Fibroblast growth factor 2 regulates cumulus differentiation under the control of the oocyte.

PURPOSE: We first assessed regulation of FGF2 expression in cumulus cells by FSH and oocyte-secreted factors during in vitro maturation (IVM). Then, we tested the hypothesis that FGF2 regulates meiotic progression, cumulus expansion, and apoptosis in cumulus-oocyte complexes (COC) undergoing IVM. METHODS: In vitro maturation of bovine COC was utilized as a model to assess regulation of FGF2 expression by FSH and oocyte-secreted factors (via microsurgical removal of the oocyte), as well as effects of graded doses of FGF2 on meiotic progression, degree of cumulus expansion, dissociation of cumulus cells, and cumulus cells apoptosis. Expression of genes regulating functional endpoints altered by FGF2 treatment was assessed in cumulus cells by real-time PCR. Cultures were replicated 4-5 times and effects of treatments were tested by ANOVA. RESULTS: FGF2 mRNA expression was increased by FSH and oocyte-secreted factors during IVM. Addition of FGF2 to the IVM medium advanced meiosis resumption, decreased the ease with which cumulus cells were dissociated, and inhibited cumulus cells apoptosis. Decreased cumulus dissociation was accompanied by decreased expression of TNFAIP6. CONCLUSIONS: This is the first study showing that FGF2 expression is regulated by the oocyte in cumulus cells. Moreover, we report novel effects of FGF2 on cumulus cell survival and extracellular matrix (ECM) quality during IVM that may favor acquisition of developmental competence and suggest physiological roles during the final steps of COC differentiation.

Effect of kit ligand on natriuretic peptide precursor C and oocyte maturation in cattle.

In vitro maturation (IVM) of oocytes in cattle is inefficient, and there is great interest in the development of approaches to improve maturation and fertilization rates. Intraovarian signalling molecules are being explored as potential additives to IVM media. One such factor is kit ligand (KITL), which stimulates the growth of oocytes. We determined if KITL enhances oocyte maturation in cattle. The two main isoforms of KITL (KITL1 and KITL2) were expressed in bovine cumulus-oocyte complexes (COC), and levels of mRNA increased during FSH-stimulated IVM. The addition of KITL to the culture medium increased the percentage of oocytes that reached meiosis II but did not affect cumulus expansion after 22 h of IVM. Addition of KITL reduced the levels of mRNA encoding natriuretic peptide precursor C (NPPC), a protein that holds oocytes in meiotic arrest, and increased the levels of mRNA encoding YBX2, an oocyte-specific factor involved in meiosis. Removal of the oocyte from the COC resulted in increased KITL mRNA levels and decreased NPPC mRNA levels in cumulus cells, and addition of denuded oocytes reversed these effects. Taken together, our results suggest that KITL enhances bovine oocyte nuclear maturation through a mechanism that involves NPPC, and that the oocyte regulates cumulus expression of KITL mRNA.

Temporal analysis of prostaglandin F2α receptor, caspase 3, and cyclooxygenase 2 messenger RNA expression and prostaglandin F2α receptor and cyclooxygenase 2 protein expression in endometrial tissue from multiparous Nelore (Bos taurus indicus) cows treated with cloprostenol sodium during puerperium.

The use of cloprostenol sodium in puerperium is questionable, as both favorable and unfavorable responses during the uterine involution process have been reported in the literature. This study is based on the hypothesis that cloprostenol sodium promotes modifications in the prostaglandin F2α receptor (FP), caspase 3 (CASP-3), and cyclooxygenase 2 (COX-2) mRNA expression that may favor the process of postpartum uterine involution in multiparous Nelore (Bos taurus indicus) females. Additionally, we aimed to describe the presence and immunolocalization of the FP and COX-2 protein in endometrial tissue at different postpartum time points in these females. Multiparous Nelore cows (n = 24) were treated with cloprostenol sodium (n = 12) or saline solution (n = 12) on postpartum Days 1 and 4 (Day 0 = birth), and endometrial biopsies were performed with a Yomann biopsy instrument and collected on Days 1, 7, 14, 28, and 42 postpartum. The mRNA expression from samples on the Days 1, 7, 14, 28, and 42 and the protein expression from samples on the Days 1, 14, 28, and 42 were then analyzed. The treated cows had altered FP and CASP-3 mRNA expression, and FP and COX-2 protein were observed in the endometrial surface epithelium, the stroma, and the glandular epithelium, with cytoplasmic immunolocalization. Although we attribute the change in CASP-3 mRNA expression to physiological phenomena, the results obtained for FP mRNA expression opens new doors for the study of hormonal protocols associated with cloprostenol sodium in the puerperium of Zebu females.

Isolation and characterization of nine polymorphic microsatellite loci in Piper solmsianum (piperaceae).

PREMISE OF THE STUDY: Nine microsatellite (simple sequence repeat [SSR]) loci were characterized for natural populations of Piper solmsianum, a potential source of bioactive secondary metabolites, and analyzed to assess the levels of genetic diversity in this species. • METHODS AND RESULTS: Based on an enriched library using the oligonucleotides (CT)8 and (GT)8, a total of 19 pairs of SSR primers were designed and nine of them were highly polymorphic after screening of 37 specimens from two populations. The number of alleles per locus ranged from one to six while the observed heterozygosity for polymorphic loci ranged from 0.000 to 0.875. • CONCLUSIONS: The SSR regions characterized were informative, and the genetic markers will be useful to assess the genetic diversity and gene flow in populations of P. solmsianum.

Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus-oocyte complexes.

Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus-oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2.

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.

Isolation and characteristics of eight novel polymorphic microsatellite loci in Lippia alba (Verbenaceae).

PREMISE OF THE STUDY: A set of eight microsatellite (simple sequence repeat [SSR]) markers for Lippia alba, an important medicinal and cosmetic plant, was developed to aid studies of genetic diversity and to define efficient strategies for breeding programs. METHODS AND RESULTS: Using a (CT)(8)- and (GT)(8)-enriched library, a total of 11 SSR loci were developed and optimized in L. alba. Of the 11 loci, eight were found to be polymorphic after screening 61 accessions from two populations. The parameters used to characterize loci were expected heterozygosity (H(e)) and number of alleles. A total of 44 alleles were identified, with an average of 5.5 alleles per loci, which were moderately to highly informative according to H(e). CONCLUSIONS: These new SSR markers have potential for informing genetic diversity, allele mining, and mapping studies and will be used to generate information for breeding programs of L. alba.

Blunted central bromocriptine-induced tachycardia in conscious, malnourished rats.

Bromocriptine-induced tachycardia, persisting after adrenalectomy, is mediated by central dopamine D2 receptor stimulation through activation of the sympathetic outflow to the heart. The present study investigated the effects of malnutrition during pregnancy on bromocriptine-induced tachycardia in adult conscious rats. Malnourished rats were obtained by feeding dams a multideficient diet (providing 8% protein) during mating and pregnancy. Birth weight was significantly reduced in malnourished rats when compared to control rats born to dams fed standard commercially diet (23% protein) during mating and pregnancy. Baseline mean aortic pressure and heart rate in malnourished rats were comparable to those of well-nourished rats. Tachycardia (33+/-9 beats/min.), but not the hypotensive response to intravenous bromocriptine (150 microg/kg) was significantly reduced in malnourished rats, compared with control rats (70+/-10 beats/min.). In malnourished rats, pretreatment with intravenous domperidone (500 microg/kg) blocked the bromocriptine-induced hypotension, without affecting the tachycardia. Neither cardiac vagal (40+/-6 beats/min.) nor sympathetic tone (76+/-6 beats/min.) was significantly altered by multideficient diet-induced malnutrition (51+/-6 and 67+/-10 beats/min., respectively). In isolated perfused heart preparations from malnourished rats, positive inotropic response to isoproterenol (10-8 to 10-4 M) was not significantly different compared to that in control rats. In summary, malnutrition during foetal life blunted the bromocriptine-induced tachycardia, an effect that could be related to central dopamine D2 receptor desensitization rather than to impairment of autonomic regulation of the heart or cardiac beta-adrenoceptor desensitization.