439
J. gen. Virol. (I98O), 5x, 439-443
Printed in Great Britain
Mouse Sperm can Horizontally Transmit Type C Viruses
( A c c e p t e d I I J u l y I980 )
SUMMARY
Washed, non-viable sperm from A K R and NZB mice contain infectious murine
type C viruses (MuLV), whereas similarly treated sperm from Balb/c and probably
C57B1/6 mice do not. Interaction of washed, viable AKR, Balb/c and NZB sperm
with tissue culture cells leads to a transfer of infectious MuLV to these cells. The
most likely mechanism is infection by virus closely associated with the sperm.
Another possible mechanism is the introduction of proviral DNA. These experiments indicate that type C viruses can be found with mouse sperm. They suggest
sperm could horizontally transmit these MuLV to animals during copulation and
to the germ line of mice during penetration of ova.
Studies involving embryo transplants and specific genetic crosses have established that
murine type C viruses (MuLV) can be endogenous to the species and inherited through the
germ cell (Gross, I97o; Barnes et al. I972; Rowe, I973). These endogenous MuLV have
been classified descriptively by their host range. The ecotropic infect and grow in mouse and
rat cells. The xenotropic can only productively infect cells heterologous to the species. They
cannot exogenously infect mouse cells (Levy, I973, 2974). The ecotropic can be further
subdivided into N- and B-tropic viruses depending on their preference for growth in N- or
B-type mouse cells (Lilly & Pincus, I973). There is molecular evidence for one to several
copies of ecotropic MuLV and six to nine copies of the xenotropic MuLV genome in the
mouse chromosome (Gelb et al. I973; Benveniste & Todaro, I974; Chattopadhyay et aL
I974). As well as transfer of mouse type C viruses by genetic means, they can also be passed
under certain conditions from the mother to the young through the milk (Law & Moloney,
I96I; Gross, I97O; Gardner et al. I979).
Since it is known that somatic cells can take up either viable or heat-killed spermatozoa
(Bendich et al. 1974, I976; Higgins et al. I975), we wished to determine whether horizontal
transmission of virus information can be achieved by this means. Experiments were designed
to establish whether mouse sperm could introduce a type C virus into cells which normally
do not harbour the virus. The results would indicate whether type C viruses could be transferred to eggs via infectious proviral D N A which might be present in the sperm head or via
infectious particles associated with the viable sperm (Bentvelzen, I974; Bendich et al. I976).
For our studies, spermatozoa were isolated from the vas deferens of AKR, Balb/c,
C57B1/6 and N Z B / J mice under sterile conditions and washed three times with Hanks'
balanced salt solution, in most experiments, the sperm were semi-purified by sedimentation
through sucrose to remove virus particles present in the seminal fluid. F o r these studies, the
sperm were diluted with a I :3 vol. (w/w) of 60 ~ sucrose in o.ot M-tris-HC1 pH 7"5. The
mixture Was then layered on 28 ml of the same sucrose solution and centrifuged at 4 °C in
a SW25. I rotor for 45 rain at 25 ooo g (Calvin et aL z973). The pellet was resuspended in
Hanks' solution and spermatozoa were counted after appropriate dilution. Aliquots were
used for incubation with tissue culture cells and for scanning electron microscopy (EM)
studies. Moreover, samples were seeded into culture dishes to determine whether the pellet
contained co-sedimenting somatic mouse cells. These cultures receiving the spermatozoa
were incubated for several weeks. In all the experiments, somatic cells were never observed.
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Table I. Recovery o f type C viruses f r o m mouse sperm preparations*
Somatic cells
Source of sperm Virus detected detected in
(mouse strain)
by EM
culture
Virus recovered from frozen
sperm
--~
Ecotropic
Xenotropic
AKR
0/4
0/4
4/4
0/4
Balb/e
0/3
o/3
0/3
0/3
NZB/J
0/2
0/2
O/I
I/It
Virus recovered from
cellsincubated with
viable sperm
4/4 N-tropic AKRMuLV from
NIH-ME and rat
embryo cells
~/3 N-tropic AKRMuLV after IdUrd
treatment of
NIH-ME
2/2 X-tropic MuLV
* Procedures employed are described in text. Each number corresponds to art individual experiment.
t Virus was not detected in the sperm preparation recovered after sucrose density-gradient centrifugation.
In one experiment the frozen NZB sperm preparation thawed so that it could not be tested.
Aliquots containing approx. IO 7 viable sperm were also frozen away at - 7 0 °C for subsequent virus assay. Since freezing kills the sperm, these samples monitored infectious virus
associated with the spermatozoa. For scanning EM, spermatozoa were fixed in 2.5%
glutaraldehyde, dehydrated in ethanol, freeze-dried, coated with gold-palladium and
evaporated using a Denton evaporation coater and viewed with a JEOL JSM-35 scanning
electron microscope. In these studies, approx. 2oo spermatozoa were examined and no
virus-like particles could be detected.
The viable sperm preparations were mixed directly for 2 h at 37 °C with cell suspensions
of N I H mouse embryo (ME), Fischer rat embryo, mink lung or human foreskin fibroblast
(HuF) cells at a ratio of 1o sperm/cell by a technique previously described (Bendich et al.
1974). The number of sperm employed was approx. 5 × lO5. The N I H - M E and rat embryo
cells were used since they contain no ecotropic A K R - M u L V genome (Rowe, 1973 ; Chattopadhyay et al. I974). The H u F and mink lung cells were employed because they lack M u L ¥
and are very sensitive for detection of mouse xenotropic virus (Levy, I975). The cells, after
receiving the sperm, were cultivated initially in plastic Petri dishes and then in flasks for up
to lO weeks in attempts to look for expression of ecotropic or xenotropic MuLV. All
cultures negative for virus were treated after passage 4 with iododeoxyuridine (IdUrd)
(3o #g/ml) for 36 h by standard techniques (Lowy et al. 1971) and overlaid with sensitive
mouse or human cells and cultured for another 3 week period. The monolayers were then
co-cultivated with the N R K - H a r v e y line, a non-virus producing MSV-transformed rat cell
line (Levy, t97I). Formation of pseudotype murine sarcoma virus (MSV), as assayed by
focus formation in rat and human cells, was used as an indication of replicating M u L V
(Levy et al. 1975). Moreover, the XC plaque assay was employed to detect infectious ecotropic MuLV (Rowe et al. I97O) and the mink S + L - assay to detect xenotropic MuLV
(Peebles, i975). The tropism of the ecotropic viruses isolated was determined by the XC
assay using N I H Swiss and Balb/c ME cells. Virus identification was confirmed by standard
neutralization assays using specific antisera for ecotropic and xenotropic virus (Levy et al.
1975).
The result of these studies are presented in Table 1. In four experiments involving A K R
mice, N-tropic A K R - M u L V was easily recovered from all frozen sperm preparations inoculated directly onto N I H - M E . A K R sperm collected after sucrose density-gradient centrifugation also contained infectious virus. Ecotropic A K R virus was also recovered from
the N I H Swiss and rat embryo cells propagated for 3 weeks after they had been incubated
with the four viable A K R sperm preparations. H u m a n cells did not produce any xenotropic
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44 I
or ecotropic MuLV either after direct inoculation of a thawed AKR sperm preparation on
to the cells or by cultivation of the HuF cells after they had been exposed to viable sperm.
When the AKR sperm preparation was pretreated with ether, or heated to 8o °C for Io
min, the transfer of infectious virus was eliminated. These treatments do not affect the
uptake of sperm by somatic cells (Bendich et al. I974, I976) but may alter the biological
properties of the sperm.
Spermatozoa removed from Balb/c mice yielded no infectious virus by direct assay of
the thawed preparations on mouse and human cell cultures. Cells propagated after incubation with the viable Balb/c sperm also did not release infectious virus. A preparation of
sperm from C57B1/6 gave a similar result, but more studies need to be done with this strain
before proving that its sperm is free of infectious MuLV. In one out of three occasions,
NIH-ME cells, receiving the viable Balb/c sperm and passed six times, yielded an N-tropic
AKR-MuLV after treatment with IdUrd. No B-tropic or xenotropic virus was detected.
Also, the same mouse cell line, not treated with IdUrd, released no infectious virus. Since
NIH-ME do not contain ecotropic virus, this observation raised the possibility that viable
Balb/c sperm penetrated the NIH mouse embryo cell and transferred the proviral DNA of
AKR-MuLV into the cell line. Nevertheless, although the sperm preparation by tissue
culture analysis was free of any detectable Balb/c cell, we are unable to rule out completely
the possibility that a few somatic cells from the vas deferens had been transferred with the
sperm fraction to the mouse cells.
Sperm from NZB mice showed a pattern of type C virus association similar to the AKR
since the frozen uncentrifuged sperm preparation contained infectious xenotropic virus.
However, the sperm collected after sucrose density-gradient centrifugation did not have
infectious MuLV. Mink lung and HuF cells exposed to both preparations of viable sperm
released xenotropic virus. Conceivably, the infection of these cells by the sucrose densityseparated sperm preparation occurred via proviral DNA, but we assume a low level of
infectious xenotropic MuLV was closely associated with the centrifuged sperm but was not
detectable following freezing and thawing. As noted above, no viable somatic cells were
detected in any NZB sperm preparations and scanning electron microscopy did not reveal
any budding particles on the representative sperm examined (Table 0.
These experiments indicate that some mouse sperm preparations, even after extensive
washing and after centrifugation through sucrose, contain infectious MuLV closely associated with the membrane of the sperm or contained within the sperm head. The studies with
Balb/c and NZB sperm also raise the possibility that transfer of proviral DNA to cells not
containing the virus may also occur, as suggested by the results with mouse mammary
tumour virus (Bentvelzen, I974). Most importantly, they suggest that MuLV could become
incorporated into the mouse germ line not only vertically via the germ cell, but also horizontally during sperm penetration of the ovum. In vitro exposure of pre-implantation mouse
embryos to Moloney leukaemia virus has led to efficient integration of this virus into the
germ line of mice (Jaenisch, I976). Likewise, in nature, if MuLV is introduced into the ovum
by sperm at the time of fertilization it could conceivably become part of the mouse germ
line. This possibility, which requires further study, could explain virus genome integration
site differences in offspring of certain genetic crosses. These experiments also indicate that
sperm transfer could be responsible for MuLV infection of both mother and offspring by
venereal epigenetic means as observed with MuLV in wild mouse strains (Gardner et al.
I979). Furthermore, they might explain germ line integration by new type C viruses which
is a rare event in nature (Todaro, I975). While transfer of virus by mating between heterologous species is highly unlikely, horizontal spread of a xenotropic virus to a species by some
other means might eventually lead to contamination of its sperm by this virus. Then, by
sperm transfer, integration into the germ line of that species could ultimately occur.
NZB males sire small
litters from
(Bielschowsky
& Bielschowsky,
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by I964) and their sperm
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44 2
Short communications
preparations c o n t a i n large quantities of type C xenotropic viruses ( F e r n a n d e s et al. I973 ;
this report). Moreover, a glycoprotein similar to M u L V gp7o has been detected in seminal
fluid, g l a n d u l a r epithelium of the testes a n d o n the surface of sperm (Del Villano & Lerner,
I976; Lerner et al. I976). In addition, reverse transcriptase-like activity has been detected
in h u m a n sperm heads a n d cell-free seminal fluid (Witkin et al. t975). These a n d other
observations cited above suggest that the association of type C viruses with sperm may have
a direct influence o n n o r m a l e m b r y o n i c development (Levy, 1978).
This research was supported by U.S. Public Health Service G r a n t s C A I3O86 a n d C A
o8748 f r o m the N a t i o n a l Cancer Institute a n d N C I C o n t r a c t No. NoI-CB-439o4. J.A.L.
was the recipient of Research Career D e v e l o p m e n t A w a r d No. 5 Ko4 CA7o99o. We t h a n k
Ms Peggy M o o d y for excellent technical assistance, Ms N. L a m p e n for the scanning elect r o n microscopy studies, a n d the late A a r o n Bendich for helpful discussions.
Cancer Research Institute
Department o f Medicine
University o f California
San Francisco, California 94143, U.S.A. and
1Laboratory o f Cell Biochemistry
Memorial Sloan-Kettering Cancer Center
New York, N.Y. IOOI, U.S.A.
J. A. LEVY
JUDY JOYNER
ELLEN BORENFREUND1
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