Effects of Physiological Factors on Follicular Development and Reproductive Output in Female European Ground Squirrels

In hibernating species like the European ground squirrel (Spermophilus citellus) reproduction is a time-constrained process. The animals have to complete mating, gestation, lactation and pre-hibernatory fattening during the short active season. Hence, ovulation and mating occur shortly after females emerge from hibernation. The prerequisite for ovulation is mature follicles. Follicular development, however, is a long-term process involving weeks or months of gonadotropin and estradiol. priming. Hibernation puts both physiological and time constraints on these processes: except during short arousal bouts, gonadal maturation is depressed in hibernating animals due to hypothermia. It would therefore be advantageous if follicular development were initiated during the previous active season. In fact, endocrine and preliminary histological data on European ground squirrels revealed that follicular maturation started in summer during late lactation. Because of the long time span required for follicular development, interactions with other activities may affect these processes. Lactation, for example, depresses LH secretion, thereby suppressing the LH-dependent processes of follicular maturation. Ecological factors and their influences on body condition may also affect gonadal maturation. As most studies on follicular development have been carried out on laboratory species or on livestock, only little is known about effects of ecological factors on these processes in free-living animals. The suggested project on female European ground squirrels, which is based on a long-term study on the same population, provides a unique opportunity to investigate follicular maturation in the field while examining ecological, physiological and behavioral parameters. The study is aimed at investigating the course of follicular development by monitoring gonadotropin and sex hormone profiles, ovarial status at critical phases of the annual cycle and reproductive output. In addition to the field study we propose an experimental approach to test the significance of specific factors. Experiments will be performed on animals living in a semi- natural enclosure within the same habitat in order to manipulate factors like lactation duration and diet while standardizing others. This type of analysis would broaden our understanding of the effects of physiological and ecological factors on follicular development (and hence reproductive output) of a free-living species that is temporally and physiologically constrained by hibernation.

The aim of this project was to document the course of follicular development and reproductive timing and success in European ground squirrels. The reproductive physiology of this hibernating species is of particular interest, since hibernation puts additional physiological and time constraints onto the normal reproductive processes. Females have to complete mating, gestation, lactation and prehibernatory fattening during the short active season and therefore produce only one litter per year. During hibernation, gonadal development is suppressed. Earlier studies have shown that females that mated shortly after emergence from hibernation had larger litters and spent more time lactating. To optimize reproductive timing it would therefore be advantageous to initiate follicular development during the previous season and gain precious time in spring. In addition to following the course of follicular development in individual females we investigated effects of female condition, age and lactational effort on these processes. Our hypothesis was that long lactation periods could delay reproduction in the following year due to their inhibitory effects on follicular growth. The study was carried out in a field enclosure within the natural habitat of the species. The ground squirrels kept in the enclosure did not differ from free-living conspecifics in their patterns of reproduction and hibernation. The secretion of estrogens and progesterone was measured throughout the season, and vaginal smears were collected to determine estrus phases. To investigate follicular development during summer, a unilateral ovarectomy was carried out in experimental females shortly before the onset of the hibernation period. The surgery took only a few minutes, and our results showed the procedure did not affect the subsequent reproductive capacity of these females. The ovarian material was analyzed with regard to the developmental stage of follicles and checked for the presence of tertiary follicles and corpora lutea. The results have provided some new insight into the control of reproduction in this species. Estradiol secretion was elevated during the mating period in spring, but peaked in July after lactation. Progesterone showed a similar pattern with maximum levels in the non-reproductive period before hibernation. These data show that ovarian activity was re-initiated after weaning and continued until the onset of hibernation. The histological analysis of the ovaries collected during summer showed intriguing results. We found different stages of development including mature Graafian follicles, and in some cases even active corpora lutea. These data demonstrate that the females go through an additional follicular cycle, with spontaneous ovulation and an active luteal phase before hibernating. The second cycle has no immediate reproductive value as males have regressed testes and are not sexually active at this time of the year. The histological results were further supported by vaginal cytology assays. A second vaginal estrus and diestrus phase occurred during summer, in line with the estradiol increases. The endocrine output of the corpus luteum which normally is important for early pregnancy, may aditionally stimulate other clusters of follicles and thereby have positive effects on the subsequent reproductive period. We have also investigated effects of age and lactation on these processes. Age had an effect in that juveniles showed less follicular development before hibernation than older individuals. Accordingly, in yearling females, estrus and mating were delayed in the subsequent spring. High maternal effort in terms of lactation duration also affected ovarian activity. Long lactating females re-initiated follicular development later than those that weaned their offspring after a shorter period. Our results demonstrate the impressive plasticity in the way these animals deal with the time and energy constraints of reproduction.