The effects of endocrine disruptors on the male germline: an intergenerational health risk.

Environmental pollution is becoming one of the major concerns of society. Among the emerging contaminants, endocrine-disrupting chemicals (EDCs), a large group of toxicants, have been the subject of many scientific studies. Besides the capacity of these compounds to interfere with the endocrine system, they have also been reported to exert both genotoxic and epigenotoxic effects. Given that spermatogenesis is a coordinated process that requires the involvement of several steroid hormones and that entails deep changes in the chromatin, such as DNA compaction and epigenetic remodelling, it could be affected by male exposure to EDCs. A great deal of evidence highlights that these compounds have detrimental effects on male reproductive health, including alterations to sperm motility, sexual function, and gonad development. This review focuses on the consequences of paternal exposure to such chemicals for future generations, which still remain poorly known. Historically, spermatozoa have long been considered as mere vectors delivering the paternal haploid genome to the oocyte. Only recently have they been understood to harbour genetic and epigenetic information that plays a remarkable role during offspring early development and long-term health. This review examines the different modes of action by which the spermatozoa represent a key target for EDCs, and analyses the consequences of environmentally induced changes in sperm genetic and epigenetic information for subsequent generations.

Cryobanking of aquatic species.

This review is focused on the applications of genome cryobanking of aquatic species including freshwater and marine fish, as well as invertebrates. It also reviews the latest advances in cryobanking of model species, widely used by the scientific community worldwide, because of their applications in several fields. The state of the art of cryopreservation of different cellular types (sperm, oocytes, embryos, somatic cells and primordial germ cells or early spermatogonia) is discussed focusing on the advantages and disadvantages of each procedure according to different applications. A special review on the need of standardization of protocols has also been carried out. In summary, this comprehensive review provides information on the practical details of applications of genome cryobanking in a range of aquatic species worldwide, including the cryobanks established in Europe, USA, Brazil, Australia and New Zealand, the species and type of cells that constitute these banks and the utilization of the samples preserved. STATEMENT OF RELEVANCE: This review compiles the last advances on germplasm cryobanking of freshwater and marine fish species and invertebrates, with high value for commercial aquaculture or conservation. It is reviewed the most promising cryopreservation protocols for different cell types, embryos and larvae that could be applied in programs for genetic improvement, broodstock management or conservation of stocks to guarantee culture production.

Molecular basis of spermatogenesis and sperm quality.

Spermatozoan quality can be evaluated in different ways, here we focus on the analysis of DNA, RNA and epigenetic status of germ cells. These characterizations also can be the bases for explaining sperm quality at other levels, so we will see how some of these molecules could affect other sperm quality markers. Moreover, we consider the possibility of using some of these molecules as predictors of sperm quality in terms of the ability to produce healthy offspring. The relevant effect of different types of RNA molecules in germ line specification and spermatogenesis and the importance of germ cell DNA integrity and a proper epigenetic pattern will be also discussed. Although most studies at this level have been performed in mammals, some information is available for fish; these recent discoveries in fish models are included. We provide a general overview on how these molecules could have a deep influence in the final sperm quality.

The relationship between ram sperm head morphometry and fertility depends on the procedures of acquisition and analysis used.

Sperm head morphometry is a parameter in the evaluation of semen that has been associated with fertility in two ways: comparing morphometric measures between predefined groups of fertility; or analyzing morphometric data by multivariate techniques to identify cell populations. We analyzed the morphometry of ram sperm head by three procedures and checked its relationship with male fertility. A Computer-Aided Sperm Morphometric Assessment procedure (CASMA), an image analysis software (NIS-Elements) in combination with an optical microscope (MO-NIS) and this image analysis software in combination with a scanning electron microscope (SEM-NIS) were used. Eight morphometric parameters were assessed: length, width, area, perimeter, ellipticity, form factor, elongation and regularity. We observed significant differences between the morphometric data of sperm head obtained with three study procedures. The CASMA procedure shows the highest values for all parameters and the SEM-NIS procedure the lowest. The analysis of a semen sample, when only the mean of morphometric parameters is used to describe the cell population, is too limited to interpret their fertilizing capacity. It is essential to analyze the complex structure of the samples by defining subpopulations by multivariate methods. With few exceptions, the means of each morphometric parameter differ between the three subpopulations analyzed in each procedure. Only the subpopulations obtained with the MO-NIS procedure showed a significant correlation with male fertility. In short, it is necessary to establish an instrumental standard for the analysis of sperm morphometry to obtain reliable results and we believe that the MO-NIS system presents these basic requirements.

Comparison of two methods for obtaining spermatozoa from the cauda epididymis of Iberian red deer.

We have compared two methods for salvaging epididymal sperm from post-mortem samples from Iberian red deer. Of each pair of testicles (29 samples), one cauda epididymis was processed by means of cuts (sperm was immediately diluted with extender) and the other was detached from the corpus and flushed from the vas deferens with 1 mL of extender. Sperm was processed for cryopreservation, and analyzed just after recovery, pre-freezing and post-thawing. Total spermatozoa recovered, contamination (concentration of epididymal cells and red blood cells (RBCs)) and quality (motility by CASA, and acrosomal status, viability and mitochondrial status by flow cytometry) were used to compare both methods. The number of recovered spermatozoa was similar for both methods. Contamination was higher for the cuts method, but when considering the final dilution before freezing, only RBCs concentration was significantly higher. Motility was similar just after extraction, but higher for both pre-frozen and post-thawed flushed sperm. Pre-freezing acrosomal status (P < 0.05) and viability (P < 0.1) were better for flushing; however post-thawing results were similar for the two methods. A clustering analysis using CASA data showed that the subpopulation pattern of motile sperm was different depending on the method, being better for flushing. With regard to yield, lower contamination (especially RBCs) and, in general, better quality results, flushing seems to be a more recommendable method for post-mortem sperm recovery. The cuts method may be more practical on certain occasions, but care must be taken in order to achieve rapid extension of the sample and to avoid contamination in order to improve sample condition.

Post mortem time and season alter subpopulation characteristics of Iberian red deer epididymal sperm.

We have studied the effect of post mortem time and season on sperm subpopulation pattern and characteristics. We used epididymal samples from free-ranging Iberian red deers harvested during the hunting season. We studied samples at different moments of the year (rut, transition period and post-rut), and at different times post mortem (up to 4 days). Sperm were extracted from the cauda epididymis and their motility was evaluated by means of a CASA system. A principal component and clustering analysis were carried out to identify subpopulations. Post mortem time caused a significant decrease in motility quality, and a general deterioration in subpopulation characteristics. We found three subpopulations the first day, and the one indicating good sperm quality decreased with post mortem time until it disappeared on the fourth day. This may indicate considerable impairment of the samples after 72 h post mortem, which could compromise their use in AI programs. With regard to season, subpopulation pattern and characteristics were better in the transition and post-rut periods. Moreover, we found one subpopulation formed by mature spermatozoa, which increased from rut to post-rut. This might be a negative fact, because samples collected after the rut may undergo hypermaturation, which possibly impairs fertility. Our results are of interest for the management of wildlife germplasm banks based on post mortem sperm recovery.

Season effect on genitalia and epididymal sperm from Iberian red deer, roe deer and Cantabrian chamois.

Seasonality deeply affects the physiology and behavior of many species, and must be taken into account when biological resource banks (BRBs) are established. We have studied the effect of seasonality on many reproductive parameters of free-ranging Iberian red deer, roe deer and Cantabrian chamois, living in Spain. Testicles from hunted animals were collected and sent to our laboratory at different times during the year. We recorded the weight and volume of testis, the weight of the epididymis and its separate parts (caput, corpus, and cauda), the weight of the sperm sample collected from the cauda epididymis, and several sperm parameters (sperm concentration, spermatozoa recovered, motility, HOS test reactivity, acrosomal status, and viability). We studied the data according to several periods, defined accordingly to each species. For red deer, we defined rut (mid-September to mid-October), post-rut (mid-October to mid-December), and non-breeding season (February). For roe deer, they were pre-rut (June), rut (July), post-rut (first fortnight of August), and non-breeding season (September). For chamois: non-breeding season (June to mid-September) and breeding season (October-November). The rut/breeding season yielded significantly higher numbers for almost all parameters. However, in the case of red deer, sperm quality was higher in the post-rut. For roe deer, testicular weight was similar in the pre-rut and in the rut, and sperm quality did not differ significantly between these two periods, although we noticed higher values in the rut. In the case of chamois, sperm quality did not differ significantly from the breeding season, but data distribution suggested that in the non-breeding season there are less males with sperm of good quality. On the whole, we find these results of interest for BRB planning. The best season to collect sperm in this species would be the breeding season. However, post-rut in red deer, pre-rut in roe deer, and non-breeding season in chamois could be used too, because of the acceptable sperm quality, despite the lower quantity salvaged. More in-depth research needs to be carried out on the quality of sperm salvaged at different times of the year in order to confirm these findings.

Sperm subpopulations in Iberian red deer epididymal sperm and their changes through the cryopreservation process.

We have applied a statistical protocol based on principal component analysis, clustering methods, and discriminant analysis for the identification of sperm subpopulations in computer-assisted sperm analysis (CASA) data. Samples were obtained from the cauda epididymis of 11 Iberian red deer and cryopreserved following a standard protocol. Motility by CASA was analyzed just after sperm recovery, just before freezing, and after thawing, and eight motility descriptors for each individual spermatozoon were recorded. Sperm viability and acrosomal status were also assessed. Subpopulation analysis was performed in four sequential steps: principal component analysis using the eight motility descriptors; nonhierarchical clustering analysis (k-means) using the first two principal components; hierarchical clustering analysis (UPGMA); and selection of the final number of clusters. Three clusters were obtained for each motility analysis: slow and nonlinear; rapid and linear; and rapid, high ALH, nonlinear. We detected variations in the clusters between treatments (initial, prefreezing and postthawed). Indeed, motility increased and linearity decreased in the prefreezing analysis. A discriminant analysis isolated three descriptors that were used again in the same statistical analysis, giving four clusters that resembled the pattern found in the first classification. We also performed a clustering analysis of the males according to prefreezing/postthawed variation of total motility, viability, and acrosomal status. The proportion of the linear subpopulations in the prefreezing treatment, in both clustering analyses, correlated positively with postthawed viability recovery. Our results show that clustering analysis of CASA data gives useful and practical information that is not obtained by conventional sperm analysis.