A Gerbil Dam's Fetal Intrauterine Position Affects the Sex Ratios of Litters She Gestates 

Physiology & Behavior, Vol. 57, No. 2, pp. 297-299, 1995 

[ Letter to Nature by Mertice M. Clark, Peter Karpiuk & Bennett G. Galef Jr. Nature v.364 19aug93 below ]

Mertice M. Clark ¹ and Bennett G. Galef, Jr.
Department of Psychology, McMaster University, Hamilton, Ontario, Canada L8S 4K1

CLARK, M. M. AND B. G. GALEF, JR. A gerbil dam's intrauterine position affects the sex ratios of litters she gestates. PHYSIOL BEHAV 57(2) 297-299, 1995.-The vaginally delivered litters of gerbil dams that, in utero, occupied intrauterine positions between two males fetuses (2M females) contain a greater proportion of sons than do the vaginally delivered litters of gerbil dams that were gestated between two female fetuses (2F females). This difference in the sex ratios of litters delivered vaginally by 2M and 2F gerbils might reflect sex-biased differences in perinatal mortality in the litters of 2M and 2F dams. However, the results of the present experiment, in which gerbil litters were delivered by caesarian section from females that developed in 2M and 2F intrauterine positions, demonstrated unequivocally that 2M female gerbils gestate male-biased litters, whereas 2F female gerbils gestate female-biased litters.

Received 23 March 1994

Intrauterine position    Sex ratio    Caesarian delivery    Cannibalism

¹ To whom requests for reprints should be addressed.

WE have recently reported (4) that the sex ratios of litters delivered vaginally by female Mongolian gerbils (Meriones unguiculatus) are influenced by the intrauterine positions that those females occupied as fetuses. When adult, female gerbils that developed between male fetuses (2M females) gave birth to litters containing a significantly greater proportion of sons than did females that developed between two female fetuses (2F females). Vandenbergh and Hewett (9) have found a similar correlation between the intrauterine positions in which mouse dams (Mrs musculus) developed and the sex ratios of their vaginally delivered litters.

The finding that the intrauterine position occupied by a female rodent affects the sex ratio of her litters is potentially important for understanding similarities between mothers and daughters in those behavioral and morphological characteristics that are affected by early exposure to androgens (6).

Female fetuses that mature between males are androgenized by testosterone crossing fetal membranes (7,12). Consequently, the adult phenotypes of 2M females differ significantly from those of 2F females that receive less intrauterine exposure to exogenous testosterone than do their 2M sisters (5,10,11,13,14).

If 2M females tend to gestate male-biased litters (and 2F females to gestate female-biased litters), daughters gestated by 2M females are more likely to occupy intrauterine positions between males than are daughters of 2F females (and, conversely, daughters gestated by 2F females are more likely to occupy intrauterine positions between female fetuses than are daughters of 2M females). A tendency of a female fetus to occupy the same intrauterine position as did its mother and, consequently, to receive a level of exposure to exogenous androgens similar to that which its mother received (4) should result in similarities in the phenotypes of mothers (either androgenized in the case of 2M females or not androgenized in the case of 2F females) and daughters (4).

However, the correlation between the intrauterine positions of dams and the sex ratios of their litters at birth described by Clark et al. (4) may not be a result of differences in the sex ratios of litters gestated by 2M and 2F females (8). If, during the perinatal period, 2M females tend to cannibalize daughters and 2F females to cannibalize sons, one would observe a correlation between the intrauterine positions occupied by dams and the sex ratios of their litters shortly after birth, even if 2M and 2F dams gestated litters having the same sex ratios. And, if the difference in sex ratios of litters delivered vaginally by 2M and 2F females observed by Clark et al. (4) were the result of sex-biased perinatal cannibalism (rather than of biases in the sex ratios of litters gestated by 2M and 2F females), then daughters of 2M females would be no more likely to mature in intrauterine positions between males than would daughters of 2F females, and there should be no concordance between mothers and daughters in the expression of those phenotypic characteristics affected by different levels of prenatal exposure to androgens.

In the present experiment, litters of 2M and 2F gerbils were delivered by caesarian section to determine whether 2M and 2F gerbils do, in fact, gestate litters with different sex ratios.

METHOD

Subjects were 10 2F and seven 2M female Mongolian gerbils that had been delivered by caesarian section on day 24 of their gestation. All subjects were descended from breeding stock acquired from Tumblebrook Farm (Brookfield, MA).

Each subject was foster reared by a gerbil dam that had given birth during the 24 h preceding caesarian delivery of the subject female she was to rear (1).

Maintenance and housing. When each subject was 60 days of age, she was placed together with a sexually proven adult male in a 35 x 30 x 15 cm polypropylene, shoebox cage.

Each breeding pair was maintained on ad lib Purina Laboratory Rodent Chow #5001 and tap water on a 12:12 h light:dark cycle (light onset 0500 h) in a temperature- and humidity-controlled colony room.

The date on which each pair first copulated was noted, with the expectation that the female would deliver 25 days after she was observed to mate. As each female entered the final week of her pregnancy, her consort was removed from her cage.

Surgical procedures. Twenty-four days following observed copulation (i.e., 1 day prior to anticipated vaginal delivery), each female that had gained weight at a rate consistent with her impregnation on the observed day of copulation was anesthetized by metofane (Janseen Pharmaceutica, Mississauga, Ontario) inhalation, her abdomen opened, her uterus externalized, and her fetuses removed. Once all the fetuses had been removed from a dam, she was sacrificed by halothane (Ayerst Laboratories, Montreal) inhalation.

An observer unaware of the intrauterine position in which dams had matured examined the anogenital distance of each caesarian-delivered fetus to determine its sex (2). The sex of a Mongolian gerbil pup on its last day of gestation is easily determined by examination of its anogenital area. However, to ensure the reliability of sex assignment, 20% of pups delivered by caesarian section were foster reared by gerbil dams that had delivered a litter vaginally within 24 h of a caesarian delivery. As we have found previously (2), no errors were made in sex assignment based on inspection of anogenital distance.

RESULTS AND DISCUSSION

As can be seen in Table 1, there was no reliable difference in the total number of pups found in the litters of 2M and 2F females delivered by caesarian section [Student's t-test, t(15) = 0.56, NS]. However, the litters of 2M females contained reliably more males than did the litters of 2F females, t(15) = 2.93, p < 0.01, and the litters of 2F females contained slightly, but not significantly more females than did the litter of 2M females, t(15) = 1.88, p < 0.07.

Table 1. Composition of Litters Delivered by Caesarian Section From 2M and 2F Mongolian Gerbils on Day 24 of Gestation

Figure 1. Mean percentage of male fetuses found in Mongolian gerbil litters gestated by 2M and 2F females and delivered by caesarian section on day 24 of pregnancy. Numbers inside histograms = N/group. Flags = + 1 SEM.

The main results of the experiment are presented in Fig. 1, which shows the mean percentage of male pups in the 17 litters delivered by caesarian section from 2M and 2F female gerbils on their 24th day of gestation. As is evident from inspection of Fig. 1, the caesarian-delivered litters of 2M gerbils contained a greater proportion of male fetuses than did the caesarian-delivered litters of 2F gerbils, t(15) = 3.36, p < 0.01.

The finding that the caesarian-delivered litters of 2M females contained a greater proportion of male fetuses than did the caesarian- delivered litters of 2F females is not consistent with the hypothesis (8) that observed differences in the sex ratios of litters delivered vaginally by 2M and 2F female gerbils (4) result from sex-biased perinatal mortality. To the contrary, the results of the present experiment demonstrate that 2M and 2F female Mongolian gerbils gestate litters containing significantly different proportions of male fetuses. The present data thus support the contention (3,4) that daughters born to 2M gerbils are more likely than are daughters of 2F gerbils to be 2M females (and, conversely, that daughters of 2F gerbils are more likely than are daughters of 2M females to be 2F females). Consequently, as Clark et al. proposed (3,4), there should be nongenetic transmission of androgen-sensitive phenotypic characteristics from one generation of female Mongolian gerbils to the next.

ACKNOWLEDGEMENTS

This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada to both M.M.C. and B.G.G.,Jr. and a McMaster University Research Board grant to B.G.G.,Jr. We thank Martin Daly for a thoughtful critique of an earlier draft.

REFERENCES

1. Clark, M. M.; Galef, B. G., Jr. Effect of uterine position on rate of sexual development in female Mongolian gerbils. Physiol. Behav. 42:15-18; 1988.

2. Clark, M. M.; Galef, B. G., Jr. Sexual segregation in the right and left horns of the gerbil uterus: "The male embryo is usually on the right and the female on the left" (Hippocrates). Dev. Psychobiol. 23:29-38; 1990.

3. Clark, M. M.; Galef, B. G., Jr. Sex-ratio and inheritance. Nature

367:327-328; 1994.

4. Clark, M. M.; Karpiuk, P.; Galef, B. G., Jr. Hormonally mediated inheritance of acquired characteristics in Mongolian gerbils. Nature 364:712; 1993.

5. Clark, M. M.; Robertson, R. K.; Galef, B. G., 1r. Intrauterine position effects on sexually dimorphic asymmetries of Mongolian gerbils: Testosterone, eye-opening and handedness. Dev. Psychobiol. 26:185-194; 1993.

6. Clark, M. M.; Spencer, C. A.; Galef, B. G., Jr. Reproductive life history correlates of early and late sexual maturation in Mongolian gerbils (Meriones unguiculatus). Anim. Behav. 34:551-560; 1986.

7. Even, M. D.; Dhar, M. G.; vom Saal, F. S. Transport of steroids between fetuses via amniotic fluid in relation to the intrauterine position phenomenon in rats. J. Reprod. Fertil. 96:709-716; 1992.

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9. Vandenbergh, J. G.; Huggett, C. L. Mother's prior intrauterine position affects the sex ratio of her offspring in house mice. Proc. Nat. Acad. Sci. USA (in press).

10. vom Saal, F. S.; Bronson, F. H. In utero proximity of female mouse fetuses to males: Effect on reproductive performance during later life. Biol. Reprod. 19:842-855; 1978.

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12. vom Saal, F. S.; Dahr, M. G. Blood flow in the uterine loop artery and loop vein is bidirectional in the mouse: Implications for transport of steroids between fetuses. Physiol. Behav. 52:163171; 1992.

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14. vom Saal, F. S.; Pryor, S.; Bronson, F. H. Effects of prior intrauterine position and housing on oestrous cycle length in adolescent mice. J. Reprod. Fertil. 62:32-37; 1981.

Nature v.364 19aug93

LETTERS TO NATURE

Hormonally mediated inheritance of acquired characteristics in Mongolian gerbils

Mertice M. Clark, Peter Karpiuk & Bennett G. Galef Jr
Department of Psychology, McMaster University, Hamilton,
Ontario L8S 4K1, Canada

THE intrauterine position relative to members of the same or opposite sex that a rodent fetus occupies affects both its morphology and behaviour when adults^1-14. Female fetuses that mature between males are androgenized by testosterone crossing fetal membranes^{15, 16}, and their phenotypes as adults differ significantly from those of sisters that received less intrauterine exposure to exogenous testosterone"^17-20. We report here that adult female Mongolian gerbils that gestated between male fetuses produce litters containing a significantly greater proportion of sons than the litters produced by those that gestated between female fetuses. Consequently, daughters delivered by dams that gestated between male fetuses are more likely to have gestated between male fetuses and be androgenized in utero than are daughters of dams that gestated between female fetuses. Female gerbils thus tend to inherit the phenotype (either androgenized or not androgenized) of their respective mothers.

In Mongolian gerbils (Meriones unguiculatus), as in other rodents, the intrauterine position that a female occupies relative to the sex of its neighbours has a profound impact on its development. Female gerbil fetuses that gestate between two male fetuses (2M females) are exposed to higher levels of testosterone than those that mature between two female fetuses (2F females)²¹, The level of exposure to androgen in utero affects both the course of development and the reproductive life history of a female gerbil. For example, androgenized, 2M female gerbils achieve puberty at a significantly greater age than their 2F sisters²² and, when adult, these late-maturing females have only half the lifetime fecundity of their early-maturing sisters²³.

Twenty-five 2M and 25 2F female gerbils were delivered by caesarian section on the last day of their normal gestation24. Each subject female and its littermates were then foster reared by a dam that had produced a litter vaginally on the same day²⁴.

We mated each of our 50 subject females with a sexually proven male when she was 67 days old. On the day of each vaginal delivery an experienced observer who was unaware of the intrauterine position that a dam had occupied as a fetus, examined the anogenital distance of each pup to determine its gender²⁵. We found a significantly greater proportion of males in litters born to 2M females (57.1 + 3.5%) than in litters born to 2F females (43.7 + 4.8%; Student's t-test, to = 2.73, d.f. 48, P < 0.01). In addition, 2M females from litters that contained a majority of females (N = 5) produced a significantly greater percentage of sons throughout their reproductive lifetimes (53.9%) than 2F females from litters that contained a majority of males (N = 4; 44.7%; Mann-Whitney U test, U= 4, P < 0.05, one-tailed). Thus intrauterine position, rather than the sex ratio of litters at birth, predicted the proportion of males in litters. This is important because although the sex ratio of a litter might be influenced by the genotype of its dam, the positions of individual fetuses within uterine horns are random²⁵ and are unaffected by maternal genotype. The finding that a female's intrauterine position (rather than the sex ratio of her natal litter) predicts the sex bias of her lifetime reproductive effort is, therefore, not consistent with the hypothesis that differences in the genotype of 2M and 2F females are transmitted to their respective daughters and cause concordance in the sex bias of litters produced by mothers and daughters.

Table 1. Proportion of daughters born to 2M and 2F dams expected to gestate in 2M and 2F intrauterine positions

The probability that a fetus will develop between two fetuses of the same or opposite sex is affected by four variables: (1) the sex ratio; (2) size of the litter of which it is a member; and the distribution either (3) within or (4) between uterine horns of the male and female fetuses in a litter.

In previous studies, we determined the number of male and female fetuses carried in the left and right uterine horns of 253 caesarian-delivered gerbil dams on their last day of pregnancy²⁵, and the sex ratios of 265 vaginally delivered litters born in our colony²⁶. The 253 caesarian-delivered dams gestated equal numbers of pups in each uterine horn, but carried a significantly greater proportion of male pups in their right uterine horns and female pups in their left uterine horn S25. We observed no deviation from chance in the distribution of male and female pups within each uterine horn. The mean and distribution of the sex ratios of caesarian and vaginally delivered gerbil litters were not significantly different²⁶.

Using these data and the present observations of the sex ratios of our sample of 50 vaginally delivered litters, we computed the expected production of 2M and 2F daughters by both 2M and 2F dams (Table 1). We found the expected probability that the daughter of a 2M mother would be a 2M female was 1.73 times greater than the expected probability that she would be a 2F female. Conversely, the expected probability that the daughter of a 2F mother would be a 2M female was only 0.60 of the expected probability that she would be a 2F female.

The effect of intrauterine position on the sex ratios of litters produced by female gerbils shows that there is a form of hormonally mediated transmission of acquired characteristics. This produces concordance between Mongolian gerbil dams and their daughters in those phenotypic characteristics that are affected by level of exposure to testosterone early in life.

Received 17 February; accepted 21 May 1993.

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ACKNOWLEDGEMENTS. We thank M. Bryden for technical assistance and D. Chiszar for his comments on earlier drafts. This research was funded by grants from the Natural Sciences and Engineering Research Council of Canada to M. M. C. and B. G. G.