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Original Articles |
Departments of Obstetrics and Gynecology, University of Illinois ((H.M.H., K.M.D., H.G.V, A.T.F.) Chicago, Illinois 60612; and University of Leicester (S.C.B.), Leicester, LE2 7LX, United Kingdom
Address all correspondence and requests for reprints to: Asgi T. Fazleabas, The University of Illinois at Chicago, Department of Obstetrics and Gynecology, 820 South Wood Street (M/C 808), Chicago, Illinois 60612-7313. E-mail: asgi{at}uic.edu
Abstract
In vitro studies indicate that glycodelin (PP14) synthesis by the human endometrium increases dramatically at the time of implantation and early pregnancy. It has been postulated that this protein may have an immunosuppressive function. Due to the limitations associated with in vivo studies in the human, this study was undertaken to study the regulation of the baboon glycodelin homolog in vivo during the menstrual cycle and early pregnancy. In nonpregnant baboons, between days 10–12 postovulation (n = 3) the mid and apical regions of the glandular epithelium showed a distinct punctate staining pattern, which increased between days 12–18 of pregnancy (n = 3). Between days 25–60 of pregnancy, staining intensity in the glandular epithelium decreased. The decrease was more apparent at the implantation site compared with the nonimplantation site. The immunostaining correlated with the synthesis of radiolabeled baboon glycodelin in explant culture. Northern blot analysis demonstrated two messenger RNA (mRNA) transcripts [1.0 and 1.7 kilobases (kb)] in the baboon uterus compared with a single 1.0-kb transcript in the human, and mRNA expression was consistent with protein localization and synthesis. The protein and mRNA expression was consistently higher in the deeper glands of the functionalis and basalis during early pregnancy. Because the increased expression of glycodelin in the baboon endometrium coincided with peak levels of CG, a simulated pregnant baboon model was used to confirm hormonal regulation. Exogenous human CG (hCG) followed by estrogen and progesterone treatment in intact and ovariectomized baboons up-regulated glycodelin expression between days 18–25 postovulation (n = 10). By day 32 postovulation (n = 3), glycodelin synthesis decreased. Estrogen and progesterone treatment in the absence of exogenous hCG did not result in an increase of glycodelin synthesis. Analysis of uterine flushings from hCG-treated animals revealed that a minimum of 7 days of hCG treatment was required for glycodelin to be detectable in the uterine lumen. These studies indicate that a posttranslationally modified glycodelin homolog is synthesized by the baboon uterus during early pregnancy and appears to be regulated directly by CG. This pattern of synthesis is comparable with that observed with in vitro studies in the human. Because glycodelin expression is associated with CG secretion, we suggest that this protein may have a functional role during implantation in the primate. Thus, the baboon may serve as a nonhuman primate model to elucidate the function of this protein in vivo.
GLYCODELIN is quantitatively the major secretory product synthesized by the human glandular epithelium during the late luteal phase and early pregnancy (1). Although originally called placental protein 14 (PP14) (2), this molecule is of endometrial origin and is immunologically identical to other proteins progesterone-dependent endometrial protein (PEP) and alpha uterine protein (AUP) described by several different investigators (see Ref.3). This protein has now been named glycodelin because of its unique carbohydrate structure (4). In addition to its presence in the human uterine endometrium, this protein has also been identified in human seminal plasma (5, 6), oviduct (7, 8), and ovary (6). In addition, hematopoietic cells of bone marrow also synthesize this protein (9).
Progesterone secretion during the luteal phase has been associated with the increase in glycodelin secretion by the glandular epithelium. However, because the serum profiles of progesterone and glycodelin in women are disparate (10), it also has been suggested that other ovarian or endometrial factors in addition to progesterone may be required for the up-regulation of its expression in the late secretory phase and early pregnancy (11). Recent studies suggest that ovarian relaxin, which is secreted by the corpus luteum in response to CG stimulation, may be one such factor (12). However, other endometrial or decidual factors, including locally synthesized relaxin cannot be ruled out (11, 12).
The biological role of glycodelin in early pregnancy is obscure. It has been associated with immunosuppression (13), transport of small hydrophobic molecules (14), and inhibition of sperm zona interactions (15). In addition, studies on endometrial regulation of glycodelin synthesis and its correlation with events in early pregnancy in the human have been limited to measurements in the peripheral circulation. Because the in vivo regulation and function of this major endometrial secretory remains to be determined, we have used our baboon model to characterize the in vivo regulation of glycodelin during the menstrual cycle and early pregnancy.
Materials and Methods
Tissue collection
Uterine tissue was obtained from adult female baboons (Papio anubis) either at hysterectomy or following endometriectomy. All experimental procedures were approved by the Animal Care Committee of the University of Illinois at Chicago.
Uterine tissue was obtained from four different groups. Comparative analyses between groups 1 and 2 delineates the direct effects of steroid hormones on the endometrium independent of the ovary. The treatment regimen described for group 4 simulates the hormonal environment in early pregnancy independent of the presence of a conceptus and allows for direct comparisons with group 3. A detailed description of each of these groups has been previously described (16).
Group 1, normally cycling female baboons (n = 9) (17), were used to study glycodelin synthesis during the menstrual cycle. Group 2, ovariectomized female baboons (18) were treated for 14 days with estradiol followed by 7 (n = 3) or 14 days (n = 2) of estradiol plus progesterone. Four animals were primed with estradiol for 14 days and treated with progesterone only for an additional 14 days. This group of animals was used to determine the regulation of glycodelin synthesis by steroid hormones in the absence of a pregnancy. Group 3, pregnant female baboons (19), in which pregnancy was verified by ultrasonography. Uterine tissue was obtained from pregnant baboons at days 12–18 (n = 3), days 22–32 (n = 9), and days 39–60 (n = 7). Group 4, simulated pregnant female baboons (20), were treated for 10 or 12 days with increasing doses of human CG (hCG) im beginning on day 6 postovulation. SILASTIC brand silicon tubing (Dow Corning, Midland, MI) implants containing estradiol or progesterone were then implanted sc as previously described (20). This treatment regimen results in hormone levels in the peripheral circulation that are within the 95% confidence intervals of pregnancy values. Tissue from hCG and steroid-treated baboons was obtained at days 18 (n = 5), 25 (n = 5), and 32 (n = 3) postovulation for direct comparison with pregnant animals. Control animals (days 18 and 25; n = 5) received only steroids and no hCG.
The uterine lumen of each animal was flushed with 10 mL sterile saline on the day of surgery. The uterus of one animal from the simulated pregnant group (day 25) group was flushed daily from days 10–23 (21). Portions of the tissue from all four groups were then taken and either fixed in Bouins solution for immunocytochemistry (19, 22), subjected to explant culture (17), or snap-frozen in liquid nitrogen for RNA extraction (23).
For purposes of comparison, luteal phase endometrial biopsies were obtained from women presenting to the reproductive endocrine service at the University of Illinois. Informed consent was obtained from all patients. Retention of a portion of the biopsy done for routine diagnostic purposes and use in this study was approved by the Institutional Review Board of the University of Illinois.
N-terminal sequence analysis
Uterine proteins (300 µg) obtained by flushing the uterus of
hCG-treated baboons (group 3; days 18 and 25) were separated on
two-dimensional polyacrylamide gels (2-D SDS-PAGE) and transferred to
polyvinylidene difluoride membranes (19, 24). The membrane was stained
with Coomassie blue, and the two proteins (Fig. 1D
, arrow) were subjected to
N-terminal sequence analysis by automated Edman degradation on an
Applied Biosystems 470 gas phase sequencer (Foster City, CA). The
sequence analysis was done at the Genetic Engineering Facility of the
University of Illinois at Urbana-Champaign.
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Endometrial tissues (75 or 150 mg) from each of the four groups were cultured in the presence or absence of [35S]methionine under serum-free conditions (17, 19). The explant culture media obtained following 24 h of culture was analyzed by fluorography or by Western blot analysis following one-dimensional (1-D) and 2-D SDS-PAGE (19).
Immunocytochemistry
Uterine tissues were immersion-fixed in Bouins solution for 24 h at room temperature, dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin (22). Before fixation, tissues from pregnant animals were separated into the implantation and nonimplantation sites by dissection (19).
Sections were cut at 6 µm on a rotary microtome. Polyclonal antibody specific for human glycodelin (7, 11) was used at a dilution of 1:750, and the immunoreactive product was visualized on ABC Vecstain kit (Vector Laboratories, Burlingame, CA) and diaminobenzadine (16). Controls consisted of preimmune serum at the same dilution or the omission of the primary antibody.
Immunoblotting
Secretory proteins (25–50 µg) in explant culture media or uterine flushings were separated by 1-D and 2-D SDS-PAGE, and proteins were transferred to nitrocellulose membranes (24). The membranes were incubated overnight in primary antibody (1:2000 dilution), and the immunoreactive product was visualized using the Bio-Rad alkaline phosphate blotting kit according to the manufacturer’s specifications (Bio-Rad, Hercules, CA).
RIA
An equilibrium RIA for glycodelin was used (22, 25). Aliquots (200 µL) of the daily uterine flushes obtained from the hCG-treated baboons were assayed in duplicate, and the concentration of glycodelin in the uterine flush was normalized against the total amount of protein in the sample. The lower and upper limits of sensitivity of the RIA were 4 ng and 250 ng, respectively.
Northern blots
Total RNA (20 µg) from nonpregnant, pregnant, and simulated pregnant baboons was separated on a 1% agarose-formaldehyde gel, and the RNA was transferred to nitrocellulose (26). The membranes were hybridized with a 32P random primer-labeled 822-bp complementary DNA insert to human glycodelin (27). The hybridization signal was visualized by exposing the membrane to Kodak X-Omat film (Eastman Kodak, Rochester, NY) with a Dupont-Cronex intensifying screen (Dupont, Wilmington, DE) at -80 C. The resulting autoradiographs were densitometrically scanned using a Molecular Dynamics PhosphoImager (Sunnyvale, CA).
Results
Identification and characterization of baboon glycodelin
Analysis of [35S]methionine-labeled proteins in
explant culture media (Fig. 1
, A-C) and uterine flushings (Fig. 1, D
and E) indicated the presence of a new group of proteins that were
synthesized by the endometrium from pregnant (Fig. 1A) and simulated
pregnant baboons treated with hCG (Fig. 1, B and D). In the absence of
hCG, these proteins were not evident (Fig. 1, C and E). N-terminal
sequence analysis of the proteins in uterine flushings (Fig. 1D, arrow) revealed a 99.5% homology to human glycodelin.
Human: M D I P Q T K Q D L E L P K L A G T W S M
Baboon: T D I P Q T K Q N L
Identification of this group of proteins as being homologous to human glycodelin in this study, allowed us to use a human polyclonal antibody and cDNA to study to expression of this protein during the menstrual cycle, pregnancy, and simulated pregnancy in the baboon.
Nonpregnant baboons (groups 1 and 2)
Figure 2 shows the
immunocytochemical localization of glycodelin in the baboon endometrium
during the menstrual cycle. A relatively faint punctated staining
pattern was evident in the mid functionalis glands during the mid and
late luteal stages of the menstrual cycle (Fig. 2, B and C). The
staining intensity in the baboon was markedly less than that seen in
the human at a comparable stage (Fig. 2C, inset).
Immunoreactive product was also evident following sequential treatment
of ovariectomized baboons with estradiol and progesterone (Fig. 2F).
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During pregnancy there was a marked up-regulation of glycodelin
synthesis by the glandular epithelium. Both the mid functionalis and
basal glands showed an increase in staining intensity (Fig. 3, A-D). This was evident as early as 2
days postimplantation (Fig. 3, A and B). The intensity of staining was
greater at the implantation site (Fig. 3, A and C) compared with the
nonimplantation site (Fig. 3, B and D). As pregnancy proceeded,
glandular regression was associated with decreased staining for
glycodelin (Fig. 3F). In contrast, a select group of decidual cells
directly below the implantation site and the luminal epithelium of the
nonimplantation site stained positively.
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). Message expression
in both the functionalis and basalis was highest at day 12
postovulation (2 days postimplantation: Fig. 4, lanes 2 and 3) and at
the implantation site on day 25 (Fig. 4, lane 6). At the end of the
first third of gestation, there was a decrease in both protein (Fig. 3F) and mRNA expression (Fig. 4, lanes 9 and 10). A higher level of
message was evident in late secretory human endometrium (Fig. 4, lane
11) compared with baboon (Fig. 4, lane 1 and Fig. 2C).
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The high levels of glycodelin in the baboon endometrium
coincided with peak levels of CG (28). To confirm that CG modulates
glycodelin synthesis during early pregnancy, we analyzed tissues from
simulated pregnant baboons (20). Strong immunoreactive product was
evident at days 18 and 25 postovulation in the CG-treated animals (Fig. 5, A and B). By day 32 (12 days after the
last hCG injection), there was a decrease in staining intensity (Fig. 5C). In the absence of hCG treatment, estradiol and progesterone did
not induce glycodelin synthesis in the basal glands (Fig. 5D). The
punctated staining pattern seen during the menstrual cycle in the mid
functionalis was still evident (data not shown).
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). mRNA expression
was highest at days 18 and 25 in the hCG-treated group (Fig. 6, lanes 1
and 2) when compared with the steroid only-treated group (Fig. 6, lanes
4–6).
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) and RIA (Fig. 7C) 7 days following
the initial hCG injection. The protein levels increased markedly on day
17 (Fig. 7A, lane 7) and remained relatively constant thereafter (Fig. 7, A and C, lanes 8–13). The 2-D Western blot (Fig. 7B) of the day 23
postovulation flush confirms that the family of proteins we had
initially identified and sequenced (Fig. 1D, arrow)
cross-react with the human polyclonal antibody and are the baboon
homolog of glycodelin.
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Our studies suggest that the baboon and human show a similar regulatory pattern for glycodelin expression. Thus, the initial synthesis is associated with progesterone-induced differentiation of the uterine endometrium. Synthesis of glycodelin in the baboon is confined to the mid functionalis glands during the mid and late secretory phase of the cycle. The marked histological change in endometrial morphology in response to pregnancy dramatically up-regulates glycodelin expression by the glandular epithelium throughout the entire uterine endometrium in the baboon.
Although the overall secretory pattern of glycodelin is similar in both the human (29, 30, 31, 32) and baboon, the electrophoretic properties are distinctly different. The baboon protein is coded for by two mRNA transcripts, and this alternate splicing could result in differences observed in both the molecular weight and isoelectric variants. The electrophoretic properties of human glycodelin during the secretory phase and early pregnancy are similar (1, 31). In contrast, the baboon secretory phase protein has a molecular weight of 21,000–24,000 and is expressed at relatively low levels. In response to pregnancy or treatment with hCG, baboon glycodelin undergoes marked changes in gene expression and posttranslational modification.
A recent study demonstrated a close temporal relationship between relaxin and glycodelin serum profiles in the human (12). Based on these studies, the authors suggested that ovarian relaxin, which increases in response to hCG in early pregnancy, may regulate the pregnancy-associated increase in endometrial glycodelin synthesis. The baboon simulated-pregnant model further supports the hypothesis that either an additional ovarian or endometrial factor induced by hCG enhances glycodelin synthesis and secretion. In our simulated-pregnant model, and in early pregnancy, CG increases relaxin production by the corpus luteum (33) and enhances the overall secretory activity of the endometrium (20). However, im injections of hCG to ovariectomized baboons also suggest that glycodelin synthesis could be regulated by CG acting directly on the endometrium. Thus, it is conceivable that CG could act directly on the endometrium to induce the local production of relaxin. Endometrial relaxin could in turn up-regulate the synthesis and expression of glycodelin in the glandular epithelial cells. We have not determined the expression of relaxin in the baboon endometrium. However, our preliminary immunochemical studies indicate that the increase in glycodelin synthesis in response to exogenous hCG or pregnancy is correlated with presence of the LH/CG receptor in the glandular epithelium (unpublished results). In addition, insulin-like growth factor binding protein-1 (IGFBP-1) synthesized by decidual cells has also been proposed as an endometrial modulator of glycodelin expression (11). It is interesting to note that when glandular expression decreased between days 50 and 60 of pregnancy, a subset of decidual cells directly below the implantation site show specific glycodelin immunoreactivity. These cells also produce high levels of IGFBP-1 (34, 35). IGFBP-1 and its ligand IGFs, (which are related to relaxin), could also modulate decidual synthesis of glycodelin. It is therefore evident from our studies that conceptus signals (i.e. CG) and the conceptus itself can directly or indirectly regulate glycodelin synthesis during early pregnancy.
The biological functions of glycodelin have yet to be elucidated. Homology with ß-lactoglobulin and retinol binding protein (27) suggest a role for the transport of the small hydrophobic molecules such as retinol to the developing fetus. However, human glycodelin does not bind retinol. In contrast, a retinol binding protein, distinct from glycodelin, is synthesized by the baboon, but not the human endometrium (26). Retinol binding protein is synthesized in the basal glands during the luteal stage, and its synthesis in the functionalis increases markedly through day 32 postovulation in pregnant and simulated pregnant baboons and declines thereafter (20, 26). The patterns of retinol binding protein and glycodelin synthesis in the pregnant baboon are very similar.
Several authors have suggested that the measurement of circulating glycodelin could provide clinicians with an estimate of uterine responsiveness to exogenous steroids (3, 36). Although initial studies appeared promising, more recent data demonstrates substantial overlap in circulating glycodelin levels in both normal and pathological states and limits its use clinically.
Most of the previous (13, 35) and current studies (4, 15) on the function of glycodelin have focused on its potential immunosuppressive function. Crude decidual extracts containing glycodelin suppressed [3H]thymidine uptake in stimulated lymphocytes (13, 37, 38). It was postulated that this effect was mediated via the suppression of interleukin-1 and interleukin-2 and its receptor (38, 39). In addition, purified glycodelin also suppressed cell lysis by natural killer cells (40). The degree of glycosylation appears to be associated with its immunosuppressive properties (4). Thus, in vitro studies suggest that glycodelin has the potential of being one of the many factors associated with the immunosuppression of the maternal response to the fetal allograft.
Although a convincing function for this protein remains to be elucidated, the dramatic increase in endometrial synthesis around the time of implantation and early pregnancy (31) and in response to CG (this study), implies that this protein may play an important role during the establishment of pregnancy in the primate. There are ethical limitations in attempting to determine the function of endometrial proteins during implantation in the human. We have established that the baboon uterus synthesizes and secretes a homologous protein in a manner similar to that observed with in vitro studies in the human. Thus, we can now use this nonhuman primate model for in vivo studies study the potential role of glycodelin during early pregnancy.
Acknowledgments
We thank Ms. Juliet Jackson for performing the RIAs.
Footnotes
1 This work was done as part of the National Cooperative Program for
Markers of Uterine Receptivity for Blastocyst Implantation and was
supported by Cooperative Agreement NIH-NICHD 29964 (to A.T.F.). 
Received October 15, 1997.
Revised December 31, 1997.
Accepted January 8, 1998.
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