104 Distribution of Gonadotropin-Releasing Hormone and Kisspeptin Neurons in the Preoptic Area and Hypothalamus During the Estrous Cycle in Cows

Abstract

Gonadal steroids hormones indirectly regulate gonadotropin-rleasing hormone (GnRH) secretion. Kisspeptin (Kp) co-expresses steroid receptors and modulates GnRH release. The objective of the study was to characterise the number and proportion of GnRH and Kp immunoreactive cells and their association in the preoptic area (POA) and hypothalamus during different phases of the oestrous cycle in cows. Daily ovarian ultrasonography was performed to detect follicle development and ovulation (Day 0) after prostaglandin treatment. On Day 5, cows were assigned randomly to the following groups: proestrus (n = 2), metestrus (n = 2) or diestrus (n = 3). Cows in the diestrus group were killed on Day 8. Cows in the proestrus and metestrus groups were given luteolytic dose of prostaglandin on Day 5.5 and Day 6 and were killed on Day 7 and 24 h after the ensuing ovulation, respectively. Cow heads were perfused with 4% paraformaldehyde via the carotid arteries to fix the brain in situ. The brain-stem (rostral portion of the POA to the mamillary body) was isolated by dissection and placed in 4% paraformaldehyde for 48 h. Following cryoprotection, the tissue block containing the POA and hypothalamus was frozen at –80°C and sectioned serially at a thickness of 50 mm using a cryostat microtome. Every 20th free-floating section was processed for double labelling using 2 sequential immuno-peroxidase reactions and ABC staining; Kp was immuno-labelled with Nickel-DAB at a dilution of 1:10,000 rabbit anti-kisspeptin (AC566, INRA, France), and GnRH was stained with DAB using 1:40,000 rabbit anti-GnRH (LR-5, Dr Benoit). The numbers of neuron cell bodies and fibres were recorded in different areas of the POA and the hypothalamus by brightfield microscopy using 10× and 40× objective lenses. Data were compared among groups by ANOVA. Major aggregations of Kp cells were localised in the mPOA, OVLT, and ARC. Overall, the number of Kp cells was higher in the metestrus v. diestrus group (719 ± 94 v. 378 ± 8; P = 0.01), but was similar to the proestrus group (558 ± 9). The number of Kp cells in the POA (mPOA, OVLT) tended to be higher in the metestrus v. diestrus group (395 ± 56 v. 147 ± 44; P = 0.06), and was intermediate in the proestrus group (206 ± 6). The number of Kp cells in the ARC did not differ among groups (metestrus 310 ± 26, diestrus 206 ± 53, proestrus 321 ± 99; P = 0.4). The number of GnRH cells bodies was not different among groups (metestrus 40 ± 3, diestrus 50 ± 9, proestrus 43 ± 8; combined; P = 0.8), and the distribution was higher in the POA (metestrus 25 ± 2, diestrus 30 ± 3, proestrus 33 ± 2) than hypothalamus. The proportion of GnRH cells in apposition to Kp fibres tended to be highest in the proestrus v. metestrus and diestrus groups (50.5 ± 1% v. 34.1 ± 9% and 31.4 ± 3%; P = 0.09). In conclusion, the number of Kp immunoreactive cells, but not GnRH cells, present in the POA and hypothalamus changed among different phases of the oestrous cycle due primarily to an increase in number of Kp cells in POA during metestrus. The proestrous phase was associated with an increase in apposition between Kp fibres and GnRH cells.