A functional study on L-type calcium channels in granulosa cells of small follicles in laying and forced molt hens

Abstract

To investigate Ca(2+) dynamics in earlier phases of follicular development we compared the resting [Ca(2+)](i) and tested the functional responses to agonist/antagonist of L-type voltage-operated calcium channels (VOCCs) in small follicles GCs from hens during oviposition (O-GCs) and forced molt (M-GCs), using the microspectrofluorimetric [Ca(2+)](i) imaging. O-GCs were obtained from prehierarchical follicles (F(6)-F(5)-F(4)<8mm). In basal and agonist/antagonist stimulated M-GCs we did not observe a change in the [Ca(2+)](i) under any of condition in all cells analyzed. Based on basal measurements we can distinguish three different patterns reflecting cells variability within O-GCs group: (a) 39% cells showed small oscillations and [Ca(2+)](i) was 108±11nM; (b) 36% cells displayed yet small oscillations and [Ca(2+)](i) was 167±14nM; (c) 25% were cells with repetitive irregular oscillations that peaked until 2 fold basal value and [Ca(2+)](i) very variable, was 248±41nM. In O-GCs L-type VOCCs stimuli displayed different effects on [Ca(2+)](i) for both treatment in three basal patterns. In our study we demonstrated: (1) at resting the [Ca(2+)](i) is low (111±5nM) in M-GCs and tend to increasing in prehierarchical O-GCs; (2) L-type Ca(2+) channels are functionally expressed in the major part of O-GCs whereas they are not activated nor inhibited in M-GCs and in a percentage of O-GCs; (3) there are three different cellular types in prehierarchical O-GCs that may be associated with increasing stages of follicular development, based on their Ca(2+) pathway. Therefore, the functional response of L-type Ca(2+) channels in cultured laying hen prehierarchical GCs may be correlated with the functional maturation phase of laying hens ovarian. We hypothesize that the L-type Ca(2+)-dependent signaling could have a critical role in the regulatory mechanisms hormone mediated in hen ovarian cycle.