Stereology of gonadotropin-releasing hormone and kisspeptin neurons in PACAP gene-deficient female mice

Abstract

The hypothalamic gonadotropin-releasing hormone (GnRH)-kisspeptin neuronal network regulates fertility in all mammals. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide isolated from the hypothalamus that is involved in the regulation of several releasing hormones and trop hormones. It is well-known that PACAP influences fertility at central and peripheral levels. However, the effects of PACAP on GnRH and kisspeptin neurons are not well understood. The present study investigated the integrity of the estrous cycle in PACAP-knockout (KO) mice. The number and immunoreactivity of GnRH (GnRH-ir) neurons in wild-type (WT) and PACAP KO female mice were determined using immunohistochemistry. In addition, the number of kisspeptin neurons was measured by counting kisspeptin mRNA-positive cells in the rostral periventricular region of the third ventricle (RP3V) and arcuate nucleus (ARC) using the RNAscope technique. Finally, the mRNA and protein expression of estrogen receptor alpha (ERα) was also examined. Our data showed that the number of complete cycles decreased, and the length of each cycle was longer in PACAP KO mice. Furthermore, the PACAP KO mice experienced longer periods of diestrus and spent significantly less time in estrus. There was no difference in GnRH-ir or number of GnRH neurons. In contrast, the number of kisspeptin neurons was decreased in the ARC, but not in the R3PV, in PACAP KO mice compared to WT littermates. Furthermore, ERα mRNA and protein expression was decreased in the ARC, whereas in the R3PV region, ERα mRNA levels were elevated. Our results demonstrate that embryonic deletion of PACAP significantly changes the structure and presumably the function of the GnRH-kisspeptin neuronal network, influencing fertility.

Keywords: GnRH; PACAP; estrogen receptor α; estrous cycle; kisspeptin.

Figure 1

Disrupted estrous cyclicity in PACAP KO mice. Representative estrous cycle diagrams illustrate the alterations in estrous cycle in WT and PACAP KO mice (A). Dot plots depict the number of estrous cycles in 4 weeks (B), the cycle length (C), and the percentage of time spent in estrus (D) and diestrus (E). Graphs show the mean ± SD for panels (C, D) and the median ± range for panels (B, E) n = 6–6, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

GnRH neurons and their arborization in different brain regions. Representative images of immunohistochemical labeling of GnRH neurons and their fibers located in the medial septum (MS), medial preoptic area (MPOA) together with organum vasculosum of lamina terminalis (OVLT), lateral hypothalamus (LH), and eminentia mediana (EM) are shown in panels (A–D) respectively (scale bar: 200 µm).

Figure 3

Number of GnRH neurons in wild-type and PACAP KO female mice. Summarized data of the number of GnRH neurons in regions of medial septum (MS), medial preoptic area (MPOA), and lateral hypothalamus (LH) from WT and PACAP KO mice are presented in panels (A–C) respectively. Experiments were performed in female mice in estrus phase. Data are presented as mean ± SD, n = 6–8.

Figure 4

GnRH-ir in wild-type and PACAP KO female mice. GnRH-ir data in regions of medial septum (MS), medial preoptic area (MPOA), lateral hypothalamus (LH), organum vasculosum of lamina terminalis (OVLT), and eminentia mediana (EM) obtained from WT and PACAP KO mice are presented in panels A–E, respectively. Experiments were performed in female mice in estrus phase. Data are presented as mean ± SD, n = 6–8.

Figure 5

Kiss1 and Esr1 mRNA expression in the RP3V of wild-type and PACAP KO female mice. Representative confocal fluorescence images depict the expression of Kiss1 mRNA in the RP3V region (panels A, E) and Esr1 mRNA-positive cells in the RP3V (panels B, F) of WT and PACAP KO mice. Nuclear counterstain with Hoechst33342 is presented in panels (C, G) while the merged image is shown in panels D, H. 3V, third ventricle. Images were taken with a 20× plan apochromat objective (scale bar: 100 µm).

Figure 6

Kiss1 and Esr1 mRNA expression in the ARC of wild-type and PACAP KO female mice. The expression of Kiss1 (panels A, E) and Esr1 (panels B, F) mRNAs in the ARC of WT and PACAP KO mice is depicted in representative confocal images. Nuclear counterstain with Hoechst33342 and the merged image are presented in panels C, G and D, H respectively. 20× magnification, scale bar: 25 µm.

Figure 7

Number of Kiss1 mRNA-positive neurons in the RP3V and ARC in wild-type and PACAP KO female mice. Summarized data of RNAscope in situ hybridization experiments are shown in dot plots. The number of Kiss1 mRNA-positive cells is shown in RP3V (A) and ARC (B) from wild-type and PACAP KO female mice (two slices per animal, six animals in both groups). Data are presented as mean ± SD, *p < 0.05.

Figure 8

ERα protein expression in the RP3V and ARC in wild-type and PACAP KO female mice. Representative brightfield images show the expression of ERα protein in the RP3V (A) and ARC (B) of a WT and a PACAP KO female mouse. 20× magnification, scale bar: 25 µm.

Figure 9

Number of ERα-immunoreactive cells in the RP3V and ARC in wild-type and PACAP KO female mice. Dot plots present the summarized data obtained by RNAscope in situ hybridization experiments showing Esr1 mRNA expression in RP3V (A) and ARC regions (C). Immunohistochemical staining demonstrating ERα protein expression in RP3V (B) and ARC regions (D). n = 6–6 animals for each group with two slices per animal. Graphs show the mean ± SD for all panels, *p < 0.05.