Direct evidence that KNDy neurons maintain gonadotropin pulses and folliculogenesis as the GnRH pulse generator

Abstract

The gonadotropin-releasing hormone (GnRH) pulse is fundamental for mammalian reproduction: GnRH pulse regimens are needed as therapies for infertile women as continuous GnRH treatment paradoxically inhibits gonadotropin release. Circumstantial evidence suggests that the hypothalamic arcuate KNDy neurons expressing kisspeptin (encoded by Kiss1), neurokinin B (encoded by Tac3), and dynorphin A serve as a GnRH pulse generator; however, no direct evidence is currently available. Here, we show that rescuing >20% KNDy neurons by transfecting Kiss1 inside arcuate Tac3 neurons, but not outside of these neurons, recovered folliculogenesis and luteinizing hormone (LH) pulses, an indicator of GnRH pulses, in female global Kiss1 knockout (KO) rats and that >90% conditional arcuate Kiss1 KO in newly generated Kiss1-floxed rats completely suppressed LH pulses. These results first provide direct evidence that KNDy neurons are the GnRH pulse generator, and at least 20% of KNDy neurons are sufficient to maintain folliculogenesis via generating GnRH/gonadotropin pulses.

Keywords: KNDy rescue; LH pulses; conditional Kiss1 KO; kisspeptin; neurokinin B.

Fig. 1.

Rescue of KNDy neurons in female global Kiss1 KO rats. (A) Kiss1 (green)- and Tac3 (magenta)-expressing cells in the mediobasal hypothalamus of three representative OVX global Kiss1 KO rats infected with AAV-Kiss1 targeted into the ARC (indicated by the dotted line). Note that a large number of Kiss1-expressing cells were found inside of the ARC of highly and moderately KNDy-rescued global Kiss1 KO rats, whereas Kiss1-expressing cells were mainly found outside of the ARC of few KNDy-rescued rats. (B) Distribution of ARC Tac3-expressing cells with (dashed lines) or without (solid lines) Kiss1 expression throughout the ARC of two representative few, moderately, and highly KNDy-rescued rats. R, the rostral part of ARC and C, the caudal part of ARC. (C) Higher magnification of the squared area in panel A showing Kiss1- and Tac3-coexpressing cells (arrowheads). Insets show a representative Kiss1- and Tac3-coexpressing cell. (D) The number of Tac3-expressing cells with or without Kiss1 expression in the ARC of few, moderately, and highly KNDy-rescued rats. Values expressed in the bar graph are mean ± SEM. Numbers in each column indicate the number of animals used. The values with different letters were significantly different from each other (P < 0.05) based on one-way ANOVA followed by Tukey's HSD test. (Scale bars, 100 μm.)

Fig. 2.

Recovery of gonadotropin synthesis/release and Gnrhr mRNA expression in the KNDy neuron-rescued female rats. (A) Plasma LH profiles in representative OVX few, moderately, and highly KNDy-rescued global Kiss1 KO rats and OVX AAV-EGFP–treated global Kiss1 KO control rats (Cont). Arrowheads indicate LH pulses identified with the PULSAR computer program. Mean LH concentration and the frequency and amplitude of LH pulses (B), the pituitary LH and FSH content (C), and the pituitary Lhb, Fshb, and Gnrhr mRNA expression (D) in the few, moderately, and highly KNDy-rescued rats and control rats. Values expressed in the bar graphs are mean ± SEM. Numbers in (or on) each column indicate the number of animals used. The values with different letters were significantly different from each other (P < 0.05) based on one-way ANOVA followed by Tukey's HSD test. Asterisk indicates statistically significant differences (P < 0.05) between the highly and moderately KNDy-rescued rats based on Welch's t test. Note that LH pulses were detected in zero out of five AAV-EGFP–treated Kiss1 KO control rats and in one out of three few KNDy-rescued rats. NA, not applicable.

Fig. 3.

Recovery of follicular development and ovarian Cyp19a1 and Lhcgr mRNA expression in the KNDy neuron-rescued female rats. (A) Ovarian weight of the few, moderately, and highly KNDy-rescued global Kiss1 KO rats and AAV-EGFP–treated global Kiss1 KO control rats (Cont). (B) A Graafian follicle (solid arrowhead) in the ovary of a representative highly KNDy-rescued rat and a non-Graafian tertiary follicle (open arrowhead) in the ovary of a representative control rat. (Scale bars, 200 μm.) (C) The diameter in each Graafian and non-Graafian tertiary follicle in the few, moderately, and highly KNDy-rescued rats and control rats. The tertiary follicle with a diameter greater than 390 µm (horizontal dashed line) was termed as Graafian. (D) The mean numbers of Graafian and non-Graafian tertiary follicles and follicular cysts in each group. (E) The ovarian Cyp19a1, Cyp17a1, Lhcgr, and Fshr gene expression in each group. Values expressed in the bar graphs are mean ± SEM. Numbers in (or on) each column indicate the number of animals used. The values with different letters were significantly different from each other (P < 0.05) based on one-way ANOVA followed by Tukey’s HSD test.

Fig. 4.

Recovery of vaginal opening (VO) and occurrence of persistent estrus in the KNDy neuron-rescued female rats. (A) Days at the VO (expressed as a percentage of the total number of animals per group). There were significant differences in the occurrence probability of the VO between the groups (P < 0.05, Kaplan–Meier analysis and log-rank test). (B) Vaginal smear pattern in a representative animal in each group. CC, cornified cells; NC, nucleated cells; and LC, leukocytes. (C) Days that animals showed CC and a mixture of NC and CC (NC/CC) in the first, second, and third weeks after the AAV-Kiss1 injection. Note that no animal showed a LC smear pattern. N/A, not applicable because animals failed to show VO.

Fig. 5.

Evaluation of conditional ARC Kiss1 KO female rats. (A) Schematic illustration of the injection of AAV-Cre into the ARC. (B) Cre-expressing cells in the ARC and AVPV of a representative OVX + low E2 AAV-Cre–treated Kiss1-floxed rats. (C) Distribution of ARC Kiss1-expressing cells throughout the ARC of two representative OVX + low E2 moderately and highly ARC Kiss1 KO rats and OVX + low E2 AAV-EGFP–treated Kiss1-floxed control rats (Cont). R, the rostral part of ARC and C, the caudal part of ARC. (D) Kiss1-expressing cells in the ARC of representative moderately and highly ARC Kiss1 KO rats and control rat. Insets show representative Kiss1-expressing cells. (E) The number of Kiss1-expressing cells in the ARC. (F) Kiss1-expressing cells in the AVPV of representative animals. (G) The number of Kiss1-expressing cells in the AVPV. Values expressed in the bar graphs are mean ± SEM. Numbers in (or on) each column indicate the number of animals used. The values with different letters were significantly different from each other (P < 0.05) based on one-way ANOVA followed by Tukey's HSD test. (Scale bars, 100 μm.)

Fig. 6.

Suppression of gonadotropin synthesis/release and Gnrhr mRNA expression in the conditional ARC Kiss1 KO female rats. (A) Plasma LH profiles in representative OVX + low E2 moderately and highly ARC Kiss1 KO rats and AAV-EGFP–treated Kiss1-floxed control rat (Cont). Arrowheads indicate LH pulses identified with PULSAR computer program. Mean LH concentration and the frequency and amplitude of LH pulses (B), the pituitary LH and FSH content (C), and the pituitary Lhb, Fshb, and Gnrhr mRNA expression (D) in the moderately and highly Kiss1 KO rats and control rats. Values expressed in the bar graphs are mean ± SEM. Numbers in each column indicate the number of animals used. The values with different letters were significantly different from each other (P < 0.05) based on one-way ANOVA followed by Tukey’s HSD test.

Fig. 7.

A positive feedback level of E2-induced surge-like increase in plasma LH levels in the conditional ARC Kiss1 KO female rats. (A) Mean plasma LH profiles in the moderately and highly ARC Kiss1 KO rats and AAV-EGFP–treated Kiss1-floxed control rats (Cont). (B) The ratios of peak to baseline of plasma LH levels. Values expressed are mean ± SEM. Numbers in each column indicate the number of animals used. No significant differences were detected in the ratio by one-way ANOVA.