(Received 10 September 1965)

402 r J. Phy8iol. (1966), 184, pp. 402-417 L I B R A R Y ) Printed in Great Britain 4MASS THE IP BETWEEN OVULATION AND THE CHA THYROID GLAND ACTIVITY THAT OCCUR DURING THE OESTROUS CYCLE IN RATS, MICE AND HAMSTERS BY K. BROWN-GRANT* From the Department of Human Anatomy, South Parks Road, Oxford (Received 10 September 1965) SUMMARY 1. When ovulation is prevented by the injection of pentobarbitone on the day of pro-oestrus in female rats, the expected increases in thyroidserum concentration ratio (T/S ratio) for 131I and in the uptake of 113I by the thyroid on the day of oestrus do not occur. 2. Ovulation induced by human chorionic gonadotrophin or ovine luteinizing hormone in rats treated with pentobarbitone at pro-oestrus is not associated with an increase in T/S ratio or 131I uptake at oestrus. 3. These results support the view that it is the neuro-endocrine changes at the hypothalamic or pituitary level that lead to ovulation rather than changes in the pattern of ovarian steroid secretion before or at ovulation that are responsible for the increased thyroid activity at oestrus in the female rat. 4. Experimental evidence of an increase in thyroid activity after ovulation in mice and hamsters also supports this view. INTRODUCTION In agreement with previous workers it was found that the activity of the thyroid gland was highest during the oestrous stage of the ovarian cycle in rats (Brown-Grant, 1962a) and it was suggested that the changes might be due to a discharge of pituitary thyrotrophic hormone (TSH) in late prooestrus at about the time of the increase in luteinizing hormone (LH) secretion that leads to ovulation. This suggestion was supported by the results obtained in a small number of rats in which ovulation was blocked by the injection of sodium pentobarbitone (Nembutal, Abbott) during late pro-oestrus and the expected increase in radio-iodine uptake by the thyroid did not occur (Brown-Grant, 1963), by observations made on rats in which spontaneous ovulation was permanently blocked by the administration * Locke Research Fellow of the Royal Society.

OVULATION AND THE THYROID 403 of testosterone during the neonatal period (Brown-Grant, 1963, 1965b), and from studies of pregnant and pseudopregnant rats (Brown-Grant, 1965a). It was not clear, however, whether the postulated discharge of TSH was directly associated with the neuroendocrine changes leading to the ovulatory discharge of LH or whether the sequence of events was hypothalamic stimulation of LH release leading to a change in ovarian steroid secretion, the steroids acting on the pituitary or hypothalamus to induce TSH discharge. If the second explanation were correct, then after suppression of endogenous LH release the injection of exogenous gonadotrophin to bring about ovulation might be expected to restore the thyroid changes. This possibility has been investigated and the negative results obtained are reported in this paper. In addition the changes occurring in thyroid gland activity during the oestrous cycle in the mouse have been reexamined and the relationship to ovulation determined. A similar study in the golden hamster is also described. METHODS Rats. Conditions of housing, lighting, diet, etc., of the virgin adult female Wistar rats from the closed colony maintained in the author's laboratory were as in previous studies (Brown- Grant, 1965a). The lights were switched on automatically at 08.00 hr and off at 22.00 hr. Vaginal smears were taken daily and only animals with a history of at least three regular cycles of 4 days' duration immediately before the experiment were used. A typical 4-day cycle consists of the day of oestrus, when a fully cornified smear is obtained; metoestrus, the next day and the first day on which leucocytes appear; dioestrus, the next day and the second day on which leucocytes are present; and pro-oestrus, the next day, when the smear contains nucleated epithelial cells only. Blockade of ovulation was produced by a single subcutaneous injection of Nembutal (sodium pentobarbitone, B.P., Veterinary Nembutal, Abbott Laboratories) at a dose level of 36 mg/kg body wt. on the day of pro-oestrus. The usual time of injection was 17.00 hr but injections were also made at other times in one series of experiments. The condition of the genital tract was carefully studied at post mortem and a note was made of the presence or absence of free fluid in the uterus, the state of the ovaries, whether grossly hyperaemic or pale, the presence of fresh corpora lutea and the state of the corpora lutea, whether pale and watery or pink, the appearance of the interstitial tissue and the presence or absence of large follicles visible to the naked eye. The description by Everett (1948) of the pro-oestrous and oestrous genital tract was of great help in the early stages of this work. The oviducts were examined under a dissecting microscope for the presence of swollen segments and eggs removed and counted if present. No animal was recorded as having ovulated unless freshly shed eggs were recovered from the oviduct. As discussed later, the appearance of the uterus and ovaries (except for the absence of large follicles) could be typical of pro-oestrus despite the presence of fresh eggs in the oviduct under certain circumstances. Blockade of ovulation was always associated in these experiments with the presence of a typical 'ballooned' fluidfilled uterus and a pale ovary with fatty interstitial tissue and visible follicles, and with the absence of a swollen segment or tubal ova on what would normally have been the day of oestrus. Vaginal smears were almost invariably fully cornified on the day of oestrus whether ovulation occurred or not, and were of no use as a guide. With these criteria it was possible to be confident that ovulation had been blocked without routine histological examination of the ovaries for the presence of ripe follicles.

404 K. BROWN-GRANT Mice. Adult virgin female albino mice bred in a closed laboratory colony were used. These animals are of the Parkes strain and are descended from animals originally obtained from the colony maintained at the National Institute for Medical Research, Mill Hill, London. The diet was the same as that of the rats (tap water plus pellets, Diet 41B from E. Dixon and Sons Ltd., Ware, said to contain 117,ug/kg iodine) and they were kept under the same conditions of temperature and lighting as the rats. Housing conditions may have profound effects on the regularity and duration of the oestrous cycle in the mouse (Parkes & Bruce, 1961). The animals used in these experiments were housed in groups of three in metal boxes 13 in x 7 in x 4i in (33 cm x 17 8 cm x 11 4 cm) deep with solidwalls andagrilledtop. Vaginal smears were taken daily. Cycles are neither as regular nor as predictable as in the rat and spontaneous pseudopregnancies do occur under these conditions. Detailed descriptions of the cycles observed are given under Results. At post-mortem the oviducts were examined for eggs as in the case of rats. Ham8ter8. The animals were a cream-coated variant of the golden hamster, (ricetu8 auratu8, obtained from the inbred colony maintained by Glaxo Research Ltd., Uxbridge, Middlesex, at about 6 weeks of age. They were housed either in solid metal boxes or in conventional grilled rat cages in groups of three to six, under the same conditions of lighting and temperature as the rats and mice. The diet was tap water and the pelleted rat diet with, in addition, various supplements of cabbage, oats and brown bread, etc., three or four times a week. Cycles were followed by daily examination for the occurrence of the profuse external 'metoestrous' vaginal discharge (Ward, 1946). This designation is correct only in that the discharge follows the period of behavioural oestrus; the day on which it is observed is the day on which sperms are found in the vaginal smear if the animals have mated, and eggs are found in the oviduct if the animals are killed on this day but not at other times. This day is better regarded as the day of oestrus as in the rat and this convention is followed here. Vaginal smears were not taken from the hamsters, but daily external examination established that two or more regular cycles had occurred before the animals were used. The oviducts were examined for tubal ova after killing as in the case of rats and mice. A88e8sment of thyroid gland activity. Uptake of radio-iodide (1831) by the gland expressed as percentage of the injected dose per gland was used exclusively in mice and hamsters. The dose was 0-5,c injected intramuscularly and the animals were killed 2-5 hr later. This method was also used for some of the experiments on rats but for the most part the T/S ratio determined 2 hr after the injection of 131I in rats pre-treated with 4-methyl-2- thiouracil to block organic binding was employed. Details have been given by Brown- Grant (1962a, 1965a). Statistical methods. Analysis of variance using Snedecor's F ratio and comparison of group means by the t test were used. 'Not significant' indicates a value for P > 0.05. All values quoted in text and in tables are means + s.e. of the mean. RESULTS The effect of pentobarbitone on ovulation and on T/S ratios in rats Altogether 101 rats were used in this experiment in four groups of 29, 21, 26 and 25 rats; analysis of variance showed that there was no significant variation in T/S ratio between the groups, and results were therefore combined. Results were obtained from 74 rats which were injected with pentobarbitone at different times on the day of pro-oestrus and the T/S ratio and the occurrence or non-occurrence of ovulation determined on the morning of the next day, that is, the predicted day of oestrus. In addition, T/S ratios were determined for nine control rats on the day of dioestrus

_~~~~~~~~~~ OVULA TION AND THE THYROID 405 (87.7 + 6.5) and for eighteen control rats (all of which had fresh eggs in the oviduct) on the day of oestrus (151-8 + 10.3). The difference between the two groups was significant (P < 0-001), as expected. The individual results obtained in the rats injected with pentobarbitone are given in Table 1. In this table values for rats in which ovulation has been blocked are given in heavy type. Considering first the effect of pentobarbitone on ovulation, this was blocked in two of twelve rats injected at 13.00 hr, six of twelve injected at 15.00 hr, nineteen of 23 injected at 17.00 hr, eight of sixteen injected at 19.00 hr and none of eleven injected at 21.00 hr. It is clear that the 'critical period', see Discussion, for LH release in this colony is between 13.00 hr. 15.00 hr. 17.00 hr. 19.00 hr 21.00 hr. 138-8 131-1 129-3 95-2 112-9 58-0 135-1 156-3 222-2 96-1* 85-6 106-9 124-2 164-5 150-1 132-6 114.0* 77-8 105.6* 170-4 134-8 102-6 198-0 100.9** 94-0 70.7** 125-3 120-2 124-9 124-4 105-9 177-8 89.3** 74-4 129-9 165-0 114-1* 94-5 96-8 65-2** 110-9 134-5 134-5 108-9 112-6 85-7 77-3** 71.1 115.5* TABLE 1. Thyroid-serum (T/S) ratios for female rats injected with pentobarbitone at the times indicated on the day of pro-oestrus and killed on the morning of the next day (day of expected oestrus). Values in heavy type are for animals which did not ovulate. * indicates an animal which ovulated but had a pro-oestrous uterus and ** an animal which ovulated but had a pro-oestrous uterus and pro-oestrous ovary as described in the text 166-9 99.7 74-8 121-9 52-4** 180-4 165.5* 65-1* 103-3 85-0 100-5 157.7** 118.2* 103-9 69-7 105-9 143-0** 111-2 104-2 86-3 96-6 99.5** 66-0 191-1 102-7 18.00 and 20.00 hr in the majority of animals and that the pentobarbitone is most effective when injected at 17.00 hr. The first comparison of T/S values that is of interest is between the 39 rats in which ovulation was not blocked (124-8 + 5.7) and the 35 in which ovulation was blocked (104-9 + 5.4). The difference is significant (P < 0-02) and it would appear that when ovulation is blocked in this way the expected high value for the T/S ratio on the morning of the day of oestrus is not obtained. However, the mean T/S ratio for all animals injected at 17.00 or 19.00 hr, and indeed the values for animals that did ovulate despite pentobarbitone as well, are minimal in the 17.00 and 19.00 hr groups (all rats: 13.00, 125-0; 15.00, 136-2; 17.00, 100-4; 19.00, 100-1; 21.00, 136-4; rats that ovulated: 13.00, 132-2; 15.00, 153-7; 17.00, 110-1; 19.00, 85-8; 21.00, 136-4). It could be argued that the low mean values for rats that did not ovulate is related to the fact that the majority of these were in the 17.00 and 19.00 hr groups and that this reflects a non-specific depression of the T/S ratio occurring within a restricted time period after the injection of pentobarbitone. This possibility was checked in two ways. First, male rats were injected with

406 K. BROWN-GRANT the same dose of pentobarbitone at 13.00, 15.00, 17.00, 19.00 or 21.00 hr or left untreated and the T/S ratios determined the next morning at the same time after pentobarbitone injection as in the experiments on female rats. The experiment was actually carried out on three separate days; analysis of variance showed no significant differences between the 3 days, between rats injected at different times, or between injected and control rats. Mean values (eight rats per group) were 161-2 + 15-2 for rats injected at 13.00 hr; 137-0 + 8*2 at 15.00; 155-4 + 9-3 at 17.00; 155-6 + 2241 at 19.00; 145X4 + 9-2 at 21.00; and 145X5 + 7X4 for control rats not receiving pentobarbitone. The mean value for the 40 pentobarbitone-treated rats was 150*9 + 6-0 and this was not significantly different from the control value. Secondly, a small group of female rats was injected with pentobarbitone at 17.00 hr on the day of metoestrus and T/S ratios determined on the morning of the next day, the day of dioestrus. The mean value for six rats was 104-9 + 14x5 and this was not significantly different from the value of 98-2 + 14-7 for six uninjected animals in dioestrus. No evidence was obtained to support the idea that pentobarbitone injected at 17.00 or 19.00 hr on the day before the T/S ratio was determined (approximately 16 hr before the injection of 131I and 18 hr before killing the animals) depressed the T/S ratio in male rats or female rats not in pro-oestrus at the time of injection. The data of Table 1, however, taken together with the value for the T/S ratio on the day of oestrus in eighteen rats not injected with pentobarbitone of 151-8 + 10-3, do suggest that there has been some effect of pentobarbitone on the T/S ratio even in the rats that ovulated. The mean value for thirty-five rats in which ovulation was blocked was, as previously stated, 104-9 + 5*4, significantly lower than the value for control animals in oestrus (P < 0 001) or for the thirty-nine pentobarbitone-treated rats that did ovulate (124.8 + 5.7, P < 0 02), but this latter value is itself significantly lower than that of the control animals (P < 0 02). Inspection of Table 1 shows that the low T/S ratios in animals which did ovulate were mainly in the groups injected at 15.00, 17.00 and especially 19.00 hr, when the blockade of ovulation was effective in 50% or more of animals. Indeed the value for T/S ratio in the twenty-one of twenty-three animals injected at 13.00 or 21.00 hr that did ovulate is 134-4 + 5-6 and this is not significantly different (P = 0.05) from the value of 151-8 for uninjected control animals in oestrus. The concentration of low values in rats that ovulated in the groups injected some time after the beginning of the 'critical period' suggested that some interference with or curtailment of the later period of LH discharge, though not sufficient to prevent follicular rupture, might have occurred and might be related to the low T/S values obtained which in turn are presumed to reflect the absence of an increase in TSH secretion. The appearance of the genital tract in the pentobarbitone-treated rats

OVULATION AND THE THYROID 40'7 provided some evidence of interference with LH discharge though not of a degree sufficient to prevent ovulation. Before the values for the T/S ratios of individual rats were known, careful notes had been made of the state of the uterus and ovaries at post-mortem and, before a possible relation to the T/S values other than with respect to the occurrence or failure of ovulation was suspected, it had become clear that rats could be assigned to one of four categories. First, all rats that failed to ovulate had the typical pro-oestrous genital tract with fluid-filled uterus and pale ovary with pale, watery corpora lutea, fatty interstitial tissue and macroscopically visible follicles. Of the rats that had ovulated, the majority had turgid, pink, fluid-free uteri, hyperaemic ovaries with creamy pink corpora lutea, clear interstitial tissue and no visible follicles. This is the typical appearance of the genital tract on the day of oestrus. Other pentobarbitone-treated rats that had ovulated, however, had the typical oestrous ovary but with persistence of the fluid-filled uterus characteristic of pro-oestrus. Animals of this group are marked with a single asterisk (*) in Table 1. Finally, other pentobarbitone-treated rats at first examination appeared not to have ovulated and to have a genital tract typical of pro-oestrus with a distended uterus and pale ovary with watery corpora lutea and fatty interstitial tissue. Closer examination of the ovary, however, showed that few or no large follicles were present and the oviducts contained typical swollen segments with the usual five or six eggs on each side. Animals of this type are marked with a double asterisk (**) in Table 1. It seems probable that rats that had ovulated and had the typical oestrous genital tract, that had ovulated but had pro-oestrous uteri, and that had ovulated but had pro-oestrous uteri and ovaries (except for the absence of follicles) represent animals exposed to progressively less LH stimulation or exposed for a shorter period of time. From the notes made before T/S values were known, pentobarbitonetreated rats that had ovulated were assigned to one of these three categories, as shown in Table 1. It is obvious that most of the atypical genital tracts were noted in rats that received pentobarbitone during or towards the end of the critical period. It is also noticeable that these are the rats which had ovulated but none the less had low T/S ratios. The mean value for the thirty-nine animals that ovulated was 124-8 + 5-7, significantly less than the value for the eighteen control oestrous rats of 151-8 + 103; the twenty-two pentobarbitone-treated rats that ovulated and had typical oestrous genital tracts had a mean T/S ratio of 141-6 + 6-1 which is not significantly different from the control value, but is significantly higher (P < 002; P < 0-01) than the value for either the eight animals that ovulated but had pro-oestrous uteri (111.8 + 9 8) or the nine animals that ovulated but had both a uterus typical of pro-oestrus and a pale, atypical ovary (95.1 + 11.7). The values for the last two groups do not differ signi-

408 K. BROWN-GRANT ficantly but each is lower (P < 0 05 and P < 0-01, respectively) than the value for uninjected control rats at oestrus. The curtailing of the period of LH secretion, while still allowing rupture of the follicles, results in a retention of the fluid formed at pro-oestrus in the uterus; this suggests a failure of progesterone secretion (Everett, 1948) and the absence of the typical macroscopic change in corpora lutea and interstitial tissue probably represents a failure of the normal effect of LH on cholesterol content (Everett, 1948). This is associated with a failure of the T/S ratio to increase and raises the possibility that it is the later period of LH action on the ovary leading to increased progestational steroid secretion which is related to TSH release. If so, replacement of endogenous by exogenous gonadotrophin in the pentobarbitone-blocked rat might restore the thyroid changes, and this possibility was tested. The effect of exogenous gonadotrophin in pentobarbitone-treated rats In the first experiments the T/S ratio was employed as an index of the level of thyroid gland activity. The control group consisted of seven rats killed on the day of oestrus with no previous treatment. All these had tubal ova and the mean T/S ratio was 167-8 + 8-4. A group of eight rats received pentobarbitone at 17.00 hr on the day of pro-oestrus and were killed the next morning; ovulation had been blocked in seven out of eight rats. The mean value for all eight rats (in all these experiments values for animals in which blockade of ovulation failed are included in the group mean to allow for a similar proportion of failures in the pentobarbitonetreated, gonadotrophin-injected group) was 1050 + 31, and this was significantly different from the value for the control group (P < 0-001). Seven rats were injected with pentobarbitone at 17.00 hr in pro-oestrus and immediately afterwards received a subcutaneous injection (at a site remote from that of pentobarbitone injection) of 15 i.u. in 0-2 ml. of isotonic saline of human chorionic gonadotrophin (HCG; 'Pregnyl', chorionic gonadotrophin, B.P., obtained from Organon Laboratories Ltd). At postmortem on the next day all seven animals had tubal ova, the uteri had no free fluid and the ovaries were hyperaemic and appeared somewhat oedematous. The ovarian weight (mg/kg body wt. + S.E. of mean) was 34*3 + 1-4 which was slightly higher but not significantly different from the value of 30-2 + 1F3 found in the group of seven control animals. The T/S ratio for the HCG-treated group, all of which had ovulated and had genital tracts characteristic of the normal oestrous rat, was 101-6 + 9-2 which was significantly less than the value for control rats (P < 0-001) and very close to the value for the pentobarbitone-treated group. When ovulation is induced by exogenous HCG in rats in which endogenous LH release (and ovulation) is blocked by pentobarbitone, the expected rise in the T/S ratio

OVULATION AND THE THYROID 409 on the day of oestrus is not restored. To exclude the possibility that the T/S ratio had in fact been increased as a result of ovulation but the effect had been obscured by an inhibitory effect of HCG, the effect of 15 i.u. of HCG in male rats injected with pentobarbitone at 17.00 hr and killed the next morning, and in female rats in metoestrus injectedwith pentobarbitone or pentobarbitone plus HCG and killed in dioestrus was determined. Pentobarbitone alone has no effect in males or metoestrous females (see previous section). Eight male rats injected with pentobarbitone alone had a mean T/S ratio of 119-1 + 8-8 and eight rats injected with pentobarbitone plus HCG gave a mean value of 1414 + 19-0; six females injected with pentobarbitone in metoestrus gave a mean value of 104-9 + 14-5; and six injected with pentobarbitone plus 15 i.u. HCG a value of 92-2 + 5-5. In neither case were the differences significant and no effects of HCG on T/S ratio were observed. The effect of pentobarbitone blockade of ovulation on the uptake of l3si by the thyroid (% injected dose in gland 2i hr after injection) reported in an earlier paper (Brown-Grant, 1963) was confirmed and the effect of ovulation induced in pentobarbitone-treated rats by either 15 i.u. of HCG or 10,ug of purified ovine LH in 1 ml. of saline injected intraperitoneally at the time of pentobarbitone injection (17.00 hr on the day of pro-oestrus) was determined. The LH preparation used was N.I.H. LH S 7, a gift from the National Institute of Health, Bethesda, U.S.A. to Professor G. W Harris. Nine control rats killed on the morning of the day of pro-oestrus had a mean uptake of 7-07 + 0-42 % and eleven rats killed on the day of oestrus (all had tubal ova) had a mean value of 10-28 + 0.55%. The difference between these two groups was highly significant (P < 0-01). Ten rats were injected with pentobarbitone and ovulation was blocked in nine of these; the mean uptake for all ten animals (5.94+0.40%) was significantly lower than that of control rats killed in oestrus (P < 0*001) but not significantly different from values found in animals killed in prooestrus. Ten rats were injected with pentobarbitone plus 10,ug LH; all had tubal ova the next morning and the genital tracts appeared quite typical of oestrus though the ovaries were heavier than those of control rats killed in oestrus (35.3 + 12 mg/l00 g body wt. as compared with 29-4 + 1-3, P < 0.01). The mean uptake was 6-04 + 0-41%, significantly less than in controls killed in oestrus (P < 0-001), but not significantly different from controls killed in pro-oestrus or rats injected with pentobarbitone alone. Eleven rats were injected with pentobarbitone plus 15 i.u. of HCG at 17.00 hr in pro-oestrus. Next day all had tubal ova and genital tracts characteristic of oestrus. Ovarian weight was greater than that of controls killed in oestrus (39.2 + 10 mg/100 g body wt., P < 0'01). Radioiodine uptake was 8-21 + 0-53 0/ which is significantly lower than (P <

410 K. BROWN-GRANT 0 02) the values for controls killed in oestrus and is not significantly different from that of controls killed in pro-oestrus. As with the experiments involving the T/S ratio, the effects of pentobarbitone alone or with LH or HCG on 131I uptake in male rats was determined. Ten control males had a mean uptake of 7 23+0-62%; eleven males injected with pentobarbitone at 17.00 hr on the previous day had a mean uptake of 7-56 + 0 60 % and ten injected with pentobarbitone plus 15 i.u. of HCG had an uptake of 7-12 + 0-50 %. In a second experiment nine pentobarbitoneinjected rats had a mean uptake of 7x65 + 0x62 %, nine rats injected with pentobarbitone plus 10 jug LH an uptake of 6'59 + 6-53 % and ten injected with pentobarbitone plus 15 i.u. HCG an uptake of 6-56 + 0-57 %. These differences were not significant. Uptake of 131I by the thyroid gland offemale mice Vaginal smears were made daily and oestrous cycles followed for between 21 and 28 days in three groups of 24, 24 and 27 mice before 131J uptakes were determined. About 15% of animals showed one or more periods of pseudopregnancy during this period, defined as the persistence of a vaginal smear containing leucocytes for six or more consecutive days. The cycle pattern was quite variable between animals but more constant for each individual mouse. The commonest cycle length was five or six days and the phases of the cycle are defined on the basis of the vaginal smear (stained with haematoxylin and eosin) as follows. Metoestrus is the first day on which leucocytes are found following the occurrence of a smear of epithelial cells only without leucocytes. Dioestrus 1 and dioestrus 2 are the next 2 days on which leucocytes are present in the smear. It was rare for a mouse showing regular cycles to have more than three successive days of leucocyte-containing smears and mice with a prolonged dioestrous period in the two cycles before the measurement of uptake were not used. The variation in cycle length between mice was usually related to the duration of the period of 'oestrogenized' smears, that is with no leucocytes. Three types of these smears were recognized: a pro-oestrous smear consisting of nucleated epithelial cells only; a pro-oestrous-oestrous smear containing both nucleated and fully cornified cells; and an oestrous smear containing cornified cells only. No difficulty was experienced in classifying smears into these three types. The names are descriptive only; as will be seen later, the type of smear obtained does not bear a constant relation to the state of the ovaries as in the rat. The commonest pattern of smear change was dioestrus 2 to pro-oestrus to oestrus to metoestrus, but all variants between dioestrus 2 to pro-oestrus to metoestrus and dioestrus 2 to pro-oestrus to pro-oestrus/oestrus to pro-oestrus/oestrus to oestrus to metoestrus were encountered, though the pattern for individual mice tended to remain

OVULATION AND THE THYROID 411 constant over the 3- to 4-week period during which smears were followed. Only animals that had at least two regular cycles of not more than 6 days' duration and not more than 3 days of consecutive smears containing leucocytes were used; on this basis l3lj uptakes were determined in sixtytwo of the seventy-five mice studied at different stages of the cycle as determined on the basis of the vaginal smear on the morning of the day the animals were killed. The oviducts were examined for tubal ova; animals killed in metoestrus did not have tubal ova, so the presence of eggs indicates that ovulation had occurred the previous night. Analysis of variance showed a just significant variation (P < 0.05) between groups and a highly significant difference (P < 0-01) between stages based on the vaginal smear pattern. Uptakes (numbers of mice in brackets) at different stages were: Metoestrus 7-66 + 0'76 % (8); dioestrus 1, 5-58 + 0 57 % (9); dioestrus 2, 5-84 + 0.95% (11); pro-oestrus 7-97 + 1.13 % (9); pro-oestrus/ oestrus 8 28 + 0 72 % (14); oestrus, 8-59 + 0 45 % (11). Uptake was highest in animals with 'oestrogenized' smears but analysis of variance for these mice showed that there was no difference between groups and no difference between the three stages. Animals with all three types of 'oestrogenized' smear were found to have tubal ova; three of nine with pro-oestrus smears; nine of fourteen with pro-oestrous/oestrous smears, and ten of eleven with oestrous smears. Classified on this basis, there was again no difference between mice from the three groups, but a significant difference (P < 0.01) between mice with or without tubal ova. The mean value for the twentyone mice that had ovulated was 9-21 + 0-42 % and for the twelve that had not 6 63 + 0 76 %, and in the t test the difference was highly significant (P < 0-01). The uptake of 1311 by mice varies during the oestrous cycle, being higher in those with 'oestrogenized'smears than in those with leucocyte-containing smears. There is no significant difference between mice with different types of 'oestrogenized' smears, but a significantly higher uptake in those that have ovulated during the night preceding the measurement of uptake than in those that have not. Observations on the golden hamster Although the hamster is reported to have an extremely stable 4-day oestrous cycle (Greenwald, 1963), this was not so for the animals used in these experiments. Of a total of eighty-four animals examined daily over a period of weeks, only fifty-one satisfied the criterion of having two or more regular 4-day cycles preceding the day on which uptake of 131I was measured. The stages of the cycle are defined as follows: oestrus is the day on which a profuse external vaginal discharge is noted; tubal ova were found in all animals killed at this stage, but at no other stage of the cycle.

412 K. BROWN-GRANT The next day is referred to as metoestrus, the day after as dioestrus and the day after that as pro-oestrus, to correspond with the terminology used in the rat. Vaginal smear changes in the hamster are complex (Ward, 1946) and single daily smears were found to be confusing and unhelpful. The classification appears reasonable in view of the macroscopic state of the ovaries on different days. At metoestrus, corpora lutea are present, follicles are not seen; at dioestrus, corpora lutea are less obvious and small follicles may be noted; at pro-oestrus, the ovaries are pale, the corpora lutea are inconspicuous, but large clear follicles are visible; and at oestrus tubal ova are present and fresh corpora lutea are found in the ovary. The uptake of 131J was determined in fifty-one animals; analysis of variance showed a significant difference between stages of the cycle. The actual values (numbers of animals in brackets) were: oestrus, 8-00 + 0-53 % (19); metoestrus, 5*57 + 0-540/ (9); dioestrus, 6-97 + 0.53% (8); pro-oestrus, 7-17 + 0.40% (15). All animals killed at oestrus had tubal ova, indicating recent ovulation, and uptake was highest in this group. None of the other animals had tubal ova and the mean uptake of 6'67 + 0.30% (32) was signifcantly lower than that of the oestrous group (P < 0.05). DISCUSSION Injection of pentobarbitone before the critical period for hypothalamic stimulation of the release of pituitary LH on the day of pro-oestrus in the rat has shown that when ovulation is blocked in this way the high thyroidserum concentration ratio for l3si normally found on the morning of the next day (the day of oestrus) is not observed. In confirmation of earlier work (Brown-Grant, 1963) the expected increase in 131J uptake by the gland on the day of oestrus was also found to be prevented when ovulation was blocked in this way. In the case of the T/S ratio, the value for animals that did not ovulate was much below that of untreated animals killed at oestrus and significantly below that of animals injected with pentobarbitone that ovulated despite this treatment. This latter group, however, had a mean T/S ratio significantly below that of the untreated animals; this did not appear to be a non-specific effect of the pentobarbitone treatment as no similar depression was found in male rats or in female rats in metoestrus treated in the same way. More detailed analysis of the results showed that it was animals in which. pentobarbitone was injected close to the critical period with ovulation occurring despite this that had low T/S ratios the next day. It was also found that animals which had ovulated, but in which a curtailment of the period of action of LH could be inferred from the persistence of uterine fluid into the oestrous stage of the cycle and the absence of the changes in ovarian appearance (other than rupture of the

OVULATION AND THE THYROID 413 follicles) characteristic of LH action (Everett, 1948), gave low T/S ratios. The association of a low T/S ratio on the day of oestrus despite the occurrence of ovulation and evidence of a deficiency in LH stimulation of the ovary and of progesterone secretion raised the possibility that the changes in TSH production and hence T/S ratio were secondary to the changes in ovarian steroid production. Experiments in which ovulation was produced by the injection of exogenous gonadotrophin in rats in which endogenous LH release was blocked by pentobarbitone did not support this view. Human chorionic gonadotrophin did not restore either T/S ratios or uptakes to normal oestrous values, nor did purified LH affect the uptake of 1311 despite the fact that these hormones were consistently successful in producing ovulation, the characteristic changes in the ovary and progesterone secretion as shown by the loss of uterine fluid in pentobarbitonetreated rats. Some other explanation is required for the failure of the T/S ratio to rise (and, by inference, of TSH secretion to increase) in some pentobarbitone-treated rats that ovulated. From the timing of the pentobarbitone injections and the state of the genital tract it appeared that these were animals in which the period of hypothalamic stimulation of LH release had been curtailed. The currently accepted view of the neuro-endocrine events involved in ovulation in the rat is that during the critical period of pro-oestrus a hypothalamic neurohumoral agent, luteinizing hormonereleasing factor (LRF), is released from the median eminence and carried in the portal vessels to the anterior pituitary where it stimulates the release of LH (Everett, 1964). There is now some evidence that production of LRF may continue beyond the period necessary to release sufficient LH to induce ovulation as the median eminence content of LRF continues to rise (Ramirez & Sawyer, 1965) and the pituitary content of LH falls after the end of the critical period (Schwarz & Caldarelli, 1965). It seems possible that during the early part of the critical period LRF releases sufficient LH to cause rupture of the follicles; pentobarbitone after this time will not block ovulation but will prevent the later periods of LRF release. The depletion of available pituitary LH at this stage may permit LRF to act as a TSH-releasing factor during the later portion of the period in which it is released. Alternatively, the abrupt fall in the median eminence content of LRF late in pro-oestrus detected by Ramirez & Sawyer (1965), though not by Chowers & McCann (1965), may mean that the neural stimulus that previously provoked LRF release now causes release of the postulated TSH-releasing factor, TRF. At present, these possibilities are no more than speculations. Indeed there is at present no satisfactory direct evidence from assays of plasma or pituitary TSH levels that there is an increased secretion of TSH at the end of or after the critical period for LH release in the female rat at pro-oestrus, though such an increase appears likely. The

414 K. BROWN-GRANT simplest explanation of the present experiments is that the increased thyroid activity at the oestrous stage of the ovarian cycle in the rat is not related to changes in circulating oestrogen, nor to changes in ovarian steroid secretion following the release of LH that leads to ovulation, but is related in some way to changes at the hypothalamic or pituitary level concerned with this ovulatory surge of LH secretion. There is, however, a possible objection to this interpretation of the results obtained when endogenous gonadotrophin is replaced by exogenous gonadotrophin in pentobarbitone-treated rats and when ovulation, but not an increase in thyroid gland activity, follows. This is that pentobarbitone is thought to interfere with some actions ofprogesterone on the hypothalamus to facilitate gonadotrophin release (see Everett, 1964). Conceivably, if the release of TSH were also due to an action of progesterone, secreted by the ovary under the influence of LH, on the hypothalamus, this too might be blocked in these experiments. This possibility cannot be excluded at present. The ideal test animal would be a rat in which ovulation could be blocked acutely by some means other than the administration of drugs or hypothalamic lesions; possibly acute exposure to continuous light might produce such an animal. A difficulty in the extension of this hypothesis to explain cyclic changes in thyroid activity in the female of other species is the reported occurrence of the highest level of thyroid activity in the mouse at the pro-oestrous stage of the cycle. There is no conclusive evidence, however, from the published reports, that the peak of thyroid activity precedes ovulation in this species and the work that has been published can be criticized on the grounds that the occurrence of regular cycles was not adequately established and that the animals were assigned to stages of the cycle on the basis of vaginal smear pictures that are not defined. Soliman & Reineke (1954) took vaginal smears for only 5 days; animals are reported as pro-oestrous, oestrous, metoestrous or dioestrous on this basis, but no description of the smears characteristic of each stage was given. It is stated that 'the ovaries were examined macroscopically with a magnifying lens, to confirm the vaginal smear readings', but no further information is provided. The uptake of 131I by the thyroid gland was highest in 'pro-oestrous' animals. In a later study (Soliman, Abdo, Soliman & Abdel Wahab, 1964) smears were taken for ten days but no mention is made as to whether regular cycles were observed or not. The mice were then classified as early pro-oestrous, late pro-oestrous, oestrous, metoestrous, early and late dioestrous but the basis for this classification is not given. It is stated that 'the ovaries were examined histologically to verify the vaginal smear picture', but no indication is given as to what ovarian appearance corresponds to what stage of the cycle as determined by the vaginal smear, and the only histological finding reported is that 'the Graafian follicles showed a tendency towards

OVULATION AND THE THYROID 415 an early development at pro-oestrus...'. Uptake of 131I is reported as being highest in animals in 'late pro-oestrus'. Boccabella & Alger (1961) took smears for five or more days and state that only animals that were cycling were used, though they do not say how they determined this. 'Only mice whose vaginal smears could be identified unequivocally as typical of one of the five stages of the oestrous cycle (pro-oestrus, early oestrus, late oestrus, metoestrus and dioestrus) were used in the experiments', but the criteria on which recognition was based are not given and no mention is made of the state of the ovaries, oviduct or uterus. They found the T/S ratio for 131J to be the highest in mice in 'pro-oestrus'. If the relation of the vaginal smear pattern to ovulation was as variable in the mice studied by these workers as it was in the animals used in the present study, then animals classified as pro-oestrous, early or late oestrous by them may all potentially have ovulated within 18 hr of the time of the thyroid measurement. A high proportion of animals that had recently ovulated in their pro-oestrous or late pro-oestrous groups could account for the high values recorded. In the present experiments it was found that high uptakes were observed in mice that had fresh tubal ova, and that thyroid activity was related to the occurrence of ovulation, as in the rat, rather than to the vaginal smear pattern (and, by inference, the circulating oestrogen level) as suggested by previous workers. The oestrous cycle of the hamster is said to be very regular and Greenwald (1963) has suggested that it may be independent of the environmental lighting conditions, as regular cycles continue in hamsters exposed to continuous light in contrast to the rat which becomes anovulatory under such conditions. Although this finding has not been confirmed and is difficult to reconcile with the report of Orsini (1963) that the time of ovulation in the hamster is altered in animals kept under conditions of reversed light and darkness, it seemed to be of some interest to determine if there was any variation in thyroid activity during the oestrous cycle in a spontaneously ovulating species where the cycle might be relatively unaffected by light. In fact considerable difficulty was experienced in obtaining hamsters showing regular cycles but a limited number of animals were tested. The results suggest that thyroid activity does vary during the ovarian cycle, that it is highest in the immediate post-ovulatory phase of the cycle and that it is related to ovulation, as in the rat and mouse. The differences were not marked, however, and the findings should be regarded as provisional until they have been confirmed by experiments on a colony where cycles are more consistent. The finding in the experiments in rats strengthens the suggestion made previously (Brown-Grant, 1963) that the variations in thyroid activity during the oestrous cycle are related to the changes at the hypothalamic 27 Physiol. 184

416 K. BROWN-GRANT or pituitary level that lead to ovulation rather than to variations in ovarian steroid hormone output during the cycle. The results obtained from mice in the present work are also consistent with this hypothesis and the observations of other workers on mice can be interpreted to support it. This association of ovulation and increased thyroid gland activity may be present in other spontaneously ovulating species whose oestrous cycles are influenced by light. Robertson & Falconer (1961) observed abrupt rises in PB1271 levels and in the rate of release of 131I from the thyroid gland coincident with oestrus and ovulation in the ewe. The ovarian cycle of the hamster may not be, and the cycle of the guinea-pig is not, influenced by environmental light (Dempsey, Myers, Young & Dennison, 1934) and in the present study and an earlier one (Brown-Grant, 1962b) increases in thyroid gland activity that may be related to ovulation were observed. The human ovulates spontaneously with no known influence of light being involved; previous studies have failed to establish any significant variation in thyroid activity during the menstrual cycle although evidence suggestive of an increase at mid-cycle have been reported (see, for instance, Pochin, 1952 and references in Brown-Grant, 1956). None of these studies was undertaken, however, with the idea of an association between ovulation and increase in thyroid gland activity in mind. With the improved methods now available for determining the time of ovulation in the human, evidence of such an association might now be obtained. Some evidence of such an association has been obtained in species less frequently studiedfor the dogfish by Olivereau (1949) and the viper by Saint Girons & Duguy (1962). These are all spontaneously ovulating animals; although early work suggests a release of pituitary TSH soon after mating in the rabbit, a species which does not ovulate spontaneously, no change in the rate of release of 131J from the thyroid gland after mating was observed (Brown- Grant, 1956). It maybe, however, that this method was not adequate to detect a transient change in thyroid activity. Recent studies have suggested that the initial hormonal changes after mating in the rabbit may be of much shorter duration than was thought to be the case (Hilliard, Hayward & Sawyer, 1964). Experiments are being carried out to investigate the possibility that very rapid but transient changes in the pituitary secretion of TSH occur in the mated female rabbit and that the general concept of an association between the neuro-endocrine changes that lead to ovulation and an increase in thyroid activity can be extended to animals that do not ovulate spontaneously. This work was supported by a grant for research expenses from the Royal Society and in part from a United States Air Force Grant (Number AF EOAR 64--3) to Professor G. W. Harris. The skilled technical assistance of Mr M. Sherwood is gratefully acknowledged.

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