Retinoids promote penis development in sequentially hermaphroditic snails.

Sexual systems are surprisingly diverse, considering the ubiquity of sexual reproduction. Sequential hermaphroditism, the ability of an individual to change sex, has emerged multiple times independently across the animal kingdom. In molluscs, repeated shifts between ancestrally separate sexes and hermaphroditism are generally found at the level of family and above, suggesting recruitment of deeply conserved mechanisms. Despite this, molecular mechanisms of sexual development are poorly known. In molluscs with separate sexes, endocrine disrupting toxins bind the retinoid X receptor (RXR), activating ectopic male development in females, suggesting the retinoid pathway as a candidate controlling sexual transitions in sequential hermaphrodites. We therefore tested the role of retinoic acid signaling in sequentially hermaphroditic Crepidula snails, which develop first into males, then change sex, maturing into females. We show that retinoid agonists induce precocious penis growth in juveniles and superimposition of male development in females. Combining RXR antagonists with retinoid agonists significantly reduces penis length in induced juveniles, while similar treatments using retinoic acid receptor (RAR) antagonists increase penis length. Transcripts of both receptors are expressed in the induced penis. Our findings therefore show that retinoid signaling can initiate molluscan male genital development, and regulate penis length. Further, we show that retinoids induce ectopic male development in multiple Crepidula species. Species-specific influence of conspecific induction of sexual transitions correlates with responsiveness to retinoids. We propose that retinoid signaling plays a conserved role in molluscan male development, and that shifts in the timing of retinoid signaling may have been important for the origins of sequential hermaphroditism within molluscs.

Obesity Is Strongly Associated With Low Testosterone and Reduced Penis Growth During Development.

CONTEXT: Growth of male genitalia represents an important marker of sexual development. Testicle size is the primary measure and little is known regards penile length changes during puberty. OBJECTIVE: This work aims to assess penis growth and testosterone levels in obese vs normal-weight children and adolescents, to evaluate a possible influence of obesity on genital development in boys, and to establish a new method for measuring penis length that allows comparison of normal-weight and overweight boys. METHODS: We assessed anthropometric and genital development in 1130 boys from birth to age 20 years. Testosterone levels were also measured. A new method for penile length measurement was employed to minimize errors when comparing obese and nonobese children. Penis length was measured with a gentle, painless, straight positioning on a centimetric ruler without stretching, which is doable from the first years of life until the end of adolescence. RESULTS: Penis length and testosterone are strongly related in children during puberty. Penile length growth is significantly decreased (by about 10%) in obese boys when compared to normal-weight boys, with concomitantly reduced testosterone levels, across puberal phases. CONCLUSION: Childhood obesity represents an important determinant of lower testosterone level and reduced penis development. A new method should be employed to improve penis measurement in normal-weight and overweight/obese boys. The possible significance of these observations for adult genital development and reproductive potential will require large longitudinal studies.

Reproductive Hormone Concentrations and Associated Anatomical Responses: Does Soy Formula Affect Minipuberty in Boys?

CONTEXT: Soy formula feeding is common in infancy and is a source of high exposure to phytoestrogens, documented to influence vaginal cytology in female infants. Its influence on minipuberty in males has not been established. OBJECTIVE: To assess the association between infant feeding practice and longitudinally measured reproductive hormones and hormone-responsive tissues in infant boys. METHODS: The Infant Feeding and Early Development study was a prospective cohort of maternal-infant dyads requiring exclusive soy formula, cow milk formula, or breast milk feeding during study follow-up. In the 147 infant boy participants, serum testosterone, luteinizing hormone, stretched penile length, anogenital distance, and testis volume were longitudinally assessed from birth to 28 weeks. We examined feeding-group differences in age trajectories for these outcomes using mixed-effects regression splines. RESULTS: Median serum testosterone was at pubertal levels at 2 weeks (176 ng/dL [quartiles: 124, 232]) and remained in this range until 12 weeks in all feeding groups. We did not observe differences in trajectories of hormone concentrations or anatomical measures between boys fed soy formula (n = 55) and boys fed cow milk formula (n = 54). Compared with breastfed boys (n = 38), soy formula-fed boys had a more rapid increase in penile length (P = .004) and slower initial lengthening of anogenital distance (P = .03), but no differences in hormone trajectories. CONCLUSION: Reproductive hormone concentrations and anatomical responses followed similar trajectories in soy and cow milk formula-fed infant boys. Our findings suggest that these measures of early male reproductive development do not respond to phytoestrogen exposure during infancy.

Penile circumference and stretched penile length in prepubertal children: A retrospective, single-center pilot study.

PURPOSE: To determine references for penile circumference according to age in prepubertal children and whether this measurement can be used as a basic penile parameter along with stretched penile length in prepubertal children. MATERIALS AND METHODS: A total of 750 children (mean age, 4.2±3.4 years) aged under 14 years without penile problems were enrolled in this study. Children with penile or testicular abnormalities were excluded. All data were gathered at the outpatient clinic by a single pediatric urologist from July 2017 to April 2020. Penile parameters (baseline and stretched penile length, penile circumference) and testicular volumes were measured by using an elastic ruler and a Prader orchidometer, respectively. RESULTS: Mean baseline and stretched penile lengths were 3.0±1.0 cm and 4.2±1.0 cm, respectively. The mean penile circumference was 4.2±0.9 cm. The stretched penile length was similar to penile circumference (p=0.425). This similarity was found for each age group except for the 0-1-year-old and 3-4-year-old age groups (p=0.001 and p=0.034, respectively). As children grow into adolescence, stretched penile length increases significantly compared to penile circumference. CONCLUSIONS: Penile circumference increased with age like stretched penile length and testicular volume in prepubertal children. Stretched penile length and penile circumference were found to be similar. This study can be used as a basic reference for penile circumference values in prepubertal children.

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.

Anatomy of the mouse penis and internal prepuce.

This paper addresses a confusing issue of preputial anatomy of the mouse. The term "internal prepuce" was used in 2013 to describe a preputial structure integral to the mouse glans penis. Subsequently in 2015 the same term was applied by another group to describe entirely different morphology, generating confusion in the literature. Because it is inappropriate to use the same term to describe entirely different structures, we take this opportunity to provide further descriptive information on the internal prepuce of the mouse employing gross dissection, analysis of serial histologic section sets, three-dimensional reconstruction, scanning electron microscopy and immunohistochemistry. For this purpose, we review and illustrate the relevant literature and provide some additional new data using standard morphological techniques including immunohistochemistry. The mouse internal prepuce is integral to the glans penis and clearly is involved in sexual function in so far as it contains a major erectile body innervated by penile nerves. The development of the mouse internal prepuce is described for the first time and related to the development of the corpus cavernosum glandis.

Characteristics of penile growth in pubertal rats and a non-invasive method to lengthen the penis.

BACKGROUND: Males with short penises may suffer from sexual dysfunction and psychological problems. However, currently, managing short penis is a huge challenge. OBJECTIVES: To explore whether inhibition of lysyl oxidase (LOX) activity (anti-LOX) combined with a vacuum device could lengthen the penis of pubertal rat. MATERIALS AND METHODS: Male rats of different ages were purchased, their exposed penile lengths and weights were measured, and protein expression and lysyl oxidase activity in the corpus cavernosum were analyzed. Fifteen-day-old rats were then purchased and divided into six groups: control, Anti-lysyl oxidase, -200 mm Hg (vacuum device under -200 mm Hg value), -200 mm Hg + Anti-lysyl oxidase, -300 mm Hg, and -300 mm Hg + Anti-lysyl oxidase groups. After the intervention duration of 7 weeks, rats' penile length was measured and erectile function was assessed. The corpus cavernosum was harvested for histopathology and molecular assessments. RESULTS: Exposed penile length and weight significantly increased with age, especially between 4 and 8 weeks. Both the protein expression and lysyl oxidase activity in corpus cavernosum were the highest at 2 weeks; however, they quickly decreased with age and slowly declined after 8 weeks. Anti-lysyl oxidase significantly increased the penile length by 10.79% over controlled rats, -200 mm Hg + Anti-lysyl oxidase lengthened it by 14.05%, and -300 mm Hg + Anti-lysyl oxidase increased it by 19.84%. Anti-lysyl oxidase significantly reduced lysyl oxidase activity to decrease pyridinoline concentration; however, it did not change desmosine (P = .28), hydroxyproline (P = .14), and total elastin (P = .06) levels. Anti-lysyl oxidase with or without a vacuum device did not diminish erectile function or impair the normal microstructure of corpus cavernosum. DISCUSSION AND CONCLUSION: The rats' penile growth peaks occurred between 4 and 8 weeks. Anti-lysyl oxidase with a vacuum device promoted penile lengthening by inhibiting pyridinoline production to induce tunica albuginea remodeling. The penile lengthening effect was more obvious in pubertal rats than the adult rats. None of the procedures decreased erectile function.

Prenatal exposure to phthalates and male reproductive system development: Results from a Canadian pregnancy cohort study.

Our objective was to determine if maternal first trimester urinary phthalate concentrations are associated with reduced penile length (PL) or width (PW) at birth in full term singletons. First trimester phthalate metabolite urinary concentrations were obtained from mothers participating in a Canadian pregnancy cohort study (MIREC). PL and PW were measured shortly after birth in the male offspring. Univariate and multivariable linear regressions were performed to study associations between maternal phthalate exposure and penile measurements, adjusting for confounders. On univariate analysis of 170 mother-infant pairs, PW showed an inverse relationship with the concentration of mono-3-carboxypropyl phthalate (MCPP-p = 0.016), which was not confirmed on multivariable analysis. On multivariable analysis controlling for infant's size and other confounders, no statistically signficant associations between phthalate metabolite concentrations and PL or PW were identified. In this population of Canadian women, there was no strong evidence to suggest an association between maternal first trimester urinary phthalates with PL or PW in term singletons.

Prenatal low-dose methyltestosterone, but not dihydrotestosterone, treatment induces penile formation in female mice and guinea pigs†.

Genital tubercle has bisexual potential before sex differentiation. Females exposed to androgen during sex differentiation show masculinized external genitalia, but the effects of different androgens on tubular urethral and penile formation in females are mostly unknown. In this study, we compared the masculinization effects of commonly used androgens methyltestosterone, dihydrotestosterone, and testosterone on the induction of penile formation in females. Our results suggested that prenatal treatment with low doses of methyltestosterone, but not same doses of dihydrotestosterone or testosterone, could induce penile formation in female mice. The minimum dose of dihydrotestosterone and testosterone for inducing tubular urethral formation in female mice was, respectively, 50 and 20 times higher than that of methyltestosterone. In vivo methyltestosterone treatment induced more nuclear translocation of androgen receptors in genital tubercles of female mice, affected Wnt signaling gene expressions, and then led to similar patterns of cell proliferation and death in developing genital tubercles to those of control males. We further revealed that low-dose methyltestosterone, but not same dose of dihydrotestosterone or testosterone, treatment induced penile formation in female guinea pigs. Exposure of female mouse genital tubercle organ culture to methyltestosterone, dihydrotestosterone, or testosterone could induce nuclear translocation of androgen receptors, suggesting that the differential effect of the three androgens in vivo might be due to the hormonal profile in mother or fetus, rather than the local genital tissue. To understand the differential role of these androgens in masculinization process involved is fundamental to androgen replacement therapy for diseases related to external genital masculinization.

Constant innervation despite pubertal growth of the mouse penis.

The sexual characteristics of the vertebrate body change under the control of sex hormones. In mammals, genitals undergo major changes in puberty. While such bodily changes have been well documented, the associated changes in the nervous system are poorly understood. To address this issue, we studied the growth and innervation of the mouse penis throughout puberty. To this end, we measured length and thickness of the mouse penis in prepubertal (3-4 week old) and adult (8-10 week old) mice and performed fiber counts of the dorsal penile nerve. We obtained such counts with confocal imaging of proximal sections of the mouse penis after paraffin embedding and antibody staining against Protein-Gene-Product-9.5 or Neurofilament-H in combination with antigen retrieval procedures. We find that the mouse penis grows roughly 1.4 times in both thickness and length. Fiber counts in the dorsal penile nerve were not different in prepubertal (1,620 ± 165 fibers per penis) and adult (1,572 ± 383 fibers per penis) mice, however. Antibody staining along with myelin staining by Luxol-Fast-Blue suggested about 57% of penile nerve fibers were myelinated. Quantification of the area of mouse somatosensory penis cortex allowed us to compare cortical magnification of the penile cortex and the whisker-barrel-cortex systems. This comparison suggested that 2 to 4 times less cortical area is devoted to a penile-nerve-fiber than to a whisker-nerve-fiber. The constant innervation of mouse penis through puberty suggests that the pubertal increase of cortical magnification of the penis is not simply a reflection of peripheral change.

Hormonal and Molecular Regulation of Phallus Differentiation in a Marsupial Tammar Wallaby.

Congenital anomalies in phalluses caused by endocrine disruptors have gained a great deal of attention due to its annual increasing rate in males. However, the endocrine-driven molecular regulatory mechanism of abnormal phallus development is complex and remains largely unknown. Here, we review the direct effect of androgen and oestrogen on molecular regulation in phalluses using the marsupial tammar wallaby, whose phallus differentiation occurs after birth. We summarize and discuss the molecular mechanisms underlying phallus differentiation mediated by sonic hedgehog (SHH) at day 50 pp and phallus elongation mediated by insulin-like growth factor 1 (IGF1) and insulin-like growth factor binding protein 3 (IGFBP3), as well as multiple phallus-regulating genes expressed after day 50 pp. We also identify hormone-responsive long non-coding RNAs (lncRNAs) that are co-expressed with their neighboring coding genes. We show that the activation of SHH and IGF1, mediated by balanced androgen receptor (AR) and estrogen receptor 1 (ESR1) signalling, initiates a complex regulatory network in males to constrain the timing of phallus differentiation and to activate the downstream genes that maintain urethral closure and phallus elongation at later stages.

Development of the human prepuce and its innervation.

Development of the human prepuce was studied over the course of 9-17 weeks of gestation in 30 specimens. Scanning electron microscopy revealed subtle surface features that were associated with preputial development, namely the appearance of epidermal aggregates that appeared to be associated with formation of the preputial fold. Transverse and sagittal sections revealed that the epidermis of the glans is considerably thicker than that of the penile shaft. We described a novel morphogenetic mechanism of formation of the preputial lamina, namely the splitting of the thick epidermis of the glans into the preputial lamina and the epidermis via the intrusion of mesenchyme containing red blood cells and CD31-positive blood vessels. This process begins at 10-11 weeks of gestation in the proximal aspect of the glans and extends distally. The process is likely to be androgen-dependent and mediated via androgen receptors strategically localized to the morphogenetic process, but signaling through estrogen receptor may play a role. Estrogen receptor alpha (ESR1) has a very limited expression in the developing human glans and prepuce, while estrogen receptor beta (ESR2) is expressed more broadly in the developing preputial lamina, epidermis and urethra. Examination of the ontogeny of innervation of the glans penis and prepuce reveals the presence of the dorsal nerve of the penis as early as 9 weeks of gestation. Nerve fibers enter the glans penis proximally and extend distally over several weeks to eventually reach the distal aspect of the glans and prepuce by 14-16 weeks of gestation.

Androgen-independent events in penile development in humans and animals.

The common view on penile development is that it is androgen-dependent, based first and foremost on the fact that the genital tubercle forms a penis in males and a clitoris in females. However, critical examination of the complex processes involved in human penile development reveals that many individual steps in development of the genital tubercle are common to both males and females, and thus can be interpreted as androgen-independent. For certain developmental events this conclusion is bolstered by observations in androgen-insensitive patients and androgen receptor mutant mice. Events in genital tubercle development that are common to human males and females include: formation of (a) the genital tubercle, (b) the urethral plate, (c) the urethral groove, (d) the glans, (e) the prepuce and (f) the corporal body. For humans 6 of 13 individual developmental steps in penile development were interpreted as androgen-independent. For mice 5 of 11 individual developmental steps were found to be androgen-independent, which were verified through analysis of androgen-insensitive mutants. Observations from development of external genitalia of other species (moles and spotted hyena) provide further examples of androgen-independent events in penile development. These observations support the counter-intuitive idea that penile development involves both androgen-independent and androgen-dependent processes.

Hot spots in fetal human penile and clitoral development.

To better understand how the human fetal penis and clitoris grows and remodels, we undertook an investigation to define active areas of cellular proliferation and programmed cell death spatially and temporally during development of human fetal external genitalia from the indifferent stage (8 weeks) to 18 weeks of gestation. Fifty normal human fetal penile and clitoral specimens were examined using macroscopic imaging, scanning electron microscopy and immunohistochemical localization for the cellular proliferation and apoptotic markers, Ki67 and Caspase-3, respectively. A number of hot spots of cellular proliferation characterized by Ki67 localization are present in the penis and clitoris especially early in development, most notably in the corporal body, glans, remodeling glanular urethra, the urethral plate, the roof of the urethral groove and the fully formed penile urethra. The 12-fold increase in penile length over 10 weeks of growth from 8 to 18 weeks of gestation based on Ki67 labelling appears to be driven by cellular proliferation in the corporal body and glans. Throughout all ages in both the developing penis and clitoris Ki67 labeling was consistently elevated in the ventral epidermis and ventral mesenchyme relative to the dorsal counterparts. This finding is consistent with the intense morphogenetic activity/remodeling in the ventral half of the genital tubercle in both sexes involving formation of the urethral/vestibular plates, canalization of the urethral/vestibular plates and fusion of the urethral folds to form the penile urethra. Areas of reduced or absent Ki67 staining include the urethral fold epithelium that fuses to form the penile tubular urethra. In contrast, the urethral fold mesenchyme is positive for Ki67. Apoptosis was rarely noted in the developing penis and clitoris; the only area of minimal Caspase-3 localization was in the epithelium of the ventral epithelial glanular channel remodeling.

Correlation between increased postpubertal phallic growth and the initiation of cranial sexual dimorphisms in male Morelet's crocodile.

While puberty is an animal commonality, little is known of its timing or process in crocodylians. Males copulate with an intromittent phallus that has a distinct glans morphology which directly interacts with the female cloaca, putatively effecting effective semen transfer and ultimately increased fecundity. Here we present, during the Morelet's crocodile lifecycle, a well-defined body length (65 cm snout-vent length) inflection point that marks a subsequent increase of phallic glans growth rates. Putatively, this postpubescent growth produces a copulatory-effective phallus. While not as robust of a trend as snout-vent length, this growth inflection concomitantly begins with a body condition index (CI = BM/SVL3 ) between 2.0 and 2.5 and is most distinct above a CI of 2.5. Also, in males, this 65 cm size threshold also aligns with the initiation of more robust growth in caniniform alveoli associated with prominent maxillary and mandibular teeth. This inflection was not observed in females, thus marking a sexual dimorphism that begins to present with the onset of puberty. This bodily manifestation of puberty other than those changes observed in the reproductive tracts is a novel observation for crocodylians and lays a foundation for further study among species of how changing endocrine signaling within sexually maturing males may also influence a broader range of secondary sex characteristics.

A critical role for estrogen signaling in penis development.

Hypospadias, a developmental defect of the penis, is one of the most common congenital malformations in humans. Its incidence has rapidly increased over recent decades, and this has been largely attributed to our increased exposure to endocrine-disrupting chemicals. Penis development is primarily an androgen-driven process; however, estrogen and xenoestrogens are known to affect penis development in both humans and mice. Here, we investigated the role of estrogen in the developing penis. Using a novel penis culture system, we showed that exogenous estrogen directly targets the developing penis in utero to cause hypospadias. In addition, we also uncovered an unexpected endogenous role for estrogen in normal postnatal penis development and showed that a loss of estrogen signaling results in a mild hypospadias phenotype, the most common manifestation of this disease in humans. Our findings demonstrated that both androgen and estrogen signaling are intrinsically required for normal urethral closure. These findings confirmed that penis development is not an entirely androgen-driven process but one in which endogenous estrogen signaling also plays a critical role.-Govers, L. C., Phillips, T. R., Mattiske, D. M., Rashoo, N., Black, J. R., Sinclair, A., Baskin, L. S., Risbridger, G. P., Pask, A. J. A critical role for estrogen signaling in penis development.

Effects of pre- and post-pubertal dihydrotestosterone treatment on penile length in 5α-reductase type 2 deficiency.

Steroid 5α-reductase type 2 deficiency (5αRD2) is a congenital disorder of sex development caused by impairment of conversion from testosterone (T) to 5α-dihydrotestosterone (DHT). DHT deficiency leads to various degrees of undervirilized external genitalia including micropenis, primarily correlated with mutations of the SRD5A2 gene that encodes 5α-reductase type 2. Four Japanese boys with isolated micropenis were diagnosed as 5αRD2 by elevated ratios of serum T/DHT, and decreased ratios of urinary 5α/5ß-reduced steroid metabolites. Genetic analyses for SRD5A2 identified that the four patients shared a hypomorphic mutation R227Q that has a residual activity related to the mild-form of 5αRD2. For prepubertal micropenis, DHT was transdermally applied to the four patients at the ages of 4-11 year, increasing a median of stretched penile lengths (SPLs) from 2.6 cm (-2.5 SD) to 4.4 cm (-0.2 SD). Nevertheless, the post-pubertal penile growth was apparently retarded, despite normal levels of T secreted from well-developed testes. The second course of DHT treatment underwent at ages of 12-18 year, but unable to normalize SPLs at a range of 6.0 to 7.0 cm (-3.4 to -2.4 SD). The prostate volumes of two patients were variable at 8.1 and 21 cm3, and a sperm cell count of one patient was normal as young adult. DHT treatment contributes to development of the penis and prostate, which are favorable for the potential fertility of 5αRD2 adults. Meanwhile, the retarded penile growth and a risk of prostate overgrowth may complicate the post-pubertal management with DHT for 5αRD2 males.

Aging and retinoid X receptor agonists on masculinization of female Pomacea canaliculata, with a critical appraisal of imposex evaluation in the Ampullariidae.

Ampullariidae are unique among gastropods in that females normally show a primordium of the copulatory apparatus (CApp). The aims of this study were (a) to quantitatively evaluate the development and growth of the female CApp with age; (b) to compare the effects of RXR and PPARγ agonists in adult females of known age and (c) to explore the effect of masculinizing RXR agonists on the expression of RXR in the CApp. It was found that the CApp grows and develops with age. A significant increase in penile sheath length (PsL) and also in a developmental index (DI) was observed in 7-8 months old females, as compared with 4-5 months old ones. A reported endogenous agonist of RXR, 9-cis retinoic acid (9cis-RA), as well as two organotin compounds, tributyltin (TBT) and triphenyltin (TPT) which have been also reported to bind to RXR, were injected and its masculinizing effects were measured. Also, the effect of a PPARγ agonist, rosiglitazone, was studied. All studied RXR agonists, but not the PPARγ agonist, were effective in increasing PsL, penile length (PL) and DI. Finally, the expression of the RXR in the CApp was studied (Western blot) in control, TBT, TPT, and 9cis-RA treated females. A significantly increased expression of RXR was only observed after 9cis-RA treatment. It is concluded that (a) development and growth of the CApp is significantly affected by female age; (b) reported RXR agonists, but not a PPARγ agonist, cause female masculinization of young females. An appraisal of previous studies of female masculinization in the Ampullariidae has also been made and it is emphasized that the masculinizing effect of aging should be considered, particularly when interpreting field data.

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.