Expression and localization of members of the thrombospondin family during final follicle maturation and corpus luteum formation and function in the bovine ovary

Abstract

The aim of this study was to characterize the expression patterns and localization of the thrombospondin family members (THBS1, THBS2) and their receptors (CD36 and CD47) in bovine ovaries. First, the antral follicles were classified into 5 groups based on the follicle size and estradiol-17beta (E2) concentration in the follicular fluid (< 0.5, 0.5-5, 5-40, 40-180 and >180 E2 ng/ml). Second, the corpus luteum (CL) was assigned to the following stages: days 1-2, 3-4, 5-7, 8-12, 13-16 and >18 of the estrous cycle and of pregnancy (month 1-2, 3-4, 6-7 and > 8). Third, the corpora lutea were collected by transvaginal ovariectomy before and 0.5, 2, 4, 12, 24, 48 and 64 h after inducing luteolysis by injecting a prostaglandin F2alpha analog. The mRNA expression of examined factors was measured by RT-qPCR, steroid hormone concentration by EIA, and localization by immunohistochemistry. The mRNA expression of THBS1, THBS2, CD36, and CD47 in the granulosa cells and theca interna was high in the small follicles and reduced in the preovulatory follicles. The mRNA expression of THBS1, THBS2, and CD47 in the CL during the estrous cycle was high, but decreased significantly during pregnancy. After induced luteolysis, thrombospondins increased significantly to reach the maximum level at 12 h for THBS1, 24 h for THBS2, and 48 h for CD36. The temporal expression and localization pattern of the thrombospondins and their specific receptors in the antral follicles and corpora lutea during the different physiological phases of the estrous cycle and induced luteolysis appear to be compatible with their inhibitory role in the control of ovarian angiogenesis.

Fig. 1.

mRNA expression of (A) THBS1, (B) THBS2, (C) CD36, and (D) CD47 in the granulosa cells (GC) and in the theca interna (TI): (E) THBS1, (F) THBS2, (G) CD36 and (H) CD47 at different stages of follicle development and maturation. Changes in the mRNA expression of the examined factors were assayed by normalization to the expression of UBQ used as the internal control. To obtain the CT (cycle threshold) difference, the data were analyzed using the ΔΔCT method [29]. Thus, ΔCP was not subtracted from the value of a control group, but from the value 40, so that high 40-ΔCP value indicated high-gene expression and vice versa. Results are presented as means ± SEM (n = 6–8 follicles/class). Different superscripts denote statistically different values (P < 0.05). Classification of follicles was performed by follicle size and estradiol-17beta concentration in the follicular fluid.

Fig. 2.

mRNA expression of (A) THBS1, (B) THBS2, (C) CD36 and (D) CD47 in the corpus luteum (CL) during the estrous cycle and pregnancy. Changes in the mRNA expression of the examined factors were assayed by normalization to the expression of UBQ used as the internal control. Data are shown as 40-ΔCP ± SEM (n = 5–6 corpora lutea/class). Different superscripts denote statistically different values (P < 0.05).

Fig. 3.

mRNA expression of (A) THBS1, (B) THBS2, (C) CD36 and (D) CD47 in the corpus luteum (CL) during induced luteolysis. Changes in the mRNA expression of the examined factors were assayed by normalization to the expression of UBQ used as the internal control. Data are shown as 40-ΔCP ± SEM (n = 5–6 corpora lutea/class). Different superscripts denote statistically different values (P < 0.05).

Fig. 4.

(A) Secondary follicle. The oocytes (OC) of follicles show weak immunostaining for THBS1 in their cytoplasm. The granulosa cells (GC) and the cells of the theca interna (TI) are negative or only weakly positive, whereas the myofibroblast cells of the theca externa (TE) and smooth muscle cells (SMC) of the blood vessels are distinctly stained. Scale bar = 25 µm. (B) Tertiary follicle. The GC and the TI are negative for THBS1. In the TE, the SMC of the blood vessels are strongly positive. Scale bar = 100 µm. (C) Tertiary follicle. The GC and the TI remain negative. Only in the mature antral follicles, the luminal layer of the granulosa cells (arrow) appears to be distinctly positive for THBS1. Scale bar = 50 µm. (D) Corpus luteum (d1–d2). In the corpus luteum (CL), the luteal cells (LC) show weak immunostaining for THBS1, whereas the endothelial cells (EC) of the capillaries are distinctly positive. Scale bar = 75 µm. (E) Corpus luteum (d5–d7). On day 5–7, immunostaining for THBS1 increased in a subpopulation of the large luteal cells (LLC+), but the small luteal cells (SLC–) stained negative and can be differentiated. The EC of the veins are distinctly positive. Scale bar = 25 µm. (F) Corpus luteum (d12–d14). The large luteal cells are negative (LLC–) or only moderately stained (LLC+), whereas the SLC remain negative. The SMC of the arterioles and the EC of the veins are distinctly positive. Scale bar = 100 µm. (G) Corpus luteum, 5 months of pregnancy. Moderate immunostaining for THBS1 is observed in many large luteal cells (LLC+). The SMC of the blood vessels and the EC of the lymph vessels stain distinctly positive for THBS1. Scale bar = 100 µm. (H) Negative control for the corpus luteum, 5 months of pregnancy. No immunostaining is visible, neither in the LLC nor in the EC. Scale bar = 75 µm.