Sexual differentiation in three unconventional mammals: spotted hyenas, elephants and tammar wallabies

The present review explores sexual differentiation in three non-conventional species: the spotted hyena, the elephant and the tammar wallaby, selected because of the natural challenges they present for contemporary understanding of sexual differentiation. According to the prevailing view of mammalian sexual differentiation, originally proposed by Alfred Jost, secretion of androgen and anti-Mullerian hormone (AMH) by the fetal testes during critical stages of development accounts for the full range of sexually dimorphic urogenital traits observed at birth. Jost's concept was subsequently expanded to encompass sexual differentiation of the brain and behavior. Although the central focus of this review involves urogenital development, we assume that the novel mechanisms described in this article have potentially significant implications for sexual differentiation of brain and behavior, a transposition with precedent in the history of this field. Contrary to the "specific" requirements of Jost's formulation, female spotted hyenas and elephants initially develop male-type external genitalia prior to gonadal differentiation. In addition, the administration of anti-androgens to pregnant female spotted hyenas does not prevent the formation of a scrotum, pseudoscrotum, penis or penile clitoris in the offspring of treated females, although it is not yet clear whether the creation of masculine genitalia involves other steroids or whether there is a genetic mechanism bypassing a hormonal mediator. Wallabies, where sexual differentiation occurs in the pouch after birth, provide the most conclusive evidence for direct genetic control of sexual dimorphism, with the scrotum developing only in males and the pouch and mammary glands only in females, before differentiation of the gonads. The development of the pouch and mammary gland in females and the scrotum in males is controlled by genes on the X chromosome. In keeping with the "expanded" version of Jost's formulation, secretion of androgens by the fetal testes provides the best current account of a broad array of sex differences in reproductive morphology and endocrinology of the spotted hyena, and androgens are essential for development of the prostate and penis of the wallaby. But the essential circulating androgen in the male wallaby is 5alpha androstanediol, locally converted in target tissues to DHT, while in the pregnant female hyena, androstenedione, secreted by the maternal ovary, is converted by the placenta to testosterone (and estradiol) and transferred to the developing fetus. Testicular testosterone certainly seems to be responsible for the behavioral phenomenon of musth in male elephants. Both spotted hyenas and elephants display matrilineal social organization, and, in both species, female genital morphology requires feminine cooperation for successful copulation. We conclude that not all aspects of sexual differentiation have been delegated to testicular hormones in these mammals. In addition, we suggest that research on urogenital development in these non-traditional species directs attention to processes that may well be operating during the sexual differentiation of morphology and behavior in more common laboratory mammals, albeit in less dramatic fashion.     

Genetic evidence for a maternal effect locus controlling genomic imprinting and growth

Crosses between two species of deer mouse (Peromyscus) yield dramatic parent-of-origin effects. Female P. maniculatus (BW) crossed with male P. polionotus (PO) produce animals smaller than either parent. PO females crossed with BW males yield lethal overgrowth that has been associated with loss-of-imprinting (LOI). Previously, we mapped two loci influencing fetal growth. These two loci, however, do not account for the LOI, nor for the dysmorphic phenotypes. Here we report that maternal genetic background strongly influences the LOI. Analyses of crosses wherein maternal genetic background is varied suggest that this effect is likely due to the action of a small number of loci. We have termed these putative loci Meil. Estimation of Meil loci number was confounded by skewed allelic ratios in the intercross line employed. We show that the Meil loci are not identical to any of the DNA methyltransferases shown to be involved in regulation of genomic imprinting.     

Non-Mendelian transmission of alleles at microsatellite loci: an example in Ixodes ricinus, the vector of Lyme disease

Microsatellite loci are usually considered to be neutral co-dominant and Mendelian markers. We undertook to study the inheritance of five microsatellite loci in the European Lyme disease vector, the tick Ixodes ricinus. Only two loci appeared fully Mendelian while the three others displayed non-Mendelian patterns that highly frequent null alleles could not fully explain. At one locus, IR27, some phenomenon seems to hinder the PCR amplification of one allele, depending on its origin (maternal imprinting) and/or its size (short allele dominance). DNA methylation, which appeared to be a possible explanation of this amplification bias, was rejected by a specific test comparing the amplification efficiency that did not differ between unmethylated and experimentally methylated DNA. The role of allele size in heterozygous individuals was then revealed from the data available on field collected ticks and consistent with the results of a theoretical approach. These observations highlight the need for prudence while inferring reproductive systems (selfing rates), parentage or even allelic frequencies from microsatellite markers, in particular for parasitic organisms for which molecular approaches often represent the only way for population biology inferences.     

Single neonatal treatment with beta-endorphin (hormonal imprinting) extremely enhances nocistatin level of cerebrospinal fluid in adult rats

In earlier experiments endorphin treatment of newborn rats caused the decrease of brain serotonin content, increasing aggressivity, enhanced sexual activity of females and changes in the binding capacity of uterine estrogen receptors at adult age, however nociceptin content of the cerebrospinal fluid was not changed. In the present experiment neonatal treatment of male and female rats was done with a single dose of 3 microg beta-endorphin and in five months old rats the level of nociceptin antagonist nocistatin was determined by radioimmunoassay in the cerebrospinal fluid. In both genders the amount of nocistatin was one magnitude higher in the endorphin treated groups. There was also a significant difference between the male and female nocistatin level in the treated and non-treated groups alike, with the advantage of females. The results call attention to the possibility of influencing pain-tolerance for life, by the pain-provoked endorphin levels during delivery.     

Meiotic and epigenetic aberrations in Dnmt3L-deficient male germ cells

The DNA methyltransferase-like protein Dnmt3L is necessary for the establishment of genomic imprints in oogenesis and for normal spermatogenesis (Bourc'his et al., 2001; Hata et al., 2002). Also, a paternally imprinted gene, H19, loses DNA methylation in Dnmt3L-/- spermatogonia (Bourc'his and Bestor, 2004; Kaneda et al., 2004). To determine the reason for the impaired spermatogenesis in the Dnmt3L-/- testes, we have carried out a series of histological and molecular studies. We show here that Dnmt3L-/- germ cells were arrested and died around the early meiotic stage. A microarray-based gene expression-profiling analysis revealed that various gonad-specific and/or sex-chromosome-linked genes were downregulated in the Dnmt3L-/- testes. In contrast, expression of retrovirus-like intracisternal A-particle (IAP) sequences was upregulated; consistent with this observation, a specific IAP copy showed complete loss of DNA methylation. These findings indicate that Dnmt3L regulates germ cell-specific gene expression and IAP suppression, which are critical for male germ cell proliferation and meiosis.     

Balance between maternal and paternal alleles sets the timing of resource accumulation in the maize endosperm

Key aspects of seed development in flowering plants are held to be under epigenetic control and to have evolved as a result of conflict between the interests of the male and female gametes (kinship theory). Attempts to identify the genes involved have focused on imprinted sequences, although imprinting is only one mechanism by which male or female parental alleles may be exclusively expressed immediately post-fertilization. We have studied the expression of a subset of endosperm gene classes immediately following interploidy crosses in maize and show that departure from the normal 2 : 1 ratio between female and male genomes exerts a dramatic effect on the timing of expression of some, but not all, genes investigated. Paternal genomic excess prolongs the expression of early genes and delays accumulation of reserves, while maternal genomic excess foreshortens the expression period of early genes and dramatically brings forward endosperm maturation. Our data point to a striking interdependence between the phases of endosperm development, and are consonant with previous work from maize showing progression from cell proliferation to endoreduplication is regulated by the balance between maternal and paternal genomes, and from Arabidopsis suggesting that this ‘phasing’ is regulated by maternally expressed imprinted genes. Our findings are discussed in context of the kinship theory.     

SEX CHROMOSOME LINKAGE OF MATE PREFERENCE AND COLOR SIGNAL MAINTAINS ASSORTATIVE MATING BETWEEN INTERBREEDING FINCH MORPHS

Assortative mating is a key aspect in the speciation process because it is important for both initial divergence and maintenance of distinct species. However, it remains a challenge to explain how assortative mating evolves when diverging populations are undergoing gene flow (e.g., during hybridization). Here I experimentally test how assortative mating is maintained with frequent gene flow between diverged head‐color morphs of the Gouldian finch (Erythrura gouldiae). Contrary to the predominant view on the development of sexual preferences in birds, cross‐fostered offspring did not imprint on the phenotype of their conspecific (red or black morphs) or heterospecific (Bengalese finch) foster parents. Instead, the mating preferences of F₁ and F₂ intermorph‐hybrids are consistent with inheritance on the Z chromosomes, which are also the location for genes controlling color expression and the genes causing low fitness of intermorph‐hybrids. Genetic associations between color signal and preference loci on the sex chromosomes may prevent recombination from breaking down these associations when the morphs interbreed, helping to maintain assortative mating in the face of gene flow. Although sex linkage of reproductively isolating traits is theoretically expected to promote speciation, social and ecological constraints may enforce frequent interbreeding between the morphs, thus preventing complete reproductive isolation.     

Phenotypic gender variation within inflorescences of the protogynous species Helleborus foetidus L. (Ranunculaceae)

In the protogynous species H. foetidus, I investigated if investment in sexual structures and seed set shows any trend with position in the flower-opening sequence. In four-flower inflorescences, stamen number decreased with flower position i.e. was highest for the earliest flower and lowest for the latest flower. Ovule number was significantly higher in the latest flower. Stamen and ovule number did not covary, indicating that there is no structural gender trade-off in this species. Seed set (i.e. percentage of ovules producing seed) did not differ between control plants and pollen-supplemented plants and the effect of pollen supplementation did not vary among positions. Seed set showed marginally significant differences between control plants and simulated-herbivory plants, but the effects of this treatment varied significantly among positions. Significant among-position variation in seed set was observed in the control plants but not the simulated-herbivory plants. Seed set of latest-opening flowers of simulated-herbivory plants was significantly higher than that of latest-opening flowers of control plants. H. foetidus support Brunet and Charlesworth's (1995) prediction that in plants with protogynous flowers, later-opening flowers should specialize as female flowers, at least under conditions of high resource availability.     

Single nucleotide polymorphisms in the imprinted bovine insulin-like growth factor 2 receptor gene (IGF2R) are associated with body size traits in Irish Holstein-Friesian cattle

The regulation of the bioavailability of insulin‐like growth factors (IGFs) is critical for normal mammalian growth and development. The imprinted insulin‐like growth factor 2 receptor gene (IGF2R) encodes a transmembrane protein receptor that acts to sequester and degrade excess circulating insulin‐like growth factor 2 (IGF‐II) – a potent foetal mitogen – and is considered an important inhibitor of growth. Consequently, IGF2R may serve as a candidate gene underlying important growth‐ and body‐related quantitative traits in domestic mammalian livestock. In this study, we have quantified genotype–phenotype associations between three previously validated intronic bovine IGF2R single nucleotide polymorphisms (SNPs) (IGF2R:g.64614T>C, IGF2R:g.65037T>C and IGF2R:g.86262C>T) and a range of performance traits in 848 progeny‐tested Irish Holstein‐Friesian artificial insemination sires. Notably, all three polymorphisms analysed were associated (P ≤ 0.05) with at least one of a number of performance traits related to animal body size: angularity, body depth, chest width, rump width, and animal stature. In addition, the C‐to‐T transition at the IGF2R:g.65037T>C polymorphism was positively associated with cow carcass weight and angularity. Correction for multiple testing resulted in the retention of two genotype–phenotype associations (animal stature and rump width). None of the SNPs analysed were associated with any of the milk traits examined. Analysis of pairwise r² measures of linkage disequilibrium between all three assayed SNPs ranged between 0.41 and 0.79, suggesting that some of the observed SNP associations with performance may be independent. To our knowledge, this is one of the first studies demonstrating associations between IGF2R polymorphisms and growth‐ and body‐related traits in cattle. These results also support the increasing body of evidence that imprinted genes harbour polymorphisms that contribute to heritable variation in phenotypic traits in domestic livestock species.     

Histone H1 affects gene imprinting and DNA methylation in Arabidopsis

Imprinting, i.e. parent-of-origin expression of alleles, plays an important role in regulating development in mammals and plants. DNA methylation catalyzed by DNA methyltransferases plays a pivotal role in regulating imprinting by silencing parental alleles. DEMETER (DME), a DNA glycosylase functioning in the base-excision DNA repair pathway, can excise 5-methylcytosine from DNA and regulate genomic imprinting in Arabidopsis. DME demethylates the maternal MEDEA (MEA) promoter in endosperm, resulting in expression of the maternal MEA allele. However, it is not known whether DME interacts with other proteins in regulating gene imprinting. Here we report the identification of histone H1.2 as a DME-interacting protein in a yeast two-hybrid screen, and confirmation of their interaction by the in vitro pull-down assay. Genetic analysis of the loss-of-function histone h1 mutant showed that the maternal histone H1 allele is required for DME regulation of MEA, FWA and FIS2 imprinting in Arabidopsis endosperm but the paternal allele is dispensable. Furthermore, we show that mutations in histone H1 result in an increase of DNA methylation in the maternal MEA and FWA promoter in endosperm. Our results suggest that histone H1 is involved in DME-mediated DNA methylation and gene regulation at imprinted loci.