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Section on Developmental and Molecular Pharmacology (C.Y.S., S.J.L., G.G., D.T.A., R.A., D.E.B., J.M.H.), Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and Division of Neonatology (S.K.M.), Children’s National Medical Center, Department of Pediatrics, George Washington University, Washington, D.C. 20010
Address all correspondence and requests for reprints to: J. M. Hill, Ph.D., Section on Developmental and Molecular Pharmacology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Building 49, 5A-38, MSC 4480, 9000 Rockville Pike, Bethesda, Maryland 20892. E-mail: jh139h{at}nih.gov
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Introduction |
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During this critical time period post implantation, there is a maternal
response to the developing embryo, resulting in an infiltration of
lymphocytes into the decidua (5). The decidual cellular milieu at this
time period is mixed and includes lymphocytes, natural killer cells,
and macrophages (6). However, one specific subtype of lymphocyte, with
a 
T cell receptor, is abundant and disproportionally high,
compared with circulating levels (7). These lymphocytes are
thought to have a specific role in the maternal-fetal interaction (7).
Lymphocytes are known to express VIP messenger RNA (mRNA) (8) and have
VIP receptors (9, 10).
The purpose of the current study was to characterize VIP mRNA, VIP peptide, and VIP binding sites in E6–E12 mouse embryos and extraembryonic tissues, identifying potential sources of VIP for growth regulation at this critical time.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
NIH-Swiss female mice (Harlan Sprague Dawley, Inc.,
Indianapolis, IN) were kept under a 12-h light, 12-h dark regimen, with
food and water available at all times. The mice received humane animal
care in compliance with the Guideline for Care and Use of Experimental
Animals. Six-week-old females (21–24 g) were mated with NIH Swiss
males for 4 h. The presence of a vaginal plug was considered day
zero of pregnancy.
RT-PCR
On days 6–12 of pregnancy, conceptuses (embryo and membranes)
and decidua were explanted from the uterus and placed in PBS. At E6 and
E7, when structures were too small to clearly dissect apart
microscopically, conceptuses were analyzed without dissection and
contained the embryo proper, membranes, trophoblast, and surrounding
decidua. From E8–E12, when the embryo could be dissected away from the
extraembryonic membranes and decidua (with adhering trophoblast,
i.e. decidua/trophoblast), these three issues were analyzed
separately. At E11–E12, the placentas were easily identified as
separate structures and were analyzed separately. Quantitative RT-PCR
was performed on the decidua, and samples were taken from that portion
of the decidua immediately adjacent to the uterine wall, for each
gestational day from E6–E17. All tissues were thoroughly rinsed in PBS
and placed in 0.5 ml RNA STAT-60 (Tel-Test, Inc.,
Friendswood, TX), with tissues from a minimum of three different
conceptuses pooled in each sample and a minimum of three samples
studied per developmental age. The tissues were homogenized in RNA
STAT-60, using disposable micropestles to avoid cross-contamination,
and were stored at -80 C. In addition, fetal brains from gestational
day 17 mice were removed, homogenized in RNA STAT-60, and stored at
-80 C.
For RNA extraction, samples were thawed on ice, 0.1 ml chloroform was added, and they were vortexed and allowed to sit on ice for 5 min. The samples were centrifuged in a microcentrifuge for 15 min at 4 C. Supernatants were transferred to clean tubes, and 0.3 ml isopropanol was added to each; samples were vortexed and frozen on dry ice for 30 min. After thawing on ice, samples were centrifuged, as before, at 4 C. Pellets were washed three times with 75% ethanol, air-dried, and resuspended in 50 µl DEPC-treated water. A 5-µl aliquot was taken for spectrophotometric determination of RNA content. The remaining sample was stored at -80 C.
Using 5 µg of total RNA, the RT reaction was performed with reverse transcriptase (Perkin-Elmer Corp., Branchburg, NJ), as recommended by the manufacturer, in a final vol of 150 µl. Each RNA sample, which was a pooled sample of at least three conceptuses, was run in duplicate. Negative controls included RT reactions performed, omitting RNA or reverse transcriptase. Each PCR reaction was repeated at least twice with different samples of the RT reaction product. Primer pairs and their mimics were designed using Oligo software (National Biosources Inc., Plymouth, MN) and were synthesized (Bio-Synthesis, Inc., Louisville, TX).
The VIP primer pair used were 5'-CAG GAA CCG GGA ACA GAC T (sense) and 5'-TAT CAG GAA TGC CAG GAA CT (antisense). Negative controls included omission of the template in the PCR reaction. A positive control was derived from tissue samples of mouse cortex that exhibited a VIP mRNA band at 20–25 PCR cycles. The negative control was E17 mouse tail RNA. Tail RNA exhibited an amplification product at 35–40 PCR cycles. Cycle tests for cortex and E17 tail were run in each PCR experiment to determine when VIP became positive.
Specifically, replicate cortex samples were removed at cycles 20, 25, 28, and 30; replicate tail samples were removed at cycles 28, 30, 35, and 40. In each experiment, there was at least a ten-cycle difference between the appearance of VIP amplification product in the positive and negative controls. Thirty cycles was designated as the negative cutoff for detection of VIP mRNA in the unknown samples. Negative controls were run at 30 and 40 cycles. Interexperiment controls included aliquots from the same sample of adult cortex and E17 tail in each series of RT reactions. Samples were also run with cyclophilin primers as an internal control for each reaction and for use for qualitative comparisons between the samples. The amplification reactions were run on 4–20% acrylamide TBE gels (Novex, San Diego, CA). Gels were stained with 0.01% ethidium bromide for 10 min and photographed.
Quantitation of mRNA
cDNA mimics with the desired sequence and an internal deletion
were constructed using PCR amplification of mouse cortex cDNA, isolated
as described, using a 5' primer homologous to two regions of the
desired gene sequences along with the appropriate 3' sequence. The 5'
deletion primers for cyclophilin were 5'-ATG GCA CAG GAG GAA AGA GCA
ATG CAG GCA AAG ACA CC; and for VIP, 5'-CAG GAA CCG GGA ACA GAC TAG CCG
GAA AGG CAG CCC TG. Products obtained from the synthesis were run on a
2% agarose gel (3 g Nusieve, 1 g agarose, 200 ml of 1 x TBE
with 10 µl ethidium bromide) in 1 x TBE with 10 µl ethidium
bromide. Bands of the appropriate molecular weight were collected into
prepared Na 45 diethylaminoethyl membranes from Schleicher & Schuell, Inc. The membranes were treated with 100 µl
high-salt NET buffer (1 M NaCl, 0.1 mM EDTA, 20
mM Tris, pH 8) and incubated at 55 C for 10 min. The buffer
was removed and saved; this step was repeated for a total of three
times (300 µl total). This was extracted three times with 300 µl
water-saturated n-butanol, precipitated with 900 µl of 95% ethyl
alcohol, and washed three times in 75% ethanol.
For quantitation of mRNA, the PCR reaction was run with dilutions of mimic primers (2 µl), with incorporation of 32P-deoxy-ATP. Each sample was also run with dilutions of a cyclophilin mimic, for internal normalization. To prevent evaporation, each sample was overlaid with 2 drops of mineral oil. The amplification reactions were separated through a 4–20% acrylamide Tris-borate EDTA gel (Novex). Gel-Marker (Research Genetics, Inc., Huntsville, AL) was run on each gel as a marker for determination of band size. Gels were stained with 0.01% ethidium bromide for 10 min and photographed, dried (Bio-Rad Laboratories, Inc., Richmond, CA), and placed on 3 M Whatman paper (Whatman International Ltd., Maidstone, UK). The gels were exposed to a Molecular Dynamics, Inc. (Sunnyvale, CA) storage phosphor screen for 18–24 h. Using ImageQuant software (Molecular Dynamics, Inc.), band intensities were measured. The location of the cross-over, from predominately mimic product to message product, was calculated after phosphoimage analysis. The samples were normalized to the cyclophilin cross-over. The cross-over values from each gestational day were compared with gestational day 17 and were expressed as the percentage of gestational day 17.
Enzyme-linked immunosorbent assay (ELISA)
At gestational days 9 and 17, conceptuses were explanted from
the uterus, within decidua (embryo, membranes, trophoblast, decidua,
and fluid), and maternal organs were removed (ovary, uterus,
cervix/para-cervix, intestine, cortex, hypothalamus, spinal cord,
and placenta) from at least five different pregnant mice. These were
quick-frozen on dry ice and stored at -80 C. The tissue was weighted
and homogenized in 3 ml of 0.1 N HCl. Samples were vortexed,
placed in boiling water for 10 min, cooled on ice, vortexed, and
centrifuged at 10,000 x g for 30 min. One-milliliter
aliquots were evaporated to dryness in vacuo. Dried samples
were stored at -80 C.
For the E6–E12 plasma samples, blood was collected by cardiac puncture into a syringe containing 15% EDTA and aprotinin (ICN Biomedicals, Inc., Aurora, OH). The samples were centrifuged at 1,600 x g for 10 min. The plasma from at least three mice was pooled, frozen on dry ice, and kept at -80 C until used. Using 0.5 ml of plasma for each gestational day, plasma extraction was performed using Sep-Pack columns, according to the manufacturer’s protocol, with 1.0% trifluoroacetic acid (HPLC grade), and eluted with 3 ml of a 60% acetonitrile (HPLC grade), 1% trifluoroacetic acid. The eluate was collected in a polypropylene tube, triturated, and divided into two polypropylene tubes. The samples were evaporated to dryness, under vacuum, and stored at -80 C until the ELISA was performed. Protein determination (Bio-Rad protein assay; Bio-Rad Laboratories, Inc., Hercules, CA) was performed on each sample before the ELISA. For the ELISA (Peninsula Laboratories, Inc., Belmont, CA), 10 mg/well of protein was added from each sample. The specificity of the ELISA is 100% for VIP (human, porcine, rat, and guinea pig) and for VIP 10–28 (human, porcine, and rat). The ELISA specificity for VIP from chicken is less than 1%; and for pituitary adenylate cyclase-activating polypeptide (PACAP)-27 (human, ovine, and rat), it is less than 0.02%. The ELISA has no cross-reactivity with substance P, endothelin-1, secretin, glucagon, galanin, somatostatin, or PACAP-38 (human, ovine, and rat). The linear range of the ELISA for VIP is 0.04–2.0 ng/ml. Concentrations of VIP were calculated per mg protein for maternal organs or per ml for maternal plasma samples. The interassay and intraassay coefficients of variation for the VIP ELISA were 4% and 8%, respectively.
Collection of human serum
Women in early gestation, at a maximum of 5 weeks from their
last menstrual period, were identified in the Obstetrics clinic at
William Beaumont Army Medical Center. Gestational dating was confirmed
in the 25 women who met this criteria (using transvaginal sonography,
at the initial visit and weekly thereafter, until 6 weeks post
conception). Seven milliliters of blood was collected in a tube
containing EDTA and aprotinin. The sample was immediately centrifuged,
and the obtained plasma was stored in a -80 C freezer until the assay
was run. An average of 5.5 samples were obtained from each patient,
with a range of 2–7.
RIA for VIP in maternal (human) serum samples was performed by Covance Laboratories, Inc. (Vienna, VA) with the use of a 125I-RIA kit (INCSTAR Corp., Stillwater, MN). Sensitivity of the RIA was 5 pg/ml. For specificity, cross-reactivity was found to be less than 0.1%, with secretin and 15 other peptides.
Immunocytochemistry (ICC)
Uteri from E9 gestations were fixed in situ with 4%
paraformaldehyde, removed, and quick-frozen on dry ice. Twenty-micron
sections were cut on a cryostat and mounted on slides. For fluorescent
ICC, sections were fixed in cold acetone for 10 min, at 4 C, on a
shaker. After rinsing, the slides were incubated in 5% donkey serum
(Jackson ImmunoResearch Laboratories, Inc., West Grove,
PA) in PBS, at room temperature for 45 min, to suppress nonspecific
binding of IgG. The slides were incubated with the primary antibody
overnight [anti-VIP, 1:500 (INCSTAR Corp.), and
anti-CD3-
(Santa Cruz Biotechnology, Inc., Santa Cruz,
CA)]. Anti-VIP is a rabbit antisera; anti-CD3- is an
affinity-purified goat polyclonal antibody to CD3-, which is present
on T lymphocytes. The slides were then incubated in 5% donkey
serum/PBS for 45 min. To permit identification of colocalized antigens,
the sections were then incubated, in the dark, in the
fluorescein-conjugated secondary antibody [lissamine
rhodamine-conjugated AffiniPure donkey antirabbit IgG for VIP, 1:200;
and CY2-conjugated AffiniPure donkey antigoat IgG for CD3, 1:100
(both from Jackson ImmunoResearch Laboratories, Inc.)]
for 45 min.
After incubation, the sections were washed, mounted in 90%
glycerol/PBS, and examined with a fluorescence microscope. For standard
ICC, the sections were pretreated with 3% H2O2
to deactivate any endogenous peroxidase activity, blocked in 1% BSA
for 1 h, and then incubated with goat polyclonal IgG anti-T cell
receptor , 1:50 (Santa Cruz Biotechnology, Inc.), at 4
C, overnight. After washing, slides were incubated with rabbit antigoat
IgG, 1:2000 (Vectastain, Vector Laboratories, Inc., Burlingham, CA), for 1 h. The sections were reacted
by using the ABC method and visualized by 3,3'-diaminobenzidine
(Sigma Chemical Co., St. Louis, MO). For dual labeling,
after washing, the slides were blocked in 1% BSA for 30 min and
incubated with the second primary antibody, rabbit anti-VIP, 1:500
(INCSTAR Corp.), at 4 C, overnight. After washing, slides
were incubated with goat antirabbit, 1:2000 (Vectastain,
Vector Laboratories, Inc.), for 1 h. The sections
were reacted by using the conventional ABC method and visualized with
Vector VIP, a peroxidase substrate kit (Vectastain,
Vector Laboratories, Inc.), which stained the
immunoreactive areas purple. The slides were then dehydrated and
coverslipped. The specificity of the antiserum was examined, by soluble
preadsorption with the peptides in question, at a final concentration
of 10-5 M (INCSTAR Corp.). VIP
immunolabeling was completely abolished by preadsorption with VIP.
Preadsorption with the following peptides resulted in no reduction of
immunostaining: (PACAP-38), secretin, gastric inhibitory polypeptide,
somatostatin, glucagon, insulin, ACTH, gastrin 34, FMFR-amide, rat
GHRF, human GHRF, peptide histidine isoleucine 27, rat pancreatic
polypeptide, motilin, peptide YY, substance P, neuropeptide Y, and
CGRP.
In vitro autoradiography
To identify VIP binding sites, in vitro
autoradiography with 125I-VIP was performed as described
previously (3, 11). In summary, a minimum of eight uteri per E were
sectioned, at 20 µm, on a cryostat, and were dried and frozen at -80
C until use. The slides were brought to room temperature and
preincubated in binding buffer (10 mM HEPES, pH 7.4, with
130 mM NaCl, 4.7 mM KCl, 5 mM
MgCl2, 5 mM MnCl2, 1 mM
EDTA, 1% BSA) for 30 min. Binding reactions were performed for 1
h at room temperature in binding buffer with 1 mg/ml bacitracin and 50
pM 125I-labeled VIP (Amersham Corp., Arlington
Heights, IL), with and without 1 µM VIP (Peninsula Laboratories, Inc.), to determine specific binding, or with 10
µM of the stable GTP analog guanylyl imidodiphosphate to
differentiate GTP-sensitive from GTP-insensitive binding sites (11).
After incubation, the slides were transferred through three 1-min
rinses of cold PBS, pH 7.4, and rapidly dried under a stream of cool
air. Sections were placed in a cassette with Hyperfilm-3H
(Amersham Corp.) for 4 days. The autoradiograms were developed in Kodak
D-19 (Eastman Kodak Co., Rochester, NY). The specific
binding was determined by subtracting the light transmittance from
brain sections (incubated with 1 µM unlabeled VIP) from
the total light transmittance.
Statistics
Statistical analysis included ANOVA for continuous variables and
Mann-Whitney U for nonparametric data [Statview 4.5 (Abacus Concepts,
Inc., Berkeley, CA)], with P < 0.05 considered
significant.
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Results |
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). By E11 or
E12, VIP mRNA was no longer evident in the decidua/trophoblast but was
detected in the embryo proper (Fig. 1). VIP mRNA was not detectable in
the extraembryonic membranes (E8–E12), E9 yolk sac, or in the placenta
(E11–E12) (data not shown). RT-PCR detected VIP mRNA in E17 fetal
brains (Fig. 1). All PCR reactions had comparable cyclophilin products
(data not shown).
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Peptide
VIP immunoreactivity was detected by ELISA in E9 conceptuses
(embryo proper, extraembryonic membranes, amniotic fluid, trophoblast,
and decidua) and was significantly elevated, compared with E17
conceptuses (15.8 ± 0.8 vs. 7.5 ± 1.7 ng/mg
protein, P < 0.01). There were no significant
differences in material organ concentrations of VIP between gestational
days 9 and 17 (Fig. 3). Maternal
intestine and cortex had the highest VIP levels among the organs
tested. Some of the maternal organs’ VIP concentrations were below the
detection of the ELISA. For gestational day 9, 1/6 uteri and 4/12
cervix/paracervix were below the limit of detection. For gestational
day 17, 1/5 ovaries and 1/5 spinal cords were below the detection of
the ELISA. Maternal plasma levels of VIP did not vary significantly
throughout gestational days 6–12 (Fig. 4). All plasma samples were within the
detection limits of the ELISA.
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VIP immunoreactivity was present in numerous scattered small cells
throughout the decidua. Fluorescent-ICC revealed VIP immunoreactivity
in decidual cells that colocalized with cells immunoreactive for the
CD3 T cell receptor (Fig. 5). In
addition, ICC demonstrated VIP immunoreactivity in cells of the decidua
that colocalized with cells immunoreactive for the TCR receptor
(Fig. 6).
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). By E9,
decidual binding sites were much less apparent; and after E10, they
were not evident. In the embryo, VIP binding sites were abundant in the
floor plate and adjacent neuroepithelium from E9, the earliest age at
which the neural tube was easily identified on autoradiograms (Fig. 7).
In the mesometrial stroma, which was undergoing decidualization, most
of the VIP binding was GTP-sensitive and completely displaced by GTP
(Fig. 8); however, most binding in the
interimplantation uterus seemed to be insensitive to GTP (Fig. 8-middle). A small region with nonspecific binding of
125I-VIP was found in the region of the ectoplacental cone
(Fig. 8, middle and bottom). In E9 and older
embryos, both GTP-sensitive and GTP-insensitive binding sites were
present.
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Discussion |
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. These
findings suggest that the VIP, which is critical to embryonic growth,
originates from maternal lymphocytes in the decidua. In the mouse, transfer of VIP from mother to embryo has been demonstrated. Previous work from our laboratory has shown that 125I-VIP, injected into the uterine vasculature, was found in the E10 mouse embryo (13). The transfer apparently is rapid, because embryos were evaluated 15 min after injection. We have been unable to detect VIP mRNA in the mouse embryo at this crucial time; however, there are abundant binding sites in the floor plate and adjacent neuroepithelium (4). Our findings suggest that the source of VIP that acts during the postimplantation growth phase of the mouse embryo is maternal in origin. After implantation, decidual invasion, and placental formation, both maternal and embryonic tissues at the implantation site are undergoing rapid growth and differentiation. This region contains maternal blood cells coexisting with extraembryonic trophoblast cells. Whether the VIP produced in these cells is released and available to the embryo at this time would be difficult to determine. However, we have shown that the peptide is present, although not expressed by the embryo, during implantation. In addition, the time period between implantation and placental development, embryonic-maternal exchange occurs primarily through the yolk sac surrounding the embryo. Several developmentally important regulatory factors and receptors are located in maternal decidual cells and embryonic trophoblast surrounding the parietal yolk sac, and these permit the transfer of nutrients to the visceral yolk sac (14). In addition, as early as the 8–10 somite stage (E8.5), vascularization of the yolk sac is continuous with the developing vasculature of the embryo. Functions of the surrounding trophoblast include transporting nutrients to the embryo, as well as transferring other molecules, including growth factors (14). Transforming growth factor-ß1 (TGF-ß1) is one growth factor known to cross from mother to fetus, because embryos with a null mutation for TGF-ß1 are able to develop normally in utero because of transport of TGF-ß1 from the mother (15). Evidence also exists that maternal PTH-related peptide, a factor with diverse actions in development, is transferred to the embryo during the early postimplantation period (16).
Previous studies suggested that the regulation of growth by VIP occurs during a narrow window of development, beginning at least at E9 in the mouse and not extending beyond E11 (1, 2). The current study demonstrates that VIP-related measures such as mRNA and binding sites are undergoing dynamic changes during this time period. In addition, the end of this period, E11, is characterized by the appearance of VIP mRNA in the embryo and the disappearance of message and binding sites from the decidua/trophoblast. Furthermore, the placenta has formed from the trophoblast by E11 and becomes the major route of embryonic nutritional exchange. Taken together, these data suggest that, before E11, VIP secreted from maternal lymphocytes reaches the embryo to act at binding sites of the primitive neuroepithelium, to regulate growth. With the formation of the placenta, dependence on yolk sac nutrition decreases, VIP production ceases in the decidua/trophoblast, and the embryo begins to synthesize VIP.
In placental formation, the decidua is invaded by trophoblast, which
results in a maternal response, with an invasion of lymphocytes.
Previous work has demonstrated that lymphocytes are able to express VIP
mRNA (8) and VIP receptors (9, 10). Ten to 15% of all cells in the
decidua are lymphocytes, and the number of T cells significantly
increases in the uterus in pregnancy. The percentage of T cell
lymphocytes at the maternal-fetal interface is 3- to 4-fold higher,
compared with the spleen, and 2-fold higher than in nonpregnant uterus.
Previous studies have demonstrated that all T lymphocytes at the
maternal-fetal interface are maternally derived (7). We have shown that
these cells, T lymphocytes, which alone have the CD3 T cell receptor,
are colocalized with VIP immunoreactivity. Furthermore, the population
of lymphocytes specific to pregnancy, T cells, colocalize with
VIP. These findings suggest that the source of VIP, at the critical
time to regulate embryonic growth, is maternal lymphocytes in decidua,
which are recruited during implantation. These findings are supported
by the surge in VIP mRNA levels in the decidua at days 6–8, the time
before and around VIP regulation of embryonic growth.
The mechanism(s) through which VIP regulates embryonic growth are not well understood. Many functions of VIP occur indirectly, through the VIP-stimulated release of trophic factors from glial cells. For example, VIP is known to stimulate the release of activity-dependent neurotrophic factor (17), protease nexin 1 (18), and several cytokines (19). In the developing embryo, VIP binds to receptors in the floor plate, which is composed of presumptive glial cells of the neural tube (20); and the bound VIP may stimulate the release of growth-regulatory factors from these cells. In addition, the VIP may stimulate cells in the decidua, to release growth regulatory peptides and/or cytokines.
An alternate hypothesis is that a VIP-like factor, synthesized by the embryo, is the endogenous growth regulator. The related peptide, PACAP with about 70% amino acid identity to VIP, binds with high affinity to the known VIP receptors, VIP1 and VIP2 (21), and is present in the E9.5 mouse embryo (22). However, previous work has shown that PACAP inhibits embryonic growth (23), which suggests that PACAP is not the endogenous ligand acting at VIP receptors to induce embryonic growth.
Mouse embryos exhibited both GTP-sensitive and GTP-insensitive VIP binding sites. Because sensitivity to GTP is characteristic of cAMP-linked receptors (24), the GTP-sensitive binding sites of the trophoblast may represent VIP binding to cloned adenyl cyclase-dependent VIP receptors, VIP1 and VIP2 (25, 26, 27). The GTP-insensitive sites of the decidua/trophoblast may represent VIP acting through adenyl cyclase-independent action implicated in the neurotrophic functions of VIP (28, 29).
This study has not determined the anatomical site(s) where VIP mRNA is localized in the E11 mouse embryo; however, a highly sensitive in situ hybridization histochemistry technique has detected VIP mRNA in cells of the hindbrain of the E11 mouse (30). In addition, we have found VIP mRNA in the E17 fetal mouse brain. In the rat, the earliest appearance of VIP mRNA was in peripheral embryonic tissues, including the sphenopalatine ganglia, as early as E16, with in situ hybridization (3), stellate ganglia at E14.5 (31), and superior cervical ganglia at E15 (32). VIP mRNA was not detected by RT-PCR in the E11 rat embryo (13) and was not apparent with in situ hybridization histochemistry in rat CNS tissues until birth (3).
Some of our findings in the mouse are different from previous work in the rat. Similar to the experience with the rat, we were unable to demonstrate VIP mRNA in the mouse embryo during the time when VIP regulates embryonic growth (E9–E11). However, VIP mRNA was demonstrated in the mouse trophoblast and decidua (E9–E11), though not apparent in the rat at corresponding gestational ages. In addition, we did not find a peak of VIP in maternal mouse plasma, in contrast to the rat (where a peak of VIP in maternal serum had been observed at the corresponding gestational age of postimplantation growth regulation) (13). The detection of increased VIP in the maternal serum of the rat may indicate spillover caused by greatly increased production, whereas the absence of a peak of VIP in maternal plasma in the mouse and human suggests that the increase in VIP is a more localized event and is limited to the decidua and extraembryonic membranes encasing the developing embryo. However, in both rat and mouse, during the critical time of postimplantation (when fetal growth is apparently regulated by VIP), the embryo does not express VIP message, thus both rely on nonembryonic sources.
Our findings suggest a dynamic interaction between the mother and the embryo, mediated by an extraembryonic neuropeptide, VIP, required for normal embryonic growth and development. The narrow window during which VIP exerts growth regulatory effects, the period between implantation and placentation, is the most vulnerable period of intrauterine development (5). The maternal-embryonic dialog, so important to development during this period, is not well understood. Further characterization of factors, like VIP, involved in this period of development may define a critical phase in the regulation of coordinated embryonic development. Such insight has potential utility in diagnosis and treatment of conditions resulting from disregulated embryonic growth, such as microcephaly or growth restriction.
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Acknowledgments |
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Footnotes |
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Received August 7, 1998.
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References |
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T cells at the maternal fetal
interface. J Immunol 149:2872–2878[Abstract]
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