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A Possible Role of the Pineal Gland in Pregnancy and Fertility
421
A Possible Role of the Pineal Gland in Pregnancy and Fertility I. NIR and N. HIRSCHMANN Department of Pharmacology and Experimental Therapeutics, Hebrew University, Schools of Medicine and Pharmacy, Jerusalem (Israel)
Until little more than a decade ago no information was available on whether the pineal gland influences gestation, or vice versa, whether pregnancy may affect pineal metabolism and function. The first observation on such an interrelationship was of decreased pineal weight in late pregnancy in rats bearing 10 or more embryos and was published in 1965 by Huang and Everitt. They considered their findings to indicate depressed pineal activity during the period studied. Later investigators focused their attention on possible metabolic changes in the pineal during pregnancy. Results obtained, however, suggested intensified metabolic activity, enhanced pineal NADH and NADPH diaphorase activity being recorded in guinea-pigs during the second half of pregnancy which disappeared after delivery, oophorectomy or hysterectomy (Vollrath and Schmidt, 1969). Moreover, histological changes observed by electron microscope in the fine structure of so-called “light” and “dark” pinealocytes during the second half of pregnancy may too serve as supportive evidence of increased pineal activity during that period (Lues, 1971). Nevertheless, there are findings to uphold the earlier suggestion of Huang and Everitt (1965) of decreased pineal activity during late pregnancy; for instance DeverEerski (1972) described decreased activity of the pineal in lactate dehydrogenase, isocitrate and other enzymes involved in glycolysis on day 15 of pregnancy. Regarding the metabolism of specific pineal indoleamines, a study carried out in pseudopregnant rats indicated that they have a different diurnal pattern of HIOMT activity although no deviation in its mean rate (Yochim and Wallen, 1974). A comprehensive study of the general metabolism and hormonal patterns of the pineal during gestation, as well as of their significance in the processes of pregnancy was considered timely. Various parameters reflecting general and indoleamine metabolism (RNA, DNA, SHT, NAT and HIOMT) were measured in the pineals of rats on days 8 and 19 of gestation, representing respectively the first and second halves of pregnancy. The values obtained were compared to, and found to be identical with, those of non-pregnant rats during dioestrus. Neither the basic pineal metabolism as reflected in nucleic acids and protein contents, nor the hormonal indoleamine appeared to be changed during pregnancy (Nir and Hirschmann, in preparation). There were, however, indications suggesting pineal implications in processes connected with gestation and fertility. Rodents exposed to continuous darkness, to short daily photoperiods (L2 : D22), or to decreased light intensity throughout the gestation period, demonstrated significantly shorter length pregnancies than controls kept in normal lighting (Ellendorf and Smidt, 1971; Mitchell and Yochim, 1970), while long daily photoperiods (L22 :
422
D2) delayed parturition (Mitchell and Yochim, 1970). However, removal of the pineal gland -which usually mediates between environmental lighting and hormone producing glands-did not abolish the effect of short photoperiods on the duration of gestation (Mitchell and Yochim, 1970) which suggests that the effect of darkness on gestation may not be mediated by the pineal gland. Neither exposure to low intensity light (Ellendorf and Smidt, 1971), blinding, nor pinealectomy (Reiter et al. 1976) prior to fertilization, had any effect on the numer of offspring, and in the case of pinealectomy, the length of gestation period (Misuno and Sensui, 1970). However, significantly fewer living foetuses were found in animals pinealectomized during pregnancy (on day 11) than in those that had undergone sham-operation(Guerra et al., 1973). Similarly confusing is the role of the pineal gland in fertility processes. While pinealectomy had no effect on the fertility index of rats (Kincl and Benagiono, 1967), blinding of hamsters markedly reduced the number of fertile matings (Reiter et al., 1976) and fertility was also reduced in rats subjected to blinding and anosmia (Reiter, 1972). These effects could be negated by pinealectomy or chronic melatonin treatment (Reiter, 1972; Reiter et al., 1976) suggesting a progonadal effect of melatonin in pregnant rodents. In contrast to the above fmdings, melatonin and arginine-vasotocin(Vaughan et al., 1976) and several polypeptides extracted from the pineal gland (Orts et al., 1977) were found to inhibit fertile matings when injected into females before copulation. It has been suggested that the pineal hormones under certain conditions act on fertility by reducing LH secretion and subsequently suppressing ovulation (Orts et al., 1977).
LH AND PROLACTIN LEVELS IN PINEALECTOMIZED PREGNANT RATS DURING THE PERINATAL PERIOD There is now much evidence to indicate that the pineal gland regulates the synthesis and release of reproductive hormones by way of its indoleamine (melatonin) and polypeptide (arginine-vasotocin) hormones, mainly by central action at the level of the hypothalamus (Minneman and Wurtman, 1976). It may also be participating in the maintenance of the endocrine balance in pregnant animals, which undergoes profound changes during the last stage of gestation. Serum prolactin and LH levels - which are very low from day 5 after conception increase several-fold during the two days before parturition (Bast and Melampy, 1972; Linkie and Niswender, 1972) accompanied by a decrease in the high pituitary levels of prolactin (Ota, et al., 1974; Nir et al., 1977), LH and FSH (Tigchelaar and Nalbandov, 1975). Furthermore, the high serum concentrations of progesterone, which are at least partly responsible for the low levels of LH in serum, fall some days before parturition (Morishige et al., 1973; McCormack and Greenwald, 1974), while blood corticosterone (Ota et al., 1974), oestradiol and oestrone (Shaikh, 1971;McCormack and Greenwald, 1974) levels rise during the same period. It would seem feasible, therefore, that the pineal gland could be involved in these changes by influencing the abrupt rise in LH, prolactin and oestrogens and the decrease in progesterone which occur before parturition, through its action on the hypothalamic-pituitary axis. We decided fvst to study the effects of pineal ablation on pituitary and serum LH and prolactin levels, and on other parameters connected with embryo development and pregnancy outcome, during the last 4 days of gestation and immediately after parturition, in rats pinealectomized after weaning and kept in regular alternations of 12 h light and 12 h
423
TABLE I SERUM AND PITUITARY LH LEVELS IN PINEALECTOMIZED RATS ON DAYS 19-22 AFTER CONCEFTION (MEAN i SEM) Group
Days after conception
Serum LH (ng RP-l/ml) Pinealectomized
Sham-operated (control) Pituitary LH b g RP-l/gland) Pinealectomized
Sham-operated (control)
19
20
21
22
93.9 f 4.24 (42) 87.6 i 3.35 (40)
111.3 i 7.40 c (31) 97.0 i 3.96 (24)
125.1 i 5.93b (34) 96.2 i 6.06 (40)
209.5 i 34.86 a d (35) 134.9 i 11.91 e (39)
805.1 f 44.2 (42) 774.1 i 34.2 (40)
779.9 i 36.8 (49) 732.0 i 51.1 (37)
919.1 (43) 892.3 (57)
945.6 (42) 836.0 (53)
i
56.2
f
35.1
64.6 i 41.9
P < 0.05; b P < 0.005: significance of difference between the pinealectomized and sham-operated groups on the same days. C P < 0.05;d P < 0.02; e P < 0.005: significance of difference within each group compared to the previous day. Number of animals in parentheses. a
darkness. The results were as follows: Elevated serum LH levels were observed in the pinealectomized rats during the last 4 days of gestation when compared to those of their sham-operated controls, although the difference reached significance only during the last two days of pregnancy. Pinealectomy also TABLE I1 SERUM AND PITUITARY PROLACTIN LEVELS IN PINEALECTOMIZED RATS ON DAYS 19-22 AFTER CONCEPTION (MEAN f SEM) Group
Days after conception
Serum prolactin (ng RP-l/ml) Pinealectomized
Shamaperated (control) Pituitary prolact in (aRP-l/gland) Pinealectomized
Sham-perated (control) '
19
20
21
22
9.19 i 0.34 (42) 9.25 i 0.20 (39)
9.92 i 0.54 (47) 9.11 f 0.31 (35)
12.23 i 0.53 (49) 11.18 i 0.53 (55)
23.09 f 2.59 bg (37) 36.44 i 4.64 g (48)
54.94 i 1.89 a (36) 61.73 i 2.35 (36)
48.33 f 1.87 c*d (30) 56.91 i 1.95 (27)
48.46
34.55 f 2.08 (28) 43.25 i 2.86 g (37)
(50)
61.86 (5 8)
f
1.89
* 2.37
P < 0 . 0 5 ; b P < O.O2;CP< 0.005;dP<0.001: significanceof differencesbetweenpinealectomized and sham-operated groups on the same days. e P < 0.02; f P < 0.005; g P < 0.001: significance of differences within each group compared to the previous day. Number of animals in parentheses. a
424
advanced to day 20 the rise in serum LH concentrations which occurred in sham-operated animals between days 21 and 22 of gestation. No differences between the pinealectomized and sham-operated rats were observed in serum LH two hours postpartum. Despite the changes in their serum LH, no deviations from the sham-operated rats were observed in pituitary LH contents of the pinealectomized group, either before or after parturition (Table I). Serum prolactin levels were less affected by pinealectomy. Except for an occasional decrease in concentration of prolactin in serum of pinealectomized rats on day 22 of gestation, no differences were noted either before or after parturition. On the other hand, pituitary contents were lowered by pinealectomy in most, though not all, experiments at all times measured (Table 11). These results indicate that normally during the last days of gestation the pineal gland has an inhibitory effect on pituitary release of LH without apparently affecting LH synthesis, whereas in the case of prolactin it mainly increases the synthesis, occasionally enhancing release from the pituitary. It could therefore be interpreted that during the last phase of pregnancy the pineal gland is involved in modifying the balance of hormones by depressing LH release and increasing the synthesis of prolactin (Nir et al., 1979). LEVELS OF GONADAL HORMONES IN PINEALECTOMIZEDRATS DURING THE FINAL STAGE OF PREGNANCY Since an interrelationship between pituitary gonadotrophins and gonadal hormones has been established, it was considered necessary to determine whether any changes analogous to those in gonadotrophins occur in the levels of gonadal hormones in pinealectomized rats during the same stage of pregnancy. As the next step, levels of blood progesterone and oestradiol were measured in pinealectomized and sham-operated control rats during the last 4 days of pregnancy. Changes in levels of gonadal hormones and the exact timing of their occurrence should indicate whether the effects exerted by the pineal substances on the pituitary hormones are primary (direct) or secondary to the changes taking place in the gonads. Pinealectomy resulted in significantly increased levels of progesterone in the serum during days 19 and 20 of gestation, but this tended to disappear towards day 21, and no differences between the pinealectomized and sham-operated rats were noted on the day preceding parturition, when very low levels of progesterone were recorded in both groups (Table 111). In contrast to progesterone, serum oestradiol showed a sharp increase during the last two days of gestation in both pinealectomized and sham-operated groups, the increase being significantly greater in the pinealectomized (Table IV). It appears, therefore, that the pineal gland could be acting as a brake to the endocrine processes, i.e., depressing progesterone and oestradiol levels while they are high, though not be accelerating their decline nor preventing their rise. Consequently, the pineal may be able to modify the levels of gonadal hormones, but not to affect the sequence of their physiological events. Concerning the mechanism of pineal involvement in the regulation of gonadal hormones in pregnant rats, a direct pineal effect through the pituitary does not seem likely since the serum progesterone levels are increased in pinealectomized rats already by day 19 of gestation, when no changes occur in serum LH and prolactin concentrations as compared to those of sham-operated animals (Nir et al., 1979).
425 TABLE 111 THE EFFECTS OF PINEALECTOMY AND/OR MELATONIN TREATMENT ON SERUM PROGESTERONE LEVELS ( d m l f SEM) IN RATS DURING LATE PREGNANCY
Group
Days after conception 19
Sham-operated
80.7 (18)
Sham-operated and melatonin-treated Pinealectomized
99.4
20 f
f
3.9
3.1
Pinealectomized and melatonin-treated a
P < 0.05 ; b P < 0.01;
C
86.1 (21)
99.7
22
21 f
f
3.1
3.6
.
60.0 (18) 65.6 (10) 68.2 (17) 63.8
f
_+
6.1 5.2
* 6.3 a,d f
4.8
(11)
13.2 f 1.3 (20) 12.7 f 0.8 (11) 12.0 f 1.1 (21) 12.9 f 0.8 (11)
P < 0.001 : compared t o the sham-operated group. d P < 0.02; e P < 0.001 : sig-
nificance of differences within each group between consecutive days. Number of animals in parentheses.
Since it was suggested (Gibori and Richards, 1978) that the placenta regulates ovarian progesterone production in the second half of pregnancy, it could be that the increased progesterone levels induced by pinealectomy may result from enhanced production of placental hormones, as has been indicated in the case of the prolactin-like luteotropin, which reaches its peak on day 18 of gestation (Kelly et al., 1975). Another possible explanation may be an effect of the pineal on adrenal steroidogenesis. It has been found that on day 22 of gestation adrenal secretion of progesterone reaches a level several-fold that produced by the ovary (Ogle and Kitay, 1977). It is possible that the well-documented enhanced adrenal
TABLE IV THE EFFECTS OF PINEALECTOMY AND/OR MELATONIN TREATMENT ON SERUM OESTRADIOL LEVELS (pg/ml f SEM) IN RATS DURING LATE PREGNANCY
Group
Days after conception 19
Sham-operated Sham-operated and melatonin-treated Pinealectomized Pinealectomized and melatonin-treated
44.7 (18)
40.1 (20)
20 f
f
4.7
2.8
42.3 (18)
53.3 (18)
f
f
4.9
8.1
21
22
62.6 f 4.5 (20) 68.5 f 3.4 (11)
62.3 (20) 59.0 (20)
80.0 (20) 73.7 (10)
84.3 (19) 76.8 (21)
f
3.9 a,d
f
4.8
f 4.9 f.
3.4
f
6.1
f
4.3
a P < 0.02; b P < 0.01: compared to the sham-operated group. f P < 0.005: compared to the sham-operated and melatonin-treated group. P < 0.005: compared to the same group on the previous day. Number of animals in parentheses.
426
function brought about by pinealectomy (Nir, 1978) could be largely responsible for the increased progesterone concentrations in pregnant pinealectomized rats. As to the effects of progesterone on prolactin metabolism in pinealectomized rats during the period preceding parturition, it has been suggested that the sharp fall of serum progesterone that takes place at that time is one of the factors inducing the sudden increase in serum prolactin levels on the last day of gestation (Simpson et al., 1973; Vermouth and Deis, 1974). The finding that higher doses of progesterone are capable of inhibiting the stimulatory action of small doses of oestrogen on pituitary prolactin levels (Meites and Turner, 1948) also confirms the possibility that progesterone may be a regulatory factor in prolactin metabolism. It is therefore possible that the elevated serum progesterone concentration occurring in pinealectomized rats during days 19 and 20 of gestation (or perhaps even earlier than that) might elicit the lowered pituitary prolactin content observed prior to parturition. In regard to the changes in oestradiol brought about by pinealectomy, it could be that the pineal hormones normally exert an inhibitory effect on oestrogen production via the placental-pituitary complex mentioned previously, or directly on the ovaries. Another possibility is that more enhanced synthesis of oestradiol is triggered by the sudden decline in serum progesterone concentration towards the end of pregnancy. It is difficult to prove this, however, as long as the source, stimulus of production, and the role of oestradiol during the second half of pregnancy, are unknown. EFFECT OF MELATONIN VERSUS THAT OF PINEALECTOMY ON LH AND PROLACTIN LEVELS As the next step we explored the role of melatonin, the pineal hormone, during this crucial period, that is to say whether melatonin is capable of reversing the effects of pinealectomy on pituitary and serum gonadotrophins as well as on serum gonadal hormones. Weekly implantations of melatonin (1 mg) into pregnant rats commencing on the day after mating brought about a decrease in serum LH levels in both sham-operated and pinealectomized animals on day 22 of gestation only. On day 21 of gestation and immediately after parturition no effect was noted in either group. In this experiment pinealectomy was found to enhance serum LH only slightly (Table V). Melatonin treatment depressed pituitary LH content in sham-operated (but not pinealectomized) animals on day 22 of gestation: the same day pinealectomy decreased pituitary LH, a phenomenon not encountered in other experiments. A sudden decline in pituitary LH content takes place postpartum in the sham-operated but not in the pinealectomized or either of the melatonin-treated groups (Table VI). This finding is consistent with that of Tigchelaar and Nalbandov (1975) who treated pregnant animals with daily subcutaneous injections of melatonin and exposed them to prolonged daily photoperiods. On the other hand, melatonin treatment brought about an increase in serum prolactin levels on day 22 of gestation (though not on day 21 or postpartum) accompanied by a respective decline in pituitary prolactin content on the same day (Table VI). In addition, as already observed, pinealectomy induced a decrease in pituitary prolactin levels not only before, but also after parturition. In spite of its effect on LH and prolactin levels, melatonin implanted weekly had no influence on either progesterone or oestradiol in serum of either sham-operated or pinealectomized rats during the prenatal period (Tables 111 and IV).
427 TABLE V SERUM AND PITUITARY LH LEVELS ON DAYS 21 AND 22 AFTER CONCEPTION AND POSTPARTUM OF RATS PINEALECTOMIZED AND/OR IMPLANTED WITH MELATONIN (MEAN f SEM) Group
Days after conception
22
21
Serum LH (ng RP-l/ml) Shamaperated Shamaperated and melatonin-treated Pinealectomized Pinealectomized and melatonin-treated Pituitagv LH (pg RP-l/lgland)
Shamaperated
Shamaperated and melatonin-treated Pinealectomized Pinealectomized and melatonin-treated a
58.7 5 (14) 52.7 * (14) 61.3 f (13) 65.6 (16)
*
426.5
(7)
Postpartum
f
5.6
76.4 f (19) 86.7 f (18) 92.8 f (11) 84.6 f (18)
5.0 5.6 3.2i
51.1
381.2 f 48.0 (9) 466.1 f 82.8 (10) 428.5 f 43.9 (11)
403.3 f 25.2 (18) 330.5 f 20.6 d (19) 298.5 f 23.4 f (19) 347.9 5 16.4 (17)
3.8 5.9
a
8.6 5.2C
245.4 f 16.3 C (19) 341.9 f 32.4 e (18) 237.5 f 33.7 (12) 342.9 f 37.1 g (18)
P < 0.01 ;b P < 0.005 ; C P < 0.001 : significance of differences within each group between consecutive
days. < 0.05; e P < 0.02; f P < 0.005: significance of differences on the same days compared to the shamoperated group. g P < 0.05 ; h P < 0.005: significance of differences on the same days between the pinealectomized and pinealectomized and melatonin-treated groups. i P < 0.05: significance of differences on the same days between the melatonin-treated, pinealectomized and shamaperated groups. Number of animals in parentheses. dP
The weights of the pituitaries were not affected by weekly implantations of melatonin (Table VII). From the' foregoing it would appear that melatonin could reverse the enhancing effect of pinealectomy on LH in the serum by inhibiting its release and probably also its synthesis. Melatonin clearly possesses a prolactin-releasing effect in pregnant rats too, but it is difficult to determine whether it may counteract the depressant effect of pinealectomy on prolactin, since in this experiment removal of the pineal was not accompanied by a decrease in the serum prolactin level. In any case it is evident that the changes in gonadotrophin levels of pregnant rats brought about by melatonin were not mediated by gonadal steroids and appear rather to be a direct effect of melatonin on the hypothalamo-pituitary axis.
428
TABLE VI SERUM AND PITUITARY PROLACXIN LEVELS ON DAYS 21 AND 22 AFTER CONCEPTION AND POSTPARTUM OF RATS PINEALECTOMIZED AND/OR IMPLANTED WITH MELATONIN (MEAN i SEM)
Group
Days ajter conception ~
Serum prolactin (ng RP-l/ml) Sham-operated Sham-operated and melatonin-treated Pinealectomized Pinealectomized and melatonin-t reat ed
Pituitary prolactin (pg RP-llgland) Sham-operated Sham-operated and melatonin-treated Pinealectomized Pinealectomized and melatonin-treated a
Postpartum
~~~~
21
22
11.1 f 0.7 (15) 10.0 f 0.6 (18)
25.0 (24) 34.3 (30)
f
3.5
f
4.9
9.6 0.8 (16) 10.1 f 0.4 (20)
29.9 (21) 33.4 (26)
i
4.2e
f
4.9 e
80.7 (15) 92.9 (18)
i
95.2 (13) 60.5 (19)
f f
8.2 b*f
89.8 (18)
82.2
f
6.5
f
8.1
i
7.5
51.2 (14) 52.1 (14)
f
6.1
76.7 (11) 68.8
9.0
(16)
72.9 (21)
5.1
17.8 i 2.1 (18) 14.8 f 0.9 (17)
12.5
15.4 i 1.3 (12) 15.2 f 1.8 (16) 98.0
C&
5.4
(19) f
6.1
C
7.1 a,f f
5.9
(18)
P < 0.05; b P < 0.02; C P < 0.01; d P < 0.005; e P < 0.001: significance of differences within each group between consecutive days. P < 0.025; g P < 0.01 : significance of differences o n the Same days compared to the sham-operated group. Number of animals in parentheses.
TABLE VII PITUITARY WEIGHTS (in mg f SEM) ON DAYS 21 AND 22 AFTER CONCEPTION AND POSTPARTUM OF RATS PINEALECTOMIZED AND/OR IMPLANTED WITH MELATONIN
Group
Sham-operated Sham-operated and melatonin-treated Pinealectomized Pinealectomized and melatonin-treated Numbers of animals in parentheses.
Days after conception 21
22
9.5 f 0.4 ( 16) 9.0 f 0.3 (20) 8.8 f 0.4 (18) 8.3 f 0.3 (22)
10.2 (14) 9.3 (21) 9.6 (17) 8.9 (18)
Postpartum
f
0.6
t
0.4
f
0.3
0.2
10.3 f 0.4 (19) 9.9 i 0.4 (17) 10.1 i 0.4 (12) 9.6 f 0.4 (18)
429
EFFECTS OF CONTINUOUS DARKNESS OR LIGHT VERSUS THOSE OF PINEALECTOMY ON LH AND PROLACTIN LEVELS Since the pineal gland is involved in mediating between environmental light and endocrine organs, we studied the effects of extreme conditions of light throughout the period of gestation on several endocrine parameters known to exhibit changes during the perinatal period. Exposure of pregnant rats to continuous darkness brought about a significant increase in serum LH levels before as well as immediately after parturition (Table VIII). Continuous light did not affect serum LH concentrations before parturition, but, like darkness, it inhibited the sudden decline in serum LH that takes place postpartum in animals kept in alternating light (Table VIII). Pituitary LH contents were not affected by any type of environmental lighting, except that a less steep decline postpartum was observed in the group kept in continuous light when compared to that kept in alternating light, while higher contents were found during the period preceding parturition in rats maintained in continuous darkness (Table VIII). Continuous darkness as well as light strongly increased serum prolactin concentrations during the preparturition period studied, but did not affect the sharp decrease following parturition (Table 1%. The serum prolactin increases were accompanied respectively by significant pituitary decreases. These differences in pituitary prolactin disappeared with the postpartum elevation, when all values became equal (Table IX).
TABLE VIII SERUM AND PITUITARY LH LEVELS ON DAYS 21 AND 22 AFTER CONCEPTION AND POSTPARTUM OF RATS KEPT IN CONTINUOUS LIGHT OR DARKNESS (MEAN f SEM) Group
Days after conception 21
Serum LH (ng RP-l/ml) Alternating light (control) Light Darkness Pituitary LH (pg RP-l/gland) Alternating light (control) Light Darkness
49.7 (29) 49.3 (27) 58.7 (25) 517.3 (27) 417.3 (30) 433.0 (27)
Postpartum
22
f
f
2.3
76.5 (14) 81.3
2.5
f
2.8
f
38.9
f
37.5
f
34.8
(20) eyh
96.7 (16)
526.4 (27) 467.4 (31) 584.3 (16)
f
6.2
f
4.7
+
5.8
&
36.1
f
32.8
f
51.7
c
45.0
2
6.4
69.9 (24) 82.6 (23)
f
8.7
f
13.6
230.7 (23) 315.3 (28) 279.9 (29)
f
21.2
f
21.6 c,f
f
16.1
(21)
a
P < 0.02; b P < 0.005; C P < 0.001: significance of differences within each group between consecutive days. d P < 0.05; e P < 0.02; f P < 0.01: significance of differences compared to controls on the same days. g P < 0.05; h P < 0.02: significance of differences on the same days between groups kept in continuous light and darkness. Number of animals in parentheses. a
430 TABLE IX SERUM AND PITUITARY PROLACTIN LEVELS ON DAYS 21 AND 22 AFTER CONCEPTION AND POSTPARTUM OF RATS KEPT IN CONTINUOUS LIGHT OR DARKNESS (MEAN * SEM) Days after conception
Group
Serum pmlactin (ng RP-l/ml) Alternating light (control)
Light Darkness Rtuitaty proloctin (pg RP-l/gland) Alternating light (control)
Light Darkness
Postpartum
21
22
8.0 i 0.6 (16) 9.8 i 0.7 b (16) 11.9 i 1.0d (17)
34.8 i 7.4a (18) 85.3 i 13.7 a d (22) 111.1 24.3 (16)
57.4 i 5.0 (18) 45.2 i 3.4 (21) 39.9 i 3.0 d (20)
31.1 i 3.1a (18) 19.6 i 1.5 (20) 21.3 i 2.8a.b (20)
*
~
10.2 i 3.0 a (24) 13.2 i 1.3 a*b (27) 10.3 i 0.9 a (21)
~~
< 0.001 : significance of differences within each group between consecutive days. P < 0.05; P < 0.01 ;d P < 0.005: significance of differences on the same days compared to controls.
aP
f
Number of animals in parentheses.
Pituitary weights were not affected by continuous darkness or light except for a slight decrease on day 21 under continuous light (Table X). It transpires, therefore, that exposure of pregnant rats to continuous darkness causes highly significant increases in serum LH and prolactin levels during the perinatal period. The increase in serum prolactin being a result of enhanced release, and that of LH of both elevated synthesis and release from the pituitary. The question remained, however, whether TABLE X PITUITARY WEIGHTS (in mg i SEMI ON DAYS 21 AND 22 AFTER CONCEPTION AND POSTPARTUM OF RATS KEPT IN CONTINUOUS LIGHT OR DARKNESS Group
Alternating light (control) Light Darkness
Days after conception 21
22
9.1 * 0.2 (28) 8.2 i 0.2 b (30) 8.7 i 0.2 (28)
8.9 i 0.3 (26) 8.4 i 0.2 (31) 8.8 0.2 (28)
Postpartum
9.1 i 0.2 (3 1) 9.3 i 0.2 a (40) 9.6 i 0.2 a (33)
a P < 0.05: significance of differences within each group between consecutive days.
b P < 0.05: significance of differences on the same days compared to controls.
Number of animals in parentheses.
43 1 TABLE XI SERUM AND PITUITARY PROLACTIN LEVELS ON DAYS 21 AND 22 AFTER CONCEPTION AND POSTPARTUM OF RATS PINEALECI'OMIZED AND/OR KEPT IN DARKNESS MEAN f SEM) Group
Days after conception
Serum prolactin (ng RP-l/ml) Sham-operated Alternating light Darkness
Pinealectomized Alternating light Darkness Pituitary prolactin (fig RP-l/gland) Sham+perated Alternating light Darkness
Pinealectomized Alternating light Darkness
Postpartum
21
22
10.6 f 0.5 (25) 19.1 f 3.3 f (16) 13.0 f 1.5 (21) 21.8 t 2.4 j (20)
44.3 f (24) 113.2 f (23) 45.6 f (19) 98.9 (17)
70.7 (29) 73.2 (17) 66.9 (25) 66.7 (21)
53.7 f 3.5 c (26) 30.7 * 4.3 d& (22) 46.2 f 3.3 d (20) 31.05. 4 . 6 d j (14)
f
3.7
f
7.0 2.5
5
7.7
7.1 19.5 d*f 8.9 d 19.9 d>h
29.8 (20) 23.8 (22) 27.2 (23) 26.8 (21)
* 2.0 f
1.0 die
f
1.3 a
f
1.8
71.8 -+ 4.9 (10) 73.1 f 5.4 (12) 69.4 2.9 b (14) 70.4 f 4.0 (11)
a P < 0.05; b P < 0.01; C P < 0.005; d P < 0.001: significance of differences within each group between consecutive days. e P < 0.01; f P < 0.005; g P < 0.001: significance of differences on the same days compared to shamoperated group kept in alternating light. h P < 0.02; i P < 0.01; j P < 0.005: significance of differences on the same days compared to pinealectomized group kept in alternating light. Number of animals in parentheses.
this darkness-induced increase in plasma gonadotrophin levels is mediated by, or independent from, the pineal gland. To this end we exposed sham-operated and pinealectomized pregnant rats to continuous darkness in order to elucidate the possible role of the pineal gland in modifying the gonadotrophin and prolactin levels during pregnancy. We are reporting here the results on prolactin changes. Studies on LH are still underway. Pinealectomy did not abolish the darkness-induced increment in serum prolactin levels during the prenatal period (Table XI). Similarly, pinealectomy had no effect on the corresponding darkness-induced decrease in pituitary prolactin levels (Table XI). Absence of the pineal does not prevent darkness-induced elevation of prolactin during the prenatal period. Darkness does not affect the weights of pituitaries; pinealectomy could not interfere with the effect of darkness on this parameter, except for some inconsistent changes (Table XII). From these results it can be understood that exposure of pregnant rats to continuous darkness affects the metabolism of prolactin by a route independent of the pineal gland.
432
TABLE XII PITUITARY WEIGHTS (in mg f SEM) ON DAYS 21 AND 22 AFTER CONCEPTION AND POSTPARTUM OF RATS PINEALECTOMIZED AND/OR KEPT IN DARKNESS Group
Days after conception
22
21
Shamapera ted Alternating light
Postpartum
8.1 f 0.2 (30) 8.2 f 0.2 (19)
Darkness Pinealectomized Alternating light
*
1.7 f 0.3 (24) 8.8 f 0.3 b (21)
Darkness
~~
~
~~
9.1 f 0.4 a (24) 10.3 0.3 (22)
8.3 0.3 (22) 8.6 f 0.2 (19)
*
~~
a P < 0.05 : significance of differences within each group between consecutive days.
P < 0.05: significance of differences on the same days between pinealectomized groups kept in darkness and alternating light. P < 0.05: significance of differences o n the same days between pinealectomized and sham-operated groups kept in darkness. Number of animals in parentheses.
Corresponding studies on the role of the pineal gland in darkness-induced changes in LH metabolism are in progress and there is no reason to assume that it would be different from that of prolactin. TABLE XIII NUMBER OF EMBRYOS AND RESOWIONS IN PINEALECTOMIZED COMPARED TO SHAMOPERATED RATS Experiment
1
2 3 4 5 6
I
8 9
Totals
Number of animals
Average embryoslrat
(4
(S)
(4
(8
51 14 21 40 31 41 41 26 14
42 15 20 40 43 38 54 34 10
8.3 8.5 5.8 9.3 8.8 9.3 8.6 9.0 8.6
8.5 8.4 8.4 9.0 8.7 9.6 8.4 9.0 8.7
4 4 4 1 8 4 4
291
296
8.6
8.8
34
Rats with resorptions
Rats with 4 or less embryos
(4
(sl
(4
(Sl
(P)
(Sl
5
2 0 1 4 10 0
2 0 1.5 2.5 3 4 3 1 2
1.5 0 2 2 3 0 2 1 0
5 1 9 1 2 2 4 0 0
1 0
3 1 6 1 4 0 1
2
1.3
24
11
0
P = pinealectomized; S = sham+paated (controls).
Resorptionsl mt
I
1 0 25
433 TABLE X N NUMBER OF RATS THAT DELIVERED ON DAYS 22 AND 23 AFTER CONCEPTION ACCORDING TO TREATMENT AND LIGHT EXPOSURE Group
Alternating light Days after conception
Darkness Days after conception
22
22
23
Lkht Days after conception
23
22
23
15 -
26
~~
Intact Sham-operat ed Pinealectomized Shamaperated and melatonin-treated Pinealectomized and melatonin-treated a
16 9 9 6 10
31 20 17 5 8
29 6 8 -
22 a
I
4a -
-
-
P < 0.02 of the ratios between days 22 and 23 after conception of the same group kept in darkness and alternating light.
CONCLUDING REMARKS Reviewing our findings it appears that the rat pineal gland may be involved in the endocrine changes which take place the perinatal period. The manner in which pineal-induced changes in the levels of circulating gonadotrophins and steroid hormones during the perinatal period affect various facets of pregnancy like duration, numbers of living fetuses and resorptions under normal conditions appears minimal. No differences reaching significance were found between the experimental and control groups in any of the aforementioned parameters (Table XIII). The only notable difference was in the duration of pregnancy. Estimated by the chi-square test a significantly larger number of deliveries occurred on day 22 than on day 23 of gestation among rats exposed to continuous darkness - sham-operated as well as pinealectomized - as compared to control groups kept in alternating light (Table XIV). Pinealectomy alone had no effect on duration of gestation, but melatonin or darkness shortened it. The role of the pineal gland in pregnancy could perhaps become more obvious should animals be exposed to conditions which drastically affect their endocrine apparatus. Under such circumstances the pineal may be able to play the role of a moderator between abnormal environmental (or perhaps even physiological) conditions and the functions of hormones in gestation. REFERENCES Bast, D. and Melampy, R.M. (1 972) Luteinizing hormone, prolactin and ovarian 20-hydroxysteroid dehydrogenase levels during pregnancy and pseudopregnancy i n the rat. Endocrinology, 9 1 : 1499- 1505. DeverEerski, V. (1972) Contribution histoenzymologique a 1'6tude du m6tabolisme de la glande pineale chez la ratte gravide. C.R. SOC.Biol. (Paris), 166: 131. Ellendorf, F. and Smidt, D. (1961) Der Einfluss unterschiedlicher Beleuchtung auf die neurosekretorische Aktivitat, Pubertat und Sexualfunktion von Mausen. J. neuro-visc. Rel., Suppl. 10, pp. 220-232.
434 Gibori, G. and Richards, J.S. (1978) Dissociation of two distinct luteotropic effects of prolactin: regulation of luteinizing hormone-receptor content and progesterone secretion during pregnancy. Endocrinology, 102: 767-774. Guerra, M.O., Silva, N.O.G., Guimarxes, C.S., Souza, J.P. and Andrade, A.T.L. (1973) PinealectomY and blindness during pregnancy in the rat. Amer. J. Obstet. Gynec., 115: 582-583. Huang, C.Y. and Everitt, A.V. (1965) The effect of pregnancy on pineal weight in the rat. J. Endocr., 32: 26 1-262. Kelly, P.A., Shiu, R.P., Robertson, M.C. and Friesen, H.G. (1975) Characterization of rat chorionic mammotropin. Endocrinology, 96: 1187-1 195. Kincl, F.A. and Benagiano, G. (1967) The failure of the pineal gland removal in neonatal animals to influence reproduction. Acta endocr., (Kbh.), 54: 189- 192. Linkie, D.M. and Niswender, G.D. (1972) Serum levels of prolactin luteinizing hormone and follicle stimulating hormone during pregnancy in the rat. Endocrinology, 90: 632-637. Lues, G. (1 97 1) Die Feinstruktur der Zirbeldruse normaler, trachtiger und experimentell beeinflusster Meerschweinchen. 2. Zellforsch., 114: 38-60. McCormack, J.T. and Greenwald, G.S. (1974) Progesterone and oestradiol 17p concentrations in the peripheral plasma during pregnancy in the mouse. J. Endocr., 62: 101-107. Meites, J. and Turner, C.W. (1948) Studies concerning the induction and maintenance of lactation. I. The mechanism controlling the initiation of lactation and parturition. Res. Bull. Missouri Agric. exp. Stat., 415: 1-65. Minnernan, K. and Wurtman, R.J. (1976) The pharmacology of the pineal gland. Ann. Rev. Pharmacol. Toxicol., 16: 33-51. Misuno, H. and Sensui, N. (1970) Lack of effects of melatonin administration and pinealectomy on the milk ejection response in the rat. Endocr. jap., 17: 417-420. Mitchell, J.A. and Yochim, J.M. (1970) Influence of environmental lighting on duration of pregnancy in the rat. Endocrinology, 87: 472-480. Morishige, W.K., Pepe, C.J. and Rothchild, 1. (1973) Serum luteinizing hormone prolactin and progesterone levels during pregnancy in the rat. Endocrinology, 92: 1527-1530. Nir, I. (1978) Non-reproductive systems and the pineal. In: I. Nir, R.J. Reiter and R.J. Wurtman (Eds.), The Pineal Gland (Supplement XI11 of J. neural Transm.), Springer-Verlag, Vienna, pp. 225-246. Nir, I., Goldhaber, G., Hirschmann, N. and Shani, J. (1977) Prolactin and luteinizing hormone levels before and after parturition in rats bearing either single or multiple embryos. J. Endocr., 73: 25 7- 262. Nir. I., Hirschmann, N., Goldhaber, G. and Shani, J. (1 979) Pinealectomy induced changes in blood and pituitary luteinizing hormone and prolactin levels during the last phase of pregnancy in rats. Neuroendocrinology, 28: 44-51. Ogle, T.F. and Kitay, J.I. (1976) Effect of pinealectomy on adrenal function in vivo and in vitro in female rats. Endocrinology, 98: 20-24. Ogle, T.F. and Kitay, J.I. (1977) Ovarian and adrenal steroids during pregnancy and the oestrous cycle in the rat. J. Endocr., 74: 89-98. Orts, R.J., Kocan, K.M. and Johnson, R.P. (1977) Antifertility properties of bovine pineal extracts: Q (Kbh.), 8 5 : Reduction of ovulation and preovulatory luteinizing hormone in the rat. A C ~endocr. 225 - 234. Ota, K., Ota, T. and Yokoyama, A. (1974) Plasma corticosterone concentrations and pituitary prolactin content in late pregnancy and their within-day fluctuations in the rat. J. Endocr., 61: 21-28. Reiter, R.J. (1972) Effects of the pineal gland on reproductive organ growth and fertility in dual sensory deprived female rats. Endocr. Exp., 6: 3-10. Reiter, R.J., Rudeen, P.K. and Vaughan, M.K. (1976) Restoration of fertility in light-deprived female hamsters by chronic melatonin treatment. J. comp. Physiol., 111: 7-13. Shaikh, A.A. (1971) Estrone and estradiol levels in the ovarian venous blood from rat during the estrous cycle and pregnancy. Biol. Repr., 51: 297-307. Simpson, A.A., Simpson, M.H. and Kulkarin, P.N. (1973) Prolactin production and lactogenesis in rats after ovariectomy in late pregnancy. J. Endocr., 63: 13-20. Tigchelaar, P.V. and Nalbandov, A.V. (1975) The effect of pineal gland on ovulation and pregnancy in the rat. Biol. Repr., 13: 461-469. Vaughan, M.K., Reiter, R.J. and Vaughan, G.M.(1976) Fertility patterns in female mice following treatment with arginine vasotocin or melatonin. Int. J. Fertil., 21: 65-68.
435
Vermouth, N.T. and Deis, R.P. (1974) Prolactin release and lactogenesis after ovariectomy in pregnant rats. Effect of ovarian horm0nes.J. Endocr., 63: 13-20. Vollrath, L. and Schmidt, D.S. (1969) Enzymhistochemische Untersuchungen an der Zirbeldruse normaler und trachtiger Meerschweinchen. Histochemie, 20: 328-337. Yochim, J.M. and Wallen, E.P. (1974) Function of the pineal gland of the rat during progestation: alterations in HIOMT rhythmicity. Biol. Repr., 10: 480-486.
DISCUSSION J.W. VISSER: Is there any difference in the parameters in stillbirth?
I. NIR: No, there is not a significant difference.
A Possible Role of the Pineal Gland in Pregnancy and Fertility I. NIR and N. HIRSCHMANN Department of Pharmacology and Experimental Therapeutics, Hebrew University, Schools of Medicine and Pharmacy, Jerusalem (Israel)
Until little more than a decade ago no information was available on whether the pineal gland influences gestation, or vice versa, whether pregnancy may affect pineal metabolism and function. The first observation on such an interrelationship was of decreased pineal weight in late pregnancy in rats bearing 10 or more embryos and was published in 1965 by Huang and Everitt. They considered their findings to indicate depressed pineal activity during the period studied. Later investigators focused their attention on possible metabolic changes in the pineal during pregnancy. Results obtained, however, suggested intensified metabolic activity, enhanced pineal NADH and NADPH diaphorase activity being recorded in guinea-pigs during the second half of pregnancy which disappeared after delivery, oophorectomy or hysterectomy (Vollrath and Schmidt, 1969). Moreover, histological changes observed by electron microscope in the fine structure of so-called “light” and “dark” pinealocytes during the second half of pregnancy may too serve as supportive evidence of increased pineal activity during that period (Lues, 1971). Nevertheless, there are findings to uphold the earlier suggestion of Huang and Everitt (1965) of decreased pineal activity during late pregnancy; for instance DeverEerski (1972) described decreased activity of the pineal in lactate dehydrogenase, isocitrate and other enzymes involved in glycolysis on day 15 of pregnancy. Regarding the metabolism of specific pineal indoleamines, a study carried out in pseudopregnant rats indicated that they have a different diurnal pattern of HIOMT activity although no deviation in its mean rate (Yochim and Wallen, 1974). A comprehensive study of the general metabolism and hormonal patterns of the pineal during gestation, as well as of their significance in the processes of pregnancy was considered timely. Various parameters reflecting general and indoleamine metabolism (RNA, DNA, SHT, NAT and HIOMT) were measured in the pineals of rats on days 8 and 19 of gestation, representing respectively the first and second halves of pregnancy. The values obtained were compared to, and found to be identical with, those of non-pregnant rats during dioestrus. Neither the basic pineal metabolism as reflected in nucleic acids and protein contents, nor the hormonal indoleamine appeared to be changed during pregnancy (Nir and Hirschmann, in preparation). There were, however, indications suggesting pineal implications in processes connected with gestation and fertility. Rodents exposed to continuous darkness, to short daily photoperiods (L2 : D22), or to decreased light intensity throughout the gestation period, demonstrated significantly shorter length pregnancies than controls kept in normal lighting (Ellendorf and Smidt, 1971; Mitchell and Yochim, 1970), while long daily photoperiods (L22 :
422
D2) delayed parturition (Mitchell and Yochim, 1970). However, removal of the pineal gland -which usually mediates between environmental lighting and hormone producing glands-did not abolish the effect of short photoperiods on the duration of gestation (Mitchell and Yochim, 1970) which suggests that the effect of darkness on gestation may not be mediated by the pineal gland. Neither exposure to low intensity light (Ellendorf and Smidt, 1971), blinding, nor pinealectomy (Reiter et al. 1976) prior to fertilization, had any effect on the numer of offspring, and in the case of pinealectomy, the length of gestation period (Misuno and Sensui, 1970). However, significantly fewer living foetuses were found in animals pinealectomized during pregnancy (on day 11) than in those that had undergone sham-operation(Guerra et al., 1973). Similarly confusing is the role of the pineal gland in fertility processes. While pinealectomy had no effect on the fertility index of rats (Kincl and Benagiono, 1967), blinding of hamsters markedly reduced the number of fertile matings (Reiter et al., 1976) and fertility was also reduced in rats subjected to blinding and anosmia (Reiter, 1972). These effects could be negated by pinealectomy or chronic melatonin treatment (Reiter, 1972; Reiter et al., 1976) suggesting a progonadal effect of melatonin in pregnant rodents. In contrast to the above fmdings, melatonin and arginine-vasotocin(Vaughan et al., 1976) and several polypeptides extracted from the pineal gland (Orts et al., 1977) were found to inhibit fertile matings when injected into females before copulation. It has been suggested that the pineal hormones under certain conditions act on fertility by reducing LH secretion and subsequently suppressing ovulation (Orts et al., 1977).
LH AND PROLACTIN LEVELS IN PINEALECTOMIZED PREGNANT RATS DURING THE PERINATAL PERIOD There is now much evidence to indicate that the pineal gland regulates the synthesis and release of reproductive hormones by way of its indoleamine (melatonin) and polypeptide (arginine-vasotocin) hormones, mainly by central action at the level of the hypothalamus (Minneman and Wurtman, 1976). It may also be participating in the maintenance of the endocrine balance in pregnant animals, which undergoes profound changes during the last stage of gestation. Serum prolactin and LH levels - which are very low from day 5 after conception increase several-fold during the two days before parturition (Bast and Melampy, 1972; Linkie and Niswender, 1972) accompanied by a decrease in the high pituitary levels of prolactin (Ota, et al., 1974; Nir et al., 1977), LH and FSH (Tigchelaar and Nalbandov, 1975). Furthermore, the high serum concentrations of progesterone, which are at least partly responsible for the low levels of LH in serum, fall some days before parturition (Morishige et al., 1973; McCormack and Greenwald, 1974), while blood corticosterone (Ota et al., 1974), oestradiol and oestrone (Shaikh, 1971;McCormack and Greenwald, 1974) levels rise during the same period. It would seem feasible, therefore, that the pineal gland could be involved in these changes by influencing the abrupt rise in LH, prolactin and oestrogens and the decrease in progesterone which occur before parturition, through its action on the hypothalamic-pituitary axis. We decided fvst to study the effects of pineal ablation on pituitary and serum LH and prolactin levels, and on other parameters connected with embryo development and pregnancy outcome, during the last 4 days of gestation and immediately after parturition, in rats pinealectomized after weaning and kept in regular alternations of 12 h light and 12 h
423
TABLE I SERUM AND PITUITARY LH LEVELS IN PINEALECTOMIZED RATS ON DAYS 19-22 AFTER CONCEFTION (MEAN i SEM) Group
Days after conception
Serum LH (ng RP-l/ml) Pinealectomized
Sham-operated (control) Pituitary LH b g RP-l/gland) Pinealectomized
Sham-operated (control)
19
20
21
22
93.9 f 4.24 (42) 87.6 i 3.35 (40)
111.3 i 7.40 c (31) 97.0 i 3.96 (24)
125.1 i 5.93b (34) 96.2 i 6.06 (40)
209.5 i 34.86 a d (35) 134.9 i 11.91 e (39)
805.1 f 44.2 (42) 774.1 i 34.2 (40)
779.9 i 36.8 (49) 732.0 i 51.1 (37)
919.1 (43) 892.3 (57)
945.6 (42) 836.0 (53)
i
56.2
f
35.1
64.6 i 41.9
P < 0.05; b P < 0.005: significance of difference between the pinealectomized and sham-operated groups on the same days. C P < 0.05;d P < 0.02; e P < 0.005: significance of difference within each group compared to the previous day. Number of animals in parentheses. a
darkness. The results were as follows: Elevated serum LH levels were observed in the pinealectomized rats during the last 4 days of gestation when compared to those of their sham-operated controls, although the difference reached significance only during the last two days of pregnancy. Pinealectomy also TABLE I1 SERUM AND PITUITARY PROLACTIN LEVELS IN PINEALECTOMIZED RATS ON DAYS 19-22 AFTER CONCEPTION (MEAN f SEM) Group
Days after conception
Serum prolactin (ng RP-l/ml) Pinealectomized
Shamaperated (control) Pituitary prolact in (aRP-l/gland) Pinealectomized
Sham-perated (control) '
19
20
21
22
9.19 i 0.34 (42) 9.25 i 0.20 (39)
9.92 i 0.54 (47) 9.11 f 0.31 (35)
12.23 i 0.53 (49) 11.18 i 0.53 (55)
23.09 f 2.59 bg (37) 36.44 i 4.64 g (48)
54.94 i 1.89 a (36) 61.73 i 2.35 (36)
48.33 f 1.87 c*d (30) 56.91 i 1.95 (27)
48.46
34.55 f 2.08 (28) 43.25 i 2.86 g (37)
(50)
61.86 (5 8)
f
1.89
* 2.37
P < 0 . 0 5 ; b P < O.O2;CP< 0.005;dP<0.001: significanceof differencesbetweenpinealectomized and sham-operated groups on the same days. e P < 0.02; f P < 0.005; g P < 0.001: significance of differences within each group compared to the previous day. Number of animals in parentheses. a
424
advanced to day 20 the rise in serum LH concentrations which occurred in sham-operated animals between days 21 and 22 of gestation. No differences between the pinealectomized and sham-operated rats were observed in serum LH two hours postpartum. Despite the changes in their serum LH, no deviations from the sham-operated rats were observed in pituitary LH contents of the pinealectomized group, either before or after parturition (Table I). Serum prolactin levels were less affected by pinealectomy. Except for an occasional decrease in concentration of prolactin in serum of pinealectomized rats on day 22 of gestation, no differences were noted either before or after parturition. On the other hand, pituitary contents were lowered by pinealectomy in most, though not all, experiments at all times measured (Table 11). These results indicate that normally during the last days of gestation the pineal gland has an inhibitory effect on pituitary release of LH without apparently affecting LH synthesis, whereas in the case of prolactin it mainly increases the synthesis, occasionally enhancing release from the pituitary. It could therefore be interpreted that during the last phase of pregnancy the pineal gland is involved in modifying the balance of hormones by depressing LH release and increasing the synthesis of prolactin (Nir et al., 1979). LEVELS OF GONADAL HORMONES IN PINEALECTOMIZEDRATS DURING THE FINAL STAGE OF PREGNANCY Since an interrelationship between pituitary gonadotrophins and gonadal hormones has been established, it was considered necessary to determine whether any changes analogous to those in gonadotrophins occur in the levels of gonadal hormones in pinealectomized rats during the same stage of pregnancy. As the next step, levels of blood progesterone and oestradiol were measured in pinealectomized and sham-operated control rats during the last 4 days of pregnancy. Changes in levels of gonadal hormones and the exact timing of their occurrence should indicate whether the effects exerted by the pineal substances on the pituitary hormones are primary (direct) or secondary to the changes taking place in the gonads. Pinealectomy resulted in significantly increased levels of progesterone in the serum during days 19 and 20 of gestation, but this tended to disappear towards day 21, and no differences between the pinealectomized and sham-operated rats were noted on the day preceding parturition, when very low levels of progesterone were recorded in both groups (Table 111). In contrast to progesterone, serum oestradiol showed a sharp increase during the last two days of gestation in both pinealectomized and sham-operated groups, the increase being significantly greater in the pinealectomized (Table IV). It appears, therefore, that the pineal gland could be acting as a brake to the endocrine processes, i.e., depressing progesterone and oestradiol levels while they are high, though not be accelerating their decline nor preventing their rise. Consequently, the pineal may be able to modify the levels of gonadal hormones, but not to affect the sequence of their physiological events. Concerning the mechanism of pineal involvement in the regulation of gonadal hormones in pregnant rats, a direct pineal effect through the pituitary does not seem likely since the serum progesterone levels are increased in pinealectomized rats already by day 19 of gestation, when no changes occur in serum LH and prolactin concentrations as compared to those of sham-operated animals (Nir et al., 1979).
425 TABLE 111 THE EFFECTS OF PINEALECTOMY AND/OR MELATONIN TREATMENT ON SERUM PROGESTERONE LEVELS ( d m l f SEM) IN RATS DURING LATE PREGNANCY
Group
Days after conception 19
Sham-operated
80.7 (18)
Sham-operated and melatonin-treated Pinealectomized
99.4
20 f
f
3.9
3.1
Pinealectomized and melatonin-treated a
P < 0.05 ; b P < 0.01;
C
86.1 (21)
99.7
22
21 f
f
3.1
3.6
.
60.0 (18) 65.6 (10) 68.2 (17) 63.8
f
_+
6.1 5.2
* 6.3 a,d f
4.8
(11)
13.2 f 1.3 (20) 12.7 f 0.8 (11) 12.0 f 1.1 (21) 12.9 f 0.8 (11)
P < 0.001 : compared t o the sham-operated group. d P < 0.02; e P < 0.001 : sig-
nificance of differences within each group between consecutive days. Number of animals in parentheses.
Since it was suggested (Gibori and Richards, 1978) that the placenta regulates ovarian progesterone production in the second half of pregnancy, it could be that the increased progesterone levels induced by pinealectomy may result from enhanced production of placental hormones, as has been indicated in the case of the prolactin-like luteotropin, which reaches its peak on day 18 of gestation (Kelly et al., 1975). Another possible explanation may be an effect of the pineal on adrenal steroidogenesis. It has been found that on day 22 of gestation adrenal secretion of progesterone reaches a level several-fold that produced by the ovary (Ogle and Kitay, 1977). It is possible that the well-documented enhanced adrenal
TABLE IV THE EFFECTS OF PINEALECTOMY AND/OR MELATONIN TREATMENT ON SERUM OESTRADIOL LEVELS (pg/ml f SEM) IN RATS DURING LATE PREGNANCY
Group
Days after conception 19
Sham-operated Sham-operated and melatonin-treated Pinealectomized Pinealectomized and melatonin-treated
44.7 (18)
40.1 (20)
20 f
f
4.7
2.8
42.3 (18)
53.3 (18)
f
f
4.9
8.1
21
22
62.6 f 4.5 (20) 68.5 f 3.4 (11)
62.3 (20) 59.0 (20)
80.0 (20) 73.7 (10)
84.3 (19) 76.8 (21)
f
3.9 a,d
f
4.8
f 4.9 f.
3.4
f
6.1
f
4.3
a P < 0.02; b P < 0.01: compared to the sham-operated group. f P < 0.005: compared to the sham-operated and melatonin-treated group. P < 0.005: compared to the same group on the previous day. Number of animals in parentheses.
426
function brought about by pinealectomy (Nir, 1978) could be largely responsible for the increased progesterone concentrations in pregnant pinealectomized rats. As to the effects of progesterone on prolactin metabolism in pinealectomized rats during the period preceding parturition, it has been suggested that the sharp fall of serum progesterone that takes place at that time is one of the factors inducing the sudden increase in serum prolactin levels on the last day of gestation (Simpson et al., 1973; Vermouth and Deis, 1974). The finding that higher doses of progesterone are capable of inhibiting the stimulatory action of small doses of oestrogen on pituitary prolactin levels (Meites and Turner, 1948) also confirms the possibility that progesterone may be a regulatory factor in prolactin metabolism. It is therefore possible that the elevated serum progesterone concentration occurring in pinealectomized rats during days 19 and 20 of gestation (or perhaps even earlier than that) might elicit the lowered pituitary prolactin content observed prior to parturition. In regard to the changes in oestradiol brought about by pinealectomy, it could be that the pineal hormones normally exert an inhibitory effect on oestrogen production via the placental-pituitary complex mentioned previously, or directly on the ovaries. Another possibility is that more enhanced synthesis of oestradiol is triggered by the sudden decline in serum progesterone concentration towards the end of pregnancy. It is difficult to prove this, however, as long as the source, stimulus of production, and the role of oestradiol during the second half of pregnancy, are unknown. EFFECT OF MELATONIN VERSUS THAT OF PINEALECTOMY ON LH AND PROLACTIN LEVELS As the next step we explored the role of melatonin, the pineal hormone, during this crucial period, that is to say whether melatonin is capable of reversing the effects of pinealectomy on pituitary and serum gonadotrophins as well as on serum gonadal hormones. Weekly implantations of melatonin (1 mg) into pregnant rats commencing on the day after mating brought about a decrease in serum LH levels in both sham-operated and pinealectomized animals on day 22 of gestation only. On day 21 of gestation and immediately after parturition no effect was noted in either group. In this experiment pinealectomy was found to enhance serum LH only slightly (Table V). Melatonin treatment depressed pituitary LH content in sham-operated (but not pinealectomized) animals on day 22 of gestation: the same day pinealectomy decreased pituitary LH, a phenomenon not encountered in other experiments. A sudden decline in pituitary LH content takes place postpartum in the sham-operated but not in the pinealectomized or either of the melatonin-treated groups (Table VI). This finding is consistent with that of Tigchelaar and Nalbandov (1975) who treated pregnant animals with daily subcutaneous injections of melatonin and exposed them to prolonged daily photoperiods. On the other hand, melatonin treatment brought about an increase in serum prolactin levels on day 22 of gestation (though not on day 21 or postpartum) accompanied by a respective decline in pituitary prolactin content on the same day (Table VI). In addition, as already observed, pinealectomy induced a decrease in pituitary prolactin levels not only before, but also after parturition. In spite of its effect on LH and prolactin levels, melatonin implanted weekly had no influence on either progesterone or oestradiol in serum of either sham-operated or pinealectomized rats during the prenatal period (Tables 111 and IV).
427 TABLE V SERUM AND PITUITARY LH LEVELS ON DAYS 21 AND 22 AFTER CONCEPTION AND POSTPARTUM OF RATS PINEALECTOMIZED AND/OR IMPLANTED WITH MELATONIN (MEAN f SEM) Group
Days after conception
22
21
Serum LH (ng RP-l/ml) Shamaperated Shamaperated and melatonin-treated Pinealectomized Pinealectomized and melatonin-treated Pituitagv LH (pg RP-l/lgland)
Shamaperated
Shamaperated and melatonin-treated Pinealectomized Pinealectomized and melatonin-treated a
58.7 5 (14) 52.7 * (14) 61.3 f (13) 65.6 (16)
*
426.5
(7)
Postpartum
f
5.6
76.4 f (19) 86.7 f (18) 92.8 f (11) 84.6 f (18)
5.0 5.6 3.2i
51.1
381.2 f 48.0 (9) 466.1 f 82.8 (10) 428.5 f 43.9 (11)
403.3 f 25.2 (18) 330.5 f 20.6 d (19) 298.5 f 23.4 f (19) 347.9 5 16.4 (17)
3.8 5.9
a
8.6 5.2C
245.4 f 16.3 C (19) 341.9 f 32.4 e (18) 237.5 f 33.7 (12) 342.9 f 37.1 g (18)
P < 0.01 ;b P < 0.005 ; C P < 0.001 : significance of differences within each group between consecutive
days. < 0.05; e P < 0.02; f P < 0.005: significance of differences on the same days compared to the shamoperated group. g P < 0.05 ; h P < 0.005: significance of differences on the same days between the pinealectomized and pinealectomized and melatonin-treated groups. i P < 0.05: significance of differences on the same days between the melatonin-treated, pinealectomized and shamaperated groups. Number of animals in parentheses. dP
The weights of the pituitaries were not affected by weekly implantations of melatonin (Table VII). From the' foregoing it would appear that melatonin could reverse the enhancing effect of pinealectomy on LH in the serum by inhibiting its release and probably also its synthesis. Melatonin clearly possesses a prolactin-releasing effect in pregnant rats too, but it is difficult to determine whether it may counteract the depressant effect of pinealectomy on prolactin, since in this experiment removal of the pineal was not accompanied by a decrease in the serum prolactin level. In any case it is evident that the changes in gonadotrophin levels of pregnant rats brought about by melatonin were not mediated by gonadal steroids and appear rather to be a direct effect of melatonin on the hypothalamo-pituitary axis.
428
TABLE VI SERUM AND PITUITARY PROLACXIN LEVELS ON DAYS 21 AND 22 AFTER CONCEPTION AND POSTPARTUM OF RATS PINEALECTOMIZED AND/OR IMPLANTED WITH MELATONIN (MEAN i SEM)
Group
Days ajter conception ~
Serum prolactin (ng RP-l/ml) Sham-operated Sham-operated and melatonin-treated Pinealectomized Pinealectomized and melatonin-t reat ed
Pituitary prolactin (pg RP-llgland) Sham-operated Sham-operated and melatonin-treated Pinealectomized Pinealectomized and melatonin-treated a
Postpartum
~~~~
21
22
11.1 f 0.7 (15) 10.0 f 0.6 (18)
25.0 (24) 34.3 (30)
f
3.5
f
4.9
9.6 0.8 (16) 10.1 f 0.4 (20)
29.9 (21) 33.4 (26)
i
4.2e
f
4.9 e
80.7 (15) 92.9 (18)
i
95.2 (13) 60.5 (19)
f f
8.2 b*f
89.8 (18)
82.2
f
6.5
f
8.1
i
7.5
51.2 (14) 52.1 (14)
f
6.1
76.7 (11) 68.8
9.0
(16)
72.9 (21)
5.1
17.8 i 2.1 (18) 14.8 f 0.9 (17)
12.5
15.4 i 1.3 (12) 15.2 f 1.8 (16) 98.0
C&
5.4
(19) f
6.1
C
7.1 a,f f
5.9
(18)
P < 0.05; b P < 0.02; C P < 0.01; d P < 0.005; e P < 0.001: significance of differences within each group between consecutive days. P < 0.025; g P < 0.01 : significance of differences o n the Same days compared to the sham-operated group. Number of animals in parentheses.
TABLE VII PITUITARY WEIGHTS (in mg f SEM) ON DAYS 21 AND 22 AFTER CONCEPTION AND POSTPARTUM OF RATS PINEALECTOMIZED AND/OR IMPLANTED WITH MELATONIN
Group
Sham-operated Sham-operated and melatonin-treated Pinealectomized Pinealectomized and melatonin-treated Numbers of animals in parentheses.
Days after conception 21
22
9.5 f 0.4 ( 16) 9.0 f 0.3 (20) 8.8 f 0.4 (18) 8.3 f 0.3 (22)
10.2 (14) 9.3 (21) 9.6 (17) 8.9 (18)
Postpartum
f
0.6
t
0.4
f
0.3
0.2
10.3 f 0.4 (19) 9.9 i 0.4 (17) 10.1 i 0.4 (12) 9.6 f 0.4 (18)
429
EFFECTS OF CONTINUOUS DARKNESS OR LIGHT VERSUS THOSE OF PINEALECTOMY ON LH AND PROLACTIN LEVELS Since the pineal gland is involved in mediating between environmental light and endocrine organs, we studied the effects of extreme conditions of light throughout the period of gestation on several endocrine parameters known to exhibit changes during the perinatal period. Exposure of pregnant rats to continuous darkness brought about a significant increase in serum LH levels before as well as immediately after parturition (Table VIII). Continuous light did not affect serum LH concentrations before parturition, but, like darkness, it inhibited the sudden decline in serum LH that takes place postpartum in animals kept in alternating light (Table VIII). Pituitary LH contents were not affected by any type of environmental lighting, except that a less steep decline postpartum was observed in the group kept in continuous light when compared to that kept in alternating light, while higher contents were found during the period preceding parturition in rats maintained in continuous darkness (Table VIII). Continuous darkness as well as light strongly increased serum prolactin concentrations during the preparturition period studied, but did not affect the sharp decrease following parturition (Table 1%. The serum prolactin increases were accompanied respectively by significant pituitary decreases. These differences in pituitary prolactin disappeared with the postpartum elevation, when all values became equal (Table IX).
TABLE VIII SERUM AND PITUITARY LH LEVELS ON DAYS 21 AND 22 AFTER CONCEPTION AND POSTPARTUM OF RATS KEPT IN CONTINUOUS LIGHT OR DARKNESS (MEAN f SEM) Group
Days after conception 21
Serum LH (ng RP-l/ml) Alternating light (control) Light Darkness Pituitary LH (pg RP-l/gland) Alternating light (control) Light Darkness
49.7 (29) 49.3 (27) 58.7 (25) 517.3 (27) 417.3 (30) 433.0 (27)
Postpartum
22
f
f
2.3
76.5 (14) 81.3
2.5
f
2.8
f
38.9
f
37.5
f
34.8
(20) eyh
96.7 (16)
526.4 (27) 467.4 (31) 584.3 (16)
f
6.2
f
4.7
+
5.8
&
36.1
f
32.8
f
51.7
c
45.0
2
6.4
69.9 (24) 82.6 (23)
f
8.7
f
13.6
230.7 (23) 315.3 (28) 279.9 (29)
f
21.2
f
21.6 c,f
f
16.1
(21)
a
P < 0.02; b P < 0.005; C P < 0.001: significance of differences within each group between consecutive days. d P < 0.05; e P < 0.02; f P < 0.01: significance of differences compared to controls on the same days. g P < 0.05; h P < 0.02: significance of differences on the same days between groups kept in continuous light and darkness. Number of animals in parentheses. a
430 TABLE IX SERUM AND PITUITARY PROLACTIN LEVELS ON DAYS 21 AND 22 AFTER CONCEPTION AND POSTPARTUM OF RATS KEPT IN CONTINUOUS LIGHT OR DARKNESS (MEAN * SEM) Days after conception
Group
Serum pmlactin (ng RP-l/ml) Alternating light (control)
Light Darkness Rtuitaty proloctin (pg RP-l/gland) Alternating light (control)
Light Darkness
Postpartum
21
22
8.0 i 0.6 (16) 9.8 i 0.7 b (16) 11.9 i 1.0d (17)
34.8 i 7.4a (18) 85.3 i 13.7 a d (22) 111.1 24.3 (16)
57.4 i 5.0 (18) 45.2 i 3.4 (21) 39.9 i 3.0 d (20)
31.1 i 3.1a (18) 19.6 i 1.5 (20) 21.3 i 2.8a.b (20)
*
~
10.2 i 3.0 a (24) 13.2 i 1.3 a*b (27) 10.3 i 0.9 a (21)
~~
< 0.001 : significance of differences within each group between consecutive days. P < 0.05; P < 0.01 ;d P < 0.005: significance of differences on the same days compared to controls.
aP
f
Number of animals in parentheses.
Pituitary weights were not affected by continuous darkness or light except for a slight decrease on day 21 under continuous light (Table X). It transpires, therefore, that exposure of pregnant rats to continuous darkness causes highly significant increases in serum LH and prolactin levels during the perinatal period. The increase in serum prolactin being a result of enhanced release, and that of LH of both elevated synthesis and release from the pituitary. The question remained, however, whether TABLE X PITUITARY WEIGHTS (in mg i SEMI ON DAYS 21 AND 22 AFTER CONCEPTION AND POSTPARTUM OF RATS KEPT IN CONTINUOUS LIGHT OR DARKNESS Group
Alternating light (control) Light Darkness
Days after conception 21
22
9.1 * 0.2 (28) 8.2 i 0.2 b (30) 8.7 i 0.2 (28)
8.9 i 0.3 (26) 8.4 i 0.2 (31) 8.8 0.2 (28)
Postpartum
9.1 i 0.2 (3 1) 9.3 i 0.2 a (40) 9.6 i 0.2 a (33)
a P < 0.05: significance of differences within each group between consecutive days.
b P < 0.05: significance of differences on the same days compared to controls.
Number of animals in parentheses.
43 1 TABLE XI SERUM AND PITUITARY PROLACTIN LEVELS ON DAYS 21 AND 22 AFTER CONCEPTION AND POSTPARTUM OF RATS PINEALECI'OMIZED AND/OR KEPT IN DARKNESS MEAN f SEM) Group
Days after conception
Serum prolactin (ng RP-l/ml) Sham-operated Alternating light Darkness
Pinealectomized Alternating light Darkness Pituitary prolactin (fig RP-l/gland) Sham+perated Alternating light Darkness
Pinealectomized Alternating light Darkness
Postpartum
21
22
10.6 f 0.5 (25) 19.1 f 3.3 f (16) 13.0 f 1.5 (21) 21.8 t 2.4 j (20)
44.3 f (24) 113.2 f (23) 45.6 f (19) 98.9 (17)
70.7 (29) 73.2 (17) 66.9 (25) 66.7 (21)
53.7 f 3.5 c (26) 30.7 * 4.3 d& (22) 46.2 f 3.3 d (20) 31.05. 4 . 6 d j (14)
f
3.7
f
7.0 2.5
5
7.7
7.1 19.5 d*f 8.9 d 19.9 d>h
29.8 (20) 23.8 (22) 27.2 (23) 26.8 (21)
* 2.0 f
1.0 die
f
1.3 a
f
1.8
71.8 -+ 4.9 (10) 73.1 f 5.4 (12) 69.4 2.9 b (14) 70.4 f 4.0 (11)
a P < 0.05; b P < 0.01; C P < 0.005; d P < 0.001: significance of differences within each group between consecutive days. e P < 0.01; f P < 0.005; g P < 0.001: significance of differences on the same days compared to shamoperated group kept in alternating light. h P < 0.02; i P < 0.01; j P < 0.005: significance of differences on the same days compared to pinealectomized group kept in alternating light. Number of animals in parentheses.
this darkness-induced increase in plasma gonadotrophin levels is mediated by, or independent from, the pineal gland. To this end we exposed sham-operated and pinealectomized pregnant rats to continuous darkness in order to elucidate the possible role of the pineal gland in modifying the gonadotrophin and prolactin levels during pregnancy. We are reporting here the results on prolactin changes. Studies on LH are still underway. Pinealectomy did not abolish the darkness-induced increment in serum prolactin levels during the prenatal period (Table XI). Similarly, pinealectomy had no effect on the corresponding darkness-induced decrease in pituitary prolactin levels (Table XI). Absence of the pineal does not prevent darkness-induced elevation of prolactin during the prenatal period. Darkness does not affect the weights of pituitaries; pinealectomy could not interfere with the effect of darkness on this parameter, except for some inconsistent changes (Table XII). From these results it can be understood that exposure of pregnant rats to continuous darkness affects the metabolism of prolactin by a route independent of the pineal gland.
432
TABLE XII PITUITARY WEIGHTS (in mg f SEM) ON DAYS 21 AND 22 AFTER CONCEPTION AND POSTPARTUM OF RATS PINEALECTOMIZED AND/OR KEPT IN DARKNESS Group
Days after conception
22
21
Shamapera ted Alternating light
Postpartum
8.1 f 0.2 (30) 8.2 f 0.2 (19)
Darkness Pinealectomized Alternating light
*
1.7 f 0.3 (24) 8.8 f 0.3 b (21)
Darkness
~~
~
~~
9.1 f 0.4 a (24) 10.3 0.3 (22)
8.3 0.3 (22) 8.6 f 0.2 (19)
*
~~
a P < 0.05 : significance of differences within each group between consecutive days.
P < 0.05: significance of differences on the same days between pinealectomized groups kept in darkness and alternating light. P < 0.05: significance of differences o n the same days between pinealectomized and sham-operated groups kept in darkness. Number of animals in parentheses.
Corresponding studies on the role of the pineal gland in darkness-induced changes in LH metabolism are in progress and there is no reason to assume that it would be different from that of prolactin. TABLE XIII NUMBER OF EMBRYOS AND RESOWIONS IN PINEALECTOMIZED COMPARED TO SHAMOPERATED RATS Experiment
1
2 3 4 5 6
I
8 9
Totals
Number of animals
Average embryoslrat
(4
(S)
(4
(8
51 14 21 40 31 41 41 26 14
42 15 20 40 43 38 54 34 10
8.3 8.5 5.8 9.3 8.8 9.3 8.6 9.0 8.6
8.5 8.4 8.4 9.0 8.7 9.6 8.4 9.0 8.7
4 4 4 1 8 4 4
291
296
8.6
8.8
34
Rats with resorptions
Rats with 4 or less embryos
(4
(sl
(4
(Sl
(P)
(Sl
5
2 0 1 4 10 0
2 0 1.5 2.5 3 4 3 1 2
1.5 0 2 2 3 0 2 1 0
5 1 9 1 2 2 4 0 0
1 0
3 1 6 1 4 0 1
2
1.3
24
11
0
P = pinealectomized; S = sham+paated (controls).
Resorptionsl mt
I
1 0 25
433 TABLE X N NUMBER OF RATS THAT DELIVERED ON DAYS 22 AND 23 AFTER CONCEPTION ACCORDING TO TREATMENT AND LIGHT EXPOSURE Group
Alternating light Days after conception
Darkness Days after conception
22
22
23
Lkht Days after conception
23
22
23
15 -
26
~~
Intact Sham-operat ed Pinealectomized Shamaperated and melatonin-treated Pinealectomized and melatonin-treated a
16 9 9 6 10
31 20 17 5 8
29 6 8 -
22 a
I
4a -
-
-
P < 0.02 of the ratios between days 22 and 23 after conception of the same group kept in darkness and alternating light.
CONCLUDING REMARKS Reviewing our findings it appears that the rat pineal gland may be involved in the endocrine changes which take place the perinatal period. The manner in which pineal-induced changes in the levels of circulating gonadotrophins and steroid hormones during the perinatal period affect various facets of pregnancy like duration, numbers of living fetuses and resorptions under normal conditions appears minimal. No differences reaching significance were found between the experimental and control groups in any of the aforementioned parameters (Table XIII). The only notable difference was in the duration of pregnancy. Estimated by the chi-square test a significantly larger number of deliveries occurred on day 22 than on day 23 of gestation among rats exposed to continuous darkness - sham-operated as well as pinealectomized - as compared to control groups kept in alternating light (Table XIV). Pinealectomy alone had no effect on duration of gestation, but melatonin or darkness shortened it. The role of the pineal gland in pregnancy could perhaps become more obvious should animals be exposed to conditions which drastically affect their endocrine apparatus. Under such circumstances the pineal may be able to play the role of a moderator between abnormal environmental (or perhaps even physiological) conditions and the functions of hormones in gestation. REFERENCES Bast, D. and Melampy, R.M. (1 972) Luteinizing hormone, prolactin and ovarian 20-hydroxysteroid dehydrogenase levels during pregnancy and pseudopregnancy i n the rat. Endocrinology, 9 1 : 1499- 1505. DeverEerski, V. (1972) Contribution histoenzymologique a 1'6tude du m6tabolisme de la glande pineale chez la ratte gravide. C.R. SOC.Biol. (Paris), 166: 131. Ellendorf, F. and Smidt, D. (1961) Der Einfluss unterschiedlicher Beleuchtung auf die neurosekretorische Aktivitat, Pubertat und Sexualfunktion von Mausen. J. neuro-visc. Rel., Suppl. 10, pp. 220-232.
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DISCUSSION J.W. VISSER: Is there any difference in the parameters in stillbirth?
I. NIR: No, there is not a significant difference.