Embryo-maternal Communication Research Articles

119 ENDOMETRIAL CO-CULTURE MODELS FOR THE IN VITRO INVESTIGATION OF EARLY EMBRYO - MATERNAL CROSSTALK IN CATTLE

Cellular and molecular changes in the endometrium during the preimplantation period are orchestrated by a complex network of molecules. The endometrium consists mainly of epithelial (EC) and stromal cells (SC) which produce a variety of active factors, such as prostaglandins (PG), in response to hormones and embryonic signals. However, it is unknown whether and how EC and SC influence each other in their reaction to these endo- and paracrine stimuli. To address this question, we measured the PG secretion of separately cultured EC and SC, and of EC+SC co-cultures in an insert system (0.2 µm membrane pore size) allowing exchange of secreted factors, but no direct contact between EC and SC. Bovine endometrial cells were isolated from cow uterus by a combination of mechanical and enzymatic procedures on Day 8 of the estrous cycle (ovarian morphology). Isolated EC and SC (90% purity) were cultured in DMEM/F12 containing 10% fetal bovine serum at 37�C in an atmosphere of 5% CO2 in air. PGE2 and PGF2α concentrations in media from separately cultured EC and SC or co-cultured EC+SC were measured by specific ELISAs after 24 h of stimulation with (i) 100 nm oxytocin (OXT); (ii) 100 ng mL–1 interferon tau (IFNT); or (iii) the respective solvent as non-stimulated control. EC responded to OXT stimulation with a decreased PGE2/PGF2α ratio, although the difference was not significant (PGE2/PGF2α ratio non-stimulated: 2.4 � 0.9; PGE2/PGF2α ratio stimulated: 1.5 � 0.8; P > 0.05 v. non-stimulated control, evaluated by t-test). No differences in the ratios of PGE2/PGF2α release into the medium were observed when comparing OXT-treated SC with non-treated SC. Incubating co-cultures of EC and SC with OXT resulted in a statistically significant, 2.5-fold decrease in the PGE2/PGF2α ratio as compared to non-stimulated cells (0.8 � 0.2 v. 2.0 � 0.7; P < 0.05; t-test). In contrast, IFNT stimulation of EC and SC co-cultures shifted PG secretion toward the pregnancy protective PGE2 (PGE2/PGF2α ratio non-stimulated: 0.99 � 0.44; PGE2/PGF2α ratio stimulated: 2.36 � 0.96; P < 0.05 v. non-stimulated control, by t-test). Conclusions: Our findings indicate that interactions between EC and SC regulate the responsiveness of the co-culture system to OXT and the pregnancy recognition molecule IFNT. We propose that co-culture of both cell types should be used for studying mechanisms of early embryo–maternal communication.

A bovine oviduct epithelial cell suspension culture system suitable for studying embryo–maternal interactions: morphological and functional characterization

We established a short-term (24 h) culture system for bovine oviduct epithelial cells (BOECs), obtained on day 3.5 of the estrous cycle and evaluated the cells with respect to morphological criteria, marker gene expression, and hormone responsiveness. BOEC sheets were isolated mechanically from the ampulla with similar yields from oviducts ipsi- and contralateral to the ovulation site (57.9 +/- 4.6 and 56.4 +/- 8.0 x 10(6) cells). BOECs showed > 95% purity and cells cultured for 24 h maintained morphological characteristics present in vivo, as determined by light microscopy, scanning electron microscopy, and transmission electron microscopy. Both secretory cells with numerous secretory granules and ciliated cells with long, well-developed, and vigorously beating kinocilia were visible. Quantitative real-time PCR failed to detect significant differences in transcript levels between ipsi-and contralateral BOECs for the majority of marker genes (estrogen receptors alpha and beta (ESR1 and ESR2), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), oviductal glycoprotein 1 (OVGP1), progesterone receptor (PGR), and tumor rejection antigen 1 (TRA1)) throughout the 24 h culture period. However, the combined data of all time points for glutathione peroxidase 4 (GPX4), a gene previously shown to be expressed at higher levels in the ipsilateral oviduct in vivo, also indicated significantly different mRNA levels in vitro. The expression of marker genes remained stable after 6 h cell culture, indicating only a short adaptation period. Western blot analysis confirmed ESR1 and PGR protein expression throughout the culture period. In agreement with cyclic differences in vivo, estradiol-17beta stimulation increased PGR transcript abundance in BOECs. Our novel culture system provides functional BOECs in sufficient quantities for holistic transcriptome and proteome studies, e.g. for deciphering early embryo-maternal communication.

Embryo-induced transcriptome changes in bovine endometrium reveal species-specific and common molecular markers of uterine receptivity

The endometrium plays a central role among the reproductive tissues in the context of early embryo-maternal communication and pregnancy. This study investigated transcriptome profiles of endometrium samples from day 18 pregnant vs non-pregnant heifers to get insight into the molecular mechanisms involved in conditioning the endometrium for embryo attachment and implantation. Using a combination of subtracted cDNA libraries and cDNA array hybridisation, 109 mRNAs with at least twofold higher abundance in endometrium of pregnant animals and 70 mRNAs with higher levels in the control group were identified. Among the mRNAs with higher abundance in pregnant animals, at least 41 are already described as induced by interferons. In addition, transcript levels of many new candidate genes involved in the regulation of transcription, cell adhesion, modulation of the maternal immune system and endometrial remodelling were found to be increased. The different expression level was confirmed with real-time PCR for nine genes. Localisation of mRNA expression in the endometrium was shown by in situ hybridisation for AGRN, LGALS3BP, LGALS9, USP18, PARP12 and BST2. A comparison with similar studies in humans, mice, and revealed species-specific and common molecular markers of uterine receptivity.

The impact of oocyte maturation media on early bovine embryonic development

Successful production of high quality blastocysts in vitro depends on the use of a culture system that ensures the acquisition of developmental competence by the maturing oocyte. It is now clear that the in vitro maturation environment has a major influence on the oocyte's ability to acquire the potential to develop into blastocysts. In this work we examine the impact of oocyte culture media on the quality of blastocysts by comparing developmental rates, cell number and their allocation to embryonic cell lineages, apoptosis, and expression of developmentally important genes. Higher total cell count and ICM:TCN ratio, which are indicative of embryo viability, were observed in embryos derived from oocyte maturation in TCM-199 supplemented with serum when compared to blastocysts derived from oocyte maturation in SOF BSA. Moreover, oocyte maturation in TCM-199 supplemented with serum-generated embryos of higher morphological quality and producing higher levels of Interferon Tau transcripts when compared to embryos derived from oocyte maturation in SOF BSA. In conclusion, the oocyte maturation regimen affected the morphological feature of blastocysts, including total cell count and allocation of cells to trophectoderm (TE) and inner cell mass (ICM) lineages and the expression profiles of genes involved in various embryo functions such as early embryonic growth, regulation of gene transcription, trophoblast differentiation and function, embryo-maternal communication, and stress response. Our results show that the oocyte culture media have strong impact on the quality of embryos produced in vitro and emphasize the need for more in depth evaluation of oocyte maturation protocols.

172 DOES ESTRADIOL-17β CAUSE CYCLE-DEPENDENT MODULATIONS OF UTERINE MILK PROTEIN IN BOVINE ENDOMETRIUM?

Uterine secretion from the endometrial glandular epithelium provides optimal conditions for early embryonic development. The uterine milk protein (UTMP), a member of the serine proteinase inhibitor superfamily, has been demonstrated to be a major progesterone-induced glycoprotein secreted by the endometrium during pregnancy. Previous transcriptomic analysis revealed that UTMP was highly abundant at estrus in the bovine endometrium (Bauersachs et al. 2005 J. Mol. Endocrinol. 34, 889–908). Here we describe a detailed characterization of UTMP mRNA expression at several time points during the bovine estrous cycle and the pre-implantation period. Simmental heifers were monitored with respect to serum progesterone (P4) and estradiol-17β (E2), and slaughtered at estrus or 3.5, 12, 15, or 18 days after estrus, or at Day 15 or 18 of pregnancy (n = 4 per group). The uterus was divided into corpus and caudal, middle and cranial parts of the ipsilateral uterine horn for sampling of intercaruncular endometrium. In addition, effects of steroid hormones were investigated by stimulating an endometrial cell culture obtained from Day 8 animals (n = 4) with physiological doses of P4 or E2. In all cases, UTMP mRNA was quantified by real-time RT-PCR. Pronounced changes of UTMP mRNA abundance were detected during the estrous cycle. Expression was highest at estrus, followed by a remarkable decrease at Day 3.5. There was no difference between pregnant and non-pregnant animals at Day 15. Cycling animals displaying a high P4 and low E2 content (P4 > 2 ng mL-1 and E2 < 1 pg mL-1) revealed a lower expression of UTMP at Day 18 compared to the pregnant heifers, whereas animals at Day 18 progressing toward estrus (P4 < 1.5 ng mL-1 and E2 > 3 pg mL-1) exceeded the mRNA expression of the pregnant group. Following stimulation with estradiol-17β, the in vitro UTMP transcripts increased significantly. These results indicate that two different interfering stimulatory events might take place. While estradiol-17β appeared to increase UTMP mRNA expression at estrus, a second factor, most probably embryo-derived or embryo-induced, is assumed to be responsible for the UTMP rise during early pregnancy. The distinct gradient from the cranial uterine horn to the corpus at estrus was less pronounced at Day 3.5 and absent at Days 12, 15, and 18, pointing toward functional implications regarding the passing gametes, particularly sperm. An antibody raised against bovine UTMP will further validate the observed mRNA regulations on the protein level. In conclusion, bovine UTMP seems to play a decisive role for precise cyclic regulation of the bovine uterine milieu and during early embryo-maternal communication. This work was supported by the DFG FOR 478.

Monozygotic Twin Model Reveals Novel Embryo-Induced Transcriptome Changes of Bovine Endometrium in the Preattachment Period1

Initiation and maintenance of pregnancy are critically dependent on an intact embryo-maternal communication in the preimplantation period. To get new insights into molecular mechanisms underlying this complex dialog, a holistic transcriptome study of endometrium samples from Day 18 pregnant vs. nonpregnant twin cows was performed. This genetically defined model system facilitated the identification of specific conceptus-induced changes of the endometrium transcriptome. Using a combination of subtracted cDNA libraries and cDNA array hybridization, 87 different genes were identified as upregulated in pregnant animals. Almost one half of these genes are known to be stimulated by type I interferons. For the ISG15ylation system, which is assumed to play an important role in interferon tau (IFNT) signaling, mRNAs of four potential components (IFITM1, IFITM3, HSXIAPAF1, and DTX3L) were found at increased levels in addition to ISG15 and UBE1L. These results were further substantiated by colocalization of these mRNAs in the endometrium of pregnant animals shown by in situ hybridization. A functional classification of the identified genes revealed several different biological processes involved in the preparation of the endometrium for the attachment and implantation of the embryo. Specifically, elevated transcript levels were found for genes involved in modulation of the maternal immune system, genes relevant for cell adhesion, and for remodeling of the endometrium. This first systematic study of maternal transcriptome changes in response to the presence of an embryo on Day 18 of pregnancy in cattle is an important step toward deciphering the embryo-maternal dialog using a systems biology approach.

Gene expression profiling of bovine endometrium during the oestrous cycle: detection of molecular pathways involved in functional changes

The endometrium plays a central role among the reproductive tissues in the context of early embryo-maternal communication and pregnancy. It undergoes typical changes during the sexual/oestrous cycle, which are regulated by the ovarian hormones progesterone and oestrogen. To identify the underlying molecular mechanisms we have performed the first holistic screen of transcriptome changes in bovine intercaruncular endometrium at two stages of the cycle--end of day 0 (late oestrus, low progesterone) and day 12 (dioestrus, high progesterone). A combination of subtracted cDNA libraries and cDNA array hybridisation revealed 133 genes showing at least a 2-fold change of their mRNA abundance, 65 with higher levels at oestrus and 68 at dioestrus. Interestingly, genes were identified which showed differential expression between different uterine sections as well. The most prominent example was the UTMP (uterine milk protein) mRNA, which was markedly upregulated in the cranial part of the ipsilateral uterine horn at oestrus. A Gene Ontology classification of the genes with known function characterised the oestrus time by elevated expression of genes, for example related to cell adhesion, cell motility and extracellular matrix and the dioestrus time by higher expression of mRNAs encoding for a variety of enzymes and transport proteins, in particular ion channels. Searching in pathway databases and literature data-mining revealed physiological processes and signalling cascades, e.g. the transforming growth factor-beta signalling pathway and retinoic acid signalling, which are potentially involved in the regulation of changes of the endometrium during the oestrous cycle.

231 TEMPORAL AND SPATIAL GENE EXPRESSION ANALYSIS OF THE BOVINE OVIDUCT EPITHELIUM

The fallopian tube plays a central role in reproduction, providing the appropriate environmental conditions for oocyte maturation as well as for sperm capacitation. Furthermore, fertilization and the first cleavage stages of embryonic development take place in the oviduct. At the molecular level, only fragmentary data are available regarding the physiological changes in the oviduct epithelium during the estrous cycle. Therefore, we performed a systematic study of gene expression changes in bovine ipsilateral oviduct epithelial cells derived from either the ampulla or the isthmus part of the oviduct at four different time points of the estrous cycle. A cDNA array consisting of approximately 400 candidate genes, primarily identified in different studies in the context of gene expression regulation in the oviduct, was designed and hybridized with 33P-labeled cDNA probes prepared from 28 different tissue samples. These tissue samples were collected from cyclic Simmental heifers at Day 0 (n = 3), Day 3.5 (n = 3), Day 12 (n = 4) and Day 18 (n = 4) of the estrous cycle. Ipsilateral epithelial cells were separately collected from ampulla and isthmus. After array evaluation (AIDA Image Analyzer, version 3.41, Raytest, Straubenhardt, Germany), the raw data were normalized to internal reference cDNAs on the arrays. Statistical analysis was done using ANOVA and the Tukey post-hoc test (GeneSpring® version 6.1, Silicon Genetics, Redwood City, CA, USA). For selected genes, differential expression was verified by real-time RT-PCR. A simplified Gene Ontology was built for the genes present on the array and a pathway analysis was performed to elucidate gene networks involved in the regulation of oviduct epithelial cell function. The expression patterns of two functional groups of genes are presented here: genes that are related to immune functions and genes of the secretory pathway or encoding secreted proteins. Messenger-RNA levels of immune-related genes were higher in epithelial cells of the ampulla compared to the isthmus part of the oviduct. This implies that certain immune functions may be differentially regulated in ampulla and isthmus. Furthermore, mRNAs of genes of the secretory pathway showed highest levels mainly in the ampulla around time of estrus, which may be explained with the increase of the secretory activity in the epithelium of the ampulla beginning at pre-estrus. In general, this study shows the importance of a separate analysis of the oviduct compartments and the influence of the estrous cycle on the expression level of a variety of genes. In the context of fertilization and early embryo-maternal communication, these results could provide an insight into the physiological changes during the estrous cycle, which are the bases for these processes. This work was supported by the Deutsche Forschungsgemeinschaft (Research Unit “Mechanisms of Embryo-Maternal Communication”; FOR 478/1).

2 IDENTIFICATION OF GENES INDUCED BY THE CONCEPTUS IN THE BOVINE ENDOMETRIUM DURING THE PRE-IMPLANTATION PERIOD

Early embryonic development, implantation, and maintenance of a pregnancy are critically dependent on an intact embryo-maternal communication. So far, only few signals involved in this dialogue have been identified. In ruminants, interferon tau (IFN�) plays a key role in the process of maternal recognition of pregnancy by exhibiting antiluteolytic activity. Even though many experimental findings indicate a pivotal role of IFN� in the context of embryo-maternal communication in ruminants, a number of other systems may be involved. To identify genes induced in the bovine endometrium by the signaling of the embryo, a combination of subtracted cDNA libraries and cDNA array hybridization was applied. Monozygotic twin pairs (n = 5) were used as the biological model. Pregnancy was created in one twin by transferring two in vitro-produced embryos on Day 7 of the estrous cycle; the other twin received a sham embryo transfer. Pregnant and nonpregnant twins were slaughtered at Day 18; endometrial tissue samples were recovered and processed for transcriptome analysis as described (Bauersachs et al. 2005 J. Mol. Endocrinol. 34, 889-908). Screening of 4608 clones of two subtracted libraries revealed 90 different up-regulated genes and mRNAs, of which almost 50% are known to be stimulated by type I interferons. Among these interferon-stimulated genes, the ISG15 system is assumed to be of particular interest, and several components were studied in more detail using in situ hybridization. The pattern of mRNA expression suggests that modification of endometrial proteins through ISG15ylation plays a fundamental role during the pre-implantation period. A classification of the identified genes based on Gene Ontologies revealed the prevalence of genes involved in regulation of gene expression, cell communication, cell growth, cell differentiation, cell proliferation, and cell adhesion, and also the prevalence of genes with immune-related functions. These results underline the intense response of the endometrium to the presence of a conceptus, culminating in the preparation of the maternal environment for embryonic implantation. Further, for eleven selected genes the expression in the endometrium was quantified by the use of real-time RT-PCR. Overall, the results of quantitative RT-PCR and array hybridization correlated very well. To our knowledge this study provides the first holistic gene expression analysis of the bovine endometrium during the pre-implantation period. The results underline the importance of IFN� as an embryo-derived pregnancy recognition signal and depict the molecular mechanisms at the mRNA level underlying the intense embryo-maternal dialog taking place at Day 18 of gestation.

211 TEMPORAL AND SPATIAL EXPRESSION PROFILE OF THE UTERINE MILK PROTEIN — A MEMBER OF THE SERINE PROTEASE INHIBITOR SUPERFAMILY — IN THE BOVINE ENDOMETRIUM

A combination of subtracted cDNA libraries and cDNA array hybridization was applied to identify differentially expressed genes in the bovine intercaruncular endometrium at estrus and diestrus. Six cyclic Simmental heifers were cycle-synchronized and slaughtered the morning after standing heat occurred (n = 3) or 12 days after estrus (n = 3), respectively. The uterus was divided into seven sections: corpus, plus the caudal, middle, and cranial parts of both uterine horns. Samples were collected from the epithelial layer of the lamina propria of the intercaruncular endometrium. Two subtracted libraries were prepared and analyzed by array hybridization with probes produced from the 42 tissue samples. More than 130 differentially expressed genes were identified. Among these genes, the mRNA for the uterine milk protein (UTMP) showed a much higher expression level at estrus versus diestrus. Furthermore, the expression level differed markedly between the uterus sections. Statistical significance was tested using ANOVA and Student Newman Keuls post-hoc test (GeneSpring® version 6.1, Silicon Genetics, Redwood City, CA, USA). There was a pronounced expression gradient from both cranial uterine horns (highest) to the corpus (lowest) at estrus. UTMP mRNA level was highest in the ipsilateral cranial horn. A more accurate quantification was done by quantitative real-time RT-PCR using the same RNA samples as for array hybridization and, in addition, samples from further time points of the estrous cycle and early pregnancy. The results obtained by array hybridization were clearly confirmed. UTMPs, also known as uterine serpins (US), were previously shown to be the major products secreted by the uteri of cattle, sheep, and pigs during pregnancy. The US are glycoproteins belonging to the serpin (serine proteinase inhibitor) superfamily of proteins. However, no inhibitory activity to any serine proteinase has been found. The ovine uterine serpin (OvUS) has been shown to inhibit a wide variety of immune responses, including mixed lymphocyte reaction, T cell-dependent antibody production, and NK (natural killer)-like activity, indicating a major role in protecting the conceptus from maternal immune reactions. The four members of the US family form a clade distinct from other serpins, which have probably adopted important biological functions in the reproductive biology of Artiodactyla. Therefore, the identified expression gradient of the UTMP mRNA at estrus may be of particular importance, e.g. for sperm selection, and deserves further investigation at the protein level and in functional studies. This work was supported by the Deutsche Forschungsgemeinschaft (Research Unit “Mechanisms of Embryo-Maternal Communication”; FOR 478/1).

329 A NOVEL SUSPENSION CULTURE SYSTEM FOR BOVINE OVIDUCT EPITHELIAL CELLS

Bovine oviduct epithelial cells (BOEC) are difficult to culture without dedifferentiation. Frequently observed changes in morphology during culture suggest that gene expression patterns are affected as well. We explored a novel short-term culture system for BOEC – suitable for co-culture experiments with embryos – and evaluated the cells with respect to morphological criteria, candidate gene expression, and hormone responsiveness. Simmental heifers were slaughtered on Day 3.5 after standing heat and BOEC were obtained by squeezing along the ampulla with forceps. The cell sheets were separated mechanically by repeated passages through syringes and pipetting, and recovered by sedimentation. Cells from the ipsi- and contralateral oviduct were cultured separately at a density of 106 cells per well in 24-well plates with 800 μL TCM-199 supplemented with 2% OCS (estrous cow serum, as used in embryo culture) or CS 3.5 (cow serum, Day 3.5 after standing heat, adequate to the cycle stage in which cells were obtained) and 0.25 mg/mL gentamicin. For cell counting, an aliquot was further disaggregated by passing 15 times through a 30-gauge needle to achieve a single-cell suspension. Culture took place at 38°C in a humidified atmosphere of 5% CO2 in air. Cells were examined by light microscopy at seeding and after 6, 12, 24, and 48 h. Cell aggregates showed a worm-like structure, displaying numerous vigorously beating cilia on their surfaces throughout the culture period. Trypan blue staining indicated that cells contained in aggregates were viable while single cells stained predominantly positive (non-viable). The purity of the epithelial cell culture was >95%, as determined by immunohistochemistry using antibodies against vimentin and cytokeratin. For electron microscopic investigations, BOEC were sampled at seeding and after 24 h in culture. Cultured BOEC showed a morphology highly similar to that of BOEC in vivo. Both secretory cells with numerous secretory granules and mitochondria, and ciliated cells with long, well developed and actively moving kinocilia were visible. RT-PCR data for candidate genes (ERα, ERβ, HMGCR, PHGPx, PR) obtained from BOEC samples at seeding and after 6, 12, 24, and 48 h in culture showed that gene expression was stable for the majority of transcripts after 6 h in culture. There was no significant difference between cells cultured with OCS or CS 3.5 and no difference between cells obtained from the ipsi- or contralateral oviduct. Estradiol-17β (E2, 10 pg/mL) or progesterone (P4, 10 ng/mL) stimulation showed that the cultured BOEC are able to respond to hormonal signals in a manner similar to their reaction in vivo (Ulbrich et al. 2003 J. Steroid. Biochem. Mol. Biol. 84, 279–89). Progesterone receptor mRNA was up-regulated by E2 and estrogen receptor β mRNA was up-regulated by P4. The culture system for bovine oviduct epithelial cells thus provides an adequate tool to investigate mechanisms of the embryo-maternal communication in cattle. This work was supported by the Deutsche Forschungsgemeinschaft (Research Unit “Mechanisms of Embryo-Maternal Communication”; FOR 478/1).

Embryo-maternal communication in bovine - strategies for deciphering a complex cross-talk.

Early embryonic development, implantation and maintenance of a pregnancy are critically dependent on an intact embryo-maternal communication. So far, only few signals involved in this dialogue have been identified. In bovine and other ruminants, interferon tau is the predominant embryonic pregnancy recognition signal, exhibiting antiluteolytic activity. However, this is just one aspect of the complex process of embryo-maternal signalling, and a number of other systems are more likely to be involved. To gain a more comprehensive understanding of these important mechanisms, integrated projects involving specialists in embryology, reproductive biotechnology and functional genome research are necessary to perform a systematic analysis of interactions between pre-implantation stage embryos and oviduct or uterine epithelial cells, respectively. State-of-the-art transcriptomic and proteomic technologies will identify reciprocal signals between embryos and their maternal environment and the respective downstream reaction cascades. For in vivo studies, the use of monozygotic twins as recipient animals provides elegant model systems, thus eliminating genetic variability as a cause of differential gene expression. In addition, suitable systems for the co-culture of oviduct epithelial or endometrium cells with the respective embryonic stages need to be established for functional validation of candidate genes potentially involved in the dialogue between embryos and their maternal environment. The knowledge of these mechanisms should help to increase the pregnancy rate following embryo transfer and to avoid embryonic losses. Candidate genes involved in embryo-maternal communication will also be used to define new quality criteria for the selection of embryos for transfer to recipients. Another application is the supplementation of embryotrophic factors or components of embryo-maternal signalling in optimized formulations, such as bioartificial matrices. As a long-term goal, signalling mechanisms identified in bovine will also be functionally evaluated in other species, including the human.

Uterine-associated serine protease inhibitors stimulate deoxyribonucleic acid synthesis in porcine endometrial glandular epithelial cells of pregnancy.

Protease inhibitors are major secretory components of the mammalian uterus that are thought to mediate pregnancy-associated events primarily by regulating the activity of proteolytic enzymes. In the present study, we examined the mitogenic potentials of two serine protease inhibitors, namely secretory leukocyte protease inhibitor (SLPI) and uterine plasmin/trypsin inhibitor (UPTI) in primary cultures of glandular epithelial (GE) cells isolated from early pregnant (Day 12) pig endometrium, using the [(3)H]thymidine incorporation assay. Purified porcine SLPI (pSPLI), porcine UPTI (pUPTI), or recombinant human SLPI (rhSLPI), all of which exhibited anti-trypsin activity, increased (p < 0.05) labeled thymidine incorporation into DNA of serum-deprived GE cells when tested at a range of 10-1000-ng/ml concentrations. Polyclonal antibodies directed against either hSLPI or pSLPI abrogated the effect of SLPI. Co-addition of pSLPI and pUPTI increased DNA synthesis in these cells to a level higher (p < 0.05) than that observed with either protease inhibitor. The glycosaminoglycan heparin, which has been previously shown to increase the anti-protease activity of SLPI, exhibited a tendency (p = 0.08) to enhance SLPI and UPTI induction of cellular DNA synthesis. Reverse transcription-polymerase chain reaction indicated that the messenger RNAs for both protease inhibitors were present in the endometrium throughout pregnancy and, within this tissue, in GE cells to a greater extent (p < 0.05) than in stromal fibroblastic cells. Results demonstrate that, in addition to their well-documented anti-protease activities, SLPI and UPTI may constitute autocrine growth promotants for the uterine epithelium. These data suggest a novel mechanism whereby locally produced protease inhibitors may modulate periimplantation events and embryo-maternal communication.

Novel insights into human endometrial paracrinology and embryo-maternal communication by intrauterine microdialysis.

The regulation of human implantation is still unknown. Evidence from mice suggests an essential role for several paracrine mediators but species differences with implantation in the human preclude the extrapolation of these concepts to humans. An intrauterine microdialysis device (IUMD), consisting of microdialysis tubing glued into a balloon catheter on one side and into a polypropylene tube on the other, allows a dynamic and accurate in-vivo measurement of uterine paracrine interactions in humans. Inserted into the uterine cavity in the form of a loop, it can be continuously perfused with saline to reveal a number of relevant cytokines and growth factors in uterine effluents of non-pregnant women in both follicular and luteal phases. These included interleukin (IL)-1alpha, IL-1beta, IL-6, leukaemia inhibitory factor (LIF), macrophage colony-stimulating factor (M-CSF), epidermal growth factor, vascular endothelial growth factor (VEGF), insulin-like growth factor binding protein-1 (IGFBP-1), prolactin, and human chorionic gonadotrophin (HCG). The source of intrauterine HCG is unclear since endometrial mRNA for the HCG beta-subunit is not revealed using reverse transcriptase polymerase chain reaction analysis. Applying urinary HCG locally via the IUMD profoundly alters endometrial secretory parameters. Prolactin, IGFBP-1, and M-CSF are significantly inhibited and VEGF is regulated in a biphasic manner involving early stimulation followed by inhibition of intrauterine levels. Use of the IUMD has thus shown that the urinary HCG preparations routinely used for ovulation induction and luteal support may directly alter endometrial function.

A factor secreted by human embryo stimulates cytokine release by uterine mast cell.

The existence of a biochemical network of embryo-maternal communication implies that various secreted molecules constitute a signal-response mechanism, important for the process of embryo implantation in mammals. Here we report the purification of a protein with an apparent molecular weight of 136 kDa, responsible for a 2000-fold increase in embryo-derived histamine-releasing factor (EHRF) activity. This protein, purified from medium from the in-vitro culture of 2-8-cell human embryos, by means of affinity chromatography, was capable of binding immunoglobulin (Ig)E as demonstrated by immunoblotting and enzyme-linked immunosorbent assays. We found EHRF was capable of inducing release of histamine and cytokines in vitro from rat uterine tissue, collected on day 4 of pregnancy (preimplantation stage of embryo development). When EHRF was used as a secretagogue, granulocyte macrophage-colony stimulating factor (GM-CSF) release increased from 3 to 55 pg/g (P < 0.01) and tumour necrosis factor-alpha (TNF-alpha) release increased from 0 to 2.1 ng/g (P < 0.01), as detected by enzyme-linked immunosorbent assay. A simple method was used to purify uterine mast cells using an IgE-Sepharose affinity chromatography column and the purity (90%) was checked with Dynabeads coupled to specific rat IgE antibody. When purified mast cells were stimulated with EHRF in the same way as the uterine explants, a similar pattern of GM-CSF and TNF-alpha release was obtained. We also describe the reverse transcription-polymerase chain reaction (RT-PCR) of GM-CSF and TNF-alpha mRNA from purified uterine mast cells. On day 4 of pregnancy only the mRNA of TNF-alpha was found and this increased after stimulation with the EHRF. In conclusion, the data presented suggest that uterine mast cells isolated during the preimplantation stage release cytokines in vitro following interaction with an embryo factor.