Published October 24, 2016
Rapid Communication: Nerve growth factor influences cleavage rate and embryo development in sheep1,2 M. Crispo,*3 P. C. dos Santos-Neto,† M. Vilariño,† A. P. Mulet,* A. de León,‡ L. Barbeito,‡ and A. Menchaca†3 *Unidad de Animales Transgénicos y de Experimentación, Institut Pasteur de Montevideo, Montevideo, Uruguay, CP 11400; †Instituto de Reproducción Animal Uruguay (IRAUy), Montevideo, Uruguay, CP12200; and ‡Laboratorio de Neurodegeneración, Institut Pasteur de Montevideo, Montevideo, Uruguay, CP 11400
ABSTRACT: Recent information about Nerve growth factor (NGF), a protein traditionally associated to the nervous system that regulates survival and maturation of developing neurons, suggests that it may exert action also on different levels in the reproductive system. The aim of this study was to evaluate the effect of NGF added during in vitro oocyte maturation, fertilization or in vitro embryo development in sheep. Nerve growth factor was supplemented to the culture medium at 0, 100, or 1,000 ng/mL, during either in vitro maturation (Exp. 1), in vitro fertilization (Exp. 2), or in vitro culture (Exp. 3). In addition, NGF mRNA expression was determined in cumulus cells and oocytes. Nerve growth factor induced early cleavage
when added during oocyte maturation or fertilization, improved embryo development when added during fertilization, and had no significant effect when added during embryo culture. In general, the effect was more evident with 100 rather than 1,000 ng/mL (P < 0.05). Expression of endogenous NGF was not detected in oocytes, and increased in cumulus cells when 1,000 ng/mL of NGF was added during fertilization, but not during maturation and embryo culture. In conclusion, the addition of NGF during oocyte maturation and fertilization affects in vitro cleavage and embryo development in sheep. We suggest a possible effect of this growth factor on oocyte maturation and mainly on the fertilization process.
Key words: blastocyst, embryo development, nerve growth factor, ovine, ovulation inducing factor © 2016 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2016.94:4447–4451 doi:10.2527/jas2016-0736 INTRODUCTION Nerve growth factor (NGF) is a protein traditionally associated to the regulation of survival and maturation of developing neurons in the peripheral nervous system and neurons in the brain during embryonic development and the early postnatal stage (Levi-Mon-
1This work was financially supported by FOCEM (MERCOSUR
Structural Convergence Fund), COF 03/11 and received additional support by PEDECIBA (UdelaR). MC, LB and AM are fellows of Sistema Nacional de Investigadores (SNI, ANII, Uruguay). 2The authors wish to thank Natalibeth Barrera, Robert Wijma and Fátima Rodríguez for the technical assistance during in vitro embryo production. 3Corresponding authors:
[email protected]; menchaca.
[email protected] Received June 13, 2016. Accepted July 21, 2016.
talcini and Hamburger, 1951). Recently, it has been shown that this growth factor has an important role in several processes (Seidel et al., 2010; Isola et al., 2011) and is recognized as a pleiotropic factor (Wang et al., 2015). Nerve growth factor has been found in follicular fluid of women undergoing in vitro embryo production, influencing follicle growth and oocyte quality (Palumbo et al., 2013). However, the information about the effect of this growth factor on oocyte maturation and fertilization is scarce. Recently, a socalled ovulation-inducing factor (OIF) protein was found in the seminal plasma of llamas– an induced ovulation species–eliciting the ovulatory response and luteal function (Adams et al., 2005; Ratto et al., 2010). This OIF induced the ovulation of large follicles in species other than llamas when it was administered by intramuscular injection (Tribulo et al., 2015). Interestingly, OIF has the same homology with human, porcine, bovine, and murine sequences of β-NGF (Ratto
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et al., 2012), concluding the authors that OIF is NGF in seminal plasma. This discovery reinforces the idea that NGF has an important role in reproductive function, health and disease. Hence, the current information places this growth factor, traditionally associated with the nervous system, as one of the renewed proteins to be assayed in its function on the reproductive biology of mammals. The objective of this study was to evaluate the local effect of NGF on maturation, fertilization, and embryo development in in vitro conditions in sheep. MATERIALS AND METHODS All procedures that include animal handling were performed in strict accordance with the recommendations in the Model code of practice for the welfare of animals: the sheep. Protocol # 001/2014 was approved by the Institutional Animal Care and Use Committee of the Fundación IRAUy, certified by the National Council of Animal Care of Uruguay. Experimental Design Three experiments were conducted on 2,465 cumulus oocyte complexes (COC) to assess the effect of different NGF concentrations supplemented during in vitro maturation (IVM), in vitro fertilization (IVF), or in vitro culture (IVC), on ovine embryo production. In Exp. 1, six replicates were conducted using immature COC maintained during 24 h in maturation media supplemented with 0 ng/mL (298 COC), 100 ng/mL (359 COC), or 1,000 ng/mL of NGF (363 COC). In Exp. 2, seven replicates were conducted on matured COC maintained during IVF in fertilization medium supplemented with 0 ng/mL (297 COC), 100 ng/mL (295 COC), or 1,000 ng/ mL of NGF (287 COC). In Exp. 3, three replicates were conducted on embryos maintained from d 2 to d 6 in culture medium supplemented with 0 ng/mL (191 embryos), 100 ng/mL (189 embryos), or 1,000 ng/mL (186 embryos) of NGF. Cleavage rate at 24 h and 48 h, development rate on d 6 (morulae and blastocyst) and hatching rate on d 8 were compared between experimental groups. Nerve growth factor mRNA expression was determined in cumulus cells and oocytes of immature and mature COC without the addition of NGF, and after fertilization in the experimental groups with 0, 100, and 1,000 ng/mL NGF. Unless indicated otherwise, chemicals were purchased from Sigma Chemical Company (St. Louis, MO). Nerve Growth Factor Recombinant human NGF was produced following the methods described (Rattenholl et al., 2001). Briefly, the plasmid pEt22b containing the proNGF gen was ex-
pressed in E. coli in 2YT medium at 37°C until reaching an optical density at 600 nm of 0.6. Protein expression was induced with 1M isopropyl thio-β-D-galactoside (IPTG). After cell lysis, the insoluble protein contained in inclusion bodies was solubilized in 6M guanidinium chloride. Protein re-solubilization was performed by successive dialysis at 8°C in 100 mM Tris-HCl (pH 9.3) buffer, 1M L-arginina, 5 mM GSH, 1 mM GSSG, 5 mM EDTA, with a final protein concentration of 100 µg/mL. After folding and solubilization, proNGF was purified by ion-exchange chromatography and subjected to trypsin digestion and further purification. The rhNGF biological activity and potency was tested in PC12 cultures (Levi et al., 1988). The range of concentrations tested was 1 to 100 ng/mL. Differentiation with production of neurites was observed with 10 ng/μL of rhNGF. The NGF solutions were prepared from a 60 ng/μL stock solution as follows: 50 μL of NGF diluted in 2,950 μL of culture media for the 1,000 ng/mL group, 5 μL of NGF + 45 μL of PBS diluted in 2,950 μL of culture media for the 100 ng/mL group, and 50 μL of PBS diluted in 2,950 μL of culture media for the 0 ng/mL group. In Vitro Embryo Production In vitro ovine embryos were produced as described in previous reports (Crispo et al., 2015). Briefly, ovaries from slaughterhouse were transported to the laboratory and COC were aspirated in recovery medium. The selected COC were placed in maturation medium for 24 h in 5% CO2 in humidified air atmosphere at 39°C. Then, expanded COC were inseminated in 100 μL drops with 1 × 106 dose of frozen-thawed spermatozoa selected by ascendant migration on a swim up method. Fertilization was performed in 5% CO2 with humidified atmosphere at 39°C for 22 h. After IVF, presumptive zygotes were transferred to development medium under mineral oil in a tri-gas chamber with 5% CO2, 5% O2, and 90% N2 in humidified atmosphere at 39°C until d 8. Real Time PCR Separate samples of oocytes and cumulus cells were obtained before and after IVM without NGF supplementation, and after IVF with 0, 100, and 1,000 ng/mL of NGF supplemented medium. For that, COC were denuded and settled in groups of 10 in TRI Reagent (8 replicates) until RNA extraction. Total RNA was isolated from those groups of 10 oocytes or zygotes, or the corresponding cumulus cells in accordance with the manufacturer protocol (Pavani et al., 2015). All the RNA obtained was reverse transcripted using RevertAid H Minus Reverse Transcriptase (Thermo Scientific, Waltham, MA) in a total volume of 20 μL fol-
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Table 1. Cleavage and development rates of in vitro produced ovine embryos after addition of 0, 100, or 1,000 ng/mL of NGF1 to COC2 in maturation or fertilization, or zygotes in culture media Cleavage rate Item Experiment 1 NGF in IVM3 0 ng/mL (n = 298) 100 ng/mL (n = 359) 1,000 ng/mL (n = 363) Experiment 2 NGF in IVF4 0 ng/mL (n = 297) 100 ng/mL (n = 295) 1,000 ng/mL (n = 287) Experiment 3 NGF in IVC5 0 ng/mL (n = 191) 100 ng/mL (n = 189) 1,000 ng/mL (n = 186)
24 h
48 h
Embryos on d 6 /oocytes
Embryos on d 6 /cleaved
Hatching/blastocysts on d 8
26.2% (78/298)a 37.0% (133/359)b 27.0% (98/363)a
69.8% (208/298) 69.9% (251/359) 66.9% (243/363)
35.2% (105/298) 34.0% (122/359) 33.1% (120/363)
50.5% (105/208) 48.6% (122/251) 49.4% (120/243)
12.5% (4/32) 20.5% (8/39) 21.9% (7/32)
33.0% (98/297)a 46.1% (136/295)b 43.6% (125/287)b
70.7% (210/297) 74.6% (220/295) 69.3% (199/287)
40.1% (119/297)a 50.5% (149/295)b 44.9% (129/287)ab
56.7% (119/210)a 67.7% (149/220)b 64.8% (129/199)ab
39.7% (31/78) 35.7% (25/70) 27.5% (19/69)
46.1% (88/191) 47.1% (89/189) 41.4% (77/186)
77.0% (147/191) 79.9% (151/189) 82.8% (154/186)
41.4% (79/191) 45.5% (86/189) 46.8% (87/186)
53.7% (79/147) 57.0% (86/151) 56.5% (87/154)
45.5% (25/55) 52.2% (35/67) 45.3% (29/64)
a,bFor
each Experiment, different superscripts within a variable show statistical differences (P < 0.05). = nerve growth factor. 2COC = cumulus oocyte complex. 3IVM = in vitro maturation. 4IVF = in vitro fertilization. 5IVC = in vitro culture. 1NGF
lowing manufacturer instructions. Real time PCR reactions were performed using 5 μL Rotor-Gene SYBR Green PCR Kit (Qiagen Inc, Valencia, CA), equimolar amounts of forward and reverse primers (0.6 μM; Integrated DNA Technologies, Coralville, IA), and 1 μL cDNA in a final volume of 10 μL. Samples were analyzed in triplicate in a 72-disk Rotor-GeneTM 6000 (Corbett Life Sciences, Sydney, Australia). Standard amplification conditions were 3 min at 95°C and 40 cycles of 5 s at 95°C and 10 s at 60°C. After determining that the efficiency of the primers was similar, the delta cycle threshold (CT) value was calculated from the difference in the CT of the gene of interest and that of the β-Actin gene. Data are presented as 2-Δ CT value (Livak and Schmittgen, 2001). All the experiments were performed in duplicate. Expression of β-actin gene was selected as endogenous reference considering previous reports of its successful use in preimplantation ovine embryos (Zacchini et al., 2011)
Statistical Analysis The effect of NGF supplementation was analyzed by logistic regression for embryo production and ANOVA for relative RNA expression using Generalized Linear Mixed Models of the Statistical Analysis System program (SAS Inst. Inc., Cary, NC). Differences were considered significant at a level of P < 0.05. RESULTS Embryo Development The supplementation of NGF affected cleavage rate when it was added to maturation or fertilization medium, and embryo development when was added to fertilization medium. Data of cleavage and development rates for each Experiment are shown in Table 1. NGF Expression No expression of NGF was detected in oocytes or cumulus cells before or after maturation in absence
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Figure 1. mRNA expression (cumulus cells corresponding to 10 cumulus oocyte complex per group) after 0, 100, or 1,000 ng/mL of exogenous nerve growth factor (NGF) supplemented during in vitro fertilization. Data is expressed as Mean ± SEM of significant differences (P < 0.05).
2-Δ CT. a,b indicate
of NGF supplementation (data not shown). In addition, no expression of NGF mRNA was found on oocytes after the addition of 0, 100, or 1,000 ng/mL NGF during IVF. However, the addition of 1,000 ng/mL of NGF during IVF showed an increase in NGF mRNA levels in cumulus cells 2.6 and 6-fold greater compared to 0 NGF and 100 ng/mL (Fig. 1). No statistical differences were found among cumulus cells with 0 and 100 ng/mL of NGF. DISCUSSION In our study, the major finding was evident when NGF was added during fertilization, advancing the cleavage and improving the embryo development, an effect that had not been demonstrated yet. Although this effect may probably be related to the oocyte and/or the cumulus cells, some evidence shows an interesting relationship between NGF and sperm that may explain this finding. NGF or OIF has been identified in seminal plasma of several species (Harper et al., 1982), produced mainly by the vesicular glands (Hofmann and Unsicker, 1982), and also by the prostate gland of guinea pigs, rats, rabbits, and bulls (Harper et al., 1982; Huang et al., 2014). Moreover, although information is scarce, NGF and its receptors have been found in sperm of some species and its function is clearly involved in sperm motility and penetration (Schachter et al., 1986; Jin et al., 2010; Li et al., 2010). Although the mechanism by which NGF supplementation during fertilization advances the em-
bryo cleavage is not explained in this study, we hypothesize that the effect of NGF found during fertilization may be associated, at least in part, to sperm function. On the other hand, the fact that the addition of 100 ng/mL during IVM also improves cleavage rate at 24 h suggests that NGF may have a role in the maturation of mammalian oocytes. This result is in agreement with previous findings reported with different approaches (Dissen et al., 1996; Barboni et al., 2002). Barboni et al. (2002) showed that the addition of 100 ng/mL of NGF to a in toto follicle sheep model cultured under hyperbaric conditions induced a marked cumulus expansion and a progressive cumulus-oocyte uncoupling similar to that produced by gonadotropins. The authors concluded that NGF had a clear stimulating effect when is added at a similar concentration to that recorded in the preovulatory follicle after GnRH administration (i.e., 135 ng/mL; Barboni et al., 2002). Because the main source of NGF during oocyte maturation in the ovulatory follicle seems to be the granulosa and theca cells (Jana et al., 2011), it is probably that in vitro culture conditions fail to provide the NGF required during this process. This hypothesis is supported by the absence of NGF mRNA expression during in vitro culture conditions (i.e., before NGF addition) found in the current study. While there are some studies reporting the NGF presence in oocytes (Shi et al., 2004; Abir et al., 2005; Jana et al., 2011), some others suggest that its presence in the oocyte depends on the estrous cycle stage and the levels may vary drastically (Jana et al., 2011). Taken together, all this information suggests that in our study the endogenous presence of NGF may be limited by the in vitro culture conditions, requiring further investigation. In conclusion, the current study demonstrates that the addition of NGF to the maturation and fertilization media during the in vitro embryo production enhances cleavage and development rate in sheep embryos. We suggest a possible local effect of this growth factor on oocyte maturation and mainly on fertilization in sheep. LITERATURE CITED Abir, R., B. Fisch, S. Jin, M. Barnnet, A. Ben-Haroush, C. Felz, G. Kessler-Icekson, D. Feldberg, S. Nitke, and A. Ao. 2005. Presence of NGF and its receptors in ovaries from human fetuses and adults. Mol. Hum. Reprod. 11:229–236. doi:10.1093/molehr/gah164 Adams, G. P., M. H. Ratto, W. Huanca, and J. Singh. 2005. Ovulationinducing factor in the seminal plasma of alpacas and llamas. Biol. Reprod. 73:452–457. doi:10.1095/biolreprod.105.040097 Barboni, B., M. Mattioli, L. Gioia, M. Turriani, G. Capacchietti, P. Berardinelli, and N. Bernabò. 2002. Preovulatory rise of NGF in ovine follicular fluid: Possible involvement in the control of oocyte maturation. Microsc. Res. Tech. 59:516–521. doi:10.1002/ jemt.10230
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