Deletion of TRPV3 and CaV3.2 T-type channels in mice undermines fertility and Ca2+ homeostasis in oocytes and eggs

Calcium (Ca2+) influx during oocyte maturation and after sperm entry is necessary to fill the internal Ca2+ stores and for complete egg activation. We knocked out the transient receptor potential vanilloid member 3 (TRPV3) and the T-type channel, CaV3.2 to determine their necessity for maintaining these functions in mammalian oocytes/eggs. Double knockout (dKO) females were subfertile, their oocytes and eggs showed reduced internal Ca2+ stores, and following sperm entry or PLCz cRNA injection, fewer dKO eggs displayed Ca2+ responses compared to wildtype (WT) eggs, which were also of lower frequency. These parameters were rescued and/or enhanced by removing extracellular Mg2+, suggesting the residual Ca2+ influx could be mediated by the TRPM7 channel, consistent with the termination of divalent-cation oscillations in dKO eggs by a TRPM7 inhibitor. In total, we demonstrated that TRPV3 and CaV3.2 mediate the complete filling of the Ca2+ stores in mouse oocytes and eggs. We also show they are required for initiating and maintaining regularly spaced-out oscillations, suggesting that Ca2+ influx through PM ion channels dictates the periodicity and persistence of Ca2+ oscillations during mammalian fertilization.

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Simultaneous Deletion of Transient Receptor Potential Vanilloid 3 and Cacna1h Undermines Ca2+ Homeostasis in Oocytes and Fertility in Mice

10.1101/443622 ◽

2018 ◽

Author(s):

Aujan Mehregan ◽

Goli Ardestani ◽

Ingrid Carvacho ◽

Rafael Fissore

Keyword(s):

Transient Receptor Potential ◽

Receptor Potential ◽

Functional Expression ◽

Egg Activation ◽

Transient Receptor Potential Vanilloid ◽

Double Knockout ◽

Knock Out ◽

Transient Receptor ◽

Mammalian Eggs ◽

Channel Currents

In mammals, calcium (Ca2+) influx fills the endoplasmic reticulum, from where Ca2+ is released following fertilization to induce egg activation. However, an incomplete index of the plasma membrane channels and their specific contributions that underlie this influx in oocytes and eggs led us to simultaneously knock out the transient receptor potential vanilloid, member 3 (TRPV3) channel and the T-type channel, CaV3.2. Double knockout (dKO) females displayed subfertility and their oocytes and eggs showed significantly diminished Ca2+ store content and oscillations after fertilization compared to controls. We also found that the cell cycle stage during maturation determines the functional expression of channels whereby they show a distinct permeability to certain ions. In total, we demonstrate that TRPV3 and CaV3.2 are required for initiating physiological oscillations and that Ca2+ influx dictates the periodicity of oscillations during fertilization. dKO gametes will be indispensable to identify the complete native channel currents present in mammalian eggs.

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Combined genetic and pharmacological inhibition of TRPV1 and P2X3 attenuates colorectal hypersensitivity and afferent sensitization

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00180.2013 ◽

2013 ◽

Vol 305 (9) ◽

pp. G638-G648 ◽

Cited By ~ 22

Author(s):

Michael E. Kiyatkin ◽

Bin Feng ◽

Erica S. Schwartz ◽

G. F. Gebhart

Keyword(s):

Transient Receptor Potential ◽

Genetic Manipulation ◽

Afferent Fiber ◽

Receptor Potential ◽

Afferent Neuron ◽

Transient Receptor Potential Vanilloid ◽

Double Knockout ◽

Inflammatory Soup ◽

Trpv1 Antagonist ◽

Transient Receptor

The ligand-gated channels transient receptor potential vanilloid 1 (TRPV1) and P2X3 have been reported to facilitate colorectal afferent neuron sensitization, thus contributing to organ hypersensitivity and pain. In the present study, we hypothesized that TRPV1 and P2X3 cooperate to modulate colorectal nociception and afferent sensitivity. To test this hypothesis, we employed TRPV1-P2X3 double knockout (TPDKO) mice and channel-selective pharmacological antagonists and evaluated combined channel contributions to behavioral responses to colorectal distension (CRD) and afferent fiber responses to colorectal stretch. Baseline responses to CRD were unexpectedly greater in TPDKO compared with control mice, but zymosan-produced CRD hypersensitivity was absent in TPDKO mice. Relative to control mice, proportions of mechanosensitive and -insensitive pelvic nerve afferent classes were not different in TPDKO mice. Responses of mucosal and serosal class afferents to mechanical probing were unaffected, whereas responses of muscular (but not muscular/mucosal) afferents to stretch were significantly attenuated in TPDKO mice; sensitization of both muscular and muscular/mucosal afferents by inflammatory soup was also significantly attenuated. In pharmacological studies, the TRPV1 antagonist A889425 and P2X3 antagonist TNP-ATP, alone and in combination, applied onto stretch-sensitive afferent endings attenuated responses to stretch; combined antagonism produced greater attenuation. In the aggregate, these observations suggest that 1) genetic manipulation of TRPV1 and P2X3 leads to reduction in colorectal mechanosensation peripherally and compensatory changes and/or disinhibition of other channels centrally, 2) combined pharmacological antagonism produces more robust attenuation of mechanosensation peripherally than does antagonism of either channel alone, and 3) the relative importance of these channels appears to be enhanced in colorectal hypersensitivity.

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