Janet L Funk

4-Vinylcyclohexene diepoxide (VCD), an occupational chemical that targets ovarian follicles and accelerates ovarian failure in rodents, was used to test the effect of early-onset reproductive senescence on mammary fibroadenoma formation. One-month female Sprague Dawley rats were dosed with VCD (80 mg/kg or 160 mg/kg) and monitored for 22 months for persistent estrus and tumor development. Only high-dose VCD treatment accelerated the onset of persistent estrus relative to controls. However, both doses of VCD accelerated mammary tumor onset by 5 months, increasing incidence to 84% (vs. 38% in controls). Tumor development was independent of time in persistent estrus, 17 β-estradiol, androstenedione and prolactin. Delay in VCD administration until after completion of mammary epithelial differentiation (3 months) did not alter tumor formation despite acceleration of ovarian senescence. VCD administration to 1-month rats acutely decreased mammary alveolar bud number and expression of β-casein, suggesting that VCD's tumorigenic effect requires exposure during mammary epithelial differentiation.

Previous work showed that retaining residual ovarian tissue protects young mice from accelerated bone loss following ovarian failure. The present study was designed to determine whether this protection is also present in aged animals. Aged (9-12 months) C57BL/6Hsd female mice were divided into: CON (vehicle), VCD (160 mg/kg; 15d), or OVX (ovariectomized). Lumbar BMD was monitored by DXA and μCT used to assess vertebral microarchitecture. BMD was not different between VCD and CON at any time point but was lower (P .05) than baseline, starting 1 month after ovarian failure in VCD and OVX mice. Following μCT analysis there were no differences between CON and VCD, but OVX mice had lower bone volume fraction, trabecular thickness, and a trend for decreased connectivity density. These findings provide evidence that retention of residual ovarian tissue may protect aged follicle-depleted mice from accelerated bone loss to a lesser extent than that observed in young mice.

Bone loss associated with menopause leads to an increase in skeletal fragility and fracture risk. Relevant animal models can be useful for evaluating the impact of ovarian failure on bone loss. A chemically induced model of menopause in which mice gradually undergo ovarian failure yet retain residual ovarian tissue has been developed using the chemical 4-vinylcyclohexene diepoxide (VCD). This study was designed to compare skeletal effects of VCD-induced ovarian failure to those associated with ovariectomy (OVX). Young (28 day) C57Bl/6Hsd female mice were dosed daily with vehicle or VCD (160 mg/kg/d, IP) for 15 days (n = 6-7/group) and monitored by vaginal cytology for ovarian failure. At the mean age of VCD-induced ovarian failure (approximately 6 wk after onset of dosing), a different group of mice was ovariectomized (OVX, n = 8). Spine BMD (SpBMD) was measured by DXA for 3 mo after ovarian failure and OVX. Mice were killed approximately 5 mo after ovarian failure or OVX, and bone architecture was evaluated by microCT ex vivo. In OVX mice, SpBMD was lower than controls 1 mo after OVX, whereas in VCD-treated mice, SpBMD was not lower than controls until 2.9 mo after ovarian failure (p 0.05). Both VCD-induced ovarian failure and OVX led to pronounced deterioration of trabecular bone architecture, with slightly greater effects in OVX mice. At the femoral diaphysis, cortical bone area and thickness did not differ between VCD mice and controls but were decreased in OVX compared with both groups (p 0.05). Circulating androstenedione levels were preserved in VCD-treated mice but reduced in OVX mice relative to controls (p 0.001). These findings support that (1) VCD-induced ovarian failure leads to trabecular bone deterioration, (2) bone loss is attenuated by residual ovarian tissue, particularly in diaphyseal cortical bone, and (3) the VCD mouse model can be a relevant model for natural menopause in the study of associated bone disorders.

PTH-related protein (PTHrP), the peptide that is responsible for most cases of hypercalcemia of malignancy, is also produced under normal circumstances by a variety of tissues. Its role and regulation at these sites are not well understood. Recently, we have shown that PTHrP is induced in the spleen during the host response to endotoxin (LPS) and that tumor necrosis factor (TNF) is a major mediator of this effect. Given the large body of in vitro evidence suggesting that PTHrP can be produced by lymphocytes and act in an autocrine loop to alter their function, studies were undertaken to determine whether lymphocytes were the cells responsible for PTHrP production in the spleen. Both constitutive and LPS-induced PTHrP messenger RNA (mRNA) levels were the same in mice lacking mature T cells (nude mice) and in mice lacking natural killer (NK) cells (due to pretreatment with antibody against NK 1.1) compared to levels in normal mice, suggesting that neither mature T cells nor NK cells were the splenic source of PTHrP. Even scid mice that lack functioning T and B cells responded to TNF with the induction of splenic PTHrP mRNA levels comparable to those in control mice. Localization of PTHrP mRNA in subfractions of rat spleens after in vivo treatment with LPS confirmed the results of the murine studies; PTHrP mRNA was barely detectable in the lymphocyte-rich single cell fraction of the spleen. In contrast, the stromal fraction of the spleen was enriched with PTHrP mRNA both in the basal state and in response to LPS. A similar pattern of distribution was seen for interleukin-6; LPS only increased mRNA levels of this TNF-inducible cytokine in the splenic stroma. In addition, mRNA for the PTH/PTHrP receptor, which decreased in response to LPS, colocalized with PTHrP mRNA in the stromal fraction of the spleen. Immunohistochemical studies identified PTHrP in two populations of splenic cells: 1) smooth muscle cells located in the splenic capsule and trabeculae and 2) a subpopulation of stromal cells located in the red pulp of the spleen, primarily in a subcapsular distribution. Consistent with the localization of PTHrP mRNA, lymphocytes in the white pulp of the spleen did not stain for PTHrP.

Previously, we reported that PTH-related protein (PTHrP) gene expression is induced in vital organs, including the liver, during endotoxemia. The liver plays a central role in the acute phase response (APR), a cytokine-mediated host defense against infection and inflammation that includes increased production of acute phase proteins and lipids by hepatocytes. Because PTHrP is thought to act locally at its site of production, in vivo studies were carried out to determine whether PTHrP could contribute to the induction of the hepatic APR. Hepatic PTHrP messenger RNA (mRNA) levels were induced acutely in rat liver in response to a near lethal dose of endotoxin. PTHrP protein, which was located by immunohistochemical staining in hepatocytes from both control and LPS-treated rats, was markedly induced in periportal hepatocytes in response to LPS treatment. Co-incident with this transient increase in PTHrP gene expression, PTH/PTHrP receptor mRNA levels were down-regulated. Administration of PTHrP(1-34), a PTH/PTHrP receptor agonist, to mice increased hepatic serum amyloid A (SAA) mRNA levels as well as circulating levels of SAA. In addition, PTHrP(1-34) increased serum triglyceride (TG) levels in rats and mice in a dose-dependent fashion. The hypertriglyceridemic effect of PTHrP(1-34) was accompanied by an increase in hepatic fatty acid synthesis. In contrast, PTHrP(7-34) amide, a receptor antagonist, had no effect on serum SAA or TG levels. These results, which provide evidence for the regulated expression of PTHrP in adult liver, suggest that PTHrP may be one additional member of the cytokine cascade produced locally in liver that can act to stimulate the hepatic acute phase response.