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

To investigate Ca2+ dynamics in earlier phases of follicular development we compared the resting [Ca2+]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 [Ca2+]i imaging. O-GCs were obtained from prehierarchical follicles (F6–F5–F4 < 8 mm). In basal and agonist/antagonist stimulated M-GCs we did not observe a change in the [Ca2+]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 [Ca2+]i was 108 ± 11 nM; (b) 36% cells displayed yet small oscillations and [Ca2+]i was 167 ± 14 nM; (c) 25% were cells with repetitive irregular oscillations that peaked until 2 fold basal value and [Ca2+]i very variable, was 248 ± 41 nM. In O-GCs L-type VOCCs stimuli displayed different effects on [Ca2+]i for both treatment in three basal patterns.In our study we demonstrated: (1) at resting the [Ca2+]i is low (111 ± 5 nM) in M-GCs and tend to increasing in prehierarchical O-GCs; (2) L-type Ca2+ 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 Ca2+ pathway. Therefore, the functional response of L-type Ca2+ 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 Ca2+-dependent signaling could have a critical role in the regulatory mechanisms hormone mediated in hen ovarian cycle.