Association between Intracellular Calcium Signaling and Tumor Recurrence in Human Non-Functioning Pituitary Adenomas.

Clinically non-functioning pituitary adenomas (CNFPAs) are the second most frequent sellar tumor among studies on community-dwelling adults. They are characterized by the absence of hormonal hypersecretion syndrome, and patients present with compressive symptoms, such as a headache and visual field defects. Immunohistochemically, most CNFPAs are of gonadotrope differentiation, with only a few of them being truly null cell adenomas. Although these tumors express receptors for one or more hypothalamic releasing hormones, to what extent this has an impact on the biological and clinical behavior of these neoplasms remains to be defined. In this research, we evaluated the basal and hypothalamic secretagogue-stimulated intracellular calcium mobilization in 13 CNFPAs, trying to correlate this response to the phenotypic features of the patients. Our results indicate that the recurrence of a CNFPA correlates positively with cellular responsiveness, as measured by spontaneous intracellular calcium activity and the ability to respond to multiple hypothalamic secretagogues. We conclude that this finding may be a useful tool for predicting the clinicopathologic behavior of CNFPAs, by testing the variation of cellular responsiveness to hypothalamic secretagogues.

Functional expression of P2Y2 receptors in mouse ovarian surface epithelium (OSE).

Ovarian surface epithelium (OSE) is a cell monolayer surrounding the ovary; it is involved in the regulation of the ovulatory process and the genesis of ovarian carcinoma. However, intercellular messengers regulating signaling events, like proliferation in the OSE, have not been completely described. Purines have emerged as novel intercellular messengers in the ovary, in which expression of purinergic receptors has been reported in different cell types. In the present work, we described the functional expression of P2Y2 receptor (P2Y2R), a purinergic receptor widely associated with cell proliferation, in the OSE. The expression of P2Y2R by immunofluorescence and RT-PCR, and its functionality by Ca2+ recording was demonstrated in primary cultured OSE. Functional expression of P2Y2R was also exhibited in situ, by recording of intracellular Ca2+ release and detection of ERK phosphorylation after injection of a selective agonist into the ovarian bursa. Furthermore, P2Y2R activation with UTPγS, in situ, induced cell proliferation at 24 h, whereas continuous stimulation of P2Y2R during a complete estrous cycle significantly modified the size distribution of the follicular population. This is the first evidence of the functional expression of purinergic P2Y2R in the OSE and opens new perspectives on the roles played by purines in ovarian physiology.

Calcium signaling and expression of voltage-gated calcium channels in the mouse ovary throughout the estrous cycle†.

The estrous cycle is an iterative change in the anatomy, endocrinology, physiology, and behavior to provide maximum fecundity. Ovarian steroid production involves gonadotropin-induced [Ca2+]i raises due in part to voltage-gated Ca2+ channels (VGCCs) whose identity and tissue distribution in situ is largely unknown. Using fluorescence Ca2+ imaging and confocal microscopy, we recorded both spontaneous and depolarization-induced Ca2+ signals in living mouse ovarian slices. They were most prominent in theca cells (TCs) and oocytes. The presence of Ca2+ channel subunits CaV 1.2, CaV 1.3, CaV 2.1, CaV 2.2, and CaV 3 was examined with immunofluorescence of ovarian sections. CaV 1.2 and CaV 1.3 (L-type Ca2+ channels) are present in the stroma, granulosa cells (GCs), and corpora lutea (CL). Intriguingly subunits that are characteristic of nerve cells are also expressed: P/Q-type (CaV 2.1; α1A) in the stroma and CL cells and N-type (CaV 2.2; α1B) in perifollicular smooth muscle cells. The expression of α1 subunits fluctuates along the estrous cycle: in metestrus-diestrus (the quiescent stage of the cycle), CL and GCs are similarly stained, while in proestrus (stage of maximal ovarian stimulation) CL staining increases relatively to GCs. Also in proestrus, CaV 3 Ca2+ channel subunits are expressed more in CL compared to GC suggesting a more significant role of Ca2+ channels. In estrus, CaV 3 subunits from mesenchymal and interfollicular stromal cells become intensely stained. Our study represents an important step in understanding the role of VGCCs in ovarian physiology and possibly in ovarian cancer and other reproductive pathologies.

GnRH-Induced Ca(2+) Signaling Patterns and Gonadotropin Secretion in Pituitary Gonadotrophs. Functional Adaptations to Both Ordinary and Extraordinary Physiological Demands.

PITUITARY GONADOTROPHS ARE A SMALL FRACTION OF THE ANTERIOR PITUITARY POPULATION, YET THEY SYNTHESIZE GONADOTROPINS: luteinizing (LH) and follicle-stimulating (FSH), essential for gametogenesis and steroidogenesis. LH is secreted via a regulated pathway while FSH release is mostly constitutive and controlled by synthesis. Although gonadotrophs fire action potentials spontaneously, the intracellular Ca(2+) rises produced do not influence secretion, which is mainly driven by Gonadotropin-Releasing Hormone (GnRH), a decapeptide synthesized in the hypothalamus and released in a pulsatile manner into the hypophyseal portal circulation. GnRH binding to G-protein-coupled receptors triggers Ca(2+) mobilization from InsP3-sensitive intracellular pools, generating the global Ca(2+) elevations necessary for secretion. Ca(2+) signaling responses to increasing (GnRH) vary in stereotyped fashion from subthreshold to baseline spiking (oscillatory), to biphasic (spike-oscillatory or spike-plateau). This progression varies somewhat in gonadotrophs from different species and biological preparations. Both baseline spiking and biphasic GnRH-induced Ca(2+) signals control LH/FSH synthesis and exocytosis. Estradiol and testosterone regulate gonadotropin secretion through feedback mechanisms, while FSH synthesis and release are influenced by activin, inhibin, and follistatin. Adaptation to physiological events like the estrous cycle, involves changes in GnRH sensitivity and LH/FSH synthesis: in proestrus, estradiol feedback regulation abruptly changes from negative to positive, causing the pre-ovulatory LH surge. Similarly, when testosterone levels drop after orquiectomy the lack of negative feedback on pituitary and hypothalamus boosts both GnRH and LH secretion, gonadotrophs GnRH sensitivity increases, and Ca(2+) signaling patterns change. In addition, gonadotrophs proliferate and grow. These plastic changes denote a more vigorous functional adaptation in response to an extraordinary functional demand.

Relationship between growth of the preovulatory follicle and its steroidogenic activity on the onset and expression of estrus behavior in CIDR-treated Bos indicus cows: an observational study.

Estrus synchronization induces cows to gather in sexually active groups (SAGs) composed of females displaying mounting activity. Although this technique promotes the enhancement of sexual behavior, there are cows in estrus (CE) that delay estrus expression and also cows not displaying estrus (CNDE) even in the presence of a preovulatory follicle (PF). To elucidate the physiological mechanisms of the delay in the onset of estrus or absence of estrus behavior, an observational study was undertaken in 17 Bos indicus cows treated with exogenous progesterone (CIDR) to synchronize estrus and to monitor follicular growth and its steroidogenic activity. After SAGs formation, cows were ovariectomized at 24, 48, and 72 h post-CIDR. Among ovariectomized groups there were only 9 CE which: 1) showed differences in the onset of estrus; 2) displayed distinctive follicular growth patterns; and 3) at 72 h produced the highest intrafollicular estradiol concentration, and showed a linear trend to increase expression of P450scc and P450arom. Comparison of CE vs. CNDE showed that: 1) both groups had progesterone levels indicative of cyclic activity, and a PF which grew at a similar rate and size; 2) CE showed a stronger association between time and growth; and 3) CE produced more intrafollicular estradiol and progesterone, together with the expression of higher levels of P450arom. Results suggest that pending on the pattern of growth of the PF and its steroidogenic potential to produce estradiol, the onset and expression of estrus behavior may be delayed probably until the establishment of the appropriate conditions to ensure ovulation.

Prolactin released in vitro from the pituitary of lactating, pregnant, and steroid-treated female or male rats stimulates prolactin secretion from pituitary lactotropes of male rats.

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.

Vesicular release of prolactin from preformed prolactin granules is stimulated by soluble factor(s) from the anterior pituitary of 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.

Cells of proopiomelanocortin lineage from the rodent anterior pituitary lack sexually dimorphic expression of neurofilaments.

Lactotrophs, gonadotrophs, thyrotrophs and somatotrophs of the rat anterior pituitary (AP) express 68-kDa neurofilaments (NF68) and other neuronal markers. NF68 expression in the AP appears to be estrogen-dependent, but its significance is unknown. The aims of this work were: (1) to establish the expression pattern of NF68 immunoreactivity in the mouse AP, and (2) discover if corticotrophs and melanotrophs from both rodent species also express NF68. Primary cultures and frozen sections of AP from sexually mature mice were immunolabeled with anti-NF68 antibodies. In separate experiments, samples were immunostained for NF68 and AP hormones. Here we report that mouse lactotrophs, gonadotrophs, thyrotrophs and somatotrophs also express NF68 in a sexually dimorphic manner. The percentages of non-expressing, weakly expressing and strongly expressing cells were similar between both rodent species, although NF68+ cells were about 50% less abundant in the mouse compared to the rat pituitary. Remarkably, our study shows for the first time that rodent pituitary cells from the proopiomelanocortin lineage nearly completely lack NF68 immunoreactivity. In this regard, they differ from the rest of the AP population. Our findings establish a foundation for experiments aimed at investigating the functional significance of estrogen-dependent regulation of NF68 expression in rodent AP cells.