Ontogeny of Adenohypophyseal Cells, Pituitary Gland Development and Structure in Adults of Astyanax Lacustris (Teleostei, Characidae): an Emerging Neotropical Fish Species

Pituitary gland morphogenesis of the adenohypophyseal (AH) cells of Astyanax lacustris are presented herein. This Characiformes species show great ecological and commercial importance, and it has been increasingly used as a biological model. The rst AH cells of A. lacustris were detected at 1 dah by the immunostaining of PRL producing cells. The morphology of the gland presented changes in shape throughout the development, starting elongated but more oval at the end. The neurohypophysis was differentiated at 3 dah, along with the identication of ACTH, MSH, TSH, and FSH producing cells. Identication of the immunoreactive cells to anti-LH, anti-SL, and anti-GH antibodies occurred at 5 dah. At 20 dah, an increase in pituitary size and the presence of the pituitary stalk were observed. At 60 dah, the pituitary already had the same shape seen in adults. The ontogeny of adenohypophyseal cells in A. lacustris corroborates the heterogeneity in the appearance of these cell types in teleosts and suggests that these hormones actively participate during the early development of this species. Our results collaborate with the understanding of the morphogenesis of the hypothalamic-pituitary-gonadal axis in South American teleosts, showing essential data for the development of future studies related to pituitary morphophysiology.


Introduction
The pituitary gland is considered the "master gland" of vertebrates (Schreibman 1986) since the hormones produced by its several adenohypophyseal (AH) cells play a crucial role on the neuroendocrine control of all endocrine axes. So, this gland is involved in the early development, growth, reproduction, metabolism adaptation to the environment, as well as to stress responses (Kawauchi and Sower 2006).
In teleost, peptides produced by the hypothalamic nuclei are transported by amyelinic bers through the neurohypophysis (NH) branches to be released adjacent to the AH cells, since sh do not have a  Schreibman 1986). Since the early development of the embryonic period is variable between different species and groups, this may result in a heterochrony in the activation of the endocrine axis (Agulleiro et al. 2006;Laiz-Carrión et al. 2003 Pandol et al. 2001Pandol et al. , 2003. The yellow tail tetra Astyanax lacustris (Chaciformes, Characidae) is a South American species widely used in sh farming and human consumption. Due its small size, easy handling, early sexual maturation, sexual dimorphism, and multiple spawning, this species has been pointed out as an emerging Neotropical sh model species. An increasing number of data has been accumulated about its biology, In this study we used Astyanax lacustris as a model species to analyze the pituitary gland development, the ontogeny of AH cells, and its topographic organization in adults. Our results provide, then, the basis for further studies related to the endocrine axis mediated by the pituitary gland in this Neotropical species.
All specimens were euthanized by benzocaine overdose. Larvae until 12 dah had the whole body xed; late larvae, juveniles, and adults had only their head xed, after jaw extraction and brain exposition. All samples were xed by immersion in Bouin's solution for 24 hours at room temperature. All experimental procedures were performed according to the procedure approved by the Ethics Committee on Animal Experiments of the Institute of Biomedical Sciences (ICB) of the University of Sao Paulo (USP), #53/2013. After xation, samples were washed in running water to remove the excess of xative solution. Head fragments were decalci ed in RDO Gold (Apex) according to the instructions of the manufacturer. Then, all samples were dehydrated in increasing concentrations of ethanol, diaphanized in xylene, and embedded in Paraplast. To further use in histochemical and immunohistochemical analyzes, serial 5 µm sections were obtained and placed on poly-L-lysine-coated slides.
For histochemistry, some sections were depara nized in xylene, rehydrated in ethanol series, and stained with Mallory trichrome. All sections were dehydrated, mounted, and analyzed using an Olympus BX51 light microscope coupled with a digital QColor5 camera (Olympus) and equipped with Image Pro-Plus digital image capture software.

Immunohistochemistry
After a previous selection, the sections with the pituitary gland were depara nized in xylene and rehydrated in ethanol series and phosphate-buffered saline (PBS, pH 7.4). Endogenous peroxidase and non-speci c binding sites blockages were performed using the solutions from the Kit Spring Reveal -Polyvalent Free Biotin-DAB (SPD-125). Sections were then incubated overnight in a humid chamber at 4°C using different polyclonal primary antibodies diluted in PBS. The primary antibodies and the dilutions used herein are detailed in Table 1. Host species: a Antibody produced in rabbit; b Antibody produced in rat. After that, sections were washed in PBS and incubated with the Complement and Conjugated solutions from the same Kit Spring Reveal, according to the instructions of the manufacturer. Sections were rewashed in PBS, revealed with 3,3'-diaminobenzidine (DAB), and counterstained with Harrys hematoxylin. Finally, slides were mounted, analyzed, and photo documented, as already described. The primary antibodies were omitted in some sections as negative control. The speci city of each antibody may also be veri ed in previous studies, in which all these antibodies have already been characterized (Table 1).

Results
All the AH cells were detected in Astyanax lacustris during the rst days after hatching, as shown in Table  2. In general, the different cell populations were found presenting a few cells during their rst appearance.
We are not able to detect the pituitary anlage (Fig. 1A). However, at 1 dah we observed a small group of cells strongly immunostained by the anti-PRL antibody, the PRL cells, which are shown as small cells with a large, central nucleus ( Fig. 1B).
The pituitary could be distinguished from the hypothalamus at 2.5 dah being a round gland ventrally located in relation to the brain (Fig. 1C). At 3 dah, the pituitary gland presented an elongated shape, where the early segregation of the pituitary regions can be seen, i.e. the glandular portion, or adenohypophysis (AH), and the nervous portion, or neurohypophysis (NH) (Fig. 1D). In parallel, we also detected the rst appearance of ACTH, MSH, TSH, and FSH-expressing cells. ACTH and MSH cells were immunostained by the same antibody, but the two groups of positive cells were seen in the anterior and posterior regions of the pituitary, respectively (Fig. 1E). On the other hand, in the central region of the pituitary gland, a reduced group cells were recognized, the TSH cells (Fig. 1F). The FSH cells, the rst type of gonadotropic cell to appear during the larval period, were immunodetected in the border of the pituitary gland (Fig. 1G).
At 5 dah, an increase in the size of the gland was noted (Fig. 1H). In addition, the three remaining AH cells, the SL, GH, and LH cells were detected at this phase. The SL cells were immunodetected in the posterior region of the pituitary gland (Fig. 1I), whereas few GH cells were detected in its central region (Fig. 1J). The LH cells (Fig. 1K) were round and more numerous than the rst detected FSH cells. The LH cells were also distributed in the central region of the pituitary gland.
From 20 dah on, the specimens were considered as juveniles. At this time, the pituitary was located on the oor of the hypothalamus where the development of the stalk was seen ( Fig. 2A). At this stage, the three AH lobes were distinguished -rostral pars distalis (RPD), proximal pars distalis (PPD) and pars intermedia (PI) -as they displayed different staining properties when submitted to the Mallory trichrome technique ( Fig. 2A). In addition, an increase in the area occupied by the NH was observed, as well as its rami cation by the three AH lobes.
In this phase, a stronger immunostaining in PRL cells was noted (Fig. 2B). The ACTH and MSH cells were elongated with an eccentric nucleus, and ACTH cells presented a granular immunostaining (Fig. 2C). We found some elongated TSH cells in the PPD lobe, where these cells were isolated or grouped (Fig. 2D). Elongated FSH producing cells were found in the PPD and RPD lobes (Fig. 2E). The SL cells were round cells with a lighter immunostaining than when they were rst detected and were seen on the periphery of PI lobe (Fig. 2F). The GH cells presented a strong labeling and were distributed throughout the PPD lobe (Fig. 2G). Finally, few LH cells were found in the PPD lobe (Fig. 2H).
Until 30 dah, the pituitary did not show marked changes in its size and shape. However, from then on, the pituitary development had a great increase in size, accompanied by an increase in each AH cell populations. At 60 dah and on (Fig. 3A), the pituitary gland showed a marked morphological change, acquiring an oval shape, similar to the morphology found in adult specimens of A. lacustris. The gland was bigger and connected to the hypothalamus by a short and thin stalk.
At the adult pituitary, the PRL cells were round cells with a large, central nucleus. A large population of PRL cells was found in the RPD lobe (Fig. 3B), especially in their border and close to blood vessels that were present in this region. On the other hand, ACTH cells were distributed between PRL cells, mostly around NH branches (Fig. 3C). These cells were found elongated with an eccentric nucleus. MSH cells were morphologically similar to the ACTH cells and were abundant in the PI lobe distributed along the NH branches (Fig. 3C). A large population of elongated TSH cells were observed in all PPD lobe extension (Fig. 3D). FSH and LH cells were distributed in the PPD lobe. FSH cell were seen in all PPD lobe (Fig. 3E), while LH cells were less numerous and distributed in the ventral region of PPD lobe, and in its border with the PI lobe (Fig. 3H). The SL cells were round cells with an eccentric nucleus located surrounding the blood vessels in the PI lobe (Fig. 3F). The GH cells maintained a strong labeling appearing in groups of rounded cells distributed throughout the PPD lobe (Fig. 3G).
The key aspects of pituitary gland development and the ontogeny of AH cell in A. lacustris are summarized in Figure 4.

Discussion
Once there are a few data related to the pituitary gland development in Neotropical sh species, this study provides a systematic and detailed description of the pituitary morphogenesis in the characid sh A. lacustris, since hatching until adult period, using histochemistry and immunohistochemistry methods. The POMC family hormones can be detected at different times, having been detected even at the time of hatching in Plecoglossus altivelis (Saga et al. 1999). In species in which these hormones were detected after hatching, this always occurs in early stages of larval development. It is known that ACTH acts on the adrenal glands promoting the release of glucocorticoids and cortisol, which, in turn, participate in important physiological processes, such as stress response, metabolism adjustment and osmoregulation (Wendellar-Bonga 1997). On the other hand, MSH promotes the dispersion of melanin granules (Agulleiro et al. 2006). Thus, the presence of these hormones seems to be related to the adaptation of the newly The thyroid hormone (TSH)-producing cells were also detected at 3 dah. Few studies have described the chronology of the appearance of these cells. TSH cells were detected in Oncorhynchus keta prior to hatching (Naito et al. 1993 TSH acts on the thyroid by regulating the synthesis of thyroxine (T4) and triiodothyronine (T3), which, in turn, regulate many physiological processes in sh (Peter 2011). TSH also acts during development, giving the body shape, head size, n and skin growth, scales, blood tissue, and liver development (Power et al. 2001). All these functions punctuate the need for TSH during the early development. The asynchrony in the appearance of FSH and LH cells in the different teleosts species reveals that these hormones are likely to be produced in distinct populations of AH cells, as occurs in C. dimerus (Pandol et al. 2006). In addition, at 20 dah in A. lacustris, the number of FSH cells was greater than the number of LH cells, which also corroborates with that assertion. The variation in the appearance and detection of these cells may still be related to the reproductive cycle, since the gonadotropic hormones produced by the pituitary gland act directly on the gonads and are still capable of controlling the production of sexual steroids (Agulleiro et al. 2006). The FSH and LH-producing cells in A. lacustris appeared before the beginning of sexual differentiation, as the gonadal differentiation occurs at 58 dah in females and at 72 dah in males, according to Adol et al. (2015). At 20 dah, a great synthesis activity of the gonadotropic hormones was observed in juveniles of A. lacustris. These results corroborate the ndings in Odontesthes bonariensis (Miranda et al. 2001) that had FSH and LH cells detected with 28 dah and 21 dah, respectively, suggesting that these hormones are produced before the gonadal differentiation (Grandi et al. 2014).
In A. lacustris, somatolactin (SL) cells were detected at 5 dah. These cells are responsible for producing somatolactin, a hormone only described in sh so far (Kawauchi and Sower 2006). These cells were also detected in D. GH-producing cells in A. lacustris were also detected at 5 dah. Immunostaining was initially observed in only a few cells. However, in the subsequent phases, the sections submitted to the anti-GH antibody presented intense immunostaining in almost the entire PPD lobe, and at 20 dah also in the PI lobe. GH was also detected at distinct times in different teleosts species. However, it was detected at the same stage of larval development as PRL-producing cells in C. dimerus (Pandol et al. 2001) and S. aurata (Villaplana et al. 2000). In addition, it has been reported in the literature that GH-producing cells were also found in PPD and PI lobes (Quesada et al. 1988 In teleosts, GH is the main hormone acting in the promotion of animal growth, being present in the rst stages of development. This is a crucial hormone for larval development, as it plays a role in energy mobilization and osmoregulation (Björnsson et al. 2002). Recent functional studies in zebra sh, using knockouts for GH, PRL, and SL, indicated that these hormones play an important role in the development and growth of the swim bladder, head, body, eyes, and melanophores (Zhu et al. 2007