EFFECT OF OSMOLALITY ON FERTILIZATION OF MOUSE AND GOLDEN HAMSTER EGGS IN VITRO
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EFFECT OF OSMOLALITY ON FERTILIZATION OF
MOUSE AND GOLDEN HAMSTER EGGS IN VITRO
H. MIYAMOTO and M. C. CHANG
Worcester Foundation for Experimental Biology,
Shrewsbury, Massachusetts 01545, U.S.A.
{Received 26th May 1972)
Summary. Fertilization of mouse and hamster eggs in vitro in media
of various calculated osmolalities was investigated. A modified Krebs\x=req-\
Ringer bicarbonate solution with bovine serum albumin was used for
the mouse gametes and a modified Tyrode's solution with heated hamster
blood serum was used for the hamster gametes. Penetration of mouse
eggs could occur in media, the osmolality of which varied from 256 to
418 mosmolal, but the optimal osmolality was between 308 and 372
mosmolal. Penetration of hamster eggs could occur in media with
osmolality varying from 232 to 452 mosmolal but the optimal osmo-
lality was from 292 to 392 mosmolal. It seems that fertilization in vitro
could be achieved better in hypertonic than hypotonic solutions and
that hamster gametes could withstand the variation of osmolality
slightly better than mouse gametes.
INTRODUCTION
Mammalian spermatozoa maintain their motility for the longest period in
media of the same osmolality (cf. osmolarity—'Materials and Methods') as
semen or blood and, in
general, hypertonic solutions are less harmful than
hypotonie ones (Blackshaw & Emmens, 1951 ; White, 1962; Mann, 1964). The
fertilizing capacity of rabbit spermatozoa also appears to be better in hypertonic
than in hypotonie solutions (Chang & Thorsteinsson, 1958).
It has been reported that the normal osmolarity of tissue culture media and
blood serum is approximately 308 mosmol (Brinster, 1965b), that the maxi¬
mum number of two-cell mouse eggs
developed into blastocysts in a medium
of 276 mosmol (Brinster, 1965a), and that the optimum osmolarity for develop¬
ment of the two- to four-cell rabbit eggs was 270 mosmol (Naglee, Maurer &
Foote, 1969). Thus, it appears that a medium of lower osmolarity than blood
serum is more favourable for development of mammalian eggs in vitro.
In attempts to define suitable conditions for fertilization of mammalian eggs
in vitro, it has been shown that the pH value of the medium affects the fertiliza¬
tion rate of hamster eggs (Bavister, 1969) and mouse eggs (Iwamatsu & Chang,
1971). No attempt has been made to elucidate the osmolality requirements for
fertilization of mammalian eggs in vitro. The purpose of this experiment was to
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Miyamoto and M. C. Chang
determine the optimum osmolality of media for fertilization of mouse and
golden hamster eggs in vitro.
MATERIALS AND METHODS
A modified Krebs-Ringer bicarbonate solution containing crystalline bovine
serum albumin was used for the mouse gametes and a modified Tyrode's
solution containing heated hamster blood serum (at 56° C for 50 min) was used
for the hamster gametes (Table 1). The media used for mouse eggs were
prepared in two ways: (1) the concentration of NaCl was varied to obtain
eleven desired osmolalities (Table 2), and (2) the concentration of five salts
(Nos 1 to 5, Table 1) was varied by adding or subtracting each salt collectively
in the same proportion as they had been in the medium shown in Table 1 ; the
concentration of other components (Nos 8 to 12, Table 1) was kept the same
as in Table 1 (Table 3).
Table 1. Composition of media used for fertilization of mouse
and hamster eggs in vitro
Concentration (mil)
No. Component
For mouse
eggs For hamster eggs
1 NaCl 119-45 (varied) 136-85 (varied)
2 KCI 4-78 2-68
3 CaCl2 1-71 1-80
4 KH2P04 1-19
5 MgS04.7H20 1*19
6 MgCl2.6H20 0-49
7 NaH2P04.H20 0-36
8 NaHC03 25-07 11-90
9 Glucose 5-55 5-55
10 Sodium pyruvate 1-00
11 Sodium lactate 21-58
12 Crystalline bovine 4 mg/ml
serum albumin
For the hamster gametes, one portion of blood serum was added to two
portions of the medium. The concentration of NaCl was varied to obtain
desired osmolalities (Table 4). The calculated osmolalities (g mol. wt/1000 g
solvent) were checked with a freezing depression Osmometer (Advanced
Instruments, Inc.) for several of these media and were recorded. Osmolality is
used in preference to osmolarity (g mol. wt/1000 ml), since osmotic pressure in a
strict sense is a function of osmolality and the difference between these terms
can be significant at high concentrations. All the media used for each experi¬
ment were freshly prepared and filtered through millipore as an aseptic
precaution.
Mature female CD-I mice were subjected to the procedure of superovulation
and their eggs were recovered according to the procedures reported elsewhere
(Edwards & Gates, 1959; Miyamoto & Chang, 1972). Mature female golden
hamsters were also subjected to a procedure of superovulation (Yanagimachi
& Chang, 1964; Yanagimachi, 1969) and killed 14 to 16 hr after the injection
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of HCG for the recovery of eggs. Sperm suspension was prepared by mincing
cauda epididymidis of a mature male CD-I mouse or a hamster in a watch-glass
containing 1 ml medium of a particular osmolality. About 0-1 to 0-5 ml of the
sperm suspension was added to the egg clot under a light-weight paraffin oil in
a watch-glass. The paraffinoil was previously equilibrated with 5% C02 in
air in the presence of a small volume of saline. After adding another 0-2 to 0-3
ml of a medium of the same osmolality, the eggs and spermatozoa were
thoroughly mixed and then incubated at 37° C in an atmosphere of 5% C02
and air. After incubation for 5 to 7 hr, the eggs were mounted on a slide, fixed
in neutral formalin and stained for the assessment of fertilization (Miyamoto &
Chang, 1972). Those eggs with an enlarged sperm head(s) or with pronuclei
and a fertilizing sperm tail(s) in the vitellus were regarded as undergoing
fertilization.
RESULTS
When the NaCl concentration in media was varied to obtain different osmolali¬
ties, the highest percentages of mouse eggs undergoing fertilization were 86 to
91% in media of 308 to 349 mosmolal. The percentages decreased sharply
(41% and 31%) in media of 281 and 418 mosmolal, and none of 194 eggs was
penetrated in media of 212 and 441 mosmolal (Table 2). When the osmolality
of media was varied by adding or subtracting salts Nos 1 to 5 collectively in the
same proportion as in Table 1, the highest percentages of mouse eggs undergoing
fertilization were 85% and 91 % at 308 to 359 mosmolal. The fertilization rate
decreased to 53% and 66% in a medium of 282 and 385 mosmolal, respectively,
but a few eggs (2% and 22%) were still penetrated in media at 230 and 411
mosmolal. However, none of 171 eggs was penetrated in media of 204 and 437
mosmolal (Table 3). From the data presented in Tables 2 and 3, it seems that
Table 2. Effect of osmolality on the fertilization of mouse eggs in
vitro by varying the concentration of NaCl*
Osmolalities
of media Total Total % eggs
(mosmolal) no. of no. of eggs undergoing
eggs undergoing fertilization
Calculated Measured^ examined fertilization (range)
212 202 113 0 0(0)
235 231 84 3 4 (0 to 10)
258 250 94 20 21 (12 to 35)
281 275 102 42 41 (26 to 60)
308 299 89 81 91 (87 to 96)
349 340 93 80 86 (67 to 95)
359 349 100 77 77 (60 to 95)
372 365 85 68 80 (50 to 93)
395 376 103 73 71 (69 to 86)
418 388 78 24 31 (4 to 52)
441 426 81 0 0(0)
* All
the other components were the same as listed in Table 1. Only the
concentration of NaCl was varied to obtain desired osmolalities—NaCl levels
used to give calculated osmolalities of 212, 235, 258, 281, 308, 349, 359, 372,
395, 418 and 441 mosmolal were 45-67, 57-24, 68-70, 80-15, 93-85, 114-52,
119-45, 125-96, 137-41, 148-87 and 160-33 mM, respectively.
t Measured with an Osmometer (Advanced Instruments, Inc.).
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Table 3. Effect of osmolality on the fertilization of mouse eggs in
vitro by varying the concentration of five salts collectively*
Osmolalities of media Total Total % eggs
(mosmolal) no. of no. of eggs undergoing
eggs undergoing fertilization
Calculated Measured^ examined fertilization (range)
204 203 83 0 0(0)
230 224 91 2 2 (0 to 5)
256 247 106 33 31 (17 to 50)
282 270 110 58 53 (26 to 67)
308 300 103 94 91 (82 to 97)
333 321 95 87 92 (80 to 100)
359 343 106 90 85 (70 to 93)
385 362 96 63 66 (31 to 88)
411 389 100 22 22 (5 to 43)
437 408 0 0(0)
*
Media were prepared by adding or subtracting salts Nos 1 to 5 collectively
in the same proportion as listed in Table 1.
f Measured with an Osmometer (Advanced Instruments, Inc.).
Table 4. Effect of osmolality on the fertilization of hamster eggs in vitro
Osmolalities of media
(mosmolal)
Calculated Total Total % eggs
no of no. of eggs undergoing
Tyrode's Serum and Measured* eggs undergoing fertilization
solution Tyrode's examined fertilization (range)
solution^
(1:2)
130 192 122 112 0 0(0)
160 212 158 105 1 1 (0 to 6)
190 232 177 103 25 24 (0 to 40)
220 252 208 103 56 54 (30 to 71)
250 272 237 102 62 61 (39 to 73)
280 292 270 112 96 86 (80 to 95)
308 311 296 131 126 96 (89 to 100)
316 316 302 137 127 93 (80 to 100)
340 332 326 105 99 94 (80 to 100)
370 352 360 110 95 86 (47 to 100)
400 372 358 107 99 93 (73 to 100)
430 392 402 93 80 86 (70 to 100)
460 412 429 110 73 66 (50 to 85)
490 432 470 114 51 45 (31 to 57)
520 452 496 112 31 28 (0 to 57)
550 472 525 92 0 0(0)
All the other components were the same as listed in Table 1. Only the concentration
of NaCl was varied to obtain desired osmolalities—NaCl levels used to give calculated
osmolalities of 130, 160, 190, 220, 250, 280, 308, 316, 340, 370,400, 430, 460, 490, 520
and 550 mosmolal were 43-85, 58-85, 73-85, 88-85, 103-85, 118-85, 132-85, 136-85,
148-85, 163-85, 178-85, 193-85, 208-85, 223-85, 238-85 and 253-85 mM, respectively.
*
Measured with an Osmometer (Advanced Instruments, Inc.).
t Calculation was based on the measured hamster serum of 315 mosmolal.
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there was little difference whether the osmolality was varied by changing the
concentration of NaCl alone or by changing the concentration of five salts
collectively.
From Table 4, it can be seen that the highest percentages of hamster eggs
undergoing fertilization were 86% to 96% when the osmolality of the modified
Tyrode's solution containing serum was 292 to 392 mosmolal. The percentages
decreased to 61 % and 66% with media of 272 and 412 mosmolal, respectively,
but a few eggs (1% and 28%) were still penetrated in media of 212 and 452
mosmolal. None of 204 eggs, however, was penetrated in media of 192 or 472
mosmolal.
310 350 430 470
Calculated mosmolals
Text-fig. 1. Effect of osmolality on the fertilization of mouse and hamster eggs in vitro.
x, Mouse eggs (by varying five salts); ·, mouse eggs (by varying NaCl); O, hamster
eggs (by varying NaCl).
From these results (Text-fig. 1), it seems that there is a wide range of osmo¬
lality in which the capacitation of both mouse and hamster spermatozoa can be
induced and the fertilization of eggs in vitro can be achieved, but hamster
gametes can withstand a wider range of osmolality (232 to 452 mosmolal) than
the mouse gametes (256 to 418 mosmolal). It seems that a proper medium with
a similar, or
slightly higher, osmolality than blood serum is better for fertiliza¬
tion in vitro than a medium of lower osmolality.
DISCUSSION
Pursley & Herman (1950) reported that for the viability of bovine spermatozoa
stored at temperatures above freezing, the osmolarity of the suspending media
could vary from 235 to 360 mosmol. The maximal motility of bovine sperma¬
tozoa has been observed in a medium which is slightly hyperosmotic to
spermatozoa (Steinbach & Foote, 1967) while the fertilizing capacity of rabbit
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H.
spermatozoa is less affected in hypertonic than in hypotonie solution (Chang
& Thorsteinsson, 1958). It seems that spermatozoa could tolerate hypertonic
better than hypotonie solutions.
The motility and metabolic activity of bovine spermatozoa were best in a
medium of osmotic pressure equivalent to that of bovine seminal plasma with
an osmolarity of 287 mosmol (Rothschild & Barnes, 1954) or bovine follicular
fluids (Salisbury, 1962). The osmolarity of bovine uterine, oviducal and folli¬
cular fluid has been recorded as 353, 350 and 287 mosmol, respectively (Olds
& VanDemark, 1957). Since spermatozoa are capacitated in the female repro¬
ductive tract and eggs are fertilized in the oviduct, one might assume that the
capacitation of bovine spermatozoa and the fertilization of bovine eggs would
occur in media of rather widely ranging osmolality. However, the chemical
compositions of a given medium, as well as its osmolality, may have some
influence on the normal activity of spermatozoa and eggs.
Rabbit eggs can be fertilized in vitro in a Krebs-Ringer bicarbonate solution
and subcultured in 50% rabbit serum in saline (Chang, 1959). They can be
fertilized in rabbit tubai fluid (Suzuki & Mastroianni, 1965) or in a chemically
defined medium with a calculated osmolarity of 406 mosmol (Brackett &
Williams, 1968). As the osmolarity of tubai fluid from oestrous rabbits is very
close to that of serum (Bishop, 1956) and fertilization of rabbit eggs in vivo
occurs at an osmolarity of 310 mosmol (Maurer, Onuma & Foote, 1970), which
is similar to that of rabbit blood serum, one may also predict that rabbit eggs
could be fertilized in vivo in vitro over a wide range of osmolalities.
or
The optimum osmolarity for the growth of Hela cells and mouse fibroblast
was 277 mosmol (Eagle, 1956) and an optimum osmolarity of 274 mosmol
was better for the survival of lymphocytes in vitro (Trowell, 1963). Although the
osmolarity of tissue culture media and blood serum is approximately 308
mosmol (Brinster, 1965b), the cleavage of eight-cell mouse eggs continued even
when the osmolarity was reduced to 90 mosmol (Whitten, 1956). Two-cell
mouse eggs could develop to blastocysts in media with an osmolarity between
200 and 354 but the optimum osmolarity was 276 mosmol (Brinster, 1965a).
Whitten & Biggers (1968) obtained blastocysts by culturing one-cell mouse
eggs in a medium with an osmolarity of 256 mosmol, while the optimum
osmolarity for the development of two-cell rabbit eggs in culture was 270
mosmol (Naglee et al., 1969). From this account, it seems that the optimum
osmolality for the culture of mammalian eggs and other body cells is lower than
that of blood serum. For the capacitation of spermatozoa and fertilization of
eggs in vitro, however, the optimum osmolality of media is higher than that of
blood serum (Text-fig. 1).
The results of this study have shown that the penetration of hamster eggs
occurred over a wider range ofosmolalities than that of mouse eggs (Text-fig. 1).
This may be due to a species difference. Since two different media were used
for these two species, such difference may be due to the composition of the
medium, especially the quantity of serum present in the medium. Since the
optimal osmolality for fertilization of mouse and hamster eggs in vitro was found
to be within 299 to 365 mosmolal and 282 to 376 mosmolal, respectively, at
37° C, the optimal osmotic pressures are 7-6 to 9-3 and 7-2 to 9-5 atmospheres.
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ACKNOWLEDGMENTS
This work was supported by grants (GM 14370, HD 03472) from the U.S.P.H.S.
and a grant from the Ford Foundation. Sincere thanks are due to Mr R. W.
High, Renal Laboratory, St Vincent Hospital, Worcester, Massachusetts, for
his help in measuring osmolalities in his laboratory and to Dr . . Davis for
reading the manuscript.
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