different ratios of PMSG and HCG on the occurrence of follicular haemorrhage THE induction of ovulation with PMSG and HCG in the rat has been studied

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1 Q. Jl exp. Physiol. (1968) 53, THE INDUCTION OF OVULATION IN IMMATURE RATS TREATED WITH PREGNANT MARE'S SERUM GONADOTROPHIN AND HUMAN CHORIONIC GONADOTROPHIN. By S. F. LUNN and E. T. BELL. From the Medical Research Council Clinical Endocrinology Research Unit, University of Edinburgh. (Received for publication 14th June 1967) Studies have been made on ovarian and uterine weight, the occurrence of follicular haemorrhage and ovulation, and the number of ova shed in the intact immature rat treated with pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG) either alone or in combination. Both PMSG and HCG when given alone were capable of inducing ovulation. This event was associated with uterine stimulation but generally occurred in the absence of marked ovarian weight increase or follicular haemorrhage. The administration of PMSG followed by HCG produced ovulation over a wide dosage range of both hormones. With low dose levels of PMSG the number of ova shed was independent of the dosage of HCG. However, this was not the case at higher dosages of PMSG, the injection of which was associated with marked ovarian weight increase and follicular hiemorrhage. The mechanism by which PMSG and HCG exert their effects on the ovary is discussed. THE induction of ovulation with PMSG and HCG in the rat has been studied by numerous investigators. Thus, Rowlands and Williams [1943] established the conditions necessary for ovulation in mature and immature hypophysectomized rats while a similar study in the intact immature animal was carried out by Rowlands [1944]. It has also been shown that the occurrence of ovulation is dependent on such factors as the body weight and age of the animal and on the schedule of hormone administration. As early as 1936, Cole reported that the effect of PMSG on the rat ovary was influenced by the age of the animal at the time of injection of the hormone and this observation has been confirmed by various investigators including Price and Ortiz [1944], Zarrow and Wilson [1961], Eckstein [1962] and Zarrow and Brown-Grant [1964]. The effect of varying the time interval between the injection of PMSG and HCG has been studied by Rowlands [1944] who found that a hr. period was optimal for the induction of ovulation. Over the last three decades the effects of PMSG and HCG on the genital tract have been extensively employed in the study of many aspects of reproductive physiology [for references see Rowlands and Parkes, 1966]. However, little information is available regarding the administration of different ratios of PMSG and HCG on the occurrence of follicular haemorrhage and ovulation or on the number of ova shed. The aim of this communication is to report such data and in addition to describe the results obtained when various dose levels of PMSG and HCG were given alone. A preliminary account of some of these experiments has been given by Lunn [1967]. VOL. LIII, No

2 13 Lunn and Bell MATERIALS AND METHODS Intact immature rats of the Wistar strain bred in a closed colony in the laboratory in Edinburgh were employed. At the commencement of each experiment the animals were days of age and weighed g. In the first experiment a total of 127 rats received a single s.c. injection of PMSG at the dose levels shown in Table I. After 93 hr. the animals were killed and the body, ovarian and uterine weights measured. In addition, the occurrence of follicular hlemorrhage and the number of ova in the oviducts were determined. The oviducts were examined in saline under a binocular dissecting microscope at a magnification of x 2. If ovulation had occurred the ova were teased out from the oviduct using triangular mounted dissecting needles. They were then counted under a No. 1 cover-slip at the same magnification, the results reported being based on the number of oviducts containing ova. In the second experiment a total of 111 rats received a single s.c. injection of HCG (Table II). The animals were killed 93 hr. later and examined as described above. The third experiment comprised 53 rats all of which received PMSG (s.c.) followed 72 hr. later by a s.c. injection of HCG (Table III). Twenty-one hours after the second injection the animals were killed and subjected to the procedures outlined above. A total of 31 rats which received no hormonal treatment acted as control. RESULTS The 31 control animals weighed 45 ±7 g. (S.D.) and their ovarian and uterine weights were 9 ±2 and 26 ±8 mg. respectively. Follicular haemorrhage and ovulation were not observed. Following hormone administration the body weights of the treated animals were similar to those of the untreated controls. 1. The Effect of PMSG Alone (Table I). - The administration of i.u. PMSG caused a significant decrease (p <.2) in ovarian weight. A TABLE I. THE EFFECT OF PMSG ALONE. Follicular Dosage Ovarian weight hemorrhage Ovulation Uterine weight (mg.) (percentage) (percentage) No. of ova (mg.) 2 1± ± ± ±32 119± ± ± 8 14± ± ± ± 3 34X8 3.±1-4 97± ± ±1.6 88± ± ±*9 8± -39 6± 2 27±21.iL^ 7± 1 21± 6 significant increase (p <.1) in this parameter was not observed until dose levels in excess of 3 13 i.u. were administered. Follicular haemorrhage was noted with 6- i.u. and above while ovulation occurred only between -78 and 6- i.u. PMSG. The lowest dose of hormone tested caused a significant fall in uterine weight (p < -5) with dosages above -39 i.u. a marked increase (p < 1) was noted.

3 Ovulation of Rats The Effect of HCG Alone (Table II). - No marked effect of HCG administration on ovarian weight was observed when compared with the results in Table I. Follicular hafmorrhage was seen only rarely while ovulation was induced with dosages ranging from 12-5 to 1 i.u. No effect Dosage *5 6-3* *78 Ovarian weight (mg.) 11±6 11±2 9±2 1±3 6±2 7±1 7±1 9±1 1±1 TABLE II. THE EFFECT OF HCG ALONE. Follicular haemorrhage Ovulation (percentage) (percentage) * No. of ova 1-8 ±96 3-5±1V2 2* ± ± 73 Uterine weight (mg.) 99±23 92±14 89±18 99±15 46±13 31±1 28± 5 26± 4 ± 3 on uterine weight was noted at low dose levels of hormone; however, marked stimulation occurred with 12-5 i.u. and above (p < 1). 3. The Effect of PMSG and HCG (Tables III-VI). - The mean ovarian weights are shown in Table III. Dose levels of PMSG ranging from -78 to 3-13 i.u. produced little or no effect. High dosages of this hormone resulted in a significant rise (p < 1) in ovarian weight. Alterations in the amount TABLE III. Dose of HCG Dose of PMSG * '56 78 THE EFFECT OF PMSG AND HCG ON OVARIAN WEIGHT (MG.) * *56 83 ±18 55±12 5±12 38±15 23± 6 14± 2 12± 2 1± 3 76±29 79±2 72 ±12 41±17 19± 7 14± 3 13± 5 12± 2 86±27 98±1 72 ±24 49±15 17± 7 1± 3 1± 2 9± 2 87±14 96±16 71±2 37 ±13 14± 3 11± 3 1± 2 1± 2 8±2 9±19 85±28 41± 13 17± 5 12± 3 12± 3 11± 2 73 ±28 74±23 78± 19 39± 7 17± 5 14± 3 11± 2 13± 4 69±17 86±14 64±17 37± 9 11± 3 1± 2 11± 2 9± 2 of HCG produced no consistent effect irrespective of the dose level of PMSG employed. In Table IV is shown the percentage of animals in which follicular hsemorrhage and ovulation occurred. The former was noted in all groups receiving, 5 or 1 i.u. PMSG. However, with low dosages of PMSG follicular haomorrhage was seen only in animals receiving high dose levels of HCG. Ovulation occurred with all dose levels of PMSG below 12-5 i.u. except when -78 i.u. PMSG was followed by 1I56 or 3-13 i.u. HCG. With 12-5 i.u. PMSG or above, larger amounts of HCG were required to induce

4 132 Lunn and Bell TABLE IV. THE EFFECT OF PMSG AND HCG ON THE OCCURRENCE OF FOLLICULAR HAEMORRHAGE AND OVULATION (PERCENTAGE). Follicular hwmorrhage Ovulation Dose of HCG, 1 _A_ _ P56 Dose of PMSG *13 1* Dose of HCG Dose of PMSC *78 TABLE V. -1 x THE EFFECT OF PMSG AND HCG ON THE NUMBER OF OVA SHED *5 6-3* ± ± 5*5 11-±8*1 4*6± '± 9-8 1*8± 4*7 18*6± ± ± *4± *1±12*7 15*2± *8±8*2 17-4±13*9 4-8±1*3 3-8± 2-2*5± ± 1-1 2*9±1-2 5*2± 5-1*5±*5 2-5± 1* 2-1± 1-2 2*± ±1*9 2*7± ±-9 3-2± 2-2*6± 1.1 2*3± ±1*7 2*3± -8 27±1*6 2*8± 1*2 2*4± 1-2 2*3± 1i 2-6± ± ± 6 2*3±1*5 3.3 ±2'3 TABLE VI. Dose of HCG Dose of PMSG *13 1*56 *78 THE EFFECT OF PMSG AND HCG ON UTERINE WEIGHT (MG.) *5 6* ±11 11± 9 97±12 19±14 99± 8 18±11 13± 9 81± 6 97± 9 87±11 91± 9 99± 6 97± 7 94± 6 73± 5 96±11 82±12 89± 7 93±1 15±15 99± 7 84± 6 96± 9 8± 3 86± 8 81±11 87±11 96±16 89± 8 87± 9 79±11 71±13 69± 7 86± 5 88±16 94± 9 83±13 75±2 76± 9 65±14 76± 8 86± 9 82±19 86±1 77± 7 74± 2 75± 7 7± ±23 58±3 6±13 58±22 68±18 41±15 48±23

5 Ovulation of Rats 133 ovulation; however, with high dosages of both hormones the percentage of animals ovulating was reduced. The mean numbers of ova shed at ovulation are shown in Table V. The ova counts were similar with 6- i.u. PMSG and below, alterations in the dosage of HCG being without effect. In rats treated with high dose levels of PMSG the ova count was generally higher and was dependent on the dosage of HCG. Thus, with 1-56 and 3-13 i.u. HCG ovulation generally did not occur; with 6- to i.u. HCG the mean ova count was generally above 1 except with 1 i.u. PMSG when ovulation was inhibited. The administration of 5 and 1 i.u. HCG was associated with superovulation when preceded by 12-5 or i.u. PMSG, but the ova count was much reduced in the case of 5 and 1 i.u. PMSG. Increasing amounts of PMSG were associated with growth of the uterus (Table VI). Thus,, 5 and 1 i.u. resulted in uteri generally weighing 9-11 mg. while with 1b i.u. the figure obtained was 7-9 mg. With -78 i.u. PMSG the uterine weight was generally less than 7 mg. and only at this dose level did HCG cause an increase in weight. DDIscusSION The results obtained in the present study have shown that the administration of PMSG alone is not capable of producing superovulation in the intact immature rat. This observation is in agreement with the findings of Rowlands [1944], but differs from the data of Zarrow and Quinn [1963]. It is of interest to note that in the results reported herein, ovulation with PMSG occurred only between -78 and 6- i.u. and that in no case did more than 4 per cent of the animals ovulate. The induction of ovulation was associated with mean uterine weights of 8-1 mg. and except at 6- i.u. with the absence of ovarian weight increase and follicular haemorrhage. Above 6- i.u. PMSG ovulation did not take place but the ovarian weight and percentage follicular haemorrhage increased, the mean uterine weight being generally greater than 1 mg. When HCG was given alone ovulation occasionally occurred between 12-5 and 1 i.u. but not at 2 i.u. The findings in relation to ovarian weight, follicular heemorrhage and uterine weight were in general similar to those with PMSG at the dosages required to induce ovulation. The dose levels of HCG required to cause release of ova and stimulation of uterine weight were some 16 and times greater than with PMSG. Most workers agree that both PMSG and HCG have biological effects which are similar to those of LH, but in addition, PMSG has FSH-like activity. Thus, by analogy with work in hypophysectomized rats [see Rowlands and Parkes, 1966], it is probable that in the present study the administration of PMSG alone caused follicular growth by a direct action on the ovary. It is also possible that ovarian cestrogen secretion stimulated the release of endogenous LH and that this resulted in ovulation [see also Quinn and Zarrow, 1965]. However, in no case did 1 per cent ovulation occur, and in addition

6 134 Lunn and Bell the number of ova shed following PMSG alone was considerably below that expected at natural cestrus. It should also be noted that overstimulation with PMSG was associated with inhibition of ovulation, increased ovarian weight, and the occurrence of follicular haemorrhage. In the hypophysectomized rat HCG does not cause follicular growth [Noble et al., 1939]. The induction of ovulation following HCG administration to the intact animal must therefore be associated with release of endogenous FSH. This could occur either by a direct action of HCG on the ovary or on the hypothalamic/hypophyseal system. The recent work of Eshkol [1967], in which the uptake of iodinated HCG by various organs in the mouse was studied, would indicate a direct effect of HCG on the ovary. This would presumably result in luteinization and steroid production which may in turn stimulate the release of endogenous FSH. When the administration of PMSG was followed 72 hr. later by an injection of HCG the ovarian weights obtained were somewhat higher than those noted with PMSG alone. However, the percentage increase due to HCG was similar at all dose levels of PMSG and was not dependent on the amount of HCG given. In animals treated with both PMSG and HCG the incidence of follicular haemorrhage was generally above 6 per cent with, 5 and 1 i.u. PMSG, and was therefore higher than with either hormone given alone. When the administration of less than 12-5 i.u. PMSG was followed by any dose of HCG the number of ova shed was similar to that with PMSG alone, but the percentage ovulation was much increased. However, although HCG administration resulted in a slight or moderate increase in ovarian weight this was not associated with any alteration in the number of ova shed. The induction of ovulation with dosages of PMSG of 12-5 i.u. or above was dependent on the amount of HCG administered. At high dosages of PMSG ovulation was inhibited unless large amounts of HCG were given; however, with high dosages of both hormones ovulation occurred only rarely. As has been emphasized previously PMSG probably stimulates follicular growth by a direct effect on the ovary. The induction of ovulation caused by the administration of HCG to the PMSG-treated rat may also be a direct effect although an intermediate action on the hypothalamic/hypophyseal system cannot be excluded. The main conclusion to emerge from the present study is that both PMSG and HCG when given alone can induce ovulation in a small proportion of animals, but only a limited number of ova are shed. In both cases ovulation generally occurs in the absence of follicular haemorrhage or marked ovarian weight increase. When HCG is given to the PMSG treated rat ovulation can be induced in the presence of both follicular haemorrhage and ovarian weight increase. Under these conditions superovulation often occurs. The present study provides no information regarding the time at which ovulation occurs following HCG administration. In view of the marked differences in ovarian weight following the hormonal treatment employed it would be of interest to study the time-relationships of secondary follicular swelling, stigma formation and ovulation using various dosages of PMSG and HCG.

7 Ovulation of Rats 135 Data of this type may well provide valuable information regarding the mechanism by which HCG induces ovulation. ACKNOWLEDGMENT The authors wish to thank Organon Laboratories Ltd. for supplies of PMSG and HCG. REFERENCES COLE, H. H. (1936). 'On the biological properties of mare gonadotrophic hormone', Am. J. Anat. 59, 299. ECKSTEIN, B. (1962). 'The development of the ovary of the rat. II. The effect of chorionic gonadotrophin and serum gonadotrophin', Acta endocr. (Kbh.) 41, 35. ESHKOL, A. (1967). In Recent Research on Gonadotrophic Hormones, p. 22. Eds. Bell, E. T. and Loraine, J. A. Edinburgh: Livingstone. LUNN, S. F. (1967). In Recent Research on Gonadotrophic Hormones, p Eds. Bell, E. T. and Loraine, J. A. Edinburgh: Livingstone. NOBLE, R. L., ROWLANDS, I. W., WARWICK, M. H. and WILLIAMS, P. C. (1939). 'Comparative effects of certain gonadotrophic extracts on the ovaries of normal and hypophysectomized rats', J. Endocrin. 1, 22. PRICE, D. and ORTIZ, E. (1944). 'The relation of age to reactivity in the reproductive system of the rat', Endocrinology 34, 215. QuINN, D. L. and ZARROW, M. X. (1965). 'Inhibition of pregnant mare's serum induced ovulation in the immature rat', Endocrinology 75, 5. ROWLANDS, I. W. (1944). 'The production of ovulation in the immature rat', J. Endocrin. 3, 384. ROWLANDS, I. W. and PARKES, A. S. (1966). 'Hypophysectomy and the gonadotrophins', In Marshall's Physiology of Reproduction, 3rd Ed., Vol. III, p. 26. Ed. Parkes, A. S. London: Longmans. ROWLANDS, I. W. and WILLIAMS, P. C. (1943). 'Production of ovulation in hypophysectomized rats', J. Endocrin. 3, 31. ZARROW, M. X. and BROWN-GRANT, K. (1964). 'Inhibition of ovulation in the gonadotrophin-treated immature rat by chlorpromazine', J. Endocrin. 3, 87. ZARROW, M. X. and QuINN, D. L. (1963). 'Superovulation in the immature rat following treatment with PMS alone and the inhibition of PMS-induced ovulation', J. Endocrin. 26, 181. ZARROW, M. X. and WILSON, E. D. (1961). 'The influence of age on superovulation in the immature rat and mouse', Endocrinology 69, 851.