Antibodies obtained by xenotransplantation of organ-cultured median eminence specifically recognize hypothalamic tanycytes

Abstract

Tanycytes are specialized ependymal cells lining the infundibular recess of the third ventricle of the cerebrum. Early and recent investigations involve tanycytes in the mechanism of gonadotropin-releasing hormone (GnRH) release to the portal blood. The present investigation was performed to obtain a specific immunological marker of tanycytes and to identify the compound(s) responsible for this labeling. After 30 days of organ culture, explants of bovine median eminence formed spherical structures mostly constituted by tanycytes. These tanycyte spheres were xenotransplanted to rats, and the antibodies raised by the host animals against the transplanted living tanycytes were used for immunochemical studies of the bovine and rat median eminence. This antiserum immunoreacted with two compounds of 60 kDa and 85 kDa present in extracts of bovine and rat median eminence. The individual immunoblotting analysis of rat medial basal hypothalami showed a decrease in the amount of the 85-kDa compound in castrated rats as compared to control rats processed at oestrus and dioestrus. The antiserum, labeled as anti-P85, when used for immunostaining of sections throughout the rat central nervous system, immunoreacted specifically with the hypothalamic tanycytes. Within tanycytes, P-85 immunoreactivity was exclusively present in the basal processes. It is suggested that the 85-kDa and 60-kDa compounds correspond to two novel proteins selectively expressed by tanycytes. The possibility that they are secretory proteins involved in GnRH release is discussed. Anti-P85 appears to be the first specific marker of hypothalamic tanycytes.

Cell organization of the rat pars tuberalis. Evidence for open communication between pars tuberalis cells, cerebrospinal fluid and tanycytes

“…A similar finding has been reported in an early study carried out in the rhesus monkey by Knowles and Kumar (1969). β Tanycytes have the capacity to transport compounds from the ventricular CSF to the perivascular space of the portal capillaries (Peruzzo et al 2004;Rodríguez et al 2005) and, consequently, to the intercellular channels of the PT; β tanycytes are also known to secrete, through their basal processes, biologically active compounds such as β-transforming growth factor, prostaglandin E2 or the specific protein p85 (Rodríguez et al 2005;Blázquez et al 2002;Ojeda et al 2008).…”

“…(2) anti-rat βTSH (NIDDK, IC-1, AFP 1274789), dilution 1:1000; (3) anti-rat α subunit (GSU, NIDDK-IC-1, AFP 66P9986), dilution 1:1000; (4) anti-α 58K, a marker of the Golgi apparatus, monoclonal (AbCam, Cambridge, UK), dilution 1:1000; (5) anti-S100 (AbCam, Cambridge, UK), dilution 1:100; (6) anti-connexin 43 (kindly provided by J.C. Saéz, Universidad Católica de Chile), dilution 1:750; (7) anti-caveolin-1, raised in rabbits, affinity purified (N-20, Santa Cruz Biotechnology, San Diego, Calif., USA), 1:200 dilution (this antibody reacts specifically with caveolin-1; Peruzzo et al 2004); (8) anti-βIV-tubulin, a marker of cilia, monoclonal (AbCam, Cambridge, UK), dilution 1:100; (9) anti-alpha catenin, raised in rabbits (Santa Cruz Biotech), dilution 1:250; (10) anti-P85, raised in rats in our laboratory (Blázquez et al 2002), dilution 1:250. Use of preimmune serum and omission of incubation in the primary antiserum during the immunostaining procedure were used as control tests and resulted in no immunostaining.…”

“…The use of anti-p85, a specific marker of tanycytes (Blázquez et al 2002), anti-βIV-tubulin and anti-S100, Fig. 5 Transmission electron microscopy of a zone of the PT similar to that boxed (rectangle) in Fig.…”

Polarized endocytosis and transcytosis in the hypothalamic tanycytes of the rat

“…The functional significance of this anatomical arrangement has been the matter of much controversy (Rodríguez 1976;Akmayev and Fidelina 1981;Flament-Durand and Brion 1985;McQueen 1994;Wittkowski 1998;García-Segura et al 1999). A solid body of evidence associates β tanycytes, and β 1 tanycytes in particular, to the mechanism of release of gonadotrophin-releasing hormone (GnRH) from the terminals of the GnRH neurons to the portal blood (Hökfelt 1973;Rodríguez et al 1982;Ojeda et al1990Ojeda et al , 1992Ojeda et al , 1997King and Rubin 1994;Ma 1998, Wittkowski 1998;Prevot et al 1999Prevot et al , 2003Blázquez et al 2002).…”

“…Tanycytes do not constitute a homogeneous cell population. In the rat, four types of tanycytes have been distinguished (Akmayev and Fidelina 1976;Rodríguez et al 1979): α 1 tanycytes line the area of the ventromedial hypothalamic nucleus and project their basal processes to this nucleus; α 2 tanycytes line the area of the arcuate nucleus and their processes are distributed within this nucleus; β 1 tanycytes line the lateral recesses in the infundibular recess and project their processes to the latero-external region of the median eminence in which they establish close contact with the gonadotrophin hormone-releasing hormone nerve fibers and end on the perivascular space of the portal capillaries located in the lateral region of the median eminence; β 2 tanycytes line the floor of the infundibular recess and their basal processes end on the portal capillaries of the medial zone of the median eminence (Rodríguez et al 1979;Flament-Durand and Brion 1985;Kozlowski and Coates 1985;Peruzzo et al 2000;Blázquez et al 2002). Tanycytes of the α 1,2 and β 1 types do not establish a barrier between the cerebrospinal fluid (CSF) and brain, whereas β 2 tanycytes do (Brightman et al 1975;Rodríguez et al 1982); similarly, α 1,2 and β 1 tanycytes express glucose transporter 1, whereas β 2 tanycytes do not (Peruzzo et al 2000).…”