Christopher S. Keator

Intrauterine infusion of BQ-610, an endothelin type A receptor antagonist, delays luteolysis in dairy heifers.

Three separate in vivo experiments were conducted to evaluate the putative role of endothelin-1 (ET-1) during luteal regression in heifers. In Experiment 1, a single intraluteal injection of 500 microg BQ-610 [(N,N-hexamethylene) carbamoyl-Leu-D-Trp (CHO)-D-Trp], a highly specific endothelin A (ETA) receptor antagonist, did not diminish the decline in plasma progesterone following a single exogenous injection of 25 mg prostaglandin F2 alpha (PGF2alpha) administered at midcycle of the estrous cycle. In Experiment 2, six intrauterine infusions of 500 microg BQ-610 given every 12 h on days 16-18 delayed spontaneous luteolysis, as evidenced by an extended elevation (P=0.054) of plasma progesterone concentration. In Experiment 3, heifers were administered six intrauterine infusions of BQ-610 or saline on days 16-19, and peripheral blood samples were collected from day 11 to 16 (before infusion), hourly on days 16-19 (during infusion), and on days 20-25 (after infusion). BQ-610 treated heifers had markedly higher (P<0.0001) levels of plasma progesterone compared with saline controls, and this effect was most notable during the infusion period (treatment by period interaction; P<or=0.05). Heifers infused with BQ-610 also had higher progesterone levels on day 21 (treatment by time interaction; P<or=0.05). Mean plasma concentrations of 13,14-dihydro-15-keto-PGF2alpha (PGFM), the primary metabolite of PGF2alpha, were measured in the samples collected hourly and were not different (P>or=0.05) between treatments. These results indicate that the in vivo antagonism of the ETA receptor can delay functional luteolysis, and supports the theory that ET-1 regulates luteal function in ruminants.

A new in vivo model for luteolysis using systemic pulsatile infusions of PGF2α

A new in vivo model for studying luteolysis was developed in sheep to provide a convenient method for collecting corpora lutea for molecular, biochemical, and histological analysis during a procedure that mimics natural luteolysis. It was found that the infusion of prostaglandin F(2α) (PGF(2α)) at 20 μg/min/h into the systemic circulation during the mid luteal phase of the cycle allowed sufficient PGF(2α) to escape across the lungs and thus mimic the transient 40% decline in the concentration of progesterone in peripheral plasma seen at the onset of natural luteolysis in sheep. Additional 1h-long systemic infusions of PGF(2α), given at physiological intervals, indicated that two infusions were not sufficient to induce luteolysis. However, an early onset of luteolysis and estrus was induced in one out of three sheep with three infusions, two out of three sheep with four infusions, and three out of three sheep with five infusions. Reducing the duration of each systemic infusion of PGF(2α) from 1h to 30 min failed to induce luteolysis and estrus even after six systemic infusions indicating that, not only are the amplitude and frequency of PGF(2α) pulses essential for luteolysis, but the actual duration of each pulse is also critical. We conclude that a minimum of five systemic pulses of PGF(2α), given in an appropriate amount and at a physiological frequency and duration, are required to mimic luteolysis consistently in all sheep. The five pulse regimen thus provides a new accurate in vivo model for studying molecular mechanisms of luteolysis.

Evidence for a potential role of neuropeptide Y in ovine corpus luteum function.

Neuropeptide Y (NPY) is a neurohormone that is typically associated with food intake, but it has also been reported to affect the production of progesterone from luteal tissue in vitro. However, NPY has not been previously immunolocalized in the ovine ovary or in the corpus luteum (CL) of any species, and the effects of this neurohormone on luteal function in vivo are not known. Thus, we performed fluorescent immunohistochemistry (IHC) to localize NPY in the ovine ovary and used avidin-biotin immunocytochemistry (ICC) to further define the intracellular localization within follicles and the CL. We then infused NPY directly into the arterial supply of the autotransplanted ovaries of sheep to determine the in vivo effect of exogenous NPY on ovarian blood flow and on the luteal secretion rate of progesterone and oxytocin. Immunohistochemistry revealed that the NPY antigen was localized to cells within the follicles and CL, in the nerve fibers of the ovarian stroma, and in the vessels of the ovarian hilus. In the follicle, the NPY antigen was localized to nerves and vessels within the theca interna layer, and strong staining was observed in the granulosal cells of antral follicles. In the CL, NPY was localized in large luteal cells and in the vascular pericytes and/or endothelial cells of blood vessels, found dispersed throughout the gland and within the luteal capsule. In vivo incremental infusions of NPY at 1, 10, 100, and 1,000 ng/min, each for a 30-min period, into the arterial supply of the transplanted ovary of sheep bearing a CL 11 d of age increased (P< or =0.05) ovarian blood flow. The intra-arterial infusions of NPY also increased (P< or =0.05) in a dose-dependent manner the secretion rate of oxytocin, which was positively correlated (P< or =0.05) with the observed increase in ovarian blood flow. The infusions of NPY had a minimal effect on the secretion rate of progesterone, and similar intra-arterial infusions of NPY into sheep with ovarian transplants bearing a CL over 30 d of age had no significant effect on ovarian blood flow or on the secretion rate of progesterone. These results suggest that NPY acts on the luteal vascular system and the large luteal cells to rapidly stimulate blood flow and the secretion of oxytocin, respectively, which collectively implies a putative role for NPY during the process of luteolysis when increasing amounts of oxytocin are secreted from the ovine CL in response to uterine pulses of prostaglandin F2alpha.

Luteotrophic and luteolytic effects of nitric oxide in sheep are dose-dependent in vivo

It has been suggested that nitric oxide (NO) acts in either an anti-luteolytic or in a luteolytic manner, but the mechanism for these opposing roles is unclear. We hypothesized that NO may act in a dose-dependent manner to regulate luteal function, whereby low concentrations of NO might stimulate luteal progesterone production (i.e. luteotrophic) and high concentrations of NO might reduce concentrations of plasma progesterone (i.e. luteolytic). To test this hypothesis we infused increasing concentrations of the fast-acting NO donor, dipropylenetriamine NONOate (DPTA), into the arterial supply of sheep with ovarian transplants bearing a corpus luteum (CL). Infusions were performed on sheep with CL 11 days of age (n=9) or over 30 days of age (n=15). We measured changes in the concentration of progesterone in ovarian venous plasma during the 1-h infusion and for 24h after the infusion, and then compared the mean concentration of progesterone between treatment groups for effects by dose and dose by period interactions. Compared with saline-treated controls (n=6), the highest dose of 1000 microg/min DPTA (n=6) reduced (P<or=0.05) the mean concentration of progesterone after the infusion. In sheep bearing a CL over 30 days of age, the 10 microg/min DPTA dose (n=3) markedly increased (P<or=0.05) the mean concentration of progesterone both during and after the infusion, whereas the 100 microg/min DPTA dose (n=3) increased (P<or=0.05) the mean concentration of progesterone only during the 1-h infusion. The mean concentration of progesterone was not different (P>0.05) in sheep infused with the lowest dose of 1 microg/min DPTA (n=6) compared with controls. We conclude that NO regulates luteal function in a dose-dependent manner in sheep in vivo.