Recipient Organization
UNIV OF HAWAII
3190 MAILE WAY
HONOLULU,HI 96822
Performing Department
Human Nutrition, Food & Animal Sciences
Non Technical Summary
The ovary produces oocytes for the propagation of offspring, and for synthesizing and secreting hormones that essential for the development of the fertilized ovum and maintenance of reproductive function. In cattle, disorders of ovary become economically significant when they adversely affect fertility and fecundity. Ovarian tumors are uncommon in domestic animals, however, they have been reported in the cattle. Incidence of ovarian tumor in cattle is about 4.3% and usually, remain undiagnosed. At the farm level, it is difficult to diagnose ovarian tumor due to the less frequent examination of the reproductive tract using ultrasonography or it also confuses with an ovarian cyst, oophoritis, and ovarian abscesses. Granulosa cell tumor (GCT) is the most commonly reported type of ovarian tumor in cattle. The GCTs are generally unilateral, and the contralateral ovary is frequently small and inactive due to the cessation of estrus cycle activity. Cattle with GCT displays irregular estrus cycles, or anestrus, thus leading to infertility. In Hawaii, the incidence of ovarian tumor in cattle is unknown. Recently, we found a case ovarian tumor in beef cattle, and yet it is unclear about the type of tumor. The aims of this research proposal are to 1) identify the types of bovine ovarian tumors using the markers for granulosa cells, epithelial cell, and theca cells, and 2) to develop the markers of ovarian tumor in the blood.Under this project, ovarian samples will be collected from slaughterhouse, markers of ovarian epithelial tumor, granulaosa cells tumor will be assessed using immunohistochemistry. The serum concentration of AMH, estradiol, and progesterone will be determined. Gene expression will be determined using RNA-Sequencing and Real time RT-PCR. At the end of the study, ovarian tumor biomarker will be established.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Goals / Objectives
The long-term goal of this proposal is to identify and characterize the types of bovine ovarian tumors existing in Hawaii. The specific objectives of this proposal are to study:Characterization of ovarian tumors in cattle using immunohistochemical methods.Establishment of ovarian tumor biomarker in serum.
Project Methods
Materials and methodsAll animal experimentations will be carried out in accordance with the guidelines and ethics of the University of Hawaii Institutional Animal Care and Use Committee (IACUC). Detail information about the animals (age, breed, parity, and body weight), ovarian activity, pregnancy status, uterine pathology will be recorded for hormonal, gene expression, and the pathophysiology of tumor formation. Ovaries from dairy and beef cattle will be collected from different slaughterhouses in Hawaii. A portion of ovarian tissues from both ovaries will be fixed in freshly prepared 4% paraformaldehyde in PBS (pH 7.4); or 10% formalin, and processed as previously described (15) for histomorphological and immunohistological analysis. A portion of ovarian tissues will be snapped frozen and stored at -80 °C for total RNA and protein isolations. Blood will be collected from jugular veins for hormonal analysis.Ovarian histomorphometric analysisOvaries will be fixed in 10% formalin for 2-3 days and will be stored in 70% ethanol until paraffin embedding. Briefly, paraffin-embedded ovaries will be serially sectioned at 5 μm thickness. Every tenth serial section will be stained with Gill's hematoxylin (Fisher Scientific) and 1% eosin Y (Sigma-Aldrich) as previously described (15). Ovarian sections will be examined for abnormalities using light microscopy blind to the pathophysiology of the ovary.Immunohistochemical analysisOvaries will be fixed in 4% paraformaldehyde in PBS, cryoprotected in 15% sucrose, embedded in optimal cutting temperature (OCT) embedding compound (Fisher Scientific) and stored at -80°C until sectioning. Ovaries will be serially sectioned at 7 µm thickness and slides will be stored at -80°C until staining. Slides will be immunostained using specific primary and secondary antibodies as previously described (15). Briefly, ovaries will be serially cryosectioned at 10 μm thickness. Sections will be subjected to antigen retrieval using 10 mmol/L sodium citrate with 0.05% Tween-20 at 95°C, blocked with avidin and biotin blocking reagents (Vector Laboratories) and normal goat serum (Vector Laboratories), and incubated with primary antibodies directed against epithelial cell marker (Pancytokeratin), granulosa cell marker (FOXL2), and cell proliferation marker (Ki67 and PCNA) and then specific secondary antibodies. Sections will be then blocked with 0.3% H202 and incubated with ABC reagent (Vector Laboratories). Immunostaining will be visualized with diaminobenzidine substrate in peroxide buffer (Roche). Sections will lightly be counterstained with hematoxylin. The following negative controls will be included in each immunostaining run: 1) primary antibody without secondary antibody, 2) primary antibody replaced by non-immune IgG with secondary antibody, 3) secondary antibody without primary antibody. Additional controls included ovarian sections from cyclic cows. Scoring of immunostaining will be done without the knowledge of the treatment groups.Analysis of gene expressionTotal RNA will be isolated from frozen tissues using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The quantity of total RNA will be determined by NanoDrop, and the quality of RNA using agarose gel electrophoresis. The synthesis of ?rst-strand cDNA will be performed by reverse transcription of 1 µg total RNA using superscript III with Oligo (DT) 20 primer (Invitrogen). Levels of mRNA expression will be measured by real-time PCR using SYBR green (Stratagene, La Jolla, CA) according to the manufacturer's protocol. Oligonucleotide primers corresponding to each gene will be designed using Primer3 software. The specificity for each primer set will be confirmed by both running the PCR products on a 2% agarose gel and analyzing the melting (dissociation) curve using the Mx Pro real-time PCR analysis program after each real-time PCR reaction as described by (15). All genes will be analyzed in duplicate and the amplification efficiency of each transcript primer set will be determined by running a standard curve. The relative abundance of the target transcripts will be normalized to the endogenous reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (15).Transcriptomic profile of control (healthy) versus nun-tumorous ovaries hen will be analyzed using the RNA-Sequence analysis as previously described (Sah et al, 2018) at the Genomics Core Facility of UH Manoa. Data obtained from RNA-Seq analysis will be validated using qPCR (16).Hormonal analysisImmediately after blood collection, the serum will be separated from each blood sample and stored at -20 °C until further analysis. The serum concentration of AMH, estradiol, and progesterone will be determined by commercial RIA and/or ELISA kits.Statistical analysisAll data will be presented as means ± SEM. One-way ANOVA will be used to test differences in levels of mRNA among the control verses tumorous ovaries. If ANOVA reveals significant effects of treatment, the means will be compared with Tukey's test, with p < 0.05 will be considered significant.