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Pregnancy; |
Unidad de Animalario,3 Fundacion Centro Nacional de Investigaciones Cardiovasculares Carlos III, 28029 Madrid, Spain
Departamento Reproduccion Animal,4 Instituto Nacional de Investigación y Tecnologia Agraria y Alimentaria, 28040 Madrid, Spain
ABSTRACT
The current study aimed to determine effects of deficiencies in nitric oxide synthase (NOS) 3 on embryo and fetal development by in vivo, noninvasive, real-time ultrasonographic assessment of phenotypic changes in Nos3-knockout pregnant mice and their wild-type counterparts. From Day 4.5 of pregnancy onwards, embryonic vesicle diameters, crown-rump lengths, and trunk diameters were obtained by serial scanning of seven adult pregnant female mice, strain B6.129P2-Nos3tm1Unc/J, N9 generation backcrossing with C57BL/6J mice, homozygous for the disruption of the endothelial NOS gene (group Nos3–/–), and 12 pregnant, wild-type C57BL/6J mice (group Nos3+/+). All the measurements increased in both genotypes throughout gestation. However, embryo length and width were significantly larger in Nos3+/+ than in Nos3–/– mice from Day 8.5, and both longitudinal and transverse diameters of the entire gestational sacs were larger in Nos3+/+ mice from Day 10.5. Assessment of the relative growth of embryos/fetuses and gestational annexes showed different patterns among Nos3–/– and Nos3+/+ mice. Throughout pregnancy, the distance between the external limit of the gestational sac and the embryo in Nos3+/+ mice diminished in longitudinal sections, or remained unaffected in transverse sections. In Nos3–/– mice, there were significant increases (P < 0.005) in the differences between embryo and gestational vesicle measurements in both longitudinal and transversal curves from Days 5.5 to 14.5, but from Day 14.5 of pregnancy onward, the changes were not significant. The results demonstrate that the processes of fetal growth retardation in the Nos3–/– mice are established from early pregnancy stages.
conceptus, early development, knockout mice, nitric oxide, pregnancy
Nitric oxide (NO) is a key mediator and regulatory agent in a wide variety of physiological functions [1]. The NO is synthesized by a family of NO synthases (NOSs), with three isoforms: neuronal constitutive (nNOS or NOS1 [type I]), inducible NOS (iNOS or NOS2 [type II]), and endothelial constitutive (eNOS or NOS3 [type III]). The NOS3-derived NO was found to be the most important vasodilator and platelet aggregation inhibitor [2]. It is now well known that NOS3 takes part in most of the female reproductive processes (reviewed in [3, 4]), acting as an important mediator of uteroplacental blood flow during pregnancy.
The study of NOS actions is currently facilitated by the existence of mouse lines deficient for each isoform (for review, see [5, 6]). Inhibition of NOS3 in Nos3-knockout mice has been found to cause decreased placental perfusion and creates alterations in the remodeling of the uterine artery [7]. Effects are found to be related to fetal growth retardation and higher mortality at the end of gestation [7, 8]. Thus, there is a reduction in the litter size and weight of neonates at delivery (confirmed by [9]), with increased perinatal losses [6]. The publications examining fetal growth [7, 8] report that differences in litter size and fetal development were established only at the last days of pregnancy (Day 17 postcoitum [p.c.]). Conversely, several studies (for review, see [3, 4]) have emphasized the role of NO and NOS, and specifically NOS3, in processes of implantation and early embryonic development [10–12]. All of these analyses were done by postmortem examination at specific gestational ages; thus, the screening of the same conceptuses in successive days throughout the pregnancy is impeded.
The current study was developed to assess the effects of deficiencies in NOS3 on fetal development. For a dynamic study of conceptus development in the same animals in successive days, in vivo monitoring of daily changes in embryonic and fetal structures during the entire gestation was performed in Nos3-knockout mice and their wild-type counterparts. The screening was carried out by real-time ultrasound imaging, a well-recognized, noninvasive, and reliable method for assessment of pregnancy and fetal development in most species, including mice [13, 14], from very early pregnancy stages (Day 4.5 p.c. in mice; Pallares and Gonzalez-Bulnes, unpublished results). From the data of another previously unpublished study (Pallares and Gonzalez-Bulnes), values of embryo vesicle and trunk diameters and crown-rump lengths of embryos and fetuses were considered to be the most representative during the entire pregnancy. These measurements can be easily made from early pregnancy stages (Day 4.5 p.c. for vesicle and 5.5 p.c. for embryo measurements) and throughout the entire gestation. Other suitable anatomical structure measurements are the skull diameter (occipito-snout length and biparietal and orbital diameters), umbilical cord diameter, and femur length, but only at later stages of gestation (from Day 11.5 onward) and with reliability determined by the position of the fetus.
All the animals were maintained at the facilities of the Fundacion Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) Animal Laboratory Unit in Madrid, Spain, which meets the requirements of the European Union for scientific procedure establishment. Aspects of pregnancies and offspring described in the literature for deficient mice had previously been found in the animals used for the trial. The experiment was carried out under project license 156/07 from the CNIC Scientific Ethic Committee. Animal manipulations were performed in accordance with Spanish policy for animal protection RD1201/05, which meets the European Union Directive 86/609 about the protection of animals used in experimentation.
Ultrasonographic Morphometry of Conceptuses Development
For this study, we used a total of seven adult, pregnant, female mice, strain B6.129P2-Nos3tm1Unc/J, N9 generation backcrossing with C57BL/6J mice, homozygous for the disruption of the endothelial NOS gene (group Nos3–/–) and 12 pregnant wild-type C57BL/6J mice (group Nos3+/+); animals were obtained from The Jackson Laboratory (Bar Harbor, ME). Females were mated in homozygosis with B6.129P2-Nos3tm1Unc/J and C57BL/6J males at a ratio of 1:1. Every morning for 4 days, appearance of estrus was determined by evaluating the presence of a vaginal plug as a result of overnight mating. The male was removed during the same morning that the vaginal plug was confirmed; this was considered as Day 0.5 of pregnancy.
Observations were performed starting at Day 3.5, estimated as mean day of implantation [15], to Day 18.5. In order to avoid an excessive number of consecutive observations, which might damage the mouse or its conceptus, the mice were randomly divided into two groups for observation, and each group was scanned on alternating days. Each mouse was manually restrained in dorsal recumbence for ultrasound scanning, without anesthesia, in order to avoid any effect on fetal development or organ function [16], mainly heart rate, during the observations. In order to diminish animal distress, ultrasound imaging was performed without shaving the abdominal hair; the coupling between the transducer and the skin was improved by abundantly wetting the abdomen with carboxymethylcellulose gel.
Scanning was performed with two different ultrasound machines: an Aloka 2500 equipped with a multifrequency (7.5–10 MHz) sectorial array probe (Aloka Co., Tokyo, Japan), and a Siemens Antares connected to a multifrequency (7.5–10 MHz) linear array probe (Siemans Medical Solutions, Erlangen, Germany). For viewing the uterine horns and fetus in transverse, frontal, or sagittal planes, ultrasound scanning was performed by placing the transducer on one flank and moving it to the opposite flank. Thus, the probe was manipulated until the largest section of each structure was obtained. Measurements were completed after the entire examination was recorded and the mouse released to avoid further stress arising from keeping the animal restrained. Subsequently, the size of the different structures was measured with built-in electronic calipers on the cine-loop. In order to avoid individual effects derived from corpulence of the fetus, the measurements from a minimum of three different conceptuses (range: 3–5 in Nos3–/– and 3–7 in Nos3+/+), from each individual pregnancy, were taken in each scanning.
Data obtained were grouped according to the day of gestation, and a statistical study was carried out by using standard linear and quadratic analyses. Predictive regression curves for each variable were calculated by the best adjustment of data in terms of their respective determination coefficients (r2). The effects of genotype on correlations between day of gestation and embryonic or fetal measurements were tested by analysis of variance for repeated measures (split-plot ANOVA). The parameter values were expressed as means ± SEM, and statistical significance was accepted for P < 0.05.
Embryonic vesicles were identified from Day 4.5 post-vaginal plug and throughout the entire pregnancy (Fig. 1). The longitudinal diameters of the embryonic vesicle increased exponentially (Fig. 2A) from the first visualization to Day 18.5, both in Nos3–/– (y = 2.2068e0.1208x; r2 = 0.674; P < 0.0005) and Nos3+/+ (y = 2.8702e0.1132x; r2 = 0.671; P < 0.0005), the diameters being significantly higher in Nos3+/+ from Day 10.5 (10.0 ± 0.3 vs. 7.7 ± 0.2; P < 0.0005). The transverse diameter of the gestational sac also increased exponentially (Fig. 2B) for both groups (Nos3–/–: y = 1.3913e0.1133x; r2 = 0.723; P < 0.0005; Nos3+/+: y = 1.4274e0.1234x; r2 = 0.652; P < 0.0005). The values of transversal diameters were higher in Nos3+/+ from Day 10.5 (5.8 ± 0.2 vs. 4.8 ± 0.1; P < 0.005).
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The crown-rump length and trunk diameter of the embryos and fetuses were measured from Day 5.5 to Day 18.5 of gestation (Fig. 3). The evolution of the crown-rump length was exponential in both groups (Nos3–/–: y = 1.5149e0.1053x; r2 = 0.776; P < 0.0005; Nos3+/+: y = 1.4274e0.1234x; r2 = 0.652; P < 0.005), like the trunk diameter (Nos3–/–: y = 0.6971e0.1089x; r2 = 0.783; P < 0.0005; Nos3+/+: y = 1.2392e0.0952x; r2 = 0.588; P < 0.0005). Differences between genotypes in both parameters were found from Day 8.5 of pregnancy (for crown-rump length: 5.5 ± 0.5 vs. 4.6 ± 0.3; P < 0.005; and for trunk diameter: 2.9 ± 0.1 vs. 2.0 ± 0.1; P < 0.005).
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From these results, subsequent analysis regarding the relative growth of embryos/fetuses and gestational annexes showed different patterns between Nos3–/– and Nos3+/+. In the control, Nos3+/+, the distance between the external limit of the gestational sac and the embryo diminished throughout the pregnancy (in longitudinal sections; Fig. 4A) or remained unaffected (in transverse sections; Fig. 4B). In the defective genotype, Nos3–/–, there was a trend to an increase throughout the gestation, both in longitudinal and transverse sections. In fact, both longitudinal and transverse curves are composed of an initial period of significant increases in the difference between embryo and gestational vesicle measurement (Days 5.5 to 14.5 p.c.; longitudinal: r2 = 0.752; P < 0.005; transverse: r2 = 0.772; P < 0.005), and a second period, from Day 14.5 of pregnancy onward, in which the changes were not significant.
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Our current data offer, for the first time, the in vivo daily screening of conceptus development in Nos3-knockout mice compared with their wild-type counterparts. These results clearly demonstrate, contrary to previous reports, that processes of fetal growth retardation described for the Nos3–/– mice are established from the early stages of pregnancy.
The comparison of growth curves between Nos3–/– and Nos3+/+mice showed differences from the very first observations. However, growth retardations in the development of the embryo and the entire gestational sac were found to reach statistical significance later, from Days 8.5 and 10.5 of pregnancy, respectively. In the mouse, at Day 8.5 of pregnancy, the development of the embryonic heart and the yolk sac blood circulation commences [17]; blood islands within the yolk sac originate both endothelial and hematopoietic cells [18]. This is a crucial period for the embryo, during which gastrulation takes place [19]. Gastrulation is the process by which the three primary germ layers (ectoderm, mesoderm, and endoderm) are established. Gastrulation also generates the extraembryonic mesoderm, which differentiates into the allantois and the mesodermal components of the chorion, amnion, and visceral yolk sac. Thus, our data confirms, in vivo, previous reports from in vitro studies regarding the importance of NO and NOS in early pregnancy. From these earlier studies, it is known that embryos with inhibition for the NOS enzyme become developmentally delayed or nonviable [10]. This fact has been related to the finding that nutrition of the embryos, before completion of the placenta, is achieved by phagocytic activity of the trophoblast [20, 21], activity which is mediated by reactive oxygen species [22]. During implantation, dilatation of uteroplacental arteries is induced by NOS3 from the extravillous trophoblast [23]. Subsequently, in the postimplantation period and during early placental development, the NO released by trophoblast NOS3 is hypothesized to be involved in tissue remodeling, immunosuppression, and vasoregulation [11], thus contributing to the maintenance of vasodilatation and to the prevention of coagulation necessary for the success of the early pregnancy [12].
Our findings, although coincidental with the studies cited above, are contradictory to previous works reporting that differences in fetal growth are only established at the end of pregnancy [7, 8]. A possible explanation for these differences may be related to the number of animals used or to the fact that these studies were only performed in late gestation (from Day 15 of pregnancy). Also, when compared postmortem, the fetal weight, and not the usual fetometric indexes (lengths and diameters of embryo/fetal head and body), were used for estimating conceptus development. From classical studies in other species, we know that the morphometric values obtained in later gestation are affected by the litter size and by individual characteristics of the fetus. These characteristics are determined both by genetic [24] and/or nutritional factors [25], with different effects on fetal weight than on fetal length or development [26, 27]. Moreover, it is essential to distinguish fetal growth from weight at term or at birth. Fetuses with the same weight may have had different growth trajectories that can only be elucidated by repeated ultrasonographic measurement throughout pregnancy [28]. These considerations may also be valid for mice. The study by Hefler et al. [8] was performed at Days 15 and 17 of pregnancy. The work of Van der Heijden et al. [7], although only giving data on embryonic weight at Day 17 and not before, reported abnormalities in the structural and cellular characteristics of the uterine artery between Days 5 and 11 of pregnancy in Nos3–/– mice, which coincides with the critical period identified in our study.
In the present study, differences between Nos3–/– and Nos3+/+ mice were not only found in the size of embryos and vesicles, but also in the growth dynamics of both structures. The comparison of the relative growth of embryos/fetuses and gestational annexes showed different patterns between Nos3–/– and Nos3+/+ mice. In the control wild-type mice, the increase of the structures throughout pregnancy is synchronous; in the mice defective for NOS3, the growth of the embryo is slower than that of its membranes and placenta for the first 14.5 days of pregnancy. In the mouse, this is the period of transition between the stages of late embryo and early fetus [29]. Possible causes for these changes in the relative size of embryos/fetuses and annexes are related to processes of intrauterine growth retardation (IUGR), preeclampsia, hydroamnios, and/or hydroallantois. However, in the current study, the accumulation of fluid may be disregarded as a cause, as it is accompanied by a significant increase of gestational sac size. Conversely, the size of the embryo vesicle decreased in Nos3–/– mice. Possible implications of IUGR and/or preeclamptic syndromes seem to be reinforced by the fact that deleterious effects were found initially in the embryo and subsequently in the annexes. Both syndromes, IUGR and pre-eclampsia, are linked. In preeclampsia, uteroplacental blood flow is compromised [30], leading to fetal growth restriction [31]. Thus, several studies indicate that a relative deficiency of placental NO synthesis is a predisposing factor for pre-eclampsia [30, 32, 33] and IUGR [34].
IUGR is a significant health concern in human medicine, being associated with mortality and morbidity during infancy and susceptibility to disease during adulthood, primarily showing coronary diseases, hypertension, and diabetes [35]. IUGR, both in humans and in animal models, has been found to develop into different types, and to differing degrees of severity. First, IUGR fetuses may be classified as "symmetrical" or "asymmetrical" (for review, see [36]). Symmetrical IUGR is characterized by a uniform reduction of the fetus and its organs from early pregnancy, and is associated with genetic and infectious factors. Asymmetrical IUGR is characterized by a reduction in size in some organs, while the remaining organs are normal. It is also associated with insufficient nutritional delivery to the fetus by maternal or placental factors during late gestation, when the requirements of the fetus are higher. Symmetrical IUGR is scarce, while asymmetrical IUGR is more abundant; thus, most studies have focused on asymmetrical IUGR. The inconveniences of research in humans led to the development of animal models, mainly rodents and sheep, for studies of maternal malnourishment and placental malfunction during late gestation [28, 36, 37].
Nevertheless, there is emerging evidence of the importance of adequate development of embryos during early pregnancy. Alterations around the time of conception, implantation, and early development may affect not only pregnancy establishment by immediate effects, but also fetal, neonatal, and adult development [38–41], leading to low birthweight, premature births [42], and, later, metabolic, nervous, and cardiovascular diseases [43]. These effects are related to genetic and infectious factors [36], but also to nutritional challenges [42, 44, 45] and, mainly, maternal body composition during early pregnancy [46], which, in cows, has been described to be influenced by weight, age, and parity [47]. Coincidentally, incidence of reproductive alterations and fetal programming in NOS3 mice has also been related to parity [9]. Linking early pregnancy, NOS3, and later fetal programming, recent studies have found that placental NOS3 concentration, mainly regulated at mid-gestation, but also during early and late gestation [48], is influenced by challenges in early gestation [49], equivalent in this current study to the fifth to eighth weeks of pregnancy in humans [50].
In conclusion, our current results demonstrate that the processes of fetal growth retardation in the Nos3–/– mouse are established from early pregnancy stages.
ACKNOWLEDGMENTS
The authors appreciatively thank Dr. J. M. Redondo for cession of the mice used in this experiment and the staff of CNIC Animal Unit for skilled technical assistance. The help from A. Vidaurrazaga (Aloka España, Madrid, Spain) and I. Cisneros (RX Cisneros Electromedicina, Madrid, Spain) in providing different ultrasound machines and probes is also acknowledged.
FOOTNOTES
1Supported by Fundacion Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) and Instituto Nacional de Investigación y Tecnologia Agraria y Alimentaria (INIA), under the collaborative project CC07-018. CNIC is supported by the Spanish Ministry of Health and Consumer Affairs and the Pro-CNIC Foundation; INIA is supported by the Spanish Ministry of Education and Science Affairs; there was no other outside funding. 
Correspondence: 2Pilar Pallares, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, Melchor Fernandez Almagro 3, 28029 Madrid, Spain. FAX: 34 91 453 12 65; e-mail: ppallares{at}cnic.es
Received: 17 October 2007.
First decision: 12 November 2007.
Accepted: 30 January 2008.
REFERENCES
This article has been cited by other articles:
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  P. Pallares, R. A Garcia-Fernandez, L. M Criado, C. A Letelier, D. Esteban, J. M Fernandez-Toro, J. M Flores, and A. Gonzalez-Bulnes Disruption of the endothelial nitric oxide synthase gene affects ovulation, fertilization and early embryo survival in a knockout mouse model Reproduction, November 1, 2008; 136(5): 573 - 579. [Abstract] [Full Text] [PDF]
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