Adult external genitalia (ExG) are the endpoints of normal sex differentiation. Detailed morphometric analysis and comparison of adult mouse ExG has revealed 10 homologous features distinguishing the penis and clitoris that define masculine vs. feminine sex differentiation. These features have enabled the construction of a simple metric to evaluate various intersex conditions in mutant or hormonally manipulated mice. This review focuses on the morphology of the adult mouse penis and clitoris through detailed analysis of histologic sections, scanning electron microscopy, and three-dimensional reconstruction. We also present previous results from evaluation of "non-traditional" mammals, such as the spotted hyena and wallaby to demonstrate the complex process of sex differentiation that involves not only androgen-dependent processes, but also estrogen-dependent and hormone-independent mechanisms.

Sexual differentiation of many brain structures and functions is dependent on levels of testosterone (T) or its metabolites during certain 'sensitive' developmental periods. If T is present during these perinatal periods, masculinization and defeminization of sexual behavior occur; also, reproductive physiology, and central nervous system morphology and function are altered. The purpose of the present study was to characterize the influence of T at specific prenatal developmental intervals on offspring reproductive morphology, physiology, locomotor activity and sexual behavior during postnatal development. To avoid complications induced by endogenous testicular activity, only females were examined. Free T was used because of its relative short half-life, so that the effects induced by its administration on a specific gestational day (GD) could be evaluated. Pregnant rats received a single subcutaneous injection of either sesame oil (controls) or 5 mg of T on GD 16, 17, 18, 19, 20, 21, or 22. Female offspring of pregnant rats exposed to T displayed significant alterations in morphology and behavior. The anogenital distance, measured at 25 days postbirth, was significantly increased if T was administered on GD 16, 17 or 18. T treatment on GD 16 or each day thereafter through GD 20 significantly delayed the normal occurrence of vaginal opening (controls at 37.5 days vs. T treatment which ranged from 38.5 to 41.4 days). Abnormal vaginal morphology (enlarged clitoris) was also observed when T was injected during a similar prenatal interval (i.e. GD 16 to GD 22). Furthermore, prenatal T treatment on GD 18 (and each day thereafter), until GD 22 significantly decreased lordotic behavior compared to control values. However, exposure to T, on any prenatal GD did not alter the animals' ability to exhibit an induced luteinizing hormone (LH) surge. These results suggest that the onset for altered reproductive morphology occurs at least as early as GD 16, whereas the onset of sexual behavior sensitivity occurs precisely at GD 18, and that the normal pattern of adult LH release in females is not altered by prenatal androgen treatment using this specific paradigm.

This report is concerned with hormone concentrations accompanying sexual maturation in a highly 'masculinized' female mammal, the spotted hyaena, Crocuta crocuta. Plasma concentrations of testosterone, androstenedione and oestrogen were determined by radioimmunoassay in a longitudinal study of 12 female and eight male hyaenas 2.5-62.5 months old. Concentrations of testosterone were significantly higher in males than in females after 26.5 months of age, but earlier measurements did not differ between sexes. Mean testosterone concentrations in adult female hyaenas (0.4-0.5 ng ml-1) were similar to those in several other female mammals that do not display a 'masculine' profile, but mean concentrations of androstenedione (2.5-5.5 ng ml-1) in female hyaenas were significantly higher than in males (1.0-2.0 ng ml-1), at most ages. Oestrogen could not be detected (less than 0.03 ng ml-1) in females until about 14 months of age and then increased (to approximately 0.13 ng ml-1) between 18 and 30 months; oestrogen remained undetectable in males. This rise in oestrogen in females corresponded to nipple enlargement and to changes in the size and elasticity of the urogenital meatus, permitting copulation and parturition through the clitoris. Gonadectomy (two males and four females) at 4-7 months resulted in nondetectable concentrations of testosterone and oestrogen and a marked attenuation in androstenedione (to approximately 0.39 ng ml-1), indicating that the gonads are the major source of these three steroids. Gonadectomy also eliminated sex differences in weight, nipple development and elasticity of the urogenital meatus.

Cataloguing gene expression during development of the genitourinary tract will increase our understanding not only of this process but also of congenital defects and disease affecting this organ system. We have developed a high-resolution ontology with which to describe the subcompartments of the developing murine genitourinary tract. This ontology incorporates what can be defined histologically and begins to encompass other structures and cell types already identified at the molecular level. The ontology is being used to annotate in situ hybridisation data generated as part of the Genitourinary Development Molecular Anatomy Project (GUDMAP), a publicly available data resource on gene and protein expression during genitourinary development. The GUDMAP ontology encompasses Theiler stage (TS) 17-27 of development as well as the sexually mature adult. It has been written as a partonomic, text-based, hierarchical ontology that, for the embryological stages, has been developed as a high-resolution expansion of the existing Edinburgh Mouse Atlas Project (EMAP) ontology. It also includes group terms for well-characterised structural and/or functional units comprising several sub-structures, such as the nephron and juxtaglomerular complex. Each term has been assigned a unique identification number. Synonyms have been used to improve the success of query searching and maintain wherever possible existing EMAP terms relating to this organ system. We describe here the principles and structure of the ontology and provide representative diagrammatic, histological, and whole mount and section RNA in situ hybridisation images to clarify the terms used within the ontology. Visual examples of how terms appear in different specimen types are also provided.

Rats were treated with testosterone propionate (TP) or oil (O) beginning in midgestation (group TTT), late gestation (group OTT), neonatally (group OOT), or were left untreated until adulthood (group OOO). At 40 days of age all animals were gonadectomized, received implants of TP in silastic capsules, and were tested in subsequent weeks for masculine copulatory behavior and ex copula phallic responses. Postmortem measures were taken of genital structures and of sexually dimorphic spinal nuclei. Males treated perinatally with androgen were unaffected or exhibited reduced masculinization except on two measures, i.e., number of penile flips and number of cells in the dorsolateral motor nucleus of the lumbar spinal cord, which reflected virilization greater than in normal males. All perinatally androgenized females showed virilization on almost every measure taken of their morphology and behavior. Relative to OOO females, the greatest increases in masculinization were evident in OOT and OTT females. No additional masculinization was detected in TTT females. Only among the TTT rats were males not reliably more masculine than females, but that usually was due to the reduced masculinization of TTT males. Four broad classes of influence characterized the effects of perinatal androgen treatment; these classes may reflect differences in the developmental courses of the variables measured.

The os penis in mice and rats is composed of a proximal intramembranous and endochondral osseous element and a distal cartilaginous, ossifying element. Female mice, but not rats, have a small os clitoridis which corresponds to the intramembranous part of the proximal element of the os penis. In mice of either sex a dense mesenchymatous formation ventral to the urethra is the anlage for the bones of the external genitalia. In the early postnatal period the proximal part of the os penis develops as bone at the outer and as cartilage at the basal end of the anlage, while in females a minute focus of ossification differentiates into the small os clitoridis without passing through a cartilaginous phase. The distal element of the os penis is formed later than the proximal rod and grows at a slower rate. Neonatal treatment with an antiandrogen inhibits the increase in size and calcification of the os penis. Neonatal castration is an even more effective inhibitor. Neonatal treatment with testosterone or dihydrotestosterone, but not with oestradiol, stimulates the growth of the bony proximal os clitoridis, but induces only a rudimentary collagenous distal element. The differences between mice and rats in the response of the tissues of the clitoris to androgenic treatment are discussed, particularly as regards the differentiation of proximal and distal elements.

Genital tubercles of 70 newborn male and female rats were transplated into the brains of unrelated infant rats. Seven other tubercles were placed subcutaneously. All female, and some male, hosts were injected with testosterone propionate. After surviving from 2-24 days,, histological study of 49 successful grafts showed survival of the urethral and balano-preputial epithelia and growth of the preputial glands, which formed secretion-filled cysts and became the major component of the graft. The fate of the mesodermal tissues witin the glands varied between remaining in an undeveloped state, with only pale fibrouw tissue and an area of granular degeneration and giant cells, and achieving an incompletely differentiatel state in which erectile tissue and the anterior process of fibrocartilage had formed and the glans had grown but the penile bone and its secondary growth cartilage failed to appear. Grafts could reach this degree of differentiation of the glands irrespective of transplantation site, attachment to the host dura, the sex of donor or host, and whether or not male hosts were given exogenous hormone.

The development of the phallus from the indifferent stage to sexual dimorphism has not been described in any marsupial. This study describes the morphological and histological changes occurring in the development of the phallus of the tammar wallaby. The development of the penis and clitoris in the tammar closely follow the most widely accepted model for the development of the same organs in eutherian mammals. The urogenital plate that is present in both sexes at birth hollows out to form a urogenital groove at approximately 70 days postpartum (p.p.). There is then greater growth of the phallus in males than in females, which results in sexual dimorphism in length approximately 100 days p.p. In males, the urogenital groove secondarily closes over at this time and fuses in the midline and by 128 days p.p. the penile urethra is fully formed. In females, the groove remains open. The clitoris changes little morphologically from the time of formation of the urogenital groove until adulthood. The pattern of development of the penis in the tammar is similar to that seen in eutherian mammals. There is strong evidence that penis development is androgen-dependent in the tammar, yet unusually it becomes sexually dimorphic at a time when androgen content of the developing testis is low.