Effects of Species Differences on Oocyte Regulation of Granulosa Cell Proliferation

<p>The role of oocytes in regulating ovulation quota between species is not fully understood. In humans, sheep, and rodents the oocyte-derived growth factors, bone morphogenetic protein 15 (BMP15) and/or growth differentiation factor 9 (GDF9) have profound effects on ovarian follicular development and ovulation quota. The aim of these studies was to compare the ability of oocytes from sheep (low ovulation-rate) and rat (poly-ovulator) to stimulate radiolabelled ³H-thymidine uptake by granulosa cells (GC) both within and between the two species, and to assess species differences of GDF9 and BMP15 in co-incubations. For these experiments, oocytes denuded of cumulus-cells (DO) were co-incubated with a fixed number of GC from either species. Rat or sheep DO stimulated ³H-thymidine uptake by GC from the same species (P<0.005). Sheep oocytes also stimulated ³H-thymidine uptake by rat GC (P<0.001) but not vice versa. To investigate this further, oocytes and GC were co-incubated with monoclonal antibodies specific to GDF9 or BMP15 or to a hydatids antigen (control). Both sheep and rat oocyte stimulation of ³H-thymidine uptake by GC was inhibited with the GDF9 antibody (P<0.05) but not the control antibody, irrespective of the species of GC. Sheep DO stimulation of rat GC was also inhibited using an antibody to BMP15 (P<0.05). However, when using the BMP15 antibody to block the effects of rat DO on rat GC, no inhibition of ³H-thymidine uptake was observed (P=0.988). The molecular forms of GDF9 and BMP15 protein in oocyte lysates and spent media were examined by Western blotting under reducing conditions. For both species, GDF9 protein was present in the mature form in both the lysate and the spend media. For sheep oocytes, BMP15 protein was present as pro-mature and monomeric mature forms in the lysate and in the spent media, whereas from rats pro-mature forms were detected in the oocyte lysate but there was minimal evidence for the mature form in the spent media. The mRNA levels of GDF9, BMP15 and several cumulus cell (CC) genes were measured at 8h intervals over a 24h incubation period. For both sheep and the rat, GDF9 and BMP15 mRNA expression levels were highly correlated (R²=0.99). In the rat, the relative mean expression level of Gdf9 mRNA was four times higher than Bmp15 mRNA, whereas in sheep the relative mean expression ratio of GDF9 to BMP15 was approximately one. For these studies, two types of incubations were performed, namely COC alone or co-incubations of DO with GC. In co-incubations of DO with GC, the relative GDF9 and BMP15 mRNA levels did not change significantly over time, whereas incubations of COC showed that the relative levels of GDF9 and BMP15 mRNA declined significantly during the incubation period. The CC genes (FSHR, LHR, KITL, CX43, AROM and CYCD2) were measured during the 24h incubation period for the COC but not the DO experiment. In rats, none of the CC genes showed any changes in mRNA levels over time except Kitl, where levels at 8h and 16h mRNA levels were significantly lower compared to those at 0h and 24h. In sheep, there were no significant mRNA changes in any of the CC genes except AROM. The level of AROM mRNA reduced to near zero by 8h and remained at low levels at both 16h and 24h. These findings suggest that, under the incubation conditions used, that the gene expression levels for most CC genes were maintained but that the oocyte growth factors alone were unable to maintain AROM gene expression in sheep. In conclusion, major differences were observed in GDF9 and BMP15 expression and regulation between rats and sheep. The lower expression levels of Bmp15 mRNA and protein are likely the reason why rat DO failed to stimulate ³H-thymidine uptake by sheep GC. The evidence suggests that oocyte-derived GDF9 is sufficient to stimulate ³H-thymidine incorporation and presumably DNA synthesis in rat GC whereas in sheep both BMP15 and GDF9 are required. These findings raise the possibility that in species with a low ovulation rate phenotype there is a higher level of GDF9 mRNA and protein synthesis and that BMP15 is a major factor in restricting ovulation quota in mammals.</p>

Effects of Species Differences on Oocyte Regulation of Granulosa Cell Proliferation

<p>The role of oocytes in regulating ovulation quota between species is not fully understood. In humans, sheep, and rodents the oocyte-derived growth factors, bone morphogenetic protein 15 (BMP15) and/or growth differentiation factor 9 (GDF9) have profound effects on ovarian follicular development and ovulation quota. The aim of these studies was to compare the ability of oocytes from sheep (low ovulation-rate) and rat (poly-ovulator) to stimulate radiolabelled ³H-thymidine uptake by granulosa cells (GC) both within and between the two species, and to assess species differences of GDF9 and BMP15 in co-incubations. For these experiments, oocytes denuded of cumulus-cells (DO) were co-incubated with a fixed number of GC from either species. Rat or sheep DO stimulated ³H-thymidine uptake by GC from the same species (P<0.005). Sheep oocytes also stimulated ³H-thymidine uptake by rat GC (P<0.001) but not vice versa. To investigate this further, oocytes and GC were co-incubated with monoclonal antibodies specific to GDF9 or BMP15 or to a hydatids antigen (control). Both sheep and rat oocyte stimulation of ³H-thymidine uptake by GC was inhibited with the GDF9 antibody (P<0.05) but not the control antibody, irrespective of the species of GC. Sheep DO stimulation of rat GC was also inhibited using an antibody to BMP15 (P<0.05). However, when using the BMP15 antibody to block the effects of rat DO on rat GC, no inhibition of ³H-thymidine uptake was observed (P=0.988). The molecular forms of GDF9 and BMP15 protein in oocyte lysates and spent media were examined by Western blotting under reducing conditions. For both species, GDF9 protein was present in the mature form in both the lysate and the spend media. For sheep oocytes, BMP15 protein was present as pro-mature and monomeric mature forms in the lysate and in the spent media, whereas from rats pro-mature forms were detected in the oocyte lysate but there was minimal evidence for the mature form in the spent media. The mRNA levels of GDF9, BMP15 and several cumulus cell (CC) genes were measured at 8h intervals over a 24h incubation period. For both sheep and the rat, GDF9 and BMP15 mRNA expression levels were highly correlated (R²=0.99). In the rat, the relative mean expression level of Gdf9 mRNA was four times higher than Bmp15 mRNA, whereas in sheep the relative mean expression ratio of GDF9 to BMP15 was approximately one. For these studies, two types of incubations were performed, namely COC alone or co-incubations of DO with GC. In co-incubations of DO with GC, the relative GDF9 and BMP15 mRNA levels did not change significantly over time, whereas incubations of COC showed that the relative levels of GDF9 and BMP15 mRNA declined significantly during the incubation period. The CC genes (FSHR, LHR, KITL, CX43, AROM and CYCD2) were measured during the 24h incubation period for the COC but not the DO experiment. In rats, none of the CC genes showed any changes in mRNA levels over time except Kitl, where levels at 8h and 16h mRNA levels were significantly lower compared to those at 0h and 24h. In sheep, there were no significant mRNA changes in any of the CC genes except AROM. The level of AROM mRNA reduced to near zero by 8h and remained at low levels at both 16h and 24h. These findings suggest that, under the incubation conditions used, that the gene expression levels for most CC genes were maintained but that the oocyte growth factors alone were unable to maintain AROM gene expression in sheep. In conclusion, major differences were observed in GDF9 and BMP15 expression and regulation between rats and sheep. The lower expression levels of Bmp15 mRNA and protein are likely the reason why rat DO failed to stimulate ³H-thymidine uptake by sheep GC. The evidence suggests that oocyte-derived GDF9 is sufficient to stimulate ³H-thymidine incorporation and presumably DNA synthesis in rat GC whereas in sheep both BMP15 and GDF9 are required. These findings raise the possibility that in species with a low ovulation rate phenotype there is a higher level of GDF9 mRNA and protein synthesis and that BMP15 is a major factor in restricting ovulation quota in mammals.</p>

PSX-A-24 Late-Breaking: Testosterone production by theca cells of large preovulatory follicles is affected by growth hormone depending on the hen age and the presence of granulosa cells

Testosterone produced by theca cells may be involved in regulating of the growth and ovulation of hen preovulatory follicles (Rangel, Gutierrez, Gen Comp Endocrinol, 203:250, 2014). In the current research, we studied effects of growth hormone (GH), a known regulator of the hen ovarian function, on in vitro testosterone production by the theca layer (TL) from the two largest yellow follicles in relation to the hen age and the presence of the granulosa layer (GL). Young hens with long clutch (YLC, 32–33 week-old, &gt;10 eggs per clutch) and old hens with short clutch (OSC, 74–76 week-old, 3–6 eggs per clutch) were used. After isolation, TL from F1 and F2 follicles (n = 8–9) was cultured for 18 h in two systems, separately or together with the corresponding GL, in the presence or absence of chicken GH (25 ng/ml). Concentrations of testosterone in the spent media were measured by ELISA. The data were analyzed by RM-ANOVA. In the case of separate TL culture, GH did not change significantly testosterone production in both follicles of YLC hens and reduced it from 338±105 to 152±52 fmol/mg tissue (P &lt; 0.05) in F1 follicles of OSC hens. When TL was cultured in the presence of GL, GH enhanced 1.8-2.6-fold (P &lt; 0.05) the secretion of testosterone in the case of F1 follicles and decreased it 1.8-2.5-fold (P &lt; 0.05) in the case of F2 follicles in both young and old hens. Regardless of the treatment, follicular size or culture system, the production of testosterone in OSC hens was 2–5 times higher than in YLC hens. The results indicate that the interaction between TL and GL changes the steroidogenic response of theca cells from preovulatory follicles to GH in young and old hens. Furthermore, testosterone production is obviously increased with reproductive aging of laying hens. The study was supported by RFBR (19-016-00216).

Varied Contribution of Phospholipid Shedding From Membrane to Daptomycin Tolerance in Staphylococcus aureus

It has been suggested that daptomycin can be inactivated by lipids released by Staphylococcus aureus and that this effect is antagonized by phenol soluble modulins (PSMs), which bind to the shed lipids. PSM production is regulated by the Agr system, and others have shown that loss of the Agr function enhances S. aureus survival in the presence of daptomycin. Here we assessed the impact of Agr function on daptomycin activity and lipid metabolism under various conditions. Daptomycin activity was evaluated against three sets of isogenic strain series with wild-type or dysfunctional Agr using static daptomycin time-kills over 24 h and against one strain pair using in vitro pharmacokinetic/pharmacodynamic (PK/PD) models simulating clinical daptomycin exposure for 48 h. We performed comprehensive lipidomics on bacterial membranes and the spent media to correlate lipid shedding with survival. In static time-kill experiments, two agr-deficient strains (SH1000- and USA300 LAC ΔagrA) showed improved survival for 8 h compared with their corresponding wild-type strains as seen in previous studies, but this difference did not persist for 24 h. However, four other agr-deficient strains (SH1001 and JE2 agr KOs) did not demonstrate improved survival compared to isogenic wild-type strains at any time in the time-kills. Lipidomics analysis of SH1000, SH1001, and SH1000- strains showed daptomycin exposure increased lipid shedding compared to growth controls in all strains with phosphatidylglycerols (PGs), lysylPGs and cardiolipins predominating. In the cell pellets, PGs and lysylPGs decreased but cardiolipins were unchanged with daptomycin exposure. The shed lipid profiles in SH1001 and SH1000- were similar, suggesting that the inability to resist daptomycin by SH1001 was not because of differences in lipid shedding. In the PK/PD model, the agr mutant SH1000- strain did not show improved survival relative to SH1000 either. In conclusion, inactivation of daptomycin by shed lipids may be dependent on genetic background, the specific agr mutations, or the techniques used to generate these KOs rather than the overall function of the Agr system, and its contribution to daptomycin tolerance seems to be varied, transient, and growth-condition dependent.

A Win–Loss Interaction on Fe0 Between Methanogens and Acetogens From a Climate Lake

Diverse physiological groups congregate into environmental corrosive biofilms, yet the interspecies interactions between these corrosive physiological groups are seldom examined. We, therefore, explored Fe0-dependent cross-group interactions between acetogens and methanogens from lake sediments. On Fe0, acetogens were more corrosive and metabolically active when decoupled from methanogens, whereas methanogens were more metabolically active when coupled with acetogens. This suggests an opportunistic (win–loss) interaction on Fe0 between acetogens (loss) and methanogens (win). Clostridia and Methanobacterium were the major candidates doing acetogenesis and methanogenesis after four transfers (metagenome sequencing) and the only groups detected after 11 transfers (amplicon sequencing) on Fe0. Since abiotic H2 failed to explain the high metabolic rates on Fe0, we examined whether cell exudates (spent media filtrate) promoted the H2-evolving reaction on Fe0 above abiotic controls. Undeniably, spent media filtrate generated three- to four-fold more H2 than abiotic controls, which could be partly explained by thermolabile enzymes and partly by non-thermolabile constituents released by cells. Next, we examined the metagenome for candidate enzymes/shuttles that could catalyze H2 evolution from Fe0 and found candidate H2-evolving hydrogenases and an almost complete pathway for flavin biosynthesis in Clostridium. Clostridial ferredoxin-dependent [FeFe]-hydrogenases may be catalyzing the H2-evolving reaction on Fe0, explaining the significant H2 evolved by spent media exposed to Fe0. It is typical of Clostridia to secrete enzymes and other small molecules for lytic purposes. Here, they may secrete such molecules to enhance their own electron uptake from extracellular electron donors but indirectly make their H2-consuming neighbors—Methanobacterium—fare five times better in their presence. The particular enzymes and constituents promoting H2 evolution from Fe0 remain to be determined. However, we postulate that in a static environment like corrosive crust biofilms in lake sediments, less corrosive methanogens like Methanobacterium could extend corrosion long after acetogenesis ceased, by exploiting the constituents secreted by acetogens.

Does miRNA Expression in the Spent Media Change During Early Embryo Development?

Distinct miRNA populations have been detected in the spent media of in-vitro culture systems. However, profiling has been limited to media conditioned with blastocyst-stage embryos. Therefore, the aim of the study was to profile extracellular miRNAs throughout the pre-implantation period in bovine embryos. To achieve this, cumulus oocyte complexes were aspirated from ovaries, in-vitro matured, fertilized, and cultured under standard laboratory procedures to the 2-cell, 8-cell, or blastocyst stage of development. At each developmental stage, 25 μl of spent in-vitro culture media was collected, pooled to 300 μl, and processed for total RNA extraction. In-vitro culture media, which never came in contact with any embryos, were additionally processed for total RNA extraction to serve as a negative control. Following hybridization on a GeneChip miRNA 4.0 array, comparative analysis was conducted between spent media and control samples. In total, 111 miRNAs were detected in the spent media samples, with 56 miRNAs identified in blastocyst spent media, 14 miRNAs shared between 8-cell and blastocyst spent media, 7 miRNAs shared between all 3 conditions, and 6 miRNAs exclusive to 2-cell spent media. miRNA-mRNA target prediction analysis revealed that the majority of genes predicted to be regulated by the miRNAs identified in the study have roles in cellular process, metabolic process, and biological regulation. Overall, the study suggest that miRNAs can be detected in the spent media of in-vitro culture system throughout the pre-implantation period and the detected miRNAs may influence genes impacting early embryo development.