Recombinant entactin promotes mouse primary trophoblast cell adhesion and migration through the Arg-Gly-Asp (RGD) recognition sequence.

In vitro culture of mouse blastocysts during the period coinciding with implantation has revealed that primary trophoblast cells can adhere and migrate in serum-free medium when provided with certain extracellular matrix components, including fibronectin and laminin. Tightly associated with laminin is the glycoprotein, entactin, that may play an important role in basement membrane assembly and cell attachment. Mouse blastocysts were studied using this in vitro model to determine whether entactin was capable of mediating trophoblast invasive activity. Although entactin has never been shown to promote cell migration, we report here that recombinant entactin supported blastocyst outgrowth in a dose-dependent manner, with a maximal effect at 20-50 micrograms/ml. The ability of trophoblast cells to adhere and migrate on entactin was specifically inhibited by anti-entactin antibody, but not by antibodies raised against laminin. The synthetic peptide, Gly-Arg-Gly-Asp-Ser-Pro, that contains the Arg-Gly-Asp (RGD) integrin recognition site, reversibly inhibited entactin-mediated blastocyst outgrowth in a dose-dependent manner, but had no effect on laminin-mediated outgrowth. The synthetic peptide, Gly-Phe-Arg-Gly-Asp-Gly-Gln, that comprises the actual RGD-containing sequence within entactin, promoted trophoblast outgrowth when immobilized on the substratum. Furthermore, a mutated recombinant entactin, altered to contain a Glu in place of Asp at the RGD site, provided no trophoblast cell adhesive activity. We conclude that entactin promotes trophoblast outgrowth through a mechanism mediated by the RGD recognition site, and that it may play an important role during invasion of the endometrial basement membrane at implantation.

Expression of heparin-binding EGF-like growth factor in term chorionic villous explants and its role in trophoblast survival.

Heparin-binding EGF-like growth factor (HBEGF) induces trophoblast extravillous differentiation and prevents apoptosis. These functions are compromised in preeclampsia. Because HBEGF is downregulated in placentas delivered by women with preeclampsia, we have examined its expression and cytoprotective activity in term villous explants. Chorionic villous explants prepared from non-pathological placentas collected by cesarean section at term were cultured at either 20% or 2% O2 and treated with the HBEGF antagonist CRM197 or recombinant HBEGF. Paraffin sections were assayed for trophoblast death, proliferation and HBEGF expression using the TUNEL method, immunohistochemistry for nuclear Ki67 expression and semi-quantitative immunohistochemistry with image analysis, respectively. Trophoblast cell death was increased significantly after 8h of culture with CRM197 or by culture for 2h at 2% O2. Exogenous HBEGF prevented cell death due to hypoxia. Proliferative capacity was not affected by culture at either 20% or 2% O2. Contrary to first trimester placenta, term trophoblasts do not elevate HBEGF expression in response to hypoxia. However, low endogenous levels of HBEGF are required to maintain survival. Therefore, HBEGF-mediated signaling significantly reduces trophoblast cell death at term and its deficiency in preeclampsia could negatively impact trophoblast survival.

Regulation of trophoblast invasion - a workshop report.

Trophoblast invasion during placental development helps to establish efficient physiological exchange between maternal and fetal circulatory systems. Trophoblast stem cells differentiate into multiple subtypes, including some that are highly invasive. Signalling to the trophoblast from decidua, uterine natural killer cells and vascular smooth muscle can regulate extravillous trophoblast differentiation. Important questions remain about how these cellular interactions promote trophoblast invasion and the signalling pathways that are involved. New and established biological models are being used to experimentally examine these interactions and the underlying molecular mechanisms.

The myelin proteolipid protein gene modulates apoptosis in neural and non-neural tissues.

Mutations of the myelin proteolipid protein gene (Plp) are associated with excessive programmed cell death (PCD) of oligodendrocytes. We show for the first time that PLP is a molecule ubiquitously expressed in non-neural tissues during normal development, and that the level of native PLP modulates the level of PCD. We analyze three non-neural tissues, and show that native PLP is expressed in trophoblasts, spermatogonia, and cells of interdigital webbing. The non-neural cells that express high levels of native PLP also undergo PCD. The level of PLP expression modulates the level of PCD because mice that overexpress native PLP have increased PCD and mice deficient in PLP have decreased PCD. We show that overexpression of native PLP causes a dramatic acidification of extracellular fluid that, in turn, causes increased PCD. These studies show that the level of native PLP modulates the amount of PCD during normal development via a pH-dependent mechanism.

Reduced expression of the epidermal growth factor signaling system in preeclampsia.

INTRODUCTION: The epidermal growth factor (EGF) signaling system regulates trophoblast differentiation, and its disruption could contribute to perinatal disease. We hypothesized that this pathway is altered in preeclampsia, a disorder associated with trophoblast apoptosis and failure to invade and remodel the uterine spiral arteries. METHODS: Six EGF family peptides and a truncated EGF receptor splice variant (p110/EGFR) were examined using immunohistochemistry in the trophoblast of placentas (N = 76) from women with preeclampsia, and compared to placentas from women of similar gestational age (GA) with preterm labor (PTL) or small for gestational age (SGA) fetuses, as well as normal term placentas. EGF, transforming growth factor-α (TGFA), and heparin-binding EGF-like growth factor (HBEGF) were evaluated using ELISA in maternal plasma from another 20 pregnancies with or without preeclampsia. Cell death was evaluated in the HTR-8/SVneo human cytotrophoblast cell line using TUNEL to evaluate the protective effects of EGF peptides. RESULTS: Trophoblast HBEGF, TGFA, and EGF were significantly reduced in preeclampsia compared to PTL and SGA, while p110/EGFR accumulated significantly on the surface of the chorionic villi (p < 0.05). Plasma EGF levels were significantly decreased in preeclamptic patients, compared to non-preeclamptic patients (p < 0.05). HBEGF, EGF, TGFA, epiregulin, and betacellulin each blocked cytotrophoblast cell death in vitro (p < 0.05). DISCUSSION: Three members of the EGF family are dysregulated in placentas with preeclampsia, whereas p110/EGFR, a potential EGF receptor antagonist, is overexpressed. These findings are consistent with the concept that disruption of the EGF signaling system contributes to aberrant trophoblast development associated with preeclampsia.

Ethanol-induced intracellular calcium mobilization rapidly alters gene expression in the mouse blastocyst.

The induction of intracellular Ca2+ release in pre-implantation mouse embryos accelerates their subsequent rate of development in vitro through a calmodulin-dependent mechanism [Stachecki J.J., Armant D.R. Transient release of calcium from inositol 1,4,5-trisphosphate-specific stores regulates mouse pre-implantation development. Development 1996; 122: 2485-2496]. To examine the hypothesis that intracellular Ca2+ signaling alters embryonic gene expression, individual transcript levels were compared by mRNA differential display before and 1 h after intracellular Ca2+ mobilization with ethanol in mouse blastocysts. Ten up-regulated and four down-regulated genes were observed, representing 3.5% of approximately 400 transcripts that were resolved. After sequencing, most of the DNA fragments appeared to be novel; however, two amplicons that increased after Ca2+ mobilization were identified as arginase and ubiquitin conjugating enzyme (E2). The up-regulation of arginase mRNA (3.5-fold after 2 h) was confirmed by reverse transcription and the polymerase chain reaction using specific oligonucleotide primers derived from the deduced mouse embryo sequence. A corresponding 2.5-fold increase in arginase enzymatic activity peaked 9 h after ethanol exposure. Increased expression of arginase and other genes may mediate the onset of rapid cell proliferation and differentiation that is induced by Ca2+ signaling during pre-implantation development.

Multiple roles for heparin-binding epidermal growth factor-like growth factor are suggested by its cell-specific expression during the human endometrial cycle and early placentation.

Embryonic expression of the epidermal growth factor (EGF) receptor as well as embryonic and steroid-dependent uterine secretion of its ligand, heparin-binding EGF-like growth factor (HB-EGF), are temporally associated with the period of blastocyst implantation. We examined the temporal cell type-specific expression of HB-EGF in human endometrium during the menstrual cycle by immunohistochemistry and in situ hybridization. Early first trimester implantation sites were also examined to determine HB-EGF protein levels in decidual and fetal tissues. In the endometrial stroma, HB-EGF protein expression increased markedly during the late proliferative phase and then decreased in the early secretory phase. By contrast, luminal and glandular epithelial cells as well as blood vessel endothelium accumulated the protein between midcycle and cycle day 20, with peak expression observed during the period of uterine receptivity for implantation. HB-EGF expression decreased dramatically at the end of the cycle, before menses. Spatiotemporal expression of HB-EGF messenger ribonucleic acid demonstrated a similar pattern. During early pregnancy, HB-EGF immunostaining was noted in the decidua and in both villous and extravillous trophoblast populations. These findings suggest that HB-EGF promotes implantation and trophoblast invasion through paracrine and autocrine signaling as cells penetrate the stroma and displace the arteriole endothelium.

Heparin-binding EGF-like growth factor modulation by antiprogestin and CG in the baboon (Papio anubis).

The objectives of this study were to determine whether antiprogestin therapy or the infusion of human CG to mimic blastocyst transit in the baboon alters heparin-binding EGF-like growth factor expression during the window of implantation. During the menstrual cycle, heparin-binding EGF-like growth factor protein accumulation in the glandular epithelium was low in the proliferative phase and increased to maximal expression on d 5 and 10 postovulation. Stromal cells accumulated high levels of heparin-binding EGF-like growth factor in the proliferative phase, which decreased by d 5 postovulation. These transitional changes in both cell types were delayed when cycling baboons were treated with the antiprogestin ZK 137.316 during the luteal phase. The treatment with human CG had no effect on expression of heparin-binding EGF-like growth factor when compared with cycling baboons on d 10 postovulation and was comparable with that observed on d 18 and 22 of pregnancy. However, the superimposition of the antiprogestin with the human CG treatment also decreased expression in the epithelial cells. In summary, heparin-binding EGF-like growth factor accumulation in the epithelial glands is under the influence of progesterone and does not seem to be influenced by the paracrine secretion of trophoblast CG.

Fucosylation events during mammalian spermatogenesis.

It will be necessary to conduct further studies to establish more precisely the localization of FT on mouse male germ cells. Antibodies to FTs are not yet available, so an immunocytochemical approach is not currently feasible. Additional cell fractionation protocols can be designed to compare plasma membrane fractions with enriched fractions of Golgi apparatus and to compare directly the activities of multiple glycosyltransferase enzymes and Golgi-specific markers in these preparations. Schachter et al. and Nyquist and colleagues have already provided experimental techniques for the isolation of Golgi fractions of good purity from rodent pachytene spermatocytes and spermatids. Ample opportunity exists, then, for a detailed analysis of the number, specificity, and localization of FT enzymes during mammalian spermatogenesis. All available data imply that these enzymes will prove to be vital components in the differentiation of cells within the seminiferous epithelium.

Intracellular signaling in the developing blastocyst as a consequence of the maternal-embryonic dialogue.

The success of blastocyst implantation is dependent on signaling between the embryo and the receptive endometrium. Intercellular signaling molecules, which include hormones, growth factors, and cytokines, have been identified that participate in the maternal-embryonic dialogue. These biologically active molecules may target uterine and/or embryonic tissues in a biochemical cascade that coordinates the two developmental programs during implantation. Two notable uterine products are calcitonin and heparin-binding epidermal growth factor-like growth factor, which are both expressed during the receptive phase of the endometrium in humans and in rodent models. We review data that demonstrate the ability of these molecules to accelerate blastocyst differentiation and delineate the respective intracellular signaling pathways that advance the embryonic developmental program. An understanding of the mediators regulating embryonic development in utero and their biochemical mechanisms of the action may provide insights for improvement of embryo culture in vitro prior to blastocyst transfer.

Improved accuracy of sperm motility assessment using a modified Micro-Cell sperm counting chamber.

OBJECTIVE: To assess the effect of modifications made in the Micro-Cell sperm counting chamber on the motility of washed human spermatozoa. DESIGN: In a 2 x 2 experimental design, human sperm samples were washed with or without protein supplementation, loaded into modified or unmodified Micro-Cell chambers, and assessed by automated semen analysis for changes associated with sperm adhesion to the glass chamber surfaces. PARTICIPANTS: Twenty-one men who were undergoing semen analysis and presented with > or = 50% motile sperm, or normal donors. MAIN OUTCOME MEASURES: Reduced motility and elevated lateral head displacement associated with sperm adhesion to glass surfaces were compared using a doubly repeated measures analysis of variance. RESULTS: Addition of protein to the sperm washing solution partially reversed the adhesion of spermatozoa to the glass of unmodified Micro-Cell chambers. Chambers manufactured to reduce cell adhesion to glass surfaces yielded the highest motility and lowest lateral head displacement, whether the sperm were washed in a solution supplemented with or without protein. CONCLUSION: These findings indicate that, as in raw semen, kinematics of washed sperm can be measured reliably in the modified Micro-Cell chamber.

The influence of chamber characteristics on the reliability of sperm concentration and movement measurements obtained by manual and videomicrographic analysis.

To assess the influence of chamber design and depth on the accuracy and precision of sperm measurements, manual counting of a standardized latex bead solution and automated sperm motility measurements were made using a Makler chamber (Sefi Medical Industries, Haifa, Israel), Neubauer hemocytometer (American Optical Company, Buffalo, NY), and a new, disposable device (Micro-Cell; Cyto Fluidics, Inc., Silver Spring, MD). Bead counts obtained with the Micro-Cell chamber or hemocytometer were not statistically different from those determined by electronic particle counting, whereas Makler chamber counts were 62% higher. Makler counts had a significantly higher standard deviation, suggesting that counts made with this device are less reproducible. Analyzing live sperm samples, the percentage of motile sperm determined using Micro-Cell and Makler chambers were similar. However, significant differences in sperm concentration and mean velocity were found. The Micro-Cell disposable chamber provided consistent and accurate data on sperm concentration, percent motility, and mean velocity. These differences in sperm measurements emphasize the importance of sampling chamber characteristics on data reliability.

The effects of in vitro cocaine exposure on human sperm motility, intracellular calcium, and oocyte penetration.

OBJECTIVE: To determine if cocaine exposure affects human sperm motility, intracellular calcium level, and fertilizing capability. DESIGN AND METHODS: Human semen samples were treated with 1 to 1,000 microM cocaine hydrochloride for up to 2 hours in vitro. Sperm motion kinematics were measured by computer-assisted semen analysis (CASA). Spermatozoan intracellular calcium was determined by laser cytometry. The sperm fertilizing capability was assessed using the zona-free hamster oocyte penetration test. RESULTS: After a short exposure (15 minutes) to cocaine, the sperm motion kinematic parameters, straight line velocity and linearity, were decreased in the high concentration groups. However, after a longer exposure (2 hours) to cocaine, the differences were no longer significant. Cocaine treatment did not alter spermatozoa intracellular calcium levels. Most importantly, human sperm treated with cocaine at a high concentration were fully capable of penetrating zona-free hamster oocytes. CONCLUSION: Human spermatozoa acutely exposed to high concentrations of cocaine initially demonstrate a decrease in two motion kinematics, straight line velocity and linearity. However, overall, cocaine exposure had no significant effects on sperm motility and fertilizing capability.

The influence of exogenous human chorionic gonadotropin cycles with spontaneous luteinizing hormone surges on the outcome of in vitro fertilization.

Thirty consecutive patients who displayed endogenous luteinizing hormone (LH) surges and reached oocyte recovery for the purpose of in vitro fertilization were reviewed (LH surge group). Another 37 patients receiving human chorionic gonadotropin (hCG) in the absence of an LH surge served as a control group (no surge-hCG group). All patients underwent ovulation induction using clomiphene citrate (CC) plus human menopausal gonadotropin (hMG). The numbers of oocytes recovered and embryos transferred were significantly lower in the LH surge group compared with the no surge-hCG group (P less than 0.05). Nevertheless, the pregnancy rate per oocyte recovery between the two groups appeared similar (20 versus 16%). Among the 30 patients with a LH surge, 9 received hCG after a LH surge was verified, whereas the others did not. Addition of hCG to the LH surge increased the numbers of oocytes recovered, embryos transferred, and the pregnancy rate (3 out of 9 versus 3 out of 21); however, this difference is not statistically significant. Furthermore, the addition of exogenous hCG to a spontaneous LH surge appears to be necessary to maximize the number of oocytes recovered, embryos transferred, and pregnancies obtained.

Spectrophotometric analysis of follicular fluid related to oocyte fertilization, embryo cleavage, and follicular fluid protein and hormone content.

Spectrophotometric absorbance patterns of follicular fluids (FF) obtained from in vitro fertilization patients were analyzed and compared with oocyte fertilization and embryo cleavage rates. Each absorbance pattern was resolved into three components: delta OD360, delta OD415, and delta OD455. A positive correlation was found between fertilization outcome and delta OD455. The absorbances were not related to embryo cleavage rates. We confirmed the presence of bilirubin and beta-carotene in FF: these pigments absorb at 455 nm and are most likely serum-derived. To explain the association between delta OD455 and fertilization, we hypothesized that the FF delta OD455 is a marker of the degree of vascularization of the follicle that could be assessed by FF protein and hormone concentrations. However, no correlation was found between the delta OD455 and these FF parameters, suggesting an alternative explanation for this association.

Spectrophotometric absorbance of follicular fluid: a predictor of oocyte fertilizing capability.

Follicular fluid volume, scoring of the oocyte-cumulus-corona-complex (OCCC), and spectrophotometric absorbance of the follicular fluid were separately compared between a group of fertilized (n = 53) and unfertilized oocytes (n = 35). Scoring of the OCCC and follicular fluid volumes was not found to be statistically different between the two groups. Spectrophotometric analysis of the follicular fluid in the visible spectrum demonstrated two peaks of maximum absorbance at 415 nm and 455 nm. The group of oocytes that fertilized was associated with follicular fluids that had significantly higher absorbances at 415 nm and 455 nm. In conclusion, follicular fluid volume and scoring of the OCCC were poor predictors of fertilizing capability; however, spectrophotometric absorbance, particularly at 455 nm, was positively correlated with oocyte fertilization.

Stimulation of cryopreserved epididymal spermatozoa of the domestic cat using the motility stimulants caffeine, pentoxifylline, and 2'-deoxyadenosine.

We have investigated the effects of caffeine, pentoxifylline, and 2'-deoxyadenosine on the motion characteristics and longevity of domestic cat spermatozoa. Freshly collected or cryopreserved domestic cat epididymal sperm were incubated with 0.01-20 mM caffeine, pentoxifylline, or 2'-deoxyadenosine for 15 minutes at 23 degrees C. The percent motility (MOT), curvilinear velocity (VCL), linearity (LIN), straight line velocity (VSL), and amplitude of lateral head displacement (ALH) were determined for each group using computer-assisted semen analysis. Freshly collected domestic cat sperm exhibited a strong forward progressive movement, and treatment with caffeine, pentoxifylline, or 2'-deoxyadenosine did not consistently alter sperm motion. Following cryopreservation, spermatozoa exhibited decreased (P < 0.05) MOT, VCL, VSL, and ALH. Caffeine and pentoxifylline increased (P < 0.05) the MOT, VSL, VCL, and ALH of cryopreserved sperm at 0.01-20 mM, in a dose-dependent manner. 2'-Deoxyadenosine also increased (P < 0.05) both VSL and VCL at 1.0 mM, and MOT, VSL, VCL, and ALH at 10 mM. All treatments shifted the percentage of nonhyperactive sperm to either a transitional or hyperactivated state. The motility indices of cryopreserved samples were examined during a 6-hour incubation to assess the effects of caffeine, pentoxifylline, and 2'-deoxyadenosine on sperm longevity. Compared to untreated control samples, the longevity of stimulated cryopreserved sperm was not reduced. These results indicate that motility stimulants may prove useful for enhancing the fertility of cryopreserved cat sperm by increasing their motility and producing hyperactivated motion.

Computer-assisted semen analysis (CASA) of epididymal sperm from the domestic cat.

Motion characteristics of epididymal sperm from domestic cats exhibiting a high (> 60%; normozoospermic; n = 21) or low (< 40%; teratozoospermic; n = 6) occurrence of structurally normal spermatozoa were correlated with morphology (MOR) using computer-assisted semen analysis (CASA). Mean values and standard errors for percent motility (MOT), curvilinear velocity (VCL), linearity (LIN), straight line velocity (VSL), and amplitude of lateral head displacement (ALH) were recorded for 3 hours. Average values for percent normal spermatozoa, MOT, VCL, VSL, and ALH were higher (P < 0.01) in samples from normozoospermic cats than from teratozoospermic cats at 0 hours, and there was no difference in motion parameters over the 3-hour incubation period in either group. Strong correlations (P < 0.01) existed between MOR and VCL, VSL, ALH, or MOT, but not LIN, upon regression analysis. We conclude that (1) motion parameters of domestic cat sperm are significantly correlated with morphology and (2) abnormal motion parameters associated with low fertility potential in other species are prevalent in samples from teratozoospermic cats. The correlation between morphology and altered sperm movement found in this study suggests that motion analysis of spermatozoa by CASA may be useful in evaluating fertilization potential in felids.

Exposure of embryonic cells to alcohol: contrasting effects during preimplantation and postimplantation development.

Alcohol is a known teratogen that causes a broad variety of developmental anomalies, including fetal growth retardation, craniofacial anomalies, and neurological disorders. The etiology of this multiple defect syndrome, known as fetal alcohol syndrome, has been studied in animal models that reproduce many of the attributes of the human disease. These studies show that ethanol is most teratogenic during organogenesis and development of the nervous system. The molecular basis of fetal alcohol effects has been further investigated using embryo and cell culture systems. Recent studies show that signal transduction pathways controlling cell proliferation are perturbed during ethanol exposure. Ethanol can induce the release of intracellular calcium stores, which stimulates the cell cycle, and it also up-regulates the expression of myc proteins associated with cell proliferation. Increased proliferation is advantageous during the preimplantation period, but ethanol interference with terminal differentiation events within developing tissues during organogenesis may underlie alcohol teratogenicity.

Protein kinase C stimulates release of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by human decidual cells.

OBJECTIVE: Increased concentrations of amniotic fluid matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 have been observed in the context of premature rupture of membranes (PROM) and microbial invasion of the amniotic cavity. However, the source of the stimuli that contribute to the accumulation of these proteins in amniotic fluid remains to be identified. The present study was conducted to investigate MMP-2, MMP-9 and TIMP-1 secretion by decidual cells in response to activated protein kinase C (PKC). METHODS: Decidual cells were isolated from term placentae, grown to confluence and incubated with control media or 10(-11) to 10(-8) mol/l concentrations of phorbol 12-myristate 13-acetate (PMA). Concentrations of MMP-2, MMP-9 and TIMP-1 in the culture supernatant were determined using sensitive and specific immunoassays. Substrate zymography was conducted to confirm MMP-9 assays. RESULTS: PMA induced a concentration-dependent stimulation of release of MMP-9 (control vs. PMA l0(-9) and 10(-8) mol/l; p < 0.01) and TIMP-1 (control vs. PMA 10(-9) and 10(-8) mol/l; p < 0.001), but not MMP-2. A direct positive correlation was observed between MMP-9 and TIMP-1 release (r = 0.645; p < 0.001). Substrate zymography confirmed increased release of MMP-9 in response to PMA (control vs. PMA 10(-8) and PMA 10(-7) mol/l; p < 0.01). CONCLUSIONS: Activation of PKC within the decidua will result in enhanced MMP-9 release, which upon activation could contribute to degradation of matrices within fetal membranes leading to PROM.