A. C. S. Castilho

Expression and Function of Fibroblast Growth Factor 10 and Its Receptor, Fibroblast Growth Factor Receptor 2B, in Bovine Follicles

Abstract Some fibroblast growth factors (FGFs) affect ovarian follicle cell growth and/or differentiation. Whereas many FGFs activate several FGF receptors, FGF7 and FGF10 primarily activate only one, FGFR2B. As FGF7 is produced by bovine theca cells and acts on granulosa cells, we tested the hypothesis that FGF10 may also play a role in folliculogenesis in cattle. Reverse transcription-polymerase chain reaction demonstrated the presence of FGF10 mRNA in the oocytes and theca cells of the antral follicles, as well as in the preantral follicles. FGF10 protein was detected by immunohistochemistry in the oocytes of the preantral and antral follicles, and in the granulosa and theca cells of the antral follicles. FGF10 expression in theca cells changed during follicle development; mRNA abundance decreased with increasing follicular estradiol concentration in healthy follicles, and was lowest in highly atretic follicles. Culturing of granulosa cells in serum-free medium revealed FSH regulation of FGF10 receptor expression. The addition of FGF10 to cultured granulosa cells decreased the level of estradiol but did not alter cell proliferation. These data support a role for FGF10 in signaling to granulosa cells from theca cells and/or the oocyte.

Expression of fibroblast growth factor 10 and its receptor, fibroblast growth factor receptor 2B, in the bovine corpus luteum.

There is evidence that several fibroblast growth factors (FGFs) are involved in growth and development of the corpus luteum (CL), but many FGFs have not been investigated in this tissue, including FGF10. The objective of this study was to determine if FGF10 and its receptor (FGFR2B) are expressed in the CL. Bovine CL were collected from an abattoir and classed as corpus hemorrhagica (stage I), developing (stage II), developed (stage III), and regressed (stage IV) CL. Expression of FGF10 and FGFR2B mRNA was measured by reverse transcription‐polymerase chain reaction (RT‐PCR). Both genes were expressed in bovine CL, and FGF10 expression did not differ between stages of CL development. FGF10 protein was localized to large and small luteal cells by immunohistochemistry. FGFR2B expression was approximately threefold higher in regressed compared to developing and developed CL (P < 0.05). To determine if FGF10 and FGFR2B expression is regulated during functional luteolysis, cattle were injected with PGF2α and CL collected at 0, 0.5, 2, 4, 12, 24, 48, and 64 hr thereafter (n = 5 CL/time point), and mRNA abundance was measured by real‐time RT‐PCR. FGF10 mRNA expression did not change during functional luteolysis, whereas FGFR2B mRNA abundance decreased significantly at 2, 4, and 12 hr after PGF2α, and returned to pretreatment levels for the period 24–64 hr post‐PGF2α. These data suggest a potential role for FGFR2B signaling during structural luteolysis in bovine CL. Mol. Reprod. Dev. 75: 940–945, 2008.

Maternal protein restriction during pregnancy affects gene expression and immunolocalization of intestinal nutrient transporters in rats

Intrauterine dietary restriction may cause changes in the functioning of offspring organs and systems later in life, an effect known as fetal programming. The present study evaluated mRNA abundance and immunolocalization of nutrient transporters as well as enterocytes proliferation in the proximal, median and distal segments of small intestine of rats born to protein-restricted dams. Pregnant rats were fed hypoproteic (6% protein) or control (17% protein) diets, and offspring rats were evaluated at 3 and 16 weeks of age. The presence of SGLT1 (sodium-glucose co-transporter 1), GLUT2 (glucose transporter 2), PEPT1 (peptide transporter 1) and the intestinal proliferation were evaluated by immunohistochemical techniques and the abundance of specific mRNA for SGLT1, GLUT2 and PEPT1 was assessed by the real-time PCR technique. Rats born to protein-restricted dams showed higher cell proliferation in all intestinal segments and higher gene expression of SGLT1 and PEPT1 in the duodenum. Moreover, in adult animals born to protein-restricted dams the immunoreactivity of SGLT1, GLUT2 and PEPT1 in the duodenum was more intense than in control rats. Taken together, the results indicate that changes in the small intestine observed in adulthood can be programmed during the gestation. In addition, they show that this response is caused by both up-regulation in transporter gene expression, a specific adaptation mechanism, and intestinal proliferation, an unspecific adaptation mechanism.

Maternal protein restriction affects gene expression and enzyme activity of intestinal disaccharidases in adult rat offspring

This study investigated the consequences of intrauterine protein restriction on the gastrointestinal tract and particularly on the gene expression and activity of intestinal disaccharidases in the adult offspring. Wistar rat dams were fed isocaloric diets containing 6% protein (restricted, n = 8) or 17% protein (control, n = 8) throughout gestation. Male offspring (n = 5-8 in each group) were evaluated at 3 or 16 weeks of age. Maternal protein restriction during pregnancy produced offspring with growth restriction from birth (5.7 ± 0.1 vs 6.3 ± 0.1 g; mean ± SE) to weaning (42.4 ± 1.3 vs 49.1 ± 1.6 g), although at 16 weeks of age their body weight was similar to control (421.7 ± 8.9 and 428.5 ± 8.5 g). Maternal protein restriction also increased lactase activity in the proximal (0.23 ± 0.02 vs 0.15 ± 0.02), medial (0.30 ± 0.06 vs 0.14 ± 0.01) and distal (0.43 ± 0.07 vs 0.07 ± 0.02 U·g-1·min-1) small intestine, and mRNA lactase abundance in the proximal intestine (7.96 ± 1.11 vs 2.38 ± 0.47 relative units) of 3-week-old offspring rats. In addition, maternal protein restriction increased sucrase activity (1.20 ± 0.02 vs 0.91 ± 0.02 U·g-1·min-1) and sucrase mRNA abundance (4.48 ± 0.51 vs 1.95 ± 0.17 relative units) in the duodenum of 16-week-old rats. In conclusion, the present study shows for the first time that intrauterine protein restriction affects gene expression of intestinal enzymes in offspring.

Experimental evidence of heparanase, Hsp70 and NF-κB gene expression on the response of anti-inflammatory drugs in TNBS-induced colonic inflammation.

AIM Etiopathogenesis of inflammatory bowel disease is unclear and results from a complex interplay of genetic, microbial, environmental and immune factors. Elucidating the mechanisms that drive IBD depends on the detailed characterization of human inflammatory mediators in animal models. Therefore, we studied how intestinal inflammation affects heparanase, NF-κB and Hsp70 gene expression in rats, and if current intestinal anti-inflammatory drugs (sulphasalazine, prednisolone and azathioprine) act on these expressions. Moreover, we investigated the relationships among these genes with colonic cytokines levels (IL-1β, TNF-α, IL-6, INF-γ and IL-10) and oxidative stress that have fundamental role in IBD. MATERIAL AND METHODS Macroscopic parameters (diarrhea, extension of lesion, colonic weight/length ratio and damage score), biochemical markers (myeloperoxidase and alkaline phosphatase activities, and glutathione, IL-1β, TNF-α, IL-6, INF-γ and IL-10 levels), gene expressions (heparanase, NF-κB and Hsp70), and microscopic evaluations (optic, electronic scanning and transmission microscopic) were performed in rats. KEY FINDINGS Expression of heparanase, Hsp70 and NF-κB and oxidative stress were increased by inflammatory process and differentially modulated by sulphasalazine, prednisolone and azathioprine treatments. Protective effects of drugs were also related to differential modulation of cytokine changes induced by inflammatory process, showing different mechanisms to control inflammation. SIGNIFICANCE Heparanase, NF-κB and Hsp70 gene expression participate in the inflammatory response induced by TNBS and represent pharmacological targets of the intestinal anti-inflammatory drugs. In addition, current drugs used to treat IBD (sulphasalazine, prednisolone and azathioprine) differentially modulate heparanase, NF-κB and Hsp70 gene expression, cytokine production and oxidative stress.

Messenger ribonucleic acid abundance of intestinal enzymes and transporters in feed-restricted and refed chickens at different ages

The effects of feed restriction and subsequent refeeding on the gene expression of intestinal enzymes and nutrient transporters at 2 ages, 7 and 35 d, were examined in different groups of broiler chickens. At each age, birds were feed restricted for 7 d (30% of ad libitum intake) followed by 3 d of refeeding ad libitum. Control groups were fed ad libitum. Total RNA of jejunal mucosa was extracted according to the Trizol protocol, and mRNA expression of sodium glucose transporter 1, glucose transporter 2, peptide transporter 1, aminopeptidase, maltase, and sucrase-isomaltase complex was obtained by reverse-transcription PCR. The expression of aminopeptidase, sodium glucose transporter 1, and peptide transporter 1 was higher in feed-restricted groups than in control groups at d 14 (181.4, 116.7, and 80.4%, respectively) and d 42 (143.5, 84.2, and 195.9%, respectively). The mRNA abundance of sucrase-isomaltase complex was higher (159.1%) only in chickens that were feed restricted from d 35 to 42. No statistically significant effect of feed restriction was observed for mRNA abundance of maltase and glucose transporter 2 at either age. After refeeding (d 17 and 45), the RNA abundance of enzymes and nutrient transporters was similar to that in the control group. Thus, this study suggests that an effect of upregulation in gene expression exists during feed restriction that disappears when feed is supplied ad libitum.

Expression of fibroblast growth factor 10 and cognate receptors in the developing bovine ovary

In the mammalian ovary, FGF10 is expressed in oocytes and theca cells and is a candidate for paracrine signaling to the developing granulosa cells. To gain insight into the participation of FGF10 in the regulation of fetal folliculogenesis, we assessed mRNA expression patterns of FGF10 and its receptors, FGFR1B and FGFR2B, in relation to fetal follicle dynamics and localized FGF10 protein in bovine fetal ovaries at different ages. Primordial, primary, secondary, and antral follicles were first observed on Days 75, 90, 150, and 210 of gestation, respectively. The levels of GDF9 and BMP15 mRNA, markers for primordial and primary follicles, respectively, increased during fetal ovary development in a consistent manner with fetal follicle dynamics. CYP17A1 mRNA abundance increased from Day 60 to Day 75 and then from Day 120 to Day 150, coinciding with the appearance of secondary follicles. FGF10 mRNA abundance increased from Day 90, and this increase was temporally associated with increases in FGFR1B mRNA abundance and in the population of primary follicles. In contrast, FGFR2B mRNA expression was highest on Day 60 and decreased thereafter. FGF10 protein was localized to oogonia and oocytes and surrounding granulosa cells at all fetal ages. The present data suggest a role for FGF10 in the control of fetal folliculogenesis in cattle.

α1-Adrenoceptors in proximal segments of tail arteries from control and reserpinised rats

It has been recently shown that the supersensitivity of distal segments of the rat tail artery to phenylephrine after chemical sympathectomy with reserpine results from the appearance of α1D-adrenoceptors. It is known that both α1A- and α1D-adrenoceptors are involved in the contractions of proximal portions of the rat tail artery. Therefore, this study investigated whether sympathectomy with reserpine would induce supersensitivity in proximal segments of the rat tail artery, a tissue in which α1D-adrenoceptors are already functional. Proximal segments of tail arteries from reserpinised rats were three- to sixfold more sensitive to phenylephrine and methoxamine than were arteries from control rats (n = 6–2; p < 0.05). The imidazolines N-[5-(4,5-Dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide hydrobromide (A-61603) and oxymetazoline, which activate selectively α1A-adrenoceptors, were equipotent in tail arteries from control and reserpinised rats (n = 4–2; p < 0.05), whereas buspirone, which activates selectively α1D-adrenoceptor, was ≈4-fold more potent in tail arteries from reserpinised rats (n = 4–6; p < 0.05). Prazosin (nonselective) and 5-methylurapidil (α1A-selective), were competitive antagonists of contractions induced by phenylephrine and were equipotent in tail arteries from control and reserpinised rats (n = 4–6). The selective α1D-adrenoceptor antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (BMY-7378) presented similar complex antagonism in tail arteries from control and reserpinised rats, with Schild slopes much lower than 1.0 (p < 0.05, n = 4–6). Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) revealed that mRNA encoding α1A-and α1B-adrenoceptors are similarly distributed in tail arteries from control and reserpinised rats, whereas mRNA for α1D-adrenoceptors is twice more abundant in the tail artery from reserpinised rats. In conclusion, the supersensitivity induced by reserpine is related only to α1D-adrenoceptors, even in tissues where this receptor subtype is already present and functional. Only the use of subtype-selective α1-adrenoceptor agonists detected the increased α1D-adrenoceptor component after reserpinisation, as the antagonists behaved similarly in tail arteries from control and reserpinised rats.

Experimental evidence of MAP kinase gene expression on the response of intestinal anti-inflammatory drugs

AIM The etiopathogenesis of inflammatory bowel disease (IBD) is unclear and further understanding of the mechanisms that regulate intestinal barrier integrity and function could give insight into its pathophysiology and mode of action of current drugs used to treat human IBD. Therefore, we investigated how intestinal inflammation affects Map kinase gene expression in rats, and if current intestinal anti-inflammatory drugs (sulphasalazine, prednisolone and azathioprine) act on these expressions. MATERIAL AND METHODS Macroscopic parameters of lesion, biochemical markers (myeloperoxidase, alkaline phosphatase and glutathione), gene expression of 13Map kinases, and histologic evaluations (optic, electronic scanning and transmission microscopy) were performed in rats with colonic inflammation induced by trinitrobenzenesulphonic (TNBS) acid. KEY FINDINGS The colonic inflammation was characterized by a significant increase in the expression of Mapk1, Mapk3 and Mapk9 accompanied by a significant reduction in the expression ofMapk6. Alterations inMapk expression induced by TNBS were differentially counteracted after treatment with sulphasalazine, prednisolone and azathioprine. Protective effects were also related to the significant reduction of oxidative stress, which was related to increase Mapk1/3 expressions, which were reduced after pharmacological treatment. SIGNIFICANCE Mapk1, Mapk3,Mapk6 and Mapk9 gene expressionswere affected by colonic inflammation induced by TNBS in rats and counteracted by sulphasalazine, prednisolone and azathioprine treatments, suggesting that these genes participate in the pharmacological response produced for these drugs.

Gene expression profile in heat-shocked Holstein and Nelore oocytes and cumulus cells

The present study determined the transcriptome profile in Nelore and Holstein oocytes subjected to heat shock during IVM and the mRNA abundance of selected candidate genes in Nelore and Holstein heat-shocked oocytes and cumulus cells (CC). Holstein and Nelore cows were subjected to in vivo follicle aspiration. Cumulus-oocyte complexes were assigned to control (38.5°C, 22h) or heat shock (41°C for 12h, followed by 38.5°C for 10h) treatment during IVM. Denuded oocytes were subjected to bovine microarray analysis. Transcriptome analysis demonstrated 127, nine and six genes were differentially expressed between breed, temperature and the breed×temperature interaction respectively. Selected differentially expressed genes were evaluated by real-time polymerase chain reaction in oocytes and respective CC. The molecular motor kinesin family member 3A (KIF3A) was upregulated in Holstein oocytes, whereas the pro-apoptotic gene death-associated protein (DAP) and the membrane trafficking gene DENN/MADD domain containing 3 (DENND3) were downregulated in Holstein oocytes. Nelore CC showed increased transcript abundance for tight junction claudin 11 (CLDN11), whereas Holstein CC showed increased transcript abundance for antioxidant metallothionein 1E (MT1E) . Moreover, heat shock downregulated antioxidant MT1E mRNA expression in CC. In conclusion, oocyte transcriptome analysis indicated a strong difference between breeds involving organisation and cell death. In CC, both breed and temperature affected mRNA abundance, involving cellular organisation and oxidative stress.