numbers above the bars are total numbers of oocytes examined. , Values with different symbols are significantly different (P < 0.01).
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Fig. 2. Survival rate of porcine oocytes at different time points of IVM following exposure to 39 °C (control) and 15 °C. Data are presented as mean ± SD of three replicates. Numbers above the bars are total numbers of oocytes examined. Values with different symbols are significantly different (P < 0.01).
Fig. 3. In contrast with other mammalian oocytes like mouse, human and bovine, porcine oocytes exhibit much higher electrondensity due to the numerous lipid vesicles (Fig. 3A and B). Besides a large volume of lipid vesicles and vacuoles with low electrondense granular contents, the porcine oocyte was characterized by short and thin microvilli projecting into a narrow subzonal space; highly electron-dense cortical granule mainly distributed centrally; clusters of tubular endoplasmic reticulum located under the plasma membrane; and the majority of mitochondria evenly distributed around lipid vesicles enclosed by smooth endoplasmic reticulum (sER). Hence, we named here this structure as mitochondria–smooth endoplasmic reticulum–lipid vesicles combination (M–E–L combination) (Fig. 3C). The M–E–L combination is a very typical structure in porcine GV stage oocytes [13]. In this combination, mitochondria with densely arranged cristae were closely adhering to sER, which enclosed the lipid vesicle (Fig. 3D), and sometimes extended to a tubular structure. In porcine oocytes, lipid vesicles divided into two categories: homogeneous lipid vesicle and heterogeneous lipid vesicle (Fig. 3E). The homogeneous lipid vesicle is characterized by even and higher electron-density than the heterogeneous lipid vesicle and generally, the two kinds of lipid vesicle are linked to each other. Compared with the control group, we were surprised to find that instead of two kinds of large lipid vesicles, many small, spherical, homogeneous lipid vesicles surrounded by vacuoles appeared in some oocytes at 3.5 h post chilling (Fig. 3F–I). And at 4 h post chilling, the sER also appeared severely fragmentated (Fig. 3J). Mitochondria exhibited a constant dysmorphosis, crenated morphology and reduction in cristae in the majority of the chilled oocytes from all time points (Fig. 3K–M). Discussion First of all, the present study confirmed Didion’s observation [7] that for porcine GV stage oocytes, 15 °C is a critical low temperature to which oocytes are sensitive. While cooled at 15 °C for 10 min, less than 50% of porcine immature oocytes remain survived, and less than 5% survived cooling to 10 °C or below. The lysis peak was occurred 2–4 h after cooling to 15 °C. It seems that the GV stage was thermally sensitive and keeping a proper temperature around this phase is crucial in achieving maximum developmental potential. The sharp decrease in survival rate in the chilled group encouraged us to observe subcellular changes occurred in the oocyte cytoplasm 2–4 h after cooling to 15 °C. We found that change
in M–E–L combination is the most evident one. Sathananthan has described the location and relation between sER and lipid vesicle in mouse and human oocyte [28], and Kruip has mentioned that sER is associated with lipid droplets and mitochondria in bovine oocyte maturated in vivo [16]. In our observation, the majority of these three organelles were organized into this special combination. In detail, M–E–L combination in porcine oocyte is comprised of a lipid vesicle with encircling sER and associated mitochondria. It is documented that the fatty acid content in pig oocytes is about three times the amount in cattle and double amount in sheep oocytes [20]. These endogenous lipid vesicles are the storage form of triglycerides (TG) [8]. As a reservoir of latent energy for oocyte maturation, TG is necessary to porcine oocyte. Sturmey et al. pointed out the role of endogenous lipid in the provision of energy during in vitro maturation of immature porcine oocytes [32] and strongly evidenced the existence of metabolic cooperation between mitochondria and endogenous lipids [33]. Lipids maybe transferred to mitochondria through sER [25]. Thus, at least, the M–E–L combination must play an indispensable role in oocyte maturation and involve ATP provision to protein synthesis, organelle migration, cytoskeletal organization, GVBD, meiotic maturation and lipid provision to membrane metabolism and self-renewal. However, 4 h after chilling the combination exhibited severe disruption and disarrangement, indicating that chilling results in irreversible damage to the most important metabolic unit in the porcine oocyte. The present study is the first to demonstrate the events of M–E–L disruption caused by chilling in the porcine immature oocyte. Chilling treatment causes two changes in lipid vesicle: the disappearance of heterogeneous lipid vesicle and the appearance of numerous small homogenous lipid globules. Isachenko has described the existence of two kind of lipid vesicle with different patterns of electron density and the bias of ER adhering to heterogeneous lipid vesicle in porcine oocyte [13]. So far, the existence of two kinds of lipid vesicle was not found in other mammalian oocyte [8,28]. Isachenko et al. deduced that homogenous lipid comes from heterogeneous lipid through the lipolysis [13]. The disappearance of heterogeneous lipid indicates that chilling treatment altered the physicochemical features of the lipid component. It was also noted that the freeze/thaw process might alter the physicochemical properties of intracellular lipids and the lipid property change became irreversible after 48 h of IVM [13]. Additionally, when COCs were cooled at 24 °C for various times in the medium with or without DMSO, the rate of spindle formation, nuclear maturation and cytoplasmic maturation were decreased [17]. It is possible that the formation of numerous small lipid globules from larger vesicles would impair surrounding organelle and cytoskeleton. Hence, This damage could be decreased by lipid removal as well as pretreatment with cytochalasin B [9,12,14,24]. ER commonly has three major functions in eukaryotic cells: protein folding, lipid and sterol biosynthesis, and storing intracellular Ca2+ and activates signaling pathway [30]. Mattioli et al. reported an increase in intracellular Ca2+ concentration of porcine GV oocytes after exposure of oocytes to a temperature of 14 °C and rewarming at 37 °C. After the addition of inhibitors of ryanodine dependent Ca2+ stores reduced chilling injuries to porcine immature oocyte [19]. It is not clear that if the segmentation of ER observed in chilled oocytes was also the one cause of intracellular Ca2+ rise. Mitochondria have a major role in metabolism. However, in our study the double-membraned mitochondria showed severe degeneration in chilled groups fixed at each time point. It is suggested that the mitochondrial membrane is different from the plasma membrane, and more sensitive to cooling than plasma membrane. Little attention has been paid to mitochondria during chilling or cryopreservation of immature oocytes. It has been reported that
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Fig. 3. Micrographs of porcine oocytes exposed to 39 °C (control) and 15 °C followed by IVM for 2 to 4 h. (A) Full view of COCs from 2 h control group. (B) Local Magnification of COCs specimen of 2 h control group. (C) Cluster of endoplasmic reticulum and mitochondria under plasma membrane in another oocyte in 2 h control group. (D) Typical feature of M–E–L combination, from 2.5 h control group. (E) Connection between two kinds of lipid vesicles. (F) Average lipid status in 3.5 h control group’s specimens. (G–I) Formation of small, spherical, homogenous lipid vesicles in 3.5 h chilled group’s specimens. (J) Architectural disruption appeared in 4 h chilled group’s specimens. (K–M) Representative ultrastructural feature of MT in chilled groups. Separately showed specimen of 2, 3, 4 h group. CC, cumulus cell, ZP, zona pellucida, Oo, oocyte, LV, lipid vesicle, Ve, vesicle. ER, endoplasmic reticulum, MT, mitochondria, HoLV, homogeneous lipid vesicle, HeLV, heterogeneous lipid vesicle, Va, vacuole. CG, cortical granule. Scale bar in (A and B) represents 10 lm and in (C–M) represents 1 lm.
the intracellular Ca2+ overload caused reduction in mitochondrial membrane potential, and potentially lead mitochondrial permeability transition in mammalian cryopreserved oocytes and embryos [1,15]. Recently, it was found that using drugs to block mitochondrial permeability transition or control the level of intracellular Ca2+ could improve the in vitro maturation rate of porcine oocytes vitrified at the GV stage [22]. The improvement in cryopreservation technology of in vitro produced porcine oocytes will facilitate studies on pig cloning and the establishment of a gene bank for transgenic pigs. However, chilling injury has been the biggest barrier to achieve successful cryopreservation of porcine oocyte, especially of immature oocyte. Because of the special features, even the most standard and successful procedures developed for other mammalian species has not been successfully applied to pig oocytes. There are only three
reports of successful production of blastocysts from porcine vitrified GV oocytes [9,10,22]. The obtained results showed that different from other mammalian oocytes, there is a special organizational pattern for cell organelles in porcine oocyte, which is vulnerable and indispensable to further development and should be specially protected during cooling and warming. According to the ultrastructural level changes observed via TEM, it is supposed that the irreversible damage to the internal functional architecture of the oocyte (especially, M–E–L combination) and adverse alteration to cellular chemical composition (e.g. lipid conversion) may partially account for the low viability of porcine oocytes after chilling at 15 °C or below. In order to understand the full mechanism and take proper measures to improve porcine oocyte cryopreservation, further investigation based on the present result would be carried out in the future.
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Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Effect of chilling on porcine germinal vesicle stage oocytes at the subcellular level q Bou Gerelchimeg, Liu Li-Qing, Zheng Zhong, Tian Jiang-Tian, Kong Qing-Ran, Song Jun, Wang Xue-Dong, Liu Zhong-Hua * College of Life Science, Northeast Agricultural University, Harbin, Heilongjiang Province 150030, China
a r t i c l e
i n f o
Article history: Received 4 December 2008 Accepted 16 April 2009 Available online 24 April 2009 Keywords: Porcine germinal vesicle stage oocyte Chilling injury Transmission electron microscope
a b s t r a c t The potential subcellular consequence of chilling on porcine germinal vesicle (GV) stage oocytes was examined. Prior to in vitro maturation (IVM), Cumulus-oocyte complexes (COCs) freshly collected from antral follicles (3–6 mm in diameter) were evenly divided into four groups and immediately incubated in PVA-TL-HEPES medium at the temperature of 39 °C (control group), 23 °C (room temperature), 15 °C and 10 °C for 10 min, respectively. Following 42 h of IVM at 39 °C, the survival rates were examined. There was no significant difference between the survival rate of 23 °C chilled group and control group (77.92 and 91.89%), but the survival rate of 15 and 10 °C chilled group were significantly decreased (46.34 and 4.81%, P < 0.01). A further experiment on15 °C group showed that most oocytes died from 2 to 4 h of IVM. In order to investigate the effects of chilling on oocytes at the subcellular level, the control and 15 °C chilled group COCs fixed at different time points of the IVM cultures (2, 2.5, 3, 3.5 and 4 h of IVM) were prepared for transmission electron microscope (TEM) observation. As the result, compared with the control group, there were two significant changes in the ultrastructural morphology of 15 °C treatment group: (1) dramatic reduction of heterogeneous lipid, (2) disorganized mitochondria–endoplasmic reticulum–lipid vesicles (M–E–L) combination. These results indicate that 15 °C is a critical chilling temperature for porcine GV stage oocyte and the alteration of cellular chemical composition and the destruction of M–E–L combination maybe responsible for chilling injury of porcine oocyte at this stage. Ó 2009 Elsevier Inc. All rights reserved.
Reproductive cells exhibit species-specific sensitivity to chilling. The porcine oocytes are excessively intolerant of low temperature compared with other mammalian oocytes. In the 1990s, Didion reported that cumulus-intact pig oocytes at the germinal vesicle stage became irreversibly damaged following exposure to 15 °C, or lower [7]; but for bovine oocytes, the irreversible damage happens at 4 °C [5]. Some calf births have been reported after transfers of embryos derived from vitrified oocytes [11,23,34,35]. However, there is no report regarding the successful production of piglet from cryopreserved porcine immature or mature oocyte. Intracellular lipid has been previously speculated to be responsible for porcine oocyte and embryo hypothermic sensitivity. Nagashima et al. [21] provided the first landmark information that intracellular lipid was associated with cooling or cryo-sensitivity of porcine embryos, and his data indicated that early cleavage stage embryos could survive after cryopreservation following delipidation. It was clear that the hypothermic tolerance of porcine oocytes and embryos was increased when their lipid content was reduced, but the mechanism remains unclear. q This study was supported by the National Natural Science Foundation of China (No.30871431). * Corresponding author. Fax: +86 0451 55190413. E-mail address: [email protected] (L. Zhong-Hua).
0011-2240/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2009.04.005
Chilling injury, a type of cell damage associated with exposure to supra zero temperature has been described in several cell types [36]. Damages associated with chilling injury include disruption of membrane [4,39], cytoskeletal organization [2,6,27,31,37,38], intracellular organelles (mitochondria, endoplasmic reticulum) [29], protein synthesis and chromosome structure [3]. Chilling injury is considered as one of key factors contributing to low survival after cryopreservation of reproductive cells, especially the oocyte and embryo [26]. A few calf births have been reported after transfers of embryos derived from vitrified immature oocytes [34,35]; however, so far, no piglet were produced from vitrified immature porcine oocytes. Researchers only succeed in producing blastocysts: Fujihira et al. reported the production of blastocysts derived from porcine vitrified GV oocytes after ICSI [9], Gupta et al. described the production of blastocysts derived from porcine vitrified GV oocytes with IVF [10], and Nakagawa et al. successfully produced blastocysts using porcine vitrified GV oocytes matured in vitro in medium containing a mitochondrial permeability transition inhibitor- ruthenium red after IVF [22]. Thus, improving our understanding in the mechanisms underlying chilling injury will certainly help in developing an effective cryopreservation method for chilled sensitive cell types including the porcine oocyte. In this study, we examined the survival rate of porcine germinal vesicle stage (GV) stage oocytes after chilling at different temper-
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atures. And we also observed the temporal cytoplasmic events at the subcellular level by transmission electron microscopy (TEM), in order to research the intrinsic cause, which induced the lethality after chilling. Materials and methods All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise indicated. Oocyte collection Ovaries were collected from slaughtered prepubertal gilts at a local abattoir, and transported to laboratory in 0.9% NaCl containing 75 lg/ml penicillin G and 50 lg/ml streptomycin at 37 °C within 2 h. Cumulus-oocyte complexes (COCs) were aspirated into PVATL-HEPES [18] from antral follicles (3–6 mm in diameter) with a 12-gauge needle connected to a 10 ml disposable syringe. Chilling treatment COCs with homogeneous cytoplasm with several layers were selected and rinsed three times in PVA-TL-HEPES at 39 °C. Then, the COCs were randomly divided into four groups in PVA-TL-HEPES medium and exposed to 39 °C (control), 23 °C (room temperature), 15 °C or 10 °C for 10 min before culture for in vitro maturation. IVM After chilling, the COCs were rinsed and cultured in TCM199 (Gibco; Invitrogen Corp., Carlsbad, CA, USA) supplemented with 0.1% (w/v) PVA, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.5 lg/ml of luteinizing hormone, 0.5 lg/ml of follicle stimulating hormone, 10 ng/ml of epidermal growth factor, 10% porcine follicular fluid, 75 lg/ml of penicillin G and 50 lg/ml of streptomycin for 42 h in a humidified atmosphere of 5% CO2 and 95% air at 39 °C. Determination of oocytes survival rate In Experiment 1, the oocyte survival rates of the four parallel groups (39, 23, 15 and 10 °C) were detected at 42 h of IVM. Furthermore, in Experiment 2, for narrowing the crucial time window of the lysing of oocyte, the survival rate of COCs in control group and 15 °C treatment group were checked at 0, 2, 4, 6 and 12 h of IVM, respectively. In order to determine the survival rate, the cumulus cells were removed from the oocytes by vortexing in PVA-TL-HEPES supplemented with 1 mg/ml hyaluronidase for 3 min. Oocytes with one of the followed criteria were considered as the survived in Experiment 1: (1) with first polar body, distinguished perivitelline space and intact membrane; (2) without polar body, but with distinguished perivitelline space, intact membrane and normal cytoplasmic transparency. For Experiment 2, the criterion is a distinguished perivitelline space, intact membrane and normal cytoplasmic transparency.
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through graded ethanol/acetone, and embedded followed by polymerization at 40 °C for 17 h, 45 °C for 24 h and 60 °C for 17 h. Ultrathin (50–70 nm) sections were cut with ultramicrotome LKB-m (LKB, Sweden). Sections were stained with uranil acetate (30 min) and Reynold lead citrate (15 min). Stained sections were observed by JEM-1200EX transmission electron microscope (JEOL, Tokyo, Japan). For those 10 groups, at least 20 COCs were examined by TEM experiment in each group. Statistical analysis One-way ANOVA on SPSS 13.0 for MicroSoftTM Windows was used to assess the differences of survival rates between groups. Duncan method was employed for pairwise comparison and followed by bonferroni correction. A difference of P < 0.01 was considered to be statistically significant. Results Survival rate of porcine GV stage oocytes treated with different temperatures As shown in Fig. 1, when porcine GV stage oocytes were exposed to 15 °C for 10 min, the survival rate significantly decreased compared to the control group (46.34% vs. 91.89%; P < 0.01) after 42 h of culture. The survival rate of oocytes exposed to room temperature (23 °C) was not significantly different from that of the control (77.92% vs. 91.89%). Since 10 °C treatment results in an extremely low survival rate (4.81%), 15 °C was chosen as the chilling temperature for further investigation. Survival rate of porcine oocytes at different time points of IVM following exposure to 39 °C (control) and 15 °C As shown in Fig. 2, the survival rate of oocytes significantly decreased during 2–4 h post chilling treatment of 15 °C for 10 min (80.15% at 2 h vs. 43.19% at 4 h, P < 0.01). Beyond 4 h, the survival rate did not significantly change. Depending on these results, oocytes at the time points of 2, 2.5, 3, 3.5 and 4 h of IVM were used to carry on TEM specimen preparation and investigation. Micrographs of porcine oocytes exposed to 39 °C (control) and 15 °C followed by IVM for 2–4 h To investigate the cryogenic effects on oocyte ultrastructure, COCs fixed at 2, 2.5, 3, 3.5 and 4 h of IVM were processed for TEM. The ultra-structures of representative oocytes are shown in
Section preparation and transmission electron microscope (TEM) observation In Experiment 3, the COCs of the control and 15 °C treatment group taken at 2, 2.5, 3, 3.5, 4 h of IVM were fixed in 1 ml fixative containing 3% glutaraldehyde and 5% paraformaldehyde (pH = 7.2). After storage in fixative at 4 °C for 1–7 days, the COCs were treated in 1% OsO4 for 2 h at room temperature, then rapidly dehydrated
Fig. 1. Survival rate of porcine GV stage oocytes treated with different temperatures. The values were assessed following 42 h of IVM. Data are presented as mean percentages ± standard deviation (mean ± SD) of seven replicates. Numbers above the bars are total numbers of oocytes examined. , Values with different symbols are significantly different (P < 0.01).
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Fig. 2. Survival rate of porcine oocytes at different time points of IVM following exposure to 39 °C (control) and 15 °C. Data are presented as mean ± SD of three replicates. Numbers above the bars are total numbers of oocytes examined. Values with different symbols are significantly different (P < 0.01).
Fig. 3. In contrast with other mammalian oocytes like mouse, human and bovine, porcine oocytes exhibit much higher electrondensity due to the numerous lipid vesicles (Fig. 3A and B). Besides a large volume of lipid vesicles and vacuoles with low electrondense granular contents, the porcine oocyte was characterized by short and thin microvilli projecting into a narrow subzonal space; highly electron-dense cortical granule mainly distributed centrally; clusters of tubular endoplasmic reticulum located under the plasma membrane; and the majority of mitochondria evenly distributed around lipid vesicles enclosed by smooth endoplasmic reticulum (sER). Hence, we named here this structure as mitochondria–smooth endoplasmic reticulum–lipid vesicles combination (M–E–L combination) (Fig. 3C). The M–E–L combination is a very typical structure in porcine GV stage oocytes [13]. In this combination, mitochondria with densely arranged cristae were closely adhering to sER, which enclosed the lipid vesicle (Fig. 3D), and sometimes extended to a tubular structure. In porcine oocytes, lipid vesicles divided into two categories: homogeneous lipid vesicle and heterogeneous lipid vesicle (Fig. 3E). The homogeneous lipid vesicle is characterized by even and higher electron-density than the heterogeneous lipid vesicle and generally, the two kinds of lipid vesicle are linked to each other. Compared with the control group, we were surprised to find that instead of two kinds of large lipid vesicles, many small, spherical, homogeneous lipid vesicles surrounded by vacuoles appeared in some oocytes at 3.5 h post chilling (Fig. 3F–I). And at 4 h post chilling, the sER also appeared severely fragmentated (Fig. 3J). Mitochondria exhibited a constant dysmorphosis, crenated morphology and reduction in cristae in the majority of the chilled oocytes from all time points (Fig. 3K–M). Discussion First of all, the present study confirmed Didion’s observation [7] that for porcine GV stage oocytes, 15 °C is a critical low temperature to which oocytes are sensitive. While cooled at 15 °C for 10 min, less than 50% of porcine immature oocytes remain survived, and less than 5% survived cooling to 10 °C or below. The lysis peak was occurred 2–4 h after cooling to 15 °C. It seems that the GV stage was thermally sensitive and keeping a proper temperature around this phase is crucial in achieving maximum developmental potential. The sharp decrease in survival rate in the chilled group encouraged us to observe subcellular changes occurred in the oocyte cytoplasm 2–4 h after cooling to 15 °C. We found that change
in M–E–L combination is the most evident one. Sathananthan has described the location and relation between sER and lipid vesicle in mouse and human oocyte [28], and Kruip has mentioned that sER is associated with lipid droplets and mitochondria in bovine oocyte maturated in vivo [16]. In our observation, the majority of these three organelles were organized into this special combination. In detail, M–E–L combination in porcine oocyte is comprised of a lipid vesicle with encircling sER and associated mitochondria. It is documented that the fatty acid content in pig oocytes is about three times the amount in cattle and double amount in sheep oocytes [20]. These endogenous lipid vesicles are the storage form of triglycerides (TG) [8]. As a reservoir of latent energy for oocyte maturation, TG is necessary to porcine oocyte. Sturmey et al. pointed out the role of endogenous lipid in the provision of energy during in vitro maturation of immature porcine oocytes [32] and strongly evidenced the existence of metabolic cooperation between mitochondria and endogenous lipids [33]. Lipids maybe transferred to mitochondria through sER [25]. Thus, at least, the M–E–L combination must play an indispensable role in oocyte maturation and involve ATP provision to protein synthesis, organelle migration, cytoskeletal organization, GVBD, meiotic maturation and lipid provision to membrane metabolism and self-renewal. However, 4 h after chilling the combination exhibited severe disruption and disarrangement, indicating that chilling results in irreversible damage to the most important metabolic unit in the porcine oocyte. The present study is the first to demonstrate the events of M–E–L disruption caused by chilling in the porcine immature oocyte. Chilling treatment causes two changes in lipid vesicle: the disappearance of heterogeneous lipid vesicle and the appearance of numerous small homogenous lipid globules. Isachenko has described the existence of two kind of lipid vesicle with different patterns of electron density and the bias of ER adhering to heterogeneous lipid vesicle in porcine oocyte [13]. So far, the existence of two kinds of lipid vesicle was not found in other mammalian oocyte [8,28]. Isachenko et al. deduced that homogenous lipid comes from heterogeneous lipid through the lipolysis [13]. The disappearance of heterogeneous lipid indicates that chilling treatment altered the physicochemical features of the lipid component. It was also noted that the freeze/thaw process might alter the physicochemical properties of intracellular lipids and the lipid property change became irreversible after 48 h of IVM [13]. Additionally, when COCs were cooled at 24 °C for various times in the medium with or without DMSO, the rate of spindle formation, nuclear maturation and cytoplasmic maturation were decreased [17]. It is possible that the formation of numerous small lipid globules from larger vesicles would impair surrounding organelle and cytoskeleton. Hence, This damage could be decreased by lipid removal as well as pretreatment with cytochalasin B [9,12,14,24]. ER commonly has three major functions in eukaryotic cells: protein folding, lipid and sterol biosynthesis, and storing intracellular Ca2+ and activates signaling pathway [30]. Mattioli et al. reported an increase in intracellular Ca2+ concentration of porcine GV oocytes after exposure of oocytes to a temperature of 14 °C and rewarming at 37 °C. After the addition of inhibitors of ryanodine dependent Ca2+ stores reduced chilling injuries to porcine immature oocyte [19]. It is not clear that if the segmentation of ER observed in chilled oocytes was also the one cause of intracellular Ca2+ rise. Mitochondria have a major role in metabolism. However, in our study the double-membraned mitochondria showed severe degeneration in chilled groups fixed at each time point. It is suggested that the mitochondrial membrane is different from the plasma membrane, and more sensitive to cooling than plasma membrane. Little attention has been paid to mitochondria during chilling or cryopreservation of immature oocytes. It has been reported that
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Fig. 3. Micrographs of porcine oocytes exposed to 39 °C (control) and 15 °C followed by IVM for 2 to 4 h. (A) Full view of COCs from 2 h control group. (B) Local Magnification of COCs specimen of 2 h control group. (C) Cluster of endoplasmic reticulum and mitochondria under plasma membrane in another oocyte in 2 h control group. (D) Typical feature of M–E–L combination, from 2.5 h control group. (E) Connection between two kinds of lipid vesicles. (F) Average lipid status in 3.5 h control group’s specimens. (G–I) Formation of small, spherical, homogenous lipid vesicles in 3.5 h chilled group’s specimens. (J) Architectural disruption appeared in 4 h chilled group’s specimens. (K–M) Representative ultrastructural feature of MT in chilled groups. Separately showed specimen of 2, 3, 4 h group. CC, cumulus cell, ZP, zona pellucida, Oo, oocyte, LV, lipid vesicle, Ve, vesicle. ER, endoplasmic reticulum, MT, mitochondria, HoLV, homogeneous lipid vesicle, HeLV, heterogeneous lipid vesicle, Va, vacuole. CG, cortical granule. Scale bar in (A and B) represents 10 lm and in (C–M) represents 1 lm.
the intracellular Ca2+ overload caused reduction in mitochondrial membrane potential, and potentially lead mitochondrial permeability transition in mammalian cryopreserved oocytes and embryos [1,15]. Recently, it was found that using drugs to block mitochondrial permeability transition or control the level of intracellular Ca2+ could improve the in vitro maturation rate of porcine oocytes vitrified at the GV stage [22]. The improvement in cryopreservation technology of in vitro produced porcine oocytes will facilitate studies on pig cloning and the establishment of a gene bank for transgenic pigs. However, chilling injury has been the biggest barrier to achieve successful cryopreservation of porcine oocyte, especially of immature oocyte. Because of the special features, even the most standard and successful procedures developed for other mammalian species has not been successfully applied to pig oocytes. There are only three
reports of successful production of blastocysts from porcine vitrified GV oocytes [9,10,22]. The obtained results showed that different from other mammalian oocytes, there is a special organizational pattern for cell organelles in porcine oocyte, which is vulnerable and indispensable to further development and should be specially protected during cooling and warming. According to the ultrastructural level changes observed via TEM, it is supposed that the irreversible damage to the internal functional architecture of the oocyte (especially, M–E–L combination) and adverse alteration to cellular chemical composition (e.g. lipid conversion) may partially account for the low viability of porcine oocytes after chilling at 15 °C or below. In order to understand the full mechanism and take proper measures to improve porcine oocyte cryopreservation, further investigation based on the present result would be carried out in the future.
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