vitro maturing bovine oocytes to transform sperm nuclei to metaphase chromosomes
Ability of in
L. R. Division
Abeydeera and K. Niwa
of Animal Science, Faculty of Agriculture, Okayama University, Okayama 700, Japan
Summary. Bovine oocytes at the germinal vesicle stage were inseminated in Brackett & Oliphant's medium with bovine serum albumin, caffeine and heparin. Eight hours after insemination, oocytes were transferred into tissue culture medium-199 containing 10% fetal calf serum and cultured for 5\p=n-\40h at 39\s=deg\Cin 5% CO2 in air. The proportions of unpenetrated and penetrated oocytes reaching metaphase II increased as the time of examination increased, reaching 70 and 65% 40 h after transfer, respectively. When oocytes were penetrated by more than four spermatozoa, meiotic maturation was greatly retarded. Sperm nuclei were decondensed in most (81%) penetrated oocytes 5 h after transfer. The decondensed sperm nuclei were recondensed and then transformed to metaphase chromosomes which were morphologically compacted at first but became slightly dispersed later. The formation of the metaphase chromosomes was observed in 86% of penetrated oocytes examined 40 h after transfer, and occurred in all metaphase II oocytes at that time. In oocytes penetrated by more than nine spermatozoa, no such transformation of sperm nuclei was observed. Well-developed male and female pronuclei were observed in only three (6%) of 51 oocytes penetrated 40 h after transfer.
Keywords: cow; oocytes; sperm penetration; sperm metaphase chromosomes; in vitro
Introduction The cytoplasm of maturing oocytes in amphibians (Gurdon, 1968; Ziegler & Masui, 1973, 1976; Masut et al, 1979) and mammals (Balakier & Czolowska, 1977; Balakier, 1978; Tarkowski & Balakier, 1980; Czolowska et al, 1984; Sorensen et al, 1985) contains a strong activity that can stimulate the transition of nuclei from other cells to a metaphase condition. The cytoplasm of maturing oocytes of Urechis (Das & Barker, 1976) and frog (Moriya & Katagiri, 1976; Elinson, 1977) can also induce the transformation of sperm nuclei to metaphase chromosomes. Although Iwamatsu & Chang (1972) observed that sperm nuclei of mammals occasionally form spindle-like structures after penetration into maturing oocytes, Clarke & Masui (1986) have clearly demon¬ strated that sperm nuclei that have penetrated mouse oocytes at prometaphase I or metaphase I of maturation are transformed directly to metaphase chromosomes. A similar phenomenon has also been observed in human oocytes penetrated in vitro at metaphase II (Schmiady et al, 1986). Niwa et al. (1991) reported that bovine oocytes at germinal vesicle (GV) stage and those under¬ going maturation are equally penetrable by spermatozoa in vitro. However, since the medium used for fertilization in that study was not suitable for maturation of oocytes, it was difficult to observe in more detail the transformation of the sperm nucleus that may occur with the progression of oocyte maturation. It is possible to overcome this problem by transferring the bovine oocytes 8 h after insemination, at the GV stage, into another medium proven to be suitable for maturation because penetration into the oocytes by spermatozoa is almost complete by this time (Niwa et al,
'Reprint requests.
1991). The present study was designed to examine whether maturing bovine oocytes possess a cytoplasmic activity that induces the transformation of sperm nuclei into metaphase chromosomes. Materials and Methods Medium Modified BO medium (Brackett & Oliphant, 1975), containing II2-0mmol NaCl 1, 402mmol KC1 1, 2-25 mmol CaCl2 1, 0-83 mmol NaH2P04 \ 0-52 mmol MgCl2 1, 37-0 mmol NaHC03 l"1, 13-9 mmol glucose 1 ', 1-25 mmol sodium pyruvate 1 ' and 31 µg potassium penicillin G ml ' but without bovine serum albumin (BSA), was used in the treatment of spermatozoa and in the fertilization of oocytes. ~
~
~
Preparation of oocytes Bovine ovaries were obtained from a local abattoir within 30 min of death and transported to the laboratory in saline at 30-32°C in a vacuum flask within 1—1 -5 h. Oocytes were aspirated from small antral follicles (3-5 mm diameter) with an 18-gauge needle fixed to a 5 ml disposable syringe. Oocytes surrounded by compact cumulus and with evenly granulated cytoplasm were washed three times in BO medium supplemented with 20mg BSA ml"1 (crystallized and lyophilized, essentially globulin free; No. A-7638, Sigma Chemical Co., St Louis, MO, USA) and 20 pg porcine intestinal mucosal heparin ml-1 (178 US Pharmacopoeia units mg"1; Sigma Chemical Co.). The washed oocytes (five to ten oocytes) were transferred into 50 µ of the same medium, which had previously been 10 mm2) and equilibrated in a C02 incubator (5% covered with warm paraffin oil in a polystyrene culture dish (35 C02 in air at 39°C) for approximately 2-3 h.
Sperm preparation and in vitro fertilization Spermatozoa were treated using the procedures described by Niwa & Ohgoda (1988). Briefly, a 0-5 ml straw of frozen semen was thawed in a water bath at 37-39°C. Spermatozoa were washed twice in BO medium supplemented with 10 mmol caffeine-sodium benzoate 1" ' (Sigma Chemical Co.); each wash was followed by centrifugation at 833 g 106 spermatozoa ml* ') was introduced into 50 µ of for 10 min. A 50 µ sample of the final sperm suspension (5-10 the medium that contained the oocytes and the mixture was cultured at 39°C in an atmosphere of 5% C02 in air. The mixture gave final concentrations of 2-5-5 106 spermatozoa ml" ', 10 mg BSA ml" \ 5 mmol caffeine 1 ' and 10 pg "
heparin ml"1.
Additional culture and examination of oocytes Eight hours after insemination, oocytes were washed three times in TCM-199 (with Earle's salts) buffered with'
25 mmol Hepes 1 ' and supplemented with 10% (v/v) heat inactivated fetal calf serum (FCS), 60 pg penicillin G ml and 100 pg streptomycin ml"1. The washed oocytes were transferred into a 100 µ drop of the same medium covered with paraffin oil and culture continued in a C02 incubator (5% C02 in air at 39°C). After 5, 10, 15 and 40 h of additional culture, oocytes were freed from cumulus cells by repeated passage through a fine pipette and placed in the centre of four vaseline spots on a glass slide. The oocytes were compressed gently with a cover-slip and fixed for 48-72 h at room temperature in 25% (v/v) acetic alcohol, stained with 1% (w/v) orcein in 45% (v/v) acetic acid as described by Ohgoda et al. ( 1988). The oocytes were then examined for evidence of oocyte nuclear maturation and for transformation of sperm nuclei in penetrated oocytes. "
"
Classification of sperm nuclear transformation The morphological changes observed in sperm nuclei were classified into the following categories (1) with decondensed chromatin, in which the sperm nucleus was swollen but retained the oblong shape of the intact spermato¬ zoon (Fig. la); (2) with recondensed chromatin, in which the swollen sperm nucleus was condensed, forming a small darkly stained mass of chromatin (Fig. la, b); (3) with metaphase chromosomes, with compact (Fig. lc) and dispersed (Fig. Id) forms or (4) with pronucleus, each accompanied by a penetrated sperm tail. When polyspermic oocytes contained sperm nuclei at different stages of transformation, they were classified according to the most prevalent
stage.
Results Most (>70%) of the oocytes were penetrated and polyspermy was common (Table 1). Oocyte maturation was evident 5 h after transfer to TCM-199, when most oocytes exhibited condensed GVs or had progressed to prometaphase I. Ten to 15 h after transfer, most oocytes were undergoing
Fig. 1. Oocytes were inseminated at the germinal vesicle stage, transferred to the maturation medium 8 h after insemination and fixed and stained at various times after transfer, (a) An oocyte penetrated 5 h after transfer showing two decondensed and recondensed (arrowheads) sperm nuclei, with penetrating sperm tails. One of the recondensed sperm nuclei is out of focus, (b) An oocyte penetrated at metaphase I (M) 5 h after transfer showing two recondensed sperm nuclei, with penetrating sperm tails, (c) An oocyte penetrated at metaphase I (M) 10 h after transfer. A sperm nucleus has been transformed into compacted metaphase chromosomes, with a penetrating sperm tail, (d) An oocyte penetrated 15 h after transfer. A sperm nucleus with a penetrating sperm tail has been transformed into metaphase chromosomes, which are slightly dispersed, (e) The same oocyte as in (d) at metaphase II (M) with first polar body (PB). 20 pm. Bars =
the first meiotic division, with 24% attaining metaphase II by 15 h. Maturation proceeded such that 62% of the oocytes reached metaphase II by 40 h. Only 4% of penetrated oocytes were activated (contained female pronuclei) by 40 h. When polyspermy was modest (one to four spermatozoa per oocyte), it appeared to have no effect on oocyte maturation. However, when more than four spermatozoa penetrated, development was delayed and fewer oocytes reached metaphase II (Table 2).
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'Oocytes examined 40 h after transfer into were
maturation medium
used.
GV:
germinal vesicle; CGV: phase I; M I: metaphase I; II; PN: pronucleus.
condensed GV; PM I: prometa¬ I: telophase I; M II: metaphase
Five hours after transfer to TCM-199, most (81%) of the sperm nuclei were decondensed (category 1), and a few (5%) had transformed to metaphase chromosomes (category 3) (Table 3). After 10 h, about half the sperm nuclei had recondensed (category 2) and half had transformed to metaphase chromosomes. This progression continued so that by 40 h, most (86%) of the penetrated oocytes contained sperm chromosomes and a few contained male pronuclei (in the oocytes with female pronuclei described above). Table 3.
Sequence of transformation of sperm nuclei in penetrated oocytes3
Time of examination (h after
transfer)
5 10 15 40
Number of oocytes
penetrated with sperm nuclei at various stages
Decondensed Recondensed Metaphase chromatin chromatin chromosomes Total
(%)
57 60 63
46(81) 2 (4) 2 (3) 1 (2)
51
Pronucleus
(%)
(%)
(%)
8(14) (45) (27) (6)
(5) (52) 44(70) 44 (86)
0(0) 0(0) 0(0) 3(6)
27 17 3
3 31
Oocytes were cultured for 5-40 h after transfer into TCM-199 containing 10% fetal calf serum
8 h after insemination.
Polyspermy did not appear to limit the transformation of sperm nuclei to metaphase chromo¬ until it reached more than nine spermatozoa per oocyte (Table 4).
somes
Table 4. Effect of number of spermatozoa to penetrate transformation of sperm nuclei3 Number of
spermatozoa to penetrate each oocyte
an
oocyte
on
Number of oocytes with sperm nuclei at the stage of
Decondensed chromatin
Recondensed chromatin
Metaphase
chromosomes
1-2
24
3 4 5-6 7-9 10-14
11 6
Pronucleus
3 0
"Oocytes examined 40 h after transfer into maturation medium were used.
Discussion The results of this study clearly indicate that bovine oocytes inseminated at the GV stage can undergo meiotic maturation in vitro when they are transferred into an appropriate culture condition (TCM-199 containing 10% FCS) after completion of penetration by spermatozoa and that the cytoplasm of the maturing oocytes has an activity that can transform sperm nuclei to metaphase chromosomes as already observed by Clarke & Masui (1986) in mouse oocytes. In a previous study, oocytes inseminated at the GV stage were continuously cultured for up to 20 h in fertilization medium (BO medium with 10mg BSA ml-1, 5mmol caffeine l"1 and 10pg heparin ml '), but meiotic maturation of the oocytes was greatly retarded and the number of spermatozoa per penetrated oocyte increased rapidly as the time after insemination increased (Niwa et al, 1991). Thus, the fact that no sperm chromosome formation was observed in that study might be attributed to the inappropriate culture conditions used for the maturation of inseminated oocytes. On the basis of previous data (Niwa et al, 1991) the present results suggest that oocytes penetrated 8 h after insemination still have intact GVs at the time of transfer into TCM-199. Since GV breakdown of bovine oocytes in vitro occurs between 4 and 8 h of culture (Sirard et al, 1989), it is clear that BO medium has retarded GV breakdown. The low numbers (21-25%) of unpenetrated and penetrated oocytes that reached metaphase II 15 h after transfer to TCM-199 could therefore be attributed to the retardation of the first step of meiosis. This retardation, however, did not inhibit the ability of the oocytes to complete maturation, because, 40 h after transfer, 70% of unpenetrated and 65% of penetrated oocytes had reached metaphase II (Table 1). The present results confirm those observed in mice by Clarke & Masui (1986), who reported that sperm penetration of maturing oocytes does not delay the first meiotic division but that polyspermy does affect oocyte maturation. In the present study, bovine oocytes that had been penetrated by more than four spermatozoa were greatly delayed in reaching metaphase II. According to Clarke & Masui (1986), the shortening and thickening of the maternal chromosomes occurred more frequently in polyspermic maturing mouse oocytes and in highly polyspermic oocytes, the chro¬ mosomes underwent drastic morphological changes. In the present study, however, it was diffi¬ cult to observe such morphological changes in the maternal chromosomes of polyspermic -
oocytes.
In general, mature oocytes are activated by sperm penetration, resume the second meiotic division and extrude the second polar body, then form a female pronucleus. In the present study, however, few penetrated oocytes had formed a female pronucleus even 40 h after transfer. This observation supports the hypothesis that sperm penetration into immature oocytes does not induce oocyte activation. This is not surprising since oocyte activation normally occurs in oocytes at meiosis (M) phase and not at an interphase state as in GV oocytes. This concept is consistent with our recent observations that bovine oocytes experimentally arrested between metaphase I and telophase I are activated by sperm penetration (Chian et al, 1992). Indeed, it is possible that oocytes that did undergo activation in our study had begun to mature during the in vitro fertiliz¬ ation incubation, and were therefore activated by the penetrating spermatozoa, although this was not experimentally verified. In contrast to most other mammalian species examined, the exception being dogs (Mahi & Yanagimachi, 1976), in bovine oocytes decondensation of sperm nuclei can be induced even before GV breakdown (Niwa et al, 1991). Furthermore, transformation of the decondensed sperm nuclei to metaphase chromosomes was clearly observed in this study. When oocytes inseminated at the GV stage were transferred into an appropriate culture medium 8 h after insemination, sperm nuclei in some penetrated oocytes were recondensed (14%) or transformed to metaphase chromosomes (5%) within 5 h of transfer. The proportion of oocytes with sperm nuclei transformed to metaphase chromosomes increased with time reaching 86% 40 h after transfer. The pattern of these morpho¬ logical changes in sperm nuclei is very similar to that observed in mouse oocytes (Clarke & Masui, 1986). In bovine oocytes, however, it was difficult to observe well separated sperm chromosomes
40 h after transfer. Although it has been reported that metaphase chromosomes derived from spermatozoa (Clarke & Masui, 1986) and other maturing oocytes or blastomeres (Balakier, 1978; Szollosi et al, 1980; Tarkowski & Balakier, 1980; Clarke & Masui, 1985) have mixed with oocyte metaphase chromosomes, such mixing of the sperm and oocyte metaphase chromosomes was not observed in bovine oocytes; both chromosomes were always observed to be separate. Clarke & Masui (1986) reported that maturing mouse oocytes, penetrated by more than four spermatozoa, never transformed any of the nuclei to metaphase chromosomes. In this study, bovine oocytes penetrated by up to nine spermatozoa transformed all of the nuclei to metaphase chromosomes, but when oocytes were penetrated by 10-14 spermatozoa, no nuclei were trans¬ formed to metaphase chromosomes. Since bovine oocytes are larger than mouse oocytes, it is expected that the former possess a much higher capacity with respect to the cytoplasmic activity than the latter. Clarke & Masui (1987) have also suggested that the transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of mouse oocytes requires nucleoplasm of the GV and even
non-GV
cytoplasmic substances, including proteins synthesized during maturation. However, the requirements of GV material and proteins for sperm nuclear transformation in bovine oocytes need to be clarified in further experiments. In the experimental conditions used, 65% of penetrated oocytes matured to metaphase II; nevertheless the formation of a male pronucleus was observed only in 6% of penetrated oocytes when they were examined 40 h after transfer. We do not consider this to result from a limitation of the amount of materials present in the cytoplasm that support sperm pronuclear development, as suggested in the previous study (Niwa et al, 1991), since the number of spermatozoa in a penetrated oocyte was limited in this study. If the male pronucleus growth factor (Thibault & Gerard, 1973) or the sperm pronucleus development factor (Yanagimachi, 1981) can function when the cytoplasm of oocytes is fully activated (Yanagimachi, 1981; Perreault et al, 1987), it is possible that the sperm nuclei incorporated into oocytes at the GV stage cannot activate the oocytes even after maturation has been completed. A
portion of this work was supported by Grant-in-Aid for Developmental Scientific
Research
(No. 03556038) from the Ministry of Education, Science and Culture of Japan. References
Balakier,
H.
(1978)
Induction of maturation in small
oocytes from sexually immature mice by fusion with meiotic and mitotic cells. Experimental Cell Research
112, 137-141. Balakier, H. & Czolowska, N. (1977) Cytoplasmic control of nuclear maturation in mouse oocytes. Experimental Cell Research 110, 466-469. Brackett, B.G. & Oliphant, G. (1975) Capacitation of rabbit spermatozoa in vitro. Biologv of Reproduction 12, 260-274. Chian, R.C., Niwa, K. & Nakahara, H. (1992) Effect of sperm penetration in vitro on completion of first meiosis by bovine oocytes arrested at various stages in culture. Journal of Reproduction and Fertility 96,
73-78.
Clarke, H.J. & Masui, Y. (1975) Inhibition by dibutyryl cyclic AMP of the transition to metaphase of oocyte nuclei and its reversal by cell fusion metaphase oocytes. Developmental Biology 108,
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to
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Clarke, H.J. & Masui,
Y. (1986) Transformation of sperm nuclei to metaphase chromosomes in the cyto¬ plasm of maturing oocytes of the mouse. Journal of Cell Biology 102, 1039-1046.
Clarke, H.J. & Masui, Y. (1987) Dose-dependent re¬ lationship between oocyte cytoplasmic volume and
transformation of sperm nuclei to metaphase chro¬ mosomes. Journal of Cell Biologv 104, 831-840. Czolowska, R., Modlinski, J.A. & tarkowski, A. (1984) Behaviour of thymocyte nuclei in non-activated and activated mouse oocytes. Journal of Cell Science 69, 19-34. Das, N.K. & Barker, C. (1976) Mitotic chromosome condensation in the sperm nucleus during postfertilization maturation division in Urechis eggs. Journal of Cell Biology 68, 155-159. Elinson, R.P. (1977) Fertilization of immature frog eggs: cleavage and development following subsequent acti¬ vation. Journal of Embryology and Experimental Morphology 37, 187-201. Gurdon, J.B. (1968) Changes in somatic cell nuclei inserted into growing and maturing amphibian oocytes. Journal of Embryology and Experimental Morphology 20, 401-414. Iwamatsu, T. & Chang, M.C. (1972) Sperm penetration in vitro of mouse oocytes at various times after maturation. Journal of Reproduction and Fertility 31, 237-247.
Mahi, CA. & Yanagimachi,
R. (1976) Maturation and sperm penetration of canine ovarian oocytes in vitro. Journal of Experimental Zoology 196, 189-196. Masui, Y., Meyerhof, P.G. & Ziegler, D.H. (1979) Con¬ trol of chromosome behaviour during progesteroneinduced maturation of amphibian oocytes. Journal of Steroid Biochemistry 11, 715-722. Moriya, M. & Katagiri, C. (1976) Microinjection of toad sperm into oocytes undergoing maturation
division. Development, Growth and Differentiation 18, 349-356. Niwa, K. & Ohgoda, O. (1988) Synergistic effect of caffeine and heparin on in-vitro fertilization of cattle oocytes matured in culture. Theriogenologv 30, 733-741. Niwa, K., Park, C-K. & Okuda, K. (1991) Penetration in vitro of bovine oocytes during maturation by frozenthawed spermatozoa. Journal of Reproduction and Fertility 91, 329-336. Ohgoda, O., Niwa, K., Yuhara, M.. Takahashi, S. & Kanoya, K. (1988) Variations in penetration rates in vitro of bovine follicular oocytes do not reflect conception rates after artificial insemination using frozen semen from different bulls. Theriogenology 29, 1375-1381. Perreault, S.D., Naish, S.J. & Zirkin, B.R. (1987) The timing of hamster nuclear decondensation and male pronucleus formation is related to sperm nuclear disulfide bond content. Biology of Reproduction 36, 239-244. Schmiady, II., Sperling, K., Kentenich, H. & Stauber, M. (1986) Prematurely condensed human sperm chro¬ mosomes after in vitro fertilization (IVF). Human Genetics 74,441-443.
Sirard, M.A., Florman, H.M., Leibfried-Rutledge, M.L., Barnes, F.L., Sims, M.L. & First, N.L. (1989) Timing
of nuclear progression and protein synthesis necess¬ ary for meiotic maturation of bovine oocytes. Biology of Reproduction 40, 1257-1263. Sorensen, R.A., Cyert, M.S. & Pederson, R.A. (1985) Active maturation-promoting factor is present in mature mouse oocytes. Journal of Cell Biologv 100, 1637-1640. Szollosi, I).. Balakier, II.. Czolowska, R. & Tarkowski, A. (1980) Ultrastructure of cell hybrids between mouse oocytes and blastomeres. Journal of Experimental Zoologv 213, 315-325. Tarkowski, A.K. & Balakier, H. (1980) Nucleocytoplasmic interactions in cell hybrids between mouse oocytes, blastomeres and somatic cells. Jour¬ nal of Embrvology and Experimental Morphology 55, 319-330. Thibault, C. & Gerard, M. (1973) Cytoplasmic and nuclear maturation of rabbit oocytes in vitro. Annales de Biologie Animale, Biochimie, Biophvsique 13, 145-156. Yanagimachi, R. (1981) Mechanisms of fertilization in mammals. In Fertilization and Embryonoic Develop¬ ment in vitro, pp. 81-182. Eds L. Mastroianni, Jr & J. D. Biggers. Plenum Press, New York. Ziegler, D.H. & Masui, Y. (1973) Control of chromo¬ some behavior in amphibian oocytes. I. The activity of maturing oocytes inducing chromosome conden¬ sation in transplanted brain nuclei. Developmental Biology 35, 283-292. Ziegler, D.H. & Masui, Y. (1976) Control of chromo¬ some behavior in amphibian oocytes. II. The effect of inhibitors of RNA and protein synthesis on the induction of chromosome condensation. Journal of Cell Biology 68, 620-628. Received 19 September 1991
Ability of in
L. R. Division
Abeydeera and K. Niwa
of Animal Science, Faculty of Agriculture, Okayama University, Okayama 700, Japan
Summary. Bovine oocytes at the germinal vesicle stage were inseminated in Brackett & Oliphant's medium with bovine serum albumin, caffeine and heparin. Eight hours after insemination, oocytes were transferred into tissue culture medium-199 containing 10% fetal calf serum and cultured for 5\p=n-\40h at 39\s=deg\Cin 5% CO2 in air. The proportions of unpenetrated and penetrated oocytes reaching metaphase II increased as the time of examination increased, reaching 70 and 65% 40 h after transfer, respectively. When oocytes were penetrated by more than four spermatozoa, meiotic maturation was greatly retarded. Sperm nuclei were decondensed in most (81%) penetrated oocytes 5 h after transfer. The decondensed sperm nuclei were recondensed and then transformed to metaphase chromosomes which were morphologically compacted at first but became slightly dispersed later. The formation of the metaphase chromosomes was observed in 86% of penetrated oocytes examined 40 h after transfer, and occurred in all metaphase II oocytes at that time. In oocytes penetrated by more than nine spermatozoa, no such transformation of sperm nuclei was observed. Well-developed male and female pronuclei were observed in only three (6%) of 51 oocytes penetrated 40 h after transfer.
Keywords: cow; oocytes; sperm penetration; sperm metaphase chromosomes; in vitro
Introduction The cytoplasm of maturing oocytes in amphibians (Gurdon, 1968; Ziegler & Masui, 1973, 1976; Masut et al, 1979) and mammals (Balakier & Czolowska, 1977; Balakier, 1978; Tarkowski & Balakier, 1980; Czolowska et al, 1984; Sorensen et al, 1985) contains a strong activity that can stimulate the transition of nuclei from other cells to a metaphase condition. The cytoplasm of maturing oocytes of Urechis (Das & Barker, 1976) and frog (Moriya & Katagiri, 1976; Elinson, 1977) can also induce the transformation of sperm nuclei to metaphase chromosomes. Although Iwamatsu & Chang (1972) observed that sperm nuclei of mammals occasionally form spindle-like structures after penetration into maturing oocytes, Clarke & Masui (1986) have clearly demon¬ strated that sperm nuclei that have penetrated mouse oocytes at prometaphase I or metaphase I of maturation are transformed directly to metaphase chromosomes. A similar phenomenon has also been observed in human oocytes penetrated in vitro at metaphase II (Schmiady et al, 1986). Niwa et al. (1991) reported that bovine oocytes at germinal vesicle (GV) stage and those under¬ going maturation are equally penetrable by spermatozoa in vitro. However, since the medium used for fertilization in that study was not suitable for maturation of oocytes, it was difficult to observe in more detail the transformation of the sperm nucleus that may occur with the progression of oocyte maturation. It is possible to overcome this problem by transferring the bovine oocytes 8 h after insemination, at the GV stage, into another medium proven to be suitable for maturation because penetration into the oocytes by spermatozoa is almost complete by this time (Niwa et al,
'Reprint requests.
1991). The present study was designed to examine whether maturing bovine oocytes possess a cytoplasmic activity that induces the transformation of sperm nuclei into metaphase chromosomes. Materials and Methods Medium Modified BO medium (Brackett & Oliphant, 1975), containing II2-0mmol NaCl 1, 402mmol KC1 1, 2-25 mmol CaCl2 1, 0-83 mmol NaH2P04 \ 0-52 mmol MgCl2 1, 37-0 mmol NaHC03 l"1, 13-9 mmol glucose 1 ', 1-25 mmol sodium pyruvate 1 ' and 31 µg potassium penicillin G ml ' but without bovine serum albumin (BSA), was used in the treatment of spermatozoa and in the fertilization of oocytes. ~
~
~
Preparation of oocytes Bovine ovaries were obtained from a local abattoir within 30 min of death and transported to the laboratory in saline at 30-32°C in a vacuum flask within 1—1 -5 h. Oocytes were aspirated from small antral follicles (3-5 mm diameter) with an 18-gauge needle fixed to a 5 ml disposable syringe. Oocytes surrounded by compact cumulus and with evenly granulated cytoplasm were washed three times in BO medium supplemented with 20mg BSA ml"1 (crystallized and lyophilized, essentially globulin free; No. A-7638, Sigma Chemical Co., St Louis, MO, USA) and 20 pg porcine intestinal mucosal heparin ml-1 (178 US Pharmacopoeia units mg"1; Sigma Chemical Co.). The washed oocytes (five to ten oocytes) were transferred into 50 µ of the same medium, which had previously been 10 mm2) and equilibrated in a C02 incubator (5% covered with warm paraffin oil in a polystyrene culture dish (35 C02 in air at 39°C) for approximately 2-3 h.
Sperm preparation and in vitro fertilization Spermatozoa were treated using the procedures described by Niwa & Ohgoda (1988). Briefly, a 0-5 ml straw of frozen semen was thawed in a water bath at 37-39°C. Spermatozoa were washed twice in BO medium supplemented with 10 mmol caffeine-sodium benzoate 1" ' (Sigma Chemical Co.); each wash was followed by centrifugation at 833 g 106 spermatozoa ml* ') was introduced into 50 µ of for 10 min. A 50 µ sample of the final sperm suspension (5-10 the medium that contained the oocytes and the mixture was cultured at 39°C in an atmosphere of 5% C02 in air. The mixture gave final concentrations of 2-5-5 106 spermatozoa ml" ', 10 mg BSA ml" \ 5 mmol caffeine 1 ' and 10 pg "
heparin ml"1.
Additional culture and examination of oocytes Eight hours after insemination, oocytes were washed three times in TCM-199 (with Earle's salts) buffered with'
25 mmol Hepes 1 ' and supplemented with 10% (v/v) heat inactivated fetal calf serum (FCS), 60 pg penicillin G ml and 100 pg streptomycin ml"1. The washed oocytes were transferred into a 100 µ drop of the same medium covered with paraffin oil and culture continued in a C02 incubator (5% C02 in air at 39°C). After 5, 10, 15 and 40 h of additional culture, oocytes were freed from cumulus cells by repeated passage through a fine pipette and placed in the centre of four vaseline spots on a glass slide. The oocytes were compressed gently with a cover-slip and fixed for 48-72 h at room temperature in 25% (v/v) acetic alcohol, stained with 1% (w/v) orcein in 45% (v/v) acetic acid as described by Ohgoda et al. ( 1988). The oocytes were then examined for evidence of oocyte nuclear maturation and for transformation of sperm nuclei in penetrated oocytes. "
"
Classification of sperm nuclear transformation The morphological changes observed in sperm nuclei were classified into the following categories (1) with decondensed chromatin, in which the sperm nucleus was swollen but retained the oblong shape of the intact spermato¬ zoon (Fig. la); (2) with recondensed chromatin, in which the swollen sperm nucleus was condensed, forming a small darkly stained mass of chromatin (Fig. la, b); (3) with metaphase chromosomes, with compact (Fig. lc) and dispersed (Fig. Id) forms or (4) with pronucleus, each accompanied by a penetrated sperm tail. When polyspermic oocytes contained sperm nuclei at different stages of transformation, they were classified according to the most prevalent
stage.
Results Most (>70%) of the oocytes were penetrated and polyspermy was common (Table 1). Oocyte maturation was evident 5 h after transfer to TCM-199, when most oocytes exhibited condensed GVs or had progressed to prometaphase I. Ten to 15 h after transfer, most oocytes were undergoing
Fig. 1. Oocytes were inseminated at the germinal vesicle stage, transferred to the maturation medium 8 h after insemination and fixed and stained at various times after transfer, (a) An oocyte penetrated 5 h after transfer showing two decondensed and recondensed (arrowheads) sperm nuclei, with penetrating sperm tails. One of the recondensed sperm nuclei is out of focus, (b) An oocyte penetrated at metaphase I (M) 5 h after transfer showing two recondensed sperm nuclei, with penetrating sperm tails, (c) An oocyte penetrated at metaphase I (M) 10 h after transfer. A sperm nucleus has been transformed into compacted metaphase chromosomes, with a penetrating sperm tail, (d) An oocyte penetrated 15 h after transfer. A sperm nucleus with a penetrating sperm tail has been transformed into metaphase chromosomes, which are slightly dispersed, (e) The same oocyte as in (d) at metaphase II (M) with first polar body (PB). 20 pm. Bars =
the first meiotic division, with 24% attaining metaphase II by 15 h. Maturation proceeded such that 62% of the oocytes reached metaphase II by 40 h. Only 4% of penetrated oocytes were activated (contained female pronuclei) by 40 h. When polyspermy was modest (one to four spermatozoa per oocyte), it appeared to have no effect on oocyte maturation. However, when more than four spermatozoa penetrated, development was delayed and fewer oocytes reached metaphase II (Table 2).
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'Oocytes examined 40 h after transfer into were
maturation medium
used.
GV:
germinal vesicle; CGV: phase I; M I: metaphase I; II; PN: pronucleus.
condensed GV; PM I: prometa¬ I: telophase I; M II: metaphase
Five hours after transfer to TCM-199, most (81%) of the sperm nuclei were decondensed (category 1), and a few (5%) had transformed to metaphase chromosomes (category 3) (Table 3). After 10 h, about half the sperm nuclei had recondensed (category 2) and half had transformed to metaphase chromosomes. This progression continued so that by 40 h, most (86%) of the penetrated oocytes contained sperm chromosomes and a few contained male pronuclei (in the oocytes with female pronuclei described above). Table 3.
Sequence of transformation of sperm nuclei in penetrated oocytes3
Time of examination (h after
transfer)
5 10 15 40
Number of oocytes
penetrated with sperm nuclei at various stages
Decondensed Recondensed Metaphase chromatin chromatin chromosomes Total
(%)
57 60 63
46(81) 2 (4) 2 (3) 1 (2)
51
Pronucleus
(%)
(%)
(%)
8(14) (45) (27) (6)
(5) (52) 44(70) 44 (86)
0(0) 0(0) 0(0) 3(6)
27 17 3
3 31
Oocytes were cultured for 5-40 h after transfer into TCM-199 containing 10% fetal calf serum
8 h after insemination.
Polyspermy did not appear to limit the transformation of sperm nuclei to metaphase chromo¬ until it reached more than nine spermatozoa per oocyte (Table 4).
somes
Table 4. Effect of number of spermatozoa to penetrate transformation of sperm nuclei3 Number of
spermatozoa to penetrate each oocyte
an
oocyte
on
Number of oocytes with sperm nuclei at the stage of
Decondensed chromatin
Recondensed chromatin
Metaphase
chromosomes
1-2
24
3 4 5-6 7-9 10-14
11 6
Pronucleus
3 0
"Oocytes examined 40 h after transfer into maturation medium were used.
Discussion The results of this study clearly indicate that bovine oocytes inseminated at the GV stage can undergo meiotic maturation in vitro when they are transferred into an appropriate culture condition (TCM-199 containing 10% FCS) after completion of penetration by spermatozoa and that the cytoplasm of the maturing oocytes has an activity that can transform sperm nuclei to metaphase chromosomes as already observed by Clarke & Masui (1986) in mouse oocytes. In a previous study, oocytes inseminated at the GV stage were continuously cultured for up to 20 h in fertilization medium (BO medium with 10mg BSA ml-1, 5mmol caffeine l"1 and 10pg heparin ml '), but meiotic maturation of the oocytes was greatly retarded and the number of spermatozoa per penetrated oocyte increased rapidly as the time after insemination increased (Niwa et al, 1991). Thus, the fact that no sperm chromosome formation was observed in that study might be attributed to the inappropriate culture conditions used for the maturation of inseminated oocytes. On the basis of previous data (Niwa et al, 1991) the present results suggest that oocytes penetrated 8 h after insemination still have intact GVs at the time of transfer into TCM-199. Since GV breakdown of bovine oocytes in vitro occurs between 4 and 8 h of culture (Sirard et al, 1989), it is clear that BO medium has retarded GV breakdown. The low numbers (21-25%) of unpenetrated and penetrated oocytes that reached metaphase II 15 h after transfer to TCM-199 could therefore be attributed to the retardation of the first step of meiosis. This retardation, however, did not inhibit the ability of the oocytes to complete maturation, because, 40 h after transfer, 70% of unpenetrated and 65% of penetrated oocytes had reached metaphase II (Table 1). The present results confirm those observed in mice by Clarke & Masui (1986), who reported that sperm penetration of maturing oocytes does not delay the first meiotic division but that polyspermy does affect oocyte maturation. In the present study, bovine oocytes that had been penetrated by more than four spermatozoa were greatly delayed in reaching metaphase II. According to Clarke & Masui (1986), the shortening and thickening of the maternal chromosomes occurred more frequently in polyspermic maturing mouse oocytes and in highly polyspermic oocytes, the chro¬ mosomes underwent drastic morphological changes. In the present study, however, it was diffi¬ cult to observe such morphological changes in the maternal chromosomes of polyspermic -
oocytes.
In general, mature oocytes are activated by sperm penetration, resume the second meiotic division and extrude the second polar body, then form a female pronucleus. In the present study, however, few penetrated oocytes had formed a female pronucleus even 40 h after transfer. This observation supports the hypothesis that sperm penetration into immature oocytes does not induce oocyte activation. This is not surprising since oocyte activation normally occurs in oocytes at meiosis (M) phase and not at an interphase state as in GV oocytes. This concept is consistent with our recent observations that bovine oocytes experimentally arrested between metaphase I and telophase I are activated by sperm penetration (Chian et al, 1992). Indeed, it is possible that oocytes that did undergo activation in our study had begun to mature during the in vitro fertiliz¬ ation incubation, and were therefore activated by the penetrating spermatozoa, although this was not experimentally verified. In contrast to most other mammalian species examined, the exception being dogs (Mahi & Yanagimachi, 1976), in bovine oocytes decondensation of sperm nuclei can be induced even before GV breakdown (Niwa et al, 1991). Furthermore, transformation of the decondensed sperm nuclei to metaphase chromosomes was clearly observed in this study. When oocytes inseminated at the GV stage were transferred into an appropriate culture medium 8 h after insemination, sperm nuclei in some penetrated oocytes were recondensed (14%) or transformed to metaphase chromosomes (5%) within 5 h of transfer. The proportion of oocytes with sperm nuclei transformed to metaphase chromosomes increased with time reaching 86% 40 h after transfer. The pattern of these morpho¬ logical changes in sperm nuclei is very similar to that observed in mouse oocytes (Clarke & Masui, 1986). In bovine oocytes, however, it was difficult to observe well separated sperm chromosomes
40 h after transfer. Although it has been reported that metaphase chromosomes derived from spermatozoa (Clarke & Masui, 1986) and other maturing oocytes or blastomeres (Balakier, 1978; Szollosi et al, 1980; Tarkowski & Balakier, 1980; Clarke & Masui, 1985) have mixed with oocyte metaphase chromosomes, such mixing of the sperm and oocyte metaphase chromosomes was not observed in bovine oocytes; both chromosomes were always observed to be separate. Clarke & Masui (1986) reported that maturing mouse oocytes, penetrated by more than four spermatozoa, never transformed any of the nuclei to metaphase chromosomes. In this study, bovine oocytes penetrated by up to nine spermatozoa transformed all of the nuclei to metaphase chromosomes, but when oocytes were penetrated by 10-14 spermatozoa, no nuclei were trans¬ formed to metaphase chromosomes. Since bovine oocytes are larger than mouse oocytes, it is expected that the former possess a much higher capacity with respect to the cytoplasmic activity than the latter. Clarke & Masui (1987) have also suggested that the transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of mouse oocytes requires nucleoplasm of the GV and even
non-GV
cytoplasmic substances, including proteins synthesized during maturation. However, the requirements of GV material and proteins for sperm nuclear transformation in bovine oocytes need to be clarified in further experiments. In the experimental conditions used, 65% of penetrated oocytes matured to metaphase II; nevertheless the formation of a male pronucleus was observed only in 6% of penetrated oocytes when they were examined 40 h after transfer. We do not consider this to result from a limitation of the amount of materials present in the cytoplasm that support sperm pronuclear development, as suggested in the previous study (Niwa et al, 1991), since the number of spermatozoa in a penetrated oocyte was limited in this study. If the male pronucleus growth factor (Thibault & Gerard, 1973) or the sperm pronucleus development factor (Yanagimachi, 1981) can function when the cytoplasm of oocytes is fully activated (Yanagimachi, 1981; Perreault et al, 1987), it is possible that the sperm nuclei incorporated into oocytes at the GV stage cannot activate the oocytes even after maturation has been completed. A
portion of this work was supported by Grant-in-Aid for Developmental Scientific
Research
(No. 03556038) from the Ministry of Education, Science and Culture of Japan. References
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