Three-dimensional ovarian organ culture as a tool to study normal ovarian surface epithelial wound repair.

Ovarian cancers are primarily derived from a single layer of epithelial cells surrounding the ovary, the ovarian surface epithelium (OSE). Ovarian surface proliferation is associated with ovulation and has been suggested to play a role in ovarian surface transformation and cancer progression. Aspects of ovarian surface repair after ovulation include proliferation, migration, and surface regeneration. To study ovarian surface repair, an organ culture system was developed that supports the proliferation, encapsulation, and repair of an artificially wounded surface. Wounded mouse ovaries embedded into an alginate hydrogel matrix have normal OSE cells as demonstrated by expression of cytokeratin 8, vimentin, N-cadherin, and a lack of E-cadherin. Normal OSE cells began proliferating and migrating around wounded surfaces after 1 d of culture. Organ cultures were propagated in medium supplemented with BSA and fetal bovine serum to determine optimal growth conditions. BSA cultured organs had OSE that proliferated significantly more than controls until d 4, whereas fetal bovine serum cultured organs had significantly more surface area encapsulated by OSE. Overall, a three-dimensional ovarian organ culture supports the growth of normal OSE in response to artificial wounding and provides a novel system for investigating wound repair as it relates to the possible role of ovulation and ovarian cancer.

The altered distribution of the steroid hormone receptors and the chaperone immunophilin FKBP52 in a baboon model of endometriosis is associated with progesterone resistance during the window of uterine receptivity.

This study examines the distribution of estrogen receptors (ESR), progesterone receptors (Pgr), and the chaperone immunophilin FKBP52 in the eutopic endometrium in a baboon model of endometriosis during the window of receptivity to determine if their aberrant distribution contributes to reduced fecundity. Endometriosis was induced by inoculation of menstrual endometrium into the peritoneal cavity. Eutopic endometrium was collected at 3, 6, 9, 12, and 15 months postinoculation. Western blot (WB) and immunohistochemical analyses were performed. Isolated endometrial stromal cells were cultured in the presence or absence of steroid hormones. In animals with endometriosis, ESR-1 (ER-alpha) decreased in endometrial stromal cells, while ESR-2 (ER-beta) was reduced in both glandular epithelial (GE) and stromal cells. Immunoreactive total Pgr was markedly diminished in the GE, which was confirmed by WB analysis. Furthermore, treatment of isolated stromal cells from baboons with endometriosis with hormones did not increase levels of PRA or PRB as in control baboons. FKBP52 was also reduced in the eutopic endometrium of baboons with endometriosis. Endometriosis results in an aberrant distribution of ESR-1, ESR-2, Pgr, and FKBP52 in the eutopic endometrium. The authors propose that a dysregulation in the paracrine signaling between the endometrial stromal and GE cells reduces the responsiveness of Pgr, creating an endometrial environment that is unsuitable for implantation.

The estrogen early response gene FOS is altered in a baboon model of endometriosis.

Endometriosis, the presence of a functional endometrium outside of the uterine cavity, is associated with infertility. In our simulated model of pregnancy in baboons with experimental endometriosis, hCG infusion fails to induce expression of the immunoregulatory protein glycodelin. To test the hypothesis that the development of endometriosis is associated with an aberrant endometrial immunological environment, we examined the expression of a series of immunoregulatory genes in endometrium from baboons with and without endometriosis. Six months following intraperitoneal inoculation with menstrual endometrium, eutopic endometrium was surgically collected between Days 9 and 11 postovulation. Control endometrium was similarly collected from disease-free animals. Total RNA was extracted, and biotinylated cDNA probes were hybridized to the SuperArray GEArray Q series Th1/Th2/Th3 cDNA array, representing 96 genes. Gene expression levels were determined using ScanAlyze and GEArray Analyzer software. Seven genes were upregulated, including JUND, FOS, CCL11, NFKB1 and others, in the endometrium from baboons with endometriosis compared with the endometrium from disease-free animals; one gene, IL1R1, was downregulated. Quantitative RT-PCR confirmed upregulation of FOS and CCL11 in endometriotic eutopic endometrium. Immunohistochemical analysis revealed altered levels and distribution of FOS protein in the eutopic endometrium of baboons with induced endometriosis. These data suggest that in an induced model of endometriosis an aberrant eutopic immunological environment results in a decreased apoptotic potential and in rapid alterations in endometrial gene expression. We propose that the reduced fecundity associated with endometriosis has a multifold etiology in spontaneous and induced disease.

Induced endometriosis in the baboon (Papio anubis) increases the expression of the proangiogenic factor CYR61 (CCN1) in eutopic and ectopic endometria.

The expression of human CYR61 (cysteine-rich, angiogenic inducer, 61; CCN1) mRNA has been previously shown to be deregulated in the endometrium of women with endometriosis. We have chosen the baboon model (Papio anubis) of induced endometriosis to clarify whether CYR61 mRNA upregulation is predisposed to an inappropriately differentiated endometrium or is deregulated as a response to the presence of ectopic lesions. In the baboon, endometrial CYR61 mRNA expression underwent moderate cyclical variation, with a significant 7.3-fold increase detected at Day 2 postmenses when compared to endometrium from the proliferative and secretory phases. The CYR61 transcript was extensively upregulated in the eutopic endometrium from all baboons with induced endometriosis, as early as 1 mo postinoculation of menstrual tissue into the peritoneal cavity. CYR61 mRNA expression then decreased throughout progression of the disease, but remained higher compared to control tissues. Ectopic endometriotic lesions showed a further increase in CYR61 mRNA, with highest expression found in red lesions. Moreover, the expression levels of CYR61 transcripts correlated significantly with those of VEGF. Immunohistochemistry revealed the presence of CYR61 protein in glandular and luminal epithelial cells as well as in blood vessels of eutopic and ectopic endometrium. As in humans, increased levels of CYR61 mRNA correlated with the development of endometriosis in baboons. The increase of CYR61 mRNA in eutopic endometrium of baboons following peritoneal inoculation with menstrual endometrium provides evidence for a feedback mechanism from resulting lesions to induce a shift in gene expression patterns in the eutopic endometrium.

In vivo infusion of interleukin-1beta and chorionic gonadotropin induces endometrial changes that mimic early pregnancy events in the baboon.

Both human chorionic gonadotropin (hCG) and IL-1beta induce changes in the endometrium that are associated with the establishment of pregnancy. We investigated the synergistic effect of these two embryonic signals on endometrial function using a baboon model of simulated pregnancy. Recombinant hCG (30 IU/d) was infused between d 6 and 10 post ovulation (PO) to mimic blastocyst transit. On the expected day of implantation (d 10 PO), IL-1beta (12 ng/d) or IL-1 receptor antagonist (IL-1Ra; 12 ng/d) was infused for an additional 5 d. Endometria were harvested on d 15 PO. Both hCG and hCG plus IL-1beta induced marked differences in the distribution of alpha-smooth muscle actin, proliferation marker Ki67, decidualization marker IGF-binding protein-1, and cyclooxygenase-1. The most marked effect of IL-1beta was the induction of IGF-binding protein-1 protein in stromal cells close to the apical surface, whereas cyclooxygenase-1 was down-regulated in the glandular epithelium. Protein arrays of uterine flushings showed significant suppression of death receptors, Fas and TNF receptor 1, in the hCG- with or without IL-1beta-treated groups, suggesting an inhibition of apoptosis. Additionally, cytotoxic T lymphocyte antigen-4, matrix metalloproteinase-3, and IL-4 were suppressed in treated animals compared with controls. However, no differences were observed in cytokine profile between hCG-treated and hCG- plus IL-1beta-treated baboons. This study confirms that in preparation for pregnancy, the primate endometrium undergoes both morphological and functional changes, which are modulated by hCG and IL-1beta, that lead to the inhibition of apoptosis and the development of an immunotolerant environment. These changes suggest that infusion of IL-1beta at the time of implantation into the nonpregnant baboon treated with hCG synergizes with hCG and mimics the early endometrial events associated with the presence of an embryo.