Effect of conspecifics on sexual maturation in female European pine voles (Pitymys subterraneus) J. Frankiewicz and A. Marchlewska-Koj Department of Genetics and Evolution, Institute of Zoology, Jagiellonian University, Krakow, Poland

Summary. Using the number of large ovarian follicles (Type 8) as an indicator of sexual maturation we found that urinary compounds released by adult males accelerated puberty while urine from females suppressed hormonal activity in juvenile female European pine voles. The release of chemosignals that delayed puberty of juvenile females was not influenced by ovarian hormones; urine from ovariectomized females was as effective as urine from unoperated animals.

Introduction

The timing of sexual maturation in rodents can be accelerated or delayed by social factors. Extensive studies on the house mouse (Mus musculus) reveal the important role that males play in hastening the onset of puberty in the female. Female mice reared in the presence of adult males attain puberty earlier than those reared in all-female groups (Vandenbergh, 1967). The acceleratory effect of the male is transmitted through olfactory stimulants released by the male with urine (see Vandenbergh, 1983). Another chemical effect is the delay of puberty by female stimulants. Drickamer (1977) showed that puberty in juvenile females can be delayed by exposure to the urine of grouped adult females. Similar chemosignal effects have been reported for microtine rodents. An accelerating effect of the male on onset of puberty in females has been reported for prairie voles, Microtus ochrogaster (Hasler & Nalbandov, 1974) and Microtus pennsylvanicus (Baddaloo & Clulow, 1981), and field voles, Microtus agrestis (Clarke & Clulow, 1973). The suppressive effect of the adult females on juvenile females is less well documented in these species. However, there is evidence that crowding of conspecifics suppresses ovarian activity in adult female pine voles, Microtus pinetorum (Schadler, 1980). The purpose of the present experiments was to test the influence of urine from males and females on the ovarian development of European pine voles, Pitymys subterraneus, during sexual development.

Materials and Methods

All the experimental pine voles came from an outbred stock colony reared in our laboratory. Animals were maintained in metal cages (40 20 25 cm) at 18-20°C, in a photoperiod schedule of 14 h light/24 h and fed with natural food (oats, wheat, apples and red beets) ad libitum. Wood shavings were provided as bedding and nesting material. Animals used for experiments were reared in sexually mixed groups until weaning at 21 days of age. After weaning young females with body weight of 9-9 ± 0-30 g (mean ± s.e.) were randomly allocated to 8 experimental groups and housed 1-2 animals per cage during the next 3 weeks. In

* Reprint requests to Dr A. Marchlewska-Koj.

Downloaded from Bioscientifica.com at 10/01/2021 04:42:04AM via free access Exp. 1, females were isolated, or caged with an adult male (not the father) or an adult female (not the mother); the adults were present in the same cage but separated by a mesh partition. In Exp. 2 the females were reared for 3 weeks in the presence of urine starting from the 21st day of life. A cage with juvenile females was connected by glass tubing with a metabolic cage in which an intact male or female, or an ovariectomized female was maintained for 24 h. Only urine reached the cage below and all faeces and food remnants were deposited in a special container. As urine donors, 18 males and 32 unoperated and 24 ovariectomized females were used. Urine donors were housed singly or as 6 animals per cage (grouped females). They were transferred to the metabolic cage once a week. Females were ovariectomized at least 4 weeks before use. All young females were killed when 42 days old. The body and uterus were weighed. Both ovaries were extirpated and fixed in Bouin's fluid, serially sectioned at 7 pm and stained with haematoxylin and eosin. The large ovarian follicles were classified in 3 separate groups, Types 6, 7 and 8 (Graafian follicles), according to diameter and to the number of granulosa cells in the largest cross-section (Pedersen & Peters, 1968). Large follicles with no cumulus cells and an oocyte surrounded by cell debris were assumed to be atretic follicles. Data were analysed by using A NOVA and Dunnett's tests (Zar, 1974).

Results

The body weights (mean + s.e.m., 13-9 ± 0-39 g) of 42-day-old females were similar in all experimental groups. Juvenile females reared in cages with mature females behind the mesh had significantly fewer Type 8 ovarian follicles than did control females (Table 1).

Table 1. Uterine weight and number of ovarian follicles in European pine vole females caged with an adult female or male

No. of ovarian follicles No. of Uterine females wt (g) Type 6 Type 7 Type 8 Atretic Total

Control 16 6-8 ± 0-37 8-7 ± 0-93 4-4 + 0-45 3-3 ± 0-42* 3-7 ± 0-58 20-1 ± 1-54 Female 13 7-5 ± 0-28 70 ± 0-67 3-8 ± 0-24 2-3 ± 0-26* 3-5 ± 0-47 15-8 ± 0-83 Male 9 8-1 + 0-58 9-3 ± 1-20 4-4 + 0-44 3-8 ± 0-36 3-7 ± 0-69 21-2 + 1-70

ANOVA NS NS NS F 4-2318 NS NS < 005 Dunnett's test *P < 005

Values are mean ± s.e.m.

The delay of puberty was more pronounced when females were exposed to urine from females (Table 2). The number of mature ovarian follicles was significantly decreased in the presence of urine ofintact and ovariectomized females. There were no differences in the reaction to the urine of singly housed and grouped females. Urine from males was associated with an increased number of ovarian follicles of Types 7 and 8 in comparison with the control group. The presence of urine from females also affected the uterine weights. Females exposed to urine from ovariectomized females had significantly smaller uteri than did controls.

Discussion

These results clearly indicate that European pine vole females are sensitive to the chemical signals of conspecifics. Urinary compounds released by adult males stimulated the hormonal system of juvenile females in such a way that there was an increase in the number of follicles ready for

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Downloaded from Bioscientifica.com at 10/01/2021 04:42:04AM via free access ovulation. We conclude that a male-acceleration of puberty effect occurs in European pine voles as it does in some other species (Vandenbergh, 1983). Using the number of large ovarian follicles as the indicator of sexual maturation (necessary because females of this species are reflex ovulators), we found that hormonal activity of pine vole females could be suppressed by the urine of conspecific adult females. The effects of chemosignals were not influenced by ovarian hormones and urine from ovariectomized females and unoperated animals was equally effective in delaying puberty. There is a strong similarity in the responses of house mice (Drickamer, Mclntosh & Rose, 1978) and European pine vole females. However, in house mice the maturation-delaying chemosignal was released only by grouped females and urine from singly reared animals did not evoke this delay effect (Drickamer, 1977). The influence of density on the release of the puberty-delaying chemosignal was confirmed in field experiments by Massey & Vandenbergh (1980). An increase in population density of house mice was correlated with the production of a substance in females that delays the onset of other female mice. In several species of rodents, the rate of sexual maturation and density of natural populations are negatively correlated (Krebs & Myers, 1974). Chemicals discharged by females could be one of the important factors limiting reproductive activity. Wild European pine voles do not occur in high-density populations (Wasilewski, 1960), perhaps explaining the fact that even urine from singly reared females delayed the onset of puberty in juvenile females. Experimental evidence provided by Mclntosh & Drickamer (1977) indicates that the maturation chemosignal is present in bladder urine of grouped and isolated female mice but the chemosignal is deactivated in the urethra of singly caged females. Such deactivation presumably does not occur in pine voles. We thank Dr J. Weiner, Jagiellonian University, for help with the statistical analysis and Dr L. C. Drickamer, Williams College, Massachusetts, for critical review of the manuscript. Sup¬ ported by Polish Academy of Sciences grant MR 11.10.

References

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