Szysiak N, Kosior-Korzecka U, longo V, Patkowski K, Greguła-Kania M, Nowakiewicz A, Bochniarz M,Junkuszew A (2025) Influence of neurokinin b, dynorphin a and kisspeptin-10 on in vitro gonadotropin secretion by anterior pituitary cells isolated from pubescent ewes. J Vet Res doi: 10.2478/jvetres-2025-0003
Objective: The aim of the study was to analyze the direct effect of the hypothalamic neuropeptides kisspeptin-10, neurokinin B, and dynorphin A on gonadotropin secretion by pituitary cells isolated from pubescent ewes.
Summary: Puberty is a multifactorial and complex process in animal development and in the case of livestock, timely attainment of sexual maturity contributes to increased reproductive efficiency, which leads to higher profitability. Studies revealed that kisspeptin, neurokinin B and dynorphin neuropeptides, collectively referred to as KNDy neuropeptides, are recognized as the key neuropeptides produced and secreted by the arcuate nucleus of the hypothalamus (ARC), and involved in the endocrine regulation of the onset of puberty. They all play roles in the endocrine regulation of the hypothalamic-pituitary-ovarian (HPO) axis in puberty. Kisspeptin-10, NKB and Dyn A had a direct impact on gonadotropin secretion by ovine pituitary cells. However, a detailed explanation of their role in gonadotropin secretion by the anterior pituitary gland in sheep and their impact on the regulation of the HPO axis during sexual maturation or in the pathomechanism of delayed puberty requires further studies.
Usage: Prepubertal ewes received 1 μL (0.7 μg) of NKB-SAP (NK3-SAP) [IT-63] or Blank-SAP (IT-21) injections aimed at the arcuate (ARC) nucleus to ablate neurons expressing NK3R.
Griesgraber MJ, Coolen LM, Onslow KM, Corey JR, Rice RE, Aerts EG, Bowdridge EC, Hardy SL, Lehman MN, Goodman RL, Hileman SM (2025) Critical role of arcuate nucleus kisspeptin and Kiss1R in regulation of the ovine luteinizing hormone surge. J Neuroendocrinol e70010. doi: 10.1111/jne.70010 PMID: 40033679
Objective: To examine the functional role of arcuate nucleus (ARC) dynorphin-containing (KNDy) and kisspeptin (Kiss) 1R-containing neurons in ovine luteinizing hormone (LH) surge secretion via injection of saporin-ligand conjugates (SAP) to ablate these neural populations.
Summary: NKB-SAP injections significantly reduced the percentage of ARC Kiss1 (~65% decrease) cells compared to control animals, and a surge-like increase of LH was prevented in ewes with the greatest degree of Kiss1 cell ablation. Kiss-SAP injections had no effect on Kiss1 cell percentage or ARC Kiss1R cell number compared to controls. However, Kiss-SAP injections consistently and robustly decreased LH surge amplitude, with 80% of Kiss-SAP-treated ewes failing to generate a surge. These results support the conclusion that KNDy neurons contribute significantly to the ovine LH surge, while ARC Kiss1R neurons appear to be necessary for a functional surge to occur in sheep.
Usage: Saporin conjugates, NKB-SAP (IT-63) and Kisspeptin-SAP (IT-102) were injected at 700 ng/μL.
Coutinho EA, Esparza LA, Steffen PH, Liaw R, Bolleddu S, Kauffman AS (2024) Selective depletion of kisspeptin neurons in the hypothalamic arcuate nucleus in early juvenile life reduces pubertal LH secretion and delays puberty onset in mice. FASEB J 28(19):e70078. doi: 10.1096/fj.202401696R PMID: 39377760
Objective: To investigate the specific kisspeptin population timing pubertal onset.
Summary: This study teases apart the contributions of different kisspeptin neural populations to the control of puberty onset, demonstrating that a majority of KNDy neurons in the arcuate nucleus are necessary for the proper timing of puberty in both sexes.
Usage: See also these studies where ~70% of ARC kisspeptin (KNDy) neurons were selectively ablated in rats using NKB-SAP (IT-63).
Thomson AC (2024) Puberty-Associated Changes in Kiss1r, MC3R, and MC4R in the Ewe. West Virgina University
Objective: To examine kisspeptin receptor (Kiss1r) mRNA expression in gonadotropin releasing hormone (GnRH) neurons and melanocortin 3 and 4 receptor (MC3R/MC4R) expression in kisspeptin neurons across pubertal development.
Summary: Kiss1r expression in GnRH neurons increased with development. In the arcuate nucleus (ARC), the proportion of kisspeptin neurons expressing MC3R, but not MC4R, also rose, though MC3R signal intensity remained unchanged. ARC Kiss1r expression did not vary. Findings support increased GnRH neuron sensitivity to kisspeptin and aMSH as part of the pubertal neurocircuitry.
Usage: Author references prior studies. Deletion of about 75% of KNDy neurons through targeted NKB-SAP (IT-63) injections significantly delayed puberty onset. Reduction of Kiss1r expression in the ARC via targeted injection of a Kisspeptin-SAP (IT-102) conjugate disrupted pulsatile LH release and blocked the LH surge.
Aerts EG (2024) Estradiol’s role in timely puberty onset in the ewe. Western Virginia Univ
Objective: To investigate the role of arcuate nucleus KNDy (kisspeptin/neurokinin B/dynorphin) neurons in regulating the timing of puberty onset and estradiol (E2) sensitivity in female sheep.
Summary: Ablation of approximately 75% of KNDy neurons using NKB-SAP in the arcuate nucleus significantly delayed puberty onset and blunted the luteinizing hormone (LH) surge, demonstrating that KNDy neurons are critical for timely puberty in ewes. However, animals retained the ability to respond to NK3R agonist senktide, suggesting upstream populations may mediate estradiol negative feedback.
Usage: NKB-SAP (IT-63) was bilaterally injected into the arcuate nucleus (1 µg/side in 1 µL) to ablate NK3R-expressing KNDy neurons and evaluate their role in pubertal timing and LH regulation.
Aerts EG, Griesgraber MJ, Shuping SL, Bowdridge EC, Hardy SL, Goodman RL, Nestor CC, Hileman SM (2024) The effect of NK3-Saporin injection within the arcuate nucleus on puberty, the LH surge, and the response to Senktide in female sheep. Biol Reprod 110(2):275-287. doi: 10.1093/biolre/ioad147 PMID: 37930247
Objective: To investigate the role of NKB-SAP (NK3-SAP) in the arcuate nucleus on the timing of puberty, the LH surge, and the response to the NK3R agonist senktide in female sheep.
Summary: This study explores how the ablation of NK3R-containing neurons in the arcuate nucleus affects puberty onset and reproductive hormone dynamics in female sheep. The findings demonstrate that NK3-SAP injections significantly delay puberty, reduce the amplitude of the LH surge, and alter the response to senktide, underscoring the critical role of NK3R-containing neurons in reproductive function.
Usage: Prepubertal ewes received 1 μL (0.7 μg) of NKB-SAP (NK3-SAP) [IT-63] or Blank-SAP (IT-21) injections aimed at the arcuate (ARC) nucleus to ablate neurons expressing NK3R.
Ivanova D, O’Byrne KT (2024) New methods to investigate the GnRH pulse generator. J Mol Endocrinol 72(2):e230079. doi: 10.1530/JME-23-0079 PMID: 38085702
Objective: To review the latest methodologies and insights into the GnRH pulse generator and its role in regulating reproductive hormone secretion.
Summary: The paper discusses recent advancements in understanding the GnRH pulse generator and its role in reproductive hormone secretion. It highlights the involvement of kisspeptin/neurokinin B/dynorphin (KNDy) neurons and the use of advanced techniques like genetic mouse models, electrophysiology, optogenetics, and calcium imaging. These findings enhance our comprehension of the KNDy network’s oscillatory behavior and its regulation by gonadal steroids, which is crucial for developing better infertility treatments.
Aquino NSS, Campideli-Santana AC, Antunes LM, Araújo-Lopes R, da Costa Silva KS, Costa Henriques P, de Oliveira Gusmão D, Bernuci MP, Szawka RE, dos Reis AM (2023) Partial loss of KNDy neurons in prenatally androgen treated female rats alters the LH secretion and ovarian morphology in a model of polycystic ovary syndrome. J Endocrine Society 7(S1):bvad114.1215. doi: 10.1210/jendso/bvad114.1215
Objective: To examine the impact of partial loss of KNDy neurons in prenatally androgen-treated female rats on luteinizing hormone (LH) secretion and ovarian morphology, as a model of polycystic ovary syndrome (PCOS).
Summary: The study found that the ablation of KNDy neurons resulted in increased LH pulse amplitude and mean LH levels without affecting pulse frequency, and partially restored the number of primordial follicles, suggesting KNDy neurons’ role in modulating LH release and ovarian reserve in PCOS.
Usage: Intra-ARC stereotaxic injections of the neurokinin-3 receptor agonist conjugated with Saporin (NKB-SAP, IT-63) to induce the lesion of KNDy neurons. Blank-SAP (IT-21) was used as a control.
Campideli-Santana AC, da Costa Silva KS, Araújo-Lopes R, Antunes LM, Szawka RE (2023) Submaximal loss of KNDy neurons accelerates and amplifies pulsatile LH secretion in female rats. J Endocrine Society 7(S1):bvad114.1214. doi: 10.1210/jendso/bvad114.1214
Objective: To investigate the impact of the submaximal loss of KNDy neurons on the pulsatile secretion of luteinizing hormone (LH) in female rats.
Summary: The study found that a partial loss of these neurons led to irregular estrous cycles and increased frequency and amplitude of LH pulses. This suggests a new role for KNDy neurons in moderating LH pulse frequency and amplitude in ovary-intact animals, with implications for early antral follicle recruitment.
Usage: Adult female rats underwent a neurochemical ablation of KNDy neurons via intra-ARC stereotaxic injections of NKB-SAP (IT-63), while control animals received Blank-SAP (IT-21).
Goodman RL, Moore AM, Onslow K, Hileman SM, Hardy SL, Bowdridge EC, Walters BA, Agus S, Griesgraber MJ, Aerts EG, Lehman MN, Coolen LM (2023) Lesions of kndy and kiss1r neurons in the arcuate nucleus produce different effects on lh pulse patterns in female sheep. Endocrinology 164(11):bqad148. doi: 10.1210/endocr/bqad148 PMID: 37776515
Objective: To test the functional role of ovine KNDy neurons in pulse generation and identify the roles of nearby Kiss1 receptor (Kiss1R)-containing cells.
Summary: Injection of NK3-SAP (NKB-SAP) ablated over 90% of the KNDy cells, Kiss-SAP lesioned about two-thirds of the Kiss1R population. This led to a significant decrease in LH pulse amplitude and altering LH pulse patterns. NK3-SAP increased the interpulse interval without affecting the regularity of LH pulses, whereas Kiss-SAP disrupted their regular hourly occurrence but not the interpulse interval. The findings suggest that KNDy neurons are critical for GnRH pulse generation in ewes, while ARC Kiss1R cells support the amplitude and regularity of these pulses, possibly as part of a positive feedback loop involving GABA or glutamate.
Usage: Saporin conjugates were injected into the arcuate nucleus. Kiss-SAP (kisspeptin54-SAP) was diluted to 700 ng/μL in PBS immediately before use. In preliminary work to test the effectiveness of Kiss-SAP, a single unilateral injection (1 μL of 700 ng/μL) of this conjugate was made in the preoptic area of 3 ewes. The contralateral side was used as control and either received no injections or Blank-SAP (1 μL of 700 ng/μL) (IT-21).
