Mammalian Sexual Differentiation Research Articles

Meerkat manners: Endocrine mediation of female dominance and reproductive control in a cooperative breeder

This article is part of a Special Issue (Hormones and Hierarchies).To gain more balanced understanding of sexual selection and mammalian sexual differentiation processes, this review addresses behavioral sex differences and hormonal mediators of intrasexual competition in the meerkat (Suricata suricatta) – a cooperative breeder unusual among vertebrates in its female aggression, degree of reproductive skew, and phenotypic divergence. Focused on the evolution, function, mechanism, and development of female dominance, the male remains a key reference point throughout. Integrated review of endocrine function does not support routine physiological suppression in subordinates of either sex, but instead a ramp up of weight, reproduction, aggression, and sex steroids, particularly androgens, in dominant females. Important and timely questions about female competition are thus addressed by shifting emphasis from mediators of reproductive suppression to mediators of reproductive control, and from organizational and activational roles of androgens in males to their roles in females. Unusually, we ask not only how inequity is maintained, but how dominance is acquired within a lifetime and across generations. Antiandrogens administered in the field to males and pregnant dominant females confirm the importance of androgen-mediated food competition. Moreover, effects of maternal endocrine milieu on offspring development reveal a heritable, androgenic route to female aggression, likely promoting reproductive priority along dominant matrilines. Integrating endocrine measures with long-term behavioral, ecological, morphological, and life-history data on normative and experimental individuals, across life stages and generations, provides better appreciation of the role of naturally circulating androgens in regulating the female phenotype, and sheds new light on the evolution of female dominance, reproductive inequity, and cooperative breeding.

A case for ovarian transdifferentiation in six adult captive masculinized lionesses (Panthera leo) in South Africa: Pathological evidence

Until recently, mammalian sex differentiation was thought to be finalized in the embryo. Development of the tubular genital tract, external genitalia, secondary sexual characteristics and sexual behavior are determined largely by the developing gonad. In the last decade, however, it has been shown that continuous sex maintenance is required throughout life. We document proposed ovary to testis transdifferentiation in six captive South African lions. These lions were phenotypically female until, as older adults, they began to develop manes, clitoromegaly and increased levels of restlessness and aggression. Similar phenotypic and behavioral changes have been reported very rarely in free-ranging lions. Five of the lions had been treated repeatedly with deslorelin until they stopped showing estrus. Serum levels of testosterone were similar to those recorded in domestic tom cats in the three lions tested. Histological examination revealed an absence of ovarian follicles and small to large scattered islands of interstitial-like cells associated, in larger lesions, with Sertoli-like cells arranged in tubular structures. Oviduct and uterus were present in the four animals in which these were sampled. To date, most studies on this phenomenon have been conducted in laboratory animals. This is the first report of this condition in felids.

Sex Maintenance in Mammals.

The crucial event in mammalian sexual differentiation occurs at the embryonic stage of sex determination, when the bipotential gonads differentiate as either testes or ovaries, according to the sex chromosome constitution of the embryo, XY or XX, respectively. Once differentiated, testes produce sexual hormones that induce the subsequent differentiation of the male reproductive tract. On the other hand, the lack of masculinizing hormones in XX embryos permits the formation of the female reproductive tract. It was long assumed that once the gonad is differentiated, this developmental decision is irreversible. However, several findings in the last decade have shown that this is not the case and that a continuous sex maintenance is needed. Deletion of Foxl2 in the adult ovary lead to ovary-to-testis transdifferentiation and deletion of either Dmrt1 or Sox9/Sox8 in the adult testis induces the opposite process. In both cases, mutant gonads were genetically reprogrammed, showing that both the male program in ovaries and the female program in testes must be actively repressed throughout the individual’s life. In addition to these transcription factors, other genes and molecular pathways have also been shown to be involved in this antagonism. The aim of this review is to provide an overview of the genetic basis of sex maintenance once the gonad is already differentiated.

Transcriptomic analysis of gene expression in normal goat ovary and intersex goat gonad.

Intersexuality is a congenital reproductive disorder that usually occurs in hornless goats, hindering breeding of goats with hornless traits and the development of the goat industry. In this study, we aimed to identify differentially expressed genes in intersex and normal goat gonads by comparing gene transcription profiles of intersex and normal goat gonads. As intersex goats are genetically based on females, we chose female goats as controls. The goats in the control group and the experimental group were both over one-year old. We evaluated the anatomical characteristics of the reproductive organs of five intersex goats using histopathological methods. The gonads were found to be ovarian and testicular types. RNA-Seq technology was used to identify differentially expressed genes in gonads and normal goat ovary tissues. Transcription analysis results were verified by qPCR. The results showed that 2,748 DEGs were upregulated and 3,327 DEGs were downregulated in intersex ovaries unlike in controls, whereas 2006 DEGs were upregulated and 2032 DEGs were downregulated in the interstitial testes. Many of these genes play important roles in mammalian sex determination and sex differentiation, such as SOX9, WT1, GATA4, DMRT1, DHH, AMH, CYP19A1 and FST. We found that many DEGs are involved in biological developmental regulation by GO and KEGG enrichment analyses, and that most genes associated with the steroid synthesis pathway were downregulated. The DEGs identified in this study may be involved in the regulation of intersex goat sex determination and differentiation, and may increase our understanding of the molecular mechanisms of mammalian sex differentiation.

Brain Sexual Differentiation and Requirement of SRY: Why or Why Not?

Brain sexual differentiation is orchestrated by precise coordination of sex steroid hormones. In some species, programming of select male brain regions is dependent upon aromatization of testosterone to estrogen. In mammals, these hormones surge during the organizational and activational periods that occur during perinatal development and adulthood, respectively. In various fish and reptiles, incubation temperature during a critical embryonic period results in male or female sexual differentiation, but this can be overridden in males by early exposure to estrogenic chemicals. Testes development in mammals requires a Y chromosome and testis determining gene SRY (in humans)/Sry (all other therian mammals), although there are notable exceptions. Two species of spiny rats: Amami spiny rat (Tokudaia osimensis) and Tokunoshima spiny rat (Tokudaia tokunoshimensis) and two species of mole voles (Ellobius lutescens and Ellobius tancrei), lack a Y chromosome/Sry and possess an XO chromosome system in both sexes. Such rodent species, prototherians (monotremes, who also lack Sry), and fish and reptile species that demonstrate temperature sex determination (TSD) seemingly call into question the requirement of Sry for brain sexual differentiation. This review will consider brain regions expressing SRY/Sry in humans and rodents, respectively, and potential roles of SRY/Sry in the brain will be discussed. The evidence from various taxa disputing the requirement of Sry for brain sexual differentiation in mammals (therians and prototherians) and certain fish and reptilian species will be examined. A comparative approach to address this question may elucidate other genes, pathways, and epigenetic modifications stimulating brain sexual differentiation in vertebrate species, including humans.

A general theory of sexual differentiation.

A general theory of mammalian sexual differentiation is proposed. All biological sex differences are the result of the inequality in effects of the sex chromosomes, which are the only factors that differ in XX vs. XY zygotes. This inequality leads to male-specific effects of the Y chromosome, including expression of the testis-determining gene Sry that causes differentiation of testes. Thus, Sry sets up lifelong sex differences in effects of gonadal hormones. Y genes also act outside of the gonads to cause male-specific effects. Differences in the number of X chromosomes between XX and XY cells cause sex differences in expression (1) of Xist, (2) of X genes that escape inactivation, and (3) of parentally imprinted X genes. Sex differences in phenotype are ultimately the result of multiple, independent sex-biasing factors, hormonal and sex chromosomal. These factors act in parallel and in combination to induce sex differences. They also can offset each other to reduce sex differences. Other mechanisms, operating at the level of populations, cause groups of males to differ on average from groups of females. The theory frames questions for further study, and directs attention to inherent sex-biasing factors that operate in many tissues to cause sex differences, and to cause sex-biased protection from disease. © 2016 Wiley Periodicals, Inc.

70 GONADAL DEVELOPMENT IN GUINEA PIG MALES (CAVIA PORCELLUS)

Sexual differentiation in mammals is an event that presents many variations between species. Because it is related to hormonal function, any imbalance in the androgens and estrogens production can lead to malformations. Because sexual differentiation occurs in different ways among various animals, the recognition of their peculiarities becomes important in order to correct reproductive handling in different species. Considering that the guinea pig is commonly used as an experimental model in the reproductive area, the goal of this work was to perform a morphological description of gonad differentiation of the male guinea pig during embryonic development. In total, 11 conceptuses with ages 25 (n = 3), 30 (n = 2), 40 (n = 2), 50 (n = 2), and 65 (n = 2) days were used for light microscopy processing. The embryos at 25 days were processed completely. For the others, the gonads were dissected. The samples were dehydrated in alcohol, embedded in paraffin, and 5-µm sections were stained with hematoxylin-eosin. In the guinea pig gonad at 25 days gestation, there was a presence of gonadal cords, formed by condensation of somatic cells, which is characteristic of an undifferentiated gonad. In addition, we observed the presence of mesonephric and paramesonephric ducts in different embryos, indicating that other genital system organs were not formed. For the 30 days of development of guinea pigs, we observed that gonadal cords were differentiated in testicular cords by invasion of mesenchymal and endothelial cells, and also composed of Sertoli cells and primordial germ cells. These cords were among a large amount of testicular mesenchyme at the 40-day group. With 50- and 65-day development samples, the gonad was completely differentiated into testicle, with the presence of spermatogonia and Sertoli cells in the seminiferous tubules, and a large amount of interstitial Leydig cells around the tubules. We conclude that gonadal differentiation in guinea pig males occurs around the middle of pregnancy, between 25 and 30 days and that, before the end of the pregnancy, at 50 days, the testicle presents morphology similar to that found in the postnatal period.

Transcriptional networks associated with 5-alpha-dihydrotestosterone in the fathead minnow (Pimephales promelas) ovary

Androgens play a significant role in regulating oogenesis in teleost fishes. The androgen dihydrotestosterone (DHT) is a potent non-aromatizable androgen involved in sexual differentiation in mammals; however, its actions are not well understood in teleost fish. To better characterize the physiological role of DHT in the fathead minnow (FHM) ovary on a temporal scale, in vitro assays for 17β-estradiol (E2) production were conducted in parallel with microarray analysis. Ovarian explants were incubated at different concentrations of DHT (10−6, 10−7, and 10−8M DHT) in three separate experiments conducted at 6, 9, and 12h. DHT treatment resulted in a rapid and consistent increase in E2 production from the ovary at all three time points. Therefore, DHT may act to shift the balance of metabolites in the steroidogenic pathway within the ovary. Major biological themes affected by DHT in the ovary in one or more of the time points included those related to blood (e.g. vasodilation, blood vessel contraction, clotting), lipids (e.g. lipid storage, cholesterol metabolism, lipid degradation) and reproduction (e.g. hormone and steroid metabolism). Gene networks related to immune responses and calcium signaling were also affected by DHT, suggesting that this androgen may play a role in regulating these processes in the ovary. This study detected no change in mRNA levels of steroidogenic enzymes (cyp19a1, star, 11βhsd, 17βhsd, srd5a isoforms), suggesting that the observed increase in E2 production is likely more dependent on the pre-existing gene or protein complement in the ovary rather than the de novo expression of transcripts. This study increases knowledge regarding the roles of DHT and androgens in general in the teleost ovary and identifies molecular signaling pathways that may be associated with increased E2 production.

Chapter Seven - The Potential Role of SRY in Epigenetic Gene Regulation During Brain Sexual Differentiation in Mammals

The brain is a sexually dimorphic organ. Little is known about molecular mechanisms underlying sexual differentiation of the brain and behavior. The classical hypothesis of brain sexual differentiation suggests that a perinatal surge of organizational sex hormones secreted from the gonad leads to irreversible changes in morphology of the brain, followed by pubertal hormones that activate neural networks to express sex-specific behavioral phenotypes. However, recent studies propose that sex hormones are not the sole factor to establish sexual dimorphism in the brain. Since mammalian sex strictly relies on sex chromosome complement, i.e., XY for males and XX for females, intrinsic genetic differences between XY and XX cells are strong candidates for the cause of sexual dimorphism. Several genes on the Y chromosome are expressed in the male brain and may act in a dominant manner. Among these Y-linked genes, the testis-determining gene Sry is of particular interest. Although SRY is known to function as a transcriptional activator triggering testicular genetic pathway, several lines of evidence suggest that it also acts as an epigenetic regulator. This chapter provides a basic overview of mammalian sex determination and brain sexual differentiation. It summarizes current evidence of brain-specific epigenetic gene regulations in mammals and other species, and explores the common features between them. Potential roles of Sry during brain sexual development are described and prospects of this research field are discussed.

Men and women, so different, so similar: observations from cross-sex hormone treatment of transsexual subjects

Sexual differentiation in mammals is largely driven by the presence of androgen in males and their absence in females. The presence of androgens induces a number of irreversible changes in males: prenatally, the genital differentiation; during puberty, the development of secondary sex characteristics - the larger facial bones, hand, feet and height in males. A large number of metabolic variables are influenced by sex hormones and consequently show difference between men and women, and this helps to explain differences in pathologies, such as cardiovascular disease, bone fractures and auto immune disease. There is some recent evidence that some sex differences in brain functions are not mediated by sex hormones, but by-products of genes located on the X and Y chromosomes. This communication reviews the results of administration of cross-sex hormone treatment to transsexual persons transitioning to the other sex. Natal males are treated with anti-androgens+oestrogens and natal females with testosterone. This provides a unique opportunity to study which metabolic functions are not irreversibly sex-differentiated but are determined by the prevailing milieu of sex steroids. The insights gained with these studies should lead to a better appreciation of the role of sex steroids in cardiovascular disease and diabetes mellitus which presently do not receive due attention.

Divergent and sex‐dimorphic expression of the paralogs of the Sox9‐Amh‐Cyp19a1 regulatory cascade in developing and adult atlantic cod (Gadus morhua L.)

The factors of the Sox9-Amh-Cyp19a1 cascade play a crucial role in the complex process of sex differentiation in mammals. The involvement of Sox9 and Cyp19a1 paralogs and the single Amh ortholog in sex differentiation and development of the gonads and the brain in Atlantic cod was examined by analyzing bimodal and sex-dimorphic gene expression patterns, respectively, during early stages and in maturing males and females. Expression of sox9a and sox9b were initiated at blastulation, and both paralogs were expressed in chondrogenic tissue in the hatched larvae. The male-specific expression of sox9a in the adult gonads supports a conserved role in testis function, while sox9b was expressed in the maturing testes and ovaries at similar levels. Amh was expressed at low, but variable, levels from late gastrulation prior to the onset of cyp19a1a and cyp19a1b expression. Male-biased amh expression was found in the maturing gonads, but the increased ovarian levels during maturation suggest a role also in females. The larval expression of cyp19a1a and cyp19a1b increased at the expected time of sex differentiation, but showed large individual variation. The ovarian expression of cyp19a1a and amh increased concomitant with increased plasma estradiol levels during vitellogenesis. The testis-specific cyp19a1b expression supports the importance of estrogen in the spermatogenesis, while abundant expression in the male and female brain is probably related to the continuous neurogenesis in fish. These divergent and sex-dimorphic expression patterns of the cod sox9 and cyp19a1 paralogs demonstrate the complexity of the genetic network regulating sexual development in fish.

GATA4 is Involved in the Gonadal Development and Maturation of the Teleost Fish Tilapia, <i>Oreochromis niloticus</i>

GATA4, a member of the GATA family, is a well-known transcription factor implicated in the regulation of sex determination and sexual differentiation in mammals. However, little is known about the possible role of GATA4 in fish reproduction. In the present study, a full-length GATA4 cDNA from the tilapia was cloned and characterized. The tilapia GATA4 gene contained an open reading frame (ORF) of 1179 nucleotides encoding a protein of 392 amino acids. Sequence alignment revealed that the tilapia GATA4 protein shared higher homology (ranging from 63.1 to 74.6%) with other vertebrates. RT-PCR analysis indicated that the GATA4 gene is expressed in the ovary, testis, liver, intestine and heart in adult tilapia. In situ hybridization was performed to examine the temporal and spatial expression patterns of GATA4 during tilapia gonadal differentiation and development. In the undifferentiated gonad, GATA4 was expressed in the somatic cells of both sexes. Subsequently, GATA4 expression persisted in the differentiated, juvenile and adult ovary and testis in tilapia. Our data indicate for the first time that GATA4 is not only necessary for the onset of gonadal differentiation, but also important for gonadal development and maturation.

Sex and hedgehog: roles of genes in the hedgehog signaling pathway in mammalian sexual differentiation

The chromosome status of the mammalian embryo initiates a multistage process of sexual development in which the bipotential reproductive system establishes itself as either male or female. These events are governed by intricate cell-cell and interorgan communication that is regulated by multiple signaling pathways. The hedgehog signaling pathway was originally identified for its key role in the development of Drosophila, but is now recognized as a critical developmental regulator in many species, including humans. In addition to its developmental roles, the hedgehog signaling pathway also modulates adult organ function, and misregulation of this pathway often leads to diseases, such as cancer. The hedgehog signaling pathway acts through its morphogenetic ligands that signal from ligand-producing cells to target cells over a specified distance. The target cells then respond in a graded manner based on the concentration of the ligands that they are exposed to. Through this unique mechanism of action, the hedgehog signaling pathway elicits cell fate determination, epithelial-mesenchymal interactions, and cellular homeostasis. Here, we review current findings on the roles of hedgehog signaling in the sexually dimorphic development of the reproductive organs with an emphasis on mammals and comparative evidence in other species.

La différenciation du sexe

Our knowledge on sex differentiation in mammals has considerably progressed during the last decennials, beginning with the discovery of the testis-determining factor. Here, the morphogenetic processes involved in the early gonadic switch will be presented, together with the major genes involved in testis and ovary formation. Existing differences between the widely used mouse model and other mammals, such as human and goat, will be highlighted.

ATRX has a critical and conserved role in mammalian sexual differentiation

BackgroundX-linked alpha thalassemia, mental retardation syndrome in humans is a rare recessive disorder caused by mutations in the ATRX gene. The disease is characterised by severe mental retardation, mild alpha-thalassemia, microcephaly, short stature, facial, skeletal, genital and gonadal abnormalities.ResultsWe examined the expression of ATRX and ATRY during early development and gonadogenesis in two distantly related mammals: the tammar wallaby (a marsupial) and the mouse (a eutherian). This is the first examination of ATRX and ATRY in the developing mammalian gonad and fetus. ATRX and ATRY were strongly expressed in the developing male and female gonad respectively, of both species. In testes, ATRY expression was detected in the Sertoli cells, germ cells and some interstitial cells. In the developing ovaries, ATRX was initially restricted to the germ cells, but was present in the granulosa cells of mature ovaries from the primary follicle stage onwards and in the corpus luteum. ATRX mRNA expression was also examined outside the gonad in both mouse and tammar wallaby whole embryos. ATRX was detected in the developing limbs, craniofacial elements, neural tissues, tail and phallus. These sites correspond with developmental deficiencies displayed by ATR-X patients.ConclusionsThere is a complex expression pattern throughout development in both mammals, consistent with many of the observed ATR-X syndrome phenotypes in humans. The distribution of ATRX mRNA and protein in the gonads was highly conserved between the tammar and the mouse. The expression profile within the germ cells and somatic cells strikingly overlaps with that of DMRT1, suggesting a possible link between these two genes in gonadal development. Taken together, these data suggest that ATRX has a critical and conserved role in normal development of the testis and ovary in both the somatic and germ cells, and that its broad roles in early mammalian development and gonadal function have remained unchanged for over 148 million years of mammalian evolution.

Conditional ablation of Gata4 and Fog2 genes in mice reveals their distinct roles in mammalian sexual differentiation

Assembly of functioning testis and ovary requires a GATA4–FOG2 transcriptional complex. To define the separate roles for GATA4 and FOG2 proteins in sexual development of the testis we have ablated the corresponding genes in somatic gonadal cells. We have established that GATA4 is required for testis differentiation, for the expression of Dmrt1 gene, and for testis cord morphogenesis. While Sf1Cre-mediated excision of Gata4 permitted normal expression of most genes associated with embryonic testis development, gonadal loss of Fog2 resulted in an early partial block in male pathway and sex reversal. We have also determined that testis sexual differentiation is sensitive to the timing of GATA4 loss during embryogenesis. Our results now demonstrate that these two genes also have non-overlapping essential functions in testis development.

Inheritable Model of Male Pseudohermaphroditism in Canine Somatic Cell Nuclear Transfer.

Normal mammalian sex differentiation depends upon three genetically controlled steps: chromosomal sex determination, gonadal differentiation, and phenotypical sex development. However, two case studies have reported anomalies in the sexual differentiation of mouse and wolf that have occurred during SCNT procedure for unknown reasons. This evokes questions about the early event of reprogramming in SCNT when the chromosomal sex is clearly determined in genetic donor cells, which can never be overcome by technical inflexibilities of sex.During our canine somatic cell nuclear transfer (SCNT) using skin fibroblasts from a male adult as a genetic donor, we report here the birth of one sex reversed cloned dog out of 6 healthy live offspring. Chromosomal, genetic, and histological analyses revealed that the sex-reversed clone has a 78 XY, SRY positive with female external genitalia, which includes an oviduct and a uterus. Interestingly enough, the bilateral testis was in the shape of a hypotrophic ovary and was positioned in the location of normal ovaries. A complete bicornuate uterus was also present. Histological analyses showed that the testis consists of a gonocyte free semiferous tubule-like structure that was stained positive with vimentin, and inhibin alpha, the sertoli cell markers.To study further whether it can be reversible or not, we recloned him, and the result was that the sex reversal condition remained. Thus we believe that we have established the cell line for canine model of inheritable male pseudohermaphroditism and its sex differentiation mechanism. However, we have not observed any opposite sex reversal cases in female genotypes. Further molecular analyses on SRY, SOX-9, MISIIR, AR showed no mutations in their full or partial sequences, which were associated with disorders of sex development in previous reports. In summary, this report describes the first SRY positive XY sex reversal with an almost complete female phenotype including clinical signs; ultrasonographic findings in the abdomen; plasma concentrations of testosterone, oestradiol and progesterone before after administration of GnRH; cytogenetic analysis; PCR analysis for presence of the SRY gene and histology of the genital tract. The results will be discussed with regard to possible causes of the sex reversal. (poster)

A Case of SRY-Positive 38,XY True Hermaphroditism (XY Sex Reversal) in a Cat

Investigation of abnormal sexual development in companion animals can allow for the elimination of inherited disorders from breeding populations while contributing to the understanding of the complex process of mammalian sexual development and differentiation. A 1-year-old mixed-breed cat, presented for neutering, was tentatively diagnosed as a male with bilateral cryptorchidism. During surgery, the surgeon identified gonads in an ovarian position and a complete bicornuate uterus. Both testicular and ovarian architecture in the gonads and Mullerian and Wolffian duct derivatives were identified histologically. The karyotype was that of a normal male (38,XY), and no causative mutation was identified in the feline SRY coding sequence amplified from genomic DNA. All features of the case were compatible with a diagnosis of SRY-positive 38,XY sex reversal, true hermaphrodite phenotype. To the authors' knowledge, this is the first report of this disorder in a domestic cat.

Morphology of Mouse External Genitalia: Implications for a Role of Estrogen in Sexual Dimorphism of the Mouse Genital Tubercle

We examined the role of androgens and estrogens in mammalian sexual differentiation by morphological characterization of adult wt and mutant mouse external genitalia. We tested the hypothesis that external genitalia development depends on androgen and estrogen action. We studied serial sections of the external genitalia of the CD-1 and C57BL6 wt strains of adult mice (Charles River Laboratories, Wilmington, Massachusetts). We recorded linear measurements of key structures in each specimen, including the urethra, erectile tissue, bone and cartilage. We used similar methodology to analyze mice mutant for estrogen receptor α (αERKO) and androgen receptor (X(Tfm)/Y) (Jackson Laboratory, Bar Harbor, Maine). Morphology in X(Tfm)/Y adult murine external genitalia was remarkably similar to that in wt females. Bone and clitoral length was similar in wt females and X(Tfm)/Y mice. Conversely the αERKO clitoris was 59% longer and bone length in αERKO females was many-fold longer than that in female wt mice or X(Tfm)/Y mutants. The αERKO clitoris contained cartilage, which is typical of the wt penis but never observed in the wt clitoris. Serum testosterone was not increased in female αERKO mice 10 days postnatally when sex differentiation occurs, suggesting that masculinization of the αERKO clitoris is not a function of androgen. Masculinization of the αERKO clitoris suggests a role for estrogen in the development of female external genitalia. We propose that normal external genital development requires androgen and estrogen action.

Behavioral toxicology in the 21st century: Challenges and opportunities for behavioral scientists: Summary of a symposium presented at the annual meeting of the Neurobehavioral Teratology Society, June, 2009

The National Research Council (NRC) of the National Academies of Science recently published a report of its vision of toxicity testing in the 21st century. The report proposes that the current toxicity testing paradigm that depends upon whole-animal tests be replaced with a strategy based upon in vitro tests, in silico models and evaluations of toxicity at the human population level. These goals are intended to set in motion changes that will transform risk assessment into a process in which adverse effects on public health are predicted by quantitative structure–activity relationship (QSAR) models and data from suites of high-throughput in vitro tests. The potential roles for whole-animal testing in this futuristic vision are both various and undefined. A symposium was convened at the annual meeting of the Neurobehavioral Teratology Society in Rio Grande, Puerto Rico in June, 2009 to discuss the potential challenges and opportunities for behavioral scientists in developing and/or altering this strategy toward the ultimate goal of protecting public health from hazardous chemicals. R. Kavlock described the NRC vision, introduced the concept of the ‘toxicity pathway’ (a central guiding principle of the NRC vision), and described the current status of an initial implementation this approach with the EPA's ToxCast® program. K. Crofton described a pathway based upon disruption of thyroid hormone metabolism during development, including agents, targets, and outcomes linked by this mode of action. P. Bushnell proposed a pathway linking the neural targets and cellular to behavioral effects of acute exposure to organic solvents, whose predictive power is limited by our incomplete understanding of the complex CNS circuitry that mediates the behavioral responses to solvents. B. Weiss cautioned the audience regarding a pathway approach to toxicity testing, using the example of the developmental toxicity of phthalates, whose effects on mammalian sexual differentiation would be difficult to identify based on screening tests in vitro. Finally, D. Rice raised concerns regarding the use of data derived from toxicity screening tests to human health risk assessments. Discussion centered around opportunities and challenges for behavioral toxicologists regarding this impending paradigm shift. Opportunities include: identifying and characterizing toxicity pathways; informing the conditions and limits of extrapolation; addressing issues of susceptibility and variability; providing reality-checks on selected positives and negatives from screens; and performing targeted testing and dose-response assessments of chemicals flagged during screening. Challenges include: predicting behavior using models of complex neurobiological pathways; standardizing study designs and dependent variables to facilitate creation of databases; and managing the cost and efficiency of behavioral assessments. Thus, while progress is being made in approaching the vision of 21st century toxicology, we remain a long way from replacing whole-animal tests; indeed, some animal testing will be essential for the foreseeable future at least. Initial advances will likely provide better prioritization tools so that animal resources are used more efficiently and effectively.