G Model
EURO 8555 1–5 European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Effect of necrostatin on mouse ovarian cryopreservation and transplantation
1 2
3 Q1 4 5 6
Jung Ryeol Lee a,b, * , Hye Won Youm a,b , Seul Ki Kim a , Byung Chul Jee a,b , Chang Suk Suh a,b , Seok Hyun Kim b a b
Department of Obstetrics and Gynaecology, Seoul National University Bundang Hospital, Seongnam, Republic of Korea Department of Obstetrics and Gynaecology, Seoul National University College of Medicine, Seoul, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 September 2013 Received in revised form 23 January 2014 Accepted 29 April 2014
Objective: To investigate the effects of necrostatin-1 (Nec-1) supplementation on vitrification, warming and transplantation of ovarian tissue. Study design: Ovaries from 4-week-old ICR mice were vitrified using a two-step procedure; ovaries were suspended in equilibration solution for 10 min, and then mixed with vitrification solution for 5 min. Ovaries were divided at random into three groups and 0 (control), 25 or 100 mM Nec-1 was added to the vitrification solution. After warming, follicular morphology and apoptosis were assessed. For each group, a sample of vitrified, warmed ovaries was autotransplanted. The same dose of Nec-1 that was added to the vitrification solution was added to each warming solution and injected intraperitoneally. Follicular morphology and apoptosis of transplanted ovaries were assessed after 2 weeks. Results: After vitrification and warming, morphological analysis revealed that the intact follicle ratio was significantly higher in the Nec-1-treated groups compared with the control group (control, 45.1%; 25 mM Nec-1, 51.7%; 100 mM Nec-1, 57.9%). The rate of apoptosis was lower in the Nec-1 treated groups compared with the control group (control, 11.2%; 25 mM Nec-1, 8.5%; 100 mM Nec-1, 7.2%). After transplantation of the vitrified, warmed ovaries, morphological analysis revealed that the intact follicle ratio was significantly higher in the Nec-1 treated groups compared with the control group (control, 43.1%; 25 mM Nec-1, 60.6%; 100 mM Nec-1, 70.7%). The rate of apoptosis was lower in the Nec-1 treated groups compared with the control group (control, 5.3%; 25 mM Nec-1, 2.5%; 100 mM Nec-1, 2.0%). Conclusions: Nec-1 supplementation during vitrification, warming and transplantation has beneficial effects on the survival of ovarian tissue. These results can help to improve ovarian tissue vitrification and transplantation protocols for fertility preservation. ã 2014 Published by Elsevier Ireland Ltd.
Keywords: Fertility preservation Ovarian tissue cryopreservation Vitrification Necrostatin
7
Introduction
8
Advances in the diagnosis and treatment of cancer have substantially improved the life expectancy of premenopausal women. However, aggressive chemotherapy or radiotherapy for cancer treatment has an adverse effect on gonadal function, and subsequently leads to a loss of fertility and premature ovarian failure. Therefore, the preservation of ovarian function in female
9 10 11 12 13
Q2
* Corresponding author at: Department of Obstetrics and Gynaecology, Seoul National University College of Medicine, 28 Yeongeon-dong, Jongno-gu, Seoul 110744, Republic of Korea. Tel.: +82 2 2072 3773/317877257; fax: +82 2 762 3599/ 317874054. E-mail addresses: [email protected] (J.R. Lee), [email protected] (S.H. Kim).
cancer survivors has become an important issue. Several options are available for fertility preservation in these women, such as cryopreservation of embryos, oocytes or ovarian tissue [1,2]. Cryopreservation of embryos and oocytes is a clinically established procedure that has been used for infertility treatment. Until recently, these procedures were considered to be the first option for fertility preservation in women. However, these procedures are preceded by controlled ovarian stimulation, which takes 2–3 weeks, resulting in a delay in cancer treatment. Cryopreservation and transplantation of ovarian tissue is one of the most promising options for fertility preservation of female cancer survivors, and has several advantages compared with other options. Ovarian stimulation is not necessary for ovarian cryopreservation, so there is no need to delay cancer treatment. In addition, this procedure is an option for single women and
http://dx.doi.org/10.1016/j.ejogrb.2014.04.040 0301-2115/ ã 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Lee JR, et al. Effect of necrostatin on mouse ovarian cryopreservation and transplantation. Eur J Obstet Gynecol (2014), http://dx.doi.org/10.1016/j.ejogrb.2014.04.040
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
G Model
EURO 8555 1–5 2 29
J.R. Lee et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 2 xxx (2014) xxx–xxx
67
prepubertal girls because a male partner is not required. To date, more than 20 live births have been reported in humans after transplantation of cryopreserved ovarian tissue [3]. However, cryopreservation of ovarian tissue is currently considered to be an experimental technique because the success rate is not appropriate for routine clinical use. The low success rate is due to damage to ovarian tissue during cryopreservation, thawing and transplantation. One of the major barriers to cryopreservation of ovarian tissue is that a large number of primordial follicles are lost when the ovarian tissue is transplanted. Ischaemic injury is a threat to tissue survival, resulting in necrosis, apoptosis and fibrotic changes soon after transplantation [4,5]. Many attempts have been made to reduce this damage, such as the addition of antioxidants [6–9], anti-apoptotic agents [10,11] or angiogenic factors [12,13] during cryopreservation and/or transplantation. However, not all of the agents studied have shown beneficial effects on tissue survival, and there have been conflicting results among reports that have studied the same agent. Traditionally, three main types of programmed cell death have been classified morphologically or biochemically: (1) apoptotic (caspase mediated or caspase independent); (2) autophagic (without the involvement of chromatin condensation); and (3) necrosis (necroptosis, paraptosis). Necroptosis is a recently discovered, regulated form of programmed necrosis that depends on the serine/threonine kinase activity of receptor-interacting protein kinase 1(RIP1) [14], and participates in the pathogenesis of diseases, including ischemic injury, neurodegeneration and viral infection [15]. The pro-necrotic function of RIP1 has been studied in various disease models using necrostatins; a series of chemical inhibitors against RIP1 activity [16]. Necrostatin-1 (Nec-1) has been reported to suppress necroptotic cell death triggered by an array of stimuli in various cell types. Nec-1-inhibitable nonapoptotic death is an important contributor to ischaemic injury in mouse models of cerebral ischaemia and myocardial infarction [17,18]. Therefore, it was hypothesized that Nec-1 supplementation may help to reduce ischaemic necrosis after ovarian transplantation. The aim of this study was to investigate the effect of Nec-1 supplementation during vitrification and transplantation of ovarian tissue.
68
Materials and methods
69
Vitrification and warming of ovarian tissue
30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
70 71 72 73 74 75 76 77
The experimental procedures performed were similar to those reported previously [11]. The animals in this study were cared for and used in accordance with the institutional guidelines established by the Animal Care and Use Committee of Seoul National University of Bundang Hospital. ICR mice (Orient Co., Seoul, Korea) were maintained under a 12-h light:12-h dark cycle at 23 C and fed ad libitum. After 1 week of adaptation, both ovaries of 4-weekold mice were resected. Ovaries were vitrified using a two-step
method involving exposure to equilibration and vitrification solutions [19]. Equilibration solution contained 20% ethylene glycol (Sigma Chemical Co., St. Louis, MO, USA), and vitrification solution was composed of 40% ethylene glycol, 18% Ficoll (Sigma) and 0.3 M sucrose (Sigma). All solutions were based on Dulbecco’s phosphate-buffered saline (DPBS; Gibco BRL, Grand Island, NY, USA) containing 20% fetal bovine serum (Gibco). Intact ovaries were initially suspended in 1 ml equilibration solution for 10 min and then mixed with 0.5 ml vitrification solution for 5 min. The ovaries were divided randomly into three groups, and 0 (control), 25 or 100 mM Nec-1 (Sigma) was added to the vitrification solution. After exposure to equilibration and vitrification solutions, ovaries were transferred immediately to 1.2-ml cryotubes (Nunc; Fisher Bioblock Scientific, Illkirch, France) and plunged into liquid nitrogen. After 2 weeks, the tissues were warmed by immersing the vials rapidly in a water bath at 37 C, and suspended serially in 0.5 M sucrose in DPBS containing 20% fetal bovine serum for 5 min, 0.25 M for 5 min, and 0 M for 10 min.
78
Morphological assessment of follicles
96
Ovarian samples were fixed in 10% buffered formalin and then embedded in a paraffin block. Paraffin-embedded ovarian sections were cut (thickness 5 mm) for routine histological examination. After deparaffinization and rehydration, the sections were stained with haematoxylin and eosin. The numbers of each type of follicle were counted for the entire cut ovarian surface (magnification 400). All follicles found in one section of each ovary were scored. Each type of follicle was categorized according to the following classification [20]:
97
(1) primordial – single layer of flattened pre-granulosa cells; (2) primary – single layer of granulosa cells; one or more being cuboidal cells; (3) secondary – two or more layers of cuboidal granulosa cells with antrum absent; and (4) antral – multiple layers of cuboidal granulosa cells with antrum present. The integrity of each follicle was evaluated using the following criteria [21]: (1) Primordial/primary follicle. G1 – spherical with even distribution of granulosa cells; G2 – granulosa cells pulled away from the edge of the follicle but oocyte still spherical; and G3 – pyknotic nuclei, mis-shapen oocyte or vacuolation. (2) Secondary/antral follicle. G1 – intact spherical follicle with evenly distributed granulosa and theca cells, small space and spherical oocyte; G2 – intact theca cell, disruption of granulosa cells and spherical oocyte; and G3 – disruption and loss of granulosa and theca cells, pyknotic nuclei and missing oocyte.
106
Fig. 1. Fluorescent TUNEL staining of vitrified, warmed ovaries for the control group, 25 mM Nec-1 treated group, 100 mM Nec-1 treated group, positive control and negative control (original magnification 100). Apoptosis was defined as staining of >30% of follicular cells.
Please cite this article in press as: Lee JR, et al. Effect of necrostatin on mouse ovarian cryopreservation and transplantation. Eur J Obstet Gynecol (2014), http://dx.doi.org/10.1016/j.ejogrb.2014.04.040
79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95
98 99 100 101 102 103 104 105
107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126
G Model
EURO 8555 1–5 J.R. Lee et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 2 xxx (2014) xxx–xxx 127
Detection of apoptotic follicles by TUNEL assay
128
Paraffin-embedded ovarian sections (thickness 5 mm) were cut and assessed for apoptosis using a commercial TUNEL assay kit (In Situ Cell Death Detection Kit, Fluorescein, Roche Applied Science, Germany). After deparaffinization and rehydration, sections were rinsed in PBS (pH 7.2) and digested using proteinase K (20 mg/ml, 37 C, 30 min) in 10 mM Tris–HCl buffer. After rinsing in DPBS, the specimens were incubated with 50 ml TUNEL reaction mixture at 37 C for 60 min in a humidified chamber in the dark, and subsequently rinsed with DPBS. Positive control slides were prepared by treating 1500 U/ml DNase I (Roche Applied Science) in 50 mM Tris–HCl (pH 7.5, including 1 mg/ml bovine serum albumin) for 10 min at room temperature to induce DNA strand breaks, prior to labelling procedures. Some ovarian tissue specimens were used as negative controls by substituting terminal deoxynucleotidyl transferase with distilled water in the reaction mixture following the protocol.
129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144
155
The slides were covered with DAPI (Vector Laboratories, Burlingame, CA, USA) to counterstain DNA. TUNEL-stained and DAPI-counterstained slides were examined under an inverted fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Green fluorescence was visualized in TUNEL-positive cells at an excitation wavelength in the range of 450–500 nm and detection in the range of 515–565 nm. DAPI reached excitation at approximately 360 nm and emitted at approximately 460 nm when bound to DNA, producing blue fluorescence in all nuclei. Only follicles with a visible nucleus were counted regardless of their type. Follicles were considered to be apoptotic if >30% of the cells were TUNEL stained (Fig. 1).
156
Ovarian autotransplantation
157
After vitrification and warming of the ovaries as described above, a sample was autotransplanted for each group into the dorsal subcutaneous space of the flank. The same dose of Nec-1 that was added to the vitrification solution (0, 25 or 100 mM) was added to each warming solution. In addition, 30 min before transplantation, the same concentration of Nec-1 was also injected intraperitoneally with a final volume of 0.3 ml. The transplanted ovarian tissues were retrieved 2 weeks later, and their gross morphology was examined. Follicular normality and apoptosis were assessed in normalappearing, intact ovaries as described above.
145 146 147 148 149 150 151 152 153 154
158 159 160 161 162 163 164 165 166
3
Fig. 2. Morphologically intact (grade I) follicle and apoptotic follicle ratios according to necrostatin-1 (Nec-1) supplementation of vitrification solution after warming.
Statistical analysis
167
Follicle stages and normality were calculated for each group. Statistical analysis was performed using analysis of variance, Student’s t-test or Kruskal Wallis test for continuous variables, and Chi-squared test or Fisher’s exact test for categorical variables, as appropriate. p < 0.05 was considered to indicate statistical significance. Statistical Package for the Social Sciences Version 18.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis.
168
Results
175
Morphological analysis following vitrification and warming found that the intact follicle ratio was significantly higher in the Nec-1 treated groups compared with the control group (control, 45.0%; 25 mM Nec-1, 51.7%; 100 mM Nec-1, 58.4%). The rate of apoptosis was lower in the Nec-1 treated groups compared with the control group (control, 11.2%; 25 mM Nec-1, 8.5%; 100 mM Nec-1, 7.2%), but the differences did not reach statistical significance (Table 1, Fig. 2). Following transplantation of the vitrified, warmed ovaries, morphological analysis showed that the intact follicle ratio was significantly higher in the Nec-1 treated groups compared with the control group (control, 43.1%; 25 mM Nec-1, 60.6%; 100 mM Nec-1, 70.7%). When follicle types were considered separately, the Nec-1
176
Table 1 Table 2 Proportions of follicles with good morphology (grade I) and apoptotic follicles Q3 Proportions of follicles with good morphology (grade I) and apoptotic follicles following necrostatin-1 (Nec-1) supplementation of vitrification solution after following necrostatin-1 (Nec-1) supplementation of vitrification and warming warming. solutions, and injection after autotransplantation of vitrified, warmed ovary.
Gross survival rate of ovaries Grade I follicles Primordial Primary Secondary Antral Total Apoptotic follicles
Controla
Nec-1 25 mMa
Nec-1100 m Ma
p
100% (35/35)
100% (30/30)
100% (29/29)
NA
77/124 (62.1%) 108/222 (48.6%)a 138/388 (35.6%)a 60/117 (51.3%) 383/851 (45.0%)a 25/223 (11.2%)
59/97 (60.8%) 115/205 (56.1%)a 157/354 (44.4%)a 69/117 (59.0%) 400/773 (51.7%)a 27/317 (8.5%)
114/166 0.341 (68.7%) 107/164 0.005 (65.2%)a 128/278 0.010 (46.0%)a 62/96 0.141 (64.6%) 411/704
EURO 8555 1–5 European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Effect of necrostatin on mouse ovarian cryopreservation and transplantation
1 2
3 Q1 4 5 6
Jung Ryeol Lee a,b, * , Hye Won Youm a,b , Seul Ki Kim a , Byung Chul Jee a,b , Chang Suk Suh a,b , Seok Hyun Kim b a b
Department of Obstetrics and Gynaecology, Seoul National University Bundang Hospital, Seongnam, Republic of Korea Department of Obstetrics and Gynaecology, Seoul National University College of Medicine, Seoul, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 September 2013 Received in revised form 23 January 2014 Accepted 29 April 2014
Objective: To investigate the effects of necrostatin-1 (Nec-1) supplementation on vitrification, warming and transplantation of ovarian tissue. Study design: Ovaries from 4-week-old ICR mice were vitrified using a two-step procedure; ovaries were suspended in equilibration solution for 10 min, and then mixed with vitrification solution for 5 min. Ovaries were divided at random into three groups and 0 (control), 25 or 100 mM Nec-1 was added to the vitrification solution. After warming, follicular morphology and apoptosis were assessed. For each group, a sample of vitrified, warmed ovaries was autotransplanted. The same dose of Nec-1 that was added to the vitrification solution was added to each warming solution and injected intraperitoneally. Follicular morphology and apoptosis of transplanted ovaries were assessed after 2 weeks. Results: After vitrification and warming, morphological analysis revealed that the intact follicle ratio was significantly higher in the Nec-1-treated groups compared with the control group (control, 45.1%; 25 mM Nec-1, 51.7%; 100 mM Nec-1, 57.9%). The rate of apoptosis was lower in the Nec-1 treated groups compared with the control group (control, 11.2%; 25 mM Nec-1, 8.5%; 100 mM Nec-1, 7.2%). After transplantation of the vitrified, warmed ovaries, morphological analysis revealed that the intact follicle ratio was significantly higher in the Nec-1 treated groups compared with the control group (control, 43.1%; 25 mM Nec-1, 60.6%; 100 mM Nec-1, 70.7%). The rate of apoptosis was lower in the Nec-1 treated groups compared with the control group (control, 5.3%; 25 mM Nec-1, 2.5%; 100 mM Nec-1, 2.0%). Conclusions: Nec-1 supplementation during vitrification, warming and transplantation has beneficial effects on the survival of ovarian tissue. These results can help to improve ovarian tissue vitrification and transplantation protocols for fertility preservation. ã 2014 Published by Elsevier Ireland Ltd.
Keywords: Fertility preservation Ovarian tissue cryopreservation Vitrification Necrostatin
7
Introduction
8
Advances in the diagnosis and treatment of cancer have substantially improved the life expectancy of premenopausal women. However, aggressive chemotherapy or radiotherapy for cancer treatment has an adverse effect on gonadal function, and subsequently leads to a loss of fertility and premature ovarian failure. Therefore, the preservation of ovarian function in female
9 10 11 12 13
Q2
* Corresponding author at: Department of Obstetrics and Gynaecology, Seoul National University College of Medicine, 28 Yeongeon-dong, Jongno-gu, Seoul 110744, Republic of Korea. Tel.: +82 2 2072 3773/317877257; fax: +82 2 762 3599/ 317874054. E-mail addresses: [email protected] (J.R. Lee), [email protected] (S.H. Kim).
cancer survivors has become an important issue. Several options are available for fertility preservation in these women, such as cryopreservation of embryos, oocytes or ovarian tissue [1,2]. Cryopreservation of embryos and oocytes is a clinically established procedure that has been used for infertility treatment. Until recently, these procedures were considered to be the first option for fertility preservation in women. However, these procedures are preceded by controlled ovarian stimulation, which takes 2–3 weeks, resulting in a delay in cancer treatment. Cryopreservation and transplantation of ovarian tissue is one of the most promising options for fertility preservation of female cancer survivors, and has several advantages compared with other options. Ovarian stimulation is not necessary for ovarian cryopreservation, so there is no need to delay cancer treatment. In addition, this procedure is an option for single women and
http://dx.doi.org/10.1016/j.ejogrb.2014.04.040 0301-2115/ ã 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Lee JR, et al. Effect of necrostatin on mouse ovarian cryopreservation and transplantation. Eur J Obstet Gynecol (2014), http://dx.doi.org/10.1016/j.ejogrb.2014.04.040
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
G Model
EURO 8555 1–5 2 29
J.R. Lee et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 2 xxx (2014) xxx–xxx
67
prepubertal girls because a male partner is not required. To date, more than 20 live births have been reported in humans after transplantation of cryopreserved ovarian tissue [3]. However, cryopreservation of ovarian tissue is currently considered to be an experimental technique because the success rate is not appropriate for routine clinical use. The low success rate is due to damage to ovarian tissue during cryopreservation, thawing and transplantation. One of the major barriers to cryopreservation of ovarian tissue is that a large number of primordial follicles are lost when the ovarian tissue is transplanted. Ischaemic injury is a threat to tissue survival, resulting in necrosis, apoptosis and fibrotic changes soon after transplantation [4,5]. Many attempts have been made to reduce this damage, such as the addition of antioxidants [6–9], anti-apoptotic agents [10,11] or angiogenic factors [12,13] during cryopreservation and/or transplantation. However, not all of the agents studied have shown beneficial effects on tissue survival, and there have been conflicting results among reports that have studied the same agent. Traditionally, three main types of programmed cell death have been classified morphologically or biochemically: (1) apoptotic (caspase mediated or caspase independent); (2) autophagic (without the involvement of chromatin condensation); and (3) necrosis (necroptosis, paraptosis). Necroptosis is a recently discovered, regulated form of programmed necrosis that depends on the serine/threonine kinase activity of receptor-interacting protein kinase 1(RIP1) [14], and participates in the pathogenesis of diseases, including ischemic injury, neurodegeneration and viral infection [15]. The pro-necrotic function of RIP1 has been studied in various disease models using necrostatins; a series of chemical inhibitors against RIP1 activity [16]. Necrostatin-1 (Nec-1) has been reported to suppress necroptotic cell death triggered by an array of stimuli in various cell types. Nec-1-inhibitable nonapoptotic death is an important contributor to ischaemic injury in mouse models of cerebral ischaemia and myocardial infarction [17,18]. Therefore, it was hypothesized that Nec-1 supplementation may help to reduce ischaemic necrosis after ovarian transplantation. The aim of this study was to investigate the effect of Nec-1 supplementation during vitrification and transplantation of ovarian tissue.
68
Materials and methods
69
Vitrification and warming of ovarian tissue
30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
70 71 72 73 74 75 76 77
The experimental procedures performed were similar to those reported previously [11]. The animals in this study were cared for and used in accordance with the institutional guidelines established by the Animal Care and Use Committee of Seoul National University of Bundang Hospital. ICR mice (Orient Co., Seoul, Korea) were maintained under a 12-h light:12-h dark cycle at 23 C and fed ad libitum. After 1 week of adaptation, both ovaries of 4-weekold mice were resected. Ovaries were vitrified using a two-step
method involving exposure to equilibration and vitrification solutions [19]. Equilibration solution contained 20% ethylene glycol (Sigma Chemical Co., St. Louis, MO, USA), and vitrification solution was composed of 40% ethylene glycol, 18% Ficoll (Sigma) and 0.3 M sucrose (Sigma). All solutions were based on Dulbecco’s phosphate-buffered saline (DPBS; Gibco BRL, Grand Island, NY, USA) containing 20% fetal bovine serum (Gibco). Intact ovaries were initially suspended in 1 ml equilibration solution for 10 min and then mixed with 0.5 ml vitrification solution for 5 min. The ovaries were divided randomly into three groups, and 0 (control), 25 or 100 mM Nec-1 (Sigma) was added to the vitrification solution. After exposure to equilibration and vitrification solutions, ovaries were transferred immediately to 1.2-ml cryotubes (Nunc; Fisher Bioblock Scientific, Illkirch, France) and plunged into liquid nitrogen. After 2 weeks, the tissues were warmed by immersing the vials rapidly in a water bath at 37 C, and suspended serially in 0.5 M sucrose in DPBS containing 20% fetal bovine serum for 5 min, 0.25 M for 5 min, and 0 M for 10 min.
78
Morphological assessment of follicles
96
Ovarian samples were fixed in 10% buffered formalin and then embedded in a paraffin block. Paraffin-embedded ovarian sections were cut (thickness 5 mm) for routine histological examination. After deparaffinization and rehydration, the sections were stained with haematoxylin and eosin. The numbers of each type of follicle were counted for the entire cut ovarian surface (magnification 400). All follicles found in one section of each ovary were scored. Each type of follicle was categorized according to the following classification [20]:
97
(1) primordial – single layer of flattened pre-granulosa cells; (2) primary – single layer of granulosa cells; one or more being cuboidal cells; (3) secondary – two or more layers of cuboidal granulosa cells with antrum absent; and (4) antral – multiple layers of cuboidal granulosa cells with antrum present. The integrity of each follicle was evaluated using the following criteria [21]: (1) Primordial/primary follicle. G1 – spherical with even distribution of granulosa cells; G2 – granulosa cells pulled away from the edge of the follicle but oocyte still spherical; and G3 – pyknotic nuclei, mis-shapen oocyte or vacuolation. (2) Secondary/antral follicle. G1 – intact spherical follicle with evenly distributed granulosa and theca cells, small space and spherical oocyte; G2 – intact theca cell, disruption of granulosa cells and spherical oocyte; and G3 – disruption and loss of granulosa and theca cells, pyknotic nuclei and missing oocyte.
106
Fig. 1. Fluorescent TUNEL staining of vitrified, warmed ovaries for the control group, 25 mM Nec-1 treated group, 100 mM Nec-1 treated group, positive control and negative control (original magnification 100). Apoptosis was defined as staining of >30% of follicular cells.
Please cite this article in press as: Lee JR, et al. Effect of necrostatin on mouse ovarian cryopreservation and transplantation. Eur J Obstet Gynecol (2014), http://dx.doi.org/10.1016/j.ejogrb.2014.04.040
79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95
98 99 100 101 102 103 104 105
107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126
G Model
EURO 8555 1–5 J.R. Lee et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 2 xxx (2014) xxx–xxx 127
Detection of apoptotic follicles by TUNEL assay
128
Paraffin-embedded ovarian sections (thickness 5 mm) were cut and assessed for apoptosis using a commercial TUNEL assay kit (In Situ Cell Death Detection Kit, Fluorescein, Roche Applied Science, Germany). After deparaffinization and rehydration, sections were rinsed in PBS (pH 7.2) and digested using proteinase K (20 mg/ml, 37 C, 30 min) in 10 mM Tris–HCl buffer. After rinsing in DPBS, the specimens were incubated with 50 ml TUNEL reaction mixture at 37 C for 60 min in a humidified chamber in the dark, and subsequently rinsed with DPBS. Positive control slides were prepared by treating 1500 U/ml DNase I (Roche Applied Science) in 50 mM Tris–HCl (pH 7.5, including 1 mg/ml bovine serum albumin) for 10 min at room temperature to induce DNA strand breaks, prior to labelling procedures. Some ovarian tissue specimens were used as negative controls by substituting terminal deoxynucleotidyl transferase with distilled water in the reaction mixture following the protocol.
129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144
155
The slides were covered with DAPI (Vector Laboratories, Burlingame, CA, USA) to counterstain DNA. TUNEL-stained and DAPI-counterstained slides were examined under an inverted fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Green fluorescence was visualized in TUNEL-positive cells at an excitation wavelength in the range of 450–500 nm and detection in the range of 515–565 nm. DAPI reached excitation at approximately 360 nm and emitted at approximately 460 nm when bound to DNA, producing blue fluorescence in all nuclei. Only follicles with a visible nucleus were counted regardless of their type. Follicles were considered to be apoptotic if >30% of the cells were TUNEL stained (Fig. 1).
156
Ovarian autotransplantation
157
After vitrification and warming of the ovaries as described above, a sample was autotransplanted for each group into the dorsal subcutaneous space of the flank. The same dose of Nec-1 that was added to the vitrification solution (0, 25 or 100 mM) was added to each warming solution. In addition, 30 min before transplantation, the same concentration of Nec-1 was also injected intraperitoneally with a final volume of 0.3 ml. The transplanted ovarian tissues were retrieved 2 weeks later, and their gross morphology was examined. Follicular normality and apoptosis were assessed in normalappearing, intact ovaries as described above.
145 146 147 148 149 150 151 152 153 154
158 159 160 161 162 163 164 165 166
3
Fig. 2. Morphologically intact (grade I) follicle and apoptotic follicle ratios according to necrostatin-1 (Nec-1) supplementation of vitrification solution after warming.
Statistical analysis
167
Follicle stages and normality were calculated for each group. Statistical analysis was performed using analysis of variance, Student’s t-test or Kruskal Wallis test for continuous variables, and Chi-squared test or Fisher’s exact test for categorical variables, as appropriate. p < 0.05 was considered to indicate statistical significance. Statistical Package for the Social Sciences Version 18.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis.
168
Results
175
Morphological analysis following vitrification and warming found that the intact follicle ratio was significantly higher in the Nec-1 treated groups compared with the control group (control, 45.0%; 25 mM Nec-1, 51.7%; 100 mM Nec-1, 58.4%). The rate of apoptosis was lower in the Nec-1 treated groups compared with the control group (control, 11.2%; 25 mM Nec-1, 8.5%; 100 mM Nec-1, 7.2%), but the differences did not reach statistical significance (Table 1, Fig. 2). Following transplantation of the vitrified, warmed ovaries, morphological analysis showed that the intact follicle ratio was significantly higher in the Nec-1 treated groups compared with the control group (control, 43.1%; 25 mM Nec-1, 60.6%; 100 mM Nec-1, 70.7%). When follicle types were considered separately, the Nec-1
176
Table 1 Table 2 Proportions of follicles with good morphology (grade I) and apoptotic follicles Q3 Proportions of follicles with good morphology (grade I) and apoptotic follicles following necrostatin-1 (Nec-1) supplementation of vitrification solution after following necrostatin-1 (Nec-1) supplementation of vitrification and warming warming. solutions, and injection after autotransplantation of vitrified, warmed ovary.
Gross survival rate of ovaries Grade I follicles Primordial Primary Secondary Antral Total Apoptotic follicles
Controla
Nec-1 25 mMa
Nec-1100 m Ma
p
100% (35/35)
100% (30/30)
100% (29/29)
NA
77/124 (62.1%) 108/222 (48.6%)a 138/388 (35.6%)a 60/117 (51.3%) 383/851 (45.0%)a 25/223 (11.2%)
59/97 (60.8%) 115/205 (56.1%)a 157/354 (44.4%)a 69/117 (59.0%) 400/773 (51.7%)a 27/317 (8.5%)
114/166 0.341 (68.7%) 107/164 0.005 (65.2%)a 128/278 0.010 (46.0%)a 62/96 0.141 (64.6%) 411/704
