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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 4 1377-1381
Copyright © 2000 by The Endocrine Society

Original Studies

Human Oviductal Gonadotropin-Releasing Hormone: Possible Implications in Fertilization, Early Embryonic Development, and Implantation1

Eva Maria Casañ, Francisco Raga, Fernando Bonilla-Musoles and Mary Lake Polan

Department of Obstetrics and Gynecology, University of Valencia School of Medicine (E.M.C., F.R., F.B.-M.), Center for Gynecology and Obstetrics (E.M.C., F.R., F.B.-M.), 46006 Valencia, Spain; and Department of Obstetrics and Gynecology, Reproductive Immunology Laboratory, Stanford University School of Medicine (E.M.C., F.R., M.L.P.), Stanford, California

Address all correspondence and requests for reprints to: Dr. Eva Maria Casañ, Pedro Aleixandre 57–7, 46006 Valencia, Spain. E-mail: cegiob{at}interbook.net


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The oviduct is host to gametes and early embryos at a critical point in their lives. It is clear that the interactions of gametes/early embryo with the maternal oviduct in an autocrine and paracrine manner provide a microenvironment that enhances fertilization, early embryonic development, and implantation. Moreover, there is considerable evidence that an extrahypothalamic GnRH may play a substantial role as a molecular autocrine/paracrine regulator in these events. Gametes and preimplantation embryos express GnRH and GnRH receptor at both messenger ribonucleic acid (mRNA) and protein levels. However, whether GnRH is produced by the human oviduct has not yet been demonstrated.

We used RT-PCR and immunohistochemical techniques to investigate GnRH mRNA and protein expression in human fallopian tubes throughout the menstrual cycle of premenopausal fertile patients. Our results, at both the mRNA and protein levels, revealed cycle-dependent production of an oviductal GnRH with expression during the luteal phase. Moreover, GnRH immunostaining was localized in the tubal epithelium during the luteal phase.

On the basis of these data, we suggest that during reproductive life, oviductal GnRH may play a substantial paracrine/autocrine role in human fertilization, early embryonic development, and implantation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE OVIDUCT in many species, including the human, occupies a central role in the establishment of pregnancy. Oviducts are regarded as being biologically active, providing an environment that sustains and enhances fertilization during early embryonic development as the embryo travels toward the uterine cavity (1, 2, 3).

Cytokines, growth factors, and their receptors have been detected in both pre- and periimplantation embryos, the fallopian tube, and uterine endometrium (1, 2, 3, 4, 5). Moreover, their roles in embryo development, endometrial preparation, and the implantation process are now well documented (1, 6, 7).

Recently, it has been shown by several groups that improved embryo morphology, development, and hatching as well as better implantation rates are obtained after embryo coculture on feeder layers of human oviductal cells and sequential oviductal-endometrial cells (6, 7, 8). Therefore, oviductal cells in the feeder layer for embryo cultures might produce factors that possess direct or indirect embryotropic activity (7, 8). However, these embryotropic factors, their regulation, and their potential function in vivo are as yet undiscovered, and their influence on the gametes/embryos has not yet been explored.

There is ample evidence that a variety of human tissues, such as the endometrium (9, 10, 11), ovary (12), testis (13), placenta (14, 15), and myometrium (10), express extrahypothalamic GnRH that is immunologically, biologically, and chemically identical to the hypothalamic hormone. Furthermore, the presence of GnRH receptor in cumulus-oocyte complexes and preimplantation embryos at different developmental stages has been previously established (10, 15, 16, 17).

Several GnRH agonists have been shown to have a direct action on these peripheral receptors both in vivo and in vitro and, consequently, to mediate a stimulatory effect on spontaneous contraction of human myometrium and fallopian tubes as well as to enhance fertilization, preimplantation embryonic development, and implantation (18, 19, 20).

Fertilization, preimplantation embryonic development, and implantation are a complex series of steps that, under normal circumstances, begin in the fallopian tube, before the blastocyst reaches the uterine cavity and attaches to the maternal endometrium. To complete this enigmatic process, there is an embryonic-maternal dialogue, in which the embryo and the maternal reproductive tract induce changes in each other to promote embryonic development and endometrial receptivity (1, 4, 7, 8, 9, 10, 11).

We hypothesized that an interaction between the embryo and the maternal reproductive tract via the GnRH system may be playing an important role during fertilization, preimplantation embryonic development, and the implantation process. Therefore, we have examined, for the first time, GnRH messenger ribonucleic acid (mRNA) and protein expression in human fallopian tubes of fertile patients during the different phases of the menstrual cycle.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue collection

Fallopian tube and endometrial samples were obtained from 24 fertile premenopausal women, aged 34–39 yr, undergoing hysterectomy for various benign gynecological reasons. Patients with endometriosis and pelvic inflammatory disease were excluded from the study. Moreover, fallopian tube and endometrial samples were obtained from 10 postmenopausal women, aged 48–53 yr, undergoing hysterectomy for various benign gynecological reasons. Written consent from the patients and approval by the institutional committee on the use of human subjects in research at Stanford University and Valencia University were obtained before collection of tissue samples for this study.

One part of the tissue was fixed with 4% paraformaldehyde; the rest was washed in 0.9% sodium chloride solution to remove the contaminating blood and directly processed for RNA extraction. Fixed tissue was embedded in paraffin, sectioned, and mounted. Twelve serial sections (6 µm) from each sample were then prepared for immunohistochemistry, and the first and last sections were stained with hematoxylin-eosin and examined with a Nikon Microphot-FXA microscope (Nikon, Tokyo, Japan). Tissue samples from premenopausal patients were classified according to time of collection and histological endometrial dating according to the method of Noyes et al. (21) into proliferative phase (n = 10) and secretory phase (n = 14). Moreover, samples were split into segments of fimbrial, ampullary, and isthmic portions of the fallopian tube.

Immunohistochemical analysis

To determine the presence of GnRH in the human fallopian tube, a double antibody technique was employed according to the avidin-biotin alkaline phosphatase technique (11). Sections were deparaffinized with xylene and quickly rehydrated through graded alcohols. Excess liquid was removed, and sections were washed with phosphate-buffered saline (PBS), pH 7.4. To block unspecific binding, sections were preincubated in humidity chambers for 30 min at room temperature in PBS containing 2% normal goat serum (Sigma, St. Louis, MO). After further washing in PBS (pH 7.4) with 0.05% Tween-20 (PBS-T: Sigma; three times for 5 min each time), sections were incubated for 90 min at 37 C with rabbit anti-[Lys8]GnRH as first antibody at a dilution of 1:100 each (Sigma). After washing in PBS-T, sections were incubated with a secondary antibody, biotinylated anti-rabbit IgG (dilution 1:800; Sigma), in humidity chambers for 90 min at room temperature (9, 11).

Immunohistochemical controls were incubated with PBS containing 2% goat serum without primary antibody (9, 11, 17, 19). To amplify the signal, sections were washed with PBS-T, and then the avidin-biotin-alkaline phosphatase staining method (Vector Laboratories, Inc., Burlingame, CA) was used. Endogenous alkaline phosphatase activity was inhibited by the addition of levamisole to the buffer used to prepare the substrate solution. Finally, slides were counterstained with 25% hematoxylin, cleared, coverslipped, and examined with a Nikon DX-DB2 camera and a Nikon Microphot-FXA microscope under x100–400 magnification. A red precipitate indicated positive staining by the primary antibody.

RNA extraction

The extraction of RNA from the tissue sample was carried out as described previously (9) with the RNA-STAT-60 reagent (Tel-Test, Friendswood, TX). Briefly, tissue samples were washed three times in PBS (Life Technologies, Inc., Grand Island, NY) to remove blood contamination. One hundred milligrams of tissue were homogenized in 1 mL RNA-STAT-60 reagent. Total RNA was separated from DNA and proteins by adding chloroform and was precipitated using isopropanol. Precipitate was washed twice times in 75% ethanol, air-dried, and rediluted in diethylpyrocarbonate (DEPC)-treated dH2O. The amount and purity of extracted RNA were quantitated by spectrophotometry in a GenQuant RNA/DNA calculator (Amersham Pharmacia Biotech, Cambridge, UK).

RT-PCR

Sequences of complementary DNA (cDNA) clone for the GnRH (22) mRNA to be detected in human oviductal samples were obtained from the GenBank database of the National Center for Biotechnology Information of the NIH (iternet address: http://www.2.ncbi.nlm.nih.gov/cgi-bin/genbank). One corresponding set of primers for GnRH was found with the help of the program OLIGO 5.0 Primer Analysis Software (National Bioscience, Plymouth, MN) and synthesized by the protein, amino acid, and nucleic acid facility (Beckman Center, Stanford University, Stanford, CA). To ensure that the detected product resulted from amplification of cDNA rather than contaminating genomic DNA, primers were designed to cross intron/exon boundaries. The primer sequence, location on the cDNA, and size of the amplified fragment were previously described by our group (9). Moreover, human ß-actin (housekeeping gene) primers that were used as an internal standard were obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA).

For the GnRH mRNA to be detected, 19 µL RT MasterMix were prepared [5 mmol/L MgCl2 solution, 10 x PCR-buffer II, 2.5 µL DEPC-treated dH2O, 1 mmol/L deoxy (d)-ATP, 1 mmol/L dCTP, 1 mmol/L dGTP, 1 mmol/L dTTP, 2.5 µmol/L oligo(deoxythymidine)16, 20 IU ribonuclease inhibitor (all from Perkin-Elmer Corp., Foster City, CA), 100 IU Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.), and 1 µg total RNA diluted in 1 µL DEPC-treated H2O] and put into a 0.5-mL thin wall PCR tube (Applied Scientific, South San Francisco, CA). RT MasterMix in PCR tubes was covered with 50 µL light white mineral oil (Sigma) and kept on ice until the RT. RT was carried out in the DNA Thermal Cycler 480 using a program with the following parameters: 42 C, 15 min; 99 C, 5 min; and 4 C, infinity. After the reaction was completed, samples were stored at -20 C until the PCR. As a negative control 1 µL DEPC-treated H2O without RNA sample was subjected to the same RT reaction.

Afterward, 5-µL aliquots from each RT product were mixed with 95 µL of the PCR-MasterMix described above with 4 µL 3' + 5' primer mix (5 µmol/L each) for GnRH, and the PCR reaction was carried out. Program parameters were: 30 cycles of 94 C for 45 s, 56 C for 45 s, and 72 C for 60 s; the reaction was terminated at 72 C for 5 min and cooled down to 4 C. After completing the PCR reaction, samples were stored at -20 C until electrophoresis.

Two percent agarose gel (Life Technologies, Inc.) electrophoresis was carried out in an H5 electrophoresis chamber. Gels were stained with ethidium bromide (Sigma). Twenty-five microliters of each PCR product and dye buffer were analyzed in parallel with a 100-bp DNA ladder (Life Technologies, Inc.) as a standard. After completion of electrophoresis, the gel blot was analyzed, and photocopies of the blot were printed on the GelDoc 2000 system (Bio-Rad Laboratories, Inc. Hercules, CA). Independent sequence analysis was performed to confirm the identity of the expected sequence and amplified cDNA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To study the presence and distribution of GnRH in the human fallopian tubes, we used RT-PCR and immunohistochemistry techniques. These techniques revealed a cycle-dependent appearance and distribution of GnRH within the fallopian tubes. Total cellular RNA, isolated and subjected to RT-PCR analysis, revealed that fallopian tube samples from patients in the luteal phase of the menstrual cycle express GnRH mRNA. Figure 1Go shows the RT-PCR products. The predicted 399-bp fragment of GnRH was observed in the fimbrial, ampullary, and isthmic segments of the fallopian tubes analyzed during the luteal phase (lanes G–I). On the other hand, we were unable to detect the GnRH mRNA in the oviductal samples studied from patients in the follicular phase (lanes D–F). In addition, negative controls without RNA sample were subjected to the same RT-PCR reaction (lane A). As positive control, GnRH mRNA from human endometrial samples from the same patients was examined and found to be positive in all of the samples studied in both the follicular and luteal phases of the menstrual cycle (Fig. 1Go, lanes B and C). Samples from postmenopausal patients (both endometrium and oviduct) were negative for GnRH mRNA. ß-Actin mRNA expression was employed as an internal standard, being detected in all samples studied, thus assuming the integrity of the RNA and the RT-PCR process.



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  		Figure 1. GnRH mRNA expression in the different phases of the menstrual cycle, A, Negative control; B, follicular phase human endometrium; C, luteal phase human endometrium; D, ithsmic portion of the fallopian tube during the follicular phase; E, ampullary portion during the follicular phase; F, fimbrial portion during the follicular phase; G, ithsmic portion of the fallopian tube during the luteal phase; H, ampullary portion during the luteal phase; I, fimbrial portion during the luteal phase.

 
Immunoreactive GnRH was identified in the oviductal samples from patients in the luteal phase using an avidin-biotin-alkaline phosphatase technique (Fig. 2CGo). We were unable to detect any staining in the samples from patients in the follicular phase of the menstrual cycle (Fig. 2BGo) or in the samples from postmenopausal patients. Furthermore, GnRH protein expression was present in the tubal epithelium of the samples studied during the luteal phase, with weak staining in the stroma (Fig. 2CGo). No differences in protein level were observed between in the various portions of the fallopian tube. In addition, no staining was detected in negative controls, from which primary antibody was omitted (Fig. 2AGo). As a positive control, GnRH was studied in human endometrial samples from the same patients and was positive in all of the samples studied in both the follicular and luteal phases of the menstrual cycle (Fig. 2Go, E and F). All experiments were performed a minimum of four times with similar results.



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  		Figure 2. GnRH immunoreactivity in sections of human fallopian tube and endometrium. A, Negative control (fallopian tube sample); B, follicular phase fallopian tube sample; C, luteal phase fallopian tube sample. Immunoreactivity was highly expressed in the tubal epithelium. D, Negative control (endometrial sample); E, follicular phase endometrial sample. Weak staining is observed in the epithelial glands. F, Luteal phase endometrial sample. Remarkable staining in both epithelial glands and stroma. Original magnification, x200.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study provides evidence at both the mRNA and protein levels that GnRH is produced in the human fallopian tube during the luteal phase of the menstrual cycle at the same time spermatozoa and oocytes are deposited communally in the oviduct to promote their union and nurture the resultant zygotes and early embryos. Furthermore, we were unable to detect the presence of oviductal GnRH during the follicular phase of the cycle or in postmenopausal samples. This is consonant with previous reports showing a dynamic pattern in GnRH mRNA (9, 23) and protein (11) expression in human endometrium throughout the menstrual cycle, with a significant increase during the secretory phase compared to the proliferative phase.

It is a well established fact that the mammalian oviduct undergoes hormonally mediated cyclical modifications that climax during the periovulatory period, thus ensuring the production of a specialized environment required to facilitate fertilization and embryonic development (7, 8, 24, 25). Moreover, the synthesis and release of many proteins in the oviduct have been recognized by isoelectric point or relative molecular mass; however, most of these proteins remain to be identified (24, 25). Therefore, the presence of immunoreactive GnRH in the epithelial cells of the human oviduct during the luteal phase may play a paracrine role in fertilization and early cleavage stage embryonic development. This hypothesis is supported by experimental evidence that GnRH has a direct stimulatory effect on both fertilization and early embryonic development (16, 17, 18, 19, 20, 26).

Previous reports in human (26) and other species (16, 18) had demonstrated that GnRH and GnRH agonist enhance in vitro fertilization. Therefore, GnRH increased zona binding ability when human sperm was incubated with these peptides (26). Moreover, oocytes fertilized in medium containing GnRH had a higher cleavage rate than controls not receiving the hormone (16). On the other hand, incubating the sperm and/or oocytes with a GnRH antagonist (16, 18, 26, 27) ablated the stimulatory effects of GnRH on in vitro fertilization. Therefore, these effects seem to be mediated by the presence of specific receptors for GnRH in both oocyte and spermatozoa (10, 26, 27, 28, 29).

The data from in vitro culture of preimplantation embryos exposed to GnRH agonist and antagonist suggest that GnRH is a crucial key in early embryonic development (18, 19). The GnRH and its agonist seem to enhance embryonic development, whereas GnRH antagonist has a detrimental effect. Further, GnRH antagonist is able to completely block early embryonic development, and the reversal of this effect by the agonist in a dose-dependent fashion suggests a specific receptor-mediated effect, rather than a nonspecific or toxic effect (19). Furthermore, our group showed that GnRH and its receptor are expressed at both the mRNA and protein levels by cultured human and mouse embryos during the preimplantation development period (17, 19). Moreover, it is a remarkable fact that GnRH mRNA and protein expression are increased in the hatching blastocyst stage compared to the morula stage (17, 19), as this hormone has been recently implicated as a possible important paracrine factor in the process of embryonic implantation (9, 11, 15, 16, 17, 18). On the other hand, the GnRH receptor was found to maintain a constant level during all developmental embryonic stages studied, reinforcing the hypothesis that the embryo communicates with the maternal tubal epithelium and endometrium through the GnRH system to promote embryonic development and endometrial receptivity (17, 18, 19).

These observations suggest that although embryonic GnRH has an autocrine role in early embryonic development, the oviductal GnRH is likely to further enhance embryonic development by a paracrine action. This is consonant with the fact that although preimplantation embryos can develop successfully in vitro, the maternal reproductive tract provides factors that, in addition, enhance embryo development and implantation (1, 2, 3, 7, 8, 15, 16, 17, 18, 19, 20). For instance, preimplantation embryos cultured in vitro lag in development compared to in vivo counterparts (1). The delay in development improves when the embryos are cultured in medium containing GnRH (15, 16, 17, 18, 19) or are cocultured with fallopian tube epithelium (8, 24, 25, 30, 31).

Mirroring the in vitro data, the clinical experience in assisted reproduction has shown that pregnancy rates are greatest with gamete intrafallopian transfer, where gametes are placed into the fallopian tube after oocyte aspiration. Pregnancy rates are next highest with zygote intrafallopian tube transfer, where fertilized zygotes are placed into the fallopian tube the day after oocyte aspiration, and are lowest with in vitro fertilization and embryo transfer, where fertilized embryos are replaced into the uterine cavity after 2–3 days (1, 30, 31, 32, 33, 34). It appears that the overall impact of GnRH analogs on reproductive medicine is still not fully understood, and most of the beneficial/detrimental effects of these molecules are only partly known. Therefore, the results of the present study as well as previous in vivo studies in both human (35) and animals (36) showing a beneficial effect of GnRH agonist on fertilization, early embryonic development, and implantation may have outstanding therapeutic potential. Nonetheless, much research remains to be performed, and future studies should confirm these data.

In conclusion, this study has demonstrated for the first time that GnRH is produced by the human fallopian tube during the luteal phase of the menstrual cycle, providing evidence that this peptide may play a substantial role in fertilization, early embryonic development, and implantation.


   Footnotes
 
1 This work was supported in part by NIH Grant HD-31575 (to M.L.P.) and FIS Grant 99/0657 (to E.M.C., F.B.M., and F.R.). Back

Received May 28, 1999.

Revised November 30, 1999.

Accepted December 15, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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P. Morales, C. Pasten, and E. Pizarro
Inhibition of In Vivo and In Vitro Fertilization in Rodents by Gonadotropin-Releasing Hormone Antagonists
Biol Reprod, October 1, 2002; 67(4): 1360 - 1365.
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