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Disruption of the endothelial nitric oxide synthase gene affects ovulation, fertilization and early embryo survival in a knockout mouse model

Fundacion Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Melchor Fernandez Almagro 3, 28029 Madrid, Spain¹ Dpto. de Medicina y Cirugia Animal, Facultad de Veterinaria, UCM, 28040 Madrid, Spain² Dpto. de Reproduccion Animal, INIA, Avda. Puerta de Hierro s/n. 28040 Madrid, Spain³ Instituto de Ciencia Animal y Tecnologia de Carnes, UACh, Casilla 567, Valdivia, Chile

Correspondence should be addressed to A Gonzalez-Bulnes; Email: bulnes{at}inia.es

	Abstract

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Two consecutive experiments determined whether disruption of the endothelial nitric oxide synthases (NOS) gene (Nos3) affects ovulation, fertilization, implantation, and embryo development. In the first trial, Nos3-knockout mice (groups Nos3^–/–) and wild-type mice (groups Nos3^+/+) showed significant differences in mean number of corpora lutea (9.7±1.2 in Nos3^–/– versus 14.2±1.2 in Nos3^+/+; P<0.01), rate of anovulation (48.3±7.3% in Nos3^–/– versus 29.7±6.3 in Nos3^+/+; P<0.05), total mean number of recovered oocytes/zygotes (4.0±1.1 in Nos3^–/– versus 10.4±1.6 in Nos3^+/+; P<0.01), and non-fertilization rate (50.7 in Nos3^–/– versus 3.3% in Nos3^+/+; P<0.001). In the second trial, implantation and early pregnancy losses in Nos3-knockout and wild-type dams were detected by real-time ultrasound imaging. The number of embryos reaching implantation was higher in Nos3^+/+ than in Nos3^–/– mice (7.5±0.4 vs 4.0±0.4; P<0.005); thereafter, embryo losses were detected between days 8.5 and 13.5, in 62.5% of the Nos3-knockout dams and, at days 10.5 and 11.5, in 16.7% of the control females (P<0.005). Thus, NO and NOS3 deficiencies affect reproductive and developmental features in the Nos3-knockout mouse model.

	Introduction

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Nitric oxide (NO), an essential mediator in numerous physiological processes (Lowenstein et al. 1994), is synthesized by three different NO synthases (NOS): neuronal constitutive (type I, NOS1), inducible NOS (type II, NOS2), and endothelial constitutive (type III, NOS3). The isoform NOS3 is involved in female reproductive function (Maul et al. 2003, Thaler & Epel 2003). Studies on the effects from the lack of NOS3 have been afforded by using Nos3-knockout mice, a strain showing elevated blood pressure, decreased heart rate, and premature death in males but not in females; so the model is widely used for cardiovascular research (http://jaxmice.jax.org/strain/002684.html). The lack of NOS3 in Nos3-knockout mice (Mashimo & Goyal 1999, Gregg 2003) has been found to cause a reduction in the number of offspring and in the weight of the neonates at delivery (Hefler et al. 2001, Longo et al. 2004, Van der Heijden et al. 2005). In the previous studies, these effects have been related to lower fetal growth and higher mortality at the end of gestation (Hefler et al. 2001, Van der Heijden et al. 2005). In such later stages of pregnancy, embryo survival and growth are mainly affected by the uterine capacity of the mother (Bennett & Leymaster 1989) and, hence, litter size and weight are highly correlated with maternal body size and weight (Van Engelen et al. 1995). Thus, offspring features are enhanced in multiparous Nos3-knockout mothers when compared with primiparous or oligoparous females (Longo et al. 2004).

However, previous studies have reported that in mice, like in other species, litter size is also determined both by the number of ovulations (Joakimsen & Baker 1977) and the incidence of embryo losses during early pregnancy (Bakker et al. 1978, Durrant et al. 1980). Several studies have emphasized the role of NO and NOS, and specifically NOS3, in processes of ovulation, fertilization, implantation, and early embryo development (Gouge et al. 1998, Purcell et al. 1999, Gagioti et al. 2000, Maul et al. 2003, Thaler & Epel 2003).

Thus, the current study aimed to determine whether deficiencies in Nos3 gene affect reproductive features of the Nos3-knockout mouse at very early stages of pregnancy. Two consecutive experiments were performed. The first trial compared the ovulation and fertilization rates of adult female Nos3-knockout and wild-type mice. The second trial compared the incidence of early pregnancy losses in pregnant Nos3-knockout and wild-type dams, determining the number of conceptuses in the same animals in successive days throughout gestation. For this dynamic study of the conceptuses, in vivo monitoring was done by using real-time ultrasound imaging; a well-recognized, non-invasive, and reliable method for the assessment of pregnancy and fetal development in most of the species, including the mouse (Brown et al. 2006, Russo et al. 2007) from very early pregnancy stages (day 4.5 p.c.; Pallares & Gonzalez-Bulnes 2008a).

	Results

Top Abstract Introduction Results Discussion Materials and Methods Declaration of interest Funding Acknowledgements References

 
Effects of NOS3 deficiencies on ovulation and fertilization rates
The size of the ovaries and the structures inside were very different between both genotypes (Fig. 1). First, the overall size of the ovary is appreciably smaller in Nos3^–/– mice; thereafter, the analysis of the structures present in the ovaries showed less ovulation sites and a higher number of anovulatory and luteinized unruptured follicles in Nos3^–/– mice. Thus, when counting and comparing these structures, there were significant differences in the mean ovulation rate and ovulatory efficiency between groups (Table 1), as reflected by the number of corpora lutea (CL: 9.7±1.2 in Nos3^–/– versus 14.2±1.2 in Nos3^+/+; P<0.01), rate of anovulation (AR: 48.3±7.3% in Nos3^–/– versus 29.7±6.3 in Nos3^+/+; P<0.05), and total recovered embryos (RE: total recovered oocytes/zygotes: 4.0±1.1 in Nos3^–/– versus 10.4±1.6 in Nos3^+/+; P<0.01).

View larger version (40K): [in this window] [in a new window]   Figure 1 Histological images of the ovaries on day 0.5 of estrous cycle in Nos3-knockout mice (Nos3–/–, left-hand side) and wild-type controls (Nos3+/+, right–hand side). It is possible to observe a smaller size of the ovary, with scarce number of ovulation sites (OS) and higher presence of anovulatory and luteinized unruptured follicles (LUF), in Nos3–/– mice.

Effects of NOS3 deficiencies on number of embryos/fetus throughout pregnancy
At first visualization of the embryos inside the gestational sacs, between days 6.5 and 8.5 after vaginal plug (Fig. 2), the number of embryos reaching implantation was higher in Nos3^+/+ than in Nos3^–/– mice (7.5±0.4 vs 4.0±0.4; P<0.005), ranging from 2 to 5 in Nos3^–/– and between 6 and 10 in Nos3^+/+.

View larger version (189K): [in this window] [in a new window]   Figure 2 Pictures 1–3 show ultrasonographic images from Nos3–/– mice, at 6.5, 9.5, and 12.5 days of pregnancy respectively; mouse gestational sacs (dotted lines) with embryos (E) inside can be differentiated. Picture 4 represents images of a normal embryo (E) and gestational losses (GL) at 11.5 days of pregnancy in a Nos3+/+ dam. Pictures 5 and 6 correspond to gestational losses (GL) occurring in Nos3–/– mice at 12.5 and 13.5 days of pregnancy; within the normal embryos, it is possible to distinguish the head (H), the liver (L), the organs inside the trunk (T), and the vertebrae (V). Picture 7 corresponds to the image of two Nos3+/+ ossificated fetuses, at 17.5 days of pregnancy, in which it is possible to differentiate the liver (L), the ribs (R), the heart (Ht), and the head (H) with the ventricles (V), the eye (Ey) and the ossification of the rostrum (Rs).

	Discussion

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The in vivo screenings performed in the current study during early pregnancy stages evidence that prolificacy in Nos3^–/– mice is hampered by variations in the rates of ovulation, fertilization, and early embryo survival when compared with their wild-type counterparts. These results clearly demonstrate, contrary to previous reports, that processes causing reduction in the number of offspring in Nos3-knockout mice are established from the early stages of pregnancy. Previous studies have reported lower growth and higher mortality of the fetuses at the end of gestation (Hefler et al. 2001, Van der Heijden et al. 2005). Possible causes for these discrepancies may be related to the strains and the number of animals used (Hefler & Gregg 2002) or to the fact that these previous studies were performed postmortem and only in late gestation, lacking data on early pregnancy. The study by Hefler et al. (2001) was performed at days 15 and 17 of pregnancy. The work of Van der Heijden et al. (2005) gave data only on embryonic features at day 17 and not before, although reported abnormalities in the structural and cellular characteristics of the uterine artery between days 5 and 11 of pregnancy in NOS3-deficient mice, which coincides with the critical period identified in our study.

Current data indicate, first, an increased anovulatory rate in non-stimulated cycles of Nos3^–/– mice. Thus, our results confirm, in vivo, previous studies in rats evidencing the influence of NO, and NOS, in the process of ovulation. In that previous study, ovulation was suppressed by the administration of NOS inhibitors and restored by the concomitant administration of an NO generator (Shukovski & Tsafriri 1994). In vitro studies (Zackrisson et al. 1996) found that NOS2 was localized in granulosa cells of the follicle while high levels of NOS3 were found in blood vessel endothelium, theca cells of the follicle and luteal cells of the corpus luteum; NOS1 has not been found in ovarian tissues. Thereafter, the knowledge of the role of NO and NOS in biological processes was broadened by the use of knockout mice. Evaluation of reproductive features in Nos3-knockout superovulated immature dams showed a lower ovulatory efficiency when compared with the controls (Jablonka-Shariff & Olson 1998). Later studies with Nos2-and Nos3-knockout mice showed that disruption of NOS2 had no effect on ovulation rate in superovulated prepubertal females; on the other hand, NOS3 deficiency had a significant negative effect (Hefler & Gregg 2002). Our results reinforce these studies, with the added value of being performed in non-stimulated cycles in mature females (a more physiological approach, since we cannot discard interactions between NO, NOS, and exogenous gonadotropins as indicated by Thaler & Epel (2003)). Current data, hence, support the hypothesis of Gregg (2003) about the role of Nos3 as an important modulator of ovulatory efficiency in mouse.

The effects of NOS3 deficiencies in ovulation may be mediated by changes in follicle development (Jablonka-Shariff et al. 1999, Tempfer et al. 2000), in the preovulatory LH surge (McCann et al. 2003), and/or the ovulatory process by itself (Thaler & Epel 2003). Thaler & Epel (2003), revising many previous studies, suggest a model in which the preovulatory LH surge induces follicle cells to activate NOS; NOS induces production of NO, which stimulates synthesis of prostaglandins PGE₂ and PGF₂ and, thus, ovulation.

In our study, we have also found fertilization failures. We can hypothesize a link between these failures and previous reports evidencing the absence or delays in meiotic maturation of the oocyte. In these studies, NOS3 was not only found in follicle and luteal cells, but also in the oocyte (Jablonka-Shariff & Olson 1997); these researchers elegantly demonstrated the existence of abnormalities in the oocyte meiotic maturation in NOS3-deficient mice (Jablonka-Shariff & Olson 1998). Other possible causes for fertilization failures suggested by results of other authors may be related to the key role of NO in egg activation via regulation of the calcium rise (Kuo et al. 2000). Calcium rise is induced by the NO provided by both the endogenous activation of NOS in the egg and the fertilizing sperm (Thaler & Epel 2003); sperm that, in our study, came from Nos3-knockout males. However, we cannot leave aside as possible causes for deficiencies in fertilization, the existence of alterations, and/or mistiming in the periovulatory processes cited above (preovulatory LH surge, prostaglandin secretion, and/or ovulatory rupture).

In the second trial of the current study, implantation and early embryo development were also found to be affected by NOS3 deficiencies. This in vivo evidence confirms previous studies on the role of NOS3 in implantation (Maul et al. 2003). In fact, the three NOS isoforms are present within the mouse implantation site; NOS2 and NOS3 being the most important (Purcell et al. 1999). NO has been also found to be produced by preimplantation embryos; this production being required for normal embryo development (Gouge et al. 1998). These authors and Gagioti et al. (2000) found that NOS3 can be found in the trophoblast, the cells that adhere to and penetrate the uterine endometrium at implantation. These findings may be related to the fact that embryos nutrition before completion of the placenta is achieved by phagocytic activity of the trophoblast (Schilesinger & Koren 1975, Pavia 1983); activity mediated by reactive oxygen species (Gagioti et al. 1996). During implantation, dilatation of uteroplacental arteries is induced by NOS3 from the extravillous trophoblast (Nanaev et al. 1995). In summary, Nos3 induces vasodilatation, angiogenesis, and phagocytosis of maternal cells occurring during the implantation process (Gagioti et al. 2000).

Finally, the current study also found that embryo survival after implantation was affected in Nos3^–/– mice, which confirms, in vivo, previous reports from in vitro studies indicating that embryos with NOS inhibition become developmentally delayed or non-viable (Gouge et al. 1998). We have to note that the daily screening of conceptus development indicated two critical points for embryo survival throughout pregnancy of Nos3^–/– mice. The first is between days 8.5 and 10.5 of pregnancy, which is coincidental with the losses found in the wild-type controls. The second is at day 13.5 of gestation. Both periods are coincidental with the results found in a previous study developed in our laboratories for the assessment of the effects of NOS3 deficiencies on embryo and fetal development, in which retardations in the development of the embryo and gestational sac were found at these stages (Pallares & Gonzalez-Bulnes 2008b). Both periods are critical in mouse embryogenesis. In the mouse, at day 8.5 of pregnancy, gastrulation commences (Boucher & Pedersen 1996). Gastrulation is the process originating the three primary germ layers (ectoderm, mesoderm, and endoderm) of the embryo and the extra-embryonic mesoderm, which differentiates into the allantoids and the mesodermal components of the chorion, amnion, and visceral yolk sac. In this period, the development of the embryo, heart, and the yolk sac blood circulation is starting (Chen & Hsu 1982); blood islands within the yolk sac will originate from endothelial and hematopoietic cells (Flamme et al. 1997). Later, around 12.5–14.5 day of pregnancy, the period of transition between the stages of late embryo and early fetus occurs (Evans & Sack 1973). In the post-implantation period and early placental development, NO and NOS3 are hypothesized to be involved in tissue remodeling, immunosuppression, and vasoregulation (Purcell et al. 1999), and thus contribute to maintaining vasodilatation and preventing coagulation, which are necessary for the success of the early pregnancy (Gagioti et al. 2000). In the current study, differences were not found between genotypes in the morphological changes around embryo death; however, this may be related to the ultrasound machine and, specifically, to the frequency of the probes used. Application of high-resolution probes like that used for ultrasound microscopy (40–70 MHz) may evidence possible differences in the development of fetoplacental circulation or embryogenesis that may be the cause of embryo losses.

In conclusion, current results indicate, by using the Nos3-knockout mouse model, that NO and NOS3 deficiencies affect the processes of ovulation, fertilization, implantation, and embryo viability during early pregnancy stages.

	Materials and Methods

Top Abstract Introduction Results Discussion Materials and Methods Declaration of interest Funding Acknowledgements References

 
Animals and husbandry
The study was performed by comparing reproductive features in mice that were homozygous for the disruption of the endothelial NOS gene (groups Nos3^–/–; strain: B6.129P2-Nos3^tm1Unc/J, N9 generation backcrossing with C57BL/6J mice) and wild-type C57BL/6J mice (groups Nos3^+/+). All the animals were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and maintained at the facilities of the CNIC Animal Laboratory Unit in Madrid, Spain. The lighting cycle was 14 h light:10 h darkness respectively (0730 h, on; 2130 h, off). Humidity was maintained around 50% and the temperature was around 21 °C. CNIC Animal Unit meets the requirements of the European Union for Scientific Procedure Establishments. The experiments were carried out under Project Licenses 156/07 and 176/07 from the CNIC Scientific Ethic Committee. Animal manipulations were performed accordingly with the Spanish Policy for Animal Protection RD1201/05, which meets the European Union Directive 86/609 about the protection of animals used in experimentation.

Experimental design
In the first trial, ovulation and fertility rates were evaluated in eight adult female Nos3-knockout mice and eight wild-type control dams ovulating in response to a treatment for synchronization of estrus and ovulation. In brief, animals were treated by injecting, on day –3, one i.p. dose of 0.5 µg of a prostaglandin analog (cloprostenol, Estrumate; Mallinckrodt Vet GmbH, Friesoythe, Germany) and one s.c. dose of 3 µg of progesterone (4-pregnen-3,20-diona; Siemsgluss Iberica, Barcelona, Spain) in saline; on day 0, dams were treated with a second i.p. injection of 0.5 µg of cloprostenol. Immediately thereafter, females were mated in homozygosis with B6.129P2-Nos3^tm1Unc/J and C57BL/6J fertile males in a ratio of 1:1. Appearance of estrus and coital behaviors was determined the following morning (day 0.5 of pregnancy), by evaluating the presence of a vaginal plug as result of overnight mating. For determining ovulation and fertility rates, females were killed by cervical dislocation; embryos/oocytes were immediately retrieved and ovaries were removed and processed thereafter.

In the second trial, the incidence of early pregnancy losses was compared in 8 pregnant Nos3-knockout and 12 pregnant wild-type dams, treated with the protocol described above. Females were also mated in homozygosis in a ratio of 1:1 and appearance of estrus was verified on the following morning (day 0.5 of pregnancy), by evaluating vaginal plugs. Determination of pregnancy, number of conceptuses, and embryo losses was performed, by ultrasonography, from day 6.5 to 18.5 of gestation.

Assessment of the number of oocytes/zygotes
The relative number of oocytes and zygotes was evaluated just after the recovery from oviduct under a stereoscopic microscope (Leica MZ12.5; Leica Microsystems GmbH, Wetzlar, Germany). In brief, recoveries were performed by thoroughly dissecting both oviducts free from adjacent tissues with fine scissors and placing them in a Petri dish (Nunclon; Nunc International, Roskilde, Denmark) containing a drop of KSOM-AA medium (MR-121-D; Specialty Media, Millipore Co., Billerica, MA, USA) with 1 M HEPES (15630-049; Gibco, Invitrogen Co.) and 300 µg/ml hyaluronidase type IV-S (H3884; Sigma Chemical Co). Immediately, both ampullae were slot lengthways with a pair of fine forceps and the recovered oocytes/embryos were counted and classified, under a light microscope (Zeiss Axio Observer D1; Carl Zeiss AG, Oberkochen, Germany), as fertilized or non-fertilized on the basis of the presence of pronuclei.

Processing of ovaries and cytomorphometry
The ovaries were placed and washed, just after removal, in PBS; immediately, the ovaries were fixed in Bouin's solution, and, thereafter, embedded in paraffin and sectioned (5 µm). Around 15–19 slides were selected (at least 8–10 sections apart) from serial sections of each ovary from each mouse. These slides were stained with hematoxylin and eosin, and the number of CL and anovulatory follicles were counted in ovarian sections using light microscopy (Olympus DX50; Olympus America Inc., Center Valley, PA, USA).

Indexes of ovulation and fertilization
The following information was recorded for each dam: number of CL, number of anovulatory follicles, total number of recovered oocytes and zygotes, number of unfertilized oocytes, and number of fertilized zygotes. The AR was obtained by dividing, in every mouse, the total number of anovulatory follicles by the total number of anovulatory follicles and CL. The rate of recovery was obtained by dividing, in every mouse, the total number of recovered oocytes and zygotes by the total number of CL. The rate of unfertilized oocytes was obtained by dividing the number of unfertilized oocytes by the total number of recovered oocytes and embryos. The rate of viability resulted from dividing the number of viable zygotes by the number of recovered oocytes and zygotes. For clarity, all rates are expressed as percentages within this article.

Ultrasonographic evaluation of conceptuses
Ultrasound scanning was performed using two different machines; an Aloka 2500 equipped with a multi-frequency (7.5–10 MHz) sectorial array probe and a Siemens Antares connected to a multi-frequency (7.5–10 MHz) linear array probe. For examination, the mice were manually restrained in dorsal recumbence. Anesthesia was avoided for preventing any effect on pregnancy (Mazze et al. 1985). In order to diminish animal distress, abdominal hair was not clipped; the presence of air between the skin and the transducer, which makes the quality of the images worse, was avoided by abundantly wetting the abdomen with carboxymethylcellulose gel. Scanning was done by placing the transducer on the abdominal wall and moving it in different directions for viewing the uterine horns and their content; when the presence of gestational structures was determined, the number of embryos/fetuses was determined by placing the transducer in one side of the abdomen and moving it to the other side.

Indexes of prolificacy and embryo losses
The following information was recorded in every scan for each dam: number of embryos, number of living embryos, and number of dead embryos by lack of heart beats and/or appearance of reabsorption processes. Every day, the presence and the number of embryo losses were obtained by determining dead embryos and/or disappearance of embryos when compared with the previous day. The rate of embryo losses was obtained, for the entire pregnancy and every day, by dividing the number of embryo losses by the total number of embryos at first scanning. For clarity, all rates are expressed as percentages within this article.

Statistical analysis
The effects of genotype on indexes of ovulation, fertilization, and prolificacy were tested by ANOVA. The effects of genotype on the presence and number of embryo losses throughout the pregnancy were determined by ANOVA for repeated measures (split-plot ANOVA). All results were expressed as mean±S.E.M. and statistical significance was accepted for P<0.05.

	Declaration of interest

Top Abstract Introduction Results Discussion Materials and Methods Declaration of interest Funding Acknowledgements References

 
There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

	Funding

Top Abstract Introduction Results Discussion Materials and Methods Declaration of interest Funding Acknowledgements References

 
Current study was developed under a collaborative project (CC07-018) between CNIC and INIA. 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.

	Acknowledgements

Top Abstract Introduction Results Discussion Materials and Methods Declaration of interest Funding Acknowledgements References

 
The authors appreciatively thank Dr J M Redondo for cession of mice used in this experiment and the staff of CNIC Animal Unit and P Aranda, A Castro and V Vera from UCM for skilled technical assistance. The help, providing different ultrasound machines and probes, from A Vidaurrazaga (Aloka España, Madrid, Spain) and I Cisneros (RX Cisneros Electromedicina, Madrid, Spain) is also acknowledged.

Received 8 May 2008
First decision 11 June 2008
Revised manuscript received 23 July 2008
Accepted 28 July 2008

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