Positional Effects and the Mammalian Uterus
Sex Change in Stoplight Parrotfish
Alternative Male Phenotypes in Plainfin Midshipman Fish
Testosterone and trade-offs between Singing, Polygyny and Parent Care in Male Birds
The goal of evolutionary behavioral analysis is to understand the source of variation in behavior within a species. Such a search must include a discussion of proximate mechanism. Proximate mechanisms begin with an understanding of how genes get translated into proteins, how such proteins build organs that produce hormones, how proteins and hormones get assembled into regulatory networks, and how such networks govern the physiological and developmental events that result in behaviors. It is at this point that the system can no longer be described in terms of genetics per se, but a complete explanation of proximate mechanisms must resort to epigenetic explanations of the development of behavior.
epi = above, genetics = the genotype
No developmental process is better characterized than the epigenetic networks that lead to sex determination and differentiation of vertebrates. The genes that initiate these epigenetic cascades are quite well characterized, as are the developmental and endocrinological machinery that serve to regulate the developing phenotype. Moreover, classic examples of neurodevelopment and behavior are triggered by the simple switches underlying sex determination:
There are many possible sex determination mechanisms in the animal kingdom and we will consider two that are found in the vertebrates:
Processes of sex determination have an organizing effect on the development of an organism. This organizing effect builds a phenotype that can respond to later acting activational effects of the sexual differentiation cascade. For example, testosterone has an organizing effect on early embryonic development that initiates the basic female and male anatomies. However, full expression of sex differences requires that testosterone also activate many aspects of the phenotype during the process of maturation.
In addition to these early and late acting effects of steroids, the maternal environment or reproductive decisions by the mother can also have a dramatic effect on the developing phenotype that bias the kind of behaviors seen during the rest of the offspring's life. Finally, some animals are not restricted to a single sex for life and they can undergo a sex change that is triggered by the environment relatively late in life. Sex determination and the behavioral phenotypes that result are due to genetic, epigenetic, and genotype-by-environment interactions.
The basic mammalian sex determination mechanism arises from the possesion of a small but important gene located on the Y-chromosome. The Y-chromosome has very few functional genes and nearly all important genes for development of early embryonic form are spread among the remaining somatic chromosome or on the X chromosome. There are indeed hundreds of genes that are necessary to construct the basic reproductive system of vertebrates during early development, but the trigger to take on a female or a male form arises from testes determining factor, the tdf gene. For years, biologists speculated that such a factor must reside on the Y chromosome and it has now been isolated in at least a few vertebrates.
The presence (XY) or absence (XX) of this gene takes the embryo down two alternative pathways -- male versus female. If the gene is absent, then the organism develops into a female with ovaries and a system of endocrine glands that regulate female reproduction and behaviors. If the gene is present, the organism develops testes rather than ovaries, and the testes in turn produce testosterone which further organizes the neuro-endocrine system.
Nearly all of the sex differences in neural development and resulting behaviors are thought to be triggered by the action of steroids (Goy and McEwen 1980).
For example, the effects of estrogen derived from aromatases action on testosterone stimulates the growth of brain regions directly linked to song production in male birds (see below). The general action of these hormones is to masculinize or defeminize regions of the brain necessary for male functions.
Other "male functions" ascribed to the organizational effects of testosterone include elaboration of the hippocampus which is related to spatial learning. Presumably the spatial learning is necessary for developing and holding a large territory. Because most male birds are territorial and females are not as territorial, males should have more processing capability in this regard.
It is not invariably the case that only males sing and only males need elaborate spatial maps. Many song birds have females with fairly elaborate song repetoires. In such species one does not find the same kind of sex differences in neuroanatomy as in species with male-only songs. Presumably, the similarities between males and females in these species result from alterations in the basic vertebrate program of organizational effects.
In addition, in some species, like the brown headed cowbird, females develop a larger hippocampus than males and this seems to be related to the spatial demands placed upon the females. Cowbirds are parasitic on the parental efforts of other bird species. They lay their eggs in other birds nests and let the host raise their offspring. Males have no need for the spatial maps that females develop during the searches for host nests. Thus, females have evolved a more elaborate hippocampus. Again, species specific sex differences in neural development presumably result from some alteration in the basic program of organizational effects which has yet to be described for the cowbirds.
In summary, the early action of testosterone or its metabolites serves to organize the brain so that it has the proper neural circuitry for later acting activational events.
With the proper neural circuitry in place, the approapriate muscle development, the correct organ systems built (e.g., sexual organs), the body is ready for the activational events that typically begin with the first breeding attempt. It is at this time that the latent sexual tendencies are awakened in the juvenile. The triggers for maturation can be genetic, or environmental. Whatever the cause or trigger, sexual maturation, requires hormones to activate physiological and neurophysiological systems. In the case of male and female vertebrates, a part of the brain called the pre-optic area begins producing a neurochemical called gonadotropin-releasing hormone (GnRH).
GnRH acts on the cells in the pituitary and the pituitary begins secreting gonadotropins. Gonadotropins act on the gonads and stimulate them to produce steroids: testosterone in males, and estrogens in females. This second wave of steroids now serve to refine the developing sexual phenotypes. Recall that the first wave of steroids during embryongenesis organized the animal and triggered development of the primary sex characters: male versus female organ systems developed. In this second wave of activational effects the secondary sexual characteristics (every other sex difference) begin to develop under the influence of these gonadal secretions.
Maternal effects are broadly defined as the impact that the mother and her phenotype has on the phenotype of the offspring. This is quite distinct from the genetic effects that a mother has on her offspring's phenotype. Lets look at the definition a little more slowly.
Just to be precise, a maternal effect includes all those attributes arising from mom that are not due to direct genetic inheritance.
Such maternal effects can be a very important source of the variation among individuals in fitness. In mammals, the environment of the womb, can have a dramatic effect on offspring behaviors, as can the quality of nursing which is the hallmark of the mammalian condition.
A female parent can impart non-genetic changes to her offspring that make them better able to cope with their environment. Rather than encode all information on how to build the offspring in the genes, placing some control into mom's descision making machinery may produce greater fitness (e.g., better progeny). The female may be able to predict the environment of the offspring. In this case she should impart some of this information as a jump start to give her offspring an edge. However, this information should not necessarily be a permanent change that genetics might entail. The offspring in its own time might need to do a similar service for its offspring and a genetic effect would last too long. What if the offspring had to do a different thing? The solution is to build a system that allows for plasticity. Below I describe a few case studies where such plasticity is imporant. The cues that the female has are easy to identify. She acts on these environmental cues and alters her offspring accordingly.
Rats and mice produce multiple offpspring in a litter, and these offspring are strung out along each of the two horns of the bicornate uterus (two - horned uterus) like a strand of pearls. All embryos are attached to the wall by a placenta, and compounds are free to circulate between the offspring and mom, and between mom and the offspring through the blood. In additon, compounds move into the amniotic fluid between embryos and are taken up by adjacent embryos. Steroids move between animals quite freely. The dose of testosterone that a male embryo produces during the early organization of the sexual reproductive system is not transmitted full strength to the female that may be next to him, but a certain amount of the hormone does reach the female offspring. Because steroids are very potent hormones, even in fairly small doses, the amount that a female receives from her prenatal brother is enough to alter her behavioral phenotype.
A female who is between two males in the uterus:
M-F-M = 2M female
has a different phenotype compared to a female that is between two females:
F-F-F = 0M female.
The most obvious external manifestation of this early androgen exposure is seen in the distance between the anus and genitals, the anogenital distance, which for M-F-M females is larger than F-F-F females. Males normally have a long anogenital distance.
The most interesting effects are seen on behavior. Female rats exposed to male androgens exhibit more mounting behavior. Female mice are more aggressive.
The effects on aggression can even be seen in 2M males (e.g., M-M-M) relative to 0M males (e.g., F-M-F). If these animals are castrated after this early exposure and then given T supplements later in life during the activational period for T, the 2M males are more aggressive than 0M males. The castration is used to remove any differences in feedback that might occur between a 2M male's gonads and brain during adult life compared to a 0M's male. By giving both types of males the same levels of exogenous hormone at maturity, the researchers ensure that the responses they see in adult animals are due to changes arising from the organizational period only (when a 2M male was between two males and he received a higher dose of T than did a 0M male that was between two females). They do not want to confoudn the comparison with differences in T production by 2M or 0M males.
In addition, the 0M males received higher doses of estrogen and this has an effect on its behaviors. What might those effects be?
Can such effects be viewed in an adaptive context?
Under conditions of crowding or stress, it might benefit a female to produce more aggressive females or males. If a female mouse (or rat) could manipulate the intrauterine position of her offspring she could impart a one generational effect on her offspring that might be advantageous for their survival or reproductive success.
references:
vom Saal, F. S., W. M. Grant, C. W. McMullen, and K. S. Laves. 1983. High fetal estrogen concentrations: correlations with increased sexual activity and decreased aggression in male mice. Science 220: 1306-1309.
Female canaries have been shown to deposit testosterone into yolk, and the amount of T that the females deposit in eggs varies with the order of laying. The hormone testosterone is lipophilic, thus it is readily put into the lipoprotein matrix during egg production in females.
The amount of testosterone put into eggs was independent of the sex of the offspring.
Such hormones have a dramatic effect on offspring dominance or social rank. Schwabl scored social rank by measuring:
Both male and female offspring experienced elevated social rank if they received an extra dose of testosterone from the mother.
reference:
Schwabl, H. Yolk is a source of maternal testosterone for developing birds. Proc. Natl. Acad. Sci. 90: 11446-11450.
Many turtles, lizards, crocodilians, and a few snakes have a form of sex determination that depends on the incubation temperature of eggs. Given that the female is responsible for the location that she buries her eggs, she can control the sex ratio of her clutch. For example, some turtles have eggs that turn into males when they are incubated at low temperatures, and females when they are incubated at high temperatures. There is also a temperature at which the eggs develop into males and females with a 50:50 ratio. This temperature is referred to as the threshold temperature. Other species of turtles produce males at low temperatures, females at intermediate temperatures, and males again at the highest temperature.
Fred Janzen produced turtle eggs and hatchlings that were incubated at:
He then released these turtles into the wild and assayed their survival. Those, hatchlings that were produced at the all male temperature or the all female temperature had higher survival than the males and females produced at the critical temperature.
If a females lay eggs in a nest that will be largely exposed to the critical temperature, those offspring will be at a severe disadvantage compared to offspring at either the all male or all female temperature. He speculates that the offspring right at the critical temperature may be incurring developmental screw ups because they are on the knife edge of becoming male or female. In contrast those that are at definitive male and female temperatures have a more "stable development." Thus, females should produce either an all male clutch or an all female clutch.
In another species of turtle (actually a box turtle), Wilhem Roosenburg has found that females that lay small eggs should lay in places were those eggs will develop into males. Small eggs do not reach a very large size at maturity, and male turtles do not have to be all that big -- even a small male can fertilize a female. There isn't a premium on male size that might arise for male-male contests. In contrast, fitness of female offspring at maturity is directly dependent on how large the turtle is at maturity -- bigger females produce more offspring. If a mother is going to produce a clutch with very large eggs, then she should lay those eggs in a warm place where they will develop into females offspring. Roosenburg speculates that a females nest site selection should be plastic depending on the size of a females eggs. If she has big eggs, perhaps because it was a good year for her, then she should produce a female biased clutch. If she has small eggs, perhaps because it was bad year, then she should produce a male biased clutch.
Sexual differentiation and the acquistion of secondary sexual characters is a two step process in which the embryo is organized by the effects of steroids, and then at maturity, the organism's development of secondary sexual characters are activated. I will describe three case studies that illustrate the development of alternative males, development of male bird song, and development of mammalian parental care. The example of the stoplight parrotfish sex change or the alternative male phenotypes in the midshipman is useful because it illustrates that distinctions between maleness and femaleness are often quite fuzzy. Development of bird song in males is illustrative of the activational effects on neural circuitry. Finally, the example of mammalian maternal care illustrates that some as complicated as female behaviors can be influenced by a single gene that affects the nervous system that seems to control a suite of maternal behaviors.
The stoplight parrotfish (Sparisoma viride) undergoes a sex change late in life. All individuals start out life as females and then later transform in to males. This species is referred to as a protogynous hermaphrodite. The twist in this little story is that some females transform into terminal phase territorial males. Other females transform into sneakers first, then they transform into terminal phase males.
When a female changes into a male (any kind), estradiol levels decrease and levels of 11-ketotestosterone increase. Females that have transformed into terminal phase males maintain very high levels of 11-ketotestosterone, whereas females that have transformed in sneakers maintain moderate levels of testosterone, and estradiol! Indeed, injections of 11-ketotestosterone causes females to transform into males by changing gonads and color.
Male Plainfin Midshipman Fish come in two alternative male phenotypes (Bass, 1996):
The difference in these secondary sexual characters is controlled by levels of testosterone:
Bass and his colleagues have found that a suite of morphological and neural changes are associated with the alternative males and seem to be regulated by either levels of testosterone:
Type I males develop:
While the sonic muscle is only found in Type I males, the neural circuits are found in both Type I males, Type II males, and females. What differs among the three is the pacemaker-motorneuron circuit that fires at a frequency that is 15 to 20 percent higher in Type I males compared to Type II males or females. The nerve cells and cell bodies of this circuit are also up to three times larger in Type I males. This song is necessary to attract females.
reference:
Bass, A. H. 1996. Shaping Brain Sexuality. American Scientist 84: 352-363.
Sex differences in passerine birds are striking in that males typically sing and females usually do not (though see exceptions noted above). Testosterone again is implicated in this fundamental difference in behavior which raises the following questions. How are the neural circuits shaped during the development of secondary sex characters?
In considering the effects of testosterone it is important to note that testosterone per se is not the potent androgen, rather testosterone gets metabolized into estrogen by the enzyme aromatase.
Estrogen treatment of females in their youth can masculinize a them and cause females to sing song. Males get their high dose from testosterone that is aromatized to estrogen. The concentrations of estrogen in the brain can be very high. It looks like the critical date for zebra finches is 3 to 10 days after hatching.
Specific regions of the brain have very high levels of aromtase and corresponding high levels of estrogen. In particular, a region adjacent to the higher vocal center (HVC) has high concentrations of estrogen. It appears that high levels of T and then E, trigger growth and proliferation of neurons in several specific areas that have been identified in the song circuit of birds.
reference:
Arnold, A. P. 1994. Critical events in the development of bird song: What can neurobiology contribute to the study of the evolution of behavior. In Behavioral Mechanisms in Evolutionary Ecology. edited by L. Real. University of Chicago Press, Chicago, pp 219-237.
Testosterone is clearly implicated in the development of male song in juvenile birds. In addition, levels of testosterone in adult birds are also correlated with levels of parental care and the tendancy to monogamous or polygynous. Species of birds that can maintain high levels of testosterone during the entire reproductive season also tend to be quite polygynous. By maintaining high levels of T, these birds keep singing and courting additional females. These males are not the best parents however, as they usually leave the female to rear the young (Wingfield et al. 1990).
To investigate these correlational pattern, Ketterson and her colleagues (reviewed in Ketterson and Nolan 1994) picked a largely monogamous species, the dark-eyed Junco, in which males and females provide parental care. They implanted half of the males with T, and the other half with sham implants. True to form, the T-implanted males sang more, ranged farther, and tried to court more females, all at the expense of parental care. The females of T-implanted males were left to carry the burden of care that normally is split between the male and the female. In the long run, such decreased care on the part of the male might be expected to cause the female to work harder and perhaps experience greater costs of reproduction.
Ketterson, E. and V. Nolan. 1994. Hormones and life histories: an integrative approach. In Behavioral Mechanisms in Evolutionary Ecology. edited by L. Real. University of Chicago Press, Chicago, pp 337-353.
Wingfield, J. C., R. E. Hegner, A. M. Dufty, Jr., and G. F. Ball. 1990. The "Challenge hypothesis": Theoretical implications for patterns of testosterone secretion, mating systems, and breeding strategies. Amer. Natur. 136:829-846.
The hormones involved in the development of maternal behaviors in mammals are linked to the hormone changes in both progesterone and estrogen. Progesterone levels are typically quite high during pregnancy, but as the date of birth approaches Progesterone levels begin to fall and Estrogen levels begin to rise. It is the synergistic action of both Progesterone and Estrogen at birth that seems to trigger maternal behaviors in female mice. Estrogen also acts synergistcally with oxytocin to stimulate milk production.
High levels of estrogen and low levels of progesterone triggers nest building increases 4-6 days prior to birth.
Simply presenting pups to virgin females or to males can induce the above maternal behaviors.
New evidence suggests a very simple neural pathway may be involved. Brown and her colleagues have succeeded in creating a mutation in a single gene, fosB, that appears to extinguish nurturing behaviors in female mice. The hypothalmus in female mice has been shown to be critical for nurturing behavior in lesion experiments. It appears that the gene products of fosB are expressed in the preoptic area, which is located in the hypothalmus.
Fos B deficient moms do not show the following nurturing behaviors:
FosB moms have normal expression of Estrogen/Progesterone, and normal olfactory abilities. They just do not nurture their young.
Brown, J. R., H. Ye, R. T. Bronson, P. Dikkes, M. E. Greenberg. 1996. A defect in nurturing in mice lacking the immediate early gene fosB. Cell 86:297-309.