Endometrial integrin expression in women with recurrent implantation failure after in vitro fertilization and its relationship to pregnancy outcome.

OBJECTIVE: To determine expression of integrins α1, α4, and αVß3 in the glandular and luminal epithelium, stroma, and cells in the blood vessel walls of the endometrium from women with recurrent implantation failure (RIF) and to determine if they are of prognostic value in determining pregnancy outcome. DESIGN: Prospective nonrandomized study. SETTING: Department of reproductive medicine. PATIENT(S): Forty-five women with RIF and six healthy fertile women were recruited. RIF was defined as the failure to conceive after the transfer of four good-quality embryos in three or more fresh or frozen cycles. INTERVENTION(S): Endometrial biopsy samples were obtained from women with RIF and control women on days LH+7-LH+9 of the cycle. Expression of integrins α1, α4, and αVß3 was determined by immunohistochemistry. MAIN OUTCOME MEASURE(S): A semiquantitative measurement of expression of each integrin protein in the luminal and glandular epithelium, stroma, and cells in the blood vessel walls was determined by H-score analysis. RESULT(S): Expression of integrins α1 and α4 was greatest in the luminal and glandular epithelial cells and the cells in the blood vessel wall, and significantly higher expression of integrins α1 and α4 was seen in the glandular epithelium compared with the luminal epithelium (H-scores: α1 293 ± 15 and 180 ± 12, α4 287 ± 14 and 191 ± 11, respectively). Expression of αVß3 in the epithelium and blood vessels was also greater than in the stroma but there was no significant difference in expression of αVß3 in glandular and luminal epithelium. No significant difference in H-scores was seen for α1, α4, and αVß3 expression in any of the endometrial compartments in tissue from women with RIF and control women. No significant difference in α1, α4, and αVß3 expression in any compartment was observed between those who achieved a clinical pregnancy after subsequent assisted conception treatment (n = 21) and those who were unsuccessful (n = 24). CONCLUSION(S): RIF, when defined as failure to achieve a clinical pregnancy after the transfer of at least four good-quality embryos in three transfer cycles, is not associated with abnormal endometrial integrin expression. In addition, the expression of integrins α1, α4, and αVß3 appears to have no prognostic value in subsequent IVF treatment.

Reelin secreted by GABAergic neurons regulates glutamate receptor homeostasis.

BACKGROUND: Reelin is a large secreted protein of the extracellular matrix that has been proposed to participate to the etiology of schizophrenia. During development, reelin is crucial for the correct cytoarchitecture of laminated brain structures and is produced by a subset of neurons named Cajal-Retzius. After birth, most of these cells degenerate and reelin expression persists in postnatal and adult brain. The phenotype of neurons that bind secreted reelin and whether the continuous secretion of reelin is required for physiological functions at postnatal stages remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: Combining immunocytochemical and pharmacological approaches, we first report that two distinct patterns of reelin expression are present in cultured hippocampal neurons. We show that in hippocampal cultures, reelin is secreted by GABAergic neurons displaying an intense reelin immunoreactivity (IR). We demonstrate that secreted reelin binds to receptors of the lipoprotein family on neurons with a punctate reelin IR. Secondly, using calcium imaging techniques, we examined the physiological consequences of reelin secretion blockade. Blocking protein secretion rapidly and reversibly changes the subunit composition of N-methyl-D-aspartate glutamate receptors (NMDARs) to a predominance of NR2B-containing NMDARs. Addition of recombinant or endogenously secreted reelin rescues the effects of protein secretion blockade and reverts the fraction of NR2B-containing NMDARs to control levels. Therefore, the continuous secretion of reelin is necessary to control the subunit composition of NMDARs in hippocampal neurons. CONCLUSIONS/SIGNIFICANCE: Our data show that the heterogeneity of reelin immunoreactivity correlates with distinct functional populations: neurons synthesizing and secreting reelin and/or neurons binding reelin. Furthermore, we show that continuous reelin secretion is a strict requirement to maintain the composition of NMDARs. We propose that reelin is a trans-neuronal messenger secreted by GABAergic neurons that regulates NMDARs homeostasis in postnatal hippocampus. Defects in reelin secretion could play a major role in the development of neuropsychiatric disorders, particularly those associated with deregulation of NMDARs such as schizophrenia.

Glutamatergic cerebellar granule neurons synthesize and secrete reelin in vitro.

In the postnatal forebrain, the extracellular matrix protein reelin is expressed and secreted by subsets of GABAergic neurons, whereas in the cerebellum reelin is detected in glutamatergic cells of the granule cell layer. Thus, various regions of the postnatal brain present different patterns of reelin expression, whose significance remains unknown. We combined immunocytochemical and pharmacological approaches to characterize the phenotypic and temporal profiles of reelin expression in dissociated cultures of cerebellar granule neurons. A single type of reelin immunoreactivity, identified by a punctate labelling, was present in the somata of the majority of neurons. This immunoreactivity was observed throughout maturation and was exclusively present in glutamatergic neurons expressing the vesicular glutamate transporter 1. Neurons containing the reelin receptors apolipoprotein E receptor 2 (Apoer2) and very low-density lipoprotein receptor (Vldlr) represented about 80% of cerebellar neurons. The vast majority of reelin-positive neurons coexpressed Apoer2, suggesting that reelin immunoreactivity resulted in part from receptor-bound reelin. Inhibition of protein synthesis with cycloheximide completely abolished reelin immunoreactivity. In contrast, blocking protein secretion with brefeldin A did not affect the proportion of punctate neurons but revealed a subpopulation of neurons characterized by a solid reelin staining. These data show for the first time that a homogeneous population of glutamatergic neurons can synthesize and secrete reelin in cerebellar granule cells in vitro.

Reelin, very-low-density lipoprotein receptor, and apolipoprotein E receptor 2 control somatic NMDA receptor composition during hippocampal maturation in vitro.

Reelin is a secreted protein that regulates brain layer formation during embryonic development. Reelin binds several receptors, including two members of the low-density lipoprotein (LDL) receptor family, the apolipoprotein E receptor 2 (ApoER2) and the very-low-density lipoprotein receptor (VLDLR). Despite the high level of expression of Reelin and ApoER2 in the postnatal brain, their functions in the adult CNS remain elusive. Here, using electrophysiological, immunocytochemical, and biochemical approaches in cultured postnatal hippocampal neurons, we show that Reelin controls the change in subunit composition of somatic NMDA glutamate receptors (NMDARs) during maturation. We found that maturation is characterized by the gradual decrease of the participation of NR1/2B receptors to whole-cell NMDAR-mediated currents. This maturational change was mirrored by a timely correlated increase of both Reelin immunoreactivity in neuronal somata and the amount of secreted Reelin. Chronic blockade of the function of Reelin with antisense oligonucleotides or the function-blocking antibody CR-50 prevented the decrease of NR1/2B-mediated whole-cell currents. Conversely, exogenously added recombinant Reelin accelerated the maturational changes in NMDA-evoked currents. The maturation-induced change in NMDAR subunits also was blocked by chronic treatment with an inhibitor of the Src kinase signaling pathway or an antagonist of the LDL receptors, but not by inhibitors of another class of Reelin receptor belonging to the integrin family. Consistent with these results, immunocytochemistry revealed that NR1-expressing neurons also expressed ApoER2 and VLDLR. These data reveal a new role for Reelin and LDL receptors and reinforce the idea of a prominent role of extracellular matrix proteins in postnatal maturation.