Interaction of photic and olfactory stimuli in mediating pineal-induced gonadal regression in adult female rats

QENERAL

AND COMPAHATIYI~

Interaction

ESDOCaI40LOGY

of Photic

Induced

Gonadal

RUSSEL

and

15, 326-333

Olfactory Regression

(1970)

Stimuli

in Mediating

in Adult

Female

J. REITER,2 SANDY SORRENTINO, AND NEIL ?tI. ELLTSON

PinealRats’

JR ..

Whereas neither blinding nor olfactory bulb removal alone appreciably alt erc~~l the reproductirc organs of adult female rats. when animals were subjected to I)otlr operations, the sexual organs were macroscopicall>and microscopically hypotrophic within 8 weeks; thr frequency of pregnancy also was reduced greatly in the doubly deprived rats. Furthermore, adrenal and pituitar. weights were c:ll:rractc~ri.~tically smaller in rats deprived of their eyes and olfactory bulbs. Pinealectomy reversed :111 the effects which follo\wd dual sensory deprivation. Xo statistically signiticant differences in the pineal Icwls of hSdros~intlol(~-O-mcth~ltrnnsfcr:~~f, :rr:ti\-it!. \V~W

Behaviorally, nasogenital relationships are relatively clearly defined. For example, the consequences of olfactory bulb lesions on mating responses in rodents have been determined (Heimcr and Iarsson, 1967; Bermant and Taylor, 1969) although even here some inaccurate generalizations have been made; these have been pointed out by Murphy a,nd Schneider (1970). In addition to the behavioral changes associated with anosmia, there arc, in rats, rathei subtle endocrine adjustment:: which alter the reproductive physiology (Kling, 1964 ; Orbach and Kling, 1966; Rritcr and Ellison. 1970) ; the induced cntlocrine changes arc ordinarily suppressive to the reproduct,ivc system. Light, deprivation also adversely influences t)he sexual behavior (Baum, 1969) and t’he growth of the reproductive organs (Wurtman et nl., 1961; Itoh et al., 1962: Hoffman and Reitcr, 1965a) of rodents. In ‘Supported by Grant * U.S.P.H.S. Career 1 K04 HD42398. a U.S.P.H.S. Postdoctoral 01.

HD-02937, Drvelopmcnt Fellow

U.S.P.H.S. Awardcc. lF024042856

this case, pincal ablation has an ameliorative effect on the gonads (Wurtman et al., 1961; Quay, 1969; Rciter and Fraschini, 1969). Since both anosmia and light, deprivation indel~entlently modulate reproduct~ivc functions. the present cxperin1ent.s were designed to test t>he influence of the combined treatments on the sexual 1)hysiology or’ adult female rats. Furthermore. the effects of the pineal gland on tltc: observed rcsponsc’s were determiilc$tl. Correlative studies on the pinenl content of the mclntonin forming enzyme. hp~lroxyintlole-(~Illr’thyltralisfera~~,~ were carried out,. VI-4TERIATS

-44KD

METHODS

Rats used in t,his study wcn~ of the SpragurDawley strain; they weighed between 150 and 183g (approximately 55 days of age) at the onset of the investigations. The animals were routinely housed two per wire-bottomed rage in tcmpersture (23 ‘- 2°C) and light (L14:DlO in hours) r*ontrolled rooms. All animals had free o(cess to food and water; some food was always placed on the floor of the cages. The details of the operations wliich were employed have been described elsewhew: orbital enucleation (Reikr et tr2., 1968). pinealwtomy (Hoffman and Reiter. 1965b) using

PINEAL

IXFLCENCE _~~~~

OF PHOTIC ..INI) AKD THE PINEAL

TABLE OLFACTORY GUNDS

OF

Body Group

and

treatment

32T

FUNCTION

1 STIMULI ADULT

ON THE BODY FEMALE RATS

weight (g)

A-

Pineal

weight HIOMT~

be)

.-__

1. I:nt,reated

10

243

+

186

1.44

*

0.71

2. Blinded

11

227

f

1.5

1.350

f

0.64

12

3. Bliuded;

pinealectomixed

4. Anosmic ,FJ. Anosmic; 6. Blinded;

piuealectomized anosmic

7. Bliuded; anosmic; .-. ‘I hlicromicromoles tissue per hour given h 1 )ata expressed as

pinealectomized of melatonin formed in parentheses. means + standard

8

239

*

7

239

+ 2.5

1.39

10 12

238 222

* 19 + 14

1 32 f

12

238

+ 14

-

40.2 (27.9 57.8 (38.5

k 18.9 zk 14.6) Z?Y24.6 * ls.aj -

49.3 (3.5.3

k 20.2 + 14.7)

54.4 (36.8

zk 19.2

-

per pineal

per

deviatious;

no siguificant’

the Seuman pinealectomy instrument (H. Neuman and Co., Skokie, Illinois), and olfactory bulb removal (Reiter et al., 1969). The operations were done with the animals anesthesized with sodium pentobarbit.al. The various groups of animals used and the number of animals per group are listed in Table 1. During the last 2 weeks of the experiment daily vaginal smears were taken by lavagc. These were air-dried and wvcre stained with toluidine blue. All animals were killed by exsanguination under cxt,her anrsthesia hcltween 1O:OO AM and 2:00 PM 8 weeks after the onscxt of the experiment. At nrc~~~psy. body. ovarian. uterine, adrenal, pituitary, and pincal weights were recorded. Tissues, except thr, pin&s. were fixed and processed for histological examination. Pinc,als were homogenized and assayed for their content of hydroxyindolc-Omt~thyltransferasc (HIOMT) using the technique of Axclrod et al. (1965) as modified by Weiss (19&Y%) Numerical data were subjected to an analysis of varinncc~. Sinccl the first study rcxvealrd that the combination of blinding and anosmia had such drastic ef’frscts on the reproductive organs, a second cxI>&ment was conducted to determine the breeding ability of these animals. Three groups of female rats wcrc used: normal rats, blinded nnosmic rats, and blinded anosmic rats that were also pinealectomized. Eight weeks after the operations, two or three experimental females were placed in clear plastic cages with two sexually expcrienced adult males. The animals were left togr,thcxr unt,il the frmalcs were oh\-iously pregnant

hour;

WEIGHTS

melatonin

I!Z 0.43

0.59

i.

15.1)

formed

differences

per between

milligram any

of pineal of the means.

or, in the case of the animals which did not breed successfully, for 30 days. No attempt was made to determine whether the females were receptive to t.he males. When the pregnant animals had delivered the pups, thr mothers were killrd with ether. A total of (94 rats wcrr utilized in the two experiments. RESULTS

Although the mean body weight of the blinded anosmic rats (Table 1, group 6) was less than that of any other group, the decrease was not statistically significant. The most obvious consequences of sensory deprivation were those exhibited by the reproductive organs. Although neither blinding nor anosmia significantly altered the sizes of the ovaries, rats that had been subject’ed to both operations had ovaries that were approximately 35% smaller (absolutely and relat’ivcly) than those of the untreated controls (Fig. 1, A). On the other hand, if rats were both eyeless and anosmic, but were also pinealectomized, the ovaries were equivalent in size to those of normal rats. A similar pattern was obscrvcd in the microscopic structure of the ovaries. Removal of the eyes (Fig. 2) or of the olfact’ory bulbs (Fig. 3) singly failed to alter the histologic appearance of the ovaries, but, when combined the gonads exhibited

NOR

BL

BL ANOS ANOS BL BL PX PX ANOSANOS PX

1

0

NOR

BL

BL ANOS ANOS BL BL PX px ANoS ““i?”

-(

changes characterized by rctcluccd follicular development and fewer and smaller corpora. lut’ea (Fig. 4). This latter finding was O~Jvious when serial s&ions of ovaries werta studied. The effects of blinding and anosmia were reversed by pincal removal (Fig. 5). Thus, the only rats that possessctl ovaries t’hat were microscopically distinguishable from the gonads of normal caolltrols ww animals that wercl tlcprivrd of two sf’nsos but, had intact, pincal glands. The inhibition of t,llc pituitary-gonadal axis wan cmphasizctl also by thr uterine, changes (Fig. I .BI. Hrrc, agaill, the> only procedure that sckusly impaired uterillc> growth was tlicx cQombinntion of visual a1111 olfactory cleprirat,ion; in this group tlic mean ut’erinc wtxiglit waq only about’ 40% of the contkol valuc~. AS with the ovaric,s-, pineal removal prtlvcantc>tl tlicl utc>rinc liypotrophic rcS~pon~c.Hi~tologic~nlly, the ut c>riof all groups n-caretrom~J:kraI)l(~ cbxccyt tlro>ci

PX.

PX ANOS AypS

: in t,heir

histt roll1 tioii foIli< phd

ova rian Aing uish

In a.ddifesic :ular 71 in this the lion-

330

REITER,

BLINDED

ANOSMIC

BLINDED,ANOSMIC,PlNX

SORRENTINO,

JR.,

AND

ELLISON

The pituitary-adrenal axis was also influenced by some of the treatments (Fig. 1,C). Removal of the eyes caused a modest depression of the absolute adrenal weigh& which was reversed by pinealectomy. Also. combined eyelessncss and anosmia led to a highly significant retardation of adrenal growth; this was likewise counteracted by ablation. Despite these gross pineal changes, microscopically it, was dificult t,o distinguish the adrenals of any one group from those of another. As with the adrenals, blinded ant1 blinded anoamic rats experienced some reduction in the growth of the pituitary glands (Fig. 1,U) ; this was not apparent in pinealectomized control groups, however, Detailed hist,ologio studies of the pituitary glands were not, conducted. The mean weights of the pine& from the four groups of nonpinealectomized rats did not differ significantly from one another (Table 1). Similarly, although there was a tendency for the pineals of blinded rat’s to have higher levels of HIOMT activity. no statistically significant differences in elthei the absolute or relative amounts of ihis enzyme were found. The second experiment was designed t,o determine tQe breeding capability of blinded anosmic female rats. Seven of 8 normal rats became pregnant after exposure t)o the males for 30 days (Table 21, Conversely, only 2 of 7 blinded anosmia rats delivered young and at least one of these animals had a smaller than normal litt’cr. If the animals were pinealectomizetl a greater peraent#agc became pregnant and the litter sizes were nearly normal ; however, the percent,age of animals that’ dclivcretl was tiot. :ts great as for untrcatrrl coiitrol rat-.

RATS

RATS

FIG. 6. 1)iagrammalic represelltarioti of 14 consecutive vaginal smears from 24 rats (8 from each of t,he three experimental groups indicated). Smears from each animal run horizontally. Smears were taken &wing the final 2 weeks of the experiment,. D = diestrus; I’ = proedrils; 13 = estrns; n,l = metest,rus.

cycles; the changes were characterized by a prolongation of t11c cycle, dicstrous smears being nirich more frequent,

I,ight deprivation in :nlult female rats usually has relatively little intlucncc on the rcproductivc system ~Browinan. 19373 , although an occasional animal may become ancstrous after long term 190 tlaysj blinding (Hoffmann, 19671. With the period of time utilized in the prerent study (56 days), none of the blinded rats esliil)ite~l any npl~rcc~iahl~~ rhange in th vngiil:tl cycle or iit tli(b size of the rc~protltic~ti~-i>ot’g:trii’. 5inti-

PINEAL

INFLYENCE

_.

Gror~p

OF III-AL

and

SENSORY

t,reatment

--1. Untreated 2. Blinded; anosmic 5. Blinded; anosmic;

pinealect.omized

DEPRIVATION

331

FCNCTION

TABLE 2 ON FREQCENCY

N

Number of pregnancies

8

7

7 9

2 6

larly, because of the rather large variations between animals in the group, there was no statistically significant increase in the pineal level of HIOMT in the blinded rats; this latter finding is uncommon (Wurtman, 1967)) however, rarely have rats been tested for their pineal content of this enzyme after such a long period of light deprivation. The impairment of reproductive functions caused by olfactory bulb removal is usually discussed in terms of the behavioral, rather than the physiological, consequences (Bermant and Taylor, 1969; Larsson, 1969; Murphy and Schneider, 1970). The present. findings, however, confirm the observations of earlier workers (Shelesnyak and Rosen, 1938; Rosen et al., 1940; Van Beugen and van der Wcrff ten Bosch, 1961) that in the rat, anosmia has only modest effects on ovarian physiology. The slight’ly reduced reproductive efficiency in the anosmic rats that were blinded and pinealectomized (Table 2, Group 3) may have been a result of changes in emotionality (Kumadaki et aI., 1967), although other explanations probably also exist. The manner in which anosmia potentiates the gonad-inhibiting influence of the pineal gland in blinded rats is unclear. ,4s in previous studies with maturing rats (Reiter et al., 1969; Reiter and Ellison, 1970), it is quite apparent, t#hat, in adult rats also, removal of the eyes combined with ablation of the olfactory bulbs leads to ma&cd hypotrophy of the genital apparatus unless the rats are pinealectomized. It is known, of course, that light deprivation enhances pineal antigonadotropic activity (Axelrod et al., 1965; Wurtman, 1967; Quay, 1969; Reiter and Fraschini, 1969) ; however, whether the reproductive effects of anosmia are mediated via the pineal gland has not l)een &ablished. Based 011 the levels of

OF PREGNANCY

IN

FEMALE

RATS

Litter sizes (mean) 6, 8, 9, 9, 10, 11, 11 (!,.l) 2, 7 (4.3) 3, 6, 7, 9, 11, 12 (8.0)

pineal HIOMT, it would appear that loss of the ability to smell does not activate the melatonin-forming enzyme within the pineal (Reiter and Ellison, 1970). However, this does not preclude the possibility that anosmia may alter the biosynthetic act,ivitp of other pineal compounds which, in turn. may be inhibitory to the reproductive: system. Another explanation is t’hat anosmia exaggerates the dark-induced gonadal illhibition of the reproductive organs by rendering the site of action of the reproductive antagonist very sensitive to the pineal substance; the loci of action of the pineal principles generally are considered t’o be within the central nervous system (Quay, 1969; Reiter and Fraschini, 1969). Fibers arising wit’hin the olfactory bulbs are widely distributed to the hypothalamus (Scott and Pfaffmann, 1967) and limbic system (Heimer, 1968) and the destruction of efferent olfactory fibers leads to widespread chemical changes within t’he brain (Pohorecky et al., 1969). Thus, it is difficult with the amount of available knowledge, to predict, where the interaction of anosmia and the pineal substance may take place to modify the neuroendocrine-gonadal axis. Another consideration is that the gonads of blinded anosmic rats underwent regression because of the stress of the combined operations. This argument seems to be negated by two fact’s: (1) when blinded anosmic rats were subjected to an additional operation, pinealectomy, the reproductive organs were normal in appearance; the logical assumption is that three operations would be more stressful than two, but the additional procedure reversed rather than enhanced the effects of eyelessness and anosmia ; (2) adrenal glands of blinded anosmic rats were significantly smaller than those of all other groups, further suggesting

sponsc brain.

after removal of frontal parts of the Actn E~~docri/~ol. 37, 470-478. WEISS, B. (1968). Discussion of the formation, mct,abolism and physiologic effects of melatonin. Advun. Pharmacol. 6, Part A, 152-155. ~VIJRTMAN, R. J. (1967). Effects of light and visual stinluli on endocrine function. I,n “Neuroendo-

crinology” Vol. II.

(I,. Martini and W. F. Ganong, pp. 19-59. Academic Press, Nt:~y WURTMAN, R.J., ROTH, W. D., ALTSCTIULE, AND WURTMAN, J. J. (1961). Interactions pineal and exposure to continuous light on weights of fcmnle rats. Acfn Endocrinol. 617-624.

rds.). York. M. I).. of the organ 36,