Pineal Regulation of Immunoreactive Luteinizing Hormone and Prolactin in Light-Deprived Female Hamsters*

Vol.

FERTILITY AND STERILITY Copyright 1974 The American Fertility Society

25,

No.

11, November 1974 Printed in U.S.A.

PINEAL REGULATION OF IMMUNOREACTIVE LUTEINIZING HORMONE AND PROLACTIN IN LIGHT-DEPRIVED FEMALE HAMSTERS* RUSSEL J, REITER, PH.D" AND LINDA Y. JOHNSON, M.S.

Department of Anatomy, The University of Texas, Health Science Center at San Antonio, San Antonio, Texas 78284

Depriving female hamsters of light, either by blinding or by restricting them to 1 or 2 hours of light per day, causes their reproductive organs to involute. I-a . This dark-mediated atrophic response is a consequence of an activated pineal gland; if hamsters are pinealectomized, light deprivation is incapable of inducing gonadal atrophy.4 Although this response is well known, relatively little has been done to determine levels of gonadotropins and prolactin in light-deprived hamsters. This paucity of information is due primarily to the lack of specific, sensitive assay methods with which to measure these hormones. Recently, Goldman and Porter5 and Donofrio et a1 6 developed radioimmunoassays for hamster luteinizing hormone (LH) and prolactin, respectively. Using their techniques, we have investigated the influence of light deprivation, pinealectomy, and pineal denervation on pituitary and plasma levels of LH and prolactin in adult female hamsters. MATERIALS AND METHODS

Sixty-two adult female hamsters (weighing 60 to 80 gm) were purchased from Lakeview Hamster Colony (Newfield, NJ). They were placed in a room illuminated for 14 hours each day; three to five animals were in each cage. The hamsters had free access to food (Wayne Received November 27,1973. *Supported in part by USPHS research grant HD-06523. RJR is a USPHS Research Career Development Awardee (1-K4-HD-42398l.

Lab-Blox) and water throughout the study. Within 1 week of their arrival, 46 of the animals were blinded by bilateral orbital enucleation. Ten of the blinded hamsters were pinealectomized and 11 were superior cervical ganglionectomized. Sixteen animals received no surgical procedures. The techniques of pinealectomy 7 and superior cervical ganglionectomy2 have been described elsewhere. All surgical procedures were carried out on animals anesthetized with sodium pentobarbital. After these procedures, all animals were maintained without further treatment for 8 weeks. After this period (during the final week of the study), 12 blinded hamsters and eight intact hamsters were given twice daily subcutaneous injections of synthetic luteinizing hormone-releasing hormone (LH-RH) (Beckman Instrument Co) in a 16% gelatin solution. Each injection contained 2 fJ-g (total of 4 fJ-g daily) ofLHRH. The injections were continued for 1 week. The control animals were given the 16% gelatin solution only. All hamsters were necropsied at the end of 9 weeks. The LH-RH was prepared for injection in the following manner. After the LHRH was dissolved in saline, it was mixed with a slightly prewarmed gelatin solution to a final concentration of 10 fJ-g LHRHlml 16% gelatin solution. After thorough mixing, the LH-RH-gelatin solution was aspirated into 1 CC disposable syringes and refrigerated until use. LH-RH solutions were prepared fresh every 5 days.

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PINEAL GLAND, LH, AND PROLACTIN

TABLE 1. Body Weight and Absolute and Relative Organ Weights of Adult Hamsters After Various Treatments Organ weightsa Ovaries

Group treatment

1. 2. 3. 4.

No. of Body hamsters wt. (gm)

Intact (control) 8 IntactandLH-RH 8 Blinded 13 Blinded and LH-RH 12 5. Blinded and pinealectomized 10 6. Blinded and ganglionectomized 11

Absolute (mg)

Uteri

Relative (mg/lOO gm body weight)

Absolute (mg)

Anterior

Relative (mg/100 gm body weight)

Absolute (mg)

Relative (mg/lOO gm body weight)

151±5 30.9 ± 1.2 20.7±0.9 335±30 233 ±21 3.91 ±0.15 3.27 ±0.17 155±3 46.9±3.6C 30.3±2.3c 346±27 233±17 4.70±0.16 3.03±0.13 176±6b 51.6 ±4.0d 28.9 ±2.0b 150 ± 18 d 89 ±15d 3.64±0.19c 2.11 ±0.17d 171±6b 52.2 ±3.5 d 30.5 ± 1.8c 144± 8 d

66 ± 4 d 3.82 ±0.24c 2.22 ±0.11 d

132±8b 26.3±1.6 20.2±1.3 352±24

276±26

4.09±0.18

3.17±0.18

146±3

249±20

4.56±0.15

3.12±0.13

28.3±1.1

19.5±1.1 365±28

aMean ± SE. bSignificantly different from intact control (J'< 0.05). CSignificantly different from intact control (J'< 0.01). dSignificantly different from intact control (J'< 0.001).

Shortly before the time of injection, the syringes were removed from the refrigerator and were allowed to come to room temperature. This was necessary since a 16% gelation solution would be too firm to inject at refrigeration temperature. The solution (2 f.Lg ofLH-RH in a volume of 0.2 ml) was deposited subcutaneously through an 18 gauge needle. The injections were given at about 8 AM and 4 PM each day. At necropsy, all hamsters were killed by decapitation. Trunk blood samples and anterior pituitary glands were collected for hormone analyses. Ovaries and uteri were weighed, preserved in Bouin's fluid, and histologically examined. Immunoreactive LH and prolactin levels were estimated using the double antibody radioimmunoassays developed by Goldman and PorterS and by Donofrio et al 6 respectively. Because rat and hamster prolactin inhibition curves were not parallel, it was necessary to express the hamster prolactin data in terms of a pool of standard hamster anterior pituitaries (SHAP).6 Data were statistically analyzed using an analysis of variance and t test. RESULTS

The blinded hamsters, unless their pineal glands were either removed or de-

nervated, weighed more than the control hamsters (Table 1). In hamsters, blinding frequently leads to an increased ... body mass. Also, the ovaries of the blinded animals were larger than those of the intact controls unless the blinded hamsters had been pinealectomized or had had their superior cervical ganglia removed. The ovaries of the intact animals treated with LH-RH also were heavier than normal. Uteri of blinded hamsters were markedly atrophic unless the animals had been pinealectomized or had had their ganglia extirpated. LH-RH treatment did not enlarge the uteri of either intact or blinded hamsters. Removal of the eyes reduced the weights of the anterior pituitary glands unless blinding was accompanied by removal or sympathetic denervation of the pineal gland (Table 1). Ovaries of blinded animals displayed a pronounced interstitial hypertrophy and a marked reduction in the number of follicles and corpora lutea (Fig. 1). LH-RH treatment of blinded hamsters for 1 week did not appreciably influence ovarian morphology. Ovaries of such animals still contained few follicles (Fig. 2). Both pinealectomy (Fig. 3) and superior cervical ganglionectomy (Fig. 4) completely reversed the structural changes of the ovary

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REITER AND JOHNSON

FIG.!' Ovary of a hamster 9 weeks after blinding. The pronounced hypertrophy of the interstitium and the reduction in tlie number of follicular elements is characteristic of ovaries of light-deprived hamsters (hematoxylin and eosin, x 30).

FIG. 2. A section of an ovary of a blinded hamster treated with synthetic LH-RH for 1 week. Only a few small follicles are present and the ovary lacks corpora lutea (hematoxylin and eosin, x 30).

FIG. 3. A histologic section of an ovary of a pinealectomized, blinded hamster. These ovaries are comparable to those of control animals maintained in long daily photoperiods (see Fig. 5). Note the sparse interstitial tissue, numerous follicles, and corpora lutea (hematoxylin and eosin, x 30).

November 1974

normally associated with blinding. In fact, the ovaries of the pinealectomized and ganglionectomized hamsters were histologically indistinguishable from those of intact control hamsters kept in long photoperiods (Fig. 5). Ovaries of intact hamsters treated with LH-RH were histologically modified. These ovaries, in addition to containing the usual number of preantral and antral follicles and corpora lutea, also possessed fresh corpora lutea as evidenced by the presence of blood clots in their central portion (Fig. 6). Compared to the normal intact hamsters, the blinded hamsters had significantly higher levels of LH within the anterior pituitary gland (Fig. 7, top). Blinded hamsters that were either pinealectomized or ganglionectomized had LH values comparable to those of the controls. Similarly, LH-RH-treated blinded hamsters also had pituitary LH levels as low as those of the intact hamsters. The administration of LH-RH to intact hamsters did not alter the amount of LH in the anterior pituitary gland. Pituitary prolactin levels were also altered by light deprivation. In this case, blinding caused a highly significant decrease in the content and concentration of prolactin within the anterior pituitary gland (Fig. 7, bottom). Again, this change was prevented in hamsters that had had their pineal glands removed or sympathetically denervated. Treatment of hamsters with LH-RH did not significantly change the levels of prolactin in the pituitary gland. Although the plasma levels of LH in blinded hamsters were about twice those in normal control hamster, the increase was not statistically significant (Fig. 8). Blinded hamsters that were either pinealectomized or superior cervical ganglionectomized had plasma titers of LH comparable to those of control hamsters. Treatment of either intact or blinded hamsters for 1 week with synthetic LH-RH caused a significant elevation ofLH in the

PINEAL GLAND, LH, AND PROLACTIN

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plasma. Plasma prolactin levels did not differ among the six experimental groups (Fig. 8). DISCUSSION

Without knowledge of hormone levels, Reiter 8 in 1969 speculated that depriving female hamsters of light leads to a hormone imbalance in which the plasma level of follicle-stimulating hormone (FSH) is depressed and the level of LH is maintained sufficiently high to cause hypertrophy of the ovarian interstitium. This idea is supported by the present results on blinded hamsters which showed that the pituitary levels ofLH are elevated and plasma titers remain at least at the level of those in control hamsters. The normal titers of LH in blinded hamsters are consistent with the ovarian morphology in these animals. Greenwald9 ,lO found that the growth of ovarian interstitial tissue depends on LH. Hypophysectomized hamsters injected with LH alone had ovaries similar to those of lightdeprived hamsters. In Greenwald's experiment, LH was present and FSH was either absent or severely depressed. In the female hamster, it appears that the pineal gland induces functional regression ofthe pituitary-ovarian axis by depressing FSH and by permitting LH to be maintained at the normal levels or even to be somewhat increased. In support of the proposed theory, Greenwald9 ,lO also pointed out that the neural mechanisms governing FSH in the hamster are extremely sensitive to exteroceptive influences. However, proof that FSH levels are altered by the pineal gland in the hamster must await the development of a sensitive and reliable assay for FSH in this species. A depression of FSH would also explain the uterine atrophy. Without FSH to cause maturation of the follicles, little estrogen would be synthesized and secreted. In the absence of estrogen, the uteri would exhibit the profound regression characteristic of the uteri of blinded

FIG. 4. Ovary of a blinded hamster whose pineal gland was sympathetically denervated. Superior cervical ganglionectomy reversed the effects of blindness on the gonads, as did pinealectomy, (hematoxylin and eosin, x 30).

FIG. 5. Ovary of an intact hamster kept in 14 hours of light and 10 hours of darkness daily (hematoxylin and eosin, x 30).

FIG. 6. Section of an ovary from an intact hamster injected with synthetic LH-RH for 1 week. In addition to containing the usual follicular elements and corpora lutea, numerous hemorrhagic corpora lutea (arrow) were found in these ovaries. The hemorrhagic corpora lutea, indicative of recent ovulation, were never observed in the ovaries of control hamsters (hematoxylin and eosin. x 30).

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REITER AND JOHNSON

400

1500

300

...L

z

>::0_

",>-e

>-'z, 5~~

1000

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~ei~ 0:0: ..

,.L

>-Z"-

0:"'4

,.l

200

>::

-'>-j!~~

~8'=

t:z .....

"-8

500

iL

100

o

NOR

NOR LH·RH

Bt

Bl LH·RH

Bt PX

Bt SCGX

600

o

3000

500

100

NOR

NOR

LH·RH

Bl

r~ Bl LH·RH

Bl

PX

Bl SCGX

FIG. 7. Pituitary LH (top) and prolactin (bottom) levels in female hamsters after various types of treatments. * = P
hamsters. In an earlier study in which both LH and FSH were injected into blinded female hamsters, follicular development was apparent, ovulation occurred, and uterine growth was stimulated. l l The effect of blinding on prolactin levels confirms a previous preliminary report from this laboratory. 6 The depression in pituitary prolactin was highly significant but it was not associated with a detectable change in the plasma titer of this hormone. The change in pituitary prolactin was doubtless a consequence of the activated pineal gland since its removal negated the effect of blinding. Because prolactin is regulated primarily by the

November 1974

prolactin inhibiting factor (PIF) from the hypothalamus,12 the pineal gland of the light-deprived female hamster may act to control either the synthesis or the release ofPIF. Darkness is known to stimulate at least one enzyme system within the pineal gland of the hamster. AntonTay and Wurtman 13 found an increased activity of the melatonin-forming enzyme,hydroxyindole-O-methyltransferase (HIOMT), in dark-exposed hamsters. Melatonin has also been shown to change the secretion rate of prolactin from the anterior pituitary glands ofrats.14 Hence, the pineal substance which regulates prolactin in the hamster may be melatonin. Morphologic studies of the reproductive organs of light-deprived hamsters have shown that pinealectomy or superior cervical ganglionectomy are equally effective in reversing the effects of darkness on the pituitary gonadal axis.4.8 The present results support these studies. Either surgical removal of the pineal gland or its sympathetic denervation in blinded hamsters resulted in LH and prolactin levels which were indistinguishable from those of intact control hamsters (Fig. 5). The functional innervation of the pineal gland in rodents is derived from the superior cervical ganglia 15 and their removal prevents darkness from influencing the enzyme activity of the organ. 16.17 Arimura et aPB have shown that female hamsters respond acutely to the intravenous injection of either natural or synthetic LH-RH as evidenced by a significant rise in plasma levels of LH within 15 minutes. The present study shows that chronic treatment (twice daily for 7 days) of hamsters with LH-RH deposited subcutaneously also releases pituitary LH. This is reflected in the significant elevation of LH in the plasma in both intact and blinded female hamsters (Fig. 1), a decrease ofLH in the pituitaries of blinded animals (Fig. 7), and recent ovulations of the intact animals (Fig. 6). According to

Vol. 25, No. 11

963

PINEAL GLAND, LH, AND PROLACTIN

8

6

!

p(O.OI

~ o

NOR NOR BL lH.RH

BL

BL

BL

lH·RH PX SCGX

o

NOR NOR lH-RH

BL

BL

BL

BL

lH-1M PX SCGX

FIG. 8. Plasma LH (left) and prolactin (right) levels in female hamsters after various types of treatment. Vertical lines at top of bars = standard errors; NOR = normal intact hamsters; LH-RH = hamsters treated with synthetic luteinizing hormonereleasing hormone; BL = blinded; PX = pinealectomized; SCGX = superior cervical ganglionectomized.

Schally and colleagues,12 LH-RH liberates both LH and FSH. In the present study there was no evidence of FSH secretion in blinded hamsters. Follicular development was not appreciabJly stimulated by LH-RH in the blinded animals (Fig. 2) and the uteri remained atrophic, indicating little or no estrogen secretion. Had both LH and FSH been stimulated by the LHRH, follicular maturation and estrogen secretion would have been expected. On the other hand, the LH-RH could have released FSH but its action at the ovaries may possibly have been inhibited. Presuming, as theorized above, that FSH is greatly depressed after blinding, the apparent inability ofLH-RH to release FSH in blinded hamsters could be related to the small residual supply of this hormone within the anterior pituitary gland. As in the rat, LH-RH had no effect on prolactin levels. 12 SUMMARY

The blinding of adult female hamsters caused atrophy of the reproductive organs, an increased level ofluteinizing hormone (LH), and a decreased level of prolactin in the anterior pituitary gland. Each of these effects in blinded hamsters

was reversed by pinealectomy or by superior cervical ganglionectomy, a procedure which sympathetically denervates the pineal gland. Chronic subcutaneous injections (2 ILg twice daily for 1 week) of synthetic luteinizing hormone-releasing hormone caused a release of pituitary LH as evidenced by the increased level of this gonadotropin in the plasma. LH-RH had no apparent influence on plasma prolactin titers or on the secretion of folliclestimulating hormone. Acknowledgment_ Kits for the double antibody radioimmlmoassays were supplied by The Rat Pituitary Hormone Distribution Program of NIAMDD_

REFERENCES 1. Hoffman RA, Reiter &T: Responses of some en-

docrine organs of female hamsters to pinealectomy and light. Life Sciences 5:1147, 1966 2. Reiter &T, Hester &T: Interrelationships of the pineal gland, the superior cervical ganglia and the photoperiod in the regulation of the endocrine systems of hamsters. Endocrinology 79:, 1168, 1966 3. Reiter &T, Sorrentino S, Jr: Reproductive effects of the mammalian pineal. Am Zool 10: 247, 1970 4. Reiter &T, Fraschini F: Endocrine aspects of the mammalian pineal gland: a review_ Neuroendocrinology 5: 219, 1969

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5. Goldman BD, Porter JC: Serum LH levels in intact and castrated golden hamsters. Endocrinology 87:676, 1970 6. Donofrio RJ, Reiter RJ, Sorrentino S, Jr, et al: A method for measurement of prolactin in the hamster by means ofradioimmunoassay. Neuroendocrinology 13:79, 1973174 7. Hoffman RA, Reiter RJ: Rapid pinealectomy in hamsters and other small rodents. Anat Rec 153:19, 1965 8. Reiter RJ:Pineal function in long term blinded male and female golden hamsters. Gen Comp Endocrinol 12:460, 1969 9. Greenwald GS: Histological transformation of the ovary of the lactating hamster. Endocrinology 77:641, 1965 10. Greenwald GS: Luteotropic complex of the hamster. Endocrinology 80: 118, 1967 11. Reiter RJ: Failure of the pineal gland to prevent gonadotrophin-induced ovarian stimulation in blinded hamsters. J Endocrinol 38:199, 1967 12. Schally AV, Arimura A, Kastin AJ: Hypothalamic regulatory hormones. Science 179: 1973

November 1974

13. Anton-Tay F, Wurlman RJ: Stimulation of hydroxyindole-O-methyltransferase activity in hamster pineal gland by blinding or continuous darkness. Endocrinology 92:1245,1968 14. Kamberi lA, Mical RS, Porter JC: Effects of melatonin and serotonin on the release of FSH and prolactin. Endocrinology 88:1288, 1971 15. Kappers JA: The mammalian pineal organ. J Neurovis Relat [Suppl] 9:140, 1969 16. Wurlman RJ, Axelrod J, Fischer JE: Melatonin synthesis in the pineal gland: effect of light mediated by the sympathetic nervous system. Science (NY) 143:1328, 1964 17. Klein DC, Weller JL: Indole metabolism in the pineal gland: a circadian rhythm in N-acetyltransferase. Science (NY) 169:1093, 1970 18. Arimura A, Debeljuk L, Schally AV: LH release by LH-releasing hormone in golden hamsters at various stages of estrous cycle. Proc Soc Exp BioI Med 140:609, 1972