Differential Regulation of Human and Mouse Myometrial Contractile Activity by FSH as a Function of FSH Receptor Density

Abstract

Previous studies from our laboratory revealed that the follicle-stimulating hormone receptor (FSHR) is expressed at low levels in nonpregnant human myometrium and that it is up-regulated in pregnant term nonlaboring myometrium; however, the physiological relevance of these findings was unknown. Herein, we examined signaling pathways stimulated by FSH in immortalized uterine myocytes expressing recombinant FSHR at different densities and showed that cAMP accumulation is stimulated in all cases but that inositol phosphate accumulation is stimulated only at high FSHR densities. Because an increase in cAMP quiets myometrial contractile activity but an increase in 1,4,5-triphosphoinositol stimulates contractile activity, we hypothesized that FSHR density dictates whether FSH quiets or stimulates myometrial contractility. Indeed, in human and mouse nonpregnant myometrium, which express low levels of FSHR, application of FSH resulted in a quieting of contractile activity. In contrast, in pregnant term nonlaboring myometrium, which expresses higher levels of FSHR, application of FSH resulted in increased contractile activity. Examination of pregnant mouse myometrium from different stages of gestation revealed that FSHR levels remained low throughout most of pregnancy. Accordingly, through mid-gestation, the application of FSH resulted in a quieting of contractile activity. At Pregnancy Day (PD) 16.5, FSHR was up-regulated, although not yet sufficiently to mediate stimulation of contractility in response to FSH. This outcome was not observed until PD 19.5, when FSHR was further up-regulated. Our studies describe a novel FSHR signaling pathway that regulates myometrial contractility, and suggest that myometrial FSHR levels dictate the quieting vs. stimulation of uterine contractility in response to FSH.

Keywords: FSH; FSH receptor; contractility; myometrium; parturition.

FIG. 1

FSHR protein expression is higher in muscle fibers and stroma of human pregnant term myometrium than in nonpregnant myometrium. Human nonpregnant myometrium (A) and nonpathological pregnant term myometrium (B) are shown stained with antibody FSHR-323 IgG2a for FSHR (brown) and counterstained with hematoxylin (blue). Myometrial muscle fibers ([M]) and endothelium of blood vessels ([V]) are labeled. A and B) Five representative samples each. Original magnification ×200; bar = 50 μm. Samples stained with nonimmune IgG2a did not exhibit staining (not shown).

FIG. 2

FSHR protein and FSHR mRNA are each up-regulated in human pregnant term nonlaboring myometrium relative to that in nonpregnant myometrium. A) FSHR protein immunostaining of muscle fibers and stroma in five samples each of nonpregnant (NP) and pregnant term nonlaboring (TNL) human myometrium was quantified as described in Materials and Methods. B) FSHR mRNA from four samples each of nonpregnant (NP) and pregnant TNL human myometrium was quantified by real-time PCR. Protein and mRNA data are mean ± SEM of the fold increase relative to those in nonpregnant myometrium. *Difference of P < 0.05 from nonpregnant myometrium.

FIG. 3

In myometrial hTERT-HM cells, FSH stimulates cAMP production at all densities of FSHR but stimulates IP3 synthesis only at high FSHR densities. hTERT-HM cells were transduced with adenovirus encoding FSHR to achieve different densities of cell surface recombinant FSHR as determined by cell surface binding of ¹²⁵I-labeled FSH. cAMP and IP3 accumulation were each measured in response to a saturating concentration of FSH as described in Materials and Methods. Control cells transduced with adenovirus encoding β-galactosidase did not reveal detectable FSH-stimulated cAMP or IP3 accumulation. Data are percentages of maximally FSH-stimulated response of each signaling pathway as a function of FSHR density.

FIG. 4

FSH quiets contractile activity in human nonpregnant myometrium and stimulates contractile activity in pregnant term nonlaboring myometrium. Relative contractile activities of strips of human nonpregnant myometrium (A) or human pregnant term nonlaboring myometrium (B) in response to increasing concentrations FSH were determined using isometric tension recordings as described in Materials and Methods. A and B) Data are mean ± SEM from four independent experiments. *Difference of P < 0.05 from no FSH addition.

FIG. 5

In the mouse, myometrial FSHR protein expression remains relatively low until late in pregnancy. Representative samples of mouse myometrium are shown from nonpregnancy (NP [A]) or at different stages of gestation (PD 7.5–19.5 [B–E]) stained with anti-rat FSHR (brown) and counterstained with hematoxylin (blue). Myometrial muscle fibers ([M]), endothelium of blood vessels ([V]), luminal epithelium ([LE]), and glandular epithelium ([GE]) are labeled. Images are representative of 6 NP, 3 PD-7.5, 3 PD-11.5, 4 PD-16.5, and 3 PD-19.5 samples. Original magnification ×200; bar = 50 μm. Samples stained with preimmune IgG did not exhibit staining (not shown).

FIG. 6

In the mouse, FSH quiets myometrial contractile activity through mid-gestation, when FSHR remains low, and stimulates contractile activity at term, when FSHR is up-regulated. Top) Staining of FSHR in mouse myometrium from 6 NP, 3 PD-7.5, 3 PD-11.5, 4 PD-16.5, and 3 PD-19.5 samples was quantified as described in Materials and Methods and shown as mean ± SEM of the fold increase relative to nonpregnant myometrium. Middle and Bottom Rows) Relative contractile activities of strips of mouse nonpregnant myometrium (NP) or mouse myometrium from different stages of gestation in response to increasing concentrations FSH were determined using isometric tension recordings as described in Materials and Methods. Data are mean ± SEM of 3 NP, 3 PD-7.5, 5 PD-11.5, 3 PD-16.5, and 5 PD-19.5 samples. *Difference of P < 0.05 from no FSH addition.