In Vitro Fertilization of Rat and Mouse Eggs by Ejaculated Sperm and the Effect of Energy Sources on In Vitro Fertilization of Rat Eggs Y. TSUNODA AND M. C . CHANG Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01 545
ABSTRACT In vitro fertilization of rat and mouse eggs by ejaculated or epididymal spermatozoa in chemically defined media was studied. Penetration rates by ejaculated sperm was very low (0 to 8% ) in the rat, but 11 to 41 % of eggs were penetrated by ejaculated sperm in the mouse. The optimal concentration of sperm for in vitro fertilization appears to be similar whether ejaculated or epididymal sperm were used. The time of sperm penetration in the mouse eggs, however, was delayed for one-half to one hour when ejaculated sperm were used. The importance of sodium pyruvate, sodium lactate and glucose in the medium containing bovine serum albumin for in vitro fertilization of rat eggs was examined. When rat eggs in cumulus clot were exposed to epididymal sperm preincubated for five hours, the presence of sodium pyruvate, sodium lactate and glucose was found to play an important role. When exposed to non-incubated epididymal sperm sodium pyruvate could be omitted without much decline of the fertilization rate. When the denuded eggs were exposed to non-incubated sperm, penetration rates were very low (0 and 5% ) in the absence of pyruvate. It appears that although lactate, pyruvate and glucose are all important for in vitro fertilization of rat eggs, pyruvate can be supplied by the follicular cells surrounding the eggs, Successful in vitro fertilization of mammalian eggs was first conducted by incubating freshly ovulated eggs with capacit,ated sperm recovered from the uterus of mated animals for the rabbit (Thibault et al., '54; Chang, '59), the hamster (Yanagimachi and Chang, '63), the mouse (Whittingham, '68) and the rat (Miyamot0 and Chang, '73b). Since the discovery that hamster eggs can be fertilized i n vitro by epididymal sperm (Yanagimachi and Chang, '64) fertilization in vitro by epididymal sperm was usually conducted for the mouse (Iwamatsu and Chang ,'69, '70) and hamster (Barros and Austin, '67; Yanagimachi, '70) in the presence of follicular fluid or serum. Although in vitro fertilization of human eggs (Edwards et al., '69) and rabbit eggs (Ericsson, '69; Okinaga, '72; Brackett and Oliphant, '75) by ejaculated sperm has been reported, the in vitro fertilization of mouse and rat eggs by ejaculated sperm has not been studied. J. EXP. ZOOL., 193: 79-86.
It has been reported that capacitation of sperm and fertilization of eggs can be achieved in a chemically defined medium in the presence of crystalline bovine serum albumin for the hamster (Bavister, '69), mouse (Toyoda et al., '71; Miyamoto and Chang, '72), guinea pig (Yanagimachi, '72) and rat (Toyoda and Chang, '74). Although the eggs of hamsters (Bavister, '69) and mice (Toyoda et al., '71) can be fertilized in media without lactate, the presence of lactate, pyruvate, glucose and especially bovine serum albumin in the medium appears to be of importance for in vitro fertilization of mouse eggs (Miyamot0 and Chang, '73a). Whether or not lactate, pyruvate and glucose play a role for fertilization of rat eggs in vitro is not known. This paper reports the efficacy of fertilization in vitro by ejaculated or epididymal sperm of rats and mice and observations concerning the importance of lactate, pyruvate and glucose in the media 79
80
Y. TSUNODA AND M. C. CHANG
for in vitro fertilization of rat eggs with or without follicular cells. MATERIALS AND METHODS
A modified Krebs-Ringer bicarbonate solution containing crystalline bovine serum albumin ( 4 mg/ml) and antibiotics was prepared according to Toyoda et al. ('71) for the mouse gametes and according to Toyoda and Chang ('74) for the rat gametes in the study of ejaculated and epididymal sperm. In the study of the effect of lactate, pyruvate and glucose on fertilization of rat eggs by epididymal sperm the concentration of NaCl was increased whenever lactate was omitted from the original concentration of 94.6 mM to 116.15 mM for about half the number of eggs tested. Ejaculated sperm were obtained in the following manner. A mature rat or mouse was held by its tail and thrown forcefully on a hard surface. Its cervical spinal column was then disconnected by pressing the neck strenuously with a pair of scissors. The animal was then placed on its back and before death about 0.1 to 0.2 ml seminal fluid with spermatozoa will be emitted from its genital orifice. Semen could usually be obtained from seven of ten animals so killed. The seminal fluid coagulated soon after emission, and the coagulum was then picked up with a pair of forceps and suspended in 0.5 ml of medium in a Falcon plastic dish covered with oil (Suspension 1 ) . Within a few minutes spermatozoa began to spread out from the coagulum and the coagulum was discarded. By experience the desired sperm concentration can be obtained by removal of the coagulum earlier or later. Epididymal sperm were obtained by cutting the edge of a n excised cauda epididymis and a drop of sperm mass was picked up with a glass needle and similarly suspended in 0.5 ml of the medium covered with oil (Suspension 1). Depending on the sperm concentration, 0.03 to 0.05 ml of the sperm suspension was placed into 0.4 ml medium (Suspension 2 ) covered with oil. In order to decrease the sperm concentration this was repeated several times (Suspensions 3 to 5 ) . These sperm suspensions were kept in a n incubator at 37°C saturated with 5% CO, in air before
the introduction of eggs. In a n attempt to achieve capacitation of sperm, the suspensions in most cases were preincubated for one hour (mouse) or five hours (rat) before the introduction of eggs. For the induction of superovulation immature female rats (20 t o 28 days old) of CD strain were subcutaneously injected with 10 IU of PMS 40 to 50 hours before an intraperitoneal injection of 10 IU of HCG. Mature CD-1 female mice weighing 21 to 30 gm were injected intraperitoneally with 5 IU of PMS 44 to 50 hours before a n intraperitoneal injection of 5 IU HCG. They were killed 14 to 18 hours after injection of HCG. The eggs in cumulus clot were dissected out and introduced to the sperm suspension. To obtain denuded eggs, the eggs in cumulus clot were treated in 0.1 % hyaluronidase in the medium for 20 to 30 minutes. After the dispersal of follicular cells attached to the eggs, the eggs were washed three times in the medium without hyaluronidase and then introduced into sperm suspensions, The preparations were then incubated at 37°C in a n incubator saturated with 5% COz in air. At various times after incubation the eggs were removed, mounted on a slide in toto, stained with 0.25% Lacmoid (Chang, '52) and examined for evidence of sperm penetration and fertilization. Those eggs which had a n enlarged sperm head(s) or male pronuclei( ei) with fertilizing sperm tail( s ) in the vitellus were considered as undergoing fertilization. The number of sperm in Suspensions 1 and 2 of every experiment was counted by means of a haemocytometer but that of other suspensions was calculated. RESULTS
In vitro fertilization of eggs by ejaculated sperm When freshly ovulated eggs in cumulus clot were exposed to preincubated ejaculated sperm i n different concentrations, very low proportions of rat eggs ( 0 to 8% ) but relatively high proportions of mouse eggs ( 11 to 41 % ) were penetrated (table 1). The optimal concentration of ejaculated sperm for in vitro fertilization appeared to be 0.10 to 0.97 million sperm/ml for the rat and 0.09 to 0.59 million sperm/
81
I N VITRO FERTILIZATION OF RAT AND MOUSE EGGS TABLE 1
In vitro fertilization o f r a t a n d m o u s e eggs b y ejaculated s p e r m No. of eem uenetrated
Animal
Sperm suspension
Rat
1 2
3 4
Mouse
1 2 3 4 5
Sperm concentration (x 106 sperm/ml)
No. of eggs examined
Total (% 1
1.11-1.97 0.10-0.97 0.021-0.063 0.002-0.009
51 93 48 54
1(2) 7(8) 5x4)
1.0-3.2 0.09-0.59
47 165
5(11) 67(41)
0.009-0.054
61
15(25)
0.0008-0.0049
35 25
0.00013-0.00044
With sperm only in the perivitelline space
With enlarged sperm head
1 1 0 0
0 4 1
0 2 1
0
0
0 2 0 0 0
0 4 0 0 0
61 15 6 5
O(0)
6(17) 5 ~ 3 )
With pronuclei
5
' R a t eggs were examined four to seven hours after exposure to sperm preincubated for five hours; mouse eggs were examined three to eight hours after exposure to sperm preincubated for one hour; five to six tests were performed for each dilution.
TABLE 2
In vitro fertilization o f rat eggs by epididymal s p e r m Sperm suspension
1 2 3 4
No. of eggs with more than one sperm
No. of eggs penetrated
( x 108 sperm/ml)
No. of eggs examined
2.5-6.1 0.16-0.28 0.015-0.025 0.0014-0.0023
37 90 78 50
Sperm concentration
Total (%)
9(24) 72(80)
O(0) O(0)
With en1a r ged sperm head
3 8 0 0
With pronuclei
Total 2 (% )
Poly-, spermic (%)
6 64
2(22) 16(22)
12(17)
0
O(0) O(0)
O(0) O(0)
0
l(11)
1 Rat eggs were examined four to seven hours after exposure to sperm preincubated for five hours; four tests were performed for each dilution. 2 Eggs with more than one sperm i n the perivitelline space are included.
ml for the mouse; above or below such concentrations, the proportions of penetrated eggs appeared to be lower in both species. When rat epididymal sperm were preincubated for five hours and used for insemination, the optimal sperm concentration for penetration (80% ) appeared to be 0.16 to 0.28 million sperm/ml and no eggs were penetrated after the third dilution of sperm where the concentration was 0.015 to 0.025 millionn sperm/ml (table 2 ) . In the rat it seems that a sperm concentration below 0.02 and/or the handling of sperm either from a n ejaculate or from the epididymis during dilution decreased the fertilizing capacity of the spermatozoa. By contrast, in the mouse the optimal concentration of ejaculated sperm for i n vitro fertilization was 0.09 to 0.59 million sperm/ml, and the sperm suspension can be diluted five times to a concentration of 130 to 440 sperm/ml with-
out loss of fertilizing capacity (table 1 ) . It thus appears that the optimal sperm concentration for in vitro fertilization is slightly lower in the mouse than in the rat and that mouse sperm can withstand handling much better than rat sperm. Moreover, the difference in fertilizing ability of ejaculated and epididymal sperm appears to be greater in the rat (tables 1, 2) than in the mouse (table 3).
Time of penetration b y ejaculated and epididymal mouse sperm I n order to determine whether there is any difference in the time of sperm penetration, ejaculated or epididymal mouse sperm (concentration 0.009 to 0.59 million/ml) were preincubated for one hour and used for insemination. When the eggs were examined from 0.5 to 3-8 hours after incubation the proportions of eggs penetrated by ejaculated (6 to 41% ) were
Y. TSUNODA AND M. C. CHANG
a o o o a ooootc tc
lower than by epididymal sperm (28 to 83% ). Moreover, the time at which sperm penetration was noted was about one-half to one hour earlier with epididymal than with ejaculated sperm. Twenty-eight to 42% of eggs were penetrated by epididyma1 sperm but only 0 to 6% eggs were penetrated by ejaculated sperm examined one-half to one hour after exposure to sperm suspension (table 3). Earlier penetration of epididymal sperm is also indicated by the fact that when examined two and one-half hours after insemination none of 11 eggs penetrated by ejaculated sperm had pronuclei, but seven (23% ) of 30 eggs penetrated by epididymal sperm had pronuclei. The proportions of eggs penetrated by more than one spermatozoon were also higher in the eggs penetrated by epididymal than by ejaculated sperm (table 3 ) . It seems that in the mouse, epididymal sperm produce a higher frequency of fertilization in vitro than ejaculated sperm.
Effect o f energy sources on in vitro fertilization o f rat eggs In order to determine the requirement of different energy sources for in vitro fertilization of rat eggs, either lactate, pyruvate or glucose was eliminated from the medium which contained bovine serum albumin and essential salts (Toyoda and Chang, '74). Sodium pyruvate and glucose contribute little osmotic pressure while sodium lactate contributes 43.10 milliosmols i n the medium (Biggers et al., '71), hence, whenever lactate was omitted the concentration of NaCl was increased from the original concentration of 94.6 mM to 116.15 mM. About half of the eggs in each test were cultured in medium with increased NaC1, but no difference was observed between the results with the two media. The results of these tests were therefore pooled. From the results presented in table 4, it can be seen that when recently ovulated rat eggs in cumulus clot were introduced to epididymal sperm suspended in the medium without lactate, pyruvate and glucose no eggs were penetrated (Medium 1). When only pyruvate (Medium 2 ) or glucose (Medium 3 ) was introduced again practically no eggs were penetrated. When only lactate was introduced (Medium 4 ) 2 to 6% of the eggs
83
IN VITRO FERTILIZATION OF RAT AND MOUSE EGGS TABLE 4
Effect o f sodium lactate ( L ) , sodium pyruvate ( P ) and glucose ( G ) i n the medium f o r in vitro fertilization of rat eggs 1 Medium 2 Epididymal spermatozoa
No. of eggs examined
No. of egg; penetrated (%)
No. of eggs penetrated by more than one sperm Total 7 (%)
Polyspermic (%)
~~
Without L, PandG5
Non-incubated 3 Preincubated
77 69
With P
Non-incubated Preincubated
102 98
With G
Non-incubated Preincubated
121 103
0
With L
Non-incubated Preincubated
150 80
0 0
With L and P
Non-incubated Preincubated Non-incubated Preincubated
71 92 162 141
With L and G
Non-incubated Preincubated
110 57
With L, P and G
Non-incubated Preincubated
127 101
With P and G5
'Rat eggs with cumulus clot were exposed to sperm suspension of 0.06 to 1.09 million/ml and each test was conducted more than three times. 2 All the media contained bovine serum albumin. 3Rat eggs were exposed to epididymal sperm prepared 5 to 30 minutes previously and examined 8 to 24 hours after incubation. 4 Rat eggs were exposed to epididymal sperm preincubated for five hours and examined three to eight hours after incubation. 5 About half the number of eggs in each test were exposed to sperm suspended in the medium with higher concentration of NaCl to compensate osmolarity. 6 Eggs with perivitelline sperm are included. 7 Eggs with more than one sperm in the perivitelline space are included.
were penetrated. In the presence of lactate and pyruvate (Medium 5) practically no eggs were penetrated but in the presence of pyruvate and glucose (Medium 6 ) 6% of the eggs were penetrated by non-incubated and none by preincubated sperm. In the presence of lactate and glucose (Medium 7) 60% of the eggs were penetrated by non-incubated but only 18% of the eggs by preincubated sperm. By contrast, in the presence of lactate, pyruvate and glucose (Medium 8 ) , 81 to 83% of the eggs were penetrated and there was no difference between non-incubated and preincubated samples. Energy sources required for i n vitro fertilization of denuded rat eggs It is known that pyruvate can be produced by the follicular cells in vitro in the presence of glucose (Donahue and Stern, '68). The present study has shown that
the presence of lactate and glucose in the medium did facilitate sperm penetration of rat eggs still surrounded by follicular cells (table 4 ) . In order to determine the requirement of pyruvate for the penetration of denuded eggs, the follicular cells surrounding the eggs were removed by the treatment of hyaluronidase, and were then introduced into the sperm suspension containing only lactate and glucose or containing lactate, pyruvate and glucose. From the results presented in table 5, it is obvious that in the absence of pyruvate, even in the presence of lactate and glucose, none or only 5% denuded eggs were penetrated but in the presence of pyruvate, lactate, and glucose 66 to 70% eggs were penetrated. This shows the importance of pyruvate for the physiological integrity of rat gametes and/or for the maintenance of normal function of the eggs.
84
Y. TSUNODA AND M. C. CHANG TABLE 5
In vitro fertilization o f rat eggs after removal of folticulur cells
Medium
With
Time of examination (hr after exposure to sperm)
7%-10.0
No. of
With
24-271h
eggs examined
eggs penetrated 2 (% 1
No. of eggs undergoing fertilization (%)
Total 3 (% 1
Polyspermic (% 1
94
5(5)
2(2)
1(20)
1(20)
39
O(0)
O(0)
O(0)
O(0)
7%-10.0
68
45(66)
44(65)
13(29)
3(7)
24-27%
27
19(70)
19(70)
1(5)
8 L,P
and G
No. of eggs with more than one sperm
No. of
7 Land G
1
~
5
)
IDenuded rat eggs were exposed to epididymal sperm suspension of 0.16 to 0.68 million/ml prepared onehalf to one hour previously and examined three to eight hours later; each test was conducted more than three times. 2 Eggs with perivitelline sperm are included. 3 Eggs with more than one sperm in the perivitelline space are included.
DISCUSSION
The motility of sperm before and after incubation with eggs was examined for each experiment. It was noticed that the motility of mouse sperm, whether ejaculated or epididymal, was in general better than that of rat sperm. There was little or no difference between epididymal and ejaculated mouse sperm after several dilutions or after a long time of incubation, although motility usually decreased after a long incubation. The motility of ejaculated rat sperm, though not very different from that of epididymal sperm in Suspensions 1 and 2, was always poor after further dilution and after incubation for a long time. They were still motile in most cases however. In view of the very low proportions of rat eggs penetrated by ejaculated sperm (table l ) , no penetration by epididymal spermatozoa diluted three times (table 2) and high proportions of mouse eggs penetrated by ejaculated or epididymal sperm (tables 1, 3 ) it is obvious that the motility is closely correlated to fertilizing capacity. On the other hand, although there was little difference between the motility of ejaculated and epididymal mouse sperm, the proportions of eggs penetrated by epididymal sperm were distinctively higher than by ejaculated sperm (table 3 ) . The inferiority of ejaculated sperm for in vitro fertilization of mouse and especially of rat eggs, as revealed in the present study, can be interpreted as due to the aging of spermatozoa in the vas deferens because a certain proportion of sperm in
a n ejaculate must come from the vas deferens. In view of the presence of a decapacitation factor in the seminal plasma (Chang, '57) it is more likely that this decapacitation factor my have a certain inhibitory effect during in vitro capacitation of spermatozoa and penetration of eggs. The delay of penetration for one-half to one hour by ejaculated mouse sperm (table 3 ) also indicates that capacitation of sperm has been inhibited by the presence of seminal plasma. It has been reported that when epididyma1 sperm were used, the optimal sperm concentration for in vitro fertilization was about 0.16 to 0.51 million sperm/ml for the rat (Niwa and Chang, '74a) and 0.10 to 0.63 million/ml for the mouse (Tsunoda and Chang, '75). These results are confirmed in the present study: the optimal sperm concentration for in vitro fertilization was about 0.10 to 0.28 million/ml for the rat and about 0.09 to 0.59 for the mouse, and there was no great difference between ejaculated and epididymal sperm. During the study of the requirement of energy sources for in vitro fertilization of rat eggs, it was noticed that the motility of sperm, freshly prepared or after incubation was not very different in the presence of either lactate, pyruvate, or glucose. But a very low proportion of eggs ( 2 to 6% ) was penetrated in the presence of lactate alone (Medium 4, table 4 ) . In the absence of the energy sources (Medium l ) , the motility was very poor especially after incubation and no eggs were penetrated. Miyamoto and Chang ('73a) have re-
IN VITRO FERTILIZATION OF RAT AND MOUSE EGGS
85
ported that in the presence of glucose, the rat (Niwa and Chang, '74b), although bovine serum albumin was the most im- certain difference was observed in the portant component for in vitro fertilization mouse eggs (Cross and Brinster, '70; Miyaof mouse eggs but addition of lactate and mot0 and Chang, '72). pyruvate facilitated the process. In the ACKNOWLEDGMENTS present study of rat eggs, bovine serum albumin was always present and yet no This work was supported by a grant (HD eggs were penetrated even in the pres- 03472) from the U. S. Public Health Servence of glucose; only in the presence of ice and a grant from the Ford Foundation. lactate, were a few rat eggs ( 2 to 6% ) One of us (M. C. C.) is a recipient of a penetrated. Thus the different require- Research Career Award (HD 18,334) from ments for in vitro fertilization between the National Institute of Child Health and rats and mice is well demonstrated. Since Human Development. Sincere thanks are a few rat eggs ( 2 to 6% ) were penetrated due to Mrs. Rose Bartke for assistance. in the presence of only lactate (Medium 4) and none in the presence of only pyruLITERATURE CITED vate (Medium 2 ) and since 18 to 60% of Barros, C., and C. R. Austin 1967 In vitro ferthe eggs were penetrated in the presence tilization and sperm acrosome reaction in hamster. J. Exp. Zool., 166: 317-324. of lactate and glucose (Medium 7) and only 0 to 6% of the eggs in the presence Bavister, B. D. 1969 Environmental factors important for in vitro fertilization in the hamster. of pyruvate and glucose (Medium 6 ) , it J. Reprod. Fert., 18: 544-545. appears that lactate probably is more im- Biggers, J. D., W. K. Whitten and D. G. Whittingportant than pyruvate for fertilization of ham 1971 Methods in Mammalian Embryology. J. C. Daniel, ed. W. H. Freeman and rat eggs with follicular cells. After the reCompany, p. 101. moval of follicular cells the penetration B. G., and G. Oliphant 1975 Capacirates were much lower in the absence of Brackett, tation of rabbit spermatozoa in vitro. Biol. pyruvate when nonincubated sperm were Reprod., 12: 260-274. used (table 5). Thus the importance of Chang, M. C. 1952 Fertilizability of rabbit ova and the effects of temperature in vitro on their pyruvate for in vitro fertilization of rat subsequent fertilization and activation in vivo. eggs is clearly demonstrated. The present J. EXP. ZOO^., 121: 351-381. study also has revealed that in the absence 1957 A detrimental effect of rabbit of pyruvate only 5% of denuded rat eggs seminal plasma on the fertilizing capacity of (table 5) but 60% of rat eggs with follicusperm. Nature, 179: 258-259. lar cells (Medium 7) were penetrated. It 1959 Fertilization of rabbit ova in vitro. Nature, 184: 466-467. is obvious that the role played by the follicular cells is dependent on the composi- Chang, M. C., A. Hanada and D. M. Hunt 1971 Fertilization of denuded rabbit eggs in vitro by tion of the medium. It is of interest to sperm recovered from the uterus or vagina. mention here that in the presence of pyruNature, 232: 343-344. vate and glucose (Medium 6 ) or lactate Cross, P. C., and R. L. Brinster 1970 In vitro development of mouse oocytes. Biol. Reprod., and glucose (Medium 7), the penetra3: 298-307. tion by non-incubated sperm was signifiR. P., and S. Stem 1968 Follicular cantly higher (p < 0.01) than preincubated Donahue, cell support of oocyte maturation: production sperm. This may indicate that rat sperm of pyruvate in vitro. J. Reprod. Fert., 17: 395can be capacitated in these media during 398. incubation with eggs but prolonged incu- Edwards, R. G., B. D. Bavister and P. C. Steptoe 1969 Early stages of fertilization in vitro of bation may lead to their deterioration. human oocytes matured in vitro. Nature, 222 : Although cumulus components were con632-635. sidered to play a role for the capacitation Ericsson, R. J. 1969 Capacitation in vitro of of hamster sperm (Gwatkin et al., '72), rabbit sperm with mule eosinophils. Nature, 221 : 568-569. there was little or no difference in the penetration rates of eggs without follicu- Fraser, L. R., P. V. Dandekar and R. A. Vaidya 1971 In vitro fertilization of tubal rabbit ova lar cells in the rabbit (Chang et al., '71; partially or totally denuded of follicular cells. Fraser et al., '71), the hamster (Miyamoto Biol. Reprod., 4: 229-233. and Chang, '72), the mouse (Iwamatsu Fukuda, Y., 0. Okada and Y. Toyoda 1972 and Chang, '70; Fukuda et al., '72) and Studies on the fertilization of mouse eggs in
86
Y. TSUNODA AND M. C. CHANG
vitro. 111. Fertilization of denuded eggs by capacitated spermatozoa. Jap. J. Anim. Reprod., 18: '73-77. Gwatkin, R. B. L., 0. F. Anderson and C. F. Hutchinson 1972 Capacitation of hamster spermatozoa in vitro: the role of cumulus components. J. Reprod. Fert., 30: 389-394. Iwamatsu, T., and M. C. Chang 1969 In vitro fertilization of mouse eggs in the presence of bovine follicular fluid. Nature, 224: 919-920. 1970 Further investigation of capacitation of sperm and fertilization of mouse eggs in vitro. J. Exp. Zool., 175: 271-282. Miyamoto, H . , and M. C. Chang 1972 Fertilization in vitro of mouse and hamster eggs after the removal of follicular cells. J. Reprod. Fert., 30: 309-312. 1973a The importance of serum albumin and metabolic intermediates for capacitation of spermatozoa and fertilization of mouse eggs in vitro. J. Reprod. Fert., 32: 193205. 1973b In vitro fertilization of rat eggs. Nature, 241: 50-52. Niwa, K., and M. C. Chang 1974a Optimal sperm concentration and minimal number of spermatozoa for fertilization in vitro of rat eggs. J. Reprod. Fert., 40: 471-474. 197413 Various conditions for the fertilization of rat eggs in vitro. Biol. Reprod., 11: 463-469. Okinaga, Y. 1972 Fertilization in vitro using
rabbit tubal ova. Jap. J. Fert. Steril., 17: 107122. Thibault, C., L. Dauzier and S. Wintenberger 1954 Etude cytologique de la fecondation in vitro de l'oeuf de l a lapine. C. R. SOC. Biol., Paris, 148: 789-790. Toyoda, Y., and M. C. Chang 1974 Fertilization of rat eggs i n vitro by epididymal spermatozoa and the development of such eggs following transfer. J. Reprod. Fert., 36: 9-22. Toyoda, Y., M. Yokoyama and T. Hosi 1971 Studies on the fertilization of mouse eggs in vitro. I. In vitro fertilization of eggs by fresh epididymal sperm. Jap. J. Anim. Reprod., 16: 147-1 51. Tsunoda, Y., and M. C. Chang 1975 Penetration of mouse eggs in vitro: optimal sperm concentration and minimal number of spermatozoa. J. Reprod. Fert. In press. Whittingham, D. G. 1968 Fertilization of mouse eggs in vitro. Nature, 220: 592-593. Yanagimachi, R. 1970 In vitro capacitation of golden hamster spermatozoa by homologous and heterologous blood sera. Biol. Reprod., 3: 147-151. 1972 Fertilization of guinea pig eggs in vitro. Anat. Rec., 174: 9-20. Yanagimachi, R., and M. C. Chang 1963 Fertilization of hamster eggs in vitro. Nature, 200: 281-282. 1964 In vitro fertilization of hamster ova. J. Exp. Zool., 156: 361-376.
ABSTRACT In vitro fertilization of rat and mouse eggs by ejaculated or epididymal spermatozoa in chemically defined media was studied. Penetration rates by ejaculated sperm was very low (0 to 8% ) in the rat, but 11 to 41 % of eggs were penetrated by ejaculated sperm in the mouse. The optimal concentration of sperm for in vitro fertilization appears to be similar whether ejaculated or epididymal sperm were used. The time of sperm penetration in the mouse eggs, however, was delayed for one-half to one hour when ejaculated sperm were used. The importance of sodium pyruvate, sodium lactate and glucose in the medium containing bovine serum albumin for in vitro fertilization of rat eggs was examined. When rat eggs in cumulus clot were exposed to epididymal sperm preincubated for five hours, the presence of sodium pyruvate, sodium lactate and glucose was found to play an important role. When exposed to non-incubated epididymal sperm sodium pyruvate could be omitted without much decline of the fertilization rate. When the denuded eggs were exposed to non-incubated sperm, penetration rates were very low (0 and 5% ) in the absence of pyruvate. It appears that although lactate, pyruvate and glucose are all important for in vitro fertilization of rat eggs, pyruvate can be supplied by the follicular cells surrounding the eggs, Successful in vitro fertilization of mammalian eggs was first conducted by incubating freshly ovulated eggs with capacit,ated sperm recovered from the uterus of mated animals for the rabbit (Thibault et al., '54; Chang, '59), the hamster (Yanagimachi and Chang, '63), the mouse (Whittingham, '68) and the rat (Miyamot0 and Chang, '73b). Since the discovery that hamster eggs can be fertilized i n vitro by epididymal sperm (Yanagimachi and Chang, '64) fertilization in vitro by epididymal sperm was usually conducted for the mouse (Iwamatsu and Chang ,'69, '70) and hamster (Barros and Austin, '67; Yanagimachi, '70) in the presence of follicular fluid or serum. Although in vitro fertilization of human eggs (Edwards et al., '69) and rabbit eggs (Ericsson, '69; Okinaga, '72; Brackett and Oliphant, '75) by ejaculated sperm has been reported, the in vitro fertilization of mouse and rat eggs by ejaculated sperm has not been studied. J. EXP. ZOOL., 193: 79-86.
It has been reported that capacitation of sperm and fertilization of eggs can be achieved in a chemically defined medium in the presence of crystalline bovine serum albumin for the hamster (Bavister, '69), mouse (Toyoda et al., '71; Miyamoto and Chang, '72), guinea pig (Yanagimachi, '72) and rat (Toyoda and Chang, '74). Although the eggs of hamsters (Bavister, '69) and mice (Toyoda et al., '71) can be fertilized in media without lactate, the presence of lactate, pyruvate, glucose and especially bovine serum albumin in the medium appears to be of importance for in vitro fertilization of mouse eggs (Miyamot0 and Chang, '73a). Whether or not lactate, pyruvate and glucose play a role for fertilization of rat eggs in vitro is not known. This paper reports the efficacy of fertilization in vitro by ejaculated or epididymal sperm of rats and mice and observations concerning the importance of lactate, pyruvate and glucose in the media 79
80
Y. TSUNODA AND M. C. CHANG
for in vitro fertilization of rat eggs with or without follicular cells. MATERIALS AND METHODS
A modified Krebs-Ringer bicarbonate solution containing crystalline bovine serum albumin ( 4 mg/ml) and antibiotics was prepared according to Toyoda et al. ('71) for the mouse gametes and according to Toyoda and Chang ('74) for the rat gametes in the study of ejaculated and epididymal sperm. In the study of the effect of lactate, pyruvate and glucose on fertilization of rat eggs by epididymal sperm the concentration of NaCl was increased whenever lactate was omitted from the original concentration of 94.6 mM to 116.15 mM for about half the number of eggs tested. Ejaculated sperm were obtained in the following manner. A mature rat or mouse was held by its tail and thrown forcefully on a hard surface. Its cervical spinal column was then disconnected by pressing the neck strenuously with a pair of scissors. The animal was then placed on its back and before death about 0.1 to 0.2 ml seminal fluid with spermatozoa will be emitted from its genital orifice. Semen could usually be obtained from seven of ten animals so killed. The seminal fluid coagulated soon after emission, and the coagulum was then picked up with a pair of forceps and suspended in 0.5 ml of medium in a Falcon plastic dish covered with oil (Suspension 1 ) . Within a few minutes spermatozoa began to spread out from the coagulum and the coagulum was discarded. By experience the desired sperm concentration can be obtained by removal of the coagulum earlier or later. Epididymal sperm were obtained by cutting the edge of a n excised cauda epididymis and a drop of sperm mass was picked up with a glass needle and similarly suspended in 0.5 ml of the medium covered with oil (Suspension 1). Depending on the sperm concentration, 0.03 to 0.05 ml of the sperm suspension was placed into 0.4 ml medium (Suspension 2 ) covered with oil. In order to decrease the sperm concentration this was repeated several times (Suspensions 3 to 5 ) . These sperm suspensions were kept in a n incubator at 37°C saturated with 5% CO, in air before
the introduction of eggs. In a n attempt to achieve capacitation of sperm, the suspensions in most cases were preincubated for one hour (mouse) or five hours (rat) before the introduction of eggs. For the induction of superovulation immature female rats (20 t o 28 days old) of CD strain were subcutaneously injected with 10 IU of PMS 40 to 50 hours before an intraperitoneal injection of 10 IU of HCG. Mature CD-1 female mice weighing 21 to 30 gm were injected intraperitoneally with 5 IU of PMS 44 to 50 hours before a n intraperitoneal injection of 5 IU HCG. They were killed 14 to 18 hours after injection of HCG. The eggs in cumulus clot were dissected out and introduced to the sperm suspension. To obtain denuded eggs, the eggs in cumulus clot were treated in 0.1 % hyaluronidase in the medium for 20 to 30 minutes. After the dispersal of follicular cells attached to the eggs, the eggs were washed three times in the medium without hyaluronidase and then introduced into sperm suspensions, The preparations were then incubated at 37°C in a n incubator saturated with 5% COz in air. At various times after incubation the eggs were removed, mounted on a slide in toto, stained with 0.25% Lacmoid (Chang, '52) and examined for evidence of sperm penetration and fertilization. Those eggs which had a n enlarged sperm head(s) or male pronuclei( ei) with fertilizing sperm tail( s ) in the vitellus were considered as undergoing fertilization. The number of sperm in Suspensions 1 and 2 of every experiment was counted by means of a haemocytometer but that of other suspensions was calculated. RESULTS
In vitro fertilization of eggs by ejaculated sperm When freshly ovulated eggs in cumulus clot were exposed to preincubated ejaculated sperm i n different concentrations, very low proportions of rat eggs ( 0 to 8% ) but relatively high proportions of mouse eggs ( 11 to 41 % ) were penetrated (table 1). The optimal concentration of ejaculated sperm for in vitro fertilization appeared to be 0.10 to 0.97 million sperm/ml for the rat and 0.09 to 0.59 million sperm/
81
I N VITRO FERTILIZATION OF RAT AND MOUSE EGGS TABLE 1
In vitro fertilization o f r a t a n d m o u s e eggs b y ejaculated s p e r m No. of eem uenetrated
Animal
Sperm suspension
Rat
1 2
3 4
Mouse
1 2 3 4 5
Sperm concentration (x 106 sperm/ml)
No. of eggs examined
Total (% 1
1.11-1.97 0.10-0.97 0.021-0.063 0.002-0.009
51 93 48 54
1(2) 7(8) 5x4)
1.0-3.2 0.09-0.59
47 165
5(11) 67(41)
0.009-0.054
61
15(25)
0.0008-0.0049
35 25
0.00013-0.00044
With sperm only in the perivitelline space
With enlarged sperm head
1 1 0 0
0 4 1
0 2 1
0
0
0 2 0 0 0
0 4 0 0 0
61 15 6 5
O(0)
6(17) 5 ~ 3 )
With pronuclei
5
' R a t eggs were examined four to seven hours after exposure to sperm preincubated for five hours; mouse eggs were examined three to eight hours after exposure to sperm preincubated for one hour; five to six tests were performed for each dilution.
TABLE 2
In vitro fertilization o f rat eggs by epididymal s p e r m Sperm suspension
1 2 3 4
No. of eggs with more than one sperm
No. of eggs penetrated
( x 108 sperm/ml)
No. of eggs examined
2.5-6.1 0.16-0.28 0.015-0.025 0.0014-0.0023
37 90 78 50
Sperm concentration
Total (%)
9(24) 72(80)
O(0) O(0)
With en1a r ged sperm head
3 8 0 0
With pronuclei
Total 2 (% )
Poly-, spermic (%)
6 64
2(22) 16(22)
12(17)
0
O(0) O(0)
O(0) O(0)
0
l(11)
1 Rat eggs were examined four to seven hours after exposure to sperm preincubated for five hours; four tests were performed for each dilution. 2 Eggs with more than one sperm i n the perivitelline space are included.
ml for the mouse; above or below such concentrations, the proportions of penetrated eggs appeared to be lower in both species. When rat epididymal sperm were preincubated for five hours and used for insemination, the optimal sperm concentration for penetration (80% ) appeared to be 0.16 to 0.28 million sperm/ml and no eggs were penetrated after the third dilution of sperm where the concentration was 0.015 to 0.025 millionn sperm/ml (table 2 ) . In the rat it seems that a sperm concentration below 0.02 and/or the handling of sperm either from a n ejaculate or from the epididymis during dilution decreased the fertilizing capacity of the spermatozoa. By contrast, in the mouse the optimal concentration of ejaculated sperm for i n vitro fertilization was 0.09 to 0.59 million sperm/ml, and the sperm suspension can be diluted five times to a concentration of 130 to 440 sperm/ml with-
out loss of fertilizing capacity (table 1 ) . It thus appears that the optimal sperm concentration for in vitro fertilization is slightly lower in the mouse than in the rat and that mouse sperm can withstand handling much better than rat sperm. Moreover, the difference in fertilizing ability of ejaculated and epididymal sperm appears to be greater in the rat (tables 1, 2) than in the mouse (table 3).
Time of penetration b y ejaculated and epididymal mouse sperm I n order to determine whether there is any difference in the time of sperm penetration, ejaculated or epididymal mouse sperm (concentration 0.009 to 0.59 million/ml) were preincubated for one hour and used for insemination. When the eggs were examined from 0.5 to 3-8 hours after incubation the proportions of eggs penetrated by ejaculated (6 to 41% ) were
Y. TSUNODA AND M. C. CHANG
a o o o a ooootc tc
lower than by epididymal sperm (28 to 83% ). Moreover, the time at which sperm penetration was noted was about one-half to one hour earlier with epididymal than with ejaculated sperm. Twenty-eight to 42% of eggs were penetrated by epididyma1 sperm but only 0 to 6% eggs were penetrated by ejaculated sperm examined one-half to one hour after exposure to sperm suspension (table 3). Earlier penetration of epididymal sperm is also indicated by the fact that when examined two and one-half hours after insemination none of 11 eggs penetrated by ejaculated sperm had pronuclei, but seven (23% ) of 30 eggs penetrated by epididymal sperm had pronuclei. The proportions of eggs penetrated by more than one spermatozoon were also higher in the eggs penetrated by epididymal than by ejaculated sperm (table 3 ) . It seems that in the mouse, epididymal sperm produce a higher frequency of fertilization in vitro than ejaculated sperm.
Effect o f energy sources on in vitro fertilization o f rat eggs In order to determine the requirement of different energy sources for in vitro fertilization of rat eggs, either lactate, pyruvate or glucose was eliminated from the medium which contained bovine serum albumin and essential salts (Toyoda and Chang, '74). Sodium pyruvate and glucose contribute little osmotic pressure while sodium lactate contributes 43.10 milliosmols i n the medium (Biggers et al., '71), hence, whenever lactate was omitted the concentration of NaCl was increased from the original concentration of 94.6 mM to 116.15 mM. About half of the eggs in each test were cultured in medium with increased NaC1, but no difference was observed between the results with the two media. The results of these tests were therefore pooled. From the results presented in table 4, it can be seen that when recently ovulated rat eggs in cumulus clot were introduced to epididymal sperm suspended in the medium without lactate, pyruvate and glucose no eggs were penetrated (Medium 1). When only pyruvate (Medium 2 ) or glucose (Medium 3 ) was introduced again practically no eggs were penetrated. When only lactate was introduced (Medium 4 ) 2 to 6% of the eggs
83
IN VITRO FERTILIZATION OF RAT AND MOUSE EGGS TABLE 4
Effect o f sodium lactate ( L ) , sodium pyruvate ( P ) and glucose ( G ) i n the medium f o r in vitro fertilization of rat eggs 1 Medium 2 Epididymal spermatozoa
No. of eggs examined
No. of egg; penetrated (%)
No. of eggs penetrated by more than one sperm Total 7 (%)
Polyspermic (%)
~~
Without L, PandG5
Non-incubated 3 Preincubated
77 69
With P
Non-incubated Preincubated
102 98
With G
Non-incubated Preincubated
121 103
0
With L
Non-incubated Preincubated
150 80
0 0
With L and P
Non-incubated Preincubated Non-incubated Preincubated
71 92 162 141
With L and G
Non-incubated Preincubated
110 57
With L, P and G
Non-incubated Preincubated
127 101
With P and G5
'Rat eggs with cumulus clot were exposed to sperm suspension of 0.06 to 1.09 million/ml and each test was conducted more than three times. 2 All the media contained bovine serum albumin. 3Rat eggs were exposed to epididymal sperm prepared 5 to 30 minutes previously and examined 8 to 24 hours after incubation. 4 Rat eggs were exposed to epididymal sperm preincubated for five hours and examined three to eight hours after incubation. 5 About half the number of eggs in each test were exposed to sperm suspended in the medium with higher concentration of NaCl to compensate osmolarity. 6 Eggs with perivitelline sperm are included. 7 Eggs with more than one sperm in the perivitelline space are included.
were penetrated. In the presence of lactate and pyruvate (Medium 5) practically no eggs were penetrated but in the presence of pyruvate and glucose (Medium 6 ) 6% of the eggs were penetrated by non-incubated and none by preincubated sperm. In the presence of lactate and glucose (Medium 7) 60% of the eggs were penetrated by non-incubated but only 18% of the eggs by preincubated sperm. By contrast, in the presence of lactate, pyruvate and glucose (Medium 8 ) , 81 to 83% of the eggs were penetrated and there was no difference between non-incubated and preincubated samples. Energy sources required for i n vitro fertilization of denuded rat eggs It is known that pyruvate can be produced by the follicular cells in vitro in the presence of glucose (Donahue and Stern, '68). The present study has shown that
the presence of lactate and glucose in the medium did facilitate sperm penetration of rat eggs still surrounded by follicular cells (table 4 ) . In order to determine the requirement of pyruvate for the penetration of denuded eggs, the follicular cells surrounding the eggs were removed by the treatment of hyaluronidase, and were then introduced into the sperm suspension containing only lactate and glucose or containing lactate, pyruvate and glucose. From the results presented in table 5, it is obvious that in the absence of pyruvate, even in the presence of lactate and glucose, none or only 5% denuded eggs were penetrated but in the presence of pyruvate, lactate, and glucose 66 to 70% eggs were penetrated. This shows the importance of pyruvate for the physiological integrity of rat gametes and/or for the maintenance of normal function of the eggs.
84
Y. TSUNODA AND M. C. CHANG TABLE 5
In vitro fertilization o f rat eggs after removal of folticulur cells
Medium
With
Time of examination (hr after exposure to sperm)
7%-10.0
No. of
With
24-271h
eggs examined
eggs penetrated 2 (% 1
No. of eggs undergoing fertilization (%)
Total 3 (% 1
Polyspermic (% 1
94
5(5)
2(2)
1(20)
1(20)
39
O(0)
O(0)
O(0)
O(0)
7%-10.0
68
45(66)
44(65)
13(29)
3(7)
24-27%
27
19(70)
19(70)
1(5)
8 L,P
and G
No. of eggs with more than one sperm
No. of
7 Land G
1
~
5
)
IDenuded rat eggs were exposed to epididymal sperm suspension of 0.16 to 0.68 million/ml prepared onehalf to one hour previously and examined three to eight hours later; each test was conducted more than three times. 2 Eggs with perivitelline sperm are included. 3 Eggs with more than one sperm in the perivitelline space are included.
DISCUSSION
The motility of sperm before and after incubation with eggs was examined for each experiment. It was noticed that the motility of mouse sperm, whether ejaculated or epididymal, was in general better than that of rat sperm. There was little or no difference between epididymal and ejaculated mouse sperm after several dilutions or after a long time of incubation, although motility usually decreased after a long incubation. The motility of ejaculated rat sperm, though not very different from that of epididymal sperm in Suspensions 1 and 2, was always poor after further dilution and after incubation for a long time. They were still motile in most cases however. In view of the very low proportions of rat eggs penetrated by ejaculated sperm (table l ) , no penetration by epididymal spermatozoa diluted three times (table 2) and high proportions of mouse eggs penetrated by ejaculated or epididymal sperm (tables 1, 3 ) it is obvious that the motility is closely correlated to fertilizing capacity. On the other hand, although there was little difference between the motility of ejaculated and epididymal mouse sperm, the proportions of eggs penetrated by epididymal sperm were distinctively higher than by ejaculated sperm (table 3 ) . The inferiority of ejaculated sperm for in vitro fertilization of mouse and especially of rat eggs, as revealed in the present study, can be interpreted as due to the aging of spermatozoa in the vas deferens because a certain proportion of sperm in
a n ejaculate must come from the vas deferens. In view of the presence of a decapacitation factor in the seminal plasma (Chang, '57) it is more likely that this decapacitation factor my have a certain inhibitory effect during in vitro capacitation of spermatozoa and penetration of eggs. The delay of penetration for one-half to one hour by ejaculated mouse sperm (table 3 ) also indicates that capacitation of sperm has been inhibited by the presence of seminal plasma. It has been reported that when epididyma1 sperm were used, the optimal sperm concentration for in vitro fertilization was about 0.16 to 0.51 million sperm/ml for the rat (Niwa and Chang, '74a) and 0.10 to 0.63 million/ml for the mouse (Tsunoda and Chang, '75). These results are confirmed in the present study: the optimal sperm concentration for in vitro fertilization was about 0.10 to 0.28 million/ml for the rat and about 0.09 to 0.59 for the mouse, and there was no great difference between ejaculated and epididymal sperm. During the study of the requirement of energy sources for in vitro fertilization of rat eggs, it was noticed that the motility of sperm, freshly prepared or after incubation was not very different in the presence of either lactate, pyruvate, or glucose. But a very low proportion of eggs ( 2 to 6% ) was penetrated in the presence of lactate alone (Medium 4, table 4 ) . In the absence of the energy sources (Medium l ) , the motility was very poor especially after incubation and no eggs were penetrated. Miyamoto and Chang ('73a) have re-
IN VITRO FERTILIZATION OF RAT AND MOUSE EGGS
85
ported that in the presence of glucose, the rat (Niwa and Chang, '74b), although bovine serum albumin was the most im- certain difference was observed in the portant component for in vitro fertilization mouse eggs (Cross and Brinster, '70; Miyaof mouse eggs but addition of lactate and mot0 and Chang, '72). pyruvate facilitated the process. In the ACKNOWLEDGMENTS present study of rat eggs, bovine serum albumin was always present and yet no This work was supported by a grant (HD eggs were penetrated even in the pres- 03472) from the U. S. Public Health Servence of glucose; only in the presence of ice and a grant from the Ford Foundation. lactate, were a few rat eggs ( 2 to 6% ) One of us (M. C. C.) is a recipient of a penetrated. Thus the different require- Research Career Award (HD 18,334) from ments for in vitro fertilization between the National Institute of Child Health and rats and mice is well demonstrated. Since Human Development. Sincere thanks are a few rat eggs ( 2 to 6% ) were penetrated due to Mrs. Rose Bartke for assistance. in the presence of only lactate (Medium 4) and none in the presence of only pyruLITERATURE CITED vate (Medium 2 ) and since 18 to 60% of Barros, C., and C. R. Austin 1967 In vitro ferthe eggs were penetrated in the presence tilization and sperm acrosome reaction in hamster. J. Exp. Zool., 166: 317-324. of lactate and glucose (Medium 7) and only 0 to 6% of the eggs in the presence Bavister, B. D. 1969 Environmental factors important for in vitro fertilization in the hamster. of pyruvate and glucose (Medium 6 ) , it J. Reprod. Fert., 18: 544-545. appears that lactate probably is more im- Biggers, J. D., W. K. Whitten and D. G. Whittingportant than pyruvate for fertilization of ham 1971 Methods in Mammalian Embryology. J. C. Daniel, ed. W. H. Freeman and rat eggs with follicular cells. After the reCompany, p. 101. moval of follicular cells the penetration B. G., and G. Oliphant 1975 Capacirates were much lower in the absence of Brackett, tation of rabbit spermatozoa in vitro. Biol. pyruvate when nonincubated sperm were Reprod., 12: 260-274. used (table 5). Thus the importance of Chang, M. C. 1952 Fertilizability of rabbit ova and the effects of temperature in vitro on their pyruvate for in vitro fertilization of rat subsequent fertilization and activation in vivo. eggs is clearly demonstrated. The present J. EXP. ZOO^., 121: 351-381. study also has revealed that in the absence 1957 A detrimental effect of rabbit of pyruvate only 5% of denuded rat eggs seminal plasma on the fertilizing capacity of (table 5) but 60% of rat eggs with follicusperm. Nature, 179: 258-259. lar cells (Medium 7) were penetrated. It 1959 Fertilization of rabbit ova in vitro. Nature, 184: 466-467. is obvious that the role played by the follicular cells is dependent on the composi- Chang, M. C., A. Hanada and D. M. Hunt 1971 Fertilization of denuded rabbit eggs in vitro by tion of the medium. It is of interest to sperm recovered from the uterus or vagina. mention here that in the presence of pyruNature, 232: 343-344. vate and glucose (Medium 6 ) or lactate Cross, P. C., and R. L. Brinster 1970 In vitro development of mouse oocytes. Biol. Reprod., and glucose (Medium 7), the penetra3: 298-307. tion by non-incubated sperm was signifiR. P., and S. Stem 1968 Follicular cantly higher (p < 0.01) than preincubated Donahue, cell support of oocyte maturation: production sperm. This may indicate that rat sperm of pyruvate in vitro. J. Reprod. Fert., 17: 395can be capacitated in these media during 398. incubation with eggs but prolonged incu- Edwards, R. G., B. D. Bavister and P. C. Steptoe 1969 Early stages of fertilization in vitro of bation may lead to their deterioration. human oocytes matured in vitro. Nature, 222 : Although cumulus components were con632-635. sidered to play a role for the capacitation Ericsson, R. J. 1969 Capacitation in vitro of of hamster sperm (Gwatkin et al., '72), rabbit sperm with mule eosinophils. Nature, 221 : 568-569. there was little or no difference in the penetration rates of eggs without follicu- Fraser, L. R., P. V. Dandekar and R. A. Vaidya 1971 In vitro fertilization of tubal rabbit ova lar cells in the rabbit (Chang et al., '71; partially or totally denuded of follicular cells. Fraser et al., '71), the hamster (Miyamoto Biol. Reprod., 4: 229-233. and Chang, '72), the mouse (Iwamatsu Fukuda, Y., 0. Okada and Y. Toyoda 1972 and Chang, '70; Fukuda et al., '72) and Studies on the fertilization of mouse eggs in
86
Y. TSUNODA AND M. C. CHANG
vitro. 111. Fertilization of denuded eggs by capacitated spermatozoa. Jap. J. Anim. Reprod., 18: '73-77. Gwatkin, R. B. L., 0. F. Anderson and C. F. Hutchinson 1972 Capacitation of hamster spermatozoa in vitro: the role of cumulus components. J. Reprod. Fert., 30: 389-394. Iwamatsu, T., and M. C. Chang 1969 In vitro fertilization of mouse eggs in the presence of bovine follicular fluid. Nature, 224: 919-920. 1970 Further investigation of capacitation of sperm and fertilization of mouse eggs in vitro. J. Exp. Zool., 175: 271-282. Miyamoto, H . , and M. C. Chang 1972 Fertilization in vitro of mouse and hamster eggs after the removal of follicular cells. J. Reprod. Fert., 30: 309-312. 1973a The importance of serum albumin and metabolic intermediates for capacitation of spermatozoa and fertilization of mouse eggs in vitro. J. Reprod. Fert., 32: 193205. 1973b In vitro fertilization of rat eggs. Nature, 241: 50-52. Niwa, K., and M. C. Chang 1974a Optimal sperm concentration and minimal number of spermatozoa for fertilization in vitro of rat eggs. J. Reprod. Fert., 40: 471-474. 197413 Various conditions for the fertilization of rat eggs in vitro. Biol. Reprod., 11: 463-469. Okinaga, Y. 1972 Fertilization in vitro using
rabbit tubal ova. Jap. J. Fert. Steril., 17: 107122. Thibault, C., L. Dauzier and S. Wintenberger 1954 Etude cytologique de la fecondation in vitro de l'oeuf de l a lapine. C. R. SOC. Biol., Paris, 148: 789-790. Toyoda, Y., and M. C. Chang 1974 Fertilization of rat eggs i n vitro by epididymal spermatozoa and the development of such eggs following transfer. J. Reprod. Fert., 36: 9-22. Toyoda, Y., M. Yokoyama and T. Hosi 1971 Studies on the fertilization of mouse eggs in vitro. I. In vitro fertilization of eggs by fresh epididymal sperm. Jap. J. Anim. Reprod., 16: 147-1 51. Tsunoda, Y., and M. C. Chang 1975 Penetration of mouse eggs in vitro: optimal sperm concentration and minimal number of spermatozoa. J. Reprod. Fert. In press. Whittingham, D. G. 1968 Fertilization of mouse eggs in vitro. Nature, 220: 592-593. Yanagimachi, R. 1970 In vitro capacitation of golden hamster spermatozoa by homologous and heterologous blood sera. Biol. Reprod., 3: 147-151. 1972 Fertilization of guinea pig eggs in vitro. Anat. Rec., 174: 9-20. Yanagimachi, R., and M. C. Chang 1963 Fertilization of hamster eggs in vitro. Nature, 200: 281-282. 1964 In vitro fertilization of hamster ova. J. Exp. Zool., 156: 361-376.
