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Sodium nitroprusside treatment during the superovulation process improves ovarian response and ovarian expression of vascular endothelial growth factor in aged female mice
Sodium nitroprusside treatment during the superovulation process improves ovarian response and ovarian expression of vascular endothelial growth factor in aged female mice Dong-Hyung Lee, M.D.,a Bo-Sun Joo, Ph.D.,b Dong-Soo Suh, M.D.,a Jong-Hun Park, M.D.,a Young-Min Choi, M.D.,c and Kyu-Sup Lee, M.D.a a
Department of Obstetrics and Gynecology, Pusan National University College of Medicine and Medical Research Institute, Pusan National University, Busan; b Center for Reproductive Medicine, Good Moonhwa Hospital, Busan; c Department of Obstetrics and Gynecology, Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Seoul, South Korea
Objective: To investigate whether sodium nitroprusside (SNP) treatment during the superovulation process improves ovarian response and oocyte developmental competence in aged female mice. Design: Controlled experimental study. Setting: Large urban medical center. Animal(s): C57BL inbred female mice of three age groups: 6 to 9, 14 to 16, and 25 to 27 weeks. Intervention(s): Female mice were co-injected intraperitoneally with SNP (1 mM or 10 mM) and pregnant mare’s serum gonadotropin (PMSG), followed by human chorionic gonadotropin injection 48 hours later and then mated with individual males. After 18 hours, zygotes were flushed and the ovaries were isolated. The control group was injected with PMSG. Main Outcome Measure(s): The number of zygotes flushed, embryo development to blastocyst stage, and vascular endothelial growth factor (VEGF) expression in ovary. Result(s): Treatment with SNP statistically significantly increased the number of flushed zygotes and blastocyst formation rate in mice aged 25 to 27 weeks, not but in mice aged less than 16 weeks compared with the control group. The SNP treatment in aged mice increased VEGF expression of the ovary in a dose-dependent manner. Conclusion(s): These results demonstrate that SNP treatment during the superovulation process improves ovarian response and oocyte developmental competence in aged female. The positive effect of SNP may be associated with increased VEGF expression. (Fertil Steril 2008;89:1514–21. 2008 by American Society for Reproductive Medicine.) Key Words: Female aging, SNP, ovarian response, oocyte developmental competence, VEGF
Advancing female age is an important contribution to an increased incidence of infertility and remains a problem in infertility treatment. Deterioration of oocyte quality and lessened endometrial receptivity are reasons for the age-related decline in fertility (1, 2). However, the finding that infertility in older women can be overcome by oocyte donation from younger women suggests that age-related infertility might be attributable to oocyte quality rather than endometrial receptivity (3, 4). It is well known that female aging is associated with decreased viability of preimplantation embryos. In mice, female aging is associated with a decreased number of ovulated Received February 12, 2007; revised and accepted May 29, 2007. Presented at the 62nd Annual Meeting of the American Society for Reproductive Medicine (ASRM); October 21–25, 2006; New Orleans, Louisiana. Supported by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (01-PJ10-PG6-01GN13-0002). Reprint request: Kyu-Sup Lee, M.D, Ph.D., Department of Obstetrics and Gynecology, Pusan National University, College of Medicine, 1-10 Amidong, Seuku, Busan, 602-739, South Korea (FAX: 82-51-248-2384; E-mail: [email protected]).
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oocytes and/or increased percentage of abnormal/degenerating oocytes (5). Chromosomal aneuploidy (6) and the injury of mitochondrial DNA (7, 8) have been known to be major causes of the age-related decline of oocyte quality. However, recent findings that dysfunction in ovarian angiogenesis results in anovulation, pregnancy loss, or polycystic ovary syndrome indicate that the angiogenesis process plays a very important role in folliculogenesis and implantation (9). Furthermore, some follicles have a more elaborate microvasculature than others, and increased vascular density is highest in mature follicles (10, 11). Angiogenesis is regulated independently in each follicle, and its regulation depends on the vascular network and permeability of vessels and the activity of angiogenic factors. Therefore, the vasculature of the follicle is thought to be a necessary factor in the delivery of hormones, hormone precursors, oxygen, and nutrients. This suggests that an active blood supply is essential for the induction of oocytes with good quality. Vascular endothelial growth factor (VEGF), a critical regulator of angiogenesis, is expressed not only in follicle but also in the stroma of the ovary, and it provides nutritional
Fertility and Sterility Vol. 89, Suppl 3, May 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
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support for primordial follicles and primary follicles (12). Thus, it is likely that VEGF regulation in the ovarian follicular phase can benefit ovarian stimulation for poor responders. Many studies have shown that enhancing VEGF expression during the follicular phase may be useful in increasing the number of predominant follicles destined for ovulation and angiogenesis. For example, direct injection of VEGF gene fragment or VEGF into the ovary increases angiogenesis and the number of follicles to be ovulated (13–15). Wulff et al. (16) showed that suppression of follicular angiogenesis by the inhibition of VEGF is associated with the inhibition of antral follicular development and results in the prevention of ovulation. In addition, blood flow indexes in the early follicular phase have been negatively co-related with the number of follicles recruited and the number of oocytes retrieved (17, 18). When hyperstimulation is performed, ovarian blood flow is significantly increased in normal responders compared with the poor responders (19). These results suggest that active blood flow is a necessary factor for an adequate folliculogenesis. Nitric oxide (NO), known to be a potent vasodilator and angiogenic factor, plays an important role in ovarian angiogenesis during folliculogenesis and ovulation, including in the determination of oocyte quality and embryo developmental competence and the inhibition of atresia and apoptosis of the growing follicles (20–22). Nitric oxide also mediates the vascular permeability of VEGF (23), and VEGF stimulates the production of NO in endothelial cells of intestine (24). We investigated whether co-injection of NO donor, sodium nitroprusside (SNP) with gonadotropin during the superovulation process improved ovarian response and oocyte developmental competence in aged female mice. MATERIALS AND METHODS This study was approved by the institutional review board of Pusan National University Hospital. Animals In all experiments, C57BL inbred mice were used, purchased from Korea Experimental Animal Center (Daegu, South Korea). Mice were maintained on a light-dark cycle, with light on at 5:00 AM and off at 7:00 PM, and with food and water available ad libitum. Superovulation One or 10 mM of SNP (Sigma, St. Louis, MO) was coinjected intraperitoneally with 5 IU of pregnant mare’s serum gonadotropin (PMSG, Sigma) to female mice of three age groups: 6 to 9 weeks, 14 to 16 weeks, and 25 to 27 weeks. Forty-eight hours after the injection of PMSG, the mice were injected with 5 IU of human chorionic gonadotropin (hCG, Sigma) and then immediately paired with an individual male. On the morning of the following day, the mice were inspected and those with a confirmed vaginal plug Fertility and Sterility
were considered to be fertilized; these mice were used in the experiment. Mice of the same age injected with PMSG without SNP were used as the control group. One-Cell Embryos Collection and Embryo Culture Female mice with a confirmed vaginal plug were killed by cervical dislocation 18 hours after the hCG injection, and the one-cell embryos were retrieved from the oviductal ampulae. Cumulus-enclosed one-cell embryos were collected and denuded by incubation for 1 minute with 1 mg/mL hyaluronidase (Sigma) in Dulbecco’s phosphate buffer saline (dPBS; GIBCO BRL, Grand Island, NY). One-cell embryos were pooled and washed three times in human tubal fluid (HTF) media supplemented with 10% human follicular fluid (hFF). Only healthy zygotes were cultured in 30-mL drops of media (HTF þ 20% hFF) under paraffin-oil at 37 C in a 5% CO2 incubator for 4 days, and the media was changed daily. The number of one-cell embryos retrieved and fragmented per mouse was counted, and embryo development to blastocyst stage was evaluated. Western Blot Analysis Proteins were extracted by mechanical homogenization of ovaries in the presence of 200 mL ice-cold lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40,1mM EDTA). The protein content of cell lysate was determined with Bradford reagent (Bio-Rad, Hercules, CA) using bovine serum albumin (BSA) as the standard. We separated 20 mg of cell lysate by SDS-PAGE and transferred to PVDF (immobilon-P) membrane (Millipore, Bedford, MD). The transfer was performed at a constant voltage of 15 V for 30 minutes. For Western blotting, the membrane was incubated antimouse VEGF (R&D Systems, Minneapolis, MN) in TBS (Sigma) containing 1% skim milk for 4 hours at room temperature. After washing three times with TBS-T (containing 0.04% Tween-20), the blotted membranes were incubated with horseradish peroxidase–conjugated goat antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 minutes at room temperature. After washing three times with TBS-T, the proteins were visualized by the enhanced chemiluminescence detection system according to the recommended procedure (Amersham Pharmacia Biotech, Piscataway, NJ). Immunnohistochemistry Serial sections (4 mm) of formalin-fixed, paraffin-embedded ovary tissues were spread on the coated-slides, and placed in an oven at 60 C for 1 hour. The slides then were deparaffinized in xylene and dehydrated in a graded series of ethanol. An immunohistochemistry for VEGF was performed with streptavidin-biotin-peroxidase complex method using rabbit antimouse VEGF polyclonal antibody (Lab Vision, Fremont, CA). The endogenous peroxidase was quenched with 0.3% hydrogen peroxide at room temperature for 5 minutes, and then the tissues were rinsed four times for 5 minutes each time in TBS. The samples were incubated with normal serum, then incubated with rabbit antimouse VEGF polyclonal 1515
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SNP improves ovarian response in older females
96 (32.8) 80 (31.9) 85 (30.7) 148 (50.5) 125 (49.8) 123 (44.6) 161 (54.9) 132 (52.5) 131 (47.5) 168 (57.3) 136 (54.2) 143 (51.8) 293 251 276 Lee. SNP improves ovarian response in older females. Fertil Steril 2008.
21.3 17.9 20.2
4-cell (%) 2-cell (%) Zygotes cultured Zygotes flushed/mouse Zygotes fragmented (%)
28 (8.7) 18 (6.7) 27 (8.9) 321 269 303 15 15 15 0 mM control 1 mM 10 mM
The results showed that there was no difference in VEGF expression in the mice aged 16 weeks who were not affected by SNP treatment. However, VEGF expression in mice aged 26 weeks, who had shown a beneficial effect with SNP
TABLE 1
The beneficial effect of SNP disappeared in mice aged 6 to 9 weeks. As shown in Table 3, the number of one-cell embryos flushed and the embryo development rate were similar, regardless of SNP treatment. Consequently, we used Western blot and immunohistochemistry to evaluate VEGF expression in ovaries of mice aged 26 weeks to find out if the effect of SNP treatment observed in old aged mice of 25 to 27 weeks was related to the expression of VEGF in their ovaries. Mice aged 16 weeks that had no confirmed positive effect with SNP were used for comparison of VEGF expression with the mice aged 26 weeks.
Zygotes flushed
We treated mice aged 25 to 27 weeks with the same doses of SNP. The number of one-cell embryos flushed was 21.7 with 1 mM SNP and 20.2 with 10 mM SNP, which was statistically significantly higher compared with the control group (15.2; P<.05). The embryo development rate to blastocyst was statistically significantly increased with SNP treatment (1 mM and 10 mM: 36.7%, 46.8%, respectively) when compared with the control group (5.5%; P<.05). A noticeably larger number of developing embryos were arrested in the control group at the two-cell or four-cell stage (Table 2).
Mice provided
RESULTS To investigate whether the stimulation of ovarian angiogenesis affects the number of oocytes and embryo development, 1 mM and 10 mM of SNP were co-injected with PMSG during superovulation into three age groups: 6 to 9 weeks, 14 to 16 weeks, and 25 to 27 weeks. First, we administered SNP to mice aged 14 to 16 weeks. The number of one-cell embryos flushed was 17.9 with 1 mM SNP, 20.2 with 10 mM SNP, and 21.3 with the control group. However, there were no statistically significant differences in the number of one-cell embryos flushed or the embryo development rate (Table 1).
SNP
Statistical Analysis All data were presented as mean standard deviation. Statistical analysis was performed using the unpaired Student’s t-test and chi-square test. P<.05 was considered statistically significant.
Effects of sodium nitroprusside (SNP) treatment on embryo development in mice aged 14–16 weeks.
Morula (%)
Blastocyst (%)
antibody at a dilution of 1:70 in PBS/BSA overnight at 4 C. After four washings with TBS of 15 minutes each, the samples were incubated with biotinylated secondary antibody (e-Bioscience, San Diego, CA) for 30 minutes at room temperature and washed three times. Then, the samples were incubated with streptavidin-peroxidase conjugate in PBS for 30 minutes at room temperature and incubated with DAB (3.30 diaminibenzidine chromogen; Sigma). Counterstaining was performed with Mayer’s hematoxylin. The results were assessed by one pathologist under a light microscope.
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11 (5.5) 101 (36.7)a 126 (46.8)a
DISCUSSION This study shows for the first time that the SNP treatment during the superovulation process improves the number of oocytes recruited and ovulated and the oocyte developmental competence in aged female, possibly via an increase of VEGF in the ovary. The sexual maturity and lifespan in the laboratory for mice is around 6 weeks and 1 year, respectively. Considering the reproductive physiology of mice, 6 to 9 weeks, 14 to 16 weeks, and 25 to 27 weeks in mice may be comparable with human teenage years, R30 years of age, and R40 years of age in humans, respectively. Furthermore, oocytes collected from 30-to-40-week-old mice are more developmentally sensitive to mitochondrial damage than oocytes from pubertal mice, resulting in significant decline of oocyte competence (8). In this respect, we think that the severe loss of fertility potential in mice over 30 weeks makes them inappropriate for evaluating the age-related decline of the number of follicles ovulated and the developmental competence of oocytes.
228 325 303 a
P< .05 (vs. control).
15 15 15 0 mM control 1 mM 10 mM
Lee. SNP improves ovarian response in older females. Fertil Steril 2008.
15 (7.6) 154 (55.8)a 158 (58.7)a 25 (12.6) 159 (57.6)a 165 (61.4)a 198 276 269
Immunohistochemistry was performed to evaluate the localized VEGF expression in the ovary induced by SNP treatment in mice aged 26 weeks; Western blot confirmed the increase in VEGF expression. The VEGF expression was usually localized in granulosa cells, stromal cells, and endothelial cells, and expression was increased after the SNP treatment. The intensity of VEGF expression was the highest in the mice treated with 10 mM of SNP; the control group showed the lowest expression of VEGF (Fig. 2). These results suggest that SNP treatment induces an increase of VEGF expression in ovarian cells.
30 (13.2) 49 (15.1) 34 (11.2)
15.2 21.7 20.2
43 (21.7) 194 (70.3)a 177 (65.8)a
Blastocyst (%) Morula (%) 4-cell (%) 2-cell (%) Zygotes cultured Zygotes flushed/mouse Zygotes fragmented (%) Zygotes flushed Mice provided SNP
Effects of sodium nitroprusside (SNP) treatment on embryo development in mice aged 25–27 weeks.
TABLE 2
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treatment, also showed a remarkable increase in a dosedependent manner (Fig. 1).
Angiogenesis is initiated early in the follicular development and plays an important role in many aspects of female reproductive physiology that require neovascularization, such as follicular growth, selection of dominant follicles, inhibition of follicular atresia, and corpus luteum formation (9, 25). Gaulden (26) proposed that a deficient microvasculature develops around the dominant follicles with aging, resulting in the hypoxia of follicles. Friedman et al. (27) reported that the increase VEGF follicular fluid concentrations in women of advanced reproductive age are due to relative hypoxia in ovarian follicles. Pellicar et al. (28) observed reduced blood flow during natural cycles around the dominant follicle in poor responders compared with controls, which suggested that women with a response to exogenous gonadotropins typical for women of advanced reproductive age have a deficient follicular microcirculation even if the issue of age is not directly addressed. In this respect, infertility such as age-related decline of ovarian response seems to be associated with inadequate follicular development that results from disturbance of follicular angiogenesis. 1517
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A critical regulator of ovarian angiogenesis is VEGF, which has the most potent angiogenic activity and vascular permeable activity (30). It plays an important role in the normal function of the ovary during follicular development (31, 32). Intrabursal injection of VEGF in the rat ovary significantly increased the number of primary and small secondary but not large secondary and preantral follicles and stimulates preantral follicle development. This result suggests that VEGF may be one of the factors that participates in the regulation of early follicular growth (14). Nitric oxide is another angiogenic factor and vascular permeable factor during ovarian angiogenesis (33, 34), and it forms a vital component of the oocyte microenvironment during folliculogenesis, ovulation, embryo development, and implantation (20–22, 35, 36). In addition, NO mediates the increased vascular permeability via VEGF (23). Due to this property, our study injected SNP during the superovulation process as a factor that may increase ovarian response to gonadotropins by promoting ovarian angiogenesis via the stimulation by VEGF. As we expected, the SNP treatment increased the expression of VEGF in the ovary.
Lee. SNP improves ovarian response in older females. Fertil Steril 2008.
265 (75.1) 250 (74.0) 246 (73.4) 301 (85.3) 292 (86.4) 282 (84.2) 314 (89.0) 299 (88.5) 288 (86.0) 353 338 335 24.2 23.3 23.1 364 140 346 0 mM control 1 mM 10 mM
15 15 15
11 (3.0) 12 (3.6) 11 (3.2)
319 (90.4) 306 (90.5) 300 (89.5)
Blastocyst (%) Morula (%) 4-cell (%) 2-cell (%) Zygotes cultured Zygotes flushed/mouse Zygotes fragmented (%) Zygotes flushed Mice provided SNP
Effects of Sodium nitroprusside (SNP) treatment on embryo development in mice aged 6–9 weeks.
TABLE 3 1518
Fraser (29) suggested that the supply of appropriate blood vessels and the maintenance of vascular permeability in the ovaries, including to follicles, are necessary for gonadotropins to have an adequate effect and for paracrine factors to sustain follicular growth and ovulation. Therefore, it is thought that the age-related decline of ovarian response or oocyte quality can be reversed if ovarian angiogenesis is stimulated in such a way that age-related reduction of ovarian blood flow is overcome.
Sodium nitroprusside has a short half life, and co-injection of SNP with PMSG at the starting time of superovulation may increase VEGF expression in early follicle. The VEGF stimulates the number of primary and small secondary follicles and preantral follicular development (14). In particular, immunohistochemical staining showed that most graafian follicles were ovulated by hCG injection, and the remaining follicles were early immature follicles. These results suggest that SNP treatment appears to enhance folliculogenesis rather than ovulatory capacity. As female age advances, the aging of the ovaries has been known to result in the gradual reduction of the number of primordial follicles and dominant follicles responding to gonadotropins, and finally the decline of oocyte quality (37). The number of follicles growing to the antral stage also reduces with age (38, 39). The number of primordial follicles steadily decreases throughout a female’s reproductive lifespan as a result of atresia and is finally depleted in menopause. We would like to postulate that the reduction of the number of follicles to be recruited and ovulated in aged females may result from the inactivation of the mechanism for the assembly of primordial follicles and the transition from primordial follicle to primary follicle rather than the depletion of primordial follicle pool. Locally produced factors including kit ligand, leukemia inhibitory factor, and keratinocyte growth
SNP improves ovarian response in older females
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FIGURE 1 Western blot analysis of vascular endothelial growth factor (VEGF) expression in the ovaries of different aged mice. The ovaries were isolated just after the flushing of one-cell embryos. (A) 16-week-old mice. (B) 26-week-old mice. Lane 1: PMSG alone (control). Lane 2: 1 mM SNP þ PMSG. Lane 3: 10 mM SNP þ PMSG.
Lee. SNP improves ovarian response in older females. Fertil Steril 2008.
factor are involved in the promotion of primordial to primary follicle transition (40). Furthermore, one report has suggested that the potential presence of a female germ-line stem cell population may provide a continual supply of primordial follicles (41). Thus, the primordial follicle pool may not be finite and may regenerate in an appropriate milieu. As shown in our study, the number of oocytes recruited from SNP-treated older mice is similar to those recruited from younger mice. It seems possible that an active blood
supply through the increase of VEGF expression prevents the follicles from atresia (42). Indeed, when the number of antral follicles was estimated to predict the ovarian reserve, the number varied in each cycle (43). These facts imply that the number of follicles to be recruited and ovulated can be different, depending on the ovarian stimulation method, even though they are in the same pool of primordial follicles. Therefore, it is possible if an adequate ovarian stimulation is given to increase ovarian angiogenesis in an older female, an efficient number of follicles develop to dominant follicles.
FIGURE 2 Immunohistochemistry of vascular endothelial growth factor (VEGF) expression in ovaries of mice aged 26 weeks. The ovaries were isolated just after the flushing of one-cell embryos. Immunostaining with anti-VEGF antibody was usually localized in granulosa cells (Gr), stromal cells (St), and endothelial cells (Ec). The immunostaining intensity was increased after sodium nitroprusside (SNP) treatment. (A) PMSG alone (control). (B) 1 mM SNP þ PMSG. (C) 10 mM SNP þ PMSG (100).
Lee. SNP improves ovarian response in older females. Fertil Steril 2008.
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The exact mechanism for the positive effect of SNP shown in aged mice remains to be elucidated. However, several possibilities can be considered. First, NO synergistically activates ovarian angiogenesis by stimulating VEGF expression. Indeed, our study showed that the SNP treatment increases VEGF expression as well as the number of oocytes and the embryo development rate. Second, the vasoactive and angiogenic activity of NO itself can have an effect on the preferential delivery of gonadotropins to the follicles. Third, exposure to NO delays oocyte aging and improves the integrity of the microtubular spindle apparatus as an antioxidant (44). Aged oocytes have a significantly increased likelihood of failed or abnormal fertilization and/or development, eventually resulting in a atresia and fragmentation. Fourth, as the possibility cannot be excluded that NO induces other factors such as matrix metalloproteinase 9 (MMP9) to stimulate neovascularization of follicles, further study is needed. In general, VEGF protein is initially absent from the granulose cells of primordial follicles and preantral follicles but becomes evident in the theca cells of antral follicles and in the granulose cells nearest in the preovulatory follicles. Vascular endothelial growth factor is expressed not only in follicles but also in the stroma of the ovary, which provides nutritional support for primordial and primary follicles (12, 29). However, in our study, VEGF expressed in granulose cells, stroma cells, and endothelial cells was increased after SNP treatment. Also, SNP treatment resulted in the increased VEGF expression in theca cells. It seems that this increased VEGF expression provides nutritional support for early follicles to be developed and ovulated. However, additional study is necessary concerning the period of VEGF expression after SNP treatment. Our study provides interesting, beneficial data toward improving assisted reproduction in age-related infertility, but we did not examine whether the SNP treatment enhances the development to blastocyst stage in vivo and the implantation rate. However, the finding that zygotes collected from SNP-injected mice increased blastocyst formation rate in vitro suggests that the same effect may occur in vivo. Therefore, further study is needed to contribute to assisted reproductive technologies. Our study shows for the first time that co-treatment of SNP with gonadotropins in aged female mice of 25 to 27 weeks during the superovulation process increases ovarian response and oocyte developmental competence at a similar level to that of pubertal mice. This beneficial effect of SNP seems to be associated with increased VEGF expression, which may result in enhanced angiogenesis during follicular development. In addition, this result implies that age-related decline of fertility might be attributable to a deterioration of oocyte quality resulting from decreased ovarian angiogenesis. This is an interesting finding in the respect that it provides a possibility that, if an adequate ovarian stimulation protocol for stimulating angiogenesis is given to the older female, efficient numbers of oocytes with a good quality and normal 1520
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embryo developmental potential can be produced. However, NO has diverse effects, depending on its dose. Therefore, before using NO clinically for ovulation induction in older women, further studies are needed to develop local or systemic administration methods and appropriate dosages without side effects.
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20. Jablonka-Shariff A, Olson LM. The role of nitric oxide in oocyte meiotic maturation and ovulation: meiotic abnormalities of endothelial nitric oxide synthase knock-out mouse oocytes. Endocrinology 1998;139:2944–54. 21. Sengoku K, Takuma N, Horikawa M, Tsuchiya K, Komori H, Sharifa D, et al. Requirement of nitric oxide for murine oocyte maturation, embryo development, and trophoblast outgrowth in vitro. Mol Reprod Dev 2001;58:262–8. 22. Chun SY, Eisenhauer KM, Minami S, Billig H, Perlas E, Hsueh AJ. Hormonal regulation of apoptosis in early antral follicles: folliclestimulating hormone as a major survival factor. Endocrinology 1996;137: 1447–56. 23. Murohara T, Horowitz JR, Silver M, Tsurumi Y, Chen D, Sullivan A, et al. Vascular endothelial growth factor/vascular permeability factor enhances vascular permeability via nitric oxide and prostacyclin. Circulation 1998;97:99–107. 24. van der Zee R, Murohara T, Luo Z, Zollmann F, Passeri J, Lekutat C, et al. Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) augments nitric oxide release from quiescent rabbit and human vascular endothelium. Circulation 1997;95:1030–7. 25. Gordon JD, Shifren JL, Foulk RA, Taylor RN, Jaffe RB. Angiogenesis in the human female reproductive tract. Obstet Gynecol Surv 1995;50:688–97. 26. Gaulden ME. Maternal age effect: the enigma of Down syndrome and other trisomic conditions. Mutat Res 1992;296:69–88. 27. Friedman CI, Danforth DR, Herbosa-Encarnacion CH, Arbogast L, Alak BM, Seifer DB. Follicular fluid vascular endothelial growth factor concentrations are elevated in women of advanced reproductive age undergoing ovulation induction. Fertil Steril 1997;68:607–12. 28. Pellicar A, Ballester MJ, Serrano MD, Mir A, Serra-Serra V, Remohi J, et al. Aetiological factors involved in the low-response to gonadotropins in infertile women with normal basal follicle stimulating hormone levels. Hum Reprod 1994;8:806–11. 29. Fraser HM. Regulation of the ovarian follicular vasculature. Reprod Biol Endocrinol 2006;4:18–26. 30. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997;18:4–25. 31. Hazzards TM, Xua F, Stouffer RL. Injection of soluble vascular endothelial growth factor receptor 1 into the preovulatory follicle disrupts ovulation and subsequent luteal function in Rhesus monkey. Biol Reprod 2002;67:1305–12.
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32. Fraser HM, Wilson H, Rudge JS, Wiegand SJ. Single injections of vascular endothelial growth factor trap block ovulation in the macaque and produce a prolonged, dose-related suppression of ovarian function. J Clin Endocrinol Metab 2005;90:1114–22. 33. Koos RD. Increased expression of vascular endothelial growth/ permeability factor in the rat ovary following an ovulatory gonadotropin stimulus: potential roles in follicular rupture. Biol Reprod 1995; 52:1426–35. 34. Powers RW, Chen L, Russell PT, Larse WJ. Gonadotropin-stimulated regulation of blood-follicle barrier is mediated by nitric oxide. Am J Physiol 1995;269:E290–8. 35. Rosselli M, Keller PJ, Dubey RK. Role of nitric oxide in the biology, physiology, and pathophysiology of reproduction. Hum Reprod Update 1998;4:3–24. 36. Tranguch S, Stuerwald N, Huet-Hudson YM. Nitric oxide synthase production and nitric oxide regulation of preimplantation embryo development. Biol Reprod 2003;68:1538–44. 37. Faddy MJ, Gosden RG, Gougeon A, Richardson SJ, Nelson JF. Accelerated disappearance of ovarian follicles in the mid-life: implications for forecasting menopause. Hum Reprod 1992;7:1342–6. 38. Faddy MJ. Follicle dynamics during ovarian aging. Mol Cell Endocrinol 2000;163:43–8. 39. Jacobs SL, Metzger DA, Dodson WC, Huney AF. Effect of age on response to human menopausal gonadotropin stimulation. J Clin Endocrinol Metab 1990;71:26–30. 40. Skinner MK. Regulation of primordial follicle assembly and development. Hum Reprod Update 2005;11:461–71. 41. Johnson J, Canning J, Kaneko T, Pru JK, Tilly JL. Germline stem cells and follicular renewal in the postnatal mammalian ovary. Nature 2004;428:145–50. 42. Gosdon RG, Telfer E, FAddy MJ. Ovarian cyclicity and follicular recruitment in unilaterally ovarirectomized mice. J Reprod Fertil 1989;87: 257–64. 43. Bancsi LFJMM, Broekmans FJM, Looman CWN, Habbema JDF, te Velde ER. Impact of repeated antral follicle counts on the prediction of poor ovarian response in women undergoing in vitro fertilization. Fertil Steril 2004;81:35–41. 44. Goud AP, Goud PT, Diamond P, Abu-Soud HM. Nitric oxide delays oocyte aging. Biochemistry 2005;44:11361–8.
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Department of Obstetrics and Gynecology, Pusan National University College of Medicine and Medical Research Institute, Pusan National University, Busan; b Center for Reproductive Medicine, Good Moonhwa Hospital, Busan; c Department of Obstetrics and Gynecology, Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Seoul, South Korea
Objective: To investigate whether sodium nitroprusside (SNP) treatment during the superovulation process improves ovarian response and oocyte developmental competence in aged female mice. Design: Controlled experimental study. Setting: Large urban medical center. Animal(s): C57BL inbred female mice of three age groups: 6 to 9, 14 to 16, and 25 to 27 weeks. Intervention(s): Female mice were co-injected intraperitoneally with SNP (1 mM or 10 mM) and pregnant mare’s serum gonadotropin (PMSG), followed by human chorionic gonadotropin injection 48 hours later and then mated with individual males. After 18 hours, zygotes were flushed and the ovaries were isolated. The control group was injected with PMSG. Main Outcome Measure(s): The number of zygotes flushed, embryo development to blastocyst stage, and vascular endothelial growth factor (VEGF) expression in ovary. Result(s): Treatment with SNP statistically significantly increased the number of flushed zygotes and blastocyst formation rate in mice aged 25 to 27 weeks, not but in mice aged less than 16 weeks compared with the control group. The SNP treatment in aged mice increased VEGF expression of the ovary in a dose-dependent manner. Conclusion(s): These results demonstrate that SNP treatment during the superovulation process improves ovarian response and oocyte developmental competence in aged female. The positive effect of SNP may be associated with increased VEGF expression. (Fertil Steril 2008;89:1514–21. 2008 by American Society for Reproductive Medicine.) Key Words: Female aging, SNP, ovarian response, oocyte developmental competence, VEGF
Advancing female age is an important contribution to an increased incidence of infertility and remains a problem in infertility treatment. Deterioration of oocyte quality and lessened endometrial receptivity are reasons for the age-related decline in fertility (1, 2). However, the finding that infertility in older women can be overcome by oocyte donation from younger women suggests that age-related infertility might be attributable to oocyte quality rather than endometrial receptivity (3, 4). It is well known that female aging is associated with decreased viability of preimplantation embryos. In mice, female aging is associated with a decreased number of ovulated Received February 12, 2007; revised and accepted May 29, 2007. Presented at the 62nd Annual Meeting of the American Society for Reproductive Medicine (ASRM); October 21–25, 2006; New Orleans, Louisiana. Supported by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (01-PJ10-PG6-01GN13-0002). Reprint request: Kyu-Sup Lee, M.D, Ph.D., Department of Obstetrics and Gynecology, Pusan National University, College of Medicine, 1-10 Amidong, Seuku, Busan, 602-739, South Korea (FAX: 82-51-248-2384; E-mail: [email protected]).
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oocytes and/or increased percentage of abnormal/degenerating oocytes (5). Chromosomal aneuploidy (6) and the injury of mitochondrial DNA (7, 8) have been known to be major causes of the age-related decline of oocyte quality. However, recent findings that dysfunction in ovarian angiogenesis results in anovulation, pregnancy loss, or polycystic ovary syndrome indicate that the angiogenesis process plays a very important role in folliculogenesis and implantation (9). Furthermore, some follicles have a more elaborate microvasculature than others, and increased vascular density is highest in mature follicles (10, 11). Angiogenesis is regulated independently in each follicle, and its regulation depends on the vascular network and permeability of vessels and the activity of angiogenic factors. Therefore, the vasculature of the follicle is thought to be a necessary factor in the delivery of hormones, hormone precursors, oxygen, and nutrients. This suggests that an active blood supply is essential for the induction of oocytes with good quality. Vascular endothelial growth factor (VEGF), a critical regulator of angiogenesis, is expressed not only in follicle but also in the stroma of the ovary, and it provides nutritional
Fertility and Sterility Vol. 89, Suppl 3, May 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
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support for primordial follicles and primary follicles (12). Thus, it is likely that VEGF regulation in the ovarian follicular phase can benefit ovarian stimulation for poor responders. Many studies have shown that enhancing VEGF expression during the follicular phase may be useful in increasing the number of predominant follicles destined for ovulation and angiogenesis. For example, direct injection of VEGF gene fragment or VEGF into the ovary increases angiogenesis and the number of follicles to be ovulated (13–15). Wulff et al. (16) showed that suppression of follicular angiogenesis by the inhibition of VEGF is associated with the inhibition of antral follicular development and results in the prevention of ovulation. In addition, blood flow indexes in the early follicular phase have been negatively co-related with the number of follicles recruited and the number of oocytes retrieved (17, 18). When hyperstimulation is performed, ovarian blood flow is significantly increased in normal responders compared with the poor responders (19). These results suggest that active blood flow is a necessary factor for an adequate folliculogenesis. Nitric oxide (NO), known to be a potent vasodilator and angiogenic factor, plays an important role in ovarian angiogenesis during folliculogenesis and ovulation, including in the determination of oocyte quality and embryo developmental competence and the inhibition of atresia and apoptosis of the growing follicles (20–22). Nitric oxide also mediates the vascular permeability of VEGF (23), and VEGF stimulates the production of NO in endothelial cells of intestine (24). We investigated whether co-injection of NO donor, sodium nitroprusside (SNP) with gonadotropin during the superovulation process improved ovarian response and oocyte developmental competence in aged female mice. MATERIALS AND METHODS This study was approved by the institutional review board of Pusan National University Hospital. Animals In all experiments, C57BL inbred mice were used, purchased from Korea Experimental Animal Center (Daegu, South Korea). Mice were maintained on a light-dark cycle, with light on at 5:00 AM and off at 7:00 PM, and with food and water available ad libitum. Superovulation One or 10 mM of SNP (Sigma, St. Louis, MO) was coinjected intraperitoneally with 5 IU of pregnant mare’s serum gonadotropin (PMSG, Sigma) to female mice of three age groups: 6 to 9 weeks, 14 to 16 weeks, and 25 to 27 weeks. Forty-eight hours after the injection of PMSG, the mice were injected with 5 IU of human chorionic gonadotropin (hCG, Sigma) and then immediately paired with an individual male. On the morning of the following day, the mice were inspected and those with a confirmed vaginal plug Fertility and Sterility
were considered to be fertilized; these mice were used in the experiment. Mice of the same age injected with PMSG without SNP were used as the control group. One-Cell Embryos Collection and Embryo Culture Female mice with a confirmed vaginal plug were killed by cervical dislocation 18 hours after the hCG injection, and the one-cell embryos were retrieved from the oviductal ampulae. Cumulus-enclosed one-cell embryos were collected and denuded by incubation for 1 minute with 1 mg/mL hyaluronidase (Sigma) in Dulbecco’s phosphate buffer saline (dPBS; GIBCO BRL, Grand Island, NY). One-cell embryos were pooled and washed three times in human tubal fluid (HTF) media supplemented with 10% human follicular fluid (hFF). Only healthy zygotes were cultured in 30-mL drops of media (HTF þ 20% hFF) under paraffin-oil at 37 C in a 5% CO2 incubator for 4 days, and the media was changed daily. The number of one-cell embryos retrieved and fragmented per mouse was counted, and embryo development to blastocyst stage was evaluated. Western Blot Analysis Proteins were extracted by mechanical homogenization of ovaries in the presence of 200 mL ice-cold lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40,1mM EDTA). The protein content of cell lysate was determined with Bradford reagent (Bio-Rad, Hercules, CA) using bovine serum albumin (BSA) as the standard. We separated 20 mg of cell lysate by SDS-PAGE and transferred to PVDF (immobilon-P) membrane (Millipore, Bedford, MD). The transfer was performed at a constant voltage of 15 V for 30 minutes. For Western blotting, the membrane was incubated antimouse VEGF (R&D Systems, Minneapolis, MN) in TBS (Sigma) containing 1% skim milk for 4 hours at room temperature. After washing three times with TBS-T (containing 0.04% Tween-20), the blotted membranes were incubated with horseradish peroxidase–conjugated goat antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 minutes at room temperature. After washing three times with TBS-T, the proteins were visualized by the enhanced chemiluminescence detection system according to the recommended procedure (Amersham Pharmacia Biotech, Piscataway, NJ). Immunnohistochemistry Serial sections (4 mm) of formalin-fixed, paraffin-embedded ovary tissues were spread on the coated-slides, and placed in an oven at 60 C for 1 hour. The slides then were deparaffinized in xylene and dehydrated in a graded series of ethanol. An immunohistochemistry for VEGF was performed with streptavidin-biotin-peroxidase complex method using rabbit antimouse VEGF polyclonal antibody (Lab Vision, Fremont, CA). The endogenous peroxidase was quenched with 0.3% hydrogen peroxide at room temperature for 5 minutes, and then the tissues were rinsed four times for 5 minutes each time in TBS. The samples were incubated with normal serum, then incubated with rabbit antimouse VEGF polyclonal 1515
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SNP improves ovarian response in older females
96 (32.8) 80 (31.9) 85 (30.7) 148 (50.5) 125 (49.8) 123 (44.6) 161 (54.9) 132 (52.5) 131 (47.5) 168 (57.3) 136 (54.2) 143 (51.8) 293 251 276 Lee. SNP improves ovarian response in older females. Fertil Steril 2008.
21.3 17.9 20.2
4-cell (%) 2-cell (%) Zygotes cultured Zygotes flushed/mouse Zygotes fragmented (%)
28 (8.7) 18 (6.7) 27 (8.9) 321 269 303 15 15 15 0 mM control 1 mM 10 mM
The results showed that there was no difference in VEGF expression in the mice aged 16 weeks who were not affected by SNP treatment. However, VEGF expression in mice aged 26 weeks, who had shown a beneficial effect with SNP
TABLE 1
The beneficial effect of SNP disappeared in mice aged 6 to 9 weeks. As shown in Table 3, the number of one-cell embryos flushed and the embryo development rate were similar, regardless of SNP treatment. Consequently, we used Western blot and immunohistochemistry to evaluate VEGF expression in ovaries of mice aged 26 weeks to find out if the effect of SNP treatment observed in old aged mice of 25 to 27 weeks was related to the expression of VEGF in their ovaries. Mice aged 16 weeks that had no confirmed positive effect with SNP were used for comparison of VEGF expression with the mice aged 26 weeks.
Zygotes flushed
We treated mice aged 25 to 27 weeks with the same doses of SNP. The number of one-cell embryos flushed was 21.7 with 1 mM SNP and 20.2 with 10 mM SNP, which was statistically significantly higher compared with the control group (15.2; P<.05). The embryo development rate to blastocyst was statistically significantly increased with SNP treatment (1 mM and 10 mM: 36.7%, 46.8%, respectively) when compared with the control group (5.5%; P<.05). A noticeably larger number of developing embryos were arrested in the control group at the two-cell or four-cell stage (Table 2).
Mice provided
RESULTS To investigate whether the stimulation of ovarian angiogenesis affects the number of oocytes and embryo development, 1 mM and 10 mM of SNP were co-injected with PMSG during superovulation into three age groups: 6 to 9 weeks, 14 to 16 weeks, and 25 to 27 weeks. First, we administered SNP to mice aged 14 to 16 weeks. The number of one-cell embryos flushed was 17.9 with 1 mM SNP, 20.2 with 10 mM SNP, and 21.3 with the control group. However, there were no statistically significant differences in the number of one-cell embryos flushed or the embryo development rate (Table 1).
SNP
Statistical Analysis All data were presented as mean standard deviation. Statistical analysis was performed using the unpaired Student’s t-test and chi-square test. P<.05 was considered statistically significant.
Effects of sodium nitroprusside (SNP) treatment on embryo development in mice aged 14–16 weeks.
Morula (%)
Blastocyst (%)
antibody at a dilution of 1:70 in PBS/BSA overnight at 4 C. After four washings with TBS of 15 minutes each, the samples were incubated with biotinylated secondary antibody (e-Bioscience, San Diego, CA) for 30 minutes at room temperature and washed three times. Then, the samples were incubated with streptavidin-peroxidase conjugate in PBS for 30 minutes at room temperature and incubated with DAB (3.30 diaminibenzidine chromogen; Sigma). Counterstaining was performed with Mayer’s hematoxylin. The results were assessed by one pathologist under a light microscope.
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11 (5.5) 101 (36.7)a 126 (46.8)a
DISCUSSION This study shows for the first time that the SNP treatment during the superovulation process improves the number of oocytes recruited and ovulated and the oocyte developmental competence in aged female, possibly via an increase of VEGF in the ovary. The sexual maturity and lifespan in the laboratory for mice is around 6 weeks and 1 year, respectively. Considering the reproductive physiology of mice, 6 to 9 weeks, 14 to 16 weeks, and 25 to 27 weeks in mice may be comparable with human teenage years, R30 years of age, and R40 years of age in humans, respectively. Furthermore, oocytes collected from 30-to-40-week-old mice are more developmentally sensitive to mitochondrial damage than oocytes from pubertal mice, resulting in significant decline of oocyte competence (8). In this respect, we think that the severe loss of fertility potential in mice over 30 weeks makes them inappropriate for evaluating the age-related decline of the number of follicles ovulated and the developmental competence of oocytes.
228 325 303 a
P< .05 (vs. control).
15 15 15 0 mM control 1 mM 10 mM
Lee. SNP improves ovarian response in older females. Fertil Steril 2008.
15 (7.6) 154 (55.8)a 158 (58.7)a 25 (12.6) 159 (57.6)a 165 (61.4)a 198 276 269
Immunohistochemistry was performed to evaluate the localized VEGF expression in the ovary induced by SNP treatment in mice aged 26 weeks; Western blot confirmed the increase in VEGF expression. The VEGF expression was usually localized in granulosa cells, stromal cells, and endothelial cells, and expression was increased after the SNP treatment. The intensity of VEGF expression was the highest in the mice treated with 10 mM of SNP; the control group showed the lowest expression of VEGF (Fig. 2). These results suggest that SNP treatment induces an increase of VEGF expression in ovarian cells.
30 (13.2) 49 (15.1) 34 (11.2)
15.2 21.7 20.2
43 (21.7) 194 (70.3)a 177 (65.8)a
Blastocyst (%) Morula (%) 4-cell (%) 2-cell (%) Zygotes cultured Zygotes flushed/mouse Zygotes fragmented (%) Zygotes flushed Mice provided SNP
Effects of sodium nitroprusside (SNP) treatment on embryo development in mice aged 25–27 weeks.
TABLE 2
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treatment, also showed a remarkable increase in a dosedependent manner (Fig. 1).
Angiogenesis is initiated early in the follicular development and plays an important role in many aspects of female reproductive physiology that require neovascularization, such as follicular growth, selection of dominant follicles, inhibition of follicular atresia, and corpus luteum formation (9, 25). Gaulden (26) proposed that a deficient microvasculature develops around the dominant follicles with aging, resulting in the hypoxia of follicles. Friedman et al. (27) reported that the increase VEGF follicular fluid concentrations in women of advanced reproductive age are due to relative hypoxia in ovarian follicles. Pellicar et al. (28) observed reduced blood flow during natural cycles around the dominant follicle in poor responders compared with controls, which suggested that women with a response to exogenous gonadotropins typical for women of advanced reproductive age have a deficient follicular microcirculation even if the issue of age is not directly addressed. In this respect, infertility such as age-related decline of ovarian response seems to be associated with inadequate follicular development that results from disturbance of follicular angiogenesis. 1517
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A critical regulator of ovarian angiogenesis is VEGF, which has the most potent angiogenic activity and vascular permeable activity (30). It plays an important role in the normal function of the ovary during follicular development (31, 32). Intrabursal injection of VEGF in the rat ovary significantly increased the number of primary and small secondary but not large secondary and preantral follicles and stimulates preantral follicle development. This result suggests that VEGF may be one of the factors that participates in the regulation of early follicular growth (14). Nitric oxide is another angiogenic factor and vascular permeable factor during ovarian angiogenesis (33, 34), and it forms a vital component of the oocyte microenvironment during folliculogenesis, ovulation, embryo development, and implantation (20–22, 35, 36). In addition, NO mediates the increased vascular permeability via VEGF (23). Due to this property, our study injected SNP during the superovulation process as a factor that may increase ovarian response to gonadotropins by promoting ovarian angiogenesis via the stimulation by VEGF. As we expected, the SNP treatment increased the expression of VEGF in the ovary.
Lee. SNP improves ovarian response in older females. Fertil Steril 2008.
265 (75.1) 250 (74.0) 246 (73.4) 301 (85.3) 292 (86.4) 282 (84.2) 314 (89.0) 299 (88.5) 288 (86.0) 353 338 335 24.2 23.3 23.1 364 140 346 0 mM control 1 mM 10 mM
15 15 15
11 (3.0) 12 (3.6) 11 (3.2)
319 (90.4) 306 (90.5) 300 (89.5)
Blastocyst (%) Morula (%) 4-cell (%) 2-cell (%) Zygotes cultured Zygotes flushed/mouse Zygotes fragmented (%) Zygotes flushed Mice provided SNP
Effects of Sodium nitroprusside (SNP) treatment on embryo development in mice aged 6–9 weeks.
TABLE 3 1518
Fraser (29) suggested that the supply of appropriate blood vessels and the maintenance of vascular permeability in the ovaries, including to follicles, are necessary for gonadotropins to have an adequate effect and for paracrine factors to sustain follicular growth and ovulation. Therefore, it is thought that the age-related decline of ovarian response or oocyte quality can be reversed if ovarian angiogenesis is stimulated in such a way that age-related reduction of ovarian blood flow is overcome.
Sodium nitroprusside has a short half life, and co-injection of SNP with PMSG at the starting time of superovulation may increase VEGF expression in early follicle. The VEGF stimulates the number of primary and small secondary follicles and preantral follicular development (14). In particular, immunohistochemical staining showed that most graafian follicles were ovulated by hCG injection, and the remaining follicles were early immature follicles. These results suggest that SNP treatment appears to enhance folliculogenesis rather than ovulatory capacity. As female age advances, the aging of the ovaries has been known to result in the gradual reduction of the number of primordial follicles and dominant follicles responding to gonadotropins, and finally the decline of oocyte quality (37). The number of follicles growing to the antral stage also reduces with age (38, 39). The number of primordial follicles steadily decreases throughout a female’s reproductive lifespan as a result of atresia and is finally depleted in menopause. We would like to postulate that the reduction of the number of follicles to be recruited and ovulated in aged females may result from the inactivation of the mechanism for the assembly of primordial follicles and the transition from primordial follicle to primary follicle rather than the depletion of primordial follicle pool. Locally produced factors including kit ligand, leukemia inhibitory factor, and keratinocyte growth
SNP improves ovarian response in older females
Vol. 89, Suppl 3, May 2008
FIGURE 1 Western blot analysis of vascular endothelial growth factor (VEGF) expression in the ovaries of different aged mice. The ovaries were isolated just after the flushing of one-cell embryos. (A) 16-week-old mice. (B) 26-week-old mice. Lane 1: PMSG alone (control). Lane 2: 1 mM SNP þ PMSG. Lane 3: 10 mM SNP þ PMSG.
Lee. SNP improves ovarian response in older females. Fertil Steril 2008.
factor are involved in the promotion of primordial to primary follicle transition (40). Furthermore, one report has suggested that the potential presence of a female germ-line stem cell population may provide a continual supply of primordial follicles (41). Thus, the primordial follicle pool may not be finite and may regenerate in an appropriate milieu. As shown in our study, the number of oocytes recruited from SNP-treated older mice is similar to those recruited from younger mice. It seems possible that an active blood
supply through the increase of VEGF expression prevents the follicles from atresia (42). Indeed, when the number of antral follicles was estimated to predict the ovarian reserve, the number varied in each cycle (43). These facts imply that the number of follicles to be recruited and ovulated can be different, depending on the ovarian stimulation method, even though they are in the same pool of primordial follicles. Therefore, it is possible if an adequate ovarian stimulation is given to increase ovarian angiogenesis in an older female, an efficient number of follicles develop to dominant follicles.
FIGURE 2 Immunohistochemistry of vascular endothelial growth factor (VEGF) expression in ovaries of mice aged 26 weeks. The ovaries were isolated just after the flushing of one-cell embryos. Immunostaining with anti-VEGF antibody was usually localized in granulosa cells (Gr), stromal cells (St), and endothelial cells (Ec). The immunostaining intensity was increased after sodium nitroprusside (SNP) treatment. (A) PMSG alone (control). (B) 1 mM SNP þ PMSG. (C) 10 mM SNP þ PMSG (100).
Lee. SNP improves ovarian response in older females. Fertil Steril 2008.
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The exact mechanism for the positive effect of SNP shown in aged mice remains to be elucidated. However, several possibilities can be considered. First, NO synergistically activates ovarian angiogenesis by stimulating VEGF expression. Indeed, our study showed that the SNP treatment increases VEGF expression as well as the number of oocytes and the embryo development rate. Second, the vasoactive and angiogenic activity of NO itself can have an effect on the preferential delivery of gonadotropins to the follicles. Third, exposure to NO delays oocyte aging and improves the integrity of the microtubular spindle apparatus as an antioxidant (44). Aged oocytes have a significantly increased likelihood of failed or abnormal fertilization and/or development, eventually resulting in a atresia and fragmentation. Fourth, as the possibility cannot be excluded that NO induces other factors such as matrix metalloproteinase 9 (MMP9) to stimulate neovascularization of follicles, further study is needed. In general, VEGF protein is initially absent from the granulose cells of primordial follicles and preantral follicles but becomes evident in the theca cells of antral follicles and in the granulose cells nearest in the preovulatory follicles. Vascular endothelial growth factor is expressed not only in follicles but also in the stroma of the ovary, which provides nutritional support for primordial and primary follicles (12, 29). However, in our study, VEGF expressed in granulose cells, stroma cells, and endothelial cells was increased after SNP treatment. Also, SNP treatment resulted in the increased VEGF expression in theca cells. It seems that this increased VEGF expression provides nutritional support for early follicles to be developed and ovulated. However, additional study is necessary concerning the period of VEGF expression after SNP treatment. Our study provides interesting, beneficial data toward improving assisted reproduction in age-related infertility, but we did not examine whether the SNP treatment enhances the development to blastocyst stage in vivo and the implantation rate. However, the finding that zygotes collected from SNP-injected mice increased blastocyst formation rate in vitro suggests that the same effect may occur in vivo. Therefore, further study is needed to contribute to assisted reproductive technologies. Our study shows for the first time that co-treatment of SNP with gonadotropins in aged female mice of 25 to 27 weeks during the superovulation process increases ovarian response and oocyte developmental competence at a similar level to that of pubertal mice. This beneficial effect of SNP seems to be associated with increased VEGF expression, which may result in enhanced angiogenesis during follicular development. In addition, this result implies that age-related decline of fertility might be attributable to a deterioration of oocyte quality resulting from decreased ovarian angiogenesis. This is an interesting finding in the respect that it provides a possibility that, if an adequate ovarian stimulation protocol for stimulating angiogenesis is given to the older female, efficient numbers of oocytes with a good quality and normal 1520
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embryo developmental potential can be produced. However, NO has diverse effects, depending on its dose. Therefore, before using NO clinically for ovulation induction in older women, further studies are needed to develop local or systemic administration methods and appropriate dosages without side effects.
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