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.

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.

Paternal inheritance of growth in fish pursuing alternative reproductive tactics

In species with indeterminate growth, age-related size variation of reproductive competitors within each sex is often high. This selects for divergence in reproductive tactics of same-sex competitors, particularly in males. Where alternative tactics are fixed for life, the causality of tactic choice is often unclear. In the African cichlid Lamprologus callipterus, large nest males collect and present empty snail shells to females that use these shells for egg deposition and brood care. Small dwarf males attempt to fertilize eggs by entering shells in which females are spawning. The bourgeois nest males exceed parasitic dwarf males in size by nearly two orders of magnitude, which is likely to result from greatly diverging growth patterns. Here, we ask whether growth patterns are heritable in this species, or whether and to which extent they are determined by environmental factors. Standardized breeding experiments using unrelated offspring and maternal half-sibs revealed highly divergent growth patterns of male young sired by nest or dwarf males, whereas the growth of female offspring of both male types did not differ. As expected, food had a significant modifying effect on growth, but neither the quantity of breeding substrate in the environment nor ambient temperature affected growth. None of the environmental factors tested influenced the choice of male life histories. We conclude that in L. callipterus growth rates of bourgeois and parasitic males are paternally inherited, and that male and female growth is phenotypically plastic to only a small degree.

Positive effect of the yellow morph on female reproductive success in the flower colour polymorphic Iris lutescens (Iridaceae), a deceptive species

The deceptive Iris lutescens (Iridaceae) shows a heritable and striking flower colour polymorphism, with both yellow- and purple-flowered individuals growing sympatrically. Deceptive species with flower colour polymorphism are mainly described in the family Orchidaceae and rarely found in other families. To explain the maintenance of flower colour polymorphism in I.lutescens, we investigated female reproductive success in natural populations of southern France, at both population and local scales (within populations). Female reproductive success was positively correlated with yellow morph frequency, at both the population scale and the local scale. Therefore, we failed to observe negative frequency-dependent selection (NFDS), a mechanism commonly invoked to explain flower colour polymorphism in deceptive plant species. Flower size and local flower density could also affect female reproductive success in natural populations. Pollinator behaviour could explain the positive effect of the yellow morph, and our results suggest that flower colour polymorphism might not persist in I.lutescens, but alternative explanations not linked to pollinator behaviour are discussed. In particular, NFDS, although an appealingly simple explanation previously demonstrated in orchids, may not always contribute to maintaining flower colour polymorphism, even in deceptive species.

Antioxidants improve vascular function in children conceived by assisted reproductive technologies: A randomized double-blind placebo-controlled trial.

AIMS Children conceived by assisted reproductive technology (ART) display vascular dysfunction. Its underlying mechanism, potential reversibility and long-term consequences for cardiovascular risk are unknown. In mice, ART induces arterial hypertension and shortens the life span. These problems are related to decreased vascular endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) synthesis. The aim of this study was to determine whether ART-induced vascular dysfunction in humans is related to a similar mechanism and potentially reversible. To this end we tested whether antioxidants improve endothelial function by scavenging free radicals and increasing NO bioavailability. METHODS AND RESULTS In this prospective double-blind placebo controlled study in 21 ART and 21 control children we assessed the effects of a four-week oral supplementation with antioxidant vitamins C (1 g) and E (400 IU) or placebo (allocation ratio 2:1) on flow-mediated vasodilation (FMD) of the brachial artery and pulmonary artery pressure (echocardiography) during high-altitude exposure (3454 m), a manoeuver known to facilitate the detection of pulmonary vascular dysfunction and to decrease NO bioavailability by stimulating oxidative stress. Antioxidant supplementation significantly increased plasma NO measured by ozone-based chemiluminescence (from 21.7 ± 7.9 to 26.9 ± 7.6 µM, p = 0.04) and FMD (from 7.0 ± 2.1 to 8.7 ± 2.0%, p = 0.004) and attenuated altitude-induced pulmonary hypertension (from 33 ± 8 to 28 ± 6 mm Hg, p = 0.028) in ART children, whereas it had no detectable effect in control children. CONCLUSIONS Antioxidant administration to ART children improved NO bioavailability and vascular responsiveness in the systemic and pulmonary circulation. Collectively, these findings indicate that in young individuals ART-induced vascular dysfunction is subject to redox regulation and reversible.

Cardiovascular dysfunction in children conceived by assisted reproductive technologies.

Epidemiological studies demonstrate a relationship between pathological events during foetal development and future cardiovascular risk and the term 'foetal programming of cardiovascular disease' has been coined to describe this phenomenon. The use of assisted reproductive technologies (ARTs) is growing exponentially and 2-5% of children are now born by this procedure. Emerging evidence indicates that ART represents a novel important example of foetal programming. Assisted reproductive technology may modify the cardiovascular phenotype in two ways: (i) ART involves manipulation of the early embryo which is exquisitely sensitive to environmental insults. In line with this concern, ART alters vascular and cardiac function in children and studies in mice show that ART alters the cardiovascular phenotype by epigenetic alterations related to suboptimal culture conditions. (ii) Assisted reproductive technology markedly increases the risk of foetal insults that augment cardiovascular risk in naturally conceived individuals and are expected to have similar consequences in the ART population. Given the young age of the ART population, it will take another 20-30 years before data on cardiovascular endpoints will be available. What is clear already, however, is that ART emerges as an important cardiovascular risk factor. This insight requires us to revise notions on ART's long-term safety and to engage on a debate on its future. There is an urgent need to better understand the mechanisms underpinning ART-induced alteration of the cardiovascular phenotype, improve the procedure and its long-term safety, and, while awaiting this aim, not to abandon medicine's fundamental principle of doing no harm (to future children) and use ART parsimoniously.

Right ventricular dysfunction in children and adolescents conceived by assisted reproductive technologies.

Assisted reproductive technologies (ART) predispose the offspring to vascular dysfunction, arterial hypertension, and hypoxic pulmonary hypertension. Recently, cardiac remodeling and dysfunction during fetal and early postnatal life have been reported in offspring of ART, but it is not known whether these cardiac alterations persist later in life and whether confounding factors contribute to this problem. We, therefore, assessed cardiac function and pulmonary artery pressure by echocardiography in 54 healthy children conceived by ART (mean age 11.5 ± 2.4 yr) and 54 age-matched (12.2 ± 2.3 yr) and sex-matched control children. Because ART is often associated with low birth weight and prematurity, two potential confounders associated with cardiac dysfunction, only singletons born with normal birth weight at term were studied. Moreover, because cardiac remodeling in infants conceived by ART was observed in utero, a situation associated with increased right heart load, we also assessed cardiac function during high-altitude exposure, a condition associated with hypoxic pulmonary hypertension-induced right ventricular overload. We found that, while at low altitude cardiac morphometry and function was not different between children conceived by ART and control children, under the stressful conditions of high-altitude-induced pressure overload and hypoxia, larger right ventricular end-diastolic area and diastolic dysfunction (evidenced by lower E-wave tissue Doppler velocity and A-wave tissue Doppler velocity of the lateral tricuspid annulus) were detectable in children and adolescents conceived by ART. In conclusion, right ventricular dysfunction persists in children and adolescents conceived by ART. These cardiac alterations appear to be related to ART per se rather than to low birth weight or prematurity.

Prevention of vascular dysfunction and arterial hypertension in mice generated by assisted reproductive technologies by addition of melatonin to culture media.

Assisted reproductive technologies (ART) induce vascular dysfunction in humans and mice. In mice, ART-induced vascular dysfunction is related to epigenetic alteration of the endothelial nitric oxide synthase (eNOS) gene, resulting in decreased vascular eNOS expression and nitrite/nitrate synthesis. Melatonin is involved in epigenetic regulation, and its administration to sterile women improves the success rate of ART. We hypothesized that addition of melatonin to culture media may prevent ART-induced epigenetic and cardiovascular alterations in mice. We, therefore, assessed mesenteric-artery responses to acetylcholine and arterial blood pressure, together with DNA methylation of the eNOS gene promoter in vascular tissue and nitric oxide plasma concentration in 12-wk-old ART mice generated with and without addition of melatonin to culture media and in control mice. As expected, acetylcholine-induced mesenteric-artery dilation was impaired (P = 0.008 vs. control) and mean arterial blood pressure increased (109.5 ± 3.8 vs. 104.0 ± 4.7 mmHg, P = 0.002, ART vs. control) in ART compared with control mice. These alterations were associated with altered DNA methylation of the eNOS gene promoter (P < 0.001 vs. control) and decreased plasma nitric oxide concentration (10.1 ± 11.1 vs. 29.5 ± 8.0 μM) (P < 0.001 ART vs. control). Addition of melatonin (10(-6) M) to culture media prevented eNOS dysmethylation (P = 0.005, vs. ART + vehicle), normalized nitric oxide plasma concentration (23.1 ± 14.6 μM, P = 0.002 vs. ART + vehicle) and mesentery-artery responsiveness to acetylcholine (P < 0.008 vs. ART + vehicle), and prevented arterial hypertension (104.6 ± 3.4 mmHg, P < 0.003 vs. ART + vehicle). These findings provide proof of principle that modification of culture media prevents ART-induced vascular dysfunction. We speculate that this approach will also allow preventing ART-induced premature atherosclerosis in humans.