/. Embryol. exp. Morph. Vol. 59, pp. 49-58, 1980
Printed in Great Britain © Company of Biologists Limited 1980
49
Sterility in mutant (tLx/tLy) male mice
III. In vitro fertilization
By JAMES McGRATH1 AND NINA HILLMAN1
From the Department of Biology, Temple University, Philadelphia
SUMMARY
Mice which are heterozygous for two complementary lethal t mutations (>6/'w32) exhibit
complete male sterility. Experiments have been conducted to determine if spermatozoa from
these heterozygous males can fertilize oviducal ova in vitro. The experiments have been
designed so that specific barriers which are present during in vivo fertilization can be sequentially
removed and the fertilizing ability of the spermatozoa tested after each barrier is eliminated.
Our results show that spermatozoa from tG/tw32 males are unable to effect fertilization even
after all of the barriers normally imposed by the female reproductive tract have been removed.
INTRODUCTION
Male mice heterozygous for two lethal but complementary t mutations,
t /t , are sterile while females are fertile. Previous investigators have noted
that intercomplement males exhibit normal mating behavior and produce near
normal numbers of spermatozoa (Bryson, 1944; Braden & GluecksohnWaelsch, 1958; Bennett & Dunn, 1967). Although the intercomplement males
contain a high percentage of morphologically abnormal spermatozoa (Bryson,
1944; Rajasekarasetty, 1951; Braden & Gluecksohn-Waelsch, 1958; Dooher &
Bennett, 1977; Nadijcka & Hillman, 1980), the number of aberrant gametes
cannot be correlated with the sterility of these animals (Rajasekarasetty, 1951;
Braden & Gluecksohn-Waelsch, 1958; Nadijcka & Hillman, 1980).
Previous reports have also noted that spermatozoa obtained from tLx/tLv
males exhibit a reduced level of motility when compared with corresponding
gametes from fertile controls (Bryson, 1944; Bennett & Dunn, 1967; McGrath
& Hillman, unreported observations). Unlike control spermatozoa which
exhibit forward motility, the mutant spermatozoa remain in place and 'twitch'.
Braden & Gluecksohn-Waelsch (1958) proposed that sterility in tLx/tLv males
results from the inability of the mutant spermatozoa to traverse the uterotubal
junction and reach the site of fertilization. Although dissimilar findings were
reported by Bennett & Dunn (1967), Olds (1970) confirmed the observation
Lx Lv
1
Authors' address: Department of Biology, Temple University, Philadelphia, Pennsylvania
19122, U.S.A.
50
J. McGRATH AND N. HILLMAN
that virtually no spermatozoa from tLx/tLv males are able to enter the female
oviduct. Moreover, Olds (1971) found that the failure of spermatozoa to reach
the site of fertilization is not the sole cause of intercomplement male sterility.
She injected spermatozoa from twl8/tw32 males into the ovarian bursae following
ovulation. Despite the close proximity of the gametes, mutant spermatozoa were
still unable to effect fertilization. From this study, however, it is not possible to
determine the factor(s) impeding fertilization. For example, it cannot be determined if the spermatozoa are unable to become capacitated, to traverse the
cumulus cells, to undergo the acrosome reaction, to penetrate the zona pellucida,
or to fuse with the vitelline membrane.
The limitations imposed by these in vivo studies can be circumvented by
studying the ability of spermatozoa from intercomplement males to fertilize
oviducal ova in vitro. We have used in vitro techniques to determine if the spermatozoa obtained from tLx/tLv (t*/tw32) males can, first, disperse the cumulus cells;
second, undergo the acrosome reaction; and third, fuse with the ovum. This
report presents the results of these studies.
MATERIALS AND METHODS
Both the balanced lethal T/t* and T/twZ2 mouse stocks were maintained by
brother-sister matings. t6/tw32 males were obtained from reciprocal crosses of
r/f6 x T/tw32 and were distinguished from their littermates by the presence of a
normal tail. Sterility was determined by mating t6/tw32 males to BALB/c females
for the duration often mating units (Bennett & Dunn, 1967). Using this mating
regimen, none of the t^/tw32 males employed in this study sired offspring.
Spermatozoa were obtained from both the cauda epididymides and vasa
deferentia of mutant (t6/twS2) and control ( + / + ; Swiss Albino) males. Spermatozoa from these regions in wild-type mice are referred to as mature spermatozoa
since they are capable of fertilizing ova either in vivo or in vitro (Bedford, 1966;
Brackett, Hall & Oh, 1978). Immature spermatozoa were obtained from the
caput epididymides of the control males. The cauda epididymides and vasa
deferentia from each male were placed together into 1 ml of modified Tyrode's
medium (Fraser & Drury, 1975). The control cauda epididymides and vasa
deferentia were gently teased to allow the spermatozoa to disperse. Since their
motility is reduced, spermatozoa from the mutant animals, and from the wildtype caput epididymides, were obtained by gently applying pressure to the
ducts.
Spermatozoan concentrations were calculated from hemocytometer counts of
aliquots of individual suspensions. Any spermatozoon evidencing spontaneous
movement was scored as motile. The percentage of immotile spermatozoa in each
suspension was determined, and the number of gametes added to the insemination dishes was adjusted to give equivalent numbers of motile spermatozoa.
Final concentrations ranged from 1-2 to 8-0 xlO 5 spermatozoa for mature
In vitro fertilization in mutant (tLx/tLy) male mice
6
51
gametes from + / + males, and from 0-45 to 4-2 x 10 spermatozoa for gametes
from either t6/twS2 males or the caput epididymides of the wild-type males.
Cumulus cell-surrounded mouse ova were obtained from F x hybrid females
(T/t" x C57BL/6J). These ova were used in fertilization studies requiring either
cumulus cell-surrounded or zona pellucida-surrounded ova (Parkening &
Chang, 1976). The hybrid females were superovulated with an intraperitoneal
injection of pregnant mare serum gonadotrophin (PMSG; 5 IU), followed 48 h
later with a second injection of human chorionic gonadotrophin (HCG; 5 IU),
(Edwards & Gates, 1959).
In contrast with the cumulus cell-surrounded and zona-surrounded ova,
denuded ova are readily fertilized in vitro regardless of their genotype (Wolf,
Inoue & Stark, 1976). Therefore, for those studies which required removal of
the zona pellucida, ova were obtained from outbred Swiss Albino females.
These females were superovulated with 10 IU PMSG and 10 IU HCG. All ova
were obtained (13-14 h post HCG injection) by puncturing the distended
ampullae of the oviducts with a pair of watchmaker's forceps. Cumulus cells
were removed from the ova by means of a brief incubation (3-5 min) in fertilization medium containing 300 IU hyaluronidase/ml (Cross & Brinster, 1973).
Zonae pellucidae were mechanically removed with a small bore glass pipette
(Wolf etal., 1976).
Following hyaluronidase treatment and zona removal, the ova were washed
through four drops of fertilization medium. These ova, as well as cumulus cellsurrounded ova, were co-incubated with the spermatozoa in 0-2 ml drops of
modified Tyrode's medium (Fraser & Drury, 1975) under silicone oil (DowCorning 200 Fluid; 50 cs viscosity). The silicone oil had been previously equilibrated with fertilization medium lacking bovine serum albumin (BSA). The
gametes were incubated for 6 h at 37 °C under an atmosphere of 5 % O2, 5 %
CO2, and 90 % N 2 . At the completion of the incubation period, the ova were
removed, washed through four drops of modified Tyrode's medium (4 mg/ml
BSA) and reincubated. Four hours later, the ova were fixed (2-5% glutaraldehyde followed by overnight fixation in cold neutral buffered formalin),
stained with aceto-lacmoid (Toyoda & Chang, 1974) and examined with phase
optics. Ova were scored as fertilized if they contained at least two pronuclei and
a spermatozoan tail (Wolf et aL, 1976).
Quantitative estimates of the number of spermatozoa from t6ftwS2 males
bound to fertilized and unfertilized eggs were performed as follows: zona-less ova
were obtained and divided into two groups. The first group was incubated for
2 h without spermatozoa. The second group was incubated for 2 h with mature
wild-type spermatozoa which had been previously incubated for 2 h in fertilization medium. At the end of this co-incubation period the fertilized ova were
removed and thoroughly washed to remove any bound spermatozoa (the absence
of spermatozoa was ascertained by a microscopic examination of a sample of the
ova). The unfertilized (group 1) and fertilized (group 2) zona-less ova were then
52
J. McGRATH AND N. HILLMAN
Table 1. In vitro fertilization of cumulus cell- and
zonae pellucidae-surrounded ova
Male genotype z6//'"32
Experiment 1
2
3
4
Total ova (%)
Male genotype + / +
No.
fertilized
No.
unfertilized
No.
fertilized
No.
unfertilized
0
0
0
0
0(0%)
39
42
26
12
119(100%)
6
20
37
28
91(93%)
0
0
4
3
7(7%)
Table 2. In vitro fertilization of zonae pellucidae-surrounded ova
Male genotype t6/tw32
Male genotype + / +
No.
fertilized
No.
unfertilized
No.
fertilized
Experiment 1
2
3
4
0
0
0
0
65
97
40
13
22
11
12
27
Total ova (%)
0(0%)
215(100%)
72(78%)
No.
unfertilized
5
3
7
5
20(22%)
separately incubated for 4 h with spermatozoa from a t6/tw32 male. Both groups
of ova were then combined (to insure identical manipulations), washed through
four drops of fertilization medium, fixed and then stained for light microscopic
analyses. Ova were classified as fertilized and unfertilized, and the number of
ova which had bound spermatozoa plus the number of bound spermatozoa/egg
were determined for each group.
Electron microscopic studies were performed on ova fixed in either 2-5 %
glutaraldehyde or picric acid-formaldehyde (Stefanini, DeMartino & Zamboni,.
1967). Ova were postfixed with 1 % osmium tetroxide, dehydrated through an
ethanol series, and embedded in Epon 812. Ultrathin serial sections were cut and
stained with uranyl acetate (Watson, 1958). The sections were examined with a
Philips 300 electron microscope.
RESULTS
In the first series of experiments, spermatozoa from the cauda epididymides
and vasa deferentia of either t6ftw32 or + / + males were incubated with cumulus
cell-surrounded ova. At the completion of the 6 h incubation period, ova which
had been co-incubated with either the mutant or wild-type spermatozoa had lost
their surrounding cumulus cells. Control ova incubated for 6 h in the absence of
In vitro fertilization in mutant (tLx/tLy) male mice
53
Table 3. In vitro fertilization of zona-less ova
Male genotype / 6 / w 3 2
Experiment 1
2
3
4
5
6
Total ova (%)
Male genotype + / +
No.
fertilized
No.
unfertilized
No.
fertilized
No.
unfertilized
0
0
0
0
0
0
0(0%)
84
58
95
28
17
32
314(100%)
22
17
77
24
55
39
234(93%)
0
0
2
14
1
0
17(7%)
Table 4. Binding of mutant spermatozoa (te/tw32) to
fertilized and unfertilized ova
Fertilized
Unfertilized
No. of ova
No. of ova with
bound sperm (%)
Mean no. of sperm
Ova with bound sperm
49
129
16(33%)
116(90%)
1-4
5-7
spermatozoa retained their cumulus cells. Thus, spermatozoa from t6/tw32 males
appear to contain qualitatively normal levels of hyaluronidase activity. This
result supports the findings by Erickson & Krzanowska (1974). Ninety-three
percent of the ova incubated with wild-type spermatozoa were fertilized. All of
those ova incubated with spermatozoa from the intercomplement males remained
unfertilized (Table 1).
In the second series of experiments, either mutant or wild-type spermatozoa
from the cauda epididymides and vasa deferentia were added to insemination
dishes which contained ova previously treated to remove their surrounding
cumulus cells. The results (Table 2) show that fertilization was effected in 78 %
of those ova incubated with wild-type spermatozoa, while all of the ova incubated with the mutant spermatozoa remained unfertilized. In the final experiment of this series, t&/tw*2 and + / + spermatozoa from the cauda epididymides
and vasa deferentia and + / + spermatozoa from the caput epididymides were
separately incubated with zona-less mouse ova. Table 3 shows that the percentage of zona-less + / + mouse ova fertilized by mature wild-type spermatozoa
was high (93 %). Conversely, none of the ova were fertilized by the mutant
spermatozoa despite the removal of all of the barriers normally present during
fertilization. In addition, none of 111 denuded ova incubated with immature
wild-type spermatozoa were fertilized.
Light microscopic analyses showed that the zona-less ova which had been
incubated with either mutant spermatozoa or immature wild-type spermatozoa
54
J. M c G R A T H AND N. HILLMAN
Fig. 1. An unwashed ovum after 6 h in vitro incubation with spermatozoa from the
cauda epididymides and vasa deferentia of a tG/tw32 male. Note the movement of
the spermatozoan tails and the normal morphology of most of the attached
spermatozoa. Arrows indicate the abnormal gametes, x 1600.
for six hours, although unfertilized, had numerous spermatozoa bound to the
egg plasma membrane (Figs 1-3). Many of these spermatozoa were 'twitching'
at the time of examination and most were morphologically normal.
In order to determine if there is specificity in the binding capacity of the
mutant gametes, spermatozoa from the intercomplement males were incubated
with either fertilized or unfertilized zona-less ova (see Methods). The results
(Table 4) show that 33 % of the fertilized ova and 90 % of the unfertilized ova
had bound mutant spermatozoa. Furthermore, of those ova which had bound
gametes, the mean number of bound spermatozoa/egg was approximately four
times greater before fertilization (5-7 spermatozoa/egg) than after fertilization
(1-4 spermatozoa/egg). Thus, spermatozoa from t^/tw32 males preferentially bind
to the plasma membranes of unfertilized mouse ova.
Since morphologically normal, motile spermatozoa from mutant males will
bind to, but not fertilize, denuded ova, it was of interest to determine whether
mutant spermatozoa undergo the acrosome reaction. At the ultrastructural
level, spermatozoa which have bound to the vitelline membrane have lost their
acrosomes. This observation does not, however, prove that a physiologically
normal acrosome reaction occurred.
In vitro fertilization in mutant (tLx/tLy) male mice
55
Fig. 2. A micrograph of a washed zona-less ovum following a 6 h incubation period
with mutant spermatozoa. The washing procedure causes the tails of the spermatozoa to be sheared from the bound heads, x 1300.
Fig. 3. A micrograph of a washed zona-less ovum that had been incubated with
immature spermatozoa from a wild-type male. Like mutant spermatozoa, the tails
of the immature gametes are lost during the washing procedure (cf. Fig. 2). Mature
wild-type spermatozoa remain intact under the same conditions, x 1300.
Ultrastructural observations of serial sections of ova with attached mutant
spermatozoa also show that while the two gametic membranes are frequently in
close apposition (Figs 4 A and B), there is no indication of extensive membrane
fusion between the two gametes. This is in contrast to the sequence of events
which normally accompanies mammalian fertilization (Yanagimachi & Noda,
1970; Anderson, Hoppe, Whitten & Lee, 1975; Noda & Yanagimachi, 1976).
With few exceptions, the mutant spermatozoa remained entirely outside the
ovum. In the exceptional cases, short cytoplasmic extensions from the egg
partially engulfed the mutant spermatozoon.
DISCUSSION
Mammalian fertilization is a complex process which requires the fertilizing
spermatozoon to: (1) acquire the ability to fertilize (mature) while in transit
through the epididymis (Bedford, 1975; Orgebin-Crist, Danzo & Davies, 1975),
(2) undergo certain biochemical changes known as 'capacitation' (Bedford, 1970;
Chang & Hunter, 1975; Chang, Austin, Bedford, Brackett, Hunter & Yanagimachi, 1977), and (3) arrive at the site of fertilization (which is ultimately the
ovum plasma membrane).
In the current series of experiments we have sequentially eliminated those
barriers which in vivo restrict the number of spermatozoa which contact the egg
56
J. McGRATH AND N. HILLMAN
4B..
Fig. 4. (A) An electron micrograph showing a portion of a denuded ovum and a
bound mutant spermatozoon. The gametes had been co-incubated for 6 h. Note
the absence of the acrosome from the spermatozoon, x 42600. (B) A higher
magnification of the area circumscribed in Fig. 4(A). Note the close association of
the gametic membranes. No membrane fusion has occurred, x 81000. AM, inner
acrosomal membrane; NM, spermatozoan nuclear membrane; PM, egg plasma
membrane.
plasma membrane. We have therefore insured that spermatozoa from
males arrive at the site of fertilization and, by so doing, have eliminated this
parameter as the primary cause for intercomplement male sterility. Our results
do not, however, allow us to determine if the sterility is caused by a failure of the
spermatozoa to mature within the epididymides or by their inability to become
capacitated.
There are several lines of evidence which indicate that tLx/tLv male sterility
results from a lack of spermatozoan maturation. Bryson (1944) and Bennett &
Dunn (1967) reported that spermatozoa from tLx/tLv males exhibit reduced
levels of motility. We have also observed that spermatozoa from the cauda
epididymides and vasa deferentia of tG/twS2t males show minimal forward progression and, in addition, their movement closely resembles the motility pattern
of immature spermatozoa obtained from the caput epididymides of + / + males.
Analogously, wild-type spermatozoa obtained from the caput region of the
epididymis will bind to, but not fertilize, zona-less mouse ova in vitro. These
similarities between the spermatozoa obtained from the cauda epididymides and
In vitro fertilization in mutant (tLx/tLy) male mice
&
57
w32
vasa deferentia of the t /t
males and the immature spermatozoa obtained
from fertile + / + males suggest that sterility results from the failure of the
mutant spermatozoa to undergo the necessary maturational events which
normally occur in the male reproductive tract.
The hypothesis that t alleles interfere with spermatozoan development can
also explain the different forms of sterility that have been noted in tLx/tLv and
tSL/tSL males. Sterility in tSL homozygotes results from the developmental
arrest of spermatids (Bennett & Dunn, 1971; Dooher & Bennett, 1974). The
present observations indicate that tLx/tLv sterility is caused by incomplete
spermatozoan maturation. The combined observations suggest that both types
of sterility are caused by a deficiency in the normal program of differentiation of
the spermatozoon. The extent or time of this deficiency in the sterile males
would be dependent upon the specific combination of the recessive tn mutations.
This research was supported by United States Public Health Service Grants nos. HD 00827
and HD 09753 and by a Biomedical Research Support Grant, SO7-RR07115. The authors
would like to thank Marie Morris and Geraldine Wileman for their technical assistance and
Dr Don P. Wolf for his advice on obtaining zona-less mouse ova.
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