Unraveling the Genetic Basis for the Rapid Diversification of Male Genitalia between Drosophila Species.

In the last 240,000 years, males of the Drosophila simulans species clade have evolved striking differences in the morphology of their epandrial posterior lobes and claspers (surstyli). These appendages are used for grasping the female during mating and so their divergence is most likely driven by sexual selection. Mapping studies indicate a highly polygenic and generally additive genetic basis for these morphological differences. However, we have limited understanding of the gene regulatory networks that control the development of genital structures and how they evolved to result in this rapid phenotypic diversification. Here, we used new D. simulans/D. mauritiana introgression lines on chromosome arm 3L to generate higher resolution maps of posterior lobe and clasper differences between these species. We then carried out RNA-seq on the developing genitalia of both species to identify the expressed genes and those that are differentially expressed between the two species. This allowed us to test the function of expressed positional candidates during genital development in D. melanogaster. We identified several new genes involved in the development and possibly the evolution of these genital structures, including the transcription factors Hairy and Grunge. Furthermore, we discovered that during clasper development Hairy negatively regulates tartan (trn), a gene known to contribute to divergence in clasper morphology. Taken together, our results provide new insights into the regulation of genital development and how this has evolved between species.

tartan underlies the evolution of Drosophila male genital morphology.

Male genital structures are among the most rapidly evolving morphological traits and are often the only features that can distinguish closely related species. This process is thought to be driven by sexual selection and may reinforce species separation. However, while the genetic bases of many phenotypic differences have been identified, we still lack knowledge about the genes underlying evolutionary differences in male genital organs and organ size more generally. The claspers (surstyli) are periphallic structures that play an important role in copulation in insects. Here, we show that divergence in clasper size and bristle number between Drosophila mauritiana and Drosophila simulans is caused by evolutionary changes in tartan (trn), which encodes a transmembrane leucine-rich repeat domain protein that mediates cell-cell interactions and affinity. There are no fixed amino acid differences in trn between D. mauritiana and D. simulans, but differences in the expression of this gene in developing genitalia suggest that cis-regulatory changes in trn underlie the evolution of clasper morphology in these species. Finally, analyses of reciprocal hemizygotes that are genetically identical, except for the species from which the functional allele of trn originates, determined that the trn allele of D. mauritiana specifies larger claspers with more bristles than the allele of D. simulans Therefore, we have identified a gene underlying evolutionary change in the size of a male genital organ, which will help to better understand not only the rapid diversification of these structures, but also the regulation and evolution of organ size more broadly.

Motile cilia of the male reproductive system require miR-34/miR-449 for development and function to generate luminal turbulence.

Cilia are cell-surface, microtubule-based organelles that project into extracellular space. Motile cilia are conserved throughout eukaryotes, and their beat induces the flow of fluid, relative to cell surfaces. In mammals, the coordinated beat of motile cilia provides highly specialized functions associated with the movement of luminal contents, as seen with metachronal waves transporting mucus in the respiratory tract. Motile cilia are also present in the male and female reproductive tracts. In the female, wave-like motions of oviductal cilia transport oocytes and embryos toward the uterus. A similar function has been assumed for motile cilia in efferent ductules of the male-i.e., to transport immotile sperm from rete testis into the epididymis. However, we report here that efferent ductal cilia in the male do not display a uniform wave-like beat to transport sperm solely in one direction, but rather exert a centripetal force on luminal fluids through whip-like beating with continual changes in direction, generating turbulence, which maintains immotile spermatozoa in suspension within the lumen. Genetic ablation of two miRNA clusters (miR-34b/c and -449a/b/c) led to failure in multiciliogenesis in murine efferent ductules due to dysregulation of numerous genes, and this mouse model allowed us to demonstrate that loss of efferent duct motile cilia causes sperm aggregation and agglutination, luminal obstruction, and sperm granulomas, which, in turn, induce back-pressure atrophy of the testis and ultimately male infertility.

Estrogens and development of the mouse and human external genitalia.

The Jost hypothesis states that androgens are necessary for normal development of the male external genitalia. In this review, we explore the complementary hypothesis that estrogens can elicit abnormal development of male external genitalia. Herein, we review available data in both humans and mice on the deleterious effects of estrogen on external genitalia development, especially during the "window of susceptibility" to exogenous estrogens. The male and female developing external genitalia in both the human and mouse express ESR1 and ESR2, along with the androgen receptor (AR). Human clinical data suggests that exogenous estrogens can adversely affect normal penile and urethral development, resulting in hypospadias. Experimental mouse data also strongly supports the idea that exogenous estrogens cause penile and urethral defects. Despite key differences, estrogen-induced hypospadias in the mouse displays certain morphogenetic homologies to human hypospadias, including disruption of urethral fusion and preputial abnormalities. Timing of estrogenic exposure, or the "window of susceptibility," is an important consideration when examining malformations of the external genitalia in both humans and mice. In addition to a review of normal human and mouse external genital development, this article aims to review the present data on the role of estrogens in normal and abnormal development of the mouse and human internal and external genitalia. Based on the current literature for both species, we conclude that estrogen-dependent processes may play a role in abnormal genital development.

Ontogeny of estrogen receptors in human male and female fetal reproductive tracts.

This paper reviews and provides new observations on the ontogeny of estrogen receptor alpha (ESR1) and estrogen receptor beta (ESR2) in developing human male and female internal and external genitalia. Included in this study are observations on the human fetal uterine tube, the uterotubal junction, uterus, cervix, vagina, penis and clitoris. We also summarize and report on the ontogeny of estrogen receptors in the human fetal prostate, prostatic urethra and epididymis. The ontogeny of ESR1 and ESR2, which spans from 8 to 21 weeks correlates well with the known "window of susceptibility" (7-15 weeks) for diethylstilbestrol (DES)-induced malformations of the human female reproductive tract as determined through examination of DES daughters exposed in utero to this potent estrogen. Our fairly complete mapping of the ontogeny of ESR1 and ESR2 in developing human male and female internal and external genitalia provides a mechanistic framework for further investigation of the role of estrogen in normal development and of abnormalities elicited by exogenous estrogens.

Hox13 is essential for formation of a sensory organ at the terminal end of the sperm duct in Ciona.

Species-specific traits are thought to have been acquired by natural selection. Transcription factors play central roles in the evolution of species-specific traits. Hox genes encode a set of conserved transcription factors essential for establishing the anterior-posterior body axis of animals. Changes in the expression or function of Hox genes can lead to the diversification of animal-body plans. The tunicate ascidian Ciona intestinalis Type A has an orange-colored structure at the sperm duct terminus. This orange-pigmented organ (OPO) is the characteristic that can distinguish this ascidian from other closely related species. The OPO is formed by the accumulation of orange-pigmented cells (OPCs) that are present throughout the adult body. We show that Hox13 is essential for formation of the OPO. Hox13 is expressed in the epithelium of the sperm duct and neurons surrounding the terminal openings for sperm ejection, while OPCs themselves do not express this gene. OPCs are mobile cells that can move through the body vasculature by pseudopodia, suggesting that the OPO is formed by the accumulation of OPCs guided by Hox13-positive cells. Another ascidian species, Ciona savignyi, does not have an OPO. Like Hox13 of C. intestinalis, Hox13 of C. savignyi is expressed at the terminus of its sperm duct; however, its expression domain is limited to the circular area around the openings. The genetic changes responsible for the acquisition or loss of OPO are likely to occur in the expression pattern of Hox13.

Depletion of gipc-1 and gipc-2 causes infertility in Caenorhabditis elegans by reducing sperm motility.

The G-protein signaling pathway plays a key role in multiple cellular processes and is well conserved in eukaryotes. Although GIPC (G-protein α subunit interacting protein (GAIP)-interacting protein, C terminus) has been studied in several model organisms, little is known about its role in Caenorhabditis elegans. In the present study, we investigated the roles of gipc-1 and gipc-2 in C. elegans. We observed that they were exclusively expressed in sperm throughout the development and that gipc-1; gipc-2 double mutants were infertile. Further examination of sperm development in gipc-1; gipc-2 mutants revealed defective sperm activation and abnormal pseudopod extension that resulted in reduced sperm motility. Moreover, major sperm protein (MSP) was abnormally segregated between spermatids and residual bodies in gipc-1; gipc-2 mutants. Our findings indicate that gipc-1 and gipc-2 are required for the proper pseudopod extension of sperm during the terminal differentiation of spermatids. During this process, the segregation of MSP into spermatids is important for ensuring normal sperm motility during fertilization.

Developmental programming and ageing of male reproductive function.

Developmental programming predisposes offspring to metabolic, behavioural and reproductive dysfunction in adult life. Evidence is accumulating that ageing phenotype and longevity are in part developmentally programmed in each individual. Unfortunately, there are few studies addressing the effects of developmental programming by maternal nutrition on the rate of ageing of the male reproductive system. This review will discuss effects of foetal exposure to maternal environmental challenges on male offspring fertility and normal ageing of the male reproductive system. We focus on several key factors involved in reproductive ageing such as decreased hormone production, DNA fragmentation, oxidative stress, telomere shortening, epigenetics, maternal lifestyle and nutrition. There is compelling evidence that ageing of the male reproductive system is developmentally programmed. Both maternal over- or undernutrition accelerate ageing of male offspring reproductive function through similar mechanisms such as decreased serum testosterone levels, increase in oxidative stress biomarkers in both the testes and sperm and changes in sperm quality. Importantly, even in adult life, exercise in male offspring of obese mothers improves adverse effects of programming on reproductive function. Maternal consumption of a low-protein diet causes transgenerational effects in progeny via the paternal line. The seminal fluid has effects on the intrauterine environment. Programming by male factors may involve more than just the sperm. Improving knowledge on developmental programming ageing interactions will improve not only male health and life span but also the health of future generations by reducing programming via the paternal line.

Evaluation of dietary dose administration as an alternative to oral gavage in the rodent uterotrophic and Hershberger assays.

The rodent uterotrophic and Hershberger assays evaluate potential estrogenic and (anti)-androgenic effects, respectively. Both US EPA and OECD guidelines specify that test substance is administered daily either by subcutaneous injection or oral gavage. However, dietary administration is a relevant exposure route for agrochemical regulatory toxicology studies due to potential human intake via crop residues. In this study, equivalent doses of positive control chemicals administered via dietary and gavage routes of administration were compared in the uterotrophic (17α-ethinyl estradiol) and Hershberger (flutamide, linuron, dichloro-2,2-bis(4-chlorophenyl) ethane; 4,4'-DDE) assays in ovariectomized and castrated rats, respectively. For all positive control chemicals tested, statistically significant changes in organ weights and decreases in food consumption were observed by both routes of test substance administration. Decreased body weight gain observed for dietary linuron and 4,4'-DDE indicated that the maximum tolerated dose was exceeded. Hershberger dietary administration resulted in a similar blood exposure (AUC24) for each positive control chemical when compared to gavage. Overall, the correlation in organ weight changes for both the uterotrophic and Hershberger assays suggest that dietary administration is an acceptable route of exposure with similar sensitivity to oral gavage dosing for evaluation of the endocrine potential of a test substance and represents a more appropriate route of test substance administration for most environmental exposure scenarios.

Farnesoid X receptor alpha (FXRα) is a critical actor of the development and pathologies of the male reproductive system.

The farnesoid-X-receptorα (FXRα; NR1H4) is one of the main bile acid (BA) receptors. During the last decades, through the use of pharmalogical approaches and transgenic mouse models, it has been demonstrated that the nuclear receptor FXRα controls numerous physiological functions such as glucose or energy metabolisms. It is also involved in the etiology or the development of several pathologies. Here, we will review the unexpected roles of FXRα on the male reproductive tract. FXRα has been demonstrated to play functions in the regulation of testicular and prostate homeostasis. Even though additional studies are needed to confirm these findings in humans, the reviewed reports open new field of research to better define the effects of bile acid-FXRα signaling pathways on fertility disorders and cancers.

Discrete Hedgehog Factor Expression and Action in the Developing Phallus.

Hypospadias is a failure of urethral closure within the penis occurring in 1 in 125 boys at birth and is increasing in frequency. While paracrine hedgehog signalling is implicated in the process of urethral closure, how these factors act on a tissue level to execute closure itself is unknown. This study aimed to understand the role of different hedgehog signalling members in urethral closure. The tammar wallaby (Macropus eugenii) provides a unique system to understand urethral closure as it allows direct treatment of developing offspring because mothers give birth to young before urethral closure begins. Wallaby pouch young were treated with vehicle or oestradiol (known to induce hypospadias in males) and samples subjected to RNAseq for differential expression and gene ontology analyses. Localisation of Sonic Hedgehog (SHH) and Indian Hedgehog (IHH), as well as the transcription factor SOX9, were assessed in normal phallus tissue using immunofluorescence. Normal tissue culture explants were treated with SHH or IHH and analysed for AR, ESR1, PTCH1, GLI2, SOX9, IHH and SHH expression by qPCR. Gene ontology analysis showed enrichment for bone differentiation terms in male samples compared with either female samples or males treated with oestradiol. Expression of SHH and IHH localised to specific tissue areas during development, akin to their compartmentalised expression in developing bone. Treatment of phallus explants with SHH or IHH induced factor-specific expression of genes associated with bone differentiation. This reveals a potential developmental interaction involved in urethral closure that mimics bone differentiation and incorporates discrete hedgehog activity within the developing phallus and phallic urethra.

Effects of androgen and oestrogen on IGF pathways controlling phallus growth.

The development of the mammalian phallus involves hormone-dependent mesenchymal-epithelial signalling mechanisms that contribute to urethral closure and regulation of phallus elongation and growth. In marsupials, most differentiation and growth of the phallus occurs post-natally, making them amenable to direct hormone treatment. Expression of IGFs, FGFs, EFNB2, MAFB, DLX5 and AP-1 mRNAs in the phallus at day 50 post-partum (pp) were altered after treatment of tammar wallaby young from day 20 to 40 pp with androgen, oestrogen or after castration at day 25 pp. However, the most interesting changes occurred in the IGF pathway genes. Androgen treatment upregulated IGF1 in female phalluses and oestrogen treatment upregulated IGF1 in male phalluses, but it was downregulated by castration. IGFBP3 was higher in female phalluses and downregulated by androgen. IGF1 expression was higher in all untreated male than in female phalluses from day 50 to 150 pp, but IGFBP3 had the reverse pattern. At day 90 pp, when urethral closure in males is progressing and male phallus growth is accelerating. IGF1 and PCNA protein were only detected in the male urorectal septum, suggesting for the first time that closure and elongation may involve IGF1 activation of cell proliferation specifically in male phalluses. These effects of sex steroids on gene expression and on the IGF1 signalling pathway in particular, suggest that the developing phallus may be especially susceptible to perturbation by exogenous hormones.

Fluoride exposure and pubertal development in children living in Mexico City.

BACKGROUND: Previous animal and ecological studies have provided evidence for an earlier sexual maturation in females in relation to fluoride exposure; however, no epidemiological studies have examined the association between fluoride exposure and pubertal development in both boys and girls using individual-level biomarkers of fluoride. Capitalizing on an ongoing Mexican birth cohort study, we examined the association between concurrent urinary fluoride levels and physical markers of pubertal development in children. METHODS: We conducted a cross-sectional study of 157 boys and 176 girls at age 10-17 years living in Mexico City. We used ion-selective electrode-based diffusion methods to assess fluoride levels in urine, adjusting for urinary specific gravity. Pubertal stages were evaluated by a trained physician. Associations of fluoride with pubertal stages and age at menarche were studied using ordinal regression and Cox proportional-hazard regression, respectively. RESULTS: In the entire sample, the geometric mean and interquartile range (IQR) of urinary fluoride (specific gravity adjusted) were 0.59 mg/L and 0.31 mg/L, respectively. In boys, our analysis showed that a one-IQR increase in urinary fluoride was associated with later pubic hair growth (OR = 0.71, 95% CI: 0.51-0.98, p = 0.03) and genital development (OR = 0.71, 95% CI: 0.53-0.95, p = 0.02). No significant associations were found in girls, although the direction was negative. CONCLUSIONS: Childhood fluoride exposure, at the levels observed in our study, was associated with later pubertal development among Mexican boys at age 10-17 years. Further research is needed to confirm these findings.

[Effect of temperature on post-diapause reproductive development in Listronotus maculicollis (Coleoptera: curculionidae)].

The annual bluegrass weevil Listronotus maculicollis requires chilling exposure to terminate reproductive diapause during overwintering, but the effects of temperature on its post-diapause development in spring remain unclear. To explore this effect, overwintering adults were transferred from cold conditions (6°C/4°C, L:D 10:14) to different warm-up temperatures at L:D 12:12. When weevils were transferred to 7, 14 and 21°C in December and late January, the sizes of male and female reproductive organs were significantly smaller at 7°C than at 14 and 21°C. When weevils were transferred to 7, 9, 11, 13 and 15°C in late January, higher temperatures facilitated the post-diapause development. In both sexes, the sizes of reproductive organs and developmental rate increased with temperature. Reproductive organs did not grow significantly at 7°C in males and at 7-9°C in females, at which the percentage of developing weevils remained low. The time required for 50% of individuals to resume development was 44, 18, 13 and 8 days at 9, 11, 13 and 15°C, respectively, in males and 19, 14 and 8 days at 11, 13 and 15°C, respectively, in females. The threshold temperature for post-diapause development was 7.8°C in males, based on which 61.7 degree-days coincided with 50% of individuals developing. Under field conditions, the percentage of male and female maturity and insemination rate were low until early March, but all reached 100% by late March.

Hormone-responsive genes in the SHH and WNT/ß-catenin signaling pathways influence urethral closure and phallus growth.

Environmental endocrine disruptors (EEDs) that affect androgen or estrogen activity may disrupt gene regulation during phallus development to cause hypospadias or a masculinized clitoris. We treated developing male tammar wallabies with estrogen and females with androgen from day 20-40 postpartum (pp) during the androgen imprinting window of sensitivity. Estrogen inhibited phallus elongation but had no effect on urethral closure and did not significantly depress testicular androgen synthesis. Androgen treatment in females did not promote phallus elongation but initiated urethral closure. Phalluses were collected for transcriptome sequencing at day 50 pp when they first become sexually dimorphic to examine changes in two signaling pathways, sonic hedgehog (SHH) and wingless-type MMTV integration site family (WNT)/ß-catenin. SHH mRNA and ß-catenin were predominantly expressed in the urethral epithelium in the tammar phallus, as in eutherian mammals. Estrogen treatment and castration of males induced an upregulation of SHH, while androgen treatment downregulated SHH. These effects appear to be direct since we detected putative estrogen receptor α (ERα) and androgen receptor (AR) binding sites near SHH. WNT5A, like SHH, was downregulated by androgen, while WNT4 was upregulated in female phalluses after androgen treatment. After estrogen treatment, WIF1 and WNT7A were both downregulated in male phalluses. After castration, WNT9A was upregulated. These results suggest that SHH and WNT pathways are regulated by both estrogen and androgen to direct the proliferation and elongation of the phallus during differentiation. Their response to exogenous hormones makes these genes potential targets of EEDs in the etiology of abnormal phallus development including hypospadias.

Systematic reviews and meta-analyses of human and animal evidence of prenatal diethylhexyl phthalate exposure and changes in male anogenital distance.

Male reproductive alterations found in animals and humans following in utero phthalate exposure include decreased anogenital distance (AGD) and other reproductive-tract malformations. The aim of this investigation was to conduct systematic reviews of human and animal evidence of the effect of in utero exposure to diethylhexyl phthalate (DEHP) on anogenital distance (AGD) in males. PubMed, Embase, and Toxline were searched for relevant human and experimental animal studies on August 15, 2016. Search results were screened for relevance, and studies that met the inclusion criteria were evaluated for quality and data extracted for analysis. Confidence in the human and animal bodies of evidence was assessed and hazard conclusions reached by integrating evidence streams. The search yielded 6 relevant human studies and 19 animal studies. Meta-analysis of 5 human observational prospective cohort studies showed that increased maternal urinary concentrations of DEHP metabolites were associated with decreased AGD in boys (-4.07 [CI, -6.49 to -1.66] % decrease per log10 rise in DEHP metabolites). Meta-analysis and meta-regression of the 19 experimental animal studies found reduced AGD with DEHP treatment, with a dose-response gradient, and with heterogeneity explained by species and strain. There is a moderate level of evidence from human investigations and a high level of data from animal studies that in utero exposure to DEHP decreases AGD. Based upon the available human and animal evidence, and consideration of mechanistic data, DEHP is presumed to be a reproductive hazard to humans on the basis of effects on AGD.

Expression patterns of Fgf8 and Shh in the developing external genitalia of Suncus murinus.

Reciprocal epithelial-mesenchymal interactions and several signalling pathways regulate the development of the genital tubercle (GT), an embryonic primordium of external genitalia. The morphology of the adult male external genitalia of the Asian house musk shrew Suncus murinus (hereafter, laboratory name: suncus) belonging to the order Eulipotyphla (the former order Insectivora or Soricomorpha) differs from those of mice and humans. However, the developmental process of the suncus GT and its regulatory genes are unknown. In the present study, we explored the morphological changes and gene expression patterns during the development of the suncus GT. Morphological observations suggested the presence of common (during the initial outgrowth) and species-specific (during the sexual differentiation of GT) developmental processes of the suncus GT. In gene expression analysis, fibroblast growth factor 8 (Fgf8) and sonic hedgehog (Shh), an indicator and regulator of GT development in mice respectively, were found to be expressed in the cloacal epithelium and the developing urethral epithelium of the suncus GT. This pattern of expression specifically in GT epithelium is similar to that observed in the developing mouse GT. Our results indicate that the mechanism of GT formation regulated by the FGF and SHH signalling pathways is widely conserved in mammals.

Maternal lactocrine programming of porcine reproductive tract development.

The lactocrine hypothesis for maternal programming of female reproductive tract development is based on the idea that non-nutritive, milk-borne bioactive factors (MbFs), delivered from mother to offspring during nursing, play a role in determining the trajectory of development with long-term consequences in the adult. Porcine female reproductive tract development is completed postnatally, and the period during which maternal support of neonatal growth derives exclusively from colostrum/milk defines a window of opportunity for lactocrine programming of reproductive tissues. Beyond nutrition, milk serves as a delivery system for a variety of bioactive factors. Porcine relaxin is a prototypical MbF. Present in colostrum at highest concentrations at birth, relaxin is transmitted into the circulation of nursing piglets where it can act on Relaxin receptors found in neonatal female reproductive tract tissues. This process is facilitated by the physiology of the maternal-neonatal dyad and the fact that the neonatal gastrointestinal tract is open to absorb macromolecules for a period of time postnatally. Age at first nursing and duration of nursing from birth are also important for porcine female reproductive tract development. These parameters affect both the quality and quantity of colostrum consumed. Disruption of lactocrine signaling by feeding milk replacer from birth altered porcine uterine, cervical, and testicular development by postnatal Day 2. Moreover, insufficient colostrum consumption in nursing piglets can impair uterine capacity to support viable litters of optimal size in adulthood. In the pig, lactocrine signaling supports neonatal organizational events associated with normal reproductive development and may program adult uterine capacity.

Comparative proteomics analysis of spermary and ovary in Hyriopsis schlegelii.

We provide the first large-scale quantitative proteomics analysis in Hyriopsis schlegelii. To investigate the proteins expressed in the gonads, a quantitative proteomics approach has been utilized to analyze differentially expressed proteins between the spermary and ovary. In this study, we identified and quantified 2416 proteins in the gonads of Hyriopsis schlegelii. Of these, 559 proteins showed significantly different expression between the spermary and ovary. Some specific proteins expressed in either the spermary or ovary were identified in Hyriopsis schlegelii. In addition, a series of proteins related to gametogenesis were also identified. Compared with previous reports, many proteins in Hyriopsis schlegelii identified here have different expression patterns between the spermary and ovary. The special hermaphroditism in Hyriopsis schlegelii may contribute to these inconsistent results. The provided proteomics data could be considered as a starting point for subsequent studies focusing on the proteins involved in sexual gland development and maturity.

Genome-wide association study of sexual maturation in males and females highlights a role for body mass and menarche loci in male puberty.

Little is known about genes regulating male puberty. Further, while many identified pubertal timing variants associate with age at menarche, a late manifestation of puberty, and body mass, little is known about these variants' relationship to pubertal initiation or tempo. To address these questions, we performed genome-wide association meta-analysis in over 11 000 European samples with data on early pubertal traits, male genital and female breast development, measured by the Tanner scale. We report the first genome-wide significant locus for male sexual development upstream of myocardin-like 2 (MKL2) (P = 8.9 × 10(-9)), a menarche locus tagging a developmental pathway linking earlier puberty with reduced pubertal growth (P = 4.6 × 10(-5)) and short adult stature (p = 7.5 × 10(-6)) in both males and females. Furthermore, our results indicate that a proportion of menarche loci are important for pubertal initiation in both sexes. Consistent with epidemiological correlations between increased prepubertal body mass and earlier pubertal timing in girls, body mass index (BMI)-increasing alleles correlated with earlier breast development. In boys, some BMI-increasing alleles associated with earlier, and others with delayed, sexual development; these genetic results mimic the controversy in epidemiological studies, some of which show opposing correlations between prepubertal BMI and male puberty. Our results contribute to our understanding of the pubertal initiation program in both sexes and indicate that although mechanisms regulating pubertal onset in males and females may largely be shared, the relationship between body mass and pubertal timing in boys may be complex and requires further genetic studies.