Oocyte-specific disruption of adrenomedullin 2 gene enhances ovarian follicle growth after superovulation.

Background: Adrenomedullin 2 (ADM2), adrenomedullin (ADM), and calcitonin gene-related peptides (α- and ß-CGRPs) signal through heterodimeric calcitonin receptor-like receptor/receptor activity-modifying protein 1, 2 and 3 (CLR/RAMP1, 2 and 3) complexes. These peptides are important regulators of neurotransmission, vasotone, cardiovascular development, and metabolic homeostasis. In rodents, ADM is essential for regulating embryo implantation, fetal-placental development, and hemodynamic adaptation during pregnancy. On the other hand, ADM2 was shown to affect vascular lumen enlargement, and cumulus cell-oocyte complex (COC) communication in rodent and bovine ovarian follicles. To investigate whether oocyte-derived ADM2 plays a physiological role in regulating ovarian folliculogenesis, we generated mice with oocyte-specific disruption of the Adm2 gene using a LoxP-flanked Adm2 transgene (Adm2 loxP/loxP) and crossed them with Zp3-Cre mice which carry a zona pellucida 3 (Zp3) promoter-Cre recombinase transgene. Results: While heterozygous Adm2 +/-/Zp3-Cre and homozygous Adm2 -/-/Zp3-Cre mice were fertile, Adm2 disruption in oocytes significantly increased the number of ovulated oocytes following a superovulation treatment. Oocyte-specific Adm2 disruption also significantly impaired the developmental capacity of fertilized eggs and decreased the size of the corpus luteum following superovulation, perhaps due to a reduction of ovarian cyclin D2-associated signaling. Conclusions: The disruption of intrafollicular ADM2 signaling leads to follicular dysfunction. These data suggested that oocyte-derived ADM2 plays a facilitative role in the regulation of hormonal response and follicle growth independent of the closely related ADM and CGRP peptides, albeit in a subtle manner.

Effect of Multiple Vaccinations with Tumor Cell-Based Vaccine with Codon-Modified GM-CSF on Tumor Growth in a Mouse Model.

Ectopic expression of codon-modified granulocyte-macrophage colony-stimulating factor (cGM-CSF) in TC-1 cells (TC-1/cGM-CSF), a model cell line for human papillomavirus (HPV)-infected cervical cancer cells, increased the expression level of GM-CSF and improved the efficacy of tumor cell-based vaccines in a cervical cancer mouse model. The number of vaccine doses required to induce a long-term immune response in a cervical cancer mouse model is poorly understood. Here, we investigated one, three, and five doses of the irradiated TC-1/cGM-CSF vaccine to determine which dose was effective in inducing a greater immune response and the suppression of tumors. Our findings showed that three doses of irradiated TC-1/cGM-CSF vaccine elicited slower tumor growth rates and enhanced survival rates compared with one dose or five doses of irradiated TC-1/cGM-CSF vaccine. Consistently, mice vaccinated with three doses of irradiated TC-1/cGM-CSF vaccine exhibited stronger interferon gamma (IFN-γ) production in HPV E7-specific CD8⁺ T cells and CD4⁺ T cells. A higher percentage of natural killer cells and interferon-producing killer dendritic cells (IKDCs) appeared in the splenocytes of the mice vaccinated with three doses of irradiated TC-1/cGM-CSF vaccine compared with those of the mice vaccinated with one dose or five doses of irradiated TC-1/cGM-CSF vaccine. Our findings demonstrate that single or multiple vaccinations, such as five doses, with irradiated TC-1/cGM-CSF vaccine suppressed the immune response, whereas three doses of irradiated TC-1/cGM-CSF vaccine elicited a greater immune response and subsequent tumor suppression.

Detection of SEA-type α-thalassemia in embryo biopsies by digital PCR.

OBJECTIVE: Accurate and efficient pre-implantation genetic diagnosis (PGD) based on the analysis of single or oligo-cells is needed for timely identification of embryos that are affected by deleterious genetic traits in in vitro fertilization (IVF) clinics. Polymerase chain reaction (PCR) is the backbone of modern genetic diagnoses, and a spectrum of PCR-based techniques have been used to detect various thalassemia mutations in prenatal diagnosis (PND) and PGD. Among thalassemias, SEA-type α-thalassemia is the most common variety found in Asia, and can lead to Bart's hydrops fetalis and serious maternal complications. MATERIALS AND METHODS: To formulate an efficient digital PCR for clinical diagnosis of SEA-type α-thalassemia in cultured embryos, we conducted a pilot study to detect the α-globin and SEA-type deletion alleles in blastomere biopsies with a highly sensitive microfluidics-based digital PCR method. Genomic DNA from embryo biopsy samples were extracted, and crude DNA extracts were first amplified by a conventional PCR procedure followed by a nested PCR reaction with primers and probes that are designed for digital PCR amplification. RESULTS: Analysis of microfluidics-based PCR reactions showed that robust signals for normal α-globin and SEA-type deletion alleles, together with an internal control gene, can be routinely generated using crude embryo biopsies after a 106-fold dilution of primary PCR products. CONCLUSION: The SEA-type deletion in cultured embryos can be sensitively diagnosed with the digital PCR procedure in clinics. The adoption of this robust PGD method could prevent the implantation of IVF embryos that are destined to develop Bart's hydrops fetalis in a timely manner. The results also help inform future development of a standard digital PCR procedure for cost-effective PGD of α-thalassemia in a standard IVF clinic.

High-resolution melting molecular signatures for rapid identification of human papillomavirus genotypes.

BACKGROUND: Genotyping of human papillomarvirus (HPV) is crucial for patient management in a clinical setting. This study accesses the combined use of broad-range real-time PCR and high-resolution melting (HRM) analysis for rapid identification of HPV genotypes. METHODS: Genomic DNA sequences of 8 high-risk genotypes (HPV16/18/39/45/52/56/58/68) were subject to bioinformatic analysis to select for appropriate PCR amplicon. Asymmetric broad-range real-time PCR in the presence of HRM dye and two unlabeled probes specific to HPV16 and 18 was employed to generate HRM molecular signatures for HPV genotyping. The method was validated via assessment of 119 clinical HPV isolates. RESULTS: A DNA fragment within the L1 region was selected as the PCR amplicon ranging from 215-221 bp for different HPV genotypes. Each genotype displayed a distinct HRM molecular signature with minimal inter-assay variability. According to the HRM molecular signatures, HPV genotypes can be determined with one PCR within 3 h from the time of viral DNA isolation. In the validation assay, a 91% accuracy rate was achieved when the genotypes were in the database. Concomitantly, the HRM molecular signatures for additional 6 low-risk genotypes were established. CONCLUSIONS: This assay provides a novel approach for HPV genotyping in a rapid and cost-effective manner.