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Department of Gynecology and Obstetrics, Reproductive Immunology Laboratory, Stanford University School of Medicine (F.R., E.M.C., J.K., Y.W., M.L.P.), Stanford, California; and the Department of Obstetrics and Gynecology, University of Valencia School of Medicine (F.R., E.M.C., F.B.-M.), and the Center for Gynecology and Obstetrics (F.R., E.M.C., F.B.-M.), 46004 Valencia, Spain
Address all correspondence and requests for reprints to: Francisco Raga, M.D., Center for Gynecology and Obstetrics, Navarro Reverter 11–1, 46004 Valencia, Spain. E-mail: cegiob{at}interbook.net
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Abstract |
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GnRH is expressed in the developing mouse embryo from morula to hatching blastocyst stages at the mRNA and protein levels. GnRH receptor mRNA is also present in the developing embryos studied.
Preimplantation embryonic development was significantly enhanced by incubation with increasing concentrations of GnRH agonist and is significantly decreased by GnRH antagonist compared with that in the control group. Moreover, GnRH antagonist (5 and 10 µM) was able to completely block embryo development. The deleterious effect of GnRH antagonist on embryo development was reversed by increasing concentrations of the agonist, as determined by the number of embryos reaching the blastocyst stage.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to the central action, a variety of tissues in several mammalian species express an extrahypothalamic GnRH that is immunologically, biologically, and chemically identical to the hypothalamic hormone (2, 3, 4, 5, 6, 7). Moreover, the presence of low affinity/high capacity binding sites for GnRH has been demonstrated in several extrapituitary organs such as the placenta, endometrium, myometrium, breast, prostate, ovary, and testis (5, 7, 8).
Preimplantation embryonic development and implantation are a complex series of steps that under normal circumstances begin even before the blastocyst reaches the uterine cavity and attaches to the endometrial epithelium after loss of the zona pellucida. To complete this enigmatic process, there is an embryonic-maternal dialogue, in which the embryo and the endometrium induce changes in each other to promote embryonic development and endometrial receptivity (9, 10).
Cytokines, growth factors, and their receptors have been detected in pre- and periimplantation embryos, the fallopian tubes, and uterine endometrium (10, 11, 12, 13). Their role in embryo development, endometrial preparation, and the implantation process has been implicated. For instance, preimplantation embryos cultured in vitro lag in development compared with their in vivo counterparts (10). Preimplantation embryonic development improves when the embryos are cocultured with other cells, such as endometrial epithelium and Vero cells (14, 15). These observations suggest that although embryos can develop successfully in vitro, the maternal reproductive tract is likely to provide other factors that further enhance embryo development and implantation.
Recent studies from our group demonstrated the presence of GnRH and its receptor at both messenger RNA (mRNA) and protein levels in human endometrium of fertile patients with a dynamic pattern, showing a rise in the midluteal phase, the time of embryonic implantation (6, 7). Furthermore, the presence of a GnRH receptor on the murine uterus and its possible paracrine role in endometrial dynamics have been elucidated (13, 16). Moreover, several reports concerning inadvertent exposure to GnRH agonist of human pregnancies during the early stages of embryonic development and implantation have been reported in in vitro fetilization patients. The clinical experience in those cases has suggested that not only are detrimental effects unlikely, but also the analog might enhance early embryonic development and implantation (17, 18).
We hypothesized that an interaction between the embryo and maternal reproductive tract via the GnRH system may be playing an important role during preimplantation embryonic development and the implantation process.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RT-PCR analysis
Single embryos from 88 h post-hCG (compacted morula; n
= 60), 95 h post-hCG (early expanded blastocyst; n = 60), and
120 h post-hCG (hatching blastocyst; n = 60) were examined by
RT followed by two rounds of nested PCR using a modification of methods
described previously (14, 20) for ß-actin (internal standard), GnRH,
and GnRH receptor mRNAs. Sequences of complementary DNA (cDNA) clones
for the mRNAs that should be detected in single embryos; ß-actin
(21), GnRH (22), and GnRH receptor (23) were obtained from the GenBank
database of the National Center for Biotechnology Information (internet
address: http://www.2.ncbi.nlm.nih.gov/cgi-bin/GenBank). The
corresponding primer sequences were constructed 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 (Stanford University Medical Center). To ensure that the
product detected resulted from amplification of cDNA rather than
contaminating genomic DNA, primers were designed to cross
intron/exon boundaries. As a negative control for specific primers,
a defined volume of culture medium in which the embryos were cultured
was subjected to the same RT-PCR reaction. The primer cDNA sequences
and the sizes of the amplified fragments are listed in Table 1
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RT Mastermix for each embryo was prepared containing 5 µM MgCl2, 10 x PCR buffer II, 1 mM of each deoxy (d)-NTP (Perkin Elmer, Foster City, CA), and 2.5 µM oligo(deoxythymidine)16 and placed in a 0.5-ml thin walled PCR tube (Applied Scientific, San Francisco, CA). RT Mastermix in each PCR tube was covered with 50 µl light white oil (Sigma Chemical Co.) and kept on ice until embryo collection. A single embryo carrying about 1 µl culture medium was aspirated with a micropipette and transferred to the PCR tube containing 17.5 µl RT Mastermix. Samples were immediately heated to 99 C for 1 min in a DNA thermal cycler 480 (Perkin Elmer) to release the total RNA and denature the proteins. Samples were cooled to 4 C, and 20 U ribonuclease inhibitor (Perkin Elmer) and 100 U Moloney murine leukemia virus reverse transcriptase (Life Technologies, Grand Island, NY) were added before starting the RT. The RT reaction in a total volume of 20 µl was carried out in the DNA thermal cycler 480 using a program with one 30-min RT cycle at 42 C, followed by 5 min at 99 C, then quenching at 4 C. Products were stored at -20 C until the subsequent PCR.
For the first PCR, 2 µl RT product from an individual embryo were added to the first PCR Mastermix to a total volume of 49.5 µl containing 2 mM MgCl2, 10 x PCR buffer II, 0.24 mM of each dNTP, and 0.24 µM 3'- and 5'-primer mixture of each specific outer pair and covered with 50 µl light white oil. PCR cycles were initiated by heading to 95 C for 5 min to denature all proteins and DNA; then at 95 C, 2.5 U AmpliTaq DNA polymerase (Perkin Elmer) were added to a total volume of 50 µl. PCR was carried out for 30 cycles of 45 sec at 94 C, 45 sec at 56 C, and 60 sec at 72 C. The reaction was terminated at 72 C for 6 min and quenched at 4 C. First round PCR products were stored at -20 C until the second round of PCR.
For the second round PCR, 5 µl of the initial PCR products were added to the second PCR Mastermix to a total volume of 99.5 µl containing 1.9 mM MgCl2, 10 x PCR buffer II, 0.2 mM of each dNTP, and 0.2 µM of a 3'- and 5'-primer mixture of each corresponding inner pair and covered with 50 µl light white oil. After completing the second round PCR (35 cycles) using the same program, samples were stored at -20 C until electrophoresis.
Two percent agarose gel (Life Technologies) electrophoresis was carried out in an H5 electrophoresis chamber. Gels were stained with ethidium bromide (Sigma Chemical Co.). Twenty-five microliters of each PCR product and dye buffer were analyzed in parallel with a 100-bp DNA ladder (Life Technologies) as a standard. After completion of electrophoresis, the gel blot was analyzed, and photocopies of the blot were printed on the GelDoc 1000 system (Bio-Rad Laboratories, Inc., Hercules, CA).
The identities of all PCR products after gel electrophoresis were confirmed by sequence analysis using the chain termination method (24, 25).
Immunohistochemical staining
Single embryos from 88 h post-hCG (compacted morula; n
= 60), 95 h post-hCG (early expanded blastocyst; n = 60), and
120 h post-hCG (hatching blastocyst; n = 60) were examined by
an avidin-biotin alkaline phosphatase technique (15) to localize at the
protein level the GnRH in the preimplantation mouse embryo. Embryos
were fixed with 4% paraformaldehyde in PBS for 30 min at 4 C in
microdrops under oil and then treated with acid Tyrode’s solution (pH
2.5) to induce permeabilization of the zona pellucida. To reduce the
nonspecific binding, 2% normal goat serum in PBS was applied to the
embryos for 30 min at room temperature, then rinsed twice in PBS (pH
7.4) with 0.05% Tween-20 (PBS-T; Sigma Chemical Co.) and
incubated for 90 min at 37 C with the primary antibody, polyclonal
rabbit anti-[Lys8]GnRH, at 1 µg/ml (Sigma Chemical Co. and Petraglia). After being rinsed with PBS-T,
embryos were incubated for 90 min at room temperature with a secondary
antibody, biotinylated antirabbit IgG, at a dilution of 1:800
(Sigma Chemical Co.). Immunohistochemical controls were
incubated with PBS containing 2% goat serum without primary antibody.
To amplify the signal, embryos 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, embryos were incubated in alkaline phosphatase substrate
solution until color become evident, then the reaction was stopped in
all the embryos simultaneously. A red precipitate indicated positive
staining by the primary antibody. Embryos in microdrops under oil were
visualized and photographed by a 35-mm camera (Olympus Corp.) attached to an inverted microscope (Olympus Corp.).
The intensity of the immunostaining was evaluated by HSCORE (15). This method provides a numeric value of the overall staining intensity and the percentage of cells per embryo stained. HSCORE is calculated by the following equation: HSCORE = (Pi (i + 1), where i is the intensity of staining with a value (obtained by two of the authors in a double blind manner) of 1 (weak), 2 (moderate), or 3 (strong), and Pi is the percentage of stained embryos in every stage for each intensity.
Embryo culture
Exp 1. Two-cell embryos (n = 785) were removed, washed
with fresh medium, and then randomly transferred to 20-µl drops (an
average of 15 embryos/drop) of modified Ham’s F-10 containing 0.3%
BSA (Sigma Chemical Co.) (19) and 0, 0.05, 0.1, 0.5, 1,
2.5, 5, and 10 µM GnRH agonist
(pGlu-His-Trp-Ser-Tyr-DHis(Bzl)-Leu-Arg-Pro-AzaGlyNH2,
Histrelin, Sigma Chemical Co.) and 0, 0.05, 0.1, 0.5, 1,
2.5, 5, and 10 µM GnRH antagonist
(NACD2Nal-D4Cphe-DTrp-Ser-Tyr-DHarg(Et2)-Leu-Arg-Pro-DAlaNH2.
Detirelix, Sigma Chemical Co.). Embryos were checked daily
using an inverted microscope (Olympus Corp.) for 3
consecutive days to evaluate the effect of GnRH agonist and antagonist
on early development of murine embryos in comparison with growth in
control medium.
Exp 2. Two-cell mouse embryos (n = 386) were cultured in medium containing 5 µM GnRH antagonist and increasing concentrations of GnRH agonist (0.1, 0.5, 1, 5, and 10 µM) for 72 h); as a control, three groups were cultured with medium only, medium with 5 µM GnRH antagonist, and medium with 10 µM GnRH agonist, respectively. Embryos were checked daily using an inverted microscope (Olympus Corp.) to evaluate embryo development in each group.
Both experiments were performed a minimum of three times with similar results.
Statistical analysis
ANOVA and 
2 test were employed to compare groups.
Tukey’s and Scheffe’s tests were applied when ANOVA revealed
statistical differences. The statistical analysis was carried out using
the Statistical Package for Social Science (SPSS, Inc.,
Chicago, IL), and P < 0.05 was considered
statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunohistochemical study
Immunoreactive GnRH was identified in all 180 mouse embryos
studied (60 at compacted morula stage, 60 at early expanded blastocyst
stage, and 60 at hatching blastocyst stage) during the preimplantation
period using an avidin-biotin-alkaline phosphatase technique. To dispel
any doubt about the specificity of the primary antibody used, two
different antibodies against GnRH were used with the same results.
Immunohistochemical localization of GnRH showed intense staining in the
different blastomeres at morula stage as well as in the trophectoderm
and inner cell mass of blastocysts (early expanded and hatching; Fig. 2).
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Embryo culture
Exp 1. The percentage of two cell mouse embryos
reaching the hatching blastocyst stage was significantly
(P > 0.05) enhanced by culture with increasing
concentrations of GnRH agonist (Fig. 3A)
and was significantly decreased (P < 0.01) by culture
with increasing concentrations of GnRH antagonist (Fig. 3B) compared
with that in the control group cultured with medium only. Embryonic
arrest occurs mostly between the two-cell and eight-cell stages.
Moreover, GnRH antagonist (5 and 10 µM) was able to
completely block embryo development, so no embryo reached the
blastocyst stage at these concentrations.
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, A and B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the principal function of GnRH is to stimulate the pituitary gland to release LH and FSH, there is evidence that an extrahypothalamic GnRH might have an important role in the reproductive process (2). A role for GnRH in modulating ovarian function has been described, involving steroidogenesis and oocyte maturation (28, 29, 30). GnRH and GnRH agonist induce resumption of meiosis in follicle-enclosed oocytes both in vivo (31) and in vitro (32). This effect is mediated through a specific receptor (33) and involves the activation effect of GnRH agonist and antagonist on preimplantation embryonic development.
Therefore, our first objective was to show that both GnRH and its receptor are expressed at the mRNA level in vitro by cultured mouse embryos during the preimplantation development period (morula to hatching blastocyst stages). Moreover, we were also able to demonstrate the presence of an immunoreactive GnRH in the different preimplantation embryos studied (morula to hatching blastocyst stages).
Our data from both RT-PCR and immunohistochemical examination demonstrate that this hormone is produced as early as the morula stage. This observation is similar to that previously reported in rhesus monkey embryos during the entire periattachment period (35).
The amount of mRNA in preimplantation embryos is not accurately known. Moreover, it has been recently show that the different blastomeres of the same embryo express different amount of mRNA for ß-actin and interleukin-1 receptor (36). This agrees with previous immunohistochemical studies in which each blastomere of morula as well as embryos in the same stage of development stain with different intensities (15). Thus, the fact that we were able to detect GnRH and GnRH receptor mRNA expression in 65–90% and 85–90%, respectively, of single embryos studied is consistent with the findings of previous studies using this method (14, 20).
Immunohistochemical examination of preimplantation mouse embryos indicates that GnRH was localized in both the inner cell mass and the trophectoderm at the blastocyst stage. This is also consistent with previous reports of the presence of immunoreactive GnRH in the cytotrophoblast of prehatched blastocyst (35) and in placental cytotrophoblast (37). In addition, we localized this hormone in the different blastomeres at the compacted morula stage.
It is remarkable that both GnRH mRNA and protein expression are increased in the hatching blastocyst stage compared with the morula stage, as this hormone has been recently implied as a possible important paracrine factor in the process of embryonic implantation (6, 7). On the other hand, the GnRH receptor was ascertained at a constant level (80–85%) in all of the 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 (10, 11, 12, 13).
Trophoblastic GnRH has been implicated as one of the primary regulators of the synthesis and secretion of hCG both in periimplantation embryos (35) and in the placenta (5). Furthermore, the role of the inner cell mass in the induction of hCG synthesis and secretion by the trophoblast of the periimplantation primate blastocyst has previously been suggested (38). The intimate cell contact between the inner cell mass and the trophoblast seems to be necessary to initiate hCG synthesis (38). This along with evidence that GnRH secretion precedes hCG secretion in periimplantation embryos (35) may explain the fact that we localized the presence of GnRH not only in the trophectoderm, but also in the inner cell mass of the mouse blastocyst. This reinforces the potentially important role of GnRH in controlling hCG synthesis and secretion not only in the placenta but also in the periimplantation embryos.
The results from our in vitro culture of preimplantation mouse embryos exposed to GnRH agonist and antagonist suggest that GnRH is a crucial key in early embryonic development. The GnRH agonist seems to enhance embryonic development, whereas the 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. This is consonant with the fact that exogenous administration of GnRH can displace the GnRH antagonist from the pituitary receptors and reestablish gonadotropin secretion, as both GnRH and GnRH antagonist compete for binding with the same receptor (39). Moreover, there is evidence that both GnRH and GnRH agonist significantly increased the cleavage rate of bovine oocytes fertilized in vitro as well as that this effect was abolished by the addition of a GnRH antagonist (40).
The direct action of both GnRH agonist and antagonist in vitro on preimplantation murine embryonic development is consistent with previous in vivo experiments in which GnRH agonist-treated mice had a higher rate of embryos reaching the hatching blastocyst stages as well as a higher pregnancy and implantation rate (30). Moreover, we have recently reported that infertile woman undergoing in vitro fertilization and embryo transfer had a significantly higher pregnancy and implantation rate if the administration of GnRH agonist was maintained during the early stages of embryonic development and implantation, suggesting a direct effect of the agonist on the embryo and maternal endometrium (41).
Mammalian embryos must emerge, or hatch from their extracellular coat, the zona pellucida, to implant in the uterus at the blastocyst stage of development (42). The exact mechanism of hatching remains controversial. However, previous reports have related PG, trypsin-like substance, plasminogen and plasmin, glutamine, and such phenomena as fluid accumulation and expansion to the mechanism of hatching (42, 43).
PGs have been implicated as remarkable participants in the hatching
process during the development of a variety of animal species (43). It
is interesting that the detrimental effect of GnRH antagonist on
preimplantation embryonic development can be completely reversed by the
agonist with development to the blastocyst stage. However, the hatching
rate was only partially reversed. This could be related to the fact
that GnRH is modulating the production and release of PGE2
and PGF2
in the trophoblast (44), so the GnRH antagonist
may be interfering the hatching process by affecting PG production.
Furthermore, it has been shown that coculture of mouse blastocysts with
human placental cells, which are known to produce GnRH, enhances the
hatching rate (45). This is also consistent with previous reports that
embryos failing to hatch in vitro secreted very low amounts
of GnRH compared with those that did hatch and attach (35).
It has also been previously demonstrated in mice that the administration of GnRH agonist in vivo significantly increases germinal vesicle breakdown and in vitro fertilization rates of these oocytes. Additionally, these fertilized oocytes developed more rapidly than the control group (30). This is consistent with our in vitro findings that embryos cultured with increasing concentrations of GnRH agonist displayed enhanced preimplantation development.
On the basis of the observations just described it is tempting to suggest that GnRH may play an important role in preimplantation mammalian embryo development, endometrial receptivity, and implantation.
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Acknowledgments |
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Footnotes |
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2 Postdoctoral research fellow supported by Ministerio de Sanidad y
Consumo grant (FIS 97/5374 and 99/0657) from the Spanish Government
(Madrid, Spain).
Received July 31, 1998.
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