Cross Fertilization In Vivo and In Vitro Between Three Species of Vesper Mice, Calomys (Rodentia, Cricetidae)
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THE JOURNAL OF EXPERIMENTAL ZOOLOGY 233:433-442(1985)
Cross Fertilization In Vivo and In Vitro Between Three Species of
Vesper Mice, Calomys (Rodentia, Cricetidae)
E.R.S. ROLDAN,A.D. VITULL0,M.S. MERANI, AND
I . VON LAWZEWITSCH
EHIGE-CONICE'I: Catedra de Histologla y Embriologia. Facultad de
Ciencias Veterinarias, Universidad de Buenos Aires. 1427-Buenos Acres
(E.R.S.R., A.D. V, 1. VL),and Catedra de Microbiologia, Facultad de
Medicina, Uniuersidad de Buenos Aires. 1121-BuenosAires (A.D.V,
M.S. M.)Argentina
ABSTRACT Cross fertilization was tested between oocytes of Calomys cal-
lidus and spermatozoa from C. callidus, C. musculinus and C.laucha by both
in vivo and in vitro insemination. After in vivo and in vitro insemination,
respectively, percentages of oocytes fertilized were 68.8 and 46.6 (C. callidus x
C. callidus), 20.3 and 12.7 (C. callidus x C. musculinus), 26.7 and 4.3 (C.
callidus x C. laucha). Thus, the percentages obtained after in vitro insemina-
tion were always lower than those obtained with in vivo insemination. It was
found that 23.9% and 44.4% of two-cell hybrid embryos were present in ovi-
ducts 30 h r after in vivo insemination of C. callidus females with C. musculi-
nus or C. laucha spermatozoa, respectively. At a later stage (56 hr
postinsemination), development did not progress further, and abnormal em-
bryos were found both at 30 and 56 hr postinsemination, suggesting some kind
of cleavage arrest or degeneration of the embryos. We suggest that fertilization
is not strictly species-specific,at least among the species we studied, but that
there are some factors that reduce the efficiency of interspecific fertilization.
Several studies on the interaction between (C. callidus) and heterologous species (C.
gametes of different species have led to the musculinus and C. laucha).
conclusion that mammalian fertilization is,
MATERIALS AND METHODS
in general, species-specific (Dickmann, '62;
Yanagimachi, '64, '72, '77, '81; Barros, '68; Animals
Hanada and Chang, '72, '76, '78; Bedford, Calomys laucha (Olfers, 1818) and C. mus-
'77; Fukuda et al., '79). One of the major sites culinus were once thought to be the same
that seems to ensure this specificity is the species (Cabrera, '61; Hershkovitz, '62). How-
zona pellucida CYanagimachi, '77). However, ever, evidence of their status as separate
this conclusion is based on experiments us- species was provided by cross-breeding ex-
ing phylogenetically distant species (mice, periments (G. de Villafafie, personal commu-
rats, hamsters, guinea-pigs, and primates, nication), by morphological and cytotaxon-
among others). Little is known about the spe- omical studies (Massoia and Fornes, '65;
cies-specificityof fertilization between closely Massoia et al., '681, and by cytogenetic anal-
related species. ysis (Pearson and Patton, '76; Gardenal et
Investigations carried out by us during the al., '77; Forcone et al., '80 Vitullo et al., '83).
last few years on animals suspected to be Thus, C. musculinus has a chromosome com-
natural reservoirs of a n Argentine endemic plement of 2n = 38, whereas C. laucha has
disease (Argentine hemorraghic fever) have 2n = 64. C. callidus was originally described
provided us the opportunity to study inter- as a subspecies of C. uenustus (Thomas, 18941,
specific fertilization in three species of vesper to which Hershkovitz ('62) gave the synonym
mice, Calomys (Rodentia, Cricetidae). C. callosus (Rengger, 1830). The distinction
In this paper we will report the results of
our experiments in which the oocytes of Cal-
omys callidus were inseminated both in vivo Dr. E.R.S. Roldan is now at the Department of Anatomy and
and in vitro with spermatozoa of homologous Reproductive Biology, University of Hawaii, Honolulu, HI 96822.
0 1985 ALAN R. LISS, INC.434 E.R.S. ROLDAN ET AL.
of separate species between C. callosus, C. followed 72 hr later by 75 IU of HCG (Endo-
venustus, and the species referred to here as corion, Elea, Buenos Aires). PMSG was given
C. callidus is fully substantiated by chromo- to animals regardless of the stage of the es-
some analysis; thus C. callosus has 2n = 36 trous cycle.
chromosomes (Pearson and Patton, '76), C.
venustus 2n = 56 (Lisanti et al., '76; Gar- Artificial insemination and in vitro
denal et al., '77; Reig, '84), and C. callidus 2n insemination
= 48 chromosomes (Vitullo et al., '82). Fur- Superovulated C. callidus females were in-
thermore, C. callidus, C. musculinus and C. seminated through the cervix under ether
laucha differ not only in chromosome num- anesthesia with 2-5 x 10' spermatozodml
ber but also in the morphology of their auto- in 0.05-0.1 ml of medium, 13-15 h r after
somes and sex chromosomes and in G- and C- HCG injection, using the technique origi-
banding patterns (Vitullo et al., '83). nally described by Dziuk and Runner ('60)
Figure 1 shows adult males of C. callidus for the common laboratory mouse. Females
(40-60 gm), C. musculinus (16-24 gm), and were killed 4.5 or 10 hr later; the oviducts
C. laucha (11-16 gm). C. callidus and C. Zau- were removed and flushed with BWW to col-
cha descended from specimens trapped in the lect the eggs.
field (El Palmar, province of Entre Rios, and For in vitro insemination, unfertilized eggs
Diego Gaynor, province of Buenos Aires, re- still surrounded by the cumulus oophorus,
spectively). The C. musculinus colony de- were obtained from the oviducts of super-
rived from animals captured in the field ovulated C. callidus females 15-17 hr after
(Laguna Larga, province of Cordoba) and injection of HCG. The eggs and cumuli were
maintained in the laboratory by De Villa- washed twice in BWW and then placed in a
faiie ('81). All animals have been bred and microdrop (100 p1) of BWW with 50% heat-
maintained for 2 years in our laboratory un- inactivated (56"C, 30 min) human serum,
der standard conditions (20 f 2°C; 12 hr which had been previously placed under min-
light-12 hr dark), and fed with laboratory eral oil in a plastic petri dish (Falcon plastics,
mouse chow supplemented weekly with grain Oxnard, CA). Spermatozoa were added to the
and fresh vegetables. More detailed infor- microdrop at a final concentration of 2-5 x
mation on these animals, and on their man- lo7 celldml. After 8-10 h r of incubation at
agement in captivity, has been reported by 37°C under air, eggs were recovered, washed
De Villafafie ('81)and Hodara et al. ('83). twice in BWW, mounted, and compressed be-
tween a slide and a coverslip supported by
Collection and examination of spermatozoa four dots of a vaseline-paraffin mixture and
Mature epididymal spermatozoa were ob- then examined with a phase-contrast micro-
tained from the caudae of mature males of scope. Some eggs were fixed in 10% neutral
the three species of Calomys. Males were formalin and stained with 2% acetic orcein
killed by cervical dislocation. Aseptically re- (Toyoda and Chang, '74). To determine the
moved epididymis was placed in a 5OO-pl drop location of spermatozoa (on the zona or within
of BWW medium (Biggers et al., '71) under the perivitelline space), the eggs were ro-
mineral oil in a petri dish and kept a t 37°C. tated by moving the coverslip in different
The cauda epidiymidis was minced with a directions.
pair of sharp scissors to allow spermatozoa to The rates of spontaneous activation and
disperse into the medium. Sperm suspen- cumulus dispersion of C. callidus eggs were
sions were only used in experiments when a t studied both in vivo and in vitro. Superovu-
least 80-90% of the spermatozoa were vigor- lated females were injected with BWW alone,
ously motile. Sperm concentrations were de- with the same insemination technique de-
termined by hemocytometer. Sperm morphol- scribed above, sacrificed 10 hr later, and eggs
ogy was studied using air-dried smears fixed were flushed from the oviducts. Unfertilized
for 20 min in 10% buffered formol, rinsed, eggs in cumulus were obtained from hor-
and stained with Giemsa as described by mone-treated females and cultured in BWW
Watson ('75). with 50% heat-inactivated human serum at
37°C for 8-10 hr. If eggs were still sur-
Induction of ovulation rounded by cumulus cells after incubation,
Each female of C. callidus was induced to they were placed in a hyaluronidase solution
superovulate by intraperitoneal injections of (50 IU/ml; Unidasa, Roux-Ocefa, Buenos
50 JS
J of PMSG (Eleagol, Elea, Buenos Aires) Aires) for 10 min before examination. EggsSPECIES-SPECIFICITY OF FERTILIZATION 435
were considered to be spontaneously acti- the hook. In C. laucha, the nucleus is grossly
vated when one pronucleus or several sub- pyriform but asymmetrical, and the tail in-
nuclei were observed in the absence of serts centrally in the flat base of the head.
spermatozoa.
Preliminary observations were made to see Interaction of gametes
if any development could occur after inter- C. callidus eggs (Fig. 8 ) were fertilized by
specific fertilization. C. callidus females were C. callidus spermatozoa a t a relatively high
inseminated with C. musculinus or C. laucha rate both in vivo (68.8%)and in vitro (46.6%)
spermatozoa and killed 30 or 56 hr later. (Table 2). C. callidus eggs rarely underwent
Eggs were recoverd by flushing the oviducts spontaneous activation in vivo, but the rate
with BWW, and examined with either a dis- was higher under in vitro conditions (Table
secting microscope or a phase microscope to 2). The cumulus oophorus did not disperse
determine their developmental status. spontaneously either in vivo or in vitro (see
below).
RESULTS Following interspecific artificial insemina-
Superovulation of C.callidus females tion, C. musculinus and C. laucha spermato-
The administration of PMSG (50 IU)and zoa ascended the female genital tract of C.
HCG (75 IU), 72 h r apart, produced good callidus up to the oviduct, where they under-
superovulation. Lower doses of HCG (436 E.R.S. ROLDAN ET AL.
SPECIES-SPECIFICITYOF FERTILIZATION 437
TABLE 2. In viuo and in uitro fertilization ofC. callidus eggs by spermatozoa ofhomologous and heterologous species
(examined 10 hr afrer artificial insemination or in uitro insemination)'
In vivo In vitro
Insemination
with No. % Eggs No. %, Eggs
spermatozoa of Females Eggs Activated Fertilized Females Eggs Activated Fertilized
Not inseminated 6' 145 0.7 - 33 84 8.3 -
C callidus 6 77 1.3 68.7 6 88 6.8 46.6
C. musculinus 6 74 0 20.3 6 79 6.3 12.7
C. laucha 5 71 1.4 26.7 5 117 8.5 4.3
'Experiments were repeated three to five times for each determination.
'Instead of sperm suspension, medium alone was injected into the female tract
'Cumulus-eggs complexes were cultured in vitro without spermatozoa.
in vitro without spermatozoa. At 4.5 hr after 17). A much higher rate (44.4%)of two-cell
insemination, some eggs were partially or embryos was obtained a t this time in females
wholly denuded, but the majority of them inseminated with C. laucha spermatozoa (Ta-
were still surrounded by a n intact cumulus. ble 3). At 56 hr after insemination, a rate of
In order to examine the early development two-cell embryos similar to that observed a t
of hybrid embryos after interspecies fertili- 30 h r was found in the C. callidus x C. mus-
zation, animals were sacrificed at 30 or 56 hr culinus cross, and only 4/50 (8%)of embryos
after insemination. When C. callidus females had progressed to the three-cell stage; in the
were sacrificed 30 h r after artificial insemi- C. callidus x C. laucha cross, although the
nation with C. musculinus sperm, 23.9%of number of one-cell eggs was higher than the
embryos had reached the two-cell stage (Fig. figure found at 30 hr, the proportion of cleav-
ing embryos (either normal or abnormal) was
similar to the fertilization rate. In this latter
cross, a sightly higher number of eggs seemed
to develop beyond the two-cell stage (Fig. 18,
Table 3).
Figs. 1-18. 1)Adult males of C. callidus (a), C. mus- Abnormalities observed 10 hr after insem-
culinus (b),and C. laucha (c). 2-7) Giemsa stained sper-
matozoa of the three species of Calomys, with (2,4,6) and ination or during in vitro incubation in-
without (3,5,7) acrosomes; 2,3: C. callidus; 4,5: C. mus- cluded spontaneous egg activation, formation
culinus; 6,": C. hucha ( x 1,000). 8-16) Interaction be- of subnuclei (Fig. 19) and supplementary
tween spermatozoa and oocytes. 8,10,11,15,16: unstained, spermatozoa in the perivitelline space (Fig.
phase contrast. 9,12-14: stained with acetic-orcein,phase
contrast. 8) Unfertilized C. callidus oocyte at 4.5 hr 20). Polyspermic fertilization was observed
postinsemination ( x 400). 9) Acrosome-reacted C. mus- in a few eggs. At 30-56 hr after insemina-
culinus spermatozoon within the matrix of the cumulus tion, some developmental abnormalities were
recovered from the oviduct of C. cullidus, 4.5 hr after observed. These included two-cell eggs with
insemination. This spermatozoon was active before being
photographed. ( ~ 4 0 0 ) 10)
. C. musculinus spermatozoon irregular blastomeres (Fig. 21), asynchroni-
attached to the zona pellucida of C. callidus oocyte cally dividing four-cell (Fig. 22) and five-cell
. C. musculinus spermatozoon in the perivitel-
( ~ 4 0 0 )11) embryos (Fig. 23), and lysis of blastomeres
line space after zona penetration ( ~ 4 0 0 )12-13)
. Swollen (Fig. 24).
C. musculinus sperm heads in C. callidus oocytes fertil-
ized in vivo (4.5 hr after insemination) ( ~ 6 3 0 ) 14)
. Male
DISCUSSION
and female pronuclei. The sperm tail is clearly visible
in close proximity to the male pronucleus ( ~ 6 3 0 ) .15) Female C. callidus superovulated with go-
Pronuclear oocyte of C. cullidus 10 hr after artificial
insemination with C. musculinus spermatozoa ( x 630).16) nadotropins at any stage of estrous cycle re-
Pronuclear C. callidus oocyte after in vitro fertilization sponded similarly to the common laboratory
with C. laucha sperm ( ~ 6 3 0 ) 17)
. Two-cell hybrid egg mouse (Gates, '711, although with greater in-
recovered 36 hr after insemination of a C. callidus fe- dividual variation. It has been reported re-
male with C. musculinus spermatozoa ( ~ 4 0 0phase; con- cently (Fleming and Yanagimachi, '80) that
trast). 18) Fourcell zygote found 56 hr after insemination
of a C. callidus female with C. laucha sperm ( ~ 4 0 0 female hamsters may also respond to gonad-
phase contrast). otropins when these are injected at any stage438 E.R.S. ROLDAN ET AL.
SPECIES-SPECIFICITY OF FERTILIZATION 439
of the estrous cycle. Although in our study
the gonadotropin dosage may seem to be
rather high for the body size of the animal,
this may be attributed, as previously re-
ported, to the quality of the hormones we
used (Roldan et al., '79).
Our success rates for in vitro fertilization
with homologous gametes of C. callidus are
low compared with those attained in most
laboratory species of rodents. As in vitro fer-
tilization rates may vary considerably with
differing experimental conditions (Niwa et
al., '80) better results could possibly be ob-
tained when the incubation conditions of the
gametes have been more precisely defined.
Similar problems may confound artificial in-
semination of C. callidus females with ho-
mologous spermatozoa. Experiments per
formed in other species have obtained rates
of fertilized eggs as high as 100% after sur-
gical insemination (Cummins and Yanagi-
machi, '82) which are well above the
fertilization rate (68.8%)we obtained. How-
ever with transcervical artificial insemina-
tion some other factors such as sperm
transport and/or retention of sperm in the
female genital tract, or timing of insemina-
tion in relation to ovulation, might have con-
tributed to the relatively low fertilization
rates we obtained.
Cross fertilization occurred between C. cal-
lidus oocytes and C. musculinus and C. lau-
cha spermatozoa. The fertilization rate was
lower in vitro (4-13%) than in vivo (20427%)
(cf. Table 21, indicating the need for further
improvement of in vitro insemination proce-
dures.
Figs. 19-24. Abnormalities found 10 hr postinsemi-
nation and in early cleavage stages of hybrid zygotes
obtained between artifical crosses of Calomys species.
19)Spontaneously activated C. callidus oocyte. Note the
presence of a large pronucleus and three small subnuclei
(after 10 hr of in vitro incubation). 20) Supplementary
spermatozoa (arrows) in the perivitelline space of C.
callidus myte 10 hr after artificial insemination with
C. musculinus sperm. 21) Twocell zygote with blasto-
meres with different sizes 30 hr after insemination. 22)
Fourcell zygote asynchronically dividing (C. callidus
ovum x C. laucha sperm) 56 hr after insemination. 23)
Five-cell asynchronically dividing egg. (C. callidus ovum
X C. laucha sperm) 56 hr after insemination. 24) Egg
showing partial lysis of blastomeres. (C.callidus ovum
x C. musculinus sperm) 56 hr after insemination.440 E.R.S. ROLDAN ET AL.
It was found that hybrid zygotes develop a t the chances for interspecies fertilization.
least to the two-cell stage. The numbers of Taking this into consideration it follows that
two-cell embryos observed 30 hr after insem- the generally assumed concept of species-
ination when C. laucha spermatozoa were specificity of fertilization should be reconsi-
used seemed high in relation to the fertiliza- dered when referring to artificial conditions.
tion rates seen 10 hr after insemination. This Hybrids should not be considered exceptions
simply may be due to the wide individual to this concept. On the contrary, we can say
variations in fertilization rates observed. that fertilization is not strictly species-spe-
Some sort of developmental arrest seemed to cific when tested in a n experimental situa-
exist at the two-cell stage in both heterolo- tion.
gous combinations because later stages were Several factors may influence the fertiliza-
not seen in the same proportion when fe- tion rates between closely related species.
males were sacrificed 56 h r after insemina- For example, some type of selection mecha-
tion. Only a small number of four- and five- nism may act in the female genital tract to
cell asynchronically dividing eggs were found reduce the number of sperm available for
at this time in the C. callidus x C. laucha fertilization. The evidence for selective mech-
crosses (Figs. 22-23, Table 3). The three spe- anisms of this sort is not clearcut (Howe and
cies show gross chromosomal differences (Vi- Black, '63; Adams, '74; Overstreet and Katz,
tullo et al., '83)and these interspecies crosses '77; Hunter, '80; Yanagimachi, '83) and
may be useful in determining the role of whether they act in Calomys must remain a
chromosomal anomalies in early develop- matter of speculation. Two interesting ques-
mental arrest. tions have been raised by Fawcett ('70): 1)
The results obtained in the present study, Are the shapes of spermatozoa "accidents of
along with others previously reported, serve evolution" or have they evolved according to
a s a basis to discuss several aspects of the their functional significance? and 2) Do these
species-specificity of fertilization. Although shape differences affect the hydrodynamic
artificial hybrids have been obtained be- characteristics of the spermatoza and in turn
tween several species of mammals, they are their swimming pattern? It was found here
considered as "exceptions" to the concept of that the three species of Calomys studied
species-specificity of fertilization (Yanagima- show considerable variation in sperm head
chi, '72, '77, '81; Barros and Leal, '82). This shape (Figs. 2-7). Whether these differences
concept has evolved from experiments in have any functional significance in sperm
which phylogenetically distant species were transport in homologous or heterologous gen-
used as model animals. Thus, heterologous ital tracts or in fertilizing ability deserves
combinations of mouse, rat, hamster, guinea- further investigation. On the other hand, the
pig, bovine, ovine, and primate gametes results of in vitro fertilization, in which sim-
showed that oocytes were rarely penetrated ilar concentrations of spermatozoa of the
by sperm of a different species (Dickmann, three species were incubated with C. callidus
'62; Barros, '68; Hanada and Chang, '72, '78; oocytes, showed definite differences in pene-
Adams, '73; Fukuda et al., '79). tration rates. So, though mechanisms acting
On the other hand, when results obtained in vivo on heterologous sperm selection can-
among the "exceptions" are more closely ex- not be discounted, other factors are evidently
amined, it is found that intra- or intergeneric acting at the level of fertilization itself even
crosses between Lagomorph species show when equivalent gamete ratios are achieved.
penetration rates of from 32%to 98%, and in As already discussed, sperm capacitation
some cases fertilization rates may approach (Yanagimachi, '77, '81) and the attachment
those seen in intraspecies inseminations (see and binding of sperm to the zona pellucida
Adams, '74 for review). In interspecies crosses (Gwatkin, '77; Bedford, '77; Schmell and Gul-
of four species of Mus, it was also observed yas, '80; Yanagimachi, '81; Fournier-Delpech
that some combinations showed lower fertil- et al., '82) are probably not species-specific.
ity in comparison with the intraspecies re- Zona recognition by specific complementary
sults (West et al., '77). Thus, though certain surface molecules of the gametes surfaces has
interspecies crosses may show reduced pene- been described (Yanagimachi, '77), but the
tration rates, complete absence of fertiliza- existence of recognition mechanisms com-
tion is by no means the case. Similar results mon to several different species has also been
were observed in the present study. Thus the postulated (Huang et al., '82). Another aspect
more closely related the species the greater to take into consideration is the possibleSPECIES-SPECIFICITYOF FERTILIZATION 441
specificity of the “zona lysin” (acrosin). penetration through the zona pellucida. J. Reprod. Fer-
Again, “species-specific”characteristics have til., 4:121-124.
Dziuk, P.J., and M. Runner (1960) Recovery of blastwyst
been recognized for this enzyme (see Ed- and induction of implantation following artificial in-
wards, ’80) but they should be considered semination of immature mice. J. Reprod. Fertil.,
cautiously until intrageneric comparisons are 1 :321-331.
made. Edwards, R.G. (1980) Conception in the Human Female.
Academic Press, London.
It is evident, anyway, in the light of the Fawcett, D.W. (1970)A comparative view of sperm ultra-
present and previous studies, that some structure. Biol. Reprod., 2[Suppl.]:90-127.
mechanism(s) is acting to reduce the effi- Fleming, A.D., and R. Yanagimachi (1980) Superovula-
ciency of penetration (or fertilization) of het- tion and superpregnancy in the golden hamster. Dev.
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Forcone, A.E., M.V. Luna, F.O. Kravetz, and J.A. Lisanti
crosses. Although the study of phylogeneti- (1980)Bandas C y G de Calomys musculinus (Rodentia,
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interactions and the underlying components, laleli, and M. Courot (1982)Acquisition of zona binding
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revealed by interaction with rat oocytes. Gamete Res.,
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subtle variations in the molecular, morpho- Fukuda, Y., M.B. Maddock, and M.C. Chang (1979) In
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ACKNOWLEDGMENTS of laboratory mouse eggs by deer mouse sperm. J. Exp.
Zool., 207481-490.
This work was supported by grants of Fun- Gardenal, C.N., N.T. Juarez, M. Gutierrez, and M.S.
dacion Emilio Ocampo, IGIA, CONICET and Sabattini (1977) Contribucion a1 conocimiento de tres
SUBCYT. The authors gratefully acknowl- especies del genero Calomys (Rodentia, Cricetidae). I.
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