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Inducible and endothelial nitric oxide synthase: genetic background affects ovulation in mice
FERTILITY AND STERILITY威 VOL. 77, NO. 1, JANUARY 2002 Copyright ©2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
REPRODUCTIVE BIOLOGY
Inducible and endothelial nitric oxide synthase: genetic background affects ovulation in mice Lukas A. Hefler, M.D.,a and Anthony R. Gregg, M.D.a,b Baylor College of Medicine, Houston, Texas
Received December 8, 2000; revised and accepted March 28, 2001. Supported in part by Erwin-SchroedingerAuslandsstipendium J1839-MED with funding by the Fonds zur Foerderung der Wissenschaftlichen Forschung (L.A.H.). Reprint requests: Anthony R. Gregg, M.D., Baylor College of Medicine, Department of Obstetrics and Gynecology, 6550 Fannin, Smith Tower—Suite 901A, Houston, Texas 77030 (FAX: 713-790-0108; E-mail: [email protected]. edu). a Department of Obstetrics and Gynecology. b Department of Molecular and Human Genetics. 0015-0282/02/$22.00 PII S0015-0282(01)02952-1
Objective: Inducible nitric oxide synthase (NOS) and endothelial NOS are involved in female reproductive physiology. We sought to investigate the influence of the inducible (Nos2) and endothelial (Nos3) NOS genes as a function of genetic background on ovulatory capacity and early embryonic development in a mouse model. Design: Observational study of genetically altered mice and their response to a superovulation protocol. Setting: Academic research institution. Animals: Wild-type mice and mice deficient for Nos2 or Nos3 were bred to C57BL/6J and 129/Sv genetic backgrounds. Intervention(s): Superovulation protocol, oocyte culture. Main Outcome Measure(s): Number of oocytes harvested, early embryonic development of zygotes, evaluation of ovarian histology. Result(s): The mean number of oocytes was significantly reduced in Nos3 deficient mice on a C57BL/6J background compared with controls. Oocytes deficient for Nos3 on a C57BL/6J background also showed reduced progression to two-cell stage embryos after 24 hours, two-cell stage embryos to blastocyst stage embryos, and survival to 48 hours. Those effects were distinctly absent in mice deficient for Nos3 on a 129/Sv background and in mice deficient for Nos2 on either genetic background. Conclusion(s): Our data show that disruption of Nos2 had no effect on ovulation in our mice. The negative effect of Nos3 deficiency on ovulatory capacity and early embryonic development is modulated by genetic background. This suggests a role for strain-specific modifier genes in these processes. (Fertil Steril威 2002;77: 147–51. ©2002 by American Society for Reproductive Medicine.) Key Words: Nitric oxide, Nos2, Nos3, ovulation, genetic background
The free radical nitric oxide (NO) is synthesized during the conversion of L-arginine to L-citrulline by the enzyme nitric oxide synthase (NOS) and is known to mediate a wide variety of physiologic functions, including neurotransmission, immune cell cytotoxicity, and regulation of the vascular tone (1–3). Three isoforms of NOS have been identified: endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) (2). In the rodent ovary, mRNA and protein expression of eNOS and iNOS, but not nNOS, has been demonstrated (4, 5). During follicular development, iNOS expression was found within granulosa cells of secondary follicles, small antral follicles, the theca cell layer, and ovarian stroma. eNOS protein was localized to blood vessels in the ovary and also to the theca
cell layer, to ovarian stroma, to the surface of oocytes, and within the corpus luteum (4, 5). After gonadotropin-induced ovulation, iNOS expression significantly decreased and was only present in the external layers of the developing corpus luteum (4 – 6). eNOS mRNA levels, however, increased and peaked in ovaries containing ovulatory follicles before declining in the luteal phase (5). Results in eNOS-deficient mice are consistent with the idea that the NO plays a physiological role in female reproduction, including pubertal maturation, estrous cyclicity, ovulation rate, early embryonic development of oocytes, and timing of menopause (7–10). The use of targeted mutagenesis in the study of gene function, however, is not without recognized pitfalls. One example is the variation in 147
phenotype as a function of genetic background or strain (11, 12). Mice with the same genetically engineered mutation can exhibit a unique phenotype on one strain but not another (13, 14). The importance of taking into account genetic background or strain and the importance of genes capable of modifying phenotypes has been stressed (11, 14). Controlling for strain-specific modifier genes, by backcrossing over several generations to obtain an almost pure genetic background, has been proposed (15). The aim of our study was to elucidate the effect of the Nos2 and Nos3 genes, encoding for iNOS and eNOS, respectively, on ovulatory capacity and early embryonic development in a mouse model. To control for possible confounding factors of background genes, we performed all our experiments on a ⬎98% pure incipient congenic genetic background of C57BL/6J and 129/Sv.
MATERIAL AND METHODS Mice and Animal Husbandry All animal experiments were approved by the internal review board at Baylor College of Medicine. Animal husbandry practices followed guidelines established by the Animal Care Committee of Baylor College of Medicine. Animals were subjected to daily 12-hour alternating periods of light and dark cycles within a humidity- and temperaturecontrolled environment. Food and water were provided ad libitum. F1 generation Nos2-deficient mice (Nos2⫺/⫺; Merck Research Laboratories, Rahway, NJ) (16) were backcrossed by natural breeding over eight generations with C57BL/6J (B6) to generate wild-type and Nos2-deficient mice on this background (B6-Nos2⫹/⫹ and B6-Nos2⫺/⫺, respectively). This was repeated similarly using the 129/Sv (129) strain to generate wild-type and Nos2-deficient mice on this background (129-Nos2⫹/⫹ and 129-Nos2⫺/⫺, respectively). Nos3-deficient mice were previously generated in our laboratory (17) and were also backcrossed by natural breeding over eight generations with C57BL/6J and 129/Sv mice to generate wild-type and Nos3-deficient mice on these strains (B6-Nos3⫹/⫹, B6-Nos3⫺/⫺, 129-Nos3⫹/⫹, and 129Nos3⫺/⫺, respectively). Thus, incipient congenic mice on a C57BL/6J and 129/Sv background were generated.
Superovulation Protocol Prepubescent (22-day-old to 26-day-old) congenic female mice, either wild type or homozygous deficient (B6Nos3⫹/⫹, B6-Nos3⫺/⫺, 129-Nos3⫹/⫹, and 129-Nos3⫺/⫺), were superovulated by intraperitoneal (IP) injection of 7.5 IU pregnant mare serum (Organon, West Orange, NJ), followed 48 hours later by an IP injection of 7.5 IU hCG (Sigma). Superovulated females were mated to a samestrain, randomly selected male that was either wild type or homozygous deficient for Nos2 or Nos3. This was done to obtain ⫹/⫹, ⫹/⫺, and ⫺/⫺ zygotes for comparison. Ran148
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dom and blinded matings between homozygous-deficient and wild-type mice allowed for zygotes with wild-type, heterozygous, or homozygous-deficient genotypes for Nos2 and Nos3, respectively. The investigator was not aware of any genotype while performing all experiments. Each stud male had sired at least one litter previously. A copulatory plug was identified, and mice were killed 18 hours after hCG administration. Oviducts were harvested and placed into M-2 medium (Cell & Molecular Technologies, Phillippsburg, NJ) containing 300 U/mL of hyaluronidase type IV from bovine testes (Sigma). Oviducts were incised, and the ovulated oocyte-cumulus complex was removed from the ampulla. Oocytes were mouth pipetted through three cycles of washing in M2 media using a glass micropipette. After washes, oocytes were placed in KSOM media (Cell & Molecular Technologies) and counted. Oocytes were then incubated at 37°C, 5% CO2. The number of oocytes and the number of zygotes that developed to two-cell and blastocyst stages were recorded after 24 and 48 hours, respectively. We defined “failure to ovulate” as no oocytes found in the oviducts at the time that the mouse was killed. The investigator (L.A.H.) performing oocyte counting was blinded to male and female mouse genotypes.
Genotyping For genotyping of Nos2-deficient mice, a polymerase chain reaction (PCR)– based strategy was used as described previously (16). Mouse genomic DNA for genotyping was obtained from Proteinase K– digested tails using phenolchloroform extraction. Polymerase chain reaction amplification with oligonucleotide primers flanking the Nos2 gene was performed. Polymerase chain reaction conditions comprised an initial denaturing step at 92°C for 5 minutes, followed by 30 cycles of 92°C for 45 seconds, 62°C for 45 seconds, and 72°C for 45 seconds and a final extension at 72°C for 5 minutes. The wild-type allele generated a 413-bp band; the mutant allele generated a 1,288-bp band. PCR products were resolved on a 1% agarose gel and stained with ethidium bromide. Nos3-deficient mice were genotyped by Southern blotting as reported elsewhere (17). Briefly, mouse genomic DNA for genotyping was obtained from Proteinase K– digested tails using phenol-chloroform extraction. Genomic DNA was digested using the restriction enzyme EcoRI (Boehringer Mannheim, Indianapolis, IN). Equal amounts of DNA (15 g) were electrophorized on a 1% agarose gel, denatured in 0.4 M NaOH for 45 minutes, and transferred overnight onto nucleic acid transfer membranes (Amersham, Piscataway, NJ) using 10⫻ SSC (0.75 M sodium chloride and 0.075 M sodium citrate, pH 7.0; Fisher Scientific, Fairlawn, NJ). The membranes were prehybridized at 64°C in a hybridization buffer containing 0.5 M of sodium phosphate (Fisher Scientific), 1 mM of ethylenediaminetetraacetic acid (J.T.
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Baker, Inc, Phillipsburg, NJ), 7% sodium dodecyl sulfate (Boehringer Mannheim), 10% PEG 8000 (Fisher), 1 g/mL of bovine serum albumin (fraction V; Sigma Corp; St. Louis; MO), and 100 g/mL of denatured salmon sperm DNA (Sigma). After 6 hours, Southern blots of genomic DNA were hybridized at high stringency with a 510-bp probe of mouse Nos3 cDNA, which had been labeled using randomprimers [␣-32P] dCTP. After hybridization for 18 hours at 64°C, membranes were washed sequentially at 64°C for 20 minutes with 5⫻ SSC, 2⫻ SSC, 1⫻ SSC, 0.5⫻ SSC, and 0.1% sodium dodecyl sulfate (Sigma), with two cycles for each stringency. The membranes were air-dried, wrapped in plastic wrap, and exposed to an autoradiography film (Amersham) for 48 hours.
FIGURE 1 Mean number of oocytes broken down by genotype and strains. Each bar with the depicted standard deviation represents one genotype on one genetic background. *A significant difference between groups (P ⬍.05).
Genotypes were determined by visual inspection of autorads. In wild-type mice, the EcoRI restriction fragment length polymorphism has a size of 9.8 kb. In Nos3⫺/⫺ mice, the EcoRI restriction fragment is 4.8 kb because of the deletion event. This difference was used to distinguish between the mutant and the wild-type allele. Hefler. NOS deficiency and ovulation in mice. Fertil Steril 2002.
Histology of Ovaries and Follicle Assessment Ovaries of superovulated females were harvested at the same time as oviducts, formalin fixed, and paraffin embedded. Routine hematoxylin– eosin staining was performed (n ⫽ 5 for each genotype and mouse strain). Primary follicles, secondary follicles, small antral follicles, large preovulatory antral follicles, and corpora lutea were identified. Small antral and large preovulatory follicles were examined.
Statistical Analysis The statistical software Sigma Stat version 2.0 (Jandel Scientific, San Rafael, CA) was used for statistical analysis. Data are presented as mean ⫾ standard deviation (SD) of the mean. After checking for normality, comparisons between groups were made by one-way ANOVA with Tukey test as multiple comparison procedure and 2 tests where appropriate. Significance was assumed at P ⬍.05.
RESULTS We subjected 132 mice to the superovulation protocol. Failure of ovarian superovulation was noted in 20 cases, independent of strain or genotype. We only analyzed ovulatory capacity for mice that responded to the superovulation protocol: B6-Nos2⫹/⫹ (n ⫽ 13), B6-Nos2⫺/⫺ (n ⫽ 14), 129-Nos2⫹/⫹ (n ⫽ 11), 129-Nos2⫺/⫺ (n ⫽ 12), B6-Nos3⫹/⫹, (n ⫽ 14), B6-Nos3⫺/⫺, (n ⫽ 10), 129-Nos3⫹/⫹ (n ⫽ 23), and 129-Nos3⫺/⫺ (n ⫽ 15). The mean number of oocytes, broken down by genotype and strains is shown in Figure 1. The ovulatory capacities of homozygous-deficient mice were compared with those of their respective wild-type controls. The ovulatory capacity of B6-Nos3⫺/⫺ mice was significantly reduced compared with that of wild-type B6Nos3⫹/⫹ mice (P ⫽.035). Interestingly, Nos3 deficiency on FERTILITY & STERILITY威
a 129/Sv background and Nos2 deficiency on either background had no effect on the number of oocytes counted. Of note, wild-type mice, (i.e., of B6-Nos2⫹/⫹, 129-Nos2⫹/⫹, B6-Nos3⫹/⫹, and 129-Nos3⫹/⫹) showed no differences in the number of ovulated oocytes (P ⫽.3). The percentage of oocytes progressing to the two-cell stage after 24 hours, the percentage of two-cell stage embryos progressing to the blastocyst stage after 48 hours, and overall survival of harvested oocytes, broken down by genotype and strain, is shown in Table 1. Of note is that genotypes in the table refer to genotypes of the embryo. The ratio of oocytes progressing to two-cell–stage embryos after 24 hours, the ratio of two-cell–stage embryos progressing to blastocyst-stage embryos after 48 hours, and survival of embryos from harvest to 48 hours was significantly reduced in B6-Nos3⫺/⫺ oocytes compared with the case of B6Nos3⫹/⫺ or B6-Nos3⫹/⫹ oocytes, respectively. No differences were found among any of the other groups. Histologic sections from ovaries of superovulated females were examined while the investigator was blinded to genotype and strain. No obvious structural or morphological differences between sections from any of the mouse strains or genotypes was identified.
DISCUSSION Our data are consistent with a role for eNOS in the regulation of ovulatory function and early embryonic development in mice. We observed this to be a function of genetic background suggesting that modifier genes are involved in these processes. Absence of functional iNOS does not seem 149
TABLE 1 Early embryonic development. Strain–genotype
Oocytes harvested (n)
Two-cell stage embryos per no. of oocytes harvested at 24 h (%)
Blastocysts per two-cell stage embryos at 48 h (%)
Blastocysts per no. of oocytes harvested at 48 h (%)
241 299 217 298 346 362 246 346 126 439 410 198
48.5 37.9 40.1 14.5 24.3 22.4 26.8a 33.9b 12.7a,b 29.0 40.7 35.7
66.1 80.8 92.2 72.5 73.4 43.5 100b 77.2a 34.6a,b 65.4 39.5 53.4
48.5 30.3 35 14.2 20.6 24.7 33.3c 26.2b 9.2b,c 21.4 16.1 18.6
B6-Nos2⫹/⫹ B6-Nos2⫹/⫺ B6-Nos2⫺/⫺ 129-Nos2⫹/⫹ 129-Nos2⫹/⫺ 129-Nos2⫺/⫺ B6-Nos3⫹/⫹ B6-Nos3⫹/⫺ B6-Nos3⫺/⫺ 129-Nos3⫹/⫹ 129-Nos3⫹/⫺ 129-Nos3⫺/⫺
Note: t-test comparisons are for B6-NOS3⫹/⫹, B6-NOS3⫹/⫺, and B6-NOS3⫺/⫺. Symbols represent significance level achieved for each comparison. a P ⬍.05. b P ⬍.01. c P ⬍.001. Hefler. NOS deficiency and ovulation in mice. Fertil Steril 2002.
to impair ovulation in response to superovulatory protocols among either C57BL6/J or SV129 strains of mice. The expression of iNOS and eNOS in the rodent ovary and the role of NO in ovulation and oocyte development is abundant. The physiological pathways, however, allowing NO to influence ovarian function are still hypothetical. A concept has been put forward describing the ability of NO to mediate the passage of inter-␣-trypsin inhibitor protein through the blood–follicle barrier. This event is thought to be critical for stabilizing the extracellular matrix of the cumulus in mature follicles (18). Nitric oxide derived from iNOS stimulated by interleukin-1 was found to be an important mediator of cell death and to act as a physiologically mediator of tissue remodeling events that occur in vivo during the ovulatory process (19). Another hypothesis considers endothelium-derived, NOdependent local vasodilatation as a trigger of hemodynamic changes associated with ovulation, for instance, increased ovarian blood flow, follicular hyperemia, and edema of the theca interna and externa (20). A deficiency of endotheliumderived NO might interfere with adequate local blood supply, secondary to prolonged or intense vasoconstriction or thromboembolic events. This may impair follicular recruitment and/or the process of ovulation itself. Our data confirm previously obtained results (10) indicating that eNOS deficiency on a C57BL/6J genetic background is associated with reduced ovulatory potential after exposure to a superovulation protocol. However, this observation is clearly straindependent. Furthermore, our data are in accordance with previously published data (21, 22) indicating the prominent role of the endothelial-derived isoform of NOS; in other words, eNOS, compared with iNOS in ovulatory processes. 150
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Elsewhere, eNOS was shown to be involved in meiotic maturation and oocyte development in vitro. To assess effects of NO deficiency on the kinetics of germinal vesicle breakdown, nuclear morphology of oocytes was evaluated (8, 10, 23). Absence of endothelial-derived NO was associated with an impaired oocyte meiotic maturation in vitro (23). These results support previous observations in vivo, indicating that endothelial-derived NO has independent functions in oocyte maturation and development (8). Furthermore, NO was indirectly linked, via a cGMP pathway, to an important regulator of early embryonic development and differentiation (24). In our series, eNOS deficiency was associated with decreased ratios of oocytes progressing to two-cell stage embryos after 24 hours, two-cell stage embryos progressing to blastocyst stage after 48 hours, and overall survival of embryos. As pointed out, these observations were strain dependent. Of note, we did not assess whether all oocytes were recovered mature by evaluating germinal vesicle breakdown and presence of polar bodies in accordance with a previously published study (10). Furthermore, we cannot prove that all recovered oocytes were fertilized. Thus, we cannot exclude that oocytes that did not develop to two-cell stage or blastocyst stage were not fertilized or immature. This has to be kept in mind when interpreting the results of our study. It is interesting to consider the functional significance of reduced blastocyst survival on overall fecundity among B6Nos3⫺/⫺ mice. Despite the statistically significant reduction in numbers, an adequate number of apparently functional blastocysts seems available for normal implantation and a normal litter size. In fact, F1 generation eNOS-deficient mice were previously shown to produce litters of expected
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size (17). This leaves open the significance of the role played by eNOS when ovulation occurs outside of superovulatory protocols. Our data support the existing evidence that eNOS plays a role in ovulation and the subsequent development of the fertilized egg. We have demonstrated the importance of mouse strain as it applies to NOS genes. Because these processes are complex and appear to be regulated by multiple genes, special consideration should be given to the genetic background when investigating ovulation and the response to ovulation using in vivo models.
9. 10. 11. 12. 13. 14. 15. 16.
Acknowledgment: F1 generation Nos2-deficient mice were generously supplied by J. S. Mudgett of Merck Research Laboratories (Rahway, NJ).
References 1. Lowenstein CJ, Dinerman JL, Snyder SH. Nitric oxide: a physiologic messenger. Ann Intern Med 1994;120:227–37. 2. Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med 1993;329:2002–12. 3. Vane JR, Anggard EE, Botting RM. Regulatory functions of the vascular endothelium. N Engl J Med 1990;323:27–36. 4. Jablonka-Shariff A, Olson LM. Hormonal regulation of nitric oxide synthases and their cell-specific expression during follicular development in the rat ovary. Endocrinology 1997;38:460 – 8. 5. Van Voorhis BJ, Moore K, Strijbos PJ, Nelson S, Baylis SA, Grzybicki D, et al. Expression and localization of inducible and endothelial nitric oxide synthase in the rat ovary. Effects of gonadotropin stimulation in vivo. J Clin Invest 1995;96:2719 –26. 6. Srivastava V, Jones BJ, Dookwah H, Hiney JK, Dees WL. Ovarian nitric oxide synthase (NOS) gene expression during peripubertal development. Life Sci 1997;61:1507–16. 7. Drazen DL, Klein SL, Burnett AL, Wallach EE, Crone JK, Huang PL, et al. Reproductive function in female mice lacking the gene for endothelial nitric oxide synthase. Nitric Oxide 1999;3:366 –74. 8. Jablonka-Shariff A, Olson LM. The role of nitric oxide in oocyte meiotic maturation and ovulation: meiotic abnormalities of endothelial
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nitric oxide synthase knock-out mouse oocytes. Endocrinology 1998; 139:2944 –54. Jablonka-Shariff A, Ravi S, Beltsos AN, Murphy LL, Olson LM. Abnormal estrous cyclicity after disruption of endothelial and inducible nitric oxide synthase in mice. Biol Reprod 1999;61:171–7. Tempfer C, Moreno RM, O’Brien WE, Gregg AR. Genetic contributions of the endothelial nitric oxide synthase gene to ovulation and menopause in a mouse model. Fertil Steril 2000;73:1025–31. Gerlai R. Gene-targeting studies of mammalian behavior: is it the mutation or the background genotype? Trends Neurosci 1996;19:177– 81. Sigmund CD. Viewpoint: are studies in genetically altered mice out of control? Arterioscler Thromb Vasc Biol 2000; 20:1425–9. Harvey M, McArthur MJ, Montgomery CA Jr, Bradley A, Donehower LA. Genetic background alters the spectrum of tumors that develop in p53-deficient mice. FASEB J 1993;7:938 – 43. Phillips TJ, Hen R, Crabbe JC. Complications associated with genetic background effects in research using knockout mice. Psychopharmacology (Berl) 1999;147:5–7. Doetschman T. Interpretation of phenotype in genetically engineered mice. Lab Anim Sci 1999;49:137– 43. MacMicking JD, Nathan C, Hom G, Chartrain N, Fletcher DS, Trumbauer M, et al. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 1995;81: 641–50. Gregg AR, Schauer A, Shi O, Liu Z, Lee CG, O’Brien WE. Limb reduction defects in endothelial nitric oxide synthase-deficient mice. Am J Physiol 1998;275:H2319 –24. Powers RW, Chen L, Russell PT, Larsen WJ. Gonadotropin-stimulated regulation of blood-follicle barrier is mediated by nitric oxide. Am J Physiol 1995;269:E290 – 8. Ellman C, Corbett JA, Misko TP, McDaniel M, Beckerman KP. Nitric oxide mediates interleukin-1-induced cellular cytotoxicity in the rat ovary. A potential role for nitric oxide in the ovulatory process. J Clin Invest 1993;92:3053– 6. Shukovski L, Tsafriri A. The involvement of nitric oxide in the ovulatory process in the rat. Endocrinology 1994;135:2287–90. Nakamura Y, Kashida S, Nakata M, Takiguchi S, Yamagata Y, Takayama H, et al. Changes in nitric oxide synthase activity in the ovary of gonadotropin treated rats: the role of nitric oxide during ovulation. Endocr J 1999;46:529 –38. Mitsube K, Mikuni M, Matousek M, Brannstrom M. Effects of a nitric oxide donor and nitric oxide synthase inhibitors on luteinizing hormone-induced ovulation in the ex-vivo perfused rat ovary. Hum Reprod 1999;14:2537– 43. Jablonka-Shariff A, Olson LM. Nitric oxide is essential for optimal meiotic maturation of murine cumulus-oocyte complexes in vitro. Mol Reprod Dev 2000;55:412–21. Gouge RC, Marshburn P, Gordon BE, Nunley N, Huet-Hudson YM. Nitric oxide as a regulator of embryonic development. Biol Reprod 1998;58:875–9.
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REPRODUCTIVE BIOLOGY
Inducible and endothelial nitric oxide synthase: genetic background affects ovulation in mice Lukas A. Hefler, M.D.,a and Anthony R. Gregg, M.D.a,b Baylor College of Medicine, Houston, Texas
Received December 8, 2000; revised and accepted March 28, 2001. Supported in part by Erwin-SchroedingerAuslandsstipendium J1839-MED with funding by the Fonds zur Foerderung der Wissenschaftlichen Forschung (L.A.H.). Reprint requests: Anthony R. Gregg, M.D., Baylor College of Medicine, Department of Obstetrics and Gynecology, 6550 Fannin, Smith Tower—Suite 901A, Houston, Texas 77030 (FAX: 713-790-0108; E-mail: [email protected]. edu). a Department of Obstetrics and Gynecology. b Department of Molecular and Human Genetics. 0015-0282/02/$22.00 PII S0015-0282(01)02952-1
Objective: Inducible nitric oxide synthase (NOS) and endothelial NOS are involved in female reproductive physiology. We sought to investigate the influence of the inducible (Nos2) and endothelial (Nos3) NOS genes as a function of genetic background on ovulatory capacity and early embryonic development in a mouse model. Design: Observational study of genetically altered mice and their response to a superovulation protocol. Setting: Academic research institution. Animals: Wild-type mice and mice deficient for Nos2 or Nos3 were bred to C57BL/6J and 129/Sv genetic backgrounds. Intervention(s): Superovulation protocol, oocyte culture. Main Outcome Measure(s): Number of oocytes harvested, early embryonic development of zygotes, evaluation of ovarian histology. Result(s): The mean number of oocytes was significantly reduced in Nos3 deficient mice on a C57BL/6J background compared with controls. Oocytes deficient for Nos3 on a C57BL/6J background also showed reduced progression to two-cell stage embryos after 24 hours, two-cell stage embryos to blastocyst stage embryos, and survival to 48 hours. Those effects were distinctly absent in mice deficient for Nos3 on a 129/Sv background and in mice deficient for Nos2 on either genetic background. Conclusion(s): Our data show that disruption of Nos2 had no effect on ovulation in our mice. The negative effect of Nos3 deficiency on ovulatory capacity and early embryonic development is modulated by genetic background. This suggests a role for strain-specific modifier genes in these processes. (Fertil Steril威 2002;77: 147–51. ©2002 by American Society for Reproductive Medicine.) Key Words: Nitric oxide, Nos2, Nos3, ovulation, genetic background
The free radical nitric oxide (NO) is synthesized during the conversion of L-arginine to L-citrulline by the enzyme nitric oxide synthase (NOS) and is known to mediate a wide variety of physiologic functions, including neurotransmission, immune cell cytotoxicity, and regulation of the vascular tone (1–3). Three isoforms of NOS have been identified: endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) (2). In the rodent ovary, mRNA and protein expression of eNOS and iNOS, but not nNOS, has been demonstrated (4, 5). During follicular development, iNOS expression was found within granulosa cells of secondary follicles, small antral follicles, the theca cell layer, and ovarian stroma. eNOS protein was localized to blood vessels in the ovary and also to the theca
cell layer, to ovarian stroma, to the surface of oocytes, and within the corpus luteum (4, 5). After gonadotropin-induced ovulation, iNOS expression significantly decreased and was only present in the external layers of the developing corpus luteum (4 – 6). eNOS mRNA levels, however, increased and peaked in ovaries containing ovulatory follicles before declining in the luteal phase (5). Results in eNOS-deficient mice are consistent with the idea that the NO plays a physiological role in female reproduction, including pubertal maturation, estrous cyclicity, ovulation rate, early embryonic development of oocytes, and timing of menopause (7–10). The use of targeted mutagenesis in the study of gene function, however, is not without recognized pitfalls. One example is the variation in 147
phenotype as a function of genetic background or strain (11, 12). Mice with the same genetically engineered mutation can exhibit a unique phenotype on one strain but not another (13, 14). The importance of taking into account genetic background or strain and the importance of genes capable of modifying phenotypes has been stressed (11, 14). Controlling for strain-specific modifier genes, by backcrossing over several generations to obtain an almost pure genetic background, has been proposed (15). The aim of our study was to elucidate the effect of the Nos2 and Nos3 genes, encoding for iNOS and eNOS, respectively, on ovulatory capacity and early embryonic development in a mouse model. To control for possible confounding factors of background genes, we performed all our experiments on a ⬎98% pure incipient congenic genetic background of C57BL/6J and 129/Sv.
MATERIAL AND METHODS Mice and Animal Husbandry All animal experiments were approved by the internal review board at Baylor College of Medicine. Animal husbandry practices followed guidelines established by the Animal Care Committee of Baylor College of Medicine. Animals were subjected to daily 12-hour alternating periods of light and dark cycles within a humidity- and temperaturecontrolled environment. Food and water were provided ad libitum. F1 generation Nos2-deficient mice (Nos2⫺/⫺; Merck Research Laboratories, Rahway, NJ) (16) were backcrossed by natural breeding over eight generations with C57BL/6J (B6) to generate wild-type and Nos2-deficient mice on this background (B6-Nos2⫹/⫹ and B6-Nos2⫺/⫺, respectively). This was repeated similarly using the 129/Sv (129) strain to generate wild-type and Nos2-deficient mice on this background (129-Nos2⫹/⫹ and 129-Nos2⫺/⫺, respectively). Nos3-deficient mice were previously generated in our laboratory (17) and were also backcrossed by natural breeding over eight generations with C57BL/6J and 129/Sv mice to generate wild-type and Nos3-deficient mice on these strains (B6-Nos3⫹/⫹, B6-Nos3⫺/⫺, 129-Nos3⫹/⫹, and 129Nos3⫺/⫺, respectively). Thus, incipient congenic mice on a C57BL/6J and 129/Sv background were generated.
Superovulation Protocol Prepubescent (22-day-old to 26-day-old) congenic female mice, either wild type or homozygous deficient (B6Nos3⫹/⫹, B6-Nos3⫺/⫺, 129-Nos3⫹/⫹, and 129-Nos3⫺/⫺), were superovulated by intraperitoneal (IP) injection of 7.5 IU pregnant mare serum (Organon, West Orange, NJ), followed 48 hours later by an IP injection of 7.5 IU hCG (Sigma). Superovulated females were mated to a samestrain, randomly selected male that was either wild type or homozygous deficient for Nos2 or Nos3. This was done to obtain ⫹/⫹, ⫹/⫺, and ⫺/⫺ zygotes for comparison. Ran148
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dom and blinded matings between homozygous-deficient and wild-type mice allowed for zygotes with wild-type, heterozygous, or homozygous-deficient genotypes for Nos2 and Nos3, respectively. The investigator was not aware of any genotype while performing all experiments. Each stud male had sired at least one litter previously. A copulatory plug was identified, and mice were killed 18 hours after hCG administration. Oviducts were harvested and placed into M-2 medium (Cell & Molecular Technologies, Phillippsburg, NJ) containing 300 U/mL of hyaluronidase type IV from bovine testes (Sigma). Oviducts were incised, and the ovulated oocyte-cumulus complex was removed from the ampulla. Oocytes were mouth pipetted through three cycles of washing in M2 media using a glass micropipette. After washes, oocytes were placed in KSOM media (Cell & Molecular Technologies) and counted. Oocytes were then incubated at 37°C, 5% CO2. The number of oocytes and the number of zygotes that developed to two-cell and blastocyst stages were recorded after 24 and 48 hours, respectively. We defined “failure to ovulate” as no oocytes found in the oviducts at the time that the mouse was killed. The investigator (L.A.H.) performing oocyte counting was blinded to male and female mouse genotypes.
Genotyping For genotyping of Nos2-deficient mice, a polymerase chain reaction (PCR)– based strategy was used as described previously (16). Mouse genomic DNA for genotyping was obtained from Proteinase K– digested tails using phenolchloroform extraction. Polymerase chain reaction amplification with oligonucleotide primers flanking the Nos2 gene was performed. Polymerase chain reaction conditions comprised an initial denaturing step at 92°C for 5 minutes, followed by 30 cycles of 92°C for 45 seconds, 62°C for 45 seconds, and 72°C for 45 seconds and a final extension at 72°C for 5 minutes. The wild-type allele generated a 413-bp band; the mutant allele generated a 1,288-bp band. PCR products were resolved on a 1% agarose gel and stained with ethidium bromide. Nos3-deficient mice were genotyped by Southern blotting as reported elsewhere (17). Briefly, mouse genomic DNA for genotyping was obtained from Proteinase K– digested tails using phenol-chloroform extraction. Genomic DNA was digested using the restriction enzyme EcoRI (Boehringer Mannheim, Indianapolis, IN). Equal amounts of DNA (15 g) were electrophorized on a 1% agarose gel, denatured in 0.4 M NaOH for 45 minutes, and transferred overnight onto nucleic acid transfer membranes (Amersham, Piscataway, NJ) using 10⫻ SSC (0.75 M sodium chloride and 0.075 M sodium citrate, pH 7.0; Fisher Scientific, Fairlawn, NJ). The membranes were prehybridized at 64°C in a hybridization buffer containing 0.5 M of sodium phosphate (Fisher Scientific), 1 mM of ethylenediaminetetraacetic acid (J.T.
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Baker, Inc, Phillipsburg, NJ), 7% sodium dodecyl sulfate (Boehringer Mannheim), 10% PEG 8000 (Fisher), 1 g/mL of bovine serum albumin (fraction V; Sigma Corp; St. Louis; MO), and 100 g/mL of denatured salmon sperm DNA (Sigma). After 6 hours, Southern blots of genomic DNA were hybridized at high stringency with a 510-bp probe of mouse Nos3 cDNA, which had been labeled using randomprimers [␣-32P] dCTP. After hybridization for 18 hours at 64°C, membranes were washed sequentially at 64°C for 20 minutes with 5⫻ SSC, 2⫻ SSC, 1⫻ SSC, 0.5⫻ SSC, and 0.1% sodium dodecyl sulfate (Sigma), with two cycles for each stringency. The membranes were air-dried, wrapped in plastic wrap, and exposed to an autoradiography film (Amersham) for 48 hours.
FIGURE 1 Mean number of oocytes broken down by genotype and strains. Each bar with the depicted standard deviation represents one genotype on one genetic background. *A significant difference between groups (P ⬍.05).
Genotypes were determined by visual inspection of autorads. In wild-type mice, the EcoRI restriction fragment length polymorphism has a size of 9.8 kb. In Nos3⫺/⫺ mice, the EcoRI restriction fragment is 4.8 kb because of the deletion event. This difference was used to distinguish between the mutant and the wild-type allele. Hefler. NOS deficiency and ovulation in mice. Fertil Steril 2002.
Histology of Ovaries and Follicle Assessment Ovaries of superovulated females were harvested at the same time as oviducts, formalin fixed, and paraffin embedded. Routine hematoxylin– eosin staining was performed (n ⫽ 5 for each genotype and mouse strain). Primary follicles, secondary follicles, small antral follicles, large preovulatory antral follicles, and corpora lutea were identified. Small antral and large preovulatory follicles were examined.
Statistical Analysis The statistical software Sigma Stat version 2.0 (Jandel Scientific, San Rafael, CA) was used for statistical analysis. Data are presented as mean ⫾ standard deviation (SD) of the mean. After checking for normality, comparisons between groups were made by one-way ANOVA with Tukey test as multiple comparison procedure and 2 tests where appropriate. Significance was assumed at P ⬍.05.
RESULTS We subjected 132 mice to the superovulation protocol. Failure of ovarian superovulation was noted in 20 cases, independent of strain or genotype. We only analyzed ovulatory capacity for mice that responded to the superovulation protocol: B6-Nos2⫹/⫹ (n ⫽ 13), B6-Nos2⫺/⫺ (n ⫽ 14), 129-Nos2⫹/⫹ (n ⫽ 11), 129-Nos2⫺/⫺ (n ⫽ 12), B6-Nos3⫹/⫹, (n ⫽ 14), B6-Nos3⫺/⫺, (n ⫽ 10), 129-Nos3⫹/⫹ (n ⫽ 23), and 129-Nos3⫺/⫺ (n ⫽ 15). The mean number of oocytes, broken down by genotype and strains is shown in Figure 1. The ovulatory capacities of homozygous-deficient mice were compared with those of their respective wild-type controls. The ovulatory capacity of B6-Nos3⫺/⫺ mice was significantly reduced compared with that of wild-type B6Nos3⫹/⫹ mice (P ⫽.035). Interestingly, Nos3 deficiency on FERTILITY & STERILITY威
a 129/Sv background and Nos2 deficiency on either background had no effect on the number of oocytes counted. Of note, wild-type mice, (i.e., of B6-Nos2⫹/⫹, 129-Nos2⫹/⫹, B6-Nos3⫹/⫹, and 129-Nos3⫹/⫹) showed no differences in the number of ovulated oocytes (P ⫽.3). The percentage of oocytes progressing to the two-cell stage after 24 hours, the percentage of two-cell stage embryos progressing to the blastocyst stage after 48 hours, and overall survival of harvested oocytes, broken down by genotype and strain, is shown in Table 1. Of note is that genotypes in the table refer to genotypes of the embryo. The ratio of oocytes progressing to two-cell–stage embryos after 24 hours, the ratio of two-cell–stage embryos progressing to blastocyst-stage embryos after 48 hours, and survival of embryos from harvest to 48 hours was significantly reduced in B6-Nos3⫺/⫺ oocytes compared with the case of B6Nos3⫹/⫺ or B6-Nos3⫹/⫹ oocytes, respectively. No differences were found among any of the other groups. Histologic sections from ovaries of superovulated females were examined while the investigator was blinded to genotype and strain. No obvious structural or morphological differences between sections from any of the mouse strains or genotypes was identified.
DISCUSSION Our data are consistent with a role for eNOS in the regulation of ovulatory function and early embryonic development in mice. We observed this to be a function of genetic background suggesting that modifier genes are involved in these processes. Absence of functional iNOS does not seem 149
TABLE 1 Early embryonic development. Strain–genotype
Oocytes harvested (n)
Two-cell stage embryos per no. of oocytes harvested at 24 h (%)
Blastocysts per two-cell stage embryos at 48 h (%)
Blastocysts per no. of oocytes harvested at 48 h (%)
241 299 217 298 346 362 246 346 126 439 410 198
48.5 37.9 40.1 14.5 24.3 22.4 26.8a 33.9b 12.7a,b 29.0 40.7 35.7
66.1 80.8 92.2 72.5 73.4 43.5 100b 77.2a 34.6a,b 65.4 39.5 53.4
48.5 30.3 35 14.2 20.6 24.7 33.3c 26.2b 9.2b,c 21.4 16.1 18.6
B6-Nos2⫹/⫹ B6-Nos2⫹/⫺ B6-Nos2⫺/⫺ 129-Nos2⫹/⫹ 129-Nos2⫹/⫺ 129-Nos2⫺/⫺ B6-Nos3⫹/⫹ B6-Nos3⫹/⫺ B6-Nos3⫺/⫺ 129-Nos3⫹/⫹ 129-Nos3⫹/⫺ 129-Nos3⫺/⫺
Note: t-test comparisons are for B6-NOS3⫹/⫹, B6-NOS3⫹/⫺, and B6-NOS3⫺/⫺. Symbols represent significance level achieved for each comparison. a P ⬍.05. b P ⬍.01. c P ⬍.001. Hefler. NOS deficiency and ovulation in mice. Fertil Steril 2002.
to impair ovulation in response to superovulatory protocols among either C57BL6/J or SV129 strains of mice. The expression of iNOS and eNOS in the rodent ovary and the role of NO in ovulation and oocyte development is abundant. The physiological pathways, however, allowing NO to influence ovarian function are still hypothetical. A concept has been put forward describing the ability of NO to mediate the passage of inter-␣-trypsin inhibitor protein through the blood–follicle barrier. This event is thought to be critical for stabilizing the extracellular matrix of the cumulus in mature follicles (18). Nitric oxide derived from iNOS stimulated by interleukin-1 was found to be an important mediator of cell death and to act as a physiologically mediator of tissue remodeling events that occur in vivo during the ovulatory process (19). Another hypothesis considers endothelium-derived, NOdependent local vasodilatation as a trigger of hemodynamic changes associated with ovulation, for instance, increased ovarian blood flow, follicular hyperemia, and edema of the theca interna and externa (20). A deficiency of endotheliumderived NO might interfere with adequate local blood supply, secondary to prolonged or intense vasoconstriction or thromboembolic events. This may impair follicular recruitment and/or the process of ovulation itself. Our data confirm previously obtained results (10) indicating that eNOS deficiency on a C57BL/6J genetic background is associated with reduced ovulatory potential after exposure to a superovulation protocol. However, this observation is clearly straindependent. Furthermore, our data are in accordance with previously published data (21, 22) indicating the prominent role of the endothelial-derived isoform of NOS; in other words, eNOS, compared with iNOS in ovulatory processes. 150
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Elsewhere, eNOS was shown to be involved in meiotic maturation and oocyte development in vitro. To assess effects of NO deficiency on the kinetics of germinal vesicle breakdown, nuclear morphology of oocytes was evaluated (8, 10, 23). Absence of endothelial-derived NO was associated with an impaired oocyte meiotic maturation in vitro (23). These results support previous observations in vivo, indicating that endothelial-derived NO has independent functions in oocyte maturation and development (8). Furthermore, NO was indirectly linked, via a cGMP pathway, to an important regulator of early embryonic development and differentiation (24). In our series, eNOS deficiency was associated with decreased ratios of oocytes progressing to two-cell stage embryos after 24 hours, two-cell stage embryos progressing to blastocyst stage after 48 hours, and overall survival of embryos. As pointed out, these observations were strain dependent. Of note, we did not assess whether all oocytes were recovered mature by evaluating germinal vesicle breakdown and presence of polar bodies in accordance with a previously published study (10). Furthermore, we cannot prove that all recovered oocytes were fertilized. Thus, we cannot exclude that oocytes that did not develop to two-cell stage or blastocyst stage were not fertilized or immature. This has to be kept in mind when interpreting the results of our study. It is interesting to consider the functional significance of reduced blastocyst survival on overall fecundity among B6Nos3⫺/⫺ mice. Despite the statistically significant reduction in numbers, an adequate number of apparently functional blastocysts seems available for normal implantation and a normal litter size. In fact, F1 generation eNOS-deficient mice were previously shown to produce litters of expected
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size (17). This leaves open the significance of the role played by eNOS when ovulation occurs outside of superovulatory protocols. Our data support the existing evidence that eNOS plays a role in ovulation and the subsequent development of the fertilized egg. We have demonstrated the importance of mouse strain as it applies to NOS genes. Because these processes are complex and appear to be regulated by multiple genes, special consideration should be given to the genetic background when investigating ovulation and the response to ovulation using in vivo models.
9. 10. 11. 12. 13. 14. 15. 16.
Acknowledgment: F1 generation Nos2-deficient mice were generously supplied by J. S. Mudgett of Merck Research Laboratories (Rahway, NJ).
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