The cell volume‐regulatory glycine transporter GLYT1 is activated following metallopeptidase‐mediated detachment of the oocyte from the zona pellucida

Independent cell volume regulation is first acquired by the oocyte in two steps that occur during meiotic maturation: (1) activation of the glycine transporter GLYT1 (Slc6a9) that mediates the intracellular accumulation of glycine to provide osmotic support in the mature egg and early preimplantation embryo, and (2) release of the oocyte from the strong attachment to its rigid extracellular matrix shell, the zona pellucida (ZP). It was recently shown that oocyte‐ZP detachment requires metallopeptidase activity that is proposed to cleave transmembrane ZP proteins connecting the oocyte to the ZP. It is unknown, however, how GLYT1 is activated. We hypothesized that oocyte‐ZP detachment precedes and may be required for GLYT1 activation. In identically treated pools of oocytes, oocyte‐ZP detachment occurred ~20 min before GLYT1 activation. In individual oocytes, GLYT1 activity was detected only in those that were mostly or fully detached. Blocking detachment using previously validated small molecule metallopeptidase inhibitors partly suppressed GLYT1 activation. However, removal of the ZP did not accelerate GLYT1 activation. This indicates that oocyte‐ZP detachment or cleavage of transmembrane ZP proteins may be required for GLYT1 to become fully activated, or alternatively that metallopeptidase activity independently affects both detachment and GLYT1 activation.

metabolic disruption that similar levels of inorganic ions would cause (Tscherner et al., 2021;Yancey, Clark, Hand, Bowlus, & Somero, 1982).Independent regulation of cell volume arises during meiotic maturation of oocytes.Acquisition of control over cell volume occurs through at least two steps.One is the activation of GLYT1.GLYT1 is quiescent in germinal vesicle (GV) stage oocytes but becomes activated over the course of several hours following the triggering of ovulation by luteinizing hormone (LH) (Tartia et al., 2009).The other step is the release of the strong attachment between the GV oocyte and its rigid glycoprotein shell, the zona pellucida (ZP) (Richard et al., 2017;Tartia et al., 2009).Before ovulation is triggered, oocytes cannot freely control their volumes since their size is mechanically constrained by the ZP.Several hours after ovulation has been triggered, however, they have detached from the ZP and have begun actively accumulating glycine to control their volumes (Richard et al., 2017;Tartia et al., 2009;Tscherner et al., 2021).Both GLYT1 activation and oocyte-ZP detachment occur almost identically when oocytes are removed from the ovarian follicle, which allows these processes to be investigated in vitro and indicates that both processes are actively inhibited in the follicle before ovulation is triggered (Richard & Baltz, 2017;Richard et al., 2017;Tartia et al., 2009).These mechanisms and cell volume regulation in oocytes and preimplantation embryos in general have been recently reviewed (Tscherner et al., 2021).
Recently, it was shown that oocyte-ZP detachment is mediated by a metallopeptidase that, when it becomes activated, is thought to cleave transmembrane ZP glycoproteins that form an attachment between the oocyte membrane and the inner ZP surface (Macaulay et al., 2023).The identity of the metallopeptidase that mediates detachment is not known, although it is likely to be a member of the M12 family of peptidases which includes the ADAM metallopeptidases (Macaulay et al., 2023).Coincident with detachment, the oocyte decreases substantially in volume (Tartia et al., 2009).It then stabilizes at a lower volume that is maintained, at least in part, by adjusting glycine concentration via GLYT1.GLYT1 is maximally active as a volume regulatory mechanism from the first metaphase (MI) oocyte through the 2-cell stage, then becomes progressively less active through the 4-to 8-cell stages after which it is inactive and no longer expressed (Richard et al., 2017;Tartia et al., 2009).
How metallopeptidase-mediated oocyte-ZP detachment and activation of GLYT1 are initiated is not fully known.Oocytes first become capable of detaching from the ZP and activating GLYT1 as they near full size during oogenesis, well before ovulation is triggered (Richard et al., 2017).However, both processes are actively suppressed in the intact antral follicle or in isolated cumulus-oocyte complexes in the presence of mural granulosa cells (Richard & Baltz, 2017).Thus, the existence of an unknown negative regulator in the preovulatory antral follicle is inferred.This suppression is released upon LH signaling in the follicle or if the oocyte is removed from the follicle (Richard & Baltz, 2017).We hypothesized that oocyte-ZP detachment could be required for subsequent GLYT1 activation, since changes in interaction with an extracellular matrix is an established mechanism of modulating downstream signal transduction in many cells (Hynes, 2009).Consistent with this hypothesis, we show here that oocyte-ZP detachment precedes GLYT1 activation, that there is a threshold level of detachment above which GLYT1 is active, and that inhibiting metallopeptidase activity partly suppresses GLYT1 activation.
Stocks were stored at −20°C.Controls contained the same concentration of DMSO as was added with each inhibitor.
mKSOM was used at 37°C in 5% CO 2 in air at 100% humidity.
Where specified, the inert trisaccharide D(+)-raffinose was added to increase osmolarity of the medium to 310 mOsM as previously described (Dawson & Baltz, 1997).repeated pipetting through a narrow bore flame-pulled Pasteur pipette in Hepes-mKSOM as previously described (Macaulay et al., 2023;Tartia et al., 2009).Oocytes were cultured in mKSOM and either allowed to spontaneously resume meiotic progression or were maintained in meiotic arrest at the GV stage with milrinone (10 µM), a selective phosphodiesterase 3 (PDE3) inhibitor.Oocytes were collected from several females and pooled.Where specified, the ZP was removed by exposure to acid Tyrode solution (pH 2.5) for ~30 s after which the oocytes were washed 3× in Hepes-mKSOM followed by 5× in mKSOM.

| Oocyte collection and culture
Standard culture was in 50 µL drops of mKSOM under filtersterilized embryo culture grade mineral oil in 35 mm culture dishes (Falcon).When using inhibitors, mineral oil was omitted and culture was done instead in 750 µL mKSOM in 4-well plates (Falcon).Culture media were pre-equilibrated for at least 2 h at 37°C in 5% CO 2 before use.Where time courses were determined, the start time (t = 0) was taken to be the initiation of oocyte culture.The time from the beginning of isolation of oocytes from the excised ovary and placing oocytes into culture was determined to be an average of 12 min (i.e., isolation began at t = −12 min).
Images of live oocytes were obtained with a Nikon E5400 camera mounted on a Zeiss Axiovert inverted microscope using a ×20 objective with Hoffman modulation contrast optics and a temperature-and atmosphere-controlled chamber.PVA was omitted from the medium to allow the oocytes to adhere to the glass bottom of the chamber so that they remained in place for the duration of imaging.Images were obtained every 2 min up to 1 h and then every 20 min to 3 h.

| GLYT1 activity assay
GLYT1 activity was measured as the rate of glycine transport into oocytes using 3 H-labeled glycine ([ 3 H]-glycine).[ 3 H]-glycine ([2-3 H]glycine; 40-60 Ci/mmole) was obtained from Perkin Elmer.Each new stock of [ 3 H]-glycine was subject to a quality control test that confirmed that transport of 1 µM [ 3 H]-glycine into mouse 1-cell embryos was inhibited by >90% in the presence of 5 mM unlabeled glycine.
When measurements were performed on single oocytes, they were incubated instead with 3 µM [ 3 H]-glycine under mineral oil.

| Oocyte-ZP detachment assay
The assay used to determine the extent of detachment of the oocyte from the ZP was previously validated and described in detail (Macaulay et al., 2023).Briefly, oocytes were exposed to hypertonic Hepes-mKSOM or mKSOM, in which osmolarity had been increased to 450 or 1000 mOsM as specified, by added NaCl.An image of each oocyte was captured immediately after transfer to hypertonic medium using a Nikon E4500 camera on an inverted microscope (see above).To determine the detachment score, a virtual radial reticle was used that was divided into six equal sextants (with each sector at 60°) and centered on the oocyte in the recorded image.
Each sector was given a score of 0 if fully attached, 0.5 if partially attached and 1 if fully detached.To maximize the number of sectors at either a score of 0 or 1 (rather than 0.5) in oocytes that were still partially attached, the reticle was rotated to adjust for this before scoring.The scores from each sector were then summed to provide an overall detachment score for each individual oocyte that ranged from 0 (fully attached) to 6 (fully detached).When oocytes were cultured or used for GLYT1 activity measurements with [ 3 H]-glycine after the detachment scores were determined, they were returned to mKSOM immediately after micrographs were obtained.

| Data analysis
Graphs were prepared with Prism 9 (GraphPad Software).Data for continuous variables were expressed as mean ± SEM. unless otherwise noted.Statistical analysis was performed with Prism, using a two tailed t-test (paired or unpaired, as specified) when comparing the means between two groups, or either one-way or two-way analysis of variance when comparing three or more groups.Either all pairs of means were compared (with Tukey's multiple comparisons test) or each mean compared to a control (Dunnett's multiple comparisons test), as specified.A p < 0.05 was considered significant.
To compare two curves where different dependent variables (GLYT1 activity and detachment score) were plotted as a function of time, the dependent variables were each transformed to the fraction of their final values.The initial values were taken to be the average at t = 0 and the final was the average at a specified time (see Section 3).
The initial value was subtracted from the value at each time point and then each was transformed to the fraction of the final value using the equation fraction = [value at time t − initial value)]/[final value − initial value].The plots were then fit to a sigmoidal curve that spanned from 0 to 1 of the form (t S )/(t S + t 0.5 S ) where t = time, S = a constant describing the steepness of the increase, and t 0.5 is the time at half-maximum (i.e., when the value of the expression = 0.5), which were all determined by the regression.Values of t 0.5 were compared by F-test.

| Time courses of oocyte-ZP detachment and GLYT1 activation in oocytes
Both oocyte-ZP detachment and GLYT1 activation are completed during the first few hours after the initiation of meiotic maturation (Macaulay et al., 2023;Tartia et al., 2009), but the temporal relationship between these two processes is unknown.If oocyte-ZP detachment is required for GLYT1 activation, then detachment would necessarily precede the initiation of GLYT1 activity, while detachment occurring after GLYT1 activation would rule out a requirement for detachment.To precisely determine the timing of GLYT1 activation relative to that of oocyte-ZP detachment, we measured the rate of glycine transport by oocytes (Figure 1a) and the extent of oocyte-ZP detachment (Figure 1b) as a function of time in identically treated groups of oocytes.GLYT1 activity began increasing at approximately 30-60 min while detachment from the ZP commenced at approximately 15-30 min.For GLYT1, there was an apparent plateau in activity from ~90-150 min after which there was a further increase to a somewhat higher plateau.
To facilitate direct comparison of these time courses, GLYT1 activity and oocyte-ZP detachment were normalized to the same scale.The most conservative normalization for GLYT1 was to the apparent plateau at 90-150 min, since that would result in the smallest difference between the two curves.Thus, both parameters were fit to sigmoidal curves with mean values at t = 0 of 0 and the mean values at t = 150 set to 1 (Figure 1c).This indicated that oocyte-

| Recovery of GLYT1 activity after osmotic shock
To determine more precisely the relationship between oocyte-ZP detachment and GLYT1 activation, both measurements should ideally be made on the same oocytes.This necessitates employing the hypertonic assay to determine the detachment score on an individual oocyte before measuring the rate of [ 3 H]-glycine transport into the same oocyte (which destroys the oocyte).However, it was not known whether such a hypertonic shock would affect subsequent GLYT1 activity.To assess this, groups of oocytes were isolated from ovaries and cultured for 90 min until substantial GLYT1 activity had developed (see Figure 1a, above) before their detachment scores and GLYT1 activity were measured.
When oocyte-ZP attachment was determined using the standard 1000 mOsM medium and then the oocytes immediately washed and transferred to medium with [ 3 H]-glycine, the measured GLYT1 activity was only approximately 50% of that measured in oocytes exposed only to isotonic (250 mOsM) medium (Figure 2a).This implied that the hypertonic shock and/or the subsequent recovery from decreased volume when 250 mOsM medium was restored suppressed GLYT1 activity in oocytes where GLYT1 had already been activated.Therefore, such an assay would be unsuitable to assess the extent of activation.It was next tested whether a short recovery period after the hypertonic shock could restore normal GLYT1 activity.Recovery periods of 5 or 10 min in 250 mOsM mKSOM medium were assessed (Figure 2b,c).However, GLYT1 activity did not recover by 10 min (although variability at 5 min resulted in there being no significant difference between 250 and 1000 mOsM).
Longer recovery periods would not be feasible, since detachment status and GLYT1 activity needed to be determined in individual oocytes at similar time points.
To attempt to address this, hypertonic medium at 450 mOsM was tested, since it was shown previously that it is possible to determine detachment scores using 450 mOsM medium (Macaulay et al., 2023).GLYT1 activity was suppressed by hypertonic shock even at 450 mOsM (Figure 2d).However, recovery periods of 5 or 10 min restored GLYT1 activity (Figure 2e,f).The measured time courses of oocyte-ZP detachment were not significantly different when either 1000 or 450 mOsM medium was used for the assay (Figure 2g).Therefore, oocyte-ZP detachment could be determined using 450 mOsM medium with a recovery period of >5 min before GLYT1 activity was measured.

| GLYT1 activity and oocyte-ZP detachment status in individual oocytes
Before comparing GLYT1 activity and oocyte-ZP detachment in the same individual oocytes, it had to be confirmed that GLYT1 activity measurements could be performed with single oocytes.GLYT1 activity has been previously measured in single preimplantation embryos (Hammer, 2000), but a 45 min incubation with [ 3 H]-glycine had been employed during which time oocyte-ZP detachment might advance significantly.Therefore, we retained a 10 min incubation but increased [ 3 H]-glycine from 1 to 3 µM, which allowed measurement of GLYT1 activity in single oocytes (Figure 3a).The time course of GLYT1 activation determined with single oocytes was not significantly different from that determined with groups of oocytes (Figure 3b).
Oocyte-ZP detachment and GLYT1 activity were then measured in the same individual oocytes as a function of time.The osmotic assay with 450 mOsM medium was used to determine the detachment score in each individual oocyte (Figure 3c).The time course of oocyte-ZP detachment was similar in this set of oocytes to those measured previously (c.f. Figure 2g).Following this, each oocyte was allowed to recover for 7.5 min, after which GLYT1 activity was measured in the same oocyte (Figure 3d).GLYT1 activity increased in these oocytes with a time course similar to that for oocytes not subjected to the osmotic assay beforehand (c.f. Figure 1b).The relationship of GLYT1 activity to the detachment score was determined by plotting GLYT1 activity as a function of the detachment score (Figure 3e,f).Oocytes with detachment scores between 0 and 3 were very infrequent (Figure 3e), likely because oocytes pass through these stages quickly, as previously shown (Macaulay et al., 2023).However, it was clear that GLYT1 activity increased in parallel with detachment score and that a significant increase occurred when oocytes reached a score of ~4 and were therefore substantially detached (Figure 3f).This is consistent with there being a threshold of detachment above which GLYT1 is active.Alternatively, it could be the result of independent processes that occur in parallel with approximately the same time courses.
However, it rules out a model in which detachment occurs significantly after GLYT1 activation.

| Dependence of GLYT1 activation on metallopeptidase activity
Since oocyte-ZP detachment occurs before GLYT1 activation and individual oocytes exhibiting GLYT1 activity seem to be substantially detached, it was possible that detachment is required for GLYT1 to be activated.We recently found that detachment is mediated by metallopeptidase activity and identified small molecule inhibitors of M12 family metallopeptidases that prevent detachment (Macaulay et al., 2023).Therefore, we tested whether the same metallopeptidase inhibitors prevented GLYT1 activation.
Groups of ~10 oocytes were cultured in mKSOM for 4 h (at which point GLYT1 is fully activated, Figure 1a) in the presence or absence of the metallopeptidase inhibitors GI254023X or batimastat.Previously, it was determined that each had an IC 50 for inhibition of oocyte-ZP detachment that was on the order of 1 µM and that complete inhibition was achieved at concentrations of ≥20 µM (Macaulay et al., 2023).It was also previously confirmed that oocytes remain viable after culture with each inhibitor as assessed by their ability to complete meiosis from the GV stage to the MII egg stage (Macaulay et al., 2023).The culture medium for this experiment contained milrinone (10 µM) to maintain meiotic arrest at the GV stage, since release from meiotic arrest alone is sufficient to induce GLYT1 activation (A.K. Tscherner and J.M. Baltz, unpublished) although maintaining meiotic arrest does not by itself prevent normal GLYT1 activation (Tartia et al., 2009).Each inhibitor significantly suppressed GLYT1 activation (Figure 4).This indicated either that blocking oocyte-ZP detachment suppressed the extent of GLYT1 activation, or that similar metallopeptidases affect each process independently.
F I G U R E 2 Recovery of GLYT1 activity in oocytes after osmotic shock.(a) Oocytes were cultured for 90 min in mKSOM (250 mOsM), by which time GLYT1 activity had developed (Figure 1).Groups of ~10 MI oocytes were then transferred to either the same osmolarity medium or hypertonic medium at 1000 mOsM.GLYT1 activity was then immediately measured by transferring the oocytes to 250 mOsM medium with 1 µM [ 3 H]-glycine for 10 min.Each point represents a single measurement of a total of five independent repeats while the bar represents the mean (±SEM).The difference just reached significance (*p = 0.0496 by t-test).(b) Same as in (a) except for a 5 min recovery period at 250 mOsM after the osmotic shock before GLYT1 activity was measured.The difference was not significant (NS, p = 0.13).(c) Same as in (b) except that the recovery period was 10 min.The difference was significant (*p = 0.041).(d) GLYT1 activity was measured as in (a) with no recovery period after exposure to hypertonic medium, except 450 mOsM hypertonic medium was used.The difference was significant (*p = 0.016).(e) Same as in (d) except with a 5 min recovery period.The difference was not significant (NS, p = 0.34).(f) Same as in (e) except that the recovery period was 10 min.The difference was not significant (NS, p = 0.66).(g) Oocyte-ZP detachment was measured as a function of time using 450 mOsM hypertonic medium.Each point for 450 mOsM represents the mean of the detachment scores for 39-51 oocytes from five independent repeats.Data for 1000 mOsM are from Figure 1b.The effect of osmolarity was not significant by two-way ANOVA (p = 0.10) while the effect of time was highly significant (p < 0.0001).ANOVA, analysis of variance; ZP, zona pellucida.

| GLYT1 activation in the absence of the ZP
It was previously found that the volume of the oocyte decreases substantially as it detaches from the ZP (Macaulay et al., 2023;Tartia et al., 2009).Therefore, it was possible that the activation of GLYT1, which is a volume-regulatory mechanism in eggs and early embryos (Steeves et al., 2003), might be activated by the decrease in volume that accompanies detachment.We first tested whether removal of the ZP would affect GLYT1 activation.As expected, GV oocytes decreased in volume rapidly after they were isolated from follicles.
The time course of the volume decrease did not differ between oocytes with the ZP intact and oocytes whose ZP had been removed by brief exposure to acid Tyrode solution (Figure 5a).Removal of the ZP also did not affect the timing or extent of GLYT1 activation (Figure 5b).Therefore, lack of attachment to the rigid ZP and loss of oocyte-ZP contact did not affect GLYT1 activation.
It was still possible, however, that the decrease in volume triggers GLYT1 activation since this occurred similarly with or without the ZP (Figure 5a).We therefore sought to impose a more rapid decrease in oocyte volume than normally occurs.ZP-free oocytes were cultured for 50 min, which is near the start of GLYT1 activation (see Figure 5b), in normal mKSOM at 250 mOsM or in mKSOM with the osmolarity raised to 310 mOsM (by adding raffinose).Culture at 310 mOsM results in oocytes with ~10%-15% smaller volume than at 250 mOsM (Collins & Baltz, 1999;Dawson et al., 1998).They were then transferred to isoosmotic mKSOM with [ 3 H]-glycine to determine GLYT1 activity.
Decreasing the volume of the oocytes during the period before the normal initiation of GLYT1 activity did not accelerate GLYT1 activation, since the oocytes cultured at 310 mOsM did not exhibit higher GLYT1 activity than those cultured at 250 mOsM (Figure 5c).Any significant acceleration should have been evident, since 50 min is immediately before higher GLYT1 activity becomes detectable (Figure 5b).
ZP proteins are secreted in a transmembrane form and retained at the surface of the GV oocyte when the ZP is solubilized by acid Tyrode exposure (Coonrod et al., 2004).The ectodomains of these transmembrane ZP proteins are apparently released via metallopeptidase activity at the time of oocyte-ZP detachment (Macaulay et al., 2023).Removal of the bulk ZP did not affect GLYT1 activation (Figure 5b), but it remained possible that the cleavage of the remaining transmembrane ZP proteins by metallopeptidase activity provides a signal to activate GLYT1.We therefore tested whether inhibiting metallopeptidase activity in ZP-free oocytes affected GLYT1 activation.GV oocytes were cultured for 4 h in mKSOM after their ZPs were removed with acid Tyrode or with their ZP intact, in the presence or absence of the previously validated (Macaulay et al., 2023) metallopeptidase inhibitors GI254023X, batimastat, or marimastat (Figure 5d).There was no significant effect of inhibitor identity or the presence versus absence of the ZP (Figure 5d).However, in each case, the presence of a metallopeptidase inhibitor significantly suppressed the development of GLYT1 activity (Figure 5d), although the decrease in the presence of inhibitor was larger (~27% decrease) for oocytes with the ZP intact than in ZPfree oocytes (~15%).This also confirmed that marimastat (Figure 5d, triangles) had a similar effect on GLYT1 activation to those of GI254023X or batimastat in ZP-intact oocytes (Figures 4 and 5d circles, squares).Taken together, these results indicate either that complete GLYT1 activation requires the cleavage of transmembrane ZP proteins that remain after the ZP is solubilized, or that similar metallopeptidases independently control oocyte-ZP detachment and affect GLYT1 activation.

| DISCUSSION
Glycine accumulation via GLYT1 is the major mechanism controlling long-term cell volume homeostasis in mature MII eggs and early preimplantation embryos through the 2-cell stage in the mouse (Steeves et al., 2003;Tscherner et al., 2021).GLYT1 is inactive in fully grown GV stage oocytes and normally becomes activated during the initial phase of meiotic maturation (Tartia et al., 2009).How GLYT1 activity is initiated was not known.It has been established that GLYT1 activation is independent of meiotic maturation and occurs with the same time course in oocytes removed from the follicle even when meiotic arrest is maintained by several independent means.These included maintenance of high intracellular cAMP in denuded oocytes using membrane-permeable cAMP analogues, phosphodiesterase inhibitors, or activators of adenylate cyclase, or within cumulus-oocyte complexes by the presence of the physiological mediator of GV arrest, natriuretic peptide precursor C (Richard & Baltz, 2017;Tartia et al., 2009).Thus, a mechanism that is independent of the well-established signaling that maintains GV arrest in the follicle is required for maintaining inactive GLYT1 in GV oocytes.
Recently, it was found that detachment from the ZP, which is a major component of the initiation of independent cell volume control in the oocyte, is mediated by metallopeptidase activity (Macaulay et al., 2023).The results reported here support the hypothesis that oocyte-ZP detachment and/or metallopeptidase activity modulates the activation of GLYT1.First, detachment preceded GLYT1 activation, which could be consistent with detachment being upstream of GLYT1 activation.Second, GLYT1 activity was only present in individual oocytes that were substantially detached from the ZP, implying that detachment may be required for GLYT1 to be activated.Third, blocking detachment by inhibiting metallopeptidase activity also partially suppressed GLYT1 activation, indicating that detachment may be required for full GLYT1 activation to occur.Alternatively, metallopeptidase activity may independently affect both detachment and GLYT1 activation.
The current model for oocyte-ZP detachment involves the cleavage of ZP glycoproteins that are in their transmembrane forms (Macaulay et al., 2023).It is known that ZP biosynthesis requires the insertion of ZP glycoproteins into the plasma membrane.A large ectodomain is then released by an unknown peptidase to selfassemble into the inner surface of the growing ZP (Jimenez-Movilla & Dean, 2011;Macaulay et al., 2023).The strong attachment of the GV oocyte to the ZP has been proposed to be mediated by a subset of ZP glycoproteins that remain in their transmembrane forms after ZP synthesis has been completed (Macaulay et al., 2023).In this model, a metallopeptidase on the oocyte surface or released into the oocyte-ZP However, whether cleavage of ZP glycoproteins can mediate signaling in oocytes is currently speculation and would need to be investigated.
The results reported here could also be consistent with alternative models.It is possible that the metallopeptidase activity is not extracellular, and instead an intracellular metallopeptidase is activated which initiates signaling that is upstream of both GLYT1 activation and oocyte-ZP detachment.This would only require that the signaling mediating detachment be more rapid that that mediating GLYT1 activation to account for the different time courses that were observed.
Removal of the soluble ZP with acid Tyrode solution did not have a discernable effect on the timing of GLYT1 activation.This could be interpreted to indicate either that a fixed period of time is required after removal of the oocyte from the follicle before GLYT1 becomes capable of being activated, or that the remaining transmembrane ZP glycoproteins (which are not removed by acid Tyrode solution) are sufficient to suppress GLYT1 activation.The actual regulation is likely complex.A more substantial inhibition of GLYT1 activation was obtained with metallopeptidase inhibitors when the intact ZP was present.However, when the soluble ZP was removed, there was still a significant, although small, inhibition of GLYT1 activation.It could be speculated that some combination of mechanical signaling by attachment to the rigid ZP and biochemical signaling via transmembrane ZP glycoproteins may contribute to suppression of GLYT1 activation.Furthermore, even with an intact ZP, metallopeptidase inhibition that completely prevented oocyte-ZP detachment still allowed GLYT1 activation to more than half its maximal level.This could reflect incomplete inhibition of metallopeptidase activity by the small molecule inhibitors used here, or it could be evidence of additional mechanisms governing the activation of GLYT1.
Further investigations will be required to determine the identity of the metallopeptidase responsible for oocyte-ZP detachment and the mechanism of its activation.Knockout or knockdown experiments would then become possible which could establish whether that specific metallopeptidase is required for GLYT1 activation and reveal whether there may be additional, parallel mechanisms governing its activation.

| CONCLUSIONS
Oocyte-ZP detachment preceded GLYT1 activation and GLYT1 was activated only in individual oocytes that were substantially or fully detached.Inhibition of metallopeptidase activity partially prevented GLYT1 activation, indicating a role for metallopeptidase activity and possibly for oocyte-ZP detachment or cleavage of transmembrane ZP proteins in the initial activation of GLYT1.These results have revealed the precise timing of two of the earliest important events that take place immediately after ovulation is triggered in the mouse -the detachment of the oocyte from its rigid ZP shell which initiates independent cell volume regulation and creates the perivitelline space that permits the positioning of the fertilizing sperm, and the activation of GLYT1, a mechanism of cell volume regulation unique to the earliest preimplantation embryo stages that is required for embryo health.
Animal protocols were approved by the University of Ottawa Faculty of Medicine Animal Care Committee and were compliant with the guidelines of the Canadian Council on Animal Care.Mice were maintained on light:dark cycles of 12 h each.They had ad libitum access to water and Teklad Global 18% protein rodent diet 2018 (Envigo).GV stage oocytes were obtained from 5-to 8-weekold female mice 44 h after superovulation had been induced by intraperitoneal injection of 5 IU equine chorionic gonadotropin (eCG; Merck, Inervet, Kirkland QC).COCs were released from excised ovaries by mincing with a razor blade and oocytes freed from the surrounding cumulus cells by ORTMAN and BALTZ | 825 [ 3 H]glycine was added to mKSOM immediately before use.Following incubation, the group of oocytes or a single oocyte was washed 7× in ice-cold Hepes-mKSOM and then transferred to a scintillation vial containing 4 mL Scintiverse BD scintillation fluid (Fisher Scientific) and vortexed.The accumulated [ 3 H]-glycine was quantified using an LS6500 liquid scintillation counter (Beckman Coulter) with a 5 min counting period.The output in CPM was converted to molar amount of [ 3 H]-glycine using standard curves constructed from serial dilutions of the [ 3 H]-glycine stock.Backgrounds determined with no added [ 3 H]-glycine were subtracted from each reading.Rates of glycine transport were reported as fmole [ 3 H]-glycine per oocyte per min per µM [ 3 H]-glycine in the incubation medium.
detachment preceded GLYT1 activation, since the detachment score reached half-maximal at t 0.5 = 34 min while half-maximal GLYT1 activation was at t 0.5 = 55 min (significantly different, p = 0.0002 by F-test).If the final maximum for GLYT1 (at t = 240 min) was instead used (Figure 1d), then t 0.5 for GLYT1 increased to 88 min (p < 0.0001).Therefore, oocyte-ZP detachment appears to precede GLYT1 activation by at least 20 min.F I G U R E 1 Relative timing of oocyte-ZP detachment and GLYT1 activation.(a) GLYT1 activity was measured by incubation of groups of ~10 oocytes with 1 µM [ 3 H]-glycine for 10 min at the indicated times after they were isolated from ovarian follicles and placed into culture.During this period, GV oocytes spontaneously exited meiotic arrest and progressed into MI.Each point represents the mean (±SEM) of five independent repeats.GLYT1 activity increased significantly with time (ANOVA, p < 0.0001) with means from 60 min and after differing significantly from t = 0 (**p = 0.002, ***p = 0.0007, ****p < 0.0001 by Dunnett's multiple comparisons test).(b) Oocyte-ZP detachment was quantified by computing the detachment score of each oocyte as a function of time defined as in (a).Each point represents the mean (±SEM) scores for 42-75 individual oocytes from five independent repeats (5-24 oocytes per repeat) at each time point.The mean detachment score increased significantly with time (ANOVA, p < 0.0001) with means from 30 min and after differing significantly from t = 0 (****p < 0.0001 by Dunnett's multiple comparisons test).(c) The time course of GLYT1 activation (a) and oocyte-ZP detachment (b) were expressed relative to their mean values at t = 150 min (set to 1.0) with the mean values at t = 0 set to 0. The horizontal dotted lines indicate 0.5 and 1.0 and the vertical dotted lines indicate the time where each curve reached half-maximal (0.5).Sigmoidal curves were fit by non-linear least-squares fits as described in the text.Points are means (±SEM) of the values for (a) and (b) normalized to this scale.(d) The time courses are presented as in (c) except GLYT1 activity was normalized to its value at t = 240 min.ANOVA, analysis of variance; ZP, zona pellucida.

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I G U R E 3 Measurement of GLYT1 activity and oocyte-ZP detachment in individual oocytes.(a) GLYT1 activity was measured in individual oocytes as a function of time in culture.Each point represents measured GLYT1 activity for a single oocyte.There were 26-35 oocytes at each time point from five independent oocyte collections.The effect of time was highly significant by one-way ANOVA (p < 0.0001).(b) The data in (a) were plotted as mean (±SEM) and compared to the data obtained using groups of ~10 oocytes replotted here from Figure 1a.A two-way ANOVA indicated no significant effect for individual versus group (NS, p = 0.09).(c) Oocyte-ZP detachment and GLYT1 activity were measured in the same individual oocytes.Each point represents the mean (±SEM) of 8-11 oocytes.A one-way ANOVA indicated a significant effect of time (p < 0.0001) with all means from t = 35 min and later significantly different than at t = 0 (**p = 0.002, ****p < 0.0001 by Dunnett's multiple comparisons test).(d) GLYT1 activity was measured in the same individual oocytes from (c) after a 7.5 min recovery period at 250 mOsM mKSOM.Each point represents the mean (±SEM) of the 8-11 oocytes.There was a significant effect of time (one-way ANOVA, p < 0.0001) and all means from t = 60 min and later were significantly different than at t = 0 (*p < 0.037, **p ≤ 0.003, ***p = 0.0002, ****p < 0.0001 by Dunnett's multiple comparisons test).(e) The data from (c) and (d) are plotted as GLYT1 activity as a function of detachment score (the line connects the means).(f) The mean (±SEM) GLYT1 activity from the data in (e) is plotted as a function of detachment score.A sigmoidal curve was fit for visualization (horizontal lines indicate maximum and minimum).The dependence of GLYT1 activity on detachment score was significant (oneway ANOVA, p < 0.0001) and GLYT1 activity was significantly higher than at t = 0 for all detachment scores of 4 or more (*p < 0.02, **p = 0.003, ***p = 0.008, ****p < 0.0001 by Dunnett's multiple comparisons test).ANOVA, analysis of variance; ZP, zona pellucida.

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I G U R E 4 Effect of inhibiting oocyte-ZP detachment on GLYT1 activation.(a) Groups of oocytes were collected form ovaries and then split into two cohorts that were cultured in the presence or absence of the M12 family metallopeptidase inhibitor GI254023X (40 µM), which prevents oocyte-ZP detachment, for 240 min after which GLYT1 activity was measured.The phosphodiesterase inhibitor, milrinone (10 µM), was present in each group to maintain meiotic arrest.The presence of GI254023X significantly suppressed GLYT1 activity (***p = 0.0004 by paired t-test).(b) Oocytes were cultured as in (a) in the presence or absence of a different M12 metallopeptidase inhibitor, batimastat (40 µM).The presence of batimastat significantly suppressed GLYT1 activity (***p = 0.0002 by paired t-test).In both graphs, each point represents the GLYT1 activity of a group of oocytes for a total of 10 groups and the bars indicate the mean (±SEM) of the independent repeats.ZP, zona pellucida.

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I G U R E 5 Effect of absence of ZP on GLYT1 activation.(a) Removing the ZP before culture did not affect the volume decrease that occurs after removal from the follicle (NS p = 0.48 for main effect of ZP by two-way ANOVA; main effect of time was highly significant, p < 0.0001).Each point is the mean (±SEM) of three independent repeats.(b) GLYT1 activity was measured in groups of oocytes with the ZP intact or ZP removed before culture.The presence versus absence of the ZP had no significant effect on GLYT1 activity (NS p = 0.987 for main effect of ZP by two-way ANOVA; main effect of time was highly significant, p < 0.0001).(c) The volumes of ZP-free oocytes were decreased during culture (50 min) by raising the osmolarity from 250 to 310 mOsM.There was no significant effect of osmolarity on GLYT1 activity (NS p = 0.52 by t-test).Bars indicate the mean (±SEM) of five independent repeats.(d) GLYT1 activity was measured in oocytes after 240 min of culture with either intact ZP (ZP+) or ZP removed before the start of culture (ZP−), each in the presence (inhibitor+) or absence (inhibitor−) of the metallopeptidase inhibitors GI254023X, batimastat, or marimastat.The symbol shape indicates the paired inhibitor and control groups.Milrinone (10 µM) was present in all groups to maintain meiotic arrest.There were N = 5 independent repeats for each inhibitor (N = 15 total).There was a significant main effect of inhibitor versus no inhibitor among the four groups (p < 0.001 by two-way ANOVA) but not of the identity of inhibitor used (p = 0. 59).The differences between treatment groups were significant where they do not share the same letter (a vs. b, p = 0.002; a vs. c, p = 0.0003; b vs. c, p < 0.0001; c vs. a,b, p = 0.0009; a vs. a, p = 0.79; b vs. b, p = 0.15, by two-way ANOVA with Tukey's multiple comparisons test).ANOVA, analysis of variance; ZP, zona pellucida.interface cleaves the remaining transmembrane ZP glycoproteins and releases the oocyte from the ZP.The activation of GLYT1 could be signaled by the loss of the extracellular domains of transmembrane ZP glycoproteins remaining after ZP removal, which would be analogous to "outside-in" signaling by other extracellular matrix-interacting proteins such as integrins(Hu & Luo, 2013).The simplest model that integrates oocyte-ZP detachment and GLYT1 activation is that the activation of a metallopeptidase that cleaves transmembrane ZP glycoproteins both releases the ZP and also produces an intracellular signal that stimulates GLYT1 activation through transmembrane transduction by the cleaved transmembrane ZP glycoproteins.