Dual suppression of follicle activation pathways completely prevents the cyclophosphamide-induced loss of ovarian reserve.

STUDY QUESTION: To what extent and how does combined administration of the follicle activation pathway suppressive agents temsirolimus (Tem) and c-terminus recombinant anti-Müllerian hormone (rAMH) protect against chemotherapy-induced ovarian reserve loss? SUMMARY ANSWER: Combined administration of Tem and rAMH completely prevents cyclophosphamide (Cy)-induced follicle depletion and protects the ovarian reserve in mice, primarily via primordial follicle (PMF) suppression of activation and to a lesser degree by reducing apoptosis. WHAT IS KNOWN ALREADY: There is conflicting evidence regarding the contributory roles of apoptosis and follicle activation in chemotherapy-induced PMF loss. Tem, a mammalian target of rapamycin (mTOR) inhibitor, reduces activity of the phosphoinositide 3-kinases-phosphatase and tensin homolog (PI3K-PTEN) pathway which provides intrinsic regulation of PMF activation. Anti-Müllerian hormone (AMH), secreted by early growing follicles, is an extrinsic regulator of PMF activation. STUDY DESIGN, SIZE, DURATION: Whole ovaries of 12-day-old mice were cultured ex vivo for 7 days in the presence of Cy ± rAMH or Tem. Eight-week-old mice were randomized into eight treatment groups: vehicle control/rAMH/Tem/Cy/Tem + rAMH/Cy + Tem/Cy + rAMH/Cy + Tem + rAMH. Twelve hours after treatment, ovaries were removed for DNA damage analysis, and 24 h after treatment either for analysis of PI3K pathway proteins or to be fixed and immunostained for analyses of proliferation and apoptosis. Three or 21 days following treatment, ovaries were fixed and sectioned for follicle counting. PARTICIPANTS/MATERIALS, SETTING, METHODS: Hematoxylin and eosin staining was used for differential follicle counts of primordial, primary, and secondary follicles in ex vivo (n = 16-18 ovaries per group) and in vivo ovaries (n = 8 mice per group). Histological analyses were carried out to measure proliferation by quantifying Ki-67-positive granulosa cells in primary follicles (n = 4 mice per group). DNA damage and apoptosis were measured by quantification of phosphorylated form of histone 2AX (γH2AX) and cleaved poly (ADP-ribose) polymerase (cPARP)-positive PMF oocytes, respectively (n = 8 mice per group). Protein extracts from whole ovaries were analyzed by western blotting. MAIN RESULTS AND THE ROLE OF CHANCE: In vivo experiments show that treatment with Cy alone caused significant loss of PMF reserve (32 ± 2.12 versus 144 ± 2.8 in control, P < 0.001), and this was significantly attenuated by treatment with either Tem (P < 0.001) or rAMH (P < 0.001). Combined cotreatment with Cy + Tem + rAMH provided complete protection of the PMF reserve, with no significant difference in numbers of PMF versus untreated animals. Similar results were demonstrated in the ex vivo experiments. Proliferation marker Ki-67 staining was significantly reduced in granulosa cells of primary follicles in the Cy + Tem + rAMH group compared with Cy alone group (after 24 h in vivo administration of Cy, 16% versus 65%, respectively; P < 0.001). Protein analysis demonstrated not significant increased phosphorylation of follicle activation proteins rpS6 and mTOR with in vivo administration of Cy alone (1.9 and 1.4 times the control ovaries, respectively), and this was reduced to below control levels in the Cy + Tem + rAMH group (P < 0.01). The Cy + Tem + rAMH combined cotreatment protected the follicle reservoir via inhibition of Cy-induced upregulation of the PI3K signaling pathway, together with replacement of AMH suppression of PMF activation with rAMH, implying a complementary effect of the two inhibitors. The DNA damage marker γH2AX was highly positive in PMF oocytes from Cy-treated ovaries 12 h after treatment, compared with controls (94% versus 59%, respectively, P < 0.001) and was significantly reduced to (69%) in Cy + Tem + rAMH cotreated ovaries (P < 0.001). However, only 22% of PMF oocytes of the Cy group showed apoptosis at 24 h, and this was significantly reduced (12%) in ovaries after treatment with Cy + Tem + rAMH (P < 0.01). This suggests that it is not possible to equate DNA damage with oocyte death, and also indicates that less than one-third of the total PMF loss can be attributed to apoptosis, implying that most of the PMF depletion results from PMF activation but that both mechanisms play a significant role. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The experimental design was limited by the selection of one time point for analysis of PMF activation and apoptosis (i.e. 24 h after Cy administration), although DNA damage was measured at 12 h after Cy administration and any impact on short-term follicle dynamics at 3 days after treatment. Protein analysis was conducted on whole ovary lysates therefore the protein changes identified cannot be localized to specific cells within the ovary. However, this complementary assay showed that there was activation in the ovary through massive reduction in the phosphorylation of key proteins in the PI3K cascade (rpS6 and mTOR), which is consistent with the sequence of events after Cy administration. WIDER IMPLICATIONS OF THE FINDINGS: Understanding the complementary nature of different follicle activation pathways and the impact of their suppression in prevention of chemotherapy-induced ovotoxic damage, as well as their involvement in DNA damage inhibition, provides an interesting direction for future research, and the potential for noninvasive pharmacological fertility preservation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a grant from the Morris Kahn Foundation. The authors declare no conflicts of interest.

Ovaries of patients recently treated with alkylating agent chemotherapy indicate the presence of acute follicle activation, elucidating its role among other proposed mechanisms of follicle loss.

OBJECTIVE: To investigate mechanisms of primordial follicle (PMF) loss in vivo in human ovaries shortly after alkylating agent (AA) chemotherapy. DESIGN: Cohort study. SETTING: Tertiary university medical center. PATIENT(S): Ninety-six women aged 15-39 years who underwent ovarian tissue cryopreservation for fertility preservation. INTERVENTION(S): Fresh ovarian tissue samples were harvested from women treated with AA (n = 24) or non-AA (n = 24) chemotherapy <6 months after treatment and age-matched untreated women (n = 48). MAIN OUTCOME MEASURE(S): Differential follicle counts, time from chemotherapy exposure, immunostaining for apoptosis (cleaved caspase-3) and FOXO3A on tissue harvested within ultrashort time intervals (4-12 days), collagen (Sirius red) and neovascularization (CD34). RESULT(S): AA-treated ovaries had significant loss of PMFs, and significant increase in absolute numbers of growing follicles compared with untreated control ovaries. The number of growing follicles was inversely correlated with time from chemotherapy. Representative staining for FOXO3A observed decreased nuclear localization in PMF oocytes in AA-treated ovaries removed within the ultrashort time interval compared with untreated ovaries. Neither significant loss of PMFs, increase in growing follicles, nor decrease in nuclear FOXO3A were observed in non-AA-treated ovaries. No increased expression of cleaved caspase-3 was seen in PMFs within the ultrashort time interval after AA or non-AA chemotherapy. Significant stromal fibrosis and neovascularization were observed in AA-treated ovaries only after follicle loss had already occurred (4-6 months). CONCLUSION(S): Follicle activation occurs in vivo in ovaries of patients treated with AA, indicating a pathologic mechanism which may contribute to chemotherapy-induced follicle loss.

Practical Applications of in Vivo and ex Vivo MRI in Toxicologic Pathology Using a Novel High-performance Compact MRI System.

Magnetic resonance imaging (MRI) is widely used in preclinical research and drug development and is a powerful noninvasive method for assessment of phenotypes and therapeutic efficacy in murine models of disease. In vivo MRI provides an opportunity for longitudinal evaluation of tissue changes and phenotypic expression in experimental animal models. Ex vivo MRI of fixed samples permits a thorough examination of multiple digital slices while leaving the specimen intact for subsequent conventional hematoxylin and eosin (H&E) histology. With the advent of new compact MRI systems that are designed to operate in most conventional labs without the cost, complexity, and infrastructure needs of conventional MRI systems, the possibility of MRI becoming a practical modality is now viable. The purpose of this study was to investigate the capabilities of a new compact, high-performance MRI platform (M2™; Aspect Imaging, Israel) as it relates to preclinical toxicology studies. This overview will provide examples of major organ system pathologies with an emphasis on how compact MRI can serve as an important adjunct to conventional pathology by nondestructively providing 3-dimensional (3-D) digital data sets, detailed morphological insights, and quantitative information. Comparative data using compact MRI for both in vivo and ex vivo are provided as well as validation using conventional H&E.

Halofuginone - the multifaceted molecule.

Halofuginone is an analog of febrifugine-an alkaloid originally isolated from the plant Dichroa febrifuga. During recent years, halofuginone has attracted much attention because of its wide range of beneficial biological activities, which encompass malaria, cancer, and fibrosis-related and autoimmune diseases. At present two modes of halofuginone actions have been described: (1) Inhibition of Smad3 phosphorylation downstream of the TGFß signaling pathway results in inhibition of fibroblasts-to-myofibroblasts transition and fibrosis. (2) Inhibition of prolyl-tRNA synthetase (ProRS) activity in the blood stage of malaria and inhibition of Th17 cell differentiation thereby inhibiting inflammation and the autoimmune reaction by activation of the amino acid starvation and integrated stress responses. This review deals with the history and origin of this natural product, its synthesis, its known modes of action, and it's various biological activities in pre-clinical animal models and in humans.

The involvement of collagen triple helix repeat containing 1 in muscular dystrophies.

Fibrosis is the main complication of muscular dystrophies. We identified collagen triple helix repeat containing 1 (Cthrc1) in skeletal and cardiac muscles of mice, representing Duchenne and congenital muscle dystrophies (DMD and CMD, respectively), and dysferlinopathy. In all of the mice, Cthrc1 was associated with high collagen type I levels; no Cthrc1 or collagen was observed in muscles of control mice. High levels of Cthrc1 were also observed in biopsy specimens from patients with DMD, in whom they were reversibly correlated with that of ß-dystroglycan, whereas collagen type I levels were elevated in all patients with DMD. At the muscle sites where collagen and Cthrc1 were adjacent, collagen fibers appeared smaller, suggesting involvement of Cthrc1 in collagen turnover. Halofuginone, an inhibitor of Smad3 phosphorylation downstream of the transforming growth factor-ß signaling, reduced Cthrc1 levels in skeletal and cardiac muscles of mice, representing DMD, CMD, and dysferlinopathy. The myofibroblasts infiltrating the dystrophic muscles of the murine models of DMD, CMD, and dysferlinopathy were the source of Cthrc1. Transforming growth factor-ß did not affect Cthrc1 levels in the mdx fibroblasts but decreased them in the control fibroblasts, in association with increased migration of mdx fibroblasts and dystrophic muscle invasion by myofibroblasts. To our knowledge, this is the first demonstration of Cthrc1 as a marker of the severity of the disease progression in the dystrophic muscles, and as a possible target for therapy.

Involvement of host stroma cells and tissue fibrosis in pancreatic tumor development in transgenic mice.

INTRODUCTION: Stroma cells and extracellular matrix (ECM) components provide the pivotal microenvironment for tumor development. The study aimed to evaluate the importance of the pancreatic stroma for tumor development. METHODS: Pancreatic tumor cells were implanted subcutaneously into green fluorescent protein transgenic mice, and stroma cells invading the tumors were identified through immunohistochemistry. Inhibition of tumor invasion by stroma cells was achieved with halofuginone, an inhibitor of TGFß/Smad3 signaling, alone or in combination with chemotherapy. The origin of tumor ECM was evaluated with species-specific collagen I antibodies and in situ hybridization of collagen α1(I) gene. Pancreatic fibrosis was induced by cerulean injection and tumors by spleen injection of pancreatic tumor cells. RESULTS: Inhibition of stroma cell infiltration and reduction of tumor ECM levels by halofuginone inhibited development of tumors derived from mouse and human pancreatic cancer cells. Halofuginone reduced the number only of stroma myofibroblasts expressing both contractile and collagen biosynthesis markers. Both stroma myofibroblasts and tumor cells generated ECM that contributes to tumor growth. Combination of treatments that inhibit stroma cell infiltration, cause apoptosis of myofibroblasts and inhibit Smad3 phosphorylation, with chemotherapy that increases tumor-cell apoptosis without affecting Smad3 phosphorylation was more efficacious than either treatment alone. More tumors developed in fibrotic than in normal pancreas, and prevention of tissue fibrosis greatly reduced tumor development. CONCLUSIONS: The utmost importance of tissue fibrosis and of stroma cells for tumor development presents potential new therapy targets, suggesting combination therapy against stroma and neoplastic cells as a treatment of choice.

Inhibition of pancreatic stellate cell activation by halofuginone prevents pancreatic xenograft tumor development.

OBJECTIVES: Most solid tumors consist of neoplastic and nonneoplastic cells and extracellular matrix components. In the pancreas, activated stellate cells (PSCs) are the source of the extracellular matrix proteins. We evaluated the significance of PSC activation in tumor establishment and development in mouse xenografts. METHODS: Xenografts were established by implanting human pancreatic cancer cells (MiaPaca-2) subcutaneously or orthotopically by injecting them into the spleen. Fibrosis was induced by cerulein. Collagen level was evaluated by Sirius red staining. Prolyl 4-hydroxylase ß and stellate cell activation-associated protein (Cygb/STAP) were determined by immunohistochemistry. RESULTS: Halofuginone inhibited subcutaneous tumor development implanted with Matrigel and reduced collagen and prolyl 4-hydroxylase ß levels. Few tumors, which developed slowly, were observed after MiaPaca-2 implantation without Matrigel. Increase in tumor number and rate of development were observed with addition of PSCs from control pancreas, and further increase was observed when the PSCs were from cerulein-treated mice. Preincubation of the PSCs with halofuginone elicited Cygb/STAP level reduction and tumor growth inhibition. More tumors developed orthotopically in cerulein-treated mice than in controls; this was prevented by halofuginone. CONCLUSIONS: Extracellular matrix production by activated PSCs is essential for tumor establishment and growth. Thus, inhibition of PSC activation is a viable means of reducing pancreatic tumor development.