Functional evaluation of the cystic fibrosis transmembrane conductance regulator in the endocervix†

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is an apical membrane chloride/bicarbonate ion channel in epithelial cells. Mutations in CFTR cause cystic fibrosis, a disease characterized by thickened mucus secretions and is associated with subfertility and infertility. CFTR function has been well characterized in vitro and in vivo in airway and other epithelia studies. However, little is known about CFTR function in the cervix in health and its contribution to cyclic regulation of fertility from endocervical mucus changes. Contributing to this research gap is the lack of information on the effect of sex steroid hormones on CFTR expression in cervical epithelial cells across the menstrual cycle. Herein, we demonstrate the hormonal regulation of CFTR expression in endocervical cells both in vitro and in vivo, and that conditionally reprogrammed endocervical epithelial cells can be used to interrogate CFTR ion channel function. CFTR activity was demonstrated in vitro using electrophysiological methods and functionally inhibited by the CFTR-specific inhibitors inh-172 and GlyH-101. We also report that CFTR expression is increased by estradiol in the macaque cervix both in vitro and in vivo in Rhesus macaques treated with artificial menstrual cycles. Estrogen upregulation of CFTR is blocked in vivo by cotreatment with progesterone. Our findings provide the most comprehensive evidence to date that steroid hormones drive changes in CFTR expression. These data are integral to understanding the role of CFTR as a fertility regulator in the endocervix.

Keywords: cervix; contraception; cystic fibrosis transmembrane conductance regulator; female reproductive tract; fertility; primates using hormone receptors; reprogramming.

Figure 1

CFTR as well as chloride and bicarbonate transporters are required for normal mucus secretion. Under normal conditions, CFTR mediated Cl⁻ ion secretion creates an electrochemical gradient that results in Na⁺ movement and water secretion along the paracellular pathway. CFTR is also a HCO₃⁻ (bicarbonate) channel that creates both an electrochemical gradient and regulates pH in the extracellular mucus necessary for mucin protein unfolding and gel formation. If CFTR is defective or inhibited, the mucus layer become dysfunctional.

Figure 2

Hormone treatments for the archived samples. The animals were treated with E2 and P4 to stimulate artificial menstrual cycles. E2-releasing implants were inserted into the animals on artificial cycle day 0 and remained in place throughout the entire hormone treatment. After 14 days of E2 priming, the animals received a P4-releasing implant. Removal of the P4 implant (leaving E2 in place) stimulated menstruation to complete the menstrual cycle.

Figure 3

Mean (+SE) transepithelial electrical resistance of CRECs treated with CFTR inhibitors Inh-172 and GlyH-101 compared with vehicle-only controls. The resistance measurements are relative to the pretreatment baseline.

Figure 4

Mean (+SE) changes in short circuit current in response to amiloride, an antagonist to the ENaC, CFTR activator forskolin and CFTR specific inhibitor Inh 172. Data represent six experiments from two different primary cell lines.

Figures 5

Representative tracing of short circuit current (Isc) over time. After cells were equilibrated and permeabilized with nystatin, amiloride (A) was added to inhibit the ENaC, decreasing Isc. The buffer was then exchanged for a chloride-free solution (B). CFTR was then interrogated with its activator (forskolin) (C) and subsequently inhibited with the known channel specific inhibitor Inh-172 (D) demonstrating subsequent increase and decrease of the Isc respectively.

Figure 6

Differentiated CRECs were treated with either no hormone control media for 9 days (“CC”), E2 (10 nM) only for 9 days (“EE”), and 7 days of E2 (10 nM) followed by 48 h of E2 + P4 medium (“EP”) (1 nM, 100 nm), or finally 7 days of E2 (10 nM) followed by no hormone control (“EC”). Mean (+SE) expression levels were normalized to RPL-32. These were additionally compared with undifferentiated cells (not taken to ALI, “UD”). ^*P < 0.001; ^**P < 0.01; ^***P < 0.05.

Figure 7

Mean (+SE) levels of CFTR transcript assayed by GeneChip analysis (upper panel) and qPCR (lower panel). [Day 0, N = 5; Day 3–4, N = 3; Day 14, N = 5; Day 15, N = 3; Day 17, N = 5; Day 21, N = 4; oophorectomized, N = 4 (qPCR only)]. P-value calculation using Fisher’s LSD test for comparison of means.

Figure 8

Immunostaining for CFTR. Immunohistochemistry (IHC) stain of CFTR at day 0 (insertion day of E2 implant), day 4 of E2 implant-only, day 14 of E2 implant-only, day 21 (E2 + 7 days of P4 implant), and in ovariectomized animals without hormone replacement. Lung and duodenum are included here as positive controls.