Cryo-scanning electron microscopy demonstrates that ice morphology is not associated with the post-thaw survival of domestic boar (Sus domesticus) spermatozoa: A comparison of directional and conventional freezing methods.

Directional freezing (in 2 or 10 ml hollow glass tubes) has been reported to improve post-thaw sperm survival parameters compared to conventional methods (in 0.5 ml straws). However, the biophysical properties that increase post-thaw survival are poorly understood. Therefore, the aim for the current study was to investigate the effect of ice morphology on the post-thaw survival of domestic boar spermatozoa directionally and conventionally cryopreserved in 0.5 ml straws. Ice morphology was quantitatively analyzed using a combination of cryo-scanning electron microscopy and Fiji Shape Descriptors. Multivariate analysis found a significant, non-linear effect (p < 0.05) of interface velocity on ice morphology, with an increase in both ice-lake size, as indicated by area and in aspect ratio, at an interface velocity of 0.2 mm/s. By contrast, post-thaw sperm survival (defined as spermatozoa with both intact plasma membranes and acrosomes) was biphasic, with peaks of survival at interface velocities of 0.2 mm/s (54.2 ± 1.9%), and 1.0 or 1.5 mm/s (56.5 ± 1.5%, 56.7 ± 1.7% respectively), and lowest survival at 0.5 (52.1 ± 1.6%) and 3.0 mm/s (51.4 ± 1.9%). Despite numerical differences in Shape Descriptors, there was no difference (p > 0.05) in the post-thaw survival between conventionally and directionally cryopreserved samples at optimal interface velocities of 1.0 or 1.5 mm/s. These findings suggest that: 1) ice morphology has little impact on post-thaw survival of boar spermatozoa, and 2) directional freezing in 0.5 ml straws (rather than 2 or 10 ml hollow glass tubes) may attenuate benefits of directional freezing.

Extracellular vesicles in the male reproductive tract of the softshell turtle.

Extracellular vesicles (EVs) are a heterogeneous group of cell-derived membranous structures comprising exosomes and microvesicles that originate from the endosomal system or are shed from the plasma membrane respectively. As mediators of cell communication, EVs are present in biological fluids and are involved in many physiological and pathological processes. The role of EVs has been extensively investigated in the mammalian male reproductive tract, but the characteristics and identification of EVs in reptiles are still largely unknown. In this review we focus our attention on EVs and their distribution in the male reproductive tract of the Chinese softshell turtle Pelodiscus sinensis, mainly discussing the potential roles of EVs in intercellular communication during different phases of the reproductive process. In softshell turtles, Sertoli-germ cell communication via multivesicular bodies can serve as a source of EVs during spermatogenesis, and these EVs interact with epithelia of the ductuli efferentes and the principal cells of the epididymal epithelium. These EVs are involved in sperm maturation, transport and storage. EVs are also shed by telocytes, which contact and exchange information with other, as well as distant interstitial cells. Overall, EVs play an indispensable role in the normal reproductive function of P. sinensis and can be used as an excellent biomarker for understanding male fertility.

Premature birth stunts early growth and is a possible driver of stress-induced maternal effects in the guppy Poecilia reticulata.

We tested the effects of gestational stress, principally in the form of alarm cue extracted from the skin of conspecifics, on reproduction in female guppies (Poecilia reticulata) and the growth and behaviour of their offspring. Offspring from mothers exposed to alarm cue exhibited stunted growth in the first few days post-partum, which appeared to be mediated by shortening of the gestation period, the length of which directly correlated with growth rate within the first 6 days post-partum. Mature offspring did not differ in behaviour or stress responses compared with controls and so the effects of maternal predation stress did not appear to persist into adulthood. A different form of gestational stress, dietary restriction, did not significantly affect offspring growth, though brood size was reduced, suggesting that the effects of predation stress were not mediated by differences in resource demand or consumption.

Breakthroughs and new horizons in reproductive biology of rare and endangered animal species.

Because of higher extinction rates due to human and natural factors, more basic and applied research in reproductive biology is required to preserve wild species and design proper strategies leading to sustainable populations. The objective of the review is to highlight recent, inspiring breakthroughs in wildlife reproduction science that will set directions for future research and lead to more successes in conservation biology. Despite new tools and approaches allowing a better and faster understanding of key mechanisms, we still know little about reproduction in endangered species. Recently, the most striking advances have been obtained in nonmammalian species (fish, birds, amphibians, or corals) with the development of alternative solutions to preserve fertility or new information about parental nutritional influence on embryo development. A novel way has also been explored to consider the impact of environmental changes on reproduction-the allostatic load-in a vast array of species (from primates to fish). On the horizon, genomic tools are expected to considerably change the way we study wildlife reproduction and develop a concept of "precision conservation breeding." When basic studies in organismal physiology are conducted in parallel, new approaches using stem cells to create artificial gametes and gonads, innovations in germplasm storage, and more research on reproductive microbiomes will help to make a difference. Lastly, multiple challenges (for instance, poor integration of new tools in conservation programs, limited access to study animals, or few publication options) will have to be addressed if we want reproductive biology to positively impact conservation of biodiversity.

Using the Koala (Phascolarctos cinereus) as a Case Study to Illustrate the Development of Artificial Breeding Technology in Marsupials: an Update.

The successful development and application of an assisted breeding program in any animal relies primarily on a thorough understanding of the fundamental reproductive biology (anatomy, physiology and behaviour) of the species in question. Surely, the ultimate goal and greatest hallmark of such a program is the efficacious establishment of a series of reliable techniques that facilitate the reproductive and genetic management of fragmented populations, both in captivity and in the wild. Such an achievement is all that more challenging when knowledge of the reproductive biology of that species is essentially rudimentary and without adequate models to compare to. Using the koala (Phascolarctos cinereus) as a case study, this chapter provides insights into the development of a concept that began as small undergraduate student project but that subsequently evolved into the first-ever successful artificial insemination of a marsupial. Apart from this historical perspective, we shall also provide a brief review of the current reproductive biology of the koala, discuss technical elements of current assisted breeding technology of this species, its application to the closely related wombat, and the potential role it might play in helping to conserve wild koala populations in the form of a live koala genome bank. There is little doubt that the unique reproductive biology and tractability of the koala has been a benefit rather than a hindrance to the success of artificial breeding in this species.

Reproductive Science as an Essential Component of Conservation Biology: New Edition.

The previous edition of this book mainly provided a snapshot of the state of the art in terms of species-specific reproductive biology and emerging technologies. The influence of environmental changes on reproductive fitness was introduced but not fully explored. The objectives of this second edition were to (1) emphasize the need for holistic and global efforts to understand and sustain reproduction in a constantly changing environment and (2) provide more knowledge in the reproductive physiology of different taxa. The first section of the book is dedicated to survival and adaptation of species in a changing environment (including chapters on environmental impacts in different taxa, as well as the role of microbiomes). The second section focuses on progress in understanding, assisting or even suppressing reproduction in wild species, keeping in mind the influence of environmental factors as well. It contains chapters from the previous edition that were updated (reproduction in elephants, koalas, marsupials, amphibians, and corals), new chapters on species such as sharks and rays, and contributions about the increasing role of reproductive manipulations, such as assisted reproduction and contraception. While the present book emphasizes the overarching issue of environmental impacts on reproduction (resulting in infertility, subfecundity, or fitness), it also highlights the challenges of maintaining wild species in captivity, including those associated with ensuring good welfare. Captive environments can influence reproduction in a multitude of ways, some unexpected, such as the selection of unwanted genetic traits, an essential dimension to be considered to ensure the success of conservation breeding programs. Lastly, new approaches, such as the use of allostatic load indexes and reproductive microbiome analyses also will be closely examined for the first time in rare and endangered species to address conservation issues.

From the Ivory Tower to Reality! Conclusions of the New Edition.

While many of the traditional scientific disciplines have developed over centuries, animal conservation is a relative newcomer. It relies on multiple specialties with different levels of expertise that, eventually, generate vast amounts of data. More specifically, conservation physiology is an emerging area that can be defined as 'an integrative scientific discipline applying physiological concepts, tools, and knowledge to characterizing biological diversity and its ecological implications; understanding and predicting how organisms, populations, and ecosystems respond to environmental change and stressors; and solving conservation problems across the broad range of taxa, including microbes, plants, and animals' (Cooke et al. 2013). Reproductive biology is more focused, given that it mainly deals with the physiology underlying the production of gametes, embryos, and offspring, and the many associated processes that control these events. However, it is integrated into the different components of conservation physiology. In bringing together the various contributors for this book, the editors' purpose was to provide readers with a new perspective about the complexity behind reproduction and the role it plays in species conservation. Chapters highlight the diversity of reproductive mechanisms across taxa, and provide insight into how they may have evolved, and likely will continue to evolve in a changing environment. To conservation physiologists, the hope is that this information will be applied to sustain populations in both natural habitats and managed facilities. Ultimately, a major goal is to forecast and mitigate negative impacts of environmental change or anthropogenic pressures on animal fitness, which will only follow once we have acquired a solid understanding of reproductive processes.

Integrated Approach Reveals Role of Mitochondrial Germ-Line Mutation F18L in Respiratory Chain, Oxidative Alterations, Drug Sensitivity, and Patient Prognosis in Glioblastoma.

Glioblastoma is the most common and malignant primary brain tumour in adults, with a dismal prognosis. This is partly due to considerable inter- and intra-tumour heterogeneity. Changes in the cellular energy-producing mitochondrial respiratory chain complex (MRC) activities are a hallmark of glioblastoma relative to the normal brain, and associate with differential survival outcomes. Targeting MRC complexes with drugs can also facilitate anti-glioblastoma activity. Whether mutations in the mitochondrial DNA (mtDNA) that encode several components of the MRC contribute to these phenomena remains underexplored. We identified a germ-line mtDNA mutation (m. 14798T > C), enriched in glioblastoma relative to healthy controls, that causes an amino acid substitution F18L within the core mtDNA-encoded cytochrome b subunit of MRC complex III. F18L is predicted to alter corresponding complex III activity, and sensitivity to complex III-targeting drugs. This could in turn alter reactive oxygen species (ROS) production, cell behaviour and, consequently, patient outcomes. Here we show that, despite a heterogeneous mitochondrial background in adult glioblastoma patient biopsy-derived cell cultures, the F18L substitution associates with alterations in individual MRC complex activities, in particular a 75% increase in MRC complex II_III activity, and a 34% reduction in CoQ10, the natural substrate for MRC complex III, levels. Downstream characterisation of an F18L-carrier revealed an 87% increase in intra-cellular ROS, an altered cellular distribution of mitochondrial-specific ROS, and a 64% increased sensitivity to clomipramine, a repurposed MRC complex III-targeting drug. In patients, F18L-carriers that received the current standard of care treatment had a poorer prognosis than non-carriers (373 days vs. 415 days, respectively). Single germ-line mitochondrial mutations could predispose individuals to differential prognoses, and sensitivity to mitochondrial targeted drugs. Thus, F18L, which is present in blood could serve as a useful non-invasive biomarker for the stratification of patients into prognostically relevant groups, one of which requires a lower dose of clomipramine to achieve clinical effect, thus minimising side-effects.

Making the most of sperm activation responses: experiments with boar spermatozoa and bicarbonate.

Attempting to extract useful and reliable information about semen quality and its fertility potential remains a difficult exercise, partly because the sperm heterogeneity within samples often renders simple statistical analyses rather meaningless. In fact, a mean and standard deviation may reflect neither the very fast swimming activities of the most active cells nor the slow and sluggish activities of others. Herein we propose that the information value within semen samples can be maximised if current knowledge about sperm activation mechanisms is exploited before undertaking the measurements. We explain, using boar semen as an example, that estimating and defining relative sperm subpopulation sizes, after activation by bicarbonate, provides a means of quantifying sperm quality. Although such estimates may indeed be related to in vivo fertility, the general approach also suggests potential new avenues that could be exploited for the elaboration of novel in vitro tests for the characterisation of toxic environmental chemicals and, indeed, to reduce the number of animals used in such testing programs.

Composition of marsupial zona pellucida: a molecular and phylogenetic approach.

The zona pellucida (ZP) is an extracellular matrix that surrounds mammalian oocytes. In eutherians it is formed from three or four proteins (ZP1, ZP2, ZP3, ZP4). In the few marsupials that have been studied, however, only three of these have been characterised (ZP2, ZP3, ZP4). Nevertheless, the composition in marsupials may be more complex, since a duplication of the ZP3 gene was recently described in one species. The aim of this work was to elucidate the ZP composition in marsupials and relate it to the evolution of the ZP gene family. For that, an in silico and molecular analysis was undertaken, focusing on two South American species (gray short-tailed opossum and common opossum) and five Australian species (brushtail possum, koala, Bennett's wallaby, Tammar wallaby and Tasmanian devil). This analysis identified the presence of ZP1 mRNA and mRNA from two or three paralogues of ZP3 in marsupials. Furthermore, evidence for ZP1 and ZP4 pseudogenes in the South American subfamily Didelphinae and for ZP3 pseudogenes in two marsupials is provided. In conclusion, two different composition models are proposed for marsupials: a model with four proteins (ZP1, ZP2 and ZP3 (two copies)) for the South American species and a model with six proteins (ZP1, ZP2, ZP3 (three copies) and ZP4) for the Australasian species.

Exploitation of Non-mammalian Model Organisms in Epigenetic Research.

Model organisms are widely used in research that is ultimately aimed at understanding the causes and consequences of human disease. It may seem counterintuitive to expect clinically useful information to be obtained from species as diverse as fishes and insects, but because fundamental biological mechanisms share evolutionary origins they transcend species barriers. Epigenetic mechanisms fulfil this expectation admirably as more and more is discovered about the basic operational rules of inheritance, which are much more elaborate than formerly thought. Only a few decades ago, although the complex interplay between genes, inheritance and the environment was recognized, it was difficult to explain. Recent discoveries about the controlling influences of gene silencing through DNA and histone methylation, the roles of so-called non-coding DNA and microRNA, and the way in which these factors respond to environmental conditions have started to shed light on these basic mechanisms. Diverse model species allow epigenetic mechanisms to be studied from different perspectives; for example, some are better suited to studies of sex determination while others may be more convenient for studying the earliest stages of organ development, growth and the influence of nutrition on future wellbeing. The rationale for including this chapter in a book that is focused on uncovering relationships between periconception nutrition in humans is to highlight the opportunities and insights that may be gained by focusing attention on studies in non-mammalian model species.

Introduction: A Brief Guide to the Periconception Environment.

Definition of the periconception period is not an exact science and is probably somewhat arbitrary. One can define it as spanning the period from the final stages of gamete maturation until formation of the embryo and the stages of embryonic development and implantation. Hence, the periconception period includes periods when spermatozoa are in the female reproductive tract, oocytes are matured and ovulated into the oviduct, fertilization occurs and the embryo undergoes development. By definition the implantation process and the early stages of placenta formation are also regarded as a part of the periconception period. In this article we highlight a few of the major advances which have transformed this topic over the last two decades. It is now clear that the fitness and wellbeing of developing mammalian embryos, including the human, are highly dependent on the health status, diet and habits of both parents especially in the months and weeks that precede the formation of oocytes and spermatozoa.

Implications of the Nagoya Protocol for genome resource banks composed of biomaterials from rare and endangered species.

The Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilisation is a multilateral legal instrument within the Convention on Biodiversity. It has now come into force, having been signed by 92 countries, 68 of which have ratified it, but notably these do not yet include the US, China, Canada and Russia. The overarching objective of the Nagoya Protocol is to prevent the unfair commercial exploitation of a country's biodiversity and it also protects traditional knowledge. Although the intentions seem reasonable and equitable, the provisions of the Nagoya Protocol will have major effects on the ability of researchers in both the commercial and non-commercial sectors to access genetic materials (which are widely defined and include almost every conceivable animal product, as well as whole animals) from around the world. It also places a heavy bureaucratic burden on researchers and their institutions, which must comply with an international standard and obtain an International Certificate of Compliance proving that all samples will be collected according to the terms of the Protocol. Herein we review of the unforeseen implications of the Nagoya Protocol in relation to biobanking and animal conservation.

Embryonic developmental plasticity in the long-snouted seahorse (Hippocampus reidi, Ginsburg 1933) in relation to parental preconception diet.

The aim of the present study was to investigate the hypothesis that parental periconception nutrition in adult seahorses affects the development and growth of their offspring. We tested the hypothesis that because seahorse embryos develop inside the male's brood pouch, manipulation of the male's diet would affect offspring growth and development independently of the female's diet. Adult males and females were fed separately with either wild-caught crustaceans or commercial aquarium diet for 1 month before conception to influence the periconception environment. Approximately 10000 offspring were obtained from four different treatment groups (Male/Wild or Male/Commercial×Female/Wild or Female/Commercial). Weights, physical dimensions and fatty acid profiles of the newborns were determined. Offspring produced when the males receiving commercial diet were mated with wild-fed females were larger (P<0.05) than those produced by wild-fed males. When both males and females were fed with commercial diet, their offspring were significantly smaller than those from the other treatment groups. When commercial diet-fed females were mated with wild-fed males, the offspring showed distortion of the snout:head length ratio. These results support the view that the preconception diet received by males and females differentially affects embryonic development.

Do sperm possess a molecular passport? Mechanistic insights into sperm selection in the female reproductive tract.

Most male mammals produce far more spermatozoa on a daily basis than is strictly necessary for reproduction and females have evolved mechanisms that prevent all but a small minority from reaching the vicinity of their oocytes. One potential explanation for the stringent selection is that females have developed these mechanisms as a way of avoiding polyspermy as well as exercising post-copulatory choice over the characteristics of the fertilizing spermatozoon. Relatively little is known about how these processes would operate, but here we use evidence from biochemical, molecular and genetic studies of sperm transport in support of a hypothesis proposing that the female reproductive tract can read and interpret a spermatozoon's 'molecular passport' or genetic signature. Such a signature would permit only a highly selected sperm population to reach and fertilize the oocyte. Moreover, the selection criteria might not only be concerned with successful fertilizing ability, but could also be tailored to suit the genetic qualities of individual females.

Validation of computer-assisted sperm-motility analysis in the amphibian Silurana tropicalis.

We have developed and validated a computer-assisted sperm-motility assessment (CASA) method for use with the emerging amphibian model Silurana tropicalis. The testicular sperm-activation method was validated by analysing activation replicate coefficients of variation, effects of tracking time settings on velocity distributions and the relative partitioning of differentially motile sperm subpopulations between matched right and left testes. Two major sperm subpopulations were identified using multivariate pattern analysis and their relative frequencies were consistent between samples from matched right and left testes and from randomly drawn subsets of six frogs sampled from the total set of 16 frogs. The power of this approach for detecting treatment effects targeting the hypothalamic-pituitary--gonadal axis was investigated by injecting a group of frogs with 100IU human chorionic gonadotrophin (hCG) 2h before sampling and comparing their sperm-subpopulation frequencies with non-injected controls. While parametric analysis across sperm samples failed to detect treatment effects, subpopulation analysis showed that hCG significantly increased the proportion of progressive and non-sinuous spermatozoa compared with controls (Chi square=6.40, DF=1, P=0.011). This demonstrated the potential value of analysing objectively measured sperm behaviour as an endpoint.

Heat-shock protein A8 restores sperm membrane integrity by increasing plasma membrane fluidity.

The constitutive 70  kDa heat-shock protein, HSPA8, has previously been shown to contribute to the long-term survival of spermatozoa inside the mammalian female reproductive tract. Here, we show that a recombinant form of HSPA8 rapidly promotes the viability of uncapacitated spermatozoa, the ability of spermatozoa to bind to oviductal epithelial cells, enhances IVF performance, and decreases sperm mitochondrial activity. Fluorescence recovery after photobleaching revealed that the repair of membrane damage is achieved by an almost instantaneous increase in sperm membrane fluidity. The ability of HSPA8 to influence membrane stability and fluidity, as well as its conserved nature among mammalian species, supports the idea that this protein protects sperm survival through membrane repair mechanisms. Free Persian abstract A Persian translation of the abstract is freely available online at http://www.reproduction-online.org/content/147/5/719/suppl/DC1.

Viable and morphologically normal boar spermatozoa alter the expression of heat-shock protein genes in oviductal epithelial cells during co-culture in vitro.

The principal aim of this study was to determine if boar spermatozoa influence the expression of four selected chaperone and heat-shock protein (HSP) genes-namely clusterin (CLU), HSP90AA1, HSPA5, and HSPA8-in oviductal epithelial cells (OECs) during in vitro co-culture. All corresponding proteins of these genes were previously identified in a sperm-interacting, 70-kDa soluble fraction derived from apical plasma membranes of OECs. The present study also sought to determine whether or not: (i) spermatozoa must directly bind to OEC for an effect on gene expression to be elicited and (ii) reproductive and nonreproductive epithelial cell types (LLC-PK1, pig kidney) respond equivalently, in terms of alterations in chaperone and HSP gene expression, during co-culture with sperm. Spermatozoa induced a significant upregulation (P < 0.05) in HSP90AA1 and HSPA5 in OECs after 3 hr, and in HSPA8 after 6 hr of co-culture when they were in direct contact with epithelial cells. Conversely, no upregulation of HSP transcription was observed when spermatozoa did not directly bind to OECs. Spermatozoa also induced a significant upregulation (P < 0.05) of the same three genes when in direct contact with LLC-PK1 cells, but the timing occurred later than with OECs. Interestingly, the extent of HSP gene upregulation induced by direct contact of spermatozoa with epithelial cells was dependent on sperm-binding index and on the viability and morphological quality of the bound sperm population. In conclusion, the upregulation of HSP genes caused by direct contact between spermatozoa and OECs, rather than nonreproductive epithelial cells, suggests HSPs could play an integral role in the modulation of sperm function in the oviductal reservoir.

Impacts of endocrine disrupting chemicals on reproduction in wildlife.

The European Environment Agency (The Weybridge + 15 (1996-2011) report. EEA Technical report, vol 2. Copenhagen, 2012) and the United Nations Environment programme together with the World Health Organisation (State of the science of endocrine disrupting chemicals-2012. Geneva, Switzerland) both recently published major and highly authoritative reviews of endocrine disrupting chemicals in the natural environment and their effects on reproduction and health in both humans and wildlife. One surprising conclusion to emerge from these reviews was that there are relatively few well documented reports of endocrine disruption (ED) in wild mammals, mainly because much of the available evidence is correlative and does not conclusively demonstrate that the chemicals in question cause the physiological and phenotypic problems attributed to them. However, based on strong evidence from studies of wild birds, reptiles, invertebrates, and laboratory animals, it is difficult to imagine that wild mammals would be the exception. This chapter is therefore included to emphasize the point that the role of reproductive science within wildlife conservation is much broader than a narrow focus on artificial breeding technologies.

The koala (Phascolarctos cinereus): a case study in the development of reproductive technology in a marsupial.

The successful development and application of an assisted breeding program in any animal relies primarily on a thorough understanding of the fundamental reproductive biology (anatomy, physiology and behaviour) of the species in question. Surely, the ultimate goal and greatest hallmark of such a program is the efficacious establishment of a series of reliable techniques that facilitate the reproductive and genetic management of fragmented populations, both in captivity and in the wild. Such an achievement is all the more challenging when the reproductive biology of that species is essentially rudimentary and without adequate reproductive models to compare to. Using the koala (Phascolarctos cinereus) as a case study, this chapter provides some personal insights into the evolution of a concept that began as a small undergraduate student project but that subsequently evolved into the first-ever successful artificial insemination of a marsupial. Apart from this historical perspective, we also provide a brief review of the current reproductive biology of the koala, discuss technical elements of current assisted breeding technology of this species, its potential application to the wombat, and the future role it might play in helping to conserve wild koala populations. There is little doubt that the unique reproductive biology and tractability of the koala has in this case been a benefit rather than a hindrance to the success of artificial breeding in this species.