Intrauterine position effects

Abstract

A review of the literature suggests that individual variability in sex-related traits may be influenced by variations in hormonal exposure during fetal development. In litter-bearing mammals, fetuses develop in utero and may be subjected to differing hormonal environments based upon the sex of neighboring fetuses. Female fetuses developing between two males tend to show masculinized anatomical, physiological and behavioral traits as adults. Female fetuses developing without adjacent males, on the other hand, tend to show more feminized traits as adults. These traits include permanently altered hormone levels, reproductive organs, aggressive behaviors, secondary sex ratios and susceptibility to endocrine disruption. This intrauterine effect is due to the transfer of testosterone from male fetuses to adjacent fetuses. While these effects have been most clearly demonstrated in mice, other rodents and swine also show intrauterine position (IUP) effects. Some of these effects are similar to the influence of prenatal stress on adult phenotypes. A few reports on human twins suggest that variability in some masculine and feminine traits may be due to intrauterine hormonal signals. IUP effects may impact a number of scientific fields of research such as endocrine disruption, toxicology, population biology, animal production and health.

Introduction

It has been disputed at what period of life the causes of variability, whatever they may be, generally act; whether during the early or late period of development of the embryo, or at the instant of conception

–Charles Darwin ‘On the Origin of Species’[31]

Explaining variability lies at the heart of biology, yet at the same time this variability must be experimentally controlled. Variability is typically controlled by standardizing the genetic background of the subjects and the environment in which they are maintained after birth. Here we review another source of variability not often recognized. This variability is not genetic or environmental in origin, but rather hormonal.

Intrauterine development in eutherian mammals allows for precise control of the fetal environment and allows the fetus to directly interact with the mother. In litter bearing mammals, pups from the same litter must share space in their mother's uterus. This space sharing results in pups from large litters developing in slightly different environments from each other. For example, a mouse or swine fetus that occupies a position at either end of a uterine horn receives more nutrient rich blood and is subsequently heavier at birth than other fetuses [6], [117], [125]. A rat fetuses located at the cervical end of the uterus receives maternal blood flow prior to fetuses in other uterine positions [42], [67]. Any fetus not located at an end of a uterus will be located between two males (2M), two females (0M), or one male and one female (1M) (Fig. 1). This intrauterine position (IUP) has significant and wide-ranging effects on the development of the fetus.

In most mammals, male fetuses produce testosterone earlier and in higher amounts than females. Females, on the other hand, produce higher amounts of estradiol later in development. Being steroids, these hormones can diffuse through the amniotic fluid between fetuses. As a result, both male and female 2M mouse fetuses (flanked by males) have higher blood concentrations of testosterone and lower blood concentrations of estradiol than 0M fetuses (flanked by females) [122]. This mechanism of hormone transfer among mouse fetuses has become fairly well understood and accepted.

In addition to diffusion through the amniotic fluid, hormones may also travel among fetuses through the bloodstream of the mother. In pregnant rats, uterine blood flows predominantly in the caudal to distal direction (i.e. from the cervix to the ovaries) [42]. Fetuses located distally to male pups will thus be subjected to higher levels of testosterone and fetuses located caudally to male pups will not be subjected to high testosterone levels. Interestingly, this uterine blood flow also results in fetuses from the same litter receiving differing levels of cocaine (and presumably other drugs) administered to their mother [67], [124]. Pregnant mice, in contrast to rats, show bi-directional blood flow through the uterus [117]. This makes a one-way movement of hormones among mouse fetuses unlikely. It is not known if the differences in circulation play some predictable role in developmental differences or IUP differences between species. However, this physiological difference between rat and mouse circulation has resulted in most investigators classifying rat fetuses by the number of males located upstream (caudal male hypothesis), whereas mice and gerbil researchers have maintained the classical 0M–2M classification (contiguous male hypothesis).

Regardless of the exact mechanism, it is clear that same-sex fetuses from the same litter will be subjected to different levels of steroid hormones. Many mammals seem to rely on this variable uterine environment to develop normally. Both gerbils and mice that do not have any littermates mature abnormally and reproduce poorly [23], [40]. IUP, therefore, serves as a source of non-genomic variability in these animals.

Sexual differentiation in mammals is largely mediated by androgens early in development. Variable levels of androgens will therefore alter this process of differentiation. 2M pups subjected to high levels of androgens will show increased masculinization and 0M pups subjected to low levels of androgens will show increased feminization. Steroid hormone transfer among fetuses not only alters development, but it increases variability among individuals from the same litter. From an experimental perspective, variability is unwanted and may mask potentially significant results. For example, IUP alters sensitivity to certain endocrine disruptors [56], [57] and therefore serves as an additional source of variation. Laboratory rodents are commonly used experimentally because of their genetic homogeneity and low variability. It is important to understand the potential implications of this IUP effect in order to control and minimize this endogenous, non-genetic, variability.

The first report of an IUP effect came at the 68th Annual Meeting of the American Society of Zoologists, in 1971. At this meeting, Clemens and Coniglio [26] reported a “positive relation between the probability of female mounting and the number of males which were present during intrauterine development. This relation may suggest that the behavior of the female is influenced by fetal testicular secretions of male sibs”. The initial work on rats was expanded in a subsequent publication [25]. It was followed up more extensively in mice [114], giving rise to a field of research that has remained active for 30 years and produced over 100 publications.

The purpose of this review is to identify areas of research where knowledge of IUP effects can reduce variability and increase the chance of detecting reproducible results. IUP has the potential to influence results in a wide variety of mammalian studies. A number of important fields that seem particularly susceptible to IUP effects will be discussed. This review will hopefully stimulate more efficient research design in these fields that may not be intuitively linked to IUP.

The recent public and scientific concern over the effect of hormone mimics in the environment has simulated a vast amount of work in the relatively new field of endocrine disruption. Similar concerns regarding low dose and mixture effects of certain toxicants have surfaced in recent years as well [52]. IUP, by altering prenatal endogenous hormone exposure, has the potential to interact with experimental manipulations of endocrine disruptors.

IUP has the ability to influence aggression, dispersal and mating in wild populations and could therefore affect studies dealing with the population biology of mammals as well as allow commercial breeders to have greater control over their animal production. IUP can also influence immunological responses as well as enzyme levels in the body and could therefore become important for general animal health and human health.

This paper reviews the literature generated in the past 30 years and is organized by specific IUP effects on physiology, morphology and behavior. Interactions between prenatal stress and IUP are also discussed.