Antagonistic selection on body size and sword length in a wild population of the swordtail fish,Xiphophorus multilineatus: Potential for intralocus tactical conflict

Alternative reproductive tactics (ARTs) have provided valuable insights into how sexual selection and life history trade‐offs can lead to variation within a sex. However, the possibility that tactics may constrain evolution through intralocus tactical conflict (IATC) is rarely considered. In addition, when IATC has been considered, the focus has often been on the genetic correlations between the ARTs, while evidence that the ARTs have different optima for associated traits and that at least one of the tactics is not at its optimum is often missing. Here, we investigate selection on three traits associated with the ARTs in the swordtail fish Xiphophorus multilineatus; body size, body shape, and the sexually selected trait for which these fishes were named, sword length (elongation of the caudal fin). All three traits are tactically dimorphic, with courter males being larger, deeper bodied and having longer swords, and the sneaker males being smaller, more fusiform and having shorter swords. Using measures of reproductive success in a wild population we calculated selection differentials, as well as linear and quadratic gradients. We demonstrated that the tactics have different optima and at least one of the tactics is not at its optimum for body size and sword length. Our results provide the first evidence of selection in the wild on the sword, an iconic trait for sexual selection. In addition, given the high probability that these traits are genetically correlated to some extent between the two tactics, our study suggests that IATC is constraining both body size and the sword from reaching their phenotypic optima. We discuss the importance of considering the role of IATC in the evolution of tactical dimorphism, how this conflict can be present despite tactical dimorphism, and how it is important to consider this conflict when explaining not only variation within a species but differences across species as well.     

Rethinking parenthood within assisted reproductive technology: The need for regulation in Nigeria

In Nigeria, reproduction is highly valued, with many people desiring to produce a child ‘in their own image and likeness’. Previously, aspiring parents often resorted to adoption. Today, the availability of assisted reproductive technologies (ARTs) has provided options other than adoption for those desiring to procreate. Through ARTs, aspirations for a family may be attained through an exchange of reproductive goods and services, and not necessarily through traditional heterosexual relationships. ARTs have altered the perception of parenthood as it exists in Nigeria, and courts face a difficult task in defining parenthood within Nigerian jurisprudence, as they can only adjudicate based on extant law. Although ARTs provide greater individual choices for fulfilling the desire to procreate, they raise a number of ethical and legal issues that evolving legal systems, such as that in Nigeria, are ill-equipped to manage. This paper describes the traditional assignment of parenthood under indigenous laws and other sources of law within the Nigerian jurisprudence. We carried out an in-depth study of the Nigerian legislative framework and found that there are no laws directly regulating parenthood, procreation and ARTs in Nigeria. We also found that the extant laws are only tangentially related and do not answer the relevant questions sufficiently well, especially concerning succession, nationality and assignment of responsibility in collaborative reproduction. We conclude by highlighting the need for and recommending a regulatory framework on ARTs with a particular focus on providing a definition for parenthood achieved through ARTs in Nigeria.     

Characterization of differentially methylated regions in 3 bovine imprinted genes: a model for studying human germ-cell and embryo development

Correct imprinting is crucial for normal fetal and placental development in mammals. Experimental evidence in animal models and epidemiological studies in humans suggest that assisted reproductive technologies (ARTs) can interfere with imprinted gene regulation in gametogenesis and early embryogenesis. Bos taurus is an agriculturally important species in which ARTs are commonly employed. Because this species exhibits a similar preimplantation development and gestation length as humans, it is increasingly being used as a model for human germ-cell and embryo development. However, in contrast to humans and mice, there is relatively little information on bovine imprinted genes. Here, we characterized the bovine intergenic IGF2-H19 imprinting control region (ICR) spanning approximately 3 kb. We identified a 300-bp differentially methylated region (DMR) approximately 6 kb upstream of the H19 promoter, containing a CpG island with CTCF-binding site and high sequence similarity with the human intergenic ICR. Additional differentially methylated CpG islands lie -6 kb to -3 kb upstream of the promoter, however these are less conserved. Both classical bisulfite sequencing and bisulfite pyrosequencing demonstrated complete methylation of the IGF2-H19 ICR in sperm, complete demethylation in parthenogenetic embryos having only the female genome, and differential methylation in placental and somatic tissues. In addition, we established pyrosequencing assays for the previously reported bovine SNRPN and PEG3 DMRs. The observed methylation patterns were consistent with genomic imprinting in all analyzed tissues/cell types. The identified IGF2-H19 ICR and the developed quantitative methylation assays may prove useful for further studies on the relationship between ARTs and imprinting defects in the bovine model.     

Epigenetic disorders and altered gene expression after use of Assisted Reproductive Technologies in domestic cattle

The use of Assisted Reproductive Technologies (ARTs) in modern cattle breeding is an important tool for improving the production of dairy and beef cattle. A frequently employed ART in the cattle industry is in vitro production of embryos. However, bovine in vitro produced embryos differ greatly from their in vivo produced counterparts in many facets, including developmental competence. The lower developmental capacity of these embryos could be due to the stress to which the gametes and/or embryos are exposed during in vitro embryo production, specifically ovarian hormonal stimulation, follicular aspiration, oocyte in vitro maturation in hormone supplemented medium, sperm handling, gamete cryopreservation, and culture of embryos. The negative effects of some ARTs on embryo development could, at least partially, be explained by disruption of the physiological epigenetic profile of the gametes and/or embryos. Here, we review the current literature with regard to the putative link between ARTs used in bovine reproduction and epigenetic disorders and changes in the expression profile of embryonic genes. Information on the relationship between reproductive biotechnologies and epigenetic disorders and aberrant gene expression in bovine embryos is limited and novel approaches are needed to explore ways in which ARTs can be improved to avoid epigenetic disorders.     

Epigenetic disorders and altered gene expression after use of Assisted Reproductive Technologies in domestic cattle

The use of Assisted Reproductive Technologies (ARTs) in modern cattle breeding is an important tool for improving the production of dairy and beef cattle. A frequently employed ART in the cattle industry is in vitro production of embryos. However, bovine in vitro produced embryos differ greatly from their in vivo produced counterparts in many facets, including developmental competence. The lower developmental capacity of these embryos could be due to the stress to which the gametes and/or embryos are exposed during in vitro embryo production, specifically ovarian hormonal stimulation, follicular aspiration, oocyte in vitro maturation in hormone supplemented medium, sperm handling, gamete cryopreservation, and culture of embryos. The negative effects of some ARTs on embryo development could, at least partially, be explained by disruption of the physiological epigenetic profile of the gametes and/or embryos. Here, we review the current literature with regard to the putative link between ARTs used in bovine reproduction and epigenetic disorders and changes in the expression profile of embryonic genes. Information on the relationship between reproductive biotechnologies and epigenetic disorders and aberrant gene expression in bovine embryos is limited and novel approaches are needed to explore ways in which ARTs can be improved to avoid epigenetic disorders.     

Artificial insemination and the assisted reproductive technologies in non-human primates

The experience with artificial insemination (AI) and the more invasive ARTs (assisted reproductive technologies) in the propagation of non-human primates (NHPs), although limited, has included representation from the Great Apes and both Old World and New World Macaques. The application of these technologies in NHPs is impacted by high cost, substantial technical requirements and the limited captive populations of available animals. A major incentive for their use would be to propagate endangered, underrepresented individuals or valuable founder animals. Detailed protocols and a substantial experience base for the ARTs are available for rhesus and cynomolgus macaques and form the basis of this review, including sperm recovery, processing and long-term storage at low temperatures, insemination techniques and timing. Controlled ovarian stimulation and subsequent oocyte recovery required for the invasive ARTs such as intracytoplasmic sperm injection (ICSI), is also described. Three recent AI reports in Old World Macaques are reviewed, along with examples of the use of the ARTs in the propagation of valuable founder animals, in the preservation of endangered macaques, and finally in the creation of neurodegenerative disease models for biomedical research purposes.     

Biogenetic ties and parent‐child relationships: The misplaced critique

According to an almost axiomatic standard in bioethics, moral commitment should ground parents’ relationship with their children, rather than biogenetic relatedness. This standard has been used lately to express skepticism about extending existing assisted reproductive treatments (ARTs) to same‐sex couples and to research into novel fertility interventions for those couples, but this skepticism is misplaced on several grounds. As a matter of access and equity, same‐sex couples seem presumptively entitled to genetic relatedness to their children as far as possible both in regard to existing ARTs and to novel ARTs under investigation. For those worried about the effects of trying to secure biogenetic relatedness for same‐sex couples, it may be noted that same‐sex couples will only ever be a fraction of the parents implicated in propping up “biologism,” as the expectation of biogenetic relatedness it is sometimes called. The cultural force of biologism would survive almost intact even if no same‐sex couples were ever to have genetically related children. It is therefore hard to see why same‐sex couples should forfeit aspirations to biogenetic relationships with their children or enjoy less subsidy for ARTs than the subsidy given to different‐sex couples. As matter of moral consistency, the full implications of the biologism critique have yet to be evaluated relative to different‐sex couples.     

Reproductive investment patterns,sperm characteristics,and seminal plasma physiology in alternative reproductive tactics of Chinook salmon (Oncorhynchus tshawytscha)

Although alternative reproductive tactics (ARTs) are common across a range of taxa, little is known about whether the different tactics have adapted to sperm competition risk. Chinook salmon, Oncorhynchus tshawytscha, have two ARTs: large males that participate in dominance‐based hierarchies for access to spawning females, known as hooknoses, and small males that attempt to sneak fertilizations during spawning events from peripheral positions, known as jacks. Jacks continually face sperm competition risk because they always spawn in the presence of another male, whereas hooknoses face relatively low sperm competition risk because other males are not always present during spawning events. Based on the sneak‐guard model of sperm competition this asymmetry in sperm competition risk predicts that jacks ought to invest significantly more into sperm‐related traits important for sperm competition success relative to hooknoses. In the present study we report on reproductive investment patterns, sperm characteristics, and seminal plasma physiology of males that exhibit ARTs in Chinook salmon. We found that jacks invest significantly more of their somatic tissue into gonads compared with hooknoses. Sperm velocity also varied significantly between the ARTs, with jacks having significantly faster sperm than hooknoses. No significant differences in seminal plasma physiology metrics related to sperm quality were detected between the ARTs. We interpret these sperm investment patterns in light of the sneak‐guard model of sperm competition that is based on differences in sperm competition risk and alternative investment possibilities among ARTs. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

The family of toxin-related ecto-ADP-ribosyltransferases in humans and the mouse

ADP-ribosyltransferases including toxins secreted by Vibrio cholera, Pseudomonas aerurginosa, and other pathogenic bacteria inactivate the function of human target proteins by attaching ADP-ribose onto a critical amino acid residue. Cross-species polymerase chain reaction (PCR) and database mining identified the orthologs of these ADP-ribosylating toxins in humans and the mouse. The human genome contains four functional toxin-related ADP-ribosyltransferase genes (ARTs) and two related intron-containing pseudogenes; the mouse has six functional orthologs. The human and mouse ART genes map to chromosomal regions with conserved linkage synteny. The individual ART genes reveal highly restricted expression patterns, which are largely conserved in humans and the mouse. We confirmed the predicted extracellular location of the ART proteins by expressing recombinant ARTs in insect cells. Two human and four mouse ARTs contain the active site motif (R-S-EXE) typical of arginine-specific ADP-ribosyltransferases and exhibit the predicted enzyme activities. Two other human ARTs and their murine orthologues deviate in the active site motif and lack detectable enzyme activity. Conceivably, these ARTs may have acquired a new specificity or function. The position-sensitive iterative database search program PSI-BLAST connected the mammalian ARTs with most known bacterial ADP-ribosylating toxins. In contrast, no related open reading frames occur in the four completed genomes of lower eucaryotes (yeast, worm, fly, and mustard weed). Interestingly, these organisms also lack genes for ADP-ribosylhydrolases, the enzymes that reverse protein ADP-ribosylation. This suggests that the two enzyme families that catalyze reversible mono-ADP-ribosylation either were lost from the genomes of these nonchordata eucaryotes or were subject to horizontal gene transfer between kingdoms.     

Impact of male alternative reproductive tactics on female costs of sexual conflict under variation in operational sex ratio and population density

Sexual conflict over mating rate is both pervasive and evolutionarily costly. For females, the lifetime reproductive fitness costs that arise through interactions with potential mates will be influenced by the frequency of such interactions, and the fitness cost of each interaction. Both of these factors are likely to be influenced by variation in operational sex ratio (OSR) and population density. Variation in OSR‐ and density‐dependent male alternative reproductive tactics (ARTs) may be particularly important if the fitness costs that females experience vary with the reproductive tactics that males express. Using a simple model, we consider several examples of OSR‐ and/or density‐dependent variation in male ARTs and the frequency of male–female interactions, and find that variation in the expression of male ARTs has the potential to augment or diminish the costs of frequent male interactions for females. Accurately documenting variation in the expression of male ARTs and associated female fitness costs will benefit future work in this area.     

Gamete and embryo isolation and culture with microfluidics

Assisted reproductive technologies (ARTs) encompass some of the most exciting modern scientific developments that tremendously impacts society at many levels. Since the beginning of ARTs, scientists have studied and critically analyzed techniques in order to find ways to improve outcomes; however, little has changed with the actual technology and equipment for embryo in vitro production (IVP). New technologic possibilities exist with the escalating advancements of microfluidic technologies. Microfluidics is based on the behavior of liquids in a microenvironment. Although a young field, substantial research demonstrates the potential of this technology in gamete and embryo isolation and culture. In this review, we briefly discuss physical principles of microfluidics and highlight previous utilization of this technology. We then present designs and outcomes for microfluidic devices utilized thus far for different steps in the IVP process: gamete isolation and processing, fertilization, and embryo culture. Finally, we discuss and speculate on future use of microfluidics for assessing embryo viability and multiparametric analysis of embryo secretions and the integration of ART stage-specific capabilities that will lead to an "IVP-lab-on-a-chip".     

Insights from intralocus tactical conflict: adaptive states,interactions with ecology and population divergence

Alternative reproductive tactics (ARTs) have improved our understanding of the evolution of adaptive variation; for instance, their study has led us to understand that the best phenotype (e.g. large and flashy) for a tactic that uses one mating behavior (e.g. court females) is often not the best phenotype (e.g. small and inconspicuous) for a tactic that uses a different mating behavior (e.g. chase and force‐copulate females). However, genetic correlations of shared traits across ARTs can constrain ARTs from reaching their optimal states, resulting in intralocus tactical conflict (IATC). While constraints on evolution in general have been well‐established and studied, there are some important implications of constraints due to intralocus tactical conflict on ARTs that have not been incorporated into the field of evolutionary ecology. Here we describe how an appreciation of IATC, including how to detect it and when to expect it, can change our perspectives in three areas: 1) adaptive states for traits associated with ARTs (e.g. growth rates, behavioural plasticity); 2) how selection due to ecological variation across populations can produce patterns of divergence between ARTS; 3) and the evolutionary stability of polymorphisms (e.g. how IATC can explain losses of one ART, and why this can lead to rapid speciation).     

Thiol redox proteomics seen with fluorescent eyes: the detection of cysteine oxidative modifications by fluorescence derivatization and 2-DE

There is increasing evidence that several reversible oxidative post-translational modifications of protein cysteines participate in cell signalling. Specific proteomic techniques are required to identify these modifications and to study their regulation in different cell processes, that are collectively known as thiol redox proteomics. Recently, fluorescence derivatization methods have been developed that enable these post-translational modifications to be studied using proteomic workflows based on two-dimensional electrophoresis, which is a relatively accessible and affordable technique. As well as enabling a large number of samples to be processed, two-dimensional electrophoresis has the advantage that it does not rely on the intensive use of mass spectrometers. This methodology allows to "visualise" redox changes in a broad context and, although identification of the modified residues is not so straightforward, complementary derivatization can overcome this drawback. Here we review the different derivatization strategies that have been employed in these studies, comparing their advantages and potential limitations. We also review the applications and results obtained, with particular emphasis on those involving (patho)physiological stimuli, thereby showing the potential of these techniques to study the thiol redox proteome.     

Assisted reproductive technologies in captive rhinoceroses

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Dihydroartemisinin: A Potential Natural Anticancer Drug

Dihydroartemisinin (DHA) is an active metabolite of artemisinin and its derivatives (ARTs), and it is an effective clinical drug widely used to treat malaria. Recently, the anticancer activity of DHA has attracted increasing attention. Nevertheless, there is no systematic summary on the anticancer effects of DHA. Notably, studies have shown that DHA exerts anticancer effects through various molecular mechanisms, such as inhibiting proliferation, inducing apoptosis, inhibiting tumor metastasis and angiogenesis, promoting immune function, inducing autophagy and endoplasmic reticulum (ER) stress. In this review, we comprehensively summarized the latest progress regarding the anticancer activities of DHA in cancer. Importantly, the underlying anticancer molecular mechanisms and pharmacological effects of DHA in vitro and in vivo are the focus of our attention. Interestingly, new methods to improve the solubility and bioavailability of DHA are discussed, which greatly enhance its anticancer efficacy. Remarkably, DHA has synergistic anti-tumor effects with a variety of clinical drugs, and preclinical and clinical studies provide stronger evidence of its anticancer potential. Moreover, this article also gives suggestions for further research on the anticancer effects of DHA. Thus, we hope to provide a strong theoretical support for DHA as an anticancer drug.     

Lifestyle impact and the biology of the human scrotum

The possession of a scrotum to contain the male gonads is a characteristic feature of almost all mammals, and appears to have evolved to allow the testes and epididymis to be exposed to a temperature a few degrees below that of core body temperature. Analysis of cryptorchid patients, and those with varicocele suggest that mild scrotal warming can be detrimental to sperm production, partly by effects on the stem cell population, and partly by effects on later stages of spermatogenesis and sperm maturation. Recent studies on the effects of clothing and lifestyle emphasize that these can also lead to chronically elevated scrotal temperatures. In particular, the wearing of nappies by infants is a cause for concern in this regard. Together all of the evidence indirectly supports the view that lifestyle factors in addition to other genetic and environmental influences could be contributing to the secular trend in declining male reproductive parameters. The challenge will be to provide relevant and targeted experimental results to support or refute the currently circumstantial evidence.     

Disinfection procedures for controlling microorganisms in the semen and embryos of humans and farm animals

Semen and embryos generated by assisted reproductive techniques (ARTs) may be contaminated with numerous microorganisms. Contamination may arise from systemic or local reproductive tract infections in donors or the inadvertent introduction of microorganisms during ARTs, and may lead to disease transmission. This review describes sanitary procedures which have been investigated to ascertain whether they are effective in rendering semen and embryos free of pathogenic microorganisms, including internationally adopted washing procedures, which can be supplemented by antibiotics and enzymatic treatments. Other methods include treatment with antibodies or ozone, photoinactivation, acidification, and the use of novel antiviral compounds. In conclusion, despite the wide range of antimicrobial procedures available, none can be recommended as a universal disinfection method for rendering semen and embryos free from all potentially pathogenic microorganisms. However, some procedures are unsuitable, as they can compromise the viability of semen or embryos. In humans, washing by the gradient centrifugation method appears to be effective for reducing the microbial population in semen and is harmless to the spermatozoa. A useful procedure for embryos involving multiple washes in sterile medium has much to commend it for the prevention of disease transmission; furthermore, it is recommended by the International Embryo Transfer Society (IETS).     

Evaluation of Blastomere Biopsy Using a Mouse Model Indicates the Potential High Risk of Neurodegenerative Disorders in the Offspring

Preimplantation genetic diagnosis (PGD), used in clinical practice, is offered to couples that may suffer from a monogenetic disorder, chromosome aneuploidy, or X-linked disease. However, blastomere biopsy, as an indispensable manipulation during the PGD procedure has not been assessed for its long term health implications. Using a mouse model, we investigated the effect of blastomere biopsy of in vitro cultured four-cell embryos on preimplantation development efficiency, postnatal growth, and physiological and behavioral activity compared with control, non-biopsied embryos. The mice generated after blastomere biopsy showed weight increase and some memory decline compared with the control group. Further protein expression profiles in adult brains were analyzed by a proteomics approach. A total of 36 proteins were identified with significant differences between the biopsied and control groups, and the alterations in expression of most of these proteins have been associated with neurodegenerative diseases. Furthermore hypomyelination of the nerve fibers was observed in the brains of mice in the biopsied group. This study suggested that the nervous system may be sensitive to blastomere biopsy procedures and indicated an increased relative risk of neurodegenerative disorders in the offspring generated following blastomere biopsy. Thus, more studies should be performed to address the possible adverse effects of blastomere biopsy on the development of offspring, and the overall safety of PGD technology should be more rigorously assessed.Preimplantation genetic diagnosis (PGD)¹ has been one of the main clinical components of assisted reproductive technologies (ARTs) since 1990 (1). At present, infertile couples experiencing recurrent miscarriage or X chromosome-linked diseases are most likely to benefit from PGD.The treatment of human infertility by ARTs has gained widespread application, but it is disconcerting to many researchers that the clinical procedures used in ARTs are rapidly outpacing the underlying science. ART procedures are generally considered to be safe, but recent studies suggest a small increase in birth defects and low birth weights in ART children (2, 3). In addition, several clinical studies have reported an increased frequency of Beckwith-Wiedemann syndrome or Angelman syndrome caused by an imprinting defect among children conceived with ARTs (4, 5). These potential risks cause serious unease and justify more serious assessments of ARTs. However, moral, ethical, and legal issues complicate assessments of the genetic quality of ART-derived human conceptions, and significant genetic and demographic differences exist among couples participating in the ARTs, so a definitive assessment of the risks associated with this technology has been difficult to achieve. Therefore, appropriate animal models provide an important tool for studying potential effects of ARTs on the health and development of mammalian embryos (6).In many ART procedures, embryos are kept for a short time in a synthetic culture medium before transfer into their recipient mothers. Animal data have demonstrated that in vitro embryo culture and related procedures may be associated with epigenetic changes, perturbed genomic imprinting, and alterations in fetal growth (7). Some evidence also suggested that the culture environment may produce specific abnormalities during fetal and postnatal development (8–10). In the studies using mouse models, more marked changes in adult physiology, including onset of hypertension, were observed (11).As with other ARTs, the protocol required for PGD necessitates embryo manipulation and culture in vitro. However, in contrast to other ARTs, PGD involves embryo biopsy of one or two blastomeres at the eight-cell stage. Some studies have shown that blastomere biopsy does not have negative effects on embryo viability (12, 13), and offspring have been produced using embryo splitting technology, which is similar to the biopsy process, in at least six different domesticated species, including mouse (14), rabbit (15), sheep (16), bovine (17), goat (18), horse (19), pig (20), and in a preliminary non-human primate study one rhesus monkey (21). However, there is still a shortage of proof to convince the public that there is no potential risk to such animals conceived by this technology.To address this issue, we developed a mouse model to study the effects of blastomere biopsy on early embryo development and on postnatal physiological phenotype and behavior. A global proteomics method was also performed to study correlative protein expression profiles in adult brains and to indicate the possibility of neural degenerative disorders in adult conceived following biopsy technology.     

Morphology and ultrastructure of the testes and spermatozoa of the South African leopard (Panthera pardus)

The survival of South African leopard (Panthera pardus) populations is at risk due to the decrease in natural habitat caused by increasing human populations, development projects and industry. The use of ex situ conservation methods such as assisted reproductive techniques (ARTs) in captive breeding programmes can be used to conserve and protect this species. However, a good understanding of the reproductive system of the species is imperative in the development of successful ARTs. This study serves to provide information on the morphology and ultrastructure of the spermatozoa and testes of P. pardus that can be used to help in the development, testing and optimization of reproductive techniques. The objectives of this study were (i) to assess the morphology and ultrastructure of the spermatozoa by means of fluorescence and electron microscopy respectively, and (ii) to examine the morphology and ultrastructure of the testicular tissue as well as of the stages of spermatogenesis using light and electron microscopy. In this study, it was noted that the morphology and ultrastructure of the spermatozoa are similar to those reported in other feline and mammalian species and that a high amount of morphologically abnormal ejaculated sperm was found to be a common occurrence in all samples obtained.