Hormonal regulation of oviductal glycoprotein 1 (OVGP1; MUC9) in the rhesus macaque cervix

Abstract

Background: Macaques are outstanding animal models for the development of new contraceptives. In women, progestin-only contraceptives often fail to block ovulation and are believed to act by altering cervix physiology. Herein, we assessed oviductal glycoprotein 1 (OVGP1) in the macaque cervix as a marker for progestogen action.

Materials: Rhesus macaques were treated with estradiol (E₂ ), E₂ plus progesterone (P), and E₂ plus levonorgestrel (LNG), a contraceptive progestin. Samples consisted of archived blocks of midcervix mucosa (epithelium and lamina propria) and fresh epithelial cells collected non-invasively by cytobrush. OVGP1 was assayed by quantitative real-time PCR and localized by immunocytochemistry.

Results: OVGP1 transcript was maximal after E₂ and reduced after treatment with E₂ + P (P < .05). LNG also reduced OVGP1 expression (P < .05). OVGP1-specific staining localized to epithelial cells, and transcript was quantifiable in cytobrush collected samples.

Conclusions: OVGP1 expression in cytobrush samples of macaque cervix provides a non-invasive indicator of contraceptive progestin action.

Keywords: cervix; cytobrush; hormonal regulation; oviductal glycoprotein; progestogen.

Figure 1

Diagram showing hormone treatments for the archived samples. On artificial cycle day 0, ovariectomized animals receive a subcutaneous capsule releasing E₂ and then after 14 days of E₂ priming; the animals received a similar capsule releasing P. Removal of the P implant (leaving E₂ in place) stimulated menstruation to complete the menstrual cycle. When animals received E₂ + LNG, the LNG implant was inserted instead of P and kept in place for 28 days (not shown).

Figure 2

Macro photograph showing a rhesus macaque cervix that has been cut along the longitudinal axis. The photograph shows the striking colliculus , which provides an excellent anatomical reference for tissue collection for excluding the squamous epithelium of the ectocervix. A black rectangle shows the portion of the cervix that provided the archived samples in this work.

Figure 3

Blots showing OVGP1 analysis by conventional PCR and Western Blot with ab 11850. (A) RT PCR analysis demonstrating probe specificity, and Western blot (B) showing antibody specificity.

Figure 4

Mean (±SE) levels of OVGP1 transcript assayed by gene array with RMA normalization (A) or by real-time PCR (B). Samples for array analysis (A) were from artificially-cycled animals only, samples for RT-qPCR (B) included treatment with LNG. Cytobrush collect cervical cells (C) were analyzed by RT-qPCR. Bars with different superscripts are significantly different (P<0.05).

Figure 5

Photographs showing IHC for OVGP1. Staining with ab 118590 is shown for both oviduct (positive control; a,b) and cervix (c–d; inset shows negative ab control). Ab 118590 staining was reduced in the secretory phase compared to the proliferative phase and completely lost after treatment with levonorgestrel (g). Staining with ab 74544 was also stronger in the proliferative phase (e; more strongly positive cells [arrows]) than secretory phase (f). Inset shows negative ab control). Cells collected by cytobrush are shown in (h) and (I).

Figure 6

Immunostaining for ESR1(a,b) and PGR (c,d). RT-qPCR analysis of ESR1 and PGR mRNA expression show significant upregulation of both ESR1 and PGR in the proliferative phase of the cycle (e).