Prior intrauterine position influences body weight in male and female mice

In two longitudinal studies, intrauterine location of male and female Rockland-Swiss mice relative to fetuses of the same and opposite sex dramatically influenced body weight. In one study, body weight of males and females that were located in utero between two female fetuses (OM animals) or between two male fetuses (2M animals) was assessed from birth to the time of weaning (25 days of age). The body weights of 2M females were indistinguishable from those of OM and 2M males on all but a few of the 25 postnatal days of life. Also, 2M females were significantly heavier than OM females from Postnatal Day 6 onward, and 2M males weighed significantly more than OM males from Postnatal Day 19 onward. In a second study, food intake and body weight of animals from different intrauterine locations were examined from 25 to 120 days of age. Regardless of prior intrauterine position, males were always heavier than females. However, prior in utero location modulated body weight in that 2M females were significantly heavier than OM females and 2M males were reliably heavier than OM males. Intrauterine position effects were observed in the absence of any appreciable influence of this variable on levels of food intake. Taken together, the results suggest that prior in utero location may influence metabolic set points involved in the regulation of body weight and fat storage.     

Paradoxical effects of maternal stress on fetal steroids and postnatal reproductive traits in female mice from different intrauterine positions

We examined effects of maternal stress on prenatal serum concentrations of testosterone and estradiol and on postnatal reproductive traits in female mice from different intrauterine positions. On Day 18 of fetal life, control females positioned in utero between two male fetuses (2M females) had higher concentrations of testosterone and lower concentrations of estradiol in serum than control female fetuses located between two females (0M females). Control females positioned between a male and a female fetus (1M females) had intermediate levels of both hormones. Prior intrauterine position in control females accounted for differences in genital morphology (length of the anogenital separation) at birth and length of estrous cycles during adulthood. Maternal stress eliminated these postnatal differences due to prior intrauterine position: all 0M, 1M, and 2M female offspring of stressed mothers exhibited postnatal traits that were indistinguishable from those of control 2M females. Maternal stress resulted in an increase of over 1 ng/ml in serum testosterone in all female fetuses; the magnitude of the increase was similar for 0M, 1M, and 2M females. There was no effect of maternal stress on serum concentrations of estradiol in 0M and 2M female fetuses. Maternal stress resulted in a dramatic change in the postnatal traits of 0M females, whereas 2M females showed no change. Since the effect of maternal stress on sex steroids was similar among fetuses from different intrauterine positions but postnatal response to maternal stress varied by intrauterine position, other components of the endocrine system may mediate effects of maternal stress on these postnatal characteristics.     

Intrauterine position affects sex-accessory biochemistry in adult female mice

Female mouse fetuses that develop in utero implanted between two male fetuses (2M females) are shown as adults to have significantly elevated (P less than 0.01) levels of beta-glucuronidase activity in their preputial glands compared to that of OM females, females that had not been contiguous to males in utero, and 1M females, females contiguous in utero to only one male (mean +/- SEM, 2M females = 23,9 +/- 2.1, 1M females = 13.3 +/- 5.2, and OM females = 7.8 +/- 2.5 Modified Sigma Units/mg frozen weight). It is hypothesized that the increased enzymatic activity in the preputial glands of 2M females could be important in releasing steroid metabolites from voided urine. These metabolites could then act as pheromones and thus explain some of the described differences in sexual behavior correlated with intrauterine position.     

Why some male Mongolian gerbils may help at the nest: testosterone, asexuality and alloparenting

In previous studies we have shown that those male Mongolian gerbils, Meriones unguiculatus, that as fetuses resided in intrauterine positions (IUPs) located between two female fetuses (2F males) have lower circulating levels of testosterone, less well-developed genital musculature, and lower reproductive success than males gestated in IUPs between two male fetuses (2M males). We have also found that such 2F males spend more time caring for conspecific young than do 2M males, and that presence of a 2F male, but not of a 2M male, with a lactating female and her litter decreases the cost to a dam of suckling one litter while gestating a second litter conceived in postpartum oestrus. Here we show that some 2F males, those with circulating levels of testosterone similar to those seen in females of their species, show no interest in females in oestrus and fail to impregnate females with which they are paired. Such 'asexual' 2F males spend 30-50% more time caring for nestlings than do sexually active 2F males. We suggest that such asexual, highly parental 2F males are incapable of direct reproduction and are obligate helpers at the nest that can contribute to their own fitness only by assisting to rear collateral kin. Copyright 2000 The Association for the Study of Animal Behaviour.     

Differential Responses to Same and Opposite Sex Odors by Adult House Mice Are Associated with Anogenital Distance

Intrauterine position (IUP) of female and male fetuses in litter-bearing mammals can affect their physiology, morphology and behavior. The relationship between anogenital distance (AGD) and IUP was used as a bioassay for the degree of exposure of female and male fetuses to hormones in utero . Based on laboratory work in several rodent species, the following predictions were made for house mice ( Mus musculus domesticus ): (1) female mice should prefer odors from males with larger AGDs because such males are more aggressive, could protect more resources, and are better parents than males with smaller AGDs; (2) male mice should prefer odors from females with smaller AGDs because these females produce more offspring and are better parents than females with larger AGDs. We also tested the prediction that within sexes, mice should avoid odors from mice with larger AGDs because such mice are more aggressive. Responses to odors in traps were used to test these predictions for house mice living in outdoor enclosures using odor-baited traps. Both predictions were confirmed. Furthermore, mice of both sexes tended to avoid odor cues from individuals of the same sex that had larger AGDs, probably to decrease chances of an aggressive encounter that could result in injury.     

Intrauterine position effects on steroid metabolism and steroid receptors of reproductive organs in male mice.

Mice differ in their adult reproductive characteristics as a function of whether they developed in utero between two male fetuses (2M males), which have higher testosterone levels, or between two female fetuses (0M males), which have higher estradiol levels. The present study was designed to further characterize biochemical parameters of 2M and 0M adult male mice. Activities of testicular steroidogenic enzymes, namely delta 5-3 beta-hydroxysteroid dehydrogenase/isomerase, 17 alpha-hydroxylase, and C17,20-lyase (C21SCC P450), were measured by means of radiometric assays and HPLC fractionation of substrate and products. Activity of 5 alpha-reductase in both seminal vesicle and prostate was measured by similar techniques. Estrogen and androgen receptor concentrations, which indicate capacity to respond to steroid hormones, were also examined in the accessory sex organs. For both seminal vesicle and prostate, 5 alpha-reductase activities were approximately 60% greater in 2M males than in 0M males, indicating greater capacity to form dihydrotestosterone from testosterone in organs from 2M mice. No significant differences were found in testicular steroidogenic enzymes between 2M and 0M animals, whereas the trend for all three activities was higher for 2M males than for 0M males. While no differences were found in estrogen receptor concentrations, 0M prostates had three times the concentration of androgen receptors (occupied receptors) compared to 2M prostates. Our findings suggest that intrauterine fetal position exerts a significant influence on subsequent adult androgen metabolism and androgen responsiveness in reproductive organs of adult male mice.     

Sex difference in whole-body androgen content in rats on fetal days 18 and 19 without evidence that androgen passes from males to females

The whole-body content of androgen (testosterone + 5 alpha-dihydrotestosterone) was significantly higher on Fetal Days 18 and 19 in male than in female rats; androgen content was equivalent in the two sexes at other fetal ages, including Days 16, 17, 20, and 21, and prior to parturition on Fetal Day 22. These results partially corroborate previous data of Weisz and Ward (Endocrinology 1980; 106:306-316), who measured testosterone in pooled plasma from rat fetuses and suggest that androgens contribute to masculine brain sexual differentiation only briefly during fetal life. No significant differences in whole-body androgen content were observed among groups of females situated in utero between 0, 1, or 2 males on each side (contiguous male model) or among groups of females with 0, 1, or 2 or more males located caudally (on the cervical side) in the same uterine horn, regardless of whether combined data from Fetal Days 17-22 or only Fetal Days 18 and 19 were considered. These results provide no evidence that androgens from males reach female fetuses in the same uterine horn.     

In utero contiguity to males does not influence morphology, behavioral sensitivity to testosterone, or hypothalamic androgen binding in CF-1 female mice.

Physiological and behavioral systems presumably influenced by prenatal exposure to testosterone (T) were compared in CF-1 female mice from known uterine positions. Anogenital distance did not differ among females that developed in utero between two females (0M), adjacent to one male (1M), or between two males (2M) at birth, at weaning on Day 21, or on Day 60 postpartum. The age of vaginal opening and mean estrous cycle length also were similar among the groups. When ovariectomized and implanted with a T-containing silastic capsule, the mean number of days of treatment required to activate male-like aggressive behavior also did not differ among the three positional classifications. Finally, androgen binding in combined hypothalamic-preoptic-septal cytosol was assessed after 8 days of T treatment, and no systematic variation in [3H]DHT binding related to uterine position was found. These results indicate that contiguity to male fetuses did not induce variation among CF-1 females in morphological, behavioral, or biochemical systems thought to be influenced by prenatal exposure to T.     

Lead exposure on critical days of fetal life affects fertility in the female mouse

Female mice were exposed to lead in utero by intravenous injection of lead chloride into the mothers at different stages of pregnancy. At a mature age the mice exposed as fetuses (F1 generation) conceived at a normal rate, but the litter size and fetal survival varied significantly. Small litters and increased numbers of fetal deaths were observed in mice exposed to lead on day 8 of intrauterine life. The live fetuses in this group were normal with respect to weight and morphological appearance. Serum levels of estradiol and progesterone, measured on day 17 of pregnancy, did not differ significantly between F1 mice of a control, unexposed group and of the group exposed to lead on day 8 of intrauterine life. Ovarian follicle counts revealed a significantly smaller number of primordial follicles in the latter group. It is suggested that the exposure to lead at a time of early organogenesis caused the observed fertility decrease by interfering with the development of the female germ cells.     

Influence of fetal position on neonatal androgen-induced sterility and sexual behavior in female rats

Two aspects of reproductive function were examined in relation to female fetus' contiguity to intrauterine male littermates. Following injection of 3.5 μg testosterone propionate (TP) on Day 3, females that had been positioned in utero between two males became sterile earlier in life than those located in utero between two females. Anogenital distances on Days 1 and 3 prior to neonatal treatment with TP were greater in females located in utero between two males than in females residing between two females in utero, suggesting that females developing between two males may have been exposed prenatally to a masculinizing substance, presumably an androgen. No reliable contribution of litter composition was apparent with respect to differentiation of female sexual behavior. Results indicate that littermate hormonal influence is present or effective and can be detected in a neonatally androgenized preparation.     

The production of and sensitivity to cues that delay puberty and prolong subsequent oestrous cycles in female mice are influenced by prior intrauterine position

Four experiments were conducted with CF-1 house mice (Mus domesticus) to examine the relationship between a female's prior intrauterine position (2M = between 2 male fetuses, 1M = next to one male fetus, 0M = not next to a male fetus) and the timing of puberty and length of subsequent oestrous cycles under a variety of housing conditions. The results of Exp. 1 confirmed that the presence of males was required for females to enter puberty and exhibit regular oestrous cycles, regardless of prior intrauterine position. When housed individually after weaning either with a male in the cage or separated by a wire mesh partition, 0M-females ovulated and mated at a younger age and had shorter post-pubertal oestrous cycles than did 2M-females (1M-females were intermediate between 0M- and 2M-females). Other females were also housed after weaning with a male or separated by a wire mesh partition from a male in a variety of social environments (4 0M-females and 1 2M-female, 4 2M-females and 1 0M-female, 5 1M-females, 3 0M-females and 3 2M-females). The objective of these different housing conditions was to determine whether prior intrauterine position influenced the transmission of and/or sensitivity to cues that delay puberty and prolong subsequent oestrous cycles. Relative to 2M-females, 0M-females both produced more potent cues and were more sensitive to the cues (again, 1M-females were intermediate between 0M- and 2M-females). Specifically, in the presence of other females, puberty was delayed and post-pubertal oestrous cycles were prolonged to the greatest extent in 0M-females. Prior intrauterine position is therefore a source of individual variation in the production of and sensitivity to cues that modulate the timing of puberty and the length of subsequent oestrous cycles in female mice. The findings suggest that prenatally-androgenized 2M-females may have a reproductive advantage over other females at high population densities.     

Fetal effects on sexual behavior and aggression in young and old female mice treated with estrogen and testosterone

During fetal life female mice (Mus musculus) that develop between two male fetuses (2M females) have higher blood concentrations of testosterone than do females that do not develop next to a male fetus (0M females). In the first experiment reported here, sexual receptivity and sexual attractiveness to males were examined in young (5 month old) and old (17 month old) ovariectomized, estrogen- and progesterone-treated 0M and 2M female mice that were placed in like-age pairs with a male. Most males inseminated the 0M female prior to inseminating the 2M female regardless of age. In addition, 0M females were more likely to exhibit lordosis when mounted than were 2M females. When the same young females were 9 months of age and the old females were 21 months of age, they were treated with testosterone and again placed together in pairs along with a sexually receptive female. Young 2M females exhibited more aggression toward the testosterone-treated female partner, and also exhibited more mounting of the receptive female, than did young 0M females. But, both old 0M and old 2M females were highly aggressive and exhibited mounting. An increase in sensitivity to the effects of testosterone on behavior thus occurs during aging in 0M females, which are relatively insensitive to testosterone in young adulthood. In contrast, when treated with estrogen and progesterone, 0M females were more attractive to males and were more sexually receptive than 2M females regardless of age.     

Male disadvantage? Fetal sex and cardiovascular responses to asphyxia in preterm fetal sheep

Clinically and experimentally male fetuses are at significantly greater risk of dying or suffering injury at birth, particularly after premature delivery. We undertook a retrospective cohort analysis of 60 female and 65 male singleton preterm fetal sheep (103-104 days, 0.7 gestation) with mean arterial blood pressure (MAP), heart rate, and carotid and femoral blood flow recordings during 25 min of umbilical cord occlusion in utero. Occlusions were stopped early if fetal MAP fell below 8 mmHg or if there was asystole for >20 s. Fetuses that were able to complete the full 25-min period of occlusion showed no differences between sexes for any cardiovascular responses. Similar numbers of occlusions were stopped early in males (mean: 21 min, n = 16) and females (mean: 23 min, n = 16); however, they showed different responses. Short-occlusion males (n = 16) showed a slower initial fall in femoral vascular conductance, followed by greater bradycardia, hypotension, and associated organ hypoperfusion compared with full-occlusion fetuses. In contrast, short-occlusion females (n = 16) showed a significantly more rapid early increase in femoral vascular conductance than the full-occlusion fetuses, followed by worsening of bradycardia and hypotension that was intermediate to the full-occlusion fetuses and short-occlusion males. Among all fetuses, MAP at 15 min of occlusion, corresponding with the time of the maximal rate of fall, was correlated with postmortem weight in males (R(2) = 0.07) but not females. In conclusion, male and female fetuses showed remarkably similar chemoreflex and hemodynamic responses to severe asphyxia, but some males did show impaired hemodynamic adaptation within the normal weight range.     

Uterine position and the activation of male sexual activity in testosterone propionate-treated female guinea pigs

Fetal female guinea pigs were delivered surgically and their position within a uterine horn relative to male fetuses was noted. When adult they were ovariectomized, injected daily with testosterone propionate, and examined for male copulatory behaviors in response to the presence of a receptive female. Females that developed contiguous to a caudal male were functionally similar to those that had been situated between two males; both exhibited enhanced levels of mounting relative to females that were located contiguous to no males (with and without the presence of a caudal male) and to females contiguous to a cephalic male. Therefore, both contiguity and positioning of the male fetus are necessary conditions for prenatal masculinization of the female guinea pig. Lastly, four animals that had resided singly in the uterus displayed the lowest levels of copulatory behaviors.     

Germline mutation rates in mice following in utero exposure to diesel exhaust particles by maternal inhalation

The induction of inherited DNA sequence mutations arising in the germline (i.e., sperm or egg) of mice exposed in utero to diesel exhaust particles (DEPs) via maternal inhalation compared to unexposed controls was investigated in this study. Previous work has shown that particulate air pollutants (PAPs) from industrial environments cause DNA damage and mutations in the sperm of adult male mice. Effects on the female and male germline during critical stages of development (in utero) are unknown. In mice, previous studies have shown that expanded simple tandem repeat (ESTR) loci exhibit high rates of spontaneous mutation, making this endpoint a valuable tool for studying inherited mutation and genomic instability. In the present study, pregnant C57Bl/6 mice were exposed to 19mg/m(3) DEP from gestational day 7 through 19, alongside air exposed controls. Male and female F1 offspring were raised to maturity and mated with control CBA mice. The F2 descendents were collected and ESTR germline mutation rates were derived from full pedigrees (mother, father, offspring) of F1 male and female mice. We found no evidence for increased ESTR mutation rates in females exposed in utero to DEP relative to control females. In contrast, a statistically significant increase in the mutation frequency of male mice exposed in utero to DEP was observed (2-fold; Fisher's exact p<0.05). Thus, maternal exposure to DEP results in increased mutation in sperm during development.     

Long-term exposure of male and female mice to 50 Hz magnetic field: effects on fertility

The effect of an extremely low frequency (ELF) magnetic field on the fertility of adult male and female Swiss mice was investigated. Adult male and female mice were exposed to a 50 Hz sinusoidal magnetic field at approximately 25 microT (rms) for 90 days before they were mated with unexposed counterparts. There were no exposure related effects on the fertility of male or female mice. The number of implantation sites, viable fetuses, and the total number of resorptions were not significantly affected in females impregnated by males exposed to the 50 Hz magnetic field as compared with the control group. The number of implantation sites, viable fetuses and the total number of resorptions in exposed females were also not statistically different from the control group. There were no significant effects on the weights of the testes, seminal vesicles, preputial gland or body weights of males exposed to 50 Hz magnetic field. Furthermore, body and uterine weights were not affected in females exposed to 50 Hz field; however, ovarian weight was significantly increased in females exposed to the same field. These results suggest that exposure of male and female mice to low frequency magnetic field had no adverse effects on fertility and reproduction in mice.     

Developmental Origins of Pregnancy Loss in the Adult Female Common Marmoset Monkey (Callithrix jacchus)

Background

The impact of the intrauterine environment on the developmental programming of adult female reproductive success is still poorly understood and potentially underestimated. Litter size variation in a nonhuman primate, the common marmoset monkey (Callithrix jacchus), allows us to model the effects of varying intrauterine environments (e.g. nutrient restriction, exposure to male womb-mates) on the risk of losing fetuses in adulthood. Our previous work has characterized the fetuses of triplet pregnancies as experiencing intrauterine nutritional restriction.

Methodology/Principal Findings

We used over a decade of demographic data from the Southwest National Primate Research Center common marmoset colony. We evaluated differences between twin and triplet females in the number of pregnancies they produce and the proportion of those pregnancies that ended in fetal loss. We found that triplet females produced the same number of total offspring as twin females, but lost offspring during pregnancy at a significantly higher rate than did twins (38% vs. 13%, p = 0.02). Regardless of their own birth weight or the sex ratio of the litter the experienced as fetuses, triplet females lost more fetuses than did twins. Females with a male littermate experienced a significant increase in the proportion of stillbirths.

Conclusions/Significance

These striking findings anchor pregnancy loss in the mother’s own fetal environment and development, underscoring a "Womb to Womb" view of the lifecourse and the intergenerational consequences of development. This has important translational implications for understanding the large proportion of human stillbirths that are unexplained. Our findings provide strong evidence that a full understanding of mammalian life history and reproductive biology requires a developmental foundation.     

MHC-mediated fetal odourtypes expressed by pregnant females influence male associative behaviour

Mice can recognize one another by individually characteristic phenotypic body odours (odourtypes) that reflect their genetic constitution at the highly polymorphic major histocompatibility complex (MHC) of genes on chromosome 17. We have shown previously that MHC-determined odours are produced by fetuses: house mice, Mus domesticus, can be trained to discriminate between genetically identical pregnant females carrying 9-18-day-old fetuses of differing MHC type. Theoretically, it should be possible for a mouse to determine the MHC type of the sire based on the odourtype of the pregnant female. In the current study we investigated whether untrained male mice show spontaneous discrimination between such pregnant mice. In experiment 1, sexually inexperienced male mice spent more time near pregnant females that carried fetuses most genetically different from the males themselves. Experiment 2, designed to evaluate possible experiential effects on this preference, tested males that were cohabiting and had impregnated a female that was either genetically identical to the test male (excepting X and Y chromosomes) or differed from him only at the MHC. Males in the former case performed virtually identically to those tested in experiment 1. In contrast males in the latter group did not display this preference. These studies reveal that among untrained male mice, fetal MHC type influences choice behaviour presumably via fetal odourtypes expressed in maternal secretions/excretions and that previous housing and/or mating experience modulates male choice. Copyright 2000 The Association for the Study of Animal Behaviour.     

Effect of position in the uterus on the fetal weight of the 21st day of pregnancy in the unilaterally ovariectomized rat

Effects of intrauterine position of the fetuses on the weight of fetal sac, fetus and placenta were investigated on the 21th day of unilaterally (right) ovariectomized pregnant Wistar strain rats. Results were obtained as follows. 1. Litter size was negatively correlated with the mean weights of fetal sac and fetus. 2. The mean weights of male fetal sac and fetus were heavier than those of female ones, while there was no difference in the weight of placenta between male and female. 3. The mean weights of male fetal sac and fetus positioning ovarian end (OV) were lighter than those of ones at the other positions. And the highest frequency of the lightest weight of live fetuses was demonstrated at OV position. 4. The mean weights of male fetal sac and fetus positioning between two males (2M) were heavier than those of ones positioning not next to male (0M). And the mean weight of 2M female fetal sac was heavier than those of 0M one. 5. The mean weights of fetal sac and fetus positioning next to resorbed fetus were heavier than those of ones positioning not next to resorbed fetus.     

Intrauterine growth restriction in rats is associated with hypertension and renal dysfunction in adulthood

Epidemiological studies have produced evidence that unfavorable intrauterine environments during fetal life may lead to adverse outcomes in adulthood. We have previously shown that a low-sodium diet, given to pregnant rats over the last week of gestation, results in intrauterine growth restriction (IUGR). We hypothesize that pups born with IUGR are more susceptible to the development of hypertension in adulthood. IUGR fetuses and rats aged 1 wk were characterized for organ growth and renal morphogenesis. The adults (12 wk) were evaluated for weight, systolic blood pressure, activity of the renin-angiotensin-aldosterone system (RAAS), and renal function; hearts and kidneys underwent a histological examination. Brain and cardiac ventricle-to-body ratios were increased in IUGR fetuses compared with age-matched controls, whereas the kidney-to-body ratio was unchanged. Systolic blood pressure was elevated in both IUGR male and female adults. Plasma aldosterone levels were not correlated with increased plasma renin activity. Moreover, urinary sodium was decreased, whereas plasma urea was elevated in both males and females, and creatinine levels were augmented only in females, suggesting a glomerular filtration impairment in IUGR. In our model of IUGR induced by a low-sodium diet given to pregnant rats, high blood pressure, alteration of the RAAS, and renal dysfunction are observed in adult life. Differences observed between male and female adults suggest the importance of gender in outcomes in adulthood after IUGR.