Characterization of raft microdomains in bovine mammary tissue during lactation: How they are modulated by fatty acid treatments.

The objective of the current study was first to characterize lipid raft microdomains isolated as detergent-resistant membranes (DRM) from mammary gland tissue, and second to determine how dietary fatty acids (FA) such as conjugated linoleic acid (CLA), 19:1 cyclo, and long-chain n-3 polyunsaturated FA affect lipid raft markers of mammary cells, and to finally establish relationships between these markers and lactation performance in dairy cows. Eight Holstein cows were used in a replicated 4 × 4 Latin square design with periods of 28 d. For the first 14 d, cows received daily an abomasal infusion of (1) 406 g of a saturated FA supplement (112 g of 16:0 + 230 g of 18:0) used as a control; (2) 36 g of a CLA supplement (13.9 g of trans-10,cis-12 18:2) + 370 g of saturated FA; (3) 7 g of Sterculia fetida oil (3.1 g of 19:1 cyclo, STO) + 399 g of saturated FA; or (4) 406 g of fish oil (55.2 g of cis-5,cis-8,cis-11,cis-14,cis-17 20:5 + 59.3 g of cis-4,cis-7,cis-10,cis-13,cis-16,cis-19 22:6, FO). Mammary biopsies were harvested on d 14 of each infusion period and were followed by a 14-d washout interval. Cholera toxin subunit B, which specifically binds to ganglioside M-1 (GM-1), a lipid raft marker, was used to assess its distribution in DRM. Infusions of CLA, STO, and FO were individually compared with the control, and significance was declared at P ≤ 0.05. Milk fat yield was decreased with CLA and FO, but was not affected by STO. Milk lactose yield was decreased with CLA and STO, but was not affected by FO. Mammary tissue shows a strong GM-1-signal enrichment in isolated DRM from mammary gland tissue. Caveolin (CAV) and flotillin (FLOT) are 2 proteins considered as lipid raft markers and they are present in DRM from mammary gland tissue. Distributions of GM-1, CAV-1, and FLOT-1 showed an effect of treatments determined by their subcellular distributions in sucrose gradient fractions. Regardless of treatments, data showed positive relationships between the yield of milk fat, protein, and lactose, and the abundance GM-1 in DRM fraction. Milk protein yield was positively correlated with relative proportion of FLOT-1 in the soluble fraction, whereas lactose yield was positively correlated with relative proportion of CAV-1 in the DRM fractions. Infusion of CLA decreased mRNA abundance of CAV-1, FLOT-1, and FLOT-2. Regardless of treatments, a positive relationship was observed between fat yield and mRNA abundance of FLOT-2. In conclusion, although limited to a few markers, results of the current experiment raised potential links between variation in specific biologically active component of raft microdomains in bovine mammary gland and lactation performances in dairy cows.

The effects of LH inhibition with cetrorelix on cumulus cell gene expression during the luteal phase under ovarian coasting stimulation in cattle.

Cumulus cells have an important role to play in the final preparation of the oocyte before ovulation. During the final phase of follicular differentiation, FSH levels are low and LH maintains follicular growth; however, it is not known if at that time LH has an influence on cumulus cells inside the follicle. In humans, LH is often inhibited to avoid a premature ovulatory LH surge. This procedure provides a tool to investigate the role of LH in follicular development. In this study, we investigated the impact of suppressing LH using the GnRH antagonist cetrorelix during an ovarian coasting stimulation protocol on the transcriptome of bovine cumulus cells (CC). Oocytes were collected twice from 6 dairy cows. For the first collection, the cows received FSH twice daily for 3 d, followed by FSH withdrawal for 68 h as a control protocol. For the second collection, the same stimulation protocol was used, but the cows also received, starting on day 2 of FSH stimulation, a GnRH antagonist once a day until recovery of the cumulus-oocyte complexes (COC). Half of the COC were subjected to in vitro maturation, fertilization, and culture to assess blastocyst rates. The other half of the COC underwent microarray analysis (n = 3 cows, 2 treatments, 6 oocyte collections) and qRT-PCR (n = 6 cows: 3 microarray cows +3 other cows, 2 treatments, 12 oocyte collections). The differential expression of specific genes was confirmed by RT-qPCR: decrease of ATP6AP2, SC4MOL, and OSTC and increase of PTGDS in the LH-inhibited condition. The global transcriptomic analysis of cumulus cells demonstrated that the inhibition of LH secretion may decrease survival and growth of the follicle. Moreover, the results suggested that LH may be important to cumulus for the maintenance of cellular mechanisms such as global RNA expression, protein and nucleic acid metabolism, and energy production. These results support the hypothesis that LH support is important during the final part of follicle maturation through its influence on the cumulus cells.

Papaverine-sensitive phosphodiesterase activity is measured in bovine spermatozoa.

Cyclic adenosine monophosphate (cAMP) plays a crucial role as a signaling molecule for capacitation, motility, and acrosome reaction in mammalian spermatozoa. It is well-known that cAMP degradation by phosphodiesterase (PDE) enzyme has a major impact on sperm functions. This study was undertaken to characterize cAMP-PDE activity in bovine spermatozoa. Total cAMP-PDE activity in cauda epididymal and ejaculated spermatozoa was 543.2 ± 49.5 and 1252.6 ± 86.5 fmoles/min/106 spermatozoa, respectively. Using different family-specific PDE inhibitors, we showed that in cauda epididymal and ejaculated spermatozoa, the major cAMP-PDE activity was papaverine-sensitive (44.5% and 57.5%, respectively, at 400 nm, papaverine is a specific inhibitor of the PDE10 family). These data are supporting the functional presence of PDE10 in bovine spermatozoa and were further confirmed by western blot to be PDE10A. Using immunocytochemistry, we showed immunoreactive signal for PDE10A present on the post-acrosomal region of the head and on the flagella of ejaculated spermatozoa. Using papaverine, we showed that it promotes tyrosine phosphorylation of sperm proteins, phosphorylation of Erk1 and Erk2, and Ca2+ release from Ca2+ store. These results suggest that PDE10 is functionally present in bovine spermatozoa and is affecting different molecular events involved in capacitation, most probably by cAMP local regulation.

Characterization of cAMP-phosphodiesterase activity in bovine seminal plasma.

The second messenger cyclic adenosine monophosphate (cAMP) has a central role in sperm physiology. Extracellular cAMP can be sequentially degraded into 5'AMP and adenosine by ecto-phosphodiesterases (ecto-PDE) and ecto-nucleotidases, a phenomenon called extracellular cAMP-adenosine pathway. As cAMP-adenosine pathway is involved in sperm capacitation, we hypothesize that extracellular PDEs are functionally present in seminal plasma. Exclusively measuring cAMP-PDE activity, total activity in bovine seminal plasma was 10.1 ± 1.5 fmoles/min/µg. Using different family-specific PDE inhibitors, we showed that in seminal plasma, the major cAMP-PDE activity was papaverine sensitive (47.5%). These data support the presence of PDE10 in bovine seminal plasma and was further confirmed by western blot. In epididymal fluid, total cAMP-PDE activity was 48.2 ± 14.8 fmoles/min/µg and we showed that the major cAMP-PDE activity was 3-isobutyl-methylxanthine insensitive and thus ascribed to PDE8 family. PDE10A mRNAs were found in the testis, epididymis, and seminal vesicles. cAMP-PDE activity is present in bovine seminal plasma and epididymal fluid. The results suggest a role for ecto-PDEs present in those fluids in the signaling pathways involved in sperm functions.

Oocyte maturation and quality: role of cyclic nucleotides.

The cyclic nucleotides, cAMP and cGMP, are the key molecules controlling mammalian oocyte meiosis. Their roles in oocyte biology have been at the forefront of oocyte research for decades, and many of the long-standing controversies in relation to the regulation of oocyte meiotic maturation are now resolved. It is now clear that the follicle prevents meiotic resumption through the actions of natriuretic peptides and cGMP - inhibiting the hydrolysis of intra-oocyte cAMP - and that the pre-ovulatory gonadotrophin surge reverses these processes. The gonadotrophin surge also leads to a transient spike in cAMP in the somatic compartment of the follicle. Research over the past two decades has conclusively demonstrated that this surge in cAMP is important for the subsequent developmental capacity of the oocyte. This is important, as oocyte in vitro maturation (IVM) systems practised clinically do not recapitulate this cAMP surge in vitro, possibly accounting for the lower efficiency of IVM compared with clinical IVF. This review particularly focuses on this latter aspect - the role of cAMP/cGMP in the regulation of oocyte quality. We conclude that clinical practice of IVM should reflect this new understanding of the role of cyclic nucleotides, thereby creating a new generation of ART and fertility treatment options.

Effects of intramuscular administration of folic acid and vitamin B12 on granulosa cells gene expression in postpartum dairy cows.

The fertility of dairy cows is challenged during early lactation, and better nutritional strategies need to be developed to address this issue. Combined supplementation of folic acid and vitamin B12 improve energy metabolism in the dairy cow during early lactation. Therefore, the present study was undertaken to explore the effects of this supplement on gene expression in granulosa cells from the dominant follicle during the postpartum period. Multiparous Holstein cows received weekly intramuscular injection of 320 mg of folic acid and 10 mg of vitamin B12 (treated group) beginning 24 (standard deviation=4) d before calving until 56 d after calving, whereas the control group received saline. The urea plasma concentration was significantly decreased during the precalving period, and the concentration of both folate and vitamin B12 were increased in treated animals. Milk production and dry matter intake were not significantly different between the 2 groups. Plasma concentrations of folates and vitamin B12 were increased in treated animals. Daily dry matter intake was not significantly different between the 2 groups before [13.5 kg; standard error (SE)=0.5] and after (23.6 kg; SE=0.9) calving. Average energy-corrected milk tended to be greater in vitamin-treated cows, 39.7 (SE=1.4) and 38.1 (SE=1.3) kg/d for treated and control cows, respectively. After calving, average plasma concentration of ß-hydroxybutyrate tended to be lower in cows injected with the vitamin supplement, 0.47 (SE=0.04) versus 0.55 (SE=0.03) for treated and control cows, respectively. The ovarian follicle ≥12 mm in diameter was collected by ovum pick-up after estrus synchronization. Recovered follicular fluid volumes were greater in the vitamin-treated group. A microarray platform was used to investigate the effect of treatment on gene expression of granulosa cells. Lower expression of genes involved in the cell cycle and higher expression of genes associated with granulosa cell differentiation before ovulation were observed. Selected candidate genes were analyzed by reverse transcription quantitative PCR. Although the effects of intramuscular injections of folic acid and vitamin B12 on lactational performance and metabolic status of animals were limited, ingenuity pathway analysis of gene expression in granulosa cells suggests a stimulation of cell differentiation in vitamin-treated cows, which may be the result of an increase in LH secretion.

Characterization of FSH signalling networks in bovine cumulus cells: a perspective on oocyte competence acquisition.

Understanding the mechanisms regulating oocyte developmental competence is essential to enhance the clinical efficiency of assisted reproduction. FSH orchestrates the acquisition of oocyte competence, both in vivo and in vitro. Multiple pathways are implicated in FSH signalling; however, their precise coordination remains unresolved. A robust system to investigate FSH signalling is oocyte in vitro maturation (IVM) and we have previously demonstrated better bovine embryo development after FSH addition for the first 6 h during IVM. Using this model, we investigated FSH signalling in cumulus through transcriptomic and pharmacological tools. We demonstrate modulation of cumulus transcriptome by FSH mainly through protein kinase A (PKA) and epidermal growth factor (EGF) pathways. Differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism and oocyte competence. FSH required rouse-sarcoma oncogene (SRC) for EGF receptor transactivation. PKA and EGF pathway crosstalk was investigated using extracellular signal-regulated kinases (ERK1/2) phosphorylation as the functional end-point. FSH enhanced ERK1/2 activation by the EGF pathway with a simultaneous diminution through PKA. More specifically, FSH increased dual specific phosphatase (DUSP1) transcripts via PKA although DUSP1 protein did not change since EGF was required to prevent degradation. Our findings implicate FSH in PKA and EGF pathway activation, which interact to maintain appropriate levels of ERK1/2 phosphorylation and eventually cumulus expansion, metabolism and steroidogenesis. Moreover, considering the implication of the EGF pathway in GDF9 and BMP15 actions, our findings suggest that FSH may have a role in modulation of the cumulus response to oocyte-secreted factors. This information has implications for improvement of IVM and hence oocyte developmental competence.

Transcriptomic analysis of cyclic AMP response in bovine cumulus cells.

Acquisition of oocyte developmental competence needs to be understood to improve clinical outcomes of assisted reproduction. The stimulation of cumulus cell concentration of cyclic adenosine 3'5'-monophosphate (cAMP) by pharmacological agents during in vitro maturation (IVM) participates in improvement of oocyte quality. However, precise coordination and downstream targets of cAMP signaling in cumulus cells are largely unknown. We have previously demonstrated better embryo development after cAMP stimulation for first 6 h during IVM. Using this model, we investigated cAMP signaling in cumulus cells through in vitro culture of cumulus-oocyte complexes (COCs) in the presence of cAMP raising agents: forskolin, IBMX, and dipyridamole (here called FID treatment). Transcriptomic analysis of cumulus cells indicated that FID-induced differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism, and oocyte competence. Functional genomic analysis revealed that protein kinase-A (PKA), extracellular signal regulated kinases (ERK1/2), and calcium (Ca(2+)) pathways as key regulators of FID signaling. Inhibition of PKA (H89) in FID-supplemented COCs or substitution of FID with calcium ionophore (A23187) demonstrated that FID activated primarily the PKA pathway which inhibited ERK1/2 phosphorylation and was upstream of calcium signaling. Furthermore, inhibition of ERK1/2 phosphorylation by FID supported a regulation by dual specific phosphatase (DUSP1) via PKA. Our findings imply that cAMP (FID) regulates cell metabolism, steroidogenesis, intracellular signaling and cumulus expansion through PKA which modulates these functions through optimization of ERK1/2 phosphorylation and coordination of calcium signaling. These findings have implications for development of new strategies for improving oocyte in vitro maturation leading to better developmental competence.

Effects of abomasal infusion of conjugated linoleic acids, Sterculia foetida oil, and fish oil on production performance and the extent of fatty acid Δ9-desaturation in dairy cows.

The purpose of this study was to determine the effects of conjugated linoleic acid (CLA), Sterculia foetida oil (STO), and fish oil (FO) on milk yield and composition, milk FA profile, Δ(9)-desaturation activity, and mammary expression of 2 isoforms of stearoyl-coenzyme A desaturase (SCD-1 and SCD-5) in lactating dairy cows. Eight multiparous Holstein cows (69 ± 13 d postpartum) were used in a double 4 × 4 Latin square design with 28-d periods. For the first 14 d of each period, cows received an abomasal infusion of (1) 406 g of a saturated fatty acid (SFA) supplement (112 g of 16:0 + 230 g of 18:0) used as a control (CTL), (2) 36 g of a CLA supplement (13.9 g of trans-10,cis-12 18:2) + 370 g of SFA, (3) 7 g of STO (3.1g of 19:1 cyclo) + 399 g of SFA, or (4) 406 g of FO (55.2 g of cis-5,-8,-11,-14,-17 20:5 + 59.3 g of cis-4,-7,-10,-13,-16,-19 22:6). Infusions were followed by a 14-d washout interval. Compared with CTL, STO decreased milk yield from 38.0 to 33.0 kg/d, and increased milk fat concentration from 3.79 to 4.45%. Milk fat concentration was also decreased by CLA (2.23%) and FO (3.34%). Milk fat yield was not affected by STO (1,475 g/d) compared with CTL (1,431 g/d), but was decreased by CLA (774 g/d) and FO (1,186 g/d). Desaturase indices for 10:0, 12:0, and 20:0 were decreased, whereas the extent of desaturation of 14:0, 16:0, 17:0, and 18:0 was not affected by CLA treatment compared with CTL. Infusion of STO significantly decreased all calculated desaturase indices compared with CTL; the 14:0 index was reduced by 80.7%. Infusion of FO decreased the desaturase indices for 10:0, 14:0, 20:0, trans-11 18:1, and 18:0. The effect of FO on the 14:0 index indicates a decrease in apparent Δ(9)-desaturase activity of 30.2%. Compared with CTL, mammary mRNA abundance of SCD-1 was increased by STO (+30%) and decreased by CLA (-24%), whereas FO had no effect. No effect was observed on mRNA abundance of SCD-5. In conclusion, abomasal infusion of CLA, STO, and FO were shown to exhibit varying and distinct effects on desaturase indices, an indicator of apparent SCD activity, and mammary mRNA abundance of SCD-1.

Cumulus cell gene expression associated with pre-ovulatory acquisition of developmental competence in bovine oocytes.

The final days before ovulation impact significantly on follicular function and oocyte quality. This study investigated the cumulus cell (CC) transcriptomic changes during the oocyte developmental competence acquisition period. Six dairy cows were used for 24 oocyte collections and received FSH twice daily over 3 days, followed by FSH withdrawal for 20, 44, 68 and 92 h in four different oestrous cycles for each of the six cows. Half of the cumulus-oocyte complexes were subjected to in vitro maturation, fertilisation and culture to assess blastocyst rate. The other half of the CC underwent microarray analysis (n=3 cows, 12 oocyte collections) and qRT-PCR (n=3 other cows, 12 oocyte collections). According to blastocyst rates, 20 h of FSH withdrawal led to under-differentiated follicles (49%), 44 and 68 h to the most competent follicles (71% and 61%) and 92 h to over-differentiated ones (51%). Ten genes, from the gene lists corresponding to the three different follicular states, were subjected to qRT-PCR. Interestingly, CYP11A1 and NSDHL gene expression profiles reflected the blastocyst rate. However most genes were associated with the over-differentiated status: GATM, MAN1A1, VNN1 and NRP1. The early period of FSH withdrawal has a minimal effect on cumulus gene expression, whereas the longest period has a very significant one and indicates the beginning of the atresia process.