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Original Studies |
-2 Macroglobulin; Localization by in Situ Hybridization and Effect on Mouse Embryo Development in Vitro1
Vincent Memorial Obstetrics and Gynecology Service, Division of Reproductive Endocrinology and Infertility, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Keith Isaacson, M.D., Vincent Memorial Obstetrics and Gynecology Service, Wang Ambulatory Care Center II, Massachusetts General Hospital, Boston, Massachusetts 02114.
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Abstract |
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-2 macroglobulin (A2M) is a 718,000-kDa broad spectrum plasma
protease inhibitor whose production by the human endometrium was
recently reported. The multifunctional A2M receptor, also known as
low-density lipoprotein receptor-related protein, was also recently
immunolocalized to the endometrial stroma. The objective of this study
was to further characterize the endometrial site of expression of A2M,
and to study its effects on mouse embryo development in
vitro, to gain some insight into the functional significance of
its endometrial production. Formalin-fixed, paraffin-embedded human endometrium from hysterectomy and endometrial biopsy specimen was used for in situ hybridization analysis, with 35S-labeled riboprobes representing subcloned A2M complementary DNA (cDNA) fragments. Duplicate sections of human endometrium were hybridized with sense and antisense probe and coated with photographic emulsion. Resultant autoradiograms were analyzed qualitatively by light- and darkfield microscopy and quantitatively by a computerized analysis of the signal intensity. Immunohistochemistry and immunoblotting for endometrial tissues were performed using an affinity-purified polyclonal antibody to human A2M. The effect of A2M on mouse embryo development was studied by exposure of one cell mouse embryo in culture to physiological concentrations of biologically active and inactive A2M.
Expression signals for A2M were more numerous and intense in the secretory endometrium, compared with proliferative endometrium. Endothelial cells lining the endometrial blood vessels seemed to be the main source of A2M expression. The A2M expression signals in secretory endothelium were 2- to 3-fold stronger than the proliferative endothelium, suggesting transcriptional activation of A2M expression in the secretory endothelium. Glandular expression was observed in secretory endometrium from two patients with endometriosis. Ectopic endometrial tissues also produced A2M. A2M at concentrations of 400–500 µmol/L significantly inhibited blastocyst development of mouse embryos in vitro.
A2M is expressed predominantly by the endometrial endothelial cells and may be involved in endometrial physiology. Physiological concentrations of A2M inhibit mouse embryo development in vitro, suggesting that endometrial production of A2M may play a role in regulating preimplantation embryo development.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-2 MACROGLOBULIN (A2M) is a 718,000-KDa
tetrameric glycoprotein molecule with a broad spectrum protease
inhibitor activity that operates by a proposed trapping model through a
conformational change in the native molecular structure (1). It
circulates at a concentration of 2 mg/mL and clears proteases via
receptor-mediated endocytosis (2). A2M also has cytokine binding
properties, which endow it with a regulatory role in cellular growth
and differentiation (3, 4). Another direct effect of A2M on cellular
function has been recently described and found to be mediated via
receptor activation and inositol as a second messenger (5). A2M has
been previously identified in human uterine effluents and was assumed
to originate from serum by selective transudation (6, 7). However,
under normal conditions, transfer of circulating A2M through the blood
vessel wall is probably limited, because of its size and acidic
isoelectric point (8). We have previously reported on the production of A2M by the human endometrium (9) and proposed that such production may account for the high concentrations observed in the uterine cavity. We have also suggested that endometrial A2M and its multifunctional receptor low-density lipoprotein receptor-related protein (LRP), which has been identified also in the endometrium (10), may have far reaching implications in endometrial physiology and in the physiology of embryo development and implantation. The objective of this study was to investigate the effect of A2M on the development of mouse embryos in vitro and to further localize the site of A2M expression within the endometrium by in situ hybridization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Restriction enzymes, RQ1- ribonuclease (RNase)-free DNase, SP6
and T7 polymerases, nucleotides, nucleic acid markers, and the
Riboprobe Gemini II Core System for subcloning were obtained from
Promega Corp. (Madison, WI). Proteinase K was purchased from Boehringer
Mannheim (Indianapolis, IN). RNase A was purchased from ICN
Biochemicals (Costa Mesa, CA). 35S-UTP (uridine
5'-triphosphate) was obtained from Dupont, NEN (Boston, MA).
Photographic NTB-2 emulsion, fixer, and Dektol developer were purchased
from Eastman Kodak (Rochester, NY). -2 macroglobulin polyclonal
antibody, lot 067, was purchased from Dako Corp. (Carpinteria, CA).
Biologically active and inactive A2M were purchased from Calbiochem (La
Jolla, CA) Chemical Company and Sigma Chemical Company (St. Louis, MO),
respectively. The Vectastain ABC kit was obtained from Vector Labs Inc.
(Burlingame, CA). Modified human tubal fluid (HTF) was purchased from
Life Technologies (Gaithersburg, MD). A2M was obtained from ATCC
(Rockville, MD). ECL reagents were purchased from Amersham, Inc.
(Arlington Heights, IL). All other chemicals were reagent grade and
were purchased from Sigma chemical and Fisher Scientific (Pittsburgh,
PA).
Tissue preparation
The use of human residual tissue was approved by the
Massachusetts General Hospital Committee on Human Studies.
Formalin-fixed paraffin-embedded tissue was obtained from the pathology
laboratory at Massachusetts General Hospital. Included were two
proliferative and four secretory endometrial biopsy specimens, as well
as five proliferative and three secretory samples from hysterectomy
specimen in which the topologic orientation of the endometrium was
preserved (Table 1
). Human liver from
autopsy material was processed in a similar fashion and used as
positive control. 4-µm-thick contiguous sections from each sample
were placed on poly-D-lysine-coated slides and baked for 30
min at 70 C. For secretory endometrial samples, dating was performed on
hematoxylin and eosin-stained sections, according to well-established
criteria (11). Duplicate sections were prepared for in situ
hybridization using RNase-free techniques. Briefly, slides were
deparaffinized in xylenes, rehydrated in descending concentrations of
ethanol (100%, 95%, 85%, 70%), and transferred to
phosphate-buffered saline containing 20 µg/mL proteinase K, for 15
min at room temperature. Slides were washed in 2x SSC buffer and
dehydrated in ascending alcohol for 1 min each (70%, 80%, 90%,
100%), then dipped in 100% chloroform, and back to 100% ethanol, and
finally in 95% ethanol. Slides were air dried and ready for in
situ hybridization.
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Human liver A2M cDNA clone pHLA2M.1 was restricted with enzymes, BamHI and EcoRI, to yield a 581-bp fragment representing sequence 547-1128 of the 4.6-kilobase clone (12). This fragment was subcloned into pGEM-3Z vector, according to the manufacturer’s instructions. 35S-labeled antisense and sense probes, specific for human A2M, were generated by transcription from T7 and SP6 promoters, respectively, of the linearized pGEM-3Z subclone using the Riboprobe Gemini II Core System. High specific activity probes were generated (108-109 cpm/µg) using an excess of [35S]UTP (urudine 5'-triphosphate) (200 Ci/mol·L). The size of the probes was confirmed by agarose/formaldehyde gel elctrophoresis, followed by ethidium bromide staining and autoradiography. The antisense probe was used to localize expression of endometrial A2M, and the sense probe was used for control hybridization.
In situ hybridization
Tissue sections were prehybridized for 2 h with 200 µL of in situ hybridization buffer containing 10% sodium thiosulfate, 10% SDS, 50% formamide, and 3.5 mol/L DTT (dithiothreitol). The cold buffer was removed, and the sections were covered with buffer containing 20,000–40,000 cpm/µL of either sense or antisense A2M riboprobe, coverslipped, and incubated overnight at 45 C in a humid chamber. The next morning, coverslips were floated off the sections by dipping slides in 2x SSC buffer, and the sections were incubated for 30 min at 37 C in a 20-µg/mL RNase A solution, to reduce nonspecific riboprobe binding. This was followed by successive washes with shaking; twice in 2x SSC for 2 min at room temperature, once in 2x SSC for 60 min at 50 C, and twice in 0.2x SSC for 60 min at 50 C. Sections were dehydrated in ascending ethanol concentrations (50%, 70%, 90%, 95%, 100%) containing 300 mmol/L ammonium acetate and air dried. In a dark room, sections were dipped in a 1:1 dilution of NTB-2 photographic emulsion and exposed for 21 days. After developing and fixation, slides were counterstained with hematoxylin, dehydrated in ascending alcohol gradient, and coverslipped with permount out of xylenes. The signals were examined under a Nikon microscope (DC Optical, Natick, MA).
Quantitative analysis of the A2M autoradiograms was performed by means
of a computer-assisted image-analysis system (Imaging Research, St.
Catharine’s, Ontario, Canada), which is described in detail elsewhere
(13). Briefly, each endometrial section was arbitrarily divided into
four quadrants and a random 100x-water immersion field, representing a
cross-section of an endometrial blood vessel, was identified in each
quadrant. The blood vessel circumference was outlined, and the number
of silver granules within the outlined area was counted. A nonspecific
count was performed on an adjacent area of equal size that did not
contain blood vessels. The specific count was obtained by subtracting
the background count from the blood vessel count. Specific counts from
each field were adjusted for surface area and averaged to yield an
in situ score for the section. Proliferative and secretory
scores were compared using the nonparametric Mann-Whitney rank test
(Table 2).
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Immunochemistry for A2M was performed using an affinity-purified polyclonal antibody and procedure described previously (9). The same antibody, at a dilution of 1:2000, was used for Western blot analysis of protein lysates from endometrial tissues. Briefly, detergent-lysates were prepared from frozen ectopic and eutopic endometrial tissues. Aliquots, containing 50 µg total proteins, were subjected to 8% SDS-PAGE for 50 min at 200 V in a cold room. Proteins were then transferred to nitrocellulose membrane, using an electroblotting apparatus, at 100 V for 90 min. After blocking nonspecific binding in 5% skim milk containing 0.1% Tween-20 and 2% BSA, blots were incubated with antibody at 4 C overnight. Specific bindings were detected by incubating the membrane with biotinylated rabbit antigoat IgG for 30 min at room temperature, followed by incubating with avidin-biotin-complex reagent for 45 min at room temperature. Thereafter, the membrane was incubated in ECL reagent for 1 min, exposed to ECL hyperfilm for 10–15 sec, and developed as usual for autoradiogram.
Preparation of embryo culture media
Bioactive A2M was supplied as lyophilized powder with preservers. To avoid unexpected effects from preservers, both forms of A2M were suspended in HTF culture medium, then centrifuged at 4 C at 3000 x g for 30 min using Model 100 microconcentrators (Amicon Inc., Beverly, MA), which eliminates molecules smaller than 100,000 mol wt. The concentrated and filtered A2M was then used to condition modified HTF containing 100 IU/mL penicillin, 100 µg/mL streptomycin, and 20 mmol/L HEPES supplemented with 0.5% BSA. All culture media were filtered through 0.22 µ filters, dispensed into culture dishes, and incubated at 37 C in an atmosphere containing 5% CO2 in air for 24 h before the culture of embryos.
Embryo recovery and culture
Four- to 7-week-old B CBAF1 female mice (Charles River, Boston, MA) were superovulated by an intraperitoneal injection of 5 IU of pregnant mare serum gonadotropin (Diosynth B.V., OSS, Holland), followed 48 h later by 5 IU of human CG (hCG). Immediately after hCG injection, females were mated with males with proven fertility of the same strain. Twenty to 22 h later, females with vaginal plugs were killed by cervical dislocation. The oviducts were excised and placed in HTF medium. The swollen ampullae were punctured under a dissection microscope, releasing the embryos, which were transferred to the HTF culture medium and cultured in a humidified atmosphere of 5% CO2 in air at 37 C for 2 h to let individual one-cell embryos disperse from the cumulus masses spontaneously. Dispersed individual one-cell embryos were pooled and distributed randomly into five experimental groups. Groups 1, 2, and 3 were exposed to bioactive A2M at concentrations of 200,400 and 500 µg/mL, respectively. Group 4 was exposed to inactive A2M at concentration of 500 µg/mL, and group 5 was exposed to HTF culture medium only. Groups of 10–30 one-cell embryos with polar bodies were cultured in 100-µL droplets under mineral oil. Embryos were observed for 5 days at 24-h intervals under a dissection microscope for cleavage and development.
Statistical analysis
The data from six consecutive embryo culture experiments were pooled. The cumulative number of embryos developing to morula and blastocyst stages at various time intervals was expressed as a percentage of the total number of fertilized one-cell embryos at initiation of culture. Comparisons between groups were made using the chi-square analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. As expected, hepatic sinusoids showed prominent expression
with the antisense riboprobes for A2M. Hybridization with sense probes
did not result in any specific signal (data not shown). All endometrial
samples showed a similar pattern of stromal expression for A2M, which
was patchy and seemed to be distributed randomly throughout the entire
thickness of the endometrium (Figs. 1 and 2). Closer inspection showed the signals
to be originating from cells lining the endometrial blood vessels.
There was no appreciable signal in the myometrium or myometrial blood
vessels. The signals from secretory samples were noted to be stronger
and more numerous, compared with those of proliferative samples. The
qualitative differences between proliferative and secretory endometrium
were confirmed by the quantitative analysis of the endothelial signals;
A2M expression in the endothelium of secretory endometrium was 2- to
3-fold higher than the proliferative endothelial expression, and this
was quite prominent in the late secretory samples (Table 2). Few glands
in two mid- to late-secretory samples expressed intense signals, and
this was confirmed with immunohistochemical analysis performed on
contiguous sections (Fig. 3). No
glandular expression of A2M was noted in any of the other secretory and
proliferative endometrial sections that showed immunostaining
characteristics for A2M previously reported (9). Immunoblotting
confirmed A2M production by native and ectopic endometrial tissues
(Fig. 4).
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After 24 h in culture, there were no appreciable differences
in development among the five experimental groups (Fig. 5A). At 72 h, there seemed to be a
significant inhibition of morula and blastocyst development in all
three groups exposed to bioactive A2M (Fig. 5, B and C). This effect
persisted at 120 h of exposure in groups 2 and 3, which were
exposed to 400 and 500 µg/mL of bioactive A2M, respectively (Fig. 5D). At 120 h of culture, group 1, containing 134 embryos, seemed
to have overcome the inhibition and achieved a 75% blastocyst
development. Groups 2 and 3, containing 209 and 300 embryos,
respectively, achieved only a 32% blastocyst development rate
(P < 0.0001, compared with the other groups). Group 4
(containing 128 embryos) and group 5 (containing 487 embryos) achieved
blastocyst development rates of 51% and 66%, respectively, which was
not significantly different from group 1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The induction of A2M expression in the secretory endothelial cells,
which do not possess estrogen and progesterone receptors (17, 18),
suggests that local nonsteroidal autocrine and paracrine influences are
instrumental in this transcriptional activation. The rare glandular
expression of A2M in the two secretory endometrial samples, which
belonged to women with endometriosis (Table 1), is consistent with the
growing evidence that the endometrial secretory profile of women with
endometriosis is altered (19). This observation also suggests a role
for local factors in induction of A2M expression in endometrial glands.
In this respect, it is interesting to note that A2M production has been
reported previously in vitro by monolayers of glandular
cells isolated from gestational endometrium (20). This suggests an
intrinsic ability of glandular elements to produce A2M, given the
proper paracrine milieu. Although IL-6 and PRL have been shown to
induce A2M expression in rat reproductive tissues (21, 22), it is
speculative whether the known production of PRL and IL-6 by the human
endometrium (23) has any regulatory effect on endometrial A2M
expression.
To the best of our knowledge, this is the first attempt to elucidate the functional significance of human endometrial A2M by studying its effect on mouse embryo development in vitro. This was justified by the fact that A2M is a highly conserved molecule throughout evolution and cross-species effects have been described previously (8, 24, 25). In this study, physiologic concentrations of A2M, which are normally present in the human uterine fluid, inhibited blastocyst development of mouse embryos in vitro. The absence of an effect with inactive human A2M (control), and the dose-response effect of the bioactive A2M, suggests a specific inhibition of blastocyst development, rather than a nonspecific or toxic effect. One possible explanation for this observed inhibition of embryo development is the binding and neutralization of embryonic cytokines by A2M. This would deprive the preimplantation embryos from the autocrine and paracrine effects of embryonic cytokines, which may be essential for further development (26). There is also the possibility that human A2M may be neutralizing embryonic proteases, which have been previously shown to be important in the process of embryo development (27). Moreover, because early embryos possess A2M receptors (28), a direct effect of A2M may also be responsible for the inhibition of blastocyst development.
It seems paradoxical that A2M would inhibit embryo development, when its endometrial production in vivo seems to peak in the mid- to late-luteal phase, at the time of blastocyst development and implantation in the human. One explanation may lie in cross-species differences in the response to A2M exposure between human and mouse embryos. Another explanation is that the human embryo enters the endometrial cavity beyond the morula stage, at which time it may have acquired the competence to overcome any inhibition by endometrial A2M. In this respect, endometrial A2M production during the window of implantation may be a mechanism to prevent the development and implantation of unfit embryos. Further studies will be needed to investigate the effect of A2M exposure at different stages of embryo cleavage.
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Footnotes |
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2 Current address: Department of Obstetrics and Gynecology, New
England Medical Center and Tufts University School of Medicine, 750
Washington Street, Boston, Massachusetts 02111.
Received February 3, 1997.
Revised July 16, 1997.
Accepted September 9, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This article has been cited by other articles:
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  S.-S. N. Siu, M.-Y. Choy, T.-N. Leung, and T.-K. Lau Lack of Site-Specific Production of Decidual Alpha-2 Macroglobulin in Human Pregnancy Reproductive Sciences, October 1, 2006; 13(7): 491 - 496. [Abstract] [PDF]
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