BackgroundOvulation and luteinization of follicles are complex biological processes initiated by the preovulatory luteinizing hormone surge. The objective of this study was to identify genes that are differentially expressed in bovine granulosa cells (GC) of ovulatory follicles.MethodsGranulosa cells were collected during the first follicular wave of the bovine estrous cycle from dominant follicles (DF) and from ovulatory follicles (OF) obtained 24 h following injection of human chorionic gonadotropin (hCG). A granulosa cell subtracted cDNA library (OF-DF) was generated using suppression subtractive hybridization and screened.ResultsDetection of genes known to be upregulated in bovine GC during ovulation, such as ADAMTS1, CAV1, EGR1, MMP1, PLAT, PLA2G4A, PTGES, PTGS2, RGS2, TIMP1, TNFAIP6 and VNN2 validated the physiological model and analytical techniques used. For a subset of genes that were identified for the first time, gene expression profiles were further compared by semiquantitative RT-PCR in follicles obtained at different developmental stages. Results confirmed an induction or upregulation of the respective mRNAs in GC of OF 24 h after hCG-injection compared with those of DF for the following genes: ADAMTS9, ARAF, CAPN2, CRISPLD2, FKBP5, GFPT2, KIT, KITLG, L3MBLT3, MRO, NUDT10, NUDT11, P4HA3, POSTN, PSAP, RBP1, SAT1, SDC4, TIMP2, TNC and USP53. In bovine GC, CRISPLD2 and POSTN mRNA were found as full-length transcript whereas L3MBLT3 mRNA was alternatively spliced resulting in a truncated protein missing the carboxy-terminal end amino acids, 774KNSHNEL780. Conversely, L3MBLT3 is expressed as a full-length mRNA in a bovine endometrial cell line. The 774KNSHNEL780 sequence is well conserved in all mammalian species and follows a SAM domain known to confer protein/protein interactions, which suggest a key function for these amino acids in the epigenetic control of gene expression.ConclusionsWe conclude that we have identified novel genes that are upregulated by hCG in bovine GC of OF, thereby providing novel insight into peri-ovulatory regulation of genes that contribute to ovulation and/or luteinization processes.Electronic supplementary materialThe online version of this article (10.1186/s12958-017-0306-x) contains supplementary material, which is available to authorized users.

Here we describe the establishment of size-selected cDNA libraries for the cloning of full-length cDNAs that were initially identified by suppression subtractive hybridization (SSH) technology as being differentially expressed. First, the SSH-cDNA fragments were used as 32P-probes to verify their level and differential pattern of expression by virtual Northern and to establish their corresponding full-length cDNA size. Second, cDNAs were separated by size on agarose gels and used to construct size-selected cDNA plasmid libraries, which were then screened by colony hybridization with the SSH-cDNA fragments. We conclude that the described approach complements SSH technology by allowing efficient cloning and characterization of the corresponding full-length cDNA from any desired cell type or species. This approach will give researchers the ability to specifically target and study differentially expressed genes in an efficient manner for functional genomic studies.

Prostaglandins are required for the ovulatory process, and their biosynthesis depends on the initial release of arachidonic acid from membrane phospholipids. We hypothesized that phospholipase A2 group IVA (PLA2G4A) expression is upregulated in granulosa cells (GC) at ovulation. We have characterized bovine PLA2G4A cDNA, and investigated its spatiotemporal regulation at the mRNA and protein levels in hCG-induced ovulatory follicles and in vitro, using forskolin-stimulated GC. Regulation of PLA2G4A mRNA expression was studied in GC obtained from bovine follicles collected at different developmental stages: small follicles (2-4 mm), dominant follicles at Day 5 (D5) of the estrous cycle, ovulatory follicles 24 h following injection of hCG, and corpus luteum at D5. PLA2G4A mRNA increased by 14-fold in GC of hCG-stimulated versus dominant follicles (P < 0.0001). Follicular walls obtained from ovulatory follicles recovered at 0, 6, 12, 18, and 24 h post-hCG injection showed an initial 16-fold increase in PLA2G4A transcript at 12 h that reached a 45-fold increase at 24 h, as compared to 0 h (P < 0.0001). Immunoblots of GC extracts showed an initial induction of the PLA2G4A protein at 18 h post-hCG, reaching a maximum at 24 h. Immunohistochemistry observations showed that PLA2G4A signal was mainly observed in mural GC compared to antral GC in hCG-stimulated follicles. Stimulation of cultured bovine GC with 10 microM of forskolin caused an increase in PLA2G4A mRNA and protein. Ovulation is associated with an LH/hCG-dependent induction of PLA2G4A in GC via the adenylyl cyclase/cAMP pathway.

In January 2021, Senegal reported the emergence of highly pathogenic avian influenza virus A(H5N1), which was detected on a poultry farm in Thies, Senegal, and in great white pelicans in the Djoudj National Bird Sanctuary. We report evidence of new transcontinental spread of H5N1 from Europe toward Africa.

Caveolins are implicated in endocytosis, cholesterol trafficking and signal transduction. A cDNA fragment corresponding to caveolin-1 (CAV1) was identified in a mRNA profiling expression study in bovine granulosa cells (GC) following human chorionic gonadotropin (hCG)-induced ovulation. Thus, we have characterized CAV1 cDNA and studied its spatio-temporal expression pattern in bovine ovarian follicles. The full-length bovine alphaCAV1 cDNA was cloned and encodes a putative 22 kDa protein. Expression of alphaCAV1 was studied in bovine GC obtained from follicles at different developmental stages: small follicles (SF: 2-4 mm), dominant follicles (DF), ovulatory follicles (OF: 24 hr post-hCG), and corpus luteum (CL). Semiquantitative RT-PCR analysis showed a 6.5-fold increase in alphaCAV1 mRNA in GC of OF versus DF (P < 0.0001), whereas CAV2 mRNA was increased by only twofold (P < 0.0007). Temporal expression of alphaCAV1 mRNA from OF recovered at 0, 6, 12, 18, and 24 hr after hCG injection showed an 8.5-fold increase of alphaCAV1 mRNA after 24 hr compared to 0 hr (P < 0.0018) whereas no significant variation was detected for CAV2. Immunoblot demonstrated an initial increase in alphaCAV1 protein level 12 hr post-hCG, reaching a maximum at 24 hr. Immunohistochemical localization of CAV1 was observed in GC of OF isolated 18 and 24 hr after hCG injection, whereas no signal was detected in GC of DF and SF. The induction of alphaCAV1 in GC of OF suggests that alphaCAV1 likely contributes to control the increase in membrane signaling that occurs at the time of ovulation and luteinization.