Oxytocin receptors in the primate ovary: molecular identity and link to apoptosis in human granulosa cells

Abstract

Background: Oxytocin (OT) is produced by granulosa cells (GCs) of pre-ovulatory ovarian follicles and the corpus luteum (CL) in some mammalian species. Actions of OT in the ovary have been linked to luteinization, steroidogenesis and luteolysis. Human IVF-derived (h)GCs possess a functional OT receptor (OTR), linked to elevation of intracellular Ca(2+), but molecular identity of the receptor for OT in human granulosa cells (hGCs) and down-stream consequences are not known.

Methods and results: RT-PCR, sequencing and immunocytochemistry identified the genuine OTR in hGCs. OT (10 nM-10 microM) induced elevations of intracellular Ca(2+) levels (Fluo-4 measurements), which were blocked by tocinoic acid (TA; 50 microM, a selective OTR-antagonist). Down-stream effects of OTR-activation include a concentration dependent decrease in cell viability/metabolism, manifested by reduced ATP-levels, increased caspase3/7-activity (P < 0.05) and electron microscopical signs of cellular regression. TA blocked all of these changes. Immunoreactive OTR was found in the CL and GCs of large and, surprisingly, also small pre-antral follicles of the human ovary. Immunoreactive OTR in the rhesus monkey ovary was detected in primordial and growing primary follicles in the infantile ovary and in follicles at all stages of development in the adult ovary, as well as the CL: these results were corroborated by RT-PCR analysis of GCs excised by laser capture microdissection.

Conclusions: Our study identifies genuine OTRs in human and rhesus monkey GCs. Activation by high levels of OT leads to cellular regression in hGCs. As GCs of small follicles also express OTRs, OT may have as yet unknown functions in follicular development.

Figure 1

Demonstration of OTR mRNA and protein.

Figure 2

OT and Ca²⁺-signaling in hGCs: composite figure.

Figure 3

Measurements of intracellular ATP level and caspase 3/7 activity.

Figure 4

Electron microscopic examination.

Figure 5

Immunohistochemistry for OTR in human ovary.

Figure 6

Example of experiments using laser microdissection (LMD) of GCs from sections of human ovaries of human pre-antral (A–C) and a large antral follicle (D–F) followed by RT–PCR.

Figure 7

Immunohistochemistry for OTR in monkey ovaries (A and B: Post-natal Day 0–1; C–E adult). (A) Sections of a monkey ovary from a post-natal animal, which contains immunoreactive small pre-antral follicles (asterisk: primordial; arrow: primary). Bar ∼15 µm. (B) Corresponding control staining to (A) (omission of antiserum). Bar ∼15 µm. (C) Adult monkey ovary: staining of GCs (GCs) in a growing antral follicle is seen, as well as staining in small pre-antral follicles (arrows). Bar ∼15 µm. (D) Control corresponding to approximately. Bar ∼15 µm. (E) As expected, luteal cells stain positive. Bar ∼25 µm.

Figure 8

Example of experiments in monkey ovary using LMD of GCs from sections of adult ovaries.