Microarray analysis identifies COMP as the most differentially regulated transcript throughout in vitro follicle growth

Abstract

In vitro follicle growth has emerged as a technology that can provide new information about folliculogenesis and serve as part of a suite of methods currently under development to assist women whose fertility is threatened by cancer treatments. Though it has been shown that in vitro-grown follicles secrete peptide and steroid hormones, much of the follicular transcriptome remains unknown. Thus, microarray analysis was performed to characterize the transcriptome and secretome of in vitro-grown follicles. One prominently regulated gene product was cartilage oligomeric matrix protein (Comp): its mRNA was upregulated during the final 4 days of culture (P < 0.05) and COMP protein could be detected in medium from individual follicles. COMP expression localized to mural granulosa cells of large antral follicles both in vitro and in vivo, with maximal expression immediately preceding ovulation in cycling and chorionic gonadotropin-primed female mice. COMP was co-expressed with two known markers of follicle maturation, inhibin β(A) and gremlin, and was expressed only in TUNEL-negative follicles. In addition to other gene products identified in the microarray, COMP has potential utility as a marker of follicle maturation.

Figure 1

Microarray analysis of in vitro-grown follicles. A: Representative gross morphology of an individual follicle encapsulated in alginate at 0, 2, 4, 5, 6, and 8 days in vitro. Asterisks denote antral cavities. Scale bar, 100 μm. Heat-map representation of results from genome-wide expression analysis of in vitro-grown follicles. K-means clustering of the scaled average intensity of the significant genes indicated seven clusters. B: Average follicle diameters from days 0 to 8 (n = 423 individual follicles). Data are expressed as mean ± standard error mean. C–E: Microarray validation. Microarray and qPCR results of Cyp17a1, Cyp19a1 (C), Inhba, Lhcgr (D), Gdf9, and Bmp15 (E) expression in cultured follicles compared to expression on day 2. For microarray P ≤ 0.01, n = 2–3. For qPCR, n = 3; *P < 0.05, **P < 0.01, ***P< 0.001, according to one-way ANOVA followed by Bonferroni's Multiple Comparison Test.

Figure 2

COMP is a secreted protein that increases significantly during in vitro follicle growth and can be detected in culture medium. A: Scaled trends of the intensity using self-organized maps (SOMs) for the genes encoding secreted proteins during ovarian follicle maturation. Solid lines indicate the average, scaled mean intensity of the group and dotted lines represent 1 standard deviation (note that group 6 contains only Gdnf). Listed is a representative gene from each group (bottom right). See Supplemental Table 2 for the complete list of genes encoding secreted proteins. B: Gene expression profiles comparing Comp derived from the microarray analysis (black) and qPCR validation (gray). *P <0.05, **P <0.01, ***P < 0.001 relative to expression at culture day 2 (n=3 independent experiments, one-way ANOVA followed by Bonferroni's Multiple Comparison Test). C: Western blot detection of VEGFA, INHBA, and COMP in conditioned media from individual follicles cultured in vitro. Images are representative of three independent experiments.

Figure 3

Ovarian COMP expression in vitro and in vivo. A: Image of a representative follicle cultured for 8 days and immunostained for COMP protein (i), counterstained with DAPI (ii), and photomerged (iii). Scale bar, 100 μm. Confocal microscopy image of mural granulosa cells within the follicle showing COMP localized to cytoplasmic and extracellular compartments (iv). Scale bar, 15 μm. B: COMP immunofluorescence of murine ovarian sections at different stages of the estrous cycle. Ovarian sections taken at diestrus, proestrus at 10:00 am, proestrus at 4:00 pm, estrus, and metestrus. Arrowheads signify COMP-positive staining within the ovarian surface epithelium. Scale bar, 200 μm. Images are representative of 2–3 independent experiments.

Figure 4

Gonadotropin regulation of COMP. A: Histological ovarian sections stained for COMP from prepubertal mice treated with 5IU eCG 12, 24, and 36hr post-injection (upper panel). Mice were injected with hCG 48hrpost-eCG, and slides were stained for COMP 3 and 6hr post-hCG injection (lower panel). Scale bar, 200 μm. Images are representative of three independent experiments. B: ELISA results for COMP in culture medium from primary granulosa cells before and after 1.5 IU hCG treatment *P < 0.05 (n=3 independent experiments).

Figure 5

COMP is a selective marker of terminally mature follicles. Histological ovarian sections stained for inhibin β_A, gremlin, and COMP from prepubertal mice treated with 5 IU eCG 36 hr post-injection. Scale bar, 400 μm. Images are representative of three independent experiments.

Figure 6

Follicles that express COMP are TUNEL-negative. Ovarian section from a prepubertal mouse 48 hr after injection with eCG immunohistochemically stained for COMP. Adjacent ovarian sections stained for TUNEL. Follicles delineated in green and red dashed lines highlight COMP-positive and TUNEL-positive staining, respectively. Scale bars, 200 μm. Images are representative of three independent experiments.