Immunogenetics (2000) 52 : 7380

DOI 10.1007/s002510000253

ORIGINAL PAPER

Dean Tatlow Robert Brownlie Lorne A. Babiuk

Philip Griebel

Differential display analysis of gene expression during the induction

of mucosal immunity

Received: 29 September 1999 / Revised: 4 August 2000 / Published online: 18 October 2000
 Springer-Verlag 2000

Abstract One approach to understanding the physiologically relevant events during the induction of an
immune response is to identify genes that are
expressed when the immune system first encounters
antigen. Such an investigation requires a naive but
fully functional immune system, and the fetal lamb
provides these conditions during the last trimester of
gestation. `Intestinal segments,' containing a jejunal
Peyer's patch, were surgically prepared in fetal lambs
(>120 days gestation) and individual `intestinal segments' were injected with either culture medium or
infectious bovine rotavirus. Peyer's patch tissue was
collected 18 h postinfection. Histology and virus culture confirmed that bovine rotavirus had infected the
mucosal epithelium. RNA was extracted from jejunal
Peyer's patch tissue and mRNA differential display
was used to identify genes expressed following rotavirus infection. Ten cDNAs were identified by differential display and these cDNAs were isolated, cloned,
and sequenced. One of the cDNAs sequenced, displayed homology to the gene encoding the sperm surface protein Sp17. Differential expression of this gene
in antigen-exposed jejunal Peyer's patches was confirmed by Northern blot and RT-PCR. The complete
sequence for sheep Sp17 mRNA was obtained from a
l cDNA library, prepared from the jejunal Peyer's
patch of a young lamb. Sp17 expression was detected

The nucleotide sequence data reported in this paper have been

submitted to the GenBank nucleotide sequence database and
have been assigned the accession number AF179926
D. Tatlow R. Brownlie L.A. Babiuk P. Griebel ())
Veterinary Infectious Disease Organization,
120-Veterinary Road, Saskatoon, Saskatchewan, S7N 5E3,
Canada
E-mail: griebelp@sask.usask.ca
Phone: +1-306-9667472
Fax: +1-306-9667478

by RT-PCR in a variety of mucosa-associated lymphoid tissues but not in primary or other secondary
lymphoid tissues. Thus, the fetal lamb model may be
appropriate for identifying genes relevant to mucosal
immunity.
Keywords Differential display Immune response
Mucosal immunity Sheep Sp17 Rotavirus

Introduction
Many of the signaling mechanisms that may potentially play a role during the induction of a mucosal
immune response have been identified (reviewed in
McGhee 1999). These studies clearly demonstrate that
the induction and regulation of an immune response is
very complex, involving a number of soluble factors,
cell-cell interactions, and intracellular signaling pathways. Furthermore, these events may be closely regulated in a temporal fashion. Both in vitro and in vivo
systems have been used to identify cellular signals that
may regulate antigen-specific activation of T and B
cells and antigen-presenting cells (Clark and Ledbetter
1994; Grewal and Flavell 1996). However, the physiological relevance of these molecules has been difficult to assess, particularly in the context of mucosa-associated lymphoid tissue (MALT).
The identification of genes expressed during the
induction of a mucosal immune response should provide significant insight regarding the mechanisms of
immune regulation. However, analyzing these events
in animal models has not been possible because the
immune system is continually exposed to environmental antigens and potential pathogens. Such an analysis
requires a naive but immune-competent immune system that can be exposed to antigen in a controlled
manner. Analyzing gene expression at specific time
points following the induction of an immune response
may then be possible.

74

The fetal lamb provides an appropriate model to

analyze gene expression during the initiation of an
immune response (reviewed in Hein 1995). The placenta of sheep ensures that the fetus develops in isolation from exogenous antigen and maternal antibody.
Also of importance is that the jejunal Peyer's patches
(PPs) appear to be fully developed during the last trimester of gestation (>120 days) (Reynolds and Morris
1983) and the jejunal PP is known to be an efficient
site for the induction of mucosal immune responses
(Mutwiri et al. 1999). Our analysis of surface immunoglobulin (sIg) expression on B cells isolated from jejunal PPs of fetal lambs confirmed that isotype switching does not occur prior to birth (unpublished data).
This observation is consistent with a naive immune
system or an immune system that cannot respond to
antigen (Clark and Ledbetter 1994). However, the gutassociated lymphoid tissue of fetal lambs appears to
be immune competent, since oral immunization of the
fetus can induce a mucosal immune response (Husband and McDowell 1975; Richardson and Conner
1972). These observations suggest that fetal lambs provide a naive but immunologically competent mucosal
immune system. To analyze immune responses in this
system, a surgical model has been developed that permits antigen delivery to the fetal jejunal PP (Mutwiri
et al. 1999). Thus, the jejunal PP of fetal lamb was
used as a model system to investigate genetic events
during the induction of a mucosal immune response.
One method that has been very useful for analyzing
gene expression in tissues is mRNA differential display (DD). This technique was originally described by
Liang and Pardee (1992) and is a rapid method of
identifying differences in mRNA expression between
variant tissues of the same genetic background. This
technique has been successfully used to identify
numerous novel and known transcripts that are differentially expressed in various biological systems. For
example, gene expression has been analyzed during
graft rejection (Wang and Feuerstein 1997), cell death
(Zhang and Zhang 1996), and malignant cell transformation (Dong et al. 1997). These investigations identified differential expression of known and novel genes
not previously associated with these specific events.
Thus, the objective of the present investigation was to
identify genes that are differentially expressed following the induction of a mucosal immune response.

Materials and methods

Animals
Suffolk sheep were obtained from the Department of Animal
and Poultry Science, University of Saskatchewan, and were
cared for according to guidelines set forth by the Canadian
Council for Animal Care. Ewes were bred during a 2-day interval following estrus synchronization, and pregnancy was confirmed by two successive ultrasound examinations at days 45
and 105 of gestation. Fetal surgeries were done between days

120130 of gestation. The fetal surgery procedure was as

described by Mutwiri and co-workers (1999). Briefly, jejunal PPs
were visible on the serosal surface as pale areas with increased
vascularity. A 4- to 5-cm length of intestine was selected anterior and posterior to the jejunal PP and the intestine was transected at these points. The mesenteric attachment and vascular
supply were maintained for each `intestinal segment' and the
ends of each segment were closed with an inverting suture. The
continuity of the intestine was re-established with an end-to-end
anastomosis and the incisions in the fetus and ewe were closed
as described previously (Smeaton et al. 1969).
Virus
Bovine rotavirus (BRV), strain C486, was selected as our model
antigen since this virus is an important enteric pathogen (Dodet
et al. 1997) and the RNA generated by this virus contains no
poly(A) signal (Estes 1996). This was important to ensure that
viral genes were not amplified by DD. BRV was grown in confluent MA-104 cells in the absence of fetal bovine serum (Gibco/BRL, Grand Island, N.Y.) and in the presence of 1 g/ml of
trypsin (Difco, Detroit, Mich.) (Babiuk et al. 1977). Cells were
cultured in minimum essential medium (MEM; Gibco/BRL) and
after 48 h, cells and supernatants were harvested together. The
cellular debris was cleared by centrifugation at 1000 g for 20 min
and the supernatant concentrated and partially purified by centrifugation through a 40% sucrose cushion at 112,700 g at 15 C
for 2.5 h. The virus pellet was resuspended in sterile distilled
water.
Rotavirus plaque assay
Plaque assays for BRV titration were performed as described
previously (Aha and Sabara 1990). Briefly, 12-well tissue culture
plates, containing confluent MA-104 cells, were washed twice
with MEM before incubating cells for 1 h with 200 l of tenfold,
serially diluted BRV. Cells were then washed once with MEM
and overlaid with 1 ml of MEM containing 2% Sephadex G-75
beads (Amersham Biotech, Baie d'Urfe, Canada), 20 mM L-glutamine (Gibco/BRL), and 25 g/ml of pancreatin (Gibco\BRL).
Plates were incubated for 48 h at 37 C before removing the
overlay and staining the cells (0.5% crystal violet, 80% methanol
in PBS) for 20 min at ambient temperature. Cells were then
washed gently with water, air-dried, and plaques were counted.
Histology
Sections of fetal intestinal tissue were fixed in 10% phosphatebuffered formalin and paraffin-embedded. Fixed sections were
stained with hematoxylin and eosin (H/E) and examined under
a light microscope.
Differential display
Sections of jejunal PP were collected 18 h postinfection and
snap-frozen in liquid nitrogen. Total RNA was isolated using
Tripure reagent (Roche Molecular Biochemicals, Laval, Canada)
according to the manufacturers protocol and the RNA was dissolved in DEPC-treated water. The concentration of RNA was
determined by absorbance at 260 nm and 50 g RNA was
digested with RNase-free DNaseI to eliminate chromosomal
DNA. DNase-treated RNA was phenol/chloroform-extracted
and ethanol-precipitated. DD was performed using an RNAimage kit from GenHunter (Nashville, Tenn.). Briefly, mRNA was
reverse-transcribed with MMLV reverse transcriptase using one
of three oligo-T primers. The resulting cDNAs were PCR-amplified using ampliTaq polymerase (Perkin-Elmer) with each
anchor primer in combination with one of the arbitrary primers

75
in the presence of 0.25 l of [a-32P]dCTP (3000 Ci/mol; Mandel,
Guelph, Canada). DNA fragments were resolved by electrophoresis on a urea 6% polyacrylamide gel and identified by
autoradiography. Bands of interest were extracted from the
sequencing gel and reamplified using the same primer set previously used in DD identification.
Cloning and sequencing
DNA fragments identified by DD were cloned into the EcoRV
site of pBluescript II KS(+) (Stratagene, La Jolla, Calif.) using
current molecular biology techniques (Sambrook et al. 1989).
Inserts were sequenced by an automated sequencing facility
(Plant Biotechnology Institute, Saskatoon, Canada).
Northern blot analysis
For Northern blot analysis, riboprobes were prepared to DNA
fragments cloned in the Bluescript vector. The DNA inserts,
flanked by T7 and T3 RNA polymerase promoters, were PCRamplified by universal primers. The purified PCR product was
then used as a template to produce antisense digoxigenin (DIG)labeled riboprobes. This template was transcribed in the presence of DIG-11-UTP (Roche Molecular Biochemicals) using
either T7 or T3 RNA polymerase according to the manufacturers instructions. The synthesized riboprobes were DNasetreated, ethanol-precipitated, and standardized before being
used.
Poly(A) RNA was prepared from total RNA by oligo(dT)
affinity chromatography (Oligotex mRNA extraction kit;
Qiagen, Mississauga, Canada). Total RNA (20 g per lane) or
poly(A) RNA (2.0 g per lane) was electrophoretically separated on a 1% denaturing agarose/formaldehyde gel and transferred to geneScreen nylon membranes (DuPont/NEN) by capillary transfer using regular methodology (Sambrook et al.
1989); fragments were fixed by UV cross-linking. Membranes
were incubated overnight with riboprobe (600 pg/ml) in NorthernMax hybridization buffer (Ambion, Austin, Tex.) at 65 C.
Blots were washed twice in 2standard sodium citrate (SSC),
0.1% SDS for 15 min at room temperature and once in 0.1SSC,
0.1% SDS for 15 min at 68 C. After washing, membranes were
processed using a DIG-luminescent detection kit (Roche Molecular Biochemicals), according to the manufacturer's instructions.
Briefly, membranes were incubated with anti-DIG-alkaline
phosphatase, washed, incubated with CDP-Star and exposed to
X-ray film for 30 min.
Construction and purification of l DNA
Jejunal PP tissue was collected from a 4-month-old, conventionally reared lamb. This tissue was selected since, in a lamb of this
age, the jejunal PP is exposed to a wide variety of environmental antigens and potential pathogens. Thus, the cDNA library
prepared from this tissue should contain most of the genes
expressed during the induction and differentiation of a mucosal
immune response. A cDNA library was prepared from this tissue using a Smart PCR cDNA Synthesis and Library Construction Kit (Clontech, Palo Alto, Calif.), following the manufacturer's instructions. The DNA, from 41011 pfu, was prepared
using a Qiagen l DNA purification kit.
RT-PCR analysis
For RT-PCR analysis, 10 g of DNaseI-treated total RNA was
reverse transcribed by Omniscript reverse transcriptase (Qiagen)
using oligo dT(18) in a total volume of 20 l for 1 h at 37 C. The
manufacturer's instructions were followed for the reaction and
the reverse transcriptase was then inactivated at 65 C for 5 min.

First strand-synthesized cDNA (2.0 l) was then used as the

template for amplification by PCR. The PCR reactions contained 50 pmol of each primer, 200 M dNTPs, and 2 units of
HotStart Taq polymerase (Qiagen) in a total volume of 50 l.
Sp17 was amplified using primers ACCAAGAAGATGTCG
ATTCC and AAACCAGTGTCCTCACTTGT. The resulting
430-bp fragment from jejunal PP RNA was cloned and
sequenced and found to have identity with the Sp17 cDNA previously sequenced. To assess Sp17 expression in different tissues,
primers to sheep b-actin (GGCATTGTCACCAACTG and
GTGTTGGCGTAGAGGTC) were included to validate RT
and PCR reactions. Amplification was carried out by an initial
incubation at 95 C for 15 min to inactivate Taq followed by 35
cycles of 94 C for 15 s, 55 C for 30 s, and 72 C for 30 s, with a
final extension at 72 C for 5 min. The resulting PCR product
(10 l) was then electrophoresed through a 1.5% agarose gel
and visualized by ethidium bromide staining. The molecular
weight markers used were l DNA-HindIII/fX-174 RF DNAHaeIII Digest (Amersham Biotech).

Results
Model for the induction of mucosal immune responses
BRV infection of young calves causes an acute infection characterized by damage to the mucosal epithelium and blunting of the intestinal villi (Varschney et
al. 1995). Thus, we chose BRV as an enteric RNA
virus that would be compatible with the analysis of
host genes expressed in the jejunal PPs following an
acute infection. Histological examination of fetal `intestinal segments' confirmed that the injection of
medium (MEM) did not alter the morphology of the
mucosal epithelium and intestinal villi (Fig. 1A). In
contrast, injection of infectious BRV resulted in villous atrophy and destruction of mucosal epithelium
(Fig. 1B). There was also increased fluid secretion
into the BRV-infected `intestinal segments' and this
fluid contained infectious virus (Table 1). We concluded from these observations that BRV was able to
infect and replicate in the mucosal epithelium of fetal
lamb intestine.
DD of mRNA expression in fetal jejunal PPs
To identify genes expressed during the induction of a
mucosal immune response, we used DD to compare
the cDNA profiles of fetal jejunal PP tissue exposed
to either BRV or MEM. A total of 30 different primer
combinations were used for the DD of up- and downregulated gene expression. Ten differentially expressed
cDNA fragments were recovered from the gels. To
reduce the frequency of false positives, we selected
cDNA fragments that were consistently differentially
expressed in duplicate control samples and duplicate
samples from the two infected `intestinal segments'
(Fig. 2). On this basis, nine cDNA fragments were
identified as upregulated and one cDNA fragment
was identified as downregulated following BRV infec-

76

Fig. 1A,B The effect of BRV infection on mucosal epithelium

in fetal `intestinal segments.' A Control `intestinal segment,' was
collected 18 h after injecting 1 ml MEM. B Infected `intestinal
segment' was collected 18 h after injecting 3.7107 pfu of BRV,
strain C486. Tissues were collected from `intestinal segments'
that were prepared in the same fetal lamb

tion. The ten cDNA fragments were reamplified and

cloned into vectors for sequencing. Nine of the cDNA
fragments had no significant homology to GenBank
database sequences. Analysis of these sequences
revealed that these cDNAs represented the 39 ends of
mRNA and did not contain an open reading frame.
One of the cDNA clones was a 460-bp fragment (Fig.
2) and the partial open reading frame present in this
fragment displayed significant sequence homology to
rabbit, mouse, baboon, and human Sp17.

Table 1 Rotavirus titer in

fetal lamb intestinal segments'
(ND no detectable viral
plaques)

Fig. 2 DD of mRNA isolated from jejunal PP tissue that was

collected from `intestinal segments' injected with either MEM or
BRV. Lanes 1, 2 control jejunal PP tissue collected from `intestinal segments' injected with 1 ml MEM; lane 3, 4 Inf#1
jejunal PP tissue collected from an `intestinal segment' that was
injected with 3.7107 pfu BRV; lanes 5, 6 Inf#2 jejunal PP tissue collected from a second `intestinal segment' that was
injected with 3.7107 pfu BRV. Primers used in DD are indicated at the top of each panel. The differentially expressed
cDNA fragment that was isolated is indicated as SP17

Intestinal
segments

Fluid collected
(ml/`intestinal segment')

BRV titer
(pfu/ml)

BRV titer
(pfu/`intestinal segment')

Infected#1a
Infected#2a
Control#1b
Control#2b

2.3
6.0
<0.2
<0.2

2.5107
2.0107
ND
ND

5.8107
1.2108
ND
ND

a
b

`Intestinal segment' injected with 3.7107 pfu BRV in 1 ml MEM

`Intestinal segment' injected with 1 ml MEM

77

Isolation of the sheep Sp17 gene from a jejunal

PP cDNA library
The full-length sequence of the sheep Sp17 open reading frame was obtained by PCR amplification using
DNA prepared from a l cDNA library to jejunal PP.
A primer corresponding to a 39 untranslated region of
Sp17 and a primer internal to the vector flanking the
59 region were used to PCR-amplify a 650-bp fragment. This fragment was cloned in the Bluescript vector and the full-length sequence of the Sp17 transcript
is shown in Fig. 3 along with the amino acid sequence
aligned with that for rabbit Sp17. The sheep Sp17 protein is 77% homologous with rabbit Sp17 and 5877%
homologous with the Sp17 protein reported for other
species (GenBank data).
Fig. 3 Nucleotide sequence of the sheep Sp17 gene isolated
from a l cDNA library to jejunal PPs collected from a conventionally reared lamb. The complete sequence and the deduced
amino acid sequence of the coding region of the sheep Sp17
gene are indicated. The amino acid sequence of the sheep and
rabbit Sp17 are aligned, and homologous amino acid sequence is
indicated in bold

Sp17 expression in testes and jejunal PP

Previous reports indicated that expression of the Sp17
gene was unique to testicular tissue (Adoyo et al.
1997; O'Rand et al. 1988). Therefore, we re-examined
the expression of the sheep Sp17 gene in testes and
jejunal PPs. A riboprobe to the 460-bp Sp17 fragment,
isolated by DD, was used to probe testicular and ovarian total RNA in Northern blots; a 600-bp riboprobe
to sheep actin was used as a control (Fig. 4A). This
analysis was consistent with previous reports that the
Sp17 gene was expressed in testes but not ovary
(Adoyo et al. 1997; O'Rand et al. 1988). Northern
blot analysis was then done to confirm that sheep
Sp17 was differentially expressed in naive, fetal jejunal
PPs and the jejunal PPs of conventionally reared
lambs. A detectable hybridization signal was observed
when the 460-bp Sp17 riboprobe was hybridized with
poly(A) RNA from lamb jejunal PPs but not fetal
jejunal PPs (Fig. 4B). No signal was detected when
using total RNA from fetal or lamb PPs (data not
shown), suggesting that the Sp17 transcript was much
more abundant in testes.

78

Fig. 4A,B Northern blot analyses using an Sp17 riboprobe generated from the cDNA fragment generated by DD. A Twenty
micrograms of total RNA from sheep ovary and testes was
hybridized with sheep actin and Sp17 riboprobes. B Two micrograms of poly(A) RNA from jejunal PPs, collected from an `intestinal segment' injected with MEM (Naive J.P.P.), and 2 g of
poly(A) RNA from jejunal PP, collected from intestine of a conventionally reared young lamb (J.P.P.) was hybridized with the
Sp17 riboprobe

Sp17 expression in lymphoid tissues

The differential expression of Sp17 in rotavirus-infected jejunal PPs of fetal lambs raised questions
regarding the expression of this gene in lymphoid tissues of animals that are exposed to a broad range of
environmental antigens and potential pathogens.
Therefore, the expression of Sp17 was determined by
RT-PCR in a variety of lymphoid tissues collected
from 4- to 6-month-old, conventionally reared lambs.
Lymphoid tissues were chosen to include a variety of
mucosa-associated lymphoid tissues (jejunal PPs, jejunum, mesenteric lymph node, retropharyngeal lymph
node), peripheral lymphoid tissues (spleen, prescapular lymph node), and primary lymphoid tissues (thymus, ileal PPs) that do not play a role during the
induction of an antigen-specific immune response. A
670-bp fragment corresponding to actin was observed
in all the reactions with the exception of controls without transcriptase thus validating RT and PCR con-

ditions (Fig. 5). Results are representative for RNA

prepared independently at least twice for each tissue.
A 450-bp fragment corresponding to Sp17 was
observed for RNA prepared from testes but not ovary
tissue or fetal jejunal PPs. The Sp17 transcript amplified from testes RNA was cloned and the sequence
was identical to the Sp17 RNA species isolated from
the jejunal PPs (data not shown). In addition, Sp17
expression was clearly observed in all mucosa-associated lymphoid tissue: jejunal PPs (lane 3), non-PP
jejunum (lane 6), the mesenteric lymph node (lane 10)
which drains the intestine, and the retropharyngeal
lymph node (lane 8) which drains the oral cavity. In
contrast, Sp17 expression was not detected in either
peripheral lymphoid tissues, such as spleen (lane 9)
and prescapular lymph node (lane 11), or primary
lymphoid tissues, such as thymus (lane 7) or ileal PPs
(lane 5). The ileal PP is developmentally distinct from
the jejunal PP and plays a major role in the generation of the preimmune B cell repertoire in ruminants
(reviewed in Griebel and Hein 1996). Thus, the RTPCR analysis revealed detectable Sp17 expression in a

79

Fig. 5 RT-PCR analysis of Sp17 gene expression in lymphoid

tissues. Lane 1 testes; lane 2 ovary; lane 3 jejunal PP tissue; lane
4 jejunal PP tissue (fetal lamb); lane 5 ileal PP tissue; lane 6
jejunum tissue; lane 7 thymus; lane 8 retropharyngeal lymph
node; lane 9 spleen; lane 10 mesenteric lymph node; lane 11
prescapular lymph node; lane 12 testes without reverse transcriptase. All lymphoid tissues, except the fetal jejunal PP, were collected from 4-month-old conventionally reared lambs

broad variety of lymphoid tissues with an apparent

specificity for mucosa-associated lymphoid tissues
involved in the generation of antigen-specific immune
responses.

Discussion
DD has been used effectively to analyze gene expression both in vitro (Dong et al. 1997; Zhang and Zhang
1996) and in vivo (Wang and Feuerstein 1997). The
present investigation used DD to analyze gene expression during the induction of a host response to an
acute BRV infection. Fetal lamb `intestinal segments',
containing jejunal PPs, were used as a model system
for a naive immune system responding to the antigenic
stimulus of a BRV infection. The isolation of ten
cDNAs from the DD indicated that BRV infection
had induced gene expression in the fetal jejunal PPs.
However, significant gene homology was identified for
only one of ten cDNAs cloned. The most likely explanation for this observation is the amplification of 39

untranslated regions, and we are currently isolating

full-length clones from cDNA libraries to identify
these genes. Alternative DD techniques, such as
restriction fragment DD (Bachem et al. 1996), may
also facilitate the isolation of more cDNA fragments
from the coding region of gene transcripts.
The full-length sequence of sheep Sp17 was
obtained (Fig. 3) and showed 77% homology with rabbit Sp17. This confirmed the identity of the cDNA
fragment isolated from DD, and cloning of Sp17 from
the jejunal PP cDNA library provided independent
confirmation of expression in this lymphoid tissue.
Furthermore, differential expression of Sp17 in naive,
fetal PPs, and antigen-exposed lamb PPs was confirmed by Northern blot (Fig. 4). These observations
contradict previous studies in the baboon that
reported Sp17 expression to be unique to testicular tissue (Adoyo et al. 1997). However, this conclusion was
based on Northern blotting and did not include an
analysis of the gut-associated lymphoid tissue or tonsil.
Our RT-PCR analysis confirmed Sp17 expression in
testes and also demonstrated expression in jejunal
PPs, non-PP intestine, and retropharyngeal lymph
node (Fig. 5). However, Northern blot analysis indicated that gene expression was probably much lower
in lymphoid tissues since poly(A) RNA was required
for detectable hybridization (Fig. 4).
The Sp17 protein is present on the sperm surface
and appears to play an important role in the binding
of spermatozoa to the zona pellucida of the ovum
(ORand et al. 1988; Yamasaki et al. 1995). Sp17 is
not an integral membrane protein but is released to
the surface membrane following the acrosomal reaction (Wen et al. 1999). The deduced amino acid
sequence of Sp17 indicates that the binding domain is
similar to a C-type lectin-binding domain and the
N-terminal region has similarity to a cAMP-dependent
protein kinase (ORand et al. 1988; Yamasaki et al.
1995). The relevance of Sp17 expression in mucosa-associated lymphoid tissue is difficult to surmise at this
time. The differential expression of Sp17 in metastatic
versus nonmetastatic squamous cell carcinoma cell
lines (Dong et al. 1997) suggested that Sp17 may play
a role in cell migration. Inflammation and the induction of an immune response are associated with
marked changes in leukocyte migration in tissues (reviewed in Griebel 1998). Thus, we hypothesize that
Sp17 expression may influence the trafficking of
immune cells in mucosa-associated lymphoid tissue.
Expression of Sp17 was analyzed in a variety of
lymphoid tissues to substantiate a possible link
between this gene and the immune system. RT-PCR
analysis confirmed differential expression of Sp17 in
lymphoid tissues that are involved in the induction of
an immune response (Fig. 5). Furthermore, Sp17
expression was restricted to mucosa-associated lymphoid tissues that are involved in the induction of specific immune responses. There was no detectable Sp17
expression in lymphoid tissues that function primarily

80

as sites of lymphopoiesis (thymus and ileal PPs),

whereas expression could be detected in lymphoid tissues that function as sites of mucosal immune
responses (jejunal PPs, jejunum, retropharyngeal and
mesenteric lymph nodes). The expression of Sp17 in
the intestinal wall (jejunum) may be consistent with
the large number of intraepithelial lymphocytes and
other immune cells present in the intestinal lamina
propria.
In conclusion, DD demonstrated the upregulation
of Sp17 expression in fetal PPs following rotavirus
infection. This observation was further substantiated
by RT-PCR detection of Sp17 in mucosa-associated
lymphoid tissues of conventionally reared lambs. The
fetal lamb thus provided a suitable model to investigate host gene expression following infection by an
enteric pathogen.
Acknowledgements We thank Dave Dixon and the staff of the
Animal and Poultry Science Department, University of Saskatchewan, for producing the bred ewes, and the Animal Care staff
at VIDO for assistance with fetal surgeries. Financial support
for this project was provided by the Alberta Agricultural
Research Institute, the Saskatchewan Health Services Utilization
and Research Commission, the Medical Research Council of
Canada, and the Human Frontier Science Program. Published
with permission of the Director of VIDO as journal series no.
274.

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