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J Pineal Res 1998; 25:229-239 Printed in the United States of America-41 rights reserved.

Copyright 0 Munksxaard. 1998 Journal of Pineal Research

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Structure of the ovine pineal gland during prenatal development

Regod6n S , Franco A, Masot J, Redondo E. Structure of the ovine pineal gland during prenatal development. J. Pineal Res. 1998; 25:229-239. 0 Munksgaard, Copenhagen

Abstract: The structure of the pineal gland of 32 clinically healthy ovine embryos at different stages of development was studied. Embryos were arranged in four age groups, each containing eight embryos (four males and four females), defined in terms of the most relevant histological features: group 1 (27 to 69 days of prenatal development), group 2 (70 to 97 days), group 3 (98 to 116 days), and group 4 (1 17 to 150 days). At around 30 days of prenatal life, according to topographic criteria, the pineal outline begins to differentiate into a dorsal evagination of the diencephalic medium line, close to the anterior and posterior commissures. The growth of the pineal is biphasic. The ontogenic-proliferative phase begins at 30 days and includes the invasion of ependymal cells and the proliferation of the pineal parenchyma cells. The hypertrophic- differentiation phase includes the volume increment of the pinealoblasts and their differentiation into pinealocytes; this occurs at around 118 days. At around 98 days, the gland acquires its definitive compact appearance due to 1) glandular growth in constant volume and 2) the obliteration of pineal recess. The glandular structure displays a parenchyma made up of pinealoblasts, interstitial cells, and cells containing pigment. The pineal stroma is structured in pseudolobes formed by reticular and collagen fiber septae, which constitute together the interstitial cell prolongation net, which is the support structure of the whole glandular cytology. Capillaries are detected all over the glandular surface, being more abundant in the medullary zone. At around 98 days of prenatal development, VIP (Vasoactive Intestinal Peptide) positive fibers, distributed around blood vessels and among pinealoblasts were detected.

Sergio Regodon, Antonio Franco, Javier Masot, and Eioy Redondo Department of Anatomy and Histology, Faculty of Veterinary Medicine, University of Extremadura, 10071 Caceres, Spain

Key words: structure - immunohistochemistry - prenatal development - sheep - pineal gland

Address reprint requests to Or. Sergio Regodon Mena, Anatomia y Embriologia, Facultad de Veterinaria, Universidad de Extremadura, E-10071 Caceres, Spain.

Received February 3, 1998; accepted June 1, 1998

introduction

The pineal gland, which in mammals develops from the diencephalic ependyma and during embryonic development is located between the anterior and the posterior commissure, has recently been the object of considerable research [Redondo et al., 1996; Franco et al., 1997; Regodh et al., 19981.

It is precisely in seasonally polyestrous species such as sheep that the pineal gland assumes great importance since it is involved in photoperiod regu- lation through secretion of melatonin [Bittman et al., 1983; Reiter, 1993; Cozzi et al., 19941. Accordingly, the aim of this study was to acquire a better under- standing of the ontogenesis of the sheep pineal gland and therefore of its potential functional significance,

by obtaining morphological data at varying stages of development, defined in terms of the most relevant structural features.

Two reasons led us to carry out a study about the pineal gland during prenatal life. The first is that few studies of these characteristics exist [Jordan, 191 1; Ariens-Kappers, 1960; Anderson, 1965; Redondo et al., 1996; Franco et al., 1997; Regod6n et al., 19981. The second reason is that any investigation on the functional aspects of the pineal gland necessarily implies wide knowledge of its morphological configuration, even of its differentiation from the diencephalic ependyma of the third ventricle [Pevet, 1977; Ueck, 19861.

The structural and immunohistochemical analy-

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sis of the ovine pineal gland during prenatal life is the aim of this paper.

Materials and methods Animals

Thirty-two clinically healthy ovine embryos at different stages of development were used for this study. Specimens were arranged in four age groups, each containing eight embryos (four males and four females), defined in terms of the most relevant histological features: group 1 (27 to 69 days of prenatal development), group 2 (70 to 97 days), group 3 (98 to 116 days), and group 4 (117 to 150 days). To obtain embryos at different stages of development, a cesarean section was performed after syn- chronization of estrus, using hormonal techniques and uterine flushing. Fluorogesterone acetate was administered 14 days before introduction of males. Then 600 IU of pregnant mare serum globulin (PMSG) (Sigma Aldrich Quimica, Madrid, Spain) was inoculated. Cesarean section was performed from day 27 following introduction of males. Ani- mals were tranquillized with an intramuscular injection of 0.5 mg/100 kg body weight of propionyl phenothiazine, and anesthesia was induced by intra- venous injection of sodium thiopental(4 g in a 20% aqueous solution). Once separated from maternal linking, embryos were euthanized by umbilical vein administration of 1 g sodium thiopental in a 20% aqueous solution.

Light microscopy

Embryo pineal glands were sliced sagittal and fron- tally after 1 hr of fixation in Carnoy’s fluid and re- fixed in 10% neutral formalin saline solution and processed by paraffin-embedding methods. Sections 4 pm thick were cut and stained with hematoxylin and eosin (HE), Masson tricromicre (MT), and Gomori’s method for reticulum fibers (GR) for rou- tine morphological examination, with Masson- Fontana (MF) for demonstration of cells containing pigment (melanin pigment) and with phosphotung- stic acid hematoxylin (PTAH) for detection of glial- type cells.

lmmunohistochemical analysis

An avidin-biotin-peroxidase complex (ABC methods) was carried out on deparaffhized pineal samples for detection of glial fibrillary acidic protein (GFAP), the main protein component of intermediate astrocyte fila- ments. Sections were incubated in diluted (1 :50) nor- mal swine serum (Dako, Madrid, Spain) for 15 min

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to reduce background and then incubated in diluted (1 : 1,000) rabbit anti-bovine GFAP (Dako, Madrid, Spain) for 3 hr at 20°C. Diluted (1 : 1,500) biotinylated swine anti-rabbit IgG (Dako, Madrid, Spain) was placed on the sections for 30 min, and sections were then incubated with diluted (1:50) ABPC reagent (Dako, Madrid, Spain) for 1 hr. After diaminobenzidine reaction, a nuclear counterstaining with hematoxylin was applied. Negative controls consisted in the sub- stitution of primary antisera for nonimmune rabbit serum. Adjacent brain tissue sections served as positive controls. VIP immunohistochemical reaction was carried out for detection of nerve fibers. VIPergic fibers are present in the sheep pineal gland [Cozzi et al., 1990, 19941 as well as in other mammals [Moller, 19921. The technique was similar to the GFAP immunoperoxidase staining procedure. Sections were incubated in diluted (1 : 1,000) rabbit anti-VIP (Dako, Madrid, Spain) for 3 hr at 20°C.

Morphometrical analysis

Numerical cell density was determined by calculat- ing the number of pinealoblasts, interstitial cells (PTAH and GFAP positive cells), and cells containing pigment. Five sections, separated from each other by a distance of roughly 50 pm, were taken for each gland. Ten fields measuring 10,000 pm2 were randomly selected per section. Fields and sections were identical for both staining techniques. Calculations were made manually by using pro- jected images. Morphometrical analysis was based on random sampling, the sample being considered satisfactory when the standard deviation was less than 5% of the mean; the formula proposed by Aherne and Dunnill [ 19821 was employed: n>(s/ O.O5x)*, where n is the number of samples, s is the standard deviation of sample, x is the sample mean, and 0.05 is the desired error.

Results

Light microscopy

Group 1 (29 to 69 days of prenatal development). At around 30 days of gestation life, the pineal outline is observed as a small dorsal evagination located in the midline of the diencephalic roof, between the habenular and posterior commissures (Fig. 1). This outline is configured as a wide lumen recess (pineal recess), covered by a pseudo- stratified epithelium of ependymal cells and communicated with the third ventricle lumen (Fig. 2). As the development advances, the neuroepithelium that constitutes the pineal outline increases its thickness as a consequence of 1 ) the immigration

Structure of ovine pineal gland

with a greater reticular fiber content and with wide, clearly defined lumina (Figs. 8,9).

The pinealoblasts are the only cellular type detected. They do not follow a defined order; only a higher cellular density in the pseudolobes detected at the periphery is outstanding. Cells with abundant pigment granules are irregularly distributed throughout the parenchyma. Their morphology is similar to that detected in group 1. The glandular stroma is highly fibrous (Fig. 10) and always denser than the mesenchyma that surrounds the glandular periphery. Group 3 (98 to 11 6 days of prenatal development). The gland appears like a compact organ with intense basophilia. The extraordinary abundance of nuclei and their proximity to each other together with the scarce quantity of cellular cytoplasm are the causes of this basophilia. The parenchyma glandular of ovoid-round morphology surrounded by a connective tissue is highly vascularized (Fig. 11). The glandular capsule shows only one differential point: the collagen fibers, whose presence was insignificant in previous groups, begins to abound.

In the glandular parenchyma, according to cellular density criteria, two clearly defined zones are differentiated: the cortical and the medullar zones. The cortical zone is characterized by a pseudolobular configuration with follicles made up of ovoid or round pinealoblasts and slightly defined intercellular limits (Fig. 12). The medullar zone, of lower cellular density, does not have the character- istic pseudolobular structure of the cortical zone.

At around 98 days of gestation, the occlusion of the pineal recess is finaIized as a result of the folding and fusion of its walls. In this obliteration process, subsidiary cavities known as cisterns remain (Fig. 13); the cistern covering, its content, and the way in which it is distributed are very similar to those described in groups 1 and 2. In addition to the pinealoblasts, a second cellular group appears: the interstitial cells. These cells display small, dense, ovoid nuclei, with uniformly distributed chromatin. These cells have a perivascular tendency and are less abundant than the pinealoblasts. As in previous groups, we detected the presence of cells with a high content of pigment granules. The identification of these cells was not possible for the reasons previously men- tioned. The stroma consists of the capsular, trabecular and pseudolobular wall connectives (Fig. 14). The only differential point with respect to the other groups is the higher collagen fiber content in all its components. The organization pattern of the vascular structures is very similar to that of the other groups. Worth noting is its higher degree of differentiation, its greater presence in the medullar zone, and the progressive reduction in caliber of the vessels as they penetrate the pineal parenchyma (Fig. 15).

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of cells from the diencephalic ependyma of the third ventricle and 2) the frequent mitosis cells, observed in the vicinity of the pineal recess lumen.

The progressive growth of the pineal outline is accompanied by a gradual reduction of the recess lumen and by an interruption of its communication with the third ventricle due to the interposition of a fine layer of mesenchyma connective tissue (Fig. 3). With further development, the glandular parenchyma presents a compact appearance, which is only interrupted by the presence of rosettes and poorly differentiated vascular structures. The rosettes (also known as cisterns) are structured with a small cir- cular section lumen, delimited by a contour of cells of identical morphology to the cells of the remain- der of the parenchyma (Fig. 4).

In this group, the pinealoblasts are the single cellular type. They are elongated cells, with centrally located nuclei, ovoid or round, with peripheral accumulation of heterochromatin, and central nucleo- lus (Fig. 4). These cells are distributed throughout the glandular parenchyma, with no defined orientation. The glandular contour is delimited by a reticular fiber connective tissue which constitutes the delimitation capsule (Fig. 5). In this capsule a small amount of elastic fibers are also detected. Vascular- connective walls begin contain strongly argyrophilic trabeculae, proceeding from the glandular capsule (Fig. 5) . Cells with pigment granules accumulation are distributed throughout the glandular surface (Fig. 6). The intensity of the argentic reaction impeded the morphological cataloguing of these cells. Group 2 (70 to 97 days of prenatal development). The glands present a compact appearance, which is re- lated with almost exclusive growth of their component cells. The invagination of the ependymal cells, proceeding from the roof of the third diencephalic ventricle, disappears due to the connective mesenchyma that interrupts communication between the recess and the third ventricle lumina (Fig. 7).

The glandular surface appears surrounded by a capsule made up of a vascular mesenchyma with isolated elastic fibers and a considerable number of reticular fibers (Fig. 8). All the capsular components are delimited by an epithelium of flat cells that rests on an basal lamina. Connective septa of identical con- stitution but with greater thickness than those detected in group 1 are located from the periphery towards the interior of the gland, forming pseudolobular structures (Fig. 8). Inside the glandular parenchyma, the presence of cisterns or reduced light rosettes stand out, generally without content and delimited by a well-defined cellular contour and a clear basal lamina (Fig. 9). The vascular structures are more abundant in the central region of the gland (Fig. 8). They show a higher degree of differentiation,

Regodon et al.

Figures 1-8.

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previous groups. The morphology and distribution of these cells are similar to those of the other groups. The cisterns or rosettes are also evident in group 4 although in lesser quantity. Two kinds of rosettes have been observed: 1) Perfectly structured rosettes, predominantly rounds forms, although elongated forms along the longitudinal axis of the gland are not infrequent. These rosettes are found in the glandular medullar zone. 2) The so-called pseudorosettes (Fig. 19), which have an irregular and disorganized appearance, with panglandular distribution and variable morphology, and are less numerous. The structural model is identical to that of group 3.

The glandular stroma and vascular structures (Figs. 20, 21) basically show a similar morphology but with marked differences with respect to the perivascular space (Fig. 20). This space was almost non-existent in previous groups, and the now considerable amount in this group impedes close con- tact between the parenchyma cells and the vascular structures.

Group 4 (1 17 to 150 days of prenatal development). The to- pography of the pineal gland situates rostrally to the cerebellum and caudally to the cerebral hemisphere (Fig. 16). Glandular development is greater than in the previous groups, so the mesenchyma connective that surrounds the gland is lesser. The whole glandular surface is surrounded by a poorly cellular capsule with a high fiber content. The capsule cells have a fusiform morphology and are identified as fibroblasts. The fibrous component is formed by a scarce amount of elastic fibers, intensely developed reticular fibers (Fig. 17), and a considerably in- creased quantity of colagen fibers, as in group 3. Within the capsular connective, vascular structures in close relation with the cerebral blood vessels and adjacent choroid plexus are detected (Fig. 16).

In general terms, the structure and topographic zonification of the glandular parenchyma are similar to those of group 3. However, the number of cells is lower in group 4. This may be due to the greater cellular volume of the pinealoblasts.

The glandular cytology is represented by pinealoblasts and interstitial cells (Fig. 18). Changes take place in the morphology of the pinealoblasts in addition to the classic round forms; some irregular and more or less elongated forms begin to be dis- cerned. These are cells with a greater cytoplasmatic volume, causing the degree of intercellular separa- tion to be higher and, consequently, cellular density is lower. The interstitial cells are basically of identical morphology to the previous group; however some differential points can be noted: 1) greater cellular numerical density per predetermined field, 2) a more marked tendency to a perivascular disposi- tion, and 3) small cytoplasmatic prolongations sug- gestive of primitive astrocytes begin to appear. The density of cells containing pigment is lower than in

Fig. I . Embryo, 30 days. Evagination of the diencephalic roof of the third ventricle. Sagittal section. H-E x220. F i g . 2. Embryo, 35 days. Pineal recess covered with polystratified of ependimary cells. Sagittal section. H-E x220. Fig. 3. Embryo, 54 days. Connective tissue interposed between the glandular outline and the diencephalic ependyma of the third ventricle. Frontal section. H-E x180. Fig. 4 . Embryo, 63 days. Presence of poorly differentiated vascular structures and cellular cisterns. Frontal section. H-E ~ 8 5 . Fig. 5. Embryo, 67 days. Cells with pigment accumulation irregularly distributed over the glandular surface. Sagittal section. M.F. x220. F i g . 6 . Embryo, 67 days. Reticular fibers with intense argyrophilia in the vascular structures both of the contour and the pineal parenchyma. Frontal section. G.R. ~ 1 8 0 . F i g . 7. Embryo, 97 days. Interposition of connective mesenchymatose tissue highly vascularized between the third ventricle and the pineal gland. Frontal section. H-E ~ 8 5 . Fig. 8. Embryo, 75 days. Intense argentic reaction of the reticular fibers of the capsula, trabeculas and vascular structures. Frontal section. G.R. ~ 1 8 0 .

lmmunohistochemical analysis

GFAP positive cells were observed in the embryonic pineal parenchyma in groups 3 and 4. In group 3 these were distributed uniformly throughout the gland. Immunopositive cells (whose appearance re- sembled that of the CNS astrocytes used as positive control) displayed small, dense, ovoid nuclei and an intensely-staining rim of cytoplasm bordering negative nuclei (Fig. 22). GFAP positive cells displayed a small number of processes, with varying diameters and arranged both longitudinally and transversally. Cell processes were interwoven amongst pinealoblasts and around blood vessels to form a limiting barrier (Fig. 23).

In group 4 embryos, GFAP positive cells were ei- ther oval or elongated in shape. Cytoplasmic processes, which were more numerous than in group 3, varied in both diameter and orientation (Fig. 24).

Numerous positive nerve fibers entered the pineal gland and travelled within connective tissue spaces, Generally VIP-immunoreactive fibers were located close to connective septa and blood vessels (Fig. 25). However VIPergic fibers possessing varicosi- ties of variable sizes were also dispersed between pinealocytes (Fig. 26).

Morphometrical analysis

Pinealoblasts with a cellular numerical density of 45f4 and 56+5 were observed in groups 1 and 2. respectively. Values of 25+2 and 36+4, for the numerical density of the cells containing pigment were observed in these groups (Table 1).

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Figures 9-16.

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two phases (the ontogenic-proliferative and the hypertrophic-differential phases), which are successive and overlapping in time. The ontogenic-proliferative phase begins at about 30 days of prenatal life, with pineal outline differentiation; it includes the invasion of the parenchyma by ependymary cells and the proliferation of the pineal cells themselves [Redon- do et al., 19961. This phase could correspond to the tubular elongation described by Calvo and Boya [1981] in the embryonic development of the rat. It could also be comparable to the results described in chickens [Calvo and Boya, 19781.

The hypertrophic-differential phase includes the increase in volume of the pinealoblasts. It occurs at about 118 days of gestation and corresponds to the morphology reorganization phase described by Calvo and Boya [1981]. In this respect, we do not agree with Quay [ 19741 who describes two phases (the morphogenic and cellular proliferation phases), which commence within the first days of embryonic age and carry on until birth. At around 98 days of gestation, the gland acquires a compact aspect due to two facts: 1) Glandular growth in almost constant volume [Calvo and Boya, 1978, 19811. The migration of the ependymary cells is responsible for this growth, mainly in group 3; in group 4, the aspect is equally compact because although there is a lower cellular density (due to the finalization of ependymary migration and to the hypertrophic pinealoblasts detected), there are fewer cisterns or rosettes. 2) The occlusion of the pineal recess, which is com- plete at about 98 days of embryonic age.

In the obliteration of the pineal recess, two phe- nomena may overlap: one is due to mechanic causes, in direct relation with the space limit pro- duced by the structures attached to the gland [Calvo and Boya, 19811; the other is due to the enlargement and folding of the epithelium in the direction of the pineal recess lumen. The abundant presence of mi- totic figures towards the recess lumen might con- firm this theory [Calvo and Boya, 1981; Redondo et al., 19961.

In the occlusion process of the pineal recess, cavities (cisterns or rosettes) remain that are considerably reduced in number at the perinatal stages (group 4). This could be a consequence of the pres- sure exerted by the adjacent cells [Jordan, 19211 or it could be due to the obliteration of lumen caused by the proliferation of wall cells [Calvo and Boya, 19811. It seems clear that these cells are always sub- sidiaries of the pineal recess lumen; they cannot be catalogued as neoformed cavities or as a parenchyma growth mechanism [Calvo and Boya, 19781.

According to our results, the cells that configure these rosettes are pinealoblasts; in this we differ from Anderson [1965] in his studies on sheep and

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In groups 3 and 4, pinealoblasts with numerical densities of 72k6 y 58+5 were detected, and cells containing pigment displayed values of 41+2 y 20+2, respectively. With regard to the interstitial cells, the numerical density for the PTAH positive cells goes from 18f2 in group 3 to 28k4 in group 4. The GFAP positive cells evolve from 9&1 in group 3 to 14+2 in group 4 (Table 1).

Discussion

According to the topographic criteria put forward by Vollrath [1979, 19811, we classify the ovine pineal gland as type A (proximal to the third ventricle).

For the differentiation of the pineal outline, we rely on topographic criteria (enlargement of diencephalic medium line close to the posterior commissure) because it cannot be histologically differentiated from the adjacent neuroepithelium from which it derives [Calvo and Boya, 1981; Ueck, 19861. The glandular outline appears at around 30 days of prenatal development. An earlier report [Jordan, 19111 describes the pineal gland development of sheep in earlier stages, i.e., at about 20 days of gestation.

From the comparative study of diverse mammal species, it can be deduced than in sheep, as in cows [Brack, 19621 and carnivores [Zach, 19601, the pineal outline appears in the first stages of development. In species such as the hamster [Sheridan and Rollag, 19831; the rat [Calvo and Boya, 1981; Ueck, 19861; and the rabbit [Romijn, 19731, there is a much later differentiation, which takes place in the final days of embryonic development.

Glandular growth at the pineal outline occurs in

~~~

Fig. 9. Embryo, 87 days. Irregular order of pinealoblasts. Cis- temal structures of reduced lumina. Sagittal section. H-E ~ 2 2 0 . Fig. 10. Embryo, 92 days. Highly fibrous glandular stroma. Sagittal section. H-E ~ 1 8 0 . Fig. 11. Embryo, 98 days. Parenchyma glandular of ovoid- round morphology surrounded by a connective tissue highly vascularized. Frontal section. H-E ~ 8 5 . Fig. 12. Embryo, 106 days. Pseudolobular configuration in the cortical zone of the pineal parenchyma delimiting follicular structures. Sagittal section. H-E ~ 2 2 0 . Fig. 13. Embryo, 115 days. Cisterns with light varied morphology, surrounded by an epithelium of pinealoblasts cells. Sag- ittal section. H-E ~ 1 8 0 . Fig. 14. Embryo, 98 days. Strongly argyrophile connective walls. Pseudolobular structures in the cortical parenchyma. Frontal section. G.R. ~ 1 8 0 . Fig. 15. Embryo, 11 5 days. Greater presence of vascular structures in the medulla zone than in the cortical. Frontal section. G.R. x180. F i g . 16. Embryo, 128 days. Pineal gland situated between the third ventricle and both cerebral hemispheres. Frontal section. H-E x85.

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Figures 17-24,

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Table 1. Numerical cell density (mean 5 S.E.IlO.OOO~m2) of pinealoblast, interstitial cells and cells containing pigment

Fig . 25. Embryo, 98 days. VIP-immunoreactive fibers located close to blood vessels. Sagittal section. ABC x380. Fig. 26. Embryo,l SO days. VIPergic fibers dispersed between pinealocytes. Sagittal section. ABC ~380.

Fig . 17. Embryo, 128 days. Pseudolobular structures made up of reticular fiber walls from the capsula. Sagittal section. G.R. x220. Fig. 18. Embryo, 124 days. PTAH positive interstitial cells distributed throughout the pineal parenchyma. Sagittal section. PTAH x380. Fig. 19. Embryo 120 days. Presence of irregular pseudorosettes in the medullar zone. Sagittal section. H-E ~ 1 8 0 . F i g . 20. Embryo, 124 days. Abundant presence of colagen fibers in the perivascular connective. Sagittal section. M.T. x180. Fig . 21. Embryo, 118 days. Numerous vascular structures towards the medullar zone. Sagittal section. G.R. ~ 1 8 0 . Fig. 22. Embryo, 98 days. GFAPpositive cells: cytoplasm sur- rounding the negative nuclear zone. Sagittal section. ABC xsoo. Fig . 23. Embryo, 112 days. Cellular processes of GFAP positive cells with different orientations. Sagittal section. ABC x380. Fig. 24. Embryo, 150 days. Cellular processes of GFAP positive cells forming a limiting barrier around blood vessels. Sag- ittal section. ABC ~ 2 2 0 .

Group 1 Group 2 Group 3 Group 4

Pinealoblast 4 5 c 4 5 6 k 5 7 2 c 6 5 8 2 5 Cells containing pigment 25 c 2 36 2 4 41 c 2 20 -c 2

Interstitial cells - 1 8 k 2 2 8 2 4 PTAH positive cells -

GFAP positive cells - - 9 c l 1 4 5 2

cow pineal, from Calvo and Boya [ 198 11 on rat pineal, and from Garcia-Mauriiio and Boya [ 1992a,b] on rabbit pineal. These authors suggest that the con- stituent cells of the rosettes are the ependymary cells of the third ventricle, non-differentiated cells, and pinealocytes type I1 (interstitial cells), respectively.

In the prenatal development of the ovine pineal gland, we observe cortex and medulla regions similar to those detected by Nesic [1962]. However, the delimitation of both regions is based on very diverse criteria: pinealocyte form [Zach 19601, distribution of cellular types [Romijn, 19731, and immunohistochemical tests for the detection of melatonin [Freund et al., 19771.

The pineal parenchyma in developing ovines is constituted by three cellular types: pinealoblasts, interstitial cells, and cells containing pigment. The pinealoblasts appear in the first stages of prenatal life and continue throughout ontogenesis. The morphology and the manner of distribution of these cells on the glandular surface are similar to the pattern described for other species of mammals [Pevet, 1977; Ueck, 19861.

Numerous terms have been used to designate the second type of pineal gland parenchyma: interstitial cells [Cozzi, 19861, type I1 pinealocytes [Pevet, 1977; Calvo and Boya, 1983, 1984; Boya and Calvo, 19841, glial cells [Anderson, 1965; Calvo et al., 1988a,b], and astrocytes [Xu Zang et al., 1985; Lbpez-Muiioz et al., 1992a,b; Boya and Calvo, 19931. The appearence of these cells is at around 98 days of gestation; this reason we can consider the sheep as a species with premature development of the pineal interstitial cells. The comparative analysis with other mammal species corroborates this view [Brack, 1962; Boya and Calvo, 19931.

Histochemical and immunological tests were performed to determinate the nature of this second cellular population. The first step was to determine the presence of glial-like cells by PTAH immunostaining. The second step was thus to determine GFAP expression; GFAP is widely considered a valid label for the detection of astrocyte development [Valentino et al., 19831 and more particularly as evidence of the presence of astrocytic cells at a certain stage of maturity [Lbpez Muiioz et al., 1992a,b].

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Comparison of the results of these two tests showed that of all glial (PTAH positive cells), only a proportion were positive to GFAP, suggesting that a certain proportion of glial cells may be immature astrocytes no expressing GFAP. The results obtained here indicate that the second cell population in developing ovine pineal gland is in fact a combina- tion of glial-astrocyte cells at varying stages of maturity [Franco et al., 19971. This hypothesis has already been advanced in rats [Schachner et al., 19843 and in carnivores [L6pez-Munoz et al., 1992a,b; Boya and Calvo, 19931.

With respect to the cells containing pigment, the intensity of the argentic reaction made it very diffi- cult to distinguish whether these cells were pinealoblasts, interstitial cells, or a new type of cells. Redondo et al. [ 19961, by means of electronic trans- mission microscopy, defends the existence in the embryonic development of ovine pineal of a third cellular population (pigment cells) different from the pinealoblasts and interstitial cells (which also contain pigment granules).

The glandular stroma is structured in pseudolobes formed by walls of reticular and colagen fibers [Calvo and Boya, 1981, 19841, which continue along the perivascular connective filling the interparenchymal space; these, together with the network of interstitial cell prolongation, make up the supporting framework of the whole glandular cytology [Lbpez- Muiioz et al., 1992a,b].

From the beginning of embryonic development, we detect a relatively abundant vascularization, re- sulting in an increase in vascular density as the development advances. There is a greater abundance of vascular structures at the medullar area, although these are of a lower caliber. This fact has been also described in adult rabbit pineal [Romijn, 19731 and in the pineal of rat embryos [Calvo and Boya, 19811. Initially, capillaries are surrounded of an almost in- existent perivascular space that reach a considerable development from 98 days onwards. It has not been possible to determine the kind of capillaries by light microscopy. Redondo et al. [1996] in an electron microscopic study of sheep pineal gland at 98 days of gestation report nonfenestrated capillaries similar to those encountered in Carnivores [Calvo et al., 1990; Boya et al., 19951.

VIP nerve fibers were observed at around 98 days of prenatal development. The localization of these fibers was similar to the those reported by Redondo et al. [1996] in the prenatal development of the ovine pineal by using electron microscopy.

Some contradictions were found regarding the functionality of the pineal gland during intrauterine life. As a counterargument to a hypothetical functional role, we report a progressive spacing between

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the pineal an the third ventricle; this would dimin- ish the hypothetical secretion of pineal products towards the cerebrospinal fluid. Arguments in favor of a hypothetical functionality involve all the cellular types that make up the ovine pineal parenchyma during ontogenesis. Redondo et al. [1996] describe in pinealoblasts a considerable development of the organoids involved in proteosyntesis. In the interstitial cells, the proximity of the vascular structures [Calvo and Boya, 1983; Boya and Calvo, 1993; Redondo et al., 19961 could be the basis of the mor- phologic support of a hypothetical functionality, relat- ing to the interchange of substances between the pineal parenchyma and the blood [Xu Zang, 19851 . This hypothetical functional role would complement the support function traditionally attributed to these cells [Lbpez-Munoz et al., 1992a,b].

If we admit the prenatal differentiation on the pigment cells as a new cellular type, different from the pinealoblasts and interstitial cells as proposed by Calvo et al. [ 1988a,b] in the postnatal development in dogs’ pineal, and Redondo et al. [1996] and Regod6n et al. [1998] in the embryonic development of the ovine pineal, we would suggest that these cells have a priority role in melanosynthesis with regard to the cells containing pigment.

Acknowledgments

The authors thank Jose Luis Sanz Rodrigo and Ma del Carmen Gonzalez Bravo of the Pathological Anatomy Unit at San Pedro de Alcantara Hospital, Caceres and GennAn Femandez Corrales of the Faculty of Veterinary Medicine, Ciceres for technical assistance.

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