Cross Fertilization In Vivo and In Vitro Between Three Species of Vesper Mice, Calomys (Rodentia, Cricetidae)


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. Eggs
SPECIES-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.
  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
                     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
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
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 stage
438   E.R.S. ROLDAN ET AL.

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-

  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 possible
SPECIES-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.
erologous gametes in several intrageneric                         Growth Differ., 22:103-112.
                                                                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,
cally distant species could disclose gross dif-                   Cricetidae). Mendeliana, 4:57-65.
ferences or similarities in heterologous                        Fournier-Delpech,S., J.L. Courtens, C.L. Pisselet, B. De-
interactions and the underlying components,                       laleli, and M. Courot (1982)Acquisition of zona binding
closely related (intrageneric) species crosses                    by ram spermatozoa during epididymal passage, as
                                                                  revealed by interaction with rat oocytes. Gamete Res.,
could contribute enormously to the study of                       5:403408.
subtle variations in the molecular, morpho-                     Fukuda, Y., M.B. Maddock, and M.C. Chang (1979) In
logical, and functional aspects of fertilization.                 vitro fertilization of two species of deer mouse eggs by
                                                                  homologous and heterologous sperm and penetration
                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.
                                                                  Estudios citogeneticos. Physis, 36[secc. C]:169-178.
edge the assistance of Adriana Kajon and                        Gates, A.H. (1971) Maximizing yield and developmental
Vida Hodara, Prof. M.C. Weissenbacher for                         uniformity of eggs. In: Methods in Mammalian Em-
encouragement, Prof. C. Barros for helpful                        bryology. J.C. Daniel Jr., ed. W.H. Freeman & Co., San
discussions, and Prof. 0. Nuiiez and Prof.                        Francisco, pp. 64-75.
                                                                Gwatkin, R.B.L. (1977) Fertilization Mechanisms in Man
O.A. Reig for critical comments on the man-                       and Mammals. Plenum Press, New York.
uscript. Thanks are also due to Dr. J.M.                        Hanada, A., and M.C. Chang (1972) Penetration of zona-
Cummins for his critical review of the man-                       free eggs by spermatozoa of different species. Biol.
uscript and the improvement of the English.                       Reprod., 6:300-309.
                                                                Hanada, A., and M.C. Chang (1976) Penetration of ham-
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Adams, C.E. (1973) Loss of eggs associated with tuba1             Reprod. Fertil., 46239-241.
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