THE AMERICAN JOURNAL OF ANATOMY 187:32-38 (1990)
Effect of Photoperiod on Pineal Gland Volume and Pinealocyte Size in the Chinese Hamster, Cricefulus griseus SHOJJ MATSUSHIMA, YUKO SAKAI, AND YOSHIKI HIRA Department of Anatomy, Asahikawa Medical College, Asahikawa, 078, Japan
ABSTRACT Male adult (200-day-old) Chinese hamsters (Cricetulus griseus) raised from weaning under either LD 16:8 or LD 8:16 were used. The pineal gland of the Chinese hamster consists of superficial (major) and deep (minor) components and a continuous, or interrupted, narrow parenchymal stalk interposed between them. The volume of the superficial pineal including the parenchymal stalk is greater under LD 16: 8 than under LD 8:16. Under both photoperiods, pinealocytes in the superficial pineal have larger nuclei and more abundant cytoplasm than those in the deep pineal. Nuclei in the superficial pineal appear pale and usually have irregular profiles, whereas those in the deep pineal appear dark and have round profiles. In the superficial pineal, pinealocyte nuclei are larger, paler, and more irregular; and, in addition, nuclear density is lower under LD 16:s than under LD 8:16. Similar, but less prominent, photoperiod-induced changes occur in the volume of the deep pineal, the size of pinealocytes, and pinealocyte nuclear morphology in the deep pineal. The results indicate that the development and differentiation of pinealocytes in both pineal portions may be advanced under long photoperiods and delayed under short photoperiods, although pinealocytes in the deep pineal may remain not fully differentiated even in adults. Since testicular weights and body weights are similar under both photoperiods, the photoperiod may exert marked influences on the development of the pineal gland without affecting reproductive activity and growth rates of animals. INTRODUCTION
Sizes of pinealocytes or pineal glands have been reported to undergo seasonal variations in many wild animals under natural conditions (golden hamster, Mogler, 1958; dormouse, Legait et al., 1975; hare, Lincoln, 1976; bat, Quay, 1976; 13-lined ground squirrel, McNulty et al., 1980; elephant seal, Griffiths and Bryden, 1981; white-footed mouse, Kachi and Quay, 1984). In these studies, the relationships between sizes of pinealocytes or pineal glands and seasons are not always consistent. Changes in photoperiod may be involved in such seasonal variations; however, the involvement of variable environmental factors other than photoperiod cannot be neglected. Influences of photoperiod on sizes of pinealocytes or pineal glands have not hitherto been investigated in animals under laboratory conditions. (C 1990 WILEY-LISS, INC.
The significance of the pineal gland in the photoperiodic regulation of reproductive function has been established in several mammalian species (for reviews, see Reiter, 1980, 1987; Hoffmann, 1981, 1985; Goldman, 1983; Bittman, 1984).The testicular development in Chinese hamsters kept under photoperiods shorter than 10 hours is markedly regressed, and this inhibitory effect of short photoperiods is not observed in ganglionectomized Chinese hamsters (unpublished observations). Thus, the pineal gland in the Chinese hamster, as in other photosensitive species, may participate in the photoperiodic control of reproductive activity. The presence of marked 24-hr rhythms in sizes of pinealocytes or pinealocyte nuclei in the Chinese hamster suggests that these features are reliable indicators of the functional state of the pineal gland in this animal (Matsushima et al., 1983; Sakai et al., 1986). It is of interest, therefore, to determine sizes of pinealocytes or pineal glands in the Chinese hamster under different photoperiodic conditions, and to study their relations to the reproductive organs. The present study was undertaken to examine whether or not sizes of pinealocytes, sizes of pineal glands, and weight of the testes differ between Chinese hamsters raised under short and long photoperiods. MATERIALS AND METHODS
Ten male Chinese hamsters (Cricetulus griseus), 200 days old, were used. The animals were housed 4-5 per clear plastic cage (dimensions 14 x 21 x 12 cm) and given a standard commercial diet and water ad libitum. The animals were kept in LD 14:lO (lights on from 0500 to 1900 hr) from birth until the time of weaning (20 days of age). Then one group of 5 hamsters was exposed to LD 16:8 (lights on from 0500 to 2100 hr) and the second group of 5 to LD 8:16 (lights on from 0900 to 1700 hr). Light was provided by cool-white fluorescent lamps, with the intensity a t the bottom of the cages being approximately 20 lux. The animal room was maintained a t a temperature of 23 ? 2°C. After weighing, the hamsters in both groups were killed by decapitation at 1300 hr, i.e., the middle of the light period, on January 26, when the animals were 200 days old. The pineal glands together with the surrounding brain tissue were rapidly removed, fixed for 20 h r in Bouin’s fluid, dehydrated in a graded series of ethanol, cleared in benzene, and embedded in paraffin. After dissecting out the brain tissue including the pineal Received April 7, 1989. Accepted July 7, 1989. Address reprint requests to Dr. S. Matsushima, Department of Anatomy, Asahikawa Medical College, Nishikagura, Asahikawa, 078, Japan.
PHOTOPERIODIC EFFECT ON CHINESE HAMSTER PINEAL
gland, both testes were removed and weighed. The embedded brain tissues were oriented so that the pineal gland could be cut transversely from the distal end. Serial sections were prepared at a thickness of 8 pm, and were stained with hematoxylin and eosin. In order to determine the volume of the pineal gland, sections of the gland were examined in an Olympus microscope equipped with a drawing tube (BH2-DA), and their outlines were drawn at a magnification of x 200; areas of the gland were measured using a semiautomatic picture analyzing system (Kontron MPOAM03). The pineal gland of the Chinese hamster is composed of two portions, a large superficial and a small deep pineal gland; a narrow parenchymal stalk composed of pinealocytes is interposed between them (Gregorek et al., 1977). The borders between the stalk and the deep pineal were generally sharp, whereas the superficial pineal often narrowed gradually toward the stalk. Thus, in this study, the stalk was included in the superficial pineal, and the volumes of the superficial and deep pineal glands were determined separately. For each animal under LD 16:8 and LD 8:16, it was found that the sum of the areas of each pineal portion on all the serial sections was the same as twenty times the area of the superficial pineal on every twentieth section or five times that of the deep pineal on every fifth section; the differences between the values obtained from every twentieth or fifth section and those from all the sections were at most 3.9%.Thus, in this study, the volume of each pineal portion was expressed as the area of the superficial pineal on every twentieth section x 20 (or the area of the deep pineal on every fifth section x 5) x section thicknessimagnification'. The nuclear profile areas of pinealocytes in the superficial and deep pineal glands were determined in the same way as used for the profile area measurements of the pineal gland. Outline drawings of pinealocyte nuclei were made at the magnification of x 1,000. In order to determine the number of nuclei sufficient to obtain accurate values for the nuclear profile areas in the superficial and deep pineal glands, the values obtained from each of 5 sets of 100 nuclei were compared with those from 500 nuclei in the respective portions of the pineal gland of each animal under LD 1 6 9 and LD 8:16. Nuclear profile areas were similar between peripheral and central regions of the middle portion of the superficial pineal; a total of 250 nuclei, 50 from each of 5 every third sections, were measured in the respective regions. Thus, 50 nuclei from peripheral and central regions in a section were combined to make a set of 100 nuclei. Every alternate section of the deep pineal was examined beginning from the level of the distal end of the pineal recess towardfthe proximal level until the total number of nuclei drawn reached 500. A set of 100 nuclei was selected from the sections of the deep pineal, proceeding from the distal to the proximal level. The differences between the mean nuclear profile areas obtained from each of the 5 sets of 100 nuclei and from the 500 nuclei were at most 5.2% and 5.6% in the superficial and deep pineal glands, respectively. Thus, in this study, the mean nuclear profile areas were obtained from 100 nuclei in each portion of each pineal gland. In order to estimate the relative size of pinealocytes,
33
the nuclear density of pinealocytes, i.e., the number of their nuclei per unit area of the superficial and deep pineal glands, was determined (Ito and Matsushima 1967). Pinealocyte nuclei per unit area of 0.01 mm2 (larger unit area) and 0.004 mm2 (smaller unit area) in the superficial and deep pineal, respectively, were counted at the magnification of x 1,000. Nuclei of cells other than pinealocytes, i.e., glia-like cells, capillary endothelial cells, and connective tissue cells, were not included for counting. In the middle portion of the superficial pineal of each animal under LD 16:8 and LD 8:16, the number of nuclei in each of 40 larger unit areas in the central and peripheral regions was found to be almost identical. In addition, the number of nuclei in 40 larger unit areas in each of 5 alternate sections in the middle portion of the superficial pineal of the above animals was similar to that in 200 unit areas in all sections; the difference between the number of nuclei in 40 unit areas and 200 unit areas was at most 2.4%. Thus, in this study, 40 larger unit areas in a section of the middle portion of the superficial pineal were randomly selected for the estimation of the nuclear density. Since the deep pineal in transverse sections usually appears as a thin ring surrounding the pineal recess, the nuclear density of the deep pineal was determined by counting the number of nuclei per smaller unit area. The nuclear density of the deep pineal was found to be 3.6 and 3.3 times greater than that of the superficial pineal in each animal under LD 16:8 and LD 8:16, respectively; the nuclear density of the deep pineal was obtained from 50 smaller unit areas in 12 or 18 serial sections of the deep pineal surrounding the distal portion of the pineal recess under LD 16% and LD 8:16, respectively. Since 40 larger unit areas correspond to 100 smaller unit areas, at least 30 smaller unit areas for each animal may be required to obtain accurate values for the nuclear density of the deep pineal. Thus, 30 smaller unit areas for each animal were randomly selected from approximately 10 serial sections of the deep pineal at the level of the distal portion of the pineal recess. Data were expressed as the mean t the standard error of the mean (S.E.M.). Since sample sizes were too small to know their distributions, statistical analysis of data was performed by the Mann Whitney U-test. RESULTS
The superficial pineal exhibits round or oval profiles in transverse sections. In oval profiles, transverse diameters usually exceed anteroposterior diameters. The pineal parenchymal stalk appears as a narrow strand of pinealocytes; it is usually seen to be composed of several pinealocytes in transverse sections. Occasional or frequent interruptions occur along the course of the parenchymal stalk; its cellular continuity is lost in such interrupted regions. The length of the interruptions varies greatly from thickness of a few sections up to 720 pm. In one exceptional animal under LD 16:8, cellular continuity was found for the entire length of the parenchymal stalk. The maximal total length of the interruptions was 976 pm; it was observed in an animal under LD 8:16. The length of the interruptions tends to be larger in animals under LD 8:16 than in those under LD 1623, but the difference is not statistically significant. A connection between the parenchymal stalk and
34
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the deep pineal was lost in each of three hamsters under LD 1 6 9 or LD 8:16, whereas in the remainder the proximal end of the parenchymal stalk was continuous with the deep pineal. Even in the latter, the pineal profile areas in transverse sections became abruptly larger from the proximal end of the parenchymal stalk toward the distal end of the deep pineal. The proximo-distal extent of the pineal gland of the Chinese hamster is fairly constant; the distance from the distal end of the superficial pineal to the proximal end of the deep pineal is 2,349 41 pm in hamsters under LD 16:8 and 2,379 33 pm in animals under LD 8:16. The profile areas of the superficial pineal in transverse sections are larger under LD 16:8 than under LD 8:16. In the largest section of the superficial pineal, anteroposterior and transverse diameters are 622 2 20 pm and 706 t 23 pm, respectively, under LD 1623, and 504 ? 26 pm and 589 12 pm, respectively, under LD 8:16; the diameters are significantly larger (anteroposterior, P ~ 0 . 0 1 6transverse, ; P50.004) under LD 16% than under LD 8:16. The volume ratio of the superficial pineal with respect to the whole organ is similar between animals under LD 16:8 and LD 8:16; the volume of the superficial pineal is, on the average, 92.6% and 90.4% that of the whole organ, under LD 16% and under LD 8:16, respectively. The volume of the superficial pineal, either on a n absolute (in mm3) or on a per-gram of body weight (in mm3 x 103/body weight in grams) basis, is markedly increased in animals under LD 16:8 as compared with that under LD 8:16 (Figs. l A , 2A). The volume of the deep pineal also shows a similar increase, although to a less striking degree (Figs. l B , 2B). In animals under both lighting regimens, pinealocyte nuclei in the superficial pineal appear pale (Fig. 3) and often show irregular profiles (Fig. 41, whereas nuclei in the deep pineal are dark and are round or oval in profile (Fig. 5). Pinealocyte nuclear size in the superficial pineal is apparently larger than that in the deep pineal (Fig. 3 vs. Fig. 5); this is commonly observed in animals exposed to LD 16:8 and LD 8:16. Pinealocyte nuclei in the superficial pineal are larger in size, paler
*
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Fig. 2. Volumes per gram of body weight of the superficial (A) and deep (B) pineal glands in animals under LD 8:16 (solid circles) and under LD 16% (open circles). "Pc0.004, **Ps0.008 compared with animals under LD 8:16.
in appearance and more irregular in shape under LD 1623 (Figs. 3,4) than under LD 8 1 6 (Fig. 7). Although less prominent, similar differences in nuclear morphology in pinealocytes of the deep pineal are observed between animals under LD 1 6 3 and LD 8:16. In the superficial and deep pineal glands, the pinealocyte nuclear area is significantly larger under LD 16:8 than under LD 8:16; the difference in the pinealocyte nuclear area between these animals is more prominent in the superficial pineal than in the deep pineal (Fig. 6). The nuclear density of pinealocytes is inversely related to the pinealocyte nuclear area. The nuclear density is greater in the deep pineal than in the superficial pineal under either photoperiod (Fig. 3 vs. Fig. 5; Fig. 8) and also greater under LD 8:16 than under LD 1 6 3 in either superficial or deep pineal gland (Fig. 3 vs. Fig. 7; Fig. 8). The difference in the nuclear density between animals under LD 16:8 and LD 8:16 is larger in the superficial pineal than in the deep pineal. The weights of both testes are similar between animals under LD 16:8 (2,458 88 mg, 47.7 2 0.6 mg/gm body weight) and under LD 8:16 (2,357 t 44 mg, 45.8 1.2 mglgm body weight). The body weights of hamsters under LD 16% (51.5 2 1.3gm) are essentially identical with those of animals under LD 8:16 (51.4 t 0.8gm).
*
*
DISCUSSION
It is reported that the pineal gland of the Chinese hamster is composed of superficial (distal) and deep (proximal) components, and a continuous, narrow parenchymal stalk composed of pinealocytes (Gregorek et al., 1977).In the Chinese hamsters used in this study, however, the parenchymal stalk is often interrupted. It is uncertain whether the cellular continuity in the parenchymal stalk is a constant feature of the materials examined by Gregorek et al. (1977). In the pineal glands of the rat (Boeckmann, 1980) and white-footed mouse (Quay, 1956),which have a shape similar to that of the Chinese hamster, the parenchymal stalk is often
PHOTOPERIODIC EFFECT ON CHINESE HAMSTER PINEAL
35
Fig. 3. Pinealocytes in the superficial pineal from an animal under LD 16:s. ~ 8 0 0 .
Fig. 5. Pinealocytes in the deep pineal from an animal under LU 16% ~ 8 0 0 .
Flg. 4. Irregularly shaped nuclei of pinealocytes in the superficial pineal from an animal under LD 1623. X 2,000.
Fig. 7.Pinealocytes in the superficial pineal from an animal under LD 8:16. x 800.
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interrupted, and a continuous parenchymal stalk is rather rare. The number of pinealocytes constituting the pineal parenchymal stalk in the Chinese hamster may differ between individual animals.
Although pinealocyte nuclear sizes in some animals show no marked regional differences in a proximodistal direction (collared lemming, Quay, 1978; guineapig, J u n g and Vollrath, 1982), pinealocyte nuclei are larger in the superficial pineal than in the deep pineal in the white-footed mouse (Quay, 19561, rat (Boeckmann, 19801, and hamster (Vollrath, 1979; the hamster used is Mesocricetus auratus, personal communication). The present results show that morphological features of pinealocytes in the superficial pineal are different from those in the deep pineal in Chinese hamsters under both long and short photoperiods. Pinealocytes in the superficial pineal have larger nuclei and more abundant cytoplasm than those in the deep pineal; pinealocyte nuclei in the superficial pineal are often of irregular shape and stain lightly in contrast to those in the deep pineal, which are round and stain deeply. In addition, long photoperiods cause enlargement of pinealocytes in both the superficial and deep pineal; however, pinealocytes of the deep pineal show less pronounced responses to long photoperiods. Pinealocytes in the mouse exhibit regional differences similar to those seen in the Chinese hamster (Hulsemann, 1967).The previous results suggest that pinealocytes of the mouse enlarge and, concomitantly, the shape of their nuclei changes from round to pleomorphic with advancing differentiation (Ito and Matsushima, 1967). From the above observations on the mouse and the present results, i t seems that the pineal gland of the
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Fi 8 Number of pinealocyte nuclei per unit area (0.01 or 0.004 mm ) of superficial (A) and deep (B) pineal glands in animals under LD 8:16 (solid circles) and under LD 16:8 (open circles). * P ~ 0 . 0 0 4 compared with animals under LD 16%.
4.
Chinese hamster consists of two different populations of pinealocytes; the pinealocytes in the deep pineal, unlike those in the superficial pineal, may not be fully differentiated cells. In the golden hamster (Reiter and Hedlund, 1976) and r a t (Bjorklund et al., 1972; Wiklund, 19741, postganglionic sympathetic fibers are abundantly distributed in the deep pineal as they are in the superficial pineal. As mentioned above, pinealocyte nuclei in these animals are smaller in the deep pineal than in the superficial pineal (Vollrath, 1979; Boeckmann, 1980). Thus, no apparent correlation may exist between the density of sympathetic innervation and size of pinealocytes in the golden hamster and rat. Sympathetic nerves are frequently observed in the superficial pineal of the Chinese hamster (Matsushima and Morisawa, 1982). A comparison of the density of sympathetic nerves between the superficial and deep pineal glands of the Chinese hamster seems important to understand the functional significance of the differences in size of pinealocytes between the two pineal portions of this animal. Differences in size of pinealocytes between peripheral (cortical) and central (medullary) regions of the superficial pineal have also been described in several mammals (Vollrath, 1979, 1981; Heidbuchel and Vollrath, 1983). In the present study, i t was found that pinealocyte sizes were similar between peripheral and central regions in the middle portion of the superficial pineal in each animal under LD 1 6 3 and LD 8:16. In addition, pinealocyte sizes obtained from the middle portion of the superficial pineal were correlated well with volumes of the superficial pineal. Thus, pinealocytes of the Chinese hamster may be uniform in size throughout the superficial pineal. The volume of the pineal gland has been determined from camera-lucida drawings of histological sections in various mammalian species (Quay, 1956, 1978; Legait et al., 1976a,b). In the Chinese hamster, the pineal volumes, expressed a s the sum of the volumes of the superficial and deep pineal in mm3 x lo3 per gram of body weight, were 3.78 k 0.12 and 2.17 2 0.08, respec-
tively, in animals under LD 16:8 and LD 8:16. When comparing these values with those obtained from the other rodent species (Legait et al., 1976a; Quay, 1978, see discussion), it may be said that the Chinese hamster has a relatively large pineal gland. A simultaneous determination of the volumes of the superficial and deep pineal glands has been carried out only on the white-footed mouse (Quay, 1956); the volumes of the anterior (deep) and posterior (superficial) pineal are 0.0197 -+ 0.0021 mm3 and 0.0894 +- 0.0068 mm3, respectively. Thus, the volume of the deep pineal is about 17% that of the whole organ. In the Chinese hamster, the deep pineal comprises less than 10%. Considerable interspecies differences may exist in the ratio between the volumes of the superficial and deep pineal. The volume of the superficial pineal is markedly larger in Chinese hamsters raised under LD 16:8 than in those under LD 8:16. In the superficial pineal, nuclei of pinealocytes are larger in size, paler in appearance, and more irregular in shape; and, in addition, nuclear density of pinealocytes is lower under LD 16% than under LD 8:16. Although less prominent, similar photoperiod-induced changes are observed in the volume of the deep pineal, and in the nuclear and cytoplasmic sizes and nuclear morphology in pinealocytes of the deep pineal. Thus, it is evident that the difference between volumes of the superficial and deep pineals of the Chinese hamster under the different photoperiods is due to the difference in size of pinealocytes. As suggested before, nuclear pleomorphism may be characteristic of differentiated pinealocytes. Thus, the postnatal development and differentiation of pinealocytes may be advanced and delayed under long and short photoperiods, respectively. Seasonal variations in pineal volumes or pinealocyte nuclear and cytoplasmic sizes have been reported to occur in some feral animals under natural environmental conditions. Pineal volumes or pinealocyte sizes are generally larger in winter or during hibernation (golden hamster, Mogler, 1958; dormouse, Legait et al., 1975; 13-lined ground squirrel, McNulty et al., 1980; elephant seal, Griffiths and Bryden, 1981). By contrast, pinealocytes of the hare are larger in size from summer to fall (Lincoln, 1976), and maximal nuclear diameters in pinealocytes of the bat are obtained in the fall (beginning of hibernation) in males and in early spring (arousal and dispersal from hibernation) in females (Quay, 1976). Additionally, pinealocyte sizes in the white-footed mouse are increased in summer and decreased in winter (Kachi and Quay, 1984). In feral animals under field conditions, it is possible that seasonal changes in a variety of environmental factors other than photoperiod may cause changes in sizes of pinealocytes and pineal glands. Thus, it is difficult to determine to what degree the photoperiod is involved in such seasonal variations. So far as we know, the present study is the first to demonstrate clearly the effects of photoperiods on sizes of pinealocytes and pineal glands in mammals under laboratory conditions; Chinese hamsters raised under long photoperiods have larger pineal glands composed of larger pinealocytes than those raised under short photoperiods. Thus, long photoperiods cause pinealocytes to enlarge. Constant light is known to induce atrophy of pinealocytes (Vollrath, 1981). Preliminary
PHOTOPERIODIC EFFECT ON CHINESE HAMSTER PINEAL
observations show that pineal volumes in 60- to 70day-old Chinese hamsters raised under LD 1 6 3 are larger than those in animals raised under photoperiods shorter or longer than 16 h r (unpublished observations). Thus, LD 16% may be the optimal photoperiod for the development of pinealocytes in the Chinese hamster. Larger pinealocytes under long photoperiods are believed to be functionally more active than smaller pinealocytes under short photoperiods. Electron microscopic and histochemical studies of such active and inactive pinealocytes are in progress in order to make clear the functional significance of these cells. Sizes of pinealocytes or pineal glands in several seasonally breeding animals are reported to increase when the gonads are regressed (golden hamster, Mogler, 1958; dormouse, Legait et al., 1975; hare, Lincoln, 1976; 13-lined ground squirrel, McNulty e t al., 1980; elephant seal, Griffths and Bryden, 19811, whereas pinealocytes of the white-footed mouse are larger when the gonads are active (Kachi and Quay, 1984). Thus, interspecies differences may exist in the relationships between pinealocyte or pineal gland sizes and reproductive function. The gonadal development in the Chinese hamster is greatly influenced by photoperiod; 60to 70-day-old Chinese hamsters raised from weaning under photoperiods longer than 14 h r have large testes (about 1.5 gm in paired weight), whereas those raised under photoperiods shorter than 10 h r have small testes (less than 0.5 gm in paired weight) (unpublished observations). Superior cervical ganglionectomy is known to block photoperiod-induced gonadal involution in some mammals (Reiter, 1980; Hoffmann, 1981). Since there are no observed effects of photoperiod on testicular development in the Chinese hamster subjected to superior cervical ganglionectomy (unpublished observations), such photoperiod-induced, testicular changes may be mediated by the pineal gland. As shown in the present study, the testes in 200-day-old Chinese hamsters raised under LD 16:8 or LD 8:16 are similar in weight (about 1.5 gm). Thus, the testicular weight in young Chinese hamsters raised under LD 8:16 may increase to reach that in animals under LD 16:8 until 200 days of age. Delayed development and subsequent regrowth of gonads have been observed in some mammalian species raised from birth or from weaning under short photoperiods (Djungarian hamster, Hoffmann, 1978; vole, Grocock, 1979; white-footed mouse, Johnston and Zucker, 1980).The present observation that sizes of pinealocytes and pineal glands are different, but testicular weights are similar, in 200day-old Chinese hamsters raised from weaning under short and long photoperiods suggests that photoperiodinduced changes in sizes of pinealocytes and pineal glands may occur without changes in reproductive activity of this animal. It is well known that in some mammals the pineal hormone melatonin is involved in the photoperiodic regulation of gonadal function (Reiter, 1980, 1987; Hoffmann, 1981,1985; Goldman, 1983; Bittman, 1984). As mentioned above, short photoperiod-induced gonadal atrophy in young Chinese hamsters is blocked by superior cervical ganglionectomy, suggesting that this gonadal change is mediated by melatonin. According to our recent observations (unpublished), sizes of pineal glands and pinealocytes are smaller in young Chinese
37
hamsters raised under LD 8:16 than in those under LD 1623. Thus, both pinealocytes or pineal glands and gonads of young Chinese hamsters are reduced in size under short photoperiods. In young Chinese hamsters, the size of pinealocytes shows a 24-hr rhythm, with its maximum a t the middle of the light period and its minimum during the second half of the dark period (Matsushima et al., 1983); this rhythm is apparently different from the rhythm in pineal melatonin synthesis in mammals. Furthermore, the 24-hr rhythm in size of pinealocytes disappears after exposure of young Chinese hamsters to constant darkness €or 7 days, suggesting that the rhythm is exogenous in nature (Sakai et al., 1986). All of these observations on young Chinese hamsters and the present results from adults may indicate that sizes of pinealocytes and pineal glands are not related to the synthesis of melatonin. From the observations on various mammalian species, it is generally supposed that pineal volume is correlated with body weight (Legait et al., 1976a,b; Vollrath, 1981). However, the present results clearly indicate that there is no correlation between pineal volume and body weight in the Chinese hamster. When comparing the body weights of the Chinese hamsters used in the present study until 200 days of age (data not shown), the body weights of the animals under LD 16:8 and LD 8:16 are similar from weaning to 40 days of age; but, between 50 and 110 days, the animals become heavier under LD 16:8 than under LD 8:16; and the body weights of the animals under the two photoperiods attain the same, maximal level around 160 days of age. Thus, the growth of the Chinese hamster appears to be temporarily inhibited under short photoperiods. Similar, but much more prominent, inhibitory effects of short photoperiods on growth rates have been reported for the Djungarian hamster (Hoffmann, 1978). From the present results, it is evident that photoperiod-induced changes in sizes of pinealocytes and pineal glands in the Chinese hamster are independent of growth of the animals. The significance of such photoperiodic influences on the pineal gland as demonstrated in the Chinese hamster remains undetermined. As a first step toward this problem, we are now examining whether or not similar photoperiodic changes occur in the pineal gland of other photosensitive and non-photosensitive animals. LITERATURE CITED Bittman, E.L. 1984 Melatonin and photoperiodic time measurement: Evidence from rodents and ruminants. In: The Pineal Gland. R.J. Reiter, ed. Raven Press, New York, pp. 155-192. Bjorklund, A., Ch. Owman, and K.A. West 1972 Peripheral sympathetic innervation and serotonin cells in the habenular region of the rat brain. 2. Zellforsch., 127570-579. Boeckmann, D. 1980 Morphological investigation of the deep pineal of the rat. Cell Tissue Res., 210;283-294. Goldman, B.D. 1983 The physiology of melatonin in mammals. In: Pineal Research Reviews, Vol. 1. R.J. Reiter, ed. Alan R. Liss, Inc., New York, pp. 145-182. Gregorek, J.C., H.R. Seibel, and R.J. Reiter 1977 The pineal complex and its relationship to other epithalamic structures. Acta Anat. (Basel), 99t425-434. Griffiths, D.J., and M.M. Bryden 1981 The annual cycle of the pineal gland of the elephant seal (Mirounga leoninu). In: Pineal Function. C.D. Matthews and R.F. Seamark, eds. ElsevieriNorth-Holland Biomedical Press, Amsterdam, pp. 57-66. Grocock, C.A. 1979 Testis development in the vole, Microtus ugrestis, subjected to long or short photoperiods from birth. J. Reprod. Fertil., 55:423-427.
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S. MATSUSHIMA ET AL.
Heidbiichel, U., and L. Vollrath 1983 Morphological findings relating to the problem of cortex and medulla in the pineal glands of rat and hamster. J. Anat., 136,723-734. Hoffmann, K. 1978 Effects of short photoperiods on puberty, growth and moult in the Djungarian hamster (Phodopus surzgorusi. J. Reprod. Fertil., 54:29-35. Hoffmann, K. 1981 Photoperiodic function of the mammalian pineal organ. In: The Pineal Organ: Photobiology-Biochronometry-Endocrinology. A. Oksche and P. Pevet, eds. ElsevieriNorth-Holland Biomedical Press, Amsterdam, pp. 123-138. Hoffmann, K. 1985 Interaction between photoperiod, pineal and seasonal adaption in mammals. In: The Pineal Gland. Current State of Pineal Research. B. Mess, CS. Rdzsas, L. Tima, and P. Pevet, eds. Elsevier Science Publishers, Amsterdam, pp. 21 1-227. Hulsemann, M. 1967 Vergleichende histologische Untersuchungen iiber das Vorkommen von Gliafasern in der Epiphysis cerebri von Saugetieren. Acta Anat. (Basel), 66249-278. Ito, T., and S. Matsushima 1967 A quantitative morphological study of the postnatal development of the pineal body of the mouse. Anat. Rec., 159:447-452. Johnston, P.G., and I. Zucker 1980 Photoperiodic regulation of reproductive development in white-footed mice (Peronzyscusleucopusi. Biol. Reprod., 22:983-989. Jung, D., and L. Vollrath 1982 Structural dissimilarities in different regions of the pineal gland of Pirbright white guinea-pigs. J. Neural Transm., 54:117-128. Kachi, T., and W.B. Quay 1984 Seasonal changes in glycogen level and size of pinealocytes of the white-footed mouse, Peromyscus leucopus: A semiquantitative histochemical study. J . Pineal Res., 1:163-174. Legait, H., M. Roux, G. Dussart, J.P. Richoux, and J.-L. Contet-Audonneau 1975 Donnees morphometriques sur la glande pineale du Loir (GZisglis) et du Lerot (Eliomys quercinus) au cours du cycle annuel. C. R. SOC.Biol., 169:132-136. Legait, H., R. Bauchot, H. Stephan, and J.-L. Contet-Audonneau 1976a Etude des correlations liant le volume de I’epiphyse aux poids somatique et encephalique chez les rongeurs, les insectivores, les chiropteres, les prosimiens et les simiens. Mammalia, 40,327-337. Legait, H., R. Bauchot, and J.-L. Contet-Audonneau 1976b Etude des correlations liant les volumes des lobes hypophysaires et de Yepiphyse au poids somatique et au poids de l’encephale chez les chiropteres. Bull. Assoc. Anat., 60:175-188. Lincoln, G.A. 1976 Seasonal changes in the pineal gland related to the reproductive cycle in the male hare, Lepus europaeus. J . Reprod. Fertil., 46:489-491. Matsushima, S., and Y. Morisawa 1982 Ultrastructural observations
on the pineal gland of the Chinese hamster, Cricetulus griseus. I. The superficial pineal. Cell Tissue Res., 222531-546. Matsushima, S., Y. Morisawa, I. Aida, and K. Abe 1983 Circadian variations in pinealocytes of the Chinese hamster, Cricetulus griseus. A quantitative electron-microscopic study. Cell Tissue Res., 228r231-244. McNulty, J.A., T.A. Dombrowski, and W.A. Spurrier 1980 A seasonal study of pinealocytes in the %lined ground squirrel, Spermophilus tridecemlineatus. Reprod. Nutr. Dev., 20,665-672, Mogler, R.K.-H. 1958 Das endokrine System des syrischen Goldhamsters (Mesocricetus auratus uuratus Waterhouse) unter Berucksichtigung des naturlichen und experimentellen Winterschlafs. Z. Morphol. Okol. Tiere., 47:267-306. Quay, W.B. 1956 Volumetric and cytologic variation in the pineal body of Peromyscus leucopus (Rodentia) with respect to sex, captivity and day-length. J. Morphol., 98r471-495. Quay, W.B. 1976 Seasonal cycle and physiological correlates of pinealocyte nuclear and nucleolar diameters in the bats, Myotis lucifugus and Myotis sodalis. Gen. Comp. Endocrinol., 29,369-375. Quay, W.B. 1978 Quantitative morphology and environmental responses of the pineal gland in the collared lemming (Dzcrostonyx groenlundicus). Am. J. Anat., 1531545-562. Reiter, R.J. 1980 The pineal and its hormones in the control of reproduction in mammals. Endocr. Rev., 1,109-131. Reiter, R.J. 1987 Mechanisms of control of reproductive physiology by the pineal gland and its hormones. In: Advances in Pineal Research, Vol. 2. R.J. Reiter and F. Fraschini, eds. John Libbey & Co., London, pp. 109-125. Reiter, R.J., and L. Hedlund 1976 Peripheral sympathetic innervation of the deep pineal gland of the golden hamsier. Experientia, 32: 1071-1072. Sakai, Y., I. Aida, and S. Matsushima 1986 Effect of continuous darkness on circadian morphological rhythms in pinealocytes of the Chinese hamster, Cricetulus griseus. Cell Tissue Res., 245:127134. Vollrath, L. 1979 Comparative morphology of the vertebrate pineal complex. In: The Pineal Gland of Vertebrates Including Man. Progress in Brain Research, Vol. 52. J.A. Kappers and P. Pevet, eds. ElsevieriNorth-Holland Biomedical Press, Amsterdam, pp. 25-38. Vollrath, L. 1981 The pineal organ. In: Handbuch der Mikroskopischen Anatomie des Menschen. Vol. VII7. A. Oksche and L. Vollrath, eds. Springer, Berlin, pp. 41-43, 67-71, 242-244. Wiklund, L. 1974 Development of serotonin-containing cells and the sympathetic innervation of the habenular region in the rat brain. A fluorescence histochemical study. Cell Tissue Res., 155:231243.
Effect of Photoperiod on Pineal Gland Volume and Pinealocyte Size in the Chinese Hamster, Cricefulus griseus SHOJJ MATSUSHIMA, YUKO SAKAI, AND YOSHIKI HIRA Department of Anatomy, Asahikawa Medical College, Asahikawa, 078, Japan
ABSTRACT Male adult (200-day-old) Chinese hamsters (Cricetulus griseus) raised from weaning under either LD 16:8 or LD 8:16 were used. The pineal gland of the Chinese hamster consists of superficial (major) and deep (minor) components and a continuous, or interrupted, narrow parenchymal stalk interposed between them. The volume of the superficial pineal including the parenchymal stalk is greater under LD 16: 8 than under LD 8:16. Under both photoperiods, pinealocytes in the superficial pineal have larger nuclei and more abundant cytoplasm than those in the deep pineal. Nuclei in the superficial pineal appear pale and usually have irregular profiles, whereas those in the deep pineal appear dark and have round profiles. In the superficial pineal, pinealocyte nuclei are larger, paler, and more irregular; and, in addition, nuclear density is lower under LD 16:s than under LD 8:16. Similar, but less prominent, photoperiod-induced changes occur in the volume of the deep pineal, the size of pinealocytes, and pinealocyte nuclear morphology in the deep pineal. The results indicate that the development and differentiation of pinealocytes in both pineal portions may be advanced under long photoperiods and delayed under short photoperiods, although pinealocytes in the deep pineal may remain not fully differentiated even in adults. Since testicular weights and body weights are similar under both photoperiods, the photoperiod may exert marked influences on the development of the pineal gland without affecting reproductive activity and growth rates of animals. INTRODUCTION
Sizes of pinealocytes or pineal glands have been reported to undergo seasonal variations in many wild animals under natural conditions (golden hamster, Mogler, 1958; dormouse, Legait et al., 1975; hare, Lincoln, 1976; bat, Quay, 1976; 13-lined ground squirrel, McNulty et al., 1980; elephant seal, Griffiths and Bryden, 1981; white-footed mouse, Kachi and Quay, 1984). In these studies, the relationships between sizes of pinealocytes or pineal glands and seasons are not always consistent. Changes in photoperiod may be involved in such seasonal variations; however, the involvement of variable environmental factors other than photoperiod cannot be neglected. Influences of photoperiod on sizes of pinealocytes or pineal glands have not hitherto been investigated in animals under laboratory conditions. (C 1990 WILEY-LISS, INC.
The significance of the pineal gland in the photoperiodic regulation of reproductive function has been established in several mammalian species (for reviews, see Reiter, 1980, 1987; Hoffmann, 1981, 1985; Goldman, 1983; Bittman, 1984).The testicular development in Chinese hamsters kept under photoperiods shorter than 10 hours is markedly regressed, and this inhibitory effect of short photoperiods is not observed in ganglionectomized Chinese hamsters (unpublished observations). Thus, the pineal gland in the Chinese hamster, as in other photosensitive species, may participate in the photoperiodic control of reproductive activity. The presence of marked 24-hr rhythms in sizes of pinealocytes or pinealocyte nuclei in the Chinese hamster suggests that these features are reliable indicators of the functional state of the pineal gland in this animal (Matsushima et al., 1983; Sakai et al., 1986). It is of interest, therefore, to determine sizes of pinealocytes or pineal glands in the Chinese hamster under different photoperiodic conditions, and to study their relations to the reproductive organs. The present study was undertaken to examine whether or not sizes of pinealocytes, sizes of pineal glands, and weight of the testes differ between Chinese hamsters raised under short and long photoperiods. MATERIALS AND METHODS
Ten male Chinese hamsters (Cricetulus griseus), 200 days old, were used. The animals were housed 4-5 per clear plastic cage (dimensions 14 x 21 x 12 cm) and given a standard commercial diet and water ad libitum. The animals were kept in LD 14:lO (lights on from 0500 to 1900 hr) from birth until the time of weaning (20 days of age). Then one group of 5 hamsters was exposed to LD 16:8 (lights on from 0500 to 2100 hr) and the second group of 5 to LD 8:16 (lights on from 0900 to 1700 hr). Light was provided by cool-white fluorescent lamps, with the intensity a t the bottom of the cages being approximately 20 lux. The animal room was maintained a t a temperature of 23 ? 2°C. After weighing, the hamsters in both groups were killed by decapitation at 1300 hr, i.e., the middle of the light period, on January 26, when the animals were 200 days old. The pineal glands together with the surrounding brain tissue were rapidly removed, fixed for 20 h r in Bouin’s fluid, dehydrated in a graded series of ethanol, cleared in benzene, and embedded in paraffin. After dissecting out the brain tissue including the pineal Received April 7, 1989. Accepted July 7, 1989. Address reprint requests to Dr. S. Matsushima, Department of Anatomy, Asahikawa Medical College, Nishikagura, Asahikawa, 078, Japan.
PHOTOPERIODIC EFFECT ON CHINESE HAMSTER PINEAL
gland, both testes were removed and weighed. The embedded brain tissues were oriented so that the pineal gland could be cut transversely from the distal end. Serial sections were prepared at a thickness of 8 pm, and were stained with hematoxylin and eosin. In order to determine the volume of the pineal gland, sections of the gland were examined in an Olympus microscope equipped with a drawing tube (BH2-DA), and their outlines were drawn at a magnification of x 200; areas of the gland were measured using a semiautomatic picture analyzing system (Kontron MPOAM03). The pineal gland of the Chinese hamster is composed of two portions, a large superficial and a small deep pineal gland; a narrow parenchymal stalk composed of pinealocytes is interposed between them (Gregorek et al., 1977). The borders between the stalk and the deep pineal were generally sharp, whereas the superficial pineal often narrowed gradually toward the stalk. Thus, in this study, the stalk was included in the superficial pineal, and the volumes of the superficial and deep pineal glands were determined separately. For each animal under LD 16:8 and LD 8:16, it was found that the sum of the areas of each pineal portion on all the serial sections was the same as twenty times the area of the superficial pineal on every twentieth section or five times that of the deep pineal on every fifth section; the differences between the values obtained from every twentieth or fifth section and those from all the sections were at most 3.9%.Thus, in this study, the volume of each pineal portion was expressed as the area of the superficial pineal on every twentieth section x 20 (or the area of the deep pineal on every fifth section x 5) x section thicknessimagnification'. The nuclear profile areas of pinealocytes in the superficial and deep pineal glands were determined in the same way as used for the profile area measurements of the pineal gland. Outline drawings of pinealocyte nuclei were made at the magnification of x 1,000. In order to determine the number of nuclei sufficient to obtain accurate values for the nuclear profile areas in the superficial and deep pineal glands, the values obtained from each of 5 sets of 100 nuclei were compared with those from 500 nuclei in the respective portions of the pineal gland of each animal under LD 1 6 9 and LD 8:16. Nuclear profile areas were similar between peripheral and central regions of the middle portion of the superficial pineal; a total of 250 nuclei, 50 from each of 5 every third sections, were measured in the respective regions. Thus, 50 nuclei from peripheral and central regions in a section were combined to make a set of 100 nuclei. Every alternate section of the deep pineal was examined beginning from the level of the distal end of the pineal recess towardfthe proximal level until the total number of nuclei drawn reached 500. A set of 100 nuclei was selected from the sections of the deep pineal, proceeding from the distal to the proximal level. The differences between the mean nuclear profile areas obtained from each of the 5 sets of 100 nuclei and from the 500 nuclei were at most 5.2% and 5.6% in the superficial and deep pineal glands, respectively. Thus, in this study, the mean nuclear profile areas were obtained from 100 nuclei in each portion of each pineal gland. In order to estimate the relative size of pinealocytes,
33
the nuclear density of pinealocytes, i.e., the number of their nuclei per unit area of the superficial and deep pineal glands, was determined (Ito and Matsushima 1967). Pinealocyte nuclei per unit area of 0.01 mm2 (larger unit area) and 0.004 mm2 (smaller unit area) in the superficial and deep pineal, respectively, were counted at the magnification of x 1,000. Nuclei of cells other than pinealocytes, i.e., glia-like cells, capillary endothelial cells, and connective tissue cells, were not included for counting. In the middle portion of the superficial pineal of each animal under LD 16:8 and LD 8:16, the number of nuclei in each of 40 larger unit areas in the central and peripheral regions was found to be almost identical. In addition, the number of nuclei in 40 larger unit areas in each of 5 alternate sections in the middle portion of the superficial pineal of the above animals was similar to that in 200 unit areas in all sections; the difference between the number of nuclei in 40 unit areas and 200 unit areas was at most 2.4%. Thus, in this study, 40 larger unit areas in a section of the middle portion of the superficial pineal were randomly selected for the estimation of the nuclear density. Since the deep pineal in transverse sections usually appears as a thin ring surrounding the pineal recess, the nuclear density of the deep pineal was determined by counting the number of nuclei per smaller unit area. The nuclear density of the deep pineal was found to be 3.6 and 3.3 times greater than that of the superficial pineal in each animal under LD 16:8 and LD 8:16, respectively; the nuclear density of the deep pineal was obtained from 50 smaller unit areas in 12 or 18 serial sections of the deep pineal surrounding the distal portion of the pineal recess under LD 16% and LD 8:16, respectively. Since 40 larger unit areas correspond to 100 smaller unit areas, at least 30 smaller unit areas for each animal may be required to obtain accurate values for the nuclear density of the deep pineal. Thus, 30 smaller unit areas for each animal were randomly selected from approximately 10 serial sections of the deep pineal at the level of the distal portion of the pineal recess. Data were expressed as the mean t the standard error of the mean (S.E.M.). Since sample sizes were too small to know their distributions, statistical analysis of data was performed by the Mann Whitney U-test. RESULTS
The superficial pineal exhibits round or oval profiles in transverse sections. In oval profiles, transverse diameters usually exceed anteroposterior diameters. The pineal parenchymal stalk appears as a narrow strand of pinealocytes; it is usually seen to be composed of several pinealocytes in transverse sections. Occasional or frequent interruptions occur along the course of the parenchymal stalk; its cellular continuity is lost in such interrupted regions. The length of the interruptions varies greatly from thickness of a few sections up to 720 pm. In one exceptional animal under LD 16:8, cellular continuity was found for the entire length of the parenchymal stalk. The maximal total length of the interruptions was 976 pm; it was observed in an animal under LD 8:16. The length of the interruptions tends to be larger in animals under LD 8:16 than in those under LD 1623, but the difference is not statistically significant. A connection between the parenchymal stalk and
34
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the deep pineal was lost in each of three hamsters under LD 1 6 9 or LD 8:16, whereas in the remainder the proximal end of the parenchymal stalk was continuous with the deep pineal. Even in the latter, the pineal profile areas in transverse sections became abruptly larger from the proximal end of the parenchymal stalk toward the distal end of the deep pineal. The proximo-distal extent of the pineal gland of the Chinese hamster is fairly constant; the distance from the distal end of the superficial pineal to the proximal end of the deep pineal is 2,349 41 pm in hamsters under LD 16:8 and 2,379 33 pm in animals under LD 8:16. The profile areas of the superficial pineal in transverse sections are larger under LD 16:8 than under LD 8:16. In the largest section of the superficial pineal, anteroposterior and transverse diameters are 622 2 20 pm and 706 t 23 pm, respectively, under LD 1623, and 504 ? 26 pm and 589 12 pm, respectively, under LD 8:16; the diameters are significantly larger (anteroposterior, P ~ 0 . 0 1 6transverse, ; P50.004) under LD 16% than under LD 8:16. The volume ratio of the superficial pineal with respect to the whole organ is similar between animals under LD 16:8 and LD 8:16; the volume of the superficial pineal is, on the average, 92.6% and 90.4% that of the whole organ, under LD 16% and under LD 8:16, respectively. The volume of the superficial pineal, either on a n absolute (in mm3) or on a per-gram of body weight (in mm3 x 103/body weight in grams) basis, is markedly increased in animals under LD 16:8 as compared with that under LD 8:16 (Figs. l A , 2A). The volume of the deep pineal also shows a similar increase, although to a less striking degree (Figs. l B , 2B). In animals under both lighting regimens, pinealocyte nuclei in the superficial pineal appear pale (Fig. 3) and often show irregular profiles (Fig. 41, whereas nuclei in the deep pineal are dark and are round or oval in profile (Fig. 5). Pinealocyte nuclear size in the superficial pineal is apparently larger than that in the deep pineal (Fig. 3 vs. Fig. 5); this is commonly observed in animals exposed to LD 16:8 and LD 8:16. Pinealocyte nuclei in the superficial pineal are larger in size, paler
*
*
Fig. 2. Volumes per gram of body weight of the superficial (A) and deep (B) pineal glands in animals under LD 8:16 (solid circles) and under LD 16% (open circles). "Pc0.004, **Ps0.008 compared with animals under LD 8:16.
in appearance and more irregular in shape under LD 1623 (Figs. 3,4) than under LD 8 1 6 (Fig. 7). Although less prominent, similar differences in nuclear morphology in pinealocytes of the deep pineal are observed between animals under LD 1 6 3 and LD 8:16. In the superficial and deep pineal glands, the pinealocyte nuclear area is significantly larger under LD 16:8 than under LD 8:16; the difference in the pinealocyte nuclear area between these animals is more prominent in the superficial pineal than in the deep pineal (Fig. 6). The nuclear density of pinealocytes is inversely related to the pinealocyte nuclear area. The nuclear density is greater in the deep pineal than in the superficial pineal under either photoperiod (Fig. 3 vs. Fig. 5; Fig. 8) and also greater under LD 8:16 than under LD 1 6 3 in either superficial or deep pineal gland (Fig. 3 vs. Fig. 7; Fig. 8). The difference in the nuclear density between animals under LD 16:8 and LD 8:16 is larger in the superficial pineal than in the deep pineal. The weights of both testes are similar between animals under LD 16:8 (2,458 88 mg, 47.7 2 0.6 mg/gm body weight) and under LD 8:16 (2,357 t 44 mg, 45.8 1.2 mglgm body weight). The body weights of hamsters under LD 16% (51.5 2 1.3gm) are essentially identical with those of animals under LD 8:16 (51.4 t 0.8gm).
*
*
DISCUSSION
It is reported that the pineal gland of the Chinese hamster is composed of superficial (distal) and deep (proximal) components, and a continuous, narrow parenchymal stalk composed of pinealocytes (Gregorek et al., 1977).In the Chinese hamsters used in this study, however, the parenchymal stalk is often interrupted. It is uncertain whether the cellular continuity in the parenchymal stalk is a constant feature of the materials examined by Gregorek et al. (1977). In the pineal glands of the rat (Boeckmann, 1980) and white-footed mouse (Quay, 1956),which have a shape similar to that of the Chinese hamster, the parenchymal stalk is often
PHOTOPERIODIC EFFECT ON CHINESE HAMSTER PINEAL
35
Fig. 3. Pinealocytes in the superficial pineal from an animal under LD 16:s. ~ 8 0 0 .
Fig. 5. Pinealocytes in the deep pineal from an animal under LU 16% ~ 8 0 0 .
Flg. 4. Irregularly shaped nuclei of pinealocytes in the superficial pineal from an animal under LD 1623. X 2,000.
Fig. 7.Pinealocytes in the superficial pineal from an animal under LD 8:16. x 800.
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interrupted, and a continuous parenchymal stalk is rather rare. The number of pinealocytes constituting the pineal parenchymal stalk in the Chinese hamster may differ between individual animals.
Although pinealocyte nuclear sizes in some animals show no marked regional differences in a proximodistal direction (collared lemming, Quay, 1978; guineapig, J u n g and Vollrath, 1982), pinealocyte nuclei are larger in the superficial pineal than in the deep pineal in the white-footed mouse (Quay, 19561, rat (Boeckmann, 19801, and hamster (Vollrath, 1979; the hamster used is Mesocricetus auratus, personal communication). The present results show that morphological features of pinealocytes in the superficial pineal are different from those in the deep pineal in Chinese hamsters under both long and short photoperiods. Pinealocytes in the superficial pineal have larger nuclei and more abundant cytoplasm than those in the deep pineal; pinealocyte nuclei in the superficial pineal are often of irregular shape and stain lightly in contrast to those in the deep pineal, which are round and stain deeply. In addition, long photoperiods cause enlargement of pinealocytes in both the superficial and deep pineal; however, pinealocytes of the deep pineal show less pronounced responses to long photoperiods. Pinealocytes in the mouse exhibit regional differences similar to those seen in the Chinese hamster (Hulsemann, 1967).The previous results suggest that pinealocytes of the mouse enlarge and, concomitantly, the shape of their nuclei changes from round to pleomorphic with advancing differentiation (Ito and Matsushima, 1967). From the above observations on the mouse and the present results, i t seems that the pineal gland of the
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Fi 8 Number of pinealocyte nuclei per unit area (0.01 or 0.004 mm ) of superficial (A) and deep (B) pineal glands in animals under LD 8:16 (solid circles) and under LD 16:8 (open circles). * P ~ 0 . 0 0 4 compared with animals under LD 16%.
4.
Chinese hamster consists of two different populations of pinealocytes; the pinealocytes in the deep pineal, unlike those in the superficial pineal, may not be fully differentiated cells. In the golden hamster (Reiter and Hedlund, 1976) and r a t (Bjorklund et al., 1972; Wiklund, 19741, postganglionic sympathetic fibers are abundantly distributed in the deep pineal as they are in the superficial pineal. As mentioned above, pinealocyte nuclei in these animals are smaller in the deep pineal than in the superficial pineal (Vollrath, 1979; Boeckmann, 1980). Thus, no apparent correlation may exist between the density of sympathetic innervation and size of pinealocytes in the golden hamster and rat. Sympathetic nerves are frequently observed in the superficial pineal of the Chinese hamster (Matsushima and Morisawa, 1982). A comparison of the density of sympathetic nerves between the superficial and deep pineal glands of the Chinese hamster seems important to understand the functional significance of the differences in size of pinealocytes between the two pineal portions of this animal. Differences in size of pinealocytes between peripheral (cortical) and central (medullary) regions of the superficial pineal have also been described in several mammals (Vollrath, 1979, 1981; Heidbuchel and Vollrath, 1983). In the present study, i t was found that pinealocyte sizes were similar between peripheral and central regions in the middle portion of the superficial pineal in each animal under LD 1 6 3 and LD 8:16. In addition, pinealocyte sizes obtained from the middle portion of the superficial pineal were correlated well with volumes of the superficial pineal. Thus, pinealocytes of the Chinese hamster may be uniform in size throughout the superficial pineal. The volume of the pineal gland has been determined from camera-lucida drawings of histological sections in various mammalian species (Quay, 1956, 1978; Legait et al., 1976a,b). In the Chinese hamster, the pineal volumes, expressed a s the sum of the volumes of the superficial and deep pineal in mm3 x lo3 per gram of body weight, were 3.78 k 0.12 and 2.17 2 0.08, respec-
tively, in animals under LD 16:8 and LD 8:16. When comparing these values with those obtained from the other rodent species (Legait et al., 1976a; Quay, 1978, see discussion), it may be said that the Chinese hamster has a relatively large pineal gland. A simultaneous determination of the volumes of the superficial and deep pineal glands has been carried out only on the white-footed mouse (Quay, 1956); the volumes of the anterior (deep) and posterior (superficial) pineal are 0.0197 -+ 0.0021 mm3 and 0.0894 +- 0.0068 mm3, respectively. Thus, the volume of the deep pineal is about 17% that of the whole organ. In the Chinese hamster, the deep pineal comprises less than 10%. Considerable interspecies differences may exist in the ratio between the volumes of the superficial and deep pineal. The volume of the superficial pineal is markedly larger in Chinese hamsters raised under LD 16:8 than in those under LD 8:16. In the superficial pineal, nuclei of pinealocytes are larger in size, paler in appearance, and more irregular in shape; and, in addition, nuclear density of pinealocytes is lower under LD 16% than under LD 8:16. Although less prominent, similar photoperiod-induced changes are observed in the volume of the deep pineal, and in the nuclear and cytoplasmic sizes and nuclear morphology in pinealocytes of the deep pineal. Thus, it is evident that the difference between volumes of the superficial and deep pineals of the Chinese hamster under the different photoperiods is due to the difference in size of pinealocytes. As suggested before, nuclear pleomorphism may be characteristic of differentiated pinealocytes. Thus, the postnatal development and differentiation of pinealocytes may be advanced and delayed under long and short photoperiods, respectively. Seasonal variations in pineal volumes or pinealocyte nuclear and cytoplasmic sizes have been reported to occur in some feral animals under natural environmental conditions. Pineal volumes or pinealocyte sizes are generally larger in winter or during hibernation (golden hamster, Mogler, 1958; dormouse, Legait et al., 1975; 13-lined ground squirrel, McNulty et al., 1980; elephant seal, Griffiths and Bryden, 1981). By contrast, pinealocytes of the hare are larger in size from summer to fall (Lincoln, 1976), and maximal nuclear diameters in pinealocytes of the bat are obtained in the fall (beginning of hibernation) in males and in early spring (arousal and dispersal from hibernation) in females (Quay, 1976). Additionally, pinealocyte sizes in the white-footed mouse are increased in summer and decreased in winter (Kachi and Quay, 1984). In feral animals under field conditions, it is possible that seasonal changes in a variety of environmental factors other than photoperiod may cause changes in sizes of pinealocytes and pineal glands. Thus, it is difficult to determine to what degree the photoperiod is involved in such seasonal variations. So far as we know, the present study is the first to demonstrate clearly the effects of photoperiods on sizes of pinealocytes and pineal glands in mammals under laboratory conditions; Chinese hamsters raised under long photoperiods have larger pineal glands composed of larger pinealocytes than those raised under short photoperiods. Thus, long photoperiods cause pinealocytes to enlarge. Constant light is known to induce atrophy of pinealocytes (Vollrath, 1981). Preliminary
PHOTOPERIODIC EFFECT ON CHINESE HAMSTER PINEAL
observations show that pineal volumes in 60- to 70day-old Chinese hamsters raised under LD 1 6 3 are larger than those in animals raised under photoperiods shorter or longer than 16 h r (unpublished observations). Thus, LD 16% may be the optimal photoperiod for the development of pinealocytes in the Chinese hamster. Larger pinealocytes under long photoperiods are believed to be functionally more active than smaller pinealocytes under short photoperiods. Electron microscopic and histochemical studies of such active and inactive pinealocytes are in progress in order to make clear the functional significance of these cells. Sizes of pinealocytes or pineal glands in several seasonally breeding animals are reported to increase when the gonads are regressed (golden hamster, Mogler, 1958; dormouse, Legait et al., 1975; hare, Lincoln, 1976; 13-lined ground squirrel, McNulty e t al., 1980; elephant seal, Griffths and Bryden, 19811, whereas pinealocytes of the white-footed mouse are larger when the gonads are active (Kachi and Quay, 1984). Thus, interspecies differences may exist in the relationships between pinealocyte or pineal gland sizes and reproductive function. The gonadal development in the Chinese hamster is greatly influenced by photoperiod; 60to 70-day-old Chinese hamsters raised from weaning under photoperiods longer than 14 h r have large testes (about 1.5 gm in paired weight), whereas those raised under photoperiods shorter than 10 h r have small testes (less than 0.5 gm in paired weight) (unpublished observations). Superior cervical ganglionectomy is known to block photoperiod-induced gonadal involution in some mammals (Reiter, 1980; Hoffmann, 1981). Since there are no observed effects of photoperiod on testicular development in the Chinese hamster subjected to superior cervical ganglionectomy (unpublished observations), such photoperiod-induced, testicular changes may be mediated by the pineal gland. As shown in the present study, the testes in 200-day-old Chinese hamsters raised under LD 16:8 or LD 8:16 are similar in weight (about 1.5 gm). Thus, the testicular weight in young Chinese hamsters raised under LD 8:16 may increase to reach that in animals under LD 16:8 until 200 days of age. Delayed development and subsequent regrowth of gonads have been observed in some mammalian species raised from birth or from weaning under short photoperiods (Djungarian hamster, Hoffmann, 1978; vole, Grocock, 1979; white-footed mouse, Johnston and Zucker, 1980).The present observation that sizes of pinealocytes and pineal glands are different, but testicular weights are similar, in 200day-old Chinese hamsters raised from weaning under short and long photoperiods suggests that photoperiodinduced changes in sizes of pinealocytes and pineal glands may occur without changes in reproductive activity of this animal. It is well known that in some mammals the pineal hormone melatonin is involved in the photoperiodic regulation of gonadal function (Reiter, 1980, 1987; Hoffmann, 1981,1985; Goldman, 1983; Bittman, 1984). As mentioned above, short photoperiod-induced gonadal atrophy in young Chinese hamsters is blocked by superior cervical ganglionectomy, suggesting that this gonadal change is mediated by melatonin. According to our recent observations (unpublished), sizes of pineal glands and pinealocytes are smaller in young Chinese
37
hamsters raised under LD 8:16 than in those under LD 1623. Thus, both pinealocytes or pineal glands and gonads of young Chinese hamsters are reduced in size under short photoperiods. In young Chinese hamsters, the size of pinealocytes shows a 24-hr rhythm, with its maximum a t the middle of the light period and its minimum during the second half of the dark period (Matsushima et al., 1983); this rhythm is apparently different from the rhythm in pineal melatonin synthesis in mammals. Furthermore, the 24-hr rhythm in size of pinealocytes disappears after exposure of young Chinese hamsters to constant darkness €or 7 days, suggesting that the rhythm is exogenous in nature (Sakai et al., 1986). All of these observations on young Chinese hamsters and the present results from adults may indicate that sizes of pinealocytes and pineal glands are not related to the synthesis of melatonin. From the observations on various mammalian species, it is generally supposed that pineal volume is correlated with body weight (Legait et al., 1976a,b; Vollrath, 1981). However, the present results clearly indicate that there is no correlation between pineal volume and body weight in the Chinese hamster. When comparing the body weights of the Chinese hamsters used in the present study until 200 days of age (data not shown), the body weights of the animals under LD 16:8 and LD 8:16 are similar from weaning to 40 days of age; but, between 50 and 110 days, the animals become heavier under LD 16:8 than under LD 8:16; and the body weights of the animals under the two photoperiods attain the same, maximal level around 160 days of age. Thus, the growth of the Chinese hamster appears to be temporarily inhibited under short photoperiods. Similar, but much more prominent, inhibitory effects of short photoperiods on growth rates have been reported for the Djungarian hamster (Hoffmann, 1978). From the present results, it is evident that photoperiod-induced changes in sizes of pinealocytes and pineal glands in the Chinese hamster are independent of growth of the animals. The significance of such photoperiodic influences on the pineal gland as demonstrated in the Chinese hamster remains undetermined. As a first step toward this problem, we are now examining whether or not similar photoperiodic changes occur in the pineal gland of other photosensitive and non-photosensitive animals. LITERATURE CITED Bittman, E.L. 1984 Melatonin and photoperiodic time measurement: Evidence from rodents and ruminants. In: The Pineal Gland. R.J. Reiter, ed. Raven Press, New York, pp. 155-192. Bjorklund, A., Ch. Owman, and K.A. West 1972 Peripheral sympathetic innervation and serotonin cells in the habenular region of the rat brain. 2. Zellforsch., 127570-579. Boeckmann, D. 1980 Morphological investigation of the deep pineal of the rat. Cell Tissue Res., 210;283-294. Goldman, B.D. 1983 The physiology of melatonin in mammals. In: Pineal Research Reviews, Vol. 1. R.J. Reiter, ed. Alan R. Liss, Inc., New York, pp. 145-182. Gregorek, J.C., H.R. Seibel, and R.J. Reiter 1977 The pineal complex and its relationship to other epithalamic structures. Acta Anat. (Basel), 99t425-434. Griffiths, D.J., and M.M. Bryden 1981 The annual cycle of the pineal gland of the elephant seal (Mirounga leoninu). In: Pineal Function. C.D. Matthews and R.F. Seamark, eds. ElsevieriNorth-Holland Biomedical Press, Amsterdam, pp. 57-66. Grocock, C.A. 1979 Testis development in the vole, Microtus ugrestis, subjected to long or short photoperiods from birth. J. Reprod. Fertil., 55:423-427.
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S. MATSUSHIMA ET AL.
Heidbiichel, U., and L. Vollrath 1983 Morphological findings relating to the problem of cortex and medulla in the pineal glands of rat and hamster. J. Anat., 136,723-734. Hoffmann, K. 1978 Effects of short photoperiods on puberty, growth and moult in the Djungarian hamster (Phodopus surzgorusi. J. Reprod. Fertil., 54:29-35. Hoffmann, K. 1981 Photoperiodic function of the mammalian pineal organ. In: The Pineal Organ: Photobiology-Biochronometry-Endocrinology. A. Oksche and P. Pevet, eds. ElsevieriNorth-Holland Biomedical Press, Amsterdam, pp. 123-138. Hoffmann, K. 1985 Interaction between photoperiod, pineal and seasonal adaption in mammals. In: The Pineal Gland. Current State of Pineal Research. B. Mess, CS. Rdzsas, L. Tima, and P. Pevet, eds. Elsevier Science Publishers, Amsterdam, pp. 21 1-227. Hulsemann, M. 1967 Vergleichende histologische Untersuchungen iiber das Vorkommen von Gliafasern in der Epiphysis cerebri von Saugetieren. Acta Anat. (Basel), 66249-278. Ito, T., and S. Matsushima 1967 A quantitative morphological study of the postnatal development of the pineal body of the mouse. Anat. Rec., 159:447-452. Johnston, P.G., and I. Zucker 1980 Photoperiodic regulation of reproductive development in white-footed mice (Peronzyscusleucopusi. Biol. Reprod., 22:983-989. Jung, D., and L. Vollrath 1982 Structural dissimilarities in different regions of the pineal gland of Pirbright white guinea-pigs. J. Neural Transm., 54:117-128. Kachi, T., and W.B. Quay 1984 Seasonal changes in glycogen level and size of pinealocytes of the white-footed mouse, Peromyscus leucopus: A semiquantitative histochemical study. J . Pineal Res., 1:163-174. Legait, H., M. Roux, G. Dussart, J.P. Richoux, and J.-L. Contet-Audonneau 1975 Donnees morphometriques sur la glande pineale du Loir (GZisglis) et du Lerot (Eliomys quercinus) au cours du cycle annuel. C. R. SOC.Biol., 169:132-136. Legait, H., R. Bauchot, H. Stephan, and J.-L. Contet-Audonneau 1976a Etude des correlations liant le volume de I’epiphyse aux poids somatique et encephalique chez les rongeurs, les insectivores, les chiropteres, les prosimiens et les simiens. Mammalia, 40,327-337. Legait, H., R. Bauchot, and J.-L. Contet-Audonneau 1976b Etude des correlations liant les volumes des lobes hypophysaires et de Yepiphyse au poids somatique et au poids de l’encephale chez les chiropteres. Bull. Assoc. Anat., 60:175-188. Lincoln, G.A. 1976 Seasonal changes in the pineal gland related to the reproductive cycle in the male hare, Lepus europaeus. J . Reprod. Fertil., 46:489-491. Matsushima, S., and Y. Morisawa 1982 Ultrastructural observations
on the pineal gland of the Chinese hamster, Cricetulus griseus. I. The superficial pineal. Cell Tissue Res., 222531-546. Matsushima, S., Y. Morisawa, I. Aida, and K. Abe 1983 Circadian variations in pinealocytes of the Chinese hamster, Cricetulus griseus. A quantitative electron-microscopic study. Cell Tissue Res., 228r231-244. McNulty, J.A., T.A. Dombrowski, and W.A. Spurrier 1980 A seasonal study of pinealocytes in the %lined ground squirrel, Spermophilus tridecemlineatus. Reprod. Nutr. Dev., 20,665-672, Mogler, R.K.-H. 1958 Das endokrine System des syrischen Goldhamsters (Mesocricetus auratus uuratus Waterhouse) unter Berucksichtigung des naturlichen und experimentellen Winterschlafs. Z. Morphol. Okol. Tiere., 47:267-306. Quay, W.B. 1956 Volumetric and cytologic variation in the pineal body of Peromyscus leucopus (Rodentia) with respect to sex, captivity and day-length. J. Morphol., 98r471-495. Quay, W.B. 1976 Seasonal cycle and physiological correlates of pinealocyte nuclear and nucleolar diameters in the bats, Myotis lucifugus and Myotis sodalis. Gen. Comp. Endocrinol., 29,369-375. Quay, W.B. 1978 Quantitative morphology and environmental responses of the pineal gland in the collared lemming (Dzcrostonyx groenlundicus). Am. J. Anat., 1531545-562. Reiter, R.J. 1980 The pineal and its hormones in the control of reproduction in mammals. Endocr. Rev., 1,109-131. Reiter, R.J. 1987 Mechanisms of control of reproductive physiology by the pineal gland and its hormones. In: Advances in Pineal Research, Vol. 2. R.J. Reiter and F. Fraschini, eds. John Libbey & Co., London, pp. 109-125. Reiter, R.J., and L. Hedlund 1976 Peripheral sympathetic innervation of the deep pineal gland of the golden hamsier. Experientia, 32: 1071-1072. Sakai, Y., I. Aida, and S. Matsushima 1986 Effect of continuous darkness on circadian morphological rhythms in pinealocytes of the Chinese hamster, Cricetulus griseus. Cell Tissue Res., 245:127134. Vollrath, L. 1979 Comparative morphology of the vertebrate pineal complex. In: The Pineal Gland of Vertebrates Including Man. Progress in Brain Research, Vol. 52. J.A. Kappers and P. Pevet, eds. ElsevieriNorth-Holland Biomedical Press, Amsterdam, pp. 25-38. Vollrath, L. 1981 The pineal organ. In: Handbuch der Mikroskopischen Anatomie des Menschen. Vol. VII7. A. Oksche and L. Vollrath, eds. Springer, Berlin, pp. 41-43, 67-71, 242-244. Wiklund, L. 1974 Development of serotonin-containing cells and the sympathetic innervation of the habenular region in the rat brain. A fluorescence histochemical study. Cell Tissue Res., 155:231243.
