Non-model species deliver a non-model result: Nutria female fetuses neighboring males in utero have lower testosterone

Highlights

• Neighboring fetuses may impact their siblings in multiple ways.

• The nutria is a rodent with a long pregnancy and precocious offspring.

• We determined intrauterine position and measured fetal testosterone by hair-testing.

• Nutria females neighboring a male in utero had lower testosterone levels.

• Non-model species are important for studying evolutionary mechanisms.

Abstract

Neighboring fetuses may impact their siblings in various respects, depending on their in utero location and sex. The effects of the intrauterine position (IUP) are widely studied in model organisms, especially laboratory bred murine strains that are characterized by short gestations and altricial offspring. In some species, the proximity to a male fetus and its higher circulating testosterone masculinizes neighboring female fetuses. In utero testosterone exposure might be manifested as higher testosterone concentrations, which contribute to a variation in morphology, reproductive potential and behavior. In this study, we examined the influence of neighboring an opposite sex fetus on testosterone levels in a feral animal model characterized by a long gestation and precocious offspring. Using necropsies of culled nutria (Myocastor coypus), we accurately determined the IUP and quantified testosterone immunoreactivity in fetal hair. We found that as expected, both male and female fetuses neighboring a male in utero had longer anogenital distance. However, females adjacent to males in utero showed lower testosterone levels than male fetuses, while testosterone levels of females without a male neighbor did not differ from those of males. This surprising result suggests an alternative mode by which local exogenous steroids may modify the local fetal environment. Our study emphasizes the importance of examining known phenomena in species with different life histories, other than the traditional murine models, to enhance our understanding of the evolutionary mechanisms that are driving sexual differentiation.

Introduction

The fetal environment is thought to be primarily shaped by maternal conditions. In polytocous (i.e., litter-bearing) mammals, individual fetuses in the litter also influence the uteral environment via space (Ibsen, 1928), vasculature (Houtsmuller and Slob, 1990; Meisel and Ward, 1981), and resources (Labov et al., 1986). The effects of fetuses on litter mates may depend on the sex, location, and proximity of each fetus (e.g., Ryan and Vandenbergh, 2002; Ward et al., 1977; Fishman et al., 2018a). Fetal intrauterine position (IUP) reflects the proximity to fetuses of the same or opposite sex (vom Saal et al., 1990). Fetal development, sexual differentiation, and maturation can be affected by the sex of neighboring fetuses, which create local hormonal environments that might drive individual differences (Fishman et al., 2018a; Ryan and Vandenbergh, 2002). The effects of IUP on both sexes are well documented in model species (reviewed by Ryan and Vandenbergh, 2002). For example, female rodent fetuses developing between two males in utero present masculinized anatomical, physiological and behavioral features in adulthood (Ryan and Vandenbergh, 2002). These females are less attractive to males and have lower reproductive success than females that developed in utero without a male neighbor (Ryan and Vandenbergh, 2002; Zielinski et al., 1992). Most IUP-related phenotypes have been attributed to testosterone concentrations and its transfer between fetuses in utero (e.g., vom Saal and Bronson, 1980; Rohde Parfet et al., 1990). Since males are presumed to have higher circulating testosterone concentrations, fetal IUP is usually defined by its proximity to a male fetus. Two main categorization systems have been used to describe the effect of proximity. Meisel and Ward (1981) suggested a directional classification, in which the effect on a fetus is dependent on the location of opposite sex fetuses (i.e., caudally or rostrally on the uterine horn), whereby uterine vasculature is responsible for the transfer of sex steroids from one fetus to another via maternal blood flow, as seen in rats (Hernández–Tristán et al., 1999; Houtsmuller and Slob, 1990; Meisel and Ward, 1981; Richmond and Sachs, 1984). vom Saal introduced a contiguity classification, based on the proximity to a male fetus, so that a fetus without a male neighbor is termed 0M; 1M means that the fetus has a single male neighbor, and 2M means that there are male neighbors on both sides of the fetus, with diffusion of androgens between fetuses via amniotic fluid or blood, as seen in mice (vom Saal, 1981).

Females are considered to be more sensitive to IUP effects than males (Ryan and Vandenbergh, 2002). However, most research on the association between IUP and fetal testosterone was conducted on a few model species, mostly lab and domesticated animals (Ryan and Vandenbergh, 2002), and showed diverse findings, which might represent different mechanisms. In mice, 2M female fetuses had higher circulating and amniotic fluid testosterone concentrations than 0M females (vom Saal et al., 1990; vom Saal and Bronson, 1980). In gerbils, circulating testosterone levels were higher in both 2M male and female fetuses, in comparison to 0M males and females (Clark et al., 1991). In rats, Houtsmuller et al. (1995) found that circulating testosterone levels were higher in 2M females than in 0M females, while Hernández–Tristán et al. (1999) did not, although male fetuses had higher testosterone concentrations than females. In pigs, males had higher circulating testosterone concentration than females, but they were not related to IUP (Wise and Christenson, 1992). Yet, it is possible that IUP classification systems that are suitable for certain species or mechanisms are not useful for others, requiring alternative approaches.

During specific stages of gestation, the effect of testosterone transfer between fetuses (Even et al., 1992) can influence the distance between the anus and genitalia (i.e., anogenital distance; AGD) through its role in perineal tissue elongation (Hotchkiss and Vandenbergh, 2005; vom Saal and Bronson, 1980). This anatomical manifestation can be hindered by antiandrogen (e.g., flutamide) administration (Clemens et al., 1978). As such, AGD has been used as a biomarker for androgen exposure in utero when verifying IUP via a caesarian section [e.g., mice (Hotchkiss and Vandenbergh, 2005; Jubilan and Nyby, 1992), rats (Hernández–Tristán et al., 1999)]. However, AGD has also been extrapolated into systems where IUP was not verified, and used as an indication of IUP postnatally (e.g., Correa et al., 2013). The AGD had been associated with morphological, physiological, behavioral and reproductive traits in mice (Hotchkiss and Vandenbergh, 2005), rats (Tobet et al., 1982; Zehr et al., 2001), pigs (Drickamer et al., 1999), rabbits (Banszegi et al., 2012), gerbils (Clark et al., 1990) and degus (Correa et al., 2013). In mice, 0M females have shorter AGD than 2M females (Ryan and Vandenbergh, 2002). In rats, females located caudally to males in utero (Hernández–Tristán et al., 1999; Houtsmuller and Slob, 1990; Meisel and Ward, 1981; Richmond and Sachs, 1984), as well as 2M females (Tobet et al., 1982) have longer AGD. In gerbils, 2M males have longer AGD than 0M males, while no differences were found in females (Clark et al., 1990), suggesting that female gerbil fetuses have a lower sensitivity to testosterone. Conversely, female rats and mice show higher androgen sensitivity than males (Ryan and Vandenbergh, 2002). An association between IUP and AGD was not found in all of the mouse and rat strains. For example, in CF-1 mice no relationship was found (Jubilan and Nyby, 1992; Nagao et al., 2004; Simon and Cologer-Clifford, 1991). Similarly, no association was found between IUP and AGD, and testosterone concentrations were not related to either IUP or AGD in Sprague-Dawley rats at the end of gestation (Hotchkiss et al., 2007).

In this study, we investigated the association between integrated testosterone levels over the last trimester of pregnancy and neighboring an opposite sex fetus in utero in the feral nutria (Myocastor coypus). This polygynous rodent is characterized by a long gestation (127–138 days) and precocious offspring. Thus, fetuses cope with the mechanical and hormonal pressures that their neighbors exert in utero for several months, while they develop. Nutria reproduction includes post gestational estrus, large litters, and receptivity year-round, making it a successful invasive species (considered one of the world's 100 worst; ISSG, 2013). We previously found that females with higher testosterone levels produce female-biased litters (Fishman et al., 2018b). Males seem to be the more ‘expensive’ sex in the last stages of pregnancy in terms of morphological development (i.e., length and weight gain), and more vulnerable, as sex ratios decline throughout the pregnancy (Fishman et al., 2018b). Severe intrauterine growth retardation was 4 times more likely in male fetuses than in females (Fishman et al., 2018b). In the last stages of pregnancy, fetuses in litters with equal sex ratios (i.e., the highest sex heterogeneity), had the highest cortisol levels (Fishman et al., 2018a), which is the main glucocorticoid produced by the nutria adrenal glands (Callard and Leathem, 1969; Wilson et al., 1964). Fetuses neighboring an opposite sex fetus also had longer trunks, regardless of sex (Fishman et al., 2018a), which might imply better lung development (Flint, 1906). Here, we explored whether females who are next to males in utero are impacted via higher testosterone levels and longer AGDs, and whether their relationship can be predicted by IUP.