Effect of endometrial cell-conditioned medium and platelet-rich plasma on the developmental competence of mouse preantral follicles: An in vitro study

Abstract

Objective: The aim of this study was to evaluate the impacts of platelet-rich plasma (PRP) and conditioned medium (CM) derived from endometrial stromal cells on mouse preantral follicle culture in a two-dimensional system to produce competent mature oocytes for fertilization.

Methods: In total, 240 preantral follicles were isolated from female mouse ovarian tissue and divided into four groups. The preantral follicles were isolated three times for each group and then cultured, respectively, in the presence of alpha minimum essential medium (control), PRP, CM, and PRP+CM. The in vitro growth, in vitro maturation, and cleavage percentage of the preantral follicles were investigated. Immunocytochemistry (IHC) was also conducted to monitor the meiotic progression of the oocytes. Additionally, the mRNA expression levels of the two folliculogenesis-related genes (Gdf9 and Bmp15) and two apoptosis-related genes (Bcl2 and Bax) were investigated using real-time polymerase chain reaction.

Results: In the PRP, CM, and PRP+CM groups, the preantral follicle maturation (evaluated by identifying polar bodies) were greater than the control group. The cleavage rate in the CM, and PRP+CM groups were also greater than the control group. IHC analysis demonstrated that in each treatment group, meiotic spindle was normal. In the PRP+CM group, the gene expression levels of Bmp15, Gdf9, and Bcl2 were greater than in the other groups. The Bax gene was more strongly expressed in the PRP and control groups than in the other groups.

Conclusion: Overall, the present study suggests that the combination of CM and PRP can effectively increase the growth and cleavage rate of mouse preantral follicles in vitro.

Keywords: Conditioned medium; Culture; Endometrial cells; Platelet-rich plasma; Preantral follicles.

Figure 1.

(A-D) Isolated preantral follicles with granulosa cell layers. (E-H) Mature oocytes (MII). (I-L) Cleaved oocytes. (A, E, I) The platelet-rich plasma (PRP). (B, F, J) The conditioned medium (CM). (C, G, K) The PRP+CM group. (D, H, L) The alpha minimum essential medium (α-MEM). Original magnification, ×400; scale bar, 100 μm. GV, germinal vesicle; MII, meiosis II; O, oocyte; G, granulosa cells; P, polar body.

Figure 2.

The maturation rate of oocytes (based on the observation of polar bodies) after in vitro growth (7 days) and in vitro maturation (24 hours). The level of oocyte maturation rate were significantly greater in all treatment groups than in the control group. PRP, platelet-rich plasma; CM, conditioned medium. Treatment group vs. control group, ^a)p<0.05; ^b)p<0.01.

Figure 3.

The survival rate of the preantral follicles was analyzed to determine the effective dose of platelet-rich plasma (PRP, A) and endometrial cell-derived conditioned medium (CM, B) using the MTT assay after 48 hours. The samples contained different concentrations of PRP and CM: 0% PRP, 5% PRP, and 10% PRP and 0% CM, 5% CM, and 10% CM. The preantral follicle survival rate was obtained three times for each group. ^a)p<0.05, ^b)p<0.01.

Figure 4.

Fluorescent images of preantral follicle culture, including the meiotic spindle positioning in the mouse oocyte. The condensed cumulus is detached from the meiosis II oocytes. The meiosis II oocyte is shown with first polar body disjunction following in vitro growth and in vitro maturation, as well as a meiotic spindle (green) and chromosomes (red) after immunofluorescence staining. The spindles with spired poles are located in the oocyte cortex. An extra meiosis II with more common wide spindle poles and chromosome adjustment is depicted. Original magnification, ×400; scale bar, 100 μm. The meiotic spindle was oriented parallel to the oocyte surface in the treatment groups (platelet-rich plasma [PRP], conditioned medium [CM], and PRP+CM).

Figure 5.

Meiotic spindle migration. The spindles are located in the oocyte cortex. The migration of the meiotic spindles from the center of the oocytes to the cortex occurred. The outer circle indicates the zona pellucida; microtubules are shown in green and chromosomes in red. PRP, platelet-rich plasma; CM, conditioned medium.

Figure 6.

mRNA expression levels of the Bmp15 (A), Gdf9 (B), Bcl2 (C), and Bax (D) genes in the meiosis II oocytes in the four groups. The mRNA expression level in the preantral follicles was obtained three times for each group. PRP, platelet-rich plasma; CM, conditioned medium. PRP+CM group vs. control group: ^a)p<0.05, ^d)p<0.01; PRP+CM group vs. PRP group: ^b)p<0.05, ^c)p<0.01.