Journal of Medical Virology 83:1623 (2011)

In Vitro Study on Vertical Transmission of the HIV-1

gag Gene by Human Sperm
Dian Wang,1 Xiang-Jin Kang,1 Lian-Bing Li,2 Qing-Dong Xie,1 Yu-Sen Gao,3 and Tian-Hua Huang1*
1

Research Center for Reproductive Medicine, Shantou University Medical College, Shantou, China
Chongqing Key Laboratory of Birth Defects and Reproductive Health, Chongqing, China
3
HuiZhou Municipal Central Hospital, Huizhou, China
2

HIV/AIDS is a major public health problem worldwide. To explore the feasibility of HIV vertical
transmission by human sperm, plasmid construction and transfection, interspecic in vitro
fertilization of zona-free hamster ova by human
sperm, uorescence in situ hybridization (FISH),
RT-PCR, and immunouorescence assay (IFA)
were carried out. The FISH signals for HIV-1 gag
DNA were observed in the nuclei and chromosomes of transfected human sperm, male pronuclei of zygotes, and nuclei of blastomeres of
two-cell embryos, indicating that the HIV-1 gag
gene could be transmitted via the sperm membrane and integrated into the sperm genome. In
contrast, human sperm carrying the target gene
achieved normal fertilization, and replication of
the sperm-mediated target gene was synchronized with the host genome. Using RT-PCR, the
positive bands for the target gene were observed
in the transfected human sperm and two-cell
embryos. These results further conrm that
the target gene can be transcribed into mRNA
in human sperm and embryonic cells. Positive
signals for the HIV-1 p24 gag protein were shown
by IFA in two-cell embryos containing the spermmediated target gene and not in the transfected
human sperm, which indicated that the spermmediated target gene could be translated to
make HIV-1 p24 gag protein in embryonic cells,
but not in sperm cells. The results provide
evidence for possible vertical transmission of
the HIV-1 gag gene to the embryo by fertilizing
sperm in vitro. J. Med. Virol. 83:1623,
2010. 2010 Wiley-Liss, Inc.
KEY WORDS: HIV-1 gag gene; human sperm;
integration; expression; vertical
transmission

INTRODUCTION
The current global situation in HIV/AIDS prevention
and treatment is a serious public health issue. In 2007,
2010 WILEY-LISS, INC.

there were approximately 33.2 million persons infected

with the virus worldwide. Nearly 80% of patients with
HIV/AIDS live in the developing world, mainly in the
countries of sub-Saharan Africa and southeast Asia
[UNAIDS, 2008]. Understanding of the route of HIV-1
transmission and its mechanism is an important
prerequisite for controlling the HIV/AIDS pandemic
[Galvin and Cohen, 2004]. HIV-1 transmission usually
involves sexual contact, needle stick, needle sharing,
unscreened blood transfusions, and perinatal transmission from mother-to-infant [Royce et al., 1997;
Galvin and Cohen, 2004]. Sexual contact is the main
route of transmission, accounting for nearly 80% of new
infections [Royce et al., 1997; Munch et al., 2007]. Men
infected with HIV are a major source of HIV-1 sexual
transmission. Men transmit the virus to their sexual
partners by semen containing cell-free or cell-associated
viruses [Haase, 2005]. During the course of infection,
the cell-free or cell-associated viruses, facilitated by
special polypeptides in the semen [Munch et al., 2007],
bind directly to the CD4 T cells or Langerhans cells
within the genital mucosal epithelium or penetrate into
the lamina propria of the genital tract, and subsequently
spread through draining lymph nodes leading to
systemic infection [Gupta et al., 2002; Miller and
Shattock, 2003; Haase, 2005; Kawamura et al., 2005;
Maher et al., 2005]. These aforementioned transmission
routes of the virus no longer represent a serious risk to
human health, as effective preventive measures have
been adopted. Since spermatozoa are the principal

Dian Wang and Xiang-Jin Kang contributed equally to this

work.
Grant sponsor: National Nature Science Foundation of China;
Grant number: 30800940; Grant sponsor: Li Ka Shing Shantou
University Foundation.
*Correspondence to: Tian-Hua Huang, Research Center for
Reproductive Medicine, Shantou University Medical College,
Shantou 515041, China. E-mail: thhuang@stu.edu.cn
Accepted 11 January 2010
DOI 10.1002/jmv.21767
Published online in Wiley Online Library
(wileyonlinelibrary.com)

Transmission of HIV-1 gag Gene by Sperm

component of semen and are able to transfer genetic

information to offspring by fertilization with ova, it is
essential to determine whether or not semen can
transmit the virus to offspring by fertilization, which
would be dened as true viral vertical transmission.
Thus, in the case of HIV infection, understanding the
potential for true vertical transmission from parents to
offspring by germ lines is of substantial importance.
Many studies have reported the presence of HIV
nucleic acid or protein in ejaculated spermatozoa
or maturing spermatids from HIV-1 seropositive men
[Bagasra et al., 1994; Nuovo et al., 1994; Muciaccia
et al., 1998, 2007]. Furthermore, Baccetti et al. [1994]
observed that HIV-1-like particles in sperm from
infected men could be transferred to oocytes through
in vitro fertilization (IVF). These results indicated that
vertical transmission of HIV from a fertilizing human
sperm to an embryo is possible. To further conrm
this, it is important to determine if HIV-1 genes can be
integrated into the genome of human sperm, and if
sperm-mediated HIV genes are able to replicate and be
functionally expressed in embryonic cells [Engelman
et al., 1991; Khiytani and Dimmock, 2002; Schroder
et al., 2002].
The HIV-1 gag gene is needed to make the structural
proteins for new virus particles as it plays an important
role in HIV pathogenesis. In order to provide further
experimental evidence for vertical transmission of the
HIV-1 gag gene from sperm to an embryo, the present
study therefore addressed aspects of the integration of
the HIV-1 gag gene into the human sperm genome,
replication, transcription, and translation of this gene in
embryonic cells.
MATERIALS AND METHODS
Ethical Approval
Mature 68-week-old female hamsters and human
sperm samples were used in the current study. The
animals were maintained under standard laboratory
conditions (12 hr light:12 hr darkness cycle). The human
sperm samples were collected from two healthy male
donors after informed consent was received. All
the protocols used in the current study were approved
by the Ethical Review Committee of Shantou University
Medical College and conformed to the National
Institutes of Health Guidelines for humane animal care
and use in research, and to the ethical guidelines of
the 1975 Declaration of Helsinki as reected in the a
priori approval by the institutions Human Research
Committee.
pIRES2-EGFP-gag Construct
The full length of HIV-1 gag DNA was PCR-amplied
from plasmid DNRF of lentivirus vector and sub-cloned
into the EcoRI and BamHI site of the plasmid pIRES2GFP to generate recombinant plasmid pIRES2-EGFPgag. The primers (Shanghai Invitrogen Biotechnology
Co., Ltd., Shanghai, China) used for PCR amplication

17

are forward, 50 -CCGGAATTCATGGGTGCGAGAGCGTC-30 with the EcoRI restriction site in 50 and reverse,
50 -CGCGGATCCTTATTGTGACGAG GGGTCG-30 with
the BamHI restriction site in 50 . The amplication
program was as follows: denaturing at 948C for 2 min,
and 35 cycles each at 948C for 30 sec, annealing at 648C
for 40 sec, and extension at 728C for 1 min, followed by a
nal extension at 728C for 5 min. Amplications under
the same conditions using plasmid DNRF and water as a
template were served as positive and negative controls,
respectively. The amplication products were analyzed
routinely by staining with ethidium bromide after
electrophoresis on 1.2% agarose gel. DNA was extracted
by a DNA purication kit (Qiagen, Guangzhou, China).
The product was then digested by EcoRI and BamHI at
378C for 20 hr and puried using a DNA purication
column. Plasmid pIRES2-EGFP was also cut by EcoRI
and BamHI at 378C for 1 hr. The puried DNA was
ligated into cut plasmid by a T4 ligase kit (Takara
Biotech, Dalian, China), and subjected to transformation into JM109 E. coli according to the manufacturers
instructions. The successful cloning was conrmed by
PCR, followed by restriction digestion mapping using
EcoRI and BamHI and DNA sequencing.
Exposure of Human Sperm to Plasmid and IVF
Semen preparation and ova collection. Motile
human sperm and hamster ova were collected and
prepared as described previously [Huang et al., 2002; Ali
et al., 2006]. Briey, motile human sperm were collected
by a swim-up method; Ca2 ionophore (Sigma, St. Louis,
MO) was used to facilitate the capacitation. The ova
were collected from golden hamsters after being superovulated with PMSG and HCG (Ningbo Hormone
Product, Ningbo, China), and sequentially treated with
0.1% hyaluronidase and 0.1% trypsin (Sigma Chemical,
Shanghai, China) to remove cumulus cells and the zona
pellucida, respectively.
Exposure of human sperm to plasmid pIRES2EGFP-gag. Fifty microliters of a mixture containing
0.75 mg recombinant plasmid and 3 mg of FuGENE1 HD
(Roche, Guangzhou, China) were made using HEPES
buffer saline (HBS) and incubated at room temperature
for 15 min. Fresh BiggersWhittemWhittingham
(BWW) medium supplemented with 0.3% bovine serum
albumin (BSA; Sigma) was used for sperm capacitation.
The human sperm were exposed to plasmid before
capacitation. Three hours later, 50 ml droplets of sperm
suspension under mineral oil (Sigma) were incubated in
a Petri dish (35
10 mm2), and kept in a CO2 incubator
(378C, 50 ml/L CO2 in air) for another 1 hr.
Insemination and post-insemination culture.
Insemination was undertaken with the sperm suspension at a concentration of 106/ml. The oocytes were kept
in the sperm suspension for 2030 min, and then
transferred and incubated in BWW medium with 0.3%
BSA under mineral oil (Sigma) for another 1 hr
to ensure sperm penetration. Then, the ova were
washed twice in ovum culture medium containing 15%
J. Med. Virol. DOI 10.1002/jmv

18

Wang et al.

heat-inactivated fetal bovine serum (FBS), and then

transferred into a Petri dish with 1.5 ml of OCM
under mineral oil for post-insemination culture, which
were separated into two groups. One group was
incubated for 24 hr until formation of two-cell embryos.
Another group was incubated for 8 hr, and then 0.5 ml of
OCM containing 0.16 mg/ml of podophyllotoxin (Sigma)
and 0.16 mg/ml of vinblastine (Sigma) was gently added
to the medium to block karyogamy and rst-cleavage
spindle formation of the ova; the nal concentration was
0.04 mg/ml for both chemicals. Sixteen hours later, many
ova had reached metaphase and their pronuclei became
invisible under the dissecting microscope, which were
selected for making chromosome slides.
Preparation of Chromosome Slides
Slides of metaphase chromosomes or the inter-phase
nuclei of embryos were further prepared with a gradual
xation-air-dry method ([Rudak et al., 1978; Kamiguchi
and Mikamo, 1986]. Briey, about 510 zygotes or twocell embryos were treated with hypotonic solution (0.9%
sodium citrate in distilled water containing 3% FBS) at
378C for 30 min, and then transferred into xative
I (methanol:acetic:water [5:1:2]). Seven minutes later,
the color of zygotes or embryos changed from brown to
white and became sub-transparent. Then, the zygotes or
embryos, together with a small amount of xative I,
were aspirated and put on a clean glass slide under a
dissection microscope, and immediately covered with
xative II (methanol:acetic [3:1]). The slides were
immediately put into a Coplin jar lled with xative II
and incubated for a minimum of 5 min. Finally, the
slides were carefully dipped into xative III (methanol:
acetic:water [3:3:1]) for 1 min and dried by blowing with
warm moist air.
Fluorescence In Situ Hybridization (FISH)
Labeling HIV-1 DNA probe with uorescein
isothiocyanate (FITC). HIV-1 gag DNA was labeled
with biotin-14-dATP by a nick translation method
(BioNickTM Labeling System; Invitrogen, Shanghai,
China) according to the manufacturers protocol. The
labeled oligonucleotides were puried by a column and
stored at 208C.
In situ hybridization. Slides containing metaphase chromosomes of human sperm or nuclei of twocell embryos were treated with 100 g/ml RNase (Sigma)
at 378C for 60 min with 200 mg/mL of pepsin (Sigma) in
0.01 M HCl at 378C for 10 min, and with 1% paraformaldehyde in 1
PBS at room temperature for 10 min in
succession. The slides were then denatured at 738C for
4 min in 70% formamide in 2
SSC. Hybridization
buffer containing 50% deionized formamide (5 ml), 10%
dextran sulfate (2.5 ml), 40 ng/L uorescein isothiocyanate (FITC) labeled HIV-1 gag probe (1.5 ml), and 500 ng/
L of sheared salmon sperm DNA (1 ml) in 2
SSC was
denatured at 738C for 8 min and put on ice immediately
and then annealed at 378C for 25 min. Hybridization
buffer (10 ml) was added to each slide, and incubated at
J. Med. Virol. DOI 10.1002/jmv

378C overnight in a moisture chamber after being

sealed perfectly with rubber cement between slide and
coverslip. The next day, after removing rubber cement,
the slides were shaken gently in 50% formamide
to remove the cover slips and pre-incubated with a
blocking buffer (10% skim milk in 4
SSC) at 378C in a
moist box for around 20 min, then incubated with FITC
(avidin FITC, blocking buffer 1:100), BAA (BAA,
blocking buffer 1:100), and FITC (avidin FITC, blocking buffer 1:100) at 378C for 30 min sequentially. The
slides were then counterstained with 0.5 ml 1
PBS
with 0.02% PI at room temperature for 15 min.
After being covered with 30 ml of 1,4-diazobicyclo(2,2,2)octane (DABCO; Sigma) and a coverslip in turn,
the slides were observed under a uorescent microscope
immediately.
Additionally, the same uorescence in situ hybridization (FISH) steps were carried out to detect the
presence of HIV-1 gag DNA in the infected human sperm
on slides. Human sperm without transfection served as
a negative control of FISH.
Nested RT PCR and PCR
Extraction of DNA from human sperm. To
obtain sperm DNA, 500 ml of sperm suspension was
mixed with an equal volume of lysis buffer containing
0.03% proteinase K and 0.2 mg/ml of RNase I, and then
incubated at 558C overnight with gentle shaking. After
incubation, 100 ml of phenol and chloroform were added
and the homogenates were vortexed gently prior to
centrifugation at 8,000g for 10 min at 108C. The aqueous
layer was transferred into a new tube and 2
vol of precooling 70% ethanol was added to the aqueous layer to
precipitate the DNA. The DNA pellet was air dried for
10 min and then dissolved with 20 ml of TE buffer.
Extraction of RNA from human sperm. To
obtain sperm RNA, a guanidinium thiocyanate
phenolchloroform extraction method was used.
Briey, 5
106 sperm/ml were treated in Trizol at room
temperature for 5 min, then mixed thoroughly with
200 ml of chloroform prior to incubation for 5 min. The
aqueous layer was mixed well with an equal volume of
isopropanol prior to incubation for 20 min to precipitate
RNA. The RNA was pelleted as above, washed twice
with 80% ethanol, and dissolved in 30 ml of diethyl
pyrocarbonate (DEPC) water. The RNA solution was
then treated with DNase I (10 U in lysis buffer) at 378C
for 30 min, and subjected to the same procedures to
pellet RNA.
RT-nest PCR using sperm RNA. The extracted
sperm RNA was reverse transcripted according to the
protocol of a RNA PCR kit (AMV) version 3.0 (Takara
Biotech). HIV-1 gag cDNA in the RT product was
detected by PCR. The primer pair for human b-actin
was used to amplify a 396 bp fragment from the
sperm RT product to be served as an internal control
of RT-PCR. Then, the product of PCR was performed
in another round of PCR amplication using primers
specic for HIV-1 p24, including forward, 50 -AGC-

Transmission of HIV-1 gag Gene by Sperm

CAAAATTACCCTATAGTGCAGA-30 and reverse, 50 -TTGTTACTT GGCTCATTGCTTCA-30 . The procedures,

conditions, and controls of PCR were the same as those of
gag PCR, as described above.
RT-nest PCR of two-cell embryos. A Cells-tocDNATM II kit (Ambion, Shanghai, China) was used to
detect the presence of the HIV-1 gag mRNA in two-cell
embryos derived from the transfected spermatozoa.
Briey, 1020 embryos which expressed GFP were
selected to perform two-step RT-PCR, according to the
manufacturers protocol. The main procedures were as
follows: at the beginning, the embryos were twice
washed immediately with cold DPBS (0.1% DEPC in
PBS), then moved into 100 ml of ice-cold cell lysis II
buffer, mixed well, and incubated at 758C for 10 min.
The sample was then put on ice for 5 min, mixed well,
incubated with DNase I (10 U in lysis buffer) at 378C for
30 min, put on ice for 8 min, followed by another 5 min
incubation at 758C to inactivate DNase I. The lysate was
directly used for reverse transcription (RT). The RT
mix was as follows: 10 ml of cell lysate (RNA), 4 ml
of dNTP mix, 2 ml of random decamers, 2 ml of 10
RT
buffer, 1 ml MLV reverse transcriptase, and 1 ml of RNase
inhibitor. For negative control of RT, nuclease-free
water was used to replace reverse transcriptase. The
conditions for the RT reaction were 428C for 20 min and
958C for 10 min. The product of RT was then followed
with two rounds of PCR (nested) to amplify the cDNA
sequence of gag and p24 with the conditions described
above. PCR products amplied with a primer pair
specic for mouse b-actin was served as an internal
control. Positive and negative controls in PCR were
conducted using plasmid and water as templates,
respectively.
PCR of sperm DNA. The extracted sperm DNA
was subjected to two rounds of PCR to detect the
presence of HIV-1 gag DNA as the conditions described
above.

19

human sperm without transfection were detected in the

same way.
Expression of HIV-1 p24 in transfected human sperm
was observed on slides with similar steps as described
above. Human sperm without transfection served as a
negative control, whereas detection of sperm protein
using anti-human sperm polyclonal antibody (made and
stored by our laboratory) and FITC-conjugated rabbit
immunoglobulin were served as a positive control of
indirect immunouorescence (IF).
RESULTS
Identication of Recombinant Plasmid
A new recombinant plasmid pIRES2-EGFP-gag construct was identied by EcoRI and BamHI digestion,
PCR, and DNA sequencing. A fragment of 1,503 bp can
be obtained after digestion of the recombinant plasmid
(Fig. 1A). Using a primer pair specic for HIV-1 gag, a
band of 1,521 bp can be PCR-amplied (Fig. 1B).
Sequencing results showed 98% identity between the
sequence of the PCR product with that of HIV-1 gag
(data not shown). The results demonstrated that the
fragment of HIV-1 gag DNA was constructed successfully into a recombinant plasmid.
Presence of gag DNA in Human Sperm

Indirect Immunouorescence (IF)

Sequence of HIV-1 gag in extractive DNA from

infected human sperm was detected by PCR and FISH.
The objective band (1,521 bp) was successfully PCRamplied from DNA extracted from transfected human
sperm, whereas no band was obtained in negative
controls in which the template was replaced with water
(Fig. 2A). The FISH signal is visible in the head of
transfected human sperm (Fig. 3A), and not in those
without transfection (Fig. 3B). These results indicated
that the fragment of HIV-1 gag DNA was successfully
integrated into the human sperm genome by transfection of a recombinant plasmid.

Two-cell embryos were collected and twice washed

carefully with 1
PBS, then xed in 4% paraformaldehyde solution for 15 min at room temperature. After
washing, the embryos were permeabilized with a
mixture of 0.5% Triton X-100, 0.5 mM MgCl2, 5 mM
EGTA, and 50 mM glycine for 15 min, washed again, and
incubated with blocking solution (0.1% phosphate buffer
with 2% fetal calf serum and 10% goat serum) at 48C for
1 hr. Subsequently, the embryos were incubated
with primary antibody (HIV-1 p24, mouse monoclonal
antibody, 1:50 in 1
PBS; Santa Cruz Biotechnology,
Inc., Santa Cruz, CA) at 378C for 2 hr, washed with 1

PBS for 3 times, and then incubated with FITCconjugated rabbit immunoglobulin (1:50 in 1
PBS;
ZSGB-BIO, Beijing, China) at 378C for 40 min. After
thrice-washing, the embryos were counterstained with
50 mg/ml of PI for 5min. The embryos were then transferred onto glass slides and observed under uorescence
microscopy. For comparison, the embryos derived from

Fig. 1. Identication of recombinant plasmid. A: Recombinant

plasmid was digested by EcoRI and BamHI: M, Marker DL15000;
lane 1, recombinant after digestion; lane 2, recombinant plasmid
without digestion. B: gag DNA was detected in recombinant plasmid by
PCR amplied with the gag-specic primers: M, Marker DL2000; lane
1, PCR product; lane 2, recombinant plasmid. Product of PCR was
routinely analyzed by electrophoresis on 1.2% agaroseTAE gel and
was visualized with ethidium bromide present in the gel (the same
below).

J. Med. Virol. DOI 10.1002/jmv

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Wang et al.

Fig. 2. Presence of HIV-1 gag DNA and its transcription in sperm

and two-cell embryos. A: gag DNA was detected from human sperm by
PCR, lane 1, Marker D2000; lane 2, sample: infected sperm, primer:
gag specic; lane 3, positive control; lane 4, negative control. B: mRNA
of gag and p24 was detected from human sperm by RT-PCR. B1: First
round PCR amplication, lane 1, Marker DL2000; lane 2, sample: RT
product of infected sperm, primer: gag specic; lane 3, sample: product
of RT(), primer: gag; lane 4, sample: RT product of infected sperm,
primer: human b-actin specic. B2: Second round PCR amplication,
lane 1, Marker DL2000; lane 2, sample: rst round PCR product of
infected sperm; lane 3, sample: rst round PCR product of RT-PCR

negative control; lane 4, negative PCR control using primers specic

for p24 for all sample in second round. C: Detection of mRNA of gag and
p24 in two-cell embryos derived from infected sperm. C1: First round
PCR amplication, lane 1, Marker DL2000; lane 2, sample: RT
product of embryos derived from infected sperm, primer: gag specic;
lane 3, sample: product of RT-PCR negative control, primer: gag
specic; lane 4, PCR negative control; lane 5, sample: RT product of
embryos from infected sperm, primer: mouse b-actin. C2: Second round
PCR amplication, lane 1, Marker DL2000; lane 2, sample: rst round
PCR product of embryos from infected sperm; lane 3, sample: rst
round PCR product of RT-PCR negative control.

Fertilization of Zona-Free Hamster Ova

With Transfected Human Sperm

amplication, a 1,521 bp band from the RT product of

infected human sperm and a 396 bp band from internal
controls was observed (Fig. 2B1 in lanes 1 and 3), and no
band in the negative control of RT and PCR (lanes 2 and
4). In the second round of amplication, a 730 bp band
was present from the rst round PCR product of
transfected human sperm, and the band was absent in
the negative control of RT and PCR (Fig. 2B2). For
embryos, only a 265 bp band of the internal control was
observed in the rst round PCR amplication (Fig. 2C1).
In the second round amplication, a 730 bp band was
amplied from the rst round PCR product of embryos
derived from transfected human sperm, and no band
was detected in the negative control of RT and PCR
(Fig. 2C2). The results showed that HIV-1 gag mRNA
was present in transfected human sperm and embryos;
HIV-1 gag mRNA in transfected human sperm was
easier to detect than in embryos.
HIV-1 P24 protein was detected in two-cell embryos
using IF; a green signal of IF was shown in embryos
derived from transfected human sperm (Fig. 4D, arrow).
Whereas no signal was observed in human sperm
exposed to plasmid for 36 hr (Fig. 4A). Likewise, a signal
was not detected in both negative controls (Fig. 4B,E).
However, a strong signal was detected in the positive
control of IF in which the sperm protein was detected
by anti-human sperm antibody and FITC-conjugated
rabbit immunoglobulin (Fig. 4C, arrow).

Oocytes (2050) were collected from each superovulated hamster. Fifty hamsters were used in
the study. The fertilization rate of zona-free hamster
ova with transfected human sperm ranged from 10% to
40%.
Integration and Replication of HIV-1 gag DNA
To detect the integration of HIV-1 gag DNA into the
genomes of sperm and embryos, FISH was carried out on
slides with chromosomes of human sperm and the
interphase nuclei of embryos. The hybridization signal
of FISH was observed in the chromosomes of transfected
human sperm (Fig. 3C), as well as in the male pronuclei
of zygotes (Fig. 3E), and in both nuclei of two-cell
embryos derived from transfected human sperm
(Fig. 3F); no signal was observed in control slides
(Fig. 3D,G).
Expression of HIV-1 p24 in Human Sperm
and Two-Cell Embryos
The mRNA of HIV-1 gag and p24 in transfected
human sperm and in two-cell embryos derived from
transfected human sperm was detected by nested RTPCR. For human sperm, in the rst round of PCR
J. Med. Virol. DOI 10.1002/jmv

Transmission of HIV-1 gag Gene by Sperm

21

Fig. 3. Presence of gag DNA in infected human sperm and its integration into genome of sperm and
embryo. A: Detection of HIV-1 gag DNA from infected human using FISH. Hybridization signal was
observed in infected sperm (arrow), the result indicated that gag DNA had been successfully transferred
into human sperm. C: Detection of HIV-1 gag DNA chromosome of human sperm using FISH.
Hybridization signal was also found in chromosome of infected human sperm (arrow), (E) in nucleus of
embryo derived from infected sperm (arrow), and occasionally, (F) in both nuclei of the two-cell embryos
(arrow). Yet, no signal can be found in negative control of them (B,D,G), magnication: 1,000
.

DISCUSSION
The transmission routes of HIV-1, including sexual
contact, needle and stick sharing, transfusions, and
perinatal transmission from mother-to-infant have been
documented. However, the vertical transmission of
HIV-1 from father to offspring via human sperm has
not been validated. Although Baccetti et al. [1994] found
that HIV-1-like particle can be transferred from infected
human sperm to oocytes through IVF, it only offered the
possibility of transmission of virus via sperm and the
exact molecular mechanism was still obscure.
The gag gene of HIV-1 is a group-specic antigen gene,
and it plays a crucial role in HIV-1 pathogenesis since it
provides the basic physical infrastructure of the virus,
and codes for p24 (the viral capsid), p6 and p7 (the
nucleocapsid proteins), and p17 (a matrix protein)

[Freed, 1998]. Thus, the HIV-1 gag gene was rst

selected in our study as a target gene to be investigated
in the present study. Human sperm were transfected
with recombinant plasmid containing the HIV-1 gag
gene, and then fertilized in vitro with zona-free hamster
ova to determine whether the HIV-1 gag gene can
integrate into human sperm genome, and whether or
not the HIV-1 gag gene integrated into the sperm
genome can replicate itself within the host genome and
express its function if introduced into embryos by
fertilization.In the present study, recombinant plasmid
pIRES2-EGFP-gag was successfully constructed and
identied by EcoRI and BamHI digestion, PCR
(Fig. 1A,B), and DNA sequencing (data not shown).
After human sperm were transfected with pIRES2EGFP-gag plasmid and fertilized with zona-free
hamster ova, the positive FISH signals for HIV-1 gag
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Wang et al.

Fig. 4. Expression of p24 in human sperm and two-cell embryos. A,B: Detection of the expression of p24
using immunouorescence (IF). No signal of IF can be observed in infected human sperm, and its negative
control without transfection. C: Strong IF signal is visible in positive control (sperm detected by antihuman sperm) of IF, counterstained with PI, magnication: 1,000
. D: IF signal (arrow) can be observed in
two-cell embryos derived from infected sperm. E: No signal in its negative control which embryo derived
from human sperm without infection, counterstained with PI, magnication: 400
.

DNA were observed in human sperm nuclei (Fig. 3A),

human sperm chromosomes (Fig. 3C), and male pronuclei of zygotes (Fig. 3E). Thus, the HIV-1 gag gene was
able to pass through the sperm membrane and integrate
into the sperm genome, and human sperm carrying the
HIV-1 gag gene was able to enter oocytes and achieve
normal fertilization. Moreover, the positive FISH
signals for HIV-1 gag DNA was found in both nuclei of
two blastomeres of two-cell embryos (Fig. 3F), suggesting that the replication of the sperm-mediated HIV-1
gag gene was synchronized with that of the host genome.
Then, the two gene copies were segregated into the two
daughter cells during the rst cleavage, which revealed
a possible replication pattern of the HIV-1 gene in the
early embryo. Using RT-PCR, the positive bands for the
HIV-1 gag gene were observed in the samples from
transfected human sperm (Fig. 2B1,B2) and two-cell
embryos containing the sperm-mediated HIV-1 gag
gene (Fig. 2C1,C2), which indicated that the HIV-1 gag
gene was able to be transcribed into mRNA in human
sperm and embryonic cells. Failure of detection of the
expression of HIV-1 p24 protein in transfected human
sperm is likely due to a lack of protein translational
system in spermatozoa [Gur and Breitbart, 2008]. IF
results clearly showed that p24 protein was present
in two-cell embryos (Fig. 4D), suggesting that the spermmediated HIV-1 gag gene was expressed at the protein
level in early embryos.
This study provided evidence to suggest the following:
(1) the HIV gene may be able to integrate into the
J. Med. Virol. DOI 10.1002/jmv

genome of human sperm, either in the nuclei or

chromosomes of sperm, and human sperms carrying
HIV-1 gag gene are able to enter oocytes and achieve
normal fertilization; (2) the gag gene can be transcribed
(producing mRNA) and then translated (producing
protein) in the early embryonic cells; and (3) the
replication of the sperm-mediated HIV-1 gag gene was
synchronized with that of the host genome, which
revealed a possible replication pattern of the HIV-1
gene in the embryo, but was different from the
replication pattern of the virus gene in its life cycle.
Overall, this study provides evidence that the HIV-1
gag gene may be transmitted vertically to the next
generation via human sperm. Replication, transcription, and translation of the HIV-1 gag gene are
maintained through at least one cycle of embryonic cell
division. Further research is necessary to conrm the
acquisition of HIV infection by vertical transmission of
HIV via human sperm by the real virus infection or the
transfection with other HIV genes. It is known that HIV1 contains 9 genes, and any single gene of the virus
cannot completely represent the entire virus in its
transmission and other biological behavior.
ACKNOWLEDGMENTS
We are very grateful to Dr. De-yi Liu from the
University of Melbourne, Dr. Golam Sabbir from the
Manitoba Institute of cell Biology (Canada), and Hongmei
Zeng from the University of Manitoba for their assistance

Transmission of HIV-1 gag Gene by Sperm

with English grammar correction. We also thank

Prof. Kailin Xu of the Xuzhou Medical College (China),
for his kindness in providing us a lentivirus vector.
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J. Med. Virol. DOI 10.1002/jmv