FOLLICLE-STIMULATING HORMONE CONTENT OF THE PITUITARY GLAND BEFORE IMPLANTATION IN THE MOUSE AND RAT

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1 FOLLICLE-STIMULATING HORMONE CONTENT OF THE PITUITARY GLAND BEFORE IMPLANTATION IN THE MOUSE AND RAT B. M. BINDON Department of Veterinary Physiology, University of Sydney, Sydney 2006, Australia (Received 1 November 1968) SUMMARY The method of Lamond & Bindon (1966) was used to measure follicle\x=req-\ stimulating hormone (FSH) in the pituitary glands of mice and rats killed at different times on each of days 1\p=n-\6 of pregnancy. The sensitivity of the method allowed detection of FSH fluctuations not previously described in these species. A significant fall in pituitary FSH occurred in both species on day 3, although it was earlier in the mouse than in the rat. This transient decline in FSH activity, interpreted as a release of thehormone, was followed in both species by a gradual increase on days 4 and 5. Ovarian weight, measured only in the mouse, increased significantly late on day 3 and is believed to reflect changes in pituitary FSH. The results indicate that implantation in the mouse and rat is initiated by altered activity of thepituitary gland during the first 3 days of pregnancy. It appears that this pituitary mechanism differs from that responsible for the events of the oestrous cycle. INTRODUCTION Recent reports from this Department (Miller, Owen & Emmens, 1968 a, 6) have been concerned with the time of oestrogen release before implantation in the mouse and rat. These studies, based on the incorporation of uridine into uterine RNA, indicate that oestrogen is produced by the ovary in increasing amounts during the first 3 days of pregnancy and thus disagree with the concept of a discrete surge of oestrogen on the day before implantation as proposed by Shelesnyak (1960). There is little evidence, in either mouse or rat, to indicate whether pituitary gonadotrophin levels fluctuate in accordance with ovarian oestrogen release, surge or otherwise. Some estimates of the gonadotrophin content of the pituitary during pregnancy have been made, but the emphasis has been on the changes occurring between early and late pregnancy or pseudopregnancy and the onset of parturition (Rothchild, 1962; Greenwald, 1966). In the present study, observations have been confined to the first 6 days of preg nancy, and to different times on these days. The changes in pituitary folliclestimulating hormone (FSH) of the mouse and rat during this period are described.

2 MATERIALS AND METHODS Animals Mice of the Quackenbush strain, aged weeks and weighing g., were used. The albino rats studied were adult females of the Wistar strain weighing g., derived from an experiment reported by Miller et al. (19686), and had been injected with [3H]uridine before killing. The mice and rats were housed separately but were maintained under uniform environmental conditions at F and lighting from to hr. each day. The animals were caged with males and examined each day for evidence of mating. The day of appearance of a vaginal plug (mice) or sperm in the vaginal smear (rats) was designated day 1 of pregnancy. Bioassay of FSH The FSH content of pooled mouse and rat pituitaries was measured by the hypo physectomized mouse uterine wt method of Lamond & Bindon (1966). Preliminary assays were conducted using pituitary homogenates from non-pregnant rats and mice in order to characterize the dose-response lines. Dose levels were then chosen so as to include the linear portion of the log dose-response line for further assays. In the pregnancy studies, the usual procedure was to assay two pituitary pools against the standard on any 1 day. Just before injection the pooled pituitary tissue was thawed, homogenized in cold 0-9% NaCl solution and serially diluted to yield the appropriate dose levels in 0-2 ml. saline. The injections (i.e. pituitary tissue and HCG) were given subcutaneously at separate sites. There were seven hypophysecto mized immature mice per dose level for both the standard and unknown, although these group sizes were occasionally reduced to six by mortality. Statistical analysis The results were analysed by standard statistical methods for parallel-line bioassays. The data were processed by an IBM 3200 computer, (C.S.I.R.O., National Standards Laboratory, Sydney) using a bioassay programme prepared by the Division of Mathematical Statistics, C.S.I.R.O. The uterine weight results were corrected by covariance for differences in weight of the test animals and were trans formed to logarithms to reduce heterogeneity of variances. For each comparison of standard and unknown the programme provided estimates of relative potency, 95 % confidence limits, Gaddum's À and a test of parallelism of the dose response lines. Experimental procedure Preliminary analysis of dose-response line for pituitary homogenates. In a single assay the FSH activity of non-pregnant mouse pituitary tissue at dose levels corresponding to 0-031, 0-125, 0-5 and 2-0 pituitaries/test animal was compared with non-pregnant rat pituitary tissue (levels of 0-006, 0-025, 0-1 and 0-4 pituitaries/ animal) and NIH-FSH-S 3 (0-31, 1-25, 5-0 or 20-0/ig./dose). Bat pituitary FSH during earlypregnancy. Groups of 7-10 rats were killed at hr or hr. on days 1-6 of pregnancy. The heads were removed and stored at 20 for 3-7 days. After thawing, the pituitary glands were removed, weighed

3 individually and again stored at 20. The weights of the pituitaries of these animals do not, therefore, represent fresh weights. The FSH activity of these pools was then examined in multiple 8-point assays at doses (in fig. wet pituitary tissue) corres ponding to 0-1, 0-2, 0-4 and 0-8 pituitary glands. The dose levels of NIH-FSH-S3 used in all assays were 1-25, 2-5, 5-0 and 10-0 fig. The same stock solutions of NIH-FSH-S3 were used in separate assays, the material being stored at 20 between assays. The ovaries from the rats from which the pituitary glands were collected were not available for study. Mouse pituitary FSH during early pregnancy. Pituitary glands from groups of mice were removed and weighed as a group at hr. and hr. on each of days 1-5 of pregnancy. In addition, further groups were killed at hr. and hr. on the evening of day 3. The pituitaries were stored at 20 until the assay. At the time of killing, the body weight and paired wet ovarian weight of each mouse were also recorded. The mice were allotted at random to the various killing times. The homogenized pituitary tissue was then assayed against the NIH-FSH-S3 standard. The dose levels (in fig.) of the homogenate corresponded in all assays to 0-2, 0-4, 0-8 or 1-6 pituitary glands per test animal. The dose levels ofthe standard were as for the rats, i.e. 1-25, 2-5, 5-0 and 10 /tg./test animal. They were again obtained from the stock NIH-FSH-S 3 solutions used for the rats. Statistical analysis of rat pituitary weights and mouse ovarian weights Both sets of data were subjected to the following procedure : covariance analysis used was to correct for differences due to the body weights in the various groups. Within group variances were then tested for homogeneity by Bartlett's test. If the variances were significantly different the data were transformed to logarithms and retested for equality. An overall analysis of variance within and between groups was then carried out to see if there was evidence of significant differences between groups. Since both sets of data were non-orthogonal it was not possible to partition the between group variance term. Therefore tests of significance between all group means were carried out as described by Snedecor (1956). RESULTS Preliminary assays of pituitary homogenates Table 1 shows the uterine weights of mice injected with four doses of NIH-FSH-S 3 or mouse or rat pituitary tissue. Comparable log dose-response lines occurred with all three preparations. Analyses of the relative potency and the FSH activity of the pituitary homogenates are also shown in this Table. FSH activity in rat pituitary glands (Table 2) The within-group variances for pituitary weights were = homogeneous (xfii] 5-65). Pituitary weights were significantly (P < 0-05) lower at hr. on day 1 and on day 3 than at hr. on day 2. All assays satisfied the requirements for validity. The larger values of À at hr. on day 3 are due to a reduced response at the highest dose of pituitary homogenate. Pituitary FSH content, with the exception ofthe level recorded at hr. on day 1,

4 hr. on day 3 and hr. on day 5, was relatively uniform in the range equivalent to fig. NIH-FSH-S 3. Compared with this level, pituitary FSH activity was significantly lower at hr. on day 1 and was reduced even further on hr. on day 3. The latter value was checked in a repeat assay with a similar result as shown in Table 2. After this transient decline late on day 3, pituitary FSH increased to a peak value equivalent to 46 fig. NIH-FSH at hr. on day 5. Table 1. Preliminary assays of follicle-stimulating hormone (FSH) in female mouse pituitary homogenates by the method of Lamond and Bindon (1966) Dose NIH-FSH-S3 Uterine Potency of pituitary tissue relative to NIH-FSH-S 3 Pituitary weight (mg.) FSH concentration (pg. NIH-FSH/ mg. pituitary) FSH content (pg. NIH-FSH/gland) Index of precision (A) Mouse pituitary glands *Dose (/tg-) Uterine Rat pituitary glands *Dose (/tg-) Dose levels refer to wet weight of pituitary tissue. Summary of analyses of relative potency Mouse pituitary glands ( )f ( ) 8-27 ( ) 0-24 t 95% confidence limits. It Uterine rat and Rat pituitary glands ( ) ( ) 41-0 ( ) 0-31 Table 2. Pituitary follicle-stimulating hormone (FSH) activity during early pregnancy Stage of pregnancy Day Time (hr.) No. of glands Mean pituitary Means+ s.e ± ll-5± ll-9± in the rat Concentration (pg. NIH-FSH- S3/mg. pituitary) 2-1 ( ) 3-3 ( ) 3-2 ( ) 3-4 ( ) 3-4 ( ) 1-3 ( ) 1-4 ( ) 2-7 ( ) 3-9 ( ) 3-8 ( ) 3-8 ( ) 2-6 ( ) 3-1 ( ) * 95% limits of error in parentheses. Pituitary FSH* Content (pg. NIH-FSH- S3/gland) 19-7 ( ) 33-7 ( ) 40-0 ( ) 38-1 ( ) 39-1 ( ) 12-0 ( ) 12-9 ( ) 32-1 ( ) 40-2 ( ) 46-4 ( ) 39-5 ( ) 28-1 ( ) 33-2 ( )

5 Mouse pituitary FSH during early pregnancy (Table 3) Since the mouse pituitary glands were not weighed individually, the significance of the changes in mean pituitary weight was not tested. All the FSH assays satisfied the requirements for statistical validity. This is reflected by uniform limits of error and values of À. The fluctuations in mouse pituitary FSH content were not as marked as those in the rats but with slight differences with regard to time, the patterns are not dis similar in the two species. Thus in the mouse, pituitary FSH content declined sig- Table 3. Follicle-stimulating hormone (FSH) activity of mouse pituitary glands during early pregnancy Stage of pregnancy No. of Mean Day Time (hr.) glands pituitary Concentration (pg. NIH-FSH- S3/mg. pituitary) 2-59 ( ) 3-21 ( ) 2-47 ( ) 2-27 ( ) 1-75 ( ) 2-49 ( ) 2-90 ( ) 2-82 ( ) 301 ( ) 2-83 ( ) 2-05 ( ) 3-37 ( ) Pituitary FSH Content (pg. NIH-FSH- S3/gland) 4-74 ( ) 6-42 ( ) 5-30 ( ) 5-25 (4' 3-56 ( ( ( (4> 7-15 ( ( ( ( ) ) ) ) ) ) ) ) ) Table 4. Changes in ovarian weight during early pregnancy of the mouse Stage of pregnancy Mean paired ovarian weight (mg.) Day Time (hr.) No. of mice (Means + s.e.) ± ll-5± ± ± Summary of overall analysis of variance of mouse ovarian weights (Data transformed to logarithms to reduce inequality of variances.) Source of variation d.f. Mean square F Between groups *** Between mice ***P <

6 nificantly between hr. on day 1 and hr. on day 3, then increased signifi cantly by hr. on day 3, and rose to a peak value at hr. on day 4. Changes in mouse ovarian weight during early pregnancy The mean paired ovarian weights during early pregnancy are shown in Table 4. Within-group variances were heterogeneous (xfii] = 28-3, P < 0-001). Log trans formation ofthe data almost equalized the variances = (xfii] 19-92, P < 0-05). An overall analysis of variance between groups was therefore carried out (Table 4). The first notable feature of the results is the sharp increase in ovarian weight between hr. and hr. on day 3. Further comparisons among the means show that, with the exception ofthe value recorded at hr. on day 3, all ovarian weights up to hr. on day 3 were significantly (P < 0-001) lower than those recorded at hr. on day 3 or later. The transient decline in ovarian weight at hr. on day 3 is also significant. Ovarian hyperaemia, particularly in the corpora lutea, accompanied the increase in ovarian weight on day 3. Histological changes in the ovaries at this time are being examined to determine if any follicular development contributes to the ovarian weight pattern. DISCUSSION Preliminary assays for FSH in rodent pituitary homogenates confirmed the reli ability of the method of Lamond & Bindon ( 1966) for this type of tissue. The fact that parallel dose-response lines were obtained with pituitary homogenates and NIH-FSH- S 3 tends to negate the recent criticism (Ross & Brown, 1967) that the response to FSH is altered by the presence of other pituitary gonadotrophins in the mixture. The simi larity between the rat FSH levels reported in the present paper and those reported by others using the Steelman & Pohley (1953) assay for FSH (Greenwald, 1966; Labhsetwar, 1967; Goldman & Mahesh, 1968) provides indirect evidence that the present method is specific for FSH. Further, the levels of FSH found for the mouse pituitary agree well with those reported recently by Lloyd, Bindon, Lamond & Meares (1968) after purification of mouse FSH by starch gel electrophoresis. The results indicate that significant changes in pituitary FSH content occur before implantation in the mouse and rat. The only comparable study in the rat is that of Greenwald (1966) who was unable to measure pituitary FSH consistently on days 1 and 4 of pregnancy due to the lower sensitivity of the Steelman & Pohley (1953) test and the fact that only one dose level of the homogenate was tested. There seem to be no previous reports of mouse pituitary FSH content during the pre-implantation period. If, as generally believed at present, lowered pituitary levels reflect release of hormones into the bloodstream, then the present results suggest a release of pituitary FSH on day 3. The earlier reduction and subsequent rise in pituitary FSH in the mouse is understandable when it is considered that implantation occurs earlier in the mouse (day 5) than the rat (day 6). Two recent reports (Greenwald, 1966; Schwartz & Talley, 1968) have been con cerned with the controversy as to whether pituitary activity during pregnancy dis plays cyclic variation similar to that ofthe normal oestrous cycle. Both investigations led to the conclusion that no cyclic variation in pituitary activity occurs during

7 pregnancy. However in neither study were pituitary hormones measured at more than one time on each day, and therefore, diurnal fluctuations would have remained undetected. The present results in the rat, together with the recent reports by Caligaris, Astrada & Taleisnik (1967) and Goldman & Mahesh (1968) allow a more detailed comparison of the pituitary FSH content during early pregnancy and the oestrous cycle. The patterns clearly differ. During the oestrous cycle pituitary FSH is reduced on the afternoon of pro-oestrus, i.e. day 4 on the pregnancy timing schedule. Significant reduction of FSH occurred late on day 3 of pregnancy. The reduction in pituitary FSH at hr. on day 1 of pregnancy is parallelled by similar results in the oestrous cycle study of Goldman & Mahesh (1968) but not that of Caligaris et al. (1967). Thus the pituitary FSH content does fluctuate during early pregnancy but this occurs at a time incompatible with that expected from changes observed in the oestrous cycle. The results should also be discussed in relation to the patterns of steroid production during early pregnancy. In the rat the release of FSH occurred too early to be associ ated with the surge of oestrogen suggested by Shelesnyak (1960). In both species release of FSH coincided with the time of completion of the rise in oestrogendependent uridine uptake by the uterus between days 2 and 3 described by Miller et at. (1968a, 6). The changes in ovarian weight recorded during early pregnancy of the mouse are worthy of comment. The increase observed late on day 3 agrees well with the pattern of FSH release. The corresponding ovarian weight pattern in the rat was not obtained in the present study. However, Greenwald (1966) showed a transient increase in ovarian weight on day 4 of pregnancy in the rat ; the ovarian weights days3 on to 5 of pregnancyin his study were 68,79 and 69 mg. respectively, with approxi mately equal limits of error. It is difficult to accept Greenwald's interpretation (1966) that such changes are without significance especially as both follicle size and the number of ovarian follicles showed identical increases on day 4. The advice of Professor C. W. Emmens is gratefully acknowledged. The author was supported by a studentship from the Australian Meat Research Committee and facilities provided by grants from the Australian Wool Board to the department. The computer programme was made available by the Division of Mathematical Statistics, C.S.I.R.O. REFERENCES Caligaris, L., Astrada, J. J. & Taleisnik, S. (1967). Pituitary FSH concentrations in the rat during the oestrous cycle. Endocrinology 81, Goldman, B. D. & Mahesh, V. B. (1968). Fluctuations in pituitary FSH during the ovulatory cycle in the rat and a possible role of FSH in the induction of ovulation. Endocrinology 83, Greenwald, G. S. (1966). Ovarian follicular development and pituitary FSH and LH content in the preg nant rat. Endocrinology 79, Labhsetwar, A. P. (1967). Differential effects of reserpine in pituitary luteinizing hormone and follicle stimulating hormone levels in the female rat. Endocrinology 81, Lamond, D. R. & Bindon, B. M. (1966). The biological assay of follicle stimulating hormone in hypo physectomized immature mice. J. Endocr. 34, Lloyd, H. M., Bindon, B. M., Lamond, D. R. & Meares, J. D. (1968). Starch gel electrophoresis of mouse pituitary gonadotrophins. J. Endocr. 40, Miller, B. G., Owen, W. H. & Emmens, C. W. (1968 a). The incorporation of tritiated uridine in the uterus and vagina of the mouse during early pregnancy. J. Endocr. 41,

8 Miller, B. G., Owen, W. H. & Emmens, C. W. (19686). Uridine incorporation in the rat genital tract during early pregnancy. J. Endocr. 42, Ross, I. P. & Brown, P. S. (1967). Specificity in assays of follicle stimulating hormone using mice. Endocrinology 81, Rothchild, I. (1962). Corpus-luteum pituitary relationship: The effect of progesterone on the folliculotropic potency of the pituitary in the rat. Endocrinology 70, Schwartz, N. B. & Talley, W. L. (1968). Daily measurement of pituitary LH content during pregnancy in the rat; Do cyclic changes persist? J. Reprod. Fert. 15, Shelesnyak, M. C. (1960). Nidation of the fertilized ovum. Endeavour 19, Snedecor, G. W. (1956). Statistical methods applied to experiments in agriculture and biology, 5th edn. p. 251, Ames, Iowa: Iowa State College Press. Steelman, S. L. & Pohley, F. M. (1953). Assay of follicle stimulating hormone based on augmentation with human chorionic gonadotrophin. Endocrinology 53,