Evaluation of biomarkers for testicular toxicity

Non-clinical safety assessment is essential during the drug development process in the pharmaceutical industry, and involves numerous, detailed in vitro and in vivo toxicology tests (general, reproductive and genetic), and safety pharmacology studies. The testis is a common organ for adverse drug effects leading to attrition of potential compounds. It would, therefore, be useful to detect testicular toxicity as early as possible in the drug development process. Histopathology is the standard method for assessing testis toxicity, but a biomarker for ‘early warning’ detection of testicular toxicity would be far more useful in non-clinical toxicology studies. The aim of this thesis was to evaluate the feasibility of this approach. It is thought that proteins can leak from seminiferous tubules into testicular interstitial fluid following testicular damage, due to either loss of integrity of the blood-testis barrier (BTB) or germ cell damage. A potential biomarker protein could, therefore, leak out of seminiferous tubules into interstitial fluid and then into blood following toxicological insult to the testis. A suitable biomarker protein must be testis specific, abundant, and not normally be present in blood. It may also need to have a low molecular weight. To investigate if proteins do leak out of seminiferous tubules following testicular damage, three known testicular toxicants which affect different aspects of the testis were used; cadmium chloride causes disruption to the blood-testis barrier and spermatogenesis, methoxyacetic acid (MAA) specifically causes a loss of pachytene spermatocytes, and 1,3-dinitrobenzene (DNB) causes Sertoli cell vacuolation and subsequent germ cell disruption. Adult male Wistar rats were treated with various doses of these toxicants to give mild and moderate responses. Samples were collected 24 hours later. Testicular damage was investigated by immunohistochemistry for well-known germ cell markers (DAZL, VASA) and using a general antibody to seminiferous tubule proteins. The integrity of the BTB was evaluated using immunofluorescent co-localisation of occludin, ZO-1, claudin-11, N-cadherin and β-catenin, and a biotin tracer. Protein leakage was investigated using analysis of interstitial fluid samples by 1D gel electrophoresis and staining with Coomassie-based dye or Western blotting for germ cell proteins and with the general antibody to seminiferous tubule proteins. Protein leakage from seminiferous tubules into interstitial fluid was observed with high dose cadmium chloride treatment. This was coincident with a loss of integrity of the BTB. No leakage was observed with MAA treatment which caused a specific loss of pachytene spermatocytes, or DNB which caused Sertoli cell vacuolation. With both treatments the BTB did not appear to be damaged suggesting that protein leakage occurs only following loss of integrity of the BTB. This was further investigated using treatments reported to specifically disrupt the BTB, namely intra-testicular administration of glycerol or transforming growth factor-β3, with samples collected 48 hours later. The damage caused was very localised, although BTB disruption with glycerol treatment caused some protein leakage. The presence of germ cell proteins in interstitial fluid samples before and after the development of the BTB during normal development was also evaluated, although most proteins of interest were not expressed in germ cells of the immature testis before BTB formation. Finally, five potential biomarker candidate proteins (ADAM3, Calpastatin, DAZL, FABP9, VASA) were selected and investigated using samples from the testicular toxicant studies. Smaller molecular weight proteins were thought to be more likely to leak out of seminiferous tubules, however, VASA, a large molecular protein (76kDa) was shown to leak into interstitial fluid following high dose cadmium chloride treatment. However, FABP9 (low molecular weight) was found to be the most promising biomarker for loss of BTB integrity. The results suggest that a biomarker could only be detected if there is a loss of integrity of the BTB and severe disruption of spermatogenesis, thus conferring no real advantage over present histopathology-based toxicity evaluations. Therefore, an automated immunohistochemistry and image analysis method was investigated as a refined method for detection of testicular toxicity at the end of a toxicology study, and shown to have promise

The Founder Strains of the Collaborative Cross Express a Complex Combination of Advantageous and Deleterious Traits for Male Reproduction

Surveys of inbred strains of mice are standard approaches to determine the heritability and range of phenotypic variation for biomedical traits. In addition, they may lead to the identification of novel phenotypes and models of human disease. Surprisingly, male reproductive phenotypes are among the least-represented traits in the Mouse Phenome Database. Here we report the results of a broad survey of the eight founder inbred strains of both the Collaborative Cross (CC) and the Diversity Outbred populations, two new mouse resources that are being used as platforms for systems genetics and sources of mouse models of human diseases. Our survey includes representatives of the three main subspecies of the house mice and a mix of classical and wild-derived inbred strains. In addition to standard staples of male reproductive phenotyping such as reproductive organ weights, sperm counts, and sperm morphology, our survey includes sperm motility and the first detailed survey of testis histology. As expected for such a broad survey, heritability varies widely among traits. We conclude that although all eight inbred strains are fertile, most display a mix of advantageous and deleterious male reproductive traits. The CAST/EiJ strain is an outlier, with an unusual combination of deleterious male reproductive traits including low sperm counts, high levels of morphologically abnormal sperm, and poor motility. In contrast, sperm from the PWK/PhJ and WSB/EiJ strains had the greatest percentages of normal morphology and vigorous motility. Finally, we report an abnormal testis phenotype that is highly heritable and restricted to the WSB/EiJ strain. This phenotype is characterized by the presence of a large, but variable, number of vacuoles in at least 10% of the seminiferous tubules. The onset of the phenotype between 2 and 3 wk of age is temporally correlated with the formation of the blood-testis barrier. We speculate that this phenotype may play a role in high rates of extinction in the CC project and in the phenotypes associated with speciation in genetic crosses that use the WSB/EiJ strain as representative of the Mus muculus domesticus subspecies

A Conserved Requirement for fbxo7 during Male Germ Cell Cytoplasmic Remodelling

Fbxo7 is the substrate-recognition subunit of an SCF-type ubiquitin E3 ligase complex. It has physiologically important functions in regulating mitophagy, proteasome activity and the cell cycle in multiple cell types, like neurons, lymphocytes and erythrocytes. Here, we show that in addition to the previously known Parkinsonian and hematopoietic phenotypes, male mice with reduced Fbxo7 expression are sterile. In these males, despite successful meiosis, nuclear elongation and eviction of histones from chromatin, the developing spermatids are phagocytosed by Sertoli cells during late spermiogenesis, as the spermatids undergo cytoplasmic remodeling. Surprisingly, despite the loss of all germ cells, there was no evidence of the symplast formation and cell sloughing that is typically associated with spermatid death in other mouse sterility models, suggesting that novel cell death and/or cell disposal mechanisms may be engaged in Fbxo7 mutant males. Mutation of the Drosophila Fbxo7 ortholog, nutcracker (ntc) also leads to sterility with germ cell death during cytoplasmic remodeling, indicating that the requirement for Fbxo7 at this stage is conserved. The ntc phenotype was attributed to decreased levels of the proteasome regulator, DmPI31 and reduced proteasome activity. Consistent with the fly model, we observe a reduction in PI31 levels in mutant mice; however, there is no alteration in proteasome activity in whole mouse testes. Our results are consistent with findings that Fbxo7 regulates PI31 protein levels, and indicates that a defect at the late stages of spermiogenesis, possibly due to faulty spatial dynamics of proteasomes during cytoplasmic remodeling, may underlie the fertility phenotype in mice

Assessing the toxicity of engineered nanomaterials in the male reproductive system: Developing an improved testing strategy for hazard assessment

Reproduction is the only certain constant within animals and maintaining the integrity of this process is key to ensuring survival of the species. However, male fertility rates in the Western world are in decline and the use of assisted reproductive techniques rising (Makarow and Hojgaard, 2010). With emerging technologies such as nanotechnologies presenting both enormous benefits alongside new risks, there is a clear need to understand and manage these potential novel threats to male reproductive health. However, the animal burden associated with such research is extreme, therefore consideration of how to reduce, refine or replace animal use in the associated developmental and reproductive toxicity (DART) testing is paramount.This research aimed to assess the toxicity of engineered nanomaterials (ENM) in the male reproductive system and develop an improved testing strategy for hazard assessment of DART. Using a panel of highly representative ENM, assays to screen for toxicity in male testicular cell lines were established and optimised in vitro. Findings were then validated for reliability and reproducibility against those generated using similar test systems with cells from alternative organs of the body. Assessment of cell and endocrine function also provided a deeper understanding of cellular responses following acute sub-lethal ENM exposure.Comparison of outcomes in vitro to in vivo was enabled by appraisal of tissues from animals exposed orally to the same ENM. Through this, a new method by which to stage tubules for histopathological analysis was developed, and for the first time a truly thorough morphological and stereological examination of tissues for markers of effect provided. Assimilation of key findings made it possible to conclude that silver ENM are reproductive toxicants and potential endocrine disruptors. The accumulated results were also used to guide development of a novel Integrated Approach to Testing and Assessment (IATA) for male reproductive toxicity from ENM

Testicular morphological and biochemical perturbations in experimental animals under antiretroviral therapy and the role of Naringenin, a bioactive flavonoid.

Doctoral Degree. University of KwaZulu-Natal, Durban.Declining male fertility is one of the neglected concerns of people living with HIV/AIDS in spite of a dual outlook of a social and a health dilemma. This issue of infertility is particularly relevant as majority of affected individuals are in their reproductive years. This thesis examines the impacts of the Fixed Dose Combination (FDC) of Highly Active Antiretroviral Therapy (HAART) Tenofovir/ Emtricitabine and Efavirenz (TDF/FTC/EFV) on the male reproductive capacity. It also explores the protective potentials of a bioactive flavonoid, Naringenin in testicular perturbations. The study was motivated by two major research questions namely: (1) what are the impacts of the recently approved first line antiretroviral therapy for adults FDC, TDF/FTC/EFV on the testes? (2) What is the role of Naringenin in alleviating testicular perturbations induced by HAART? Previous studies point to the negative impacts of the older generation of FDC of HAART on the semen quality and histomorphometry of the testes following a long-term use. The study addresses both the long-term and short-term use of antiretroviral drugs as observed in pre-exposure prophylaxis (PrEP) and post exposure prophylaxis (PEP). The research assesses the impacts of the drugs on the reproductive capability as well. Findings from this study support the argument that the negative effects of the drugs were consequent upon both the short-term and long-term use. To illustrate this hypothesis, the study was conducted in two distinct phases. The first phase which lasted a total of 28 days considered the duration of PEP or PrEP. The second phase which lasted a total of ten weeks captured all the stages of spermatogenesis in rats. In this phase male Sprague Dawley rats were exposed to fertile females after the treatments. The study thus advances an understanding of the mechanism of HAART-induced testicular injury. A therapeutic dose of TDF/FTC/EFV adjusted for animal weight was aministered on a total of 48 animals randomly divided into 6 equal groups each with a different treatment as follows; Group A: Control (Distilled water); Group B: HAART (TDF/FTC/EFV), Group C: Naringenin, 40 mg/kg; Group D: Naringenin, 80 mg/kg; Group E: HAART + Naringenin, 40 mg/kg; Group F: HAART+ Naringenin, 80 mg/kg. At the end of each phase, harvested testes were subjected to histomorphometry and ultrastructural analysis. The caudal epididymis was assessed for semen parameters and sperm mitochondrial DNA (mtDNA) fragmentation. Biochemical parameters such as serum levels of reproductive hormones (Luteinizing hormone and Testosterone) and intratesticular antioxidant enzyme activities were assayed. Contrary to prior beliefs, this research reveals that the immediate effects following short-term use of HAART are far more deleterious. This finding is consequent upon the significant drop in the sperm count (p˂0.001) and sperms with normal morphology (p˂0.001) compared to (p˂0.01) in the long-term. Histomorphometric analysis also revealed a significantly shrunken seminiferous tubule following a short-term use. These outcomes were associated with an increase in the mtDNA fragmentation in group B when compared to control (p˂0.05). Naringenin reversed abnormalities in groups E and F, displaying better semen parameters in both count and motility. Serum levels of testosterone were altered in both phases. The overall effects of all these changes were observed in the pregnancy rate which reduced in group B when compared to all the other groups. This study established that HAART has deleterious effects on the testicular microanatomy and function. These effects may impact on steroidogenesis and ultimately spermatogenesis. It consequently impairs fertility while Naringenin promises to be a potential complimentary adjuvant especially in the short term therapies. Keywords: HAART, semen parameters, reproductive hormones, testicular ultrastructure, 3 beta hydroxysteroid dehydrogenase

Mitochondrial Dynamics and Mitophagy during Male Germline Development

Mitochondrial fusion and fission (mitochondrial dynamics) and mitophagy are well-established mitochondrial quality control mechanisms that safeguard cellular homeostasis. However, their role during development remains poorly understood. In this thesis, we establish the role of mitochondrial dynamics and mitophagy during the development of the male germline (spermatogenesis). Spermatogenesis is one of biology’s most complex and lengthy differentiation processes, transforming spermatogonial stem cells into highly specialized sperm cells capable of fertilization. This elaborate differentiation program requires multiple transitions in mitochondrial morphology and extensive degradation of mitochondria, making it an attractive model system for investigating mitochondrial dynamics and mitophagy in vivo. Indeed, the field of mitochondrial dynamics has a long history with spermatogenesis. The first mitochondrial dynamics gene, Fuzzy onions (Fzo), was discovered in 1997 to mediate mitochondrial fusion during Drosophila spermatogenesis. However, the role of mitochondrial dynamics during mammalian spermatogenesis remained unknown for nearly two decades after discovery of Fzo. To address this gap in knowledge, we investigate mitochondrial dynamics and mitophagy during mammalian spermatogenesis. We uncover essential roles for mitochondrial fusion (Chapter 2), mitochondrial fission (Chapter 3), and mitophagy (Chapter 4) during spermatogenesis and show that each of these mitochondrial quality control mechanisms regulates a distinct stage of germ cell development. Our analyses reveal requirements for mitochondrial fusion, fission, and mitophagy that correspond to the mitochondrial and metabolic needs of the developing germ cells. We also investigate the role of mitochondrial fusion and fission in regulating subcellular mitochondrial domains upon fusion of a skeletal muscle stem cell with a myofiber (Chapter 5). Thus, the work presented in this thesis characterizes the in vivo role of mitochondrial dynamics in two systems: male germline development and skeletal muscle regeneration. However, we focus on the role of mitochondrial dynamics and mitophagy during male germline development.</p

Germ Cell Tumor

The book aims to provide an overview of current knowledge regarding germ cell tumors. It deals with the clinical presentations, treatment modalities, the biology and genetics of germ cell tumors in children and adults. Most chapters are focused on testicular germ cell tumors whose incidence has been increasing in young males. Included are reviews on the pathogenesis, risk factors, diagnosis and treatment regimens applied to precursor, pre-invasive lesions as well as to seminomatous and non-seminomatous germ cell tumors of the testes. In addition, a review is included on the diagnosis and current management options for intracranial germ cell tumors in children. Authors have also contributed articles on the genetics and epigenetics of germ cell tumor development in humans and in the mouse model system. This book will be of interest to scientists, physicians and lay readers wishing to review recent developments in the field of germ cell cancers

SPAG16 is a Bifunctional Gene Regulating Male Fertility

SPAG16 is the murine orthologue of Chlamydomonas reinhardtii PF20, a protein known to be essential to the structure and function of the “9 + 2” axoneme. The “9 + 2” axoneme provides the cytoskeletal core of all eukaryotic motile cilia and flagella. In Chlamydomonas, the Pf20 gene encodes a single protein present in the central pair of the axoneme. Loss of Pf20 prevents central pair assembly and results in flagellar paralysis. The murine Spag16 gene encodes two proteins. While 71 kDa SPAG16L is found in all murine cells with motile cilia or flagella, 35 kDa SPAG16S transcript and protein are detected only in male germ cells, suggesting a unique role distinct from general axoneme formation. Transgenic mouse studies published previously by our lab have shown that abrogation of both SPAG16 isoforms causes arrest of spermatogenesis, and the mutant allele is not transmitted to offspring by chimeric males. Mice homozygous for a knock-out of SPAG16L alone are infertile, but show no abnormalities in spermatogenesis. The defects seen in chimeric Spag16 mutant mice, unaccounted for by loss of SPAG16L, indicate a possible role for SPAG16S in the specialized process of male germ cell development. Our results demonstrate that SPAG16S is predominantly found in specific regions within the nucleus of round spermatids. These nuclear sub-domains also contain SC35, a known marker of nuclear speckles enriched in pre-mRNA splicing factors. Putative interaction partners of SPAG16S are also shown to play critical roles in the peri-nuclear region during the round spermatid transition to the condensation and elongation stage of spermiogenesis, the final specialization point in sperm development. The distinct localization of SPAG16S at this critical juncture, its interaction with discretely localized proteins at a critical temporal junction in spermatogenesis, and its ability to modulate SPAG16L expression, suggest that SPAG16S plays an important role in the gene expression machinery of male germ cells, and represents an evolutionary distinction in axoneme gene function