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Morphological features and microtubular changes in vitrified ovine oocytes
Theriogenology xxx (xxxx) xxx
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Morphological features and microtubular changes in vitrified ovine oocytes Elisa Serra 1, Sergio Domenico Gadau*, 1, Fiammetta Berlinguer, Salvatore Naitana, Sara Succu Department of Veterinary Medicine, University of Sassari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 July 2019 Received in revised form 31 October 2019 Accepted 9 November 2019 Available online xxx
Cryobanking of oocytes collected from prepubertal donors may supply a virtually unlimited number of female gametes for both basic research and commercial applications. Prepubertal oocytes show some structural and functional limitations compared to the adult ones that may impair their ability to recover damages from cryopreservation. In oocytes, the meiotic spindle is acutely sensitive to temperature deviation, but capable of regeneration following cryopreservation. In the present work, we studied the effects of vitrification and post-warming incubation on the microtubular cytoskeleton and the tubulin post-translational modifications (tyrosination and acetylation) in prepubertal and adult oocytes. Obtained results showed that prepubertal oocytes are more affected by vitrification-induced injuries than adult ones. In fact, prepubertal oocytes showed more severe alterations of the meiotic spindle conformation and a higher percentage of parthenogenetic activation compared to adult ones. Moreover, in the adult oocytes the equilibrium between tyrosinated and acetylated a-tubulin was restored after 4 h of post-warming incubation. Diversely, in prepubertal oocytes the imbalance between tyrosinated and acetylated a-tubulin was increased during post-warming incubation. Our study shows that prepubertal oocytes react differently to the insults provoked by vitrification compared to adult oocytes, showing an impaired ability to recover from vitrification-induced injuries. In the evaluation of oocyte ability to recover from vitrification-induced injuries, tubulin post-translational modifications represent an important indicator for assessing oocyte quality. © 2019 Elsevier Inc. All rights reserved.
Keywords: Ovine oocytes Vitrification Tubulin post-translational modifications Microtubular network
1. Introduction The demand for accelerated genetic gain of farm animals has promoted the use of prepubertal animals as a source of oocytes for in vitro embryo production (IVP). Different studies on the development of the prepubertal ovine ovary [1e3] have evidenced that at 4 weeks of age lambs could be excellent donors, when the maximal numbers of oocytes are available [4]. Thus, large numbers of ovaries of prepubertal ruminants can be easily obtained from the slaughterhouses and many oocytes can be incorporated into an IVP system. Moreover, female gamete banking through oocyte cryopreservation may simplify the management of genetic resources and improve basic research. For these reasons, the cryopreservation
* Corresponding author. Department of Veterinary Medicine, University of Sassari, Via Vienna 2, 07100, Italy. E-mail address: [email protected] (S.D. Gadau). 1 Serra Elisa and Gadau Sergio Domenico contributed equally to this work.
of oocytes collected from prepubertal donors is considered as a tool to supply a virtually unlimited number of female gametes for both basic research and commercial applications. However, prepubertal oocytes show some structural and functional limitations compared to the adult ones, such as small size, defective coupling between cumulus cells and oocytes, decrease in amino acid uptake, reduced protein synthesis and energy metabolism, and low developmental competence [5e9]. One of the most frequent problems during the maturation of prepubertal oocytes is linked to the inclination of them to chromosomal segregation errors, due to defects in the functioning of the microtubular network involved in spindle formation [10]. The cytoskeleton network of the oocyte has a peculiar arrangement of microtubules and plays an important role during the maturation and the fertilization of the oocyte as well as in the early embryonic development [11]. The complex operations that take place during the formation of the meiotic spindle, its coupling to the chromosomes and their consequent distribution are inherent
https://doi.org/10.1016/j.theriogenology.2019.11.007 0093-691X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Serra E et al., Morphological features and microtubular changes in vitrified ovine oocytes, Theriogenology, https:// doi.org/10.1016/j.theriogenology.2019.11.007
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in the microtubular network dynamic [12]. Microtubules are hollow cylinders made up by 13 protofilaments formed by association “head-to-tail” of a/b tubulin heterodimers [13]. Many of the functions that microtubules play in the cell (cell division, cell shape maintenance, movement of cytoplasmic organelles, etc.) are regulated by their post-translational modifications (PTMs) which affect the carboxy-terminal of a (mainly) and b-tubulin. Each of these PTMs may coexist in the same microtubule, displaying affinity for a whole series of associated and motor proteins, giving rise to that complex architecture that goes by the name of “tubulin code” [14e17]. In a previous work [18] we found that the most abundant tubulin PTMs in the meiotic spindle of ovine oocytes are the tyrosinated and the acetylated ones, and that prepubertal and adult oocytes show several major differences in their tubulin PTMs. Several studies have shown that the meiotic spindle is acutely sensitive to temperature deviation [19]. Exposure to cryoprotectants and to the cold temperatures lead to the disruption of microtubules and the change of the meiotic spindle morphology of mature oocytes [20e26]. It also emerged that the meiotic spindle is capable of regeneration following cryopreservation [24,27]. However, the window of opportunity for spindle recovery is not unlimited. The optimal time for post-warming recovery seems to vary depending on the method of cryopreservation, initial oocyte quality, donor age, method of spindle assessment and species [28e32]. Leaving oocytes for longer than “optimal time” will result in progressive loss of bipolar spindle structure coincident with chromosome displacement. Starting from these premises, the aim of this work was to investigate the efficiency of prepubertal ovine oocytes in restoring the injuries on the meiotic spindle caused by vitrification by evaluating the changes in tubulin PTMs during the post-warming recovery time. These changes were evaluated by comparing adult and prepubertal oocytes, representing a model of high and low developmental competence, respectively [6,7,9,33]. This comparative analysis is essential to establish reference data that could indicate important mechanisms involved in the acquisition of developmental competence and in the different susceptibility to cryopreservation procedures.
as previously described [18]. Briefly, collected ovaries were transported from the commercial slaughterhouse to the laboratory within 1e2 h in Dulbecco Phosphate Buffered Saline (PBS) with antibiotics at 27 C. After being washed in PBS fresh medium, the ovaries were sliced using a micro-blade and the follicle content was released in medium TCM199 (with Earle’s salts and bicarbonate, HEPES 25 mmol, penicillin 0.1 g/L, streptomycin 0.1 g/L and 0.1% (w/ v) polyvinylalcohol-PVA). Cumuluseoocyte complexes (COCs) selected for in vitro culture, were matured in TCM 199 medium (supplemented with 10% heat-treated oestrus sheep serum (OSS), 1 IU/mL of FSH and 1 IU/mL of LH, 100 mM cysteamine and 8 mg/mL of pyruvate) in standard conditions (5% CO2 in air at 39 C for 22 h). After in vitro maturation, COCs were denuded of cumulus cells mechanically and only those at metaphase II (MII), that is, showing the extrusion of the first polar body, were selected. The oocytes were collected in a common pool and randomly assigning for vitrification (adult n ¼ 388; prepubertal n ¼ 312) or examined as CTR (adult n ¼ 164; prepubertal n ¼ 187). 2.3. Oocyte vitrification and warming Vitrification was performed following the minimum essential volume method, according to Succu et al. [34]. Briefly, after maturation a group of 5 MII oocytes were equilibrated in base medium (BM, PBS without Caþþ and Mgþþ plus 20% fetal calf serum) at 39 C for 2 min. Then, oocytes were equilibrated in a solution containing 7.5% (v/v) dimethyl sulfoxide (DMSO) and 7.5% (v/v) ethylene glycol (EG) in BM for 3 min and transferred to vitrification solution (VS) containing 16.5% (v/v) DMSO, 16.5% (v/v) EG and 0.5 M trehalose in BM. Finally, oocytes were loaded in cryotop devices (Kitazato Ltd., Tokyo, Japan) and directly plunged into liquid nitrogen within 20e25 s. For warming to a biological temperature, the content of cryotop was transferred from liquid nitrogen into 200 mL drops of 1.25 M trehalose in BM for 1 min at 38 C. To promote removal of intracellular cryoprotectants, oocytes were transferred stepwise into 200 mL drops of decreasing trehalose solutions (0.5, 0.25 and 0.125 M trehalose in BM) for 30 s before being equilibrated for 10 min in BM. 2.4. Oocytes immunostaining
2. Materials and methods All chemicals in this study were obtained from Sigma Chemical CO. (St. Louis, MO, USA) unless stated otherwise. 2.1. Experimental design A total of 1051 (adult: sheep 4e6 years old, n ¼ 552; prepubertal: lamb 30e40 days old, n ¼ 499) in vitro matured oocytes were used for this study. After vitrification and warming, oocytes were incubated for 6 h in PBS/20% FCS in 5% CO2 in air at 39 C. At specific time points of post-warming incubation (0, 2, 4 and 6 h), vitrified/ warmed oocytes were retrieved from the culture system and processed by indirect immunofluorescence for a-total tubulin detection with the aim to evaluate: i) the morphology of meiotic spindle (length, width and area); ii) spindle conformation and chromosomal organization; iii) parthenogenetic activation rate. Moreover, the pattern for both tyrosinated and acetylated a-tubulin were analyzed in all experimental groups. For each experimental procedure a pool of both adult and prepubertal in vitro matured oocytes, not vitrified, were used as a control group (CTR). 2.2. Oocytes collection and in vitro maturation The oocytes collection and in vitro maturation were performed
Immunostaining was performed following the methodology illustrated previously [18]. Briefly, fixed warmed and CTR oocytes have been processed through serial incubations (1 h per antibody at 37 C) with the following primary antibodies: anti-total a-tubulin (monoclonal, clone DM1A, 1:1000); anti-tyrosinated a-tubulin (monoclonal, clone TUB-1A2, 1:1000); anti-acetylated a-tubulin (monoclonal, clone 6-11B-1, 1:1000). After rinsing with PBS/2% FCS, cells were incubated for 1 h at 37 C with secondary anti-mouse fluorescein isothiocyanate-conjugated antibodies (FITC-AlexaFluor 488, Thermo Fisher Scientific, Waltham, MA, USA). Finally, oocytes were mounted on a glass slide in a 4 ml drop of medium containing 50% glycerol, 2.5 mg/ml sodium azide and 1 mg/ml Hoechst 33342 (Sigma Chemical CO. (St. Louis, MO, USA), using wax cushions to avoid compression of samples. 2.5. Confocal analysis and quantification The analysis of immunolabelled sections were performed with a confocal laser scanning microscope from Leica (TCS SP5 DMI 6000CS, Leica Microsystems GmbH, Wetzlar, Germany), equipped with Ar/He/Ne lasers, using a 40/60X oil objective. The sections were analyzed by sequential excitation. FITC was excited at 488 nm and emission was detected between 510 and 550 nm. Nuclear counterstaining was performed using Hoechst 33342 (1:5000),
Please cite this article as: Serra E et al., Morphological features and microtubular changes in vitrified ovine oocytes, Theriogenology, https:// doi.org/10.1016/j.theriogenology.2019.11.007
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excited at 358 nm, and emission in the blue range, at 461 nm. Parameters related to fluorescence intensity were maintained at constant values during all image acquisitions (laser energy 26%, Sequential Settings 1: PMT1 gain 649-PMT2 gain 482; Sequential Setting 2: PMT1 gain 625-PMT2 gain 589; offset 0; pinhole size: 68). Quantitative analysis of fluorescence intensity was performed using the Leica LAS AF Lite image analysis software package (Leica Microsystems GmbH, Wetzlar, Germany), following the procedures standardized by Gadau [35]. Briefly, the pictures were captured once, moving on the Z axis, we reached the equatorial plane. On each photo, transformed into gray scale, once channel 1 (related to Hoechst blue) was turned off, a region of interest (ROI) were manually drawn on a circumscribed area, that was around the meiotic spindle. So that, the software could automatically read the pixel average value on the channel 2 (FITC, AlexaFluor 488) subtracting the value of the background from it. Moreover, the mean values of pixel were recorded and submitted to statistical analysis. Qualitative analysis (parthenogenetic activation, spindle conformation and chromosomal organization) was performed using the LAS AF Lite software observing each oocyte individually and recording the pattern observed. The valuation of chromosomal configuration allows to highlight the spontaneously activated
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oocytes after culture. Were considered as activated by spontaneous parthenogenesis the oocytes showing: a) metaphase II-telophase II transition; b) chromatin decondensation c) one or more pronuclei formation. 2.6. Statistical analyses Differences in spindle conformation, chromosomal organization and parthenogenetic activation rates between groups and at different time points were analyzed using the chi square test (c2). After analysis for homogeneity of variance by Levene’s test, statistical differences into adult and prepubertal on spindle morphology and tubulins fluorescence intensity were evaluated by ANOVA one way Analysis of Variance. While statistical differences between adult and prepubertal oocytes on tubulins fluorescence intensity at each time point (0, 2, 4, 6 h) were evaluated using General Linear Model method (GLM) where: Y ¼ m þ species (adult vs prepubertal) þ time þ species x time. The species and time points were considered as fixed factors. All results are expressed as mean ± S.E.M. Statistical analyses were performed using the statistical software Minitab® 18.1 (2017 Minitab) and a probability of P 0.05 was considered the minimum level of significance.
Fig. 1. Effects of vitrification on spindle morphology in vitrified/warmed oocytes (adult: A, B, C; prepubertal: D, E, F) during different time points of prolonged incubation. Different letters indicate statistical differences (A: P ¼ 0.0000; B: P ¼ 0.4375; C: P ¼ 0.0001; D: P ¼ 0.0964; E: P ¼ 0.921; F: P ¼ 0.173).
Please cite this article as: Serra E et al., Morphological features and microtubular changes in vitrified ovine oocytes, Theriogenology, https:// doi.org/10.1016/j.theriogenology.2019.11.007
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Table 1 Spindle conformation and chromosomal organization in vitrified/warmed adult oocytes during different time points of prolonged incubation. In vitro matured adult oocytes are used as control. Within the same column, different letters indicate statistical differences between control and vitrified group at each time point. c2 test: spindle conformation: P ¼ 0.004; chromosomal organization: P ¼ 0.001. Spindle Conformation %
Chromosomal Organization%
Group
Oocytes
Normal
Abnormal
Absent
Normal
Abnormal
CTR Vitrified
60 35 34 33 33
68.3 a 22.9 b,c 38.2 c 45.5 a,c 51.5 a,c
28.3 a 57.2 b,c 52.9 c 42.4 a,c 39.4 a,c
3.3 a 20.0 b,c 8.8 c 12.1 a,c 9.1 a,c
61.7 a 22.9 b,c 26.5 b,c 45.5 c,d 45.5 c,d
38.3 a 77.1 b,c 73.5 b,c 54.5 c,d 54.5 c,d
0h 2h 4h 6h
Table 2 Spindle conformation and chromosomal organization in vitrified/warmed prepubertal oocytes during different time points of prolonged incubation. In vitro matured prepubertal oocytes are used as control. Within the same column, different letters indicate statistical differences between control and vitrified group at each time point. c2 test: spindle conformation: P ¼ 0.0000; chromosomal organization: P ¼ 0.0000.
Group CTR vitrified
0h 2h 4h 6h
Spindle Conformation %
Chromosomal Organization %
Oocytes
Normal
Abnormal
Absent
Normal
Abnormal
50 32 34 33 33
70.6a 12.5 b 4.2 c,d 22.7 c,d 5.3 d,e
29.4a 87.5 b 58.3 c,d 54.5 c,d 47.4 d,e
e e 37.5 c,d 22.7 c,d 47.4 d,e
62.7a 15.6 b 4.2 b 18.2 b 5.3 b
37.3a 84.4 b 95.8 b 81.8 b 94.7 b
3. Results 3.1. The morphology of the meiotic spindle In adult, the meiotic spindle occupied a larger area and was wider in CTR (151.1 ± 7.9 mm2 and 14.9 ± 0.7 mm width) than vitrified/warmed oocytes (0 h ¼ 101.4 ± 13.8 mm2; 2 h ¼ 91.1 ± 11.0 mm2; 4 h ¼ 87.4 ± 9.4 mm2; 6 h ¼ 94.8 ± 9 mm2; 0 h ¼ 12.6 ± 1.2 mm; 2 h ¼ 10.6 ± 0.9 mm; 4 h ¼ 10.6 ± 0.8 mm; 6 h ¼ 10.1 ± 0.8 mm; P ¼ 0.0001). No differences were found in pole-to-pole length (P ¼ 0.4375) between CTR and vitrified/warmed oocytes and during the post-warming incubation period (Fig. 1).
In prepubertal no differences were found between CTR and vitrified/warmed oocytes in all parameters studied (area P ¼ 0.6700; length pole-to-pole P ¼ 0.9210; width P ¼ 0.1730). No differences were found (P ¼ 0.1610) between adult and prepubertal vitrified oocytes in all parameters considered.
3.2. Spindle configuration and chromosomal organization In adult (Table 1), the rate of abnormal meiotic spindles increased following vitrification and post-warming culture raising from 28.3% in CTR oocytes to 57.2% in vitrified ones immediately after warming (P ¼ 0.004). Thereafter, this percentage tended to decrease during post-warming prolonged incubation and at 4 h and 6 h of incubation (respectively 42.4% and 39.4%) there were no differences between vitrified and CTR groups. Chromosomal organization reflected the trend of spindle morphology, but the rate of abnormalities was higher in vitrified oocytes (0 h ¼ 77.1%; 2 h ¼ 73.5%; 4 h ¼ 54.5%; 6 h ¼ 54.5%) compared to CTR ones (38.3%) during all time points of post-warming incubation (P ¼ 0.001). In prepubertal (Table 2), a similar pattern was observed. However, while the rate of abnormal meiotic spindles in prepubertal CTR oocytes did not differ from adult ones (29.4%), in prepubertal vitrified oocytes it reached the 87.5% at warming. This value was higher compared to CTR prepubertal oocytes (P < 0.01) and to vitrified/warmed adult oocytes at the same time point (P < 0.01; Fig. 2, panel A). Thereafter, the percentage of
Fig. 2. Effects of vitrification on spindle configuration (A) and chromosomal organization (B) during different rime points of post-warming incubation. Asterisk indicate statistical differences between adult and prepubertal oocytes at each time point (A: 0 h P ¼ 0.008; 2 h P ¼ 0.002; 6 h P ¼ 0.000. B: 2 h P ¼ 0.027; 4 h P ¼ 0.037; 6 h P ¼ 0.002). Pictures are representative for normal (a), abnormal (b) and absent (c) spindle configuration and chromosomal organization.
Please cite this article as: Serra E et al., Morphological features and microtubular changes in vitrified ovine oocytes, Theriogenology, https:// doi.org/10.1016/j.theriogenology.2019.11.007
E. Serra et al. / Theriogenology xxx (xxxx) xxx Table 3 Parthenogenetic activation in vitrified/warmed adult and prepubertal oocytes during different time points of post-warming incubation. In vitro matured adult and prepubertal oocytes are used as control. In each column different lowercase letters indicate statistical differences between control and vitrified oocytes at each time point: c2 test: adult: P ¼ 0.098; prepubertal: P ¼ 0.001). Different uppercase letters indicate statistical difference between control and vitrified groups c2 test: CTR adult vs CTR p prepubertal P ¼ 0.013; adult 2 h vs prepubertal 2 h P ¼ 0.006; adult 4 h vs prepubertal 4 h P ¼ 0.012; adult 6 h vs prepubertal 6 h P ¼ 0.001.
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incubation compared to control ones (P ¼ 0.001) reaching 50% at 6 h. The parthenogenetic activation rate was higher in CTR prepubertal oocytes compared to adult ones (P ¼ 0.013; Table 3) Furthermore, in prepubertal oocytes the activation rates increased significantly during post-warming incubation compared to adult ones (P ¼ 0.0000; Fig. 3).
Parthenogenetic Activation Groups CTR 0h 2h 4h 6h
Adult aA
(n ¼ 61) 1.6% 12.5% b (n ¼ 40) bA 12.8% (n ¼ 39) 17.5% bA (n ¼ 40) bA 13.2% (n ¼ 38)
Prepubertal 14.0% 24.0% 40.0% 44.0% 50.0%
aB
(n ¼ 59) (n ¼ 42) (n ¼ 40) bB (n ¼ 39) bB (n ¼ 38) b
3.4. Tubulin post translational modification in vitrified/warmed prepubertal and adult ovine oocytes during post-warming incubation
bB
normal spindles was not restored during post-warming incubation. At 2 h (P ¼ 0.002) and at 6 h (P ¼ 0.000) the percentage of abnormal spindle was higher compared to adult oocytes at the same time points after vitrification/warming. Chromosomal organization reflected the trend of spindle morphology (P ¼ 0.0000; Fig. 2, panel B), showing significant differences between prepubertal and adult vitrified oocytes at 2 h, 4 h and 6 h during post-warming incubation (2 h P ¼ 0.027; 4 h P ¼ 0.037; 6 h P ¼ 0.002).
In both adult and prepubertal oocytes, fluorescence intensity of tyrosinated tubulin (Fig. 4 and Fig. 6, respectively) and of acetylated tubulin (Figs. 5 and 7, respectively) were significantly higher in vitrified/warmed oocytes compared to CTR ones (P ¼ 0.0000) during all time points of post-warming incubation. In adult oocytes, however, the level of fluorescence intensity decreased after 4e6 h of culture (P ¼ 0.0000). In adult oocytes, tyrosinated tubulin showed a higher fluorescence intensity compared to acetylated tubulin at 2 h postwarming incubation (P ¼ 0.0041; Fig. 9, panel A), thereafter no differences were found between fluorescence intensity of the tubulin PTMs. In prepuberal oocytes (Fig. 9, panel B), the imbalance between tubulin PTMs increases significantly during post-warming
3.3. Parthenogenetic activation rate As shown in Table 3, in adult ovine oocytes the parthenogenetic activation rate was significantly higher in vitrified/warmed oocytes compared to CTR ones (CTR vs 0 h: P ¼ 0.0240; CTR vs 2 h: P ¼ 0.0220; CTR vs 4 h: P ¼ 0.0040; CTR vs 6 h: P ¼ 0.0190). In prepubertal oocytes (Table 3), parthenogenetic activation increased in vitrified/warmed oocytes during all time points of post-warming
Fig. 3. Parthenogenetic activation in vitrified/warmed oocytes during different time points of post-warming incubation. Asterisk indicate statistical differences between adult and prepubertal oocytes at each time point (P ¼ 0.0000). Representative images of parthenogenetic activation: oocyte in metaphase II-telophase II transition (on the left) and one pronucleus formation (on the right).
Fig. 4. Evaluation of tyrosinated a-tubulin fluorescence intensity in vitrified/warmed adult oocytes during post-warming incubation. In the histogram, different letters indicate statistical differences at each time point (P ¼ 0.0000). Bar ¼ 10 mm.
Please cite this article as: Serra E et al., Morphological features and microtubular changes in vitrified ovine oocytes, Theriogenology, https:// doi.org/10.1016/j.theriogenology.2019.11.007
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Fig. 5. Evaluation of acetylated a-tubulin fluorescence intensity in vitrified/warmed adult oocytes during incubation post-warming. In the histogram, different letters indicate statistical differences at each time point (P ¼ 0.0000). Bar ¼ 10 mm.
incubation (P ¼ 0.0002). No difference was found in the fluorescence intensity of tyrosinated tubulin (Fig. 8) between adult and prepubertal oocytes (P ¼ 0.1110). On the contrary, fluorescence intensity of acetylated tubulin (Fig. 8), was higher in adult compared to prepubertal oocytes at 4 h and 6 h of post-warming incubation (P ¼ 0.0000). 4. Discussion Results obtained in the present study showed that the vitrification and warming procedures cause significant alterations in meiotic spindle and chromosome configuration, and that these alterations are more severe in prepubertal compared to adult oocytes. At warming, vitrified adult oocytes showed a reduction in size of the meiotic spindle and a significant increase in the rate of abnormal spindle and chromosome configuration compared to fresh IVM oocytes. Prepubertal oocytes, while not showing significant alteration in spindle size, showed higher rates of abnormalities in its configuration and in chromosomal organization compared to adult and fresh prepubertal IVM oocytes. These differences were associated with differences in tubulin PTMs during the post-warming recovery time. The extreme vulnerability of MII oocytes to cryoinjury is related, among the other factors, to the presence of the meiotic spindle, whose apparatus is acutely sensitive to temperature deviation [19]. Previous studies showed that vitrification causes partial or total
Fig. 6. Immunofluorescence underlined changes in tyrosinated a-tubulin fluorescence intensity in vitrified/warmed prepubertal oocytes during incubation post-warming. In the histogram, different letters indicate statistical differences at each time point (P ¼ 0.0000). Bar ¼ 10 mm.
depolymerization of the microtubules and disorganization of the structure in ovine [20], equine [36], and human oocytes [28,37]. A reduction in meiotic spindle size has also been described in humans [38] and mice [39] oocytes placed in contact with the cryoprotectants. Changes in spindle morphology following vitrification have been related to the subsequent failure of fertilization and embryonic development [36,40,41]. Some authors, however, have described the recovery of alterations of the meiotic spindle after 1e3 h of in vitro post-warming culture whose success depends, on the recovery time, on the vitrification procedures and on the species [42,43]. This de novo reassembly of the spindle microtubules is a requirement for correct chromosome alignment and segregation after fertilization [44]. Already several authors had highlighted different structural and functional limitations in prepubertal ovine oocytes regarding small size, defective coupling between cumulus cells and oocytes, reduced protein synthesis and energy metabolism [5e9]. Moreover, in these oocytes it occurs frequently defects in the functioning of the microtubular network involved in spindle formation with chromosomal segregation errors [10]. In the present study, in vitrified adult oocytes the meiotic spindle recovered its typical barrel conformation during in vitro post-warming incubation, while in the vitrified prepubertal oocytes no signs of recovery were found, and the rates of structural anomalies even increased during postwarming incubation hours.
Please cite this article as: Serra E et al., Morphological features and microtubular changes in vitrified ovine oocytes, Theriogenology, https:// doi.org/10.1016/j.theriogenology.2019.11.007
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Fig. 7. Evaluation of acetylated a-tubulin fluorescence intensity in vitrified/warmed prepubertal oocytes during incubation post-warming. In the histogram, different letters indicate statistical differences at each time point (P ¼ 0.0001). Bar ¼ 10 mm.
Fig. 8. Effects of vitrification on tyrosinated a-tubulin (panel A) and acetylated atubulin (panel B) fluorescence intensity between vitrified/warmed adult and prepubertal oocytes during incubation post-warming. Asterisk indicate statistical differences between adult and prepubertal oocytes at each time point (A: P ¼ 0.111; B: P ¼ 0.0000).
This difference between prepubertal and adult oocytes in the ability of the spindle microtubules to reassembly after vitrification/ warming could be attributed to the observed differences in tubulin PTMs. In a previous study, we showed that in adult in vitro matured oocytes there are no differences between the fluorescence quantification of acetylated and tyrosinated a-tubulin [18]. On the other hand, in prepubertal oocytes the acetylated tubulin showed a higher fluorescence intensity compared to the tyrosinated tubulin. Predominant levels of tubulin acetylation in prepubertal oocytes may be correlated with excessive increase of microtubule stability, altered spindle morphology and defects in chromosome alignment [45]. In the present study, fluorescence intensity of tubulin PTMs was higher in vitrified/warmed prepubertal and adult oocytes compared to their fresh counterpart. However, during postwarming incubation adult oocytes showed a balance in the fluorescence quantification of the expressed tubulin PTMs. On the contrary, in prepubertal oocytes the fluorescence intensity of acetylated tubulin decreased during post-warming incubation, being lower compared to adult oocytes and leading to an imbalance in the fluorescence quantification of the expressed tubulin PTMs. The acetylated tubulin can form microtubule populations capable to withstand low temperatures [46]. The higher intensity of this tubulin PTMs in adult oocytes may indicate that the structure of the meiotic spindle is more stable than in prepubertal oocytes, a necessary requirement while awaiting fertilization. Moreover, in
adult oocytes the balance in tubulin PTMs could be related to their ability to reassembly the spindle microtubules after vitrification and warming and ultimately to restore their developmental competence during post-warming incubation, as previously described [32]. On the contrary, prepubertal oocytes, showing an imbalance in tubulin PTMs, are not able to reassemble their meiotic spindle after vitrification/warming and show a lack of developmental competence after vitrification [21,22]. In the present study, vitrified/warmed prepubertal oocytes also showed very high rates of parthenogenetic activation compared to fresh controls and adult oocytes. Parthenogenetic activation is given by an electrical or chemical stimulus that simulates the entry of the sperm into the egg cell. This signal induces the mobilization of Ca2þ from the endoplasmic reticulum with the consequent triggering of the pathways of completion of meiosis, with the transition from metaphase II to telophase II, the decondensation of chromatin, the formation of the first pronucleus and the first cell division [47]. The chromatin reorganization and nuclear membrane reconstruction depend on the Maturation Promoting Factor (MPF) activity that turns out to be higher in MII stage and then decreases after a few hours from oocyte activation [48]. It was noted that, in prepubertal ovine, the MPF activity is lower in vitrified oocytes after 2 h of post-warming incubation compared to control oocytes [21]. On the contrary, in adult ovine, there is a resumption of the kinase activity of MPF in vitrified oocytes [20]. Therefore, the high
Please cite this article as: Serra E et al., Morphological features and microtubular changes in vitrified ovine oocytes, Theriogenology, https:// doi.org/10.1016/j.theriogenology.2019.11.007
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posttranslational modifications may therefore represent a further parameter to evaluate oocyte quality and cryotolerance. Funding Financing of doctoral courses aimed at training highly specialized human capital - P.O.R. SARDINIA F.S.E. 2007e2013. Declaration of competing interest No conflict of interest declared. References
Fig. 9. Effects of vitrification on tubulin PTMs in vitrified/warmed adult (A) and prepubertal (B) oocytes during incubation post-warming. Asterisk indicate statistical differences between the fluorescence intensity of acetylated and tyrosinated a-tubulin at each time point (A: P ¼ 0.0041; B: P ¼ 0.0002).
rates of parthenogenetic activation observed during our experiments in vitrified prepubertal oocytes may be related to a low MPF activity. The high percentages of parthenogenetic activation in prepubertal oocytes indicate a high sensitivity to the damages associated with vitrification/warming and an impair functioning of the recovery mechanisms. 5. Conclusions In conclusion, our study showed that prepubertal and adult oocytes react differently to the insults provoked by the vitrification procedures. After vitrification/warming, the prepubertal oocytes show important anomalies in the meiotic spindle with high rates of parthenogenetic activation. Furthermore, they were not able to repair those damages during post-warming incubation. In contrast, adult oocytes were able to recover, at least partially, their functionality after 4 h of post-warming incubation, and this finding supports results shown in a previous study [32]. This ability was associated in adult oocyte with a balance in the fluorescence quantification of tubulin PTMs. Prepubertal oocyte, on the other hand, showed a strong imbalance between the tyrosinated and the acetylated tubulin during post-warming recovery time. The balance between these two tubulin PTMs could be essential for maintaining the functional and structural integrity of the ovine oocyte. An indepth study of the microtubular cytoskeleton and its
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Morphological features and microtubular changes in vitrified ovine oocytes Elisa Serra 1, Sergio Domenico Gadau*, 1, Fiammetta Berlinguer, Salvatore Naitana, Sara Succu Department of Veterinary Medicine, University of Sassari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 July 2019 Received in revised form 31 October 2019 Accepted 9 November 2019 Available online xxx
Cryobanking of oocytes collected from prepubertal donors may supply a virtually unlimited number of female gametes for both basic research and commercial applications. Prepubertal oocytes show some structural and functional limitations compared to the adult ones that may impair their ability to recover damages from cryopreservation. In oocytes, the meiotic spindle is acutely sensitive to temperature deviation, but capable of regeneration following cryopreservation. In the present work, we studied the effects of vitrification and post-warming incubation on the microtubular cytoskeleton and the tubulin post-translational modifications (tyrosination and acetylation) in prepubertal and adult oocytes. Obtained results showed that prepubertal oocytes are more affected by vitrification-induced injuries than adult ones. In fact, prepubertal oocytes showed more severe alterations of the meiotic spindle conformation and a higher percentage of parthenogenetic activation compared to adult ones. Moreover, in the adult oocytes the equilibrium between tyrosinated and acetylated a-tubulin was restored after 4 h of post-warming incubation. Diversely, in prepubertal oocytes the imbalance between tyrosinated and acetylated a-tubulin was increased during post-warming incubation. Our study shows that prepubertal oocytes react differently to the insults provoked by vitrification compared to adult oocytes, showing an impaired ability to recover from vitrification-induced injuries. In the evaluation of oocyte ability to recover from vitrification-induced injuries, tubulin post-translational modifications represent an important indicator for assessing oocyte quality. © 2019 Elsevier Inc. All rights reserved.
Keywords: Ovine oocytes Vitrification Tubulin post-translational modifications Microtubular network
1. Introduction The demand for accelerated genetic gain of farm animals has promoted the use of prepubertal animals as a source of oocytes for in vitro embryo production (IVP). Different studies on the development of the prepubertal ovine ovary [1e3] have evidenced that at 4 weeks of age lambs could be excellent donors, when the maximal numbers of oocytes are available [4]. Thus, large numbers of ovaries of prepubertal ruminants can be easily obtained from the slaughterhouses and many oocytes can be incorporated into an IVP system. Moreover, female gamete banking through oocyte cryopreservation may simplify the management of genetic resources and improve basic research. For these reasons, the cryopreservation
* Corresponding author. Department of Veterinary Medicine, University of Sassari, Via Vienna 2, 07100, Italy. E-mail address: [email protected] (S.D. Gadau). 1 Serra Elisa and Gadau Sergio Domenico contributed equally to this work.
of oocytes collected from prepubertal donors is considered as a tool to supply a virtually unlimited number of female gametes for both basic research and commercial applications. However, prepubertal oocytes show some structural and functional limitations compared to the adult ones, such as small size, defective coupling between cumulus cells and oocytes, decrease in amino acid uptake, reduced protein synthesis and energy metabolism, and low developmental competence [5e9]. One of the most frequent problems during the maturation of prepubertal oocytes is linked to the inclination of them to chromosomal segregation errors, due to defects in the functioning of the microtubular network involved in spindle formation [10]. The cytoskeleton network of the oocyte has a peculiar arrangement of microtubules and plays an important role during the maturation and the fertilization of the oocyte as well as in the early embryonic development [11]. The complex operations that take place during the formation of the meiotic spindle, its coupling to the chromosomes and their consequent distribution are inherent
https://doi.org/10.1016/j.theriogenology.2019.11.007 0093-691X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Serra E et al., Morphological features and microtubular changes in vitrified ovine oocytes, Theriogenology, https:// doi.org/10.1016/j.theriogenology.2019.11.007
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in the microtubular network dynamic [12]. Microtubules are hollow cylinders made up by 13 protofilaments formed by association “head-to-tail” of a/b tubulin heterodimers [13]. Many of the functions that microtubules play in the cell (cell division, cell shape maintenance, movement of cytoplasmic organelles, etc.) are regulated by their post-translational modifications (PTMs) which affect the carboxy-terminal of a (mainly) and b-tubulin. Each of these PTMs may coexist in the same microtubule, displaying affinity for a whole series of associated and motor proteins, giving rise to that complex architecture that goes by the name of “tubulin code” [14e17]. In a previous work [18] we found that the most abundant tubulin PTMs in the meiotic spindle of ovine oocytes are the tyrosinated and the acetylated ones, and that prepubertal and adult oocytes show several major differences in their tubulin PTMs. Several studies have shown that the meiotic spindle is acutely sensitive to temperature deviation [19]. Exposure to cryoprotectants and to the cold temperatures lead to the disruption of microtubules and the change of the meiotic spindle morphology of mature oocytes [20e26]. It also emerged that the meiotic spindle is capable of regeneration following cryopreservation [24,27]. However, the window of opportunity for spindle recovery is not unlimited. The optimal time for post-warming recovery seems to vary depending on the method of cryopreservation, initial oocyte quality, donor age, method of spindle assessment and species [28e32]. Leaving oocytes for longer than “optimal time” will result in progressive loss of bipolar spindle structure coincident with chromosome displacement. Starting from these premises, the aim of this work was to investigate the efficiency of prepubertal ovine oocytes in restoring the injuries on the meiotic spindle caused by vitrification by evaluating the changes in tubulin PTMs during the post-warming recovery time. These changes were evaluated by comparing adult and prepubertal oocytes, representing a model of high and low developmental competence, respectively [6,7,9,33]. This comparative analysis is essential to establish reference data that could indicate important mechanisms involved in the acquisition of developmental competence and in the different susceptibility to cryopreservation procedures.
as previously described [18]. Briefly, collected ovaries were transported from the commercial slaughterhouse to the laboratory within 1e2 h in Dulbecco Phosphate Buffered Saline (PBS) with antibiotics at 27 C. After being washed in PBS fresh medium, the ovaries were sliced using a micro-blade and the follicle content was released in medium TCM199 (with Earle’s salts and bicarbonate, HEPES 25 mmol, penicillin 0.1 g/L, streptomycin 0.1 g/L and 0.1% (w/ v) polyvinylalcohol-PVA). Cumuluseoocyte complexes (COCs) selected for in vitro culture, were matured in TCM 199 medium (supplemented with 10% heat-treated oestrus sheep serum (OSS), 1 IU/mL of FSH and 1 IU/mL of LH, 100 mM cysteamine and 8 mg/mL of pyruvate) in standard conditions (5% CO2 in air at 39 C for 22 h). After in vitro maturation, COCs were denuded of cumulus cells mechanically and only those at metaphase II (MII), that is, showing the extrusion of the first polar body, were selected. The oocytes were collected in a common pool and randomly assigning for vitrification (adult n ¼ 388; prepubertal n ¼ 312) or examined as CTR (adult n ¼ 164; prepubertal n ¼ 187). 2.3. Oocyte vitrification and warming Vitrification was performed following the minimum essential volume method, according to Succu et al. [34]. Briefly, after maturation a group of 5 MII oocytes were equilibrated in base medium (BM, PBS without Caþþ and Mgþþ plus 20% fetal calf serum) at 39 C for 2 min. Then, oocytes were equilibrated in a solution containing 7.5% (v/v) dimethyl sulfoxide (DMSO) and 7.5% (v/v) ethylene glycol (EG) in BM for 3 min and transferred to vitrification solution (VS) containing 16.5% (v/v) DMSO, 16.5% (v/v) EG and 0.5 M trehalose in BM. Finally, oocytes were loaded in cryotop devices (Kitazato Ltd., Tokyo, Japan) and directly plunged into liquid nitrogen within 20e25 s. For warming to a biological temperature, the content of cryotop was transferred from liquid nitrogen into 200 mL drops of 1.25 M trehalose in BM for 1 min at 38 C. To promote removal of intracellular cryoprotectants, oocytes were transferred stepwise into 200 mL drops of decreasing trehalose solutions (0.5, 0.25 and 0.125 M trehalose in BM) for 30 s before being equilibrated for 10 min in BM. 2.4. Oocytes immunostaining
2. Materials and methods All chemicals in this study were obtained from Sigma Chemical CO. (St. Louis, MO, USA) unless stated otherwise. 2.1. Experimental design A total of 1051 (adult: sheep 4e6 years old, n ¼ 552; prepubertal: lamb 30e40 days old, n ¼ 499) in vitro matured oocytes were used for this study. After vitrification and warming, oocytes were incubated for 6 h in PBS/20% FCS in 5% CO2 in air at 39 C. At specific time points of post-warming incubation (0, 2, 4 and 6 h), vitrified/ warmed oocytes were retrieved from the culture system and processed by indirect immunofluorescence for a-total tubulin detection with the aim to evaluate: i) the morphology of meiotic spindle (length, width and area); ii) spindle conformation and chromosomal organization; iii) parthenogenetic activation rate. Moreover, the pattern for both tyrosinated and acetylated a-tubulin were analyzed in all experimental groups. For each experimental procedure a pool of both adult and prepubertal in vitro matured oocytes, not vitrified, were used as a control group (CTR). 2.2. Oocytes collection and in vitro maturation The oocytes collection and in vitro maturation were performed
Immunostaining was performed following the methodology illustrated previously [18]. Briefly, fixed warmed and CTR oocytes have been processed through serial incubations (1 h per antibody at 37 C) with the following primary antibodies: anti-total a-tubulin (monoclonal, clone DM1A, 1:1000); anti-tyrosinated a-tubulin (monoclonal, clone TUB-1A2, 1:1000); anti-acetylated a-tubulin (monoclonal, clone 6-11B-1, 1:1000). After rinsing with PBS/2% FCS, cells were incubated for 1 h at 37 C with secondary anti-mouse fluorescein isothiocyanate-conjugated antibodies (FITC-AlexaFluor 488, Thermo Fisher Scientific, Waltham, MA, USA). Finally, oocytes were mounted on a glass slide in a 4 ml drop of medium containing 50% glycerol, 2.5 mg/ml sodium azide and 1 mg/ml Hoechst 33342 (Sigma Chemical CO. (St. Louis, MO, USA), using wax cushions to avoid compression of samples. 2.5. Confocal analysis and quantification The analysis of immunolabelled sections were performed with a confocal laser scanning microscope from Leica (TCS SP5 DMI 6000CS, Leica Microsystems GmbH, Wetzlar, Germany), equipped with Ar/He/Ne lasers, using a 40/60X oil objective. The sections were analyzed by sequential excitation. FITC was excited at 488 nm and emission was detected between 510 and 550 nm. Nuclear counterstaining was performed using Hoechst 33342 (1:5000),
Please cite this article as: Serra E et al., Morphological features and microtubular changes in vitrified ovine oocytes, Theriogenology, https:// doi.org/10.1016/j.theriogenology.2019.11.007
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excited at 358 nm, and emission in the blue range, at 461 nm. Parameters related to fluorescence intensity were maintained at constant values during all image acquisitions (laser energy 26%, Sequential Settings 1: PMT1 gain 649-PMT2 gain 482; Sequential Setting 2: PMT1 gain 625-PMT2 gain 589; offset 0; pinhole size: 68). Quantitative analysis of fluorescence intensity was performed using the Leica LAS AF Lite image analysis software package (Leica Microsystems GmbH, Wetzlar, Germany), following the procedures standardized by Gadau [35]. Briefly, the pictures were captured once, moving on the Z axis, we reached the equatorial plane. On each photo, transformed into gray scale, once channel 1 (related to Hoechst blue) was turned off, a region of interest (ROI) were manually drawn on a circumscribed area, that was around the meiotic spindle. So that, the software could automatically read the pixel average value on the channel 2 (FITC, AlexaFluor 488) subtracting the value of the background from it. Moreover, the mean values of pixel were recorded and submitted to statistical analysis. Qualitative analysis (parthenogenetic activation, spindle conformation and chromosomal organization) was performed using the LAS AF Lite software observing each oocyte individually and recording the pattern observed. The valuation of chromosomal configuration allows to highlight the spontaneously activated
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oocytes after culture. Were considered as activated by spontaneous parthenogenesis the oocytes showing: a) metaphase II-telophase II transition; b) chromatin decondensation c) one or more pronuclei formation. 2.6. Statistical analyses Differences in spindle conformation, chromosomal organization and parthenogenetic activation rates between groups and at different time points were analyzed using the chi square test (c2). After analysis for homogeneity of variance by Levene’s test, statistical differences into adult and prepubertal on spindle morphology and tubulins fluorescence intensity were evaluated by ANOVA one way Analysis of Variance. While statistical differences between adult and prepubertal oocytes on tubulins fluorescence intensity at each time point (0, 2, 4, 6 h) were evaluated using General Linear Model method (GLM) where: Y ¼ m þ species (adult vs prepubertal) þ time þ species x time. The species and time points were considered as fixed factors. All results are expressed as mean ± S.E.M. Statistical analyses were performed using the statistical software Minitab® 18.1 (2017 Minitab) and a probability of P 0.05 was considered the minimum level of significance.
Fig. 1. Effects of vitrification on spindle morphology in vitrified/warmed oocytes (adult: A, B, C; prepubertal: D, E, F) during different time points of prolonged incubation. Different letters indicate statistical differences (A: P ¼ 0.0000; B: P ¼ 0.4375; C: P ¼ 0.0001; D: P ¼ 0.0964; E: P ¼ 0.921; F: P ¼ 0.173).
Please cite this article as: Serra E et al., Morphological features and microtubular changes in vitrified ovine oocytes, Theriogenology, https:// doi.org/10.1016/j.theriogenology.2019.11.007
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Table 1 Spindle conformation and chromosomal organization in vitrified/warmed adult oocytes during different time points of prolonged incubation. In vitro matured adult oocytes are used as control. Within the same column, different letters indicate statistical differences between control and vitrified group at each time point. c2 test: spindle conformation: P ¼ 0.004; chromosomal organization: P ¼ 0.001. Spindle Conformation %
Chromosomal Organization%
Group
Oocytes
Normal
Abnormal
Absent
Normal
Abnormal
CTR Vitrified
60 35 34 33 33
68.3 a 22.9 b,c 38.2 c 45.5 a,c 51.5 a,c
28.3 a 57.2 b,c 52.9 c 42.4 a,c 39.4 a,c
3.3 a 20.0 b,c 8.8 c 12.1 a,c 9.1 a,c
61.7 a 22.9 b,c 26.5 b,c 45.5 c,d 45.5 c,d
38.3 a 77.1 b,c 73.5 b,c 54.5 c,d 54.5 c,d
0h 2h 4h 6h
Table 2 Spindle conformation and chromosomal organization in vitrified/warmed prepubertal oocytes during different time points of prolonged incubation. In vitro matured prepubertal oocytes are used as control. Within the same column, different letters indicate statistical differences between control and vitrified group at each time point. c2 test: spindle conformation: P ¼ 0.0000; chromosomal organization: P ¼ 0.0000.
Group CTR vitrified
0h 2h 4h 6h
Spindle Conformation %
Chromosomal Organization %
Oocytes
Normal
Abnormal
Absent
Normal
Abnormal
50 32 34 33 33
70.6a 12.5 b 4.2 c,d 22.7 c,d 5.3 d,e
29.4a 87.5 b 58.3 c,d 54.5 c,d 47.4 d,e
e e 37.5 c,d 22.7 c,d 47.4 d,e
62.7a 15.6 b 4.2 b 18.2 b 5.3 b
37.3a 84.4 b 95.8 b 81.8 b 94.7 b
3. Results 3.1. The morphology of the meiotic spindle In adult, the meiotic spindle occupied a larger area and was wider in CTR (151.1 ± 7.9 mm2 and 14.9 ± 0.7 mm width) than vitrified/warmed oocytes (0 h ¼ 101.4 ± 13.8 mm2; 2 h ¼ 91.1 ± 11.0 mm2; 4 h ¼ 87.4 ± 9.4 mm2; 6 h ¼ 94.8 ± 9 mm2; 0 h ¼ 12.6 ± 1.2 mm; 2 h ¼ 10.6 ± 0.9 mm; 4 h ¼ 10.6 ± 0.8 mm; 6 h ¼ 10.1 ± 0.8 mm; P ¼ 0.0001). No differences were found in pole-to-pole length (P ¼ 0.4375) between CTR and vitrified/warmed oocytes and during the post-warming incubation period (Fig. 1).
In prepubertal no differences were found between CTR and vitrified/warmed oocytes in all parameters studied (area P ¼ 0.6700; length pole-to-pole P ¼ 0.9210; width P ¼ 0.1730). No differences were found (P ¼ 0.1610) between adult and prepubertal vitrified oocytes in all parameters considered.
3.2. Spindle configuration and chromosomal organization In adult (Table 1), the rate of abnormal meiotic spindles increased following vitrification and post-warming culture raising from 28.3% in CTR oocytes to 57.2% in vitrified ones immediately after warming (P ¼ 0.004). Thereafter, this percentage tended to decrease during post-warming prolonged incubation and at 4 h and 6 h of incubation (respectively 42.4% and 39.4%) there were no differences between vitrified and CTR groups. Chromosomal organization reflected the trend of spindle morphology, but the rate of abnormalities was higher in vitrified oocytes (0 h ¼ 77.1%; 2 h ¼ 73.5%; 4 h ¼ 54.5%; 6 h ¼ 54.5%) compared to CTR ones (38.3%) during all time points of post-warming incubation (P ¼ 0.001). In prepubertal (Table 2), a similar pattern was observed. However, while the rate of abnormal meiotic spindles in prepubertal CTR oocytes did not differ from adult ones (29.4%), in prepubertal vitrified oocytes it reached the 87.5% at warming. This value was higher compared to CTR prepubertal oocytes (P < 0.01) and to vitrified/warmed adult oocytes at the same time point (P < 0.01; Fig. 2, panel A). Thereafter, the percentage of
Fig. 2. Effects of vitrification on spindle configuration (A) and chromosomal organization (B) during different rime points of post-warming incubation. Asterisk indicate statistical differences between adult and prepubertal oocytes at each time point (A: 0 h P ¼ 0.008; 2 h P ¼ 0.002; 6 h P ¼ 0.000. B: 2 h P ¼ 0.027; 4 h P ¼ 0.037; 6 h P ¼ 0.002). Pictures are representative for normal (a), abnormal (b) and absent (c) spindle configuration and chromosomal organization.
Please cite this article as: Serra E et al., Morphological features and microtubular changes in vitrified ovine oocytes, Theriogenology, https:// doi.org/10.1016/j.theriogenology.2019.11.007
E. Serra et al. / Theriogenology xxx (xxxx) xxx Table 3 Parthenogenetic activation in vitrified/warmed adult and prepubertal oocytes during different time points of post-warming incubation. In vitro matured adult and prepubertal oocytes are used as control. In each column different lowercase letters indicate statistical differences between control and vitrified oocytes at each time point: c2 test: adult: P ¼ 0.098; prepubertal: P ¼ 0.001). Different uppercase letters indicate statistical difference between control and vitrified groups c2 test: CTR adult vs CTR p prepubertal P ¼ 0.013; adult 2 h vs prepubertal 2 h P ¼ 0.006; adult 4 h vs prepubertal 4 h P ¼ 0.012; adult 6 h vs prepubertal 6 h P ¼ 0.001.
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incubation compared to control ones (P ¼ 0.001) reaching 50% at 6 h. The parthenogenetic activation rate was higher in CTR prepubertal oocytes compared to adult ones (P ¼ 0.013; Table 3) Furthermore, in prepubertal oocytes the activation rates increased significantly during post-warming incubation compared to adult ones (P ¼ 0.0000; Fig. 3).
Parthenogenetic Activation Groups CTR 0h 2h 4h 6h
Adult aA
(n ¼ 61) 1.6% 12.5% b (n ¼ 40) bA 12.8% (n ¼ 39) 17.5% bA (n ¼ 40) bA 13.2% (n ¼ 38)
Prepubertal 14.0% 24.0% 40.0% 44.0% 50.0%
aB
(n ¼ 59) (n ¼ 42) (n ¼ 40) bB (n ¼ 39) bB (n ¼ 38) b
3.4. Tubulin post translational modification in vitrified/warmed prepubertal and adult ovine oocytes during post-warming incubation
bB
normal spindles was not restored during post-warming incubation. At 2 h (P ¼ 0.002) and at 6 h (P ¼ 0.000) the percentage of abnormal spindle was higher compared to adult oocytes at the same time points after vitrification/warming. Chromosomal organization reflected the trend of spindle morphology (P ¼ 0.0000; Fig. 2, panel B), showing significant differences between prepubertal and adult vitrified oocytes at 2 h, 4 h and 6 h during post-warming incubation (2 h P ¼ 0.027; 4 h P ¼ 0.037; 6 h P ¼ 0.002).
In both adult and prepubertal oocytes, fluorescence intensity of tyrosinated tubulin (Fig. 4 and Fig. 6, respectively) and of acetylated tubulin (Figs. 5 and 7, respectively) were significantly higher in vitrified/warmed oocytes compared to CTR ones (P ¼ 0.0000) during all time points of post-warming incubation. In adult oocytes, however, the level of fluorescence intensity decreased after 4e6 h of culture (P ¼ 0.0000). In adult oocytes, tyrosinated tubulin showed a higher fluorescence intensity compared to acetylated tubulin at 2 h postwarming incubation (P ¼ 0.0041; Fig. 9, panel A), thereafter no differences were found between fluorescence intensity of the tubulin PTMs. In prepuberal oocytes (Fig. 9, panel B), the imbalance between tubulin PTMs increases significantly during post-warming
3.3. Parthenogenetic activation rate As shown in Table 3, in adult ovine oocytes the parthenogenetic activation rate was significantly higher in vitrified/warmed oocytes compared to CTR ones (CTR vs 0 h: P ¼ 0.0240; CTR vs 2 h: P ¼ 0.0220; CTR vs 4 h: P ¼ 0.0040; CTR vs 6 h: P ¼ 0.0190). In prepubertal oocytes (Table 3), parthenogenetic activation increased in vitrified/warmed oocytes during all time points of post-warming
Fig. 3. Parthenogenetic activation in vitrified/warmed oocytes during different time points of post-warming incubation. Asterisk indicate statistical differences between adult and prepubertal oocytes at each time point (P ¼ 0.0000). Representative images of parthenogenetic activation: oocyte in metaphase II-telophase II transition (on the left) and one pronucleus formation (on the right).
Fig. 4. Evaluation of tyrosinated a-tubulin fluorescence intensity in vitrified/warmed adult oocytes during post-warming incubation. In the histogram, different letters indicate statistical differences at each time point (P ¼ 0.0000). Bar ¼ 10 mm.
Please cite this article as: Serra E et al., Morphological features and microtubular changes in vitrified ovine oocytes, Theriogenology, https:// doi.org/10.1016/j.theriogenology.2019.11.007
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Fig. 5. Evaluation of acetylated a-tubulin fluorescence intensity in vitrified/warmed adult oocytes during incubation post-warming. In the histogram, different letters indicate statistical differences at each time point (P ¼ 0.0000). Bar ¼ 10 mm.
incubation (P ¼ 0.0002). No difference was found in the fluorescence intensity of tyrosinated tubulin (Fig. 8) between adult and prepubertal oocytes (P ¼ 0.1110). On the contrary, fluorescence intensity of acetylated tubulin (Fig. 8), was higher in adult compared to prepubertal oocytes at 4 h and 6 h of post-warming incubation (P ¼ 0.0000). 4. Discussion Results obtained in the present study showed that the vitrification and warming procedures cause significant alterations in meiotic spindle and chromosome configuration, and that these alterations are more severe in prepubertal compared to adult oocytes. At warming, vitrified adult oocytes showed a reduction in size of the meiotic spindle and a significant increase in the rate of abnormal spindle and chromosome configuration compared to fresh IVM oocytes. Prepubertal oocytes, while not showing significant alteration in spindle size, showed higher rates of abnormalities in its configuration and in chromosomal organization compared to adult and fresh prepubertal IVM oocytes. These differences were associated with differences in tubulin PTMs during the post-warming recovery time. The extreme vulnerability of MII oocytes to cryoinjury is related, among the other factors, to the presence of the meiotic spindle, whose apparatus is acutely sensitive to temperature deviation [19]. Previous studies showed that vitrification causes partial or total
Fig. 6. Immunofluorescence underlined changes in tyrosinated a-tubulin fluorescence intensity in vitrified/warmed prepubertal oocytes during incubation post-warming. In the histogram, different letters indicate statistical differences at each time point (P ¼ 0.0000). Bar ¼ 10 mm.
depolymerization of the microtubules and disorganization of the structure in ovine [20], equine [36], and human oocytes [28,37]. A reduction in meiotic spindle size has also been described in humans [38] and mice [39] oocytes placed in contact with the cryoprotectants. Changes in spindle morphology following vitrification have been related to the subsequent failure of fertilization and embryonic development [36,40,41]. Some authors, however, have described the recovery of alterations of the meiotic spindle after 1e3 h of in vitro post-warming culture whose success depends, on the recovery time, on the vitrification procedures and on the species [42,43]. This de novo reassembly of the spindle microtubules is a requirement for correct chromosome alignment and segregation after fertilization [44]. Already several authors had highlighted different structural and functional limitations in prepubertal ovine oocytes regarding small size, defective coupling between cumulus cells and oocytes, reduced protein synthesis and energy metabolism [5e9]. Moreover, in these oocytes it occurs frequently defects in the functioning of the microtubular network involved in spindle formation with chromosomal segregation errors [10]. In the present study, in vitrified adult oocytes the meiotic spindle recovered its typical barrel conformation during in vitro post-warming incubation, while in the vitrified prepubertal oocytes no signs of recovery were found, and the rates of structural anomalies even increased during postwarming incubation hours.
Please cite this article as: Serra E et al., Morphological features and microtubular changes in vitrified ovine oocytes, Theriogenology, https:// doi.org/10.1016/j.theriogenology.2019.11.007
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Fig. 7. Evaluation of acetylated a-tubulin fluorescence intensity in vitrified/warmed prepubertal oocytes during incubation post-warming. In the histogram, different letters indicate statistical differences at each time point (P ¼ 0.0001). Bar ¼ 10 mm.
Fig. 8. Effects of vitrification on tyrosinated a-tubulin (panel A) and acetylated atubulin (panel B) fluorescence intensity between vitrified/warmed adult and prepubertal oocytes during incubation post-warming. Asterisk indicate statistical differences between adult and prepubertal oocytes at each time point (A: P ¼ 0.111; B: P ¼ 0.0000).
This difference between prepubertal and adult oocytes in the ability of the spindle microtubules to reassembly after vitrification/ warming could be attributed to the observed differences in tubulin PTMs. In a previous study, we showed that in adult in vitro matured oocytes there are no differences between the fluorescence quantification of acetylated and tyrosinated a-tubulin [18]. On the other hand, in prepubertal oocytes the acetylated tubulin showed a higher fluorescence intensity compared to the tyrosinated tubulin. Predominant levels of tubulin acetylation in prepubertal oocytes may be correlated with excessive increase of microtubule stability, altered spindle morphology and defects in chromosome alignment [45]. In the present study, fluorescence intensity of tubulin PTMs was higher in vitrified/warmed prepubertal and adult oocytes compared to their fresh counterpart. However, during postwarming incubation adult oocytes showed a balance in the fluorescence quantification of the expressed tubulin PTMs. On the contrary, in prepubertal oocytes the fluorescence intensity of acetylated tubulin decreased during post-warming incubation, being lower compared to adult oocytes and leading to an imbalance in the fluorescence quantification of the expressed tubulin PTMs. The acetylated tubulin can form microtubule populations capable to withstand low temperatures [46]. The higher intensity of this tubulin PTMs in adult oocytes may indicate that the structure of the meiotic spindle is more stable than in prepubertal oocytes, a necessary requirement while awaiting fertilization. Moreover, in
adult oocytes the balance in tubulin PTMs could be related to their ability to reassembly the spindle microtubules after vitrification and warming and ultimately to restore their developmental competence during post-warming incubation, as previously described [32]. On the contrary, prepubertal oocytes, showing an imbalance in tubulin PTMs, are not able to reassemble their meiotic spindle after vitrification/warming and show a lack of developmental competence after vitrification [21,22]. In the present study, vitrified/warmed prepubertal oocytes also showed very high rates of parthenogenetic activation compared to fresh controls and adult oocytes. Parthenogenetic activation is given by an electrical or chemical stimulus that simulates the entry of the sperm into the egg cell. This signal induces the mobilization of Ca2þ from the endoplasmic reticulum with the consequent triggering of the pathways of completion of meiosis, with the transition from metaphase II to telophase II, the decondensation of chromatin, the formation of the first pronucleus and the first cell division [47]. The chromatin reorganization and nuclear membrane reconstruction depend on the Maturation Promoting Factor (MPF) activity that turns out to be higher in MII stage and then decreases after a few hours from oocyte activation [48]. It was noted that, in prepubertal ovine, the MPF activity is lower in vitrified oocytes after 2 h of post-warming incubation compared to control oocytes [21]. On the contrary, in adult ovine, there is a resumption of the kinase activity of MPF in vitrified oocytes [20]. Therefore, the high
Please cite this article as: Serra E et al., Morphological features and microtubular changes in vitrified ovine oocytes, Theriogenology, https:// doi.org/10.1016/j.theriogenology.2019.11.007
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posttranslational modifications may therefore represent a further parameter to evaluate oocyte quality and cryotolerance. Funding Financing of doctoral courses aimed at training highly specialized human capital - P.O.R. SARDINIA F.S.E. 2007e2013. Declaration of competing interest No conflict of interest declared. References
Fig. 9. Effects of vitrification on tubulin PTMs in vitrified/warmed adult (A) and prepubertal (B) oocytes during incubation post-warming. Asterisk indicate statistical differences between the fluorescence intensity of acetylated and tyrosinated a-tubulin at each time point (A: P ¼ 0.0041; B: P ¼ 0.0002).
rates of parthenogenetic activation observed during our experiments in vitrified prepubertal oocytes may be related to a low MPF activity. The high percentages of parthenogenetic activation in prepubertal oocytes indicate a high sensitivity to the damages associated with vitrification/warming and an impair functioning of the recovery mechanisms. 5. Conclusions In conclusion, our study showed that prepubertal and adult oocytes react differently to the insults provoked by the vitrification procedures. After vitrification/warming, the prepubertal oocytes show important anomalies in the meiotic spindle with high rates of parthenogenetic activation. Furthermore, they were not able to repair those damages during post-warming incubation. In contrast, adult oocytes were able to recover, at least partially, their functionality after 4 h of post-warming incubation, and this finding supports results shown in a previous study [32]. This ability was associated in adult oocyte with a balance in the fluorescence quantification of tubulin PTMs. Prepubertal oocyte, on the other hand, showed a strong imbalance between the tyrosinated and the acetylated tubulin during post-warming recovery time. The balance between these two tubulin PTMs could be essential for maintaining the functional and structural integrity of the ovine oocyte. An indepth study of the microtubular cytoskeleton and its
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Please cite this article as: Serra E et al., Morphological features and microtubular changes in vitrified ovine oocytes, Theriogenology, https:// doi.org/10.1016/j.theriogenology.2019.11.007