Richard M. Rohan

Arteriolar and venular patterning in retinas of mice selectively expressing VEGF isoforms

The murine VEGF gene is alternatively transcribed to yield the VEGF(120), VEGF(164), and VEGF(188) isoforms, which differ in their potential to bind to heparan sulfate and neuropilin-1 and to stimulate endothelial growth. Here, their role in retinal vascular development was studied in mice selectively expressing single isoforms. VEGF(164/164) mice were normal, healthy, and had normal retinal angiogenesis. In contrast, VEGF(120/120) mice exhibited severe defects in vascular outgrowth and patterning, whereas VEGF(188/188) mice displayed normal venular outgrowth but impaired arterial development. It is noteworthy that neuropilin-1, a receptor for VEGF(164), was predominantly expressed in retinal arterioles. These findings reveal distinct roles of the various VEGF isoforms in vascular patterning and arterial development in the retina.

Human intraovarian interleukin-1 (IL-1) system: highly compartmentalized and hormonally dependent regulation of the genes encoding IL-1, its receptor, and its receptor antagonist.

To delineate the scope of the human intraovarian IL-1 system we used a solution hybridization/RNase protection assay to test for expression of the genes encoding IL-1, its type I receptor (IL-1R), and its receptor antagonist (IL-1RA). IL-1 transcripts were not detected in whole ovarian material from days 4 or 12 of an unstimulated menstrual cycle but transcripts (IL-1 beta much greater than IL-11 alpha) were detected in preovulatory follicular aspirates from gonadotropin-stimulated cycles. Concurrently obtained peripheral monocytes did not contain IL-1 beta transcripts but macrophage-depleted follicular aspirates did, thus implicating the granulosa cells as the site of IL-1 expression. IL-1R transcripts were detected in RNA from whole ovaries and follicular aspirates but not in RNA from peripheral monocytes. IL-1RA transcripts were detected in whole ovarian material as well as in macrophage-free follicular aspirates. Cultured human granulosa and theca cells did not contain mRNA for IL-1 beta or IL-1RA but did contain mRNA for IL-1R. Treatment of cell cultures with forskolin (25 microM) induced IL-1 beta transcripts in granulosa but not theca cells. Forskolin also increased the basal levels of IL-1R transcripts in both granulosa and theca cells but did not induce IL-RA transcripts in either cell type. Taken together, these findings reveal the existence of a complete, highly compartmentalized, hormonally dependent intraovarian IL-1 system replete with ligands, receptor, and receptor antagonist.

The midcycle increase in ovarian glucose uptake is associated with enhanced expression of glucose transporter 3. Possible role for interleukin-1, a putative intermediary in the ovulatory process.

This study characterizes the rat ovary as a site of hormonally dependent glucose transporter (Glut) expression, and explores the potential role of interleukin (IL)-1, a putative intermediary in the ovulatory process, in this regard. Molecular probing throughout a simulated estrous cycle revealed a significant surge in ovarian Glut3 (but not Glut1) expression at the time of ovulation. Treatment of cultured whole ovarian dispersates from immature rats with IL-1beta resulted in upregulation of the relative abundance of the Glut1 (4.5-fold) and Glut3 (3.5-fold) proteins as determined by Western blot analysis. Other members of the Glut family (i.e., Gluts 2, 4, and 5) remained undetectable. The ability of IL-1 to upregulate Glut1 and Glut3 transcripts proved time-, dose-, nitric oxide-, and protein biosynthesis-dependent but glucose independent. Other ovarian agonists (i.e., TNF alpha, IGF-I, interferon-gamma, and insulin) were without effect. Taken together, our findings establish the mammalian ovary as a site of cyclically determined Glut1 and Glut3 expression, and disclose the ability of IL-1 to induce the ovarian expression as well as translation of Glut1 and Glut3 (but not of Gluts 2, 4, or 5). Our observations also establish IL-1 as the first known regulator of Glut3, the most efficient Glut known to date. In so doing, IL-1, a putative component of the ovulatory process, may be acting to meet the increased metabolic demands imposed on the growing follicle and the ovulated cumulus-enclosed oocyte.

Intraovarian regulation: peptidergic signaling systems.

Intraovarian regulation, an evolving field, is now at a crossroad. Although a number of putative intraovarian regulators appear to be of import to ovarian physiology, none has thus far been demonstrated to be indispensable to in vivo ovarian function. That notwithstanding, it is already clear that optimal gonadotropin hormonal action is highly contingent upon the input of tissue-based regulatory principles. It is with a strong sense of excitement that future work in this evolving area is anticipated.

Detection and in vivo hormonal regulation of rat ovarian type I and type II interleukin-I receptor mRNAs: increased expression during the periovulatory period.

Objective: To study the expression, localization, and in vivo hormonal regulation of type I and type II interleukin-1 (IL-1) receptors in the rat ovary. Methods: Segments of the cDNAs for rat type I and type II IL-1 receptors were cloned and used as probes in RNase protection assays and in situ hybridization. Tissues obtained from immature rats and hormonally treated rat ovaries were examined. Results: Type I IL-1 receptor (IL-1R(1)) was ubiquitously expressed in rat tissues, including, granulosa cells prepared from immature ovaries, whereas type II IL-1 recetor (IL-1R(2)) expression was restricted to macrophage, thymus, and lung. Hypophysectomy and subsequent treatment with FSH and/or diethylstilbestrol did not alter significantly the abundance of IL-1R(1) transcripts in the whole overy. However, the relative amount of ovarian IL-1R(1) transcripts increased 7.3-fold 6 hours after the administration of hCG to pregnant mare serun gonadotropin—primed immature rats. During this time, IL-1R(1) mRNA was localized primarily in the granulosa cells. The increased expression of IL-1R(1) persisted 24 hours after hCG administration but declined to baseline by 48 hours. Ovarian expression of IL-1R(2) mRNA was observed only before ovulation in amounts that were approximately 70-fold lower than IL-1R(1). Donclusion: The increased intraovarian expression of IL-1R(1) in granulosa cells during the periovulatory period implies that this cell type has a heightened receptivity to IL-1 and provides further indirect evidence that this cytokine is involved in the ovulatory process.

Simplified riboprobe purification using translucent straws as gel tubes

Gel purification of radioactive riboprobes enhances the quality of the ribonuclease protection assay. A simple and effective method for riboprobe purification is described. The method uses acrylamide gels in plastic tubes to achieve electrophoretic separation of the RNA polymerase products.

Rat ovarian insulin-like growth factor binding protein-4 : A hormone-dependent granulosa cell-derived antigonadotropin

Objective: To assess the in vivo regulation of ovarian insulin-like growth factor binding protein-4 (GFBP-4) mRNA expression by gonadotropins and estrogen. Methods: Whole varian RNA, obtained from two models of follicular development, was extracted and analyzed by Northern blotting. Immature rats were treated with pregnant mare senum gonadotropin (PMSG) followed 48 hours later with hCG, or alternatively were hypophysectomized and treated with FSH and/or diethylstilbestrol (DES). Localization of IGFBP-4 expression was assessed in the former study by in situ hybridization. Finally, the ability of human IGFBP-4 to antagonize FSH-stimulated progesterone accumulation was assessed in vitro. Results: The ovarian content of IGFBP-4 transcripts increased threefold (P < .05) at 12 hours after PMSG but was near baseline at 24 and 48 hours. The abundance of IGFBP-4 mRNA increased (P < .05) again at 6 and 24 hours after hCG. The expression of IGFBP-4 was localized to granulosa cells of prenatral (untreated) and small antral (12 hours after PMSG) follicles. No IGFBP-4 expression was noted in large (gonadotropin-primed) antral follicles. Hypophysectomy increased (P < .05) the ovarian content of IGBP-4 mRNA by 1.5-fold, an effect further enhanced (1.8-fold; P < .05) by the provision of FSH and DES. In vitro studies revealed the ability of increasing concentrations (0.01-1 μg/mL) of recombinant human IGFBP-4 to inhibit the FSH-supported accumulation of progesterone. Conclusion: Increased expression after administration of PMSG, hCG, and FSH/DES suggests that IGFBP-4 is a dynamic and hormonally responsive component of the ovarian cycle. The lack of expression in preovulatory follicles and its antigonadotropic actions in vitro imply that the attenuated expression of IGFBP-4 may constitute a requirement for successful follicular maturation.

In vivo hormonal regulation of insulin-like growth factor binding protein-5 mRNA in the immature rat ovary.

Objective: Despite the potential importance of insulin-like growth factor binding protein-5 (IGFBP-5) to follicular development, the hormonal regulation of this antigonadotropic IGFBP has not been investigated. Therefore, it was the objective of this study to eludicate the role of gonadotropins and estrogen in the in vivo regulation of IGFBP-5 mRNA expression. Methods: Two models of follicular development in immature rats were used. Specifically, rats were hypophysectomized and treated with FSH and/or diethylstilbestrol (DES). Alternatively, terminal follicular development was induced in intact immature rats by pregnant mare serum gonadotropin (PMSG) and hCG. The IGFBP-5 mRNA in whole ovarian RNA was assayed by Northern blot hybridization. Localization of expression in PMSG and hCG-stimulated ovaries was further assessed by in situ hybridization. Results: Expression of IGFBP-5 mRNA was increased in ovaries from hypophysectomized rats. Treatment with FSH and/or DES did not alter the abundance of this mRNA. Treatment with PMSG induced a transient increase in IGFBP-5 expression that was localized in a subset of α-inhibin-negative follicles. At later times after PMSG, IGFBP-5 expression persisted in the surface epithelium but was not detected in large preovulatory follicles. In vitro studies affirmed the antigonadotropic action of IGFBP-5. Conclusion: In vivo expression of IGFBP-5 in the rat ovary is moderated by hormonal treatment both in terms of total expression and follicular localization.

Characterization and Hormonal Regulation of Granulosa Cell-Derived Insulin-Like Growth Factor Binding Protein-4

Objective: Because of the potential importance of insulin-like growth factor binding protein-4 (IGFBP-4) to ovarian physiology and the obvious limitations imposed by in vivo-exclusive experimental paradigms, we set out to delineate the characteristics and hormonal regulation of granulosa cell-derived IGFBP-4 under in vitro circumstances. Methods: Granulosa cells obtained by follicular puncture of the ovaries from diethylstilbestrol-primed intact immature rats were subjected to culture for up to 72 hours. Insulin-like growth factor binding protein-4 mRNA extracted from culture was subjected to Northern blot hybridization. Data normalization was assured by reprobing with the hamster Chinese hamster ovary B (CHOB) cDNA, and the IGFBP-4/CHOB ratio was calculated. Conditioned culture media were subjected to Western ligand blot before and after immunoprecipitation with a rat IGFBP-4-directed polyclonal antiserum (αB104). Results: Immunoprecipitation studies revealed granulosa cell-derived IGFBP-4 to be composed of a major 24-kDa species as well as a relatively minor 27-kDa moiety. Given cultures of untreated granulosa cells from immature estrogen-treated rats, transcripts corresponding to IGFBP-4 displayed an initial temporary decline culminating in a 6-hour nadir (a decrease of 67%; P < .05) followed by relatively prompt recovery (within 24 hours) to levels comparable to those noted at the outset of the culture (time 0). However, additional (albeit statistically insignificant) increments were noted at the 48-hour (but not 72-hour) time point. Treatment of granulosa cells with increasing concentration of FSH resulted in decrements of up to 30% (P < .05) in the steady-state levels of IGFBP-4 transcripts. A modest, biphasic, time-dependent response was noted for IGFBP-4 transcripts after treatment with high-dose FSH (100 ng/mL), an effect characterized by 24-and 48-hour increments (51% [P < .05] and 26% [P = .052] over untreated controls, respectively) and a 72-hour decrement (25%; P = .16). The concurrent provision of the C19 aromatase substrate androstenedione (10-7 mol/L) to the culture medium from 72 hours enhanced the inhibitor effect of FSH (100 ng/mL) for a maximal decrement in IGFBP-4 transcripts of 49% (P < .05). Treatment with insulin-like growth factor (IGF)-I produced limited inhibition (up to 26%) of the steady-state levels of IGFBP-4 transcripts (P < .05). Conclusion: Findings indicate the existence of heterogenously-sized IGFBP-4 species, of which the 27-kDa (as distinct from the 27-kDa) IGFBP-4 moiety constitutes a relatively minor component. The steady-state levels of granulosa cell-derived IGFBP-4 transcripts display relatively limited regulation in response to treatment with either FSH or IGF-I.