The "Ram Effect": A "Non-Classical" Mechanism for Inducing LH Surges in Sheep

Abstract

During spring sheep do not normally ovulate but exposure to a ram can induce ovulation. In some ewes an LH surge is induced immediately after exposure to a ram thus raising questions about the control of this precocious LH surge. Our first aim was to determine the plasma concentrations of oestradiol (E2) E2 in anoestrous ewes before and after the "ram effect" in ewes that had a "precocious" LH surge (starting within 6 hours), a "normal" surge (between 6 and 28h) and "late» surge (not detected by 56h). In another experiment we tested if a small increase in circulating E2 could induce an LH surge in anoestrus ewes. The concentration of E2 significantly was not different at the time of ram introduction among ewes with the three types of LH surge. "Precocious" LH surges were not preceded by a large increase in E2 unlike "normal" surges and small elevations of circulating E2 alone were unable to induce LH surges. These results show that the "precocious" LH surge was not the result of E2 positive feedback. Our second aim was to test if noradrenaline (NA) is involved in the LH response to the "ram effect". Using double labelling for Fos and tyrosine hydroxylase (TH) we showed that exposure of anoestrous ewes to a ram induced a higher density of cells positive for both in the A1 nucleus and the Locus Coeruleus complex compared to unstimulated controls. Finally, the administration by retrodialysis into the preoptic area, of NA increased the proportion of ewes with an LH response to ram odor whereas treatment with the α1 antagonist Prazosin decreased the LH pulse frequency and amplitude induced by a sexually active ram. Collectively these results suggest that in anoestrous ewes NA is involved in ram-induced LH secretion as observed in other induced ovulators.

Fig 1. Schematic representation of the experimental design showing the time of collection of the samples that were used in the different experiments to measure oestradiol concentrations (Exp 1 and 2), and LH concentration (All experiments).

Double time lines indicate that the samples were collected every 15 min.

Fig 2. Changes in oestradiol concentrations before and after ram introduction in anoestrous ewes who presented LH surges at different time after ram introduction.

"precocious": ewes who presented a LH surge within 4 hours after male introduction; "normal": ewes presented a LH surge between 16 and 28 hours after the introduction of rams; "late": ewes who did not present a surge before 56h. A exp. 1, B exp. 2 year 1, C exp. 2 year 2. Data are expressed as Mean ± SEM. * p<0.03, **p<0.01 compared to “normal” at the same time.

Fig 3. Changes in LH concentrations before and after insertion of a 3 cm silastic oestradiol implant for 3 hours. (small E2 study).

Data are expressed as Mean ± SEM **p<0.01 compared to other times.

Fig 4. Photographs illustrating neurons expressing Fos and TH proteins in the A1 (A, C) and A7 (B, D) nucleus of a female exposed to a ram. C and D are details of the area shown in the rectangle.

Small blue arrows: Fos alone; black arrows: Fos protein in a TH neuron; arrow heads: TH without Fos protein. Scale bar = 160μm for A and B and 40μm for C and D.

Fig 5. Box Plot representations of the density of Fos-IR neurons (A), the number of TH-IR cells counted (B), the proportion of Fos-IR cells also containing TH-IR (C) and proportion of TH-IR cells also containing Fos-IR (D) in the different noradrenergic nuclei (A1, A2, A5, LC-A7) in adult Ile de France ewes exposed to a ram (hatched boxes) or to a control situation (empty boxes).

The bottom and top of the boxes are the first and third quartiles, the red square inside the boxes is the median and the end of the whiskers are the minimum and maximum of all the data; * p<0.02, ** p<0.01 compared to the control group. Because of the differences in the number, density and proportions among the different nuclei the scales are different for the different nuclei.

Fig 6. Schematic representation of the localization of the tip of dialysis probes used in the “pharmacological study”.

Numbers correspond to the different animals. ac: anterior commissure, fx: fornix, Int Caps: Internal Capsule, LV: lateral ventricle, MPOA: medial preoptic area, oc: optic chiasma, S: septum, SON: supraoptique nucleus, 3V: 3^rd ventricle.

Fig 7. Effect of infusion with noradrenaline (100 ng/mL) on LH secretion induced in anoestrous Ile de France ewes by exposure to ram fleece.

A: Representative LH profiles in 2 ewes; B: Box Plot representations of the effect on pulse frequency, C: Box Plot representations of the effect on pulse amplitude. The thick horizontal lines depict the median. ** Different from noradrenaline before the stimulation p<0.003, ◊ different from ringers after stimulation p<0.02.

Fig 8. Effect of infusion with Prazosin (100 μg/mL) on LH secretion induced in anoestrous Ile de France ewes by exposure to a ram.

A: Representative LH profiles in 2 ewes; B: Box Plot representations of the effect on pulse frequency, C: Box Plot representations of the effect on pulse amplitude. * Different from before the stimulation p<0.02, ** different from before the stimulation p<0.003, ◊ Prazosin different from ringer p<0.04.