THE INTRAUTERINE POSITION EFFECT: A NOVEL SOURCE OF NON-GENOMIC VARIATION IN LITTER BEARING MAMMALS
Hillary V. Jay
Barnard College of Columbia University
New York, NY
My review of previous studies investigating the intrauterine position effect suggests that variation in sexually dimorphic traits between male and female rodents may be related to the position of these rodents during gestation. Because of the unique bicornate, or two-horned, architecture of the maternal rodent’s womb, developing fetuses may be exposed to varying levels of sex hormones, most importantly testosterone, depending on the sexes of the fetuses immediately neighboring it on either side. Fetuses developing between two males (2M) may exhibit masculinized morphological, physiological and behavioral traits throughout adulthood, including a higher sensitivity to testosterone and an effect on birth ratios. Likewise, fetuses developing between two females (0M) may exhibit more feminized morphological, physiological and behavioral traits throughout adulthood. In mice, the intrauterine position effect can be explained by the diffusion of testosterone from the amniotic sacs of male fetuses to those of adjacent fetuses. In rats, the effect is better explained by the maternal rat’s one way caudal (cervix) to distal (ovaries) blood flow. Given the ubiquitous use of lab rodents in modern scientific studies, it is important to fully understand and control for distinctions introduced by the intrauterine position effect. This newfound source of variation may point to a shortcoming in our current sexual classification system, suggesting that more specific categories be designed before any studies can be considered “controlled.”
Introduction—————————————————————————————————In litter-bearing mammals, hormone levels of individual pups are significantly affected by the sex of adjacently developing fetuses, and these altered prenatal hormones can have significant effects later in life. This is known as the intrauterine position effect. The intrauterine position effect should be considered as an important source of non-genomic variation in the rodent population, one with broad ramifications, morphologically, physiologically and behaviorally. Given the widespread use of lab rodents in the modern scientific process, it is critical that we acknowledge and attempt to control for this type of variation, which has the potential to alter interpretations of data for a variety of fields. In 2012, scientists in the United Kingdom employed the use of over 3 million lab mice and 260,000 lab rats. This accounted for over 83 percent of their experimental animal use that year (Blunt 2014). The United States office of Technology Assessment has estimated that between 17 and 23 million animals are used for scientific research each year. Of those animals, about 95 percent are rats and mice bred specifically for science (U.S. Congress 1986) Together, the magnitude of the intrauterine position effect, combined with the widespread use of lab rodents, suggest that it may no longer be sufficient to characterize rodents as simply “male” or “female.” Rather, there may be benefit in the development of more specific categories relating to the prenatal neighbors of affected rodents.
Sexual Differentiation of Reproductive Organs—————————————————-—————————————Because it is important to understand the intrauterine position effect in context, a primer on sexual differentiation may be useful. All mammals begin with their twenty-third set of chromosomes as XX or XY. The presence of a Y chromosome will eventually assign “maleness” through the action of the SRY gene, so named for the “sex-determining region of the Y chromosome.” In the absence of the Y chromosome and its SRY gene, the default developmental cascade will be towards the female. In fact, individuals with a Y chromosome but an inactive SRY gene will develop phenotypically female. The XX individual’s undifferentiated “bipotential” precursor gonads, which have the ability to later become either male or female gonads, will develop into ovaries. Her primordial germ cells are swallowed by follicles on the surface of the developing ovary, ensuring their later release into the body cavity for capture by the fallopian tubes. Female development also involves the presence of the Müllerian duct, which in the absence of male hormones, will develop into uterine tubes, the uterus, cervix and upper vagina (McCarthy 2012, Sizonenko 2012)
However, in the presence of the SRY gene, the female default will be de-railed in favor of male development. The SRY gene codes for a protein known as Testis Determining Factor (TDF.) TDF is a transcription factor that initiates a domino effect of precisely controlled gene expression. The developing gonadal tissue will curl up, forming tubules encasing the primordial germ cells. These germ cells will eventually produce sperm, working with a system of ducts to coordinate the elimination of sperm from the body. As the male gonads develop, they will quickly begin producing androgens, like testosterone, and anti-Müllerian hormone. This ensures the destruction of the female inducing Müllerian ducts and the survival of the Wolffian ducts, which will eventually develop into the epididymis and the vas deferens. Subsequent hormonal cascades will tie up any loose ends, forming the penis and scrotum (McCarthy 2012, Sizonenko 2012).
Sexual Differentiation of the Brain————————————————————————————————-Such an explanation covers physical determination, and leads to the next important component of sexual determination: brain sexuality. It is a common oversimplification that testosterone is the primary male sex hormone while estrogen is the primary female sex hormone. The reality is far less well defined. Testosterone is a member of the androgen class of steroid hormones, of which dihydrotestosterone is the most potent. Estrogens are another class of steroidal hormones, of which estradiol is the most powerful. As it is the precursor to both estradiol and dihydrotestosterone, testosterone is a key mediator in brain sexual chemistry (fig. 1.) The enzyme aromatase synthesizes estradiol from testosterone via aromatic conversion of its A-ring. Dihydrotestosterone is synthesized via 5α-reduction. Functionally, estradiol and dihydrotestosterone will only respond to their own dedicated receptors in the brain, estrogen receptor (ER) and androgen receptor (AR,) respectively.
Historically, androgens have been considered the primary class of hormones involved in brain sexuality. Eventually, scientists observed some peculiar effects, which lead to the Aromatization Hypothesis of sexual differentiation in the brain (McEwen et al. 1977, Kawata 2013). These studies showed that female rat pups responded with masculinization to treatment with testosterone, but responded not as strongly when treated with its more potent brother, dihydrotestosterone. More suspiciously, the most masculine effects were observed in females treated with estradiol. The discovery that there is a high density of estradiol receptors (ER) in brain regions responsible for reproductive behavior, and that in those same regions, there are high levels of aromatase, shed light on the mysterious phenomenon (McCarthy 2012).
The key lies in a combination of high aromatase concentrations and dense estradiol receptors. When the pups were exposed to testosterone, this testosterone was aromatized by the elevated aromatase, becoming estradiol, and finally received by the dense estradiol receptors in the sexual region of their brains. When the pups were just given estradiol, intensified effects were observed because the researchers were effectively taking a short-cut, skipping the aromatization step. However, when dihydrotestosterone was administered, little effect was observed because dihydrotestosterone would have no relevance to the estradiol receptors. Furthermore, dihydrotestosterone’s limited effect and lack of androgen receptors only reinforce the thought that estradiol is the primary hormone responsible for brain masculinization.
At first, this seems to put female brains in a precarious position. How is it that the high levels of circulating maternal estrogens do not masculinize females too? The answer is a steroid binding blood-hormone known as alpha-fetoprotein. Alpha-fetoprotein is highly attracted to estradiol, but has little affinity for testosterone. Thus, it is able to bind and inactivate when in the presence of estradiol, like a sponge, never disturbing the testosterone. When the testes of developing males are producing high levels of testosterone during development, alpha-fetoprotein levels are also elevated in both male and female fetuses. So, maternal estrogens in the blood will bind to the alpha-fetoprotein, effectively deactivating themselves and ensuring that no estradiol reaches the brain to induce masculinization. In this way, female pups remain female. In males, alpha-fetoprotein still sucks up maternal estrogens in the blood stream, but their testes are also producing elevated testosterone. That testosterone will flow undisturbed through the bloodstream, reaching the male brain, where it will be aromatized into estradiol, inducing masculinization (McCarthy 2012). Because alpha- fetoprotein is only present in the bloodstream, and not the brain, it cannot deactivate estradiol produced in brain tissue. Essentially, alpha-fetoprotein acts as a selective vacuum, sequestering unwanted maternal estradiol in the bloodstream, ensuring that it does not induce aimless, arbitrary masculinization in female pups (fig. 2).
To confirm that prenatal estrogens induce masculinization and de-feminization and that alpha-fetoprotein is responsible for protecting the female brain from the effects of estrogen, Bakker et al. (2006) conducted a study using alpha-fetoprotein mutant, female mice. These mutant mice produced no alpha-fetoprotein, leaving them presumably vulnerable to all maternal estrogens. When observed, the brain and behaviors of mutant females were masculinized and defeminized, confirming that alpha-fetoprotein is necessary to protect the female’s brain from maternal estrogens. Next, the researchers blocked the mutant female’s estrogen production with an aromatase inhibitor. The result was a female fetus with no circulating alpha-fetoprotein or estrogen. Fetuses from the estrogen-blocked group exhibited a feminine phenotype, proving that prenatal estrogens defeminize and masculinize the brain (Bakker et al. 2006)
However, there are exceptions to the Aromatization Hypothesis. It appears that the neural systems that mediate male aggression in rodents are still thought to be controlled by the interaction of testosterone and neuronal androgen receptors, not estradiol levels (Miller and Hay 2004). This finding suggests that the Aromatization Hypothesis may be more physiologically than behaviorally relevant.
Clearly, prenatal hormones play an enormous role in the proper development of rodents. Thus any factor significantly altering these fetal hormone levels should also be of interest. In litter bearing mammals, studies show that hormonal levels of individual pups are significantly affected by the sex of their neighbors, and that those altered prenatal hormones can have significant and wide-reaching effects later in life. Any study employing a comparison between male and female rodents must recognize that all male rodents are not equally masculine, and all female rats are not equally feminine. Thus, the intrauterine position effect is a key source of non-genomic variation, and must be incorporated into our sex classification systems and research design.
Structure of the bicornate uterus—————————————————————————————————-Rodents are an especially valuable organism for research of the intrauterine position effect because of the way the developing litter is organized in utero. The key is their bicornate uterus (fig.3). A maternal rodent’s womb is organized in a U-shape, with the base of the U opening into the cervix and vagina, and the upturned ends ending at the ovaries. Each side of the U is termed a “uterine horn.” On either side of the U, the right and left uterine horns, developing male and female fetuses will randomly organize, so that they either develop between two males (2M), or two females (0M), or one of each sex (1M.) A special case exists at the both the caudal and distal ends of the uterine horn. These four individuals only have neighbors to one side, with either the cervix (caudal) or an ovary (distal) occupying the other side (Vandenbergh 2009).
In mice, fetuses developing in closer proximity to male mice may be exposed to elevated levels of testosterone. Early in development, male pups produce relatively high concentrations of testosterone to direct their own development (Vreeburg et al. 1983) Females, however produce very small amounts of androgens and estrogens during prenatal development, only producing increased levels of estrogens during puberty (Vandenbergh 2009). The testosterone produced by a male fetus, only intended for its own masculinization, may also affect surrounding fetuses, male or female. The end result is a gradient of testosterone exposure; with 2M individuals being exposed to the most testosterone, and 0M individuals being exposed to the least. Because testosterone is a lipophilic steroid hormone, it can diffuse through amniotic membranes into surrounding amniotic fluid. Male individuals will have higher levels of testosterone in their amniotic sacs, and this testosterone can travel down a diffusion gradient into the amniotic sacs of neighboring pups. Females are especially sensitive, because of their naturally low testosterone exposure, thus, a masculinizing effect can occur. Likewise, affected males already producing their own testosterone may then be exposed to excess additional androgen, maxing out masculinization (Vomsaal et al. 1990, Vandenbergh 2009).
However, because of their unique uterine blood flow, rats do not adhere to the same pattern. The mouse uterus has bi-directional blood-flow, so circulating testosterone is balanced (Vom Saal and Dhar 1992). But rats exhibit a one-way, caudal to distal blood flow (cervix to ovaries.) This means that pups located distally, or downstream, from a male will be exposed to more testosterone than those located caudally, or upstream, from the male in question (fig. 4) (Meisel and Ward 1981, Houtsmuller et al. 1994, Gorodeski et al. 1995). Just as in the mouse model, developing rats exposed to more testosterone because of their distal position will be masculinized. Likewise, developing rats exposed to less testosterone because of their caudal position will exhibit less masculine characteristics.
Regardless of the mechanism by which this testosterone gradient is established, either through diffusion or one-way blood-flow, the end result is significant variation in the amount of testosterone that each pup is exposed to during critical stages of sexual differentiation. These differing concentrations are likely responsible for the variation in masculinity seen within litters.
Although the intrauterine position effect and the resultant variation in masculinity may appear to obscure proper development, it is important to dispel such a misconception. In fact, many litter-bearing mammals appear to depend upon the intrauterine position effect for normal development. In studies of rodents without any littermates, it has been found that those individuals matured abnormally and reproduced poorly as adults (Gandelman and Graham 1986a, b, Clark et al. 1997). In this way, it appears that intrauterine position is a critical foundation for the non-genomic variation and normal development of litter-bearing mammals.
Methods to Determine Intrauterine Position—————————————————————————————-—-Intrauterine position has broad consequences for the physiological, morphological and behavioral future of affected developing fetuses, so a simple way to ascertain a rat pup’s location in utero is important. Caesarean section is effective, but invasive and disruptive. Thus the anogenital distance index has evolved, as a much less intrusive method of ascertaining a pup’s prior position.
Anogenital distance is the space as measured between the anus and genital papilla of the rodent to be studied. Because anogenital distance is sexually dimorphic, with more masculinized rodents typically exhibiting longer anogenital distances, and because ruler measurement is non-invasive, it is a popular method used to determine prior androgen exposure. To control for variation in body size, a common method of dividing the rodent’s anogenital distance measurement by its weight produces the anogenital distance index (Ryan and Vandenbergh 2002).
Anogenital distance varies not only between sexes, but within sexes; female rodents exposed to higher levels of androgen in utero (2M or distal) exhibited longer anogenital distances than their less masculine sisters. Thus, anogenital distance measurement is a minimally invasive way to determine prior androgen exposure and intrauterine position (Vandenbergh and Huggett 1995, Hurd et al. 2008).
However, there are drawbacks to the anogenital distance index as well. Research has shown that certain environmental endocrine disruptors may also have an effect on anogenital distance. One such study provides evidence that a maternal rodent exposed to these environmental chemicals may pass them onto her unborn pups, introducing a second variable in the development of anogenital distance (Liu et al. 2014). Because of this, maternal exposure to endocrine disruptors should be considered before conclusions about prior intrauterine position are made based solely upon the anogenital distance index
Morphological Effects—————————————————————————————————The intrauterine position has been extensively linked to the morphology of adult rodents, with both male and female 2M rodents exhibiting masculinized physical features. As was noted before, intrauterine position affects anogenital distance by varying androgen exposure. Increased testosterone exposure, because of proximity to a male fetus, is correlated with longer anogenital distances. For reasons unexplained, possibly relating to differences in metabolism, stress response or aggression, body weight is also affected. 2M individuals typically exhibit heavier body weights than 0M mice of either sex (Kinsley et al. 1986). Reproductive organs are also, predictably, affected. 2M male mice have larger seminal vesicles and smaller prostates than 0M male mice (Nonneman et al. 1992). Correspondingly, 2M male rats have heavier testes than 0M male rats (Vanderhoeven et al. 1992).
Such morphological variation means that any study hoping to use lab rodents to compare male and female morphology must recognize the variation introduced by the intrauterine position effect. Groups labeled simply as “male” and “female” are outdated and insufficiently specific. Without narrower categories, which incorporate variable fetal androgen exposure, future studies run the risk of producing misleading results, hampered by hidden variables.
Physiological Effects—————————————————————————————————-Physiologically, the intrauterine position effect has even broader application. On a hormonal level, it is important to clarify the effect of intrauterine position upon adult circulating testosterone levels. Although fetal 2M mice show higher testosterone levels in both plasma and amniotic fluid, elevated baseline testosterone levels are not maintained throughout adult life (Vom Saal and Bronson 1980). However, testosterone sensitivity is affected throughout adulthood. Adult 2M female mice and distal female rats are more sensitive to testosterone injection than 0M or caudal females (Gandelman et al. 1977, Brand et al. 1990, Houtsmuller et al. 1994). This is evidenced by faster and more intense aggressive and sexual responses to supplemental testosterone.
Enzymatically, intrauterine position has been correlated with 5α-reductase activity. Recall 5α-reductase as the enzyme involved in converting testosterone into dihydrotestosterone, the hormone responsible for embryological development of male external genitalia and the adult development of secondary sex characteristics. 0M male mice have lower 5α-reductase activity, which disrupts their circulating levels of dihydrotestosterone (Ryan and Vandenbergh 2002). Given the cascading effect of hormones, low dihydrotestosterone has enormous ramifications for androgen activity and the eventual development of secondary sexual characteristics.
Metabolically, 2M individuals show increased levels of various enzymes associated with elevated metabolism (Ryan and Vandenbergh 2002). Thus, intrauterine position must also play a role in the metabolism of affected individuals. And given that certain toxins have sexual dimorphism in their metabolic rates, it fits that intrauterine position, by affecting androgen sensitivity, may also alter an individual’s ability to process certain toxins. In a study involving alcohol, juvenile 2M male rats showed less interest in ethanol when their mothers had been given ethanol during pregnancy (Mankes et al. 1992). This decreased preference may be explained by discrepancies in the ability of 0M and 2M pups to metabolize alcohol.
Intrauterine position also has an effect on secondary sex ratios, which over generations, has a unique influence on population dynamics. Adult 2M female mice give birth to a higher proportion of male offspring, producing litters that are approximately sixty percent male and forty percent female. And because the litters of 2M female mothers are mostly male, any females in that litter are more likely to be 2M. Likewise, 0M female mothers give birth to a higher proportion of female offspring, with litters that are roughly 60 percent female and 40 percent male. Thus, their predominantly female litters will give rise to more 0M female pups. 1M mothers, predictably, have traditional one to one ratios, with litters that are fifty percent male and fifty percent female (Vandenbergh and Huggett 1995). Essentially, this means that 0M mothers are more likely to have 0M daughters, and 2M mothers are more likely to have 2M daughters. In this way, intrauterine position may be a form of non-genomic maternal-line inheritance.
Reproductive physiology is also largely affected by intrauterine position. In female individuals, intrauterine position affects the age at onset of puberty and the reproductive potential of the affected rodent. In rats, a shorter anogenital distance is associated with earlier vaginal opening and earlier first estrus (Zehr et al. 2001). Female mice from 0M positions also show earlier vaginal opening and first estrus (Vom Saal et al. 1981). Pregnancy is also affected, with shorter anogenital distances being associated with increased pregnancy odds (Drickamer 1996). 2M females stop producing young earlier than their 0M counterparts, and produce fewer viable litters throughout their limited fertile years (Vom Saal and Moyer 1985).
Even more so than morphology, a rodent’s physiology is impacted by its exposure to androgens in utero, as mediated by its intrauterine position. The future testosterone sensitivity, secondary sex-ratios, and enzymatic, metabolic and reproductive functioning are all affected by the position of a rodent during intrauterine development and subsequent androgen exposure. Thus, as with morphology, any study striving to incorporate a comparison of male and female physiology may be inconclusive if it does not further define its sexual categorization and control for the variation introduced by intrauterine position.
Behavioral Effects—————————————————————————————————Although evident in both the morphology and physiology of adult rodents, the intrauterine position effect was originally noticed in a behavioral context. The effect was first discovered by scientists hoping to explain differences in sexual mounting behavior among females. Properly feminized female rats express lordosis behavior during mating, as a way of signaling their receptivity to copulation. The lordosis position is measured by the significance of the arch in a female’s back when mounted by her male conspecific (fig. 5.) 0M female mice show increased lordosis response (Rines and Vonsaal 1984). Likewise, female rats located distally from two or more male rats show decreased lordosis response (Houtsmuller and Slob 1990). And further supporting the theory that 2M individuals are more sensitive to testosterone, females treated with testosterone are more likely to show mounting behavior if they developed as 2M or distally to males (Brand et al. 1990).
Less conclusive research has examined the male behavioral response to intrauterine position effects. Recall that anogenital distance is a sexually dimorphic measurement of the space between a rodent’s anus and genital papilla, and that a longer anogenital distance is associated with masculinization. It was reported that male mice are more attracted to the odors of female mice with short anogenital distances (Drickamer et al. 2001). They are more likely to choose a 0M female mouse for mating (Brand et al. 1993) and they are also more likely to attack that same 0M female if she rejects his sexual advance (Vom Saal and Bronson 1978).
Territoriality is another important behavior that appears to be affected by intrauterine position. Males often mark territory with urine, a testosterone dependent behavior. A male individual will use his urine for marking more when in the presence of a 2M female than in the presence of a 0M female, indicating his awareness of the higher testosterone levels in the 2M female (Palanza et al. 1995). He may be sensing her masculinity. Urine marking behavior is even seen in 2M females themselves, but more rarely in 0M females (Vom Saal and Bronson 1980). When placed in field settings, both male and female 2M individuals preserve much larger home ranges than their 0M siblings (Zielinski et al. 1992, Godsall et al. 2014). In the same setting, 2M males show a higher incidence of novelty seeking than 0M males (Laviola et al. 2003). Anogenital distance is also positively related to dispersal rate in male mice, meaning that males with longer anogenital distances, who were thus more likely to have been 2M and exposed to increased testosterone, dispersed more than those with shorter anogenital distances (Drickamer 1996).
In general, male individuals exhibit more aggressive behavior than females. 2M female mice initiate more fights than 0M females (Quadagno et al. 1987). Combining the effects of increased adult testosterone sensitivity with aggression, it was found that when given injections of testosterone, 2M female mice show higher incidence of chasing, biting, and fight initiation than 0M mice. The 2M individuals also show an aggressive response after a shorter duration of testosterone exposure than the 0M females (Gandelman et al. 1977)
Intrauterine position may also affect nutritional preference behaviors. Rodents typically exhibit sexual dimorphism in their preference for high-sugar food sources, with females being more interested in concentrated saccharin solutions than males (Valenste et al. 1967a, Valenste et al. 1967b). 0M female mice prefer more concentrated saccharin solutions than their 2M counterparts (Bushong and Mann 1994).
Just as with morphology and physiology, the behavioral effects of intrauterine position are expansive and significant. Together, these three components account for an individual’s entire body system. Nearly every aspect of a rodent’s functioning is affected by its fetal androgen exposure, from body size to puberty to nutrition preferences and territorial behavior. Recall the research done by Bushong and Mann (1994) on rodent saccharin preference. Bushong and Mann found that although saccharin preference is a sexually dimorphic characteristic, with females showing greater interest, 0M female mice have a more significant preference for concentrated saccharin solutions than 2M females. By recognizing the intrauterine position effect, Bushong and Mann were able to magnify the distinction between male and female, sharpening their focus to discover greater subtlety in a sexually dimorphic character. Consider the possibility of a study incorporating sexually dimorphic preference for saccharin solutions, but not acknowledging the intrauterine position effect. If the randomly selected female rodents happened to be mostly 2M, their results would likely show a less significant difference between male and female preference. However, if the researchers incorporated intrauterine position by separating 0M and 2M female participants, their study would remain controlled and produce more reliable results. Thus, given the pervasive significance of the intrauterine position, choosing to ignore these effects may impede our ability to achieve maximum scientific accuracy.
Effects on Population Dynamics————————————————————————————————-When combined with the effects of stress hormones and behavioral ramifications, inherited birth ratios take on an even more salient dimension. Stressed mothers, often due to environmental pressures like population density and lack of nutrients, exhibit higher circulating testosterone levels (Ward and Weisz 1984). These stressed mothers also give birth to more 2M-like females, with litters that are disproportionately biased towards 2M characteristics, like lower fecundity and higher dispersal (Zielinski et al. 1991) This may be a result of higher circulating maternal testosterone levels mimicking the effects of testosterone diffusion from male pups to neighboring female littermates. These 2M-like females will produce less young and venture from their original population, relieving population density. And because of birth-ratio inheritance, the second generation 2M mothers will produce more 2M daughters, only intensifying the environmental relief effects (Vandenbergh and Huggett 1995). Potentially, as environmental stress returns to normal, maternal testosterone levels may normalize and the population will regain normal 0M/2M ratios. Likewise, if population density is extremely low, abundant resources may lower stress and circulating testosterone levels. This might then cause mothers to produce predominantly female litters, with more 0M females, who may then go on to produce additional 0M females. 0M females are less dispersive and more fertile, so they might increase population density until the environment reaches capacity, at which point the cycle would potentially begin again with environmental stress elevating maternal testosterone.
The Intrauterine Position Effect in Wild Organisms—————————————————————————————————Of course, studying organisms in an artificial environment is not a perfect measurement of the phenomenon in question. Field studies of organisms in their natural environments– the environments in which they evolved — are critical for a heightened understanding of the intersection of physiology, morphology and behavior. To investigate whether intrauterine position was an important factor in wild behavior, wild house mice were captured and allowed to mate before being released onto grassy highway islands. In this study, caesarean section was used to determine intrauterine position, rather than anogenital distance. Once the pups reached adulthood, 2M and 0M females and 1M males were transported to the highway islands, chosen because released mice emigrate from the islands at a low rate, and many of the islands include environments favored by wild house mice. The mice were monitored over seven weeks. It was observed that the 2M females had home ranges forty percent larger than 0M females (Zielinski et al. 1992).
In another study, intrauterine position was determined through anogenital distance measurement and mice were observed in large outdoor enclosures over a seven-month period. It was found that female mice with shorter anogenital distances were more likely to reproduce, and experienced more pregnancies. Observations also revealed that male mice with longer anogenital distances showed increased aggression and maintained larger home ranges (Drickamer 1996).
Together, these two studies strongly suggest that the behavioral effects of intrauterine position are preserved in the wild. The effects’ perpetuation in both a lab and field setting only further legitimizes the role of intrauterine position, supporting the need for its inclusion in research design.
Conclusion—————————————————————————————————Here I demonstrated the considerable and wide-ranging effects of intrauterine position upon the morphological, physiological and behavioral characteristics of rodents throughout life. All areas of an individuals’ life appear to be affected, including reproductive potential and the composition of second-generation litters, meaning that the intrauterine position of one individual may have the ability to influence aspects of many generations to come.
Given such significant ramifications, it is surprising that the sexual classification system for the common lab rodent model has yet to incorporate intrauterine position into most experimental interpretation. Studies focusing on or incorporating intersexual differences must be wary about relying upon the simple male versus female classification system. Much research has indicated that there are gradations of morphological, physiological and behavioral sexuality. It may not be sufficient to classify two same-sex lab rodents of different intrauterine positions as simply “male” or “female.” Their intrauterine position should be controlled for to ensure that a maximum level of scientific accuracy is achieved, either by only including male and female rodents of the same intrauterine position, or by creating additional distinct groups for rodents’ of each position. Of course, this increased specificity would potentially come at a cost of both researcher time and funding. Superfluous spending can inhibit scientific progress, but incorporating the intrauterine position into a more explicit rodent sex-categorization would be anything but superfluous; time and funding seems a reasonable trade for the possibility of increased precision. Unawareness of such variation may only impede achievement of the utmost scientific accuracy.
Bakker, J., C. De Mees, Q. Douhard, J. Balthazart, P. Gabant, J. Szpirer, and C. Szpirer. 2006. Alpha-fetoprotein protects the developing female mouse brain from masculinization and defeminization by estrogens. Nature Neuroscience 9:220–226.
Blunt, D. 2014. Annual Statistics of Scientific Procedures on Living Animals, Great Britain 2013. 9781474103244, Home Office, London, United Kingdom.
Brand, T., E. J. Houtsmuller, and A. K. Slob. 1990. ANDROGENS AND THE PROPENSITY FOR ADULT MOUNTING BEHAVIOR IN THE FEMALE WISTAR RAT.
Brand, T., E. J. Houtsmuller, and A. K. Slob. 1993. NEONATAL PROGRAMMING OF ADULT PARTNER PREFERENCE IN MALE-RATS.
Bushong, M. E., and M. A. Mann. 1994. GENDER AND INTRAUTERINE POSITION INFLUENCE SACCHARIN PREFERENCE IN MICE. Hormones and Behavior 28:207–218.
Clark, M. M., J. M. Vonk, and B. G. Galef. 1997. Reproductive profiles of adult Mongolian gerbils gestated as the sole fetus in a uterine horn. Physiology & Behavior 61:77–81.
Drickamer, L. C. 1996. Intra-uterine position an anogenital distance in house mice: Consequences under field conditions (vol 51, pg 925, 1996). Animal Behaviour 52:223–223.
Drickamer, L. C., A. S. Robinson, and C. A. Mossman. 2001. Differential responses to same and opposite sex odors by adult house mice are associated with anogenital distance. Ethology 107:509–519.
Gandelman, R., and S. Graham. 1986a. DEVELOPMENT OF THE SURGICALLY PRODUCED SINGLETON MOUSE FETUS. Developmental Psychobiology 19:343–350.
Gandelman, R., and S. Graham. 1986b. SINGLETON FEMALE MOUSE FETUSES ARE SUBSEQUENTLY UNRESPONSIVE TO THE AGGRESSION-ACTIVATING PROPERTY OF TESTOSTERONE. Physiology & Behavior 37:465–467.
Gandelman, R., F. S. Vomsaal, and J. M. Reinisch. 1977. CONTIGUITY TO MALE FETUSES AFFECTS MORPHOLOGY AND BEHAVIOR OF FEMALE MICE. Nature 266:722–724.
Godsall, B., T. Coulson, and A. F. Malo. 2014. From physiology to space use: energy reserves and androgenization explain home-range size variation in a woodland rodent. Journal of Animal Ecology 83:126–135.
Gorodeski, G. I., L. A. Sheean, and W. H. Utian. 1995. SEX-HORMONE MODULATION OF FLOW VELOCITY IN THE PARAMETRIAL ARTERY OF THE PREGNANT RAT. American Journal of Physiology-Regulatory Integrative and Comparative Physiology 268:R614-R624.
Hernandez-Tristan, R., M. L. Leret, and D. Almeida. 2006. Effect of intrauterine position on sex differences in the gabaergic system and behavior of rats. Physiology & Behavior 87:625–633.
Houtsmuller, E. J., J. Juranek, C. E. Gebauer, A. K. Slob, and D. L. Rowland. 1994. MALES LOCATED CAUDALLY IN THE UTERUS AFFECT SEXUAL-BEHAVIOR OF MALE-RATS IN ADULTHOOD. Behavioural Brain Research 62:119–125.
Houtsmuller, E. J., and A. K. Slob. 1990. MASCULINIZATION AND DEFEMINIZATION OF FEMALE RATS BY MALES LOCATED CAUDALLY IN THE UTERUS. Physiology & Behavior 48:555–560.
Hurd, P. L., A. A. Bailey, P. A. Gongal, R. H. Yan, J. J. Greer, and S. Pagliardini. 2008. Intrauterine position effects on anogenital distance and digit ratio in male and female mice. Archives of Sexual Behavior 37:9–18.
Kawata, M. 2013. Nurture: Effects of Intrauterine Position on Behaviour. Journal of Neuroendocrinology 25:422–423.
Kinsley, C., J. Miele, C. K. Wagner, L. Ghiraldi, J. Broida, and B. Svare. 1986. PRIOR INTRAUTERINE POSITION INFLUENCES BODY-WEIGHT IN MALE AND FEMALE MICE. Hormones and Behavior 20:201–211.
Laviola, G., S. Macri, S. Morley-Fletcher, and W. Adriani. 2003. Risk-taking behavior in adolescent mice: psychobiological determinants and early epigenetic influence. Neuroscience and Biobehavioral Reviews 27:19–31.
Liu, C., X. Xu, and X. Huo. 2014. Anogenital distance and its application in environmental health research. Environmental Science and Pollution Research 21:5457–5464.
Mankes, R., R. Lefevre, and C. Calvano. 1992. Prenatal ethanol exposure significantly effects alcohol preference in masculinized male offspring of LE rats. Alcoholism 16.
McCarthy, M. M. 2012. Sexual Differentiation of Brain and Behavior. Handbook of Neuroendocrinology:393–413.
McEwen, B. S., I. Lieberburg, C. Chaptal, and L. C. Krey. 1977. AROMATIZATION — IMPORTANT FOR SEXUAL DIFFERENTIATION OF NEONATAL RAT-BRAIN. Hormones and Behavior 9:249–263.
Meisel, R. L., and I. L. Ward. 1981. FETAL FEMALE RATS ARE MASCULINIZED BY MALE LITTERMATES LOCATED CAUDALLY IN THE UTERUS. Science 213:239–242.
Miller, V., and M. Hay. 2004. Principles of Sex-Based Differences in Physiology. Elsevier B.V. , Amsterdam, The Netherlands.
Nonneman, D. J., V. K. Ganjam, W. V. Welshons, and F. S. V. Saal. 1992. INTRAUTERINE POSITION EFFECTS ON STEROID-METABOLISM AND STEROID-RECEPTORS OF REPRODUCTIVE-ORGANS IN MALE-MICE. Biology of Reproduction 47:723–729.
Palanza, P., S. Parmigiani, and F. S. V. Saal. 1995. URINE MARKING AND MATERNAL AGGRESSION OF WILD FEMALE MICE IN RELATION TO ANOGENITAL DISTANCE AT BIRTH. Physiology & Behavior 58:827–835.
Quadagno, D. M., C. McQuitty, J. McKee, L. Koelliker, G. Wolfe, and D. C. Johnson. 1987. THE EFFECTS OF INTRAUTERINE POSITION ON COMPETITION AND BEHAVIOR IN THE MOUSE. Physiology & Behavior 41:639–642.
Rines, J. P., and F. S. Vonsaal. 1984. FETAL EFFECTS ON SEXUAL-BEHAVIOR AND AGGRESSION IN YOUNG AND OLD FEMALE MICE TREATED WITH ESTROGEN AND TESTOSTERONE. Hormones and Behavior 18:117–129.
Ryan, B. C., and J. G. Vandenbergh. 2002. Intrauterine position effects. Neuroscience and Biobehavioral Reviews 26:665–678.
Sizonenko, P. C. 2012. Human Sexual Differentiation. Reproductive health. Geneva Foundation for Medical Education and Research Geneva, Switzerland.
U.S. Congress, O. o. T. A. 1986. Alternatives to Animal Research, Testing, and Education. Washington, DC.
Valenste, E. S., V. C. Cox, and Kakolews.Jw. 1967a. FURTHER STUDIES OF SEX DIFFERENCES IN TASTE PREFERENCES WITH SWEET SOLUTIONS. Psychological Reports 20:1231-&.
Valenste, E. S., Kakolews.Jw, and V. C. Cox. 1967b. SEX DIFFERENCES IN TASTE PREFERENCE FOR GLUCOSE AND SACCHARIN SOLUTIONS. Science 156:942-&.
Vandenbergh, J. G. 2009. Effects of intrauterine position in litter-bearing mammals.
Vandenbergh, J. G., and C. L. Huggett. 1995. THE ANOGENITAL DISTANCE INDEX, A PREDICTOR OF THE INTRAUTERINE POSITION EFFECTS ON REPRODUCTION IN FEMALE HOUSE MICE. Laboratory Animal Science 45:567–573.
Vanderhoeven, T., R. Lefevre, and R. Mankes. 1992. EFFECTS OF INTRAUTERINE POSITION ON THE HEPATIC-MICROSOMAL POLYSUBSTRATE MONOOXYGENASE AND CYTOSOLIC GLUTATHIONE-S-TRANSFERASE ACTIVITY, PLASMA SEX STEROIDS AND RELATIVE ORGAN WEIGHTS IN ADULT MALE AND FEMALE LONG-EVANS RATS. Journal of Pharmacology and Experimental Therapeutics 263:32–39.
Vom Saal, F. S., and F. H. Bronson. 1978. IN UTERO PROXIMITY OF FEMALE MOUSE FETUSES TO MALES EFFECT OF REPRODUCTIVE PERFORMANCE DURING LATER LIFE. Biology of Reproduction 19:842–853.
Vom Saal, F. S., and F. H. Bronson. 1980. SEXUAL CHARACTERISTICS OF ADULT FEMALE MICE ARE CORRELATED WITH THEIR BLOOD TESTOSTERONE LEVELS DURING PRE NATAL DEVELOPMENT. Science (Washington D C) 208:597–599.
Vom Saal, F. S., and M. G. Dhar. 1992. Blood flow in the uterine loop artery and loop vein is bidirectional in the mouse: implications for transport of steroids between fetuses. Physiology & Behavior 52:163–171.
Vom Saal, F. S., and C. L. Moyer. 1985. PRENATAL EFFECTS ON REPRODUCTIVE CAPACITY DURING AGING IN FEMALE MICE. Biology of Reproduction 32:1116–1126.
Vom Saal, F. S., S. Pryor, and F. H. Bronson. 1981. EFFECTS OF PRIOR INTRA UTERINE POSITION AND HOUSING ON ESTROUS CYCLE LENGTH IN ADOLESCENT MICE. Journal of Reproduction and Fertility 62:33–38.
Vomsaal, F. S., D. M. Quadagno, M. D. Even, L. W. Keisler, D. H. Keisler, and S. Khan. 1990. PARADOXICAL EFFECTS OF MATERNAL STRESS ON FETAL STEROIDS AND POSTNATAL REPRODUCTIVE TRAITS IN FEMALE MICE FROM DIFFERENT INTRAUTERINE POSITIONS. Biology of Reproduction 43:751–761.
Vreeburg, J. T. M., J. O. Groeneveld, P. E. Post, and M. P. Ooms. 1983. CONCENTRATIONS OF TESTOSTERONE AND ANDROSTERONE IN PERIPHERAL AND UMBILICAL VENOUS PLASMA OF FETAL RATS. Journal of Reproduction and Fertility 68:171–175.
Ward, I. L., and J. Weisz. 1984. DIFFERENTIAL-EFFECTS OF MATERNAL STRESS ON CIRCULATING LEVELS OF CORTICOSTERONE, PROGESTERONE, AND TESTOSTERONE IN MALE AND FEMALE RAT FETUSES AND THEIR MOTHERS. Endocrinology 114:1635–1644.
Zehr, J. L., S. E. Gans, and M. K. McClintock. 2001. Variation in reproductive traits is associated with short anogenital distance in female rats. Developmental Psychobiology 38:229–238.
Zielinski, W. J., J. G. Vandenbergh, and M. M. Montano. 1991. EFFECTS OF SOCIAL STRESS AND INTRAUTERINE POSITION ON SEXUAL PHENOTYPE IN WILD-TYPE HOUSE MICE (MUS-MUSCULUS). Physiology & Behavior 49:117–123.
Zielinski, W. J., F. S. Vomsaal, and J. G. Vandenbergh. 1992. THE EFFECT OF INTRAUTERINE POSITION ON THE SURVIVAL, REPRODUCTION AND HOME RANGE SIZE OF FEMALE HOUSE MICE (MUS-MUSCULUS). Behavioral Ecology and Sociobiology 30:185–191.