Modulation of the baboon (Papio anubis) uterine endometrium by chorionic gonadotrophin during the period of uterine receptivity

Abstract

This study was undertaken to determine the modulation of uterine function by chorionic gonadotrophin (CG) in a nonhuman primate. Infusion of recombinant human CG (hCG) between days 6 and 10 post ovulation initiated the endoreplication of the uterine surface epithelium to form distinct epithelial plaques. These plaque cells stained intensely for cytokeratin and the proliferating cell nuclear antigen. The stromal fibroblasts below the epithelial plaques stained positively for alpha-smooth muscle actin (alphaSMA). Expression of alphaSMA is associated with the initiation of decidualization in the baboon endometrium. Synthesis of the glandular secretory protein glycodelin, as assessed by Western blot analysis, was markedly up-regulated by hCG, and this increase was confirmed by immunocytochemistry, Northern blot analysis, and reverse transcriptase-PCR. To determine whether hCG directly modulated these uterine responses, we treated ovariectomized baboons sequentially with estradiol and progesterone to mimic the hormonal profile of the normal menstrual cycle. Infusion of hCG into the oviduct of steroid-hormone-treated ovariectomized baboons induced the expression of alphaSMA in the stromal cells and glycodelin in the glandular epithelium. The epithelial plaque reaction, however, was not readily evident. These studies demonstrate a physiological effect of CG on the uterine endometrium in vivo and suggest that the primate blastocyst signal, like the blastocyst signals of other species, modulates the uterine environment prior to implantation.

Figures

Figure 1

Diagrammatic illustration of the experimental design for hCG infusion into normally cycling baboons (A) and ovariectomized baboons (B). The steroid treatment regimen for ovariectomized baboons is based on an idealized 28-day menstrual cycle (10). Day 0 in this group is 30 days after ovariectomy. E2, estradiol; P, progesterone.

Figure 2

Morphology (A and B) and immunolocalization of αSMA (C and D) in cycling baboons treated with hCG. Note the distinct epithelial plaques that are associated with the luminal epithelium (arrow; A) and the localization of αSMA in stromal fibroblasts below the plaques (St; C) in hCG-treated animals. The controls treated with heat-inactivated CG showed no response in either of the two cell types (B and D). [Final magnification ×77 (A and B) and ×45 (C and D).]

Figure 3

Morphology (A and B) and immunolocalization of αSMA (C and D) in ovariectomized baboons. Note the absence of distinct epithelial plaques in the hCG-treated baboon (A). In contrast, the induction of αSMA in stromal cells (St; C) was comparable to that seen in cycling baboons treated with hCG. Ovariectomized baboons treated with heat-inactivated CG showed no response in either cell type (B and D). (Final magnification ×45.)

Figure 4

Immunocytochemical localization of glycodelin in cycling baboons treated with hCG. Treatment with hCG markedly up-regulates the staining for glycodelin in the glandular epithelial cells (A). A limited amount of immunoreactivity is evident in the perinuclear region of the epithelial cells in the heat-inactivated controls (B). (Final magnification ×74.)

Figure 5

Western blot of explant culture medium. Protein from conditioned media after 24 h of culture was immunoreacted with a polyclonal antibody to human glycodelin. Treatment with hCG markedly increased the synthesis and the molecular size of glycodelin (lanes 3–6). Endometrial explants from the heat-inactivated controls secreted a low molecular weight form of glycodelin (lanes 7–10) comparable to that seen normally at day 10 PO (lane 2). Lane 1 is the human protein (0.25 μg) purified from first-trimester cytosolic extracts.

Figure 6

Western blot of explant culture medium (lanes 2–5) and uterine flushings (lanes 6 and 7) from ovariectomized baboons. Note that treatment with bioactive hCG (lanes 2, 3, and 6) resulted in the secretion of the higher molecular weight form of glycodelin both in vivo (lane 6) and in vitro (lanes 2 and 3). Only the lower molecular weight form was evident in explant culture media (lanes 4 and 5) but not in uterine flushings (lane 7) obtained from the heat-inactivated controls (lanes 4 and 5). Endometrial explant culture medium obtained at early pregnancy (day 14) is shown for comparison (lane 1).

Figure 7

(A) RT-PCR for glycodelin transcripts in early pregnant (lanes 1 and 2), hCG-treated (lanes 3 and 4), and heat-inactivated hCG-treated (lanes 5 and 6) cycling baboons (A). Lanes 7 and 8 represent ovariectomized baboons treated with hCG or heat- inactivated hCG, respectively. Note that two major transcripts (383 and 279 bp) are amplified during pregnancy and after hCG infusion. The 213-bp transcript is the histone 3.3 internal standard that was coamplified with the glycodelin primers. (B) The bar graphs represent the mean values of the densitometric scans of each transcript shown in A and expressed as a ratio to histone 3.3. Note that in response to CG either in pregnancy (bars 1) or with hCG infusion (bars 2) the higher molecular weight transcript (383 bp; black bars) predominates. The response after ovariectomy is attenuated, but the higher molecular weight form is evident in animals treated with bioactive hCG (lane 7 in A and 4 in B). The gray bars represent the low molecular weight (279 bp) transcript.