Fully grown germinal vesicle stage mouse oocytes remain arrested in meiotic prophase I until ovulation. This arrest is maintained by cGMP produced in cumulus granulosa cells surrounding the oocyte. Recently, it was found that cGMP production in cumulus cells depends on NPR2 guanylate cyclase activated by its ligand natriuretic peptide precursor C (NPPC). It is assumed that cGMP reaches the oocyte through gap junctions that couple cumulus granulosa cells to each other and to the oocyte. Previous work identified two main types of gap junctions in the follicle, connexin-43 gap junctions (GJA1 protein) between granulosa cells and connexin-37 gap junctions (GJA4) between cumulus cells and the oocyte. However, it had not been established that both types are required for meiotic arrest mediated by NPPC/NPR2 signaling. To investigate this, we used connexin mimetic peptides (CMPs) that specifically disrupt gap junction isoforms within cumulus-oocyte complexes (COCs) and isolated antral follicles in culture. We furthermore developed a punctured antral follicle preparation to permit CMP access to the antral cavity in an otherwise intact follicle. CMP directed against connexin-43 (Cx43 CMP) overcame NPPC-mediated meiotic arrest in both isolated COCs and antral follicles. Cx37 CMP, in contrast, had no effect when present in the medium, but released oocyte arrest in the presence of NPPC when microinjected into the perivitelline space near the oocyte surface in COCs. This is consistent with both connexin isoforms being required for meiotic arrest and with the reported localization of connexin-43 throughout the cumulus cells and connexin-37 at the oocyte surface.

Mouse embryos employ a unique mechanism of cell volume regulation in which glycine is imported via the GLYT1 transporter to regulate intracellular osmotic pressure. Independent cell volume regulation normally becomes active in the oocyte after ovulation is triggered. This involves two steps: the first is the release of the strong adhesion between the oocyte and zona pellucida (ZP) while the second is the activation of GLYT1. In fully-grown oocytes, release of adhesion and GLYT1 activation also occur spontaneously in oocytes removed from the follicle. It is unknown, however, whether the capacity to release oocyte-ZP adhesion or activate GLYT1 first arises in the oocyte after ovulation is triggered or instead growing oocytes already possess these capabilities but they are suppressed in the follicle. Here, we assessed when during oogenesis oocyte-ZP adhesion can be released and when GLYT1 can be activated, with adhesion assessed by an osmotic assay and GLYT1 activity determined by [ H]-glycine uptake. Oocyte-ZP adhesion could not be released by growing oocytes until they were nearly fully grown. Similarly, the amount of GLYT1 activity that can be elicited in oocytes increased sharply at the end of oogenesis. The SLC6A9 protein that is responsible for GLYT1 activity and Slc6a9 transcripts are present in growing oocytes and increased over the course of oogenesis. Furthermore, SLC6A9 becomes localized to the oocyte plasma membrane as the oocyte grows. Thus, oocytes acquire the ability to regulate their cell volume by releasing adhesion to the ZP and activating GLYT1 as they approach the end of oogenesis. J. Cell. Physiol. 232: 2436-2446, 2017. © 2016 Wiley Periodicals, Inc.

Antibody-drug conjugates (ADCs) are likely to make a significant contribution in the treatment of acute lymphoblastic leukemia (ALL) by combining the cytotoxicity of chemotherapy with the specificity of monoclonal antibodies. CD22, an endocytic receptor expressed by the majority of B cells, is an excellent target for ADCs. Inotuzumab ozogamicin (INO) is an ADC that consists of a cytotoxic moiety (derivative of calicheamicin) attached to a humanized monoclonal anti-CD22 antibody. As a single agent, INO, was shown to be effective with an objective response rate of 50% in the treatment of relapsed and refractory CD22 positive ALL patients. Clinical trials investigating the combination of INO with the conventional chemotherapies are ongoing. This review summarizes the clinical potential of INO in treatment of relapsed and refractory ALL, based on currently available data in the literature.

Radiolabeled compounds that are substrates for transmembrane transporters can be used to study transport and metabolism in mammalian oocytes and preimplantation embryos. Because even very small amounts of radioisotopes can be detected, these techniques are feasible to use with only a few oocytes or embryos, even down to the level of single oocytes or embryos. Here, we describe the methods for determining the transport and accumulation of radiolabeled compounds into oocytes and preimplantation embryos and the determination of the rate of saturable transport via specific transporters in the plasma membrane.