Vasopressin gene products are colocalised with corticotrophin‐releasing factor within neurosecretory vesicles in the external zone of the median eminence of the Japanese macaque monkey (Macaca fuscata)

Arginine vasopressin (AVP), when released into portal capillaries with corticotrophin‐releasing factor (CRF) from terminals of parvocellular neurones of the hypothalamic paraventricular nucleus (PVH), facilitates the secretion of adrenocorticotrophic hormone (ACTH) in stressed rodents. The AVP gene encodes a propeptide precursor containing AVP, AVP‐associated neurophysin II (NPII), and a glycopeptide copeptin, although it is currently unclear whether copeptin is always cleaved from the neurophysin and whether the NPII and/or copeptin have any functional role in the pituitary. Furthermore, for primates, it is unknown whether CRF, AVP, NPII and copeptin are all colocalised in neurosecretory vesicles in the terminal region of the paraventricular CRF neurone axons. Therefore, we investigated, by fluorescence and immunogold immunocytochemistry, the cellular and subcellular relationships of these peptides in the CRF‐ and AVP‐producing cells in unstressed Japanese macaque monkeys (Macaca fuscata). Reverse transcription‐polymerase chain reaction analysis showed the expression of both CRF and AVP mRNAs in the monkey PVH. As expected, in the magnocellular neurones of the PVH and supraoptic nucleus, essentially no CRF immunoreactivity could be detected in NPII‐immunoreactive (AVP‐producing) neurones. Immunofluorescence showed that, in the parvocellular part of the PVH, NPII was detectable in a subpopulation (approximately 39%) of the numerous CRF‐immunoreactive neuronal perikarya, whereas, in the outer median eminence, NPII was more prominent (approximately 52%) in the CRF varicosities. Triple immunoelectron microscopy in the median eminence demonstrated the presence of both NPII and copeptin immunoreactivity in dense‐cored vesicles of CRF‐containing axons. The results are consistent with an idea that the AVP propeptide is processed and NPII and copeptin are colocalised in hypothalamic‐pituitary CRF axons in the median eminence of a primate. The CRF, AVP and copeptin are all co‐packaged in neurosecretory vesicles in monkeys and are thus likely to be co‐released into the portal capillary blood to amplify ACTH release from the primate anterior pituitary.


| INTRODUC TI ON
The hypothalamic neuroendocrine response to stress is an important survival mechanism in vertebrates. Corticotrophin-releasing factor (CRF), a 41-amino-acid peptide, potently stimulates adrenocorticotrophic hormone (ACTH) secretion from the anterior pituitary when released into the portal capillaries of the median eminence (ME) from terminals of parvocellular neurones of the hypothalamic paraventricular nucleus (PVH). [1][2][3] The release of arginine vasopressin (AVP) into portal capillary plasma results in a stronger ACTH response than that obtained with CRF alone, because of the synergistic action of CRF and AVP to induce ACTH release. 4-10 A population of CRF neurones in the PVH can co-secrete AVP, and both peptides are secretagogues for ACTH in mammals, including rats 11 and humans. 12 Furthermore, the expression of the AVP gene in CRF neurones is increased under stressed conditions. 13,14 Translation of the AVP gene results in the formation of a propeptide which is cleaved to release the AVP hormone, but which also contains the AVP-associated neurophysin (NP) II, and a glycopeptide copeptin. [15][16][17] The NPII has the important function of binding/stabilising the AVP, 18 although the function of the copeptin and when and if it is proteolytically cleaved from the NPII is currently unclear. 19 In many mammals, including rats and humans, ultrastructural immunocytochemistry demonstrates that CRF and AVP are co-packaged into the same neurosecretory vesicles (NSVs) in the CRF-positive ( + ) and AVP + subpopulation of fibres in the ME, indicating that they are almost certainly co-released into the portal capillary blood. 20,21 The concentration of AVP in the pituitary portal vessels increases when animals are stressed or are subjected to adrenalectomy. 22 Although, in unstressed mice, very few parvocellular neurones are doubly positive for CRF and AVP, severe stress or adrenalectomy significantly increases the ratio of double-positive cells (to half). 23 However, at least in primates, it is unknown whether CRF, AVP and the AVP-associated NPII and copeptin can all be detected in NSVs in both the cell bodies and terminal region of the CRF neurone axons. Furthermore, any clear evidence for the function of the secreted copeptin is currently lacking. 19 The present study provides the first evidence in the hypothalamus of unstressed Japanese macaque monkeys (Macaca fuscata) that CRF, AVP, and the AVP-associated NPII and copeptin are all co-packaged within the NSVs, and therefore that the AVP precursor protein undergoes proteolysis during the passage of NSVs from the CRH perikarya to the ME.

| RNA extraction and reverse transcriptionpolymerase chain reaction (RT-PCR)
One male and two female monkeys were anaesthetised with an overdose of sodium pentobarbital (50-90 mg kg -1 body weight), transcardially perfused with physiological saline, and killed via blood loss.
The hypothalamus and cerebral cortex (temporal lobe) were quickly removed and frozen with powdered dry ice and stored at − 80ºC until RNA extraction. Brains were sectioned at 100 µm thickness with a cryostat (CM3050S; Leica Microsystems, Wetzlar, Germany).
Sections were then dissected at − 20ºC into 2-3 mm square tissue fragments containing either the PVH or temporal cortex. Total RNA was extracted using an illustra RNAspin Mini RNA isolation kit (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) in accordance with the manufacturer's instructions. The concentration of total RNA was measured using a NanoDrop Lite (Thermo Fisher Scientific, Waltham, MA, USA). First-strand cDNA was synthesised from 200 ng of total RNA with random primers using Omniscript RT kit (Qiagen, Valancia, CA, USA). The RT-PCR conditions for target genes and GAPDH as an internal control are shown in the Supporting information (Table S1). The PCR amplicons were electrophoresed on eminence of a primate. The CRF, AVP and copeptin are all co-packaged in neurosecretory vesicles in monkeys and are thus likely to be co-released into the portal capillary blood to amplify ACTH release from the primate anterior pituitary.

K E Y W O R D S
corticotrophin-releasing factor, Japanese macaque monkey (Macaca fuscata), median eminence, paraventricular nucleus of the hypothalamus, vasopressin 2% agarose gels. RT-PCR studies were repeated three times using independently extracted RNA samples from different animals.
Consistent results were obtained from each run.

| Tissue preparation for microscopic analysis
Two male and two female monkeys were anaesthetised with an overdose of sodium pentobarbital (50-90 mg kg -1 body weight), and transcardially perfused with physiological saline followed by
After blocking nonspecific binding with 1% normal goat serum and 1% bovine serum albumin (BSA) in PBST for 30 minutes at room temperature, the sections were incubated with primary rabbit antisera directed against CRF (dilution 1:20 000) or copeptin (dilution 1:10 000) and with a mouse monoclonal anti-AVP-NPII antibody (dilution 1:1000) for 4-5 days at 4ºC. The rabbit polyclonal antiserum against rat/human copeptin, which was generated by us, has been characterised previously via western analysis in rats. 24 The copeptin 7-14 (ATQLDGPA) fragment, used as an antigen to produce our copeptin antiserum, is well conserved in mammals. It is identical in rats, 25 25 Thus, our antiserum appears to be appropriate for use in various mammalian species including primates. The mouse monoclonal NPII (PS41) antibody has previously shown to be specific for AVP neurones in rodents. 27,28 The rabbit polyclonal antiserum against rat/human CRF (PBL rC70), which was kindly donated by Wylie Vale, has been characterised elsewhere. 29 It was directed against the rat form of CRF, which is identical to the human form, and has also been characterised in non-human primates. 30 Alexa Fluor 546-linked anti-mouse immunoglobulin G (IgG) (Molecular Probes, Carlsbad, CA, USA) and Alexa Fluor 488-linked anti-rabbit IgG (Molecular Probes) were used for detection at 1:1000 dilution. Immunostained sections were viewed by confocal laser scanning microscopy (FluoView 1000; Olympus, Tokyo, Japan). The antibodies used in the study are shown in Table 1. The co-expression ratio of the immunoreactivity for CRF/NPII was also determined in sections of the ME. For each of the four to five 30-µm thick sections per animal (n = 4 monkeys; two females, two males), we analysed at 400× magnification, unit areas (50 × 50 μm 2 ) to determine CRF/NPII immunoreactivity. In total, 74 unit areas in the external layer and 60 unit areas in the internal layer from 17 sections were analysed. The optical density of the immunostaining of CRF (green), NPII (magenta) and the merged images (green, magenta and white) were all determined by preparing black-white images that were converted from each immunofluorescence micrograph using ImageJ, version 1.52a (National Institutes of Health, Bethesda, MD, USA). The optical density of the background labelling was estimated by comparison with similar areas of the control sections (see Supporting information, Figure S1). Each threshold optical density was determined by normalising the data to those of the control sections. The CRF/NPII-immunoreactive-fibre pixel density was semi-quantitated as the average pixel density in both the external and internal layers of the ME. The percentage of co-expression ratio was calculated in accordance with the formula: [pixel density of CRF immunoreactivity (green) + pixel density of NPII-immunoreactivity (magenta) -pixel density of the merged image (including all green,

| Post-embedding immunoelectron microscopy
Slices of PFA + glutaraldehyde-fixed tissues containing the ME were  Table 1.

| Statistical analysis
The data are presented as the mean ± SEM and were analysed using Statcel4 (version 4, OMS publishing, Tokyo, Japan). Statistical analysis of the number of CRF-and NPII-specific gold particles was assessed via Student's t test.

| RE SULTS
The mRNAs were also detected in the cerebral cortex. Almost equivalent amounts of GAPDH cDNA were amplified from RNA preparations among these tissues, which showed that no significant RNA degradation had occurred and a proper RT was obtained in the present study. Figure 1 shows the topographic distribution of the neuronal components of the PVH, supraoptic nucleus, and ME derived from immunofluorescence labelling for CRF (left; green) and AVPassociated NPII (right; magenta) derived from coronal sections of the macaque hypothalamus. Neurones expressing NPII are referred to as 'AVP(NPII)' neurones.
In the PVH, a difference in size between neurones in the putative 'parvo'-and 'magnocellular' divisions of the PVH was not apparent in these primates; in both parts, the immunostained neuronal perikarya appeared to be of similar size. In the magnocellular part of the PVH (PaM), essentially no CRF immunoreactivity could be detected in the AVP(NPII)-immunoreactive neurones (Figure 2A; arrows). By contrast, in the medial parvocellular part of the PVH (PaPV), numerous neurones were CRF-immunoreactive and 39.0 ± 0.6% of the CRF + perikarya contained detectable AVP(NPII) immunoreactivity ( Figure 2B; arrows). Virtually all AVP(NPII)-immunoreactive neurones in the PVH also contained copeptin immunoreactivity ( Figure 3). The CRF + and AVP(NPII) + neurones were also more intermingled with magnocellular neurones in the monkey than in rodent PVH, as reported previously. 30,32 In the SON, no neurones exhibited both AVP(NPII) and CRF immunoreactivity (Figure 1). The immunofluorescence labelling for CRF and AVP(NPII) in the macaque ME shows the topographic distribution of the specific staining as reported previously ( Figure 4). 33,34 The internal and external zones were separated by a region in which the numerical density of immunoreactive processes was much lower (Figures 4 and 5A). A dense plexus of CRF + fibres was present in the external layer of the ME as reported previously, 33 and analysis of the immunoreactivity suggests that 52.3 ± 1.5% of CRF + fibres co-expressed AVP(NPII) (Figure 5A, arrowheads). By contrast, in the internal layer of the ME, AVP(NPII) + fibres were the most prominent, and only 2.8 ± 0.3% fibres contained both AVP(NPII) and CRF immunoreactivity ( Figure 5A).

Controls in which the primary antiserum was omitted showed no
immunostaining in the ME (see Supporting information, Figure S1). Double-immunofluorescence for AVP(NPII) and the AVP-associated copeptin showed that, as expected, most AVP(NPII) + fibres in the ME also exhibited copeptin immunoreactivity in both the internal and external layers ( Figure 5B). In the zone between the internal and external zones, CRF immunoreactivity was most marked (Figure 5A, merged) and, as in the PaPV, in only approximately half of the CRFimmunoreactive fibres could AVP(NPII) or copeptin immunoreactivity also be detected ( Figure 5B, merged).
Electron microscopy showed that, in the external layer of the ME, the diameter of an NSV was approximately 100 nm as expected for axons derived from parvocellular neurones in the PVH ( Figure 6). 28,31 Double-immunoelectron microscopy for CRF/AVP(NPII) ( Figure 6) showed that, in the varicosities where both immunoreactivity was present, some individual vesicles were labelled by gold particles of both 10 nm and 15 nm, demonstrating the vesicular colocalisation of both CRF and AVP(NPII) ( Figure 6A,B,B'). In some neighbouring terminals, only CRF single-positive vesicles could be detected ( Figure 6A, asterisks, and C,C'). Quantification showed that the number of CRFspecific gold particles per NSV was 5.2 ± 0.5 (n = 1875 NSVs) and that F I G U R E 2 Double-label immunofluorescence for corticotrophinreleasing factor (CRF) and vasopressin (AVP)-associated neurophysin (NPII) in the paraventricular nucleus of the macaque hypothalamus (PVH). PaM, paraventricular hypothalamic nucleus, magnocellular part (A); PaPV, paraventricular hypothalamic nucleus, parvocellular part, ventral division (B). Immunoreactivity against CRF (green) and AVP(NPII) (magenta) is merged on the right (merge). Arrowheads in (B) indicate perikarya doubly immunopositive for CRF and AVP(NPII) in the PaPV. Scale bars = 50 µm Double-labelling also showed that, in some AVP(NPII) + varicosities, NSVs were also positive for copeptin ( Figure 7A,B,B'). However, no immunoreactivity for either AVP(NPII) or copeptin could be detected in many neighbouring varicosities ( Figure 7A, asterisk, C,C'). Triple immunoelectron microscopy for CRF/AVP(NPII)/copeptin confirmed that the NSVs in certain neurosecretory axons contained CRF, AVP(NPII) and copeptin immunoreactivity together ( Figure 8A,B,B'), and triple-immunopositive vesicles were frequently seen. Neighbouring fibres often contained only CRF single-positive NSVs ( Figure 8A, asterisk, C,C').
Very few gold particles were associated with other intra-varicosity organelles, such as the mitochondria, and background labelling over glial and endothelial elements was also extremely low. Immunoelectron microscopy studies were repeated independently at least three times using different monkeys and always produced similar results.

| D ISCUSS I ON
This is the first report of the subcellular colocalisation of CRF, the AVP-associated NPII and copeptin (and therefore also AVP) in NSVs F I G U R E 3 Double-label immunofluorescence for copeptin and vasopressin (AVP)-associated neurophysin (NPII) in the paraventricular nucleus of the macaque hypothalamus (PVH). PaM, paraventricular hypothalamic nucleus, magnocellular part (A); PaPV, paraventricular hypothalamic nucleus, parvocellular part, ventral division (B). Immunoreactivity against copeptin (green) and AVP(NPII) (magenta) is merged on the right (merge). The identical staining pattern between AVP(NPII) and copeptin at the cell body level is observed in the monkey PVH. Scale bars = 50 µm F I G U R E 4 Low magnification images of macaque median eminence. Double-label immunofluorescence for corticotrophin-releasing factor (CRF) and vasopressin (AVP)-associated neurophysin (NPII) in the median eminence shows topographic distribution of the immunoidentified peptides as previously reported. Immunoreactivity against CRF (green) and AVP(NPII) (magenta) is merged on the right (merge). InL, internal layer of the median eminence; ExL, external layer of the median eminence. Scale bars = 100 µm Merge AVP(NPII) CRF InL ExL in the hypothalamic parvocellular CRF-secreting system of a primate. Our RT-PCR analysis indicates the expression of both CRF and AVP mRNAs in the monkey PVH. Earlier immunocytochemical studies have also reported that CRF and AVP can be localised to the same parvocellular neurones in the rat 20 and mouse, 23 as well as in the human PVH. 12,21,35 However, although it appears likely that CRF and the other AVP precursor components (NPII and copeptin) are copackaged within the same NSV in the primate hypothalamus, there has thus far been no anatomical evidence.
In our unstressed monkeys, in the medial part of the PVH where the parvocellular perikarya from which axons project directly to the ME are located, approximately 61% of CRF + perikarya did not contain immunodetectable amounts of NPII or copeptin (approximately 39% contained immunodetectable NPII). The internal zone of the ME contained only a few CRF + fibres as expected. In the pre-terminal region between the internal and external zones of the ME, only a minority of the CRF-immunoreactive fibres contained AVP(NPII) or copeptin immunoreactivity and the overall amount of immunoreactivity was less marked, suggesting that there were fewer NSVs in the axons at this point. In the external zone of the ME close to the hypothalamic-pituitary capillaries, all three peptides (CRF, AVP and copeptin) were located in about half of the axons (approximately 52% of the CRF-immunoreactive tissue was also NPIIimmunoreactive). In other adjacent axons containing CRF + vesicles, no AVP(NPII) or copeptin could be detected. These observations are consistent with reports that, in unstressed rodents, only a minority of the CRF neurones produce significant amounts of the AVP precursor, although that either stress or adrenalectomy activates the production of AVP (and therefore the AVP precursor molecule) in the CRF neurones. 20,23 Our findings suggest that the difference in the extent to which AVP, NPII and copeptin are present in the CRF and magnocellular systems relates primarily to the proportion of the neurones that express the AVP gene, rather than any differences in the rate of processing of the AVP precursor. In the internal zone of the ME through which the magnocellular axons pass en route to the posterior pituitary, the AVP precursor has clearly been processed sufficiently to reveal prominent AVP(NPII) and copeptin immunoreactivity in almost all of the fibres.
Both magnocellular AVP and parvocellular CRF/AVP neurones express the prohormone convertases, PC1 and PC2 in rodents. 36,37 In CRF/AVP neurones, the expression of PC1 is selectively regulated by glucocorticoids, although adrenalectomy has no effect on PC1 or PC2 levels in magnocellular neurones in rats. 37 Our finding that AVP(NPII) and copeptin immunoreactivity is somewhat more prominent in the terminal parts of the CRF-immunoreactive axons in the external ME than in their PVN perikarya could reflect the progressive proteolysis of the AVP/NPII/copeptin precursor in the CRF/AVP axons of the unstressed monkeys.
In monkeys, the axons of magnocellular AVP neurones project to the posterior pituitary via the internal zone of the ME, whereas other AVP-producing parvocellular neurones of the PVH have axons that project throughout the brain including the external zone of the ME and into the spinal cord as in other mammals. 32,[38][39][40][41] In many studies, AVP has been reported to potentiate the ACTH secretory activity of CRF at the level of the ME. [7][8][9] The expression of AVP in the parvocellular CRF neurones is increased as a result of both adrenalectomy and stress. 42 In the unstressed rat ME, approximately 44% of the CRF-immunoreactive axons and terminals were stained for AVP. By contrast, in the adrenalectomised rat ME, almost all of the CRF-immunoreactive terminals showed strong staining for AVP, suggesting that adrenalectomy causes the transformation of a subpopulation of CRF + /AVPaxons F I G U R E 5 Double-label immunofluorescence for corticotrophinreleasing factor (CRF) (A) or copeptin (B) and vasopressin (AVP)-associated neurophysin (NPII) in the macaque median eminence. Immunoreactivity against CRF (A; green) or copeptin (B; green) and AVP(NPII) (magenta) is merged in the right (overlap white). The outlined areas in merged images are enlarged. Arrowheads indicate tissue doubly immunopositive for CRF or copeptin and AVP(NPII). InL, internal layer of the median eminence; ExL, external layer of the median eminence. Scale bars = 50 µm (and 10 µm in enlarged images)  20 ; the monkeys that we studied were not stressed, and so it is unsurprising that, in many CRF neurones, the AVP gene was not expressed to a detectable extent.

InL
In the magnocellular system, any clear evidence for a function of secreted copeptin is currently lacking. 19 However, experiments seeking a possible physiological role for either NPII or copeptin have so far been confined to the systemic circulation and a potential role for either NPII or copeptin in the anterior pituitary has yet to be investigated. Our results demonstrate that CRF, AVP(NPII) F I G U R E 6 Double immunoelectron microscopy for vasopressin (AVP)-neurophysin (NPII) and corticotrophin-releasing factor (CRF) in the external zone of the macaque median eminence. In some varicosities, numerous neurosecretory vesicles were doubly immunopositive for CRF (signalled by 10-nm gold particles) and AVP(NPII) (signalled by 15-nm gold particles). The outlined area in (A) is enlarged in (B) and (C). By contrast, in other neighbouring varicosities, no AVP(NPII) immunoreactivity was detected (C).

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
AO and SM performed PCR experiments. AO, NK, SM, YU and HS performed immunofluorescence experiments. NK, JFM and HS performed immunoelectron microscopy analyses. YU, JFM and TS interpreted the data and provided the advice and equipment. HS wrote the paper with assistance from JFM. NK and AO contributed equally to this study. HS supervised the whole study. All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.