Mol. Cells 2014; 37(12): 865-872
Published online November 10, 2014
https://doi.org/10.14348/molcells.2014.0145
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: aihao131@sina.com (HA); xuexiaoou@sina.com (XX)
Premature ovarian failure (POF) is a long-term adverse effect of chemotherapy treatment. However, current available treatment regimens are not optimal. Emerging evidence suggests that bone marrow-derived mesenchymal stem cells (BMSCs) could restore the structure and function of injured tissues, but the homing and restorative effects of BMSCs on chemotherapy injured ovaries are still not clear. In this study, we found that granulosa cell (GC) apoptosis induced by cisplatin was reduced when BMSCs were migrated to granulosa cells (GCs)
Keywords bone marrow-derived mesenchymal stem cells, cisplatin, homing, intravenous injection, premature ovarian failure
Chemotherapy regimens cause reproduction damage in young females (M?rse et al., 2013). Premature ovarian failure (POF) is a long-term adverse effect of chemotherapy treatment, which leads to increased risk of infertility and degenerative health conditions, such as cardiovascular disease, osteoporosis, and cognitive impairment (De Vos et al., 2004; Lana et al., 2010; Maclaran and Panay, 2011; Tannock et al., 2004). Therefore, the preservation of fertility and gonadal function has become an important issue for cancer survivors of reproductive age. It has been widely accepted that hormone replacement therapy (HRT), offered to women with POF, could alleviate estrogen deficiency symptoms and minimize the risk of complication (Maclaran and Panay, 2011). However, HRT has been shown to increase the risk of breast cancer (Antoine et al., 2013). Other options, such as ovarian tissue, oocyte and embryo cryo-preservation have been applied (Donnez and Dolmans, 2013). But, then again, these available treatment regimens are still not optimal. Therefore, it would be desirable to explore improved therapeutic strategies for treating POF.
Recently, stem cell therapy has been supported as a potential and alternative therapeutic modality, offering the possibility of repairing and restoring the normal function of injured tissues (Agung et al., 2006; Bi et al., 2007; Lee et al., 2005; Muscari et al., 2013). The bone marrow stem cell is the best-studied transplanted stem cell, which contains multiple cell types, such as mesenchymal stem cells (MSCs) and hematopoietic stem cells. MSCs are characterized as adherent cells that has the capability to differentiate into fibroblasts, adipocytes, chondrocytes and osteocytes
Previous studies have revealed that bone marrow stem cells can restore ovarian function and generate immature oocytes in female mice (Ghadami et al., 2012; Lee et al., 2007; Selesniemi et al., 2009). It has also been demonstrated that BMSCs can contribute in recovering ovarian structure and function, which were injured by cyclophosphamide (Abd-Allah et al., 2013; Fu et al., 2008; Kilic et al., 2013). In recent years, several other mesenchymal phenotypes, such as adipose-derived MSCs, umbilical cord MSCs, and amniotic fluid MSCs have been referred as a therapeutic potential for chemotherapy induced ovarian damage (Lai et al., 2013; Liu et al., 2012; Takehara et al., 2013; Wang et al., 2013).
However, selecting an effective method for stem cell delivery is a critical point for achieving a successful cell based therapy. Previous studies have demonstrated that directly injecting BMSCs into the chemotherapy injured ovaries can improve ovarian function and structure (Fu et al., 2008; Kilic et al., 2013). In contrast to cellular orthotopic injection, intravenous injection of BMSCs is a simple, quick and less invasive strategy. More importantly, intravenous injection is specifically more beneficial for chemotherapeutic toxicities that affect various organs. However, there are only a few studies about the homing of BMSCs into the ovaries and the therapeutic potential of intravenous injection for ovaries injured by cisplatin. A current study suggested that intravenous injection of MSCs containing Y chromosomes could migrate into the ovaries and improve ovarian function (Abd-Allah et al., 2013), however, the specific distribution mechanisms of BMSCs in the ovaries were not clear. Therefore, the purpose of the present study is to evaluate the homing and distribution of BMSCs in ovaries injured by cisplatin after intravenous injection, and to evaluate its restorative effects on ovarian structure and function.
Immature (3-week old) and young (10-week old) female Sprague-Dawley rats were used in the experiments. All animal experiments were approved by the Institutional Animal Committee of Liaoning Medical University.
BMSCs were obtained from the 10-week-old rats (n = 10). Bone marrow was flushed out from tibias using DMEM/F12 (1:1) medium (Hyclone, USA) and centrifuged at 1,000 rpm for 5 min. BMSCs were cultured with a medium, consisting of DMEN/F12 (1:1) medium and 10% fetal bovine serum (Hyclone), and humidified in a cell culture incubator containing 5% CO2 at 37°C. BMSCs were detached with 0.25% trypsin-EDTA (Hyclone), which was replanted in other flasks with 1:2 ratios at 80% confluence. Third passage BMSCs were used in all experiments.
The surface marker expression of BMSCs was assessed by flow cytometry (Beckman Coulter). BMSCs were suspended (1 × 106 cells/ml) and stained with FITC- or R-phycoerythrin (PE)-conjugated monoclonal antibodies, such as CD29 (Biolegend), CD34 (Santa Cruz), CD45 (eBioscience), CD90 (Biolegend).
The recombinant adenovirus vector with enhanced green fluorescent protein (EGFP) gene (Ad-EGFP) was purchased from SinoGenoMax (China). BMSCs were seeded in 96-well plates, cultured for 24 h, and exposed to Ad-EGFP with a multiplicity of infection (MOI) of 50, 100, 150, 200 and 400 for 12 h. After 48 h, BMSCs were fixed with 4% paraformaldehyde; and the viability and transfection efficiency of the cells were evaluated by an inverted fluorescence microscope (Olympus IX71, Japan).
Immature female rats were intraperitoneally injected with pregnant mare serum gonadotropin (Sigma, USA) to stimulate follicle growth. After 48 h, bilateral ovaries were removed under aseptic conditions. Then, the adipose and connective tissues of the ovaries were removed and washed twice with PBS solution. GCs were isolated under an anatomical microscope and single cell suspensions were obtained. The cells were washed three times and centrifuged at 1,000 rpm for 5 min. The GCs were cultured with a medium, consisting of a DMEN/F12 (1:1) medium and 10% fetal bovine serum with 5% CO2 at 37°C. First passage GCs were used in all experiments.
The follicle stimulating hormone receptor (FSHR) of the GCs was assessed by immunocytochemistry. GCs were seeded in 24-well plates (CORNING, USA), cultured for 48 h, fixed with 4% paraformaldehyde for 30 min, and washed twice with PBS solution. Then, the GCs were incubated with 3% H2O2 for 10 min at room temperature to eliminate endogenous catalase activities. Subsequently, the GCs were incubated in a humidified chamber for one hour at 37°C with FSHR primary monoclonal antibodies (1:100, Santa Cruz). Negative control was conducted with PBS alone. After incubating with Polymer Helper (ZSBIO, China) for 20 min, polyperoxidase-anti-rabbit IgG (ZSBIO, China) were added for 20 min at 37°C. Then, color developed with the 3,3′-Diaminobenzidine (DAB) chromogen. After washing with tap water, the samples were determined by an Olympus IX71 microscope.
The toxicity of cisplatin (Qilu Pharmaceutical Ltd. China) to GCs was assessed by Cell Counting Kit-8 (CCK-8, Dojindo Company, Japan). The GCs were seeded at a density of 104 cells in 96-well plates, cultured for 72 h, and exposed to various cisplatin concentrations (0, 1.0, 5.0 and 7.5 mg/L). After 48 h, 100 microliters of medium, consisting of DMEN/F12 (1:1) medium and 10% CCK-8 without fetal bovine serum, was added to each well and incubated for 4 h. Then, OD value was assessed by a microplate reader (Awareness Technology). After the supernatant was removed, the GCs were fixed with 4% paraformaldehyde for 30 min and stained with crystal violet (Sigma, USA).
The GCs were divided into three groups: normal control group, cisplatin (5 mg/L) group, and cisplatin (5 mg/L) co-cultured with BMSCs group. The GCs (2.5 × 105 cells/well) were seeded in 6-well plates and cultured for 48 h. In the cisplatin and cisplatin co-cultured with BMSCs groups, GCs were cultured with 2 ml medium, which included 5 mg/L of cisplatin. In the cisplatin co-cultured with BMSCs group, BMSCs were seeded on the upper side of the Transwell chambers (0.4 m, CORNING) at a density of 2.5 × 105 cells/well. After 48 h, the GCs were collected and assessed by flow cytometry (Beckman Coulter) with an Annexin V/PI apoptosis detection kit (eBioscience, USA).
The migration of BMSCs was evaluated by Transwell Permeable Supports (CORNING) with 8.0 μm pore filters (R?ster et al., 2005). The GCs were divided into two groups: normal control group and cisplatin (5 mg/L) group. The GCs (1 × 105/well) were seeded in 24-well plates and cultured for 24 hours. In the cisplatin group, the GCs were incubated with a 500 μl medium and cisplatin (5 mg/L). After 48 h, 200 μl of medium, containing BMSCs (5 × 104 cells), was added on the upper side of the Transwell chambers (8.0 μm, CORNING). The plates were incubated for 6 and 24 h. BMSCs were removed from the upper side of the filters; BMSCs at the lower side of filters were fixed with 4% paraformaldehyde for 20 min, and stained with crystal violet. The number of migrated BMSCs was determined by counting five random fields per well using an Olympus IX71 microscope.
The animals were divided into three groups (10 rats/group): normal control group (group 1), cisplatin-induced POF group (group 2) and BMSCs treatment group (group 3). To establish the POF model, the rats were intraperitoneally injected with a daily dose of cisplatin at 2 mg/kg of body weight for six days. BMSCs (4 × 106 cells/rat) labeled with EGFP in 0.6 ml PBS were injected via the tail vein on the seventh day. At day 15 and 30, five rats from each group were narcotized; the blood and organs were collected. The serum was isolated, and stored at ?80°C for hormone test. The ovaries and other organs were dissected immediately, and were fixed in 4% paraformaldehyde for follicle counting, GFP-labeled BMSCs tracking and TUNEL.
Blood was collected from postcava and centrifuged at 3,000 rpm for 10 min to separate the serum. The level of estradiol (E2) was measured with an ARCHITECT Estradiol Reagent Kit (Abbott, USA).
The left ovary of each rat was fixed with 4% paraformaldehyde for 24 h, embedded in paraffin, cut into sections (5 μm), and stained with hematoxylin and eosin (HE) ? to evaluate follicle growth. Preantral follicles contained less than five layers of GCs and lacked antral vesicles. Antral follicles contained more than five layers of GCs and antral vesicles. The antral follicles and corpus were counted in the biggest section; which was across the ovarian hilum. The sections were examined and photographed with the Olympus BX-51 light microscope (Olympus, Japan).
The organs were fixed in 4% paraformaldehyde for 24 h, dehydrated and embedded in paraffin. Sections (5 μm) were deparaffinized, rehydrated and high pressured for 2 min in a citrate buffer (pH 6.0) to retrieve antigenicity. Then, the samples were incubated with 3% H2O2 for 10 min at room temperature. The samples were incubated in a humidified chamber for one hour at 37°C with EGFP primary monoclonal antibodies (1:100, Santa Cruz). Negative control was conducted with PBS alone. After incubating with Polymer Helper (ZSBIO, China) for 20 min, polyperoxidase-anti-rabbit IgG (ZSBIO, China) were added for 20 min at 37°C. Then, color developed with the 3,3′-Diaminobenzidine (DAB) chromogen and was counter-stained with hematoxylin for 20 s. After washing with tap water, the samples were dehydrated and photographed with the Olympus BX51 microscope.
Ovarian granulosa cell apoptosis was measured by TUNEL apoptosis assay kit (Promega, USA), according to manufacturer’s instructions. The ovarian sections, across ovarian hilum and nuclei of apoptotic cells, were stained dark brown. The sections were examined and photographed with the Olympus BX-51 light microscope.
To calculate the statistical differences between the groups, the SPSS 19.0 statistical package (Chicago, USA) was used. One-way ANOVA was used to determine the significant differences among the groups. All values were presented as mean ± standard deviation. In general,
After isolating from the bone marrow and 48 h of adherent growth, the BMSCs morphologically resembled fibroblasts. The morphology of third passage BMSCs was consensus (Fig. 1A). Surface antigens of the BMSCs were characterized by flow cytometry. Most of the BMSCs were CD29+, CD90+ and CD34+, CD45+ (Fig. 1C). To trace the fate of the BMSCs
After isolating the GCs from the ovarian follicles, the cells showed adherent growth and morphologically resembled round or oval cells (Fig. 2A). The morphology of the first passage GCs was consensus, containing only a few polygonal cells. The GCs were characterized by immunocytochemistry. The round or oval cells expressed as intracellular follicle stimulating hormone receptor (FSHR), while the polygonal cells were negative (Fig. 2C).
To determine the effects of the viability and inhibition of GCs induced by cisplatin, the viability of the GCs were measured by CCK-8 assay. Significant cytotoxic effects and cell growth inhibition on the GCs were shown by cisplatin; the GCs were sparse and irregular (Fig. 2B). Compared with the control group, the viability of GCs with high doses of cisplatin (5.0 and 7.5 mg/L) significantly decreased, compared to normal conditions (
The migration ability of BMSCs was assessed by Transwell assay. After 24 h, an abundant number of BMSCs migrated to GCs that were injured by cisplatin, through polycarbonate filters (Fig. 4B). Only a few BMSCs migrated to the normal GCs (Fig. 4A). Statistical analysis of the number of BMSCs revealed that GCs injured by cisplatin promoted BMSCs to migrate after 6 and 24 h (Fig. 4C).
30 days after injecting BMSCs, the E2 serum in the BMSCs group increased compared with the POF group (
All stages of follicular development (primary, secondary, and antral follicles) were observed in the rat control group for 30 days (Fig. 6A). Specimens that were taken from the POF group had only a few primordial follicles with degenerated growth and antral follicles (Fig. 6B). All types of follicles, including the primary, secondary and antral, and corpus luteum were apparently normal in the BMSCs treatment group (Figs. 6C and 6D). In addition, the antral follicles in the treatment group were healthy (Figs. 6E and 6F).
The statistical analysis of the antral follicular count revealed that the increased dosage of cisplatin resulted to the decreased amount of follicular count. However, in the BMSCs treatment group, a significant increase in healthy antral follicles and corpus lutea count was detected, compared with the cisplatin injection group (Fig. 5B); no significant change was observed after 15 days (
Following the transplantation of BMSCs, ovaries displayed EGFP-positive BMSCs on the 15th day. Interestingly, the number of BMSCs in the ovarian hilum and ovarian medulla was greater than in the ovarian cortex (Figs. 7A?7F). We also found BMSCs surrounding the follicles; but there were no BMSCs in the ovarian follicle and corpus lutea. 30 days after injecting BMSCs, we found that BMSCs survived and proliferated in the ovary (Figs. 7G?7I). BMSCs were also found in other organs, such as the heart, kidneys, liver and adipose tissues near the ovaries (Figs. 7J?7L). However, the number of BMSCs in the ovaries and kidneys were greater than in the liver and heart.
More interesting, most of the BMSCs were distributed along the blood vessels.
The granulosa cell apoptosis was detected by TUNEL. There were no apoptotic GCs in the healthy antral follicles (Fig. 8A). An abundant number of apoptotic GCs were observed in the atretic follicles that did not contain oocytes (Figs. 8B and 8C). Atretic follicles were found in all groups; and the number of healthy antral follicles in the BMSCs group was greater than in the POF group after 30 days (healthy antral follicular count 5.60 ± 1.14 in BMSCs group versus 2.60 ± 1.52 in POF group;
The ability of BMSCs to self-renew and regenerate pushed the trial to evaluate their role in fertility preservation. So far, no ideal marker for BMSCs has been identified. However, it has been identified that BMSCs expressed CD29, CD90, CD105 and negative expressions of hematopoietic cell-surface antigens, such as CD45, CD34, CD14 (Ohishi and Schipani, 2010). In our study, BMSCs were identified to express CD90 and CD29, but not CD45 and CD34. In addition to their fibroblast-shaped morphology and adherence to plastic, it was possible to conclude that the cell population used in our study was an enriched culture of BMSCs. Since homing of BMSCs is inefficient and many BMSCS were trapped in the lung following systemic administration, it is imperative that we trace the fate of the injected cells. We transfected BMSCs with Ad-EGFP and yielded the highest percentage of GFP-expression cells (99%) without affecting cell viability (Bosch et al., 2006; Yuan et al., 2011). A classic method in labeling cells is with Ad-EGFP; which expresses fluorescent proteins that are helpful in gaining insights on the homing and engraftment of BMSCs.
Cisplatin is one of the most effective chemotherapeutic agents, but injury may occur at higher dosages. It has been reported that cisplatin caused ovarian failure during clinical usage and non-clinical investigations (Ai et al., 2012; Iorio et al., 2014; Nozaki et al., 2009). Cisplatin induced granulosa cell apoptosis or necrosis
In the present study, BMSCs that migrated to granulosa cells injured by cisplatin
The present study found that the infusion of BMSCs could restore ovarian function. 30 days after administering BMSCs, ovarian function and structure improved based on results; which showed increased estradiol levels and follicle population. The mechanisms that enable BMSCs to induce beneficial effects in the ovaries are still controversial. It has been proposed that BMSCs can differentiate into fibroblasts, adiopocytes, chondrocytes, and osteocytes
It has been identified that follicular growth needed the systemic regulation by hormones and intraovarian regulation by cytokines, growth factors, and intracellular proteins (Matsuda et al., 2011). Granulosa cells, in particular, play an important role in follicular development and maintenance (Maruo et al., 1999). In our study, BMSCs reduced the apoptosis rate of cisplatin injured GCs
Therefore, the migration capabilities of BMSCs to injured tissue sites and its secretion function, allows BMSCs to exert their restorative actions. Moreover, studies have shown that BMSCs not only protects the ovaries from cisplatin-induced injuries, but also protects and restores other organs that were injured by cisplatin, such as kidneys and the bone marrow (Bi et al., 2007; Frenette et al., 2013). These results suggest that systemically delivered BMSCs are fit for protecting and restoring multiple organs injured by chemotherapeutic toxicities.
In conclusion, the present study demonstrated that through intravenous injection, BMSCs home to the ovaries and distribute mainly in the ovarian hilum and medulla; a small number of BMSCs engraft in the ovarian cortex; and no BMSCs were observed in the follicles and corpus lutea. In addition, ovarian function and structure was restored, after intravenously injecting BMSCs. Therefore, intravenously injecting BMSCs is an efficient treatment method for chemotherapy-induced POF.
Mol. Cells 2014; 37(12): 865-872
Published online December 31, 2014 https://doi.org/10.14348/molcells.2014.0145
Copyright © The Korean Society for Molecular and Cellular Biology.
Jiabin Liu1,2,3, Haiying Zhang1, Yun Zhang2, Nan Li2, Yuku Wen2, Fanglei Cao2, Hao Ai1,2,3,*, and Xiaoou Xue4,*
1Liaoning Medical University, Jinzhou, Liaoning 121001, China, 2Department of Gynecology and Obstetrics, Third Affiliated Hospital of Liaoning Medical University, Jinzhou, Liaoning 121001, China, 3Key laboratory of Follicular Development and Reproductive Health of Liaoning Province, Liaoning Medical University, Jinzhou, Liaoning 121001, China, 4Dongzhimen Hospital Affiliated to Beijing University of China Medicine, Beijing 100029, China
Correspondence to:*Correspondence: aihao131@sina.com (HA); xuexiaoou@sina.com (XX)
Premature ovarian failure (POF) is a long-term adverse effect of chemotherapy treatment. However, current available treatment regimens are not optimal. Emerging evidence suggests that bone marrow-derived mesenchymal stem cells (BMSCs) could restore the structure and function of injured tissues, but the homing and restorative effects of BMSCs on chemotherapy injured ovaries are still not clear. In this study, we found that granulosa cell (GC) apoptosis induced by cisplatin was reduced when BMSCs were migrated to granulosa cells (GCs)
Keywords: bone marrow-derived mesenchymal stem cells, cisplatin, homing, intravenous injection, premature ovarian failure
Chemotherapy regimens cause reproduction damage in young females (M?rse et al., 2013). Premature ovarian failure (POF) is a long-term adverse effect of chemotherapy treatment, which leads to increased risk of infertility and degenerative health conditions, such as cardiovascular disease, osteoporosis, and cognitive impairment (De Vos et al., 2004; Lana et al., 2010; Maclaran and Panay, 2011; Tannock et al., 2004). Therefore, the preservation of fertility and gonadal function has become an important issue for cancer survivors of reproductive age. It has been widely accepted that hormone replacement therapy (HRT), offered to women with POF, could alleviate estrogen deficiency symptoms and minimize the risk of complication (Maclaran and Panay, 2011). However, HRT has been shown to increase the risk of breast cancer (Antoine et al., 2013). Other options, such as ovarian tissue, oocyte and embryo cryo-preservation have been applied (Donnez and Dolmans, 2013). But, then again, these available treatment regimens are still not optimal. Therefore, it would be desirable to explore improved therapeutic strategies for treating POF.
Recently, stem cell therapy has been supported as a potential and alternative therapeutic modality, offering the possibility of repairing and restoring the normal function of injured tissues (Agung et al., 2006; Bi et al., 2007; Lee et al., 2005; Muscari et al., 2013). The bone marrow stem cell is the best-studied transplanted stem cell, which contains multiple cell types, such as mesenchymal stem cells (MSCs) and hematopoietic stem cells. MSCs are characterized as adherent cells that has the capability to differentiate into fibroblasts, adipocytes, chondrocytes and osteocytes
Previous studies have revealed that bone marrow stem cells can restore ovarian function and generate immature oocytes in female mice (Ghadami et al., 2012; Lee et al., 2007; Selesniemi et al., 2009). It has also been demonstrated that BMSCs can contribute in recovering ovarian structure and function, which were injured by cyclophosphamide (Abd-Allah et al., 2013; Fu et al., 2008; Kilic et al., 2013). In recent years, several other mesenchymal phenotypes, such as adipose-derived MSCs, umbilical cord MSCs, and amniotic fluid MSCs have been referred as a therapeutic potential for chemotherapy induced ovarian damage (Lai et al., 2013; Liu et al., 2012; Takehara et al., 2013; Wang et al., 2013).
However, selecting an effective method for stem cell delivery is a critical point for achieving a successful cell based therapy. Previous studies have demonstrated that directly injecting BMSCs into the chemotherapy injured ovaries can improve ovarian function and structure (Fu et al., 2008; Kilic et al., 2013). In contrast to cellular orthotopic injection, intravenous injection of BMSCs is a simple, quick and less invasive strategy. More importantly, intravenous injection is specifically more beneficial for chemotherapeutic toxicities that affect various organs. However, there are only a few studies about the homing of BMSCs into the ovaries and the therapeutic potential of intravenous injection for ovaries injured by cisplatin. A current study suggested that intravenous injection of MSCs containing Y chromosomes could migrate into the ovaries and improve ovarian function (Abd-Allah et al., 2013), however, the specific distribution mechanisms of BMSCs in the ovaries were not clear. Therefore, the purpose of the present study is to evaluate the homing and distribution of BMSCs in ovaries injured by cisplatin after intravenous injection, and to evaluate its restorative effects on ovarian structure and function.
Immature (3-week old) and young (10-week old) female Sprague-Dawley rats were used in the experiments. All animal experiments were approved by the Institutional Animal Committee of Liaoning Medical University.
BMSCs were obtained from the 10-week-old rats (n = 10). Bone marrow was flushed out from tibias using DMEM/F12 (1:1) medium (Hyclone, USA) and centrifuged at 1,000 rpm for 5 min. BMSCs were cultured with a medium, consisting of DMEN/F12 (1:1) medium and 10% fetal bovine serum (Hyclone), and humidified in a cell culture incubator containing 5% CO2 at 37°C. BMSCs were detached with 0.25% trypsin-EDTA (Hyclone), which was replanted in other flasks with 1:2 ratios at 80% confluence. Third passage BMSCs were used in all experiments.
The surface marker expression of BMSCs was assessed by flow cytometry (Beckman Coulter). BMSCs were suspended (1 × 106 cells/ml) and stained with FITC- or R-phycoerythrin (PE)-conjugated monoclonal antibodies, such as CD29 (Biolegend), CD34 (Santa Cruz), CD45 (eBioscience), CD90 (Biolegend).
The recombinant adenovirus vector with enhanced green fluorescent protein (EGFP) gene (Ad-EGFP) was purchased from SinoGenoMax (China). BMSCs were seeded in 96-well plates, cultured for 24 h, and exposed to Ad-EGFP with a multiplicity of infection (MOI) of 50, 100, 150, 200 and 400 for 12 h. After 48 h, BMSCs were fixed with 4% paraformaldehyde; and the viability and transfection efficiency of the cells were evaluated by an inverted fluorescence microscope (Olympus IX71, Japan).
Immature female rats were intraperitoneally injected with pregnant mare serum gonadotropin (Sigma, USA) to stimulate follicle growth. After 48 h, bilateral ovaries were removed under aseptic conditions. Then, the adipose and connective tissues of the ovaries were removed and washed twice with PBS solution. GCs were isolated under an anatomical microscope and single cell suspensions were obtained. The cells were washed three times and centrifuged at 1,000 rpm for 5 min. The GCs were cultured with a medium, consisting of a DMEN/F12 (1:1) medium and 10% fetal bovine serum with 5% CO2 at 37°C. First passage GCs were used in all experiments.
The follicle stimulating hormone receptor (FSHR) of the GCs was assessed by immunocytochemistry. GCs were seeded in 24-well plates (CORNING, USA), cultured for 48 h, fixed with 4% paraformaldehyde for 30 min, and washed twice with PBS solution. Then, the GCs were incubated with 3% H2O2 for 10 min at room temperature to eliminate endogenous catalase activities. Subsequently, the GCs were incubated in a humidified chamber for one hour at 37°C with FSHR primary monoclonal antibodies (1:100, Santa Cruz). Negative control was conducted with PBS alone. After incubating with Polymer Helper (ZSBIO, China) for 20 min, polyperoxidase-anti-rabbit IgG (ZSBIO, China) were added for 20 min at 37°C. Then, color developed with the 3,3′-Diaminobenzidine (DAB) chromogen. After washing with tap water, the samples were determined by an Olympus IX71 microscope.
The toxicity of cisplatin (Qilu Pharmaceutical Ltd. China) to GCs was assessed by Cell Counting Kit-8 (CCK-8, Dojindo Company, Japan). The GCs were seeded at a density of 104 cells in 96-well plates, cultured for 72 h, and exposed to various cisplatin concentrations (0, 1.0, 5.0 and 7.5 mg/L). After 48 h, 100 microliters of medium, consisting of DMEN/F12 (1:1) medium and 10% CCK-8 without fetal bovine serum, was added to each well and incubated for 4 h. Then, OD value was assessed by a microplate reader (Awareness Technology). After the supernatant was removed, the GCs were fixed with 4% paraformaldehyde for 30 min and stained with crystal violet (Sigma, USA).
The GCs were divided into three groups: normal control group, cisplatin (5 mg/L) group, and cisplatin (5 mg/L) co-cultured with BMSCs group. The GCs (2.5 × 105 cells/well) were seeded in 6-well plates and cultured for 48 h. In the cisplatin and cisplatin co-cultured with BMSCs groups, GCs were cultured with 2 ml medium, which included 5 mg/L of cisplatin. In the cisplatin co-cultured with BMSCs group, BMSCs were seeded on the upper side of the Transwell chambers (0.4 m, CORNING) at a density of 2.5 × 105 cells/well. After 48 h, the GCs were collected and assessed by flow cytometry (Beckman Coulter) with an Annexin V/PI apoptosis detection kit (eBioscience, USA).
The migration of BMSCs was evaluated by Transwell Permeable Supports (CORNING) with 8.0 μm pore filters (R?ster et al., 2005). The GCs were divided into two groups: normal control group and cisplatin (5 mg/L) group. The GCs (1 × 105/well) were seeded in 24-well plates and cultured for 24 hours. In the cisplatin group, the GCs were incubated with a 500 μl medium and cisplatin (5 mg/L). After 48 h, 200 μl of medium, containing BMSCs (5 × 104 cells), was added on the upper side of the Transwell chambers (8.0 μm, CORNING). The plates were incubated for 6 and 24 h. BMSCs were removed from the upper side of the filters; BMSCs at the lower side of filters were fixed with 4% paraformaldehyde for 20 min, and stained with crystal violet. The number of migrated BMSCs was determined by counting five random fields per well using an Olympus IX71 microscope.
The animals were divided into three groups (10 rats/group): normal control group (group 1), cisplatin-induced POF group (group 2) and BMSCs treatment group (group 3). To establish the POF model, the rats were intraperitoneally injected with a daily dose of cisplatin at 2 mg/kg of body weight for six days. BMSCs (4 × 106 cells/rat) labeled with EGFP in 0.6 ml PBS were injected via the tail vein on the seventh day. At day 15 and 30, five rats from each group were narcotized; the blood and organs were collected. The serum was isolated, and stored at ?80°C for hormone test. The ovaries and other organs were dissected immediately, and were fixed in 4% paraformaldehyde for follicle counting, GFP-labeled BMSCs tracking and TUNEL.
Blood was collected from postcava and centrifuged at 3,000 rpm for 10 min to separate the serum. The level of estradiol (E2) was measured with an ARCHITECT Estradiol Reagent Kit (Abbott, USA).
The left ovary of each rat was fixed with 4% paraformaldehyde for 24 h, embedded in paraffin, cut into sections (5 μm), and stained with hematoxylin and eosin (HE) ? to evaluate follicle growth. Preantral follicles contained less than five layers of GCs and lacked antral vesicles. Antral follicles contained more than five layers of GCs and antral vesicles. The antral follicles and corpus were counted in the biggest section; which was across the ovarian hilum. The sections were examined and photographed with the Olympus BX-51 light microscope (Olympus, Japan).
The organs were fixed in 4% paraformaldehyde for 24 h, dehydrated and embedded in paraffin. Sections (5 μm) were deparaffinized, rehydrated and high pressured for 2 min in a citrate buffer (pH 6.0) to retrieve antigenicity. Then, the samples were incubated with 3% H2O2 for 10 min at room temperature. The samples were incubated in a humidified chamber for one hour at 37°C with EGFP primary monoclonal antibodies (1:100, Santa Cruz). Negative control was conducted with PBS alone. After incubating with Polymer Helper (ZSBIO, China) for 20 min, polyperoxidase-anti-rabbit IgG (ZSBIO, China) were added for 20 min at 37°C. Then, color developed with the 3,3′-Diaminobenzidine (DAB) chromogen and was counter-stained with hematoxylin for 20 s. After washing with tap water, the samples were dehydrated and photographed with the Olympus BX51 microscope.
Ovarian granulosa cell apoptosis was measured by TUNEL apoptosis assay kit (Promega, USA), according to manufacturer’s instructions. The ovarian sections, across ovarian hilum and nuclei of apoptotic cells, were stained dark brown. The sections were examined and photographed with the Olympus BX-51 light microscope.
To calculate the statistical differences between the groups, the SPSS 19.0 statistical package (Chicago, USA) was used. One-way ANOVA was used to determine the significant differences among the groups. All values were presented as mean ± standard deviation. In general,
After isolating from the bone marrow and 48 h of adherent growth, the BMSCs morphologically resembled fibroblasts. The morphology of third passage BMSCs was consensus (Fig. 1A). Surface antigens of the BMSCs were characterized by flow cytometry. Most of the BMSCs were CD29+, CD90+ and CD34+, CD45+ (Fig. 1C). To trace the fate of the BMSCs
After isolating the GCs from the ovarian follicles, the cells showed adherent growth and morphologically resembled round or oval cells (Fig. 2A). The morphology of the first passage GCs was consensus, containing only a few polygonal cells. The GCs were characterized by immunocytochemistry. The round or oval cells expressed as intracellular follicle stimulating hormone receptor (FSHR), while the polygonal cells were negative (Fig. 2C).
To determine the effects of the viability and inhibition of GCs induced by cisplatin, the viability of the GCs were measured by CCK-8 assay. Significant cytotoxic effects and cell growth inhibition on the GCs were shown by cisplatin; the GCs were sparse and irregular (Fig. 2B). Compared with the control group, the viability of GCs with high doses of cisplatin (5.0 and 7.5 mg/L) significantly decreased, compared to normal conditions (
The migration ability of BMSCs was assessed by Transwell assay. After 24 h, an abundant number of BMSCs migrated to GCs that were injured by cisplatin, through polycarbonate filters (Fig. 4B). Only a few BMSCs migrated to the normal GCs (Fig. 4A). Statistical analysis of the number of BMSCs revealed that GCs injured by cisplatin promoted BMSCs to migrate after 6 and 24 h (Fig. 4C).
30 days after injecting BMSCs, the E2 serum in the BMSCs group increased compared with the POF group (
All stages of follicular development (primary, secondary, and antral follicles) were observed in the rat control group for 30 days (Fig. 6A). Specimens that were taken from the POF group had only a few primordial follicles with degenerated growth and antral follicles (Fig. 6B). All types of follicles, including the primary, secondary and antral, and corpus luteum were apparently normal in the BMSCs treatment group (Figs. 6C and 6D). In addition, the antral follicles in the treatment group were healthy (Figs. 6E and 6F).
The statistical analysis of the antral follicular count revealed that the increased dosage of cisplatin resulted to the decreased amount of follicular count. However, in the BMSCs treatment group, a significant increase in healthy antral follicles and corpus lutea count was detected, compared with the cisplatin injection group (Fig. 5B); no significant change was observed after 15 days (
Following the transplantation of BMSCs, ovaries displayed EGFP-positive BMSCs on the 15th day. Interestingly, the number of BMSCs in the ovarian hilum and ovarian medulla was greater than in the ovarian cortex (Figs. 7A?7F). We also found BMSCs surrounding the follicles; but there were no BMSCs in the ovarian follicle and corpus lutea. 30 days after injecting BMSCs, we found that BMSCs survived and proliferated in the ovary (Figs. 7G?7I). BMSCs were also found in other organs, such as the heart, kidneys, liver and adipose tissues near the ovaries (Figs. 7J?7L). However, the number of BMSCs in the ovaries and kidneys were greater than in the liver and heart.
More interesting, most of the BMSCs were distributed along the blood vessels.
The granulosa cell apoptosis was detected by TUNEL. There were no apoptotic GCs in the healthy antral follicles (Fig. 8A). An abundant number of apoptotic GCs were observed in the atretic follicles that did not contain oocytes (Figs. 8B and 8C). Atretic follicles were found in all groups; and the number of healthy antral follicles in the BMSCs group was greater than in the POF group after 30 days (healthy antral follicular count 5.60 ± 1.14 in BMSCs group versus 2.60 ± 1.52 in POF group;
The ability of BMSCs to self-renew and regenerate pushed the trial to evaluate their role in fertility preservation. So far, no ideal marker for BMSCs has been identified. However, it has been identified that BMSCs expressed CD29, CD90, CD105 and negative expressions of hematopoietic cell-surface antigens, such as CD45, CD34, CD14 (Ohishi and Schipani, 2010). In our study, BMSCs were identified to express CD90 and CD29, but not CD45 and CD34. In addition to their fibroblast-shaped morphology and adherence to plastic, it was possible to conclude that the cell population used in our study was an enriched culture of BMSCs. Since homing of BMSCs is inefficient and many BMSCS were trapped in the lung following systemic administration, it is imperative that we trace the fate of the injected cells. We transfected BMSCs with Ad-EGFP and yielded the highest percentage of GFP-expression cells (99%) without affecting cell viability (Bosch et al., 2006; Yuan et al., 2011). A classic method in labeling cells is with Ad-EGFP; which expresses fluorescent proteins that are helpful in gaining insights on the homing and engraftment of BMSCs.
Cisplatin is one of the most effective chemotherapeutic agents, but injury may occur at higher dosages. It has been reported that cisplatin caused ovarian failure during clinical usage and non-clinical investigations (Ai et al., 2012; Iorio et al., 2014; Nozaki et al., 2009). Cisplatin induced granulosa cell apoptosis or necrosis
In the present study, BMSCs that migrated to granulosa cells injured by cisplatin
The present study found that the infusion of BMSCs could restore ovarian function. 30 days after administering BMSCs, ovarian function and structure improved based on results; which showed increased estradiol levels and follicle population. The mechanisms that enable BMSCs to induce beneficial effects in the ovaries are still controversial. It has been proposed that BMSCs can differentiate into fibroblasts, adiopocytes, chondrocytes, and osteocytes
It has been identified that follicular growth needed the systemic regulation by hormones and intraovarian regulation by cytokines, growth factors, and intracellular proteins (Matsuda et al., 2011). Granulosa cells, in particular, play an important role in follicular development and maintenance (Maruo et al., 1999). In our study, BMSCs reduced the apoptosis rate of cisplatin injured GCs
Therefore, the migration capabilities of BMSCs to injured tissue sites and its secretion function, allows BMSCs to exert their restorative actions. Moreover, studies have shown that BMSCs not only protects the ovaries from cisplatin-induced injuries, but also protects and restores other organs that were injured by cisplatin, such as kidneys and the bone marrow (Bi et al., 2007; Frenette et al., 2013). These results suggest that systemically delivered BMSCs are fit for protecting and restoring multiple organs injured by chemotherapeutic toxicities.
In conclusion, the present study demonstrated that through intravenous injection, BMSCs home to the ovaries and distribute mainly in the ovarian hilum and medulla; a small number of BMSCs engraft in the ovarian cortex; and no BMSCs were observed in the follicles and corpus lutea. In addition, ovarian function and structure was restored, after intravenously injecting BMSCs. Therefore, intravenously injecting BMSCs is an efficient treatment method for chemotherapy-induced POF.
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