ANS Annals of Neurosciences 0972-7531 0976-3260 Indian Academy of Neurosciences 06130303 10.5214/ans.0972.7531.2006.130303 Biochemical characteristics of mammalian pineal gland BIOCHEMICAL ALTERATION IN THE AMOUNTS OF TOTAL PROTEIN, MONOAMINE-OXIDASE, ADRENALINE AND NORADRENALINE IN THE PINEAL GLAND OF RHINOPOMA KINNEARI WROUGHTON (MICROCHIROPTERA: MAMMALIA) DURING VARIOUS DEVELOPMENTAL AND REPRODUCTIVE STATES. Bhatnagar Sunita * * Lal SB ** Lecturer in Zoology MDM College Kota, India Ph. No.: 0294–2426007 Assoc. Prof Deptt. of Zoology M LS University, Udaipur 313 001 India Sunita Bhatnagar Lecturer in Zoology, MDM College Kota India 7 2006 13 3 72 75 Copyright © 2006, Annals of Neurosciences 2006

Biochemical characteristics of mammalian pineal gland have been studied in a number of mammalian species under normal and experimental conditions. However, only a few species have been investigated biochemically and from the point of view of developmental specific profiles. Total protein, MAO, adrenaline and noradrenaline were biochemically estimated. Maximum protein. 7.20 mg.% was found in the pineal gland of juvenile male. The estimate for juvenile female was 8.9 mg.%. However, in adult females and pregnant bats the percentage of estimated protein was significantly higher. The most significant feature was a dramatic drop in total protein of lactating females, which was 0.27 mg.%. MAO is an important enzyme, which is concerned with the metabolism of biogenic amines. Maximum amount of MAO was computed in the pineal gland of juvenile female whereas minimum value was recorded for pregnant bats. Adrenaline and noradrenaline amounts in the pineal gland of male and female bats exhibited sexual differences as well as that of the developmental and reproductive states.

 Bats Pineal gland Biochemistry Introduction

Pineal biochemical secretions have been shown to display alteration that are age-related; and which are characteristic of a particular type of physiological or developmental state (1). A variety of enzymes NAT, HIOMT, MAO have been biochemically quantitated in the pineal gland of rat, cow and rabbit (2). An evolutionary shift has been suggested to occur from the cholinergic innervations seen in pineal gland of lower invertebrates to the adrenergic innervations observed in mammalian pineal gland. This sympathetic innervation is thought to depend on nor-adrenaline which is a neuro transmitter. (3–6)

Very little is known about the biochemical repertoire of the pineal gland of bats (7–13) The literature relating to biochemical milieu of pineal gland of chiropteran with reference to the age, sex, developmental and reproductive state is scant and fragmented.

The present studies with Rhinopoma kinneari were initiated to monitor the biochemical characteristics of the pineal gland with regard to total protein, MAO, adrenaline and nor-adrenaline during various developmental and reproductive state.

 Materials and Methods

Rhinopoma kinneari were collected from their roosting sites near Jagdish Temple, Udaipur (Raj.) India (latitude 24/34 N, longitude 73/42 E). Bats were sexed and categorized on the basis of weight and reproductive state. They were sacrificed by cervical dislocation between 11 a.m. to 12 noon. The brain was removed. The pineal gland was carefully excised. Ten pineal glands of identical developmental and reproductive state were 'pooled' and homogenised in 1.0 ml of chilled 0.9 per cent physiological saline. The homogenates was cryocentrifuged at 3000 r. p.m. for 20 min. The supernatant was decanted and used for biochemical estimation of the total protein according to the method of Henri etal, MAO according to the method of Green and Haughton, adrenaline and noradrenaline according to the method of Von Euler and Hamberg (14–16). The data were analyzed using a one-way ANOVA (17)

 Results

Considerable variations were observed in the profile of total protein MAO, adrenaline and nor-adrenaline in the male and female during various developmental and reproductive states (Table 1). For the sake of clarity each chemical species assayed biochemically is narrated as under:

1. Total Protein

A significant difference (P <0.01) was observed in the total protein content of pineal gland between juvenile male and adult male stage. Whereas in juvenile female-adult female and adult female-pregnant stage pineal gland, the difference was not significant. Differences occurred in the pineal gland total protein of pregnant and lactating bats.

A significant decrease was observed in total protein content of juvenile to adult pineal gland of male bats. This was also true for parous and lactating bats. However, the total protein in juvenile female was at par with the adult female and with the pregnant stage.

In case of male maximum pineal gland protein content was computed for juvenile stage (7.20 mg%). In case of juvenile female maximum pineal gland protein was 8.9 mg%. Minimum pineal gland total protein of lactating females was 0.272 mg%.

 2. Monoamine Oxidase

A significant difference (P<0.01) was observed in case of monomine oxidase contents of pineal gland of juvenile and adult males. However in case of female, significant differences were observed between juvenile to adult and from adult to pregnant stages. No significant variation in total MAO could be discerned in pregnant and lactating bats.

A significant incremental trend of MAO in the pineal gland was observed from juvenile to adult stage of male. On the other hand a significant decrease in MAO content of pineal gland was observed in females from juvenile to pregnant stage, juvenile to adult female; adult female to pregnant. However, increase was insignificant in case of pregnant and lactating females.

Maximum MAO content in the pineal gland was observed in case of juvenile female 3.95 U/min/mg protein, while in adult female it was 2.77 U/min/mg protein. In case of male highest value of MAO was observed in adult male 1.738 U/min/mg protein. Minimum content of MAO occurred in the pineal gland of male at juvenile stage i.e. 0.087 U/min/mg protein. In case of pregnant females, MAO amount was 0.212 U/min/mg protein.

 3. Adrenaline

Adrenaline content in pineal gland of juvenile and adult male and female. In case of male the difference was significant (p<0.01) whereas, in case of female, it was not significant. However, a significant difference in the adrenaline content of pineal gland was observed between adult to pregnant stage of the female. The values of adrenaline content for pregnant stage was comparable with the values computed for lactating bats.

Similarly, the adrenaline content of juvenile female was at par with the adult female.

In both male and female, the adrenaline content of pineal gland in various stages displayed a decremental trend. Significant reduction in adrenaline amounts were noted in case of male from juvenile to adult stages; whereas in case of female significant reduction in adrenaline content of pineal gland were observed from adult to pregnant stage.

Juvenile males and females were observed to contain the maximum amount of adrenaline stage lowest value adrenaline was record in lactating stage.

 4. Nor-Adrenaline

A significant difference (P<0.01) was observed in the noradrenaline content of pineal gland between juvenile and adult stage of males. Similarly, in case of females, the difference of nor-adrenaline amount was also significant between juvenile and adult stage. In pregnant and lactating bats significant differences were observed in the nor-adrenaline content of their pineal gland.

Significant decremental trend in the level of nor-adrenaline was observed in the pineal gland of male and female.

Maximum level of nor-adrenaline was observed in the juvenile males (0.002377 μg) and juvenile female (0.002076 μg) minimum level was recorded for adult male (0.00165 μg).

 Discussion

The major thrust of the present study was to highlight the variation in total protein, monoamine oxidase, adrenaline, and noradrenaline in the pineal gland of an insectivorous - microchiropteran Rhinopoma kinneari from different developmental to reproductive states. In case of pineal gland of male maximum protein was observed at juvenile stage (7.20 mg%). Similarly, in case of females, juvenile bats. Both these values were significant at 1 percent C.D. However the pineal gland protein of adult female pregnant bats was also significantly higher than the adult male. In lactating bat a sudden but significant ebb in the total protein (0.272 mg%) was computed.

The maximum pineal protein at juvenile stage explains the presence of maximum tryptophan, which may be a factor responsible, for the delay in gonadal development and the concomitant maturational changes.

It is well known that in case of female a large number of physiological and biochemical activities occur which are significantly different from the males. Increase in total protein in the pineal gland in all stages except lactation phase may be related to this variation, The lowest protein content in lactation stage is observed in the present study seems to indicate that release from the, major physiological responsibility of bearing the concepts and sustaining its growth, development, maturation and birth may have some relationship. These data also indicate the differential kinetics with which protein are biosynthesized or bio-degraded during the aforesaid stages. Many proteins may have structural role. Others may act as enzymes in the diverse metabolic pathways.

Thus, very limited literature is available on these aspects for other placentals in the pineal gland of mole, some pinealocytes were observed to contain large quantities of yellow orange, auto-fluorescent material (6,12). This was believed to be the protein which contains a high percentage of tryptophan(l). Investigated that daily fluctuation occurs in the metabolism of protein in attempt to determine the relationship between circadian changes in the pineal metabolic rate. The levels of protein in mature male rat a peak level is reached about 4 p.m.

MAO is an important enzyme which is concerned with the metabolism of biogenic amines, Maximum amount of MAO was found in the pineal gland of juvenile female bats, Whereas minimum quantities was computed in the pineal gland homogenates of pregnant bats.

The maximum pineal MAO at juvenile stage may be due, to the fact that at this stage most of the serotonin is metabolized by MAO and nor-adrenaline which may be a factor responsible to delay the gonadal maturity (18). The lowest MAO amount is observed in pregnant bats which may mean that serotonin and nor-adrenaline are not biosynthesized to lowest quantity of MAO. Ralph also suggested that pineal gland secretions are inversely related to gonadal activity(19). Reiter observed MAO granules in the pinealocytes (12).

Thus, the activity of MAO was observed to be high in the pineal gland of rats, cow and rabbit (18,20,21). Sympathetic innervation of pineal gland in rats, cats and rabbits caused a fall in MAO concentration(20,21). Wurtman et. al, suggested that MAO in the pineal gland of rat may metabolize serotonin to 5-hydroxy-indole acetaldehyde. They did not observe any age related changes(22). This is at variance with the present study. It is interesting to record the postulate of Bausher that MAO in the pineal gland of rabbit occurs in two variant forms which is designated as MAO A and MAO B (23–26).

Adrenaline and nor-adrenaline concentration in the pineal gland of male and female Rhinopoma kinneari exhibited sexual differences as well as with the developmental and reproductive states. Lerner observed high concentrations of adrenaline in the pineal gland than in other parts of the; brain(27).

In Rhinopoma kinneari maximum quantities of adrenaline and nor-adrenaline was computed in the pineal gland of juvenile female, whereas minimum quantity was observed at lactating female stage. The maximum pineal adrenaline and nor-adrenaline at juvenile female stage may be regulated by MAO) and could serve as a factor responsible to delay the gonadal maturity. On the other hand lowest adrenaline and nor-adrenaline, concentration in lactating females may be due to the fact that neonate may be anchored to mammary nipples for nursing, This may delay and stop follicular development and ovulation in females. Nor-adrenaline has been found in relatively large amounts in the rat pineal gland, concentration of this catecholamine shows a diurnal rhythm during the day it falls to one-third its peak nocturnal level. Essentially all of the nor-adrenaline in the pineal gland is located within the sympathetic nerve endings (13). Further the sympathetic nerves in rabbit, cat, sheep, cow, pig and hamster, store mostly nor-adrenaline, while those in the rat, and guinea pig contain large amounts of serotonin. Although further meaningful comparisons about adrenaline and nor-adrenaline profile with other placentals, are not feasible due to lack of information, it is tempting to record the information, that elevated production of melatonin by the pineal gland during darkness is due primarily to the release of nor-adrenaline from the postganglionic nerve terminals within the pineal gland. Once released, this acts on beta-adrenergic receptors of the pinealocyte membrane to promote cAMP production and eventually melatonin synthesis. Melatonin production at night is governed by electrical signals received by the pineal gland from the SCN (28)

Thus, the present study on the biochemical repertoire of pineal gland of Rhinopoma kinneari clearly indicates sex related differences in the profiles of total protein, adrenaline, noradrenaline and MAO. It also shows that there is a significant shift in the amounts of these in male and female bats as they transit from juvenile to adult, pregnant and lactating stage.

 Reference NirI HirschmannN SulmanFG Diurnal rhythms of pineal nucleic acids and protein Neuroendocrinoiogy 1971 1 271 77 WurtmanRJ AxelrodJ The formation, metabolism, physiologic effect of melatonin in marnais KapperJA SchadeJP In: Structure and function of Epiphysis cerebri Amsterdan Elsevier 1963 52029 WolfDE PotterLT RichardsonKC AxelrodJ Localizing tritiated norepinephrine in sympathetic axons by electron microscopic autoradiography Science 1962 138 440 42 OwmanCh Localization of neural and parenchymal monoamines under normal and experimental conditions in the mammalian pineal gland Prog Brain Res 1965 10 423 53 WurtmanRJ Effects of light and visual stimuli on endocrine function GangonWF MartiniL Neuroendocrinology New York Academic Press 1966 PevetP JuillardMT SmithAR KapperJA The pineal gland of the mole (Talpa Europaea L.). A florescence histo chemical study Cell Tiss Res 1976 165 297 306 BhatnagarKP The vampire pinealocyte : Some ultrastructural observations Splechtra/Hilgers Fortschrifte der zoology/progress in Zoology Band. Trends invertebrate morphology Gustav Fischer Verlay Stuttagard New York 1989 334 BhatnagarKP FrahmHD StephanH The megachiropteran pineal organ: a comparative morphological and volumetric investigation with special emphasis on the remarkably large pineal of Dobsonia pradatrix J Anat 1990 168 143 66 BhatnagarKP The ultra structure of the mammalian pinealocytes. A systematic investigation Microscopy Research Tech 1992 21 85 115 BhatnagarS LallSB The pineal gland biochemical analysis and importance Adv Pharmacat Toxicol 2004 5 5960 BhatnagarS LallSB Morphometric and histochemical alterations in the pineal gland of Rhinopoma kinneari during various developmental and reproductive states Ann Neurosci 2005 12 12 26 ReiterRJ The Pineal gland regulator of regulators Prog Psychobiol Physcol Psychol 1980 9 32357 WurtmanRJ The effect of light on the human body Sci Amer 1975 6977 HenriSobel Segalove: Total protein estimation: trichloracetic acid method (modified) Proc Soc Exp Biol Med 1956 92 748 GreenAl HaughtonTM A colorimetrie method for the determination of monamine oxidase Biochem J 1961 78 172 75 Von EeulerHambergSpectrophotometric determination of catecholamines in biological tissues Hawk's Physiological Chemistry Ed OserBL Mc Graw Hill Book Co 1984 302303 FedererWT Experimental Design Theory and Application New Delhi Oxford and IBM Publication Co. 1955 WurtmanRJ AxelrodJ PhilipsLS Melatonin synthesis in the pineal gland: Control by flight Science 1963 142 1071 73 RalphCL Cytology of the pineal gland : Changes produced by various treatments J Neural Trans 1978 13 Suppl. 2545 SynderSH FischerJ AxelrodJ Evidence for the presence of monamine oxidase by the sympathetic nerve endings Biochem Pharmacol 1965 14 363 65 HokansonR OwmanC Pineal dopa decarboxylase and monoamine oxidase, activities as related to the monoamine stores J Neurochem 1966 13 597 605 WurtmanRJ AxelrodJ KellyDE The Pineal New York Academic Press 1968 BauchotLP Identification of A and B forms of monoamine oxidase in the iris, ciliary body, superior cervical ganglion, and pineal gland of albino rabbits Invest Ophthal 1976 15 529 37 PevetPJ Correlation between pineal gland and sexual cycle Doctoral thesis, Netherlands Cenerai Institute for Brain Res Amsterdam 1976 1 198 PevetP KapperJA VouteAM Morphologic evidence for differentiation of pinealocytes from photoreceptor cells in the adult noctule bat (Nyctatus noctula Schreber) Cell Tissue Res 1977 182 99 109 PevetP KapperJ VouteAM The pineal gland of nocturnal mammals. The pinealocyte of the bat, Nyctatus Noctula Schreber J Neural Trans 1977 40 47 68 LemerAB WilliamsRH Text book of Endocrinology Philadelphia Pennasylvania Saunders 1962 880 ReiterRJ Pineal indoles : Production Secretion and actions In : Neuroendocrine perspectives MaleodRM EE Muller Amsterdam Elsevier 1984 34577 Appendix I Anova of Biochemical estimate of total protein, monoamine oxidase, adrenaline and noradrenaline in the male and female Rhinopoma kinneari during various developmental and reproductive states
Source df Total protein (mg%) Monoamine oxidase U/min/mg protein Adrenaline (μg) Noradrenaline
SS MS f cal SS MS f cal SS MS f cal SS MS f cal
Stages 5 295.2 59.04 57.5438* 62.4616 12.4923 60.9975** 0.0000178 0.0000085 24.5380** 0.0000179 0.0000035 28.8582**
Arror 24 24.64 1.026 4.9153 0.2048 0.0000035 9.00000014 0.00000298 0.00000012
Total 29 319.84 67.3769 0.000021386 0.000020896

* Significance difference at 1%, * Significance difference at 5%,

 Biochemical estimates of total protein, monoamine oxidase, adrenaline and noradrenaline in pineal gland of male and female Rhinopoma kinneari during various developmental and reproductive stage States
S.No. Stages Mean total protein N=5 (mg%)** Mean monoamine oxidase U/min/mg protein** N=5 Mean adrenaline (ug)** N= 5 Mean noradrenaline N=5(ug)**
1. Juvenile male pineal gland 7.200 a 0.087 a 0.003877 a 0.002377 a
2. Adult male pineal gland 3.500 b 1.738 b 0.00243 b 0.000165 b
3. Juvenile female pineal gland 8.900 a 3.954 c 0,004042 a 0.002076 a
4. Adult female pineal gland 8.000 a 2.775 d 0.003915 a 0.001246 b
5. Pregnant female pineal gland 8.480 a 0.212 e 0.002583 b 0.001712 b
6. Lactating female pineal gland 0.272 b 0.432 e 0.002234 bc 0.000672 c
Standard error 0.4529 0.2023 0.0001707 0.0001575
Critical difference 5% 1.322 0.5904 0.0004985 0.0004599
Critical difference 1% 1.79 0.8010 0.0006762 0.0006240
Coefficient of variance(%) 16.718 29.5236 12.006166 25.633641

** - Significant at 1% * - Significant at 5%

Values within a columns followed by the same letters are not significantly different.