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Is buffering capacity the principal role of the jelly coat in Bufo arenarum fertilization?
Camp. Biochem. Physiol. Vol. IOSA, No. 3, pp. 533-537, 1993 Printed
0300-9629/93$6.00+ 0.00 0 1993Pergamon Press Ltd
in Great Britain
IS BUFFERING CAPACITY THE PRINCIPAL ROLE OF THE JELLY COAT IN BUFO ARENARUM FERTILIZATION? TERESAM. FONOVICHDESCHROEDER, LIDIA GAUNAand ANA M. PECI&NDED’ANGELO* LIBIQUIMA, Departamento de Quimica, Facultad de Ingenieria, Universidad National de1 Comahue, Buenos Aires 1400, 8300-Neuqutn, Argentina (Fax 54-943-23609) (Received
30 June 1992; accepted 25 September 1992)
Abstract-l. Dejellied B&o arenarum oocytes can be fertilized in Ringer-Phosphate buffer with the same efficiency as jellied (control) oocytes. 2. Ringer-Phosphate buffer at pH = 7.4 not only provides buffering capacity but also the ApH necessary for the acrosomal reaction. 3. The use of Ringer-TRIS buffer at pH = 7.4 does not render as good as Ringer-Phosphate buffer results, in terms of fertilization percentages. 4. Insemination of oocytes in Ringer-TRIS buffer interferes with early development.
INTRODUCTION
that fertilization would not take place in amphibians in the absence of the “diffusible factor”, a low molecular weight compound present in the jelly coat (Barbieri, 1982; Cabada, 1975). Also, the cervical mucus of mammals and the jelly coat of amphibians have been considered as analogous in connection with the fertilization process. On the other hand, it has been accepted that triggering of the acrosome reaction occurs as a result of some alterations in the sperm plasma membrane permeability, due to sperm contact with a component of the jelly in the sea urchin (Shapiro et al., 1980). For many years it has been stated
Domino and Garbers (1988) reported that the fucase-sulfate-rich glycoconjugate (FSG) that induces
the acrosome reaction elicits some signal transduction mechanisms. Though these findings would confirm glycoconjugate as one of the female coordinating factors essential for fertilization, these authors consider the fact that high external pH as well as A23 187 (Summers et al., 1976) and the monovalent cation H+ exchanger, nigericin, also induce the acrosome reaction; strong evidence against this theory. Those previous findings and the absolute requirement of calcium led Domino and Garbers (1988) to conclude that although increases in cyclic AMP and 1,4,5-IP, accumulation are associated with the induction of the acrosome reaction, there is no evidence that those second messengers are required for the event. In addition Chang (1984) demonstrated that both the acrosome reaction and in vitro fertilization can be achieved in mammals without the participation of *To whom all correspondence
should be addressed.
any specific substance from the female reproductive tissue and biological fluids. The sperm acrosome reaction involves H+ efflux and other ion movements (Shapiro et al., 1980) and a rise in intracellular pH also occurs soon after fertilization in the sea urchin eggs, just at the time of the appearance of an outward current (David et al., 1988). The events following fertilization fall into two categories: early events taking place within 2 min after fertilization, and late events beginning 2-5 min after fertilization. The increase in intracellular pH is the linkage between early and late events (Campisi and Scandella, 1980). In order to elucidate whether the principal role of the amphibian jelly is related to the maintenance of an adequate pH during fertilization, we perform fertilization assays of dejellied Bufo arenarum oocytes in buffered Ringer solutions. Our results are consistent with a non essential role of the jelly coat in fertilization; moreover it can be successfully replaced by a Ringer-Phosphate buffer. MATERIALS
AND METHODS
Bufo arenarum oocytes were obtained as reported previously (Fonovich de Schroeder and PechCn de D’Angelo, 1991) and sperm suspensions (about 106-10’ cells/ml) were prepared by dilacerating the testes in Ringer solution. The jelly suspension was obtained according to Stewart-Savage and Grey (1984) by exposing the oocyte strings in Ringer solution to UV light for short periods of time and then centrifuging them at 11,000g for 20 min. Proteins from the jelly were measured by the method of Lowry et al. (1951). The pH of sperm suspension, jelly suspension and Ringer solution were measured 533
534
TERESA
M.
FONOVICH DE SCHROEDERet al.
with an Orion pH meter (Orion Research Inc., U.S.A.) Oocytes were dejellied using 0.01 M Thioglycolic acid treatment (neutralized with COrNa,) and washed with either Ringer-Phosphate (0.05 M) or Ringer-TRIS (0.1 M) pH = 7.4 before being inseminated. Inseminations were carried out in duplicate in Petri dishes, about 50 oocytes in each. Jellied as well as dejellied oocytes were inseminated in the presence of 1 ml of Ringer solution, Ringer-Phosphate buffer or Ringer-TRIS buffer, with or without the addition of 20-30 mg of jelly suspension, by adding two drops of concentrated sperm suspension. The oocytes were examined under a stereoscopic microscope and soon after fertilization the insemination medium was replaced by Ringer solution. RESULTS
Figure 1A shows the fertilization percentages obtained when jellied (control) and dejellied oocytes were inseminated in the presence of Ringer solution, with or without the addition of jelly suspension. We did not find significant differences between jellied samples as well as the dejellied ones (with or without the addition of jelly). Dejellied oocytes could never be fertilized in Ringer solution even if jelly suspension was added to the insemination medium. In Fig. 1B the results from inseminating jellied (control) and dejellied oocytes in Ringer-Phosphate buffer are represented. Dejellied oocytes were able to fertilize to a similar extent as control ones, and the addition of jelly suspension did not improve the results. Dejellied oocytes were also able to fertilize at similar levels than control ones in Ringer-TRIS buffer (Fig. 1C). No significant differences were found between jellied (control) oocytes inseminated
loo (A) Ringer 1
0
in different Ringer mediums (Ringer, RingerPhosphate or Ringer-TRIS) and the addition of jelly suspension did not cause any increase in the fertilization rate. Embryos were examined again under the stereoscopic microscope the moment they reached the “muscular response” stage. The results were expressed as: % Muscular resp. stage embryos =
N embryos at muscular resp. stage x 100. (1) N fertilized oocytes
A significant difference was found between the percentage of embryos that reached the “muscular response” stage when they were fertilized in Ringer solution and the control ones fertilized in Ringer-Phosphate buffer (P c 0.01). Significant differences were also found between fertilization of jellied (control) oocytes carried out in Ringer solution and in the presence or absence of jelly suspension (P < 0.005) (Fig. 2). Ooytes fertilized in Ringer-Phosphate buffer reached the “muscular response” stage independently of the treatment they received (control or dejellied ones, with or without jelly addition, Fig. 2B), while a significant difference was found between dejellied and control oocytes fertilized in Ringer-TRIS buffer with the addition of jelly suspension (P < 0.05). Also a low percentage of embryos in the “muscular response” stage was found in dejellied oocytes fertilized in Ringer-TRIS buffer without jelly addition, though it was not significantly different from the one obtained for its control. Table 1 shows the pH (Means f SD) of the sperm and jelly suspensions as well as the ones for
B) Ringer - Phospate
(C) Ringer - TRIS
1
C- C+ D- D+
C- C+ D- D+
C-‘C+ D- D+
Fig. 1. Fertilization percentages of in vitro inseminated, jellied (control) or dejellied Bufo arenarum oocytes; with (+) or without (-) the addition of jelly suspension; carried out in different insemination media: (A) Rinaer. (B) Rinser-Phosnhate buffer, (C) Rinser-TRIS buffer. Results are the Means + SD of duplicate sambles and areexpressed as a percentage of the oocytes present in the insemination medium which arrived at the 4-8 cells stage. Similar results were obtained from other experiments. C+, Control with jelly suspension addition; C - , Control without jelly suspension addition; D + , dejellied with jelly suspension addition. D-, dejellied without jelly suspension addition. Student’s t-test: (a) P < 0.0001 between Cand D- as well as between C+ and D+ inseminated in Ringer solution. N = 50.
Jelly coat and fertilization
:B) Ringer - Phospab
C- C+ D- D+
535 C) Ringer - TRIS
C- C+ D- D+
Fig. 2. Embryos in muscular response stage percentages of in vitro inseminated, jellied (Control) or dejellied Bufo nrenarum oocytes; with (+) or without (-) the addition of jelly suspension; carried out in different insemination media: (A) Ringer, (B) Ringer-Phosphate buffer, (C) Ringer-TRIS buffer. Results are the means f SD of duplicate samples and are expressed as percentage of the fertilized oocytes that arrived to the muscular response stage. Similar results were obtained from other experiments. C+, control with jelly suspension addition; C-, control without jelly suspension addition; D+, dejellied with jelly suspension addition; D -, dejellied without jelly suspension addition. Student’s r-test: (a) P < 0.01 between C- inseminated in Ringer solution and C- inseminated in Ringer-Phosphate buffer; (b) P < 0.005 between C- and Cf inseminated in Ringer solution; (c) P < 0.05 between C+ and D+ inseminated in Ringer-TRIS buffer. N = 30-40.
Ringer-Phosphate and Ringer-TRIS buffers used for the in vitro fertilization assays. A significant difference was found between sperm suspension pH and the other parameters. DISCUSSION
Our results not only demonstrate that fertilization can take place in amphibians in the absence of any component of the jelly, but they are also in agreement with being its principal role to provide adequate pH and ionic strength as well as buffering capacity. Although some authors have established that the “egg water”, prepared by extracting the diffusible factor of the jelly with distilled water, had buffering capacity (Cabada, 1975), the importance of this capacity had not been determined. Not only does the fertilized egg need a buffer to support the H+ exchange which occurs 2-5 min after fertilization, but this buffer capacity at a higher pH could also be the key for the acrosomal reaction (Fig. 3). The occurrence of this reaction is related to a rise in cyclic AMP concentration and 1,4,5-Inositol trisphosphate (IP,) accumulation (Domino and Garbers, 1988). Table 1. pH of the sperm suspension, the jelly suspension and the buffered Ringer solutions used for in vitro insemination
Medium N PH Sperm suspension 3 6.88* 0.29 Jelly suspension 5 7.31& 0.29 Ringer-Phosphate buffer 3 7.40* 0.01 Rinser-TRIS buffer 3 7.39+ 0.01 Values are means + SD; ANOVA: P < 0.05 between tested media.
Although the fucose-sulfate-rich glycoconjugate (FSG) that induces the acrosome reaction also elicits the signal transduction mechanisms described, the fact that high external pH can also induce the acrosome reaction and the findings of Morand et al. (1988) on the effects of pH on adenylate cyclase activity; lead us to suggest that these mechanisms can be elicited just by the ApH we found between jelly or buffered Ringer solutions and sperm suspensions (see Table 1). The events that allow the reacted sperm to fertilize the oocyte involves its binding to a specific receptor (Vaquier, 1980), eliciting the generation of intracellular second messengers: IP,, the one responsible for calcium release from cytoplasmic stores (Crossley et al., 1988; Miyazaki, 1988) and diacylglycerol (DAG), whose principal role is to activate protein kinase C. Free cytoplasmic calcium then participates in exocytosis of cortical alveoli (Iwamatsu, 1989), a process in which egg proteases induce the detachment of the vitelline layer from the egg plasma membrane, to form the fertilization membrane (Fig. 3). Ringer-Phosphate proved to be the more appropriate buffer tested, in providing the same conditions in the inseminating medium as the ones provided by the jelly coat (in control oocytes), resulting in a percentage of fertilization quite similar to the control ones fertilized in Ringer solution. The addition of jelly suspension to the insemination medium did not improve the results in any case. The lack of response in dejellied oocytes inseminated in Ringer solution with the addition of jelly suspension (Fig. lA), is probably due to the formation of jelly clumps that trap large
TEREsaM. FONOVICH DE
536
SCHROEDER
et al.
Acroaomal reactlo
0 time
-
Inward current Outward current 15-30
2-5
Fig. 3. Scheme showing some biochemical aspects of fertilization, especially the ion movements which occur soon after the oocyte becomes fertilixed. PM, plasma membrane; VM, vitelline membrane; CG, cortical granule; R, receptor; FM, fertilization membrane; CaB, calcium binding protein; GP, G protein; PhLC, phospholipase C; PKC, protein lcinase C.
aggregates of sperm (Stewart-Savage and Grey, 1984). Ringer-TRIS buffer did not give as good development results as Ringer-Phosphate buffer. It has been reported that the loss in receptor Quinuclidinyl-benzilate (QNB) binding in TRIS but not in Phosphate buffer in rat brain homogenate (Abe et al., 1985; David et al., 1988), described alterations produced by TRIS on the electrical properties of the unfertilized sea urchin eggs. A profound study on TRIS effects on mechanisms of signal transduction in relation to the plasma membrane, will be necessary to explain the mechanism by which it interferes with early development. Though Bufo arenartun dejellied oocytes can be fertilized in Ringer-Phosphate buffer (162 mOsm/l) to the same extent as jellied oocytes in Ringer solution (22.8 mOsm/l), the fertilization rate of the former abruptly fell when the buffer was replaced after 1 hr instead of 15 min after insemination (percentage fertilization = 1.66 & 2.35). The reason for this restriction in exposure time must be the need for an appropriate ionic strength and osmolarity, in order to keep the cells in the required physiological conditions. Acknowledgements-This
work was supported in part by
CONICET (Argentina). Teresa M. Fonovich de Schroeder is the holder of a fellowship from CONICET.
REFERENCES Abe K., Kogure K., Arai H. and Nakano M. (1985) Ascorbate induced lipid peroxidation results in loss of receptor binding in TRIS, but not in phosphate buffer. Implications for the involvement of metal ions. Eiochem. Int. 11, 341-348. Barbieri F. D. (1982) Biochemical mechanisms of fertilization. In Physiopathology of Hypophysial Disturbances and Diseases of Reproduction,
pp. 235-250. Alan R. Liss, New
York. Cabada M. 0. (1975) Some female coordinating factors in amphibian fertilization. Deu. Growth Dr@?r. 17, 187-195. Campisi J. and Scandella C. J. (1980) Bulk membrane fluidity increases after fertilization or partial activation of sea urchin eggs. J. biol. Chem. 255, 5411-5419. Crossley I., Swann K., Chambers E. and Whitaker M. (1988) Activation of sea urchin eggs by inositol phosphates is independent of external calcium. Biochem. J. 252, 257-262. Chang M. C. (1984) The meaning of sperm capacitation. A historical perspective. J. Androl. 5, 45-50. David C., Halliwell J. and Whitaker M. (1988) Some properties of the membrane currents underlying the fertilization potential in sea urchin eggs. J. Physiol. 402, 139-154.
Domino S. E. and Garbers D. L. (1988) The fucose-sulfate glycoconjugate that induces an acrosome reaction in spermatozoa stimulates inositol 1,4,5-trisphosphate accumulation. J. biol. Chem. 263, 690-695. Fonovich de Schroeder T. M. and Pechen de D’Angelo A. M. (1991) Dieldrin effects on phospholipid and phosphoinositide metabolism in Bufo arenarum oocytes. Comp. Biochem. Physiol. 9W,
287-292.
Jelly coat and fertilization Iwamatsu T. (1989) Exocytosis of cortical alveoli and its initiation time in medaka eggs induced by microinjection of various agents. Dev. Growth Differ. 31, 39-44. Lowry 0. H., Rosebrough N. J., F&r A. L. and Randall R. S. (1951) Protein measurement with the Folin nhenol reagent. J. hiol. Chem. 193, 265-275. Miyazaki S-I. (1988) Inositol 1,4,5-Trisphosphate-induced calcium release and guanine nucleotide-binding proteinmediated periodic calcium rises in Golden hamster eggs. J. Cell Biol. 106, 345-353. Morand C., Remesy C. and Demigne C. (1988) Modulation of glucagon effects by changes in extracellular pH and calcium. Biochim. biophys. Acta 968, 192-202.
537
Shapiro 8. M., Schackmann R. W., Gabel C. A., Foerder C. A. and Farance M. L. (1980) Molecular alterations in gamete surfaces during fertilization and early developLent. Cell surface 1271149. Stewart-Savage J. and Grey R. D. (1984) Fertilization of investment-free Xenopus eggs. Exp. Cell Res. 154, 639642. Summers R. G., Talbot P., Keough E. M., Hylander B. L. and Franklin L. E. (1976) Ionophore A23187 induces acrosome reactions in sea urchin and guinea pig spermatozoa. J. exp. Zool. 196, 381-385. Vacquier V. D. (1980) The adhesion of sperm to sea urchin Cell surface 151-158.
0300-9629/93$6.00+ 0.00 0 1993Pergamon Press Ltd
in Great Britain
IS BUFFERING CAPACITY THE PRINCIPAL ROLE OF THE JELLY COAT IN BUFO ARENARUM FERTILIZATION? TERESAM. FONOVICHDESCHROEDER, LIDIA GAUNAand ANA M. PECI&NDED’ANGELO* LIBIQUIMA, Departamento de Quimica, Facultad de Ingenieria, Universidad National de1 Comahue, Buenos Aires 1400, 8300-Neuqutn, Argentina (Fax 54-943-23609) (Received
30 June 1992; accepted 25 September 1992)
Abstract-l. Dejellied B&o arenarum oocytes can be fertilized in Ringer-Phosphate buffer with the same efficiency as jellied (control) oocytes. 2. Ringer-Phosphate buffer at pH = 7.4 not only provides buffering capacity but also the ApH necessary for the acrosomal reaction. 3. The use of Ringer-TRIS buffer at pH = 7.4 does not render as good as Ringer-Phosphate buffer results, in terms of fertilization percentages. 4. Insemination of oocytes in Ringer-TRIS buffer interferes with early development.
INTRODUCTION
that fertilization would not take place in amphibians in the absence of the “diffusible factor”, a low molecular weight compound present in the jelly coat (Barbieri, 1982; Cabada, 1975). Also, the cervical mucus of mammals and the jelly coat of amphibians have been considered as analogous in connection with the fertilization process. On the other hand, it has been accepted that triggering of the acrosome reaction occurs as a result of some alterations in the sperm plasma membrane permeability, due to sperm contact with a component of the jelly in the sea urchin (Shapiro et al., 1980). For many years it has been stated
Domino and Garbers (1988) reported that the fucase-sulfate-rich glycoconjugate (FSG) that induces
the acrosome reaction elicits some signal transduction mechanisms. Though these findings would confirm glycoconjugate as one of the female coordinating factors essential for fertilization, these authors consider the fact that high external pH as well as A23 187 (Summers et al., 1976) and the monovalent cation H+ exchanger, nigericin, also induce the acrosome reaction; strong evidence against this theory. Those previous findings and the absolute requirement of calcium led Domino and Garbers (1988) to conclude that although increases in cyclic AMP and 1,4,5-IP, accumulation are associated with the induction of the acrosome reaction, there is no evidence that those second messengers are required for the event. In addition Chang (1984) demonstrated that both the acrosome reaction and in vitro fertilization can be achieved in mammals without the participation of *To whom all correspondence
should be addressed.
any specific substance from the female reproductive tissue and biological fluids. The sperm acrosome reaction involves H+ efflux and other ion movements (Shapiro et al., 1980) and a rise in intracellular pH also occurs soon after fertilization in the sea urchin eggs, just at the time of the appearance of an outward current (David et al., 1988). The events following fertilization fall into two categories: early events taking place within 2 min after fertilization, and late events beginning 2-5 min after fertilization. The increase in intracellular pH is the linkage between early and late events (Campisi and Scandella, 1980). In order to elucidate whether the principal role of the amphibian jelly is related to the maintenance of an adequate pH during fertilization, we perform fertilization assays of dejellied Bufo arenarum oocytes in buffered Ringer solutions. Our results are consistent with a non essential role of the jelly coat in fertilization; moreover it can be successfully replaced by a Ringer-Phosphate buffer. MATERIALS
AND METHODS
Bufo arenarum oocytes were obtained as reported previously (Fonovich de Schroeder and PechCn de D’Angelo, 1991) and sperm suspensions (about 106-10’ cells/ml) were prepared by dilacerating the testes in Ringer solution. The jelly suspension was obtained according to Stewart-Savage and Grey (1984) by exposing the oocyte strings in Ringer solution to UV light for short periods of time and then centrifuging them at 11,000g for 20 min. Proteins from the jelly were measured by the method of Lowry et al. (1951). The pH of sperm suspension, jelly suspension and Ringer solution were measured 533
534
TERESA
M.
FONOVICH DE SCHROEDERet al.
with an Orion pH meter (Orion Research Inc., U.S.A.) Oocytes were dejellied using 0.01 M Thioglycolic acid treatment (neutralized with COrNa,) and washed with either Ringer-Phosphate (0.05 M) or Ringer-TRIS (0.1 M) pH = 7.4 before being inseminated. Inseminations were carried out in duplicate in Petri dishes, about 50 oocytes in each. Jellied as well as dejellied oocytes were inseminated in the presence of 1 ml of Ringer solution, Ringer-Phosphate buffer or Ringer-TRIS buffer, with or without the addition of 20-30 mg of jelly suspension, by adding two drops of concentrated sperm suspension. The oocytes were examined under a stereoscopic microscope and soon after fertilization the insemination medium was replaced by Ringer solution. RESULTS
Figure 1A shows the fertilization percentages obtained when jellied (control) and dejellied oocytes were inseminated in the presence of Ringer solution, with or without the addition of jelly suspension. We did not find significant differences between jellied samples as well as the dejellied ones (with or without the addition of jelly). Dejellied oocytes could never be fertilized in Ringer solution even if jelly suspension was added to the insemination medium. In Fig. 1B the results from inseminating jellied (control) and dejellied oocytes in Ringer-Phosphate buffer are represented. Dejellied oocytes were able to fertilize to a similar extent as control ones, and the addition of jelly suspension did not improve the results. Dejellied oocytes were also able to fertilize at similar levels than control ones in Ringer-TRIS buffer (Fig. 1C). No significant differences were found between jellied (control) oocytes inseminated
loo (A) Ringer 1
0
in different Ringer mediums (Ringer, RingerPhosphate or Ringer-TRIS) and the addition of jelly suspension did not cause any increase in the fertilization rate. Embryos were examined again under the stereoscopic microscope the moment they reached the “muscular response” stage. The results were expressed as: % Muscular resp. stage embryos =
N embryos at muscular resp. stage x 100. (1) N fertilized oocytes
A significant difference was found between the percentage of embryos that reached the “muscular response” stage when they were fertilized in Ringer solution and the control ones fertilized in Ringer-Phosphate buffer (P c 0.01). Significant differences were also found between fertilization of jellied (control) oocytes carried out in Ringer solution and in the presence or absence of jelly suspension (P < 0.005) (Fig. 2). Ooytes fertilized in Ringer-Phosphate buffer reached the “muscular response” stage independently of the treatment they received (control or dejellied ones, with or without jelly addition, Fig. 2B), while a significant difference was found between dejellied and control oocytes fertilized in Ringer-TRIS buffer with the addition of jelly suspension (P < 0.05). Also a low percentage of embryos in the “muscular response” stage was found in dejellied oocytes fertilized in Ringer-TRIS buffer without jelly addition, though it was not significantly different from the one obtained for its control. Table 1 shows the pH (Means f SD) of the sperm and jelly suspensions as well as the ones for
B) Ringer - Phospate
(C) Ringer - TRIS
1
C- C+ D- D+
C- C+ D- D+
C-‘C+ D- D+
Fig. 1. Fertilization percentages of in vitro inseminated, jellied (control) or dejellied Bufo arenarum oocytes; with (+) or without (-) the addition of jelly suspension; carried out in different insemination media: (A) Rinaer. (B) Rinser-Phosnhate buffer, (C) Rinser-TRIS buffer. Results are the Means + SD of duplicate sambles and areexpressed as a percentage of the oocytes present in the insemination medium which arrived at the 4-8 cells stage. Similar results were obtained from other experiments. C+, Control with jelly suspension addition; C - , Control without jelly suspension addition; D + , dejellied with jelly suspension addition. D-, dejellied without jelly suspension addition. Student’s t-test: (a) P < 0.0001 between Cand D- as well as between C+ and D+ inseminated in Ringer solution. N = 50.
Jelly coat and fertilization
:B) Ringer - Phospab
C- C+ D- D+
535 C) Ringer - TRIS
C- C+ D- D+
Fig. 2. Embryos in muscular response stage percentages of in vitro inseminated, jellied (Control) or dejellied Bufo nrenarum oocytes; with (+) or without (-) the addition of jelly suspension; carried out in different insemination media: (A) Ringer, (B) Ringer-Phosphate buffer, (C) Ringer-TRIS buffer. Results are the means f SD of duplicate samples and are expressed as percentage of the fertilized oocytes that arrived to the muscular response stage. Similar results were obtained from other experiments. C+, control with jelly suspension addition; C-, control without jelly suspension addition; D+, dejellied with jelly suspension addition; D -, dejellied without jelly suspension addition. Student’s r-test: (a) P < 0.01 between C- inseminated in Ringer solution and C- inseminated in Ringer-Phosphate buffer; (b) P < 0.005 between C- and Cf inseminated in Ringer solution; (c) P < 0.05 between C+ and D+ inseminated in Ringer-TRIS buffer. N = 30-40.
Ringer-Phosphate and Ringer-TRIS buffers used for the in vitro fertilization assays. A significant difference was found between sperm suspension pH and the other parameters. DISCUSSION
Our results not only demonstrate that fertilization can take place in amphibians in the absence of any component of the jelly, but they are also in agreement with being its principal role to provide adequate pH and ionic strength as well as buffering capacity. Although some authors have established that the “egg water”, prepared by extracting the diffusible factor of the jelly with distilled water, had buffering capacity (Cabada, 1975), the importance of this capacity had not been determined. Not only does the fertilized egg need a buffer to support the H+ exchange which occurs 2-5 min after fertilization, but this buffer capacity at a higher pH could also be the key for the acrosomal reaction (Fig. 3). The occurrence of this reaction is related to a rise in cyclic AMP concentration and 1,4,5-Inositol trisphosphate (IP,) accumulation (Domino and Garbers, 1988). Table 1. pH of the sperm suspension, the jelly suspension and the buffered Ringer solutions used for in vitro insemination
Medium N PH Sperm suspension 3 6.88* 0.29 Jelly suspension 5 7.31& 0.29 Ringer-Phosphate buffer 3 7.40* 0.01 Rinser-TRIS buffer 3 7.39+ 0.01 Values are means + SD; ANOVA: P < 0.05 between tested media.
Although the fucose-sulfate-rich glycoconjugate (FSG) that induces the acrosome reaction also elicits the signal transduction mechanisms described, the fact that high external pH can also induce the acrosome reaction and the findings of Morand et al. (1988) on the effects of pH on adenylate cyclase activity; lead us to suggest that these mechanisms can be elicited just by the ApH we found between jelly or buffered Ringer solutions and sperm suspensions (see Table 1). The events that allow the reacted sperm to fertilize the oocyte involves its binding to a specific receptor (Vaquier, 1980), eliciting the generation of intracellular second messengers: IP,, the one responsible for calcium release from cytoplasmic stores (Crossley et al., 1988; Miyazaki, 1988) and diacylglycerol (DAG), whose principal role is to activate protein kinase C. Free cytoplasmic calcium then participates in exocytosis of cortical alveoli (Iwamatsu, 1989), a process in which egg proteases induce the detachment of the vitelline layer from the egg plasma membrane, to form the fertilization membrane (Fig. 3). Ringer-Phosphate proved to be the more appropriate buffer tested, in providing the same conditions in the inseminating medium as the ones provided by the jelly coat (in control oocytes), resulting in a percentage of fertilization quite similar to the control ones fertilized in Ringer solution. The addition of jelly suspension to the insemination medium did not improve the results in any case. The lack of response in dejellied oocytes inseminated in Ringer solution with the addition of jelly suspension (Fig. lA), is probably due to the formation of jelly clumps that trap large
TEREsaM. FONOVICH DE
536
SCHROEDER
et al.
Acroaomal reactlo
0 time
-
Inward current Outward current 15-30
2-5
Fig. 3. Scheme showing some biochemical aspects of fertilization, especially the ion movements which occur soon after the oocyte becomes fertilixed. PM, plasma membrane; VM, vitelline membrane; CG, cortical granule; R, receptor; FM, fertilization membrane; CaB, calcium binding protein; GP, G protein; PhLC, phospholipase C; PKC, protein lcinase C.
aggregates of sperm (Stewart-Savage and Grey, 1984). Ringer-TRIS buffer did not give as good development results as Ringer-Phosphate buffer. It has been reported that the loss in receptor Quinuclidinyl-benzilate (QNB) binding in TRIS but not in Phosphate buffer in rat brain homogenate (Abe et al., 1985; David et al., 1988), described alterations produced by TRIS on the electrical properties of the unfertilized sea urchin eggs. A profound study on TRIS effects on mechanisms of signal transduction in relation to the plasma membrane, will be necessary to explain the mechanism by which it interferes with early development. Though Bufo arenartun dejellied oocytes can be fertilized in Ringer-Phosphate buffer (162 mOsm/l) to the same extent as jellied oocytes in Ringer solution (22.8 mOsm/l), the fertilization rate of the former abruptly fell when the buffer was replaced after 1 hr instead of 15 min after insemination (percentage fertilization = 1.66 & 2.35). The reason for this restriction in exposure time must be the need for an appropriate ionic strength and osmolarity, in order to keep the cells in the required physiological conditions. Acknowledgements-This
work was supported in part by
CONICET (Argentina). Teresa M. Fonovich de Schroeder is the holder of a fellowship from CONICET.
REFERENCES Abe K., Kogure K., Arai H. and Nakano M. (1985) Ascorbate induced lipid peroxidation results in loss of receptor binding in TRIS, but not in phosphate buffer. Implications for the involvement of metal ions. Eiochem. Int. 11, 341-348. Barbieri F. D. (1982) Biochemical mechanisms of fertilization. In Physiopathology of Hypophysial Disturbances and Diseases of Reproduction,
pp. 235-250. Alan R. Liss, New
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