J. Embryol. exp. Morph. Vol. 31, 3, pp. 621-634, 1974
Printed in Great Britain
621
Artificial insemination
of deermice (Peromyscus maniculatus) with
sperm from other rodent species
By MICHAEL B. MADDOCK 1 AND WALLACE D. DAWSON
From the Department of Biology, University of South Carolina
SUMMARY
Deermice (Peromyscus maniculatus) were hormonally induced to ovulate and artificially
inseminated with sperm of 13 other rodent species varying in taxonomic distinction from
species to family level. These were compared with inter se and sham inseminated controls.
Embryos were recovered at various stages from 5 h to 10 days. Hybrid embryos failed to
develop fully in all crosses except those within the same species group. Closely allied interspecies group crosses produced implantation embryos, but intersubgeneric and intergeneric crosses did not develop beyond early cleavage. Interfamilial hybrid embryos could
not be obtained.
INTRODUCTION
The cricetid rodent genus Peromyscus (deermice and allies) affords a particularly valuable model for species hybrid studies because of its many forms
which exhibit varying, and often subtle, degrees of relationship, and because
of the relative ease with which they can be maintained in the laboratory.
Additionally, hormone-induced ovulation and implantation, and artificial
insemination methods developed with common laboratory animals (rats, mice,
hamsters) can be employed. These techniques make possible hybridizations
which could not be accomplished through natural mating, and permit the
study of developmental interplay between genotypes from divergent sources.
The rationale of the present study was to inseminate female deermice (P.
maniculatus) with sperm of progressively more distantly related species. It was
anticipated that the extent of ontogenetic development would be negatively
correlated with phylogenetic divergence, and that given a specific degree of
taxonomic distinction, developmental end points could be predicted. Additionally, sensitive ontogenetic stages should be recognized, and some evolutionary
implications could be drawn.
1
Author's address: Department of Biology, University of South Carolina, Columbia,
S.C., 29208, U.S.A.
622
M. B. MADDOCK AND W. D. DAWSON
MATERIALS AND METHODS
Young (40- to 90-day-old) virgin female deermice were induced to ovulate
with intraperitoneal injections of 15i.u. pregnant mare serum gonadotrophin
(PMS) followed two days later by lOi.u. of human chorionic gonadotrophin
(HCG). Ovulation occurred about 13 h after the second injection.
Epididymal sperm from rodents killed one hour previously were suspended
in reconstituted 9 % skim milk at concentrations in excess of 106/mm3 (Dziuk
& Runner, 1960). Sperm counts and motility were confirmed visually. Artificial
insemination was accomplished through the cervix using a syringe with a
blunted needle. Insemination was timed to coincide with ovulation.
Mice from each cross were autopsied at intervals from 5 to 96 h after
insemination. Fallopian tubes and uterine horns were flushed with physiological
saline, and ova and/or pre-implantation embryos recovered. In crosses where
development proceeded to the blastocyst stage, mice were examined for implantation sites between the 7th and 10th day, where day 0 was the day of
insemination.
One mg medroxyprogesterone acetate (MPA), as DepoProvera (Upjohn),
was injected subcutaneously at both 5 and 24 h post-insemination to facilitate
egg transport. Additional daily injections of 1 mg MPA were given to some
mice together with 0-025 mg estradiol in 0-025 cm3 sesame oil on day 5 postinsemination, to induce implantation of blastocysts.
Freshly recovered ova and embryos were examined and photographed under
phase microscopy at x 100. Representative specimens were fixed, stained and
mounted for further study.
Sperm donors represented 13 other rodent species differentiated from P.
maniculatus at species to family level, with six intermediate categories (Table 1).
P. maniculatus, P. polionotus, Mesocricetus auratus, Meriones unguiculatus,
Rattusnorvegicus and Mus musculus were from domesticated or semi-domesticated
stocks, while wild captured animals of the other species were used.
RESULTS
In the P. maniculatus inter se inseminated controls, 49 (83 %) of 59 mice
inseminated and 140 (81 %) of 172 ova recovered were fertilized. The
criteria for fertilization in each instance were: (1) sperm penetration of ova;
(2) cleavage; or (3) blastocyst development and implantation (Fig. 1). None
of the 40 ova recovered from the milk sham inseminated control showed
gynogenesis at 48 h. These and numerous additional observations indicate
that spontaneous cleavage (fragmentation) is not a common occurrence in
unfertilized deermouse ova during the first two days. Therefore any cleavage
observed at 48 h was assumed to result from fertilization rather than gynogenesis.
623
Artificial insemination of deermice
Table 1. Comparisons o/Peromyscus maniculatus
and sperm donor species
Sperm donor species
Level of
Diploid
taxonomic chromosome
differentiation
number
P. maniculatus
(deermouse)
P. polionotus
Species
(oldfield mouse)
P. leucopus
\
(woodmouse) I
Species group
P. gossypinus j
(cottonmouse)J
P. truei
Species group
(pinyon mouse)
P. floridanus
Subgenus
(Florida mouse)
Reithrodontomys humulisGenus
(harvest mouse)
Ochrotomys nuttalli
Genus
(golden mouse)
Sigmodon hispidus -\
(cotton rat)
I
Geographic line
Oryzomys palustris j
(rice rat)
J
Mesoericetus auratus Tribe
(golden hamster)
Meriones unguieulatus Subfamily
(Mongolian gerbil)
Rattus norvegicus
N
(laboratory Norway
rat)
Mus museulus
(laboratory house
mouse)
,
Usual
adult
wt (g)
Estimated time of
most recent common
ancestry (108 yrs)
48
002
(Pleistocene-Wisconsin)
48
48
0-4
(Pleistocene-Sangamon)
48
10
(Mid-Pleistocene)
20
(Pleistocene-Nebraskan)
40
(Pliocene)
70
(Pliocene)
48
48
42
52
52
20
(Miocene)
56
44
20-30
44
20-30
f42
Family
40
25-35
20-30
Inseminations with sperm from other species are compared with the controls
in Tables 2 and 3. Each type of insemination within the genus Peromyscus
produced some examples of development through first cleavage, but development to blastocyst and beyond was restricted to crosses within the maniculatus
and leucopus species groups. Inseminations with P. polionotus sperm produced
a high proportion of fertilized ova (62 %) and all embryos examined had
normal morphology (Fig. 2). This was expected and consistent with the occurrence of viable, fertile hybrids in laboratory matings of these species (Dice,
1933; Dawson, 1965).
P. leucopus and P. gossypinus are closely related species of the leucopus
species group, and are interfertile in laboratory crosses (Dice, 1937). Hybrids
of either of these two leucopus group species with P. maniculatus were expected
V)
EMB
31
624
M. B. MADDDOCK AND W. D. DAWSON
Fig. 1. Early embryonic development of P. maniciilatus. (A) Fertilized ovum with
pronuclei and sperm tail. (B-D) Cleavage. (E) Morula. (F) Blastocyst in zona.
(G) Section of uterus showing implanted blastocyst. (H) Uterus with fourconceptuses.
Artificial insemination of deermice
625
Table 2. Fertility of deermice artificially inseminated
with sperm from other rodents
Sperm source
P. maniculatus
P. polionotus
P. leucopus
P. gossypinus
P. truei
P. floridamis
Reithrodontomys
Ochrotomys
Sigmodon
Oryzomys
Mesocricetus
Men'ones
Mus
Rattus
Percentage of
inseminated mice
successfully
fertilized
83
83
17
29
30
10
17
0
8
33
11
0
0
0
Percentage
of eggs
fertilized
5-48 h
62 (85)*
58 (48)
15(46)
87(15)
52 (46)
13(30)
8(12)
0(26)
3(25)
15(27)
9(40)
0(9)
0(29)
0(15)
* Total number of eggs in sample given in parentheses.
to show similar development. Earlier attempts at laboratory matings of P.
maniculatus x P. leucopus produced no progeny (Dice, 1933), although there
are unconfirmed reports of adult hybrids in nature. Chang, Pickworth &
McGaughey (1969) obtained pre-implantation embryos by artificial insemination, and, in one instance, observed an 11-day-old implanted embryo.
Insemination with P. leucopus sperm gave a lower frequency of fertilized
ova than expected (Table 2). However, some representatives of various stages
from sperm penetration to implantation were observed. These had normal
morphology. Sperm quality (motility and concentration) was not good in the
limited number of P. leucopus we had available, and this probably contributed
to the reduced proportion of fertilized ova. Some P. maniculatus x P. gossypinus
embryos showed normal cleavage, but in other instances irregular shaped
blastocysts were observed (Fig. 3). A single implantation site was seen in one
mouse, indicating that hybrids of P. gossypinus as well as P. leucopus are capable
of initiating post-implantation development. Another study (Dawson, Mintz,
Maddock & Lewin, 1972) has shown that occasional P. maniculatus-leucopus
hybrids produced by artificial insemination may be live-born but non-viable;
however, development is usually interrupted before 15 days post-insemination.
P. truei also differs from P. maniculatus at the species group level, but the
truei group is usually considered more remotely related to P. maniculatus than
the leucopus group (Hooper & Musser, 1964). Moreover, P. truei are substantially larger than P. maniculatus. Six experimental matings attempted by
39-2
Rattus
Milk sham
Mus
59
12
30
24
30
20
6
7
12
6
18
6
5
5
6
49
10
5
7
9
2
1
0
1
2
2
0
0
0
—
mice
fertile
mice
inseminated
32 (53)
16(24)
4(19)
12(13)
18 (31)
3(4)
0(10)
0(6)
1 (10)
4(18)
2(35)
0(9)
0(25)
0(11)
0(40)
18(26)
8(16)
3(27)
0(1)
6(14)
1(25)
1(2)
0(20)
0(25)
0(9)
1(5)
—
0(4)
0(4)
0
Cleavage
(2-8 cell)
0
0
0
0
—
—
—
—
—
0(1)
0(1)
KD
0
3(6)
4(8)
Morula
A
43 (43)
8 (10)
1(7)
0
0
0
—
—
—
—
—
—
—
—
—
Blastocyst
Development observed
Sperm
penetration
1
44
—
1*
1
—
—
—
—
—
—
—
—
—
—
—
Implantation
sites
172
58
54
16
46
30
12
26
35
27
40
9
29
15
40
and
embryos
\J V C*.
A V Q
Number
* Does not include 16 implantations reported elsewhere (Dawson et al. 1972). Numbers in parentheses
represent total eggs observed at equivalent time.
Reithrodon tomys
Ochrotomys
Sigmodon
Oryzomys
Mesocricetus
Meriones
P. floridanus
P. maniculatus
P. polionotus
P. leucopus
P. gossypinus
P. truei
Sperm source
Nnmhpr
Nnmhpr
Table 3. Embryonic development in artificially inseminated deermice
Total
140
36
9
14
24
4
1
0
1
4
3
0
0
0
—
ova or
embryos
fprtilp
z
o
d
p
b
z
o
d
0
X
x
•fcd
OS
Artificial insemination of deermice
627
Fig. 2. Early embryonic development of P. mamculatus-polionotus F1 hybrids
produced by artificial insemination. (A). Fertilized ovum with pronuclei. (B-D).
Cleavage. (E). Morula. (F). Blastocyst in zona.
Dice (1933) between P. mcmiculatus and P. truei were sterile. About half of
the ova which we recovered from inseminations with P. truei sperm had been
fertilized (Table 2), and the first cleavage appeared normal. However, the
second cleavage was abortive (Fig. 4A-D), and no development beyond the
four-cell stage was observed.
Inseminations with P. floridanus sperm gave a small number of fertile ova.
The first cleavage was normal, but irregular cleavage followed (Fig. 4E-F).
628
M. B. MADDOCK AND W. D. DAWSON
Fig. 3. (A) Fertilized ovum of P. maniculatus inseminated with P. leucopus sperm
showing sperm penetration and pronuclei. (B) Two-cell hybrid embryo of P.
maniculatus inseminated with P. gossypinus. (C-D) Embryos of P. maniculatusgossypinus hybrids showing irregular cleavage. (E) Morula of P. maniculatusgossypinus hybrid. (F) Single conceptus of P. maniciilatus-gossypinus hybrid.
One 6-cell embryo was observed. No previous experimental hybridization has
been attempted with this species, but Dice (1933) had demonstrated that other
intersubgeneric crosses in Peromyscus do not yield progeny.
Reithrodontomys (harvest mice) and Peromyscus are immediately allied
genera within the Peromyscine line (Hooper & Musser, 1964). In a limited
number of observations, one P. maniculatus ovum had been penetrated by
a R. humulis sperm, but no subsequent cleavage was seen (Table 2).
Artificial insemination ofdeer mice
629
Fig. 4. (A-C) Sperm penetration and cleavage of P. maniculatus-truei hybrid
embryos. (D-F) Sperm penetration and cleavage of P. maniculatus-florhkinus
hybrid embryos. Note excess nucleoli and irregular cleavage in F.
Ochrotomys nuttalli (golden mouse) formerly was classified under Peromyscus
as a monotypic subgenus, but its chromosome number and phallic morphology
evidence its distinctness from Peromyscus (Blair, 1942; Hooper, 1958; Patton
& Hsu, 1967). The generic status of Ochrotomys is further supported in our
study by the failure of any of 26 P. maniculatus ova recovered to be fertilized
by O. nuttalli sperm. Golden mouse sperm differs markedly from other New
World cricetids in both morphology and motility (Fig. 5E), as was noted also
by Hirth (1960).
630
M. B. MADDOCK AND W. D. DAWSON
Fig. 5.(A). Reithrodontomys humulis sperm penetration of P. manicitlatus. (B-D).
Abortive and apparently normal first cleavage of P. maniculatus-Oryzomys palustris
embryos. (E). Ochrotomys nuttalli sperm. (F). P. maniculatus sperm.
Although North American species of Sigmodon (cotton rats) and Oryzomys
(rice rats) occur, these genera represent a distinct cricetid line with neotropical
affinities. Insemination of deermice with sperm from these rat-size rodents
produced some abortive first cleavage, and two regular two-cell embryos were
observed - one with Sigmodon and one with Oryzomys (Fig. 5).
Insemination with hamster {Mesocricetus) sperm gave two instances of first
cleavage. In one case a regular two-cell stage was observed, and each blastomere
Artificial insemination of deermice
631
indicated regular organization of a mitotic apparatus for the second division.
An irregular two-cell embryo was observed in the same mouse. Probable sperm
penetration was observed in an ovum from another mouse.
Inseminations of deermice with sperm from other Old World myomorphs
(Meriones, Rattus, and Mus) gave entirely negative results (Table 2).
DISCUSSION
There are numerous reports (Gray, 1953) of viable mammalian species
hybrids, including classic cases such as mule and cattalo, but few examples
of prenatally interrupted hybrid development have been investigated. These
examples were reviewed by Chang & Hancock (1967). They are limited to
goat x sheep, mink x ferret, and artificial crosses among various species of
rabbits and hares. Goats (Copra hircus) inseminated by sheep (Ovis aries)
customarily give rise to conceptuses which may survive to midgestation, but
development in the reciprocal cross is limited to early cleavage.
Various crosses among domestic rabbits, European and American hares,
and cottontails by artificial insemination do not progress beyond the blastocyst
stage (Chang & Hancock, 1967). Mink (Mustela visori) sperm may fertilize
ferret (Mustela furo) eggs which undergo cleavage and occasionally develop
to blastocyst and implant abnormally (Chang, 1965).
These studies imply that taxonomic relationship per se is not a reliable
indicator of the extent of embryonic development in species hybrids since
(1) an intergeneric hybrid (goat x sheep) may develop further than an intrageneric one (ferret x mink), and (2) reciprocal crosses differ significantly.
However, within a restricted taxonomic grouping (Leporidae), crosses between
more closely related genera (Oryctolagus x Lepus) apparently give better
percentages of fertilization and greater development than those between the
more distinctly related Oryctolagus and Syhilagus (Chang & Hancock, 1967).
It is noteworthy that in most instances development fails at or before implantation. Only in goat-sheep hybrids were fetuses formed.
The interspecific inseminations of Peromyscus gave a spectrum ranging from
completely viable hybrids among species of the same species group, to failure
of sperm penetration at the subfamily level (Fig. 6). As in lagomorph and
mustelid hybridizations, when reproductive failure occurs, it was more likely
to affect preimplantation embryos. Only in P. maniculatus x P. leucopus (or
P. gossypinus) did limited fetal organization occur, analogous to goat x sheep.
It is reasonable to generalize that if inseminations between mammalian species
of the same or a closely related genus fail to produce living offspring, the
reproductive block most likely occurs between first cleavage and implantation.
The probability of normal morphogenesis diminishes rapidly with increasing
phylogenetic separation.
Crosses between Peromyscus subgenera or closely related genera occasionally
632
M. B. MADDOCK AND W. D. DAWSON
0
5
1 'I-
Pcromy.scu.s
mamvulutits
(female)
X
(sperm donor)
Sperm
penetration
X
X
X
Cleavane,
2-cell
Clcnvaye.
4-cell
8-eell
Morula
Blastocyst
Implantation
Fetal
development
Live birth
Viability
Fig. 6. Maximum development of embryos of P. maniculatus artificially
inseminated with sperm from various species.
initiated cleavage, but this often was abnormal and rarely progressed beyond
a few divisions. The concept of Dice (1940) that Peromyscus subgeneric crosses
will not produce viable hybrids is reinforced.
A wide variety of agents can induce sperm capacitation (Yanagimachi, 1970).
Species specificity of capacitation was not indicated by our investigation. If
capacitation is requisite to egg penetration in these rodents, conditions of the
P. maniculatus genital tract apparently are sufficient to induce it.
The inseminations in this study were all in vivo with zona pellucida intact.
Artificial insemination of deermice
633
Observation that some deermouse eggs were penetrated by sperm of other
cricetid genera indicates that neither the zona nor vitelline block is particularly
specific here. Yanagimachi (1972) proposed that the vitelline surface in zonafree murid (rat and mouse) eggs is strongly species specific, whereas the zonafree hamster egg, which can be penetrated by guinea-pig spermatozoa, is not.
However, Hanada & Chang (1972) reported that a low percentage of zonafree mouse eggs were penetrated by rat or hamster sperm, and that rat eggs
are fertilized by hamster sperm. Additionally, they obtained penetration of
zona-free hamster eggs by rat sperm in 8-26 % of the attempts, and high
percentages of zona-free rat and hamster eggs penetrated by mouse sperm. The
possibility exists that both the zona and vitelline blocks in cricetids (hamsters
and deermice) show less specificity than in murids, particularly mouse.
Wild deermice reproduce at the rate of approximately two generations per
year. Various lines of evidence indicate that the leucopus and maniculatus
species groups became distinct in the Sangamon interglacial (ca. 5xl0 5 yrs
ago). From this it may be deduced that a million generations of separation
from a common ancestral gene pool is sufficient to evolve strong genetic
isolating mechanisms.
The authors gratefully acknowledge the generous assistance of Mr Read Lewin. Portions
of this research were supported by grant HD 06754 from the Institute of Child Health and
Human Development, U.S. Public Health Service.
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HIRTH,
{Received 5 October 1973)