Effect of the Photoperiod and Administration of Melatonin on the Pars Tuberalis of Viscacha Lagostomus maximus maximusAn Ultrastructural Study.код для вставкиСкачать
THE ANATOMICAL RECORD 293:871–878 (2010) Effect of the Photoperiod and Administration of Melatonin on the Pars Tuberalis of Viscacha (Lagostomus maximus maximus): An Ultrastructural Study EDITH PEREZ ROMERA,1* FABIAN MOHAMED,2 TERESA FOGAL,3 SUSANA DOMINGUEZ,2 RAMÓN PIEZZI,3 AND LUIS SCARDAPANE2 1 Cátedra de Anatomı́a Humana, Bioquı́mica y Farmacia, Universidad Nacional de San Luis, San Luis, Argentina 2 Cátedra de Histologı́a y Embriologı́a, Facultad de Quı́mica, Bioquı́mica y Farmacia, Universidad Nacional de San Luis, San Luis, Argentina 3 Instituto de Histologı́a y Embriologı́a (IHEM), Universidad Nacional de Cuyo, Consejo Nacional de Investigaciones (CONICET), Argentina ABSTRACT The pituitary pars tuberalis (PT) is a glandular zone exhibiting welldeﬁned structural characteristics. Morphologically, it is formed by speciﬁc secretory cells, folliculostellate cells, and migratory cells coming from the pars distalis. The purpose of this work was to investigate differences in speciﬁc cellular characteristics in the PT of viscachas captured in summer (long photoperiod) and winter (short photoperiod), as well as the effects of chronic melatonin administration in viscachas captured in summer and kept under long photoperiod. In summer, the PT-speciﬁc cells exhibited celllike characteristics with an important secretory activity and a moderate amount of glycogen. In winter, the PT-speciﬁc granulated cells showed ultrastructural variations with signs of a reduced synthesis activity. Also, PT showed a high amount of glycogen and a great number of cells in degeneration. After melatonin administration, the ultrastructural characteristics were similar to those observed in winter, but the amount of glycogen was higher. These results suggest possible functional implications as a result of morphological differences between long and short photoperiods, and are in agreement with the variations of the pituitary-gonadal axis, probably in response to the natural photoperiod changes through the pineal melatonin. The ultrastructural differences observed in PT, after melatonin administration, were similar to those observed in the short photoperiod, thus supporting the hypothesis that these cytological changes are induced by melatonin. C 2010 Wiley-Liss, Inc. Anat Rec, 293:871–878, 2010. V Key words: Lagostomus maximus maximus; pars tuberalis; ultrastructure; photoperiod; melatonin Grant sponsor: Universidad Nacional de San Luis; Contract grant number: Proyecto 22/Q603 CyT. *Correspondence to: Edith Perez Romera, Cátedra de Anatomı́a Humana, Facultad de Quı́mica, Bioquı́mica y Farmacia, Universidad Nacional de San Luis, Av. Ejército de los Andes 950–2 Piso, (5700) San Luis, Argentina. Fax: 54-2652-422644/ 430224. E-mail: firstname.lastname@example.org C 2010 WILEY-LISS, INC. V Received 25 June 2009; Accepted 15 October 2009 DOI 10.1002/ar.21083 Published online in Wiley InterScience (www.interscience.wiley. com). 872 ROMERA ET AL. INTRODUCTION The pituitary pars tuberalis (PT) is a glandular zone exhibiting well-deﬁned structural characteristics (Dellmann et al., 1974; Fitzgerald, 1979; Gross, 1984; Stoeckel and Porte, 1984). Its abundant irrigation and the presence of cells containing dense secretory vesicles demonstrate its important secretory activity. In all the studied mammals, the PT is a thin cellular sheath surrounding the pituitary stalk and extending along the basal surface of the median eminence. Morphologically, it is constituted by speciﬁc secretory cells (the so-called PT-speciﬁc cells), folliculostellate cells and migratory cells coming from the pars distalis (PD) (Oota and Kurosomi, 1966; Cameron and Foster, 1972; Stoeckel and Porte, 1984). The viscacha pituitary PT exhibits a parenchyma with a well-deﬁned histoarchitecture and a well-developed capillary plexus, probably belonging to the hypothalamus-pituitary portal system. The capillaries extend all along the pituitary zone. The PT cells are arranged longitudinally in cords and separated by blood capillaries. Observation with the transmission electron microscope reveals the presence of speciﬁc granulated cells (PT-speciﬁc cells), agranulated cells and folliculostellate cells. Two types of speciﬁc granulated cells can be distinguished: cells with large secretory granules ranging from 150 to 500 nm (PT-speciﬁc cells Type I), and cells with small secretory granules between 65 and 200 nm (PT-speciﬁc cells Type II). Agranulated cells are distributed along the entire PT. Folliculostellate cells are arranged in follicles. The plasma membrane exhibits microvilli that project into the lumen, which displays colloidal-like material. All the described cellular types exhibit deposits of cytoplasmic glycogen. Numerous nerve endings in contact with the plasma membrane are observed in the secretory cells (Perez Romera et al., 2005). The PT is characterized by the presence of melatonin receptors. Numerous studies have shown that the highest density of melatonin receptors has been found in the pituitary PT and in a projection from this region extending over the anteroventral PD, known as zona tuberalis (ZT) (Skinner and Robinson, 1995) of all the studied animals (Vanecek et al., 1987; Williams and Morgan 1988; Masson-Pevet et al., 1996; Morgan and Williams, 1996), including humans (Von Gall C et al., 2002; Wu YH et al., 2006). The PT-speciﬁc cells constitute the melatonin-responsive cell group (Morgan et al., 1991, 1994). Studies carried out in some species such as the hibernating garden door mouse (Dellmann et al., 1974) and the hedgehog (Rütten et al., 1988) have demonstrated circannual morphological changes in the PT-speciﬁc cells. Wittkowski et al. (1984) examined the ultrastructural aspects of the pituitary PT of the Djungarian hamsters maintained under long and short photoperiods, and Böckers et al. (1995) studied the effects of melatonin in the same species. Both studies showed changes in the ultrastructure of PT-speciﬁc cells. In our laboratory, previous studies have been conducted to determine the structural and ultrastructural characteristics of pituitary PD (Mohamed et al., 1995; 2000), pars intermedia (Scardapane, 1990) and PT of the viscacha (Perez Romera et al., 2005). However, studies on the inﬂuence of the photoperiod and melatonin administration on the PT have not been carried out in this species. TABLE 1. Environmental conditions during summer and winter H P (mm) T ( C) Summer (January–February) Winter (June-July) 10.93 100 22.5 6.11 11 10.25 The viscacha (Lagostomus maximus maximus) is a subterranean rodent belonging to the family Chinchillidae, living in the central zone of Argentina. These herbivorous animals, nocturnally active, emerge from their burrows during periods of darkness from dawn to dusk to feed on the surrounding vegetation (Llanos and Crespo, 1952). It has a seasonal reproductive cycle (Fuentes et al., 1991), with important participation of the pineal gland (Dominguez et al., 1987; Cernuda-Cernuda et al., 2003). Its reproductive activity occurs during the long days of summer and early autumn whereas on short winter days, these animals experience an important testicular regression. These changes at the central level affect the testicular and epididymal histophysiology (Muñoz et al., 1997, 2001; Aguilera Merlo et al., 2005). This study was performed to investigate differences in the ultrastructural characteristics of the pituitary PT of viscachas captured and killed in summer (long photoperiod, maximal gonadal activity) and winter (short photoperiod, minimal gonadal activity), as well as to determine the effects of chronic melatonin administration in viscachas captured in summer and kept under long photoperiod, in order to provide new data about the PT and its relation with the pineal-pituitary-gonadal axis in this photoperiod-dependent seasonal reproduction model. MATERIALS AND METHODS Animals and Tissue Preparation Seasonal study. Twelve adult male viscachas (Lagostomus maximus maximus) weighing 4–8 kg were captured in their natural habitat (six in summer; February–March and six in winter; July–August), near San Luis, Argentina (33 20’south latitude and 760 m altitude). Values of solar irradiation expressed as heliophany (H) and mean values of precipitations (P) and temperature (T) were provided by the Servicio Meteorológico Nacional San Luis (Table 1). The animals were captured at night (between 24:00 and 05:00 hr) using traps placed in their burrows and taken to the laboratory. Then, the viscachas were anesthetized with Nembutal (pentobarbital) and quickly decapitated (between 07:00 and 08:00 hr). The skull was opened, and the basal hypothalamus and pituitary gland were rapidly dissected out en bloc and ﬁxed by immersion. The specimens were ﬁxed in Karnovsky’s ﬂuid (Karnovsky, 1965), post-ﬁxed in 1% osmium tetroxide 2 hr at 4 C, washed in phosphate buffer, pH 7.2–7.4, dehydrated in acetone and embedded in Spurr plastic resin. Consecutive 1 lmthick sections were stained with 1% toluidine blue for morphological orientation. For electron microscopy, ultrathin sections were cut with a Porter-Blum ultramicrotome, contrasted with lead citrate and uranyl acetate (Millonig, 1961). The ultrastructural characteristics of PT were studied in detail under a Siemens Elmiscop I PHOTOPERIOD, MELATONIN AND PT OF VISCACHA electron microscope, and micrographs were captured for morphometric analysis. The reproductive condition of viscachas was carefully assessed on the basis of observations by light microscopy of testes. All male viscachas captured in winter were in the gonadal regression period. These results were similar to those previously found in our laboratory (Muñoz, 1998). The experimental design was approved by the local Ethics Committee and was in agreement with the guidelines of the National Institutes of Health (NIH, USA) for the use of experimental animals. Melatonin administration. Eight adult male viscachas captured during the month of February (summer) were used. The rodents were kept in isolated boxes with free access to water and food at 20 C 2 C. They were maintained under long photoperiods with a controlled light regimen (14L:10D, lights-on from 10:00 to 24:00 hr). The experimental group received two daily subcutaneous injections of melatonin (Sigma, 100 lg/kg body weight in oil solution) at 09:00 hr (lights-off), and 17:00 hr (lights-on), for 9 weeks. The control group received only the diluent. The animals were anesthetized with Nembutal (pentobarbital) and killed by decapitation at 08:00 hr (lights-off). Tissues were treated with the same histological techniques used for the seasonal study. The experimental design was carried out according to protocols previously used in viscachas in our laboratory (Scardapane et al., 1983; Muñoz, 1998; Mohamed et al., 2000; Filippa et al., 2005; Filippa and Mohamed, 2006a,b, 2008). In addition, in both groups, the histological study of the testes was carried out to conﬁrm the effect of melatonin on the reproductive status. In the melatonintreated viscachas an inhibitory effect of this hormone on the spermatogenic activity was observed. These results were similar to those previously found in this rodent (Muñoz, 1998). Morphometric analysis. Glycogen: A computerassisted image analysis system was used to measure the cytoplasmic area occupied by the glycogen in PT cells. The images were captured by a Siemens Elmiscop I electron microscope, scanned at resolutions yielding 800 dpi and processed with Image Pro Plus 5.0 software under control of a Pentium IV computer. The software allowed the following processes: automatic analogous adjust, thresholding, background subtraction, distance calibration, and area measuring. The image was displayed on a color monitor, and the cytoplasmic glycogen areas were measured. Before counting, the distance calibration was performed in lm, considering the magniﬁcation of the electronic micrographs and the number of pixels per inch. Twenty-ﬁve electronic micrographs were analyzed in each PT for the morphometric study. Finally, 300 micrographs were used for the seasonal study and 200, for the melatonin study. The percentage of cytoplasmic glycogen area P (% P GA) was calculated P using the formula % GA ¼ Ag/ At 100, where PAg was the sum of the cytoplasmic glycogen area and At was the sum of the cellular cytoplasmic area. The number of cells with degenerative processes was determined in the same micrographs previously described for the glycogen determination. The results were expressed as percentage of degenerating cells (%DC). 873 Statistical analysis. The results were expressed as % means standard error of the mean (SEM) for all data sets. Differences between summer-winter groups and experimental-control groups were evaluated using unpaired Student’s t test. A probability of less than 0.05 was assumed to be signiﬁcant. RESULTS Pars Tuberalis in Long Photoperiod (Summer) The ultrastructural study showed that both PT-speciﬁc cells types (Type I and II) exhibited an eccentric nucleus with regular edges and ﬁnely dispersed chromatin. They showed large round, oval and elongated mitochondria, rough endoplasmic reticulum, Golgi complex and glycogen particles which were scattered throughout the cytoplasm (Fig. 1b,c). Agranulated cells had nuclei with smooth contours of the nuclear membrane and ﬁnely distributed chromatin. A voluminous cytoplasm exhibited numerous oval and elongated mitochondria, Golgi complex and scarce rough endoplasmic reticulum, phagosomes and glycogen deposits (Fig. 1a). The cytoplasmic glycogen area was 2.7% 0.28%. Folliculostellate cells were characterized by dark and irregularly shaped nuclei, moderately condensed chromatin and elongated mitochondria. Pars Tuberalis in Short Photoperiod (Winter) The ultrastructural analysis showed that the PT-speciﬁc granulated cells Type I exhibited a cytoplasm with a great amount of large round and elongated mitochondria, a well-developed and often expanded rough endoplasmic reticulum and secretory granules with heterogeneous electronic density (Fig. 2b). They frequently exhibited great cytoplasmic vacuolization and irregularly shaped nuclei with clotted chromatin (Figs. 1e, 2d). Besides, a higher percentage of cells with degenerative processes (%DC) was observed in winter (17.6% 0.13%) in relation to summer (2.93% 0.08%, P < 0.001; Figs. 1e, 2c). The PT-speciﬁc granulated cells Type II showed light nuclei with smooth contours. The amount of mitochondria and rough endoplasmic reticulum was markedly reduced. Most of these cells exhibited a scarce amount of secretory granules (Fig. 2a). High amounts of glycogen either aggregated in clusters or scattered throughout the cytoplasm were observed in many cells (Fig. 1f). The cytoplasmic glycogen area was 6.32% 0.3%, showing signiﬁcant differences in relation to summer (P < 0.001). Few changes in the ultrastructural appearance of the agranulated cells were detected. Phagosomes, however, were more abundant (Fig. 1d). Apparent morphological differences of the folliculostellate cells were not found. Pars Tuberalis: Melatonin Administration The PT of the control group exhibited cell-like characteristics with an important secretory activity, a moderate amount of glycogen (3.5% 0.15%) and low %DC (3.13% 0.07%). These ultrastructural characteristics were similar to those observed in summer (Fig. 3a,b). In melatonin-treated animals, the PT-speciﬁc granulated cells showed ultrastructural differences similar to those observed in winter, with signs of a reduced 874 ROMERA ET AL. Fig. 1. (a–c) Electronic micrographs of pituitary pars tuberalis (PPT) of viscacha captured in the long photoperiod (summer). The glandular cells exhibit regular nuclei (N) of smooth edges with ﬁnely distributed chromatin. g, glycogen particles; m, mitochondria. (d–f) PPT of viscacha captured in the short photoperiod (winter). d. Note the abundant accumulation of lysosome-like bodies (arrow); g, glycogen. e. degen- erating PT-speciﬁc cell. This image is frequent in winter. *, dilated cisternae of rough endoplasmic reticulum. f. Cell with numerous deposits of glycogen aggregated in clusters (arrowhead). Magniﬁcation: a, d ¼ 2,000. Scale Bar ¼ 5 lm; b, e ¼ 2,500. Scale Bar ¼ 4 lm; c ¼ 5,000. Scale Bar ¼ 2 lm; f ¼ 4,000. Scale Bar ¼ 2.5 lm. PHOTOPERIOD, MELATONIN AND PT OF VISCACHA 875 Fig. 2. (a–d) Images of PT of viscacha captured in winter (short photoperiod). a. I, type I PT-speciﬁc cell; II, type II PT-speciﬁc cell. b. junction complex (arrow) among type I PT-speciﬁc cell and folliculostellate cells (F). c. follicular cavity ﬁlled with amorphous electron dense material. c. Cells in advanced state of degeneration. d. vacuoles formed by the great dilation of rough endoplasmic reticulum (*). N, irregular nucleus with condensed chromatin. Magniﬁcation: a ¼ 5,000. Scale Bar ¼ 2 lm; b, c ¼ 4,000. Scale Bar ¼ 2.5 lm; d ¼ 8,000. Scale Bar ¼ 1.25 lm. synthesis activity. The morphometric study of the cytoplasmic glycogen area in melatonin-treated animals showed a signiﬁcant increase in relation to the control group 7.5% 0.3% (P < 0.001). Nevertheless, the PT showed a higher amount of glycogen particles than in winter (Fig. 3b,c). Besides, the %DC was signiﬁcantly higher in relation to the control (17.2% 0.12%, P < 0.001). from the neuroendocrine neurons of the hypothalamus, suggesting a probable modulation of PT secretory cells on the PD glandular cells (Wittkowski et al., 1999). Morgan et al. (1992) studied the synthesis and secretion of proteins by using [35S] methionine in primary cultures of ovine PT speciﬁc cells. These authors showed that the accumulation of these proteins was enhanced by stimulation of PT cells with forskolin, and this effect was blocked by melatonin. Dellmann et al. (1974) carried out a comparative ultrastructural study of the PT of several species and observed regressive changes in the secretory cells of the garden door mouse during hibernation. This hibernating species showed a decrease in the secretory granules, a decrease in the rough endoplasmic reticulum and an aggregation of increasing amounts of glycogen granules. These ﬁndings suggested that the photoperiod might have some inﬂuence on the PT histophysiology. Wittkowski et al. (1984) studied the inﬂuence of the photoperiod on the PT ultrastructure of the Djungarian DISCUSSION Studies on different species have shown that the PT is a well-developed pituitary area with an important secretory activity, made evident by the presence of secretory granules and abundant irrigation (Oota and Kurosomi, 1966; Dellmann et al., 1974; Fitzgerald, 1979; Perez Romera et al., 2005). The secretory cells are in close contact with the primary plexus capillaries of the portal hypothalamic-pituitary system, together with nerve endings of ﬁbers coming 876 ROMERA ET AL. hamster and found alterations in the speciﬁc secretory cells of the animals exposed to short photoperiods. In the present work, the ultrastructural aspects of the viscacha PT were studied during the long (summer) and short (winter) photoperiods and after melatonin-chronic administration. The PT of viscacha has two types of PTspeciﬁc cells: Type I and Type II. These cells do not react with antibodies directed against the PD hormones (Perez Romera et al., 2005). Both cell types showed ultrastructural differences in relation to the photoperiod length. In winter, the PT speciﬁc cells type I showed great dilation of the endoplasmic reticulum. The abundant amount of cytoplasmic vacuoles and the presence of secretory granules in clusters observed in a great number of cells in different degradation stages are indicative of a local cytoplasmic degeneration known as crinophagy (Farquhar, 1977). The PT-speciﬁc cells type II showed signs of a reduced synthesis activity in winter. The marked glycogen increase indicated an energy reserve state, probably due to a decrease in the cellular activity. The PT of viscacha showed a higher number of lysosome-like bodies in winter in relation to summer, unlike the Djungarian hamster, which showed a decrease in the number of dense lysosome-like bodies in winter (Wittkowski et al., 1984). The changes are also comparable to those reported by an investigation of the hedgehog (Rütten et al., 1988). Chronic melatonin administration induced variations in PT of viscacha similar to those observed in winter. These ultrastructural characteristics found in viscacha are suggestive of functional differences of the PT in summer and in winter, and after the administration of melatonin. The high afﬁnity of melatonin receptors found in the PT-speciﬁc cells of several species (Vanecek et al., 1987; Williams and Morgan, 1988; Masson-Pevet et al., 1996; Morgan and Williams, 1996) suggested that the pineal melatonin might be involved in these differences. In other seasonal breeders, pronounced photoperioddriven seasonal changes occur in the levels of prolactin secretion, and these effects are thought to be mediated by the pineal hormone melatonin, which acts as a humoral indicator of the photoperiod. Therefore, factors released by the PT might regulate the activity of lactotrophs in the PD (Wittkowski et al., 1992; Morgan et al., 1996). Hazlerigg et al. (1996) investigated this hypothesis using a range of co-culture and medium-conditioning experiments on primary cultures of ovine PT and PD cells. They reported that PT cells secreted an unidentiﬁed factor that is a potent stimulus of prolactin secretion by PD cells. Lafarque et al. (1998) reported that the active factor/s should have a molecular weight higher than 30 kDa. Melatonin might directly regulate the synthesis and liberation of this unidentiﬁed factor, which acts on the PD cells, mainly the lactotrophs, thus regulating the synthesis and liberation of prolactin (Wittkowski et al., 1999). This hypothesis is structurally supported by the studies on the hypothalamus–pituitary disconnected ram. These studies suggest that photoperiodic modulation of prolactin Fig. 3. a. Characteristic aspect of pars tuberalis of viscacha maintained under long photoperiod (control). PT-speciﬁc cell with typical smooth outlines of nucleus (N). The cytoplasm shows a well-developed rough endoplasmic reticulum, mitochondria and secretory granules. (b, c) Characteristic aspect of pars tuberalis of melatonin-treated viscachas. b. Nucleus of secretory cell (N) is irregularly formed and shows deep invaginations. High amounts of glycogen aggregated in clusters and scattered throughout the cytoplasm (arrow). c. Typical aspect of secretory cells of PT. Cytoplasm ﬁlled with dense accumulation of glycogen particles (arrow); N, nucleus. Magniﬁcation: a ¼ 5,000. Scale Bar ¼ 2 lm; b ¼ 5,500. Scale Bar ¼ 1.82 lm; c ¼ 4,000. Scale Bar ¼ 2.5 lm. PHOTOPERIOD, MELATONIN AND PT OF VISCACHA secretion can occur independently from the hypothalamus, presumably due to melatonin direct effects on the anterior pituitary (Lincoln and Clarke, 1994, 1995). It has also been demonstrated in most species that the PT-speciﬁc cells express the common alpha chain of glycoprotein hormones (Böckers et al., 1994; Stoeckel et al., 1994) and thyrotrophin beta chain (TSHb). The PT TSHpositive cells were indeed different from PD TSH-positive cells (Sakai et al., 1999; Wittkowski et al., 1999; Bockman et al., 1997). Recent studies have demonstrated that the TSHb secreted by PT controls the expression of type II and type III thyroid hormone deiodinase (Dio2) and (Dio3) in the ependymal cell layer of the infundibular recess (EC) via TSH receptors (TSHr) (Nakao et al., 2008). Melatonin-dependent regulation of thyroid hormone levels in the mediobasal hypothalamus appears to involve TSH in mammals. There is clear evidence in mice that TSH participates in this photoperiodic signal transduction (Ono et al., 2008). Hanon et al. (2008) demonstrated in Soay sheep that the TSH-expressing cells of the PT play an ancestral role in seasonal reproductive control in vertebrates. In mammals, this role provides the missing link between the pineal melatonin signal and thyroid-dependent seasonal biology. It has recently been suggested that melatonin affects the expression of Dio2 and Dio3 in EC due to the action on the receptor MT1 localized in the PT. (Yasuo et al., 2009). Unfried et al. (2009) postulated a model depicting the autocrine/paracrine pathway of TSH derived from the PT. Melatonin acts through the MT1 receptor and activates expression of the TSHr in the PT. The expression of TSHb mRNA in the PT is inhibited by melatonin and the molecular clockwork. Thyroid-stimulatin hormone acts retrogradely in the EC in which it activates Dio2 expression via phosphorylated cAMP response element-binding protein (pCREB) signaling. DIO2 converts T4 into T3, which facilitates the release of gonadotropin-releasing hormone (GnRH) into the hypothalamopituitary portal system. This mechanism might be important for ﬁne-tuning of seasonal reproduction. Previous studies have demonstrated that the viscacha showed maximal pineal activity in winter and minimal activity in summer (Dominguez et al, 1987; Fuentes et al, 2003). Also, it exhibited in its photoperiod-dependent reproductive cycle maximal gonadal activity in summer and minimal in winter (Fuentes et al., 1991; Muñoz et al., 1997, 2001). In PD, the LH gonadotrophs, somatotrophs, corticotrophs, thyrotrophs, and lactotrophs showed lower cellular activity in winter. In addition, LH gonadotrophs, corticotrophs, somatotrophs, and lactotrophs decreased their activity after melatonin administration (Filippa et al., 2005; Filippa and Mohamed, 2006a,b, 2008; Filippa V, 2008). In this work, we demonstrate that seasonal ultrastructural differences occur in the PT of the viscacha and that melatonin-chronic administration induces variations in the PT similar to those occurring during the short photoperiod (winter). These results are in agreement with the variations of the pituitary-gonadal axis, probably in response to the changes in the natural photoperiod through the pineal melatonin. In addition, our results allow us to propose the viscacha (Lagostomus maximus maximus) as a new and interesting model for future studies of the PT and its participation in the complex mechanism of reproduction regulation through the environmental signals. 877 ACKNOWLEDGEMENT The authors wish to thank Mr. J. Arroyuelo and Mr. N. Perez for their technical participation. LITERATURE CITED Aguilera-Merlo C, Muñoz E, Dominguez S, Scardapane L, Piezzi R. 2005. Epididymis of viscacha (Lagostomus maximus maximus): morphological changes during the annual reproductive cycle. Anat Rec A Discov Mol Cell Evol Biol 282A:83–92. Böckers TM, Bockman J, Fauteck JD, Kreutz MR, Bock R, Wittkowski W. 1994. Pars tuberalis-speciﬁc cells in the ovine pituitary do express the common a-chain of glycoprotein hormones: an in situ hybridization and immunocytochemical study. Eur J Endocrinol 131:540–546. Böckers TM, Niklowitz P, Bockmann J, Fauteck JD, Wittkowski W, Kreutz MR. 1995. Daily melatonin injections induce cytological changes in pars tuberalis speciﬁc cells similar to short photoperiod. J Neuroendocrinol 7:607–613. Bockmann J, Böckers TM, Winter C, Wittkowski W, Winterhoff H, Deufel T, Kreutz MR. 1997. Thyrotropin expression in hypophyseal pars tuberalis-speciﬁc cells is 3,5,30 -triiodothyronine, thyrotropin-releasing hormone, and pit-1 independent. Endocrinology 138:1019–1028. Cameron E, Foster CL. 1972. Some light and electron microscopical observations on the pars tuberalis of the pituitary gland of the rabbit. J Endocrinol 54:505–511. Cernuda-Cernuda R, Piezzi RS, Dominguez S, Alvarez-Urı́a M. 2003. Cell populations in the pineal gland of the viscacha (Lagostomus maximus maximus). Seasonal variations. Histol Histopathol 18:827–836. Dellmann HD, Stoeckel ME, Hindelang-Gertner C, Porte A, Stutinsky F. 1974. A comparative ultrastructural study of the pars tuberalis of various mammals, the chicken and the newt. Cell Tissue Res 148:313–329. Dominguez S, Piezzi R S, Scardapane L, Guzmán JA. 1987. A light and electron microscopic study of the pineal gland of the vizcacha (Lagostomus maximus maximus). J Pineal Res 4:211–219. Farquhar MG. 1977. Secretion and crinophagy in prolactin cells. In: Dellmann D, Johnson JA, Klachko DM. Comparative endocrinology of prolactin. Plenum: New York, p 37–94. Filippa V. 2008. Histoﬁsiologı́a de la pars distalis hipoﬁsiaria del Lagostomus maximus maximus: relación con el fotoperı́odo, edad y sexo. Doctoral Thesis, Biblioteca Central Universidad Nacional de San Luis, San Luis, Argentina. Filippa V, Mohamed F. 2006a. ACTH cells of pituitary pars distalis of viscachas (Lagostomus maximus maximus): immunohistochemical study in relation to season, sex, and growth. Gen Comp Endocrinol 146:217–225. Filippa V, Mohamed F. 2006b. Immunohistochemical study of somatotrophs in pituitary pars distalis of viscachas (Lagostomus maximus maximus) in relation to the gonadal activity. Cell Tissues Organs 184:188–197. Filippa V, Mohamed F. 2008. Immunohistochemical and morphometric study of pituitary pars distalis thyrotrophs of male viscacha (Lagostomus maximus maximus): Seasonal variations and effect of melatonin and castration. Anat Rec (Hoboken) 291:400–409. Filippa V, Penissi A, Mohamed F. 2005. Seasonal variations of gonadotropins in the pars distalis male viscachas pituitary. Effect of chronic melatonin treatment. Eur J Histochem 49:291–300. Fitzgerald KT. 1979. The structure and function of the pars tuberalis of the vertebrate adenohypophysis. Gen Comp Endocrinol 37:383–399. Fuentes LB, Caravaca N, Pelzer LE, Scardapane L, Piezzi RS, Guzmán JA. 1991. Seasonal variations in the testis and epididimys of viscacha (Lagostomus maximus maximus). Biol Reproduction 45:493–497. Fuentes LB, Moller M, Muñoz E, Calderón C, Pelzer L. 2003. Seasonal variations in the expression of the mRNA encoding b1-adrenoceptor and AA-NAT enzyme, and in the AA-NAT activity in the 878 ROMERA ET AL. pineal gland of vizcacha (Lagostomus maximus maximus). Correlation with serum melatonin. Biol Rhythm Res 34:193–206. Gross DS. 1984. The mammalian hypophyseal pars tuberalis: a comparative immunocytochemical study. Gen Comp Endocrinol 56:283–298. Hanon EA, Lincoln GA, Fustin JM, Dardente H, Masson-Pevet M, Morgan PJ, Hazlerigg DG. 2008. Ancestral TSH mechanism signals summer in a photoperiodic mammal. Curr Biol 18:1147– 1152. Hazlerigg DG, Hastings MH, Morgan PJ. 1996. Production of a prolactin releasing factor by the ovine pars tuberalis. J Neuroendocrinol 8:489–492. Karnovsky MJ. 1965. A formaldehyde-glutaraldehyde ﬁxate of high osmolarity for use in electron microscopy. J Cell Biol 27:137–8A. Lafarque M, Oliveros L, Aguado L. 1998. Efecto de secreciones de pars tuberalis de la adenohipoﬁsis sobre la liberación de prolactina desde pars distalis. Medicina 58:36–40. Lincoln GA, Clarke IJ. 1994. Photoperiodically-induced cycles in the secretion of prolactin in hypothalamo-pituitary disconnected rams: evidence for translation of the melatonin signal in the pituitary gland. J Neuroendocrinol 6:251–260. Lincoln GA, Clarke IJ. 1995. Evidence that melatonin acts in the pituitary gland through a dopamine-independent mechanism to mediate effects of daylength on the secretion of prolactin in the ram. J Neuroendocrinol 7:637–643. Llanos AC, Crespo JA. 1952. Ecologia de la vizcacha (Lagostomus maximus maximus) en el nordeste de la provincia de Entre Rios. Rev Invest Agric 10:5–95. Masson-Pevet M, Gauer F, Recio J. 1996. Melatonin receptors, pars tuberalis and photoperiodic response. Front Horm Res 21:84–89. Millonig GA. 1961. A modiﬁed procedure for lead staining of thin sections. J Biophys Biochem Cytol 11:736–739. Mohamed F, Fogal T, Dominguez S, Scardapane L, Guzmán J, Piezzi RS. 1995. Ultrastructure of pars distalis of viscacha (Lagostomus maximus maximus). Pituitary cellular types. Biocell 19:133–140. Mohamed F, Fogal T, Dominguez S, Scardapane L, Guzmán J, Piezzi RS. 2000. Colloid accumulations in the pituitary pars distalis of viscacha (Lagostomus maximus maximus). A numeric study in relation to season, sex and growth. Anat Rec 258:252–261. Morgan PJ, Barrett P, Davidson G, Lawson W. 1992. Melatonin regulates the synthesis and secretion of several proteins by pars-tuberalis cells of the ovine pituitary. J Neuroendocrinol 4:557–563. Morgan PJ, Barrett P, Howell HE, Helliwell R. 1994. Melatonin receptors: localization, molecular pharmacology and physiological signiﬁcance. Neurochem Int 24:101–146. Morgan PJ, King TP, Lawson W, Slater D, Davidson G. 1991. Ultrastructure of melatonin-responsive cells in the ovine pars tuberalis. Cell Tissue Res 263:529–534. Morgan PJ, Webster CA, Mercer JG, Ross AW, Hazlerigg DG, MacLean A, Barrett P. 1996. The ovine pars tuberalis secretes a factor(s) that regulates gene expression in both lactotropic and nonlactotropic pituitary cells. Endocrinology 137:4018–4026. Morgan PJ, Williams LM. 1996. The pars tuberalis of the pituitary: a gateway for neuroendocrine output. Rev Reprod 1:153–161. Muñoz E. 1998. Estudio estacional del compartimiento tubular e intersticial del testı́culo de vizcacha (Lagostomus maximus maximus): papel de la melatonina en la ciclicidad reproductiva y su relación con el fotoperı́odo. Doctoral Thesis, Biblioteca Central Universidad Nacional de San Luis, San Luis, Argentina. Muñoz E, Fogal T, Dominguez S, Scardapane L, Guzmán J, Piezzi R. 1997. Seasonal changes of the Leydig cells of viscacha (Lagostomus maximus maximus). A ligth and electron microscopy study. Tissue Cell 29:119–128. Muñoz E, Fogal T, Dominguez S, Scardapane L, Piezzi R. 2001. Ultrastructural and morphometric study of the Sertoli cell of the viscacha (Lagostomus maximus maximus) during the annual reproductive cycle. Anat Rec 262:176–185. Nakao N, Ono H, Yamamura T, Anraku T, Takagi T, Higashi K, Yasuo S, Katou Y, Kageyama S, Uno Y, Kasukawa T, Iigo M, Sharp PJ, Iwasawa A, Suzuki Y, Sugano S, Niimi T, Mizutani M, Namikawa T, Ebihara S, Ueda HR, Yoshimura T. 2008. Thyrotrophin in the pars tuberalis triggers photoperiodic response. Nature 452:317–322. Ono H, Hoshino Y, Yasuo S, Watanabe M, Nakane Y, Murai A, Ebihara S, Korf HW, Yoshimura T. 2008. Involvement of thyrotropin in photoperiodic signal transduction in mice. Proc Natl Acad Sci USA 105:18238–18242. Oota Y, Kurosomi K. 1966. Electron microscopic studies on the pars tuberalis of the rat hypophysis. Arch Histol Jpn 27:501–520. Perez Romera E, Mohamed F, Filippa V, Fogal T, Dominguez S, Scardapane L, Piezzi RS. 2005. Ultrastructural and immunocytochemical studies of the viscacha (Lagostomus maximus maximus) pituitary pars tuberalis. Anat Rec A Discov Mol Cell Evol Biol 284A:431–438. Rütten A, Hewing M, Wittkowski W. 1988. Seasonal ultrastructural changes of the hypophyseal pars tuberalis in the hedgehog (Erinaceus europaeus L.) Acta Anat 133:217–223. Sakai T, Sakamoto S, Ijima K, Matsubara K, Kato Y, Inoue K. 1999. Characterization of TSH-positive cells in foetal rat pars tuberalis that fail to express Pit-1 factor and thyroid hormone beta2 receptors. J Neuroendocrinol 11:187–193. Scardapane L. 1990. Aspectos histoﬁsiológicos de la pars intermedia hipoﬁsiaria del Lagostomus maximus maximus. Doctoral Thesis, Biblioteca Central Universidad Nacional de San Luis, San Luis, Argentina. Scardapane L, Lucero J B, Dominguez S, Piezzi RS, Guzmán JA. 1983. Effect of chronic administration of melatonin on viscacha pars intermedia (Lagostomus maximus maximus). Com Biol 2:183–188. Skinner DC, Robinson JE. 1995. Melatonin-binding sites in the gonadotroph enriched zona tuberalis of ewes. J Reprod Fertility 104:243–250. Stoeckel ME, Hindelang C, Klein MJ, Poissonnier M, Felix JM. 1994. Expression of the alpha-subunit of glycoprotein homones in the pars tuberalis-speciﬁc glandular cells in rat, mouse and guinea-pig. Cell Tissue Res 278:617–624. Stoeckel ME, Porte A. 1984. Ultrastructure and development of the pars tuberalis of the mammals. In: Motta PM, editor. Ultrastructure of endocrine cells and tissues. Nijhoff: Boston. p 29–38. Unfried C, Ansari N, Yasuo S, Korf HW, von Gall C. 2009. Impact of melatonin and molecular clockwork components on the expression of thyrotropin beta chain (Tshb) and the Tsh receptor in the mouse pars tuberalis. Endocrinology 150:4653–4662. Vanecek J, Pavlik A, Illnerova H. 1987. Hypothalamic melatonin receptor sites revealed by autoradiography. Brain Res 435(1–2): 359–362. Von Gall C, Stehle JH, Weaver DR. 2002. Mammalian melatonin receptors: molecular biology and signal transduction. Cell Tissue Res 309:151–162. Williams LM, Morgan PJ. 1988. Demonstration of melatonin-binding sites on the pars tuberalis of the rat. J Endocrinol 119:Rl–R3. Wittkowski W, Bockman J, Kreutz MR, Böckers TM. 1999. Cell and molecular biology of the pars tuberalis of the pituitary. Int Rev Cytol 185:157–194. Wittkowski W, Hewing M, Hoffmann K, Bergmann M, Fechner J. 1984. Inﬂuence of photoperiod on the structure of the hypophyseal pars tuberalis of the Djungarian hamster, Phodopus sungorus. Cell Tissue Res 238:213–216. Wittkowski W, Schulze-Bonhage A, Böckers T. 1992. The pars tuberalis of the hypophyis: a modulador of the pars distalis? Acta Endocrinol (Copenh) 126:285–290. Wu YH, Zhou JN, Balesar R, Unmehopa U, Bao A, Jockers R, Van Heerikhuize J, Swaab DF. 2006. Distribution of MT1 melatonin receptor immunoreactivity in the human hypothalamus and pituitary gland: colocalization of MT1 with vasopressin, oxytocin, and corticotrophin releasing hormone. J Comp Neurol 499:897–910. Yasuo S, Yoshimura T, Ebihara S, Korf HW. 2009. Melatonin transmits photoperiodic signals through the MT1 melatonin receptor. J Neurosci 29:2885–2889.