We have previously shown LH surges induced by physiological estradiol levels are invariably accompanied by robust and sustained GnRH surges in the ewe. Such an increase, however, has not been observed consistently during the preovulatory LH surge. In the present study, we examined GnRH secretion in Suffolk and Ile de France ewes during the preovulatory period using a method for pituitary portal blood collection which allows simultaneous portal and jugular blood samples to be taken at frequent intervals for up to 48 h. Ewes were sampled either during the mid-late luteal phase (n = 8) or follicular phase (n = 20). During the follicular phase, a robust increase in GnRH secretion occurred at the onset of the LH surge in 11 of 12 ewes sampled during the LH surge. The GnRH increase in most ewes was a massive surge, reaching values averaging 40-fold greater than baseline and extending well beyond the end of the preovulatory LH surge. In the single ewe not exhibiting a GnRH surge during the LH surge, postmortem inspection indicated blood was probably not sampled from the pituitary portal vessels. In the early follicular phase, GnRH-pulse frequency was greater than that observed in the luteal phase and, within the follicular phase, GnRH-pulse frequency increased further and amplitude decreased as the surge approached. These data demonstrate GnRH secretion leading up to ovulation in the ewe is dynamic, beginning with slow pulses during the luteal phase, progressing to higher frequency pulses during the follicular phase and invariably culminating in a robust surge of GnRH. The LH surge, however, ends despite continued elevation of GnRH.

Proteins present in and secreted into the lumen of various regions of the stallion epididymis were characterized qualitatively and quantitatively by two-dimensional electrophoresis. Using this proteomic approach, 201 proteins were found in the lumen and 117 were found that were secreted by the epithelium in various parts of the organ. Eighteen proteins made up 92.6% of the total epididymal secretory activity, lactoferrin (41.2%) and clusterin (24.8%) being the most abundant. Procathepsin D, HE1/CTP (cholesterol transfer protein), GPX (glutathione peroxidase), beta-N-acetyl-hexosaminidase, and PGDS (prostaglandin D2 synthase) were the other major compounds secreted. The most abundant proteins found in the luminal fluid were albumin and the secreted proteins: lactoferrin, PGDS, GPX, HE1/CTP, and hexosaminidase. Three main secretory epididymal regions were identified from the protein pattern, i.e., regions E0-E2, E3-E5, and E6-E9. Region E0-E2 was characterized by the secretion of clusterin (53%), PGDS (44%), and GPX (6%). Region E3-E5 had the highest number of secreted proteins, the highest protein concentrations (60-80 mg/ml), and the highest spermatocrit value (85%). Lactoferrin (60% in E4), clusterin (29% in E3), hexosaminidase (10% in E3), and procathepsin D (6.9% in E4) were the most abundant proteins in this region. Region E6-E9, in which few region-specific secreted compounds were found, was characterized by a high quantity of lactoferrin in the luminal fluid (2-14 mg/ml). Comparison between the secretion of the major proteins and their concentrations in the lumen throughout the organ showed that the behavior of each protein is specific, in particular for the three isoforms of clusterin.

In ovariectomized ewes, an injection of oestrogen initially inhibits the tonic secretion of LH, and then induces a large release of LH similar to the preovulatory surge in intact ewes. The pattern of hypothalamic secretion of gonadotrophin-releasing hormone (GnRH) into the pituitary portal blood during this biphasic response to oestrogen was investigated in conscious, unrestrained, ovariectomized adult Ile-de-France ewes during the breeding season. The ewes were ovariectomized and implanted with cannulae for portal blood collection on the same day. Seven days later, portal and peripheral blood samples were collected simultaneously every 5 min for 25 h. The ewes were injected with oestradiol-17 beta (25 micrograms i.v. and 25 micrograms i.m.) 6.25 h after the start of sampling. GnRH and LH were measured by radioimmunoassay in portal and jugular plasma samples respectively. A clear pulsatile pattern of LH secretion was observed before the oestradiol injection in all the ewes, followed by the typical biphasic decrease (negative feedback) and increase (positive feedback) in mean concentrations. The sampling period was divided, for analysis, into pretreatment, negative feedback and positive feedback phases. Before injection with oestradiol, the GnRH pulses were clearly defined in portal blood and were always synchronized with LH pulses in the peripheral circulation. The frequency was 5.9 +/- 0.6 pulses/6 h (mean +/- S.E.M.), and the amplitude was 31.6 +/- 7.6 pmol/l. During negative feedback, both the frequency (4.2 +/- 0.5 pulses/6 h, P less than 0.01) and amplitude (15.2 +/- 4.6 pmol/l, P less than 0.05) of the GnRH pulses decreased.(ABSTRACT TRUNCATED AT 250 WORDS)