Reproductive physiology of the female short-tailed field vole (Microtus agrestis) with special reference to ovarian function

The basic reproductive physiology of the vole has been little studied in the past, although its population dynamics have been extensively investigated. In this thesis the reproductive physiology of the adult female is considered.
The work was stimulated by a hypothesis put forward by Chitty and Austin (1957); that 3-4 day oestrous cycles (vaginal and behavioural) occurred in females living apart from strangers, but constant cornification and heat was present if strangers were mixed together - the former implied spontaneous ovulation every few days; the latter implied induced ovulation.
The results of Chitty and Austin could not be reproduced, however; no regular 3-4 day vaginal smear changes were found in any females. Once vaginal perforation had become established, just over half the individuals had no leucocytes present in the smears; a smear that was predominantly cornified and/or nucleated was present for most of the time. No corpora lutea were found in the ovaries of any females. It thus became apparent that, regardless of the social environment, a state of induced ovulation was probably present.
Seven out of 11 adult females ovulated when placed, for 24 hours, with a vasectomised male - this confirmed that ovulation could be induced by the male, presumably by coitus, (mounting of 6 out of the 7 females that ovulated was seen during the 60-90 minute period of observation). Neither sexual excitement in females tested, that were not mounted, nor stimulation of the cervix with a glass rod, caused ovulation. From analysis of smears taken from females just prior to testing with males, it was found that mating could occur if either nucleated and/or cornified cells were present, but not in the presence of leucocytes.
Other environmental factors were investigated to ascertain whether or not spontaneous ovulation occurred under other conditions. Neither reduced light regimes, nor a known balanced diet permitted spontaneous ovulation. Light, however, greatly affected reproductive development and function, as shown by uterine weight, time of perforation, and follicle size - complete gonad development took place when animals were subjected to 14 or more hours of light; but there seemed to be more or less complete inhibition when subjected to 12 or less hours light per day. Food collected from the natural environment, given to laboratory animals, did not permit normal reproductive organ or body growth.
After environmental factors thought most likely to affect ovulation had been tested, an investigation of females in the natural environment was undertaken. Two groups of wild animals were caught, and caged in the wild for 1 month - one group was caged from late July to late August; the second, from late August to late September, No healthy corpora lutea were found in any individuals killed one month after capture. Thus, no evidence of spontaneous ovulation occurring in wild animals was obtained. All evidence indicated that the vole never ovulated under normal circumstances unless coitus occurred. Smear patterns (and mating tests) implied that virgin voles remained on heat for long periods of time.
In order to obtain some insight into whether uterine weights and smear patterns were related in any way to ovarian hormonal levels, voles were ovariectomised and given either no hormone replacement therapy, or variable doses of oestrogen, or a combination of oestrogen and progesterone. After ovariectomy vaginal closure took place within a few days, a marked drop in uterine weight also resulted. Oestrogen replacement appeared to inhibit these effects. Females given 0.01 andmu;gs. of oestrogen per day had variable smears although cornified cells were normally present; 1 andmu;g. of oestrogen per day invariably resulted in smears with nucleated epithelial cells. The mean uterine weight of the latter females was greater than that of females given 0.01 andmu;gs. of oestrogen. Thus, it appeared that nucleated epithelial smears and heavier uteri were related to higher oestrogen levels. Progesterone invariably induced a leucocytic invasion, followed by thin smears, and mucification of the vaginal epithelium; the size of the vaginal opening was also reduced.
Once it had been established that the vole was an induced ovulator, a reinvestigation of ovarian histology was considered to be necessary, as this had previously been likened to that of the mouse. It seemed plausible that if the vole's ovarian physiology differed from the mouse, so would its morphology.
A Graafian follicle and various stages of atresia were described. By killing groups of voles at approximate 2-hour intervals up to 18-20 hours after mating, morphological changes before and after ovulation were determined. Ovulation occurred about 9 hours after coitus; the latter appeared to affect all Graafian follicles and most, but not all of these, ovulated. There did not appear to be any marked preovulatory swelling.
Ovarian changes during pregnancy and pseudopregnancy were then studied. There were two waves of corpora luteal growth during pregnancy; the first took place within the first 3 days and the second during the latter half of pregnancy. Graafian follicles had reappeared by day 3 of pregnancy and were present throughout its length. Corpora lutea of pseudopregnancy were initially similar to those of pregnancy, but by day 8 some signs of degeneration were seen. Degenerative changes were very marked by day 10. Concurrent with luteal regression, there was a marked increase in Graafian follicle size.
The vaginal smears of pregnant and pseudopregnant animals were initially leucocytic. A thin smear then developed and mucification of the vaginal epithelium took place at about the same time. Thin smears remained until term in the pregnant animals, but disappeared after day 8 or 9 in the pseudopregnant females; this, therefore, indicated the length of pseudopregnancy in the vole.
No spontaneous ovulation occurred postpartum; thus no corpora lutea of lactation were formed. During this period, the corpora lutea of pregnancy degenerated quickly; the smears were variable but nucleated cells were usually predominant. It was found that mating could occur throughout the lactation period. No marked delay in implantation, following mating, took place at this time.
In the final chapter some aspects of the hypothalamo-hypophyseal control of ovulation were considered. Electrophysiological studies indicated that the anterior hypothalamus was most intimately concerned with the ovulatory response; electrical stimulation above or posterior to the pituitary region resulted in ovulation in very few individuals.
Intravenous injections of a crude sheep median eminence extract invariably resulted in some ovarian stimulation, ovulation occurred in most individuals; intraperitoneal injections did not appear to have such potent effects. Intraperitoneal injections of 0.6 andmu;gs. or more of luteinizing hormone resulted in ovulation in most individuals; 0.3 andmu;gs. of N.I.H. - LH - S or less had no effect. Intravenous injections of LH had a similar dose/response result. Ovulation occurred between 8 and 12 hours after a 2.5 andmu;g LH intraperitoneal injection.
This study has shown, therefore, that the vole, M.agrestis, appears to be a typical induced ovulator - no corpora lutea were ever found in virgin females subjected to various external environmental conditions, nor did ovulation occur spontaneously postpartum. Smear patterns suggested that the vole remained constantly on heat for long periods of time. Coitus usually occurred when perforate adult females were tested with virile males; this almost invariably resulted in ovulation. Electrical stimulation of the anterior hypothalamus, intravenous injection of extracts of median eminence, and 0.5 - 0.6 andmu;gs. or above of LH given either intravenously or intraperitoneally, to adult perforate virgin voles, usually brought about ovulation.