Hormones and Reproduction of Vertebrates, Volume 5: Mammals

Table of Contents

Cover image

Front Matter

Copyright

Dedication

Preface

Preface

Contributors

Chapter 1. Sexual Differentiation of the Mammalian Brain

1. Introduction

2. Historical Overview

3. The Organizational/Activational Hypothesis of Sexual Differentiation

4. Advances in Mechanisms

5. Summary and Future Directions

Chapter 2. Neuroendocrine Control of Gonadotropins in Mammals

1. Introduction to Gonadotropins

2. Gonadotropin-Releasing Hormone (GnRH)

3. Gonadotropin-Releasing Hormone (GnRH) Receptors

4. Conclusions

Chapter 3. Endocrine and Paracrine Regulation of Mammalian Spermatogenesis

1. Overview of Spermatogenesis

2. Regulation of Stem Spermatogonia Via Growth Factors and Paracrine Interactions

3. Endocrine Regulation of Spermatogenesis

4. Future Directions

Chapter 4. The Endocrinology of the Mammalian Ovary

1. Introduction

2. Embryogenesis of the ovary

3. The ovarian cycle

4. Ovarian lifespan and reproductive aging

5. Ovarian events and reproductive cycles of selected mammals

6. Future research needs

Chapter 5. Hormones and Pregnancy in Eutherian Mammals

1. Introduction

2. The Menstrual Cycle, Luteolysis, and Pregnancy in Primates (Homo Sapiens and Macaca Mulatta)

3. Estrous Cycles, Luteolysis, Pseudopregnancy, Delayed Implantation, and Pregnancy in Subprimate Mammals

4. Summary and Conclusions

5. Future Research

Chapter 6. The Comparative Physiology of Parturition in Mammals: Hormones and Parturition in Mammals

1. Introduction

2. What determines gestation length?

3. How are fetal maturation and parturition synchronized?

4. How does the fetus signal the initiation of labor?

5. How are the uterotonic mechanisms activated?

6. Conclusions

Chapter 7. Stress and Reproduction in Mammals

1. Introduction

2. Effects of Food Restriction

3. Effects of Stress on Immunity and Reproduction

4. Effects of Other Stressors

5. Role of HPA Activation (Glucocorticoids and CRH)

6. Implications for Wildlife

7. Conclusions

Chapter 8. Behavioral Neuroendocrinology of Reproduction in Mammals

1. Introduction

2. Description of Male and Female Sexual Behavior (MSB/FSB)

3. The Role of Gonadal Steroids in Sexual Behavior

4. Neural Circuitry Regulating Sexual Behavior

5. Steroid Receptors and Sexual Behavior

6. Ligand-Independent Activation of Steroid Receptors

7. Relevant Neurotransmitters and Neuropeptides

8. Gonad-Independent Sexual Behavior

9. Genetics of Sexual Behavior

10. Neuroendocrinology of Human Sexual Behavior

11. Summary

Chapter 9. Pheromones and Reproduction in Mammals

1. Introduction

2. Social Odors and Physiology

3. Social Odors and Behavior

4. Conclusions

Chapter 10. Reproductive Endocrinology of Prototherians and Metatherians

1. The Prototherians and Metatherians: The ‘Other’ Mammals

2. Endocrine Control of Sexual Differentiation in Monotremes and Marsupials

3. The Ovarian Cycle in Monotremes and Marsupials

4. Control of Ovarian Cycles in Monotremes and Marsupials

5. Hormonal Regulation of Birth in Monotremes and Marsupials

6. Embryonic Diapause and Gestational Developmental Arrest

7. Regulation of the Testicular Cycle in Monotremes and Marsupials

8. Control of Testicular Function in Monotremes and Marsupials

9. Summary and Conclusions

Chapter 11. Hormones and Reproductive Cycles in Rodents

1. Introduction

2. Food Availability

3. Photoperiod

4. Water Availability

5. Temperature

6. Social Cues

7. Small Rodent Population Cycles

8. Other Ecological Factors Affecting Reproduction

9. Conclusions

Chapter 12. Hormones and Reproductive Cycles in Bats

1. Introduction

2. Bat Reproductive Cycles

3. Reproductive Delays in Bats

4. Hormones and Reproduction in Bats

5. Hormones and Reproduction in Male Bats

6. Hormones and Reproduction of Female Bats

7. Environmental Factors and Additional Hormones Affecting Reproduction in Bats

8. Conclusions and Future Directions

Chapter 13. Hormones and Reproductive Cycles in Primates

1. Overview of the Primates

2. Testicular Function and ITS Neuroendocrine Control

3. Ovarian Function and ITS Neuroendocrine Control

4. Puberty

5. Pregnancy

6. Lactation

7. Reproductive Aging

8. Sexual Behavior

9. Environmental Influences on Reproduction

10. Conclusions and Future Directions

Chapter 14. Endocrine-disrupting Chemicals (EDCs) in Mammals

1. Introduction

2. Endocrine-Disrupting Chemicals that Affect Reproduction

3. Mechanisms of Endocrine-Disrupting Chemical (EDC) Action

4. Adult vs. Developmental Effects

5. Levels of Xenoestrogens Relative to Endogenous Estrogens in Utero

6. Endpoints

7. Conclusion

Color Plates

Index

Index

Front Matter

Hormones and Reproduction of Vertebrates, Volume 5—Mammals

Hormones and Reproduction of Vertebrates

Volume 5: Mammals

David O. Norris

Department of Integrative Physiology, University of Colorado, Boulder, Colorado

Kristin H. Lopez

Department of Integrative Physiology, University of Colorado, Boulder, Colorado

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Dedication

Richard Evan Jones

This series of five volumes on the hormones and reproduction of vertebrates is appropriately dedicated to our friend and colleague of many years, Professor Emeritus Richard Evan Jones, who inspired us to undertake this project. Dick spent his professional life as a truly comparative reproductive endocrinologist who published many papers on hormones and reproduction in fishes, amphibians, reptiles, birds, and mammals. Additionally, he published a number of important books including The Ovary (Jones, 1975, Plenum Press), Hormones and Reproduction in Fishes, Amphibians, and Reptiles (Norris and Jones, 1987, Plenum Press), and a textbook, Human Reproductive Biology (Jones & Lopez, 3rd edition 2006, Academic Press). Throughout his productive career he consistently stressed the importance of an evolutionary perspective to understanding reproduction and reproductive endocrinology. His enthusiasm for these subjects inspired all with whom he interacted, especially the many graduate students he fostered, including a number of those who have contributed to these volumes.

Preface

Hormones and Reproduction of Vertebrates Preface to the Series

Every aspect of our physiology and behavior is either regulated directly by hormones or modified by their actions, as exemplified by the essential and diverse roles of hormones in reproductive processes. Central to the evolutionary success of all vertebrates are the regulatory chemicals secreted by cells that control sexual determination, sexual differentiation, sexual maturation, reproductive physiology, and reproductive behavior. To understand these processes and their evolution in vertebrates, it is necessary to employ an integrated approach that combines our knowledge of endocrine systems, genetics and evolution, and environmental factors in a comparative manner. In addition to providing insight into the evolution and physiology of vertebrates, the study of comparative vertebrate reproduction has had a considerable impact on the biomedical sciences and has provided a useful array of model systems for investigations that are of fundamental importance to human health. The purpose of this series on the hormones and reproduction of vertebrates is to bring together our current knowledge of comparative reproductive endocrinology in one place as a resource for scientists involved in reproductive endocrinology and for students who are just becoming interested in this field.

In this series of five volumes, we have selected authors with broad perspectives on reproductive endocrinology from a dozen countries. These authors are especially knowledgeable in their specific areas of interest and are familiar with both the historical aspects of their fields and the cutting edge of today’s research. We have intentionally included many younger scientists in an effort to bring in fresh viewpoints. Topics in each volume include sex determination, neuroendocrine regulation of the hypothalamus–pituitary–gonadal (HPG) axis, separate discussions of testicular and ovarian functions and control, stress and reproductive function, hormones and reproductive behaviors, and comparisons of reproductive patterns. Emphasis on the use of model species is balanced throughout the series with comparative treatments of reproductive cycles in major taxa.

Chemical pollution and climate change pose serious challenges to the conservation and reproductive health of wildlife populations and humans in the twenty-first century, and these issues must be part of our modern perspective on reproduction. Consequently, we have included chapters that specifically deal with the accumulation of endocrine-disrupting chemicals (EDCs) in the environment at very low concentrations that mimic or block the critical functions of our reproductive hormones. Many authors throughout the series also have provided information connecting reproductive endocrinology to species conservation.

The series consists of five volumes, each of which deals with a major traditional grouping of vertebrates: in volume order, fishes, amphibians, reptiles, birds, and mammals. Each volume is organized in a similar manner so that themes can be easily followed across volumes. Terminology and abbreviations have been standardized by the editors to reflect the more common usage by scientists working with this diverse assembly of organisms we identify as vertebrates. Additionally, we have provided indices that allow readers to locate terms of interest, chemicals of interest, and particular species. A glossary of abbreviations used is provided with each chapter.

Finally, we must thank the many contributors to this work for their willingness to share their expertise, for their timely and thoughtful submissions, and for their patience with our interventions and requests for revisions. Their chapters cite the work of innumerable reproductive biologists and endocrinologists whose efforts have contributed to this rich and rewarding literature. And, of course, our special thanks go to our editor, Patricia Gonzalez of Academic Press, for her help with keeping us all on track and overseeing the incorporation of these valuable contributions into the work.

David O. Norris and Kristin H. Lopez

Preface

Preface to Hormones and Reproduction, Vol. 5 Mammals

The vast majority of literature on the reproductive endocrinology of mammals is focused on laboratory mice, laboratory rats, and humans. In this volume, we have made a conscious effort to present a more comparative approach to the reproductive endocrinology of mammals. This volume therefore offers a phylogenetically broad view of mammalian reproduction while incorporating the most recent biochemical and endocrine research. We have devoted specific chapters to the treatment of wild species of a wide range of mammalian groups including monotremes and marsupials, rodents, bats, and primates. Notably missing are the marine mammals for which there are not sufficient data to provide a complete picture of reproductive endocrinology. Also not given separate treatment are the large terrestrial carnivores for which studies of reproductive endocrinology are limited mostly to captive zoo animals, nor have we chosen to feature domestic species such as sheep, cattle, dogs, and cats that are covered in the veterinary literature.

This volume is organized in the same format as the other volumes in the Series, beginning with treatments of sex determination, neuroendocrine regulation of the hypothalamus-pituitary-gonad axis, and specific functions of the ovaries and testes. Pregnancy and parturition are covered in separate chapters, whereas lactation and reproductive behaviors are discussed throughout many of the chapters. The effects of stress hormones and pheromones are featured, followed by discussions of the reproductive cycles of selected mammalian groups. The final chapter discusses the disruption of endocrine activities in humans and other mammals as a consequence of widespread environmental exposures to estrogenic chemicals, and implications for future reproduction of wild mammalian populations and of our own species.

Contributors

Karen L. Bales

University of California, Davis, CA, USA

Fuller W. Bazer

Texas A&M University, Texas, TX, USA

Joseph M. Beeman

University of Missouri, Columbia, MO, USA

Kunwar P. Bhatnagar

University of Louisville, Louisville, KY, USA

T.R. Brown

Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA

Wilson C.J. Chung

University of Colorado, Boulder, CO, USA

Caroline M. Hostetler

University of California, Davis, CA, USA

J.P. Jarow

Johns Hopkins School of Medicine, Baltimore, MD, USA

Graham Jenkin

Monash Institute of Medical Research (MIMR), Clayton, Victoria, Australia

Desiree L. Krebs-Kraft

University of Maryland School of Medicine, Baltimore, MD, USA

Amitabh Krishna

Banaras Hindu University, Varanasi, India

Kristin H. Lopez

University of Colorado at Boulder, Boulder, CO, USA

Bronwyn M. McAllan

The University of Sydney, Sydney, NSW, Australia

Margaret M. McCarthy

University of Maryland School of Medicine, Baltimore, MD, USA

Sam Mesiano

Case Western Reserve University, Cleveland, OH, USA

Susan C. Nagel

University of Missouri, Columbia, MO, USA

Bridget A. Niebruegge

University of Missouri, Columbia, MO, USA

David O. Norris

University of Colorado at Boulder, Boulder, CO, USA

Toni R. Pak

Loyola University Stritch School of Medicine, Maywood, IL, USA

Jin Ho Park

University of Massachusetts, Boston, MA, USA

Katherine E. Pelch

University of Missouri, Columbia, MO, USA

Aras Petrulis

Georgia State University, Atlanta, GA, USA

Marilyn B. Renfree

The University of Melbourne, Victoria, Australia

Emilie F. Rissman

University of Virginia School of Medicine, Charlottesville, VA, USA

Julienne N. Rutherford

University of Illinois at Chicago, Chicago, IL, USA

Wendy Saltzman

University of California, Riverside, CA, USA

Geoff Shaw

The University of Melbourne, Victoria, Australia

Roger Smith

The University of Newcastle, NSW, Australia

Thomas E. Spencer

Texas A&M University, Texas, TX, USA

Suzette D. Tardif

University of Texas Health Science Center, San Antonio, TX, USA

Lynda Uphouse

Texas Woman’s University, Denton, TX, USA

Stacey R. Winkeler

University of Missouri, Columbia, MO, USA

W.W. Wright

Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA

I. Ross Young

Monash University, Clayton, Victoria, Australia

Barry R. Zirkin

Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA

Chapter 1. Sexual Differentiation of the Mammalian Brain

Desiree L. Krebs-Kraft and Margaret M. McCarthy

University of Maryland School of Medicine, Baltimore, MD, USA

Summary

Sexual differentiation of the mammalian brain requires the activity of gonadal hormones. The organizational/activational hypothesis of sexual differentiation postulates that the organizational effects of these gonadal hormones on brain development occur early in life, during a sensitive period, whereas activational effects in adulthood ensure appropriate and timely sex-specific behaviors. Testosterone (T), or estradiol (E 2) produced by aromatization of T, is responsible for masculinization of the brain and the expression of normal male reproductive behavior in adulthood. In the absence of early exposure to gonadal hormones, the brain is, by default, feminized and mediates normal female reproductive behavior in adulthood. In this chapter we will review the role of apoptosis, cell genesis and synaptogenesis in establishing neuroanatomical sex differences. We will also look at how these processes are regulated by γ-aminobutyric acid (GABA), glutamate, prostaglandins, focal adhesion kinase, paxillin, and kisspeptin to act as the mechanisms of hormone action on sexual differentiation of the brain.

1. Introduction

An individual’s sex is among the most salient of biological cues for determining both intrinsic physiology and behavior and external responses from conspecifics. In mammals, sex is determined genetically, with the homogametic sex being female (XX) and the heterogametic sex being male (XY). The phenotype of the undifferentiated gonad undergoes sex determination as a result of the presence or absence of the SRY gene (sex-determining region of the Y chromosome). This gene codes for testis-determining factor (TDF), which will initiate a gene expression cascade that ultimate leads to development of a testis (Koopman, Gubbay, Vivian, Goodfellow, & Lovell-Badge, 1991). In the absence of this gene, the undifferentiated gonadal anlage becomes an ovary. The remaining sexual characteristics are a byproduct of gonadal secretions; therefore, this process is referred to as sex determination and the phenotype of the body, including the brain, is the product of sexual differentiation. This is not to say that genetics plays no additional role in physiology or behavior, but, for endpoints that are directly relevant to reproduction, the importance of gonadal hormones and signaling peptides cannot be overstated.

Successful reproduction requires more than functioning gonads and a viable reproductive tract. The animal must also display the appropriate behaviors in response to the appropriate conditions, including but not limited to the presence of a sexually receptive partner with which to mate. In the overwhelming majority of animals, expression of this behavior is constrained by internal variables, such as viable sperm in males or temporal relation to ovulation in females, as well as environmental variables including the availability of food, shelter, and nesting sites, which may be directly related to the season. The brain is critical to integrating both the internal and external variables to achieve optimal coordination of physiology and behavior. The optimality of this coordination varies between males and females because of the constraints of internal fertilization and requirements for extensive postnatal maternal care by mammalian females. Sexual differentiation in response to gonadal hormones ensures that the brain and the body are in sync; in other words, that brain sex matches gonadal sex. The two critical endpoints are the control of gonadotropin (GTH) secretion by the anterior pituitary, which is pulsatile in males but exhibits a midcycle ovulation-inducing surge in females, and sexual behavior. Male rodents exhibit mounting and thrusting toward sexually receptive females, whereas females adopt a sexually receptive posture referred to as lordosis that permits access by the male. The sex-specific control of GTH secretion and sexual behavior are both results of hormonally mediated sexual differentiation of the brain, and the mechanisms by which these occur are the topic of this review.

2. Historical Overview

2.1. Phoenix, Goy, Gerall, and Young: 1959

Every field has its icons and for the discipline of behavioral neuroendocrinology there is no question that a single paper published in Endocrinology in 1959 by trainees in the laboratory of William Young at the University of Kansas (Phoenix, Goy, Gerall, & Young, 1959) has determined the direction of research for the past 50 years. This seminal paper brought coherence to what had previously been a disparate collection of observations about hormonal effects on the reproductive behavior of males and females. The authors proposed a novel hypothesis, that early hormonal actions predisposed adult behavior, and then conducted a series of careful experiments in guinea pigs ( Cavia porcellus) that tested the predictions stemming from this hypothesis. Now codified as the organizational/activational hypothesis of hormone action, the authors demonstrated that treatment of pregnant guinea pigs with testosterone (T) masculinized their female offspring such that they displayed male sexual behavior in adulthood, but only when these females were treated with T as adults. Females whose brains were organized in a masculinized fashion did not display maletypic behavior as adults if treated with estradiol (E 2) and progesterone (P 4), the hormones that normally activate female sexual behavior. This demonstrated the two-step actions of hormones to organize responses early in development that must then be acted upon in adulthood (Figure 1.1). Ironically, the notion that hormones would act on the brain to organize its development and thereby permanently determine adult behaviors was not well accepted at the time, as demonstrated by the authors’ repeated reference to a hormone-responsive ‘tissue,’ with mention of the brain only as a potential candidate. Regardless, the organizational/activational hypothesis, as a sturdy framework, has withstood many challenges and provides a strong foundation against which contrasting or alternative interpretations can be tested. This hypothesis continues to provide the benefits of dogma by serving as a guiding principle (Becker et al., 2005) as well as a yielding doctrine for more modern synthesis (McCarthy & Konkle, 2005).

2.2. Modern Views—Where Science and Society Collide

Sexual differentiation of the brain is a fascinating biological phenomenon of tremendous interest in its own right and also as a valuable heuristic model for brain development. Several important discoveries about the basic principles establishing phenotypic variation in neural function have arisen directly from the study of hormonally mediated sexual differentiation. Unlike many other corners of biology, however, there is also an important social or cultural component to work on sex differences in the mammalian brain. This contributes to an exaggerated emphasis on rudimentary findings in simple organisms as being relevant for the human condition and a greater need for precision and restraint in reaching conclusions. Everyone is ready and willing to believe that men and women are different, but it is how they differ and what this means that are the sources of considerable contention. Discussion of this topic could fill volumes and consensus would still not be reached. None-the-less, it is worth asking whether the organizational/activational hypothesis applies to humans ( Homo sapiens) and sex differences in human behavior. The very short answer is that we do not know. This is in part because we do not do experiments on humans and therefore resort to relying on Mother Nature to provide so-called ‘natural experiments.’ These include infants or children exposed to abnormal or heterotypic steroid levels because of medical treatments or disease states, genetic mutations, and exposures to endocrine-disrupting chemicals (EDCs) (see Chapter 14, this volume) resulting in excessive steroid production, select hormone insensitivity, or endocrine disruption. Study of these individuals is consistent with some developmental organizational effects of steroids on adult human behavior that are relevant to reproduction, but, in both this and other arenas, the magnitude and significance of the effects remains highly controversial (Berenbaum, 1999 and Hines, 2002). The tendency to overinterpret results from animal studies as relevant to humans is nowhere greater than in the area of cognition. The notion that ‘boys are better at math and girls are better at language’ is as frequently and thoughtlessly repeated as ‘an apple a day keeps the doctor away’. Animal studies are often considered to provide the unbiased-by-culture proof of sex differences in cognition, and yet we have no animals in which to examine either mathematics or language. In fact, when animal studies purporting sex differences in cognition, predominantly spatial learning, are carefully examined, there is much more smoke than fire (see McCarthy & Konkle, 2005), and the same is true for many common cognitive assessments in humans (Spelke, 2005). Does this mean there are no sex differences in the human brain? Certainly not, and indeed the growth trajectories of male and female human brains are markedly different (Lenroot et al., 2007), as are many neurological endpoints. But it is important that appropriate perspective is applied when evaluating both animal and human studies.

3. The Organizational/Activational Hypothesis of Sexual Differentiation

There are several seminal events that built on the original Phoenix report that have influenced the course of the field ever since. One of the earliest was the realization that the guinea pig, the animal model used by Phoenix and colleagues, is not the best species in which to address these questions due to its small litter size, long gestation, and precocial young. The fact that the seminal organizational events occurred prenatally both confounded investigation by the hormones of pregnancy and precluded the ability to manipulate males and females independently. The laboratory rat soon replaced the guinea pig as an ideal species for study due to it large litter size, short gestation, and the observation that the sensitive period for sexual differentiation began prenatally but extended well into the postnatal period as well.

3.1. Basic Principles of Hormone Action

Steroid hormones are lipid-soluble, which allows them to cross the extracellular membrane to the inside of the cell. Classical models of steroid action suggest that steroid hormones bind to specific nuclear receptor proteins once inside the cell. In the absence of steroid hormones, the nuclear steroid receptors in various target tissues are associated with chaperone proteins, such as heat-shock proteins, in a transcriptionally inactive state (DeFranco, Ramakrishnan, & Tang, 1998). These chaperone proteins keep the receptor in a high affinity state for binding of receptor ligands. Binding of steroid hormone to its cognate nuclear receptor causes the inhibitory heat-shock proteins to dissociate and the receptor to become activated. Steroid receptor activation leads to receptor dimerization and facilitates the ability of estrogen receptors (ERs) and androgen receptors (ARs) to bind to specific DNA sequences, called hormone response elements, on the promoters of target genes (Figure 1.2). Before interacting with DNA, the steroid receptors may bind to other proteins, forming part of a transcriptional complex. This complex consists of multiple proteins called coactivators or corepressors that affect DNA transcription and are recruited to promote or inhibit the expression of target genes (Enmark and Gustafsson, 1999, He et al., 1999, Roy et al., 1999, Klinge et al., 2000, Osborne et al., 2000, Nilsson et al., 2001 and Heinlein and Chang, 2002). As a result of this principal mechanism of action, steroid receptors are considered nuclear transcription factors and members of a large superfamily of nuclear receptors (Beato & Klug, 2000).

Steroid hormone receptors may have multiple isoforms. For example, E 2 binds to two ER isoforms, ERα and ERβ. The existence of multiple ER isoforms allows cells to selectively regulate responses to E 2. In contrast, the AR has only one form for both T and 5α-dihydrotestosterone (DHT) binding, but their effects on gene expression can be quite distinct.

In addition to the slow ‘genomic’ actions of steroid hormones that take hours or days for final changes in protein expression to occur, there are also rapid actions of steroids. For example, E 2 administration increases cyclic-3’,5’-adenosine monophosphate (cAMP) in the uterus within 15 seconds (Szego & Davis, 1967). The ‘nongenomic’ actions of steroid hormones occur rapidly (milliseconds to minutes), and are initiated at the plasma membrane, resulting in the activation of kinases and signal transduction pathways, including calcium influx, within target cells (Figure 1.2). The rapid effects of steroid hormones can be mediated by direct binding to a specific binding site on the target cell, through the classical intracellular steroid receptors, and by distinct nonclassical membrane receptors, such as a G-protein-coupled receptor (Michels & Hoppe, 2008). The discovery of a novel G-protein-coupled membrane ER, GPR30 (Prossnitz, Arterburn, & Sklar, 2007), which is unrelated to nuclear ERs and localized to the endoplasmic reticulum, is another substrate for rapid estrogen signaling (Revankar et al., 2005 and Thomas et al., 2005).

Thus, it is apparent that steroid hormones can directly or indirectly regulate gene transcription. Steroid hormones that bind to classical steroid nuclear receptors cause binding of AR and ER to DNA sequences on promoters of genes, therefore directly affecting DNA transcription to promote or inhibit the expression of target genes (Enmark and Gustafsson, 1999, He et al., 1999, Roy et al., 1999, Klinge, 2000, Osborne et al., 2000, Nilsson et al., 2001 and Heinlein and Chang, 2002). The traditional nuclear steroid receptors can also indirectly regulate gene transcription by serving as signal transduction proteins that interact with various signaling pathways in the cytoplasm of a cell. In particular, ER can activate kinases, enzymes that phosphorylate and thereby activate other proteins. This sets in motion a signaling cascade that eventually ends in the nucleus with the induction of gene transcription (Singh, Setalo, Guan, Frail, & Toran-Allerand, 2000). A number of second messengers are produced, including cAMP and the inositol phospholipids. In order for membrane receptors to influence the expression of genes, the signal transduction pathway must reach the nucleus (Aronica, Kraus, & Katzenellenbogen, 1994). Ultimately, the increased calcium released by inositol triphosphate (IP 3) and cAMP influences gene expression by activating cAMP-response-element-binding protein (CREB), protein kinase A (PKA) (Aronica et al., 1994; Zhou et al., 1996 and Abraham et al., 2004), mitogen-activated protein kinase (MAPK) (Watters, 1998, Singh et al., 1999 and Kuroki et al., 2000), and phosphatidylinositol 3-kinase (PI3K) (Singh et al., 1999 and Znamesnsky et al., 2003). The discovery of the myriad ways in which steroids can impact on cell function greatly expanded the potential mechanisms by which steroid-mediated sexual differentiation of the brain could occur, with both classic ‘genomic’ and nonclassic ‘rapid effects’ identified to date.

3.2. Sensitive Periods

A common principle in brain development is the restricted time course of critical events that must unfold in a scripted order to provide the template for subsequent phases of the maturational process to be successful. For example, in the visual system there is a period of uncoordinated excitation in the retina followed by activity-dependent pruning of synapses in the visual cortex to generate patterns of neuronal input that provide for the selective response to particular stimuli. If visual input to the retina is restricted during a critical developmental window, the ability to generate the appropriate pattern of synaptic input is forever lost.

Similarly, early in development there is a sensitive or critical period for sexual differentiation of the brain (MacLusky and Naftolin, 1981, Arnold and Gorski, 1984 and Nevison et al., 1997). A critical period also can be thought of as a limited window of development in which an event can occur because of enhanced sensitivity to an environmental stimulus, which may be endogenous or exogenous (Figure 1.1). During the last days of gestation in the rat, as early as embryonic day 18, and as early as the first trimester of pregnancy in primates, the testes of the developing male begin to produce significant quantities of T (Weisz and Ward, 1980 and Rhoda et al., 1984). This timed exposure to T is necessary to induce a phenotypic male brain and the formation of the secondary sex characteristics, including the epididymis, vas deferens, and male genitalia (Jost, 1947). From embryonic day 18 until around postnatal day 10, exogenous treatment of female rodents with T can override development as a female and induce a phenotypic male brain. For instance, a single injection of T within the first week of life disrupts normal cyclicity in female rodents during adulthood (Barraclough, 1961 and Barraclough and Gorski, 1962). After postnatal day 10, treatment of female rodents with T has no effect on brain development because it has been permanently changed to be phenotypically female (MacLusky and Naftolin, 1981 and Arnold and Gorski, 1984), signaling the end of the sensitive period.

Sensitive periods of steroid hormone effects on brain organization are species-specific. In rats ( Rattus norvegicus), the sensitive period spans the late gestation/early postnatal period (Goldman & Gorski, 1971); however, the sensitive period in more precocious mammals is earlier in gestation. The sensitive period occurs at midgestation in guinea pigs (Goy, Bridson, & Young 1964), during the first trimester in monkeys (Herman, Jones, Mann, & Wallen, 2000), and midgestation to postnatal development in sheep (Clarke et al., 1976, Wood et al., 1995 and Foster et al., 2002). Even within one species the timing of the sensitive period may vary as a function of the endpoint. In rats and mice ( Mus musculus), the timing for sexual differentiation of GTH secretion is earlier than that for sexual behavior (Diaz, Fleming, & Rhees, 1995), illustrating the point that different neural substrates are modified to produce distinct outcomes.

The critical period for sexual differentiation of the brain is early in development and usually perinatal, meaning around birth. However, there is also evidence for an important effect of the elevated hormones associated with puberty on the final differentiation of the brain (Arnold and Breedlove, 1985, Romeo, 2003, Sisk and Foster, 2004, Sisk and Zehr, 2005 and Wallen, 2005). The absence of gonadal hormones during adolescent brain development in Syrian hamsters ( Mesocricetus auratus) significantly reduces T-induced mounting and intromissions in adulthood (Schulz et al., 2004). The presence of pubertal gonadal hormones is also important for defeminization in Syrian hamsters. Specifically, males that are castrated prior to puberty and treated with estrogens and P 4 show equal levels of lordosis to females hormonally primed in adulthood (Schulz et al., 2003). Gonadal hormones also organize agonistic behaviors, such as flank marking and aggression, and anxiety-related behaviors, such as locomotor activity in the open field, during puberty in males and in some cases females (Sisk & Zehr, 2005). It is still unclear whether adolescence serves as a sensitive period for steroid-dependent remodeling of the brain, and further experiments are needed to elucidate this possibility.

3.3. The Role of Aromatization

As noted above, initial studies on sexual differentiation of the brain involved T treatment of pregnant guinea pigs. Unforeseen at the time was an important difference between rats and guinea pigs: the critical role of androgens vs. estrogens. That E 2 is the masculinizing hormone in rodents was codified by the aromatization hypothesis (DonCarlos et al., 1995, Roselli and Klosterman, 1998 and Vinader-Caerols et al., 2000), which reconciled the divergent observations that exogenous administration of E 2 was as or more effective than T at masculinizing behavior, that the critical brain regions expressed ERs, and that the synthetic enzyme, E 2 synthase or aromatase (P450 aro), the enzyme responsible for the conversion of T to E 2, was highly active in the neonatal brain (McCarthy, 2008). An additional critical piece of the puzzle was the determination that the steroid-binding globulin, α-fetoprotein, preferentially binds estrogens in rodents, thereby allowing T to freely enter cells where it is converted to E 2 (Soloff et al., 1976 and MacLusky et al., 1979). Only decades later did it become apparent that androgens mediate sexual differentiation of the primate ( Macaca mulatta) brain (Wallen, 2005), presumably including humans, suggesting that guinea pigs may be a far better model than rodents for understanding the biological basis of gender biases in humans. Determining whether this is true depends on whether the cellular mechanisms establishing sex differences in the brain are common in primates and rodents. Antecedent to that is determining the cellular mechanisms of sexual differentiation in rodents. Understanding the cellular mechanisms of organizing effects of hormones first requires a complete characterization of the behavioral and neuroanatomical phenomena that are the subjects of organization.

3.4. Masculinization, Feminization, and Defeminization

Sexual differentiation of the brain is a developmental process whereby gonadal steroids act during a perinatal sensitive period on the undifferentiated neural substrate to permanently alter it so that ultimately brain phenotype will match gonadal phenotype. Recent studies of potential contributions of chromosomal sex to brain phenotype have further confirmed the supremacy of steroids when it comes to reproductively related endpoints (Arnold, Rissman, & De Vries, 2003; Arnold et al., 2004). Central to the process of differentiation is the conversion of testicular-derived T to E 2 within neurons. Most sexually dimorphic areas of the brain contain substantial levels of both P450 aro (CYP19) and high densities of ER (MacLusky et al., 1994, DonCarlos et al., 1995, DonCarlos, 1996, Pfaff and Keiner, 1973, Roselli et al., 1984 and Roselli and Resko, 1993). Maternal estrogens are sequestered in the peripheral circulation of the fetus by α-fetoprotein, which has very little affinity for androgens, allowing the testicular T to reach and influence fetal target tissues, including the brain. Treatment with T, but not the nonaromatizable androgen DHT, mimics many of the effects of estrogens, and normal masculinization of the brain is prevented subsequent to disruption of P450 aro during the sensitive period (McEwen et al., 1977, Vreeburg et al., 1977, Mong et al., 1999 and Amateau and McCarthy, 2002a; 2002b; Bakker, Honda, & Balthazart, 2002; Bakker, Honda, Harada, & Balthazart, 2002). This basic principle that the male brain is masculinized by local conversion of E 2 is elucidated by the aromatization hypothesis (Naftolin et al., 1975) and expanded on by others (McEwen et al., 1977 and Vreeburg et al., 1977). More recently, advances have been made regarding the mechanisms by which E 2 exerts a masculinizing influence on the brain.

Studies attempting to elucidate the mechanistic basis of sex differences in the brain benefit from a robust and reliable endpoint: sexual behavior in the albino laboratory rat. Whether an adult rat responds to sexual advances from another adult rat with a male response (mounting, intromitting, and ejaculating) or a female response (lordosis) is entirely dependent on two variables: (1) the hormonal milieu during a perinatal sensitive window that must match (2) the hormonal milieu of the adult. For male behavior, the neonate must be exposed to a critical level of neuronal E 2 matched with a critical level of circulating T as an adult. For female behavior, the neonate must not be exposed to a critical level of neuronal E 2 matched with a threshold level of E 2 sequential with P 4 in the adult. Male sexual behavior in the laboratory rat is opportunistic and readily expressed whenever a receptive female is present. Female sexual behavior is physiologically constrained to be expressed only in proximity to ovulation. A fascinating but mechanistically unexplained distinction in male and female sexual behavior is the impact of experience. Males improve with practice and if T is removed (via castration), will continue to exhibit high levels of copulatory behavior that only gradually extinguishes over a period of months. Females, by contrast, get it right the first time and every time and will only exhibit lordosis under the proper hormonal umbrella. If steroids are eliminated, so is the behavior. In terms of ultimate causation, the adaptive basis for the distinction is obvious: there is no benefit but is possible cost to females mating outside the window of opportunity for conception, whereas males maximize fitness by never missing an opportunity to share the wealth. Proximately, however, this dichotomy in the nature of the plasticity attendant to both behaviors is suggestive of distinct and separate neuronal circuitries, be they physically or merely functionally so.

So how does the brain mediate these distinct strategies in males and females? A useful framework for investigating mechanistic questions of sexual differentiation is the operationally defined and distinct processes of masculinization, feminization, and defeminization. Masculinization refers to an active developmental process initiated by gonadal steroids during the perinatal sensitive period followed by expression of normal male copulatory behavior in adulthood. Feminization is essentially what happens in the absence of masculinization, meaning that it is the default pathway leading to expression of lordosis under the proper hormonal conditions in adulthood. Defeminization is distinct from but normally occurs in tandem with masculinization and refers to the process whereby the ability to express female sexual behavior is lost. Defeminization appears to be exclusively regulated by E 2, whereas masculinization involves both estrogens and androgens (Whalen and Edwards, 1967, Vreeburg et al., 1977 and Auger et al., 2002).

3.5. Brain vs. Behavior

There are multiple sexually dimorphic brain regions as a result of the organizational effects of gonadal hormones. Regional volumetric sex differences have been found in the preoptic area (POA), spinal nucleus of the bulbocavernosus (SNB), bed nucleus of the stria terminalis (BNST), anteroventral periventricular nucleus (AVPV), and the medial amygdala (mAMG) of the laboratory rat. The sexually dimorphic nucleus of the POA (SDN-POA) (Gorski, Gordon, Shryne, & Southam, 1978), the SNB (Nordeen et al., 1985 and Freeman et al., 1996), the BNST (Guillamon et al., 1988 and Hines et al., 1992), and the mAMG (Mizukami et al., 1983 and Cooke et al., 1999) are larger in males than in females. The volume of the sex difference in the SDN-POA is so robust that it is visible to the naked eye, being approximately 3–5 times larger in males than in females (Gorski et al., 1978). In contrast, the AVPV is larger in females than males (Simerly et al., 1985, Murakami and Arai, 1989 and Sumida et al., 1993). Gonadal hormones establish these sex differences in volume by affecting the number of cells in each brain region. While the direction of the sex difference varies by brain region, in all cases the mechanism by which the volumetric difference is achieved is the same: differential cell death. The SNB, SDN-POA, and BNST are larger in males than females because gonadal hormones promote cell survival. Males and females begin with the same number of neurons, but many of these die in females due to the lack of trophic support from T and/or E 2 at a critical time in development (Gorski, Gordon, Shryne, & Southam, 1978, Nordeen et al., 1985, Guillamon et al., 1988, Freeman et al., 1996 and Forger et al., 2004). Conversely, in the AVPV, E 2 promotes cell death, resulting in a smaller volume nucleus in males (Murakami and Arai, 1989, Simerly, 1989 and Sumida et al., 1993). There are also sex differences in the connectivity between brain regions. Specifically, the projection from the BNST to the AVPV contains a greater number of galanin and GABAergic fibers in males than females (Gu and Simerly, 1997, Ibanez et al., 2001, Polston and Simerly, 2003 and Polston et al., 2004). In contrast, the number of substance P fibers projecting from the BNST to the POA is greater in females than males (Polston and Simerly, 2003 and Polston et al., 2004). Importantly, these projections form a functional circuit controlling sexually dimorphic GTH secretion (Gu and Simerly, 1997, Herbison, 1998 and De Vries and Simerly, 2002) and are organized by prenatal exposure to gonadal hormones (Simerly, 2002).

In addition to volumetric and cell number differences induced by early hormone exposure in the brain, there are also robust sex differences in synaptic patterning.

Perinatal exposure to T exerts region-specific effects on dendritic spines and branching in the developing hypothalamus. The arcuate nucleus (ARC) of males contains twice the number of axosomatic and half the number of axodendritic spine synapses as that of females (Matsumoto & Arai, 1983). Early T exposure decreases the dendritic spine density and axodendritic spine synapses in the ARC (Mong et al., 1996, Mong and McCarthy, 1999 and Mong et al., 2001). Perinatal E 2 exposure has the opposite effect in the POA. Males have two to three times more dendritic spines than females, and treating neonatal females with E 2 will produce the male pattern of dendritic spines and permit the expression of T-induced male sexual behavior in adulthood (Amateau & McCarthy, 2002a; 2004). In males, the ventromedial nucleus (VMN) is only slightly larger than in females but contains more than three times as many dendritic spine and shaft synapses as that of females (Matsumoto & Arai, 1983). These sex differences in synaptic patterning are detected as early as postnatal day three and are still present at postnatal day 100 (Matsumoto and Arai, 1986 and Pozzo-Miller and Aoki, 1991), which is consistent with the organizational effects of gonadal hormones. The nature of the sex difference in the synaptic pattern is fundamentally different in the VMN vs. the POA and ARC. In the latter two nuclei there are greater numbers of dendritic spine synapses per unit dendrite, meaning that the density of synapses has increased. In the VMN, males have overall more dendritic spine synapses because the dendrites are longer and branch more frequently, but the density of spine synapses per unit dendrite is the same in males and females (Mong et al., 2001 and Todd et al., 2007). The way in which this difference in synaptic pattern impacts on neuronal function is entirely unknown.

Neonatal exposure to gonadal hormones also affects cell morphology of astrocytes in sexually dimorphic brain regions. Testosterone or its metabolized product, E 2, increases the complexity of astrocytes in the ARC toward a more stellate morphology with more primary processes and branches (Mong et al., 1999 and Mong and McCarthy, 1999). There is no evidence of ERs in astrocytes of the ARC (Mong & McCarthy, 1999). Instead, E 2 indirectly promotes astrocyte differentiation by enhancing the synthesis and release of GABA in neighboring neurons (Mong, Nunez, & McCarthy, 2002). These changes in astrocyte complexity are correlated with a reduction in the formation of dendritic spine synapses in males (Mong et al., 2001). Males also have more complex astrocytes in the POA than females (Amateau & McCarthy, 2002b); however, enhanced astrocyte differentiation in the POA is correlated with greater formation of dendritic spine synapses in males, the opposite of that seen in the ARC. The question of how hormonal modulation of astrocyte complexity modulates neuronal complexity is an important area for future study.

The GTHs provide an excellent example of neurochemical sex differences that are a result of organizational effects of gonadal hormones in the brain. Neurons expressing gonadotropin-releasing hormone (GnRH) are located in the POA, BNST, and ARC of the hypothalamus of many species (e.g., there are no GnRH neurons in the ARC of rats and mice). Gonadotropin-releasing hormone is the regulator of the pulsatile release of luteinizing hormone (LH) from the anterior pituitary in both males and females and there are no obvious sex differences in the number, distribution, or morphology of GnRH neurons. The GTHs regulate reproduction in females through a massive surge in LH at the appropriate time in the cycle to induce ovulation. Estradiol regulates the synchronized firing of GnRH neurons to induce the LH surge via a positive feedback mechanism in females but not males (Becu-Villalobos & Libertun, 1995). The masculinized brain does not respond to E 2 with positive feedback and there is therefore no LH surge (Diaz et al., 1995). A proposed mechanism for this sex difference in the LH surge is enhanced excitatory glutamatergic input onto GnRH neurons in females (Becu-Villalobos & Libertun, 1995), and this input appears to originate in the AVPV, which is larger in females than males (Gu & Simerly, 1997).

While anatomical sex differences are clearly important, it is also possible that parameters less easily measured but equally important, such as excitability, are different in males and females. In addition to electrophysiological measures, the activity of neurons can be assessed indirectly via markers of cell activity such as the immediate early gene, c-fos, or the phosphorylated form of CREB, pCREB. Males have enhanced levels of c-fos and pCREB in the immature hypothalamus during early postnatal development (Auger et al., 2001 and Olesen and Auger, 2005) and the sex difference in pCREB is the result of neonatal exposure to T (Auger et al., 2001). Estradiol enhances the depolarizing action of GABA in the developing hypothalamus by increasing the magnitude of depolarization-induced calcium influx and extending the developmental duration of depolarizing GABA. This delays the onset of inhibitory GABA and provides the brain with critical excitation during development (Nunez et al., 2005 and Perrot-Sinal et al., 2007). The functional significance of enhanced and extended depolarizing GABA early in development is an important topic for future research.

Aggressive behavior in adulthood is linked to concentrations of androgens during sensitive periods of development. For example, in-utero exposure to T induces organizational effects in the brain that are later expressed in terms of aggressive behavior in male and female hyenas (Glickman et al., 1992 and Glickman et al., 2005). These same females also express male-like characteristics during play-fighting and other sexually dimorphic behaviors (Glickman et al., 1992; 2005). Those females exposed to the greatest amounts of T in utero are likely to be the most aggressive (Glickman et al., 1992; 2005). Although T declines in females more than in males upon reaching maturity, the hormone’s early impact means a lifelong tendency toward aggressive behavior (Glickman et al., 1992; 2005). The underlying neural circuits for aggressive behavior in hyenas are unclear; however, the brain regions associated with aggression in rodents include the medial POA and anterior hypothalamic areas, the medial BNST, and the mAMG (Albert et al., 1992 and Ferris et al., 1997), all regions that exhibit a high degree of sexual dimorphism.

3.6. Challenges to the Dogma

A scientific principle is only called a dogma when it is being challenged. The organizational/activational hypothesis as dogma has proved highly effective for providing form and substance to various challenges, which range from the questioning of the generalities of specific details to paradigm shifts in the way we view the origin and meaning of sex differences in behavior. Many challenges are really just modifications or qualifications to the strength of the predictions made by the dogma. For instance, while the organizational/activational hypothesis infers that early hormone effects are permanent, it has been known since prior to the articulation of the hypothesis that there is some degree of plasticity in adult sexual behavior. Treatment of females with high levels of androgens for an extended period of time will induce some degree of maletypic behavior (Sodersten, 1984), and the converse is also true in males (Olster & Blaustein, 1988), but the sensitivity to heterotypic hormones (i.e., T in females) is much less than for homotypic hormones (i.e., E 2 in females). These observations do not negate the dogma; they merely modify it to not be so absolute. Two more substantive challenges are the importance of genetics, and the notion that some organizational effects are designed to make the sexes more similar as opposed to different. These challenges also do not negate the central tenets of the organizational/activational hypothesis but they do provide a richer and more nuanced view of sex differences in behavior.

Decades of study on hormonal induction of sex differences in the brain have established the profound influences of these signaling molecules on brain development, but these studies have been confounded in that all mammalian male neurons have an XY genotype and all female neurons have an XX genotype. This simple fact had been largely ignored, or, if acknowledged, set aside as unaddressable in part because we did not know the genetic basis of testis development. The discovery of SRY, the sex-determining region of the Y chromosome, in the late 1990s provided the tools needed for separation of genetic sex from gonadal sex. This single gene initiates an expression cascade that leads to the production of a testis (Lovell-Badge, 1992). If SRY is absent or the SRY protein dysfunctional, the gonadal anlage will become an ovary. This discovery allowed for generation of mice that have the sry gene on an autosome, thereby allowing for genetic crosses that produce an XX animal with testes or an XY animal with ovaries. When combined with XX animals with an ovary and XY animals with a testis, these constitute what has come to be known as the four core genotypes (Arnold et al., 2004). The comparison of an XX female with an XY female allows for detection of genetic effects while comparison of an XX female with an XX male allows for detection of hormonal effects (Figure 1.3). Initial studies of this model unambiguously supported the dogma that early hormonal effects determine adult reproductive physiology and behavior (De Vries et al., 2002). However, when investigation strayed beyond those behaviors directly relevant to reproduction, a number of interesting examples of genetic sex being a pertinent variable emerged. Behavioral endpoints influenced by genetic sex include aggression (Gatewood et al., 2006) and habit formation (Quinn, Hitchcott, Umeda, Arnold, & Taylor, 2007), and neuroanatomical endpoints include vasopressin innervation of the forebrain (De Vries et al., 2002) and the number of dopamine-expressing neurons in the brainstem (Dewing et al., 2006). This is not to say that hormones are without effect on these endpoints, but rather that hormones cannot explain all the variance. Several critical questions remain. What are the genes on the X or Y chromosome that contribute to sex differences in brain and behavior? How do hormones and genetics interact to produce the final phenotype? What is the relative importance of genes vs. gonads for various endpoints? With the advent of the four core genotypes, progress can be made on this interesting front.

When considering the biological basis of sex differences in the brain, the emphasis is, quite naturally, on sex differences. However, there are times when this emphasis appears misplaced or may even distort the functional interpretation of observed differences. Arginine vasopressin (AVP) has emerged as a critical regulator of complex social behaviors, in particular those that involve lasting recognition between two individuals; i.e., pair bonding and parenting (Lim & Young, 2006). The strength of a pair bond and the degree of parenting behavior varies enormously between males and females and across species. Many mammalian species show little pair bonding, or it may be essential for the female to recognize and remember former mates, whereas it is inconsequential for the male. Likewise, in many mammalian species the care of offspring is entirely in the realm of the female, with only modest input from the male, at best. Voles have proven particularly useful in comparing and contrasting the various mating systems as one species will exhibit strong pair bonding and parenting by both sexes, whereas another closely related species exhibits polygamy and unisex parenting (Carter et al., 1992 and Insel and Hulihan, 1995). The patterning of vasopressin innervation is highly sexually dimorphic in most rodents, and has been found to vary between species of vole as a function of reproductive strategy (De Vries & Miller, 1998; Lonstein, Rood, & De Vries, 2005; Lim & Young, 2006). Clarity was brought to this confusing collection of observations by the theoretical advance that, in monogamous species showing biparental behavior, one of the organizational hormonal effects is to induce pair bonding capacity in males (A. DeVries, M. DeVries, Taymans, & Carter, 1996). Thus, in this case, males are more like females in order to maximize their own reproductive success.

A conceptually similar interpretation has been proposed for sex differences in the hippocampus. Reports of hormonal effects on adult learning and memory extend back as far as 1926 (Ball, 1926) and remain an intensely studied topic to this day. However, the concept of hormonal modulation of learning and memory is frequently equated with sexual differentiation of learning and memory (McCarthy & Konkle, 2005). Emphasis is on the hippocampus since this is a critical brain region for spatial learning and animal models cannot be exploited to examine the two major areas of learning of interest in humans: verbal and mathematical learning. Conclusions regarding sex differences in spatial learning, which rely predominantly on the Morris water maze, have been challenged of late by the assertion that what researchers are really detecting is a sex difference in learning strategy and/or stress reactivity during the testing paradigm (Johnston and File, 1991, Perrot-Sinal et al., 1996, Barros and Ferigolo, 1998, Kanit et al., 2000, Bielajew et al., 2003, Beiko et al., 2004 and Jonasson, 2005). Similar arguments have been made for sex differences in learning in humans (Spelke, 2005). This raises the general question: is the hippocampus sexually dimorphic and, if so, is it subject to sexual differentiation along the lines of the organizational/activational hypothesis? The best answer at the moment is that it is unclear. The central tenet of the organizational/activational hypothesis is that steroid levels are elevated in one sex or the other during the critical period for sexual differentiation. This has been established clearly for reproductively relevant brain regions, such as the POA and hypothalamus, but does not hold true for the cortex or hippocampus (Amateau, Alt, Stamps, & McCarthy, 2004). When tissue levels of E 2 are quantified in these telencephalic brain regions, the levels are the same in male and female rats. Converging evidence in the adult is consistent with a view that neurons or astrocytes of some brain regions, in particular the hippocampus, are capable of de novo steroidogenesis of E 2 from cholesterol (Prange-Kiel et al., 2003 and Hojo et al., 2004). This also may be true in the developing hippocampus, with the female brain synthesizing E 2 locally in order to equalize levels with those of the male, who generates E 2 from peripherally derived T (Nunez & McCarthy, 2008). This provocative hypothesis has not been rigorously tested but, if upheld, would be another example of one sex adapting strategies to be similar in physiology to the other.

4. Advances in Mechanisms

4.1. Cell Birth and Cell Death

Perinatal exposure to gonadal hormones can permanently organize the volume of a brain region by influencing cell genesis, cell survival, or cell migration. To date, there is limited evidence suggesting that neonatal exposure to gonadal hormones promotes cell proliferation in the brain (Zhang, Konkle, Zup, & McCarthy, 2008). There