Maturation and Sperm Penetration of Canine Ovarian Oocytes In Vitro CHERRIE A N N MAHI AND RYUZO YANAGIMACHI Department of Anatomy a n d Reproductive Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii
ABSTRACT Canine ovarian oocytes were cultured in a medium consisting of TC medium 199, fetal calf serum and antibiotics. Ninety-nine percent of the apparently healthy oocytes were i n the germinal vesicle (dictyate) stage when recovered from the ovaries; 25% of them reached metaphase I or I1 by 72 hours of culture. Washed ejaculated spermatozoa were added to BWW medium containing oocytes which had either been removed directly from the follicles or which had been cultured for 24-72 hours. The earliest acrosome reaction and zona penetration by spermatozoa were seen at seven hours after insemination. Seventy-four percent of the oocytes examined between 11 and 24 hours after insemination showed evidence of zona penetration by spermatozoa. Neither the condition of the oocyte vitellus nor the stage of nuclear maturation influenced the incidence of zona penetration. Decondensing sperm nuclei were found in the vitellus of 27% of the oocytes which had not been cultured and in the vitellus of 20 % of those which had been cultured for 24-72 hours and were in various stages of maturation. These results indicate that (1) canine ovarian oocytes can be matured i n vitro, (2) the spermatozoa require capacitation which takes approximately seven hours in vitro and (3) maturation of the oocytes is not required for sperm passage through the zona pellucida or entry into the vitellus nor for sperm nuclear decondensation.
The interval between estrous periods in bitches averages seven months (Hancock and Rowlands, '49) and ovulation is difficult to predict or detect (Phemister et al., '73). Therefore if fertilization in dogs is to be studied routinely, a system must be devised which does not depend on natural ovulation. Mammalian ovarian oocytes can mature in vitro when liberated from Graafian follicles and placed in appropriate culture media (Pincus and Enzmann, '35; Chang, '55a,b; for review see Donahue, '72; Biggers, '73). Edwards et al. ('69) and Cross and Brinster ('70) proved that human and mouse oocytes matured in vitro can be fertilized in vitro. We postulated that canine oocytes could be matured in vitro in order to obtain material for studying fertilization in this species. The structure of both fertilized and unfertilized canine oocytes was described in detail by Van der Stricht ('23), but the fertilization process has not been observed. Doak et al. ('67) found that canine spermatozoa survive for extended periods (6-11 J. EXP. ZOOL., 196: 189-196.
days) within the uteri of estrous bitches; however the capacitation process has not been described for canine spermatozoa nor has the need for capacitation been established. The dog differs from most mammals in that oocytes are ovulated in the germinal vesicle (dictyate) stage (Van der Stricht, '23). Since mating can take place from 2-3 days before to a week after ovulation (Evans and Cole, '31; Phemister et al., '73), it seems quite possible for canine spermatozoa and immature oocytes to meet in the oviducts. We felt that it would be extremely interesting to determine whether these dictyate oocytes could be penetrated by spermatozoa and if so, whether the sperm nuclei decondense and form male pronuclei. MATERIALS A N D METHODS
Maturation of ovarian oocytes Ovaries were obtained from 75 bitches at random stages of the estrous cycle either by autopsy following euthanasia in a decompression chamber at the Hawaiian
189
190
CHERRIE A N N MAHI A N D RYUZO YANAGIMACHI
a medium devised by Biggers et al., (‘71) for the culture of mouse ova. No attempt was made to remove the corona or cumulus cells at this time. Freshly ejaculated semen was collected in a plastic bag from a mature fertile stud dog by manual stimulation; then the spermatozoa were washed twice by centrifugation in 5-10 ml of BWW medium and resuspended in 1-2 ml of fresh BWW medium. The dense sperm suspension (0.01 ml) was then mixed with the oocytes. The final concentration of spermatozoa was 1-5 X lo6 spermlml. The inseminated oocytes were then cultured under oil in an organ culture dish at 37°C with an atmorphere of 5% C 0 2 in air. At various intervals after the start of incubation, a drop of medium (without oocytes) was transferred to a slide and the free-swimming spermatozoa were examined for evidence of the acrosome reaction with a phase-contrast microscope ( X 400). Then some of the oocytes were removed from culture, pipetted to remove corona cells and mounted on a slide so that the condition of the spermatozoa on the zona surface and the incidence of zona penetration could be determined. The oocytes were then fixed and stained to reveal their state of nuclear maturation and the presence of swelling sperm heads.
Humane Society or surgically during hysterectomy at a local veterinary hospital. Within one-half hour following removal of the ovaries, as many antral follicles as possible were punctured with a needle to release the oocytes with corona and cumulus cells into a sterile culture medium consisting of 80% TC medium 199 (Hank’s solution base; Difco Labs, Detroit, Michigan) and 20 % heat-inactivated fetal calf serum (Grand Island Biol. Co., Grand Island, New York) with an additional 2 mg/ml NaHC03, 100 unitslml K-penicillin G and 50 pg/ml streptomycin sulphate (Sigma Chem. Co., St. Louis, Missouri). Oocytes with corona and cumulus cells were rinsed three times in culture medium then placed with 0.5 ml of fresh culture medium under mineral oil in a plastic organ culture dish (Falcon Plastics, Oxnard, California) and incubated at 37°C in an anaerobic jar with an atmosphere of 5% C 0 2 in air. Following various periods of culture, oocytes were removed, pipetted to remove corona cells (the corona and cumulus are resistant to hyaluronidase) and slightly compressed between a slide and a coverslip supported at four corners by dots of a Vaseline-paraffin mixture (lo:]). The vitellus of canine oocytes is extremely opaque (fig. 1) due to the presence of a large quantity of lipid; therefore the oocytes were fixed overnight in acetic acid and ethanol (1:3) and stained with aceto-carmine (0.25% carmine in 45% acetic acid) to reveal cytological details.
RESULTS
Maturation o f ovarian oocytes When oocytes were examined immediately after recovery from the ovaries, 65% of them were found to be degenerate. Table 1 shows the incidence of maturation in culture of the apparently healthy oocytes. At the beginning of incubation, all but one of the oocytes examined were in the germinal vesicle stage (fig. 2). The single exception was at anaphase I. As the length
Sperm capacitation and penetration of mature and immature oocytes In order to study sperm capacitation and oocyte penetration by spermatozoa in vitro, oocytes were transferred after 0-72 hours of culture to 0.5 ml of BWW medium,
TABLE I
Canine oocyte maturation i n vitro Incubation time (hours)
0 24 48 72
Number oocytes examined
90
51 111
177
Germinal vesicles
Germinal vesicle breakdown
Metaphase I & I1
%
%
%
98.9 82.4 66.7 52.5
1.1 17.6 33.3 47.5
0 5.9 22.5 25.4
MATURATION AND PENETRATION OF CANINE OOCYTES
191
TABLE 2
Canine oocytes penetrated by spermatozoa in vitro: examined between I 1 and 24 hours after insemination Total number of oocytes examined
Oocytes with germinal vesicles
Oocytes with spermatozoa penetrating the zona
Oocytes with swelling sperm nuclei i n the vitellus
%
%
9%
Oocytes not cultured
77
98.9
77.6
27.3
Oocytes cultured 24-72 hours
240
61.7
73.0
19.7
8). Some oocytes examined 24 hours after insemination contained sperm nuclei in advanced stages of decondensation or transforming into male pronuclei (fig. 9). There was little difference in the frequency of penetration of the zona pellucida in cultured and uncultured oocytes (table 2). In fact spermatozoa could penetrate the zona of any oocyte including those which were badly degenerated. All but one of the uncultured oocytes were still in the germinal vesicle stage when examined (fig. 10) and 27.3% contained sperm nuclei in advanced stages of decondensation (fig. l l ) . The oocytes that had been cultured for 24-72 hours prior to Sperm capacitation and penetration of insemination were at various stages of mature and immature ooc ytes maturation and 19.7% contained swelling A total of 317 uncultured and cultured sperm nuclei. There was no difference oocytes were inseminated in vitro. The in the degree of decondensation of the earliest acrosome reaction and zona pene- sperm nuclei in oocytes which had germtration were seen at seven hours after in- inal vesicles and those which did not. semination. At this time, many of the sperDISCUSSION matozoa displayed extremely vigorous The results of the present study indicate motility and swam in a pattern strikingly similar to the “activated movement” of that canine ovarian oocytes are capable capacitated hamster (Yanagimachi, ’70) of initiating nuclear maturation when and guinea pig (Yanagimachi, ’72) sperma- cultured in vitro. The proportion of the tozoa. Large numbers of spermatozoa at- oocytes reaching metaphase I1 was low, tached to the corona cells, but the corona but it could be increased with further redid not disperse. Of the 269 living oocytes finements in the culture procedures. A n examined between 11 and 24 hours after interesting finding was that uncultured insemination, 199 ( 7 4 % ) had sperm heads dictyate oocytes and cultured oocytes unwithin either the zona pellucida (fig. 6), dergoing maturation were equally penethe perivitelline space (fig. 7) or both. Of trable by spermatozoa in vitro. The zonae 310 oocytes fixed and examined during the pellucidae of these oocytes as well as same period, 6 7 (21.6%) had one or more those of badly degenerated oocytes were swelling sperm nuclei in the vitellus (fig. penetrable by spermatozoa, indicating that
of incubation was increased to 72 hours, the percentage of oocytes showing breakdown of the germinal vesicle increased to 47.5% and the percentage advancing to metaphase increased to 25.4 % . Because the polar body was often lost during the fixation procedure, it was often difficult to distinguish metaphase I from metaphase I1 in the lateral view of the spindle (fig. 3) but in the polar view (figs. 4, 5) the reduction in chromosome number made it possible to recognize metaphase 11. The first polar body was seen in some instances, especially when the corona was not completely removed.
192
CHERRIE A N N MAHI AND RYUZO YANAGIMACHI
zona penetration by canine spermatozoa is independent of the condition of the oocyte vitellus. The ability of spermatozoa to cross the zona pellucida appears to develop with time. We observed that the spermatozoa spent at least seven hours in the medium or around the oocytes before they underwent acrosome reactions and began to penetrate the zona pellucida. This fact suggests that (1) canine spermatozoa require capacitation before they are able to penetrate the zona pellucida and (2) the capacitation is accomplished at least i n some spermatozoa in seven hours under the in vitro conditions employed. The relatively low percentage of inseminated oocytes containing swelling sperm nuclei (table 2) may be indicative of the low percentage of oocytes that were actually healthy and capable of responding to the penetrating spermatozoa. Atresia overtakes the vast majority of canine follicular oocytes and is not always easily detectable in early stages (Andersen and Simpson, ’73). The finding that dictyate oocytes contained swelling sperm nuclei was particularly surprising since Yanagimachi and Usui (‘72) found that hamster oocytes were not capable of decondensing sperm nuclei until after the breakdown of the germinal vesicle. Similar findings have been reported by Iwamatsu and Chang (‘72) for the mouse and by Barros and Munoz (’73) for the hamster. Unlike most mammals, the dog ovulates dictyate oocytes (Van der Stricht, ’23). Van der Stricht (‘23) found that spermatozoa could enter canine oocytes at any time after ovulation and in at least one case, a spermatozoon was clearly detected within a n oocyte i n the dictyate stage. However, he noted that the sperm nucleus did not go through transformation until the oocyte completed the meiotic divisions. Holst and Phemister (‘71) stated that fertilization i n the dog is “not possible” until after shedding of the first polar body which occurs on the third to seventh day of estrus (Evans and Cole, ’31). However the only criterion for fertilization in their study was cleavage of the oocytes. Perhaps they based their conclusion on the fact that the eggs of other mammals are not normally fertilized until after formation of the first polar body. Gier (’50) and Doak et al. (‘67) have also
stated that fertilization in the dog does not occur until after the completion of maturation, but neither of these papers presented any evidence. The results presented here support Van der Stricht’s conclusion that canine oocytes may be penetrated at any time following ovulation (Van der Stricht, ’23). Overstreet and Bedford (‘74) likewise found that the zona pellucida of rabbit oocytes could be readily penetrated by spermatozoa regardless of maturation. However they observed that the spermatozoa could not attach to or penetrate the vitellus of dictyate oocytes. Earlier workers have also noted that follicular rat and rabbit oocytes are not capable of fertilization i n vivo until after the resumption of meiosis (Noyes, ’52; Chang, ’55a,b). In the present study, the vitellus of dictyate oocytes was not only penetrable by spermatozoa, but in contrast to Van der Stricht’s observations, was also capable of decondensing sperm nuclei. Van der Stricht obtained his oocytes from naturally mated animals and fixed them immediately. It is possible that the in vitro system employed here has altered the eggs so that they can decondense sperm nuclei earlier than they could i n vivo. This seems unlikely since similar in vitro systems were used by Iwamatsu and Chang (‘72) and by Yanagimachi and Usui (‘72) who found that dictyate oocytes of the mouse and hamster were not able to decondense sperm nuclei. Syngamy and cleavage were not observed in this study possibly because the penetrated oocytes were not incubated long enough and the BWW medium used is not suitable for extended culture of the oocytes. Studies are continuing to determine the optimum medium for in vitro sperm capacitation and one which is suitable for both oocyte culture and fertilization so that we can observe the formation of the male and female pronuclei and subsequent events. ACKNOWLEDGMENTS
This work was supported by grants from the Population Council, the Ford Foundation and the NIH-USPHS (HD-03402). We would like to thank the officers and staff of the Hawaiian Humane Society, especially Mr. Tom Kekoa, for their cooperation i n this project and also Drs. Howard Furu-
MATURATION AND PENETRATION OF CANINE OOCYTES
moto and Jea Hyung Lee of Care Animal Hospital for donating the ovaries of bitches undergoing hysterectomy. We should also like to thank Drs. B. J. Rogers and B. Bavister for their assistance in the preparation of the manuscript. LITERATURE CITED Andersen, A. C., and M. E. Simpson 1973 The Ovary and Reproductive Cycle ofthe Dog (Beagle). Geron-X, Los Altos. 290 PP. Barros, C., and G. Munoz 1973 Sperm-egg interaction i n immature hamster oocytes. J. Exp. ZJO~.,186: 73-78. Biggers, J. D. 1973 Oogenesis and ovum maturation. In: The Regulation of Mammalian Reproduction. s. J. Segal, R. Crozier, P. A. Corfman, and P. G. Conliffe, eds. C. C. Thomas, Springfield, pp. 273-283. Biggers, J. D., W. K. Whitten and D. G. Whittingham 1971 The culture of mouse embryos in uitro. In: Methods of Mammalian Embryology. J. C. Daniel, ed. Freeman & Co., San Francisco, pp. 86-1 16, table 6-5. Chang, M. C. 1955a The maturation of rabbit oocytes i n culture and their maturation, activation, fertilization and subsequent development in the fallopian tubes. J . Exp. Zool., 128: 379406. 1955b Fertilization and normal development of follicular oocytes in the rabbit. Science, 121 : 8 6 7 4 6 9 . Cross, P. C., and R. L. Brinster 1970 In uitro development of mouse oocytes. Biol. Reprod., 3: 298-307. Doak, R. L., A. Hall and H. E. Dale 1967 Longevity of spermatozoa in the reproductive tract of the bitch. J. Reprod. Fert., 13: 5 1 5 8 . Donahue, R. P. 1972 The relation of oocyte maturation to ovulation i n mammals. In: Oogenesis. J. D. Biggers and A. W. Schuetz, eds. University Park Press, Baltimore, pp. 4 1 3 4 3 8 . Edwards, R. G., B. D. Bavister and P. C. Steptoe
193
1969 Early stages of fertilization in uitro of human oocytes matured in uitro. Nature, 221 : 632-635. Evans, H. M., and H. H. Cole 1931 An introduction to the study of the oestrous cycle in the dog. Mem. Univ. Calif., 9 : 65-1 19. Gier, H. T. 1950 Early embryology of the dog. Anat. Rec., 108: 5 6 1 5 6 4 . Hancock, J. L., and I. W. Rowlands 1949 The physiology of reproduction in the dog. Vet. Rec., 61 : 771-776. Holst, P. A., and R. D. Phemister 1971 The prenatal development of the dog: preimplantatioii events. Biol. Reprod., 5: 194-206. Iwamatsu, T., and M. C. Chang 1972 Sperm penetration i n uitro of mouse oocytes at various times during maturation. J. Reprod. Fert., 31 : 237-247. Noyes, R. W. 1952 Fertilization of follicular ova. Fert. Steril., 3: 1-12. Overstreet, J. W., and J. M. Bedford 1974 Comparison of the penetrability of the egg vestments in follicular oocytes, unfertilized and fertilized ova of the rabbit. Devel. Biol., 41: 185-192. Phemister, R. D., P. A. Holst, J. S. Spano and M. L. Hopwood 1973 Time of ovulation in the beagle bitch. Biol. Reprod., 8: 74-82. Pincus, G., and E. V. Enzmann 1935 The comparative behavior ofmammalian eggs in uiuo and in uitro. I. The activation of ovarian eggs. J. Exp. Med., 62: 665-675. V a n der Stricht, 0. 1923 Etude comparee des ovules des mammiferes aux differentes periodes de l’ovogenese, d’apres les travaux du Laboratoire d’Histologie et d’Embryologie de 1’Universite de Gand. Arch. Biol., Paris, 33: 229-300. Yanagimachi, R. 1970 The movement of golden hamster spermatozoa before and after capacitation. J. Reprod. Fert., 23: 193-196. 1972 Fertilization of guinea pigs eggs in uitro. Anat. Rec., 174: 9-20. Yanagimachi, R., and N. Usui 1972 The appearance and dissappearance of factors involved i n sperm decondensation in the hamster egg. J . Cell Biol., 55: 293a.
PLATE 1 EXPLANATION OF FIGURES
194
1
Living canine ovarian oocyte before culture. Note opaque vitellus and thick corona. co, cumulus oophorus; cr, corona radiata; z, zona pellucida. X 150.
2
Germinal vesicle of a n oocyte fixed and stained before culture. X 750.
3
Lateral view of the metaphase I or I1 spindle i n an oocyte fixed and stained after 72 hours of culture. X 1,400.
4
Polar view of the chromosomes at metaphase I i n a n oocyte fixed and stained after 48 hours of culture. x 1,050.
5
Polar view of the chromosomes at metaphase I1 i n a n oocyte fixed and stained after 48 hours of culture. X 1,100.
6
Canine spermatozoon penetrating the zona pellucida (z) of a living oocyte, seven hours after insemination. X 1,150.
7
A spermatozoon whose head has passed through the zona pellucida (z), 12 hours after insemination. X 1,000.
8
Sperm nucleus decondensing in the cytoplasm of a n oocyte fixed and stained 24 hours after insemination. Arrow indicates sperm tail. X 1,000.
9
Sperm nucleus in advanced state of decondensation and beginning to transform into a male pronucleus i n the cytoplasm of a n oocyte fixed and stained 24 hours after insemination. Arrow indicates sperm tail. X 1,000,
10
Dictyate oocyte with a swollen sperm head (arrow) in the cytoplasm. g, germinal vesicle. X 500.
11
Higher magnification of the sperm nucleus indicated by a n arrow i n figure 10. Note advanced state of decondensation. Arrow indicates sperm tail. X 1,500.
MATURATION A N D PENETRATION OF CANINE OOCYTES Cherrie Ann Mahi and Ryuzo Yanagimachi
PLATE 1
ABSTRACT Canine ovarian oocytes were cultured in a medium consisting of TC medium 199, fetal calf serum and antibiotics. Ninety-nine percent of the apparently healthy oocytes were i n the germinal vesicle (dictyate) stage when recovered from the ovaries; 25% of them reached metaphase I or I1 by 72 hours of culture. Washed ejaculated spermatozoa were added to BWW medium containing oocytes which had either been removed directly from the follicles or which had been cultured for 24-72 hours. The earliest acrosome reaction and zona penetration by spermatozoa were seen at seven hours after insemination. Seventy-four percent of the oocytes examined between 11 and 24 hours after insemination showed evidence of zona penetration by spermatozoa. Neither the condition of the oocyte vitellus nor the stage of nuclear maturation influenced the incidence of zona penetration. Decondensing sperm nuclei were found in the vitellus of 27% of the oocytes which had not been cultured and in the vitellus of 20 % of those which had been cultured for 24-72 hours and were in various stages of maturation. These results indicate that (1) canine ovarian oocytes can be matured i n vitro, (2) the spermatozoa require capacitation which takes approximately seven hours in vitro and (3) maturation of the oocytes is not required for sperm passage through the zona pellucida or entry into the vitellus nor for sperm nuclear decondensation.
The interval between estrous periods in bitches averages seven months (Hancock and Rowlands, '49) and ovulation is difficult to predict or detect (Phemister et al., '73). Therefore if fertilization in dogs is to be studied routinely, a system must be devised which does not depend on natural ovulation. Mammalian ovarian oocytes can mature in vitro when liberated from Graafian follicles and placed in appropriate culture media (Pincus and Enzmann, '35; Chang, '55a,b; for review see Donahue, '72; Biggers, '73). Edwards et al. ('69) and Cross and Brinster ('70) proved that human and mouse oocytes matured in vitro can be fertilized in vitro. We postulated that canine oocytes could be matured in vitro in order to obtain material for studying fertilization in this species. The structure of both fertilized and unfertilized canine oocytes was described in detail by Van der Stricht ('23), but the fertilization process has not been observed. Doak et al. ('67) found that canine spermatozoa survive for extended periods (6-11 J. EXP. ZOOL., 196: 189-196.
days) within the uteri of estrous bitches; however the capacitation process has not been described for canine spermatozoa nor has the need for capacitation been established. The dog differs from most mammals in that oocytes are ovulated in the germinal vesicle (dictyate) stage (Van der Stricht, '23). Since mating can take place from 2-3 days before to a week after ovulation (Evans and Cole, '31; Phemister et al., '73), it seems quite possible for canine spermatozoa and immature oocytes to meet in the oviducts. We felt that it would be extremely interesting to determine whether these dictyate oocytes could be penetrated by spermatozoa and if so, whether the sperm nuclei decondense and form male pronuclei. MATERIALS A N D METHODS
Maturation of ovarian oocytes Ovaries were obtained from 75 bitches at random stages of the estrous cycle either by autopsy following euthanasia in a decompression chamber at the Hawaiian
189
190
CHERRIE A N N MAHI A N D RYUZO YANAGIMACHI
a medium devised by Biggers et al., (‘71) for the culture of mouse ova. No attempt was made to remove the corona or cumulus cells at this time. Freshly ejaculated semen was collected in a plastic bag from a mature fertile stud dog by manual stimulation; then the spermatozoa were washed twice by centrifugation in 5-10 ml of BWW medium and resuspended in 1-2 ml of fresh BWW medium. The dense sperm suspension (0.01 ml) was then mixed with the oocytes. The final concentration of spermatozoa was 1-5 X lo6 spermlml. The inseminated oocytes were then cultured under oil in an organ culture dish at 37°C with an atmorphere of 5% C 0 2 in air. At various intervals after the start of incubation, a drop of medium (without oocytes) was transferred to a slide and the free-swimming spermatozoa were examined for evidence of the acrosome reaction with a phase-contrast microscope ( X 400). Then some of the oocytes were removed from culture, pipetted to remove corona cells and mounted on a slide so that the condition of the spermatozoa on the zona surface and the incidence of zona penetration could be determined. The oocytes were then fixed and stained to reveal their state of nuclear maturation and the presence of swelling sperm heads.
Humane Society or surgically during hysterectomy at a local veterinary hospital. Within one-half hour following removal of the ovaries, as many antral follicles as possible were punctured with a needle to release the oocytes with corona and cumulus cells into a sterile culture medium consisting of 80% TC medium 199 (Hank’s solution base; Difco Labs, Detroit, Michigan) and 20 % heat-inactivated fetal calf serum (Grand Island Biol. Co., Grand Island, New York) with an additional 2 mg/ml NaHC03, 100 unitslml K-penicillin G and 50 pg/ml streptomycin sulphate (Sigma Chem. Co., St. Louis, Missouri). Oocytes with corona and cumulus cells were rinsed three times in culture medium then placed with 0.5 ml of fresh culture medium under mineral oil in a plastic organ culture dish (Falcon Plastics, Oxnard, California) and incubated at 37°C in an anaerobic jar with an atmosphere of 5% C 0 2 in air. Following various periods of culture, oocytes were removed, pipetted to remove corona cells (the corona and cumulus are resistant to hyaluronidase) and slightly compressed between a slide and a coverslip supported at four corners by dots of a Vaseline-paraffin mixture (lo:]). The vitellus of canine oocytes is extremely opaque (fig. 1) due to the presence of a large quantity of lipid; therefore the oocytes were fixed overnight in acetic acid and ethanol (1:3) and stained with aceto-carmine (0.25% carmine in 45% acetic acid) to reveal cytological details.
RESULTS
Maturation o f ovarian oocytes When oocytes were examined immediately after recovery from the ovaries, 65% of them were found to be degenerate. Table 1 shows the incidence of maturation in culture of the apparently healthy oocytes. At the beginning of incubation, all but one of the oocytes examined were in the germinal vesicle stage (fig. 2). The single exception was at anaphase I. As the length
Sperm capacitation and penetration of mature and immature oocytes In order to study sperm capacitation and oocyte penetration by spermatozoa in vitro, oocytes were transferred after 0-72 hours of culture to 0.5 ml of BWW medium,
TABLE I
Canine oocyte maturation i n vitro Incubation time (hours)
0 24 48 72
Number oocytes examined
90
51 111
177
Germinal vesicles
Germinal vesicle breakdown
Metaphase I & I1
%
%
%
98.9 82.4 66.7 52.5
1.1 17.6 33.3 47.5
0 5.9 22.5 25.4
MATURATION AND PENETRATION OF CANINE OOCYTES
191
TABLE 2
Canine oocytes penetrated by spermatozoa in vitro: examined between I 1 and 24 hours after insemination Total number of oocytes examined
Oocytes with germinal vesicles
Oocytes with spermatozoa penetrating the zona
Oocytes with swelling sperm nuclei i n the vitellus
%
%
9%
Oocytes not cultured
77
98.9
77.6
27.3
Oocytes cultured 24-72 hours
240
61.7
73.0
19.7
8). Some oocytes examined 24 hours after insemination contained sperm nuclei in advanced stages of decondensation or transforming into male pronuclei (fig. 9). There was little difference in the frequency of penetration of the zona pellucida in cultured and uncultured oocytes (table 2). In fact spermatozoa could penetrate the zona of any oocyte including those which were badly degenerated. All but one of the uncultured oocytes were still in the germinal vesicle stage when examined (fig. 10) and 27.3% contained sperm nuclei in advanced stages of decondensation (fig. l l ) . The oocytes that had been cultured for 24-72 hours prior to Sperm capacitation and penetration of insemination were at various stages of mature and immature ooc ytes maturation and 19.7% contained swelling A total of 317 uncultured and cultured sperm nuclei. There was no difference oocytes were inseminated in vitro. The in the degree of decondensation of the earliest acrosome reaction and zona pene- sperm nuclei in oocytes which had germtration were seen at seven hours after in- inal vesicles and those which did not. semination. At this time, many of the sperDISCUSSION matozoa displayed extremely vigorous The results of the present study indicate motility and swam in a pattern strikingly similar to the “activated movement” of that canine ovarian oocytes are capable capacitated hamster (Yanagimachi, ’70) of initiating nuclear maturation when and guinea pig (Yanagimachi, ’72) sperma- cultured in vitro. The proportion of the tozoa. Large numbers of spermatozoa at- oocytes reaching metaphase I1 was low, tached to the corona cells, but the corona but it could be increased with further redid not disperse. Of the 269 living oocytes finements in the culture procedures. A n examined between 11 and 24 hours after interesting finding was that uncultured insemination, 199 ( 7 4 % ) had sperm heads dictyate oocytes and cultured oocytes unwithin either the zona pellucida (fig. 6), dergoing maturation were equally penethe perivitelline space (fig. 7) or both. Of trable by spermatozoa in vitro. The zonae 310 oocytes fixed and examined during the pellucidae of these oocytes as well as same period, 6 7 (21.6%) had one or more those of badly degenerated oocytes were swelling sperm nuclei in the vitellus (fig. penetrable by spermatozoa, indicating that
of incubation was increased to 72 hours, the percentage of oocytes showing breakdown of the germinal vesicle increased to 47.5% and the percentage advancing to metaphase increased to 25.4 % . Because the polar body was often lost during the fixation procedure, it was often difficult to distinguish metaphase I from metaphase I1 in the lateral view of the spindle (fig. 3) but in the polar view (figs. 4, 5) the reduction in chromosome number made it possible to recognize metaphase 11. The first polar body was seen in some instances, especially when the corona was not completely removed.
192
CHERRIE A N N MAHI AND RYUZO YANAGIMACHI
zona penetration by canine spermatozoa is independent of the condition of the oocyte vitellus. The ability of spermatozoa to cross the zona pellucida appears to develop with time. We observed that the spermatozoa spent at least seven hours in the medium or around the oocytes before they underwent acrosome reactions and began to penetrate the zona pellucida. This fact suggests that (1) canine spermatozoa require capacitation before they are able to penetrate the zona pellucida and (2) the capacitation is accomplished at least i n some spermatozoa in seven hours under the in vitro conditions employed. The relatively low percentage of inseminated oocytes containing swelling sperm nuclei (table 2) may be indicative of the low percentage of oocytes that were actually healthy and capable of responding to the penetrating spermatozoa. Atresia overtakes the vast majority of canine follicular oocytes and is not always easily detectable in early stages (Andersen and Simpson, ’73). The finding that dictyate oocytes contained swelling sperm nuclei was particularly surprising since Yanagimachi and Usui (‘72) found that hamster oocytes were not capable of decondensing sperm nuclei until after the breakdown of the germinal vesicle. Similar findings have been reported by Iwamatsu and Chang (‘72) for the mouse and by Barros and Munoz (’73) for the hamster. Unlike most mammals, the dog ovulates dictyate oocytes (Van der Stricht, ’23). Van der Stricht (‘23) found that spermatozoa could enter canine oocytes at any time after ovulation and in at least one case, a spermatozoon was clearly detected within a n oocyte i n the dictyate stage. However, he noted that the sperm nucleus did not go through transformation until the oocyte completed the meiotic divisions. Holst and Phemister (‘71) stated that fertilization i n the dog is “not possible” until after shedding of the first polar body which occurs on the third to seventh day of estrus (Evans and Cole, ’31). However the only criterion for fertilization in their study was cleavage of the oocytes. Perhaps they based their conclusion on the fact that the eggs of other mammals are not normally fertilized until after formation of the first polar body. Gier (’50) and Doak et al. (‘67) have also
stated that fertilization in the dog does not occur until after the completion of maturation, but neither of these papers presented any evidence. The results presented here support Van der Stricht’s conclusion that canine oocytes may be penetrated at any time following ovulation (Van der Stricht, ’23). Overstreet and Bedford (‘74) likewise found that the zona pellucida of rabbit oocytes could be readily penetrated by spermatozoa regardless of maturation. However they observed that the spermatozoa could not attach to or penetrate the vitellus of dictyate oocytes. Earlier workers have also noted that follicular rat and rabbit oocytes are not capable of fertilization i n vivo until after the resumption of meiosis (Noyes, ’52; Chang, ’55a,b). In the present study, the vitellus of dictyate oocytes was not only penetrable by spermatozoa, but in contrast to Van der Stricht’s observations, was also capable of decondensing sperm nuclei. Van der Stricht obtained his oocytes from naturally mated animals and fixed them immediately. It is possible that the in vitro system employed here has altered the eggs so that they can decondense sperm nuclei earlier than they could i n vivo. This seems unlikely since similar in vitro systems were used by Iwamatsu and Chang (‘72) and by Yanagimachi and Usui (‘72) who found that dictyate oocytes of the mouse and hamster were not able to decondense sperm nuclei. Syngamy and cleavage were not observed in this study possibly because the penetrated oocytes were not incubated long enough and the BWW medium used is not suitable for extended culture of the oocytes. Studies are continuing to determine the optimum medium for in vitro sperm capacitation and one which is suitable for both oocyte culture and fertilization so that we can observe the formation of the male and female pronuclei and subsequent events. ACKNOWLEDGMENTS
This work was supported by grants from the Population Council, the Ford Foundation and the NIH-USPHS (HD-03402). We would like to thank the officers and staff of the Hawaiian Humane Society, especially Mr. Tom Kekoa, for their cooperation i n this project and also Drs. Howard Furu-
MATURATION AND PENETRATION OF CANINE OOCYTES
moto and Jea Hyung Lee of Care Animal Hospital for donating the ovaries of bitches undergoing hysterectomy. We should also like to thank Drs. B. J. Rogers and B. Bavister for their assistance in the preparation of the manuscript. LITERATURE CITED Andersen, A. C., and M. E. Simpson 1973 The Ovary and Reproductive Cycle ofthe Dog (Beagle). Geron-X, Los Altos. 290 PP. Barros, C., and G. Munoz 1973 Sperm-egg interaction i n immature hamster oocytes. J. Exp. ZJO~.,186: 73-78. Biggers, J. D. 1973 Oogenesis and ovum maturation. In: The Regulation of Mammalian Reproduction. s. J. Segal, R. Crozier, P. A. Corfman, and P. G. Conliffe, eds. C. C. Thomas, Springfield, pp. 273-283. Biggers, J. D., W. K. Whitten and D. G. Whittingham 1971 The culture of mouse embryos in uitro. In: Methods of Mammalian Embryology. J. C. Daniel, ed. Freeman & Co., San Francisco, pp. 86-1 16, table 6-5. Chang, M. C. 1955a The maturation of rabbit oocytes i n culture and their maturation, activation, fertilization and subsequent development in the fallopian tubes. J . Exp. Zool., 128: 379406. 1955b Fertilization and normal development of follicular oocytes in the rabbit. Science, 121 : 8 6 7 4 6 9 . Cross, P. C., and R. L. Brinster 1970 In uitro development of mouse oocytes. Biol. Reprod., 3: 298-307. Doak, R. L., A. Hall and H. E. Dale 1967 Longevity of spermatozoa in the reproductive tract of the bitch. J. Reprod. Fert., 13: 5 1 5 8 . Donahue, R. P. 1972 The relation of oocyte maturation to ovulation i n mammals. In: Oogenesis. J. D. Biggers and A. W. Schuetz, eds. University Park Press, Baltimore, pp. 4 1 3 4 3 8 . Edwards, R. G., B. D. Bavister and P. C. Steptoe
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1969 Early stages of fertilization in uitro of human oocytes matured in uitro. Nature, 221 : 632-635. Evans, H. M., and H. H. Cole 1931 An introduction to the study of the oestrous cycle in the dog. Mem. Univ. Calif., 9 : 65-1 19. Gier, H. T. 1950 Early embryology of the dog. Anat. Rec., 108: 5 6 1 5 6 4 . Hancock, J. L., and I. W. Rowlands 1949 The physiology of reproduction in the dog. Vet. Rec., 61 : 771-776. Holst, P. A., and R. D. Phemister 1971 The prenatal development of the dog: preimplantatioii events. Biol. Reprod., 5: 194-206. Iwamatsu, T., and M. C. Chang 1972 Sperm penetration i n uitro of mouse oocytes at various times during maturation. J. Reprod. Fert., 31 : 237-247. Noyes, R. W. 1952 Fertilization of follicular ova. Fert. Steril., 3: 1-12. Overstreet, J. W., and J. M. Bedford 1974 Comparison of the penetrability of the egg vestments in follicular oocytes, unfertilized and fertilized ova of the rabbit. Devel. Biol., 41: 185-192. Phemister, R. D., P. A. Holst, J. S. Spano and M. L. Hopwood 1973 Time of ovulation in the beagle bitch. Biol. Reprod., 8: 74-82. Pincus, G., and E. V. Enzmann 1935 The comparative behavior ofmammalian eggs in uiuo and in uitro. I. The activation of ovarian eggs. J. Exp. Med., 62: 665-675. V a n der Stricht, 0. 1923 Etude comparee des ovules des mammiferes aux differentes periodes de l’ovogenese, d’apres les travaux du Laboratoire d’Histologie et d’Embryologie de 1’Universite de Gand. Arch. Biol., Paris, 33: 229-300. Yanagimachi, R. 1970 The movement of golden hamster spermatozoa before and after capacitation. J. Reprod. Fert., 23: 193-196. 1972 Fertilization of guinea pigs eggs in uitro. Anat. Rec., 174: 9-20. Yanagimachi, R., and N. Usui 1972 The appearance and dissappearance of factors involved i n sperm decondensation in the hamster egg. J . Cell Biol., 55: 293a.
PLATE 1 EXPLANATION OF FIGURES
194
1
Living canine ovarian oocyte before culture. Note opaque vitellus and thick corona. co, cumulus oophorus; cr, corona radiata; z, zona pellucida. X 150.
2
Germinal vesicle of a n oocyte fixed and stained before culture. X 750.
3
Lateral view of the metaphase I or I1 spindle i n an oocyte fixed and stained after 72 hours of culture. X 1,400.
4
Polar view of the chromosomes at metaphase I i n a n oocyte fixed and stained after 48 hours of culture. x 1,050.
5
Polar view of the chromosomes at metaphase I1 i n a n oocyte fixed and stained after 48 hours of culture. X 1,100.
6
Canine spermatozoon penetrating the zona pellucida (z) of a living oocyte, seven hours after insemination. X 1,150.
7
A spermatozoon whose head has passed through the zona pellucida (z), 12 hours after insemination. X 1,000.
8
Sperm nucleus decondensing in the cytoplasm of a n oocyte fixed and stained 24 hours after insemination. Arrow indicates sperm tail. X 1,000.
9
Sperm nucleus in advanced state of decondensation and beginning to transform into a male pronucleus i n the cytoplasm of a n oocyte fixed and stained 24 hours after insemination. Arrow indicates sperm tail. X 1,000,
10
Dictyate oocyte with a swollen sperm head (arrow) in the cytoplasm. g, germinal vesicle. X 500.
11
Higher magnification of the sperm nucleus indicated by a n arrow i n figure 10. Note advanced state of decondensation. Arrow indicates sperm tail. X 1,500.
MATURATION A N D PENETRATION OF CANINE OOCYTES Cherrie Ann Mahi and Ryuzo Yanagimachi
PLATE 1
