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. 2017 Mar 21;114(12):E2514-E2523.
doi: 10.1073/pnas.1618917114. Epub 2017 Mar 7.

Mechanisms Regulating Angiogenesis Underlie Seasonal Control of Pituitary Function

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Free PMC article

Mechanisms Regulating Angiogenesis Underlie Seasonal Control of Pituitary Function

Jennifer Castle-Miller et al. Proc Natl Acad Sci U S A. .
Free PMC article

Erratum in

Abstract

Seasonal changes in mammalian physiology, such as those affecting reproduction, hibernation, and metabolism, are controlled by pituitary hormones released in response to annual environmental changes. In temperate zones, the primary environmental cue driving seasonal reproductive cycles is the change in day length (i.e., photoperiod), encoded by the pattern of melatonin secretion from the pineal gland. However, although reproduction relies on hypothalamic gonadotrophin-releasing hormone output, and most cells producing reproductive hormones are in the pars distalis (PD) of the pituitary, melatonin receptors are localized in the pars tuberalis (PT), a physically and functionally separate part of the gland. How melatonin in the PT controls the PD is not understood. Here we show that melatonin time-dependently acts on its receptors in the PT to alter splicing of vascular endothelial growth factor (VEGF). Outside the breeding season (BS), angiogenic VEGF-A stimulates vessel growth in the infundibulum, aiding vascular communication among the PT, PD, and brain. This also acts on VEGF receptor 2 (VEGFR2) expressed in PD prolactin-producing cells known to impair gonadotrophin secretion. In contrast, in the BS, melatonin releases antiangiogenic VEGF-Axxxb from the PT, inhibiting infundibular angiogenesis and diminishing lactotroph (LT) VEGFR2 expression, lifting reproductive axis repression in response to shorter day lengths. The time-dependent, melatonin-induced differential expression of VEGF-A isoforms culminates in alterations in gonadotroph function opposite to those of LTs, with up-regulation and down-regulation of gonadotrophin gene expression during the breeding and nonbreeding seasons, respectively. These results provide a mechanism by which melatonin can control pituitary function in a seasonal manner.

Keywords: VEGF; angiogenesis; melatonin; pituitary gland; season.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Angiogenesis in the pituitary is seasonally dependent. (A) Endothelial staining in the PT/infundibulum and quantification of vessel loops in the summer (i.e., NBS) and winter (i.e., BS). (B) Endothelial proliferation (PCNA/CD31 double-positive cells) in the NBS and BS. (C) Total VEGF-A levels in the PT/stalk in the BS and NBS (not significantly different; P > 0.05). (D) VEGF-Axxxb–specific ELISA on protein extracted from pituitaries of animals killed in the BS or NBS. (E) Proportion of VEGF-A that was VEGF-Axxxb in the BS and NBS. (F) Staining of the melatonin receptor (green) and VEGF-A (red) in different regions of the pituitary; costaining (found in PT and vascular loops) is shown as yellow (***P < 0.01 and ***P < 0.001 vs. BS). (Scale bar: 50 µm.)
Fig. S1.
Fig. S1.
(A) cDNA amplified from the pituitary PD and PT from ewes by primers that detect exon 8a- and exon 8b-containing isoforms (Fig. 1). Both bands were purified and sequenced. (B) Chromatogram of sequence of PCR products. Upper band (Left) shows splicing from exon 7 to exon 8a. Lower band shows splicing from exon7 to exon 8b. (C) MT1 receptor was colocalized with VEGF-Axxxb staining, but not glial cells or endothelium (Scale bar: 20 μm.)
Fig. 2.
Fig. 2.
VEGF-A isoforms levels are regulated by melatonin periodicity in the PT. (A) PT cells in culture were isolated from pituitaries of sheep and VEGF-Axxxb measured by ELISA. Cells from winter sheep (BS, blue) were treated with melatonin for 16 h each day for 6 d; cells from summer sheep (NBS, red) were treated for 8 h each day with melatonin. (B) Levels of panVEGF-A were also measured from these cells. (C) VEGF-Axxxb levels were measured from sheep PT cells incubated with the incongruous melatonin exposure for the time from which they were harvested (cells from summer sheep were given a winter melatonin regimen; those from winter sheep were given a summer melatonin regimen). (D) Levels of panVEGF-A from cells treated as in C. (E) VEGF-A isoform mRNA expression in PT cells from the BS (winter) after 6 d of treatment with BS and NBS melatonin regimens. (F) VEGF-A isoform mRNA expression in PT cells from the NBS (summer) after 6 d of treatment with BS and NBS melatonin regimens. Boxes show positions of the primers used to amplify the cDNA (***P < 0.001 vs. control, +++P < 0.001 vs. BS regimen).
Fig. S2.
Fig. S2.
(A) PT cells were isolated from sheep in BS and stained for the melatonin receptor, VEGF-A, and Hoechst (Fig. 2). (B) VEGF levels from cells cultured in the absence of melatonin. There were no significant differences between VEGF-A levels from PT cells taken from the winter (BS, blue), or summer (NBS, red) ewes. (C) VEGF levels did not alter over 6 d in primary culture (Scale bar: 20 μm.)
Fig. 3.
Fig. 3.
VEGFR2 is up-regulated in the PD during the summer (i.e., NBS). (A) Colocalization of VEGFR2 (green) and glial-type FSCs (red) in the BS (winter) and NBS (summer). (B) VEGFR2 (green) expression in LTs (red) in the BS and NBS. (C) VEGFR2 (green) and endothelial cells stained by isolectin B4 (IB4, red) in both seasons. (D) Proliferating (PCNS, green) endothelial (IB4, red) cells were stained, and colocalization was quantified. (E) VEGF-Axxxb expression was detected in the PD in the winter (BS) but not in the summer (NBS); VEGF-Axxx expression was detected in the summer (NBS) but not in the winter (BS). (F) Quantification of the expression of VEGF-A isoforms. (G) ELISA quantification of the amount of VEGF-Axxxb in the BS and NBS. (H) ELISA quantification of the amount of total VEGF-A in the two seasons (*P < 0.05 and **P < 0.01; ns, nonsignificant at P > 0.05 vs. BS). (Scale bar: 50 µm.)
Fig. 4.
Fig. 4.
VEGF-A mediates prolactin release from the PD in an isoform-dependent manner. (A) VEGFR2 expression in cultured prolactin positive cells (i.e., LTs) of the PD. (B) Prolactin secretion following treatment with TRH (positive control), melatonin (Mel, negative control), and medium (control) in PD cells cultured during the NBS (summer) and BS (winter). (C) Prolactin secretion in PD cells from sheep killed in the summer (NBS) after rhVEGF-A165a treatment was greater than that from winter (BS) sheep. (D) Prolactin mRNA expression in PD cells taken from ewes in the summer (NBS) following a summer (NBS) regimen of rhVEGF-A165a (8 h on, 16 h off) was greater than that of cells taken from the same animals following a winter (BS) rhVEGF-A165a regimen (16 h on, 8 h off). (E) In cells taken from ewes in the BS (winter), prolactin was induced only if VEGF-A was given in an NBS (summer) regimen. (F) Conditioned media from summer (NBS) PT cells treated with a summer (NBS) melatonin regimen (red) induced prolactin production. This was blocked by an antibody to VEGF-Axxxa. (G) Prolactin mRNA in summer (NBS) cells was induced in the presence of conditioned media from summer (NBS) regimen PT cells, and this was blocked by an anti-VEGF-Axxxa antibody (**P < 0.01 and ***P < 0.001 vs. BS). (Scale bar: 20 µm.)
Fig. S3.
Fig. S3.
(A) Pars distalis cells from NBS treated with rhVEGF-A165a in a BS regimen (red) do not produce prolactin (Fig. 4). In contrast, cells from the BS treated with rhVEGF-A165a in an NBS manner (blue) did produce prolactin. (B) Conditioned media from PT cells treated with an NBS melatonin regimen had no effect on FSH production in PD cells, irrespective of which season the cells were from. (C) BS cells treated with BS, but not with NBS, PT-conditioned media produced FSH mRNA. (D) NBS cells treated with CM from PT cells exposed to a BS regimen of melatonin produced FSH. This was blocked by incubation with an antibody to VEGF-Axxxb (αVEGF-Axxxb; *P < 0.05 vs. untreated).
Fig. 5.
Fig. 5.
Working model for a melatonin-induced, VEGF-A isoform-dependent intrapituitary regulation of seasonal physiology. In this model, the duration of nocturnal melatonin secretion induces differential synthesis and release of proangiogenic and antiangiogenic isoforms of VEGF-A in the PT region of the ovine pituitary and in the vascular loops that connect this tissue with the infundibulum. (A) In the short days of winter (BS), the long duration of nocturnal melatonin exposure up-regulates the secretion of the antiangiogenic isoform VEGF-A164b at the expense of the proangiogenic isoform VEGF-A164a, resulting in reduced angiogenesis, reduced density of VEGF receptors in endocrine and FS cells of the PD, suppression of prolactin secretion, and no inhibition of the gonadotrophic axis. (B) In contrast, during the long days of summer (NBS), the short duration of nocturnal melatonin exposure up-regulates the secretion of the proangiogenic isoform VEGF-A164a at the expense of the antiangiogenic isoform VEGF-A164b, leading to increased angiogenesis, increased density of VEGF receptors in endocrine and FS cells of the PD, stimulation of prolactin secretion, and inhibition of the gonadotrophic axis.

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