Date of Graduation

2015

Document Type

Thesis

Degree Type

MS

College

Davis College of Agriculture, Natural Resources and Design

Department

Animal and Nutritional Sciences

Committee Chair

Robert L Goodman

Committee Co-Chair

Robert A Dailey

Committee Member

Stanley M Hileman

Abstract

The neural mechanisms underlying the onset of puberty are not well understood. In sheep, this process involves an increase in GnRH release due to decreased sensitivity to estrogen feedback. Because GnRH neurons do not express the relevant estrogen receptors, this pathway must contain interneurons. Neurons in the arcuate nucleus (ARC) of the hypothalamus that co-express kisspeptin (Kiss), neurokinin B, and dynorphin (DYN), i.e. KNDy neurons, play a critical role. Based on previous data, the overall hypothesis is that the prepubertal hold in GnRH/LH release results from reduced Kiss stimulation and heightened DYN inhibition in response to estrogen negative feedback. Two experiments were performed to address this idea. The first experiment tested the hypothesis that Kiss expression would be increased and DYN expression decreased in response to ovariectomy (OVX). Prepubertal ewe lambs were randomly assigned to three groups: OVX (n=5), OVX and treated with estrogen (OVX+E) (n=5), or left ovary-intact (n=4). As hypothesized, numbers of Kiss-immunoreactive cells in the ARC were increased by OVX (207.5 +/- 5.6 cells/section) compared to either ovary-intact (110.6 +/- 2.7 cells/section) or OVX+E (48.4 +/- 2.4 cells/section) groups and were significantly higher in ovary-intact compared to OVX+E females. Contrary to the hypothesis, very few DYN-immunopositive cells were found in the ARC of any treatment group, even though they were readily evident in control hypothalamic tissue concurrently assessed from adult ewes collected during the luteal phase (29.8 +/- 3.9 cells/section). Although data regarding DYN were not consistent with the hypothesis, a role for DYN cannot be ruled out as the lack of staining could be reflective of high release rates of the peptide. Thus, a second study was performed to test the hypothesis that blockade of the kappa-opioid receptor (kappa- OR) (DYN's cognate receptor) would increase LH secretion in steroid-treated females of a prepubertal, but not postpubertal, age. Thus the effects of norbinaltorphimine (nor-BNI), a kappa- OR antagonist, on LH secretion were compared between OVX+E prepubertal ewes (n=6) and OVX+E postpubertal ewes. Prepubertal OVX+E lambs were infused ICV (60 ul/h) with artificial cerebral spinal fluid (aCSF), as a control, or nor-BNI (60 nmol/h) for 3h. As hypothesized, administration of nor-BNI increased LH pulse frequency (3.5 +/- 0.6 pulses/3 h) and mean LH (6.3 +/- 0.9 ng/mL) compared to aCSF (frequency: 1.7 +/- 0.5 pulses/3 h; mean: 3.2 +/- 0.7 ng/mL). Nor-BNI or aCSF was infused for 4h in these same OVX+E ewes at a postpubertal age. There were no significant differences in LH pulse frequency, mean LH, or LH pulse amplitude between treatments. These data demonstrate that inhibition by DYN is at least partly responsible for the prepubertal suppression of GnRH/LH secretion by estrogen. Based on these data, it is suggested that increased stimulation by Kiss and reduced inhibition by DYN play important roles in the escape from estrogen negative feedback that underlies the transition to puberty in the female lamb.

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