CHANGES IN LEVELS OF FOLLICLE STIMULATING HORMONE AND LUTEINIZING HORMONE IN THE BOVINE PITUITARY GLAND AT OVULATION

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1 CHANGES IN LEVELS OF FOLLICLE STIMULATING HORMONE AND LUTEINIZING HORMONE IN THE BOVINE PITUITARY GLAND AT OVULATION A. M. RAKHA and H. A. ROBERTSON The Division of Agricultural Biochemistry, Department of Biological Chemistry, University of Aberdeen, Scotland (Received 30 July 1964) SUMMARY A significant (P < 0\m=.\05)drop in follicle stimulating hormone (FSH) and luteinizing hormone (LH) content of the pituitary was shown to occur in the cow during the period 0\p=n-\18hr. after the onset of oestrus. Calculation of the ratio of LH to FSH in the pituitary during the ovulatory period, suggests that this ratio varied and hence that the ratio of LH to FSH secreted may also vary. INTRODUCTION Although the classical concept of the functions of follicle stimulating hormone (FSH) and the luteinizing hormone (LH) was that they were solely responsible for the growth of the follicle and for ovulation respectively, it has now become apparent that FSH and LH are secreted together by the pituitary although it is not yet clear whether the ratio of FSH to LH secreted at different stages of the cycle varies. It is ofinterest, therefore, to study the relative changes ofthe levels of these hormones in the tissues and body fluids of animals throughout the oestrous cycle. In animals other than man, most of the work carried out has been confined to studying changes in the pituitary concentration, while in man most of the work has included determi nations in urinary extracts. Many ofthe investigations so far carried out have involved the measurement of 'total gonadotrophic' activity rather than the determination of the individual levels of FSH and LH. During the oestrous cycle it is fairly well established that the gonadotrophin content of the pituitary varies. In domestic animals a decrease in the gonadotrophin content of the pituitary at oestrus has been shown in the cow (Smirnova, 1945) and in the ewe (Kammlade, Welch, Nalbandov & Norton, 1952). Using biological assay procedures which differentiate between FSH and LH Santolucito, Clegg & Cole (1960) and Robertson & Hutchinson (1962) have shown that in the ewe both the FSH and LH levels in the pituitary fall during oestrus. No conclusions were drawn about changes in the ratio of FSH to LH. The present investigation was carried out to determine whether similar changes in the levels of FSH and of LH occurred in the bovine pituitary just before ovulation and to determine whether such changes were associated with a change in the ratio of these gonadotrophins.

2 MATERIALS AND METHODS Twenty-five virgin heifers, aged months and with a dressed carcass weight of lb. when killed, were used. Twenty-two were Friesians, two were Ayrshires and one was a Shorthorn. All animals were first examined by rectal palpation to verify that their reproductive organs were mature and normal, a minimum of three oestrous cycles were then followed in each animal in order to establish regularity of cycling. A heifer was considered to be in oestrus when she stood to be mounted. These investigations were combined with an examination of the vestibule, vagina and the external os of the cervix since impending oestrus can generally be detected by such an examination hr. before the external signs of heat (Bane & Rajakoski, 1961). The animals were divided into five groups for slaughter: (1) on the 15th day, (2) the 18th day, (3) the onset of oestrus, (4) the end of oestrus (18-20 hr.), (5) after ovulation (25-40 hr.). The timing of the cycle was taken from the beginning of oestrus; day 1 of the cycle was the day on which the animal came into oestrus. Animals which had a slightly longer or shorter cycle than anticipated at the time of slaughter, were killed according to the timing of the physiological events that took place. At slaughter the pituitary gland was removed, trimmed and weighed, the neural lobe and the infundibular stem removed and the adenohypophysis weighed. The anterior lobe was frozen on dry ice and then freeze-dried. The dried anterior lobes of each group were pooled, ground to a fine powder and stored in a desiccator. Assays All assays were carried out by a (2 + 1) design on Sprague-Dawley female rats. The animals were weighed before the start of an assay and each experimental group (eight animals) balanced to minimize within-group variation in body weight. The FSH and LH determinations were run as single assays. The potency of the prepara tions was calculated by the method of Gaddum (1953). (1) FSH was determined by the augmentation method of Steelman & Pohley (1953); every animal receiving 20 i.u. human chorionic gonadotrophin (HCG). The standard doses used were 0-1 and 0-2 mg. NIH-FSH-S-1, saline extracts of 75 mg. of pituitary powder represented the 'unknown'. The total dose was 3 ml. (6x0-5 ml.). (2) LH was determined by the depletion of ovarian ascorbic acid in pseudopregnant rats (Parlow, 1961). The standard doses used were 1-4 and 2-8 pig. NIH- LH-B-1, a saline extract of 0-2 mg. (15th day, 18th day and onset of oestrous groups), 0-4 mg. (after-ovulation group) or 0-6 mg. (end of oestrus group) pituitary powder represented the 'unknown'. All preparations were given as a single intraperitoneal injection of 0-5 ml. and the animals killed 4 hr. later. Ascorbic acid was determined by the method of Maickel (1960). Since a single dried pituitary weighed mg. and a minimum of approxi mately 450 mg. (6 x 75 mg.) was required for the estimation of FSH and LH at one dose level (five test animals), it was decided not to attempt to estimate the gonado trophin content of each pituitary individually but to use bulked samples for each

3 group. The weight of pituitary tissue used including the amount required to establish the dose level was approximately 900 mg. It would therefore have been possible to repeat either of these determinations should the first assay have been non-valid. It was considered that the size of the sample (five animals per group) would enable a reasonably reliable mean value to be obtained for each stage of the cycle. This decision infers that the lack of precision of the estimate which would have occurred had an attempt been made to assay each pituitary individually using fewer test animals, i.e. by reducing the number from eight to two test animals per dose, would have been such that the estimates would have been almost valueless. RESULTS The lengths of the oestrous cycles of the experimental animals were very uniform. Ninety-two cycles were observed for the 25 heifers before slaughter. The mean length was 20-8 ± 0-4 (s.e.) days. Under the conditions of this experiment, there was no correlation between the weight of the anterior lobe of the pituitary, fresh or freeze-dried, and the stage of the cycle. The results of the FSH and LH estimations are shown in Table 1 and in Fig. 1. Table 1. The ratio of LH to FSH activities in the pituitary of different stages of the reproductive cycle Time of slaughter of animals (day of oestrous cycle) 15th day 18th day Onset of oestrus End of oestrus After ovulation LH activity of pituitary in terms of fig. NIH-LH-B-1/mg. pituitary (dried wt.) A 14-9 ( ) ( ) ( ) ( ) ( ) 0-17 the cow at FSH activity of pituitary in terms of fig. NIH-FSH-S-1/mg. Ratio: pituitary (dried wt.) À LH/FSH 1-73 ( ) ( ) ( ) ( ) ( ) The values suggest that a significant drop (P < 0-05) occurs in the levels of both FSH and LH during the period 0-18 hr. after the onset of oestrus. From ratios of LH to FSH for the five groups it would appear that the ratio not remain constant. LH : FSH in the pituitary may DISCUSSION The most significant finding was the decrease in the levels of both FSH and LH by 27 and 61 %, respectively, sometime between the onset and the end of oestrus indicating that FSH as well as LH plays a role in the induction of ovulation in cattle. A similar fall in both FSH and LH has been observed in sheep (Santolucito et al. 1960; Robertson & Hutchinson, 1962). Although the results cannot be evaluated statistically the change in the LH : FSH ratio during the period of release is possibly a real one. The 'total gonadotrophic' activity of ovine and bovine female pituitaries cannot be estimated by the assay based upon the increase of mouse uterine weight, since the slope of the response curve is too flat (H. A. Robertson, J. S. M. Hutchinson & A. M. Rakha, unpublished observations). Brown & Billiewicz (1962) showed that

4 with mixtures of ovine LH and ovine FSH the response of the mouse uterus declined series of when the percentage of LH in the mixture rose above 20 %. In the present pituitaries, LH as a percentage of the total gonadotrophin content varied between 16-8 and 86-5%. H œ En K se 3. Oestrus Day of cycle hr. t 1 End Ovulation Fig. 1. Changes in pituitary gonadotrophin levels of cows at ovulation in terms of fig. of the standard preparation/mg. dried pituitary. It seems likely that the release of the gonadotrophins occurs early in the 0-18 hr. period after the onset of oestrus. Hansel & Trimberger (1951) blocked ovulation in cattle by administering atropine at the onset of oestrus, and Robertson & Rakha (1964) in sheep have shown by blockade with chlorpromazine that the neural stimulus to the hypothalamus which leads to ovulation occurs within 4 hr. of the onset of oestrus. The authors are indebted to Mr T. Begg and Miss H. Kenley for technical assistance, to Parke, Davis and Co. Ltd for a generous supply of HCG and the Endocrine Study Section of NIH for the standard pituitary hormone preparations. REFERENCES Bane, A. & Rajakoski, E. (1961). The bovine oestrous cycle. Cornell Vet. 51, Brown, P. S. & Billiewicz, W. Z. (1962). The response of immature mice to mixtures of gonadotrophins. J. Endocrin. 24, Gaddum, J. H. (1953). Simplified mathematics for bioassays. J. Pharm. Pharmacol. 5, Hansel, W. & Trimberger, G. W. (1951). Atropine blockage of ovulation in the cow and its possible significance. J. Anim. Sei. 10, Kammlade, W. G., Welch, J. A., Nalbandov, A. V. & Norton, H. W. (1952). Pituitary activity of sheep in relation to the breeding season. J. Anim. Sei. 11, Maickel, R. P. (1960). A rapid procedure for the determination of adrenal ascorbic acid. Application of the Sullivan and Clarke method to tissue analysis. Analyt. Biochem. 1, Parlow, A. F. (1961). Bioassay of pituitary luteinising hormone by depletion of ovarian ascorbic acid. In Human pituitary gonadotrophins, pp Ed. A. Albert. Illinois: Thomas.

5 Robertson, H. A. & Hutchinson, J. S. M. (1962). The levels of FSH and LH in the pituitary of the ewe in relation to follicular growth and ovulation. J. Endocrin. 24, Robertson, H. A. & Rakha, A. M. (1964). The timing of the neural stimulation of the hypothalamus which leads to ovulation in the sheep. Proc Vth Int. Congre. Anim. Reprod., Trento, 3, Santolucito, J. A., Clegg, M. T. & Cole, H. H. (1960). Pituitary gonadotrophins in the ewe at different stages of the oestrus cycle. Endocrinology, 66, Smirnova, E. I. (1945). Cyclic changes in the content of gonadotrophic and thyrotrophic hormones in the anterior lobe of the pituitary. Bull. exp. Biol. Med. 19, Steelman, S. L. & Pohley, F. M. (1953). Assay of the follicle stimulating hormone based on the augmenta tion with HCG. Endocrinology, 53,