This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nemansky, M. Articles by Blithe, D. L. Search for Related Content PubMed PubMed Citation Articles by Nemansky, M. Articles by Blithe, D. L. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CHORIONIC GONADOTROPIN PROGESTERONE

Human Endometrial Stromal Cells Generate Uncombined -Subunit from Human Chorionic Gonadotropin, Which Can Synergize with Progesterone to Induce Decidualization

Unit of Glycobiology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Diana L. Blithe, Ph.D., Contraception and Reproductive Health Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 61E, Room 8B13, Bethesda, Maryland 20892. E-mail: BlitheD{at}hd01.nichd.nih.gov

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
During the secretory phase of the menstrual cycle, endometrial stromal cells differentiate into decidual cells, which play a crucial role in implantation and maintenance of pregnancy. In this and our previous study, we demonstrate that glycoprotein hormone free -subunit potentiates progesterone-mediated decidualization of human endometrial stromal cells in vitro. Although addition of intact hCG to cultures resulted in stimulatory activity, its potency was 20-fold less than that of -subunit. However, in the present study we show that decidualizing endometrial cells actively generate uncombined -subunit by dissociating hCG. The amount of dissociated -subunit could fully account for the stimulatory activity observed with hCG. Active dissociation of hCG was dependent on the presence of endometrial cells and did not occur in conditioned medium, excluding involvement of a stable secreted factor such as a protease. In addition to dissociated - and ß-subunits, minor amounts of ß-core and -fragments were detected as degradation products during active dissociation. We also observed an increase in ß-immunoreactivity that coeluted with hCG on size-exclusion gel chromatography, indicating that a portion of the still dimeric hCG may have been nicked in the dissociation process. However, using an assay with specificity for nicked hCG, we showed that dissociation of hCG was not produced from a pool of preexisting nicked hCG. These findings more firmly establish the concept that gonadotropin hormone free -subunit plays a role in the regulation of human endometrial cell differentiation. In addition, identification of the various products formed by incubation of hCG with decidualizing cells yielded insight into the mechanism of hCG degradation, and may explain some activity previously ascribed to hCG.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
UTERINE endometrial stromal cells differentiate into decidual cells during the luteal (secretory) phase of the menstrual cycle and continue this process throughout pregnancy. Decidualization plays a crucial role in implantation and maintenance of pregnancy. If pregnancy does not occur, the decidua is shed, and new growth of the endometrium begins (1, 2). During the process of decidualization, endometrial stromal cells change their morphology from a fibroblastic spindle-like appearance to a more spherical shape. Differentiation is characterized by the production of specific hormones (i.e. PRL and IGFBP-1), cytokines, extracellular matrix components, neuropeptides, and various enzymes, including arylsulfatase, enkephalinase, proteases, and protease inhibitors (2, 3, 4, 5, 6). Progesterone (P), a steroid hormone secreted during the luteal phase of the menstrual cycle and during pregnancy, is generally considered to be the key inducing factor for the decidualization process. However, the use of newly developed systems to culture human endometrial stromal cells in vitro has revealed that additional endocrine, paracrine, and autocrine factors modulate decidualization (4, 5, 7, 8, 9, 10, 11, 12, 13, 14). We have recently reported that free glycoprotein hormone -subunit acts synergistically with P to induce decidualization of human endometrial stromal cells, thereby establishing a novel role for free -subunit in human reproduction (15).

Glycoprotein hormone -subunit is common to the heterodimeric hormones CG, LH, FSH, and TSH. In addition to being combined with a hormone-specific ß-subunit, is secreted as a free molecule by the pituitary during the normal menstrual cycle (16) and by the placenta throughout pregnancy (17, 18). The free form of -subunit is differently glycosylated from the combined form (19, 20). Glycan structures on secreted free -subunit prevent it from combining with ß-subunits that are encountered after secretion (20, 21), thus ensuring a population of free molecules.

Until recently, the free glycoprotein hormone -subunit had been ignored as a bioactive molecule, mostly due to its lack of activity at receptors for heterodimeric hormones (22). However, evidence is mounting that free -subunit can function independently from the heterodimeric hormones as an endocrine and paracrine factor, and that the target cells for activity include those that ultimately produce PRL as a secretory product (15, 23, 24, 25, 26, 27, 28, 29). It is our hypothesis that free -subunit is a factor involved in processes of growth and differentiation, possibly analogous to its structural homologs nerve growth factor, platelet-derived growth factor, and transforming growth factor-ß, which all share a cysteine knot motif (30, 31).

Glycoprotein hormones have been reported to have stimulatory effects on PRL production and endometrial cell differentiation (10, 23, 32, 33, 34). Some studies proposed that the stimulation was mediated by receptors specific for heterodimeric hormones (35, 36). However, in other studies it was implied that the stimulatory effects may in reality be caused by the presence of uncombined -subunits in their preparations (23, 25, 26). In this paper, we have compared the activity of hCG and -subunit in the stimulation of human endometrial cell differentiation in vitro. Although addition of intact hCG produced stimulatory activity, hCG was less potent than its free -subunit. Moreover, decidualizing endometrial cells were found to actively generate uncombined -subunit by dissociation of hCG, and the amount of dissociated -subunit could fully account for the stimulatory activity of the hCG preparation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
hCG (CR125), hCG (CR119), and hCGß (CR119) were obtained from Drs. S. Birken and R. Canfield through the Center for Population Research. hCG -subunit preparations were assessed for purity by SDS-PAGE, protein analysis, and immunoassay (37). For experiments comparing the effects of hCG with those of -subunit, highly purified hCG (hCG-DB: 21,700 IU/mg) was prepared from crude hCG (Diosynth, Oss, The Netherlands) by chromatography on Sephadex G-100 and diethylaminoethyl-Sephacel (Pharmacia, Uppsala, Sweden) as described previously (37). The hCG-DB preparation contained very little contamination with uncombined -subunit (<0.3%, wt/wt). P was obtained from Sigma Chemical Co. (St. Louis, MO) and was dissolved in ethanol before use: the final concentration of ethanol in the medium was 0.005%. The same concentration of ethanol was added to medium that did not contain P.

Immunoassays

Intact hCG was assayed using a monoclonal antibody, A03C9 (Monoclonal Antibodies, Sunnyvale, CA), with cross-reactivities for - and ß-subunits of 1.3% and 0.3%, respectively. Free ß-subunit was assayed using a polyclonal antiserum, SB6 (38). Its cross-reactivity with hCG was established to be less than 0.6%. Free was assayed using a monoclonal antibody with specificity for the uncombined subunit (BioMerica, Newport Beach, CA). The cross-reactivity of intact hCG with the free monoclonal antibody was less than 0.1% (39, 40). ß-Core was assayed by RIA using purified ß-core and a polyclonal antiserum with less than 1% cross-reactivity with hCG ß-subunit (41). Nicked hCG was assayed by immunoradiometric assay using monoclonal antibodies B151/B207 with specificity for nicked hCG. The maximal cross-reactivity of the nicked hCG assay with intact hCG was 14% (O’Connor, J. F., and S. Birken, personal communication). PRL levels in media were assayed by specific homologous RIA (42) using human PRL standards and antibodies obtained from the National Hormone and Pituitary Program (NIDDK).

Tissue collection and cell culture

Human endometrial tissue was obtained by Pipelle endometrial biopsy within 1–3 days of the LH surge (OvuQuick, Quidel Corp., San Diego, CA). Normal female volunteers (age, 21–45 yr) had regular menstrual cycles and were not taking hormonal medications. The protocol was approved by the institutional review board for human subjects research of the NICHD, and informed consent was obtained. Biopsies were histologically dated by the Pathology Department, NIH Clinical Center, using the criteria of Noyes et al. (43).

Stromal cells were isolated from endometrial tissue using a modification of the method of Fleming et al. (44). Tissue was transported, minced, and digested with 0.25% collagenase-CLS1 (Worthington Biochemical Corp., Freehold, NJ) as described previously (15). The cell suspension was centrifuged (200 x g) for 5 min, and the pellet was washed with PBS. Cell clumps were dispersed by gentle pipetting in 2 mL 0.1% trypsin-0.05% ethylenediamine tetraacetate (Advanced Biotechnologies, Columbia, MD) and incubating at 37 C for 5 min. The cell suspension was diluted with MEM (15 mL), filtered through a 105-µm pore size sieve, and collected in a T-175 culture flask. Cells were incubated at 37 C in a humidified atmosphere of 95% air-5% CO₂ for 20–30 min. Unattached cells were removed, and attached cells were washed with PBS. Cells were detached by the addition of 3 mL trypsin-ethylenediamine tetraacetate and incubation for 5 min at 37 C. Detached cells were diluted in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10% charcoal-treated FBS (CoCalico Biologicals, Reamstown, PA), insulin (8 µg/mL; Eli Lilly Co., Indianapolis, IN), and 1% antibiotic-antimycotic solution (10,000 U/mL penicillin, 10 mg/mL streptomycin, and 25 µg/mL fungizone; Advanced Biotechnologies). Cells were plated (150,000–200,000 cells/well) into 12-well plates (Costar, Cambridge, MA).

Cells were grown to confluence, with fresh medium added at 48-h intervals (1 mL/well). The serum concentration was reduced to 2% 48 h before beginning hormone treatment and was maintained at 2% throughout the experiment. In experiments comparing hCG with stimulation, wells containing hCG-DB and P (50 ng/mL) were incubated in parallel to wells containing plus P or P alone. One- and two-way ANOVAs were used to analyze the time course, and post-hoc tests were performed and interpreted with the Student-Newman-Keuls adjustment for multiple comparisons. Unpaired Student’s t tests were performed for comparisons at timed intervals.

To compare whether the degree of decidualization influenced the dissociation of hCG, fibroblastic cells (confluent stromal cells with no prior exposure to P) and partially decidualized cells (at least 14-day incubation in medium containing P) from the same endometrial preparation were used. Each condition was performed either in triplicate or in six wells, as indicated. Media from each well were changed every 48 h (or as indicated) and stored at -20 C until assayed.

Size-exclusion gel chromatography

Size-exclusion gel chromatography was performed on a column (1.6 x 100 cm) of Sephadex G-100 (superfine; Pharmacia LKB Biotechnology, Piscataway, NJ) eluted in 0.1 mol/L ammonium acetate (pH 7.4) at 4 C with a flow rate of 5 mL/h. Fractions of 2 mL were collected into tubes containing 2 mg BSA and assayed for intact hCG, free ß-subunit, free -subunit, and ß-core. Samples of fresh or conditioned media were concentrated (15–20 times) before application on the column using Microcon-3 concentrators (Amicon, Beverly, MA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Stimulation of decidualization

Stromal cells were obtained from endometrial biopsies performed during the periovulatory phase of the menstrual cycle. To compare the potential activity of hCG on cell differentiation with the stimulatory activity of -subunit, confluent stromal cells were cultured in medium supplemented with P (50 ng/mL) alone or P plus either (0.2 or 1 ng/mL) or hCG (4 or 20 ng/mL; Fig. 1). Concentrations of hCG and were selected with the following rationale. Based on mol wt, a level of 4 ng/mL hCG is approximately equivalent on a molar basis to 1 ng/mL of -subunit. Furthermore, although the hCG-DB preparation initially had no detectable contamination, -subunit was actively generated by dissociation of hCG in the presence of cells (see below). During the 48-h incubation period, cell cultures containing 4 ng/mL hCG produced about 0.2 ng/mL dissociated -subunit, and cultures with 20 ng/mL hCG produced 1 ng/mL uncombined . In Fig. 1, each bar represents the mean PRL concentration (±SEM) of triplicate wells incubated for 48 h in medium containing the various hormone combinations. PRL production (nanograms per mL/2 days) increased throughout the 30 days of exposure to each condition. PRL production was not observed in control cells that were not exposed to P, hCG, or throughout the time period (<1 ng/mL·2 days). In the absence of P, we observed no significant stimulation of PRL by hCG (data not shown) and a very slight effect of , as shown previously (15). Cells incubated in the presence of either P plus hCG or P plus exhibited higher PRL production than cells incubated with P alone; however, the stimulatory activity of -subunit was greater than that of parallel doses of hCG. The data in Fig. 1 compared stimulatory effects of the different agents on cells derived from a single endometrial preparation; however, similar results were found with endometrial cells obtained from other individuals (n = 3).

View larger version (23K): [in this window] [in a new window]   Figure 1. Comparison of and hCG stimulation of PRL production from human endometrial cells in culture. Confluent stromal cells were cultured in medium with no additions (control), P (50 ng/mL) alone, or P together with (0.2 or 1 ng/mL) or hCG (4 or 20 ng/mL). Media were collected every 48 h and stored at -20 C until assayed for PRL. Each bar represents the mean PRL concentration (±SEM of triplicate wells) in 48 h media of the indicated condition at the given time. Control cells produced PRL levels of 0.5, 0.7, 0.9, and 0.1 ng/mL·2 days on days 0, 12, 24, and 30, respectively. Two-way ANOVA was used to analyze the effect of treatment with plus P vs. treatment with hCG plus P compared to production from wells containing P alone. Unpaired Student’s t tests were used for comparisons at the indicated time intervals. *, P < 0.05; **, P < 0.01. N.B., The 4 ng/mL hCG preparation will contain 0.2 ng/mL after incubation for 48 h. Likewise, 20 ng/mL hCG will contain 1 ng/mL at the time of collection.

View larger version (14K): [in this window] [in a new window]   Figure 2. Dose-response curve of PRL production from cells treated with P alone and P plus increasing doses of or hCG. Human endometrial stromal cells were induced to decidualize in culture with varying concentrations of (•) or hCG () together with P (50 ng/mL). The PRL production shown was assayed in medium collected over a 96-h period (days 27–30 after initiation of stimulation) and is expressed relative to stimulation with P alone (). The dashed line (X) shows the PRL stimulation caused by the presence of -subunit generated through dissociation of the hCG preparations during their incubation with the cells. Each point represents the mean of triplicate wells.

Active generation of -subunit from hCG

To characterize the dissociation of hCG, endometrial cells (either fibroblastic or partially decidualized) were incubated with medium containing hCG (100 ng/mL) plus P. The production of dissociated -subunit was measured by RIA using the free -specific monoclonal antibody. Generation of uncombined -subunit by dissociation of hCG appeared to be linear in time for at least 96 h (Fig. 3). Fibroblastic stromal cells actively dissociated hCG at a 4- to 6-fold higher rate than spontaneous dissociation of hCG that occurred in the absence of cells. No -subunit was produced from cells incubated without hCG. Cells that were partially decidualized were able to dissociate hCG at a higher rate (1.6-fold) than fibroblastic stromal cells.

View larger version (15K): [in this window] [in a new window]   Figure 3. Generation of uncombined -subunit from dissociation of hCG: influence of the presence or absence of cells and their state of decidualization. Media containing hCG (100 ng/mL) plus P were incubated for the given period of time and then assayed for dissociated -subunit. The following conditions were used: and •, hCG in the presence of confluent cells; and , hCG in the presence of conditioned media but in the absence of cells; and , hCG incubated in the absence of cells; and , no hCG, just cells. Each point represents the mean ± SEM of six wells. Upper panel, Fibroblastic cells. Lower panel, Decidualizing cells.

To confirm that the increasing immunoreactivity was indeed due to generation of uncombined -subunit rather than to exposure of epitopes by partial degradation of hCG, column chromatography was performed on hCG-containing medium before and after incubation with decidualized cells (Fig. 4). Fractions were collected and assayed for intact hCG, ß-subunit, -subunit, and ß-core fragment. Molar ratios of intact hCG and of its various products were calculated relative to the amount of intact hCG in the starting preparation (Table 1). The starting hCG preparation contained no free -subunit; however, it was found to include a small amount (3.8%) of free ß-subunit (Fig. 4, upper panel). No hCG dissociation was observed during the preparation of the samples or during column chromatography. After incubation in the presence of decidualized cells for 72 h, the medium contained 19% less intact hCG (Fig. 4, lower panel). The increased -subunit immunoreactivity (+15.5%) was indeed due to the appearance of uncombined -subunit (eluting at fraction 60). Dissociation of hCG was also confirmed by the increase in free ß-subunit (+19.9%; fractions 46–48). Concurrently, minor amounts of degradation products, ß-core (+1.6%; fraction 71) and -fragment (+2.5%; fractions 74–76) were detected in the incubated medium. Also, an increase in ß-immunoreactivity (+5.9%) was observed to coelute with intact hCG (fraction 42), indicating that a part of the still dimeric hCG was nicked, thereby exposing an epitope that was recognized by the free ß-specific antibody.

View larger version (19K): [in this window] [in a new window]   Figure 4. Sephadex G-100 column chromatography of media containing an hCG preparation before (upper panel) and after (lower panel) incubation with endometrial stromal cells. The hCG preparation was incubated for 72 h in the presence of decidualizing stromal cells. Both the starting preparation and the incubated media were subjected to column chromatography as described in Materials and Methods. Fractions of 2 mL were collected and assayed for the presence of intact hCG (•), free ß-subunit (), free -subunit (), and ß-core ().

View larger version (16K): [in this window] [in a new window]   Figure 5. Relative concentrations of intact hCG (black bars) and nicked hCG (open bars) from unconditioned (0 h) and conditioned (96 h) media. Media containing P plus hCG (100 ng/mL) were conditioned in the presence of fibroblastic or decidualizing cells as described in Materials and Methods. The amounts of intact and nicked hCG are expressed relative to the intact hCG concentration in unconditioned (0 h) media. Each bar represents the mean ± SEM of six wells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

hCG functions as a luteotropic agent in early pregnancy, maintaining the production of P from the corpus luteum until this function is taken over by the placenta (46, 47), and may regulate functions in other tissues throughout pregnancy (48). hCG and other glycoprotein hormones have been reported to stimulate PRL production and endometrial cell differentiation (10, 23, 32, 33, 34). Some investigators have suggested that these effects may be mediated by receptors specific for heterodimeric hormones (35, 36); however, other studies indicate that the stimulatory effects of dimeric hormones are caused by uncombined -subunits in the hormone preparations (23, 25, 26). In the present study we have examined the roles of hCG and -subunit in stimulation of PRL production from human endometrial stromal cells. In the absence of P, we observed no significant stimulation of PRL by hCG. Addition of hCG to P-containing medium resulted in stimulation of cell decidualization and PRL production, but hCG was 20-fold less potent than -subunit. Moreover, cultured cells were found to actively generate uncombined -subunit from hCG, which could fully account for the observed stimulation. The stimulatory activity of -subunit is physiologically significant, occurring at doses typically found in sera of women during the normal menstrual cycle (10^-11–10^-10 mol/L). A dose-dependent, saturable, biphasic stimulation pattern was found, suggesting that activity of free may be mediated through distinct -subunit-specific membrane receptors.

Our results indicate that the stimulatory effect of hCG on endometrial cell differentiation most likely does not result from intact hCG itself, as it can be fully accounted for by uncombined -subunit generated from hCG in the presence of cells. Thus, decidualizing activity attributed to intact gonadotropins may need to be reexamined in light of the bioactivity of -subunit for two reasons. Firstly, levels of -subunit contamination in hormone preparations may be high; some purified preparations of gonadotropins contained substantial amounts of uncombined (24, 39, 40). Secondly, as illustrated by this study, uncombined -subunit can be generated from intact dimeric hormone, and the -subunit may act through independent mechanisms to produce distinct effects. Theoretically, the actions of gonadotropins and free -subunit may occur simultaneously, as they may be mediated through separate specific receptors and second messenger systems.

The pathways through which hCG is dissociated and degraded are not fully understood. Degradation plays a role in regulating active hormone levels in the circulation that determine action of the hormone in vivo. In this study we evaluated the stability of the hCG dimer. Using size-exclusion gel chromatography and immunoassays specific for intact hCG, dissociated -subunit, dissociated ß-subunit, ß-core, and nicked hCG, respectively, we were able to follow the dissociation and degradation of hCG. The presence of endometrial cells (either fibroblastic or decidualized) considerably enhanced the dissociation rate of hCG compared to spontaneous dissociation in the absence of cells, indicating involvement of a factor(s) that actively influences the process. Active dissociation of hCG could not be mimicked by conditioned medium from decidualized cells, indicating that this factor is either not stable or, more likely, is associated with the cells themselves. Our findings are consistent with those of previous studies showing that an assisted process of hCG subunit assembly and dissociation occurs in cells and can be mimicked in vitro by agents such as protein disulfide isomerase, which reduce and oxidize disulfide bonds of ß-subunit (49). Moreover, the - and ß-subunits of hCG are stabilized by a segment of the ß-subunit (the "seat-belt" region) that wraps around the -subunit and is covalently linked by a disulfide bond that must be reduced before dissociation can occur (30).

Exposure of hCG to cells in culture led primarily to dissociation into intact subunits, but some further degradation to smaller sized -fragments and ß-core fragment was observed. In general, it is believed that ß-core is a product of metabolism of hCG or ß-subunit in the kidney. During pregnancy, considerable amounts of hCG, free - and ß-subunits, and ß-core are found in the urine (50); however, there is no detectable ß-core in serum (51, 52), suggesting that ß-core derives from renal degradation of hCG ß-subunit. Furthermore, ß-core has been detected in the urine of normal human subjects infused with hCG (53), and studies in rats have demonstrated the production of ß-core in kidney tissue and the excretion of ß-core in urine after injection of hCG and ß-subunit (54). In the light of these data, it was surprising to find generation of ß-core by nonrenal cells, indicating that production of ß-core may occur in other tissues as well.

ß-Core and -fragments may be produced by nicking of the respective intact subunits. This view is supported by the appearance of free ß-immunoreactivity that coeluted with intact hCG on gel chromatography and was not present in the original hCG preparation. Apparently, an epitope that is recognized by the free ß-subunit-specific antibody was exposed on hCG by nicking of the still dimeric molecule. This nicked molecule could represent an intermediate state for hCG dissociation and degradation, as nicking of hCG, especially of its ß-subunit, has been suggested to play an important role in these processes (55, 56, 57). However, using immunoassays that specifically discriminate between nicked and intact hCG, we did not detect nicked hCG above the level attributable to cross-reactivity with intact hCG, nor did we observe an increase in nicked hCG during the dissociation process. This rules out the possibility that dissociation proceeds from a preexisting pool of nicked hCG. Furthermore, a secreted stable nicking enzyme, such as a protease, did not seem to be involved in the degradation of hCG, as conditioned medium did not produce the same effect as the presence of cells. These findings are consistent with studies of hCG in blood, where no role for a protease could be found in its dissociation process (58). Our observations do not rule out the possibility that dissociation of hCG proceeds concurrently with nicking of the dimer or a possible nicking enzyme that might be associated with the cell surface. Nonetheless, endometrial stromal cells in culture were able to actively dissociate hCG into its subunits and successively use the generated -subunit to further induce their own decidualization.

	Acknowledgments

 
The authors thank Dr. Lawrence Nelson and Ms. Lorene Kimzey, R.N., for their help with endometrial biopsies, and Ms. Paulette O’Connell for her excellent assistance with tissue culture experiments. Drs. John O’Connor and Steven Birken are gratefully acknowledged for performing assays for nicked hCG.

Received July 11, 1997.

Revised October 14, 1997.

Accepted October 22, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Kearns M, Lala PK. 1983 Life history of decidual cells: a review. Am J Reprod Immunol Microbiol. 3:78–82.
2. Gurpide E, Tabanelli S, Tang B. 1992 Human endometrial stromal cells. In: Genazzani AR, Petraglia F, eds. Hormones in gynecological endocrinology. Oxford: Pergamon Press; 717–724.
3. Schatz F, Papp C, Toth-Pal E, et al. 1994 Protease and protease inhibitor expression during in vitro decidualization of human endometrial stromal cells. Ann NY Acad Sci. 734:33–42.[Medline]
4. Huang JR, Tseng L, Bischof P, Janne O. 1987 Regulation of prolactin production by progestin, estrogen, and relaxin in human endometrial stromal cells. Endocrinology. 121:2011–2017.[Abstract]
5. Zhu HH, Huang JR, Mazella J, Rosenberg M, Tseng L. 1990 Differential effects of progestin and relaxin on the synthesis and secretion of immunoreactive prolactin in long term culture of human endometrial stromal cells. J Clin Endocrinol Metab. 71:889–899.[Abstract]
6. Irwin JC, Kirk D, King RJB, Quigley MM, Gwatkin RBL. 1989 Hormonal regulation of human endometrial stromal cells in culture: an in vitro model for decidualization. Fertil Steril. 52:761–768.[Medline]
7. Giudice LC, Dsupin BA, Irwin JC. 1992 Steroid and peptide regulation of insulin-like growth factor-binding proteins secreted by human endometrial stromal cells is dependent on stromal differentiation. J Clin Endocrinol Metab. 75:1235–1241.[Abstract]
8. Irwin JC, Utian WH, Eckert RL. 1991 Sex steroids and growth factors differentially regulate the growth and differentiation of cultured human endometrial stromal cells. Endocrinology. 129:2385–2392.[Abstract]
9. Irwin JC, de las Fuentes L, Dsupin BA, Giudice LC. 1993 Insulin-like growth factor regulation of human endometrial stromal cell function: coordinate effects of insulin-like growth factor binding protein-1, cell proliferation and prolactin secretion. Regul Pept. 48:165–177.[CrossRef][Medline]
10. Tang B, Gurpide E. 1993 Direct effect of gonadotropins on decidualization of human endometrial stromal cells. J Steroid Biochem Mol Biol. 47:115–121.[CrossRef][Medline]
11. Frank GR, Brar AK, Cedars MI, Handwerger S. 1994 Prostaglandin E₂ enhances human endometrial stromal differentiation. Endocrinology. 134:258–263.[Abstract]
12. Kariya M, Kanzaki H, Takakura K, et al. 1991 Interleukin-1 inhibits in vitro decidualization of human endometrial stromal cells. J Clin Endocrinol Metab. 73:1170–1174.[Abstract]
13. Takuya I, Kanzaki H, Imai K, et al. 1994 Bestatin, a potent aminopeptidase-N inhibitor, inhibits in vitro decidualization of human endometrial stromal cells. J Clin Endocrinol Metab. 79:171–175.[Abstract]
14. Ferrari A, Petraglia F, Gurpide E. 1995 Corticotropin releasing factor decidualizes human endometrial stromal cells in vitro. Interaction with progestin. J Steroid Biochem Mol Biol. 54:251–255.[CrossRef][Medline]
15. Moy E, Kimzey LM, Nelson LM, Blithe DL. 1996 Glycoprotein hormone -subunit functions synergistically with progesterone to stimulate differentiation of cultured human endometrial stromal cells to decidualized cells: a novel role for free -subunit in reproduction. Endocrinology. 137:1332–1339.[Abstract]
16. Hagen C, McNatty KP, McNeilly AS. 1976 Immunoreactive - and ß-subunits of luteinizing hormone in human peripheral blood and follicular fluid throughout the menstrual cycle, and their effect on the secretion rate of progesterone by human granulosa cells in tissue culture. J Endocrinol. 69:33–46.[Abstract]
17. Ashitaka Y, Nishimura R, Futamura K, Ohashi M, Toyo S. 1974 Serum and chorionic tissue concentrations of human chorionic gonadotropin and its subunits during pregnancy. Endocrinol Jpn. 21:547–550.[Medline]
18. Reuter AM, Gaspard UJ, Deville JL, Vrindts-Gevaert Y, Franchimont P. 1980 Serum concentrations of human chorionic gonadotropin and its alpha and beta subunits. Clin Endocrinol (Oxf). 13:305–317.[Medline]
19. Blithe DL. 1990 Carbohydrate composition of the -subunit of human choriogonadotropin (hCG) and the free molecules produced in pregnancy: most free and some combined hCG molecules are fucosylated. Endocrinology. 126:2788–2799.[Abstract]
20. Blithe DL. 1990 N-linked oligosaccharides on free interferes with its ability to combine with human chorionic gonadotropin- subunit. J Biol Chem. 265:21951–21956.[Abstract/Free Full Text]
21. Blithe DL, Iles RK. 1995 The role of glycosylation in regulating the glycoprotein hormone free -subunit and free ß-subunit combination in the extraembryonic coelomic fluid of early pregnancy. Endocrinology. 136:903–910.[Abstract]
22. Canfield RE, Morgan FJ, Kammerman S, Bell JJ, Agnosto GM. 1971 Studies of human chorionic gonadotropin. Recent Prog Horm Res. 27:121–164.
23. Begeot M, Hemming FJ, Dubois PM, Combarnous Y, Dubois MP, Aubert ML. 1984 Induction of pituitary lactotrope differentiation by luteinizing hormone subunit. Science. 226:566–568.[Abstract/Free Full Text]
24. Blithe DL, Richards RG, Skarulis MC. 1991 Free molecules from pregnancy stimulate secretion of prolactin from human decidual cells: a novel function for free in pregnancy. Endocrinology. 129:2257–2259.[Abstract]
25. Stewart EA, Rein MS, Friedman AJ, Zuchowski L, Nowak RA. 1994 Glycoprotein hormones and their common alpha-subunit stimulate prolactin production by explant cultures of human leiomyoma and myometrium. Am J Obstet Gynecol. 170:677–683.[Medline]
26. Stewart EA, Jain P, Penglase MD, Friedman AJ, Nowak RA. 1995 The myometrium of postmenopausal women produces prolactin in response to human chorionic gonadotropin and -subunit in vitro. Fertil Steril. 64:972–976.[Medline]
27. Kendall SK, Samuelson LC, Saunders TL, Wood RL, Camper SA. 1995 Targeted disruption of the pituitary glycoprotein hormone -subunit produces hypogonadal and hypothyroid mice. Genes Dev. 9:2007–2019.[Abstract/Free Full Text]
28. Oguchi A, Tanaka S, Yamamoto K, Kikuyama S. 1996 Release of -subunit of glycoprotein hormones from the bullfrog pituitary: possible effect of -subunit on prolactin cell function. Gen Comp Endocrinol. 102:141–146.[CrossRef][Medline]
29. Van Bael A, Denef C. 1996 Evidence for a trophic action of the glycoprotein hormone -subunit in rat pituitary. J Neuroendocrinol. 8:99–102.[CrossRef][Medline]
30. Lapthorn AJ, Harris DC, Littlejohn A, et al. 1994 Crystal structure of human chorionic gonadotropin. Nature. 369:455–461.[CrossRef][Medline]
31. Wu H, Lustbader JW, Liu Y, Canfield RE, Hendrickson WA. 1994 Structure of human chorionic gonadotropin as 2.6 Å resolution from MAD analysis of the selenomethionyl protein. Structure. 2:544–558.
32. Tang B, Guller S, Gurpide E. 1994 Mechanism of human endometrial stromal cells decidualization. Ann NY Acad Sci. 734:19–25.[Medline]
33. Han SW, Lei ZM, Sanfilippo JS, Rao ChV. Human chorionic gonadotropin as a new regulator of human endometrial stromal cells differentiation into decidua [Abstract]. Proc of the 77th Annual Meet of The Endocrine Soc. 1995; p 311.
34. Crosignani PG, Carena Maini M, Negri E, Ragni G. 1991 Human prolactin release induced by follicle stimulating hormone, luteinizing hormone and human chorionic gonadotropin. Hum Reprod. 6:1070–1073.[Abstract/Free Full Text]
35. Han SW, Lei ZM, Rao ChV. Homologous down-regulation of luteinizing hormone/chorionic gonadotropin receptors by a post-transcriptional mechanism during the differentiation of human endometrial stromal cells into decidua [Abstract]. Proc of the 10th Int Congr of Endocrinol. 1996; p 842.
36. Han SW, Lei ZM, Rao ChV. 1996 Up-regulation of cyclooxygenase-2 gene expression by chorionic gonadotropin during the differentiation of human endometrial stromal cells into decidua. Endocrinology. 137:1791–1797.[Abstract]
37. Blithe DL, Nisula BC. 1985 Variations in the oligosaccharides on free and combined subunits of human chorionic gonadotropin in pregnancy. Endocrinology. 117:2218–2228.[Abstract]
38. Vaitukaitis JL, Braunstein GD, Ross GT. 1972 A radioimmunoassay which specifically measures human chorionic gonadotropin in the presence of human luteinizing hormone. Am J Obstet Gynecol. 113:751–758.[Medline]
39. Thotakura NR, Bahl OP. 1985 Highly specific and sensitive hybridoma antibodies against the alpha subunit of glycoprotein hormones. Endocrinology. 117:1300–1308.[Abstract]
40. Whitcomb RW, Sangha JS, Schneyer AL, Crowley WF. 1988 Improved measurement of free alpha subunit of glycoprotein hormones by assay with use of a monoclonal antibody. Clin Chem. 34:2022–2025.[Abstract/Free Full Text]
41. Akar AH, Wehmann RE, Blithe DL, Blacker C, Nisula BC. 1988 A radioimmunoassay for the core fragment of the human chorionic gonadotropin ß-subunit. J Clin Endocrinol Metab. 66:538–545.[Abstract]
42. Sinha YN, Selby FW, Lewis UJ, Vanderlaan WP. 1973 A homologous radioimmunoassay for human prolactin. J Clin Endocrinol Metab. 36:509–516.[Medline]
43. Noyes RW, Hertig AT, Rock J. 1950 Dating the endometrial biopsy. Fertil Steril. 1:3–25.
44. Fleming H, Namit C, Gurpide E. 1980 Estrogen receptors in epithelial and stromal cells of human endometrium in culture. J Steroid Biochem. 12:169–174.[CrossRef][Medline]
45. Kendall SK, Saunders TL, Jin L, et al. 1991 Targeted ablation of pituitary gonadotropes in transgenic mice. Mol Endocrinol. 5:2025–2036.[Abstract]
46. Caspo AI, Pulkkinen MO, Ruttner B, Savauge JP, Wiest WG. 1972 The significance of the human corpus luteum in pregnancy maintenance. Am J Obstet Gynecol. 112:1061–1067.[Medline]
47. Yen SSC, Jaffe RB. 1991 Reproductive endocrinology, 3rd ed. Philadelphia: Saunders; 888–981.
48. Rodway MR, Rao ChV. 1995 A novel perspective on the role of human chorionic gonadotropin during pregnancy and in gestational trophoblastic disease. Early Pregnancy: Biol Med. 1:176–187.
49. Huth JR, Perini F, Lockridge O, Bedows E, Ruddon RW. 1993 Protein folding and assembly in vitro parallel intracellular folding and assembly. J Biol Chem. 268:16472–16482.[Abstract/Free Full Text]
50. Schroeder HR, Halter CM. 1983 Specificity of human ß-choriogonadotropin assays for the hormone and for an immunoreactive fragment present in urine during normal pregnancy. Clin Chem. 29:667–671.[Abstract/Free Full Text]
51. Franchimont P, Gaspard U, Reuter A, Heynen G. 1972 Polymorphism of protein and polypeptide hormones. Clin Endocrinol (Oxf). 1:315–336.[Medline]
52. Good A, Ramos-Uribe M, Ryan RJ, Kempers RD. 1977 Molecular forms of human chorionic gonadotropin in serum, urine, and placental extracts. Fertil Steril. 28:846–850.[Medline]
53. Wehmann RE, Nisula BC. 1981 The metabolic and renal clearance rates of purified human chorionic gonadotropin. J Clin Invest. 68:184–194.
54. Lefort GP, Stolk JM, Nisula BC. 1986 Renal metabolism of the ß-subunit of human choriogonadotropin in the rat. Endocrinology. 119:924–931.[Abstract]
55. Cole LA, Kardana A, Park SY, Braunstein GD. 1993 The deactivation of hCG by nicking and dissociation. J Clin Endocrinol Metab. 76:704–710.[Abstract]
56. Kardana A, Cole LA. 1994 Human chorionic gonadotropin ß-subunit nicking enzymes in pregnancy and cancer patient serum. J Clin Endocrinol Metab. 79:761–767.[Abstract]
57. Kagimoto A, Sakakibara R, Fukushima N, Ikeda N, Ishiguro M. 1995 The occurrence of nicked human chorionic gonadotropin (hCG) by a thermolytic endoprotease. Biol Pharm Bull. 18:810–817.[Medline]
58. Sancken U, Bahner D. 1995 The effect of thermal instability of intact human chorionic gonadotropin (ihCG) on the application of its free ß-subunit (free ßhCG) as a serum marker in Down syndrome screening. Prenat Diagn. 15:731–738.[Medline]

This article has been cited by other articles:

				J.-M. Krause, P. Berger, J. Roig, V. Singh, and W. E. Merz Rapid Maturation of Glycoprotein Hormone Free {alpha}-Subunit (GPH{alpha}) and GPH{alpha}{alpha} Homodimers Mol. Endocrinol., October 1, 2007; 21(10): 2551 - 2564. [Abstract] [Full Text] [PDF]

				U.-H. Stenman, A. Tiitinen, H. Alfthan, and L. Valmu The classification, functions and clinical use of different isoforms of HCG Hum. Reprod. Update, November 1, 2006; 12(6): 769 - 784. [Abstract] [Full Text] [PDF]

				J. Kang, P. Chapdelaine, P.Y. Laberge, and M.A. Fortier Functional characterization of prostaglandin transporter and terminal prostaglandin synthases during decidualization of human endometrial stromal cells Hum. Reprod., March 1, 2006; 21(3): 592 - 599. [Abstract] [Full Text] [PDF]

				I. Rothchild The Yolkless Egg and the Evolution of Eutherian Viviparity Biol Reprod, February 1, 2003; 68(2): 337 - 357. [Abstract] [Full Text] [PDF]

				P. Narayan, J. Gray, and D. Puett Yoked Complexes of Human Choriogonadotropin and the Lutropin Receptor: Evidence that Monomeric Individual Subunits Are Inactive Mol. Endocrinol., December 1, 2002; 16(12): 2733 - 2745. [Abstract] [Full Text] [PDF]

				C.J.P. Jones and A.T. Fazleabas Ultrastructure of epithelial plaque formation and stromal cell transformation by post-ovulatory chorionic gonadotrophin treatment in the baboon (Papio anubis) Hum. Reprod., December 1, 2001; 16(12): 2680 - 2690. [Abstract] [Full Text] [PDF]

				G. Untergasser, H. Rumpold, E. Plas, M. Witkowski, and P. Berger Seminal Plasma Factors Induce in Vitro PRL Secretion in Smooth Muscle Cells of the Human Prostate J. Clin. Endocrinol. Metab., November 1, 2001; 86(11): 5577 - 5584. [Abstract] [Full Text] [PDF]

				Y. Pohnke, R. Kempf, and B. Gellersen CCAAT/Enhancer-binding Proteins Are Mediators in the Protein Kinase A-dependent Activation of the Decidual Prolactin Promoter J. Biol. Chem., August 27, 1999; 274(35): 24808 - 24818. [Abstract] [Full Text] [PDF]

				A. T. Fazleabas, K. M. Donnelly, S. Srinivasan, J. D. Fortman, and J. B. Miller Modulation of the baboon (Papio anubis) uterine endometrium by chorionic gonadotrophin during the period of uterine receptivity PNAS, March 2, 1999; 96(5): 2543 - 2548. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nemansky, M. Articles by Blithe, D. L.