- Email: [email protected]
Sperm binding, in vitro fertilization, and in vitro embryonic development of bovine oocytes fertilized with spermatozoa incubated with norepinephrine
Animal Reproduction Science 96 (2006) 1–9
Sperm binding, in vitro fertilization, and in vitro embryonic development of bovine oocytes fertilized with spermatozoa incubated with norepinephrine A.L. Way a,∗ , G.J. Killian b a b
Lock Haven University, Health Science, Clearfield Campus, 201 University Dr., Clearfield, PA 16830, USA Almquist Research Center, Penn State University, University Park, PA 16802, USA Received 9 June 2005; accepted 19 October 2005 Available online 21 November 2005
Abstract The final stages of sperm maturation, fertilization, and early embryonic development occur within the oviduct and are essential for successful reproduction in mammals. Norepinephrine was previously identified in native bovine oviductal fluid and its in vitro effects on bull sperm capacitation and the acrosome reaction have been determined. It was unknown how physiological concentrations of norepinephrine influence sperm binding, fertilization, and embryo development. Therefore, the objective of this study was to determine if preincubating bovine spermatozoa with physiological concentrations of norepinephrine prior to insemination of bovine oocytes would improve sperm–oocyte binding, fertilization, and embryonic development in vitro. Norepinephrine, in concentrations representing those measured in bovine oviductal fluid, was used to treat bovine spermatozoa prior to insemination. Spermatozoa incubated in norepinephrine were used to inseminate bovine oocytes matured in vitro, and oocytes were evaluated for sperm binding and fertilization. Additional experiments were conducted to evaluate how early in the co-incubation period oocytes were fertilized by spermatozoa pre-incubated with norepinephrine, and to test the developmental competence of those oocytes fertilized with norepinephrine-treated sperm. Sperm binding to the zona pellucida was reduced by preincubation with norepinephrine. Rates of fertilization and embryo development did not increase as a result of pre-incubating spermatozoa with norepinephrine, but as early as 4 h after insemination, spermatozoa treated with 20 ng/ml norepinephrine fertilized more oocytes than spermatozoa incubated in medium alone. Interestingly, this concentration of norepinephrine was found to capacitate spermatozoa in previous studies. These data suggest that oocytes fertilized by spermatozoa incubated in 20 ng/ml norepinephrine fertilize
∗
Corresponding author. Tel.: +1 814 768 3431; fax: +1 814 768 3452. E-mail address: [email protected] (A.L. Way).
0378-4320/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2005.10.006
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A.L. Way, G.J. Killian / Animal Reproduction Science 96 (2006) 1–9
earlier in vitro than sperm pre-incubated in medium alone, and provide additional support for the role of norepinephrine in sperm capacitation and the acrosome reaction. © 2005 Elsevier B.V. All rights reserved. Keywords: Bovine; Spermatozoa; Oviduct; Fertilization
1. Introduction Identifying components of oviductal fluid (ODF) that influence in vivo fertilization and early embryonic development will provide insight into the physiological processes that take place within the oviductal environment. Identification of components that improve fertilization rates and embryo development in vitro may be of significant economic value to areas of animal agriculture that rely on reproductive technology. Epinephrine is routinely added to culture dishes during in vitro insemination of bovine oocytes as it is thought to aid in fertilization (Hasler et al., 1995). The concentrations used are based on in vitro assays conducted in rodents (Leibried and Bavister, 1982), however, its addition at insemination makes it unclear as to whether the catecholamine is affecting the spermatozoa, the oocytes or both. There have been no investigations into the effects of norepinephrine (NE) on bovine in vitro fertilization and embryo development. Mouse spermatozoa pre-incubated in NE were able to penetrate significantly more ova than control spermatozoa (Stanger, 1983). However, the concentrations of catecholamines used in these studies were higher than the picogram quantities present in murine plasma (Lucot et al., 2005) or in bovine ODF (Way et al., 2001), suggesting the possibility that the effects observed were pharmacological rather than physiological. There are no reports describing the effects of physiological concentrations of NE on the events surrounding fertilization and embryo development in the bovine. Norepinephrine was shown to stimulate capacitation of bovine spermatozoa in vitro (Way and Killian, 2002). Because capacitation is a prerequisite for sperm–oocyte binding and fertilization, it is reasonable to ask whether spermatozoa treated with NE are capable of binding to the zona pellucida and fertilizing in vitro-matured oocytes, and whether the developmental potential of embryos created with NE-treated spermatozoa is affected. The objective of this study was to determine if pre-incubating bovine spermatozoa with physiological concentrations of NE prior to insemination of bovine oocytes would improve sperm–oocyte binding, fertilization, and embryonic development in vitro. 2. Materials and methods 2.1. Oocyte collection and maturation Bovine ovaries (approximately 4.5 kg) were harvested at an abbatoir and placed into 1 l of Dulbecco’s PBS (Life Technologies, Grand Island, NY) (35 ◦ C) prior to transport back to the laboratory. The temperature of the ovaries during transport ranged from 25 to 30 ◦ C. Once in the laboratory, ovaries were rinsed with 35–37 ◦ C tap water and soaked for 15 min in a solution of 1% Chlorhexiderm (AJ Buck, Owings Mills, MD) and 1% 7X Cleaning Solution (ICN Biomedicals, Costa Mesa, CA) in 35–37 ◦ C tap water (Hasler et al., 1995). After a final rinse in 35–37 ◦ C tap water, ovaries were rinsed with Dulbecco’s PBS (39 ◦ C). Oocytes were obtained
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from visible follicles by aspiration with an 18 g needle attached to a 10 cm3 syringe. The aspirate was poured into an Em-Con filter (Immuno Systems, Spring Valley, WI) and rinsed with 1 l of low bicarbonate HEPES (Bavister et al., 1983). The contents of the filter were distributed among 8–12, 60 mm × 15 mm sterile petri dishes. In order to obtain sufficient oocytes to assess sperm binding, fertilization, and embryo culture, oocytes were selected based on the presence of at least one layer of intact cumulus cells for the sperm–oocyte binding experiments, and two or more intact cumulus cell layers for in vitro fertilization and embryo culture. This criteria for oocyte selection was based on previous experience with oocyte quality for in vitro fertilization, and data reported by Hasler et al. (1995) where embryo production was improved if oocytes with multiple layers of cumulus cells are used. Oocytes for sperm binding were considered acceptable if they contained at least one complete layer of compact cumulus as the purpose of these oocytes was to assess the ability of spermatozoa to bind, not the oocyte to be capable of fertilization and embryo development. Oocytes were matured in vitro by incubating cumulus–oocyte complexes in medium M199 containing 10% fetal calf serum, LH (0.01 units/ml), FSH (0.01 units/ml), and penicillin (100 units/ml)/streptomycin (100 g/ml) for 22–24 h at 39 ◦ C in 5% CO2 /air (v/v; Hasler et al., 1995). After maturation oocytes were prepared for sperm binding, fertilization, and embryo culture experiments as described below. 2.2. Sperm preparation Semen was collected from three mature Holstein dairy bulls (Bos taurus) by artificial vagina and washed twice by centrifugation in 10 ml modified Tyrode’s medium (MTM, Parrish et al., 1988). Spermatozoa (5 × 107 ml) were incubated in MTM alone (control) and 20, 80, and 400 ng/ml NE at 39 ◦ C in 5% CO2 /air (v/v). These concentrations were chosen to represent physiological concentrations of NE found in ODF (Way et al., 2001). The 20 ng/ml NE treatment was found to induce the most acrosome reactions in vitro (Way and Killian, 2002). After 2 h of incubation spermatozoa were separated from their incubation medium using percoll gradient centrifugation. Briefly, 2 ml of a 90% percoll solution in MTM (v/v) were placed in the bottom of a 15 ml polypropylene tube and 2 ml of a 45% percoll solution in MTM (v/v) were gently overlaid on top of it. For each treatment, 1 ml of sperm suspension was layered onto the 45% solution and centrifuged for 30 min at 700 × g. The pelleted spermatozoa were recovered, assessed for motility, and suspended in fertilization medium (Bavister et al., 1983). 2.3. Sperm–oocyte binding In vitro-matured oocytes were vortexed for 2 min to remove cumulus cells, washed twice in low bicarbonate HEPES medium and placed 25-well in Nunclon 4-well culture dishes (Fisher Scientific, Pittsburgh, PA) containing 0.5 ml fertilization medium. Oocytes must be denuded before fertilization in order to be able to quantitate sperm bound to the zona after insemination (Way et al., 1997). Oocytes were inseminated with 6.25 × 104 spermatozoa from the MTM, 20 ng/ml NE, 80 ng/ml NE, or 400 ng/ml NE sperm treatments, for a final concentration of 1.25 × 105 spermatozoa/ml. Heparin (2 g) was added to each well at the time of insemination (Hasler et al., 1995). Oocytes and spermatozoa were co-incubated for 18 h at 39 ◦ C in 5% CO2 /air (v/v). After co-incubation oocytes were washed once in low bicarbonate HEPES medium and placed, 10 per slide, under a coverslip mounted with paraffin wax and petroleum jelly at each
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corner. The coverslip was lowered over the oocytes until they burst, and the cytoplasm was rinsed away with low bicarbonate HEPES medium. The zonae pellucidae and any spermatozoa bound to them were stained with Hoechst fluorescent dye 33342 (Sigma, St. Louis, MO). The number of spermatozoa bound to each zona pellucida was determined using fluorescence microscopy (Way et al., 1997). 2.4. In vitro fertilization and embryo culture In vitro-matured oocytes were washed twice in low bicarbonate HEPES medium, placed in fertilization medium with 2 g heparin and inseminated as described above. After 18 h of coincubation oocytes were vortexed to remove cumulus cells and accessory spermatozoa according to the method of Hasler et al. (1995), and washed twice in low bicarbonate HEPES medium. Fertilization and embryo development were assessed independently of each other using two separate pools of oocytes. This was to eliminate the inaccurate assessment of parthenogenic embryos as originating from fertilized oocytes. Oocytes designated for evaluation of fertilization were placed 10 per slide under a coverslip mounted at the corners with paraffin wax and petroleum jelly. The coverslip was gently lowered over the oocytes and adhered to the slide with rubber cement. Oocytes were fixed in acid alcohol for 24 h and stained with aceto-orcein (Sirard et al., 1988). The presence of two pronuclei in the cytoplasm of the oocyte indicated normal fertilization. An additional study was undertaken in which oocytes were prepared for insemination as described above, and fertilized with spermatozoa pre-incubated for 2 h in 20 ng/ml NE or MTM. At 4, 8, 12, and 16 h of co-incubation, oocytes were removed from both treatments, vortexed to removed accessory spermatozoa, washed and placed in fresh fertilization medium. All oocytes were incubated for a total of 16 h before being washed, fixed in acid alcohol and stained with aceto-orcein. This study was designed to determine whether fertilization occurs earlier in the co-incubation period for sperm treated with 20 ng/ml NE. Oocytes designated for embryo culture were vortexed and washed as described above and presumptive zygotes were placed in 4-well culture dishes containing synthetic oviduct fluid (SOF; Tervit et al., 1972) minus glucose and with the addition of Eagle’s Basal Medium essential and Minimum Essential Medium non-essential amino acids (Walker et al., 1996; Wagtendonk-de Leeuw van et al., 2000). Each well contained 0.5 ml SOF and up to 25 presumptive zygotes. The dishes were placed in a humidified modular incubator chamber (Billups-Rothenberg Inc., Del Mar, CA) purged with a gas mixture of 5% O2 /5% CO2 /90% N2 (v/v/v). Embryonic development was assessed on day 4 of culture for cleavage and development, day 8 for development to the blastocyst stage, and day 11 for final assessment of development to the blastocyst and hatched blastocyst stages. 3. Statistical analysis Each experiment was repeated four times and data from each experiment were pooled. Approximately 40–50 oocytes per treatment for sperm binding, 80–90 oocytes per treatment for fertilization, and 90–100 oocytes per treatment for embryo development were evaluated in each replicate. Analysis of variance using a general linear model was performed using mean number of spermatozoa bound per zona pellucida for each treatment in the sperm–oocyte binding experiments, and a weighted mean based on the number of oocytes per treatment in the fertilization and embryo experiments. Least square means comparisons were used to assess sperm binding and weighted least square means were used to analyze fertilization data. Data on embryo
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Fig. 1. Mean number of spermatozoa bound per zona pellucida ± S.E.M. for spermatozoa incubated in MTM, 20 ng/ml NE, 80 ng/ml NE, and 400 ng/ml NE prior to insemination. Binding of MTM-treated spermatozoa to the zona pellucida was significantly different (*) from all of the NE treatments (P < 0.05). There were no differences among the NE treatments.
development were analyzed using least square means to determine the effect of each treatment on embryonic cleavage and development to the blastocyst stage (SAS 6.12 for Windows, Statistical Analysis Systems, 1989). The significance level for all tests was P < 0.05. 4. Results Incubating spermatozoa in all concentrations of NE prior to insemination significantly reduced sperm binding compared to the MTM control (P < 0.05, Fig. 1). There were no significant differences among the concentrations of NE for sperm binding, and NE did not improve fertilization or embryonic development over the control at any concentration used in this study. The only differences observed for fertilization were between the 20 and 80 ng/ml NE treatments (Fig. 2a). Fertilization was lower for spermatozoa incubated in 20 ng/ml NE compared to spermatozoa incubated in 80 ng/ml NE (P = 0.04). There were no differences among any of the treatments for cleavage, development to blastocyst, or blastocyst hatching (Fig. 2b). When evaluating fertilization throughout the co-incubation period for oocytes inseminated with 20 ng/ml NE and MTM-treated spermatozoa, significant differences were observed only at the 4 h time point (Fig. 3). Spermatozoa treated with 20 ng/ml NE fertilized more oocytes after 4 h of co-incubation than spermatozoa incubated in MTM alone (P < 0.05). The 4 h time point was the only time at which differences could be observed. 5. Discussion Previous experiments demonstrated that 20 ng/ml NE capacitated bovine spermatozoa after 2 h of incubation (Way and Killian, 2002), a concentration representative of NE measured in ODF (Way et al., 2001). This observation led to the present experiments, which were designed to determine the effects of treating spermatozoa with physiological concentrations of NE on sperm binding, fertilization, and embryonic development. In this study NE significantly reduced the number of spermatozoa that bound to the zona pellucida of each oocyte as compared to control spermatozoa. Given that NE was
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Fig. 2. (a) Mean percentage ± S.E.M. of oocytes fertilized. (b) Mean percentage ± S.E.M. of oocytes cleaved (open bar), developed to blastocyst (left diagonal bar), and hatched (right diagonal bar). Oocytes were inseminated with spermatozoa incubated in MTM, 20 ng/ml NE, 80 ng/ml NE, and 400 ng/ml NE prior to insemination. Bars with same superscripts (a and b) were not significantly different from each other. Fertilization was lower for spermatozoa incubated in 20 ng/ml NE compared to spermatozoa incubated in 80 ng/ml NE (P = 0.04). There were no statistically significant differences between treatments for cleavage, development to blastocyst, or hatching.
found to induce the acrosome reaction in spermatozoa in a previous study (Way and Killian, 2002), this result is not surprising as capacitated, but not acrosome-reacted spermatozoa are capable of binding to the zona pellucida (Bleil and Wassarman, 1983; Florman and First, 1988a,b).
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Fig. 3. Mean percentage ± S.E.M. of oocytes fertilized by spermatozoa incubated in 20 ng/ml NE (open bar) and MTM (filled bar) prior to fertilization. Gametes were co-incubated for 4, 8, 12, and 16 h. Spermatozoa pre-incubated in 20 ng/ml norepinephrine fertilized significantly more oocytes than sperm pre-incubated in medium alone, but this difference was only evident at the 4 h time point (* P < 0.05).
It is possible that sperm binding was decreased for spermatozoa incubated in 20 ng/ml NE because there were more capacitated spermatozoa at the time of insemination in this group as compared to MTM or other NE treatments. Spermatozoa incubated in 20 ng/ml NE prior to insemination may have fertilized oocytes earlier in the 18 h co-incubation period than spermatozoa incubated in the other treatments and initiated the zona block to polyspermy, ultimately preventing more spermatozoa from binding to the zona pellucida. This possibility does not extend to the higher concentrations of NE (80 and 400 ng/ml), which did not capacitate spermatozoa in the previous study. These higher concentrations of NE may be inhibiting capacitation. Catecholamines in concentrations higher than that found in blood may be acting as chelators (Hexum, 1977; Fagan and Racker, 1977) and/or phosphodiesterase inhibitors (Goren and Rosen, 1972; Ain-Shoka and Buckner, 1978) and have been shown to affect sperm function in vitro in other species (Mrsny and Meizel, 1979, 1980; Dobrzynska et al., 2004). Capacitation with 20 ng/ml NE occurs for a discrete time period (Way and Killian, 2002) suggesting that capacitated spermatozoa incubated with 20 ng/ml NE would not remain functionally capacitated throughout the 18 h co-incubation period. The number of spermatozoa capacitated with heparin increases over time during incubation, making capacitated spermatozoa available for fertilization for a minimum of 6 h and possibly longer during the co-incubation period. This may mean that more oocytes will be fertilized by heparin–capacitated spermatozoa than by NE–capacitated spermatozoa simply because capacitated spermatozoa continue to be available for a longer period of time during the co-incubation. However, capacitation with NE occurs within 2 h of incubation and at levels comparable to capacitation with heparin after 4 h, reducing the preparatory time required of spermatozoa before they can fertilize ova. This reduction in capacitation time and
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its presence in native oviductal fluid make it a viable alternative to heparin capacitation, and a more reasonable choice than epinephrine which is not present in oviductal fluid (Way and Killian, 2002). Capacitated spermatozoa are fragile, short-lived cells (Chang and Hunter, 1975; Yanagimachi, 1981) and any spermatozoa that were not capacitated with NE by 2 h of incubation likely did not participate in fertilization of the oocytes. If spermatozoa capacitated with 20 ng/ml NE were able to fertilize oocytes and initiate the block to polyspermy, they would represent the population of spermatozoa to participate in fertilization, rather than spermatozoa incubated in the 20 ng/ml NE treatment that may respond to heparin that was added upon insemination. In those spermatozoa, capacitation would be delayed for several hours if they follow the normal pattern of capacitation with heparin in vitro (Parrish et al., 1988). Data from the experiment in which oocytes were removed from co-incubation over the course of 16 h suggests that sperm treated with 20 ng/ml NE are capacitated and fertilizing oocytes earlier in the co-incubation period than sperm in medium alone (Fig. 3). This supports the reduction in sperm binding observed for those oocytes inseminated with 20 ng/ml NE-treated spermatozoa, and supports the hypothesis that it is spermatozoa capacitating with NE that are fertilizing the oocytes in that treatment. For all treatments except 20 ng/ml NE, the embryo cleavage rate was lower than the fertilization rate, while the percentages of oocytes fertilized (75.11 ± 2.13%) and cleaved (75.08 ± 3.09%) were almost identical for the 20 ng/ml NE treatment (Fig. 2). This interesting difference between the 20 ng/ml NE treatment and the other treatments cannot be assessed statistically because two separate populations of oocytes were used in the fertilization and embryo culture experiments. It appears that spermatozoa incubated in MTM and higher concentrations of NE were capable of fertilization, but may not have been as capable of initiating the events leading to cleavage as spermatozoa treated with 20 ng/ml NE. Even though fertilization rates were higher in the other treatments, embryonic development was not improved. Essentially all of the oocytes that were fertilized by spermatozoa incubated with 20 ng/ml NE were capable of cleaving, and embryonic development did not differ between any of the treatments. Studies with murine zygotes suggest that NE is taken up by the early embryo, and may enhance cleavage (Burden and Lawrence, 1973; Buznikov, 1991). Although initial examination of these data suggests that NE does not improve fertilization or embryo development, further work is needed to determine the physiological impact of NE on spermatozoa, oocytes, and the early embryo. Acknowledgements The authors thank the staff at the Almquist Research Center for technical assistance. This project was supported by grant 96-35203-3428 from the US Department of Agriculture, training grant GM 08619 from the National Institute of Health, and funding from the National Association of Animal Breeders. References Ain-Shoka, A.A., Buckner, C.K., 1978. Studies on the inhibition of beta-adrenergic receptor agonists of cyclic AMP phosphodiesterase activity of rat heart. Arch. Int. Pharm. 232, 269–278. Bavister, B.D., Leibfried, M.L., Lieberman, G., 1983. Development of preimplantation embryos of the golden hamster in defined medium. Biol. Reprod. 28, 235–247. Bleil, J.D., Wassarman, P.M., 1983. Sperm–egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein. Dev. Biol. 95, 317–324.
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Burden, H.W., Lawrence Jr., I.E., 1973. Presence of biogenic amines in early rat development. Am. J. Anat. 136, 251–257. Buznikov, G.A., 1991. The biogenic amines as regulators of early (pre-nervous) embryogenesis: new data. Adv. Exp. Med. Biol. 296, 33–48. Chang, M.C., Hunter, R.H.F., 1975. Capacitation of mammalian sperm: biological and experimental aspects. In: Hamilton, D.W., Greep, R.O. (Eds.), Handbook of Physiology, Endocrinology V. American Physiological Society, Washington DC, pp. 339–351. Dobrzynska, M.M., Baumgartner, A., Anderson, D., 2004. Antioxidants modulate thyroid hormone-and noradrenalineinduced DNA damage in human sperm. Mutagenesis 19, 325–330. Fagan, J.B., Racker, E., 1977. Reversible inhibition of (Na+ , K+ ) ATPase by Mg2+ , adenosine triphosphate and K+ . Biochem. 16, 152–158. Florman, H.M., First, N.L., 1988a. The regulation of acrosomal exocytosis, 1. Sperm capacitation is required for the induction of acrosome reactions by the bovine zona pellucida in vitro. Dev. Biol. 128, 453–463. Florman, H.M., First, N.L., 1988b. The regulation of acrosomal exocytosis, 2. The zona pellucida-induced acrosome reactions of bovine spermatozoa is controlled by extrinsic positive regulatory elements. Dev. Biol. 128, 464–473. Goren, E.N., Rosen, O.M., 1972. Inhibition of a cyclic nucleotide phosphodiesterase from beef heart by catecholamines and related compounds. Molec. Pharm. 8, 380–384. Hasler, J.F., Henderson, W.B., Hurtgen, P.J., Jin, Z.Q., McCauley, A.D., Mower, S.A., Nedy, B., Shuey, L.S., Stokes, J.E., Trimmer, S.A., 1995. Production, freezing and transfer of bovine IVF embryos and subsequent calving results. Theriogenology 43, 141–152. Hexum, T., 1977. The effect of catecholamines on transport (Na, K) adenosine triphosphatase. Biochem. Pharm. 26, 1221–1227. Lucot, J.B., Jackson, N., Bernatova, I., Morris, M., 2005. Measurement of plasma catecholamines in small samples from mice. J. Pharmacol. Toxicol. Methods 52, 274–277. Mrsny, R.J., Meizel, S., 1979. Inhibition of hamster sperm acrosome reaction by inhibitors of Na-K ATPase. J. Cell. Biol. 83, 206a. Mrsny, R.J., Meizel, S., 1980. Evidence suggesting a role for cyclic nucleotides in acrosome reactions of hamster sperm in vitro. J. Exp. Zool. 211, 153–157. Parrish, J.J., Susko-Parrish, J.L., Winer, M.A., First, N.L., 1988. Capacitation of bovine sperm by heparin. Biol. Reprod. 38, 1171–1180. SAS Institute Inc., 1989–1996. SAS for Windows Version 6.12. Gary, NC. Sirard, M.A., Parrish, J.J., Ware, C.B., Leibfried-Rutledge, M.L., First, N.L., 1988. The culture of bovine oocytes to obtain developmentally competent embryos. Biol. Reprod. 39, 546–552. Stanger, J.D., 1983. The effect if catecholamines and their antagonists on the fertilization of cumulus-free mouse ova in vitro at a suboptimal spermatozoal density. Gamete Res. 7, 111–122. Tervit, H.R., Whittingham, D.G., Rowson, L.E.A., 1972. Successful culture in vitro of sheep and cattle ova. J. Reprod. Fertil. 10, 493–497. Wagtendonk-de Leeuw van, A.M., Mullaart, E., de Roos, A.P.W., Merton, J.S., den Dass, J.H.G., Kemp, B., De Ruigh, L., 2000. Effects of different reproduction techniques: AI, MOET, or IVP, on health and welfare of bovine offspring. Theriogenology 53, 575–597. Walker, S.K., Hill, J.L., Kleeman, D.O., Nancarrow, C.D., 1996. Development of ovine embryos in synthetic oviductal fluid containing amino acids at oviductal concentrations. Biol. Reprod. 55, 703–708. Way, A.L., Schuler, A.M., Killian, G.J., 1997. Influence of bovine ampullary and isthmic oviductal fluid on sperm–egg binding and fertilization in vitro. J. Reprod. Fertil. 109, 95–101. Way, A.L., Barbato, G.F., Killian, G.J., 2001. Identification of norepinephrine in bovine oviductal fluid by high performance liquid chromatography. Life Sci. 70, 567–576. Way, A.L., Killian, G.J., 2002. Capacitation and induction of the acrosome reaction in bull spermatozoa with norepinephrine. J. Androl. 23, 352–357. Yanagimachi, R., 1981. Mechanisms of fertilization in mammals. In: Mastroianni Jr., L., Biggers, B.D. (Eds.), Fertilization and Embryonic Development. Plenum Press, New York.
Sperm binding, in vitro fertilization, and in vitro embryonic development of bovine oocytes fertilized with spermatozoa incubated with norepinephrine A.L. Way a,∗ , G.J. Killian b a b
Lock Haven University, Health Science, Clearfield Campus, 201 University Dr., Clearfield, PA 16830, USA Almquist Research Center, Penn State University, University Park, PA 16802, USA Received 9 June 2005; accepted 19 October 2005 Available online 21 November 2005
Abstract The final stages of sperm maturation, fertilization, and early embryonic development occur within the oviduct and are essential for successful reproduction in mammals. Norepinephrine was previously identified in native bovine oviductal fluid and its in vitro effects on bull sperm capacitation and the acrosome reaction have been determined. It was unknown how physiological concentrations of norepinephrine influence sperm binding, fertilization, and embryo development. Therefore, the objective of this study was to determine if preincubating bovine spermatozoa with physiological concentrations of norepinephrine prior to insemination of bovine oocytes would improve sperm–oocyte binding, fertilization, and embryonic development in vitro. Norepinephrine, in concentrations representing those measured in bovine oviductal fluid, was used to treat bovine spermatozoa prior to insemination. Spermatozoa incubated in norepinephrine were used to inseminate bovine oocytes matured in vitro, and oocytes were evaluated for sperm binding and fertilization. Additional experiments were conducted to evaluate how early in the co-incubation period oocytes were fertilized by spermatozoa pre-incubated with norepinephrine, and to test the developmental competence of those oocytes fertilized with norepinephrine-treated sperm. Sperm binding to the zona pellucida was reduced by preincubation with norepinephrine. Rates of fertilization and embryo development did not increase as a result of pre-incubating spermatozoa with norepinephrine, but as early as 4 h after insemination, spermatozoa treated with 20 ng/ml norepinephrine fertilized more oocytes than spermatozoa incubated in medium alone. Interestingly, this concentration of norepinephrine was found to capacitate spermatozoa in previous studies. These data suggest that oocytes fertilized by spermatozoa incubated in 20 ng/ml norepinephrine fertilize
∗
Corresponding author. Tel.: +1 814 768 3431; fax: +1 814 768 3452. E-mail address: [email protected] (A.L. Way).
0378-4320/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2005.10.006
2
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earlier in vitro than sperm pre-incubated in medium alone, and provide additional support for the role of norepinephrine in sperm capacitation and the acrosome reaction. © 2005 Elsevier B.V. All rights reserved. Keywords: Bovine; Spermatozoa; Oviduct; Fertilization
1. Introduction Identifying components of oviductal fluid (ODF) that influence in vivo fertilization and early embryonic development will provide insight into the physiological processes that take place within the oviductal environment. Identification of components that improve fertilization rates and embryo development in vitro may be of significant economic value to areas of animal agriculture that rely on reproductive technology. Epinephrine is routinely added to culture dishes during in vitro insemination of bovine oocytes as it is thought to aid in fertilization (Hasler et al., 1995). The concentrations used are based on in vitro assays conducted in rodents (Leibried and Bavister, 1982), however, its addition at insemination makes it unclear as to whether the catecholamine is affecting the spermatozoa, the oocytes or both. There have been no investigations into the effects of norepinephrine (NE) on bovine in vitro fertilization and embryo development. Mouse spermatozoa pre-incubated in NE were able to penetrate significantly more ova than control spermatozoa (Stanger, 1983). However, the concentrations of catecholamines used in these studies were higher than the picogram quantities present in murine plasma (Lucot et al., 2005) or in bovine ODF (Way et al., 2001), suggesting the possibility that the effects observed were pharmacological rather than physiological. There are no reports describing the effects of physiological concentrations of NE on the events surrounding fertilization and embryo development in the bovine. Norepinephrine was shown to stimulate capacitation of bovine spermatozoa in vitro (Way and Killian, 2002). Because capacitation is a prerequisite for sperm–oocyte binding and fertilization, it is reasonable to ask whether spermatozoa treated with NE are capable of binding to the zona pellucida and fertilizing in vitro-matured oocytes, and whether the developmental potential of embryos created with NE-treated spermatozoa is affected. The objective of this study was to determine if pre-incubating bovine spermatozoa with physiological concentrations of NE prior to insemination of bovine oocytes would improve sperm–oocyte binding, fertilization, and embryonic development in vitro. 2. Materials and methods 2.1. Oocyte collection and maturation Bovine ovaries (approximately 4.5 kg) were harvested at an abbatoir and placed into 1 l of Dulbecco’s PBS (Life Technologies, Grand Island, NY) (35 ◦ C) prior to transport back to the laboratory. The temperature of the ovaries during transport ranged from 25 to 30 ◦ C. Once in the laboratory, ovaries were rinsed with 35–37 ◦ C tap water and soaked for 15 min in a solution of 1% Chlorhexiderm (AJ Buck, Owings Mills, MD) and 1% 7X Cleaning Solution (ICN Biomedicals, Costa Mesa, CA) in 35–37 ◦ C tap water (Hasler et al., 1995). After a final rinse in 35–37 ◦ C tap water, ovaries were rinsed with Dulbecco’s PBS (39 ◦ C). Oocytes were obtained
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from visible follicles by aspiration with an 18 g needle attached to a 10 cm3 syringe. The aspirate was poured into an Em-Con filter (Immuno Systems, Spring Valley, WI) and rinsed with 1 l of low bicarbonate HEPES (Bavister et al., 1983). The contents of the filter were distributed among 8–12, 60 mm × 15 mm sterile petri dishes. In order to obtain sufficient oocytes to assess sperm binding, fertilization, and embryo culture, oocytes were selected based on the presence of at least one layer of intact cumulus cells for the sperm–oocyte binding experiments, and two or more intact cumulus cell layers for in vitro fertilization and embryo culture. This criteria for oocyte selection was based on previous experience with oocyte quality for in vitro fertilization, and data reported by Hasler et al. (1995) where embryo production was improved if oocytes with multiple layers of cumulus cells are used. Oocytes for sperm binding were considered acceptable if they contained at least one complete layer of compact cumulus as the purpose of these oocytes was to assess the ability of spermatozoa to bind, not the oocyte to be capable of fertilization and embryo development. Oocytes were matured in vitro by incubating cumulus–oocyte complexes in medium M199 containing 10% fetal calf serum, LH (0.01 units/ml), FSH (0.01 units/ml), and penicillin (100 units/ml)/streptomycin (100 g/ml) for 22–24 h at 39 ◦ C in 5% CO2 /air (v/v; Hasler et al., 1995). After maturation oocytes were prepared for sperm binding, fertilization, and embryo culture experiments as described below. 2.2. Sperm preparation Semen was collected from three mature Holstein dairy bulls (Bos taurus) by artificial vagina and washed twice by centrifugation in 10 ml modified Tyrode’s medium (MTM, Parrish et al., 1988). Spermatozoa (5 × 107 ml) were incubated in MTM alone (control) and 20, 80, and 400 ng/ml NE at 39 ◦ C in 5% CO2 /air (v/v). These concentrations were chosen to represent physiological concentrations of NE found in ODF (Way et al., 2001). The 20 ng/ml NE treatment was found to induce the most acrosome reactions in vitro (Way and Killian, 2002). After 2 h of incubation spermatozoa were separated from their incubation medium using percoll gradient centrifugation. Briefly, 2 ml of a 90% percoll solution in MTM (v/v) were placed in the bottom of a 15 ml polypropylene tube and 2 ml of a 45% percoll solution in MTM (v/v) were gently overlaid on top of it. For each treatment, 1 ml of sperm suspension was layered onto the 45% solution and centrifuged for 30 min at 700 × g. The pelleted spermatozoa were recovered, assessed for motility, and suspended in fertilization medium (Bavister et al., 1983). 2.3. Sperm–oocyte binding In vitro-matured oocytes were vortexed for 2 min to remove cumulus cells, washed twice in low bicarbonate HEPES medium and placed 25-well in Nunclon 4-well culture dishes (Fisher Scientific, Pittsburgh, PA) containing 0.5 ml fertilization medium. Oocytes must be denuded before fertilization in order to be able to quantitate sperm bound to the zona after insemination (Way et al., 1997). Oocytes were inseminated with 6.25 × 104 spermatozoa from the MTM, 20 ng/ml NE, 80 ng/ml NE, or 400 ng/ml NE sperm treatments, for a final concentration of 1.25 × 105 spermatozoa/ml. Heparin (2 g) was added to each well at the time of insemination (Hasler et al., 1995). Oocytes and spermatozoa were co-incubated for 18 h at 39 ◦ C in 5% CO2 /air (v/v). After co-incubation oocytes were washed once in low bicarbonate HEPES medium and placed, 10 per slide, under a coverslip mounted with paraffin wax and petroleum jelly at each
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corner. The coverslip was lowered over the oocytes until they burst, and the cytoplasm was rinsed away with low bicarbonate HEPES medium. The zonae pellucidae and any spermatozoa bound to them were stained with Hoechst fluorescent dye 33342 (Sigma, St. Louis, MO). The number of spermatozoa bound to each zona pellucida was determined using fluorescence microscopy (Way et al., 1997). 2.4. In vitro fertilization and embryo culture In vitro-matured oocytes were washed twice in low bicarbonate HEPES medium, placed in fertilization medium with 2 g heparin and inseminated as described above. After 18 h of coincubation oocytes were vortexed to remove cumulus cells and accessory spermatozoa according to the method of Hasler et al. (1995), and washed twice in low bicarbonate HEPES medium. Fertilization and embryo development were assessed independently of each other using two separate pools of oocytes. This was to eliminate the inaccurate assessment of parthenogenic embryos as originating from fertilized oocytes. Oocytes designated for evaluation of fertilization were placed 10 per slide under a coverslip mounted at the corners with paraffin wax and petroleum jelly. The coverslip was gently lowered over the oocytes and adhered to the slide with rubber cement. Oocytes were fixed in acid alcohol for 24 h and stained with aceto-orcein (Sirard et al., 1988). The presence of two pronuclei in the cytoplasm of the oocyte indicated normal fertilization. An additional study was undertaken in which oocytes were prepared for insemination as described above, and fertilized with spermatozoa pre-incubated for 2 h in 20 ng/ml NE or MTM. At 4, 8, 12, and 16 h of co-incubation, oocytes were removed from both treatments, vortexed to removed accessory spermatozoa, washed and placed in fresh fertilization medium. All oocytes were incubated for a total of 16 h before being washed, fixed in acid alcohol and stained with aceto-orcein. This study was designed to determine whether fertilization occurs earlier in the co-incubation period for sperm treated with 20 ng/ml NE. Oocytes designated for embryo culture were vortexed and washed as described above and presumptive zygotes were placed in 4-well culture dishes containing synthetic oviduct fluid (SOF; Tervit et al., 1972) minus glucose and with the addition of Eagle’s Basal Medium essential and Minimum Essential Medium non-essential amino acids (Walker et al., 1996; Wagtendonk-de Leeuw van et al., 2000). Each well contained 0.5 ml SOF and up to 25 presumptive zygotes. The dishes were placed in a humidified modular incubator chamber (Billups-Rothenberg Inc., Del Mar, CA) purged with a gas mixture of 5% O2 /5% CO2 /90% N2 (v/v/v). Embryonic development was assessed on day 4 of culture for cleavage and development, day 8 for development to the blastocyst stage, and day 11 for final assessment of development to the blastocyst and hatched blastocyst stages. 3. Statistical analysis Each experiment was repeated four times and data from each experiment were pooled. Approximately 40–50 oocytes per treatment for sperm binding, 80–90 oocytes per treatment for fertilization, and 90–100 oocytes per treatment for embryo development were evaluated in each replicate. Analysis of variance using a general linear model was performed using mean number of spermatozoa bound per zona pellucida for each treatment in the sperm–oocyte binding experiments, and a weighted mean based on the number of oocytes per treatment in the fertilization and embryo experiments. Least square means comparisons were used to assess sperm binding and weighted least square means were used to analyze fertilization data. Data on embryo
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Fig. 1. Mean number of spermatozoa bound per zona pellucida ± S.E.M. for spermatozoa incubated in MTM, 20 ng/ml NE, 80 ng/ml NE, and 400 ng/ml NE prior to insemination. Binding of MTM-treated spermatozoa to the zona pellucida was significantly different (*) from all of the NE treatments (P < 0.05). There were no differences among the NE treatments.
development were analyzed using least square means to determine the effect of each treatment on embryonic cleavage and development to the blastocyst stage (SAS 6.12 for Windows, Statistical Analysis Systems, 1989). The significance level for all tests was P < 0.05. 4. Results Incubating spermatozoa in all concentrations of NE prior to insemination significantly reduced sperm binding compared to the MTM control (P < 0.05, Fig. 1). There were no significant differences among the concentrations of NE for sperm binding, and NE did not improve fertilization or embryonic development over the control at any concentration used in this study. The only differences observed for fertilization were between the 20 and 80 ng/ml NE treatments (Fig. 2a). Fertilization was lower for spermatozoa incubated in 20 ng/ml NE compared to spermatozoa incubated in 80 ng/ml NE (P = 0.04). There were no differences among any of the treatments for cleavage, development to blastocyst, or blastocyst hatching (Fig. 2b). When evaluating fertilization throughout the co-incubation period for oocytes inseminated with 20 ng/ml NE and MTM-treated spermatozoa, significant differences were observed only at the 4 h time point (Fig. 3). Spermatozoa treated with 20 ng/ml NE fertilized more oocytes after 4 h of co-incubation than spermatozoa incubated in MTM alone (P < 0.05). The 4 h time point was the only time at which differences could be observed. 5. Discussion Previous experiments demonstrated that 20 ng/ml NE capacitated bovine spermatozoa after 2 h of incubation (Way and Killian, 2002), a concentration representative of NE measured in ODF (Way et al., 2001). This observation led to the present experiments, which were designed to determine the effects of treating spermatozoa with physiological concentrations of NE on sperm binding, fertilization, and embryonic development. In this study NE significantly reduced the number of spermatozoa that bound to the zona pellucida of each oocyte as compared to control spermatozoa. Given that NE was
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Fig. 2. (a) Mean percentage ± S.E.M. of oocytes fertilized. (b) Mean percentage ± S.E.M. of oocytes cleaved (open bar), developed to blastocyst (left diagonal bar), and hatched (right diagonal bar). Oocytes were inseminated with spermatozoa incubated in MTM, 20 ng/ml NE, 80 ng/ml NE, and 400 ng/ml NE prior to insemination. Bars with same superscripts (a and b) were not significantly different from each other. Fertilization was lower for spermatozoa incubated in 20 ng/ml NE compared to spermatozoa incubated in 80 ng/ml NE (P = 0.04). There were no statistically significant differences between treatments for cleavage, development to blastocyst, or hatching.
found to induce the acrosome reaction in spermatozoa in a previous study (Way and Killian, 2002), this result is not surprising as capacitated, but not acrosome-reacted spermatozoa are capable of binding to the zona pellucida (Bleil and Wassarman, 1983; Florman and First, 1988a,b).
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Fig. 3. Mean percentage ± S.E.M. of oocytes fertilized by spermatozoa incubated in 20 ng/ml NE (open bar) and MTM (filled bar) prior to fertilization. Gametes were co-incubated for 4, 8, 12, and 16 h. Spermatozoa pre-incubated in 20 ng/ml norepinephrine fertilized significantly more oocytes than sperm pre-incubated in medium alone, but this difference was only evident at the 4 h time point (* P < 0.05).
It is possible that sperm binding was decreased for spermatozoa incubated in 20 ng/ml NE because there were more capacitated spermatozoa at the time of insemination in this group as compared to MTM or other NE treatments. Spermatozoa incubated in 20 ng/ml NE prior to insemination may have fertilized oocytes earlier in the 18 h co-incubation period than spermatozoa incubated in the other treatments and initiated the zona block to polyspermy, ultimately preventing more spermatozoa from binding to the zona pellucida. This possibility does not extend to the higher concentrations of NE (80 and 400 ng/ml), which did not capacitate spermatozoa in the previous study. These higher concentrations of NE may be inhibiting capacitation. Catecholamines in concentrations higher than that found in blood may be acting as chelators (Hexum, 1977; Fagan and Racker, 1977) and/or phosphodiesterase inhibitors (Goren and Rosen, 1972; Ain-Shoka and Buckner, 1978) and have been shown to affect sperm function in vitro in other species (Mrsny and Meizel, 1979, 1980; Dobrzynska et al., 2004). Capacitation with 20 ng/ml NE occurs for a discrete time period (Way and Killian, 2002) suggesting that capacitated spermatozoa incubated with 20 ng/ml NE would not remain functionally capacitated throughout the 18 h co-incubation period. The number of spermatozoa capacitated with heparin increases over time during incubation, making capacitated spermatozoa available for fertilization for a minimum of 6 h and possibly longer during the co-incubation period. This may mean that more oocytes will be fertilized by heparin–capacitated spermatozoa than by NE–capacitated spermatozoa simply because capacitated spermatozoa continue to be available for a longer period of time during the co-incubation. However, capacitation with NE occurs within 2 h of incubation and at levels comparable to capacitation with heparin after 4 h, reducing the preparatory time required of spermatozoa before they can fertilize ova. This reduction in capacitation time and
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its presence in native oviductal fluid make it a viable alternative to heparin capacitation, and a more reasonable choice than epinephrine which is not present in oviductal fluid (Way and Killian, 2002). Capacitated spermatozoa are fragile, short-lived cells (Chang and Hunter, 1975; Yanagimachi, 1981) and any spermatozoa that were not capacitated with NE by 2 h of incubation likely did not participate in fertilization of the oocytes. If spermatozoa capacitated with 20 ng/ml NE were able to fertilize oocytes and initiate the block to polyspermy, they would represent the population of spermatozoa to participate in fertilization, rather than spermatozoa incubated in the 20 ng/ml NE treatment that may respond to heparin that was added upon insemination. In those spermatozoa, capacitation would be delayed for several hours if they follow the normal pattern of capacitation with heparin in vitro (Parrish et al., 1988). Data from the experiment in which oocytes were removed from co-incubation over the course of 16 h suggests that sperm treated with 20 ng/ml NE are capacitated and fertilizing oocytes earlier in the co-incubation period than sperm in medium alone (Fig. 3). This supports the reduction in sperm binding observed for those oocytes inseminated with 20 ng/ml NE-treated spermatozoa, and supports the hypothesis that it is spermatozoa capacitating with NE that are fertilizing the oocytes in that treatment. For all treatments except 20 ng/ml NE, the embryo cleavage rate was lower than the fertilization rate, while the percentages of oocytes fertilized (75.11 ± 2.13%) and cleaved (75.08 ± 3.09%) were almost identical for the 20 ng/ml NE treatment (Fig. 2). This interesting difference between the 20 ng/ml NE treatment and the other treatments cannot be assessed statistically because two separate populations of oocytes were used in the fertilization and embryo culture experiments. It appears that spermatozoa incubated in MTM and higher concentrations of NE were capable of fertilization, but may not have been as capable of initiating the events leading to cleavage as spermatozoa treated with 20 ng/ml NE. Even though fertilization rates were higher in the other treatments, embryonic development was not improved. Essentially all of the oocytes that were fertilized by spermatozoa incubated with 20 ng/ml NE were capable of cleaving, and embryonic development did not differ between any of the treatments. Studies with murine zygotes suggest that NE is taken up by the early embryo, and may enhance cleavage (Burden and Lawrence, 1973; Buznikov, 1991). Although initial examination of these data suggests that NE does not improve fertilization or embryo development, further work is needed to determine the physiological impact of NE on spermatozoa, oocytes, and the early embryo. Acknowledgements The authors thank the staff at the Almquist Research Center for technical assistance. This project was supported by grant 96-35203-3428 from the US Department of Agriculture, training grant GM 08619 from the National Institute of Health, and funding from the National Association of Animal Breeders. References Ain-Shoka, A.A., Buckner, C.K., 1978. Studies on the inhibition of beta-adrenergic receptor agonists of cyclic AMP phosphodiesterase activity of rat heart. Arch. Int. Pharm. 232, 269–278. Bavister, B.D., Leibfried, M.L., Lieberman, G., 1983. Development of preimplantation embryos of the golden hamster in defined medium. Biol. Reprod. 28, 235–247. Bleil, J.D., Wassarman, P.M., 1983. Sperm–egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein. Dev. Biol. 95, 317–324.
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