Glycodelin in reproduction

Abstract

To achieve a successful pregnancy in humans, sperm is required for capacitation, followed by binding to and entry into an oocyte. Maternal endometrial epithelial cells (EECs) prepare the appropriate implantation environment through regulation of immune cells and endometrial cells. After acquiring endometrial receptivity, a successful pregnancy consists of complex and finely regulated steps involving apposition, adhesion, invasion, and penetration. Glycodelin is a secretory glycoprotein that affects cell proliferation, differentiation, adhesion, and motility. Glycodelin has four glycoforms (glycodelin‐A, ‐S, ‐F. and ‐C); differences in glycosylation affect each characteristic function. Glycodelin has a unique temporospatial pattern of expression, primarily in the reproductive tract where glycodelin is mid‐secretory phase‐dominant. Recent studies have demonstrated that glycodelin protein has the potential to regulate various processes, including immunosuppression, fertilization, and implantation. This review details the orchestrated regulation of successful pregnancy by glycodelin as well as a discussion of the basic characteristics of glycodelin.

Basic characteristics of glycodelin

After the initial identification of glycodelin in amniotic fluid [1], glycodelin was given various names by several investigators, such as placental α2‐globulin [1], chorionic α2‐microglobulin, α‐uterine protein, placental protein (PP14), progestogen‐dependent endometrial protein (PEP), pregnancy‐associated endometrial α2‐globulin (α2‐PEG), and progesterone‐associated endometrial protein (PAEP) [2]. Currently, the Human Genome Organization uses PAEP and glycodelin as the official symbol and preferred term amongst the list of recommended names, respectively.

The glycodelin gene is located at 9q34.3 and the mRNA is comprised of 7 exons. Several splicing variants of glycodelin mRNA have been reported in the female and male reproductive organs and an endometrial cell line [3, 4, 5]; however, the differentiation of physiologic functions is still unknown. Although the full length of glycodelin mRNA encodes 180 amino acids and the predicted molecular mass of glycodelin protein is about 18–19 kDa, on SDS‐PAGE and Western blotting, the glycodelin band is located at 50–60 kDa, because of glycosylation modification and homodimeric action.

Temporospatial expression of glycodelin

Glycodelin has a limited spatial pattern of expression. Glycodelin expression in mammary glands [6] and endometrium is well described, and this secretory protein is regulated by progesterone. In addition to secretory and decidualized endometrium [7, 8], glycodelin is expressed in bone marrow [9, 10], ovaries [11], fallopian tubes [12], and seminal vesicles [13, 14]. The unique distribution of glycodelin suggests the relationship between glycodelin function and immunologic and hormonal regulation of reproduction.

Based on differentiation of the attached glycosylation modification pattern, glycodelin can be divided into four glycoforms (glycodelin‐A, ‐S, ‐F, and, ‐C). The postfix represents the field to act for each glycoform. In the male reproductive tract, glycodelin‐S is secreted from seminal vesicles to the seminal fluid [4, 13, 14]. In the female genital tract, glycodelin‐A is mainly expressed in EECs [15, 16] and secreted into the uterine fluid [17] and amniotic fluid [7, 18]. Granulosa cells secrete glycodelin‐F into the follicular fluid and glycodelin‐C is detected in cumulus cells [19].

Progesterone‐induced glycodelin‐A also has a characteristic temporal pattern of expression. During the proliferative phase, glycodelin has not been immunohistochemically detected in the endometrium; however, 4–5 days after ovulation glycodelin expression can be gradually detected, with a peak 10 days after ovulation [20]. During human pregnancy glycodelin is expressed in decidualized EECs or amniotic fluid, peaking at 10–18 weeks of gestation [7, 21]. Glycodelin in EECs is secreted to the uterine cavity. Glycodelin can only be detected in uterine flush during the mid‐secretory phase [22]. Moreover, glycodelin can be measured in serum. The concentration of serum glycodelin is quite low compared to the concentration of glycodelin in the endometrium or a uterine flush, but can be detected throughout the menstrual cycle. After ovulation, the serum glycodelin level gradually increases, with a peak in the menstrual phase [15]. These characteristic temporal patterns of expression are in response to the secretion of progesterone. During anovulatory cycles, increasing glycodelin expression can not be measured [23]. Glycodelin expression is increased by the administration of micronized progesterone for anovulatory cycles in women [24]; however, glycodelin levels in women with premature ovarian insufficiency undergoing oocyte donation and embryo transfer are much lower compared to women with normal menstrual cycles [25]. Together, this evidence indicates that the expression of glycodelin is regulated not only by progesterone, but also by another complex system, involving hCG [26] and relaxin [27].

In addition to hormonal regulation, the expression of glycodelin is regulated by one of the histone modifications, histone acetylation. The balance of two enzymes, histone acetyltransferase and histone deacetylase, regulates a part of gene transcription. Histone deacetylase inhibitors (HDACIs), such as trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), increase histone acetylation and induce expression of glycodelin mRNA and protein [28]. Unique glycodelin temporospatial expression is based on hormonal and epigenetic regulation.

Glycodelin in the immune response

Pregnancy is a type of semi‐allograft implantation; therefore, suppression of maternal immune response is important to protect the combination of embryo and endometrial tissue for establishment of human pregnancy.

Okamoto et al. [29] demonstrated that glycodelin‐A suppresses the cytotoxicity of natural killer cells in the first study to point out the relationship between glycodelin and immune cells. Subsequently, numerous studies have clarified the role of glycodelin in regulating the immune system during pregnancy (Fig. 1).

Figure 1.

Glycodelin in successful pregnancy. 1 Glycodelin‐A (GdA) suppresses the cytotoxicity of natural killer cells. 2 Th2‐dominant Th1/Th2 balance is induced by GdA‐mediated apoptosis of Th1 cells, and increased secretion of Th2 cytokines. 3 Sperm morphology dependent glycodelin‐S (GdS) binding to sperm. 4 GdS prevents capacitation by inhibiting albumin‐induced cholesterol efflux. 5 Capacitation of sperm by replacement of GdS to GdA in the uterine cervix. 6 GdF (and GdA) binds to sperm and inhibits the progesterone‐induced acrosome reaction. 7 GdA suppresses the binding between sperm and oocyte. 8 Replacement of GdF/GdA to Glycodelin‐C (GdC) derived from cumulus oocyte complex (COC) induces acrosome reaction. 9 GdA induces secretion of progesterone and hCG from trophoblast. 10 GdA transdifferentiates EECs. 11 Adhesion ability of EECs against embryo is up‐regulated by GdA. 12 Increased expression of GdA accelerates motility of EECs and thereby assists embryo penetration

Glycodelin‐A stimulates human choriogonadotropin (hCG) production by trophoblast and induces progesterone secretion from trophoblast [30, 31]; hCG treatment increases Fas ligand (FasL) expression in endometrial epithelial and stromal cells [32]. Treatment with glycodelin‐A induces apoptosis of Th1 cells more than Th2 cells [33], probably due to the expression of Fas receptors in Th1 cells more abundantly than Th2 cells [33, 34]. The glycodelin‐A‐induced cell apoptosis has been mediated by activation of caspase‐3, ‐8, and ‐9 [33].

Natural killer cells occupy 70 % of the leukocyte population within the uterus. Lee et al. [35] demonstrated that glycodelin‐A induces the secretion of Th2 cytokines, such as interleukin‐6 and ‐13, and GM‐CSF from natural killer cells. IL‐6 and GM‐CSF acts on blastocysts through stimulation of trophoblast cell motility and differentiation. These observations indicate that glycodelin‐A indirectly engages the regulation of Th2‐dominant Th1/Th2 balance for successful pregnancy.

Glycodelin in sperm reaction

After ejaculation into the vagina, glycodelin‐S quickly binds to spermatozoa, depending on the morphology of the sperm [36]. Teratospermia has a low fertilization ratio, which likely accounts for poor accessibility of glycodelin‐S to spermatozoa. Glycodelin‐S inhibits albumin‐induced cholesterol efflux from spermatozoa [37], and thereby prevents capacitation, which is triggered by cholesterol decrement in the sperm plasma membrane. During the peri‐ovulation phase, glycodelin‐A is not abundant in the uterine mucus and, therefore, glycodelin‐S in the seminal plasma has easy access to spermatozoa without binding competition to glycodelin‐A. Albumin is less in seminal plasma and abundant in the uterine mucus. Prevention of capacitation against moving spermatozoa in the uterine cavity by a concentration gradient of albumin is meaningful because fertilization is allowed a few hours after capacitation [38]. After removal of glycodelin‐S, cholesterol efflux and capacitation occur in sperm.

In the fallopian tube, glycodelin‐A and glycodelin‐F are expressed. Glycodelin‐F, which binds to sperm, inhibits the progesterone‐induced acrosome reaction [39]. Elevated progesterone after ovulation induces the acrosome reaction and likely prevents multiple fertilization because acrosome‐reacted sperm lose the ability to bind to the zona pellucida [40]. Binding of glycodelin‐F to capacitated sperm is important to maintain the acrosome‐unreacted state until apposition of the sperm and oocyte. Glycodelin‐A attached to sperm supports the zona pellucida‐induced acrosome reaction. Because of this glycodelin‐A function, sperm can start the acrosome reaction effectively and immediately after binding to zona pellucida. Glycodelin‐F and glycodelin‐A attached to sperm are replaced by glycodelin‐C in cumulus cells. Oehlinger et al. [41] demonstrated that glycodelin‐A suppresses the binding between sperm and oocyte and it is believed as the multiple pregnancy system; however, the balance between the blockade of sperm–oocyte binding by glycodelin‐A and glycodelin‐F, and the replacement of functional glycodelin‐A and glycodelin‐F by glycodelin‐C effectively regulate the successful singleton pregnancy.

Glycodelin in implantation

Endometrial receptivity peaks in the mid‐secretory phase called the ‘implantation window’. Glycodelin is induced by progesterone, which is abundantly secreted after ovulation, and increases hCG secretion from blastocyst [30, 31]; thereafter, the secreted hCG induces glycodelin secretion from EECs [26]. Through this cyclic mechanism, glycodelin expression is accelerated during the mid‐secretory phase. In association with glycodelin [15, 16], the expression of endometrial receptivity marker proteins have been changed through the implantation window, such as leukemia inhibitory factor [28, 42], integrin αvβ3 [43], trophinin [44], and MUC‐1 [45] in EECs. MUC‐1 is the cell surface mucin and is thought to act as an anti‐adhesion molecule by covering binding sites of adhesion molecules. Both the increased expression of trophinin, which is a homodimeric adhesion molecule, and the decreased expression of MUC‐1 regulate adhesion between the embryo and EECs.

In contrast, glycodelin affects EEC adhesion against embryos [5]. Transfection of glycodelin cDNA lacking a putative N‐terminal signal peptide into an EEC line (Ishikawa cells) resulted in increased adhesion ability between EECs and embryo models. In culturing Ishikawa cells, glycodelin secretion to culture media is undetectable [46]. These results indicate that glycodelin plays a role to regulate EEC adhesion to the oocyte through an intracellular signal transduction pathway, but not by acting as a secretory protein.

In cultured EEC line (Ishikawa cells), treatment with the combination of estradiol and progesterone (E2P4) or SAHA, one of the HDACIs, induces glycodelin expression at the mRNA and protein levels [28]. In this in vitro study, hormone‐ or HDACI‐induced glycodelin has caused transdifferentiation of an EEC line, observed by flattened and wide‐spread morphological changes and up‐regulation of glycogen synthesis. Furthermore these alterations have been completely abrogated by glycodelin gene silencing using small interference RNA (siRNA) [28]. This study shows the possibility that glycodelin is involved in endometrial receptivity before oocyte apposition.

For fertilized oocytes, human implantation is a complex process, involving apposition and adhesion onto EECs followed by invasion and penetration into EECs. For successful pregnancy, adhesion between the oocyte and EECs is required after apposition. Glycodelin induced by E2P4 or SAHA has enhanced adhesion ability [5], while glycodelin in culture media can not be measured in Ishikawa cells [46]. Furthermore, transfection of glycodelin cDNA lacking the N‐terminal putative signal peptide has also enhanced adhesion ability, and the enhancement of adhesions by stimulation of E2P4 or SAHA can be completely abrogated by glycodelin siRNA transfection [5]. Taken together, glycodelin regulates EEC adhesion ability against oocytes via an intracellular signal transduction pathway without action as a secretory protein.

Increased glycodelin expression is timed to endometrial receptivity, whereas glycodelin‐A in culture media has suppresses invasion of immotile cytotrophoblast cells and the choriocarcinoma cell line, JEG‐3, by down‐regulation of the extracellular signal‐regulated kinase (ERK)/c‐Jun signaling pathway. Furthermore, the suppressive mechanism by glycodelin‐A has been mediated by interaction with sialic acid binding immunoglobulin‐like lectins (Siglec)‐6 [47]. A member of the Siglec protein family is known as the cell–cell interaction protein in immune cells and Siglec‐6 is a trophoblast‐specific protein.

In an in vitro implantation assay using Ishikawa cells and spheroids of JAR cells (choriocarcinoma cell line) for an embryo model, spreading away onto the Ishikawa monolayer and occupying the space by JAR cells mimics invasion of trophoblasts. In E2P4‐ or SAHA‐treated Ishikawa cells, JAR spheroids has spread more widely than control Ishikawa cells [48]. This phenomenon is probably due to acceleration of Ishikawa cell motility by stimulation of ovarian steroid hormones or HDACI [49]. EEC motility is accelerated or suppressed by transfection of glycodelin cDNA or siRNA, respectively [49]. These studies indicate that glycodelin of EECs affect trophoblast invasion regulating the balance between suppression of trophoblast cell motility (paracrine pathway) and acceleration of EEC cell motility (intracellular pathway).

Overexpression of glycodelin in Ishikawa cells has resulted in reduction of EEC proliferation through up‐regulation of p21, p27, and p16 and thereby G1/S stop [50]. Furthermore, progesterone‐induced inhibition of Ishikawa cell growth has been attenuated by glycodelin siRNA transfection [50]. Glycodelin has a possible potential to assist trophoblast invasion and penetration through EEC apoptosis and suppression of EEC proliferation.

Summary

After glycosylation and dimerization, glycodelin protein acts as a multiple‐regulator (immunosuppression, fertilization, and implantation) and a director to orchestrate the complex, step‐by‐step process of fertilization and implantation. There remain a number of unknown functions and mechanisms to be elucidated; however, collective evidence of glycodelin function and regulation should be applied for reproductive medicine, such as infertility, recurrent miscarriage, and anti‐conception.

Acknowledgments

We are grateful to Rika Shibata for her secretarial work. This work was supported in part by grants‐in‐aid for scientific research C 24592484 from the Ministry of Education, Science, Sports, and Culture of Japan.