School Of Veterinary Medicine
Non Technical Summary
Age-related testicular degeneration is a common cause of progressive infertility in stallions that can end in sterility. Resulting economic losses in the equine breeding industry are substantial and stem from losses of breeding fees, increased management costs and loss of valuable male genetics. To date, the underlying causes of the disease remain poorly understood and, as a result, testicular degeneration is largely characterized by the appearance of a common set of progressive clinical signs including an increase in softness and a decrease in size of the testis and a decline in semen quality. In large part due to our lack of understanding of the disease, there is no satisfactory treatment. Xenografting is a novel technique developed by us that involves grafting of small pieces of testicular tissue from a donor species (in this case the stallion) under the back skin of host mice. We have shown that xenografting of both normal and cryptorchid stallion testicular tissue onto mice results in the reconstitution of horse spermatogenesis within the grafts on the mice. This technique will now allow us to test a variety of hypotheses relating to equine testicular function and disease without having to perform experiments on horses. Instead, small pieces of testicular tissue recovered following castration or from biopsy sampling of intact testicles can be grafted and experiments can be performed on the host mouse. We will use this technique to study the underlying cause of and to explore potential treatments for testicular degeneration in stallions. Testicular degeneration is a common and progressive problem that affects many stallions as they age. At the least, it results in reduced fertility and at the worst, it can end in complete sterility for a stallion. We have used testis tissue xenografting to show that, in very severe cases of testicular degeneration, the testis itself, and not the extratesticular environment (e.g., an abnormal hormonal environment) is defective. We will now graft testicular tissue affected by more mild degrees of testicular degeneration to determine at what point in the disease process the testis becomes irreparable. If the testis itself is defective, then this transfer should have no effect on the condition of the grafted tissue. However, if the testis is normal and the extratesticular environment of the stallion is the problem, then the transfer should result in at least some improvement in the condition of the testicle. We then will expand on this study by administering a variety of treatments that are commonly used in clinical practice to the host mice carrying degenerate equine testicular tissue and will determine if any of these treatments benefit the condition. Xenografting will allow us to answer many questions involving this important disease for relatively little expense and without having to perform experiments on horses. The results of these experiments should provide us with valuable information on the basic mechanisms underlying testicular degeneration and also will provide practitioners with valuable information on both effective and ineffective treatment modalities for the disease.
Animal Health Component
Research Effort Categories
Goals / Objectives
Objective 1: To apply testis tissue xenografting to the study of the pathophysiology of idiopathic testicular degeneration (ITD) in stallions. Specific Aim 1a: To evaluate changes in spermatogenic efficiency in ITD seen in response to modulation of the endocrine environment. Hypothesis: Supplementation of species-specific exogenous gonadotropins will not rescue the ITD phenotype. Specific Aim 1b: To rescue the ITD phenotype by co-grafting of healthy stallion testicular tissue. Hypothesis: Paracrine effects provided by co-grafted, healthy testicular tissue may benefit the affected testis. Specific Aim 1c: To determine whether the primary defect in ITD, regardless of the stage of the disease, lies within the testis or with the extratesticular environment. Hypothesis: The primary defect in stallions affected to any degree with ITD lies within the testis. Objective 2: To test the efficacy of commonly-used treatments for testicular disease. Specific Aim 1. To evaluate changes in spermatogenic efficiency in ITD seen in response to common clinical treatment modalities. Hypothesis: If ITD is tissue autonomous, then nonspecific treatments designed to modify the extratesticular environment or drive function of the diseased testis will be ineffective, particularly during the later stages of the disease.
General Methods: Grafting procedure: Experiments will be performed using horses as donor species. Small fragments of testis tissue (1 mm3) obtained from castrations, biopsies or post-mortem samples will be grafted under the back skin of anesthetized male NCR nu/nu mice through small incisions as previously described (Honaramooz et al., 2002). Up to 8 grafts will be placed on each host mouse. The mice will be castrated at the time of grafting. Unless otherwise stated, all experiments will be replicated in a minimum of three to five donors. Comparable tissue pieces will be fixed at the time of grafting to serve as a reference point for graft development. Analysis of the grafted testes: The host mice will be killed at time points between 2 and 8 months post grafting. The skin tissue containing the grafts will be dissected and grafts from all experiments will be fixed and processed and stained for histology. In each graft, all seminiferous tubule cross-sections will be examined for the status of testicular maturation (seminiferous tubule and lumen diameter), and spermatogenesis (most advanced germ cell type). The number of spermatogonia/ gonocytes per tubular cross section and per 100 Sertoli cells will be counted during the pre-meiotic stage of spermatogenesis. Mouse will be considered as the experimental unit. The data obtained from grafts from a single mouse will be pooled and the average of the results from all the mice in an experimental group will be analyzed. Seminal vesicles from all recipient mice will be weighed as an indication of secretion of bioactive testosterone by the xenografts Statistical Analysis: Where appropriate, a Chi-square or a Student's t-test will be performed to compare two groups. When comparing more than one group, ANOVA will be used. Data will be expressed as means +/- SEM and p < 0.05 will be considered significant. Experiment 1.1: Supplementation of species-specific exogenous gonadotropins. Mice carrying xenografts from stallions affected with testicular degeneration will be begun on a course of twice weekly injections of equine FSH and hCG. Untreated animals will serve as controls. All grafts will be analyzed 35 weeks after grafting. Experiment 1.2: Co-grafting Samples of testicular tissue from a minimum of 3-5 stallions affected with severe ITD will be grafted onto mice together and in contact with testicular samples from normal prepubertal colts. We will determine if contact with the normal testicular tissue has a beneficial effect on the development of the diseased tissue. Control mice will carry normal and TD grafts that are not in physical contact. Experiment 1.3: Early stage TD. We will xenograft testicular tissue from stallions with more mild TD to determine if, at earlier stages of the disease, the testicular tissue can be rescued. Experiment 2.1: Treatment of host mice. Mice carrying xenografts of testicular tissue affected by TD will be treated with a variety of agents that currently are used in clinical practice to treat stallions with TD. We will determine if any of these treatments affects outcome of the grafts.