Connexin Hemichannels with Prostaglandin Release in Anabolic Function of Bone to Mechanical Loading

Mechanical stimulation, such as physical exercise, is essential for bone formation and health. Here, we demonstrate the critical role of osteocytic Cx43 hemichannels in anabolic function of bone in response to mechanical loading. Two transgenic mouse models, R76W and Δ130-136, expressing dominant-negative Cx43 mutants in osteocytes were adopted. Mechanical loading of tibial bone increased cortical bone mass and mechanical properties in wild-type and gap junction-impaired R76W mice through increased PGE2, endosteal osteoblast activity, and decreased sclerostin. These anabolic responses were impeded in gap junction/hemichannel-impaired Δ130-136 mice and accompanied by increased endosteal osteoclast activity. Specific inhibition of Cx43 hemichannels by Cx43(M1) antibody suppressed PGE2 secretion and impeded loading-induced endosteal osteoblast activity, bone formation and anabolic gene expression. PGE2 administration rescued the osteogenic response to mechanical loading impeded by impaired hemichannels. Together, osteocytic Cx43 hemichannels could be a potential new therapeutic target for treating bone loss and osteoporosis.

Asynchronous Embryo Transfer Followed by Comparative Transcriptomic Analysis of Conceptus Membranes and Endometrium Identifies Processes Important to the Establishment of Equine Pregnancy

Preimplantation horse conceptuses require nutrients and signals from histotroph, the composition of which is regulated by luteal progesterone and conceptus-secreted factors. To distinguish progesterone and conceptus effects we shortened the period of endometrial progesterone-priming by asynchronous embryo transfer. Day 8 embryos were transferred to synchronous (day 8) or asynchronous (day 3) recipients, and RNA sequencing was performed on endometrium and conceptuses recovered 6 and 11 days later (embryo days 14 and 19). Asynchrony resulted in many more differentially expressed genes (DEGs) in conceptus membranes (3473) than endometrium (715). Gene ontology analysis identified upregulation in biological processes related to organogenesis and preventing apoptosis in synchronous conceptuses on day 14, and in cell adhesion and migration on day 19. Asynchrony also resulted in large numbers of DEGs related to ‘extracellular exosome’. In endometrium, genes involved in immunity, the inflammatory response, and apoptosis regulation were upregulated during synchronous pregnancy and, again, many genes related to extracellular exosome were differentially expressed. Interestingly, only 14 genes were differentially expressed in endometrium recovered 6 days after synchronous versus 11 days after asynchronous transfer (day 14 recipient in both). Among these, KNG1 and IGFBP3 were consistently upregulated in synchronous endometrium. Furthermore bradykinin, an active peptide cleaved from KNG1, stimulated prostaglandin release by cultured trophectoderm cells. The horse conceptus thus responds to a negatively asynchronous uterus by extensively adjusting its transcriptome, whereas the endometrial transcriptome is modified only subtly by a more advanced conceptus.

Asynchronous Embryo Transfer Followed by Comparative Transcriptomic Analysis of Conceptus Membranes and Endometrium Identifies Processes Important to the Establishment of Equine Pregnancy

Pre-implantation horse conceptuses require nutrients and signals from histotroph, the composition of which is regulated by luteal progesterone and conceptus-secreted factors. To distinguish progesterone and conceptus effects we shortened the period of endometrial progesterone-priming by asynchronous embryo transfer. Day 8 embryos were transferred to synchronous (day 8) or asynchronous (day 3) recipients, and RNA sequencing was performed on endometrium and conceptuses recovered 6 and 11 days later (embryo days 14 and 19). Asynchrony resulted in many more differentially expressed genes (DEGs) in conceptus membranes (3473) than endometrium (715). Gene ontology analysis identified upregulation in biological processes related to organogenesis and preventing apoptosis in synchronous conceptuses on day 14, and in cell adhesion and migration on day 19. Asynchrony also resulted in large numbers of DEGs related to ‘extracellular exosome’. In endometrium, genes involved in immunity, the inflammatory response, and apoptosis regulation were upregulated during synchronous pregnancy and, again, many genes related to extracellular exosome were differentially expressed. Interestingly, only 14 genes were differentially expressed in endometrium recovered 6 days after synchronous versus 11 days after asynchronous transfer (day 14 recipient in both). Among these, KNG1 and IGFBP3 were consistently up-regulated in synchronous endometrium. Furthermore bradykinin, an active peptide cleaved from KNG1, stimulated prostaglandin release by cultured trophectoderm cells. The horse conceptus thus responds to a negatively asynchronous uterus by extensively adjusting its transcriptome, whereas the endometrial transcriptome is modified only subtly by a more advanced conceptus.

PSII-33 Differences in prostaglandin release in cows with high vs low circulating concentrations of pregnancy associated glycoproteins

Embryonic mortality (EM) is a major factor limiting pregnancy rates in cattle and occurs early (< day 28) or late (≥ day 28) during gestation. However, causes and mechanisms associated with late EM remain unknown. Despite negative connotations related to pregnancy, prostaglandin F2α (PGF) is capable of being released by the bovine uterus between days 28 and 32 of gestation. Therefore, the objective was to evaluate differences in PGF release between cows with high circulating concentrations of pregnancy associated glycoproteins (PAG) vs low PAG following an oxytocin (OT) challenge. Increased concentrations of PAG are associated with pregnancy success. At day 29 of gestation, pregnant cows were divided into high (n = 10) and low (n = 10) PAG groups and received either 100 I. U. of oxytocin intramuscularly (OT) or saline (control). Blood samples were collected every 30 minutes beginning 1 hour before and continuing for 4 hours after treatment. Prostaglandin F2α metabolite (PGFM), progesterone (P4), estradiol (E2) and PAG concentrations were determined. Peak concentration of PGFM occurred 2 hours after OT injection (average high PAG group peak: 345.6 ± 73.6 pg/mL; low PAG group peak: 326.4 ± 61.4 pg/mL, P > 0.05) and returned to baseline levels by 4 hours. No correlations were observed between PAG and PGFM concentrations (P > 0.05). There was no difference in initial or final PGFM concentrations between high or low PAG groups and control animals (P > 0.05). Furthermore, there was no difference in P4 or E2 concentration between treatment and control animals (P > 0.05) despite a luteolytic release of PGF2α. In summary, cows with high vs low PAG concentrations at day 30 of gestation have a similar PGFM response to OT challenge. This project supported by Agriculture and Food Research Initiative Competitive Grant no. 2017-67015-26457 from USDA-NIFA.