Anatomy of the hypothalamic–pituitary axis and buccal lobe in the holocephalan Callorhinchus callorynchus

Anatomy of the hypothalamic–pituitary axis and buccal lobe in the holocephalan Callorhinchus callorynchus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Anatomy of the hypothalamic–pituitary axis and buccal lobe in the holocephalan Callorhinchus callorynchus Camila Harillo, Cynthia A. Awruch, Marcelo Santo, Gabriela C. López, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9052503/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Chimaeras (Holocephali) occupy a pivotal phylogenetic position for understanding the evolution of hypothalamic–pituitary organization in vertebrates; however, the structural organization of the hypothalamic–pituitary axis and the anatomical relationships with the buccal lobe (BL) remain incompletely characterized. We present an anatomo-histological analysis of the chimaera Callorhinchus callorynchus using classical histology, immunohistochemistry, and RT-PCR. The pituitary gland displays clear regionalization into the rostral pars distalis , proximal pars distalis , and neurointermediate lobe. The BL is connected to the posterior telencephalon by paired canals containing a prominent blood vessel and bundles of neuropeptidergic fibers. Gonadotropin-releasing hormone (GnRH)-immunoreactive nerve cell bodies and fibers were identified in the posterior telencephalon using two antisera, with fibers projecting to the proximal pars distalis of the pituitary and the BL. Secretoneurin (SN)-immunoreactive neuronal somata and fibers were observed in the posterior telencephalon and hypothalamus, and SN-immunoreactive cells were also present within the pituitary and BL. Cholecystokinin (CCK)-immunoreactive neuronal elements were detected in the hypothalamus, while CCK-immunoreactive cells were present in the pituitary, and a subset of fibers extending into the BL. RT-PCR analyses revealed the presence of fshb and lhb transcripts in the pituitary and BL, with a stronger apparent signal in the BL. These observations provide anatomical evidence for direct neuropeptidergic innervation of both the pituitary and BL and indicate that the BL is associated with gonadotropin subunit gene expression in C. callorynchus . This new anatomical framework of the hypothalamus–pituitary–BL axis identifies two new pathways: direct GnRH and CCK innervation, and local SN-mediated autocrine/paracrine signaling. Holocephali Chimaera GnRH SN CCK Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction In vertebrates, the hypothalamus and the pituitary gland play key roles in integrating internal and environmental signals that regulate reproduction (Trudeau and Somoza 2020 ). Comparative studies of the vertebrate hypothalamic–pituitary (HP) axis reveal a largely conserved anatomical organization and hormonal signaling pathways (Dufour et al. 2020 ; Trudeau & Somoza 2020 ; Santiago-Andres et al. 2021 ). However, a major gap remains in our understanding of the HP axis in chondrichthyans (Awruch 2013 ; Trudeau and Somoza 2020 ), making claims of evolutionary conservation tentative. Chondrichthyans diverged from bony vertebrates approximately 450 Ma ago (Inoue et al. 2010 ) and are represented today by two extant subclasses, Elasmobranchii (sharks, rays, and skates) and Holocephali (chimaeras). Owing to their long evolutionary history and distinctive brain characteristics, which differ markedly from both tetrapod and teleost lineages (Northcutt 1977 ; Smeets et al. 1983 ; Trudeau and Somoza 2020 ; Fontaine et al. 2022 ), chondrichthyans represent an important model for comparative studies of the HP axis. Nevertheless, detailed anatomical studies of the HP axis in this group remain scarce and are largely restricted to sharks and rays, leaving chimaeras poorly studied (Holmes and Ball 1974 ; Northcutt 1977 ; Smeets et al. 1983 ; Dodd and Dodd 1985 ; Santos-Durán et al. 2015 ; 2022 ). The pituitary gland is classically subdivided into the adenohypophysis, the pars intermedia (which is reduced or even absent in mammals), and the neurohypophysis (Page 2006). In chondrichthyans, as in teleosts, pituitary anatomy differs because the adenohypophysis is subdivided into the rostral pars distalis (RPD), proximal pars distalis (PPD), and a well-developed pars intermedia (Trudeau and Somoza 2020 ; Fontaine et al. 2022 ). In these groups, the pars intermedia is interdigitated by neurohypophyseal nerve terminals and termed the neuro-intermediate lobe (NIL; Meurling 1962 ). Chondrichthyans also possess distinctive glands: the ventral lobe (VL) in elasmobranchs, which is ventrally attached to the PPD, and the buccal lobe (BL) in chimaeras, located in the roof of the oral cavity and separated from the brain by a cartilaginous barrier (Smeets et al. 1983 ). Although the functions of the VL and BL remain poorly understood, available evidence indicates that luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are produced mainly in the VL of elasmobranchs (Quérat et al. 2001 ), and crude BL extracts from the chimaera Hydrolagus colliei show gonadotropin-like activity (Dodd et al. 1982 ). The preoptic area (POA), positioned immediately rostral to the hypothalamus (Puelles & Rubenstein 2015 ), plays a central role in the regulation of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) through the presence of gonadotropin-releasing hormone (GnRH)-expressing neurons (Muñoz-Cueto et al. 2020 ; Zohar et al. 2022 ). In tetrapods, GnRH fibers project to the median eminence (ME), where GnRH is released into the hypothalamic pituitary portal system and transported to the pituitary (Page 2006; Yin and Gore 2010 ). Along with other neurohormones and neurotransmitters, GnRH plays a central role in controlling the synthesis and secretion of LH and FSH by the adenohypophysis, thereby supplying critical endocrine signals to the gonads (Trudeau and Somoza 2020 ). Early studies proposed a portal system linking the hypothalamus and pituitary in elasmobranchs (Green 1951 ; Meurling 1960 ), and later observations supported a direct vascular connection between the ME and the pituitary (Holmes and Ball 1974 ; Dodd 1983 ; Smeets et al. 1983 ). However, no direct vascular connection has been demonstrated between the hypothalamus and either the VL in elasmobranchs (Dodd 1983 ) or the BL in holocephalans (Sathyanesan 1965 ; Jasiński and Gorbman 1966 ). Teleost fish illustrate divergence in HP communication systems. During evolution, they lost the ME, and GnRH neurons instead project to and enter the pars distalis, directly controlling gonadotropin (GtH) cells. Teleost GnRHs were therefore proposed as primary regulators of GtHs, with a stronger effect on LH than FSH cells (Zohar et al. 2022 ). However, recent studies using gene mutations of GnRH and GnRH receptors in zebrafish and medaka have questioned the essential role of GnRHs in reproductive regulation (Trudeau 2022 ; Zohar et al. 2022 ). By contrast, secretoneurin (SN), derived from selective processing of secretogranin-2 (Scg2), induces LH release in goldfish (Blázquez et al. 1998 ) and in mouse LβT2 cells (Zhao et al. 2011 ). Secretoneurin is of interest because only 10% of zebrafish pairs with double scg2a/scg2b frameshift mutations spawn (Mitchell et al. 2020 ), and SNa injection robustly activates the hypothalamus-pituitary-gonadal axis and induces ovulation in wild-type females (Peng et al. 2025). Likewise, recent studies have shown that cholecystokinin (CCK) also contributes to the regulation of FSH expression and secretion in medaka (Uehara et al. 2024 ) and zebrafish (Hollander-Cohen et al. 2024 ). CRISPR-mediated frameshift mutations in CCK precursors and a CCK receptor in medaka demonstrated that CCK is a critical regulator of ovarian and testicular development through FSH-mediated actions (Uehara et al. 2024 ). Whether these emerging examples represent a conserved vertebrate pathway requires further investigation in more ancient lineages. Chondrichthyans do not conform to either the typical tetrapod or teleost hypothalamic pituitary systems. In several elasmobranch and holocephalan species, GnRH fibers are detected throughout much of the brain, whereas GnRH cell bodies are concentrated in the terminal nerve, telencephalon, and midbrain (Lovejoy et al. 1992 ; Sherwood and Lovejoy 1993 ; D’Antonio et al. 1995 ; Forlano et al. 2000 ; Moeller and Meredith 2010 ; Masini et al. 2008 ). Evidence indicates that GnRH fibers terminate on blood vessels in the ventrocaudal telencephalon and are not associated with the chondrichthyan ME (Sherwood and Lovejoy 1993 ; D’Antonio et al. 1995 ). The prevailing hypothesis is that GnRH reaches the VL or BL via general circulation (Dodd 1983 ; Sherwood and Lovejoy 1993 ; D’Antonio et al. 1995 ). Beyond this, the neuropeptide-mediated hypothalamic regulation of pituitary function in chondrichthyans remains poorly understood, and several key questions persist (Trudeau and Somoza 2020 ; Santiago-Andres et al. 2021 ). Given these gaps in understanding in elasmobranchs and holocephalans, the present study provides a comprehensive anatomical and histological description of GnRH, SN, and CCK distributions in the HP axis and BL of the chimaera Callorhinchus callorynchus . This species belongs to one of the three extant holocephalan families, Callorhinchidae, which comprises three species worldwide (Finucci et al. 2021 ). Callorhinchidae has gained prominence in evolutionary studies following the sequencing of the Callorhinchus milii genome, which suggests an unusually low evolutionary rate among studied vertebrates (Venkatesh et al. 2005 , 2014 ). Material and methods Sampling . The project was authorized by the Secretaría de Pesca of the province of Chubut (permit N° 06/2023-DCPyA-SsP-SP) and the Institutional Committee for the Care and Use of Study Animals (CICUAE-CENPAT N° 11). Fourteen C. callorynchus (nine females and five males) were caught by angling by recreational fishermen along the shore of Puerto Madryn City, Argentina, between December 2021 to April 2022. Sex, total length (TL, measured from the rostral appendix to the beginning of the superior lobule of the caudal fin, cm), total weight (TW, g), and clasper calcification in males (assessed manually as either fully calcified or not) were recorded. According to Bernasconi et al. ( 2015 ), C. callorynchus are considered adults when they reach 47 cm TL for females, and 43 cm TL with fully calcified claspers for males. The whole brain was dissected in situ immediately following capture. RNA isolation and cDNA synthesis . The brains were dissected from two females and two males. The pituitary gland and the saccus vasculosus (SV) were dissected together, and the BL separately. Samples were preserved in RNAlater (Invitrogen™, USA) until total RNA extraction with the TransZol reagent (TransGen Biotech, China). After quantification and purification with DNase I (Invitrogen™, USA), RNA samples were reverse transcribed into cDNA by using M-MLV reverse transcriptase (Invitrogen™, USA), RNaseOUT (Invitrogen™, USA), and Oligo dT universal adaptor primer, following the manufacturer's protocol. Design of primers for RT-PCR . Different consensus primers were designed for the β-subunits of LH and FSH. For LHβ, the sequences of C. milii (XM_042345119), Pristis pectinata (XM_052042827.1), Scyliorhinus canicula (AJ310345), Chiloscyllium plagiosum (XM_043679302.1), Stegostoma fasciatum (XM_048521342.1), Rhincodon typus (XM_020512596), and Carcharodon carcharias (XM_041177731) were aligned to look for conserved regions. The same strategy was performed for FSHβ, aligning the sequences of C. milii (HQ174783), Amblyraja radiata (XM_033038641), P. pectinata (XM_052029671.1), S. canicula (AJ310344), and S. fasciatum (XM_048546119.1; Table 1 ). The PCR amplifications were performed with GoTaq DNA Polymerase (Promega, USA) with a program including an initial heat denaturation step at 94°C for 3 min, followed by 35 cycles of 30s at 94°C, 30s at 54°C, 45s at 72°C, and a final extension of 4 min at 72°C. The products were visualized on 1.5% agarose gel electrophoresis, isolated with the ADN PuriPrep-GP kit (Inbio Highway, Argentina), and sent for sequencing to Macrogen (Seoul, South Korea). Table 1 Primers used to amplify and sequence the luteinizing hormone and follicle-stimulating hormone β-subunits of C. callorynchus . Gene Sense Antisense Expected amplicon (bp) Tm (°C) lhb GACTCGACACTTCTGCC AAGTCTGGTCGGATGCTCTGC 315 56.8 fshb GTGGAAAAGGAAGAGTGTGG CGGTATTGCACATTCCAC 235 50.2 Histology . The brains were fixed in situ in Bouin’s solution, transported to the laboratory, and maintained at 4°C for 24 h. All brains were fixed with the cartilaginous skull base attached to prevent possible loss of the BL. Once fixed, brains were transferred to 70% alcohol and stored at 4°C until histological processing. Brains were sectioned at 5 µm in the coronal or sagittal plane for immunohistochemistry, mounted on 3-aminopropyltriethoxysilane–coated slides, deparaffinized in xylol, and rehydrated through graded alcohols. For immunohistochemistry, brains were sectioned coronally or sagittally at 5 µm and mounted on slides previously treated with 3-aminopropyltriethoxysilane. Sections were deparaffinized in xylol and rehydrated through a graded alcohol series. Sections were then washed in 10 mM phosphate-buffered solution (PBS). Endogenous peroxidase activity was blocked by incubating the slides with hydrogen peroxide 3% for 30 min at room temperature. After being washed with PBS, sections were incubated in 3% normal bovine serum albumin for 35 min at room temperature to avoid non-specific reactions, followed by incubation with the primary antiserum overnight at 22°C in a humidity chamber. Slides were then washed in PBS and incubated for 30 min with biotinylated anti-mouse/anti-rabbit immunoglobulins (Bio SB, Santa Barbara, USA). Sections were washed with PBS, and a final incubation was done with Streptavidin conjugated to Horseradish Peroxidase (Bio SB, Santa Bárbara, USA) for 35 min at 22°C. After being washed with PBS, the slides were exposed to a 3,30-diaminobenzidine tetrahydrochloride solution (DAKO, Santa Clara, USA) for a maximum of 3 min under the microscope. The reaction was then stopped with distilled water. Following all processes, the slides were rehydrated and mounted. The antisera used were PBL#45 and PBL#49 (generously provided by Dr. W. Vale, the Salk Institute, USA), which have cross-reactivity against different GnRH variants (Somoza et al. 2002 ), used in a final dilution of 1:500 (Stefano et al. 2000 ). Two negative controls were performed: one by suppressing the antisera and another by preadsorbing the antisera with 1 µM of the mammalian variant of GnRH overnight at 4°C. For labeling SN cells and fibers, an anti-SN antiserum was used at a final dilution of 1:500. This antiserum was raised against the 15-mer highly conserved central core of goldfish SNs (YTPQKLATLQSVFEE) and has been extensively validated in different fish species (Zhao et al. 2006 ; Canosa et al. 2011 ; Pouso et al. 2015 ; Tao et al. 2018 ). Two negative controls were performed: one by omitting the primary antiserum and the other by preadsorption with 10 µM full-length C. milii SN (TNEIVEEQYTPQSLATLESAFQELGKYTGAY; 98% purity) synthesized at the University of Ottawa, BioEngineering and Therapeutic Solutions Centre ( https://www.beatsresearch.com/ ) overnight at 4°C. The CCK neurons and fibers were labeled using a rabbit anti-antiserum against desulphated cholecystokinin octapeptide (T-4254, BMA Biomedicals, Switzerland) at a final dilution of 1:1000. Two negative controls were performed: one by omitting the primary antiserum and the other by preadsorption with 1 µM CCK-8 (Bachem). Results A schematic drawing of the C. callorynchus brain, pituitary gland, and BL is shown in Fig. 1 . This figure illustrates the telencephalon, comprising the anterior telencephalic bulbs, and the posterior telencephalon, which connects to the hypothalamus immediately caudal to the optic chiasm. The hypothalamus surrounding the third ventricle (3V) contains the following circumventricular organs: the rostro-lateral paraventricular organ, the ME, the dorso-caudal posterior recess organ, and the caudal SV. The pituitary gland is ventrally attached to the hypothalamus, and the BL, a glandular structure, is distinguished under the cartilage. The ME, a thin and basal region of the hypothalamus, is formed by the ependymal layer of the 3V and a fibrous layer (Fig. 2 a, b, c). Beneath the fibrous layer lies a circulatory network supported by connective tissue (Fig. 2 c). Caudal to the ME, the SV contains coronet cells and blood vessels (Fig. 2 a, b, d). The pituitary gland is a dorsoventrally elongated structure, exhibiting a clear cellular regionalization: the RPD, the PPD, and the NIL (Fig. 2 a, b). The RPD consists of follicular bodies of various sizes situated within a network of capillaries and supporting connective tissue (Fig. 3 a, b). These follicular bodies are composed of a stratified epithelium, with their lumens intricately connected. Two types of glandular cells can be identified: basophil cells in contact with the lumen and acidophil cells in contact with the capillaries (Fig. 3 c). Both cell types are columnar with basal and rounded nuclei. The luminal content of the follicular bodies exhibits a PAS-positive reaction (Fig. 3 d). The PPD presents follicular bodies intricately arranged in cords surrounded by sinusoidal blood vessels (Fig. 4 a, b). Collagen and unidentified fibers penetrate the dorsal region of the PPD (Fig. 4 b). Hematoxylin–eosin staining reveals three cell types in the follicular bodies: basophilic, acidophilic, and chromophobe cells. Basophilic cells are fusiform and are typically located in the central region of the cell cords (Fig. 4 c). Acidophil cells are cylindrical with their cytoplasm projected towards the lumens and/or sinusoidal vessels (Fig. 4 c). Chromophobe cells are rounded in shape with a large nucleus and a small cytoplasm (Fig. 4 c). The luminal content of the follicular bodies also exhibits a PAS-positive reaction (Fig. 4 d). The NIL is the largest area of the pituitary gland; it is organized in cell cords with several layers of large cells surrounding large sinusoidal blood vessels (Fig. 5 a, b). Small capillaries and collagen fibers are distinguished alongside the NIL cords (Fig. 5 b). The NIL dorsal region presents uncharacterized fibers (Fig. 5 b). Three types of stained cells were observed in this region: basophilic, acidophilic, and chromophobe cells. The basophilic cells were found in the dorsal area of the NIL; they have large nuclei and are projected toward the blood vessels (Fig. 5 c). The ventral region is mostly composed of acidophil cells which are in contact with sinusoidal blood vessels; they are cylindrical with an oval and central nucleus (Fig. 5 d). Chromophobe cells are distributed throughout the NIL; they are small with a round nucleus and small cytoplasm and located in the middle of the cords (Fig. 5 c, d). The BL, a gland situated under the cartilage, comprises follicular bodies intricately connected by lumens and surrounded by collagen and non-characterized fibers (Fig. 6 a, b). These follicular bodies are formed by acidophil cells arranged around the lumens (Fig. 6 c). These cells are surrounded by larger basophil cells (Fig. 6 c). Luminal content exhibits a PAS-positive reaction (Fig. 6 d). The BL is connected to the posterior telencephalon by channels, one on each side, that pass through the cartilage just anterior to the optic chiasma containing a large blood vessel surrounded by collagen fibers (Fig. 7 a, b, c). In the brain, the same GnRH immunoreactive (ir) neuronal cell bodies and fibers were detected using two different antisera: PBL#45 (Fig. 8 ) and PBL#49 (data not shown). These neurons were arranged as a chain of nerve cell bodies and fibers located in the posterior telencephalon (Fig. 8 a, b). GnRH-ir neuronal somata were bipolar, narrow, and elongated along the anteroposterior axis, reaching a maximum length of approximately 100 µm. These cell bodies and fibers extended from the telencephalic bulb to the optic chiasm, ventral to the telencephalic ventricle. Fibers with GnRH-ir project posteriorly, passing above the optic chiasm and extending along both sides of the third ventricle, running in different directions. In the ME, fibers ran either parallel to the third ventricle or ventrally toward the pituitary. GnRH-ir fibers also reached cells in both the PPD and the BL (Fig. 8 c, d). These fibers were arranged as a compact, almost indistinguishable bundle converging onto cells. GnRH-ir nerve cell bodies and fibers were additionally detected in the midbrain. Each of the channels connecting the telencephalon and the BL also contained GnRH-ir fibers, which were also observed entering the BL (Fig. 7 b-e). Neuronal cell bodies and fibers displaying SN-ir were detected in the posterior telencephalon (Fig. 9 a, b), showing a distribution like that of GnRH-ir elements. SN-ir structures were also observed in the paraventricular nucleus, the ME (Fig. 9 c), and the posterior recess organ (Fig. 9 d). SN-ir neuronal somata were smaller than GnRH-ir cells, measuring approximately 20 µm in diameter and displaying an oval shape. SN-ir cells were also detected in the pituitary gland and the BL, exhibiting considerable variation in size and morphology (Fig. 10 a-e). Some SN-ir fibers were additionally observed in the NIL (Fig. 10 d). Neuronal cell bodies and fibers displaying CCK-ir were evident in the hypothalamic posterior recess (Fig. 11 a, b). These unipolar neurons had their somata positioned close to the ventricular lining of the posterior recess. A single primary process emerged from each soma, projecting perpendicularly away from the ventricular surface. CCK-ir neuronal somata measured approximately 15 µm in diameter and displayed an oval morphology. CCK-ir cells were also detected in the PPD (Fig. 11 c) and the NIL (Fig. 11 d), showing variation in size and shape. CCK-ir fibers were observed reaching the BL (Fig. 11 e). In all cases, negative controls showed no immunoreactivity for GnRH, SN, or CCK (Supplementary S1-S4), and no sex differences were observed. In all samples analyzed, fshb and lhb were detected by RT-PCR (Fig. 12 ). The amplified PCR fragments exhibited 100% identity with those of C. milii (Supplementary Figs. 5 and 6). Both GtH subunits showed a relatively higher expression in the BL than in the pituitary gland, and a relatively higher expression of lhb was evident in the pituitary gland of both sexes compared to fshb (Fig. 12 a, b). Discussion We report the anatomical locations of key neuropeptides involved in the control of GtHs, GnRH, SN, and CCK in C. callorynchus , revealing a new perspective on the communication between the holocephalan hypothalamus, pituitary gland, and BL. The general anatomy of the hypothalamus and pituitary gland resembles that in other studied chondrichthyans (Sathyanesan 1965 ; Meurling 1967a ; 1967b ; Holmes and Ball 1974 ; Northcutt 1977 ). All circumventricular organs of the hypothalamus that we have observed in C. callorynchus are like those described in elasmobranchs by Rodríguez-Moldes ( 2011 ). The pituitary gland is organized into three clearly distinguished regions, the RPD, the PPD, and the NIL, like those previously described in H. colliei and Chimaera monstrosa (Sathyanesan 1965 ; Jasiński and Gorbman 1966 ; Meurling 1967b ). The BL is like to that described in H. colliei and C. monstrosa (Sathyanesan 1965 ; Meurling 1967b ). Although the BL in holocephalans and the VL in elasmobranchs are regarded as the primary structures regulating reproduction (Dodd et al. 1982 ; Dodd 1983 ), most studies on chondrichthyans have not included these glands, possibly because they are overlooked due to their anatomical location. The question now is how the brain regulates the synthesis and secretion of GtHs and other hormones in chondrichthyans. According to Meurling’s descriptions, both elasmobranchs and holocephalans possess a hypothalamo–hypophyseal portal blood system similar to that of tetrapods (Meurling 1960 ; 1967a ; 1967b ). The current description of vascularization in the HP axis indicates that C. callorynchus also exhibits a portal-like system, like that described in H. colliei and C. monstrosa (Sathyanesan 1965 ; Jasiński and Gorbman 1966 ; Meurling 1967b ). The present results demonstrate the presence of GnRH-ir neurons and fibers in the brain, as well as GnRH-ir fibers in the pituitary gland and the BL. A strip of GnRH-ir neuronal cell bodies is present along the posterior telencephalon, as in other vertebrates (Amano et al. 1991 ; Muske and Moore 1994 ; Kim et al. 1995 ; Okubo and Nagahama 2008 ; Zohar et al. 2022 ). Immunoreactive GnRH fibers showed a hypothalamic distribution similar to that reported for Squalus acanthias , particularly at the level of the third ventricle (D’Antonio et al. 1995 ). However, in C. callorynchus , the fiber density observed in the ME was considerably lower than that of immunoreactive fibers observed by D’Antonio et al. ( 1995 ). In the pituitary gland of C. callorynchus , GnRH-ir fibers reach the PPD, surround blood vessels, and likely terminate in proximity to pituitary cells. Importantly, GnRH-ir fibers reach the BL, ending in close contact with glandular cells. These results provide a morphological basis for the regulation of pituitary hormone synthesis and secretion in this species, as GnRH is known to control the release of multiple pituitary hormones in other vertebrates (Marchant et al. 1989 ; Chang et al. 1990 ; Villalobos et al. 1997 ). For example, according to Yamaguchi (2015), the PPD of C. milii contains growth hormone-producing cells; therefore, the GnRH released from these fibers could regulate the synthesis and/or release of GtHs and GH. The presence of GnRH-ir fibers ending at the PPD and the BL suggests that the synthesis and release of the GtHs may be controlled by direct glandular innervation. This challenges the hypothesis that the BL is regulated exclusively by GnRH delivered via the general circulation, as previously proposed for H. colliei . In this context, the association of these fibers with blood vessels could explain the high levels of GnRH in circulation found in H. colliei by Sherwood and Lovejoy ( 1993 ). Neuronal cell bodies exhibiting SN-ir were evident in the posterior telencephalon of C. callorynchus . These cells may be homologous to the SN neurons located in the POA of teleosts (Canosa et al. 2011 ; Pouso et al. 2015 ). The SN-ir fibers reaching the NIL resemble those found in goldfish (Canosa et al. 2011 ) and zebrafish (Peng et al. 2025), which co-express isotocin (the fish equivalent of oxytocin), and in the electric fish Brachyhypopomus gauderio , which co-expresses isotocin and vasotocin (fish equivalent to vasopressin; Pouso et al. 2015 ). In rats, SN and oxytocin are co-stored in large dense-core vesicles and may be co-released into the circulation (Mahata et al. 1993 ). Thus, the relationship between nonapaptides and SN co-expression in magnocellular cells is ancient and must have emerged in an ancestral gnathosome. The SN-ir cells identified in the RPD of C. callorynchus may be equivalent to those in the goldfish RPD that co-localized with PRL (Zhao et al. 2010 ). In the electric fish, these cells were found in both the RPD, where they co-localized with PRL, and some cells in the PPD (Pouso et al. 2015 ). In the case of zebrafish, using the same anti-SN antiserum, cells in the RPD were SN-ir, strong SN-ir fibers were found in the PPD and RPD and SN-ir was not detected in FSH and LH cells (Peng et al. 2025). It is known that in these regions, cells expressing hormones such as PRL, LH, and GH are present; therefore, it is possible that SN could be co-expressed in some of them and/or exerts autocrine/paracrine effects on them (Zhao et al. 2010 ; Pouso et al. 2015 ; Mitchell et al. 2020 ). The presence of SN-ir cells in the BL suggests a potential local role for SN and/or its release from this gland. In goldfish, the SN in RPD lactotrophs is released upon stimulation by GnRH and acts in a paracrine manner to stimulate LH release from the PPD (Zhao et al. 2006 ). Additionally, GnRH may stimulate SN release from the murine LβT2 gonadotroph cell line, which in turn may exert an autocrine stimulatory effect that enhances LH secretion (Zhao et al. 2011 ). These findings raise the possibility that SN may control LH by an autocrine/paracrine manner not only in the pituitary gland but also in the BL of C. callorynchus . In the present study, CCK-ir neuronal cell bodies and fibers were found in the hypothalamic posterior recess and, in the pituitary gland, CCK-ir cells were identified in the PPD and the NIL, which can be related to an autocrine/paracrine regulation of ACTH and α-MSH cells, as seen in mammals (Rehfeld 2017 ). Hashimoto and Kimura ( 1986 ) were among the first to report the stimulatory effects of CCK on LH and, to a lesser extent, on FSH secretion in rats. Early studies in rhesus monkeys (Schreihofer et al. 1993 ) and goldfish (Himick et al. 1993 ) also indicated that CCK may exert stimulatory effects on LH. In contrast, high doses in rats were found to be inhibitory to LH, with no effects on FSH (Vijayan et al. 1979 ). These and other studies indicate that CCK from multiple sources (e.g., neuronal, paracrine, hormonal) has the potential to regulate the GtHs. In zebrafish, the cckr transcript was found to be highly expressed in FSH cells and, at lower levels, in LH cells of both sexes (Hollander-Cohen et al. 2021 ). In zebrafish, a GnRH3 analog and CCK preferentially activate calcium-dependent secretion in LH and FSH cells, respectively (Hollander-Cohen et al. 2024 ). More recently, CCK has been shown to specifically stimulate FSH cells via CCK2Rb receptors, thereby regulating ovarian and testicular development in Japanese medaka (Uehara et al. 2024 ). Similar results were also reported in zebrafish (Mizrahi et al. 2025 ). Thus, the possibility that CCK may regulate FSH or LH differentially in either the BL or pituitary of C. callorynchus remains an open question. In this context, based on observations in mammals and teleosts, effects of CCK on LH or FSH could be either stimulatory or inhibitory. This is the first report demonstrating that both fshb and lhb mRNAs are synthesized in the pituitary of C. callorynchus as in other vertebrates, but also in the BL. Previous studies in H. colliei by Dodd et al. ( 1982 ) suggested that the principal gonadotropin-like activity resides in the BL, with lesser activity present in the pituitary. These conclusions were derived from bioassays in which quail and chick testes were exposed to various freeze-dried H. colliei pituitary extracts, which inevitably contained GtHs along with other pituitary hormones, peptides, and additional regulators of gonadal steroidogenesis. Sampling and replication were limited, precluding firm conclusions regarding the primary source of holocephalan GtHs. Nevertheless, both GtHs were produced outside the pituitary gland, which has long been assumed in elasmobranchs (Quérat et al. 2001 ; Arimura et al. 2024 ). However, before the present study, this had not been empirically tested in holocephalans. By determining the differential distributions of GnRH, SN, and CCK immunoreactivity in C. callorynchus we propose that the regulation of the pituitary and BL is complex and multimodal. The present findings expand the anatomical framework for understanding HP communication in holocephalans. Our results demonstrate that fshb and lhb transcripts are detectable not only in the pituitary but also, at comparatively higher expression levels, in the BL, with both structures receiving neuropeptidergic inputs. Specifically, GnRH-ir fibers reach the PPD and BL, while CCK-ir fibers also enter the BL. In addition, SN-ir is present in hypothalamic regions, pituitary tissue, and BL cells, suggesting the potential for local interactions within these endocrine structures. Although the present study does not assess hormone release, receptor expression, or functional outcomes, the anatomical relationships described here are consistent with the possibility that GtH-related processes in holocephalans are influenced by multiple routes of neuropeptidergic signaling. These observations provide a structural basis for future studies aimed at determining how neural, paracrine, and endocrine mechanisms contribute to the regulation of reproduction in early diverging jawed vertebrates. Declarations Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding Declaration : This work was supported by the Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación (PICT 2017–4507, Argentina) to CAA and institutional funding to GMS. Correspondence: Gustavo M. Somoza ( [email protected] ) Author Contribution Conceptualization: CAA, VLT, GMS; Histology: CH, MS, GCL, FAV; Immunocytochemistry: CH, GCL, MRP; Molecular Biology: CH, MRP; Writing-original draft preparation: CH, CAA, GMS; Writing-review and editing: All authors. All authors have read and agreed to the published version of the manuscript. Acknowledgement The authors would like to acknowledge Juan Francisco Solana and Franco Cristiani for their assistance with fish sampling. This work was supported by the Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación (PICT 2017-4507, Argentina) to CAA and institutional funding to GMS. 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Pérez","email":"","orcid":"","institution":"Instituto Tecnológico de Chascomús (CONICET-UNSAM)","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"R.","lastName":"Pérez","suffix":""},{"id":605816108,"identity":"88187203-ea3c-491f-acc0-a291deda973d","order_by":5,"name":"Fabricio A. Vigliano","email":"","orcid":"","institution":"Facultad de Ciencias Veterinarias. Universidad Nacional de Rosario","correspondingAuthor":false,"prefix":"","firstName":"Fabricio","middleName":"A.","lastName":"Vigliano","suffix":""},{"id":605816109,"identity":"931518da-0ee7-4513-89fc-627f9e6f8cdc","order_by":6,"name":"Vance L. Trudeau","email":"","orcid":"","institution":"University of Ottawa","correspondingAuthor":false,"prefix":"","firstName":"Vance","middleName":"L.","lastName":"Trudeau","suffix":""},{"id":605816111,"identity":"5789e16f-3a5b-4718-85f1-9b76cca6a145","order_by":7,"name":"Gustavo M. Somoza","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYPACGx4QyUyKljQeBjYStRxmIF6LfHt34sMfNedlzOWbD34uYKiVMzjA/OwDPi0GZ85uNpA4dpvHso0tWXoGw3FjgwNsxjPwapHI3SZhwHabx+AYjxkzD8OxxJkNDMb4HTYDqCXh3zmgFv5vUC3sn/F75gZQy8G2AyBb2IBaahL7GXjw2wLyi2FjXzJQS5qxNI/BAWN+Zp5i/A5r79348Mc3O3uDw4cffuapqJNjY2/fjN9haJYeJjFCgaCORPWjYBSMglEwEgAAF5hAp2MJlZsAAAAASUVORK5CYII=","orcid":"","institution":"Instituto Tecnológico de Chascomús (CONICET-UNSAM)","correspondingAuthor":true,"prefix":"","firstName":"Gustavo","middleName":"M.","lastName":"Somoza","suffix":""}],"badges":[],"createdAt":"2026-03-06 16:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9052503/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9052503/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104753582,"identity":"c40347c9-2d54-4ef4-a2cc-0fd21833fda7","added_by":"auto","created_at":"2026-03-16 21:01:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":746554,"visible":true,"origin":"","legend":"\u003cp\u003eSagittal schematic drawing of the \u003cem\u003eC. callorynchus \u003c/em\u003ebrain, pituitary gland and buccal lobe. The following structures are distinguished: telencephalic bulbs (TB), posterior telencephalon (pT), optic chiasma (Ch), midbrain, hypothalamus (H), paraventricular organ (pV), posterior recess organ (pR), median eminence (ME), third ventricle (3V), \u003cem\u003esaccus vasculosus \u003c/em\u003e(SV), pituitary gland (P), hindbrain, cartilage (C) and buccal lobe (BL).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9052503/v1/bb770a3c7d3aff9cdafa5f64.png"},{"id":104782430,"identity":"9b8198e4-65e9-4f29-bb66-7aab0b73232b","added_by":"auto","created_at":"2026-03-17 07:57:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1647587,"visible":true,"origin":"","legend":"\u003cp\u003eSagittal sections of hypothalamus-pituitary area. [a]Sagittal scheme, see details in Fig. 1. [b]Hypothalamic areas: Median eminence (ME), third ventricle (3V), and \u003cem\u003esaccus vasculosus \u003c/em\u003e(SV). Pituitary gland areas: rostral \u003cem\u003epars distalis \u003c/em\u003e(RPD), proximal \u003cem\u003epars distalis \u003c/em\u003e(PPD), and neurointermedial lobe (NIL). Hematoxylin-eosin. Scale bar: 1 mm. [c] Median eminence structure: ependymal layer (arrowhead), fibrous layer (arrow), and blood vessels (bv). Masson's trichrome. Scale bar: 50 µm. [d] \u003cem\u003eSaccus vasculosus \u003c/em\u003eshowing coronet cells (arrowheads), and blood vessels. Masson's trichrome. Scale bar: 20 µm.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9052503/v1/87eb82a9565fbbcc4193ebd7.png"},{"id":104753583,"identity":"f873f4e0-5b81-447e-a864-d82b9b878382","added_by":"auto","created_at":"2026-03-16 21:01:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1634992,"visible":true,"origin":"","legend":"\u003cp\u003eSagittal section of the rostral \u003cem\u003epars distalis \u003c/em\u003e(RPD). [a] Sagittal scheme, see details in Fig. 1. [b] Follicular bodies, composed of a stratified epithelium surrounding a lumen (asterisk) and blood vessels (bv). Haematoxylin-eosin. Scale bar: 100 µm. [c] Cellular layers of the follicular bodies: Basophilic (arrow) and acidophilic cells (arrowhead). Haematoxylin-eosin. Scale bar: 50 µm. [d] Lumen exhibiting a PAS-positive reaction in purple (asterisk). Periodic acid-Schiff. Scale bar: 200 µm.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9052503/v1/1481cfd798ce573b98b6d2b8.png"},{"id":104783097,"identity":"c4df4143-d8d0-4abf-b139-c37369e4df4f","added_by":"auto","created_at":"2026-03-17 07:58:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1517428,"visible":true,"origin":"","legend":"\u003cp\u003eSagittal section of the proximal \u003cem\u003epars distalis \u003c/em\u003e(PPD). [a] Sagittal scheme, see details in Fig. 1. [b] The follicular bodies (white arrow) surrounded by blood vessels (bv). Fibers reaching the PPD: collagen fibers in green (outline arrowhead) and uncharacterized fibers with no stain (white arrowhead). Neurointermedial lobe (NIL). Masson's trichrome. Scale bar: 500 µm. [c]Types of cells comprising follicular bodies: basophilic (arrow), acidophilic (arrowhead), and chromophobe (outline arrow) cells. Lumens (asterisk). Haematoxylin-eosin. Scale bar: 50 µm. [d] The content of the lumen exhibits a PAS-positive reaction in purple (asterisks). Periodic acid-Schiff. Scale bar: 50 µm.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9052503/v1/e911f754c1d53080e2cebaf3.png"},{"id":104783399,"identity":"e05ad6f0-1aad-46a3-8cca-4d9bc2a155f3","added_by":"auto","created_at":"2026-03-17 07:58:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1880298,"visible":true,"origin":"","legend":"\u003cp\u003eSagittal section of the neurointermedial lobe (NIL). [a] Sagittal scheme, see details in Fig. 1. [b] Cell cords within small capillaries (white arrow) surrounded by sinusoidal blood vessels (bv), collagen fibers in green (outline arrowhead), and uncharacterized fibers (white arrowhead). Masson's trichrome. Scale bar: 100 µm. [c] The cellular layers of the NIL: basophilic cell (arrow) and chromophobe cell (outline arrow). Haematoxylin-eosin. Scale bar: 50 µm. [d] The cellular layers of ventral NIL: acidophilic cells (arrowhead) and chromophobe cells (outline black arrow). Haematoxylin-eosin. Scale bar: 50 µm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9052503/v1/274fbcf4730f2b9c7c14bfdb.png"},{"id":104753585,"identity":"4b23bb60-9b86-435a-928f-a20ffd2c5af1","added_by":"auto","created_at":"2026-03-16 21:01:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1646992,"visible":true,"origin":"","legend":"\u003cp\u003eSagittal section of the buccal lobe (BL). [a] Sagittal scheme, see details in Fig. 1. [b] The BL situated under the cartilage is surrounded by unidentified (white arrowhead) and collagen fibers in blue (outline arrowhead). Masson's trichrome. Scale bar: 1 m. [c] The cellular layers of the follicular bodies: basophilic (arrow) and acidophilic cells (arrowhead). Haematoxylin-eosin. Scale bar: 50 µm. [d] The lumen's content of the follicular bodies with PAS-positive reaction in purple (asterisk). Periodic acid-Schiff. Scale bar: 200 µm.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-9052503/v1/56daa3c32aef97edeb4a46a7.png"},{"id":104753586,"identity":"9583f7d1-fbf4-41db-a233-7458ee26978d","added_by":"auto","created_at":"2026-03-16 21:01:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":876270,"visible":true,"origin":"","legend":"\u003cp\u003eFrontal sections of the buccal lobe (BL). [a] Sagittal scheme, see details in Fig. 1. [b] The BL section with the channel crossing the cartilage with blood vessels (bv) arriving at the gland. Haematoxylin-eosin. Scale bar: 1 mm. [c] The BL section with a channel crossing the cartilage with uncharacterized (white arrowhead) and collagen fibers stained in blue (outline arrowhead). Masson's trichrome. Scale bar: 1 mm. [d] GnRH-immunoreactive (ir) fibers (arrowheads) in the channel. Scale bar: 20 µm [e] GnRH-ir fibers (arrowheads) reaching the BL. Scale bar: 20 µm\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-9052503/v1/e297a1a4f8a3ea67547cbe01.png"},{"id":104783396,"identity":"bcac6e94-80e4-4d81-86ed-98908b25b53f","added_by":"auto","created_at":"2026-03-17 07:58:50","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2821328,"visible":true,"origin":"","legend":"\u003cp\u003e[a] Sagittal scheme, see details in Fig. 1. [b] Sagittal section of the posterior telencephalon showing cell bodies (arrows) and fibers (arrowheads) immunoreactive (ir) to GnRH. Scale bar: 200 µm. [c] Sagittal section of the proximal \u003cem\u003epars distalis \u003c/em\u003eshowing GnRH-ir fibers (arrowheads). Scale bar: 20 µm [d] Sagittal section of the buccal lobe showing GnRH-ir fibers (arrowheads) reaching BL cells. Scale bar: 20 µm\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-9052503/v1/085308e2c6ab9ff58039037d.png"},{"id":104753587,"identity":"1ee1a1b7-c782-48e5-bd1f-91dcbdf49ade","added_by":"auto","created_at":"2026-03-16 21:01:05","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":978346,"visible":true,"origin":"","legend":"\u003cp\u003e[a] Sagittal scheme, see details in Fig. 1. [b] Sagittal section of the posterior telencephalon showing cell bodies (arrows) and fibers (arrowheads) immunoreactive (ir) to secretoneurin (SN). Scale bar: 200 µm. [c]Sagittal section of the paraventricular organ (pV) and median eminence (ME) showing SN-ir cell bodies (arrow) and fibers (arrowheads). Scale bar: 100 µm. [d] Sagittal section of the posterior recess organ showingSN-ir cell bodies (arrow) and fibers (arrowhead). Scale bar: 50 µm.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-9052503/v1/d550db9cfcc65f5714b6306f.png"},{"id":104783340,"identity":"bad97faf-3bbc-41f5-93b5-05f64c292140","added_by":"auto","created_at":"2026-03-17 07:58:41","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":2048317,"visible":true,"origin":"","legend":"\u003cp\u003e[a] Sagittal scheme, see details in Fig. 1. [b] Sagittal section of the rostral \u003cem\u003epars distalis \u003c/em\u003eshowing immunoreactivity to SN cell bodies (arrow). Scale bar: 50 µm. [c] Sagittal section of the proximal \u003cem\u003epars distalis \u003c/em\u003eshowing ir-SN cell bodies (arrow). Scale bar: 50 µm. [d]Sagittal section of the NIL presenting SN-ir cell bodies (arrow) and fibers (arrowhead). Scale bar: 20 µm [e] Buccal lobe shows SN-ir cell bodies (arrow). Scale bar: 100 µm.\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-9052503/v1/23d772c6b2da9bbfcc8968c9.png"},{"id":104783424,"identity":"9534dc7d-223c-48e4-bdf6-1e7f1252fc17","added_by":"auto","created_at":"2026-03-17 07:58:55","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":1239604,"visible":true,"origin":"","legend":"\u003cp\u003e[a] Sagittal scheme, see details in Fig. 1. [b] Sagittal section of the posterior recess organ showing immunoreactivity to CCK cell bodies (arrow) and fibers (arrowhead). Scale bar: 50 µm. [c] Sagittal section of the proximal \u003cem\u003epars distalis \u003c/em\u003eshowing CCK-ir cell bodies (arrow). Scale bar: 50 µm. [d] Sagittal section of the NIL showing CCK-ir cell bodies (arrow). Scale bar: 50 µm. [e] Sagittal section of the buccal lobe showing CCK-ir fibers (arrowheads) close to BL cells. Scale bar: 20 µm\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-9052503/v1/2d7af68d53caae7ddf6486d3.png"},{"id":104753590,"identity":"5434f750-e184-41cf-ac9e-fa7915f3d646","added_by":"auto","created_at":"2026-03-16 21:01:05","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":518124,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of \u003cem\u003efshb\u003c/em\u003e [a] and \u003cem\u003elhb \u003c/em\u003e[b] \u0026nbsp;detected by RT-PCR in the buccal lobe (BL) and the pituitary gland (Pit). The arrowheads show the 300 bp molecular weight ladder.\u003c/p\u003e","description":"","filename":"Figure12.png","url":"https://assets-eu.researchsquare.com/files/rs-9052503/v1/ae5c5aa794799b24c85afbb3.png"},{"id":107479909,"identity":"2150fa80-ac6a-441d-b296-53f2bbb60300","added_by":"auto","created_at":"2026-04-22 01:59:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18483203,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9052503/v1/d74f0202-dd46-445c-9fc2-862c84ad76f6.pdf"},{"id":104753591,"identity":"cdd40178-f693-430a-8e50-938625a11965","added_by":"auto","created_at":"2026-03-16 21:01:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8039283,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9052503/v1/09cdebaaa1066a1fa86837ca.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anatomy of the hypothalamic–pituitary axis and buccal lobe in the holocephalan Callorhinchus callorynchus ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn vertebrates, the hypothalamus and the pituitary gland play key roles in integrating internal and environmental signals that regulate reproduction (Trudeau and Somoza \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Comparative studies of the vertebrate hypothalamic\u0026ndash;pituitary (HP) axis reveal a largely conserved anatomical organization and hormonal signaling pathways (Dufour et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Trudeau \u0026amp; Somoza \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Santiago-Andres et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, a major gap remains in our understanding of the HP axis in chondrichthyans (Awruch \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Trudeau and Somoza \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), making claims of evolutionary conservation tentative. Chondrichthyans diverged from bony vertebrates approximately 450 Ma ago (Inoue et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and are represented today by two extant subclasses, Elasmobranchii (sharks, rays, and skates) and Holocephali (chimaeras). Owing to their long evolutionary history and distinctive brain characteristics, which differ markedly from both tetrapod and teleost lineages (Northcutt \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1977\u003c/span\u003e; Smeets et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Trudeau and Somoza \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Fontaine et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), chondrichthyans represent an important model for comparative studies of the HP axis. Nevertheless, detailed anatomical studies of the HP axis in this group remain scarce and are largely restricted to sharks and rays, leaving chimaeras poorly studied (Holmes and Ball \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Northcutt \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1977\u003c/span\u003e; Smeets et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Dodd and Dodd \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Santos-Dur\u0026aacute;n et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe pituitary gland is classically subdivided into the adenohypophysis, the pars intermedia (which is reduced or even absent in mammals), and the neurohypophysis (Page 2006). In chondrichthyans, as in teleosts, pituitary anatomy differs because the adenohypophysis is subdivided into the rostral \u003cem\u003epars distalis\u003c/em\u003e (RPD), proximal \u003cem\u003epars distalis\u003c/em\u003e (PPD), and a well-developed pars intermedia (Trudeau and Somoza \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Fontaine et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In these groups, the \u003cem\u003epars intermedia\u003c/em\u003e is interdigitated by neurohypophyseal nerve terminals and termed the neuro-intermediate lobe (NIL; Meurling \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1962\u003c/span\u003e). Chondrichthyans also possess distinctive glands: the ventral lobe (VL) in elasmobranchs, which is ventrally attached to the PPD, and the buccal lobe (BL) in chimaeras, located in the roof of the oral cavity and separated from the brain by a cartilaginous barrier (Smeets et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). Although the functions of the VL and BL remain poorly understood, available evidence indicates that luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are produced mainly in the VL of elasmobranchs (Qu\u0026eacute;rat et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), and crude BL extracts from the chimaera \u003cem\u003eHydrolagus colliei\u003c/em\u003e show gonadotropin-like activity (Dodd et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1982\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe preoptic area (POA), positioned immediately rostral to the hypothalamus (Puelles \u0026amp; Rubenstein \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), plays a central role in the regulation of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) through the presence of gonadotropin-releasing hormone (GnRH)-expressing neurons (Mu\u0026ntilde;oz-Cueto et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zohar et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In tetrapods, GnRH fibers project to the median eminence (ME), where GnRH is released into the hypothalamic pituitary portal system and transported to the pituitary (Page 2006; Yin and Gore \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Along with other neurohormones and neurotransmitters, GnRH plays a central role in controlling the synthesis and secretion of LH and FSH by the adenohypophysis, thereby supplying critical endocrine signals to the gonads (Trudeau and Somoza \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEarly studies proposed a portal system linking the hypothalamus and pituitary in elasmobranchs (Green \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1951\u003c/span\u003e; Meurling \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1960\u003c/span\u003e), and later observations supported a direct vascular connection between the ME and the pituitary (Holmes and Ball \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Dodd \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Smeets et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). However, no direct vascular connection has been demonstrated between the hypothalamus and either the VL in elasmobranchs (Dodd \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1983\u003c/span\u003e) or the BL in holocephalans (Sathyanesan \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1965\u003c/span\u003e; Jasiński and Gorbman \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1966\u003c/span\u003e). Teleost fish illustrate divergence in HP communication systems. During evolution, they lost the ME, and GnRH neurons instead project to and enter the pars distalis, directly controlling gonadotropin (GtH) cells. Teleost GnRHs were therefore proposed as primary regulators of GtHs, with a stronger effect on LH than FSH cells (Zohar et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, recent studies using gene mutations of GnRH and GnRH receptors in zebrafish and medaka have questioned the essential role of GnRHs in reproductive regulation (Trudeau \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zohar et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). By contrast, secretoneurin (SN), derived from selective processing of secretogranin-2 (Scg2), induces LH release in goldfish (Bl\u0026aacute;zquez et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) and in mouse LβT2 cells (Zhao et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Secretoneurin is of interest because only 10% of zebrafish pairs with double \u003cem\u003escg2a/scg2b\u003c/em\u003e frameshift mutations spawn (Mitchell et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and SNa injection robustly activates the hypothalamus-pituitary-gonadal axis and induces ovulation in wild-type females (Peng et al. 2025). Likewise, recent studies have shown that cholecystokinin (CCK) also contributes to the regulation of FSH expression and secretion in medaka (Uehara et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and zebrafish (Hollander-Cohen et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). CRISPR-mediated frameshift mutations in CCK precursors and a CCK receptor in medaka demonstrated that CCK is a critical regulator of ovarian and testicular development through FSH-mediated actions (Uehara et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Whether these emerging examples represent a conserved vertebrate pathway requires further investigation in more ancient lineages.\u003c/p\u003e \u003cp\u003eChondrichthyans do not conform to either the typical tetrapod or teleost hypothalamic pituitary systems. In several elasmobranch and holocephalan species, GnRH fibers are detected throughout much of the brain, whereas GnRH cell bodies are concentrated in the terminal nerve, telencephalon, and midbrain (Lovejoy et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Sherwood and Lovejoy \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; D\u0026rsquo;Antonio et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Forlano et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Moeller and Meredith \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Masini et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Evidence indicates that GnRH fibers terminate on blood vessels in the ventrocaudal telencephalon and are not associated with the chondrichthyan ME (Sherwood and Lovejoy \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; D\u0026rsquo;Antonio et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). The prevailing hypothesis is that GnRH reaches the VL or BL via general circulation (Dodd \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Sherwood and Lovejoy \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; D\u0026rsquo;Antonio et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Beyond this, the neuropeptide-mediated hypothalamic regulation of pituitary function in chondrichthyans remains poorly understood, and several key questions persist (Trudeau and Somoza \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Santiago-Andres et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven these gaps in understanding in elasmobranchs and holocephalans, the present study provides a comprehensive anatomical and histological description of GnRH, SN, and CCK distributions in the HP axis and BL of the chimaera \u003cem\u003eCallorhinchus callorynchus\u003c/em\u003e. This species belongs to one of the three extant holocephalan families, Callorhinchidae, which comprises three species worldwide (Finucci et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Callorhinchidae has gained prominence in evolutionary studies following the sequencing of the \u003cem\u003eCallorhinchus milii\u003c/em\u003e genome, which suggests an unusually low evolutionary rate among studied vertebrates (Venkatesh et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cb\u003eSampling\u003c/b\u003e. The project was authorized by the Secretar\u0026iacute;a de Pesca of the province of Chubut (permit N\u0026deg; 06/2023-DCPyA-SsP-SP) and the Institutional Committee for the Care and Use of Study Animals (CICUAE-CENPAT N\u0026deg; 11). Fourteen \u003cem\u003eC. callorynchus\u003c/em\u003e (nine females and five males) were caught by angling by recreational fishermen along the shore of Puerto Madryn City, Argentina, between December 2021 to April 2022. Sex, total length (TL, measured from the rostral appendix to the beginning of the superior lobule of the caudal fin, cm), total weight (TW, g), and clasper calcification in males (assessed manually as either fully calcified or not) were recorded. According to Bernasconi et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), C. \u003cem\u003ecallorynchus\u003c/em\u003e are considered adults when they reach 47 cm TL for females, and 43 cm TL with fully calcified claspers for males. The whole brain was dissected \u003cem\u003ein situ\u003c/em\u003e immediately following capture.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA isolation and cDNA synthesis\u003c/b\u003e. The brains were dissected from two females and two males. The pituitary gland and the \u003cem\u003esaccus vasculosus\u003c/em\u003e (SV) were dissected together, and the BL separately. Samples were preserved in RNAlater (Invitrogen\u0026trade;, USA) until total RNA extraction with the TransZol reagent (TransGen Biotech, China). After quantification and purification with DNase I (Invitrogen\u0026trade;, USA), RNA samples were reverse transcribed into cDNA by using M-MLV reverse transcriptase (Invitrogen\u0026trade;, USA), RNaseOUT (Invitrogen\u0026trade;, USA), and Oligo dT universal adaptor primer, following the manufacturer's protocol.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDesign of primers for RT-PCR\u003c/b\u003e. Different consensus primers were designed for the β-subunits of LH and FSH. For LHβ, the sequences of \u003cem\u003eC. milii\u003c/em\u003e (XM_042345119), \u003cem\u003ePristis pectinata\u003c/em\u003e (XM_052042827.1), \u003cem\u003eScyliorhinus canicula\u003c/em\u003e (AJ310345), \u003cem\u003eChiloscyllium plagiosum\u003c/em\u003e (XM_043679302.1), \u003cem\u003eStegostoma fasciatum\u003c/em\u003e (XM_048521342.1), \u003cem\u003eRhincodon typus\u003c/em\u003e (XM_020512596), and \u003cem\u003eCarcharodon carcharias\u003c/em\u003e (XM_041177731) were aligned to look for conserved regions. The same strategy was performed for FSHβ, aligning the sequences of \u003cem\u003eC. milii\u003c/em\u003e (HQ174783), \u003cem\u003eAmblyraja radiata\u003c/em\u003e (XM_033038641), \u003cem\u003eP. pectinata\u003c/em\u003e (XM_052029671.1), \u003cem\u003eS. canicula\u003c/em\u003e (AJ310344), and \u003cem\u003eS. fasciatum\u003c/em\u003e (XM_048546119.1; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The PCR amplifications were performed with GoTaq DNA Polymerase (Promega, USA) with a program including an initial heat denaturation step at 94\u0026deg;C for 3 min, followed by 35 cycles of 30s at 94\u0026deg;C, 30s at 54\u0026deg;C, 45s at 72\u0026deg;C, and a final extension of 4 min at 72\u0026deg;C. The products were visualized on 1.5% agarose gel electrophoresis, isolated with the ADN PuriPrep-GP kit (Inbio Highway, Argentina), and sent for sequencing to Macrogen (Seoul, South Korea).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers used to amplify and sequence the luteinizing hormone and follicle-stimulating hormone β-subunits of \u003cem\u003eC. callorynchus\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSense\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAntisense\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eExpected\u003c/p\u003e \u003cp\u003eamplicon (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTm\u003c/p\u003e \u003cp\u003e(\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003elhb\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGACTCGACACTTCTGCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAAGTCTGGTCGGATGCTCTGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e315\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e56.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003efshb\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGTGGAAAAGGAAGAGTGTGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCGGTATTGCACATTCCAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e235\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e50.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHistology\u003c/b\u003e. The brains were fixed \u003cem\u003ein situ\u003c/em\u003e in Bouin\u0026rsquo;s solution, transported to the laboratory, and maintained at 4\u0026deg;C for 24 h. All brains were fixed with the cartilaginous skull base attached to prevent possible loss of the BL. Once fixed, brains were transferred to 70% alcohol and stored at 4\u0026deg;C until histological processing. Brains were sectioned at 5 \u0026micro;m in the coronal or sagittal plane for immunohistochemistry, mounted on 3-aminopropyltriethoxysilane\u0026ndash;coated slides, deparaffinized in xylol, and rehydrated through graded alcohols.\u003c/p\u003e \u003cp\u003eFor immunohistochemistry, brains were sectioned coronally or sagittally at 5 \u0026micro;m and mounted on slides previously treated with 3-aminopropyltriethoxysilane. Sections were deparaffinized in xylol and rehydrated through a graded alcohol series. Sections were then washed in 10 mM phosphate-buffered solution (PBS). Endogenous peroxidase activity was blocked by incubating the slides with hydrogen peroxide 3% for 30 min at room temperature. After being washed with PBS, sections were incubated in 3% normal bovine serum albumin for 35 min at room temperature to avoid non-specific reactions, followed by incubation with the primary antiserum overnight at 22\u0026deg;C in a humidity chamber. Slides were then washed in PBS and incubated for 30 min with biotinylated anti-mouse/anti-rabbit immunoglobulins (Bio SB, Santa Barbara, USA). Sections were washed with PBS, and a final incubation was done with Streptavidin conjugated to Horseradish Peroxidase (Bio SB, Santa B\u0026aacute;rbara, USA) for 35 min at 22\u0026deg;C. After being washed with PBS, the slides were exposed to a 3,30-diaminobenzidine tetrahydrochloride solution (DAKO, Santa Clara, USA) for a maximum of 3 min under the microscope. The reaction was then stopped with distilled water. Following all processes, the slides were rehydrated and mounted. The antisera used were PBL#45 and PBL#49 (generously provided by Dr. W. Vale, the Salk Institute, USA), which have cross-reactivity against different GnRH variants (Somoza et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), used in a final dilution of 1:500 (Stefano et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Two negative controls were performed: one by suppressing the antisera and another by preadsorbing the antisera with 1 \u0026micro;M of the mammalian variant of GnRH overnight at 4\u0026deg;C. For labeling SN cells and fibers, an anti-SN antiserum was used at a final dilution of 1:500. This antiserum was raised against the 15-mer highly conserved central core of goldfish SNs (YTPQKLATLQSVFEE) and has been extensively validated in different fish species (Zhao et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Canosa et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Pouso et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Tao et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Two negative\u003c/p\u003e \u003cp\u003econtrols were performed: one by omitting the primary antiserum and the other by preadsorption with 10 \u0026micro;M full-length \u003cem\u003eC. milii\u003c/em\u003e SN (TNEIVEEQYTPQSLATLESAFQELGKYTGAY; 98% purity) synthesized at the University of Ottawa, BioEngineering and Therapeutic Solutions Centre (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.beatsresearch.com/\u003c/span\u003e\u003cspan address=\"https://www.beatsresearch.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e overnight at 4\u0026deg;C. The CCK neurons and fibers were labeled using a rabbit anti-antiserum against desulphated cholecystokinin octapeptide (T-4254, BMA Biomedicals, Switzerland) at a final dilution of 1:1000. Two negative controls were performed: one by omitting the primary antiserum and the other by preadsorption with 1 \u0026micro;M CCK-8 (Bachem).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA schematic drawing of the \u003cem\u003eC. callorynchus\u003c/em\u003e brain, pituitary gland, and BL is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. This figure illustrates the telencephalon, comprising the anterior telencephalic bulbs, and the posterior telencephalon, which connects to the hypothalamus immediately caudal to the optic chiasm. The hypothalamus surrounding the third ventricle (3V) contains the following circumventricular organs: the rostro-lateral paraventricular organ, the ME, the dorso-caudal posterior recess organ, and the caudal SV. The pituitary gland is ventrally attached to the hypothalamus, and the BL, a glandular structure, is distinguished under the cartilage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe ME, a thin and basal region of the hypothalamus, is formed by the ependymal layer of the 3V and a fibrous layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b, c). Beneath the fibrous layer lies a circulatory network supported by connective tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Caudal to the ME, the SV contains coronet cells and blood vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b, d). The pituitary gland is a dorsoventrally elongated structure, exhibiting a clear cellular regionalization: the RPD, the PPD, and the NIL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe RPD consists of follicular bodies of various sizes situated within a network of capillaries and supporting connective tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). These follicular bodies are composed of a stratified epithelium, with their lumens intricately connected. Two types of glandular cells can be identified: basophil cells in contact with the lumen and acidophil cells in contact with the capillaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Both cell types are columnar with basal and rounded nuclei. The luminal content of the follicular bodies exhibits a PAS-positive reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe PPD presents follicular bodies intricately arranged in cords surrounded by sinusoidal blood vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Collagen and unidentified fibers penetrate the\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003edorsal region of the PPD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Hematoxylin\u0026ndash;eosin staining reveals three cell types in the follicular bodies: basophilic, acidophilic, and chromophobe cells. Basophilic cells are fusiform and are typically located in the central region of the cell cords (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Acidophil cells are cylindrical with their cytoplasm projected towards the lumens and/or sinusoidal vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Chromophobe cells are rounded in shape with a large nucleus and a small cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The luminal content of the follicular bodies also exhibits a PAS-positive reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eThe NIL is the largest area of the pituitary gland; it is organized in cell cords with several layers of large cells surrounding large sinusoidal blood vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). Small capillaries and collagen fibers are distinguished alongside the NIL cords (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The NIL dorsal region presents uncharacterized fibers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Three types of stained cells were observed in this region: basophilic, acidophilic, and chromophobe cells. The basophilic cells were found in the dorsal area of the NIL; they have large nuclei and are projected toward the blood vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). The ventral region is mostly composed of acidophil cells which are in contact with sinusoidal blood vessels; they are cylindrical with an oval and central nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Chromophobe cells are distributed throughout the NIL; they are small with a round nucleus and small cytoplasm and located in the middle of the cords (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe BL, a gland situated under the cartilage, comprises follicular bodies intricately connected by lumens and surrounded by collagen and non-characterized fibers (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). These follicular bodies are formed by acidophil cells arranged around the lumens (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). These cells are surrounded by larger basophil cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Luminal content exhibits a PAS-positive reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). The BL is connected to the posterior telencephalon by channels, one on each side, that pass through the cartilage just anterior to the optic chiasma containing a large blood vessel surrounded by collagen fibers (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b, c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the brain, the same GnRH immunoreactive (ir) neuronal cell bodies and fibers were detected using two different antisera: PBL#45 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) and PBL#49 (data not shown). These neurons were arranged as a chain of nerve cell bodies and fibers located in the posterior telencephalon (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea, b). GnRH-ir neuronal somata were bipolar, narrow, and elongated along the anteroposterior axis, reaching a maximum length of approximately 100 \u0026micro;m. These cell bodies and fibers extended from the telencephalic bulb to the optic chiasm, ventral to the telencephalic ventricle. Fibers with GnRH-ir project posteriorly, passing above the optic chiasm and extending along both sides of the third ventricle, running in different directions. In the ME, fibers ran either parallel to the third ventricle or ventrally toward the pituitary. GnRH-ir fibers also reached cells in both the PPD and the BL (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec, d). These fibers were arranged as a compact, almost indistinguishable bundle converging onto cells. GnRH-ir nerve cell bodies and fibers were additionally detected in the midbrain. Each of the channels connecting the telencephalon and the BL also contained GnRH-ir fibers, which were also observed entering the BL (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb-e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNeuronal cell bodies and fibers displaying SN-ir were detected in the posterior telencephalon (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea, b), showing a distribution like that of GnRH-ir elements. SN-ir structures were also observed in the paraventricular nucleus, the ME (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec), and the posterior recess organ (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed). SN-ir neuronal somata were smaller than GnRH-ir cells, measuring approximately 20 \u0026micro;m in diameter and displaying an oval shape. SN-ir cells were also detected in the pituitary gland and the BL, exhibiting considerable variation in size and morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea-e). Some SN-ir fibers were additionally observed in the NIL (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNeuronal cell bodies and fibers displaying CCK-ir were evident in the hypothalamic posterior recess (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003ea, b). These unipolar neurons had their somata positioned close to the ventricular lining of the posterior recess. A single primary process emerged from each soma, projecting perpendicularly away from the ventricular surface. CCK-ir neuronal somata measured approximately 15 \u0026micro;m in diameter and displayed an oval morphology. CCK-ir cells were also detected in the PPD (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003ec) and the NIL (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003ed), showing variation in size and shape. CCK-ir fibers were observed reaching the BL (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn all cases, negative controls showed no immunoreactivity for GnRH, SN, or CCK (Supplementary S1-S4), and no sex differences were observed.\u003c/p\u003e \u003cp\u003eIn all samples analyzed, \u003cem\u003efshb\u003c/em\u003e and \u003cem\u003elhb\u003c/em\u003e were detected by RT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e). The amplified PCR fragments exhibited 100% identity with those of \u003cem\u003eC. milii\u003c/em\u003e (Supplementary Figs.\u0026nbsp;5 and 6). Both GtH subunits showed a relatively higher expression in the BL than in the pituitary gland, and a relatively higher expression of \u003cem\u003elhb\u003c/em\u003e was evident in the pituitary gland of both sexes compared to \u003cem\u003efshb\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe report the anatomical locations of key neuropeptides involved in the control of GtHs, GnRH, SN, and CCK in \u003cem\u003eC. callorynchus\u003c/em\u003e, revealing a new perspective on the communication between the holocephalan hypothalamus, pituitary gland, and BL. The general anatomy of the hypothalamus and pituitary gland resembles that in other studied chondrichthyans (Sathyanesan \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1965\u003c/span\u003e; Meurling \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1967a\u003c/span\u003e; \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1967b\u003c/span\u003e; Holmes and Ball \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Northcutt \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). All circumventricular organs of the hypothalamus that we have observed in \u003cem\u003eC. callorynchus\u003c/em\u003e are like those described in elasmobranchs by Rodr\u0026iacute;guez-Moldes (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The pituitary gland is organized into three clearly distinguished regions, the RPD, the PPD, and the NIL, like those previously described in \u003cem\u003eH. colliei\u003c/em\u003e and \u003cem\u003eChimaera monstrosa\u003c/em\u003e (Sathyanesan \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1965\u003c/span\u003e; Jasiński and Gorbman \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1966\u003c/span\u003e; Meurling \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1967b\u003c/span\u003e). The BL is like to that described in \u003cem\u003eH. colliei\u003c/em\u003e and \u003cem\u003eC. monstrosa\u003c/em\u003e (Sathyanesan \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1965\u003c/span\u003e; Meurling \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1967b\u003c/span\u003e). Although the BL in holocephalans and the VL in elasmobranchs are regarded as the primary structures regulating reproduction (Dodd et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Dodd \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1983\u003c/span\u003e), most studies on chondrichthyans have not included these glands, possibly because they are overlooked due to their anatomical location. The question now is how the brain regulates the synthesis and secretion of GtHs and other hormones in chondrichthyans. According to Meurling\u0026rsquo;s descriptions, both elasmobranchs and holocephalans possess a hypothalamo\u0026ndash;hypophyseal portal blood system similar to that of tetrapods (Meurling \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1960\u003c/span\u003e; \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1967a\u003c/span\u003e; \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1967b\u003c/span\u003e). The current description of vascularization in the HP axis indicates that \u003cem\u003eC. callorynchus\u003c/em\u003e also exhibits a portal-like system, like that described in \u003cem\u003eH. colliei\u003c/em\u003e and \u003cem\u003eC. monstrosa\u003c/em\u003e (Sathyanesan \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1965\u003c/span\u003e; Jasiński and Gorbman \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1966\u003c/span\u003e; Meurling \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1967b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe present results demonstrate the presence of GnRH-ir neurons and fibers in the brain, as well as GnRH-ir fibers in the pituitary gland and the BL. A strip of GnRH-ir neuronal cell bodies is present along the posterior telencephalon, as in other vertebrates (Amano et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Muske and Moore \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Okubo and Nagahama \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zohar et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Immunoreactive GnRH fibers showed a hypothalamic distribution similar to that reported for \u003cem\u003eSqualus acanthias\u003c/em\u003e, particularly at the level of the third ventricle (D\u0026rsquo;Antonio et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). However, in \u003cem\u003eC. callorynchus\u003c/em\u003e, the fiber density observed in the ME was considerably lower than that of immunoreactive fibers observed by D\u0026rsquo;Antonio et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). In the pituitary gland of \u003cem\u003eC. callorynchus\u003c/em\u003e, GnRH-ir fibers reach the PPD, surround blood vessels, and likely terminate in proximity to pituitary cells. Importantly, GnRH-ir fibers reach the BL, ending in close contact with glandular cells.\u003c/p\u003e \u003cp\u003eThese results provide a morphological basis for the regulation of pituitary hormone synthesis and secretion in this species, as GnRH is known to control the release of multiple pituitary hormones in other vertebrates (Marchant et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Chang et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Villalobos et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). For example, according to Yamaguchi (2015), the PPD of \u003cem\u003eC. milii\u003c/em\u003e contains growth hormone-producing cells; therefore, the GnRH released from these fibers could regulate the synthesis and/or release of GtHs and GH. The presence of GnRH-ir fibers ending at the PPD and the BL suggests that the synthesis and release of the GtHs may be controlled by direct glandular innervation. This challenges the hypothesis that the BL is regulated exclusively by GnRH delivered via the general circulation, as previously proposed for \u003cem\u003eH. colliei\u003c/em\u003e. In this context, the association of these fibers with blood vessels could explain the high levels of GnRH in circulation found in \u003cem\u003eH. colliei\u003c/em\u003e by Sherwood and Lovejoy (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1993\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNeuronal cell bodies exhibiting SN-ir were evident in the posterior telencephalon of \u003cem\u003eC. callorynchus\u003c/em\u003e. These cells may be homologous to the SN neurons located in the POA of teleosts (Canosa et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Pouso et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The SN-ir fibers reaching the NIL resemble those found in goldfish (Canosa et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and zebrafish (Peng et al. 2025), which co-express isotocin (the fish equivalent of oxytocin), and in the electric fish \u003cem\u003eBrachyhypopomus gauderio\u003c/em\u003e, which co-expresses isotocin and vasotocin (fish equivalent to vasopressin; Pouso et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In rats, SN and oxytocin are co-stored in large dense-core vesicles and may be co-released into the circulation (Mahata et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Thus, the relationship between nonapaptides and SN co-expression in magnocellular cells is ancient and must have emerged in an ancestral gnathosome.\u003c/p\u003e \u003cp\u003eThe SN-ir cells identified in the RPD of \u003cem\u003eC. callorynchus\u003c/em\u003e may be equivalent to those in the goldfish RPD that co-localized with PRL (Zhao et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In the electric fish, these cells were found in both the RPD, where they co-localized with PRL, and some cells in the PPD (Pouso et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In the case of zebrafish, using the same anti-SN antiserum, cells in the RPD were SN-ir, strong SN-ir fibers were found in the PPD and RPD and SN-ir was not detected in FSH and LH cells (Peng et al. 2025). It is known that in these regions, cells expressing hormones such as PRL, LH, and GH are present; therefore, it is possible that SN could be co-expressed in some of them and/or exerts autocrine/paracrine effects on them (Zhao et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Pouso et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Mitchell et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The presence of SN-ir cells in the BL suggests a potential local role for SN and/or its release from this gland. In goldfish, the SN in RPD lactotrophs is released upon stimulation by GnRH and acts in a paracrine manner to stimulate LH release from the PPD (Zhao et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Additionally, GnRH may stimulate SN release from the murine LβT2 gonadotroph cell line, which in turn may exert an autocrine stimulatory effect that enhances LH secretion (Zhao et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). These findings raise the possibility that SN may control LH by an autocrine/paracrine manner not only in the pituitary gland but also in the BL of \u003cem\u003eC. callorynchus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn the present study, CCK-ir neuronal cell bodies and fibers were found in the hypothalamic posterior recess and, in the pituitary gland, CCK-ir cells were identified in the PPD and the NIL, which can be related to an autocrine/paracrine regulation of ACTH and α-MSH cells, as seen in mammals (Rehfeld \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Hashimoto and Kimura (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1986\u003c/span\u003e) were among the first to report the stimulatory effects of CCK on LH and, to a lesser extent, on FSH secretion in rats. Early studies in rhesus monkeys (Schreihofer et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) and goldfish (Himick et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) also indicated that CCK may exert stimulatory effects on LH. In contrast, high doses in rats were found to be inhibitory to LH, with no effects on FSH (Vijayan et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1979\u003c/span\u003e). These and other studies indicate that CCK from multiple sources (e.g., neuronal, paracrine, hormonal) has the potential to regulate the GtHs. In zebrafish, the \u003cem\u003ecckr\u003c/em\u003e transcript was found to be highly expressed in FSH cells and, at lower levels, in LH cells of both sexes (Hollander-Cohen et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In zebrafish, a GnRH3 analog and CCK preferentially activate calcium-dependent secretion in LH and FSH cells, respectively (Hollander-Cohen et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). More recently, CCK has been shown to specifically stimulate FSH cells via CCK2Rb receptors, thereby regulating ovarian and testicular development in Japanese medaka (Uehara et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Similar results were also reported in zebrafish (Mizrahi et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Thus, the possibility that CCK may regulate FSH or LH differentially in either the BL or pituitary of \u003cem\u003eC. callorynchus\u003c/em\u003e remains an open question. In this context, based on observations in mammals and teleosts, effects of CCK on LH or FSH could be either stimulatory or inhibitory.\u003c/p\u003e \u003cp\u003eThis is the first report demonstrating that both \u003cem\u003efshb\u003c/em\u003e and \u003cem\u003elhb\u003c/em\u003e mRNAs are synthesized in the pituitary of \u003cem\u003eC. callorynchus\u003c/em\u003e as in other vertebrates, but also in the BL. Previous studies in \u003cem\u003eH. colliei\u003c/em\u003e by Dodd et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1982\u003c/span\u003e) suggested that the principal gonadotropin-like activity resides in the BL, with lesser activity present in the pituitary. These conclusions were derived from bioassays in which quail and chick testes were exposed to various freeze-dried \u003cem\u003eH. colliei\u003c/em\u003e pituitary extracts, which inevitably contained GtHs along with other pituitary hormones, peptides, and additional regulators of gonadal steroidogenesis. Sampling and replication were limited, precluding firm conclusions regarding the primary source of holocephalan GtHs. Nevertheless, both GtHs were produced outside\u003c/p\u003e \u003cp\u003ethe pituitary gland, which has long been assumed in elasmobranchs (Qu\u0026eacute;rat et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Arimura et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, before the present study, this had not been empirically tested in holocephalans.\u003c/p\u003e \u003cp\u003eBy determining the differential distributions of GnRH, SN, and CCK immunoreactivity in \u003cem\u003eC. callorynchus\u003c/em\u003e we propose that the regulation of the pituitary and BL is complex and multimodal. The present findings expand the anatomical framework for understanding HP communication in holocephalans. Our results demonstrate that \u003cem\u003efshb\u003c/em\u003e and \u003cem\u003elhb\u003c/em\u003e transcripts are detectable not only in the pituitary but also, at comparatively higher expression levels, in the BL, with both structures receiving neuropeptidergic inputs. Specifically, GnRH-ir fibers reach the PPD and BL, while CCK-ir fibers also enter the BL. In addition, SN-ir is present in hypothalamic regions, pituitary tissue, and BL cells, suggesting the potential for local interactions within these endocrine structures. Although the present study does not assess hormone release, receptor expression, or functional outcomes, the anatomical relationships described here are consistent with the possibility that GtH-related processes in holocephalans are influenced by multiple routes of neuropeptidergic signaling. These observations provide a structural basis for future studies aimed at determining how neural, paracrine, and endocrine mechanisms contribute to the regulation of reproduction in early diverging jawed vertebrates.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003ch2\u003eFunding \u003cstrong\u003eDeclaration\u003c/strong\u003e:\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Agencia Nacional de Promoci\u0026oacute;n de la Investigaci\u0026oacute;n, el Desarrollo Tecnol\u0026oacute;gico y la Innovaci\u0026oacute;n (PICT 2017\u0026ndash;4507, Argentina) to CAA and institutional funding to GMS.\u003c/p\u003e\n\u003cp\u003eCorrespondence: Gustavo M. Somoza ([email protected])\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eConceptualization: CAA, VLT, GMS; Histology: CH, MS, GCL, FAV; Immunocytochemistry: CH, GCL, MRP; Molecular Biology: CH, MRP; Writing-original draft preparation: CH, CAA, GMS; Writing-review and editing: All authors. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors would like to acknowledge Juan Francisco Solana and Franco Cristiani for their assistance with fish sampling. This work was supported by the Agencia Nacional de Promoci\u0026oacute;n de la Investigaci\u0026oacute;n, el Desarrollo Tecnol\u0026oacute;gico y la Innovaci\u0026oacute;n (PICT 2017-4507, Argentina) to CAA and institutional funding to GMS.\u003c/p\u003e\n\u003ch2\u003eData Availability Statement\u003c/h2\u003e\n\u003cp\u003e(DAS) Data supporting this study are included within the article and/or supporting materials\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmano M, Oka Y, Aida K, Okumoto N, Kawashima S, Hasegawa Y (1991) Immunocytochemical demonstration of salmon GnRH and chicken GnRH-II in the brain of masu salmon, \u003cem\u003eOncorhynchus masou\u003c/em\u003e. J Comp Neurol 314:587\u0026ndash;597. ttps://doi.org/10.1002/cne.903140313\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArimura S, Kwok M, Wong S, Inoue R, Kawano M, Shimoyama K, Fujimori C, Tokunaga K, Takagi W, Hyodo S (2024) Functional characterization of follicle-stimulating hormone and luteinizing hormone receptors in cloudy catshark, \u003cem\u003eScyliorhinus torazame\u003c/em\u003e. Gen Comp Endocrinol 354:114542. ttps://doi.org/10.1016/j.ygcen.2024.114542\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAwruch CA (2013) Reproductive endocrinology in chondrichthyans: The present and\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ethe future Gen Comp Endocrinol 192:60\u0026ndash;70. ttps://doi.org/10.1016/j.ygcen.2013.05.021\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBernasconi J, Cubillos L, Acu\u0026ntilde;a E, Perier R, Di Gi\u0026aacute;como E (2015) Growth, maturity and mortality of cockfish, \u003cem\u003eCallorhinchus callorynchus\u003c/em\u003e, in San Mat\u0026iacute;as Gulf, Northern Patagonia, Argentina. Rev Biol Mar Oceanogr 50:283\u0026ndash;298. ttps://doi.org/10.4067/s0718-19572015000300007\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBl\u0026aacute;zquez M, Bosma PT, Chang JP, Docherty K, Trudeau VL (1998) γ-Aminobutyric acid up-regulates the expression of a novel secretogranin-II messenger ribonucleic acid in the goldfish pituitary. Endocrinology 139:4870\u0026ndash;4880. ttps://doi.org/10.1210/endo.139.12.6339\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCanosa LF, Lopez GC, Scharrig E, Lesaux-Farmer K, Somoza GM, Kah O, Trudeau VL (2011) Forebrain mapping of secretoneurin-like immunoreactivity and its colocalization with isotocin in the preoptic nucleus and pituitary gland of goldfish. 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J Neuroendocrinol 34:1\u0026ndash;13. ttps://doi.org/10.1111/jne.13069\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-and-tissue-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ctre","sideBox":"Learn more about [Cell and Tissue Research](https://link.springer.com/journal/441)","snPcode":"441","submissionUrl":"https://submission.springernature.com/new-submission/441/3","title":"Cell and Tissue Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Holocephali, Chimaera, GnRH, SN, CCK","lastPublishedDoi":"10.21203/rs.3.rs-9052503/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9052503/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChimaeras (Holocephali) occupy a pivotal phylogenetic position for understanding the evolution of hypothalamic\u0026ndash;pituitary organization in vertebrates; however, the structural organization of the hypothalamic\u0026ndash;pituitary axis and the anatomical relationships with the buccal lobe (BL) remain incompletely characterized. We present an anatomo-histological analysis of the chimaera \u003cem\u003eCallorhinchus callorynchus\u003c/em\u003e using classical histology, immunohistochemistry, and RT-PCR. The pituitary gland displays clear regionalization into the rostral \u003cem\u003epars distalis\u003c/em\u003e, proximal \u003cem\u003epars distalis\u003c/em\u003e, and neurointermediate lobe. The BL is connected to the posterior telencephalon by paired canals containing a prominent blood vessel and bundles of neuropeptidergic fibers. Gonadotropin-releasing hormone (GnRH)-immunoreactive nerve cell bodies and fibers were identified in the posterior telencephalon using two antisera, with fibers projecting to the proximal \u003cem\u003epars distalis\u003c/em\u003e of the pituitary and the BL. Secretoneurin (SN)-immunoreactive neuronal somata and fibers were observed in the posterior telencephalon and hypothalamus, and SN-immunoreactive cells were also present within the pituitary and BL. Cholecystokinin (CCK)-immunoreactive neuronal elements were detected in the hypothalamus, while CCK-immunoreactive cells were present in the pituitary, and a subset of fibers extending into the BL. RT-PCR analyses revealed the presence of \u003cem\u003efshb\u003c/em\u003e and \u003cem\u003elhb\u003c/em\u003e transcripts in the pituitary and BL, with a stronger apparent signal in the BL. These observations provide anatomical evidence for direct neuropeptidergic innervation of both the pituitary and BL and indicate that the BL is associated with gonadotropin subunit gene expression in \u003cem\u003eC. callorynchus\u003c/em\u003e. This new anatomical framework of the hypothalamus\u0026ndash;pituitary\u0026ndash;BL axis identifies two new pathways: direct GnRH and CCK innervation, and local SN-mediated autocrine/paracrine signaling.\u003c/p\u003e","manuscriptTitle":"Anatomy of the hypothalamic–pituitary axis and buccal lobe in the holocephalan Callorhinchus callorynchus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-16 21:00:55","doi":"10.21203/rs.3.rs-9052503/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-13T13:44:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-01T10:25:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-31T10:27:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"93533062064727052384519925082770721012","date":"2026-03-29T14:26:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"161912550705295516333108790351778389353","date":"2026-03-13T17:38:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-13T17:27:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-11T07:26:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-11T07:25:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell and Tissue Research","date":"2026-03-06T16:10:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-and-tissue-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ctre","sideBox":"Learn more about [Cell and Tissue Research](https://link.springer.com/journal/441)","snPcode":"441","submissionUrl":"https://submission.springernature.com/new-submission/441/3","title":"Cell and Tissue Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"462a0fa0-a2ed-4300-853c-699f354d7ab4","owner":[],"postedDate":"March 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T11:39:23+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-16 21:00:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9052503","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9052503","identity":"rs-9052503","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}