Granulosa Cells Improved Mare Oocyte Cytoplasmic Maturation by Providing Collagens

Abstract

Assisted reproductive technology has important clinical applications and commercial values in the horse industry. However, this approach is limited largely by the low efficiency of oocyte in vitro maturation (IVM), especially cytoplasmic maturation. To improve the efficiency of mare oocyte IVM, we evaluated the effects of co-culture with cumulus-oocyte complexes (COCs) and granulosa cells (GCs) from follicles with small (<15 mm) and large diameters (>35 mm). Our results showed that oocyte nucleus maturation was not significantly improved by co-culturing with GCs. Interestingly, the cytoplasmic maturation of oocytes, defined by the distribution of cortical granules and mitochondria, as well as reactive oxygen species (ROS) levels, improved dramatically by co-culture with GCs, especially those derived from small follicles. Moreover, GCs promoted cumulus cell expansion by upregulating the expression of BMP15 in oocytes. To determine the mechanism underlying the effects of GCs, the transcriptomes of GCs from large and small follicles were compared. Expression levels of COL1A2, COL6A1, and COL6A2 were significantly higher in GCs from small follicles than in those from large follicles. These three genes were enriched in the extracellular matrix proteins-receptor interaction pathway and were involved in the regulation of collagens. Taken together, our results suggest that co-culture with GCs is beneficial to oocyte cytoplasmic maturation, and the increased expression of COL1A2, COL6A1, and COL6A2 improve the mare oocyte IVM system via the regulation of collagen.

Keywords: BMP15; collagens; cytoplasm maturation; granulosa cells; mare; oocyte.

FIGURE 1

Effects of GC co-culture on mare oocyte in vitro nuclear maturation. (A) Nuclear staining of oocytes in GV, GVBD, and MII stages. Scale bar = 40 µm. (B) Percentages of GV stage mare oocytes derived from the CONTROL, LFGC + O, and SFGC + O groups after 4 h of culture in vitro (30 oocytes per group). (C) Percentages of GVBD stage mare oocytes derived from the CONTROL, LFGC + O, and SFGC + O groups after 4 h of culture in vitro (30 oocytes per group). (D) Percentages of MII stage mare oocytes derived from the CONTROL, LFGC + O, and SFGC + O groups after 36 h of culture in vitro (76 oocytes per group). Data are shown as means ± SEM.

FIGURE 2

Effects of GC co-culture on the cortical granule distribution of mare MII oocytes. (A) Cortical granule staining with RNA-FITC in mare oocytes after in vitro maturation was detected by confocal microscopy. Scale bar = 20 µm. (B) Percentages of distributions classified as peripheral, cortical, homogeneous, and abnormal for cortical granules in mare MII oocytes derived from the CONTROL, LFGC-O, and SFGC-O groups (59 oocytes per group). Data are shown as means ± SEM. *p < 0.05 and **p < 0.01.

FIGURE 3

Effects of GC co-culture on mitochondrial distributions in mare MII oocytes. (A) Mitochondria stained with MitoTracker Red in mare MII oocytes after in vitro maturation were detected using confocal microscopy. Scale bar = 20 µm. (B) Percentages of homogeneous, peripheral, and atrophic distributions of mitochondria in mare MII oocytes derived from the CONTROL, LFGC-O, and SFGC-O groups (76 oocytes per group). Data are shown as means ± SEM. *p < 0.05 and **p < 0.01.

FIGURE 4

Effects of GC co-culture on ROS production in mare MII oocytes. (A) Representative fluorescence images were obtained from mare MII oocytes stained with DCFH-DA. Scale bar = 150 μm. (B) Relative fluorescence intensity of DCFH-DA in mare MII oocytes derived from the CONTROL, LFGC-O, and SFGC-O groups (30 oocytes per group). Average optical intensity was measured using ImageJ. Data are expressed as means ± SEM. *p < 0.05, **p < 0.01.

FIGURE 5

Effects of GC co-culture on cumulus cell expansion and BMP15 expression in mare MII oocytes. Morphology of COCs matured without GCs [CONTROL, (A)] or with LFGCs [LFGC + O, (B)], or SFGCs [SFGC + O, (C)] for 36 h was examined. (D) The degree of cumulus cell expansion was assessed after IVM (20 oocytes per group). Scale bar = 150 μm. (E) Immunofluorescence detection of BMP15 in MII oocytes derived from the CONTROL, LFGC + O, and SFGC + O groups (30 oocytes per group). (F) Average optical intensity was measured using ImageJ. Values are presented as means ± SEM. *p < 0.05, **p < 0.01.

FIGURE 6

mRNA expression profiles of granulosa cells and validation of differentially expressed mRNAs between granulosa cells from small and large follicles. (A) Volcano map displaying DEGs between the granulosa cells from small follicles and those from large follicles. (B) KEGG pathway analysis of common DEGs. (C–E) Relative expression levels of COL1A2, COL6A1, and COL6A2 in large and small follicle–derived granulosa cells were evaluated using q-PCR. (F) Speculative model for the beneficial effect of GCs on the cytoplasmic maturation of mare oocytes in vitro.