Comparison of growth characteristics of in vitro cultured granulosa cells from geese follicles at different developmental stages

Abstract

Granulosa cells (GCs) are essential components of follicles and are involved in regulating the process of follicles development. However, comparative studies on GCs isolated from different staged follicles have not been conducted in goose. The aim of the present study was to identify the growth characteristics of goose GCs from pre-hierarchical (6–10 mm) and hierarchical (F4–F2, F1) follicles. Our results showed that the three cohorts of cells had different tolerance to collagenase and had noticeable morphological differences. The F1 granulosa layers were fully digested by 0.1% collagenase, while higher concentration (0.3%) was used for both F4–F2 and pre-hierarchical granulosa layers. In the state of suspension, the diameter of F1 individual cell was larger than the other two cohorts. However, after adhering to the culture plate, cells of F1 just had changes in the diameter accompanied by small bright spots, while both pre-hierarchical and F4–F2 GCs proliferated rapidly with spreading and irregularly shaped voids. Furthermore, all attached cells could be stained by the follicle-stimulating hormone receptor antibody. Analyses of both growth curve and the mRNA expression profiles of genes related to cellular proliferation, apoptosis, and steroidogenesis suggested that three cohorts of in vitro cultured GCs had different physiological viability and functions. Taken together, the present study not only revealed differences of the growth characteristics among three cohorts of goose GCs from pre-hierarchical, F4–F2 and F1 follicles, but also optimized the in vitro culture system of geese different staged GCs.

Exploration of the effects of goose TCs on GCs at different follicular stages using a co-culture model

“…Pre-hierarchical, hierarchical (F4-F2) and F1 follicles [23] from each ovary were dissected and washed with ice-cold sterile phosphate buffered saline (PBS, pH 7.4, Solarbio). Tweezers were used to peel away the connective tissue, and then an approximate 0.5-2.0 cm slit was cut with a surgical blade across from the stalk.…”

“…However, there are few reports on the specific mechanisms of TCs effects on GCs at different developmental stages of follicle. Therefore, we developed a noncontact transwell co-culture system of GCs (with TCs at same stage) from goose (an important and relatively low-yielding poultry species) pre-hierarchical, hierarchical (F4-F2) and F1 follicle stages [23]; fetal bovine serum (FBS) medium was used in the models for long-term culture [24,25]. The aim was to explore the dynamic influences of TCs on GCs at different follicular development stages in vivo.…”

Expression of FOXL2 and RSPO1 in Hen Ovarian Follicles and Implication of Exogenous Leptin in Modulating Their mRNA Expression in In Vitro Cultured Granulosa Cells

“…For mRNA expression analysis, we performed RT-qPCR using a CFX 96TM Real-Time PCR Detection System (Bio-Rad, USA), as described previously [4] with some modifications. Briefly, we used a 15 µL reaction mixture containing 1.5 µL of cDNA template, 6.5 µL of 2 × SYBR Premix Ex Taq II (Takara, Dalian, China), and 0.4 µL each of the forward and reverse primers.…”

“…The PCR reaction conditions were as follows: (1) an initial denaturation at 95 • C for 30 s, followed by (2) 40 cycles of 95 • C for 5 s and (3) primer-specific annealing temperature for 30 s (see Table 1). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta actin (ACTB) were used as reference genes [4,43]. For quality control and threshold cycle (Ct) calibration, we included a no template control (nuclease-free water instead of cDNA) and a negative control (without reverse transcriptase) in all technical replicates of PCR assays, as per our laboratory's protocol.…”

“…These growing ovarian follicles are usually classified on the basis of their size (for example: 3-5 or 6-9 mm) or their color (as large white follicles or small yellow follicles). Classically, the ovarian follicles are mainly categorized as prehierarchical (≤9 mm in diameter) and hierarchical (>9 mm in diameter; designated as F5-F1: F5 < F4 < F3 < F2 < F1) follicles [4].…”