Indian Journal of Experimental Biology Vol. 38, October 2000, pp. 974-981

Profile of organ weights and plasma concentrations of melatonin, estradiol and progesterone during gestation and post-parturition periods in female

Indian palm squirrel Funambulus pennanti

K S Bishnupuri & C Haldar*

Department of Zoology, Banaras Hindu University, Yaranasi 221 005, India

Received 7 September / 999; revised 26 July 2000

Todate, report about the role of pineal gland in maintaining the normal physiology of gestation is scanty. Present study is the first of its kind giving a detai l profile of organ weights and plasma concentration of melatonin, estradiol and progesterone to suggest a possible role of pineal gland in maintaining normal physiology during gestati on and post­parturition periods of female Indian palm squirrel F. pennanti. lnspite of, inverse pineal-gonadal/melatonin-steroids interrelationship in adult (non-pregnant) females, the present results study suggest a direct relationship of pineal gland activity with ovarian steroids especiall y during the ge:; tation period. The inverse rel ationship of melatonin and ovarian steroids is again establi shed after parturition and maintained th roughout the life. Thus the pineal gland (acti vity as judged by its weight, biochemical contents i.e. protein and cholesterol and plasma melatonin level) maintained ovarian/uterine physiology and regulated plasma concentrations of estradiol and progesterone during gestation and post-parturiti on periods. It is suggested that the pineal gland and its hormone melatonin pl ay an important role to mai ntain the normal physiology of gestation and the post-partum recovery in Indian palm sc_uirrel F.pennanti.

Until little more than a decade ago, no information was available on whether the pineal gland influences gestation or vice versa and whether pregnancy may affect pineal metabolism and function . But, there are some indications suggesting pineal implications in processes related with gestation 1 and fertilit /" . Rodents exposed to continuous darkness, to short daily photoperiods or to decreased light intensity throughout the gestation peri od demonstrated significantly shorter pregnancies than controls kept in normal lighting4

'5

, while long tdail y photoperiod de layed parturition5

. However, removal of the pineal gland, which usually mediates between environmental lighting and endocrine glands did not abo li sh ~ h e

effect of short photoperiod on the duration of gestation5

, which suggests that the effect of darkness on gestat ion may not be mediated by the pineal gland .

Similarly, confusing is the ro le of pineal g land in fertility processes. While pinealectomy had no effec t on the fertility index of rats6

, blinding of hamsters markedly reduced the number of fertil e mating2 and ferti lity was also reduced in rats subjected to blind ing and ansomia'. These effects could be negated by

. I I . ? ' pmea ectomy or chronic me atontn treatment-..

*Correspondent author

suggesting a progonadal effect of melatonin in pregnant rodents.

Tilldate, not a single report is available explaining the changes occurring in pineal gland during gestation and post-partuntiOn periods re lated with ovarian/uterine physiology and plasma concentration of estradiol and progesterone. The pineal gland we ight, its prote in and cholesterol content can be used as markers, there fore, bes ides the plasma melatonin level they were analysed to judge the pineal gland activity . Further, the gestation period is a very sensitive period of femal e reproducti ve cycle and the adrenal phys iology refl ec ts directly the stress condition for animals, hence, adrenal g land weight analysi s during these periods was also taken into consideration. To throw a li ght on post-partum recovery process, all these parameters were ana lysed even after parturition in female Indi an palm squirre ls.

Materials and Methods Pregnant female Indian palm squirre ls Funambulus

pennanti- having body weight ( 120 ± Sg) were co llec ted from the vic inity of Varanasi (Lat. 25° 18' N; Long. 83° I ' E) during March and April. T he pregnancy was initially tested by feeling externa lly the bu lging of uteru s wi th implanted embryo and

BISHNUPURI AND HALDAR: PINEAL GLAND DURING PREGNANCY IN SQUIRREL 975

finally with Preg-test (Intercare Pharmaceuticals, Calcutta). All the pregnant females were kept in separate wire net cages (25"x25"x30" in size) individually in an animal room fully exposed to ambient condition of light, temperature and humidity. They were provided with water soaked gram (Cicer arietinum) and water ad libitum. For determination of total gestation period, the days starting from initial implantation till date of parturition was found to be -45±2. With extensive observation and experiencing the size of implanted embryo intoto (by feeling externally), the pregnant females were divided into II groups i.e. GP3, GP6, GP9, GP12, GP15, GP20, GP25, GP30, GP35, GP40, GP45 (GP: gestation period followed by numerical representing the days: Table I; Figs I and 2). Three groups of delivered females i.e. AP5, APlO, AP20 (AP: after parturition followed by numerical representing the days: Fig 3) were taken into consideration to note post-parturition condition and to define the phenomenon of post­partum recovery. One group of non-pregnant (NP) females was used to note comparative changes occurring in organ weights and plasma concentrations of melatonin, estradiol and . progesterone during gestation and post-parturition periods. Different groups of pregnant females were sacrificed by decapitation at night (2200 - 2300 hrs). The maternal pineal, ovary, uterus and adrenal gland were dissected out and weighed. The pineal gland was collected on ice and then processed for biochemical analyses of protein and cholesterol content. The blood of sacrificed females was collected in heparinised tubes and centrifuged at 3000 x g to collect the plasma. The plasma of all the experimental animals was stored at -20°C till the hormonal analysis.

For biochemical analyses, the pineal gland after washing in normal saline (0.9% NaCI) homogenised in a micro glass homogenizer. Pineal protein content was estimated spectrophotometrically by Folin­Ciocalteau reagent7

, while the cholesterol content with the help of Leibermann-Berchard reagent8

.

For hormonal analyses, the plasma melatonin content of respective animals was estimated following the radioimmunoassay method9

•10

. The

plasma concentrations of estradiol and progesterone were estimated by commercial radioimmunoassay kits; Estradiol (125I) coated RIA kit from Orion Diagnostics, Espoo, Finland and progesterone (1251) radioimmunoassay kit from Binax, Portland, MAINE, USA. The validation data of above hormonal assays are given in Table 2.

Results In comparison to non-pregnant (NP) females the

body weight of pregnant females increased gradually during the gestation period and then decreased after parturition (Fig. 4). No change in adrenal gland weight was noted during gestation and post­parturition periods (Fig. 4). Following a peak value of ovarian weight at initial implantation of embryo, it decreased gradually with advancement of pregnancy and after parturition again exhibited a gradual increase (Fig. 5) . In comparison to NP females, uterine weight of pregnant females increased gradually with fetal development till parturition and then decreased sharply after parturition (Fig. 5). The plasma levels of estradiol and progesterone in pregnant females increased gradually during gestation period. After parturition, plasma level of progesterone decreased sharply while estradiol decreased gradually (Fig. 6). In comparison to NP females the pineal gland weight and plasma melatonin level of pregnant females attained a lowest value at initial implantation of embryo and increased gradually during gestation period and even after parturition (Fig. 7). Similarly, the pineal protein content after attaining a lowest value at initial implantation of embryo exhibited

Table 2-Hormonal assay validation data

Hormone

Melatonin

Estradiol

Progesterone

Intra-assay percision (CY%)

9

5.97

7 .3

Inter-assay Sensitivity precision (CV%)

15 10 pg/ml

8. 13 0 .817 pg/ml

8.6 0 .8 ng/ml

Table !-Size of embryo (in toto) at different gestation periods

Gestation period (in days) 3 6 9 12 15 20 25 30 35 40 45 Average size of embryo (in toto) in 0.4 0.5 0 .7 0.9 1.1 1.3 1.7 2. 3 3.5 3.8 4 .0 long axis (em)

976 INDIAN J EXP BIOL, OCTOBER 2000

Fig. 1-Size of embryo (i n toto) : NP: non-pregnant, GP: ges tati on period and numeri cals representing the days.

Fig. 2-ln ulero age of Ic tus (GP: gestation period and numericals representing the days) .

Fig. 3-Post-part um uteru s (AP: after parturition period and numericals representing the days).

BJSHNUPURI AND HALDAR: PINEAL GLAND DURING PREGNANCY IN SQUIRREL 977

Discussion increas ing trend during pregnancy and even after parturition, however, no change in pineal cholesterol content was noted during gestation and post­parturition periods (Fig. 8).

The reproductive act ive phase in tropics for seasonally breeding rodent is marked with avai lability of food and favourable environmental conditions

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Status of females

Fi g. 4-Changes in hotly and ad renal gland weight during gestation and after parturiti on (N P: non-pregnant ; GP: gestati on period lollowed by day~: AP: after parturition fo ll owed by days: One way ANOYA: for body weight, P<O.OOI and for adrenal gland weight, ?>0.2) .

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Status of females

Fig. 5-Changes in ovarian and uterine weight during gestation period and after parturition (NP: non-pregnant; GP: gestation period followed by days ; AP: aft er parturition followed by days: One way ANOV A: Ovarian weight, P <0.00 I and Uterine weight, P<O.OO I).

978 INDIAN J EXP BIOL, OCTOBER 2000

(moderate temperature and humid ity) with sufficient place for their movement and mating. T hese factors are available for Indian subtropics during February to Apri l. Hence, maximum mating of Indian palm

squirrels occurred during February and March . A

large number of pregnant females could be noted during March and April in nature. Quite less number of pregnant females were also during August and

September, when the above eco-socio factors were moderately available in a tropical country. After

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20

Fig. 6-Changes in pl asma levels of estradiol and progesterone during geSiation period and after parturition (NP: non-pregnant; GP: gestation period followed by days; AP: after parturition fo llowed by days: One way ANOY A, P for estradiol and progesterone <0.00 I).

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Status of females

Fig. 7-Changes in pineal gland weight and pl asma level of melatonin during gestation period and after parturition (NP: non-pregnant ; GP: gestation period followed by days; AP: after parturition fell owed by days: One way ANOY A, P for pineal gland weight and pl asma mel atonin <0.00 I).

BISHNUPURI AND HALDAR: PINEAL GLAND DURING PREGNANCY IN SQUIRREL 979

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Status of females

Fig. 8-Changes in pineal protein and cholesterol content during ges tation period and after parturition (NP: non-pregnant; GP: gestation period followed by days; AP: after parturition followed by days: One way ANOV A, pineal protein, P<O.OO I and pineal cholesterol , P>0.2)

successful mating, fertilization of ovum with sperm occurs and finally the uterine wall is being implanted with embryos (number of implanted embryo: 1-5 in

case of Indian palm squirrel). During gestation period, gradual thickening of uterine wall (to maintain the developing embryos) and accumulation of fat in lower abdominal area (to fulfil the demand of lactation) increases the female's body weight. After parturition, uterus again starts to shrink to maintain its normal physiology and finally to make itse lf suitable for next implantation of embryos. Hence, the uterine weight decreased gradual ly after parturition in these squirre ls. The accumu lated fat in the lower abdominal area gets metabolized to fulfil the energetic demand of lactation, hence female ' s body weight decreased gradually after parturition as well. A sudden decrease in female's body weight was also registered just after parturition due to delivery of pups, which shared maximally their weight with female's body during pregnancy. The loss of body weight in female squirrels in weaning and feeding of young ones favoured the idea of Whittier and Crews 11

suggesting that females bear the maximum cost of reproduction in all vertebrates.

A peak ac tivity of ovarian physiology was required for successful mating and possible implantation of

embryos, hence a maximum ovarian weight was noted at the beginning of gestation period. Following implantation of embryo, ovarian weight decreased gradually till parturition and after parturition, it increased gradually ti ll next breeding (reproducti ve active phase) to attain normal ovarian physiology. The ovarian steroidogenesis i.e. synthesis and release of estradiol and progesterone marked by their plasma concentrations in squi rre ls increased gradually during gestation period and attained hi ghes t value at parturition . After parturition, the plasma progesterone level declined sharply, as it was required to maintain the pregnancy only. However, the plasma estradiol level declined gradually as it was required to maintain the normal physiology of gestation as well as to facilitate lactation 111 delivered females. The maintenance of uteri ne physiology is a progesterone dependent phenomenon, hence a higher plasma leve l of progesterone and increased uterine weight was also required to protect and maintain the implanted embryos.

Several authors todate reported the inverse re lationship of pineal gland activity with ovari an weight and plasma levels of ovari an steroids in normal female rodents 12

·". Pineal gland activity was inversely corre lated wi th reproductive activity of

980 INDIAN J EXP BIOL, OCTOBER 2000

Indian palm squirre l14• The present study for the first

time suggested a direc t re lationship of pineal gland acttvtty with pl asma leve ls of estradiol and progesterone espec ially during the gestation per iod. The pineal gland acti vity as judged by its weight , protein content and plasma melatonin level was lowest at the time of implantati on of embryos during the reproducti ve acti ve phase of Indi an palm squirrel.

Foll owing implantation, along with gradual increase in plasma levels of es tradiol and progesterone, gradual increase in pineal gland acti vity was registered throughout the gestation period (from init ial implantation to date of parturiti on). Therefore, it may be sugges ted that high level of plasma melatonin and ovari an steroids (a direct re lati onship) is required fo r the maintenance of normal phys iology of gestation. Interestingly, after parturiti on, the inverse re lationship of melatonin and ovarian steroids is again establi shed (i.e . high level of plasma melatonin with low level of plasma estradiol and progesterone) and maintained throughout the life . The exogenous melatonin treatment 14 and photoperiodic alterations 15 leads to change in pineal gland acti vity before 30'11 day of gestation resulting in resorpt ion of implanted embryo 15

, while after 30'11 day of gestati on it did not interfe re with the maintenance of pregnancy

but altered the neonatal growth and sexual development 16

• The change in pineal gland acti vity following exogenous melatonin treatment 14 and di fferent photopereiodic exposures 15 disturbs the hormonal profiles of estradi ol and proges::erone · during early gestati on period 14

'15 and finally results in

major changes of female's ability to maintain ovarian/uterine functi on, thereby causing resorpti on of implanted embryos. Later in pregnancy, the placenta may support the ovary/uterus to some extent, so that the effects of the changes in pineal gland acti vity on plasma levels of estradiol and progesterone are inconsequential, hence female deli vers pups normally but with altered growth and

sexual maturation15• It is now well establi shed that

the receptors of gonadal steroids (especially estradiol and testosterone) were present in the pineal17

,

whereas the melatonin receptors were present in the gonads18

• Hence it may be possible that the gonadal steroids and pineal melatonin regulates the plasma levels of each other. Further, the receptors of

melatonin in pars-tuberali s show seasonal variation following which the steroid hormone level changes in

plasma19. Surprisingly, when the plasma estradiol

level increased during gestation, it also enhanced the pineal gland acti vity and faci litated the synthesis and re lease of melatonin rather than inhibiting it as it does in normal female rodents12

•13

.

Therefore, it may be concluded that inspite of melatonin-steroids inverse re lationship in adult (non­pregnant) females, a direct relationship is maintained between pl asma levels of melatonin and ovarian steroid especiall y during pregnancy (starting fro m date of implantation till date of parturition) for this rodent. After parturiti on, the inverse melatonin­steroid re lati onship is again establi shed to enable these rodents to recover themselves for consequenti al reproductive cyc le (pos t-partum recovery) . Thus, the pineal gland and its hormone melatonin play an important role to mainta in the normal physiology of gestat ion.

The reproducti ve acti vity of thi s rodent needs further an elaboration as it shows a typical post­partum recovery. On the bas is of present study we may divide thi s post-partum recovery phase of female into 6 important phys iological steps, which take about 2 months time in total. It begins with (a) gradual increase in plasma progesterone level fo ll owing its sharp decline during parturiti on fo ll owed by, (b) gradual decrease in uterine weight , (c) an increase in ovarian weight after a week or two, (d) gradual decrease in body weight due to metaboli zation of fa t in lower abdominal area, (e) an increase in pl asma estradiol level fo ll owing its gradual decrease after parturition, and (f) an increase in pineal gland activity (i.e. pineal gland weight, pineal prote in content and plasma leve l of melatonin).

Acknowledgement Authors thank to UGC, New Delhi for financial

support.

References I Huang C Y & Everitt A V, The effect of pregnancy on pi neal

weight in the rat, J Endocrinology, 32 ( 1965) 26 1. 2 Reiter R J, Evidence for refractoriness of the pituitary­

gonadal axis to the pineal gland in the golden hamsters and its possible implications in annual reproductive rhythms. A nat Res, 173 ( 1972) 365.

3 Reiter R J, Blask D E, Johnson L Y, Rudeen P K, Yauglan N K & Wari ng P J, Melatonin inhibition of the reproduction in the male hamster: Its dependency on time of day of administrati on and an impact on sympathetically innervated pineal gland , Neuroendocrinology, 22 (1976) I 07.

BISHNUPURI AND HALDAR: PINEAL GLAND DURING PREGNANCY IN SQUIRREL 981

4 Ellendorf F & Smidt D, Der einfluss unterschiedlicher beleuchtung auf die neurosekretorische aktivatat, pubertal and sexual funktion von mausen, 1 Neuro- Vise Rel ( suppl), 10 (1961 ) 220.

5 Mitchell J A & Yochim J M, Influence of environmental li ghting on duration of pregnancy in the rat, Endocrinology, 87 (1970) 472.

6 Kincl F A & Benagiano G, The failure of the pineal gland removal in neonatal animals to influence reproduction, Acta Endocrinologica ( Kbh ). 54 ( 1967) 189.

7 Lowry 0 H, Rosenbrough N J, Farr A L & Randall R J, Protein measurements with the Folin-phenol reagent, 1 Bioi Chem, 193 (1951 ) 265.

8 Stadman T C, Preparation and assay of cholesterol and ergosterol , Methods in Enzymology, Vol 3 (Academi c Press, New York) 1957,392.

9 Roll ag M D & Niswender G D, Radioimmunoassay of serum concentrations of melatonin in sheep exposed to different light regimes, Endocrinology, 98 (1976) 482.

I 0 Attanasio A, Borelli P & Gupta D, Circadian rhythms in serum melatoni n from in fancy to adolescence, 1 C/in Endocrinol Metab, 61 ( 1985) 388.

II Whittier J M & Crews D, Seasonal reproduction: Pattern and control, Hormones and reproduction in fish es, amphibians and reptiles, Plennum Press, New York, (1987) 385.

12 Reiter R J, Interacti on of photoperiod, pineal and seasonal reproduction as examplitied by findings in the hamster, in The Pineal and reproduction , (Karger, Basel), 1978, 169.

13 Carter D S & Goldman B D, Antigonadal effect of ti med melatonin infusion in pinealectomized male Djungarian hamsters (Phodopus sungorus sungorus): Duration is the critical parameter, Endocrinology, 11 3 ( 1983) 126 1.

14 Bishnupuri KS , Maternal pineal physiology and fe tal development in Indian palm squirrel F. Pennanti, Ph D thesis, Department of Zoology, Banaras Hindu University, Varanasi, 1999.

15 Bishnupuri K S & Haldar C, Impact of photoperiodic exposures during late gestation and lactation peri ods on the pineal and reproductive physiology of the Indian palm squirrel F. pennanti, 1 Reprod. Fer/, 11 8(2000) 295 .

16 Bishnupuri K S & Haldar C, Maternal photoperiodic exposures alter the neonatal growth, pineal functions and sexual development of the Indian palm squirrel F. pennanti, 1 Neural Transm I 06 ( 1999) 869.

17 Haldar C & Gupta D, Sex and age dependent nature of the cytoplasmic 5a-dihyclrotestosterone (DHT) binding sites/receptors, 1 Pin Res, 814 ( 1990) 289.

18 Ayre E A, Wang Z P, Brown G M & Pang S F, Locali zation and characterization of [ 125 1] iodomelatonin binding sites in duck gonads, 1 Pin Res, 17 ( 1994) 39.

19 Skene D J, Masson-Pevet M & Pevet P, Seasonal changes in melatonin binding sites in the pars tuberali s of the European hamster and the effect of testosterone manipulation , Endocrinology, 132 (1993) 1682.