N-acetylcysteine improves function and follicular survival in mice ovarian grafts through inhibition of oxidative stress.

ARTICLE IN PRESS Reproductive BioMedicine Online (2014) ■■, ■■–■■

w w w. s c i e n c e d i r e c t . c o m w w w. r b m o n l i n e . c o m

ARTICLE

N-acetylcysteine improves function and follicular survival in mice ovarian grafts through inhibition of oxidative stress Monireh Mahmoodi a, Malek Soleimani Mehranjani a,*, Seyed Mohammad Ali Shariatzadeh a, Hussein Eimani b, Abdulhussein Shahverdi b a

Department of Biology, Faculty of Science, Arak University, Arak 381-5688138, Iran; b Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran 166-5659911, Iran * Corresponding author. E-mail address: [email protected] (M Soleimani Mehranjani). Authors’ roles: MSM, MM and SMAS, HE, AS were responsible for study concept and design; MM was responsible for experimental procedures; MM and MSM were responsible for analysis of results and manuscript preparation; and MSM was responsible for manuscript revision. Dr Malek Soleimani Mehranjani obtained his MSc in histology at the Tarbiat Modares University, Iran, and his PhD at Sheffield University, UK. He returned to Arak University in Iran in 1999 and jointly founded the Reproductive Biology centre there. He has carried out many relevant studies as Associate Professor ever since. His major areas of interest are histological, pathological and clinical evaluation of the reproductive system, especially the ovary. He has published extensively in the field of environmental toxicants, focusing on their disturbing effects on the reproductive system along with challenging different therapeutic approaches.

Abstract The effect of N-acetylcysteine (NAC) on mouse ovary heterotopic autotransplantation was investigated. Mice (age 4–5 weeks) were divided into the following groups: control; autograft plus NAC (150 mg/kg daily intraperitoneal injection) and autograft plus saline (n = 6 per group). Groups were treated from 1 day before until 7 days after transplantation. After 28 days, ovary compartments were estimated stereologically. Plasma malondialdehyde, progesterone, oestradiol concentrations and the percentage of apoptotic follicles were measured to evaluate the rate of oxidative stress and ovarian graft function. The mean total volume of ovary, cortex and the number of follicles was significantly higher (all P < 0.001) in the autografts plus NAC group compared with the saline group. In the autografts plus NAC group, the mean percentage of apoptotic follicles (P < 0.001) and malondialdehyde concentration (P < 0.001 day 7; P < 0.05 day 28) were significantly lower, whereas oestradiol concentration was significantly higher (P < 0.05) compared with the saline group. Although NAC cannot compensate the above parameters to the control level, it considerably improves follicular survival and development and also the structure and function of transplanted ovaries, through reducing oxidative stress and apoptosis. © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.rbmo.2014.09.013 1472-6483/© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Monireh Mahmoodi, Malek Soleimani Mehranjani, Seyed Mohammad Ali Shariatzadeh, Hussein Eimani, Abdulhussein Shahverdi, N-acetylcysteine improves function and follicular survival in mice ovarian grafts through inhibition of oxidative stress, Reproductive BioMedicine Online (2014), doi: 10.1016/ j.rbmo.2014.09.013

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M Mahmoodi et al.

KEYWORDS: autotransplantation, ischaemia reperfusion, mice, N-acetylcysteine, ovary

Introduction Transplantation of ovarian tissue is used as a promising technology for preserving fertility in cancer patients undergoing chemotherapy and radiotherapy whose reproductive potential is threatened (Kim, 2010; Skaznik-Wikiel et al., 2011). The most important challenge in ovarian tissue transplantation is the survival rate and the functional longevity of the ovary (Callejo et al., 2002). During the neovascularisation of the transplanted ovarian tissue, an initial ischaemia followed by a reperfusion period occurs, leading to an excessive production of reactive oxygen species (ROS). This causes endothelial injury, increases microvascular permeability and tissue swelling, as well as starting the inflammatory responses through activation of adhesion molecules and enhanced cytokine production (Damous et al., 2009; Khan et al., 2004). The production of ROS is also directly involved in the oxidative damage of cellular macromolecules, such as nucleic acids, proteins and lipids in ischaemic tissues (Usta et al., 2008). All of these events can lead to a massive follicular loss in the transplanted ovarian tissue (Damous et al., 2009; Gao et al., 2013; Hirayama et al., 2011; Kim, 2010; Skaznik-Wikiel et al., 2011). Therefore, the application of antioxidants to reduce the production of free radicals can be considered as an important drug target to confront the consequences of ischaemia and reperfusion injury after ovarian tissues transplantation. N-acetylcysteine (NAC) has been extensively studied as a potent antioxidant in several studies (Danilovic et al., 2011; Inci et al., 2007; Usta et al., 2008). It is an aminothiol, a precursor of intracellular cysteine and reduced glutathione (Inci et al., 2007; Sun, 2010), which easily penetrates the cell membranes (Usta et al., 2008). Loss of the cell antioxidant capacity is mainly caused by a decrease in glutathione reservoir, its precursor cysteine, or both. It has been shown that NAC reduces oxidative stress through the scavenging of free oxygen radicals or up-regulating antioxidant systems, such as superoxide dismutase or enhancing the catalytic activity of glutathione peroxidase (Cuzzocrea et al., 2000; Nitescu et al., 2006; Zafarullah et al., 2003). Moreover, NAC can also prevent apoptosis and promote cell survival (Zafarullah et al., 2003). It has also been used in many therapeutic applications in different disorders, such as infertility in patients with clomipheneresistant polycystic ovary syndrome, endothelial dysfunction, inflammation and lung fibrosis (Cuzzocrea et al., 2000; Millea, 2009; Zafarullah et al., 2003). In addition, the protective effect of NAC on ischaemia-reperfusion injuries of tissues like testis, myocardium, brain, spinal cord, kidney, liver and ovary has been also reported (Araujo et al., 2005; Glantzounis et al., 2004; Khan et al., 2004; Nitescu et al., 2006; Usta et al., 2008). Considering all the beneficial effects of NAC, along with the fact that it has low cell toxicity and few side-effects (Usta et al., 2008), the aim of this study was to investigate the effects of NAC, as an antioxidant and antiapoptosis agent, on reducing the ischaemia reperfusion injury after heterotopic autologus ovarian transplantation, for the first time, as well as evaluating its effect on improving the ovarian graft

survival rate and function, follicular development and preservation of follicular pool.

Materials and methods Animals All animal procedures were approved by the Animal Care and Use Committee at Royan Institute on 10 July 2010. Female Naval Medical Research Institute (NMRI) mice (4–5 weeks) were purchased from Pasteur Institute (Tehran, Iran), and kept in the animal house of Arak University under light and temperature controlled conditions in light–dark cycles for 12 h at 21 ± 2°C with free access to enough food and water. We used the NMRI mice because other studies have also shown that NMRI mice are suitable for ovarian tissue transplantation (Abedi et al., 2014; Abtahi et al., 2014; Eimani et al., 2009, 2011). Mice were randomly divided into three groups (six animals per group): control (mice without ovariectomy or grafting); autograft plus saline; and autograft plus NAC (treated with 150 mg/kg intraperitoneal NAC).

Ovarian autotransplantation and N-acetylcysteine treatment The animals were anaesthetized with an intraperitoneal injection of ketamine (100 mg/kg, ketamine 10%; Alfasan,Woerden, the Netherlands) and xylazine (10 mg/kg, xylazine 2%; Alfasan, Woerden, the Netherlands). Dorsal areas of each mouse were shaved and the skin was aseptically cleaned immediately before surgery. Ovariectomy was carried out through bilateral incisions on each side of the spinal column in the dorsal body wall. A small incision (0.5 cm) was made along the right and left gluteus superficialis muscle fibres, and the excised ovaries were autografted immediately into the gluteal muscles as the transplantation site as multiple reports have documented the effect of intramuscular transplantation on increasing follicular development and survival rate and fast vascularization (Abir et al., 2011; Dath et al., 2010; Friedman et al., 2012; Li et al., 2010; Soleimani et al., 2008, 2010). In addition, Israely et al. (2003) compared the efficacy of two transplantation sites (subcutaneous and intramuscular), which found that intramuscular grafts recovered and resembled normal ovaries; however, most of the subcutaneous grafts remained impaired and necrotic (Israely et al., 2003). Finally, the muscle and skin incisions were closed with absorbable suture (Hurchromic 5/0; Iran) and non-absorbable suture (Supalon 5/0; Iran) under aseptic conditions, respectively. In the autografts plus NAC group, mice were injected with NAC (150 mg/kg daily intraperitoneal injection; SigmaAldrich, China), 1 day before surgery until 7 days after transplantation; in the autograft plus saline group, intraperitoneal injections of physiological saline were carried out in the same manner. A dose of 150 mg/kg of NAC was used based on doses used in the studies by Khan et al. (2004) on cerebral


ARTICLE IN PRESS N-acetylcysteine improves ovary function in grafts ischaemia and Inci et al. (2007) on lung transplantation in rats. After 28 days, the mice were anaesthetized and the ovarian grafts were recovered from the transplantation site.

Stereological study The recovered ovarian tissues were fixed in Bouin’s solution for 24 h and were dehydrated in ascending concentrations of ethanol (70–100%) by an automated tissue processor (Leica, Histokinette; Germany) then embedded in paraffin and blocked. The ‘isector’ method was used to obtain isotropic uniform random sections. For this purpose, spherical modules filled with paraffin were chosen to embed each ovary. These modules were rotated in a random manner and serially cut into 5- and 20-µm thick sections. This was followed by systematic random sampling to select 8 to 12 sections from each ovary, which were in turn stained using the haematoxylin and eosin (Merck, Darmstadt, Germany) method (Howard and Reed, 1998; Karbalay-Doust and Noorafshan, 2012; Soleimani Mehranjani et al., 2010). Finally the ovarian tissue was examined stereologically.

Estimation of the volume of ovary, cortex and medulla

3

Estimation of the number of follicles In order to estimate the number of follicles, a combination of an unbiased sampling frame and the optical disector design was used to sample the tissue. Following systematic random sampling, an average of 12 sections were selected out of 20-µm thick sections, which were then studied by microscope (Olympus, Japan, BX51) with ×100 oil immersion objective and a high numerical aperture (NA: 1.4). The microscope stage was moved in an equal distance to select microscopic fields. To measure the movement of the stage in the z-axis, a microcator (ND 221 B; Heidenhain, Traunreut, Germany) connected to a computer and microscope (Olympus BX51,Tokyo, Japan) was used. At each microscopic field, 5 µm from the top and bottom of the sections was ignored as a guard area against the cutting artifacts (Karbalay-Doust and Noorafshan, 2012; Myers et al., 2004; Soleimani Mehranjani et al., 2010). An unbiased counting frame superimposed on the monitor was used to sample the nucleoli profiles of the oocytes. Different types of follicles were identified based on the Myers et al. (2004) classification. The selected nucleoli of the oocytes were those that were placed inside or partially inside the sampling frame having no contact with the exclusion lines of the frame. About 100–120 oocytes per ovary were analysed and the numerical density (Nv), defined as the number of the cells in the unit volume of the ovary, was estimated by: n

To estimate the total volume of ovary, cortex and medulla, an average of 12 sections were randomly selected from 5-µm thick sections. The sections were studied using the microscope (Olympus, Japan, BX51) with 4× magnification. The resulting points from the randomly superimposed probe on the images were then counted. Using Cavalieri method, the total volume of the ovary was estimated: n

Vtotal ovary = ∑ P × a ( P ) × t i =1

n

∑ P is the total number of points superimposed

In which

i =1

on the image of ovarian sections, a (p) stands for the corresponding area of each point, and (t) denotes the distance between the sampled sections and the section thickness. The volume density for each ovary compartment was calculated as follows: n

∑p

Cortex

VVcortex =

∑p

total

∑P

total

the total number of is counted points and

i =1

cortex

=

i =1 n

h× ∑P ×a f n=1

Where ΣQ is the total number of the counted follicles in the disector height (h), a/f is the area of each frame and ΣP denotes the total number of the frames counted in all microscopic fields. The total number of the follicles was estimated through multiplying the numerical density (Nv) by the total volume of the ovary, (Ntotal = NV × Vtotal) (Howard and Reed, 1998; Karbalay-Doust and Noorafshan, 2012; Soleimani Mehranjani et al., 2010). To estimate the mean percentage of ovaries containing corpus luteum in both saline- and NAC-treated groups, the obtained sections from each graft were examined, and any ovary that was observed to contain the corpus luteum in its relevant sections was counted as one. On the basis of the number of ovaries containing the corpus luteum among the other ovaries in each group, the mean percentage of grafts possessing corpus luteum was estimated.

Estimation of the volume of oocyte and its nuclei

n

Where n

V

i =1 n

i =1

∑P

N

∑Q

is the total number of superimposed points on the

i =1

cortex. In turn, the volume density (Vv) was multiplied by the total volume of ovary to estimate the volume of cortex and medulla (Howard and Reed, 1998; Karbalay-Doust and Noorafshan, 2012; Noorafshan et al., 2013; Soleimani Mehranjani et al., 2010).

The unbiased stereological technique of the nucleator was used to estimate the mean volumes of oocytes and their nuclei. The optical disector procedure was used for sampling in conjunction with an unbiased counting frame. An average of 12 sections from 20-µm thick sections was randomly selected. The nucleus was the target sample and the selected follicles were then studied by the Olympus microscope with 100× magnification. For each sampled oocyte, an isotropic direction is generated from a random point within the nucleolus


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(for practical purposes, the nucleolus centre), and the distances in each direction out from the point to the boundary of either the nucleus (to estimate the nuclear volume), and/ or the oocyte membrane (to estimate the oocyte volume) are recorded. In the absence of nucleoli, the virtual point was considered the centre of the nucleus. In this study, to estimate the volume precisely, distance measurements are usually carried out in four systematic random directions from the unique reference point. The mean volume is estimated through: VN =

4π 3 4π n 3 ⋅ ln = ∑ ln,i 3 3n i=1

Where ln refers to the distances from the sampling point to the edge of the particle (such as the nucleus or the cell) (Calado et al., 2003; Myers et al., 2004; Noorafshan et al., 2013).

the slides were incubated for 60 min at 37°C in a humidified dark chamber, incubation with Converter-POD at 37°C for 30 min then followed. A series of tissue sections were also incubated in the reaction buffer without TdT as a negative control. As a positive control, a set of sections of mice thymus gland was used (Abtahi et al., 2014). Thymus was chosen as the positive control owing to the high frequency and distribution of apoptotic cells in lymphoid tissues such as thymus. The samples were stained with diaminobenzidine substrate (Roche, Germany) for 10 min, followed by counterstaining with haematoxylin, washed with distilled water, dehydrated, mounted with entellan (Merck, Germany), and finally examined by light microscope (Olympus, Japan, BX51). Oocytes and follicular cells containing stained nuclei (dark brown) were considered as TUNEL-positive. If more than 10% of the follicular cells were TUNEL-positive, the follicle was considered apoptotic (Yang et al., 2008).

Hormonal assay Zona pellucida thickness Quantitative measurement of the zona pellucida thickness, which plays an important role during oogenesis, fertilization and preimplantation development (Wassarman, 2008), has a positive predictive value in relation to embryonic development after graft recovery and IVF. To estimate the mean thickness of zona pellucida, an average of 12 sections was randomly selected out of 5-µm thick sections and was studied using ×100 oil immersion objective. Random superimposition of the specific line grid (three parallel lines) on the sampled fields served the purpose of identifying the measurement sites. To measure zona pellucida thickness, the orthogonal intercept method was used. In this method, the length of a perpendicular line extended from the inner membrane to the outer surface of zona pellucida at each intercept of the line of the grid with zona membrane considered as orthogonal intercept (oi). An average of 110 measurements were made to calculate the harmonic mean thickness of the zona pellucida as follows (Ferrando et al., 2003): Harmonic mean layer thickness = 8 3π × harmonic mean of orthogonal intercepts

Where harmonic mean = number of measurements/sum of the reciprocal of orthogonal intercepts lengths (oi) = number 1 1 1 ⎛ 1 ⎞ + + + +… ⎟ . ⎠ oi1 oi2 oi3 oi4

After 28 days, blood samples were collected and centrifuged (3000 g, 5 min), and serum was analysed in duplicates with the P4 kit (DRG Progesterone ELISA kit, EIA-1561; DRG Instruments GmbH, Marburg, Germany), with a sensitivity of 0.045 ng/ml and an assay range of 0–40 ng/ml and the oestradiol kit (DRG Estradiol ELISA kit, EIA-2693; DRG Instruments GmbH, Marburg, Germany), with a sensitivity of 9.714 pg/ml and assay range of 9.7–2000 pg/ml, according to the manufacturer’s instructions.

Measurement of malondialdehyde concentration The level of malondialdehyde (MDA), a lipid peroxidation product that is usually used to evaluate oxidative stress, was measured in blood serum samples collected on days 7 and 28 after transplantation, using the thiobarbituric acid method according to the kit manufacturer’s instructions (NWLSS™ NWKMDA01; Vancouver, Canada). Butylated hydroxyl toluene (10 µl), serum (250 µl), and/or calibrators, acid reagent (250 µl) and two-thiobarbituric acid were added and vortexed vigorously. The samples were then incubated at 60°C for 60 min and their absorbance was recorded in 532 nm with a spectrophotometer (T80+; PG Instruments Ltd, London, UK). The MDA concentration was calculated based on the absorption standard curve.

Vaginal cytology

of measurements/ ⎜⎝

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay Detection of DNA fragmentation was performed by TdT (terminal deoxynucleotidyl transferase) dUTP nick-end labelling (TUNEL), according to the kit manufacturer’s manual (In Situ Cell Death Detection Kit; Roche, Germany). Briefly, the sections were deparaffinized, rehydrated and then incubated with 3% hydrogen peroxide (H2O2; Merck, Germany) for 10 min, followed by digestion with 20 µg/ml proteinase K for 30 min at 37°C. The TUNEL reaction mixture was added, and

The vaginal cytology, representing the resumption of cyclic ovarian activity, was assessed 7 days after surgery until the appearance of the first typical estrous profile (cornified epithelial cells) and then weekly afterwards until the time mice were killed. Vaginal smear of each mouse was observed immediately under a light microscope (×100) in order to be classified either as pro-estrous, estrous, metestrous or diestrous (Byers et al., 2012).

Statistical analysis The results were analysed by one-way analysis of variance and Tukey’s test, using the Statistical Package for Social


ARTICLE IN PRESS N-acetylcysteine improves ovary function in grafts Sciences (SPSS) version 16/0 (SPSS Inc., USA), and the means were considered significantly different at P < 0.05.

Results

5 volume of ovary and cortex were also significantly higher in the grafts treated with NAC compared with grafts treated with saline (P < 0.001). The mean volume of medulla did not differ between both grafted groups (Table 1).

Ovarian histology

The number of follicles

The ovary recovery rate in both transplanted groups was 100%. Through macroscopic observations, the size of the ovaries reduced in both transplanted groups compared with the controls, and this was more prominent in the autograft plus saline group. Microscopic observations of ovaries in both transplanted groups revealed follicles in different development stages; however, the density of different follicles and corpus luteum in the autograft plus NAC group was higher than those in the autograft plus saline group. A comparison of the density of the stromal cells (tissue integrity) showed no difference among NAC-treated grafts and the controls, and they were all morphologically normal with no sign of fibrosis (Figure 1).

The mean number of primordial, primary, pre-antral, antral and the mean total number of follicles were significantly lower in both saline- and NAC-treated grafts compared with the control group (P < 0.001). The mean number of primordial, primary (P < 0.001), pre-antral, antral (P < 0.01), and also the total number of follicles (P < 0.001) were significantly higher in grafts treated with NAC compared with the groups treated with saline (Table 2). A total of 33.33% of the ovaries in the saline-treated grafts and 66.67% in the NAC-treated grafts were detected to contain the corpus luteum.

The volume of ovary, cortex and medulla

The mean volumes of oocytes and their nuclei in the primordial, primary, pre-antral and antral follicles showed no significant difference in any of the groups (Tables 3 and 4). In addition, no significant difference was found in the mean thickness of zona pellucida in the pre-antral and antral

Although the mean total volume of ovary, cortex and medulla showed a highly significant reduction in both grafted groups compared with the control group (P < 0.001), the mean total

Volume of oocytes and their nuclei and zona pellucida thickness

Figure 1 Microscopic images of mice ovary tissues in different groups 28 days after autotransplantation. (A) The control group: follicles are observed in different development stages; (B) autograft plus saline group: fewer follicles are observed in different development stages; (C, c) autograft plus N-acetylcysteine group: a considerable number of different primordial, growing, antral and preovulatory follicles are observed compared with B. Arrows showing the different types of follicles; and (D) another cross-section of the ovaries in the autograft plus N-acetylcysteine group demonstrating the corpus luteum (*). Scale bar = 100 µm.


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M Mahmoodi et al. Table 1 Comparison of the mean total volume of ovary, cortex and medulla (mm3) in different groups of mice, 28 days after heterotopic ovarian tissue transplantation and treatment with N-acetylcysteine (150 mg/kg intraperitoneal injection). Group

Volume of ovary

Volume of cortex

Volume of medulla

Control Autograft plus saline Autograft plus N-acetylcysteine

1.92 ± 0.08a 1.09 ± 0.12b 1.37 ± 0.12c

1.74 ± 0.08a 1.06 ± 0.11b 1.35 ± 0.12c

0.19 ± 0.01a 0.02 ± 0.01b 0.02 ± 0.01b

Values are means ± SD. The means with different code letter are considered significantly different (one way ANOVA, Tukey’s test; all P < 0.001).

Table 2 Comparison of the mean number of primordial, primary, pre-antral and antral follicles and the mean total number of follicles in different groups of mice, 28 days after heterotopic ovarian tissue transplantation and treatment with N-acetylcysteine (150 mg/kg intraperitoneal injection). Group

Primordial follicles

Primary follicles

Pre-antral follicles

Antral follicles

Total


1879.50 ± 89.70a 977.67 ± 131.87b 1479.33 ± 120.83c

572.5 ± 33.04a 302.00 ± 25.42b 503.83 ± 36.18c

307.00 ± 25.35a 173.33 ± 14.42b 208.00 ± 9.42c

117.83 ± 8.06a 53.17 ± 10.44b 72.33 ± 12.53c

2876.83 ± 103.93a 1506.17 ± 132.25b 2263.50 ± 117.06c

Values are means ± SD. The means with different code letter are considered significantly different (one way analysis of variance and Tukey’s test); a versus b,c all P < 0.001, b versus c P < 0.001 for primordial, primary and total follicles and P < 0.01 for pre-antral and antral follicles.

Table 3 Comparison of the mean oocyte volume in different types of follicles, in different groups of mice, 28 days after heterotopic ovarian tissue transplantation and N-acetylcysteine treatment (150 mg/kg intraperitoneal injection). Group


Oocyte volume (µm3) Primordial follicles

Primary follicles


Antral follicles

1795.97 ± 31.27 1786.40 ± 58.56 1843.75 ± 94.39

3973.31 ± 769.26 3819.19 ± 535.93 3973.91 ± 659.69

80022.82 ± 3799.90 78560.33 ± 2475.71 79887.36 ± 1927.37

154789.90 ± 3872.09 151231.75 ± 2324.30 154519.38 ± 3118.93

Values are means ± SD. One way analysis of variance and Tukey’s test showed no statistically significant differences.

Table 4 Comparison of the mean volume of oocyte nucleus in different types of follicles, in different groups of mice, 28 days after heterotopic ovarian tissue transplantation and N-acetylcysteine treatment (150 mg/kg intraperitoneal injection). Group


Oocyte nucleus volume (µm3) Primordial follicles

Primary follicles


Antral follicles

534.06 ± 58.50 519.31 ± 55.39 537.80 ± 64.80

724.05 ± 49.87 748.27 ± 44.66 778.20 ± 39.70

2747.35 ± 129.31 2659.85 ± 147.45 2663.44 ± 213.19

6635.49 ± 72.50 6531.96 ± 142.28 6600.15 ± 195.95


follicles in both grafted groups compared with control group (Table 5).

the percentage of apoptotic follicles between the NACtreated and the control groups (Figures 2 and 3).

Apoptosis assay

Concentration of malondialdehyde

The mean percentage of apoptotic follicles was significantly higher in the saline-treated group (11.29 ± 2.78) compared with the control (1.65 ± 0.64) and the NAC-treated groups (2.38 ± 0.54) (P < 0.001). No significance difference was found in

The concentration of MDA was significantly higher on days 7 (P < 0.001) and 28 (P < 0.01) after transplantation in the saline-treated group compared with the control group. The concentration of MDA was significantly lower in the


ARTICLE IN PRESS N-acetylcysteine improves ovary function in grafts Table 5 Comparison of the mean thickness of zona pellucida (µm) in pre-antral and antral follicles, in different groups of mice, 28 days after heterotopic ovarian tissue transplantation and N-acetylcysteine treatment (150 mg/kg intra peritoneal injection). Group

Control Autograft plus saline Autograft pluls N-acetylcysteine

Thickness of zona pellucida Pre-antral follicles

Antral follicles

12.07 ± 0.34 11.97 ± 0.39 12.00 ± 0.50

17.61 ± 0.42 16.95 ± 0.24 17.12 ± 0.62


NAC-treated group after 7 (P < 0.001) and 28 days (P < 0.05) after transplantation compared with the saline-treated group. No significant difference in the concentration of MDA was detected between the NAC-treated group and controls at 7 and 28 days after transplantation (Table 6).

Hormone assay The level of progesterone and oestradiol hormones were significantly lower in both saline (P < 0.001) and NAC-treated (P < 0.01) groups when compared with the control group. No significant difference was found in the level of progesterone in the NAC-treated grafts and saline treated grafts. Oestradiol concentrations were significantly higher in the NAC group compared with the saline-treated grafts (P < 0.05) (Table 7).

Vaginal cytology The recovery rate of the estrous cycle was 100% in both transplanted groups. Cytological vaginal examination of autografted mice revealed cornified epithelial cells 9–12 days after transplantation. However, the estrous cycle initiated more rapidly in the NAC-treated grafts compared with the saline treated grafts (P < 0.01) (Table 7). A significant difference in the initiation of the estrous cycle was found in both saline and NACtreated groups compared with the control group (P < 0.001).

Discussion In the present study, the effects of NAC on mouse ovarian tissue after heterotopic autotransplantation were investigated. The results indicated that treatment with NAC, although not to the level of control, significantly improved the structure and function of mice ovarian grafts as well as the follicular survival and development through preventing oxidative stress and apoptosis. The animals in this study underwent bilateral ovariectomy to remove both ovaries to prevent any ovarian hormonal secretions for a better interpretation of vaginal cytology; increase in gonadotropins as a result of ovariectomy also provides a better condition for follicle development (Commin et al.,

7 2012). The NAC injection was carried out constantly from 1 day before until 7 days after transplantation to gain more effect (Khan et al., 2004; Mei et al., 2009). As various doses of NAC have been used in previous studies on ischaemia reperfusion injury in different tissue transplantations (Danilovic et al., 2011; Inci et al., 2007; Mei et al., 2009; Santiago et al., 2008), a dose of 150 mg/kg was chosen, which has been shown to be most effective in reducing ischaemia-reperfusion injury (Inci et al., 2007; Khan et al., 2004). In this study, the recovery rate of the grafts transplanted into the back muscle was 100% and ovary stroma tissue and morphology was normal, particularly in the NAC-treated group compared with the control group. Also, no sign of fibrosis was visible, which was consistent with the results obtained by Dath et al., 2010. Previous studies have also shown that the stroma cells and extracellular matrix of the ovary play an important role in tissue integrity, normal function of the ovary and follicular development and survival (Commin et al., 2012; Soleimani et al., 2011). Therefore, it seems that NAC, as an antioxidant and antiapoptosis factor, has been able to prevent degeneration and atresia of stroma, which in turn preserves the entirety of follicles and enhances their development. As our results indicated, the volume of ovary, cortex and medulla decreased considerably, as did the number of follicles in the autografted groups compared with the control groups, which confirms previous reports (Hemadi et al., 2009; Kim et al., 2002). This could be because in the first days of transplantation, granulosa cells and oocytes, particularly in developing follicles, undergo apoptosis as a result of ischaemiareperfusion injury induced by free radicals and lipid peroxidation; this can lead to the degeneration and atresia of follicles and disruption in folliculogenesis and oogenesis, which eventually decreases the ovary volume and size and also the number of follicles (Commin et al., 2012; Demeestere et al., 2009; Hemadi et al., 2009; Soleimani et al., 2011; Wang et al., 2012). Meanwhile, treatment with NAC resulted in a significantly higher volume of ovary and cortex and also the number of follicles compared with the saline-treated grafts through preventing initial tissue degeneration and follicle atresia as a result of its ability to delay or inhibit apoptosis of ovary cells in the time of hypoxia and also to reduce the ischaemia-reperfusion injury through its antioxidant ability and also increasing the glutathione reservoir. In this study, NAC treatment resulted in significantly higher plasma level of oestradiol hormone, one of the most important steroid hormones (Greenfeld et al., 2007; Li et al., 2010) compared with the saline treated group. As the antral follicles secrete oestradiol, the rise in the plasma level of oestradiol confirms the effect of NAC in the ovary endocrine function recovery (Callejo et al., 2002; Li et al., 2010); this could also be as a result of an increase in the number of antral follicles after NAC treatment. In the present study, the plasma MDA level, an indicator of lipid peroxidation and tissue injury, was considerably higher after transplantation; however, NAC treatment resulted in lower MDA level compared with the saline-treated group (Sapmaz et al., 2003; Usta et al., 2008). Other studies have also demonstrated that NAC injection after kidney (Danilovic et al., 2011) or heart (Mei et al., 2009) transplantation decreases the MDA level. This highlights the antioxidant ability of NAC, which in turn prevents lipid peroxidation and reduces injuries resulting from oxidative stress.


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Figure 2 Histomorphological evaluation of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining in ovarian grafts 28 days after transplantation. (A) Control group: little sign of apoptosis was detected; (B) salinetreated group; (C) N-acetylcysteine-treated group: few apoptotic cells were observed in the stroma and follicles. Arrows indicate TUNEL-positive follicular cells (dark brown nucleus). Scale bar = 100 µm. Table 6 Comparison of the concentration of malondialdehyde (MDA, µM) in different groups of mice, 7 and 28 days after heterotopic ovarian tissue transplantation and N-acetylcysteine treatment (150 mg/kg intraperitoneal injection). Group


Malondialdehyde 7 days after transplantation

28 days after transplantation

3.74 ± 0.61a 7.50 ± 0.89b 4.31 ± 0.85a

3.45 ± 0.69a 4.94 ± 0.32b 3.56 ± 0.89a

Values are means ± SD. The means with different code letter are considered significantly different (one way analysis of variance and Tukey’s test): for control versus autograft plus saline P < 0.001 at 7 days and P < 0.01 at 28 days; for autograft plus saline versus autograft plus N-acetylcysteine P < 0.001 at 7 and P < 0.05 at 28 days after transplantation.

In this study, the mean percentage of TUNEL-positive follicular cells was significantly lower after NAC treatment compared with the saline-treated group. This finding is in accordance with previous reports on the effect of NAC on inhibiting apoptosis (Khan et al., 2004; Zafarullah et al., 2003). Survival and development of ovarian follicles after NAC treatment could be a result of its performance in reducing oxidative stress and preventing apoptosis through inhibiting the induction of pre-inflammatory cytokines such as tumour necrosis factor alpha and interleukin-1 beta, inducible nitric oxide synthase and nitric oxide production (Khan et al., 2004), which play an important role in ischaemia-reperfusion injury. An important role is played by NAC in scavenging oxygen free radicals and also increasing intracellular glutathione concentrations (Araujo et al., 2005). On the other hand, NAC reduces the activities of the nuclear factor kappa B, vascular cell adhesion molecule 1, intercellular adhesion molecule 1 and E-selectin, which cause inflammatory reactions of ischaemic tissue (Araujo et al., 2005; Nitescu et al., 2006; Zafarullah et al., 2003). Therefore, it can be said that NAC may inhibit ischaemia-reperfusion injury through its anti-inflammatory and antioxidant effect as well as through inhibition of apoptosis

Figure 3 Comparison of apoptosis rate in different groups of mice 28 days after heterotopic ovarian autotransplantation and treatment with N-acetylcysteine (150 mg/kg intraperitoneal injection). Values presented as mean ± SD and the different code letters are considered significantly different (one way analysis of variance and Tukey’s test); a versus b P < 0.001. NAC = N-acetylcysteine.

and direct reduction of functional protein of thiols on the surface of the cells (Araujo et al., 2005; Danilovic et al., 2011; Khan et al., 2004; Mei et al., 2009). This, altogether, leads to a more rapid recovery rate of the estrous cycle and ovarian function, which has also been concluded by other investigators who have demonstrated the beneficial effect of NAC on faster recovery and better function of kidney grafts in human (Danilovic et al., 2011) and heart isografts in rat (Mei et al., 2009). The potential benefit of other antioxidants, such as allopurinol (Abedi et al., 2014), vitamin E (Abir et al., 2011; Nugent et al., 1998), melatonin (Friedman et al., 2012; Hemadi et al., 2009), ascorbic acid (Kim et al., 2004) and melatonin and oxytetracycline (Sapmaz et al., 2003), have also been reported during ovarian tissue transplantation. Despite the significant effect of NAC in improving the structure and function of mice ovarian grafts and follicular survival and development, it could not compensate for the consequences of ischaemic reperfusion injury after transplantation, such as the decrease in the volume of ovary,


ARTICLE IN PRESS N-acetylcysteine improves ovary function in grafts

9

Table 7 Comparison of the mean level of progesterone and oestradiol and the mean recovery time of the estrous cycle in different groups of mice, 28 days after heterotopic ovarian tissue transplantation and N-acetylcysteine treatment (150 mg/kg intraperitoneal injection). Group

Progesterone (ng/ml)

Oestradiol (pg/ml)

Starting day of estrous cycle


2.81 ± 0.94a 0.80 ± 0.13b 1.58 ± 0.37b

43.25 ± 5.68a 26.21 ± 4.311b 34.24 ± 5.01c

8.17 ± 0.75a 11.50 ± 0.55b 10.17 ± 0.75c

Values are means ± SD. The means with different code letter are considered significantly different (one way analysis of variance and Tukey’s test): for control versus autograft plus saline P < 0.001. The difference between control and autograft plus N-acetylcysteine in the level of progesterone and oestradiol P < 0.01, and for the estrous cycle P < 0.001. A significant difference was observed in the level of oestradiol (P < 0.05) and the initiation of estrous cycle (P < 0.01) between the autografts.

cortex, medulla, number of follicles, level of progesterone and oestradiol hormones, to the control level; however, considering the severe injury that ovary undergoes during the first days of ischaemia before neovascularization, this is to some extent predictable, but further studies with modified doses, administration routes of NAC or even periods of treatment may improve the obtained results. In addition to the use of antioxidants, it should also be mentioned that host treatment with gonadotrophins before and after grafting can improve follicular survival in human (Abir et al., 2011) and mice (Imthurn et al., 2000) through stimulating vascularization and reduction of apoptosis. This study opens new windows to researchers to evaluate the mechanism of NAC performance in more detail and suggests the therapeutic applications of NAC in ovary tissue transplantation; however, further examinations are needed to translate our results to clinical trials of human ovarian transplantation, along with suitable modifications (e.g. using targeted delivery systems such as oral administration), which provide an easier route of administration in human patients.

Acknowledgements The authors are very grateful to the Arak University (grant number 90/12774) for its financial support.

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