Mouse Oocytes Acquire Mechanisms That Permit Independent Cell Volume Regulation at the End of Oogenesis

Abstract

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.

Initiation of cell volume regulation and unique cell volume regulatory mechanisms in mammalian oocytes and embryos

“…Transport of glycine by GLYT1 is also found in early human preimplantation embryos (Hammer et al, 2000). SLC6A9 is expressed in oocytes and cleavage stage embryos at the transcript and protein levels but is no longer present in morulae or blastocysts (Richard et al, 2017; Van Winkle & Campione, 1996).…”

“…The oocyte‐ZP attachment is quite strong. This has been most vividly demonstrated using hypertonic shock, which revealed that the rigid ZP actually will deform substantially and buckle into a flattened concave structure rather than losing its attachment to the oocyte (Richard et al, 2017; Tartia et al, 2009). Thus, before meiotic maturation begins, the oocyte is unable to control its cell volume which is instead determined by the size of its ZP, which is a spherical, rigid shell that has a high tensile strength and thus cannot be deformed by forces normally produced by cells (Green, 1987).…”

“…Simultaneously, the oocyte detaches from the ZP (Figure 3a). This proceeds gradually through a transitional state of partial attachment over the course of the first few hours of meiotic maturation and then to full detachment (Richard et al, 2017; Tartia et al, 2009). Detachment is actively inhibited within the preovulatory ovarian follicle (Richard & Baltz, 2017), but the inhibitory signal and the mechanism of detachment remain unknown.…”

“…The mechanism of GLYT1 activation remains unknown. It does not involve synthesis of GLYT1 protein, since SLC6A9 is already present in fully grown GV oocytes, having been synthesized during oocyte growth and trafficked to the plasma membrane as the oocyte nears full size (Richard et al, 2017). Instead, it is likely that GLYT1 is actively suppressed within the antral follicle in the ovary, since, if an oocyte is removed from its follicle, GLYT1 activity initiates spontaneously (Richard & Baltz, 2017; Tartia et al, 2009).…”

Biomechanics and mechanical signaling in the ovary: a systematic review

“…These changes in tensile strength and intrafollicular pressure are generated by the action of smooth muscle cells and by osmotic fluid shifts. Regulation of the intracellular osmotic pressure is accomplished by a glycine transporter (GLYT1) encoded by the solute carrier family 6, member 9 (Slc6a9) gene in mice [79]. The oocytes are able to regulate their cell volume by releasing adhesions between the zona pellucida and the oocyte by a sharp increase in GLYT1 activity [79].…”