The horse genome project - sequence based insights into male reproductive mechanisms

The growing knowledge on physiology, cell biology and biochemistry of the reproductive organs has provided many insights into molecular mechanisms that are required for successful reproduction. Research directed at the investigation of reproduction physiology in domestic animals was hampered in the past by a lack of species-specific genomic information. The genome sequences of dog, cattle and horse have become publicly available in 2005, 2006 and 2007 respectively. Although the gene content of mammalian genomes is generally very similar, genes involved in reproduction tend to be less conserved than the average mammalian gene. The availability of genome sequences provides a valuable resource to check whether any protein that may be known from human or mouse research is present in cattle and/or horse as well. Currently there are more than 200 genes known that are involved in the production of fertile sperm cells. Great progress has been made in the understanding of genetic aberrations that lead to male infertility. Additionally, the first genetic mechanisms are being discovered that contribute to the quantitative variation of fertility traits in fertile male animals. Here, I will review some selected aspects of genetic research in male fertility and offer some perspectives for the use of genomic sequence information.

Male reproductive pattern in a polygynous ungulate with a slow life-history: the role of age, social status and alternative mating tactics

According to life-history theory age-dependent investments into reproduction are thought to co-vary with survival and growth of animals. In polygynous species, in which size is an important determinant of reproductive success, male reproduction via alternative mating tactics at young age are consequently expected to be the less frequent in species with higher survival. We tested this hypothesis in male Alpine ibex (Capra ibex), a highly sexually dimorphic mountain ungulate whose males have been reported to exhibit extremely high adult survival rates. Using data from two offspring cohorts in a population in the Swiss Alps, the effects of age, dominance and mating tactic on the likelihood of paternity were inferred within a Bayesian framework. In accordance with our hypothesis, reproductive success in male Alpine ibex was heavily biased towards older, dominant males that monopolized access to receptive females by adopting the 'tending' tactic, while success among young, subordinate males via the sneaking tactic 'coursing' was in general low and rare. In addition, we detected a high reproductive skew in male Alpine ibex, suggesting a large opportunity for selection. Compared with other ungulates with higher mortality rates, reproduction among young male Alpine ibex was much lower and more sporadic. Consistent with that, further examinations on the species level indicated that in polygynous ungulates the significance of early reproduction appears to decrease with increasing survival. Overall, this study supports the theory that survival prospects of males modulate the investments into reproduction via alternative mating tactics early in life. In the case of male Alpine ibex, the results indicate that their life-history strategy targets for long life, slow and prolonged growth and late reproduction.

Endocrine disrupting compounds: can they target the immune system of fish?

Endocrine disruption, in particular disruption by estrogen-active compounds, has been identified as an important ecotoxicological hazard in the aquatic environment. Research on the impact of endocrine disrupting compounds (EDCs) on wildlife has focused on disturbances of the reproductive system. However, there is increasing evidence that EDCs affect a variety of physiological systems other than the reproductive system. Here, we discuss if EDCs may be able to affect the immune system of fish, as this would have direct implications for individual fitness and population growth. Evidence suggesting an immunomodulatory role of estrogens in fish comes from the following findings: (a) estrogen receptors are expressed in piscine immune organs, (b) immune gene expression is modulated by estrogen exposure, and (c) pathogen susceptibility of fish increases under estrogen exposure.

Complex genital system of a haplogyne spider (Arachnida, Araneae, Tetrablemmidae) indicates internal fertilization and full female control over transferred sperm

The female genital organs of the tetrablemmid Indicoblemma lannaianum are astonishingly complex. The copulatory orifice lies anterior to the opening of the uterus externus and leads into a narrow insertion duct that ends in a genital cavity. The genital cavity continues laterally in paired tube-like copulatory ducts, which lead into paired, large, sac-like receptacula. Each receptaculum has a sclerotized pore plate with associated gland cells. Paired small fertilization ducts originate in the receptacula and take their curved course inside the copulatory ducts. The fertilization ducts end in slit-like openings in the sclerotized posterior walls of the copulatory ducts. Huge masses of secretions forming large balls are detectable in the female receptacula. An important function of these secretory balls seems to be the encapsulation of spermatozoa in discrete packages in order to avoid the mixing of sperm from different males. In this way, sperm competition may be completely prevented or at least severely limited. Females seem to have full control over transferred sperm and be able to express preference for spermatozoa of certain males. The lumen of the sperm containing secretory balls is connected with the fertilization duct. Activated spermatozoa are only found in the uterus internus of females, which is an indication of internal fertilization. The sperm cells in the uterus internus are characterized by an extensive cytoplasm and an elongated, cone-shaped nucleus. The male genital system of I. lannaianum consists of thick testes and thin convoluted vasa deferentia that open into the wide ductus ejaculatorius. The voluminous globular palpal bulb is filled with seminal fluid consisting of a globular secretion in which only a few spermatozoa are embedded. The spermatozoa are encapsulated by a sheath produced in the genital system. The secretions in females may at least partly consist of male secretions that could be involved in the building of the secretory balls or play a role in sperm activation. The male secretions could also afford nutriments to the spermatozoa.

Intraspecific sexual selection on a speciation trait, male coloration, in the Lake Victoria cichlid Pundamilia nyererei

The haplochromine cichlids of Lake Victoria constitute a classical example of explosive speciation. Extensive intra– and interspecific variation in male nuptial coloration and female mating preferences, in the absence of postzygotic isolation between species, has inspired the hypothesis that sexual selection has been a driving force in the origin of this species flock. This hypothesis rests on the premise that the phenotypic traits that underlie behavioural reproductive isolation between sister species diverged under sexual selection within a species. We test this premise in a Lake Victoria cichlid, by using laboratory experiments and field observations. We report that a male colour trait, which has previously been shown to be important for behavioural reproductive isolation between this species and a close relative, is under directional sexual selection by female mate choice within this species. This is consistent with the hypothesis that female choice has driven the divergence in male coloration between the two species. We also find that male territoriality is vital for male reproductive success and that multiple mating by females is common.

Sequential female assessment drives complex sexual selection on bower shape in a cichlid fish

In many animals, sexual selection on male traits results from female mate choice decisions made during a sequence of courtship behaviors. We use a bower-building cichlid fish, Nyassachromis cf. microcephalus, to show how applying standard selection analysis to data on sequential female assessment provides new insights into sexual selection by mate choice. We first show that the cumulative selection differentials confirm previous results suggesting female choice favors males holding large volcano-shaped sand bowers. The sequential assessment analysis reveals these cumulative differentials are the result of selection acting on different bower dimensions during the courtship sequence; females choose to follow males courting from tall bowers, but choose to engage in premating circling with males holding bowers with large diameter platforms. The approach we present extends standard selection analysis by partitioning the variances of increasingly accurate estimates of male reproductive fitness and is applicable to systems in which sequential female assessment drives sexual selection on male traits.

17Beta-estradiol affects the response of complement components and survival of rainbow trout (Oncorhynchus mykiss) challenged by bacterial infection

Research on the endocrine role of estrogens has focused on the reproductive system, while other potential target systems have been less studied. Here, we investigated the possible immunomodulating role of 17beta-estradiol (E2) using rainbow trout (Oncorhynchus mykiss) as a model. The aims of the study were to examine a) whether estrogens can modulate immune gene transcription levels, and b) whether this has functional implications for the resistance of trout towards pathogens. Trout were reared from fertilization until 6 months of age under (1) control conditions, (2) short-term E2-treatment (6-month-old juveniles were fed a diet containing 20 mg E2/kg for 2 weeks), or c) long-term E2-treatment (twice a 2-h-bath-exposure of trout embryos to 400 mug 17beta-estradiol (E2)/L, followed by rearing on the E2-spiked diet from start-feeding until 6 months of age). Analysis of plasma estrogen levels indicated that the internal estrogen concentrations of E2-exposed fish were within the physiological range and analysis of hepatic vitellogenin mRNA levels indicated that the E2 administration was effective in activating the endogenous estrogen receptor pathway. However, expression levels of the hepatic complement components C3-1, C3-3, and Factor H were not affected by E2-treatment. In a next step, 6-month-old juveniles were challenged with pathogenic bacteria (Yersinia ruckeri). In control fish, this bacterial infection resulted in significant up-regulation of the mRNA levels of hepatic complement genes (C3-1, C3-3, Factor B, Factor H), while E2-treated fish showed no or significantly lower up-regulation of the complement gene transcription levels. Apparently, the E2-treated trout had a lower capacity to activate their immune system to defend against the bacterial infection. This interpretation is corroborated by the finding that survival of E2-treated fish under bacterial challenge was significantly lower than in the control group. In conclusion, the results from this study suggest that estrogens are able to modulate immune parameters of trout with functional consequences on their ability to cope with pathogens.

Interference of endocrine disrupting chemicals with aromatase CYP19 expression or activity, and consequences for reproduction of teleost fish

Many natural and synthetic compounds present in the environment exert a number of adverse effects on the exposed organisms, leading to endocrine disruption, for which they were termed endocrine disrupting chemicals (EDCs). A decrease in reproduction success is one of the most well-documented signs of endocrine disruption in fish. Estrogens are steroid hormones involved in the control of important reproduction-related processes, including sexual differentiation, maturation and a variety of others. Careful spatial and temporal balance of estrogens in the body is crucial for proper functioning. At the final step of estrogen biosynthesis, cytochrome P450 aromatase, encoded by the cyp19 gene, converts androgens into estrogens. Modulation of aromatase CYP19 expression and function can dramatically alter the rate of estrogen production, disturbing the local and systemic levels of estrogens. In the present review, the current progress in CYP19 characterization in teleost fish is summarized and the potential of several classes of EDCs to interfere with CYP19 expression and activity is discussed. Two cyp19 genes are present in most teleosts, cyp19a and cyp19b, primarily expressed in the ovary and brain, respectively. Both aromatase CYP19 isoforms are involved in the sexual differentiation and regulation of the reproductive cycle and male reproductive behavior in diverse teleost species. Alteration of aromatase CYP19 expression and/or activity, be it upregulation or downregulation, may lead to diverse disturbances of the above mentioned processes. Prediction of multiple transcriptional regulatory elements in the promoters of teleost cyp19 genes suggests the possibility for several EDC classes to affect cyp19 expression on the transcriptional level. These sites include cAMP responsive elements, a steroidogenic factor 1/adrenal 4 binding protein site, an estrogen-responsive element (ERE), half-EREs, dioxin-responsive elements, and elements related to diverse other nuclear receptors (peroxisome proliferator activated receptor, retinoid X receptor, retinoic acid receptor). Certain compounds including phytoestrogens, xenoestrogens, fungicides and organotins may modulate aromatase CYP19 activity on the post-transcriptional level. As is shown in this review, diverse EDCs may affect the expression and/or activity of aromatase cyp19 genes through a variety of mechanisms, many of which need further characterization in order to improve the prediction of risks posed by a contaminated environment to teleost fish population.

A novel open channel blocker of GABA-A receptors

GABA-A receptors are chloride ion channels composed of five subunits, mediating fast synaptic and tonic inhibition in the mammalian brain. 19 different subunit isoforms have been identified, with the major receptor type in mammalian adult brain consisting of α1, β2, and γ2 subunits. GABA-A receptors are the target of numerous sedating and anxiolytic drugs such as benzodiazepines. The currently known endogenous ligands are GABA, neurosteroids and the endocannabinoid 2- arachidonoyl glycerol (2-AG). The pharmacological properties of this chloride ion channel strictly depend on receptor subunit composition and arrangement. GABA-A receptors bind and are inhibited by epileptogenic agents such as picrotoxin, and cyclodiene insecticides such as dieldrin. We screened aromatic monovalent anions with five-fold symmetry for inhibition of GABA-A receptors. One of the anions, PCCPinhibited currents elicited by GABA with comparable potency as picrotoxin. This inhibition showed all characteristics of an open channel block. The GABA-A receptor ion channel is lined by residues from the M2 membrane-spanning segment. To identify important residues of the pore involved in the interaction with the blocking molecules PCCP-, a mutation scan was performed in combination with subsequent analysis of the expressed mutant proteins using electrophysiological techniques. In a second project we characterised a light-switchable modulator of GABA-A receptors based on propofol. It was my responsibility to investigate the switching kinetics in patch clamp experiments. After its discovery in 1980, propofol has become the most widely used intravenous general anaesthetic. It is commonly accepted that the anaesthesia induced by this unusually lipophilic drug mostly results from potentiation of GABA induced currents. While GABA-A receptors respond to a variety of ligands, they are normally not sensitive towards light. This light sensitivity could be indirectly achieved by using modulators that can be optically switched between an active and an inactive form. We tested an azobenzene derivative of propofol where an aryldiazene unit is directly coupled to the pharmacophore. This molecule was termed azopropofol (AP2). The effect of AP2 on Cl- currents was investigated with electrophysiological techniques using α1β2γ2 GABA-A receptors expressed in Xenopus oocytes and HEK-cells. In the third project we wanted to investigate the functional role of GABA-A receptors in the liver, and their possible involvement in cell proliferation. GABA-A receptors are also found in a wide range of peripheral tissues, including parts of the peripheral nervous system and non-neural tissues such as smooth muscle, the female reproductive system, liver and several cancer tissues. However their precise function in non neuronal or cancerous cells is still unknown. For this purpose we investigated expression, localization and function of the hepatocytes GABA-A receptors in model cell lines and healthy and cancerous hepatocytes.

Reproductive parasitism: male and female responses to conspecific and heterospecific intrusions at spawning in a mouth-brooding cichlid Ophthalmotilapia ventralis

A rare form of alternative reproductive behaviour without simultaneous parasitic spawning was observed in Ophthalmotilapia ventralis, a lekking mouth-brooding cichlid from Lake Tanganyika. Floater males attempted to sneak opportunistically into the territory to actively court the female, while the owner (bourgeois male) defended the territory against other potential intruders. Floater males had more body fat than territory owners and generally higher condition factors. In field experiments, the response of bourgeois males and courted females was tested towards floaters and egg predators (a catfish Synodontis multipunctatus) present in the territories. Territory owners responded aggressively particularly to floaters, and female responsiveness to bourgeois male courtship tended to decline when floaters were present. The potential influence of reproductive parasitism on sexual selection in mouth-brooding cichlids is discussed.

Effect of sibling competition and male carotenoid supply on offspring condition and oxidative stress

Early developmental conditions have major implications for an individual's fitness. In species where offspring are born simultaneously, the level of sibling competition for food access is intense. In birds, high sibling competition may subject nestlings to decreased growth rate as a result of limited food and increased levels of oxidative stress through high metabolic activity induced by begging behaviors. We manipulated the level of sibling competition in a natural population of great tits and assessed the consequences for nestling body condition and resistance to oxidative stress. In a full factorial design, we both augmented brood size to increase sibling competition and supplemented the male parents with physiological doses of carotenoids thereby doubling the natural carotenoid intake, aiming at increasing the males' investment in current reproduction and thereby decreasing sibling competition. Nestling body mass was reduced by the brood enlargement and enhanced by the carotenoid supplementation of fathers. Nestling resistance to oxidative stress, measured as total antioxidant defenses in whole blood, was not influenced by the treatments. Because nestlings experience high metabolic activities, an absence of an effect of sibling competition on free radicals production seems unlikely. Nestling body mass decreased and resistance to oxidative stress tended to increase with initial brood size, and hence these correlational effects suggest a trade-off between morphological growth and development of the antioxidant system. However, the result of the experimental treatment did not support this trade-off hypothesis. Alternatively, it suggests that nestling developed compensatory mechanisms that were not detected by our antioxidant capacity measure.

Habitat-specific demography and source-sink dynamics in a population of Siberian jays

P>1. There are a number of models describing population structure, many of which have the capacity to incorporate spatial habitat effects. One such model is the source-sink model, that describes a system where some habitats have a natality that is higher than mortality (source) and others have a mortality that exceeds natality (sink). A source can be maintained in the absence of migration, whereas a sink will go extinct. 2. However, the interaction between population dynamics and habitat quality is complex, and concerns have been raised about the validity of published empirical studies addressing source-sink dynamics. In particular, some of these studies fail to provide data on survival, a significant component in disentangling a sink from a low quality source. Moreover, failing to account for a density-dependent increase in mortality, or decrease in fecundity, can result in a territory being falsely assigned as a sink, when in fact, this density-dependent suppression only decreases the population size to a lower level, hence indicating a 'pseudo-sink'. 3. In this study, we investigate a long-term data set for key components of territory-specific demography (mortality and reproduction) and their relationship to habitat characteristics in the territorial, group-living Siberian jay (Perisoreus infaustus). We also assess territory-specific population growth rates (r), to test whether spatial population dynamics are consistent with the ideas of source-sink dynamics. 4. Although average mortality did not differ between sexes, habitat-specific mortality did. Female mortality was higher in older forests, a pattern not observed in males. Male mortality only increased with an increasing amount of open areas. Moreover, reproductive success was higher further away from human settlement, indicating a strong effect of human-associated nest predators. 5. Averaged over all years, 76% of the territories were sources. These territories generally consisted of less open areas, and were located further away from human settlement. 6. The source-sink model provides a tool for modelling demography in distinct habitat patches of different quality, which can aid in identifying key habitats within the landscape, and thus, reduce the risk of implementing unsound management decisions.