Does kisspeptin exert a local modulatory effect on bovine ovarian steroidogenesis?

Abstract

Kisspeptin, a hypothalamic neuropeptide encoded by the KISS1 gene, has a pivotal role in promoting GnRH secretion in mammals. Kisspeptin and its receptor (KISS1R) are also expressed in certain peripheral tissues including gonads, suggesting intra-gonadal roles. Such actions at the level of the bovine ovary have not been explored previously. The current aims were to determine whether KISS1 and its receptor (KISS1R) are expressed in the bovine ovary and whether kisspeptin or a kisspeptin antagonist can modulate ovarian steroid production by cultured ovarian cells. Granulosa (GC) and theca interna (TC) were collected from antral follicles (3-18 mm) categorized into five class sizes. Early, mid and regressing corpora lutea (CL) were also collected for RT-qPCR analysis of KISS1 and KISS1R expression. Bovine TC and GC cultured under both non-luteinizing (serum-free) and luteinizing (serum-supplemented) conditions were treated for 4 days with kisspeptin-10 (10-10-10-6M) or kisspeptin antagonist (p234; 10-10-10-6M), alone and in combination with either FSH (GC), LH (TC) or forskolin (luteinized GC/TC). Steroid secretion (GC: oestradiol, progesterone; TC: androstenedione, progesterone; luteinized GC/TC: progesterone) was measured by ELISA and viable cell number determined by neutral red uptake assay. KISS1 and KISS1R transcripts were detected in TC, GC and CL with significant differences between follicle categories and CL stages. However, neither kisspeptin-10 nor kisspeptin antagonist affected steroid secretion or viable cell number in any of the four ovarian cell culture models. As such, the hypothesis that kisspeptin has a direct intra-ovarian role to modulate follicular or luteal steroidogenesis, or cell proliferation/survival, is not supported.

Figure 1

Comparison of the relative abundance of mRNA transcripts for (A) KISS1 and (B) KISS1R (GPR54) in different bovine endocrine tissues including adrenal gland (A, n = 6), pituitary gland (P, n = 6), testis (T, n = 6), corpus luteum (CL, n = 13), granulosa cells (GC, n = 38), and theca cells (TC, n = 43) and bars indicate s.e.m.; means without a common letter are significantly different (P < 0.05).

Figure 2

Comparison of the relative abundance of mRNA transcript for (A) KISS1 and (B) KISS1R in GC and TC from ovarian follicles classified according to size. Follicles in the largest size category were subdivided on the basis of their intrafollicular oestrogen to progesterone ratio (E2:P4 >1 or E2:P4 <1) shown in panel (C). Values are means and bars indicate s.e.m.; summarized ANOVA results are shown. Open bars (GC) without a common letter are significantly different (P < 0.05); Expression levels in TC did not differ between different size categories (P > 0.05). *P < 0.05 TC group vs corresponding GC group.

Figure 3

The expression of (A) KISS1 and (B) KISS1R in CL tissue at growing (G, n = 4), mid-luteal (M, n = 5) and regressing (R, n = 4) stages. Values are means and bars indicate s.e.m.; results of pairwise comparisons are shown.

Figure 4

Lack of effect of kisspeptin-10 (left) and kisspeptin antagonist (right) on basal and LH-induced production of (A, B) A4 and (C, D) P4 by non-luteinized TC; lower panels (E, F) show viable cell number at the end of the culture. Values are means and bars indicate s.e.m. (n = 6 independent batches of cells); two-way ANOVA results are shown.

Figure 5

Lack of effect of kisspeptin-10 (left) and kisspeptin antagonist (right) on basal and FSH-dependent production of (A, B) E2 and (C, D) P4 by non-luteinized bovine GC; lower panels (E, F) show viable cell number at the end of the culture. Values are means, and bars indicate s.e.m. (n = 3 independent batches of cells); two-way ANOVA results are shown.

Figure 6

Lack of effect of kisspeptin-10 (A, B) and kisspeptin antagonist (C, D) on basal and FSK-stimulated production of P4 by luteinized bovine TC (A, C) and on viable cell number at the end of the culture (B, D). Values are means, and bars indicate s.e.m. (n = 5–6 independent batches of cells); two-way ANOVA results are shown.

Figure 7

Lack of effect of kisspeptin-10 and kisspeptin antagonist on basal and FSK-stimulated production of P4 by luteinized bovine GC (A, C) and on viable cell number at the end of the culture (B, D). Values are means, and bars indicate s.e.m. (n = 3 independent batches of cells); two-way ANOVA results are shown.