@article{ahern_martins_florez_ross_huisman_cushman_shuping_nestor_desaulniers_white_et al._2024, title={Development of KISS1 knockout pigs is characterized by hypogonadotropic hypogonadism, normal growth, and reduced skatole}, ISSN={["1529-7268"]}, DOI={10.1093/biolre/ioae140}, abstractNote={Abstract Kisspeptin is a major regulator of gonadotropin secretion in pigs. Previously, CRISPR/Cas9 knockout of KISS1 was used to develop a mosaic parental line of pigs to generate offspring that would not need castration due to loss of kisspeptin. The current goal was to characterize growth and reproductive development of F1 pigs from this parental line. Body weights, gonadotropin concentrations and gonadal development were measured from birth through development (boars to 220 d of age, n = 42; gilts to 160 d of age, n = 36). Testosterone, skatole, and androstenone were also measured in boars. Blood samples were collected by jugular venipuncture for quantification of serum hormones, gonadal tissues collected for gross morphology and histology, and a fat biopsy collected (boars) for skatole and androstenone analysis. Body weight did not differ with genotype. There were no differences between KISS1+/+ and heterozygote KISS1+/− animals for most parameters measured. Gonadotropin concentrations were reduced in KISS1−/− boars and gilts compared with KISS1+/+ and KISS1+/− animals (P < 0.05). Concentrations of testosterone in serum and both androstenone and skatole in adipose were less in KISS1−/− boars than in KISS1+/+ and KISS1+/− boars (P < 0.05). Hypogonadism was in all KISS1−/− gilts and boars. These data indicate that knocking out KISS1 causes hypogonadotropic hypogonadism but does not negatively affect growth in pigs. Only one KISS1 allele is needed for normal gonadotropin secretion and gonadal development, and accumulation of compounds in adipose leading to boar taint.}, journal={BIOLOGY OF REPRODUCTION}, author={Ahern, Daniel F. and Martins, Kyra and Florez, Julio M. and Ross, Caitlin E. and Huisman, Abe and Cushman, Robert A. and Shuping, Sydney L. and Nestor, Casey C. and Desaulniers, Amy T. and White, Brett R. and et al.}, year={2024}, month={Oct} }
 @article{griesgraber_onslow_shuping_bowdridge_hardy_aerts_coolen_nestor_hileman_lehman_et al._2023, title={Role of Kndy and Arcuate Kiss1r-Containing Neurons in the Preovulatory Luteinizing Hormone Surge and Puberty Onset of Female sheep}, volume={101}, ISSN={["1525-3163"]}, DOI={10.1093/jas/skad281.284}, abstractNote={Abstract
               Neurons within the arcuate nucleus (ARC) of the hypothalamus containing kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons) have an important role in regulating the pulsatile secretion of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH). In sheep, kisspeptin neurons also contribute to the LH surge, as kisspeptin receptor (Kiss1r) antagonist administration reduces surge amplitude by 50% and KNDy neurons are likely involved, based on increased Fos expression at the time of the surge. However, the extent to which kisspeptin acts within the ARC regulate the GnRH/LH surge remains unclear. Thus, herein we tested the hypothesis that deletion of KNDy or ARC Kiss1r-containing neurons would impair the LH surge. Adult female sheep received bilateral injections targeting the ARC of NKB-saporin (NKB-SAP, n = 8), kisspeptin-saporin (Kiss-SAP, n = 10), or blank-saporin (Blank-SAP, n = 7) as a control. In other work, NKB-SAP lesioned over 90% of ovine KNDy neurons, while Kiss-SAP lesioned 67% of Kiss1r-containing cells without affecting KNDy or GnRH cell number. Ewes were also ovariectomized and a subcutaneous silastic estradiol (E2) implant was inserted at the time of neurosurgery. Two artificial luteal phases were simulated with progesterone-containing CIDRs, immediately followed by E2 treatment via implants to induce an LH surge. Blood samples were collected every two to four hours over two days and analyzed for LH via radioimmunoassay. LH surge amplitude in six of eight NKB-SAP ewes (49.5 ± 11.7 ng/mL) was significantly reduced compared with Blank-SAP control ewes (156.7 ± 20.2 ng/mL, p = 0.0001), a reduction similar to that produced by treatment with a Kiss1r antagonist. Nine of ten Kiss-SAP treated ewes displayed little to no increase of LH at the time of the expected surge (16.6 ± 5.3 ng/mL, p < 0.0001). Lesion effectiveness is currently being assessed by RNAscope, however all Kiss-SAP animals examined to date have significantly reduced ARC Kiss1r cell numbers except a single ewe which exhibited a normal LH surge. Based on these data, we propose that in ewes, KNDy neurons contribute to, but are not required for, the LH surge. In contrast, ARC Kiss1r-containing cells are essential for a functional LH surge. Given these results, we are currently assessing the role of ARC Kiss1r neurons in ovine puberty using a similar approach. Our data to date shows that time to puberty onset is similar for Kiss-SAP, Blank-SAP, and non-surgical control animals as measured by an increase in progesterone (p = 0.35). Blood samples to detect LH pulses and the LH surge are currently being analyzed, as are ARC Kiss1r cell numbers.}, journal={JOURNAL OF ANIMAL SCIENCE}, author={Griesgraber, Max J. and Onslow, Kayla M. and Shuping, Sydney and Bowdridge, Elizabeth and Hardy, Steven and Aerts, Eliana and Coolen, Lique and Nestor, Casey and Hileman, Stanley and Lehman, Michael and et al.}, year={2023}, month={Nov}, pages={235–236} }
 @article{aerts_griesgraber_shuping_bowdridge_hardy_goodman_nestor_hileman_2023, title={The effect of NK3-Saporin injection within the arcuate nucleus on puberty, the LH surge, and the response to Senktide in female sheep}, volume={10}, ISSN={["1529-7268"]}, url={https://doi.org/10.1093/biolre/ioad147}, DOI={10.1093/biolre/ioad147}, abstractNote={Abstract
               The timing of puberty onset is reliant on increased gonadotropin-releasing hormone (GnRH). This elicits a corresponding increase in luteinizing hormone (LH) due to a lessening of sensitivity to the inhibitory actions of estradiol (E2). The mechanisms underlying the increase in GnRH release likely involve a subset of neurons within the arcuate (ARC) nucleus of the hypothalamus that contain kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons). We aimed to determine if KNDy neurons in female sheep are critical for: timely puberty onset; the LH surge; and the response to an intravenous injection of the neurokinin-3 receptor (NK3R) agonist, senktide. Prepubertal ewes received injections aimed at the ARC containing blank-saporin (control, n = 5) or NK3-saporin (NK3-SAP, n = 6) to ablate neurons expressing NK3R. Blood samples taken 3/week for 65 days following surgery were assessed for progesterone to determine onset of puberty. Control ewes exhibited onset of puberty at 33.2 ± 3.9 days post sampling initiation, whereas 5/6 NK3-SAP treated ewes didn’t display an increase in progesterone. After an artificial LH surge protocol, surge amplitude was lower in NK3-SAP ewes. Finally, ewes were treated with senktide to determine if an LH response was elicited. LH pulses were evident in both groups in the absence of injections, but the response to senktide vs saline was similar between groups. These results show that KNDy cells are necessary for timely puberty onset and for full expresson of the LH surge. The occurrence of LH pulses in NK3-SAP treated ewes may indicate a recovery from an apulsatile state.}, journal={BIOLOGY OF REPRODUCTION}, author={Aerts, Eliana G. and Griesgraber, Max J. and Shuping, Sydney L. and Bowdridge, Elizabeth C. and Hardy, Steven L. and Goodman, Robert L. and Nestor, Casey C. and Hileman, Stanley M.}, year={2023}, month={Oct} }
 @article{harlow_griesgraber_seman_shuping_sommer_griffith_hileman_nestor_2022, title={The impact of undernutrition on KNDy (kisspeptin/neurokinin B/dynorphin) neurons in female lambs}, volume={34}, ISSN={["1365-2826"]}, url={https://doi.org/10.1111/jne.13135}, DOI={10.1111/jne.13135}, abstractNote={AbstractUndernutrition limits reproduction through inhibition of gonadotropin‐releasing hormone (GnRH)/luteinizing hormone (LH) secretion. Because KNDy neurons coexpress neuropeptides that play stimulatory (kisspeptin and neurokinin B [NKB]) and inhibitory (dynorphin) roles in pulsatile GnRH/LH release, we hypothesized that undernutrition would inhibit kisspeptin and NKB expression at the same time as increasing dynorphin expression. Fifteen ovariectomized lambs were either fed to maintain pre‐study body weight (controls) or feed‐restricted to lose 20% of pre‐study body weight (FR) over 13 weeks. Blood samples were collected and plasma from weeks 0 and 13 were assessed for LH by radioimmunoassay. At week 13, animals were killed, and brain tissue was processed for assessment of KNDy peptide mRNA or protein expression. Mean LH and LH pulse amplitude were lower in FR lambs compared to controls. We observed lower mRNA abundance for kisspeptin within KNDy neurons of FR lambs compared to controls with no significant change in mRNA for NKB or dynorphin. We also observed that FR lambs had fewer numbers of arcuate nucleus kisspeptin and NKB perikarya compared to controls. These findings support the idea that KNDy neurons are important for regulating reproduction during undernutrition in female sheep.}, number={6}, journal={JOURNAL OF NEUROENDOCRINOLOGY}, publisher={Wiley}, author={Harlow, KaLynn and Griesgraber, Max J. and Seman, Andrew D. and Shuping, Sydney L. and Sommer, Jeffrey R. and Griffith, Emily H. and Hileman, Stanley M. and Nestor, Casey C.}, year={2022}, month={May} }
 @article{merkley_shuping_sommer_nestor_2021, title={Evidence That Agouti-Related Peptide May Directly Regulate Kisspeptin Neurons in Male Sheep}, volume={11}, ISBN={2218-1989}, DOI={10.3390/metabol1030138}, number={3}, journal={METABOLITES}, author={Merkley, Christina M. and Shuping, Sydney L. and Sommer, Jeffrey R. and Nestor, Casey C.}, year={2021}, month={Mar} }
 @article{harlow_renwick_shuping_sommer_lents_knauer_nestor_2021, title={Evidence that pubertal status impacts kisspeptin/neurokinin B/dynorphin neurons in the gilt(dagger)}, volume={105}, ISSN={["1529-7268"]}, DOI={10.1093/biolre/ioab189}, abstractNote={Abstract
               Puberty onset is a complex physiological process, which enables the capacity for reproduction through increased gonadotropin-releasing hormone and subsequently luteinizing hormone secretion. While cells that coexpress kisspeptin, neurokinin B (NKB), and dynorphin in the hypothalamic arcuate nucleus are believed to govern the timing of puberty, the degree to which kisspeptin/NKB/dynorphin (KNDy) neurons exist and are regulated by pubertal status remains to be determined in the gilt. Hypothalamic tissue from prepubertal and postpubertal, early follicular phase gilts was used to determine the expression of kisspeptin, NKB, and dynorphin within the arcuate nucleus. Fluorescent in situ hybridization revealed that the majority (>74%) of arcuate nucleus neurons that express mRNA for kisspeptin coexpressed mRNA for NKB and dynorphin. There were fewer arcuate nucleus cells that expressed mRNA for dynorphin in postpubertal gilts compared to prepubertal gilts (P < 0.05), but the number of arcuate nucleus cells expressing mRNA for kisspeptin or NKB was not different between groups. Within KNDy neurons, mRNA abundance for kisspeptin, NKB, and dynorphin of postpubertal gilts was the same as, less than, and greater than, respectively, prepubertal gilts. Immunostaining for kisspeptin did not differ between prepubertal and postpubertal gilts, but there were fewer NKB immunoreactive fibers in postpubertal gilts compared to prepubertal gilts (P < 0.05). Together, these data reveal novel information about KNDy neurons in gilts and support the idea that NKB and dynorphin play a role in puberty onset in the female pig.}, number={6}, journal={BIOLOGY OF REPRODUCTION}, author={Harlow, KaLynn and Renwick, Allison N. and Shuping, Sydney L. and Sommer, Jeffrey R. and Lents, Clay A. and Knauer, Mark T. and Nestor, Casey C.}, year={2021}, month={Dec}, pages={1533–1544} }
 @article{harlow_renwick_shuping_sommer_knauer_nestor_2020, title={Effects of genetic selection for early puberty on the hypothalamic-pituitary-ovarian axis in gilts}, volume={98}, ISSN={["1525-3163"]}, DOI={10.1093/jas/skaa054.368}, abstractNote={Abstract
               Puberty onset in gilts is an awakening of the hypothalamic-pituitary-ovarian axis that is the result of reduced estradiol-negative feedback at the level of the hypothalamus which yields elevated gonadotropin secretion from the anterior pituitary. Given the importance of hypothalamic kisspeptin and neurokinin B (NKB) signaling for the onset of puberty in other species, the objective of this study was to determine if gilts selected for early pubertal onset (SELECT) would display measurable differences within the hypothalamus (i.e. increased expression of kisspeptin and NKB) and within the ovary (i.e. increased ovarian mass) compared to age-matched and weight-matched gilts (CONTROL) that achieve puberty 20 days later than SELECT gilts. Gilts were sacrificed at three timepoints: Timepoint A, both groups were determined to be prepubertal (n=6/group), Timepoint B, SELECT gilts were determined to be pubertal and CONTROL gilts were determined to be prepubertal (n=6/group), and Timepoint C, both groups were determined to be pubertal (n=6/group). All animals were euthanized, heads were perfused with 8 L of 4% paraformaldehyde, and ovaries were harvested. Brain tissue was removed post-fixation, submerged in fixative for 24 hrs followed by 20% sucrose until sectioned for immunohistochemistry. Ovarian mass tended (p≤0.10) to be greater for SELECT gilts on the right ovary (4.34 vs. 3.67 g) and the left ovary (4.49 vs. 3.68 g) when compared to CONTROL (Timepoints A and C), and at Timepoint B right ovary mass from SELECT gilts was heavier than CONTROL gilts (p< 0.05; 7.22 vs. 4.65 g). Hypothalamic immunohistochemistry for kisspeptin and NKB revealed differences in neuronal fiber density between both groups at various timepoints. Therefore, we conclude that gilts genetically selected for early puberty do so via changes within the hypothalamus that increase gonadotropin secretion and, in turn, stimulate ovarian growth to ultimately advance the timing of puberty onset.}, journal={JOURNAL OF ANIMAL SCIENCE}, author={Harlow, KaLynn and Renwick, Allison and Shuping, Sydney and Sommer, Jeff and Knauer, Mark and Nestor, Casey}, year={2020}, month={Nov}, pages={212–212} }