We have previously shown that soluble factor(s) in conditioned media (CM) from the central and peripheral regions of the anterior pituitary (AP) gland of lactating rats promoted the in vitro dose-related release of prolactin (PRL) from pituitary glands of male rats. In the present experiments we sought to determine whether CM from rats in different physiological states provoked similar effects (like those of lactating rats), and the nature of the factors, whether 23K PRL or other variants of the hormone, were responsible for these effects. Stimulatory effects were induced by CM from pregnant females and steroid-treated castrated males or females, but not from untreated castrated rats, intact males, or by a PRL standard. More potent effects occurred with CM from APs of early- than from mid- or late-lactating rats, and from rats unsuckled for 8 or 16 h than from those unsuckled for 32 h. With respect to the nature of factor(s) responsible for these effects, immunoprecipitation of PRL from the CM of lactating females and of steroid-treated, castrated males eliminated, whereas dephosphorylation or deglycosylation of CM of lactating rats greatly increased its effects upon PRL release. Also, electrophoretic analysis and Western blotting of the CM proteins under native and denaturing conditions revealed a variety of PRL variants, ranging from 14 to <90 kDa, in CM from lactating rats, and the main effects on PRL release were provoked by the 23- to 46-kDa PRL variants. These results indicate that specific effects upon male rat lactotropes may be exerted by PRL variants released from APs of lactating and non-lactating rats.
This study demonstrates that conditioned media (CM) from the anterior pituitary gland (AP) of lactating rats contains soluble factors that promote in vitro prolactin (PRL) release from the pituitary glands of male rats. CM-induced PRL release was confirmed by polyacrylamide gel electrophoresis, ELISA and bioassay. In cultured AP cells challenged with CM, increased intracellular staining with the dye FM1-43 was observed, suggesting vesicular PRL release and subsequent endocytosis. The percentage and hormone content of PRL-containing cells but not of growth hormone-containing cells increased in cultured male AP cells when exposed to CM. When the release of PRL, prelabeled with [3H] leucine for 30 min to 24 h was examined, no stimulatory effect of CM was observed, suggesting that released PRL originates from hormone synthesized more than 24 h earlier. Accordingly, the PRL content of mature granules from male pituitary tissues decreased after CM treatment. These findings were confirmed by electron microscopy immunogold PRL labeling. Treatment with inhibitors of protein synthesis or vesicle trafficking between the endoplasmic reticulum and the Golgi complex did not prevent the stimulatory effect of CM on PRL release. However, blockage of traffic to the plasma membrane completely abolished the effect of CM. These results suggest that CM from the AP of lactators contains soluble factor(s) capable of inducing rapid vesicular release of PRL in the male AP, which originates from preformed, mature granules by mechanisms independent of protein synthesis.
Angiogenesis is essential for the growth and maturation of the ovarian follicle and its transition into the corpus luteum. In addition to the main proangiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), follicular fluid (FF) contains the hormone prolactin (PRL), which is known to promote angiogenesis in vivo. Here, we show that FF from large follicles, which contains twice the PRL level of FF from small follicles, stimulates endothelial cell proliferation to a greater extent than the latter, and that immunoneutralization of PRL prevents FF from stimulating endothelial cell proliferation. Notably, the FF increases the expression of the short and long PRL receptor isoforms in endothelial cells, and a purified PRL standard stimulates endothelial cell proliferation but only after the cells have been pretreated with FF. However, purified PRL activates the JAK2/STAT3 pathway in endothelial cells in the absence of pretreatment with FF. In summary, PRL present in the FF stimulates the proliferation of endothelial cells. This effect likely involves the upregulation of the short and long PRL receptor isoforms and is independent of PRL-induced JAK2/STAT3 signaling.
PRL secretion exerted by variants of the PRL in presence of hypothalamic hormones. PRL variants (4,22) are secreted under different physiological conditions (4,(16)(17)(18)(23)(24)(25), and interactions with other pituitary cells (4,7,11,16) and hypothalamic hormones (3,10,12,(26)(27)(28)(29)(30) are known to occur in different circumstances. For instance, the lactotrophs of lactating rats from the central AP region, which is the region surrounding the neurointermediate pituitary lobe (23,31,32), are bigger, secrete more PRL than those of the peripheral AP region, and after a short period of suckling, become more sensitive to the PRL-stimulatory agents TRH and angiotensin II. Moreover, they become unresponsive to dopamine and interact with lactotrophs in the periph-Background: Previous studies used western blotting to show that prolactin (PRL) released from the adenohypophysis (AP) of lactating rats in vitro contains size variants from 7-14 kDa to 70-97 kDa. These variants when eluted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and incubated with AP lactotrophs from male rats and rats in other conditions, promoted the selective stimulation and/or inhibition of the in vitro release of PRL variants. Objectives: In the present study, we determined the regulatory effects of dopamine (DA), thyrotropin-releasing hormone (TRH), and oxytocin (OT) on release of PRL variants from AP lactotrophs. Materials and Methods: Primary cultures of lactotrophs from lactating rats, which were non-suckled (NS) for 6 h or suckled (S) for 15 min after NS, were incubated with PRL variants that were electroeluted from SDS-PAGE gels along with different doses of DA (0.5, 1.0, 1.5 µM), TRH (0.1, 1.0, 10 µM), or OT (0.1, 1.0, 10 µM). The secretion of PRL from the lactotrophs was determined by enzyme-linked immunosorbant assay. Results: The results showed stimulatory and/or inhibitory effects of DA, TRH, and OT on the release of PRL variants from AP lactotrophs both by the presence of PRL variants. Conclusions: These results indicate that PRL variants are released from AP lactotrophs, and, in concert with hypothalamic hormones, they regulate the release of PRL from lactating rat APs.
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