Physiology and Behavior, Vol. 10, pp. 65-67, Brain Research Publications Inc., 1973. Printed in U.S.A.

Endocrine Control of the Maternal Pheromone in the Postpartum Female Rat

MICHAEL LEON 3 AND HOWARD MOLTZ

Department o f Psychology, University o f Chicago Chicago, Illinois 60637

(Received 17 July 1972)

LEON M. AND H. MOLTZ. Endocrine control of the maternal pheromone in the postpartum female rat. PHYSIOL. BEHAV. 10(1 ) 65-67, 1973.- The maternal pheromone in the postparturient female rat was shown to be under prolactin control. Ergocornine, a drug known to inhibit discharge of the tropin completely eliminated the pheromone, while prolactin replacement restored the pheromone. Removal of adrenal and ovarian hormones does not interfere with the emission of the attractant.

Maternal pheromone Prolactin Mother litter interaction Lactation Adrenal steroids Maternal behavior

Ovarian steroids

LEON AND MOLTZ [4] demonstrated recently that 16-day lactating female rats characteristically release an odor capable of attracting 16-day-old young. This odor, subsequently called a maternal pheromone, was found to be litter general rather than litter specific, insofar as 16-day young characteristically approach all lactating females 16 days postpartum. A second series of studies [5] revealed the existence of a striking synchrony in the development and subsequent disappearance of the pheromone. Specific- ally, it was shown that the female first begins to release the pheromone at about 14-days postpartum, coincident with the age at which the young first become responsive to it, and then ceases to release the pheromone at about 27 days postpartum which corresponds to the age at which the young cease to be attracted to it. The adaptive value of this temporal coincidence may lie in the fact that the young begin to leave the nest when about 2 weeks of age but are not weaned until they reach about 4 weeks of age. Since it is only during this 14-day period that the pheromone is released and in turn approached, what perhaps is being witnessed is a pheromonal bond that serves periodically to reunite mother and young, allowing nursing to continue until weaning.

But what of the physiological mechanisms governing the maternal pheromone? We can speculate that they might be hormonal in nature since the period immediately preceding initiation of emission is a period of high endocrine output during which ovarian and adrenal agents are actively discharged, as is prolactin from the adenohypophysis. Prolactin, in particular, seemed to us promising as a governing mechanism, for not only is it characteristically

present at high titers throughout the first half of the postpartum period I l l but it is released in response to pup stimulation [3, 8, 14], stimulation Moltz and Leon [5] found important in controlling pheromonal emission. Accordingly, the role of prolactin was investigated along with the role of ovarian and adrenal steroids in the control of the pheromone. To this end, one group of puerperal females was ovariectomized, a second adrenalectomized, and a third subjected to the combined operation. The more likely possibility that prolactin alone might be involved in pheromonal emission was explored through the use of the drug, ergocornine, a drug found to inhibit the release of hypophyseal prolactin both in vivo [15, 16, 17, 18] and in vitro [6, 12, 20].

METHOD

We used 840 Wistar pups each 16-days old, born in our laboratory. All litters were routinely reduced to six in number shortly after birth. Each pup was tested in the olfactory discrimination apparatus described previously by Leon and Moltz [4]. Briefly, this apparatus was designed to permit approach across a triangular open field from a start box to either of two goal boxes, each containing a stimulus female. Forced air flowed into the goal compartments from a central valve and from there made to pass across an open field to a start box.

The stimulus animals were primiparous lactating females and nuUiparae of comparable chronological age. Each had been isolated for three hr prior to being placed in the goal compartments. A single pup, separated from its mother for

This research was supported in part by NIH Grant No. HD06872 to H.M. 2We would like to thank Sue Lerner for her assistance and Michael Numan for his helpful advice. 3 Present address: Department of Psychology, McMaster University, Hamilton, Ontario, Canada.

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66 LEON AND MOLTZ

the same three-hr period was then placed in the start box and allowed 15 min for a choice. After each pup registered a choice, or after 15 min had elapsed, it was removed from the apparatus and the absorbent paper covering the open field and start box was replaced. After three pups of a given litter were tested, the apparatus was washed and the goal box positions of the stimulus females were reversed. Each combination of nulliparous female and mother was used to test twelve, 16-day-old pups, with each pup tested but once.

RESULTS

Experiment l

Here we were interested in determining whether adrenal and ovarian hormones, either respectively or in synergy, play a role in governing pheromonal emission. To this end 4 groups, each consisting of 10 females, were formed. The first group was bilaterally adrenalectomized on the day of parturition, the second bilaterally ovariectomized, and the third subjected to the combined operation. A fourth group of intact females served as a control. The drinking water of those animals subjected to adrenal surgery was 0.9% saline.

Since we expected lactational failure following the combined operation, all females, regardless of group membership, had their litters rotated daily with those of a battery of donor females of comparable postparturient age. Every litter was weighed daily and observations of maternal behavior were taken. At 16 days postpartum, each female was paired in our olfactory discrimination apparatus with a nulliparous female and tested for the presence of the pheromone, using colony young, 16-days old.

Reference to Table 1 indicates that neither adrena- lectomy, ovariectomy, nor even the combined operation interfered with the emission of the pheromone; as is evident, the mothers in each of these groups were chosen significantly over the nulliparous test females with which they were paired. In addition, it might be noted that all females displayed normal maternal behavior. Indeed, each was observed to lick her young, retrieve, build a nest, and assume a nursing posture, even when, as in the case of the adrenalectomized ovariectomized groups, lactation was suppressed.

Experiment 2

Here we were interested in testing the possible involve- ment of prolactin in the emission of the maternal p h e r o m o n e . Accordingly, 10 primiparous females, beginning on the day of parturition, were given daily injections of 0.5 mg ergocornine hydrogen maleate (kindly supplied by Sandoz Pharmaceutical Co.) dissolved in 0.15 cc of 70% ethanol. This particular dosage was chosen because it was shown previously to interfere with prolactin release as measured by the decidual cell reaction in lactating females [ 11 ].

To be certain that prolactin as such was involved in the ergocornine induced inhibition of the pheromone, still another group of 10 females was used. These females also received daily injections of the drug at a dosage level of 0.5 rag. In addition, however, they were given 30 IU of prolactin (kindly supplied by the Endocrinology Study Section of NIH) in 0.2 cc physiological saline twice daily.

As in Experiment 1, all females had their litters rotated each day with those of a battery of donor females of

comparable postparturient age. Also, the pups were weighed daily and routine observations were made of maternal behavior. Testing was carried out at 16 days postpartum, using, once again, colony young, 16-day-old.

Reference to Table 1 reveals unmistakably the effects of prolactin inhibition. The injection of ergocornine sup- pressed entirely the emission of the maternal pheromone, as evidence by the fact that such females were not chosen over their nulliparous test partners. In contrast, when prolactin was administered along with ergocornine, a significant preference, indicative of pheromone release, was once again exhibited.

Here too, it is worthy of note that all mothers, regardless of treatment, behaved in perfect maternal fashion, including, to be sure, those injected with ergocornine alone. As was expected, these females showed lactational inhi- bition.

The results of one additional group should be discussed at this point, a group (N=I0) adrenalectomized and ovariectomized on the day of parturition and then given twice daily injections of 30 IU of prolactin. The reason such females were included was due to our concern over the data obtained from the adrenalectomized ovariectomized group of Experiment 1. To be sure, these data revealed a significant preference, indicating that the combined opera- tion had by no means eliminated the pheromone. Yet, the preference was not as striking as in either the adrena- lectomized or the ovariectomized females. Considering the fact that steroidal hormones exert a positive feedback effect on prolactin release [2, 9, 10, 13], it was thought that the combined operation, in totally removing these hormones, might have decreased prolactin output and, consequently, decreased pheromonal emission. Accord- ingly, it was reasoned that daily injections of prolactin into adrenalectomized ovariectomized females should increase their attractiveness, relative at least to their non-injected counterparts. This is precisely what occurred-more young approached the injected than the non-injected doubly- operated females, as Table 1 reveals.

DISCUSSION

The present studies demonstrate conclusively that the m a t e r n a l p h e r o m o n e is under prolactin control. Ergocornine, a drug known to inhibit discharge of the tropin, completely eliminates the pheromone, whereas removal of adrenal and ovarian hormones does not.

That prolactin proved critical in governing pheromonal emission is in accord with the in vivo profile this hormone presents [ 1 ], a profile that shows high and sustained plasma levels throughout the two-week period preceding the appearance of the pheromone [5]. in accord as well is the fact that the continued release of prolactin following parturition depends on stimulation from the young [3, 8, 14], and that such stimulation also governs pheromonal emission [7]. The suggestion of prolactin as a mediating agent seems obvious.

But along another dimension, however, the role of prolactin is not obvious. How, specifically, does this hypophyseal agent function to control the pheromone? Does it function simply in pheromonal emission or does it function in pheromonal synthesis as well? And is the involvement direct, or is there still another mediating factor?

With respect to this last question, we had thought that

M A T E R N A L P H E R O M O N E 67

T A B L E I

CHOICE BEHAVIOR OF 16-DAY-OLD YOUNG TO MOTHERS

Number choosing Number choosing mothers Nulliparous females No choice Significance

Intact Control 110 4 6 <0.001 Ergocornine 53 65 7 >0.05 Ergocornine

and Prolactin 88 13 19 <0.001 Adrenalectomized I 01 17 2 <0.001 Ovariect omized 106 8 6 <0.001 Adrenalectomized-

Ovariectomized 82 24 14 <0.01 Adrenalectomized-

Ovariectomized and Prolactin 95 8 17 <0.001

the p h e r o m o n e migh t be d e p e n d e n t o n lac ta t ion , t ha t , in o the r words, some milk b y - p r o d u c t or milk me tabo l i t e was involved. Given t he role of p ro lac t in in the lac togenic process [ 19] , th is idea was appeal ing. It p roved , however , to be w i thou t suppor t . In t he p resen t s tudy , for example , the females of our ad r ena l ec t om i zed ova r i ec tomized group did no t m a i n t a i n l ac ta t ion , yet t hey p roduced t he

p h e r o m o n e . In the s tudy by Moltz and Leon 17], the re were females t h a t lac ta ted con t inuous ly , and yet , even at 16-days p o s t p a r t u m , failed to p roduce the p h e r o m o n e . However , to resolve conclus ively t he ques t i on of lac ta t iona l invo lvement , to ta l ly m a m m e c t o m i z e d females mus t be run u n d e r each of our expe r imen t a l cond i t ions .

REFERENCES

1. Amenomori, Y., C. L. Chen and J. Meites. Serum prolactin levels in rats during different reproductive states. Endo- crinology 86: 511-516, 1970.

2. Chert, C. L. and J. Meites. Effects of estrogen and progesterone on serum and pituitary prolactin levels in ovariectomized rats. Endocrinology 8 6 : 5 0 3 - 5 1 0 , 1970.

3. Grosvenor, C. E. Evidence that exteroceptive stimuli can release prolactin from the pituitary gland of the lactating rat. Endocrinology 76: 340-342, 1965.

4. Leon, M. and H. Moltz. Maternal Pheromone: Discrimination by pre-weanling albino rats. Physiol. Behav. 7: 265-267, 1971.

5. Leon, M. and H. Moltz. The development of the pheromonal bond in the albino rat. Physiol. Behav. 8: 683-686 , 1972.

6. Lu, R. H., F. Koch and J. Meites. Direct inhibition by ergocornine of pituitary prolactin release. Endocrinology 89: 229-233, 1971.

7. Moltz, H. and M. Leon. Stimulus control of the maternal pheromone. Physiol. Behav. 10: 69 -71 , 1973.

8. Moltz, H., R. Levin and M. Leon. Prolactin in the postpartum rat: Synthesis and release in the absence of suckling stimulation. Science 163: 1083-1084, 1969.

9. Nicoll, C. S. and J. Meites. Estrogen stimulation of prolactin production by rat adenohupophysis in vitro. Endocrinology 70: 272-277, 1962.

10. Nicoll, C. S. and J. Meites. Prolactin secretion in vitro: effects of gonadal and adrenal cortical steroids. Proc. Soc. exp. Biol. Med. 117: 579-583 , 1964.

11. Numan, M., N. Leon and H. Moltz. Interference with prolactin release and the maternal behavior of female rats. Horm. Behav. 3: 29-38 , 1972.

12. Pasteels, J. L. and F. Ectors. Mode of action of ergocornine on the secretion of prolactin. Archs Int. Pharmacodyn. ThOr. 186: 195-196, 1970.

13. Rather, A., P. K. Talwalker and J. Meites. Effect of estrogen administration in vivo on prolactin release of rat pituitary in vivo. Proc. Soc. exp. Biol. Med. 112: 12-15 , 1963.

14. Sar, M. and J. Meites. Effects of suckling on pituitary release of prolactin, GH, and TSH in postpartum lactating rats. Neuroen- docrinology. 4: 25-31 , 1969.

15. Shaar, C. J. and J. A. Clemens. Inhibition of lactation and prolactin secretion in rats by ergot alkaloids. Endocrinology. 90: 285-288, 1972.

16. Shelesnyak, M. D. Ergotoxine inhibition of deciduoma formation and its reversal by progesterone. Am. J. Physiol. 179 :301-304 , 1954.

17. S h e l e s n y a k , M. D. Some experimental studies on ovaqmplantation in the rat. Recent Prog. Horm. Res. 13: 269-322, 1957.

18. Shelesnyak, M. D. Maintenance of gestation in ergotoxine- treated rats by exogenous prolactin. Acta Endocr. 27: 99-109 , 1958.

19. Sulman, F. G. Hypothalamic Control of Lactation. New York: Springer-Verlag, 1970.

20. Wuttke, W., E. Cassell and J. Meites. Effects of ergocornine on serum prolactin to LH and on hypothalamic content of PIF and LRF. Endocrinology. 88: 737-741 , 1971.