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 Morgan1988; 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.
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
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 μm-thick 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 electron microscope, and micrographs were captured for morphometric analysis.
Table 1. Environmental conditions during summer and winter
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.
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 μg/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 melatonin-treated 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).
Glycogen: A computer-assisted 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 μm, 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 (% GA) was calculated using the formula % GA = ∑Ag/∑At × 100, where ∑Ag 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).
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.
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 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).
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 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 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 PT-specific 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 photoperiod-driven 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 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 TSH-positive 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 hypothalamo-pituitary 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.
The authors wish to thank Mr. J. Arroyuelo and Mr. N. Perez for their technical participation.