We previously proposed that an endothelin-angiotensin-atrial natriuretic peptide system may contribute to inducing ovulation of mature bovine follicles by modulating follicular secretion of steroids and prostaglandins (PGs). Thus, this study aimed to determine the real-time changes in the local release of angiotensin II (Ang II), endothelin (ET), atrial natriuretic peptide (ANP), PGF(2alpha), and steroid hormones from bovine mature follicles during the periovulatory period in vivo. Seven cows were treated for superovulation using FSH and PGF(2alpha) injections. Two dialysis capillary membranes per follicle were surgically implanted into the theca layer of mature follicles and connected to a microdialysis system (MDS). Fractions of the perfusate were collected from Day -1 (Day 0 = LH surge) to Day 3. Five out of seven treated cows were normally ovulated, and the newly formed corpora lutea were observed at the end of the experiment. In these five ovulated cows, the release of estradiol, androstenedione, and progesterone in the theca layer increased (P < 0.05) synchronously with the LH surge. Acute increases in PGF(2alpha) and Ang II concentrations in the ovarian venous plasma (OVP) were observed at 24-48 h after the peak of the LH surge, when multiple ovulations were expected to occur. The follicular Ang II release was low during the pre-LH surge period and rose (P < 0.05) at the beginning of the increase in the LH surge. On the other hand, ET-1 release dropped (P < 0.05) when plasma LH started to increase. However, no clear changes in ANP concentration in the MDS perfusate and plasma were observed. The above local changes in Ang II, PGF(2alpha), as well as steroid hormones were not observed in cows (n = 2) that did not show an LH surge and ovulation. The present results demonstrate for the first time the local release of Ang II, ET-1, and ANP from the bovine mature follicle in real-time in vivo and show that Ang II and PGF(2alpha) concentrations in the OVP acutely increase around the time of ovulation. The overall results support the concept of a local functional ET-Ang-ANP system in the bovine mature follicle that may be involved in the ovulatory process.

The corpus luteum (CL) offers the opportunity to study high proliferative processes during its development and degradation processes during its regression. We examined the mRNA expression of matrix metalloproteases (MMP)-1, MMP-2, MMP-9,

The aim of this study was to test if the opioid antagonist naloxone has a beneficial effect on normalization of oxytocin (OT) release during repeated milking of cows in unfamiliar surroundings. One control milking without naloxone treatment in all cows was performed in the familiar parlour. For four successive evening milkings, cows were transported to, and milked in, the operating theatre of the research station without (control group) or with naloxone administration (1 mg/kg BW) (naloxone group) before milking. After cessation of spontaneous milk flow, but not before 3 min of milking, vaginal stimulation was applied for 2 min. After milk flow ceased again, 10 IU of OT was injected intravenously to remove the remaining milk including residual milk. Milk flow was recorded continuously and blood samples were collected via a jugular vein cannula at 1-min intervals from 1 min before the start of milking until i.v. injection of OT. The inhibition of milk ejection and its normalization during repeated milking in unfamiliar surroundings was not influenced by naloxone treatment. Concentrations of cortisol and β-endorphin during control milking and all relocations were similar in the naloxone and control groups, although their concentrations were higher after relocations than in the control. Therefore, a role of endogenous opioids in the inhibition of milk ejection in unfamiliar surroundings could not be demonstrated. In addition, the effect of exogenous ACTH1–24 (8 IU, i.v.) on the release of cortisol related to the response of cows milked in unfamiliar surroundings was studied. Cows with totally inhibited milk ejection in response to vaginal stimulation during milking after first relocation had numerically, but not significantly lower cortisol levels (8·8±3·4 ng/ml; AUC/min) in response to ACTH than did cows with at least partial milk ejection (38·7±12·9 ng/ml). Thus animals with a higher adrenal response to ACTH seemed to have less severe inhibition of milk ejection.

There is evidence that angiotensin II (Ang II) and endothelin-1 (ET-1) may interact in an additive or synergistic way during luteal regression. The aim of the study was to investigate real time changes in luteal tissue of angiotensin and endothelin system members in mRNA expression, tissue concentrations, tissue localization, and ACE (angiotensin converting enzyme) antagonist application after prostaglandin F(2alpha) (PG) induced (days 8-12) luteal regression in cow. Corpora lutea (CL) were collected by transvaginal ovaryectomy before and 2, 4, 12, 24, 48, and 64 hr (n = 5/time point) after PG injection. ACE mRNA expression (RT-PCR) increased continuously and peaked at 12, 24 hr; ECE-1 (endothelin converting enzyme) peaked at 12 hr, and both peptides in tissue (Ang II and ET-1) increased significantly and peaked at 24 hr. The expression of receptors for Ang II (AT1R and AT2R) did not change in contrast to ET receptors (ETR-A and ETR-B), which were up-regulated. Localization in tissue revealed very weak staining for Ang II and ET-1 before PG application followed by a clear increase of staining predominantly in large luteal cells, but also in endothelial cells. In two experiments, the attempt was made to block ACE by the antagonist captopril with two different doses. In both experiments with captopril, progesterone levels were not significantly different from controls. Ang II alone seems to be not essential for functional luteolysis in bovine system. In conclusion, the results suggest that both Ang II and ET-1 are in parallel up-regulated during luteal regression and may act as vasoconstrictors during functional luteolysis, but also as apoptosis inducer during functional/structural luteolysis.

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