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.
Bone morphogenetic protein 15 (BMP15) and members of the fibroblast growth factor (FGF) family are expressed by the oocyte and are involved in the control of cumulus cell function. We tested the hypothesis that FGF17, alone or combined with BMP15 in the maturation medium, enhances cumulus expansion, meiosis progression, embryonic development, and expression of mRNA encoding key genes regulating expansion (prostaglandin-endoperoxide synthase 2 [PTGS2], hyaluronan synthase 2 [HAS2], tumor necrosis factor-stimulated gene 6 [TNFAIP6], and pentraxin 3 [PTX3]) and markers of oocyte developmental competence (phosphofructokinase [PFKP], gremlin [GREM1], versican [VCAN], and the genomic progesterone receptor [nPR]) in cumulus cells. Fibroblast growth factor 17 and BMP15 increased the percentage of fully expanded cumulus-oocyte complexes (COCs), but there was no additive effect when both were combined. Neither FGF17 nor BMP15 altered the percentage of oocytes reaching meiosis II at the end of COC culture or cleavage and blastocyst rates after IVF. However, embryo quality, as assessed by the number of cells in the inner cell mass, was improved by the combination of FGF17 with BMP15. Fibroblast growth factor 17 alone did not alter gene expression in cumulus cells at the end of IVM, whereas BMP15 increased PTGS2 and PTX3 mRNA levels. The combination of FGF17 and BMP15 increased nPR mRNA abundance in cumulus cells but did not change the expression of other markers of developmental competence. This study provides novel evidence that FGF17 enhances cumulus expansion in bovine COCs submitted to IVM and that the supplementation of the IVM medium with FGF17 and BMP15 may improve embryo quality.
The objective of this experiment was to evaluate the effects of glucose infusion on serum concentrations of glucose, insulin, and progesterone (P4), as well as mRNA expression of hepatic CYP2C19 and CYP3A4 in nonlactating, ovariectomized cows in adequate nutritional status. Eight Gir × Holstein cows were maintained on a low-quality Brachiaria brizantha pasture with reduced forage availability, but they individually received, on average, 3 kg/cow daily (as fed) of a corn-based concentrate from d -28 to 0 of the experiment. All cows had an intravaginal P4-releasing device inserted on d -14, which remained in cows until the end of the experiment (d 1). On d 0, cows were randomly assigned to receive, in a crossover design containing 2 periods of 24h each (d 0 and 1), (1) an intravenous glucose infusion (GLUC; 0.5 g of glucose/kg of BW, over a 3-h period) or (2) an intravenous saline infusion (SAL; 0.9%, over a 3-h period). Cows were fasted for 12h before infusions, and they remained fasted during infusion and sample collections. Blood samples were collected at 0, 3, and 6h relative to the beginning of infusions. Liver biopsies were performed concurrently with blood collections at 0 and 3h. After the last blood collection of period 1, cows received concentrate and returned to pasture. Cows gained BW (16.5 ± 3.6 kg) and BCS (0.08 ± 0.06) from d -28 to 0. Cows receiving GLUC had greater serum glucose and insulin concentrations at 3h compared with SAL cohorts. No treatment effects were detected for serum P4 concentrations, although mRNA expression of CYP2C19 and CYP3A4 after the infusion period was reduced for cows in the GLUC treatment compared with their cohorts in the SAL treatment. In conclusion, hepatic CYP3A4 and CYP2C19 mRNA expression can be promptly modulated by glucose infusion followed by acute increases in circulating insulin, which provides novel insight into the physiological mechanisms associating nutrition and reproductive function in dairy cows.
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.
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.
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.
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 oocytesecreted 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.
The majority of mammalian oocytes destined for in vitro maturation (IVM) have not undergone all molecular and structural changes necessary for competence acquisition to support the fertilization and early embryogenesis. In this context, different methods able to provide a transient arrest of meiosis resumption have been tested in order to improve the in vitro developmental potential of oocytes. Based on that, our study aimed to evaluate the effect of temporary meiosis inhibition using roscovitine on gene expression in sheep oocytes and cumulus cells. For this, cumulus-oocyte complexes (COCs) were cultured for 6 h in modified TCM199 medium with (Rosco) and without (Control) 75 M roscovitine. Subsequently, they were in vitro matured for a further 18 h in inhibitor-free TCM199 medium supplemented with gonadotropins. At 0, 6 and 24 h of culture, nuclear status of oocytes and expression of selected genes were evaluated by Hoescht staining and qRT-PCR, respectively. The analysis of oocyte chromatin organization revealed that roscovitine efficiently inhibited the meiosis of sheep oocytes for 6 h and its action was completely reversed after 18 h of in vitro maturation in inhibitor-free medium. Besides, no detrimental effect on cumulus expansion was observed. The expression profile of most investigated genes in cumulus cells (PTX3, GREM1, GLUT1, PTGS2, ALK5, ALK6) and oocytes (ZAR1, NLRP5, SOD1, BMP15, GDF9) was similar between Control and Rosco treatments and the ratio BCL2/BAX was maintained in both cell types even in the presence of roscovitine. These results indicate that reversible meiotic arrest promoted by roscovitine, at the concentration and exposure time studied, neither impaired nor improved the expression of investigated genes in sheep oocytes and cumulus cells. Moreover, the efficiency of temporary meiotic arrest and the absence of deleterious effect on COCs suggest that roscovitine provides a useful method for transportation or manipulation of sheep oocytes at onset of maturation. However, further investigations are necessary to evaluate the benefits of roscovitine treatment on in vitro development of sheep embryos and its effects on cellular ultrastructure.
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