MOLECULAR REPRODUCTION AND DEVELOPMENT 2640-46 (1990)
Effect of Follicle Cells on the Acrosome Reaction, Fertilization, and Developmental Competence of Bovine Oocytes Matured In Vitro Y. FUKUI Department of Meat Animal Reproduction, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan The role of follicle cells in the ABSTRACT acrosome reaction of frozen-thawed bovine spermatozoa, in vitro fertilization, cleavage, and development in vitro was investigated. Cumulus-oocyte complexes were cocultured and matured in vitro with additional granulosa cells for 24 hr. Immediately before in vitro insemination, the oocytes were divided into three types with different follicle cells: denuded and corona- and cumulus-enclosed oocytes. The proportion of live, acrosome-reacted spermatozoa significantly increased at 3 and 6 hr after insemination in all types of oocytes. However, the mean proportion of live, acrosome-reacted spermatozoa that inseminated cumulus-enclosed oocytes at 6 hr after insemination was significantly higher than that of spermatozoa inseminating denuded oocytes (18.3% and 13.3%, respectively). The frequency of in vitro fertilization was significantly higher for cumulus-enclosed oocytes (65.4%) than for denuded and corona-enclosed oocytes (30.8% and 39.4%, respectively). Cumulus-enclosed oocytes when cocultured with oviduct epithelial cells also had significantly higher rates of cleavage (two- to eight-cell, 59.8%; eight-cell, 22.4%) and blastocyst formation (7.7%) than denuded and corona-enclosed oocytes. No eightcell embryos or more advanced stages of embryonic development were observed in either denuded or corona-enclosed oocytes without the coculture. The present results indicate that cumulus cells at fertilization play an important role in inducing the acrosome reaction and promoting a high fertilization rate, cleavage, and development into blastocysts in vitro.
Key Words: In vitro fertilization, Embryonic development, Cattle ~
Fukui and Sakuma, 1980; Critser et al., 1986b; Leibfried-Rutledge et al., 1987; Grad1 et al., 1988). In inducing not only nuclear maturation but also cytoplasmic maturation leading to preimplantation embryonic development, it has been clearly demonstrated that follicle cells, especially the cumulus cells surrounding immature oocytes, play a central role in developmental competence in sheep (Staigmiller and Moor, 1984) and cattle (Goto et al., 1988; Shioya et al., 1988; Sirard et al., 1988). However, the role of follicle cells in in vitro fertilization and subsequent oocyte development has not yet been clearly identified, especially in relation to the acrosome reaction of spermatozoa occurring during in vitro insemination. Although the acrosome reaction can occur independently of the eggs (Yanagimachi, 19811, it is probably the egg investments that induce the acrosome reaction in fertilizing spermatozoa (Florman and Storey, 1982; Crozet, 1984; Cummins and Yanagimachi, 1986; Uto et al., 19881. Cumulus cells andlor additional granulosa cells are needed to complete oocyte maturation (Critser et al., 1986a,b; Lu et al., 1987; Thibault et al., 1987; Xu et al., 1987; Fukui and Ono, 1988; Fukui et al., 19891, but it has been reported that cumulus cells are not essential for in vitro fertilization (Moore and Bedford, 1978). Recent studies on in vitro fertilization in pigs (Cheng, 19851, sheep (Fukui et al., 1988a,b), and cattle (Lu et al., 1987; Xu et al., 1987)have indicated that cumulus cells of in vitro-matured oocytes when denuded or partly removed by pipetting or by hyaluronidase treatment showed improved sperm penetration, but the effect has not been clarified, and the timing of the acrosome reaction of bovine spermatozoa in vitro associated with different somatic cells in follicles has not been investigated. The present study was conducted to clarify the exact role of follicle cells (corona radiata and cumulus
~~
INTRODUCTION Bovine oocytes matured and fertilized in vitro seem to be unable to acquire developmental competence unless cocultured with follicle cells in the presence of gonadotropins and estradiol (Leibfried and First, 1979;
@ 1990 WILEY-LISS, INC.
Received March 6,1989; accepted September 25, 1989. Address reprint requests to Dr. Yutaka Fukui, Department of Meat Animal Reproduction, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080, Japan.
IN VITRO FERTILIZATION OF BOVINE OOCYTES TABLE 1. Concentrations of Media Used for In
Vitro Fertilization Ingredient NaCl KC1
NaHCO, NaH,PO, 2H,O Na-Lactate" CaC1, MgCl, 6H,O Phenol red
Units mM mM mM mM mM mM mM w/ml mM mM mM
Capacitation Washing Fertilization (pH 7.4) (pH 7.4) (pH 7.8) 114.0
114.0
3.2 25.0 0.4
3.2
3.2
2.0
25.0
10.0
0.4 10.0
0.4 10.0
0.5
2.0 0.5 10.0 10.0
2.0 0.5
-
10.0
HEPES 5.0 Na-pyruvate 1.0 0.5 Glucose 12.5 5.0 Bovine serum albuminb mdml 6.0 3.0 "Sixty percent sodium salt. bFatty-acid-freeprepared from fraction V
114.0
10.0 10.0 0.5 5.0
6.0
cells) in the acrosome reaction of frozen-thawed bovine spermatozoa in vitro, in vitro fertilization, and subsequent developmental capacity in a coculture with or without bovine oviduct epithelial cells (BOEC) in bovine oocytes matured in vitro. MATERIALS AND METHODS Oocyte Maturation Ovaries were obtained from Holstein cows and heifers killed a t a local abattoir and were transported in a saline solution (0.9% NaCl) a t 30-35°C to the laboratory within 1hr. Cumulusoocyte complexes were collected from follicles 2-5 mm in diameter with an 18gauge needle attached to a 10 ml disposable syringe. Only oocytes with unexpanded cumulus oophorus and evenly granulated cytoplasm were cultured in 30 mm plastic dishes (Sterillin, Middlesex, England; 20-40 oocytes/dish) containing 2 ml tissue culture medium (TCM) 199 (Wittaker M.A. Bioproducts, Walkersville, MD, pH 7.4, Earle's salt with sodium bicarbonate and L-glutamine) supplemented with 15% (v/v) heat-inactivated (56"C, 30 min) estrous cow serum (ECS). ECS used in this study was obtained from cows at the time of artificial insemination during the "standing estrus" a t the University Farm and pooled as a stock to be used throughout the study. The medium was also supplemented with granulosa cells (5 x 10' cells/ml) collected from antral follicles of about 10 mm in diameter, dissected by the method of Moor and Trounson (1977), and washed (500g,5 min) three times with TCM 199 containing 5% (v/v) ECS and 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid (HEPES; Sigma Chemical Co., St. Louis, MO). The oocytes were statically cultured for 24 hr at 39°C in an atmosphere of 5% CO, in air and 95% humidity.
In Vitro Fertilization The media used for in vitro fertilization are shown in Table 1. Three modifications of Tyrode's medium
41
(TALP) as described by Bavister and Yanagimachi (1977) were prepared for sperm capacitation (capacitation medium), for washing oocytes (washing medium), and for a fertilization medium. Ca2+was not present in the capacitation medium. Four chemicals (HEPES, Napyruvate, glucose, and bovine serum albumin) were added immediately before use of the media. All media were used within 1 week and were stored at 4°C. Frozen straws (0.5 ml in size) of semen from a Japanese black bull were thawed at 35-37°C and were prepared for sperm capacitation. The thawed semen (0.2 ml) was placed under 1ml of capacitation medium in conical tubes for a swim-up procedure (Parrish et al., 198613; Lu et al., 1987). The top 0.8 ml of the medium was then collected after 1 hr of incubation a t 39°C and pooled. The medium containing spermatozoa was washed twice (500g, 10 min) with capacitation medium. The final pellet of semen was resuspended in the medium to the concentration of 50 x 10' spermatozoa/ ml. An equal volume of a 200 pg/ml heparin (Sigma Chemical Co.) solution was added to the semen suspension to yield spermatozoa and heparin concentrations of 25 x 10" cells/ml and 100 pg/ml, respectively. The heparin-treated spermatozoa were incubated for 15 min at 39°C in 5% COP in air (Lu et al., 1987). Oocytes cultured for 24 hr were washed three times with a washing medium (Table 1)by pipetting and divided into the following three types of oocytes (Fig. 1) having different kinds of follicle cells: 1) those completely devoid of follicle cells (denuded oocytes), 2) those surrounded by one to three layers of corona cells (corona-enclosed oocytes), and 3) those surrounded by expanding cumulus cells (cumulus-enclosed oocytes). In each group of oocytes, a 2 pl aliquot of the washing medium containing five oocytes was placed into 45 pl drops of fertilization medium (Table 1)under sterile oil (Nakarai Chemical Co., Tokyo, Japan). Three microliters of the heparin-treated semen suspension were then added to the oocytes to give a final concentration of 1.5 x 10' celldml. After in vitro insemination, oocytes and spermatozoa were incubated for 18-20 hr a t 39°C under 5% CO, in air. In experiment 1, live and acrosome-reacted spermatozoa inseminating the three different types of oocytes were examined. Following in vitro insemination (0hr), spermatozoa cocultured with the different oocytes in the droplets were prepared for smears at 3 hr intervals over a 24 hr period (0,3,6, 9, 12, 15, 18,21, and 24 hr) using the simplified triple-stain method (Kusunoki et al., 1984, 1985). Light brown postacrosomal regions with unstained acrosomal regions indicated spermatozoa that were alive and in which the acrosome reaction had taken place. The proportions of live, acrosome-reacted spermatozoa were calculated for the 100-200 spermatozoa per smear, and the results were compared over four repeated trials to determine the effect of follicle cells surrounding oocytes. For each test, oocytes were fixed with acetoalcohol (1:3)and stained with 1%
42
Y.FUKUI
Fig. 1. Three types of bovine oocytes used for in vitro fertilization following 24 hr in culture: A, denuded; B, corona-enclosed;C, cumulus-enclosed oocytes.
acetoorcein to observe sperm penetration and pronucleus formation. Experiment 2 was carried out to examine the effect of follicle cells on’in vitro fertilization in the three types of oocytes. A total of 808 oocytes were examined over seven trials for evidence of fertilization after fixation and staining as in experiment 1.The only oocytes considered as being normally fertilized were those having female and male pronuclei, with a residual sperm-tail visible in the cytoplasm. In experiment 3, to examine developmental capacity following in vitro fertilization, the three types of oocytes were separately cultured with or without BOEC. As was described previously (Fukui and Ono, 1988; Fukui et al., 19891, BOEC were collected by flushing the oviducts of cows and heifers with a newly formed corpus luteum with 10 ml Dulbecco’s phosphate-buffered saline containing 10% (v/v) ECS per oviduct. The flushed fluid was kept a t 39°C for 10 min, and supernatant solution was carefully removed. After resuspension with TCM 199 containing 10% (vlv) ECS and 35 ng/ml insulin (Sigma Chemical Co.), a 0.5 ml aliquot of the cell suspension was placed into four-well dishes (Nunclon, Intermed, Copenhagen, Denmark) without any enzyme treatments. After movement was apparent in the ciliated cells from oviducts and after monolayers (>50% confluent) had formed (within 48 hr), about 10 embryos were placed in each well containing BOEC or only medium and were cultured for 7 days at 39°C under 5% C02 in air. On the third day of culture, cleavage (two- to eight-cell) was examined,
and the numbers of morulae and expanded blastocysts were recorded after 7 days in culture. The mean proportions of live spermatozoa and live, acrosome-reacted spermatozoa over the four trials at the nine observation times a t 3 hr intervals (0-24 hr) were analyzed by Student’s t test (experiment 1).The frequencies (mean 2 S.E.M.) of fertilization for each type of oocytes (experiment 2) in the seven trials were compared by Duncan’s new multiple range test after angular transformation (Steel and Torrie, 1960). For examining cleavage and development (experiment 3),a factorial study (3 x 2: three types of oocytes, with or without BOEC) was designed. Proportions of eight-cell embryos, cleaved (two- to eight-cell) embryos, and developed embryos (morulae and blastocysts) were subjected to least-square methods for analysis of variance after angular transformation (Steel and Torrie, 1960). Duncan’s new multiple range test (Steel and Torrie, 1960) was also used to compare the cleavage and development rates.
RESULTS Experiment 1: Proportions of Acrosome-Reacted Spermatozoa Over Time During In Vitro Insemination With Oocytes Surrounded by Different Follicle Cells As is shown in Table 2, during the 24 hr observation period, the mean proportion of live spermatozoa decreased gradually without significant differences among the types of oocytes except between the groups
IN VITRO FERTILIZATION OF BOVINE OOCYTES
43
TABLE 2. Mean (2S.E.M.) Proportions of Live Spermatozoa and Acrosome-Reacted Spermatozoa During In Vitro Insemination With Bovine Oocytes Surrounded by Different Follicle Cells Times (hr) following insemination (c/o) Oocvtes
Spermatozoa
Denuded
Live Live and acrosomereacted Live
Coronaenclosed
Live and acrosomereacted Cumulus- Live enclosed Live and acrosomereacted
6 0 3 49.5 f 3.9 49.2 2 2.9 44.3 f 3.0 7.0 f 1.0 10.32 1.2 13.3t 0.7"
9 12 15 18 21 24 31.0 f 2.6 28.5 f 2.8 23.0 2 1.2 18.0 2 2.7 17.3 ? 0.6 13.0 f 1 .3 ~. 7.0 1.6 6.0 2 1.6 6.0 -c 0.6 4.0 f 0.4 2.3 0.3 2.0 f 0.0
55.3 f 3.1 50.0 2 1.2 50.8 f 3.1
39.5 f 3.1 33.5 f 3.1 26.8 2 1.2 24.6 f 2.7 16.8 t 1.8 15.8 +- 1.8
*
*
5.0 f 0.8 15.7 2 0.3 15.0f 1.5".b 9.3 2 1.3 51.5 f 2.3 45.9 2 2.2
49.0
* 3.3
5.5 f 0.9 13.8 2 0.9 18.3f l.gb
7.3 f 2.3
6.5 -t 0.8
4.5 2 0.8
2.5 f 0.6
2.5 ? 0.3
36.4 f 2.2 32.8 f 3.1 29.8 t- 2.7 24.9 t 2.1 18.8 f 2.1 15.8 +- 1.5 8.8-+ 1.8 ~
8.5 2 0.6
7.0 2 1.0 ~~
5.3 f 0.8 4.0f 0.9
2.850.5 ~
a,bFigureswith differentsuperscripts in each column are significantly different (P< 0.001).
of corona- and cumulus-enclosed oocytes at the 3 hr Experiment 3: Cleavage and Development of observation (P < 0.05).However, the spermatozoa inOocytes Surrounded by Different Follicle Cells seminating the denuded oocytes tended t o decline in and Cocultured With or Without Bovine Oviduct viability more rapidly than the other two groups. In the Epithelial Cells three types of oocytes, the mean proportion of live, acAnalysis of variance in a factorial design showed rosome-reacted spermatozoa significantly increased (P that the cell types surrounding oocytes were signifi< 0.01) at 3 hr after insemination, and proportions cantly (P < 0.001) different in regard to cleavage and were significantly higher ( P < 0.01) a t 6 hr than a t 0, 3, and 9 hr and other times of observations. It was development into blastocysts. The proportions of eightcell embryos (P < 0.025) and the total number of shown that the mean proportion of live, acrosome-recleaved embryos ( P < 0.05) had a significant correlaacted spermatozoa became significantly lower ( P < 0.01)9 hr after insemination in all groups. The effect of tion to the cell types and BOEC. It was clearly shown that BOEC was highly effective for cleavage (P < follicle cells on the mean proportion of live, acrosome0.001) and further development (P < 0.001)of in vitro reacted spermatozoa inseminating three different fertilized eggs. No eight-cell embryos or more advanced types of oocytes was similar except between the cumustages of embryonic development were observed in both lus-enclosed (18.38)and denuded (13.3%)oocytes at 6 denuded and corona-enclosed oocytes when they were hr, when a significant difference ( P < 0.01) was obcultured in the absence of BOEC (Table 4).Even with served. No other significant differences were found. the coculture of the two types of oocytes in BOEC, the The timing of sperm penetration and the formation rate of cleavage ( P < 0.01) and development into blasof female and male pronuclei in the three types of tocysts (P < 0.05)was significantly lower than that oocytes was similar; spermatozoa with a swollen head observed in cumulus-enclosed oocytes cocultured in the and a residual tail were observed at 3 and 6 hr, and the presence of BOEC. The highest rates of cleavage (eightformation of two pronuclei was observed a t 9 and 12 hr cell, 22.4%; total cleaved embryos, 59.8'%), developafter insemination. ment into blastocysts (7.7%), and combined development into morulae and blastocysts (10.4%) were Experiment 2 In Vitro Fertilization of Oocytes observed in cumulus-enclosed oocytes cocultured with Surrounded by Different Follicle Cells BOEC. A total of 808 oocytes in seven trials were examined DISCUSSION for fertilizability (Table 3). The cumulus-enclosed The importance of the cumulus cells surrounding imoocytes had the highest frequency (mean 2 S.E.M.) of 4.8%) with significant differ- mature oocytes for maturation competence has been fertilization (65.4% ences ( P < 0.001) observed when compared to both de- clearly recognized with regard to the subsequent fernuded (30.8& 5.9%) and corona-enclosed (39.4% 2 tilizability and developmental capacity in sheep (Staig6.3%) oocytes. Comparing the frequency of fertilization miller and Moor, 1984)and cattle (Critser et al., 1986b; between corona-enclosed oocytes and denuded oocytes, Thibault et al., 1987; Shioya et al., 1988).The presence the former was significantly (P < 0.05) higher than the of additional granulosa cells with cumulus-oocyte complexes is of further importance for developmental comlatter.
*
Y.FUKUI
44
TABLE 3. Effect of the Follicle Cells Surrounding Oocytes on In Vitro Fertilization of Bovine Oocytes
Matured In Vitro
No. Percent No. of of oocytes (mean S.E.M.) of
trials Oocytes 7 Denuded Corona-enclosed 7 Cumulus-enclosed 7 a vs. b P < 0.05.
*
examined
oocytes fertilized
280 274 254
30.8 2 5.9" 39.4 +- 6.3b 65.4 & 4.8'
a,b vs. c
P < 0.001.
petence in maturing ovine and bovine oocytes (Staigmiller and Moor, 1984; Critser et al., 1986a; Lu et al., 1987; Lutterbach et al., 1987; Thibault et al., 1987; XU et al., 1987; Fukui and Ono, 1988,1989). In the present study, all oocytes with compacted cumulus cells were cocultured with additional granulosa cells for in vitro maturation. After 24 hr in culture, the oocytes were then washed by pipetting to divide them into the three types of oocytes differing in their surrounding follicle cells (denuded and corona- and cumulus-enclosed oocytes). It has been established that the acrosome reaction in inseminating mammalian spermatozoa can occur through contact with egg investments, such as follicular fluid (Yanagimachi, 1969; Iwamatsu and Chang, 1969; Lenz et al., 1982), cumulus cells (Bavister, 1982; Yanagimachi et al., 1983; Cummins and Yanagimachi, 19861,and zonae pellucidae (Florman and Storey, 1982; Cross et al., 1988; Uto et al., 1988). Since Lenz et al. (1982, 1983) reported their findings that glycosaminoglycans, such as heparin, chondroitin sulfate A, and so on, in bovine follicular fluid enhance the incidence of capacitation and the acrosome reaction in bovine spermatozoa, follicular fluid (Fukui et al., 1983; Sanbuissho and Threlfall, 1989) or heparin (Parrish et al., 1985, 1988) has been utilized to increase the rate of sperm penetration and fertilization in vitro. The present results indicate two important points. First, cumulus cells are necessary a t the time of in vitro insemination to maximize the incidence of the acrosome reaction in frozen-thawed bovine spermatozoa and to enhance the frequencies of fertilization, cleavage, and development into blastocysts in vitro. Second, the times of the maximum incidence of live, acrosome-reacted spermatozoa were at 3 and 6 hr after insemination independently of the type of follicle cells. The significant increases in the frequency of live, acrosome-reacted spermatozoa at 3 and 6 hr after insemination coincided with sperm penetration into the oocytes. Concerning the importance of cumulus cells, Gwatkin et al. (1972, 1974) reported similar findings in hamster and mouse spermatozoa, demonstrating that in the capacitation process cumulus cells play a key role with spermatozoa interacting closely with the cu-
mulus oophorus. Recently, Lu et al. (1987) and Fukui et al. (1989) removed the cumulus cells surrounding bovine oocytes matured in vitro by either gentle pipetting or hyaluronidase treatment before in vitro insemination in order to increase sperm penetration. The present study, however, indicates that to increase sperm penetration the expanding cumulus cells should be attached onto the oocytes for maximizing the acrosome reaction of bovine spermatozoa and fertilization and subsequent development in vitro. Completely denuded oocytes, however, have also shown some ability to induce the acrosome reaction and to result in fertilizability, although the frequencies were less than those in cumulus-enclosed oocytes. These results suggest that the zonae pellucidae of matured bovine oocytes also have the ability to induce the acrosome reaction. Since the existence of the ability has been clearly established for the zonae pellucidae of mature, unfertilized hamster (Cherr et al., 1986; Uto et al., 1988), rabbit (O'Rand and Fisher, 1986), cattle (Florman and Bobcock, 19881, and human (Cross et al., 1988) eggs, it can be surmised that a zona pellucida component induces the acrosome reaction in bovine oocytes. A receptor, called ZP3, has been identified in solubilized mouse zonae pellucidae as an inducer of the acrosome reaction (Wassarman et al., 1986; cited by Wassarman, 1987). Florman and Babcock (1988) obtained a 30-35% rate of acrosome reaction in bovine spermatozoa following heparin treatment (10 p,g/ml) with soluble extracts of bovine zona pellucida. The rather lower frequencies (1318%)of live, acrosome-reacted spermatozoa observed in the present study, despite the higher dose of heparin (100 pg/ml), are probably due to the presence of glucose in the capacitation medium, considering the reports (Parrish et al., 1985, 1986a) showing that glucose inhibits or delays the effect of heparin on bovine sperm capacitation. To obtain satisfactory results in fertilization in vitro when glucose is present in the medium, as in this study, a higher dose of heparin likely is required. The present study has clearly demonstrated that coculture with BOEC is very effective for cleavage and development into blastocysts in vitro following fertilization. This has been confirmed in previous studies (Eyestone et al., 1987; Eyestone and First, 1988, 1989; Fukui and Ono, 1988, 1989; Lu et al., 19881, although other types of somatic cells, such as fibroblasts (Kuzan and Wright, 19821, trophoblastic vesicles (Camous et al., 1984), and cumulus cells (Kajihara et al., 1987; Goto et al., 19881, have also been employed successfully. The highest rate of development into blastocysts in the present study was 7.7%, but previous studies in which different bulls were used have shown higher results (12-17%) (Fukui and Ono, 1988, 1989). Further research on improved culture systems will be necessary to decrease the great loss of embryos occurring in the present study from 65% a t the time of fertilization to 22% at the eight-cell stage, and in other studies, such
IN VITRO FERTILIZATION OF BOVINE OOCYTES
45
TABLE 4. Effects of the Follicle Cells Surrounding Oocytes and Coculture With Bovine Oviduct Epithelial Cells (BOEC) on In Vitro Development of Bovine Oocytes Matured and Fertilized In Vitro
No. of embryos developed into (%) Oocytes Denuded
Coronaenclosed
No. of oocytes cultured with or withoutBOEC With 308
Two-cell 25
Four-cell 31
Without 241
9
0
With 316
24
57
Without 308
16
2
Eight-cell 9 (2.9)' 0 (O.OP 36 (11.4)b 0 (O.O)d
Cumulusenclosed
With 326
36
86
Without 251
39
39
73 (22.4)" 8 (3.2)'
Total cleaved (two- to eight-cell) 65 (21.1)" 9 (3.7)d 117 (37.0Ib 18 (~5.8)~ 195 (59.8)" 86 (34.3)b
Morulae 1 0
5 0
9 0
Blastocysts 3 (1.0)C 0 (O.OP 12 (3.8)B 0
(O.OP
25 (7.71-4 3 (1.2F
Morulae + blastocysts 4 (1.3)" 0 (O.OP 17 (5.4)" 0 (O.OP 34 (10.4)* 3 (1.2)C
"-d*A-DFigures with different superscripts in each column are different (a-d, P < 0.01; A-D, P < 0.05).
as those of Fayrer-Hosken et al. (1988) (penetration, 91%;cleavage, 38%;with the half of the embryos reaching the four- to eight-cell stage) and Eyestone and First (1989) (cleavage, 76%; development into morulae and blastocysts, 22%). In conclusion, for the induction of the acrosome reaction in frozen-thawed bovine spermatozoa and for improved in vitro fertilization and developmental capacity with in vitro-matured oocytes, eggs should be surrounded a t fertilization by expanding cumulus cells. The oocytes completely denuded of follicle cells have shown a slight ability to induce the acrosome reaction of spermatozoa and be fertilized in vitro, but the developmental capacity was limited even when cocultured with BOEC.
ACKNOWLEDGMENTS The author thanks Tomoo Sonoyama, Hitoshi Mochizuki, Yasushi Tamukai, and Hidenori Izumi for their assistance in collecting ovaries and oocytes. This work was partly supported by a Grant-in-Aid for Scientific Research (62560255)from the Ministry of Education, Science and Culture of Japan.
REFERENCES Bavister BD (1982): Evidence for a role of postovulatory cumulus components in supporting fertilizing ability of hamster spermatozoa. J Androl 3:365-372. Bavister BD, Yanagimachi R (1977): The effects of sperm extract and energy sources on the motility and acrosome reaction of hamster sperm in vitro. Biol Reprod 16:228-237. Camous S, Heyman Y, Meziou W, MBn6zo Y (1984): Cleavage beyond the block stage and survival after transfer of early bovine embryos cultured with trophoblastic vesicles. J Reprod Fertil 72:479-485. Cheng WTK (1985): "In Vitro Fertilization of Farm Animal Oocytes." PhD Thesis, Cambridge University. Cherr GN, Lambert H, Meizel S, Katz DF (1986): In vitro studies of the golden hamster sperm acrosome reaction: Completion on the zonae pellucidae and induction by homologous soluble zonae pellucidae. Dev Biol 114119-131. Critser ES, Leibfried-Rutledge ML, Eyestone WH, Northey DL, First
NL (1986a): Acquisition of developmental competence during maturation in vitro. Theriogenology 25:150 (abstract). Critser ES, Leibfried-Rutledge ML, First NL (1986b): Influence of cumulus cell association during in vitro maturation of bovine oocytes on embryonic development. Biol Reprod (Suppl. 1):192. Cross NL, Morales P, Overstreet JW, Hanson FW (1988): Induction of acrosome reactions by the human zona pellucida. Biol Reprod 38: 235-244. Crozet N (1984): Ultrastructural aspects of in vivo fertilization in the cow. Gamete Res 10241-251. Cummins JM, Yanagimachi R (1986): Development of ability to penetrate the cumulus oophorus by hamster spermatozoa capacitated in vitro, in relation to the timing of the acrosome reaction. Gamete Res 15:187-212. Eyestone WH, First NL (1988): Co-culture of bovine embryos with oviductal tissue. Proc 11th Int Congr Anim Reprod A1 4471. Eyestone WH, First NL (1989): Co-culture of early cattle embryos to the blastocyst stage with oviductal tissue or in conditioned medium. J Reprod Fertil 85:715-720. Eyestone WH, Vignieri J, First NL (1987):Co-culture of early bovine embryos with oviductal epithelium. Theriogenology 27:228 (abstract). Fayrer-Hosken RA, Younis AI, Brackett BG, McBride CE, Harper KM, Keefer CL, Cabaniss DC (1988): Pregnancy after laparoscopic oviductal transfer of in vitro matured and in vitro fertilized bovine oocytes. Biol Reprod 38 [Suppl 11:72. Florman HM, Babcock DF (1988): Changes in intracellular ICa" 1 and pH accompany the zona pellucida-induced acrosome reaction of bovine spermatozoa. Biol Reprod 38lSuppl 11:60. Florman HM, Storey BT (1982): Mouse gamete interactions: The zona pellucida is the site of the acrosome reaction leading to fertilization in vitro. Dev Biol 91:121-130. Fukui Y, Fukushima M, Ono H (1983): Fertilization in vitro of bovine oocytes after various sperm procedures. Theriogenology20:651-660. Fukui Y,Glew AM, Gandolfi F, Moor RM (1988a):Ram-specific effects in in-vitro fertilization and cleavage of sheep oocytes matured in vitro. J Reprod Fert 82:337-340. Fukui Y,Glew AM, Gandolfi F, Moor RM (1988b): In vitro culture of sheep oocytes matured and fertilized in vitro. Theriogenology 29: 883-891. Fukui Y, Ono H (1988): In vitro development to blastocyst of in vitro matured and fertilized bovine oocytes. Vet Rec 122:282. Fukui Y, Ono H (1989):Effects of sera, hormones and granulosa cells added to culture medium for in-vitro maturation, fertilization, cleavage and development of bovine oocytes. J Reprod Fertil 8 6 501-506.
46
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Fukui Y, Sakuma Y (1980):Maturation of bovine oocytes cultured in vitro: Relation to ovarian activity, follicular size and the presence or absence of cumulus cells. Biol Reprod 22:669-673. Fukui Y, Urakawa M, Sasaki C, Chikamatsu N, Ono H (1989): Development to the late morula or blastocyst stage following in vitro maturation and fertilization of bovine oocytes. Anim Reprod Sci 18:139-148. Goto K, Kajihara Y, Kosaka S, Koba M, Nakanishi Y, Ogawa K (1988): Pregnancies after co-culture of cumulus cells with bovine embryos derived from in-vitro fertilization of in-vitro matured follicular oocytes. J Reprod Fertil 83:753-758. Grad1 E, Lutterbach A, Brem C (1988): Influence of follicle cell coculture and 17-p-estradiol on in vitro maturation and fertilization of cattle oocytes. Proc 11th Int Congr Anim Reprod A1 3:329. Gwatkin RBL, Andersen OF, Hutchinson CF (1972):Capacitation of hamster spermatozoa in vitro: The role of cumulus components. J Reprod Fertil30:389-394. Gwatkin RBL, Andersen OF, Williams DT (1974): Capacitation of mouse spermatozoa in vitro: Involvement of epididymal secretions and cumulus oophorus. J Reprod Fertil41:253-256. Iwamatsu T, Chang MC (1969):In vitro fertilization of mouse eggs in the presence of bovine follicular fluid. Nature 224:919-920. Kajihara Y, Goto K, Kosaka S, Nakanishi Y, Ogawa K (1987):In vitro fertilization of bovine follicular oocytes and their development up to hatched blastocysts in vitro. Jpn J Anim Reprod 33:173-180. Kusunoki H, Fujisaki H, Kato S, Kanda S (1985): Identification of acrosome-reactedbull spermatozoa by a simplified triple-stain technique. J p n J Anim A1 Res 7:9-11 (in Japanese). Kusunoki H, Yasui T, Kato S, Kanda S (1984): Identification of acrosome-reacted goat spermatozoa by a simplified triple-stain technique. Jpn J Zootech Sci 552332-837 (in Japanese). Kuzan FB, Wright RW (1982): Observations on the development of bovine morulae on various cellular and noncellular substrata. J Anim Sci 54:811-816. Leibfried L, First NL (1979): Characterization of bovine follicular oocytes and their ability to mature in vitro. J Anim Sci 48:7686. Leibfried-Rutledge ML, Critser ES, Eyestone WH, Northey DL, First NL (1987): Developmental potential of bovine oocytes matured in vitro or in vivo. Biol Reprod 36:376-383. Lenz RW, Ax RL, Grimek HJ, First NL (1982): Proteoglycan from bovine follicular fluid enhances on acrosome reaction in bovine spermatozoa. Biochem Biophys Res Commun 106:1092-1098. Lenz RW, Ball GD, Lohse JK, First NL, Ax RL (1983): Chondroitin sulfate facilitates a n acrosome reaction in bovine spermatozoa as evidenced by light microscopy, electron microscopy and in vitro fertilization. Biol Reprod 28:683-690. Lu KH, Gordon I, Chen HB, Gallagher M, McGovern M (1988):Birth of twins after transfer of cattle embryos produced by in vitro techniques. Vet Rec 122:539-540. Lu KH, Gordon I, Gallagher M, McGovern M (1987): Pregnancy established in cattle by transfer of embryos derived from in vitro fertilisation of oocytes matured in vitro. Vet Rec 121:259-260. Lutterbach A, Koll RA, Brem G (1987):In vitro maturation of bovine
oocytes in coculture with granulosa cells and their subsequent fertilization and development. Zuchthygiene 22:145-150. Moor RM, Trounson A 0 (1977): Hormonal and follicular factors affecting maturation of sheep oocytes in vitro and their subsequent developmental capacity. J Reprod Fertil 49:lOl-109. Moore HDM,Bedford J M (1978):An in vitro analysis of factors influencing fertilization of hamster eggs. Biol Reprod 19879-885. ORand MG, Fisher SJ (1986):Localization of zona binding sites and specificity of the acrosome reaction in rabbit sperm. J Cell Biol 103:367 (abstract). Parrish J J , Susko-Parrish JL, First NL (1985): Effect of heparin and chondroitin sulfate on the acrosome reaction and fertility of bovine sperm in vitro. Theriogenology 24:537-549. Parrish J J , Susko-Parrish JL, First NL (1986a): Capacitation of bovine sperm by oviduct fluid or heparin is inhibited by glucose. J Androl 7:22. Parrish J J , Susko-Parrish JL, Leibfried-Rutledge ML, Critser ES, Eyestone WH, First NL (1986b):Bovine in vitro fertilization with frozen-thawed sperm. Theriogenology 25591-600. Parrish J J , Susko-Parrish JL, Winer MA, First NL (1988):Capacitation of bovine sperm by heparin. Biol Reprod 38:1171-1180. Sanbuissho A, Threlfall WR (1989):The effect of estrous cow serum on the in vitro maturation and fertilization of the bovine follicular oocyte. Theriogenology 31:693-699. Shioya Y, Kuwayama M, Fukushima M, Iwasaki S, Hanada A (1988): In vitro fertilization and cleavage capacity of bovine follicular oocytes classified by cumulus cells and matured in vitro. Theriogenology 30:489-496. Sirard MA, Parrish JJ, Ware CB, Leibfried-Rutledge ML, First NL (1988): The culture of bovine oocytes to obtain developmentally competent embryos. Biol Reprod 39546-552. Staigmiller RB, Moor RM (1984):Effect of follicle cells on the maturation and developmental competence of ovine oocytes matured outside the follicle. Gamete Res 9221-229. Steel RGD, Torrie J H (1960): “Principles and Procedures of Statistics.” New York McGraw-Hill Book Co. Thibault C, Szollosi D, G6rard M (1987):Mammalian oocyte maturation. Reprod Nutr Dev 27:865-896. Uto N, Yoshimatsu N, Lopata A, Yanagimachi R (1988): Zona-induced acrosome reaction of hamster spermatozoa. J Exp Zoo1 248: 113-120. Wassarman PM (1987): The biology and chemistry of fertilization. Science 235:553-560. Xu KR, Greve T, Callesen H, Hyttel P (1987):Pregnancy resulting from cattle oocytes matured and fertilized in vitro. J Reprod Fertil 81501-504. Yanagimachi R (1969):In vitro acrosome reaction and capacitation of golden hamster spermatozoa by bovine follicular fluid and its fractions. J Exp Zoo1 170:269-280. Yanagimachi R (1981): Mechanisms of fertilization in mammals. In Mastoianni L, Biggers J (eds): “Fertilization and Embryonic Development in Vitro.” New York: Plenum Publishers, pp 81-182. Yanagimachi R, Kamiguchi Y, Sugawara S, Mikamo K (1983): Gametes and fertilization in the Chinese hamster. Gamete Res 8:97117.
Effect of Follicle Cells on the Acrosome Reaction, Fertilization, and Developmental Competence of Bovine Oocytes Matured In Vitro Y. FUKUI Department of Meat Animal Reproduction, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan The role of follicle cells in the ABSTRACT acrosome reaction of frozen-thawed bovine spermatozoa, in vitro fertilization, cleavage, and development in vitro was investigated. Cumulus-oocyte complexes were cocultured and matured in vitro with additional granulosa cells for 24 hr. Immediately before in vitro insemination, the oocytes were divided into three types with different follicle cells: denuded and corona- and cumulus-enclosed oocytes. The proportion of live, acrosome-reacted spermatozoa significantly increased at 3 and 6 hr after insemination in all types of oocytes. However, the mean proportion of live, acrosome-reacted spermatozoa that inseminated cumulus-enclosed oocytes at 6 hr after insemination was significantly higher than that of spermatozoa inseminating denuded oocytes (18.3% and 13.3%, respectively). The frequency of in vitro fertilization was significantly higher for cumulus-enclosed oocytes (65.4%) than for denuded and corona-enclosed oocytes (30.8% and 39.4%, respectively). Cumulus-enclosed oocytes when cocultured with oviduct epithelial cells also had significantly higher rates of cleavage (two- to eight-cell, 59.8%; eight-cell, 22.4%) and blastocyst formation (7.7%) than denuded and corona-enclosed oocytes. No eightcell embryos or more advanced stages of embryonic development were observed in either denuded or corona-enclosed oocytes without the coculture. The present results indicate that cumulus cells at fertilization play an important role in inducing the acrosome reaction and promoting a high fertilization rate, cleavage, and development into blastocysts in vitro.
Key Words: In vitro fertilization, Embryonic development, Cattle ~
Fukui and Sakuma, 1980; Critser et al., 1986b; Leibfried-Rutledge et al., 1987; Grad1 et al., 1988). In inducing not only nuclear maturation but also cytoplasmic maturation leading to preimplantation embryonic development, it has been clearly demonstrated that follicle cells, especially the cumulus cells surrounding immature oocytes, play a central role in developmental competence in sheep (Staigmiller and Moor, 1984) and cattle (Goto et al., 1988; Shioya et al., 1988; Sirard et al., 1988). However, the role of follicle cells in in vitro fertilization and subsequent oocyte development has not yet been clearly identified, especially in relation to the acrosome reaction of spermatozoa occurring during in vitro insemination. Although the acrosome reaction can occur independently of the eggs (Yanagimachi, 19811, it is probably the egg investments that induce the acrosome reaction in fertilizing spermatozoa (Florman and Storey, 1982; Crozet, 1984; Cummins and Yanagimachi, 1986; Uto et al., 19881. Cumulus cells andlor additional granulosa cells are needed to complete oocyte maturation (Critser et al., 1986a,b; Lu et al., 1987; Thibault et al., 1987; Xu et al., 1987; Fukui and Ono, 1988; Fukui et al., 19891, but it has been reported that cumulus cells are not essential for in vitro fertilization (Moore and Bedford, 1978). Recent studies on in vitro fertilization in pigs (Cheng, 19851, sheep (Fukui et al., 1988a,b), and cattle (Lu et al., 1987; Xu et al., 1987)have indicated that cumulus cells of in vitro-matured oocytes when denuded or partly removed by pipetting or by hyaluronidase treatment showed improved sperm penetration, but the effect has not been clarified, and the timing of the acrosome reaction of bovine spermatozoa in vitro associated with different somatic cells in follicles has not been investigated. The present study was conducted to clarify the exact role of follicle cells (corona radiata and cumulus
~~
INTRODUCTION Bovine oocytes matured and fertilized in vitro seem to be unable to acquire developmental competence unless cocultured with follicle cells in the presence of gonadotropins and estradiol (Leibfried and First, 1979;
@ 1990 WILEY-LISS, INC.
Received March 6,1989; accepted September 25, 1989. Address reprint requests to Dr. Yutaka Fukui, Department of Meat Animal Reproduction, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080, Japan.
IN VITRO FERTILIZATION OF BOVINE OOCYTES TABLE 1. Concentrations of Media Used for In
Vitro Fertilization Ingredient NaCl KC1
NaHCO, NaH,PO, 2H,O Na-Lactate" CaC1, MgCl, 6H,O Phenol red
Units mM mM mM mM mM mM mM w/ml mM mM mM
Capacitation Washing Fertilization (pH 7.4) (pH 7.4) (pH 7.8) 114.0
114.0
3.2 25.0 0.4
3.2
3.2
2.0
25.0
10.0
0.4 10.0
0.4 10.0
0.5
2.0 0.5 10.0 10.0
2.0 0.5
-
10.0
HEPES 5.0 Na-pyruvate 1.0 0.5 Glucose 12.5 5.0 Bovine serum albuminb mdml 6.0 3.0 "Sixty percent sodium salt. bFatty-acid-freeprepared from fraction V
114.0
10.0 10.0 0.5 5.0
6.0
cells) in the acrosome reaction of frozen-thawed bovine spermatozoa in vitro, in vitro fertilization, and subsequent developmental capacity in a coculture with or without bovine oviduct epithelial cells (BOEC) in bovine oocytes matured in vitro. MATERIALS AND METHODS Oocyte Maturation Ovaries were obtained from Holstein cows and heifers killed a t a local abattoir and were transported in a saline solution (0.9% NaCl) a t 30-35°C to the laboratory within 1hr. Cumulusoocyte complexes were collected from follicles 2-5 mm in diameter with an 18gauge needle attached to a 10 ml disposable syringe. Only oocytes with unexpanded cumulus oophorus and evenly granulated cytoplasm were cultured in 30 mm plastic dishes (Sterillin, Middlesex, England; 20-40 oocytes/dish) containing 2 ml tissue culture medium (TCM) 199 (Wittaker M.A. Bioproducts, Walkersville, MD, pH 7.4, Earle's salt with sodium bicarbonate and L-glutamine) supplemented with 15% (v/v) heat-inactivated (56"C, 30 min) estrous cow serum (ECS). ECS used in this study was obtained from cows at the time of artificial insemination during the "standing estrus" a t the University Farm and pooled as a stock to be used throughout the study. The medium was also supplemented with granulosa cells (5 x 10' cells/ml) collected from antral follicles of about 10 mm in diameter, dissected by the method of Moor and Trounson (1977), and washed (500g,5 min) three times with TCM 199 containing 5% (v/v) ECS and 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid (HEPES; Sigma Chemical Co., St. Louis, MO). The oocytes were statically cultured for 24 hr at 39°C in an atmosphere of 5% CO, in air and 95% humidity.
In Vitro Fertilization The media used for in vitro fertilization are shown in Table 1. Three modifications of Tyrode's medium
41
(TALP) as described by Bavister and Yanagimachi (1977) were prepared for sperm capacitation (capacitation medium), for washing oocytes (washing medium), and for a fertilization medium. Ca2+was not present in the capacitation medium. Four chemicals (HEPES, Napyruvate, glucose, and bovine serum albumin) were added immediately before use of the media. All media were used within 1 week and were stored at 4°C. Frozen straws (0.5 ml in size) of semen from a Japanese black bull were thawed at 35-37°C and were prepared for sperm capacitation. The thawed semen (0.2 ml) was placed under 1ml of capacitation medium in conical tubes for a swim-up procedure (Parrish et al., 198613; Lu et al., 1987). The top 0.8 ml of the medium was then collected after 1 hr of incubation a t 39°C and pooled. The medium containing spermatozoa was washed twice (500g, 10 min) with capacitation medium. The final pellet of semen was resuspended in the medium to the concentration of 50 x 10' spermatozoa/ ml. An equal volume of a 200 pg/ml heparin (Sigma Chemical Co.) solution was added to the semen suspension to yield spermatozoa and heparin concentrations of 25 x 10" cells/ml and 100 pg/ml, respectively. The heparin-treated spermatozoa were incubated for 15 min at 39°C in 5% COP in air (Lu et al., 1987). Oocytes cultured for 24 hr were washed three times with a washing medium (Table 1)by pipetting and divided into the following three types of oocytes (Fig. 1) having different kinds of follicle cells: 1) those completely devoid of follicle cells (denuded oocytes), 2) those surrounded by one to three layers of corona cells (corona-enclosed oocytes), and 3) those surrounded by expanding cumulus cells (cumulus-enclosed oocytes). In each group of oocytes, a 2 pl aliquot of the washing medium containing five oocytes was placed into 45 pl drops of fertilization medium (Table 1)under sterile oil (Nakarai Chemical Co., Tokyo, Japan). Three microliters of the heparin-treated semen suspension were then added to the oocytes to give a final concentration of 1.5 x 10' celldml. After in vitro insemination, oocytes and spermatozoa were incubated for 18-20 hr a t 39°C under 5% CO, in air. In experiment 1, live and acrosome-reacted spermatozoa inseminating the three different types of oocytes were examined. Following in vitro insemination (0hr), spermatozoa cocultured with the different oocytes in the droplets were prepared for smears at 3 hr intervals over a 24 hr period (0,3,6, 9, 12, 15, 18,21, and 24 hr) using the simplified triple-stain method (Kusunoki et al., 1984, 1985). Light brown postacrosomal regions with unstained acrosomal regions indicated spermatozoa that were alive and in which the acrosome reaction had taken place. The proportions of live, acrosome-reacted spermatozoa were calculated for the 100-200 spermatozoa per smear, and the results were compared over four repeated trials to determine the effect of follicle cells surrounding oocytes. For each test, oocytes were fixed with acetoalcohol (1:3)and stained with 1%
42
Y.FUKUI
Fig. 1. Three types of bovine oocytes used for in vitro fertilization following 24 hr in culture: A, denuded; B, corona-enclosed;C, cumulus-enclosed oocytes.
acetoorcein to observe sperm penetration and pronucleus formation. Experiment 2 was carried out to examine the effect of follicle cells on’in vitro fertilization in the three types of oocytes. A total of 808 oocytes were examined over seven trials for evidence of fertilization after fixation and staining as in experiment 1.The only oocytes considered as being normally fertilized were those having female and male pronuclei, with a residual sperm-tail visible in the cytoplasm. In experiment 3, to examine developmental capacity following in vitro fertilization, the three types of oocytes were separately cultured with or without BOEC. As was described previously (Fukui and Ono, 1988; Fukui et al., 19891, BOEC were collected by flushing the oviducts of cows and heifers with a newly formed corpus luteum with 10 ml Dulbecco’s phosphate-buffered saline containing 10% (v/v) ECS per oviduct. The flushed fluid was kept a t 39°C for 10 min, and supernatant solution was carefully removed. After resuspension with TCM 199 containing 10% (vlv) ECS and 35 ng/ml insulin (Sigma Chemical Co.), a 0.5 ml aliquot of the cell suspension was placed into four-well dishes (Nunclon, Intermed, Copenhagen, Denmark) without any enzyme treatments. After movement was apparent in the ciliated cells from oviducts and after monolayers (>50% confluent) had formed (within 48 hr), about 10 embryos were placed in each well containing BOEC or only medium and were cultured for 7 days at 39°C under 5% C02 in air. On the third day of culture, cleavage (two- to eight-cell) was examined,
and the numbers of morulae and expanded blastocysts were recorded after 7 days in culture. The mean proportions of live spermatozoa and live, acrosome-reacted spermatozoa over the four trials at the nine observation times a t 3 hr intervals (0-24 hr) were analyzed by Student’s t test (experiment 1).The frequencies (mean 2 S.E.M.) of fertilization for each type of oocytes (experiment 2) in the seven trials were compared by Duncan’s new multiple range test after angular transformation (Steel and Torrie, 1960). For examining cleavage and development (experiment 3),a factorial study (3 x 2: three types of oocytes, with or without BOEC) was designed. Proportions of eight-cell embryos, cleaved (two- to eight-cell) embryos, and developed embryos (morulae and blastocysts) were subjected to least-square methods for analysis of variance after angular transformation (Steel and Torrie, 1960). Duncan’s new multiple range test (Steel and Torrie, 1960) was also used to compare the cleavage and development rates.
RESULTS Experiment 1: Proportions of Acrosome-Reacted Spermatozoa Over Time During In Vitro Insemination With Oocytes Surrounded by Different Follicle Cells As is shown in Table 2, during the 24 hr observation period, the mean proportion of live spermatozoa decreased gradually without significant differences among the types of oocytes except between the groups
IN VITRO FERTILIZATION OF BOVINE OOCYTES
43
TABLE 2. Mean (2S.E.M.) Proportions of Live Spermatozoa and Acrosome-Reacted Spermatozoa During In Vitro Insemination With Bovine Oocytes Surrounded by Different Follicle Cells Times (hr) following insemination (c/o) Oocvtes
Spermatozoa
Denuded
Live Live and acrosomereacted Live
Coronaenclosed
Live and acrosomereacted Cumulus- Live enclosed Live and acrosomereacted
6 0 3 49.5 f 3.9 49.2 2 2.9 44.3 f 3.0 7.0 f 1.0 10.32 1.2 13.3t 0.7"
9 12 15 18 21 24 31.0 f 2.6 28.5 f 2.8 23.0 2 1.2 18.0 2 2.7 17.3 ? 0.6 13.0 f 1 .3 ~. 7.0 1.6 6.0 2 1.6 6.0 -c 0.6 4.0 f 0.4 2.3 0.3 2.0 f 0.0
55.3 f 3.1 50.0 2 1.2 50.8 f 3.1
39.5 f 3.1 33.5 f 3.1 26.8 2 1.2 24.6 f 2.7 16.8 t 1.8 15.8 +- 1.8
*
*
5.0 f 0.8 15.7 2 0.3 15.0f 1.5".b 9.3 2 1.3 51.5 f 2.3 45.9 2 2.2
49.0
* 3.3
5.5 f 0.9 13.8 2 0.9 18.3f l.gb
7.3 f 2.3
6.5 -t 0.8
4.5 2 0.8
2.5 f 0.6
2.5 ? 0.3
36.4 f 2.2 32.8 f 3.1 29.8 t- 2.7 24.9 t 2.1 18.8 f 2.1 15.8 +- 1.5 8.8-+ 1.8 ~
8.5 2 0.6
7.0 2 1.0 ~~
5.3 f 0.8 4.0f 0.9
2.850.5 ~
a,bFigureswith differentsuperscripts in each column are significantly different (P< 0.001).
of corona- and cumulus-enclosed oocytes at the 3 hr Experiment 3: Cleavage and Development of observation (P < 0.05).However, the spermatozoa inOocytes Surrounded by Different Follicle Cells seminating the denuded oocytes tended t o decline in and Cocultured With or Without Bovine Oviduct viability more rapidly than the other two groups. In the Epithelial Cells three types of oocytes, the mean proportion of live, acAnalysis of variance in a factorial design showed rosome-reacted spermatozoa significantly increased (P that the cell types surrounding oocytes were signifi< 0.01) at 3 hr after insemination, and proportions cantly (P < 0.001) different in regard to cleavage and were significantly higher ( P < 0.01) a t 6 hr than a t 0, 3, and 9 hr and other times of observations. It was development into blastocysts. The proportions of eightcell embryos (P < 0.025) and the total number of shown that the mean proportion of live, acrosome-recleaved embryos ( P < 0.05) had a significant correlaacted spermatozoa became significantly lower ( P < 0.01)9 hr after insemination in all groups. The effect of tion to the cell types and BOEC. It was clearly shown that BOEC was highly effective for cleavage (P < follicle cells on the mean proportion of live, acrosome0.001) and further development (P < 0.001)of in vitro reacted spermatozoa inseminating three different fertilized eggs. No eight-cell embryos or more advanced types of oocytes was similar except between the cumustages of embryonic development were observed in both lus-enclosed (18.38)and denuded (13.3%)oocytes at 6 denuded and corona-enclosed oocytes when they were hr, when a significant difference ( P < 0.01) was obcultured in the absence of BOEC (Table 4).Even with served. No other significant differences were found. the coculture of the two types of oocytes in BOEC, the The timing of sperm penetration and the formation rate of cleavage ( P < 0.01) and development into blasof female and male pronuclei in the three types of tocysts (P < 0.05)was significantly lower than that oocytes was similar; spermatozoa with a swollen head observed in cumulus-enclosed oocytes cocultured in the and a residual tail were observed at 3 and 6 hr, and the presence of BOEC. The highest rates of cleavage (eightformation of two pronuclei was observed a t 9 and 12 hr cell, 22.4%; total cleaved embryos, 59.8'%), developafter insemination. ment into blastocysts (7.7%), and combined development into morulae and blastocysts (10.4%) were Experiment 2 In Vitro Fertilization of Oocytes observed in cumulus-enclosed oocytes cocultured with Surrounded by Different Follicle Cells BOEC. A total of 808 oocytes in seven trials were examined DISCUSSION for fertilizability (Table 3). The cumulus-enclosed The importance of the cumulus cells surrounding imoocytes had the highest frequency (mean 2 S.E.M.) of 4.8%) with significant differ- mature oocytes for maturation competence has been fertilization (65.4% ences ( P < 0.001) observed when compared to both de- clearly recognized with regard to the subsequent fernuded (30.8& 5.9%) and corona-enclosed (39.4% 2 tilizability and developmental capacity in sheep (Staig6.3%) oocytes. Comparing the frequency of fertilization miller and Moor, 1984)and cattle (Critser et al., 1986b; between corona-enclosed oocytes and denuded oocytes, Thibault et al., 1987; Shioya et al., 1988).The presence the former was significantly (P < 0.05) higher than the of additional granulosa cells with cumulus-oocyte complexes is of further importance for developmental comlatter.
*
Y.FUKUI
44
TABLE 3. Effect of the Follicle Cells Surrounding Oocytes on In Vitro Fertilization of Bovine Oocytes
Matured In Vitro
No. Percent No. of of oocytes (mean S.E.M.) of
trials Oocytes 7 Denuded Corona-enclosed 7 Cumulus-enclosed 7 a vs. b P < 0.05.
*
examined
oocytes fertilized
280 274 254
30.8 2 5.9" 39.4 +- 6.3b 65.4 & 4.8'
a,b vs. c
P < 0.001.
petence in maturing ovine and bovine oocytes (Staigmiller and Moor, 1984; Critser et al., 1986a; Lu et al., 1987; Lutterbach et al., 1987; Thibault et al., 1987; XU et al., 1987; Fukui and Ono, 1988,1989). In the present study, all oocytes with compacted cumulus cells were cocultured with additional granulosa cells for in vitro maturation. After 24 hr in culture, the oocytes were then washed by pipetting to divide them into the three types of oocytes differing in their surrounding follicle cells (denuded and corona- and cumulus-enclosed oocytes). It has been established that the acrosome reaction in inseminating mammalian spermatozoa can occur through contact with egg investments, such as follicular fluid (Yanagimachi, 1969; Iwamatsu and Chang, 1969; Lenz et al., 1982), cumulus cells (Bavister, 1982; Yanagimachi et al., 1983; Cummins and Yanagimachi, 19861,and zonae pellucidae (Florman and Storey, 1982; Cross et al., 1988; Uto et al., 1988). Since Lenz et al. (1982, 1983) reported their findings that glycosaminoglycans, such as heparin, chondroitin sulfate A, and so on, in bovine follicular fluid enhance the incidence of capacitation and the acrosome reaction in bovine spermatozoa, follicular fluid (Fukui et al., 1983; Sanbuissho and Threlfall, 1989) or heparin (Parrish et al., 1985, 1988) has been utilized to increase the rate of sperm penetration and fertilization in vitro. The present results indicate two important points. First, cumulus cells are necessary a t the time of in vitro insemination to maximize the incidence of the acrosome reaction in frozen-thawed bovine spermatozoa and to enhance the frequencies of fertilization, cleavage, and development into blastocysts in vitro. Second, the times of the maximum incidence of live, acrosome-reacted spermatozoa were at 3 and 6 hr after insemination independently of the type of follicle cells. The significant increases in the frequency of live, acrosome-reacted spermatozoa at 3 and 6 hr after insemination coincided with sperm penetration into the oocytes. Concerning the importance of cumulus cells, Gwatkin et al. (1972, 1974) reported similar findings in hamster and mouse spermatozoa, demonstrating that in the capacitation process cumulus cells play a key role with spermatozoa interacting closely with the cu-
mulus oophorus. Recently, Lu et al. (1987) and Fukui et al. (1989) removed the cumulus cells surrounding bovine oocytes matured in vitro by either gentle pipetting or hyaluronidase treatment before in vitro insemination in order to increase sperm penetration. The present study, however, indicates that to increase sperm penetration the expanding cumulus cells should be attached onto the oocytes for maximizing the acrosome reaction of bovine spermatozoa and fertilization and subsequent development in vitro. Completely denuded oocytes, however, have also shown some ability to induce the acrosome reaction and to result in fertilizability, although the frequencies were less than those in cumulus-enclosed oocytes. These results suggest that the zonae pellucidae of matured bovine oocytes also have the ability to induce the acrosome reaction. Since the existence of the ability has been clearly established for the zonae pellucidae of mature, unfertilized hamster (Cherr et al., 1986; Uto et al., 1988), rabbit (O'Rand and Fisher, 1986), cattle (Florman and Bobcock, 19881, and human (Cross et al., 1988) eggs, it can be surmised that a zona pellucida component induces the acrosome reaction in bovine oocytes. A receptor, called ZP3, has been identified in solubilized mouse zonae pellucidae as an inducer of the acrosome reaction (Wassarman et al., 1986; cited by Wassarman, 1987). Florman and Babcock (1988) obtained a 30-35% rate of acrosome reaction in bovine spermatozoa following heparin treatment (10 p,g/ml) with soluble extracts of bovine zona pellucida. The rather lower frequencies (1318%)of live, acrosome-reacted spermatozoa observed in the present study, despite the higher dose of heparin (100 pg/ml), are probably due to the presence of glucose in the capacitation medium, considering the reports (Parrish et al., 1985, 1986a) showing that glucose inhibits or delays the effect of heparin on bovine sperm capacitation. To obtain satisfactory results in fertilization in vitro when glucose is present in the medium, as in this study, a higher dose of heparin likely is required. The present study has clearly demonstrated that coculture with BOEC is very effective for cleavage and development into blastocysts in vitro following fertilization. This has been confirmed in previous studies (Eyestone et al., 1987; Eyestone and First, 1988, 1989; Fukui and Ono, 1988, 1989; Lu et al., 19881, although other types of somatic cells, such as fibroblasts (Kuzan and Wright, 19821, trophoblastic vesicles (Camous et al., 1984), and cumulus cells (Kajihara et al., 1987; Goto et al., 19881, have also been employed successfully. The highest rate of development into blastocysts in the present study was 7.7%, but previous studies in which different bulls were used have shown higher results (12-17%) (Fukui and Ono, 1988, 1989). Further research on improved culture systems will be necessary to decrease the great loss of embryos occurring in the present study from 65% a t the time of fertilization to 22% at the eight-cell stage, and in other studies, such
IN VITRO FERTILIZATION OF BOVINE OOCYTES
45
TABLE 4. Effects of the Follicle Cells Surrounding Oocytes and Coculture With Bovine Oviduct Epithelial Cells (BOEC) on In Vitro Development of Bovine Oocytes Matured and Fertilized In Vitro
No. of embryos developed into (%) Oocytes Denuded
Coronaenclosed
No. of oocytes cultured with or withoutBOEC With 308
Two-cell 25
Four-cell 31
Without 241
9
0
With 316
24
57
Without 308
16
2
Eight-cell 9 (2.9)' 0 (O.OP 36 (11.4)b 0 (O.O)d
Cumulusenclosed
With 326
36
86
Without 251
39
39
73 (22.4)" 8 (3.2)'
Total cleaved (two- to eight-cell) 65 (21.1)" 9 (3.7)d 117 (37.0Ib 18 (~5.8)~ 195 (59.8)" 86 (34.3)b
Morulae 1 0
5 0
9 0
Blastocysts 3 (1.0)C 0 (O.OP 12 (3.8)B 0
(O.OP
25 (7.71-4 3 (1.2F
Morulae + blastocysts 4 (1.3)" 0 (O.OP 17 (5.4)" 0 (O.OP 34 (10.4)* 3 (1.2)C
"-d*A-DFigures with different superscripts in each column are different (a-d, P < 0.01; A-D, P < 0.05).
as those of Fayrer-Hosken et al. (1988) (penetration, 91%;cleavage, 38%;with the half of the embryos reaching the four- to eight-cell stage) and Eyestone and First (1989) (cleavage, 76%; development into morulae and blastocysts, 22%). In conclusion, for the induction of the acrosome reaction in frozen-thawed bovine spermatozoa and for improved in vitro fertilization and developmental capacity with in vitro-matured oocytes, eggs should be surrounded a t fertilization by expanding cumulus cells. The oocytes completely denuded of follicle cells have shown a slight ability to induce the acrosome reaction of spermatozoa and be fertilized in vitro, but the developmental capacity was limited even when cocultured with BOEC.
ACKNOWLEDGMENTS The author thanks Tomoo Sonoyama, Hitoshi Mochizuki, Yasushi Tamukai, and Hidenori Izumi for their assistance in collecting ovaries and oocytes. This work was partly supported by a Grant-in-Aid for Scientific Research (62560255)from the Ministry of Education, Science and Culture of Japan.
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