To clarify signal transduction pathways mediating putative gene expression of transcription factor SP1 (selective promoter factor 1 or specificity protein 1) by GnRH or GHRH porcine anterior pituitary monolayer cultures were exposed for various time periods to GnRH, GHRH, activators of adenylate cyclase (AC) or proteinkinase C (PKC), and mRNA levels of SP1, LHbeta-subunit, and GH were determined by multiplex RT-PCR. In many experiments LH and GH release were measured as well for comparison. Another approach was to inactivate AC, PKC, or proteinkinase A (PKA) by specific inhibitors, MDL, GFX, and H89, respectively. Postnatally (4 weeks) SP1 mRNA level was maximally increased by GnRH, GHRH and both by activation of AC or PKC after 2 h of exposure. Two-hour stimulation of SP1 mRNA levels by dbcAMP was totally blocked by H89, while this inhibitor not clearly blocked GHRH stimulated SP1 mRNA levels. Stimulation of LHbeta mRNA by GnRH was suppressed by inactivation of AC or of PKC but not by inactivation of PKA. Inactivation of AC or PKA but not of PKC inhibited GHRH induced GH mRNA. Already at day 50 of fetal life (and likewise day 80) SP1 mRNA levels were stimulated by GHRH or activation of AC, but not by GnRH or activation of PKC. The results are consistent with the notion that SP1 plays an important role 1) in conferring GnRH responsiveness to the LHbeta-subunit gene by mediating the actions of both AC and PKC and 2) in conferring GHRH responsiveness to the GH gene through activation of the AC probably PKA pathway. Furthermore, the data are in line with the view that the GHRH/AC/SP1/GH pathway develops earlier during fetal life than the GnRH/PKC/SP1/LHbeta pathway.

Information on how supplementation of high-yield dairy cows with rumen-protected fat affects fertility in cattle herds is scarce. Here, Holstein-Friesian heifers (n = 84) received a supplement consisting of either rumen-protected conjugated linoleic acid (CLA; cis-9,trans-11-CLA and trans-10,cis-12-CLA) or stearic acid 18 : 0 (SA) on top of an isocaloric grass silage diet. Two supplementation doses were used (100 and 200 g d–1). Blood and follicular fluid were collected at the start and end of the supplementation period for analysis of cholesterol, insulin-like growth factor (IGF), and nonesterified fatty acids (NEFA), and for fatty acid profiling. Although cholesterol, IGF, and NEFA levels did not differ among experimental groups, lipid profiles in blood and follicular fluid were affected in a dose-dependent manner by both supplements. After 45 days of supplementation, oocytes were collected by ovum pick-up (OPU). The mRNA relative abundance of target genes (IGF1r, GJA1, FASN, SREBP1, and SCAP) was analysed in single in vitro- (24 h IVM) and in vivo-matured (collected by OPU 20 h after GnRH injection) oocytes and in vitro-produced blastocysts (Day 8) by qPCR (n = 6/group). Lipid profiling of individual oocytes from the CLA-supplemented (n = 37) and the SA-supplemented (n = 50) was performed by desorption electrospray ionization mass spectrometry (DESI-MS). Oocytes from the CLA-supplemented (n = 413) and the SA-supplemented (n = 350) groups were used for assessing maturation and blastocysts development rates. In immature oocytes, CLA supplementation led to an increase of triacylglycerol 52 : 3 [TAG (52 : 3)] and TAG (52 : 2), squalene, palmitic acid 16 : 0, and oleic acid 18 : 1, and decreased abundance of TAG (56 : 3), TAG (50 : 2) and TAG (48 : 1). In vitro-matured oocytes showed different lipid profiles, with increased abundances of TAG (52 : 3), and TAG (52 : 2) as well as phosphatidylinositol 34 : 1 [Plo (34 : 1)], whereas phosphatidylglycerol (34 : 1) [PG (34 : 1)] and palmitic acid 16 : 0 were less abundant in in vitro-matured oocytes. SCAP was significantly down-regulated in in vitro-matured oocytes from supplemented heifers compared with their in vivo-matured counterparts. Maturation (CLA = 74% v. SA = 67%) and blastocyst rates (CLA = 22.4% v. SA = 12.7%) were different among experimental groups. One-way ANOVA and the Tukey-Kramer test were applied for a multiple comparison of means (P-value ≤ 0.05 was considered as statistically significant). In conclusion, we demonstrate here that fatty acid monitoring along different compartments (i.e. blood system, follicular fluid, and intra-oocyte) after rumen-protected fat supplementation of dairy heifer diet reveals nutritional footprints on oocyte quality and embryo development. These results demonstrate the close relationship between nutrition and cattle herd's fertility and, at the same time, support the role of the bovine model for understanding nutritional-dependent fertility impairments.

Oocyte culture in the presence of the nonspecific competitive phosphodiesterase inhibitor caffeine has been reported to increase developmental capacity of oocytes in different mammalian species. Here, we evaluated the effects of caffeine supplementation during the final phase of in vitro maturation (IVM) on developmental rates and blastocyst cell numbers. Bovine ovaries were collected from a local abattoir. A total of 1142 cumulus-oocyte-complexes were obtained by slicing. Cumulus-oocyte complexes were either in vitro matured for 24 h (Standard) or matured for 20 h followed by additional culture for 6 h in fresh IVM medium supplemented with 10 mM caffeine (Caffeine 6 h). In vitro fertilization was performed for 19 h using frozen-thawed sperm from 2 different bulls. After IVF, presumptive zygotes were cultured in vitro for 8 days until the blastocyst stage. Cleavage and blastocyst rates were evaluated 3 and 8 days after IVF, respectively. Expanded blastocysts from the different treatments were submitted to differential staining. SAS/STAT software (SAS Institute Inc., Cary, NC, USA) was used to evaluate cleavage and blastocyst rates using the Glimmix procedure and blastocyst cell numbers were compared using the linear model procedure. Cleavage rates were lower using caffeine for bull B and blastocyst production decreased for bull A. Caffeine treatment increased inner cell mass (ICM) number for bull B and decreased trophectoderm (TE) and total cell numbers for bull A. However, similar TE and total cells were obtained for bull B (Table 1; P < 0.05). Results show that developmental competence can be affected by caffeine supplementation at the final phase of IVM probably due to oocyte-sperm interaction changes. Table 1. In vitro developmental competence of oocytes cultured with caffeine at the end of IVM