RESEARCH ARTICLE Molecular Reproduction & Development 81:918–927 (2014)
Supplementation of Bovine Embryo Culture Medium With L-Arginine Improves Embryo Quality via Nitric Oxide Production ~ PRISCILA DI PAULA BESSA SANTANA,1* THIAGO VELASCO GUIMARAES SILVA,1 NATHALIA NOGUEIRA DA COSTA,1 1 2 BRUNO BARAUNA DA SILVA, TIMOTHY FREDERICK CARTER, MARCELA DA SILVA CORDEIRO,3 MARTINS DA SILVA,4 SIMONE DO SOCORRO DAMASCENO SANTOS,1 ANDERSON MANOEL HERCULANO,5 BRUNO JOSE DOS SANTOS MIRANDA1 PAULO ROBERTO ADONA,6 OTAVIO MITIO OHASHI1, AND MOYSES 1
~o-Poço, Para , Brazil Federal Rural University of Amazon, Capita Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada 3 , Abaetetuba, Para , Brazil Federal Institute of Education, Science and Technology of Para 4 , Bele m, Para , Brazil Laboratory of Parasitology, Institute of Biological Science, Federal University of Para 5 , Bele m, Para , Brazil Laboratory of Neuroendocrinology, Institute of Biological Science, Federal University of Para 6 , Londrina, Parana , Brazil University of Northern Parana 2
SUMMARY Nitric oxide (NO) is a cell-signaling molecule that regulates a variety of molecular pathways. We investigated the role of NO during preimplantation embryonic development by blocking its production with an inhibitor or supplementing in vitro bovine embryo cultures with its natural precursor, L-arginine, over different periods. Endpoints evaluated included blastocyst rates, development kinetics, and embryo quality. Supplementation with the NO synthase inhibitor N-Nitro-L-arginine-methyl ester (L-NAME) from Days 1 to 8 of culture decreased blastocyst (P < 0.05) and hatching (P < 0.05) rates. When added from Days 1 to 8, 50 mM L-arginine decreased blastocyst rates (P < 0.001); in contrast, when added from Days 5 to 8, 1 mM L-arginine improved embryo hatching rates (P < 0.05) and quality (P < 0.05) as well as increased POU5F1 gene expression (P < 0.05) as compared to the untreated control. Moreover, NO levels in the medium during this culture period positively correlated with the increased embryo hatching rates and quality (P < 0.05). These data suggest exerts its positive effects during the transition from morula to blastocyst stage, and that supplementing the embryo culture medium with L-arginine favors preimplantation development of bovine embryos.
Mol. Reprod. Dev. 81: 918927, 2014. ß 2014 Wiley Periodicals, Inc. Received 31 March 2014; Accepted 1 August 2014
Corresponding author: Priscila Di Paula Bessa Santana Institute of Biological Science Bele m Federal University of Para PA, Brazil. 66075-900. E-mail: [email protected]
Grant sponsor: Foundation of research state (FAPESPA); support of Para Grant sponsor: National council of science and technology (CNPq); Grant sponsor: University of northern (UNOPAR) Parana
Published online 18 September 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22387
INTRODUCTION Nitric oxide (NO), a free radical with important physiological roles, can be derived from artificial NO donors such as sodium nitroprusside (SNP) and 2,20 -(hydroxynitrosohydrazino)
ß 2014 WILEY PERIODICALS, INC.
Abbreviations: D#, culture day #; L-NAME, N-Nitro-L-arginine-methyl ester; NO, nitric oxide; NOS, nitric oxide synthase.
L-ARGININE IMPROVES
bisethanamine (NONOate) (Scatena et al., 2005), but is more commonly synthesized from the amino acid L-arginine by nitric oxide synthase (NOS) (Bruckdorfer, 2005). Each of the three NOS isoformsone inducible (iNOS) and two constitutive (cNOS), neuronal and endothelial, respectively (Bruckdorfer, 2005)are utilized in a tissue-dependent manner according to the physiological function of NO in the particular location. Given its lipophilic nature, NO readily diffuses through the cell membrane into the cytosol, where it binds to guanylate cyclase and promotes the generation of cGMP (Bruckdorfer, 2005). NO can also interact with several proteins via S-nitrosylation, a post-translational modification that modulates the activity of oxidases, reductases, kinases, and GTPases (Hess and Stamler, 2012), thereby influencing cell-signaling pathways such as apoptosis, differentiation, and cell division (Wang, 2012). Within the reproductive system, NO is reported to play roles in a variety of physiological mechanisms, including oocyte maturation, parthenogenetic activation (Chmelikova et al., 2010; Jeseta et al., 2012), embryo implantation (Harris et al., 2008), and neuroendocrine modulation, such as the activity and secretion of GnRH (Bellefontaine et al., 2011). In the uterine tube, for example, NO production was selectively upregulated by estradiol in a select period, resulting in modified epithelial cell ciliary activity and smooth muscle relaxation in the isthmus, which are consistent with roles for NO in important events such as oocyte maturation and gamete transport (Lapointe et al., 2006; Yilmaz et al., 2011). Consistent with this model, inhibition of NO production impaired the in vitro progression of bovine oocytes to metaphase II (Schwarz et al., 2010) and decreased the percentage of acrosome-reacted bovine sperm (O’Flaherty et al., 2004). The role of NO on pre-implantation embryo development has also been investigated, although mainly in humans and mice. For instance, in vivo-derived mouse embryos produced NO (Gouge et al., 1998); inhibiting this production impaired embryonic development beyond the two-cell stage (Kim et al., 2004; Manser et al., 2004). High NO concentrations, on the other hand, also resulted in the developmental arrest of in vitro-produced murine embryos at the two-cell stage (Chen et al., 2001; Gouge et al., 1998), which coincides with the period of embryonic genome activation in this species (Latham and Schultz, 2001). Hence, while there is no consensus on the exact role of NO during embryo development, these reports together suggest that the amount of NO produced may be critical, possibly playing different roles depending upon the stage, such as that of embryonic genome activation (Chen et al., 2001; Kim et al., 2004; Tranguch et al., 2003). Previous studies have taken advantage of artificial NO donors to ascertain the role of this signaling molecule on in vitro embryo development (Gouge et al., 1998; Chen et al., 2001; Tranguch et al., 2003; Manser et al., 2004; Manser and Houghton, 2006; Tesfaye et al., 2006). While a few studies have addressed the role of NO during of bovine preimplantation embryo development (Tranguch et al., 2003; Kim et al., 2004; Tesfaye et al., 2006), none have
Mol. Reprod. Dev. 81:918–927 (2014)
EMBRYO QUALITY
used the natural precursor, L-arginine. We therefore used L-arginine supplementation to investigate the role of NO production at two key bovine embryo development stages, namely embryonic genome activation and blastocyst formation.
RESULTS Inhibition of NO Production at Specific Stages of Embryo Development Negatively Impacts Blastocyst Formation We first investigated the effects of inhibiting NO production with the addition of 10 mM N-Nitro-L-arginine-methyl ester (L-NAME) (Tesfaye et al., 2006) either before or after Day 4 (D4), or throughout the in vitro culture period. Inhibitor treatment had no effect on cleavage rates (data not shown). Inhibiting NO production during the first half of the culture period (N1-4) yielded blastocyst development rates similar to those in the control group (P > 0.05) (Fig. 1), with only a slight decrease in hatching rates (P > 0.05) (Fig. 2A).
Figure 1. Effect of the NOS inhibitor L-NAME on bovine embryo development. L-NAME (10 mM) was added to the medium at different periods of in vitro culture to investigate the effect of NO on bovine embryo development. A: Experimental design: Following in vitro fertilization (IVF), in vitro culture medium was supplemented with L-NAME from D1 to D4 (N1-4), D4 to D8 (N4-8), or D1 to D8 (N1-8) of culture. Arrows denote the time of L-NAME addition. Medium was replaced for all treatments on D4 of culture. The control group was cultured without inhibitor. B: D8 blastocyst rates. NOS inhibition from D1 to D8 of culture reduced blastocyst rates (P < 0.05). Values are expressed as mean standard deviation. Different letters denote significant differences among treatments.
919
Molecular Reproduction & Development
SANTANA
ET AL.
from D1 to D8 of in vitro culture. This period was chosen because L-NAME-dependent NO inhibition throughout the culture period negatively impacted embryo kinetics and quality. Culture medium supplemented with 1 or 10 mM L-arginine had no effect on embryo development, yielding similar blastocyst rates as well as embryo development kinetics and embryo quality scores as those observed in the control group (P > 0.05) (Figs. 3 and 4). Conversely, 50 mM L-arginine impaired embryo development, specifically reducing blastocyst formation (P < 0.001) and good-quality embryo rates (P < 0.05) when compared to the control group (Figs. 3 and 4). The highest L-arginine concentration also completely inhibited blastocyst hatching (P < 0.001).
Effect of L-Arginine Supplementation from D5 to D8 of Embryo Culture The above results clearly suggested an effect of NO on the kinetics of embryo development. This prompted us to investigate the role of L-arginine on blastocyst formation, specifically supplementing the in vitro culture medium from D5 to D8. While 1 mM L-arginine did not affect blastocyst rates, it did improve blastocyst hatching rates (P < 0.05) and quality (P < 0.001) compared to results in the control group (Figs. 5 and 6). Total cell number in hatched
Figure 2. Effect of the NOS inhibitor L-NAME on D8 bovine embryo development kinetics and quality. In vitro culture medium was supplemented with L-NAME from D1 to D4 (N1-4), D4 to D8 (N4-8), or D1 to D8 (N1-8) of culture. A: Inhibition of NO production decreased hatched blastocyst rates in the N4-8 and N1-8 groups (P < 0.05) as compared to untreated controls. B: Embryo quality decreased with NOS inhibition in the N1-8 group (P 0.05) as compared to untreated controls. Values are expressed as mean standard deviation. Bars with different capital letters denote significant differences among experimental groups. Bars with different lowercase letters denote significant differences among categories within an experimental condition.
Conversely, while inhibition during the second half of the culture period (N4-8) did not affect blastocyst rates (Fig. 1), it did reduce hatched-blastocyst rates (P < 0.05) compared to untreated controls (Fig. 2A). Inhibition during the entire culture period (N1-8) was detrimental to embryo production, reducing all blastocyst and hatched-blastocyst rates as well as negatively affecting blastocyst quality (P < 0.05) (Fig. 2A,B).
Effect of L-Arginine Supplementation on Embryo Development Culture medium was supplemented with different concentrations of L-arginine, a natural precursor of nitric oxide,
920
Figure 3. Effect of L-arginine on in vitro bovine embryo development.
A: Experimental design: Following in vitro fertilization (IVF), zygotes were cultured in medium supplemented with 1, 10, or 50 mM L-arginine. Arrows denote the addition of L-arginine to the in vitro culture medium. Medium was replaced on D4 for all treatments. The control group was cultured without L-arginine. B: D8 blastocyst rates were significantly lower with the addition of 50 mM L-arginine as compared to all other treatments (P < 0.001). Values are expressed as mean standard deviation. Different letters denote significant differences among treatments.
Mol. Reprod. Dev. 81:918–927 (2014)
L-ARGININE IMPROVES
EMBRYO QUALITY
Figure 5. Effect of L-arginine addition to the in vitro culture medium on
bovine blastocyst formation. A: Experimental design: Following in vitro fertilization (IVF), L-arginine (Arg-1 mM) was added from D5 to D8 of culture (as indicated by the arrow). The control group was cultured without L-arginine. B: D8 blastocyst rates. Values are expressed as mean standard deviation.
Figure 4. Effect of different concentrations of L-arginine on in vitro bovine embryo development. Zygotes were cultured in medium supplemented with 1, 10, or 50 mM L-arginine. A: D8 development kinetics. B: Embryo quality. Fifty millimolar L-arginine inhibited blastocyst hatching (P < 0.001) and depressed embryo quality (P < 0.001). Different capital letters denote significant differences among experimental groups. Different lowercase letters denote significant differences among categories within an experimental condition.
period. When NO metabolite values were normalized to the number of embryos in the particular droplet, culture in L-arginine containing medium contained a 3-fold increase (P < 0.05) in metabolite abundance per embryo as compared to the control group. Remarkably, NO production per embryo, as assessed by the concentration of NO metabolites in the cultured medium, positively correlated to blastocyst hatching rates (R2 ¼ 92.8%, P < 0.05) and the percentage of good-quality (grade 1) embryos (R2 ¼ 90.7%, P < 0.05) (Fig. 7).
Gene Expression Analysis blastocysts was not different (P > 0.05) between L-arginine and control treatments (192.6 22.0 vs 196.72 33.1, respectively).
Measurement of NO Production Without L-arginine supplementation, there was a slight increase in NO production on D5D8 as compared to D1D5 of in vitro culture, which corresponds to the culture period immediately preceding and leading to blastocyst formation (Table 1). Addition of 1 mM L-arginine to the culture medium from D5 to D8 of in vitro culture resulted in a 2.4-fold increase (P < 0.05) in NO production as compared to the untreated control group for the same culture
Mol. Reprod. Dev. 81:918–927 (2014)
The relative expression of POU5F1 transcript was quantified in in vitro-cultured bovine embryos as a proxy to assess embryo quality (Niemann and Wrenzycki, 2000; Yao et al., 2009). POU5F1 expression was increased 0.87-fold by the presence of L-arginine when compared with the control group cultured in the absence of L-arginine (P < 0.05) (Fig. 8).
DISCUSSION Previous studies had shown that L-NAME, a NOS inhibitor, was able to block in vitro murine (Barroso et al., 1998; Gouge et al., 1998; Chen et al., 2001; Kim et al., 2004; Manser et al., 2004) and bovine (Tesfaye et al., 2006)
921
Molecular Reproduction & Development
SANTANA
ET AL.
TABLE 1. Concentration of NO Metabolites in the Culture Medium of D8 In Vitro-Produced Bovine Embryos Concentration of nitrate (NO2)/ nitrite (NO3) (mM)
Control, D1-D5b Control, D5-D8b c L-arginine, D5-D8
Total
Per embryoa
1.68 0.53A 2.10 0.93A,B 5.07 1.89B
0.13 0.08A 0.40 0.04B
The results are average of at least 3 independent experiments. Mean standard deviations are shown. Analyzed by ANOVA (Tukey; P < 0.05). A,B Different capital-letter superscripts denote significant differences (P < 0.05) within a column. a Control D1-D5 and D5-D8 correspond to the measurement of NO production in the culture medium from D1 to D5 or D5 to D8 of culture, without L-arginine supplementation. b 1 mM L-arginine was added to the culture medium from D5 to D8 of in vitro culture. c Total concentration of metabolites divided by the number of blastocysts produced in that droplet of medium was used for analysis.
Figure 6. Effect of L-arginine addition during D5 to D8 of in vitro
culture on bovine embryo development. A: D8 development kinetics. B: Embryo quality. Supplementation with L-arginine (Arg-1 mM) increased hatching rates (P < 0.05) and improved blastocyst quality (P < 0.001) as compared to the control group. Values expressed as mean standard deviation. Different capital letters denote significant differences among experimental groups. Different lowercase letters denote significant differences among categories within an experimental condition.
embryo development. This prompted us to investigate the role of NO production during specific periods of in vitro bovine embryo culture, focusing on the periods of embryonic genome activation and blastocyst formation. We found that L-NAME adversely affected blastocyst hatching rates and embryo quality when added starting at the stage immediately following embryonic genome activation (D4D8). Moreover, NOS inhibition decreased blastocyst rates when performed during the entire embryo culture period (D1D8). These results contrast with previous reports showing that using the same concentration of L-NAME (10 mM) during the entire culture period completely arrested in vitro bovine and murine embryo development
922
on culture D9 and D4, respectively (Chen et al., 2001; Tesfaye et al., 2006). A direct comparison between studies is not feasible, however, given that different embryo culture media and conditions were usednamely that different concentrations of amino-acid substrates in each media are available for NO synthesis, and differences in the overall conditions of each in vitro culture system can influence the endpoint results of these studies. In addition, our study specifically determined that inhibition of NO synthesis during the early culture period, e.g., before genome activation, did not affect later development, except for a slight decline in blastocyst hatching rates. We thus speculated that any detrimental effect occurring early on could be later overcome if the source of inhibition was removed. Altogether, these results support the notion that NO production during bovine preimplantation embryogenesis is required mainly during the period immediately following genome activation. We also showed that supplementing bovine embryo culture medium with L-arginine, a natural NO synthesis precursor, improved blastocyst hatching rates and embryo quality. The positive correlation with increased NO levels in the culture medium suggests that L-arginine exerts its positive effects via enhanced NO production and that NO is important for preimplantation bovine embryos. L-arginine supplementation did not, however, increase blastocyst rates in our study, as previously shown for murine zygotes (Kim et al., 2004). A caveat that prevents the direct comparison between our model and the murine study is that L-arginine-supplemented modified Whitten’s culture medium was directly microinjected into the cytoplasm of murine fertilized oocytes (5 hr after insemination). Microinjected L-arginine concentrations higher than 1 mM caused embryo degeneration (Kim et al., 2004), which agrees with the decreased embryo development rates and quality observed in our study when 10 and 50 mM L-arginine was used. We therefore speculate that supplementation
Mol. Reprod. Dev. 81:918–927 (2014)
L-ARGININE IMPROVES
EMBRYO QUALITY
Figure 7. Correlation between per-embryo NO-metabolite production and blastocyst hatching rates or embryo quality. A: Hatching rate. B: Embryo quality. NO2/NO3 production was calculated from the total concentration of NO2/NO3 per culture droplet divided by the total number of blastocysts in that droplet. The results are an average of at least three independent experiments. Quantity of metabolites in the D8 culture medium was considered an indicator of NO production by the embryos. NO production was positively correlated with blastocyst hatching rates (R2 ¼ 92.8%, P < 0.05) and the percentage of good-quality (grade 1) embryos (R2 ¼ 90.7%, P < 0.05).
rather than microinjection of L-arginine is not sufficient to improve blastocyst formation, although it can impact developmental kinetics and embryo-quality parameters through the regulation of metabolism. Notably, there is a concentration threshold above which L-arginine becomes potentially cytotoxic. Indeed, overproduction of NO may result in the accumulation of peroxynitrite, a potent oxidative agent (Riobo et al., 2001), providing a plausible mechanism by which excess NO may be damaging embryos. Our results are consistent with tight regulation of the arginine/NO system during specific stages of bovine embryo development. This finding is consistent with earlier
Figure 8. Relative abundance of POU5F1 transcripts from bovine embryos cultured in the absence or presence of L-arginine (Arg-1 mM) from D5 to D8 of culture. Medium supplementation with L-arginine yielded an increase (P < 0.05) in POU5F1 expression. Asterisk denotes a significant difference.
Mol. Reprod. Dev. 81:918–927 (2014)
observations regarding the differential expression of amino acid transporter proteins and nitric oxide synthases, which respectively regulate L-arginine uptake and its conversion to NO at different stages of embryo development (Van Winkle and Campione, 1990; Van Winkle, 2001; Tesfaye et al., 2006). These expression profiles also corroborate with our observation that L-arginine added only from D5 to D8 of culture improved blastocyst hatching and embryo quality, whereas continuous exposure from D1 to D8 did not. Thus, our results strongly support a model wherein the morula and blastocyst stages are critically dependent upon NO metabolism. Increased NO production in our embryo culture system also correlated with increased blastocyst hatching rates. This is particularly important given how critical the hatching process is for subsequent development and implantation (Petersen et al., 2005; Bazer et al., 2009; Seshagiri et al., 2009). One possible mechanism for NO-enhanced hatching may be through prostaglandin synthesis, which promotes mouse blastocyst hatching (Huang et al., 2004). For example, NO may stimulate cyclooxygenase activity, possibly via S-nitrosylation (Cuzzocrea and Salvemini, 2007). Indeed, cyclooxygenase expression increased precisely at the morula and blastocyst stages of in vitroproduced bovine embryos (Saint-Dizier et al., 2011). Another possible source of the L-arginine enhanced hatching rates could be through mechanisms activated by L-arginine uptake, e.g., cationic amino acid transporters, and its effects on osmolality (Van Winkle, 2001). In this regard, osmotic modifications induced by L-arginine uptake could further enhance blastocoel expansion and, eventually, blastocyst hatching. Finally, a recent study showed that among all the amino acids, arginine and leucine were able to induce mTOR (mammalian target of rapamicyn) activity in mice, which activates the downstream signaling cascade
923
Molecular Reproduction & Development
regulating implantation (Gonzalez et al., 2012). While the L-arginine/NO system has been extensively studied in murine and human models (Gao et al., 2009; Kim et al., 2011a,b,c), little is known about how it functions in the bovine; therefore, extrapolation of such pre-implantation processes is difficult and entirely speculative given the physiological differences in implantation mechanisms among the cited species (Bazer et al., 2010). In an attempt to identify possible targets for the L-arginine/NO effects on embryo quality, we evaluated expression of the POU5F1 gene, a marker for pluripotency in bovine blastocysts (Niemann and Wrenzycki, 2000; Yao et al., 2009). POU5F1 is a member of the POU transcription factor family found to be highly expressed in pluripotent stem cells (Shi and Jin, 2010), and its expression is commonly lower in nuclear-transfer embryos as compared to in vitro fertilization-derived embryos, which correlates with developmental failure (Boiani et al., 2002; Aston et al., 2010). We observed that the presence of L-arginine seemed to stimulate POU5F1 expression, suggesting that the pluripotent state of the embryonic cells is heightened and thus results in improved developmental kinetics and embryo quality. As POU5F1 is also a key, differentially regulated factor between the trophoblast and inner cell mass of bovine embryos (Madeja et al., 2013), we speculate that the L-arginine/NO system could exert different effects in these two compartments. Altogether, the findings of the present study provide evidence that NO can interact with different signaling events in order to regulate preimplantation development of bovine embryos cultured in vitro. Specifically, our results demonstrate a relationship between NO and blastocyst hatching, as well as embryo quality and development, and further show that this relationship can be modified by the addition of L-arginine to the embryo culture medium, especially during the morula-to-blastocyst transition, Future studies should address how L-arginine/NO mechanisms affect bovine embryo metabolism and cell differentiation.
MATERIALS AND METHODS Reagents All chemicals were purchased form Sigma Chemical Company (St. Louis, MO), unless otherwise stated. Both L-arginine and L-NAME were dissolved in water according to the manufacturer’s recommendation.
In Vitro Embryo Production Cumulus-oocyte complexes (COCs) were recovered by follicular aspiration from abattoir ovaries, and compact COCs were selected for in vitro maturation in tissue culture medium (TCM) 199 supplemented with 2.2 g/L sodium bicarbonate, 11 mg/mL pyruvate, 50 mg/mL gentamicin, 10% fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY), 0.5 mg/mL follicle-stimulating hormone (Folltropin) (Bioniche Animal Health, Belleville, Ont., Canada), and
924
SANTANA
ET AL.
5.0 mg/mL luteinizing hormone (Lutropin) (Bioniche Animal Health). COCs were incubated for 24 hr. Frozen-thawed bull (Bos taurus taurus) semen was prepared for in vitro fertilization (IVF) by density-gradient centrifugation in Percoll. Spermatozoa were diluted to a concentration of 2 106 cells/mL in Tyrode’s albuminlactate-pyruvate (TALP) medium (Parrish et al., 1988). Expanded COCs were then placed into sperm-containing 80-ml IVF droplets and co-incubated for 24 hr. Following IVF, groups of 20 presumptive zygotes were transferred to 100-ml droplets of SOF medium (Holm et al., 1999) supplemented with 5% FBS, 50 mg/mL gentamicin, and 6 mg/mL bovine serum albumin (BSA) for in vitro culture. In this study, embryos were not co-cultured with granulosa cells due to their potential interference with NO measurements. All in vitro maturation, fertilization, and culturing were undertaken in a 5% CO2 atmosphere in humidified air at 38.58C.
Experimental Design Three experiments were designed in order to investigate if NO is involved in bovine embryo development. The goal of the first experiment was to inhibit NO production before and after D4 of embryo culture. L-NAME (Tesfaye et al., 2006), a NO synthesis inhibitor, was added to the embryo culture medium at a final concentration of 10 mM from D1 to D4 (N1-4), D4 to D8 (N4-8), or D1 to D8 (N1-8) of in vitro culture. A control group was cultured without the inhibitor. For all groups, the culture medium was replaced on D4. The second experiment was conducted to address the effect of L-arginine on embryo development. For this purpose, the in vitro culture medium was supplemented with 1, 10, or 50 mM L-arginine from D1 to D8 of the culture period. The control group had no L-arginine supplementation. In the third experiment, the L-arginine concentration providing the best results in the second experiment (1 mM) was added from D5 to D8 of the in vitro culture period to assess its effects upon blastocyst formation and total number of cells. Before D5, all groups were cultured in control culture medium (without supplementation). Measurements of NO production were performed before and after induction with L-arginine. For all experiments, embryos were assessed for cleavage rates on D2 of culture and for blastocyst rates, embryo quality, and development kinetics on D8 of culture.
Embryo Endpoint Evaluations Cleavage and blastocyst rates were evaluated on D2 and D8 of the culture period, respectively (the day of in vitro fertilization was set as D0). Blastocyst rates were calculated based on the number of oocytes fertilized. According to the criteria established by Stringfellow and Seidel (1998), blastocysts were classified for developmental kinetics and morphological quality using an inverted fluorescence microscope (Eclipse TE 300, Nikon Instruments Inc., Melville, NY). Hatched blastocysts displayed no zona pellucida and a large trophoblast diameter. Early blastocysts displayed a
Mol. Reprod. Dev. 81:918–927 (2014)
L-ARGININE IMPROVES
thick zona pellucida and a smaller trophoblast diameter when compared expanded blastocysts, which had thinning zona pellucidae and an increasing number of cells. Embryos were graded based upon their morphological quality. Grade 1 or very-good-quality embryos had an inner cell mass with symmetrical or few very slightly asymmetrical blastomeres of a light appearance. Grade 2 or fair-quality embryos had an inner cell mass with very slightly asymmetrical blastomeres. And, Grade 3 or poor-quality embryos had an inner cell mass with widespread, marked asymmetry, and a darker appearance. Following morphological evaluation, hatched blastocysts were fixed in 1% formol-saline solution, and stained with the DNA dye Hoechst 33342 (5 mg/mL) in phosphatebuffered saline. Total cell number was counted using a fluorescence microscope (Eclipse 50i, Nikon Instruments Inc.).
Measurement of NO Production Given its short half-life, NO was quantified indirectly based on its nitrite and nitrate (NO3/NO2) metabolite production (Flora-Filho and Zilberstein, 2000). For this purpose, droplets of medium from D8 of culture were stored at 208C for later analysis using the Griess colorimetric method (Ricart-Jane et al., 2002). Griess reagent is composed of 2% (w/v) sulphanilamide and 0.2% (w/v) N-(1-naphthyl) ethylene-diamine in deionized water. These components react with NO metabolites in a test solution, yielding a purple azo-dye product with a peak absorbance at 540 nm. Readings were performed using a microplate reader (BIO-RAD 450, Bio-Rad Laboratories, Hercules, CA). For quantification, we generated a standard curve with sodium nitrate/nitrite solution diluted in in vitro culture medium with concentrations ranging from 0 to 100 mM. To avoid quantification bias, the medium used for setting the standard curve was aliquoted from exactly the same source medium used for in vitro culture. The amount of nitric oxide produced per embryo was calculated by dividing total metabolite concentration by the total number of blastocysts cultured in the corresponding droplet.
Gene Expression Analysis of In Vitro-Produced Bovine Embryos For relative gene expression quantification, ten sets of embryos per group were used in a total of three replicates. The embryos were frozen in RNAlater1 solution (Ambion, Life Technology, Carlsbad, CA). Aliquots were kept frozen at 808C until RNA extraction, which was performed using Trizol1 reagent (Invitrogen, Molecular Research Center, Cincinnati, OH), according to the manufacturer’s instructions. RNA samples were resuspended in 10 mL of diethylpyrocarbonate (DEPC) H2O (Amresco, Cochran, GA) and kept frozen at 808C until needed. Reverse-transcriptase PCR was performed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Auckland, New Zealand), according to the manufacturer’s protocol.
Mol. Reprod. Dev. 81:918–927 (2014)
EMBRYO QUALITY
Real-time PCR was performed in an ABI PRISM 7500 Real Time PCR system (Applied Biosystems), using the primers previously reported by Costa et al. (Costa et al., 2013) for histone H2a.1 (forward, 50 -GTCGTGGCAAGCAAGGAG/reverse, 50 -GATCTCGGCCGTTAGGTACTC) and the POU transcription factor family, POU5F1, also known as OCT-4 (forward, 50 -GGTGTTCAGCCAAACGACTATC/reverse, 50 -TCTCTGCCTTGCATATCTCCTG). Each 20-mL PCR reaction mixture consisted of 10 mL of SYBR Green PCR Master Mix (Applied Biosystems), 0.2 mM of each forward and reverse primer, and 10 mL of a 1:20-diluted cDNA sample. The following cycling conditions were applied for initial amplification: 958C for 10 min, and 45 cycles at 68C for 1 min. The PCR specificity was verified by checking the corresponding dissociation curves. Optimal primer concentration was 0.2 mM for POU5F1 and 0.1 mM for histone H2a.z, a previously described endogenous control for embryos (Vigneault et al., 2007). The amplification efficiencies of each gene were calculated using the DDCT method, selecting only values between 1.9 and 2.0 (Livak and Schmittgen, 2001). To ensure equal experimental conditions, the same reagents were used to amplify all genes with assays run simultaneously using the same PCR plate. The relative quantity of target transcripts was corrected based upon the quantity of H2a.z.
Statistical Analysis All experiments were performed starting with 30 oocytes per treatment. Within experiments and an oocyte batch, all treatments were tested in parallel, with at least four replicates per experiment performed. Data for cleavage and blastocyst rates, as well as development kinetics, embryo quality, total number of cells, and NO production were analyzed by ANOVA with Tukey’s post-hoc test applied when significant differences were identified. Regression analysis was performed to evaluate the correlation between NO production and development kinetics or embryo quality. Analysis of data for relative gene expression was performed using the t-test. SigmaPlot1 11.0 software was used for analysis, with the significance level set at P < 0.05.
ACKNOWLEDGMENTS This study was supported by Foundation of research state (FAPESPA), National council of support of Para science and technology (CNPq), and University of northern (UNOPAR). The authors thank Drs. Edilene Oliveira Parana ^nio Loureiro Maue s for providing da Silva and Luıs Anto generous access to laboratory equipment.
REFERENCES Aston KI, Li GP, Hicks BA, Sessions BR, Davis AP, Rickords LF, Stevens JR, White KL. 2010. Abnormal levels of transcript abundance of developmentally important genes in various stages of preimplantation bovine somatic cell nuclear transfer embryos. Cell Reprogram 12:2332.
925
Molecular Reproduction & Development
Barroso RP, Osuamkpe C, Nagamani M, Yallampalli C. 1998. Nitric oxide inhibits development of embryos and implantation in mice. Mol Hum Reprod 4:503507. Bazer FW, Spencer TE, Johnson GA, Burghardt RC, Wu G. 2009. Comparative aspects of implantation. Reproduction 138:195209. Bazer FW, Wu G, Spencer TE, Johnson GA, Burghardt RC, Bayless K. 2010. Novel pathways for implantation and establishment and maintenance of pregnancy in mammals. Mol Hum Reprod 16:135152. Bellefontaine N, Hanchate NK, Parkash J, Campagne C, de Seranno S, Clasadonte J, d’Anglemont de Tassigny X, Prevot V. 2011. Nitric oxide as key mediator of neuron-to-neuron and endothelia-to-glia communication involved in the neuroendocrine control of reproduction. Neuroendocrinology 93: 7489. Boiani M, Eckardt S, Scholer HR, McLaughlin KJ. 2002. Oct4 distribution and level in mouse clones. consequences for pluripotency. Genes Dev 16:12091219. Bruckdorfer R. 2005. The basics about nitric oxide. Mol Aspects Med 26:331. Chen HW, Jiang WS, Tzeng CR. 2001. Nitric oxide as a regulator in preimplantation embryo development and apoptosis. Fertil Steril 75:11631171. Chmelikova E, Jeseta M, Sedmikova M, Petr J, Tumova L, Kott T, Lipovova P, Jilek F. 2010. Nitric oxide synthase isoforms and the effect of their inhibition on meiotic maturation of porcine oocytes. Zygote 18:235244. Costa NN, Cordeiro MS, Silva TV, Sastre D, Santana PPB, Sa AL, Sampaio RV, Santos SS, Adona PR, Miranda MS, Ohashi OM. 2013. Effect of triiodothyronine on developmental competence of bovine oocytes. Theriogenology 80:295301. Cuzzocrea S, Salvemini D. 2007. Molecular mechanisms involved in the reciprocal regulation of cyclooxygenase and nitric oxide synthase enzymes. Kidney Int 71:290297. Flora-Filho R, Zilberstein B. 2000. Æxido nıtrico: O simples mensageiro percorrendo a complexidade. Metabolismo, sıntese e funçes. Rev Ass Med Brasil 46:265271. Gao H, Wu G, Spencer TE, Johnson GA, Bazer FW. 2009. Select nutrients in the ovine uterine lumen. III. Cationic amino acid transporters in the ovine uterus and peri-implantation conceptuses. Biol Reprod 80:602609. Gonzalez IM, Martin PM, Burdsal C, Sloan JL, Mager S, Harris T, Sutherland AE. 2012. Leucine and arginine regulate trophoblast motility through mTOR-dependent and independent pathways in the preimplantation mouse embryo. Dev Biol 361:286300. Gouge RC, Marshburn P, Gordon BE, Nunley W, Huet-Hudson YM. 1998. Nitric oxide as a regulator of embryonic development. Biol Reprod 58:875879. Harris LK, McCormick J, Cartwright JE, Whitley GS, Dash PR. 2008. S-nitrosylation of proteins at the leading edge of migrating
926
SANTANA
ET AL.
trophoblasts by inducible nitric oxide synthase promotes trophoblast invasion. Exp Cell Res 314:17651776. Hess DT, Stamler JS. 2012. Regulation by S-nitrosylation of protein post-translational modification. J Biol Chem 287: 44114418. Holm P, Booth PJ, Schmidt MH, Greve T, Callesen H. 1999. High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins. Theriogenology 52:683700. Huang JC, Wun WS, Goldsby JS, Matijevic-Aleksic N, Wu KK. 2004. Cyclooxygenase-2-derived endogenous prostacyclin enhances mouse embryo hatching. Hum Reprod 19:29002906. Jeseta M, Marin M, Tichovska H, Melicharova P, Cailliau-Maggio K, Martoriati A, Lescuyer-Rousseau A, Beaujois R, Petr J, Sedmikova M, Bodart JF. 2012. Nitric oxide-donor SNAP induces Xenopus eggs activation. PLoS One 7:e41509. Kim BH, Kim CH, Jung KY, Jeon BH, Ju EJ, Choo YK. 2004. Involvement of nitric oxide during in vitro fertilization and early embryonic development in mice. Arch Pharm Res 27:8693. Kim J, Burghardt RC, Wu G, Johnson GA, Spencer TE, Bazer FW. 2011a. Select nutrients in the ovine uterine lumen. IX. Differential effects of arginine, leucine, glutamine, and glucose on interferon tau, ornithine decarboxylase, and nitric oxide synthase in the ovine conceptus. Biol Reprod 84:11391147. Kim JY, Burghardt RC, Wu G, Johnson GA, Spencer TE, Bazer FW. 2011b. Select nutrients in the ovine uterine lumen. VII. Effects of arginine, leucine, glutamine, and glucose on trophectoderm cell signaling, proliferation, and migration. Biol Reprod 84:6269. Kim JY, Burghardt RC, Wu G, Johnson GA, Spencer TE, Bazer FW. 2011c. Select nutrients in the ovine uterine lumen. VIII. Arginine stimulates proliferation of ovine trophectoderm cells through MTOR-RPS6K-RPS6 signaling cascade and synthesis of nitric oxide and polyamines. Biol Reprod 84:7078. Lapointe J, Roy M, St-Pierre I, Kimmins S, Gauvreau D, MacLaren LA, Bilodeau JF. 2006. Hormonal and spatial regulation of nitric oxide synthases (NOS) (neuronal NOS, inducible NOS, and endothelial NOS) in the oviducts. Endocrinology 147: 56005610. Latham KE, Schultz RM. 2001. Embryonic genome activation. Front Biosci 6:D748759. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402408. Madeja ZE, Sosnowski J, Hryniewicz K, Warzych E, Pawlak P, Rozwadowska N, Plusa B, Lechniak D. 2013. Changes in subcellular localisation of trophoblast and inner cell mass specific transcription factors during bovine preimplantation development. Dev Biol 13:32. Manser RC, Houghton FD. 2006. Ca2þ-linked upregulation and mitochondrial production of nitric oxide in the mouse preimplantation embryo. J Cell Sci 119:20482055.
Mol. Reprod. Dev. 81:918–927 (2014)
L-ARGININE IMPROVES
Manser RC, Leese HJ, Houghton FD. 2004. Effect of inhibiting nitric oxide production on mouse preimplantation embryo development and metabolism. Biol Reprod 71:528533. Niemann H, Wrenzycki C. 2000. Alterations of expression of developmentally important genes in preimplantation bovine embryos by in vitro culture conditions: Implications for subsequent development. Theriogenology 53:2134. O’Flaherty C, Rodriguez P, Srivastava S. 2004. L-Arginine promotes capacitation and acrosome reaction in cryopreserved bovine spermatozoa. Biochim Biophys Acta 1674:215221. Parrish JJ, Susko-Parrish J, Winer MA, First NL. 1988. Capacitation of bovine sperm by heparin. Biol Reprod 38:11711180. Petersen CG, Mauri AL, Baruffi RL, Oliveira JB, Massaro FC, Elder K, Franco JG, Jr. 2005. Implantation failures. success of assisted hatching with quarter-laser zona thinning. Reprod Biomed Online 10:224229. Ricart-Jane D, Llobera M, Lopez-Tejero MD. 2002. Anticoagulants and other preanalytical factors interfere in plasma nitrate/nitrite quantification by the Griess method. Nitric Oxide 6:178185. Riobo NA, Clementi E, Melani M, Boveris A, Cadenas E, Moncada S, Poderoso JJ. 2001. Nitric oxide inhibits mitochondrial NADH: Ubiquinone reductase activity through peroxynitrite formation. Biochem J 359:139145. Saint-Dizier M, Guyader-Joly C, Charpigny G, Grimard B, Humblot P, Ponter AA. 2011. Expression of enzymes involved in the synthesis of prostaglandin E2 in bovine in vitro-produced embryos. Zygote 19:277283. Scatena R, Bottoni P, Martorana GE, Giardina B. 2005. Nitric oxide donor drugs: an update on pathophysiology and therapeutic potential. Expert Opin Investig Drugs 14:835846. Schwarz KR, Pires PR, de Bem TH, Adona PR, Leal CL. 2010. Consequences of nitric oxide synthase inhibition during bovine oocyte maturation on meiosis and embryo development. Reprod Domest Anim 45:7580. Seshagiri PB, Sen Roy S, Sireesha G, Rao RP. 2009. Cellular and molecular regulation of mammalian blastocyst hatching. J Reprod Immunol 83:7984.
Mol. Reprod. Dev. 81:918–927 (2014)
EMBRYO QUALITY
Shi G, Jin Y. 2010. Role of Oct4 in maintaining and regaining stem cell pluripotency Stem Cell Res Ther 1:39. Stringfellow DA, Seidel SM. 1998. Manual of the International Embryo Transfer Society: A procedural guide and general information for the use of embryo transfer technology emphasizing sanitary procedures. IETS: Savoy, Illinois: p 172. Tesfaye D, Kadanga A, Rings F, Bauch K, Jennen D, Nganvongpanit K, Holker M, Tholen E, Ponsuksili S, Wimmers K, Montag M, Gilles M, Kirfel G, Herzog V, Schellander K. 2006. The effect of nitric oxide inhibition and temporal expression patterns of the mRNA and protein products of nitric oxide synthase genes during in vitro development of bovine pre-implantation embryos. Reprod Domest Anim 41:501509. Tranguch S, Steuerwald N, Huet-Hudson YM. 2003. Nitric oxide synthase production and nitric oxide regulation of preimplantation embryo development. Biol Reprod 68:15381544. Van Winkle LJ. 2001. Amino acid transport regulation and early embryo development. Biol Reprod 64:112. Van Winkle LJ, Campione AL. 1990. Functional changes in cationpreferring amino acid transport during development of preimplantation mouse conceptuses. Biochim Biophys Acta 1028: 165173. Vigneault C, Gilbert I, Sirard MA, Robert C. 2007. Using the histone H2a transcript as an endogenous standard to study relative transcript abundance during bovine early development. Mol Reprod Dev 74:703715. Wang Z. 2012. Protein S-nitrosylation and cancer Cancer Lett 320:123129. Yao N, Wan PC, Hao ZD, Gao FF, Yang L, Cui MS, Wu Y, Liu JH, Liu S, Chen H, Zeng SM. 2009. Expression of interferon-tau mRNA in bovine embryos derived from different procedures. Reprod Domest Anim 44:132139. Yilmaz O, Calka J, Bukowski R, Zalecki M, Wasowicz K, Jaroszewski JJ, Markiewicz W, Bulbul A, Ucar M. 2011. Nitric oxide in the bovine oviduct: Influence on contractile activity and nitric oxide synthase isoforms localization. Theriogenology 77: 13121327.
927
Supplementation of Bovine Embryo Culture Medium With L-Arginine Improves Embryo Quality via Nitric Oxide Production ~ PRISCILA DI PAULA BESSA SANTANA,1* THIAGO VELASCO GUIMARAES SILVA,1 NATHALIA NOGUEIRA DA COSTA,1 1 2 BRUNO BARAUNA DA SILVA, TIMOTHY FREDERICK CARTER, MARCELA DA SILVA CORDEIRO,3 MARTINS DA SILVA,4 SIMONE DO SOCORRO DAMASCENO SANTOS,1 ANDERSON MANOEL HERCULANO,5 BRUNO JOSE DOS SANTOS MIRANDA1 PAULO ROBERTO ADONA,6 OTAVIO MITIO OHASHI1, AND MOYSES 1
~o-Poço, Para , Brazil Federal Rural University of Amazon, Capita Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada 3 , Abaetetuba, Para , Brazil Federal Institute of Education, Science and Technology of Para 4 , Bele m, Para , Brazil Laboratory of Parasitology, Institute of Biological Science, Federal University of Para 5 , Bele m, Para , Brazil Laboratory of Neuroendocrinology, Institute of Biological Science, Federal University of Para 6 , Londrina, Parana , Brazil University of Northern Parana 2
SUMMARY Nitric oxide (NO) is a cell-signaling molecule that regulates a variety of molecular pathways. We investigated the role of NO during preimplantation embryonic development by blocking its production with an inhibitor or supplementing in vitro bovine embryo cultures with its natural precursor, L-arginine, over different periods. Endpoints evaluated included blastocyst rates, development kinetics, and embryo quality. Supplementation with the NO synthase inhibitor N-Nitro-L-arginine-methyl ester (L-NAME) from Days 1 to 8 of culture decreased blastocyst (P < 0.05) and hatching (P < 0.05) rates. When added from Days 1 to 8, 50 mM L-arginine decreased blastocyst rates (P < 0.001); in contrast, when added from Days 5 to 8, 1 mM L-arginine improved embryo hatching rates (P < 0.05) and quality (P < 0.05) as well as increased POU5F1 gene expression (P < 0.05) as compared to the untreated control. Moreover, NO levels in the medium during this culture period positively correlated with the increased embryo hatching rates and quality (P < 0.05). These data suggest exerts its positive effects during the transition from morula to blastocyst stage, and that supplementing the embryo culture medium with L-arginine favors preimplantation development of bovine embryos.
Mol. Reprod. Dev. 81: 918927, 2014. ß 2014 Wiley Periodicals, Inc. Received 31 March 2014; Accepted 1 August 2014
Corresponding author: Priscila Di Paula Bessa Santana Institute of Biological Science Bele m Federal University of Para PA, Brazil. 66075-900. E-mail: [email protected]
Grant sponsor: Foundation of research state (FAPESPA); support of Para Grant sponsor: National council of science and technology (CNPq); Grant sponsor: University of northern (UNOPAR) Parana
Published online 18 September 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22387
INTRODUCTION Nitric oxide (NO), a free radical with important physiological roles, can be derived from artificial NO donors such as sodium nitroprusside (SNP) and 2,20 -(hydroxynitrosohydrazino)
ß 2014 WILEY PERIODICALS, INC.
Abbreviations: D#, culture day #; L-NAME, N-Nitro-L-arginine-methyl ester; NO, nitric oxide; NOS, nitric oxide synthase.
L-ARGININE IMPROVES
bisethanamine (NONOate) (Scatena et al., 2005), but is more commonly synthesized from the amino acid L-arginine by nitric oxide synthase (NOS) (Bruckdorfer, 2005). Each of the three NOS isoformsone inducible (iNOS) and two constitutive (cNOS), neuronal and endothelial, respectively (Bruckdorfer, 2005)are utilized in a tissue-dependent manner according to the physiological function of NO in the particular location. Given its lipophilic nature, NO readily diffuses through the cell membrane into the cytosol, where it binds to guanylate cyclase and promotes the generation of cGMP (Bruckdorfer, 2005). NO can also interact with several proteins via S-nitrosylation, a post-translational modification that modulates the activity of oxidases, reductases, kinases, and GTPases (Hess and Stamler, 2012), thereby influencing cell-signaling pathways such as apoptosis, differentiation, and cell division (Wang, 2012). Within the reproductive system, NO is reported to play roles in a variety of physiological mechanisms, including oocyte maturation, parthenogenetic activation (Chmelikova et al., 2010; Jeseta et al., 2012), embryo implantation (Harris et al., 2008), and neuroendocrine modulation, such as the activity and secretion of GnRH (Bellefontaine et al., 2011). In the uterine tube, for example, NO production was selectively upregulated by estradiol in a select period, resulting in modified epithelial cell ciliary activity and smooth muscle relaxation in the isthmus, which are consistent with roles for NO in important events such as oocyte maturation and gamete transport (Lapointe et al., 2006; Yilmaz et al., 2011). Consistent with this model, inhibition of NO production impaired the in vitro progression of bovine oocytes to metaphase II (Schwarz et al., 2010) and decreased the percentage of acrosome-reacted bovine sperm (O’Flaherty et al., 2004). The role of NO on pre-implantation embryo development has also been investigated, although mainly in humans and mice. For instance, in vivo-derived mouse embryos produced NO (Gouge et al., 1998); inhibiting this production impaired embryonic development beyond the two-cell stage (Kim et al., 2004; Manser et al., 2004). High NO concentrations, on the other hand, also resulted in the developmental arrest of in vitro-produced murine embryos at the two-cell stage (Chen et al., 2001; Gouge et al., 1998), which coincides with the period of embryonic genome activation in this species (Latham and Schultz, 2001). Hence, while there is no consensus on the exact role of NO during embryo development, these reports together suggest that the amount of NO produced may be critical, possibly playing different roles depending upon the stage, such as that of embryonic genome activation (Chen et al., 2001; Kim et al., 2004; Tranguch et al., 2003). Previous studies have taken advantage of artificial NO donors to ascertain the role of this signaling molecule on in vitro embryo development (Gouge et al., 1998; Chen et al., 2001; Tranguch et al., 2003; Manser et al., 2004; Manser and Houghton, 2006; Tesfaye et al., 2006). While a few studies have addressed the role of NO during of bovine preimplantation embryo development (Tranguch et al., 2003; Kim et al., 2004; Tesfaye et al., 2006), none have
Mol. Reprod. Dev. 81:918–927 (2014)
EMBRYO QUALITY
used the natural precursor, L-arginine. We therefore used L-arginine supplementation to investigate the role of NO production at two key bovine embryo development stages, namely embryonic genome activation and blastocyst formation.
RESULTS Inhibition of NO Production at Specific Stages of Embryo Development Negatively Impacts Blastocyst Formation We first investigated the effects of inhibiting NO production with the addition of 10 mM N-Nitro-L-arginine-methyl ester (L-NAME) (Tesfaye et al., 2006) either before or after Day 4 (D4), or throughout the in vitro culture period. Inhibitor treatment had no effect on cleavage rates (data not shown). Inhibiting NO production during the first half of the culture period (N1-4) yielded blastocyst development rates similar to those in the control group (P > 0.05) (Fig. 1), with only a slight decrease in hatching rates (P > 0.05) (Fig. 2A).
Figure 1. Effect of the NOS inhibitor L-NAME on bovine embryo development. L-NAME (10 mM) was added to the medium at different periods of in vitro culture to investigate the effect of NO on bovine embryo development. A: Experimental design: Following in vitro fertilization (IVF), in vitro culture medium was supplemented with L-NAME from D1 to D4 (N1-4), D4 to D8 (N4-8), or D1 to D8 (N1-8) of culture. Arrows denote the time of L-NAME addition. Medium was replaced for all treatments on D4 of culture. The control group was cultured without inhibitor. B: D8 blastocyst rates. NOS inhibition from D1 to D8 of culture reduced blastocyst rates (P < 0.05). Values are expressed as mean standard deviation. Different letters denote significant differences among treatments.
919
Molecular Reproduction & Development
SANTANA
ET AL.
from D1 to D8 of in vitro culture. This period was chosen because L-NAME-dependent NO inhibition throughout the culture period negatively impacted embryo kinetics and quality. Culture medium supplemented with 1 or 10 mM L-arginine had no effect on embryo development, yielding similar blastocyst rates as well as embryo development kinetics and embryo quality scores as those observed in the control group (P > 0.05) (Figs. 3 and 4). Conversely, 50 mM L-arginine impaired embryo development, specifically reducing blastocyst formation (P < 0.001) and good-quality embryo rates (P < 0.05) when compared to the control group (Figs. 3 and 4). The highest L-arginine concentration also completely inhibited blastocyst hatching (P < 0.001).
Effect of L-Arginine Supplementation from D5 to D8 of Embryo Culture The above results clearly suggested an effect of NO on the kinetics of embryo development. This prompted us to investigate the role of L-arginine on blastocyst formation, specifically supplementing the in vitro culture medium from D5 to D8. While 1 mM L-arginine did not affect blastocyst rates, it did improve blastocyst hatching rates (P < 0.05) and quality (P < 0.001) compared to results in the control group (Figs. 5 and 6). Total cell number in hatched
Figure 2. Effect of the NOS inhibitor L-NAME on D8 bovine embryo development kinetics and quality. In vitro culture medium was supplemented with L-NAME from D1 to D4 (N1-4), D4 to D8 (N4-8), or D1 to D8 (N1-8) of culture. A: Inhibition of NO production decreased hatched blastocyst rates in the N4-8 and N1-8 groups (P < 0.05) as compared to untreated controls. B: Embryo quality decreased with NOS inhibition in the N1-8 group (P 0.05) as compared to untreated controls. Values are expressed as mean standard deviation. Bars with different capital letters denote significant differences among experimental groups. Bars with different lowercase letters denote significant differences among categories within an experimental condition.
Conversely, while inhibition during the second half of the culture period (N4-8) did not affect blastocyst rates (Fig. 1), it did reduce hatched-blastocyst rates (P < 0.05) compared to untreated controls (Fig. 2A). Inhibition during the entire culture period (N1-8) was detrimental to embryo production, reducing all blastocyst and hatched-blastocyst rates as well as negatively affecting blastocyst quality (P < 0.05) (Fig. 2A,B).
Effect of L-Arginine Supplementation on Embryo Development Culture medium was supplemented with different concentrations of L-arginine, a natural precursor of nitric oxide,
920
Figure 3. Effect of L-arginine on in vitro bovine embryo development.
A: Experimental design: Following in vitro fertilization (IVF), zygotes were cultured in medium supplemented with 1, 10, or 50 mM L-arginine. Arrows denote the addition of L-arginine to the in vitro culture medium. Medium was replaced on D4 for all treatments. The control group was cultured without L-arginine. B: D8 blastocyst rates were significantly lower with the addition of 50 mM L-arginine as compared to all other treatments (P < 0.001). Values are expressed as mean standard deviation. Different letters denote significant differences among treatments.
Mol. Reprod. Dev. 81:918–927 (2014)
L-ARGININE IMPROVES
EMBRYO QUALITY
Figure 5. Effect of L-arginine addition to the in vitro culture medium on
bovine blastocyst formation. A: Experimental design: Following in vitro fertilization (IVF), L-arginine (Arg-1 mM) was added from D5 to D8 of culture (as indicated by the arrow). The control group was cultured without L-arginine. B: D8 blastocyst rates. Values are expressed as mean standard deviation.
Figure 4. Effect of different concentrations of L-arginine on in vitro bovine embryo development. Zygotes were cultured in medium supplemented with 1, 10, or 50 mM L-arginine. A: D8 development kinetics. B: Embryo quality. Fifty millimolar L-arginine inhibited blastocyst hatching (P < 0.001) and depressed embryo quality (P < 0.001). Different capital letters denote significant differences among experimental groups. Different lowercase letters denote significant differences among categories within an experimental condition.
period. When NO metabolite values were normalized to the number of embryos in the particular droplet, culture in L-arginine containing medium contained a 3-fold increase (P < 0.05) in metabolite abundance per embryo as compared to the control group. Remarkably, NO production per embryo, as assessed by the concentration of NO metabolites in the cultured medium, positively correlated to blastocyst hatching rates (R2 ¼ 92.8%, P < 0.05) and the percentage of good-quality (grade 1) embryos (R2 ¼ 90.7%, P < 0.05) (Fig. 7).
Gene Expression Analysis blastocysts was not different (P > 0.05) between L-arginine and control treatments (192.6 22.0 vs 196.72 33.1, respectively).
Measurement of NO Production Without L-arginine supplementation, there was a slight increase in NO production on D5D8 as compared to D1D5 of in vitro culture, which corresponds to the culture period immediately preceding and leading to blastocyst formation (Table 1). Addition of 1 mM L-arginine to the culture medium from D5 to D8 of in vitro culture resulted in a 2.4-fold increase (P < 0.05) in NO production as compared to the untreated control group for the same culture
Mol. Reprod. Dev. 81:918–927 (2014)
The relative expression of POU5F1 transcript was quantified in in vitro-cultured bovine embryos as a proxy to assess embryo quality (Niemann and Wrenzycki, 2000; Yao et al., 2009). POU5F1 expression was increased 0.87-fold by the presence of L-arginine when compared with the control group cultured in the absence of L-arginine (P < 0.05) (Fig. 8).
DISCUSSION Previous studies had shown that L-NAME, a NOS inhibitor, was able to block in vitro murine (Barroso et al., 1998; Gouge et al., 1998; Chen et al., 2001; Kim et al., 2004; Manser et al., 2004) and bovine (Tesfaye et al., 2006)
921
Molecular Reproduction & Development
SANTANA
ET AL.
TABLE 1. Concentration of NO Metabolites in the Culture Medium of D8 In Vitro-Produced Bovine Embryos Concentration of nitrate (NO2)/ nitrite (NO3) (mM)
Control, D1-D5b Control, D5-D8b c L-arginine, D5-D8
Total
Per embryoa
1.68 0.53A 2.10 0.93A,B 5.07 1.89B
0.13 0.08A 0.40 0.04B
The results are average of at least 3 independent experiments. Mean standard deviations are shown. Analyzed by ANOVA (Tukey; P < 0.05). A,B Different capital-letter superscripts denote significant differences (P < 0.05) within a column. a Control D1-D5 and D5-D8 correspond to the measurement of NO production in the culture medium from D1 to D5 or D5 to D8 of culture, without L-arginine supplementation. b 1 mM L-arginine was added to the culture medium from D5 to D8 of in vitro culture. c Total concentration of metabolites divided by the number of blastocysts produced in that droplet of medium was used for analysis.
Figure 6. Effect of L-arginine addition during D5 to D8 of in vitro
culture on bovine embryo development. A: D8 development kinetics. B: Embryo quality. Supplementation with L-arginine (Arg-1 mM) increased hatching rates (P < 0.05) and improved blastocyst quality (P < 0.001) as compared to the control group. Values expressed as mean standard deviation. Different capital letters denote significant differences among experimental groups. Different lowercase letters denote significant differences among categories within an experimental condition.
embryo development. This prompted us to investigate the role of NO production during specific periods of in vitro bovine embryo culture, focusing on the periods of embryonic genome activation and blastocyst formation. We found that L-NAME adversely affected blastocyst hatching rates and embryo quality when added starting at the stage immediately following embryonic genome activation (D4D8). Moreover, NOS inhibition decreased blastocyst rates when performed during the entire embryo culture period (D1D8). These results contrast with previous reports showing that using the same concentration of L-NAME (10 mM) during the entire culture period completely arrested in vitro bovine and murine embryo development
922
on culture D9 and D4, respectively (Chen et al., 2001; Tesfaye et al., 2006). A direct comparison between studies is not feasible, however, given that different embryo culture media and conditions were usednamely that different concentrations of amino-acid substrates in each media are available for NO synthesis, and differences in the overall conditions of each in vitro culture system can influence the endpoint results of these studies. In addition, our study specifically determined that inhibition of NO synthesis during the early culture period, e.g., before genome activation, did not affect later development, except for a slight decline in blastocyst hatching rates. We thus speculated that any detrimental effect occurring early on could be later overcome if the source of inhibition was removed. Altogether, these results support the notion that NO production during bovine preimplantation embryogenesis is required mainly during the period immediately following genome activation. We also showed that supplementing bovine embryo culture medium with L-arginine, a natural NO synthesis precursor, improved blastocyst hatching rates and embryo quality. The positive correlation with increased NO levels in the culture medium suggests that L-arginine exerts its positive effects via enhanced NO production and that NO is important for preimplantation bovine embryos. L-arginine supplementation did not, however, increase blastocyst rates in our study, as previously shown for murine zygotes (Kim et al., 2004). A caveat that prevents the direct comparison between our model and the murine study is that L-arginine-supplemented modified Whitten’s culture medium was directly microinjected into the cytoplasm of murine fertilized oocytes (5 hr after insemination). Microinjected L-arginine concentrations higher than 1 mM caused embryo degeneration (Kim et al., 2004), which agrees with the decreased embryo development rates and quality observed in our study when 10 and 50 mM L-arginine was used. We therefore speculate that supplementation
Mol. Reprod. Dev. 81:918–927 (2014)
L-ARGININE IMPROVES
EMBRYO QUALITY
Figure 7. Correlation between per-embryo NO-metabolite production and blastocyst hatching rates or embryo quality. A: Hatching rate. B: Embryo quality. NO2/NO3 production was calculated from the total concentration of NO2/NO3 per culture droplet divided by the total number of blastocysts in that droplet. The results are an average of at least three independent experiments. Quantity of metabolites in the D8 culture medium was considered an indicator of NO production by the embryos. NO production was positively correlated with blastocyst hatching rates (R2 ¼ 92.8%, P < 0.05) and the percentage of good-quality (grade 1) embryos (R2 ¼ 90.7%, P < 0.05).
rather than microinjection of L-arginine is not sufficient to improve blastocyst formation, although it can impact developmental kinetics and embryo-quality parameters through the regulation of metabolism. Notably, there is a concentration threshold above which L-arginine becomes potentially cytotoxic. Indeed, overproduction of NO may result in the accumulation of peroxynitrite, a potent oxidative agent (Riobo et al., 2001), providing a plausible mechanism by which excess NO may be damaging embryos. Our results are consistent with tight regulation of the arginine/NO system during specific stages of bovine embryo development. This finding is consistent with earlier
Figure 8. Relative abundance of POU5F1 transcripts from bovine embryos cultured in the absence or presence of L-arginine (Arg-1 mM) from D5 to D8 of culture. Medium supplementation with L-arginine yielded an increase (P < 0.05) in POU5F1 expression. Asterisk denotes a significant difference.
Mol. Reprod. Dev. 81:918–927 (2014)
observations regarding the differential expression of amino acid transporter proteins and nitric oxide synthases, which respectively regulate L-arginine uptake and its conversion to NO at different stages of embryo development (Van Winkle and Campione, 1990; Van Winkle, 2001; Tesfaye et al., 2006). These expression profiles also corroborate with our observation that L-arginine added only from D5 to D8 of culture improved blastocyst hatching and embryo quality, whereas continuous exposure from D1 to D8 did not. Thus, our results strongly support a model wherein the morula and blastocyst stages are critically dependent upon NO metabolism. Increased NO production in our embryo culture system also correlated with increased blastocyst hatching rates. This is particularly important given how critical the hatching process is for subsequent development and implantation (Petersen et al., 2005; Bazer et al., 2009; Seshagiri et al., 2009). One possible mechanism for NO-enhanced hatching may be through prostaglandin synthesis, which promotes mouse blastocyst hatching (Huang et al., 2004). For example, NO may stimulate cyclooxygenase activity, possibly via S-nitrosylation (Cuzzocrea and Salvemini, 2007). Indeed, cyclooxygenase expression increased precisely at the morula and blastocyst stages of in vitroproduced bovine embryos (Saint-Dizier et al., 2011). Another possible source of the L-arginine enhanced hatching rates could be through mechanisms activated by L-arginine uptake, e.g., cationic amino acid transporters, and its effects on osmolality (Van Winkle, 2001). In this regard, osmotic modifications induced by L-arginine uptake could further enhance blastocoel expansion and, eventually, blastocyst hatching. Finally, a recent study showed that among all the amino acids, arginine and leucine were able to induce mTOR (mammalian target of rapamicyn) activity in mice, which activates the downstream signaling cascade
923
Molecular Reproduction & Development
regulating implantation (Gonzalez et al., 2012). While the L-arginine/NO system has been extensively studied in murine and human models (Gao et al., 2009; Kim et al., 2011a,b,c), little is known about how it functions in the bovine; therefore, extrapolation of such pre-implantation processes is difficult and entirely speculative given the physiological differences in implantation mechanisms among the cited species (Bazer et al., 2010). In an attempt to identify possible targets for the L-arginine/NO effects on embryo quality, we evaluated expression of the POU5F1 gene, a marker for pluripotency in bovine blastocysts (Niemann and Wrenzycki, 2000; Yao et al., 2009). POU5F1 is a member of the POU transcription factor family found to be highly expressed in pluripotent stem cells (Shi and Jin, 2010), and its expression is commonly lower in nuclear-transfer embryos as compared to in vitro fertilization-derived embryos, which correlates with developmental failure (Boiani et al., 2002; Aston et al., 2010). We observed that the presence of L-arginine seemed to stimulate POU5F1 expression, suggesting that the pluripotent state of the embryonic cells is heightened and thus results in improved developmental kinetics and embryo quality. As POU5F1 is also a key, differentially regulated factor between the trophoblast and inner cell mass of bovine embryos (Madeja et al., 2013), we speculate that the L-arginine/NO system could exert different effects in these two compartments. Altogether, the findings of the present study provide evidence that NO can interact with different signaling events in order to regulate preimplantation development of bovine embryos cultured in vitro. Specifically, our results demonstrate a relationship between NO and blastocyst hatching, as well as embryo quality and development, and further show that this relationship can be modified by the addition of L-arginine to the embryo culture medium, especially during the morula-to-blastocyst transition, Future studies should address how L-arginine/NO mechanisms affect bovine embryo metabolism and cell differentiation.
MATERIALS AND METHODS Reagents All chemicals were purchased form Sigma Chemical Company (St. Louis, MO), unless otherwise stated. Both L-arginine and L-NAME were dissolved in water according to the manufacturer’s recommendation.
In Vitro Embryo Production Cumulus-oocyte complexes (COCs) were recovered by follicular aspiration from abattoir ovaries, and compact COCs were selected for in vitro maturation in tissue culture medium (TCM) 199 supplemented with 2.2 g/L sodium bicarbonate, 11 mg/mL pyruvate, 50 mg/mL gentamicin, 10% fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY), 0.5 mg/mL follicle-stimulating hormone (Folltropin) (Bioniche Animal Health, Belleville, Ont., Canada), and
924
SANTANA
ET AL.
5.0 mg/mL luteinizing hormone (Lutropin) (Bioniche Animal Health). COCs were incubated for 24 hr. Frozen-thawed bull (Bos taurus taurus) semen was prepared for in vitro fertilization (IVF) by density-gradient centrifugation in Percoll. Spermatozoa were diluted to a concentration of 2 106 cells/mL in Tyrode’s albuminlactate-pyruvate (TALP) medium (Parrish et al., 1988). Expanded COCs were then placed into sperm-containing 80-ml IVF droplets and co-incubated for 24 hr. Following IVF, groups of 20 presumptive zygotes were transferred to 100-ml droplets of SOF medium (Holm et al., 1999) supplemented with 5% FBS, 50 mg/mL gentamicin, and 6 mg/mL bovine serum albumin (BSA) for in vitro culture. In this study, embryos were not co-cultured with granulosa cells due to their potential interference with NO measurements. All in vitro maturation, fertilization, and culturing were undertaken in a 5% CO2 atmosphere in humidified air at 38.58C.
Experimental Design Three experiments were designed in order to investigate if NO is involved in bovine embryo development. The goal of the first experiment was to inhibit NO production before and after D4 of embryo culture. L-NAME (Tesfaye et al., 2006), a NO synthesis inhibitor, was added to the embryo culture medium at a final concentration of 10 mM from D1 to D4 (N1-4), D4 to D8 (N4-8), or D1 to D8 (N1-8) of in vitro culture. A control group was cultured without the inhibitor. For all groups, the culture medium was replaced on D4. The second experiment was conducted to address the effect of L-arginine on embryo development. For this purpose, the in vitro culture medium was supplemented with 1, 10, or 50 mM L-arginine from D1 to D8 of the culture period. The control group had no L-arginine supplementation. In the third experiment, the L-arginine concentration providing the best results in the second experiment (1 mM) was added from D5 to D8 of the in vitro culture period to assess its effects upon blastocyst formation and total number of cells. Before D5, all groups were cultured in control culture medium (without supplementation). Measurements of NO production were performed before and after induction with L-arginine. For all experiments, embryos were assessed for cleavage rates on D2 of culture and for blastocyst rates, embryo quality, and development kinetics on D8 of culture.
Embryo Endpoint Evaluations Cleavage and blastocyst rates were evaluated on D2 and D8 of the culture period, respectively (the day of in vitro fertilization was set as D0). Blastocyst rates were calculated based on the number of oocytes fertilized. According to the criteria established by Stringfellow and Seidel (1998), blastocysts were classified for developmental kinetics and morphological quality using an inverted fluorescence microscope (Eclipse TE 300, Nikon Instruments Inc., Melville, NY). Hatched blastocysts displayed no zona pellucida and a large trophoblast diameter. Early blastocysts displayed a
Mol. Reprod. Dev. 81:918–927 (2014)
L-ARGININE IMPROVES
thick zona pellucida and a smaller trophoblast diameter when compared expanded blastocysts, which had thinning zona pellucidae and an increasing number of cells. Embryos were graded based upon their morphological quality. Grade 1 or very-good-quality embryos had an inner cell mass with symmetrical or few very slightly asymmetrical blastomeres of a light appearance. Grade 2 or fair-quality embryos had an inner cell mass with very slightly asymmetrical blastomeres. And, Grade 3 or poor-quality embryos had an inner cell mass with widespread, marked asymmetry, and a darker appearance. Following morphological evaluation, hatched blastocysts were fixed in 1% formol-saline solution, and stained with the DNA dye Hoechst 33342 (5 mg/mL) in phosphatebuffered saline. Total cell number was counted using a fluorescence microscope (Eclipse 50i, Nikon Instruments Inc.).
Measurement of NO Production Given its short half-life, NO was quantified indirectly based on its nitrite and nitrate (NO3/NO2) metabolite production (Flora-Filho and Zilberstein, 2000). For this purpose, droplets of medium from D8 of culture were stored at 208C for later analysis using the Griess colorimetric method (Ricart-Jane et al., 2002). Griess reagent is composed of 2% (w/v) sulphanilamide and 0.2% (w/v) N-(1-naphthyl) ethylene-diamine in deionized water. These components react with NO metabolites in a test solution, yielding a purple azo-dye product with a peak absorbance at 540 nm. Readings were performed using a microplate reader (BIO-RAD 450, Bio-Rad Laboratories, Hercules, CA). For quantification, we generated a standard curve with sodium nitrate/nitrite solution diluted in in vitro culture medium with concentrations ranging from 0 to 100 mM. To avoid quantification bias, the medium used for setting the standard curve was aliquoted from exactly the same source medium used for in vitro culture. The amount of nitric oxide produced per embryo was calculated by dividing total metabolite concentration by the total number of blastocysts cultured in the corresponding droplet.
Gene Expression Analysis of In Vitro-Produced Bovine Embryos For relative gene expression quantification, ten sets of embryos per group were used in a total of three replicates. The embryos were frozen in RNAlater1 solution (Ambion, Life Technology, Carlsbad, CA). Aliquots were kept frozen at 808C until RNA extraction, which was performed using Trizol1 reagent (Invitrogen, Molecular Research Center, Cincinnati, OH), according to the manufacturer’s instructions. RNA samples were resuspended in 10 mL of diethylpyrocarbonate (DEPC) H2O (Amresco, Cochran, GA) and kept frozen at 808C until needed. Reverse-transcriptase PCR was performed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Auckland, New Zealand), according to the manufacturer’s protocol.
Mol. Reprod. Dev. 81:918–927 (2014)
EMBRYO QUALITY
Real-time PCR was performed in an ABI PRISM 7500 Real Time PCR system (Applied Biosystems), using the primers previously reported by Costa et al. (Costa et al., 2013) for histone H2a.1 (forward, 50 -GTCGTGGCAAGCAAGGAG/reverse, 50 -GATCTCGGCCGTTAGGTACTC) and the POU transcription factor family, POU5F1, also known as OCT-4 (forward, 50 -GGTGTTCAGCCAAACGACTATC/reverse, 50 -TCTCTGCCTTGCATATCTCCTG). Each 20-mL PCR reaction mixture consisted of 10 mL of SYBR Green PCR Master Mix (Applied Biosystems), 0.2 mM of each forward and reverse primer, and 10 mL of a 1:20-diluted cDNA sample. The following cycling conditions were applied for initial amplification: 958C for 10 min, and 45 cycles at 68C for 1 min. The PCR specificity was verified by checking the corresponding dissociation curves. Optimal primer concentration was 0.2 mM for POU5F1 and 0.1 mM for histone H2a.z, a previously described endogenous control for embryos (Vigneault et al., 2007). The amplification efficiencies of each gene were calculated using the DDCT method, selecting only values between 1.9 and 2.0 (Livak and Schmittgen, 2001). To ensure equal experimental conditions, the same reagents were used to amplify all genes with assays run simultaneously using the same PCR plate. The relative quantity of target transcripts was corrected based upon the quantity of H2a.z.
Statistical Analysis All experiments were performed starting with 30 oocytes per treatment. Within experiments and an oocyte batch, all treatments were tested in parallel, with at least four replicates per experiment performed. Data for cleavage and blastocyst rates, as well as development kinetics, embryo quality, total number of cells, and NO production were analyzed by ANOVA with Tukey’s post-hoc test applied when significant differences were identified. Regression analysis was performed to evaluate the correlation between NO production and development kinetics or embryo quality. Analysis of data for relative gene expression was performed using the t-test. SigmaPlot1 11.0 software was used for analysis, with the significance level set at P < 0.05.
ACKNOWLEDGMENTS This study was supported by Foundation of research state (FAPESPA), National council of support of Para science and technology (CNPq), and University of northern (UNOPAR). The authors thank Drs. Edilene Oliveira Parana ^nio Loureiro Maue s for providing da Silva and Luıs Anto generous access to laboratory equipment.
REFERENCES Aston KI, Li GP, Hicks BA, Sessions BR, Davis AP, Rickords LF, Stevens JR, White KL. 2010. Abnormal levels of transcript abundance of developmentally important genes in various stages of preimplantation bovine somatic cell nuclear transfer embryos. Cell Reprogram 12:2332.
925
Molecular Reproduction & Development
Barroso RP, Osuamkpe C, Nagamani M, Yallampalli C. 1998. Nitric oxide inhibits development of embryos and implantation in mice. Mol Hum Reprod 4:503507. Bazer FW, Spencer TE, Johnson GA, Burghardt RC, Wu G. 2009. Comparative aspects of implantation. Reproduction 138:195209. Bazer FW, Wu G, Spencer TE, Johnson GA, Burghardt RC, Bayless K. 2010. Novel pathways for implantation and establishment and maintenance of pregnancy in mammals. Mol Hum Reprod 16:135152. Bellefontaine N, Hanchate NK, Parkash J, Campagne C, de Seranno S, Clasadonte J, d’Anglemont de Tassigny X, Prevot V. 2011. Nitric oxide as key mediator of neuron-to-neuron and endothelia-to-glia communication involved in the neuroendocrine control of reproduction. Neuroendocrinology 93: 7489. Boiani M, Eckardt S, Scholer HR, McLaughlin KJ. 2002. Oct4 distribution and level in mouse clones. consequences for pluripotency. Genes Dev 16:12091219. Bruckdorfer R. 2005. The basics about nitric oxide. Mol Aspects Med 26:331. Chen HW, Jiang WS, Tzeng CR. 2001. Nitric oxide as a regulator in preimplantation embryo development and apoptosis. Fertil Steril 75:11631171. Chmelikova E, Jeseta M, Sedmikova M, Petr J, Tumova L, Kott T, Lipovova P, Jilek F. 2010. Nitric oxide synthase isoforms and the effect of their inhibition on meiotic maturation of porcine oocytes. Zygote 18:235244. Costa NN, Cordeiro MS, Silva TV, Sastre D, Santana PPB, Sa AL, Sampaio RV, Santos SS, Adona PR, Miranda MS, Ohashi OM. 2013. Effect of triiodothyronine on developmental competence of bovine oocytes. Theriogenology 80:295301. Cuzzocrea S, Salvemini D. 2007. Molecular mechanisms involved in the reciprocal regulation of cyclooxygenase and nitric oxide synthase enzymes. Kidney Int 71:290297. Flora-Filho R, Zilberstein B. 2000. Æxido nıtrico: O simples mensageiro percorrendo a complexidade. Metabolismo, sıntese e funçes. Rev Ass Med Brasil 46:265271. Gao H, Wu G, Spencer TE, Johnson GA, Bazer FW. 2009. Select nutrients in the ovine uterine lumen. III. Cationic amino acid transporters in the ovine uterus and peri-implantation conceptuses. Biol Reprod 80:602609. Gonzalez IM, Martin PM, Burdsal C, Sloan JL, Mager S, Harris T, Sutherland AE. 2012. Leucine and arginine regulate trophoblast motility through mTOR-dependent and independent pathways in the preimplantation mouse embryo. Dev Biol 361:286300. Gouge RC, Marshburn P, Gordon BE, Nunley W, Huet-Hudson YM. 1998. Nitric oxide as a regulator of embryonic development. Biol Reprod 58:875879. Harris LK, McCormick J, Cartwright JE, Whitley GS, Dash PR. 2008. S-nitrosylation of proteins at the leading edge of migrating
926
SANTANA
ET AL.
trophoblasts by inducible nitric oxide synthase promotes trophoblast invasion. Exp Cell Res 314:17651776. Hess DT, Stamler JS. 2012. Regulation by S-nitrosylation of protein post-translational modification. J Biol Chem 287: 44114418. Holm P, Booth PJ, Schmidt MH, Greve T, Callesen H. 1999. High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins. Theriogenology 52:683700. Huang JC, Wun WS, Goldsby JS, Matijevic-Aleksic N, Wu KK. 2004. Cyclooxygenase-2-derived endogenous prostacyclin enhances mouse embryo hatching. Hum Reprod 19:29002906. Jeseta M, Marin M, Tichovska H, Melicharova P, Cailliau-Maggio K, Martoriati A, Lescuyer-Rousseau A, Beaujois R, Petr J, Sedmikova M, Bodart JF. 2012. Nitric oxide-donor SNAP induces Xenopus eggs activation. PLoS One 7:e41509. Kim BH, Kim CH, Jung KY, Jeon BH, Ju EJ, Choo YK. 2004. Involvement of nitric oxide during in vitro fertilization and early embryonic development in mice. Arch Pharm Res 27:8693. Kim J, Burghardt RC, Wu G, Johnson GA, Spencer TE, Bazer FW. 2011a. Select nutrients in the ovine uterine lumen. IX. Differential effects of arginine, leucine, glutamine, and glucose on interferon tau, ornithine decarboxylase, and nitric oxide synthase in the ovine conceptus. Biol Reprod 84:11391147. Kim JY, Burghardt RC, Wu G, Johnson GA, Spencer TE, Bazer FW. 2011b. Select nutrients in the ovine uterine lumen. VII. Effects of arginine, leucine, glutamine, and glucose on trophectoderm cell signaling, proliferation, and migration. Biol Reprod 84:6269. Kim JY, Burghardt RC, Wu G, Johnson GA, Spencer TE, Bazer FW. 2011c. Select nutrients in the ovine uterine lumen. VIII. Arginine stimulates proliferation of ovine trophectoderm cells through MTOR-RPS6K-RPS6 signaling cascade and synthesis of nitric oxide and polyamines. Biol Reprod 84:7078. Lapointe J, Roy M, St-Pierre I, Kimmins S, Gauvreau D, MacLaren LA, Bilodeau JF. 2006. Hormonal and spatial regulation of nitric oxide synthases (NOS) (neuronal NOS, inducible NOS, and endothelial NOS) in the oviducts. Endocrinology 147: 56005610. Latham KE, Schultz RM. 2001. Embryonic genome activation. Front Biosci 6:D748759. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402408. Madeja ZE, Sosnowski J, Hryniewicz K, Warzych E, Pawlak P, Rozwadowska N, Plusa B, Lechniak D. 2013. Changes in subcellular localisation of trophoblast and inner cell mass specific transcription factors during bovine preimplantation development. Dev Biol 13:32. Manser RC, Houghton FD. 2006. Ca2þ-linked upregulation and mitochondrial production of nitric oxide in the mouse preimplantation embryo. J Cell Sci 119:20482055.
Mol. Reprod. Dev. 81:918–927 (2014)
L-ARGININE IMPROVES
Manser RC, Leese HJ, Houghton FD. 2004. Effect of inhibiting nitric oxide production on mouse preimplantation embryo development and metabolism. Biol Reprod 71:528533. Niemann H, Wrenzycki C. 2000. Alterations of expression of developmentally important genes in preimplantation bovine embryos by in vitro culture conditions: Implications for subsequent development. Theriogenology 53:2134. O’Flaherty C, Rodriguez P, Srivastava S. 2004. L-Arginine promotes capacitation and acrosome reaction in cryopreserved bovine spermatozoa. Biochim Biophys Acta 1674:215221. Parrish JJ, Susko-Parrish J, Winer MA, First NL. 1988. Capacitation of bovine sperm by heparin. Biol Reprod 38:11711180. Petersen CG, Mauri AL, Baruffi RL, Oliveira JB, Massaro FC, Elder K, Franco JG, Jr. 2005. Implantation failures. success of assisted hatching with quarter-laser zona thinning. Reprod Biomed Online 10:224229. Ricart-Jane D, Llobera M, Lopez-Tejero MD. 2002. Anticoagulants and other preanalytical factors interfere in plasma nitrate/nitrite quantification by the Griess method. Nitric Oxide 6:178185. Riobo NA, Clementi E, Melani M, Boveris A, Cadenas E, Moncada S, Poderoso JJ. 2001. Nitric oxide inhibits mitochondrial NADH: Ubiquinone reductase activity through peroxynitrite formation. Biochem J 359:139145. Saint-Dizier M, Guyader-Joly C, Charpigny G, Grimard B, Humblot P, Ponter AA. 2011. Expression of enzymes involved in the synthesis of prostaglandin E2 in bovine in vitro-produced embryos. Zygote 19:277283. Scatena R, Bottoni P, Martorana GE, Giardina B. 2005. Nitric oxide donor drugs: an update on pathophysiology and therapeutic potential. Expert Opin Investig Drugs 14:835846. Schwarz KR, Pires PR, de Bem TH, Adona PR, Leal CL. 2010. Consequences of nitric oxide synthase inhibition during bovine oocyte maturation on meiosis and embryo development. Reprod Domest Anim 45:7580. Seshagiri PB, Sen Roy S, Sireesha G, Rao RP. 2009. Cellular and molecular regulation of mammalian blastocyst hatching. J Reprod Immunol 83:7984.
Mol. Reprod. Dev. 81:918–927 (2014)
EMBRYO QUALITY
Shi G, Jin Y. 2010. Role of Oct4 in maintaining and regaining stem cell pluripotency Stem Cell Res Ther 1:39. Stringfellow DA, Seidel SM. 1998. Manual of the International Embryo Transfer Society: A procedural guide and general information for the use of embryo transfer technology emphasizing sanitary procedures. IETS: Savoy, Illinois: p 172. Tesfaye D, Kadanga A, Rings F, Bauch K, Jennen D, Nganvongpanit K, Holker M, Tholen E, Ponsuksili S, Wimmers K, Montag M, Gilles M, Kirfel G, Herzog V, Schellander K. 2006. The effect of nitric oxide inhibition and temporal expression patterns of the mRNA and protein products of nitric oxide synthase genes during in vitro development of bovine pre-implantation embryos. Reprod Domest Anim 41:501509. Tranguch S, Steuerwald N, Huet-Hudson YM. 2003. Nitric oxide synthase production and nitric oxide regulation of preimplantation embryo development. Biol Reprod 68:15381544. Van Winkle LJ. 2001. Amino acid transport regulation and early embryo development. Biol Reprod 64:112. Van Winkle LJ, Campione AL. 1990. Functional changes in cationpreferring amino acid transport during development of preimplantation mouse conceptuses. Biochim Biophys Acta 1028: 165173. Vigneault C, Gilbert I, Sirard MA, Robert C. 2007. Using the histone H2a transcript as an endogenous standard to study relative transcript abundance during bovine early development. Mol Reprod Dev 74:703715. Wang Z. 2012. Protein S-nitrosylation and cancer Cancer Lett 320:123129. Yao N, Wan PC, Hao ZD, Gao FF, Yang L, Cui MS, Wu Y, Liu JH, Liu S, Chen H, Zeng SM. 2009. Expression of interferon-tau mRNA in bovine embryos derived from different procedures. Reprod Domest Anim 44:132139. Yilmaz O, Calka J, Bukowski R, Zalecki M, Wasowicz K, Jaroszewski JJ, Markiewicz W, Bulbul A, Ucar M. 2011. Nitric oxide in the bovine oviduct: Influence on contractile activity and nitric oxide synthase isoforms localization. Theriogenology 77: 13121327.
927
