In Vivo Disruption of Leptin Receptor Function Impairs the Establishment and Viability of Endometriosis-Like Lesions

Abstract

There has been an increased interest in the role of the metabolic regulator, leptin, in several reproductive processes including endometriosis. Leptin peritoneal fluid levels are elevated in women with endometriosis, and have been shown to be inversely proportional to disease extent, suggesting a potential role for leptin in disease establishment. Similar to other cytokines, the exact role of leptin in the establishment of ectopic lesions is incompletely understood. Unfortunately there are a limited number of in vivo models to test the function of specific cytokines in the development of ectopic lesions. Mutant mouse models have been used effectively to elucidate function of genes/proteins related to human disease. Thus, we sought to investigate the effect of leptin signaling disruption on the successful establishment and proliferation of ectopic endometrium derived from either wild type (WT) or leptin receptor mutant (Leprdb-nonfunctional receptor) mice and hosted by their WT and/or Leprdb sisters. Animal studies utilizing syngeneic WT and leptin receptor deficient mice. Female Leprdb and control mice 6-8 wks of age were ovariectomized and received a 8mm silastic 17β-estradiol (E2) implant. Uteri were removed 96 hrs later, incised longitudinally, and endometrium was excised, minced, and inserted into the peritoneal cavity through a dorsolateral incision. Leprdb and control mice received uterine tissue from the opposite genotype of the same litter, and were monitored for 14d. Animals were then euthanized, ectopic endometrial lesions were removed, fixed in 4% PFA, paraffin embedded, serial sectioned, and examined for histological evidence of viable glandular and stromal epithelial components (H&E), vascularization (VEGF), and proliferation (phospho-histone 3-PH3). Similarly, WT C57BL6 mice 6-8 wks of age underwent ovariectomy and received an E2 implant. Female siblings were primed with 10 IU PMSG. Their uteri were removed 41 hrs later, the endometrium was processed as previously described, and inserted into the peritoneal cavity. Treatment groups included: 60μM leptin-peptide receptor antagonist (LPA-2), 60μM nonfunctional scrambled peptide (LPA2-sc), DMSO 0.0005% (vehicle), and PBS (control). Treatments consisted of daily IP injections initiated 1d prior to tissue transfer and continued for 13d. Ectopic endometrial lesions were processed as previously described. Ectopic lesions of Leprdb mice (hosting control tissue) displayed a gross purulent, necrotic appearance and significantly fewer viable glands with disorganized and apoptotic epithelium compared to control hosts hosting Leprdb tissue. In the WT model, the histological appearance and IHC staining (PH3 and VEGF) of the native uterus was similar in all treatment groups. Ectopic lesions of the LPA-2 group were characterized by uniform pallor, reduced gross vascularization, minimal viable glandular epithelium and diffuse fibrosis, and no significant PH3 staining compared to the other groups. Disruption of leptin signaling in the peritoneum impairs the establishment and proliferation of endometriosis-like lesions in a syngeneic mouse model. The absence of a functional leptin receptor results in the failure of ectopic endometrium to proliferate. These results suggest that the functional leptin receptor of the host plays a significant role and is a necessary component for early lesion development.