Molecular Human Reproduction - current issue

Expression of epithelial cadherin in the human male reproductive tract and gametes and evidence of its participation in fertilization

2008-11-05

Epithelial cadherin (E-cadherin) has been involved in several calcium-dependent cell–cell adhesion events; however, its participation in gamete interaction has not been fully investigated. Our results have demonstrated expression of E-cadherin mRNA in the human male reproductive tract showing higher levels in the caput, corpus and cauda epididymis than in the testis. The mature 122 kDa E-cadherin was detected in epididymal protein extracts and was localized in the epithelial cells from the three epididymal regions. Moreover, the 86 kDa E-cadherin ectodomain was found in cauda epididymal and seminal plasma. Western immunoblotting of human sperm protein extracts allowed the identification of four E-cadherin forms (122, 105, 97 and 86 kDa). The protein was localized in the acrosomal region of intact spermatozoa, remained associated with the head of acrosome-reacted cells and was also detected on the oocyte surface. A similar localization was determined for other proteins of the adhesion complex (β-catenin and actin). Spermatozoa incubated with anti-E-cadherin antibodies showed impaired binding to homologous zona pellucida (ZP); in addition, presence of these antibodies inhibited the penetration of human spermatozoa to ZP-free hamster oocytes. The results presented here describe the expression of E-cadherin in the male reproductive tract and gametes and strongly suggest its involvement in adhesion events during human fertilization. The identification of proteins involved in gamete interaction will contribute to the understanding of the molecular basis of fertilization and help in the diagnosis and treatment of infertility.

Validation of preimplantation genetic diagnosis by PCR analysis: genotype comparison of the blastomere and corresponding embryo, implications for clinical practice

2008-11-05

The aim of this study was to validate the overall preimplantation genetic diagnosis (PGD)–PCR procedure and to determine the diagnostic value. Genotyped embryos not selected for embryo transfer (ET) and unsuitable for cryopreservation after PGD were used for confirmatory analysis. The PGD genotyped blastomeres and corresponding embryos were compared, and morphology was scored on Day 4 post fertilization. To establish the validity of the PGD–PCR procedure and the diagnostic value, misdiagnosis rate, false-negative rate and negative predictive value were calculated. Moreover, comparison on the validity was made for the biopsy of one or two blastomeres. For the total embryo group (n = 422), a misdiagnosis rate of 7.1% and a false-negative rate of 3.1% were found. The negative predictive value was 96.1%. Poor morphology Day 4 embryos (Class 1) were over-represented in the embryo group in which the blastomere genotype was not confirmed by the whole embryo genotype. The misdiagnosis rate of Class 1 embryos was 12.5% and the false-negative rate 17.1%. Exclusion of these embryos resulted in a misdiagnosis rate of 6.1%, a false-negative rate of 0.5% and a negative predictive value of 99.3%. The two blastomere biopsies revealed a significant higher positive predictive value, lowering the misdiagnosis rate, whereas the negative predictive value remained the same. In conclusion, the PGD–PCR procedure is a valid diagnostic method to select unaffected embryos for ET. The misdiagnosis and false-negative rates decrease by rejecting Class 1 embryos for ET. The biopsy of a second blastomere improves the positive predictive value, lowering the misdiagnosis rate.

Identification and characterization of human embryonic poly(A) binding protein (EPAB)

2008-11-05

Transcriptional silencing that begins with oocyte maturation persists during the initial mitotic divisions of the embryo. Gene expression during this period largely depends on the translational activation of maternal mRNAs by cytoplasmic polyadenylation and requires an embryonic poly(A) binding protein (EPAB). EPAB has been identified in Xenopus and mouse, where it is expressed exclusively in oocytes and early embryos until zygotic genome activation (ZGA) when it is replaced by the somatic cytoplasmic poly(A) binding protein (PABPC1). EPAB plays a central role in the regulation of maternal mRNA activation by preventing deadenylation and promoting translation. In this study, we identified and characterized the human EPAB ortholog. Human EPAB is a 619 amino acid protein with 77% identity and 84% similarity to mouse EPAB. Human EPAB mRNA is detected in ovaries, testes and several somatic tissues including pancreas, liver and thymus. Similar to the observations in Xenopus and mouse, human EPAB is the predominant poly(A) binding protein in immature (germinal vesicle) and mature (metaphase II) oocytes, and it is replaced by PABPC1 following ZGA, which occurs at 4- to 8-cell stage in human. Our findings suggest that the unique translational regulatory pathways that control gene expression during oogenesis and early embryo development may be common between model organisms and humans.

The role of T-lymphoma invasion and metastasis inducing protein 1 in early pregnancy in mice

Ma, H.-L., Zhang, T., Meng, J., Qin, Z.-Y., Du, F., Wang, Q.-Y., Wei, S.-L. — 2008-11-05

T-lymphoma invasion and metastasis inducing protein 1 (Tiam1) is involved in tumorigenesis processes, including cell migration, adhesion and invasion, proteolysis, cytoskeleton reorganization, cell morphological transformation and intracellular signaling. These processes are also critical for embryo implantation, although the role of Tiam1 during embryo implantation remains poorly understood. The aim of this study was to investigate the spatio-temporal expression of Tiam1 in endometria of mice comparing early pregnancy and non-pregnancy. Fluorescent quantitative-PCR and immunohistochemical analysis showed that the expression of Tiam1 mRNA and protein in endometria of pregnant mice was higher than that of non-pregnant mice, and gradually increased from Day 1 of pregnancy reaching a maximum level on Day 5 and then declining on Days 6 and 7. Immunohistochemistry showed that Tiam1 protein was present in luminal epithelium from Days 3–5 of pregnancy and in gland epithelium from Days 4 to 6 and enhanced significantly in stromal cells on Day 5, regarded as the ‘implantation window’ period. The number of implantation sites was greatly decreased by the intrauterine injection with anti-Tiam1 polyclonal antibody in the early morning of the Day 4 of pregnancy. These findings suggest that Tiam1 might play an important role in embryo implantation in mice.

Increased levels of pregnancy-associated plasma protein-A2 in the serum of pre-eclamptic patients

2008-11-05

Pregnancy-associated plasma protein-A and -A2 (PAPP-A and -A2) are proteases that cleave insulin-like growth factor-binding proteins (IGFBPs), resulting in local activation of IGF signaling pathways. Here, we examined PAPP-A and -A2 mRNA and protein levels in placenta and maternal sera from women with pre-eclampsia and compared them with samples from uncomplicated pregnancy. PAPP-A2 but not PAPP-A mRNA and protein were elevated in pre-eclamptic placenta (P < 0.01). PAPP-A2 is normally produced in placental syncytiotrophoblast cells and maternal decidua. PAPP-A2 in syncytiotrophoblast cells was dramatically increased in pre-eclampsia. Maternal serum concentrations of PAPP-A2 but not PAPP-A were also significantly elevated in pre-eclampsia as compared with uncomplicated pregnancy. mRNA levels of IGFBP5, a specific substrate for PAPP-A2 protease activity, were also significantly increased, suggesting a potential role for IGFBP5 in fetal and placental growth suppression during pre-eclampsia. However, IGFBP5 protein levels were not increased in placenta from pre-eclampsia, possibly due to cleavage by up-regulated PAPP-A2. These data might imply that PAPP-A2 may be up-regulated in pre-eclamptic pregnancy to compensate for IGFBP5-mediated suppression of the IGF pathway, although final birthweights are still low in pre-eclamptic pregnancy.

Relaxin signalling in primary cultures of human myometrial cells

Heng, K., Ivell, R., Wagaarachchi, P., Anand-Ivell, R. — 2008-11-05

In myometrium of pigs and rats, though not humans, relaxin appears to mediate an inhibition of spontaneous and oxytocin-induced contractility, presumably acting through a G-protein coupled receptor (RXFP1) to generate cAMP. In humans, circulating relaxin is highest in the first trimester, including the time of implantation, when transitory uterine quiescence could help a blastocyst to implant. We investigated whether relaxin can activate adenylate cyclase in primary human myometrial cells from non-pregnant tissue, and we show that relaxin is able to stimulate the generation of cAMP in a manner, which is dependent upon a tyrosine phosphorylation activity, as in the endometrium. We identified transcripts for the relaxin receptor RXFP1 as full-length variants, though a minor splice variant missing exon 2 was also present in low amounts. These cells also express transcripts encoding RXFP2, the receptor for the closely related hormone, INSL3. Although able to respond to relaxin at high concentrations, this receptor does not appear to function by contributing to the cAMP production in human myometrial cells, nor does INSL3 act as a functional agonist or antagonist of relaxin action. In conclusion, the inability of relaxin to inhibit contractility in human myometrial cells would appear to be due to events downstream of simple cAMP generation.

Calpain5 expression is decreased in endometriosis and regulated by HOXA10 in human endometrial cells

Penna, I., Du, H., Ferriani, R., Taylor, H. S. — 2008-11-05

Calpains have been implicated in the regulation of apoptosis. Here, we identified Calpain5 as a target of HOXA10 transcriptional regulation in endometrial cells as well as its aberrant regulation in endometriosis. Histologically confirmed biopsies of endometriosis were obtained from 20 women. Eutopic endometrium was collected by endometrial biopsy from 30 controls and from the 20 subjects with endometriosis. First trimester decidual samples were obtained from five subjects at the time of pregnancy termination. Immunohistochemistry was used to identify Calpain5 expression. Calpain5 was expressed in endometrial stromal and glandular cells throughout the menstrual cycle and in decidua. Calpain5 protein expression was decreased in both stromal and glandular cells from women with endometriosis compared with that of fertile controls. Human endometrial stromal and epithelial cell lines were transfected with pcDNA/HOXA10, HOXA10 siRNA or respective controls. Quantitative real-time RT–PCR was performed to determine expression of HOXA10 and Calpain5 in each group. Transfection of HESC cells with an HOXA10 expression construct led to increased Calpain5 expression, whereas transfection with siRNA resulted in decreased expression. In conclusion, Calpain5 expression is regulated by HOXA10. Calpain5 expression was decreased in endometriosis likely as a result of decreased HOXA10 expression. Decreased apoptosis in endometrial cells may promote the development of endometriosis through a pathway involving HOXA10, Calpain5 and caspase.