Can we induce spermatogenesis in the domestic cat using an in vitro tissue culture approach?

The reduced number of animals in most wild felid populations implies a loss of genetic diversity. The death of juveniles, prior to the production of mature sperm, represents a loss of potential genetic contribution to future populations. Since 2011 mouse testicular organ culture has introduced an alternative mechanism to produce sperm in vitro from immature tissue. However, extension of this technology to other species has remained limited. We have used the domestic cat (Felis catus) as a model for wild felids to investigate spermatogenesis initiation and regulation, with the mouse serving as a control species. Testicular tissue fragments were cultured in control medium or medium supplemented with knockout serum replacement (KSR), AlbuMax, beta-estradiol or AlbuMax plus beta-estradiol. Contrary to expectations, and unlike results obtained in mouse controls, no germ cell differentiation could be detected. The only germ cells observed after six weeks of culture were spermatogonia regardless of the initial stage of tubule development in the donor tissue. Moreover, the number of spermatogonia decreased with time in culture in all media tested, especially in the medium supplemented with KSR, while AlbuMax had a slight protective effect. The combination of AlbuMax and beta-estradiol led to an increase in the area occupied by seminiferous tubules, and thus to an increase in total number of spermatogonial cells. Considering all the media combinations tested the stimulus for felid germ cell differentiation in this type of system seems to be different from the mouse. Studies using other triggers of differentiation and tissue survival factors should be performed to pursue this technology for the genetic diversity preservation in wild felids.

High glucose levels affect spermatogenesis: an in vitro approach.

Besides known factors that may cause male infertility, systemic diseases such as diabetes mellitus may further exacerbate a decline in male fertility. This metabolic disease, clinically characterised by a hyperglycaemic phenotype, has devastating consequences in terms of human health, with reproductive dysfunction being one of the associated clinical complications. Nonetheless, the mechanisms responsible for such alterations are still poorly understood due to the multiplicity of factors involved in the induced pathophysiological changes. With this in mind, we focused on the main mediator of diabetes-associated alterations and performed an in vitro approach to address the effects of high glucose conditions on spermatogenesis, avoiding other confounding in vivo factors. Mouse (5 days post partum) testis fragments were cultured on agar gel stands at a gas-liquid interface with either 5, 25 or 50mM D-glucose for 3 weeks. Stereological analysis revealed that high D-glucose levels increased Sertoli cell number (P<0.05) and decreased tubular luminal area (P<0.01), suggesting an impairment of this somatic cell type. Moreover, higher proliferative activity in a TM4 Sertoli cell line exposed to high D-glucose was found (P<0.05) without compromising cell viability (P>0.05), further suggesting altered Sertoli cell maturation. Overall, high D-glucose concentrations may lead to impairment of Sertoli cell function, which, given their significant role in spermatogenic control, may compromise male fertility.

Dioxin-induced acute cardiac mitochondrial oxidative damage and increased activity of ATP-sensitive potassium channels in Wistar rats.

The environmental dioxin 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is classified as a Group 1 human carcinogen and teratogenic agent. We hypothesize that TCDD-induced oxidative stress may also interfere with mitochondrial ATP-sensitive potassium channels (mitoKATP), which are known to regulate and to be regulated by mitochondrial redox state. We investigated the effects of an acute treatment of male Wistar rats with TCDD (50 µg/kg i.p.) and measured the regulation of cardiac mitoKATP. While the function of cardiac mitochondria was slightly depressed, mitoKATP activity was 52% higher in animals treated with TCDD. The same effects were not observed in liver mitochondria isolated from the same animals. Our data also shows that regulation of mitochondrial ROS production by mitoKATP activity is different in both groups. To our knowledge, this is the first report to show that TCDD increases mitoKATP activity in the heart, which may counteract the increased oxidative stress caused by the dioxin during acute exposure.

Type 2 diabetic and Alzheimer's disease mice present similar behavioral, cognitive, and vascular anomalies.

Type 2 diabetes (T2D) is considered a major risk factor for Alzheimer's disease (AD). To elucidate the links between both pathological conditions, we compared behavioral and cognitive functions, cerebral amyloid-ß peptide (Aß) levels and vasculature integrity of 11-month-old T2D and AD mice. For this purpose, we performed behavioral tests (open field, object recognition, Y-maze, and elevated plus maze tests), ELISA to assess plasma markers of endothelial/vascular dysfunction, spectrophotometric assays to evaluate cerebral vascular permeability and enzymatic activities, and immunohistochemistry for the assessment of Aß levels. Both T2D and AD showed similar behavioral and cognitive anomalies characterized by increased fear and anxiety and decreased learning and memory abilities. Interestingly, both groups of animals presented increased plasma markers of endothelial/vascular dysfunction and permeability of cerebral vasculature and impaired mitochondrial enzymatic activities. In addition, a significant increase in Aß levels was observed in the cortex and hippocampus of T2D mice. These results support the notion that T2D predisposes to cerebrovascular alterations, cognitive decline, and development of AD.

Effects of different storage protocols on cat testis tissue potential for xenografting and recovery of spermatogenesis.

The loss of genetic diversity due to premature death of valuable individuals is a significant problem in animal conservation programs, including endangered felids. Testis tissue xenografting has emerged as a system to obtain spermatozoa from dead immature animals, however protocols to store this tissue before xenografting are still lacking. This study focused on testis tissue cryopreservation and storage from the domestic cat (Felis catus) classified as "pre-pubertal" and "pubertal" according to spermatogenesis development. Grafts from testis tissue cryopreserved with DMSO 1.4M, recovered after 10 weeks xenografting, presented seminiferous tubules with no germ cells. On the contrary, testis tissue from pre-pubertal animals preserved in ice-cold medium for 2 to 5 days presented no loss of viability or spermatogenic potential, while the number of grafts of pubertal cat testis tissue with germ cells after 10 weeks of xenografting decreased with increasing storage time. Nevertheless, even grafts from pre-pubertal cat testis tissue presented lower anti-DDX4 and anti-BOULE staining (proteins necessary for the meiosis completion), when compared with adult cat testis. Finally, a strong correlation found between testis weight and xenograft outcome may help choose good candidates for xenografting.

Differential effects of p,p'-DDE on testis and liver mitochondria: implications for reproductive toxicology.

The release of environmental contaminants can contribute to impaired male fertility. The bioenergetics of isolated liver mitochondria have been used as a toxicological indicator, an inexpensive first line model to screen possible effects of several substances. Here we report the effects of 2,2-bis(4-chlorophenyl)-1,1-dichloro-ethylene (DDE) on the bioenergetical parameters of testicular mitochondria. A significant decrease in repolarization potential (after a phosphorylative cycle), state 3 respiration and uncoupled respiration, with a concomitant increase in lag phase was found, demonstrating a decrease in mitochondrial function. Importantly, there was also a clear increase in maximum potential in DDE-treated testis mitochondria, which was not mirrored by more commonly used liver mitochondria. Indeed, comparative studies showed that testis and liver mitochondria have strikingly different sensitivities and patterns of response to DDE, indicating that testis mitochondria should be used as a primary toxicological model for a proper evaluation of putative effects of environmental toxicants on the bioenergetics of spermatogenesis and male fertility.

Xenografting as a tool to preserve endangered species: outcomes and challenges in model systems.

The use of testis tissue xenografting as a valuable tool to rescue endangered and genetically valuable individuals that die young or otherwise fail to produce sperm has been the subject of much interest. Although the technique has been successfully applied to a wide variety of species, little is known about what determines the outcome. Furthermore, to improve the applicability of xenografting, new methods to preserve and transport testis tissue from valuable animals are emerging. However, one major issue remains: the application of xenografting implies the development of subsequent ART techniques to produce offspring from the recovered material. This paper focuses on these three aspects of testis tissue xenografting as a tool for rescuing endangered and valuable genetic pools.

Mitochondrial functionality in reproduction: from gonads and gametes to embryos and embryonic stem cells.

BACKGROUND: Mitochondria are multitasking organelles involved in ATP synthesis, reactive oxygen species (ROS) production, calcium signalling and apoptosis; and mitochondrial defects are known to cause physiological dysfunction, including infertility. The goal of this review was to identify and discuss common themes in mitochondrial function related to mammalian reproduction. METHODS: The scientific literature was searched for studies reporting on the several aspects of mitochondrial activity in mammalian testis, sperm, oocytes, early embryos and embryonic stem cells. RESULTS: ATP synthesis and ROS production are the most discussed aspects of mitochondrial function. Metabolic shifts from mitochondria-produced ATP to glycolysis occur at several stages, notably during gametogenesis and early embryo development, either reflecting developmental switches or substrate availability. The exact role of sperm mitochondria is especially controversial. Mitochondria-generated ROS function in signalling but are mostly described when produced under pathological conditions. Mitochondria-based calcium signalling is primarily important in embryo activation and embryonic stem cell differentiation. Besides pathologically triggered apoptosis, mitochondria participate in apoptotic events related to the regulation of spermatogonial cell number, as well as gamete, embryo and embryonic stem cell quality. Interestingly, data from knock-out (KO) mice is not always straightforward in terms of expected phenotypes. Finally, recent data suggests that mitochondrial activity can modulate embryonic stem cell pluripotency as well as differentiation into distinct cellular fates. CONCLUSIONS: Mitochondria-based events regulate different aspects of reproductive function, but these are not uniform throughout the several systems reviewed. Low mitochondrial activity seems a feature of 'stemness', being described in spermatogonia, early embryo, inner cell mass cells and embryonic stem cells.

Testicular mitochondrial alterations in untreated streptozotocin-induced diabetic rats.

Diabetes-induced complications are associated with mitochondrial dysfunction and increasing evidence suggests that diabetes has an adverse effect on male reproductive function. The STZ-induced diabetic rat was used as an animal model for the type 1 form of the disease with the aim of determining its effects in spermatogenesis and testicular mitochondrial function. Several aspects of mitochondrial function were measured, including respiratory and electric potential function, as well as mitochondrial calcium loading capacity. Additionally oxidative stress production, antioxidant levels and possible apoptotic alterations were also evaluated. We observed that diabetic animals present alterations in spermatogenesis in both the testis and epidydimus. However, and surprisingly, the overall results in mitochondrial parameters failed to reveal severe testicular mitochondrial dysfunction in diabetic animals, with the exception of a decrease in calcium load. Taken together, results suggest that in animal models that mimic untreated type 1 diabetes the severe effects of the condition on spermatogenesis are not directly mitochondrial-mediated.

Comparison between different markers for sperm quality in the cat: Diff-Quik as a simple optical technique to assess changes in the DNA of feline epididymal sperm.

The majority of wild felids, as well as some domestic cats, have low sperm concentration in their ejaculates, and a high proportion of abnormal spermatozoa. We have employed several possible semen quality markers to further characterize cat epididymal sperm. Methods included possible apoptotic reporters, such as the annexin V assay to monitor exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane, as well as cell integrity; and the TUNEL assay to quantify DNA breaks. Sperm surface ubiquitination, another putative marker of sperm quality, was also monitored. The annexin V assay revealed a high percentage of sperm with PS exposure, and the TUNEL assay pointed to high levels (13+/-12%) of sperm with DNA breaks. Correlations were found between apoptotic markers (but not ubiquitination) and semen parameters. In parallel to this analysis, cat sperm morphology was evaluated using the Diff-Quik optical stain, which has been used in human reproduction laboratories. Several types of abnormalities could be characterized with this method. Remarkably, head staining abnormalities detected using the Diff-Quik staining method were strongly correlated with, and could accurately predict, sperm DNA defects detected in the same sample using the TUNEL assay. We therefore suggest that sperm morphology analysis using Diff-Quik could be used in field conditions to assess sperm status, due to the simplicity of the procedure and the equipment involved.