Kisspeptin neuron projections to oxytocin neurons are not necessary for parturition in the mouse

All experimental procedures were approved by the University of Otago Animal Ethics Committee and were carried out in accordance with the New Zealand Animal Welfare Act (1999) and associated guidelines.

Female adult C57/B6J, Kiss-Cre⁺ and Kiss-Cre⁻ mice (de Croft et al. 2012) were group-housed in the University of Otago animal facility under controlled conditions (12 h light, 12 h dark cycle: lights on at 0600 h, 22 ± 1 °C) with free access to food and water. Mice had their estrous cycles monitored daily using vaginal cytology to examine the appearance of the epithelial cells. On proestrus, females were placed overnight in a cage with a male. Mating was considered to be successful when a vaginal plug was visualized on the following morning and this was recorded as day 1 of gestation (G1). For mice that gave birth, the day of birth was recorded as day 1 post-partum (PP1).

Mice were anesthetized with sodium pentobarbitone (10 mg kg⁻¹, IP) and transcardially perfused with 25 ml of 4% paraformaldehyde (PFA). The brains were removed, post-fixed in 4% PFA for 1 h and then in 30% sucrose/0.1 M phosphate buffer for 24–48 h, and frozen. Three series of 30 µm coronal sections were cut on a freezing microtome. Fluorescent immunohistochemistry for kisspeptin and oxytocin in the SON/PVN involved incubating sections from non-pregnant (NP, no specific day of the estrous cycle), day 7 (G7), 14 (G14) and 19 pregnant (G19) and day 7 lactating (PP7) C57/B6J mice in a cocktail of polyclonal rabbit anti-kisspeptin-10 (1:25,000, AC 564, A. Caraty, INRA, France, RRID:AB_2622231; Franceschini et al. 2006) and monoclonal mouse anti-oxytocin antiserum (1:5000, MAB5296 Millipore, MA, USA, RRID:AB_2157626; Dabrowska et al. 2011) in incubation solution (Tris-buffered saline (TBS) containing 0.3% Triton X-100, 0.25% bovine serum and 2% normal goat serum) for 48 h at 4 °C on an orbital shaker. Sections were then incubated in a cocktail of Alexa Fluor goat anti-rabbit 568 (1:500, A11036, Molecular Probes, OR, USA) and Alexa Fluor goat anti-mouse 488 (1:500, A11029, Molecular Probes, OR, USA) in incubation solution for 90 min. Sections were mounted onto gelatin-coated slides and cover-slipped using Vectashield Hardset mounting medium (H-1400, Vector Laboratories, Burlingame, CA, USA).

Z-series stacks (one section per area per mouse) were captured at 0.5 µm intervals through the SON and PVN using a Nikon A1R multiphoton confocal microscope under a 40 × oil-immersion objective and three-dimensional images were reconstructed using Nikon NIS Elements AR imaging software (Nikon Instruments Inc. Melville, NY, USA). More specific areas were also scanned using boxes of fixed-size (106 μm × 26.5 μm) in the SON (above the optic chiasm, where oxytocin neuron cell bodies predominantly reside (Pirnik et al. 2004)) and PVN; one box medially next to the third ventricle (the parvocellular division of the PVN), and the other 150 µm more lateral to the third ventricle, to incorporate the magnocellular division of the PVN (− 0.82 mm posterior from Bregma, based on the cytoarchitecture as described previously (Biag et al. 2012)). The fraction of kisspeptin signal-containing voxels to total voxels in the region of interest was calculated using IMARIS Image Analysis Software 9.8 (Oxford Instruments, Zurich, Switzerland) and expressed as a percentage of the area counted. The regions of interest were also analyzed for close appositions between kisspeptin fibers and oxytocin neurons using the spot counter in IMARIS. A surface was applied to the oxytocin neurons and spots were created for the kisspeptin immuno-positive fibers. Close appositions were defined as any spot from kisspeptin fibers contacting the surface of an oxytocin cell body or proximal dendrite. The number of oxytocin neurons within each box were counted in each area (SON; 2.58 ± 0.41) and did not differ between box 1 and box 2 in the PVN (3.64 ± 0.43 and 4.88 ± 0.48, respectively). Kisspeptin fiber density and appositions did not differ between box 1 and box 2 either. Therefore, the results were averaged for presentation.

Double-label fluorescent immunohistochemistry for kisspeptin and synaptophysin in the SON and PVN sections was carried out as described above, except sections were incubated in a cocktail of polyclonal rabbit anti-kisspeptin-10 (1:25,000, AC 564, A. Caraty, INRA, France, RRID:AB_2622231) and mouse anti-synaptophysin antiserum (1:1000, MAB368, Millipore, RRID:AB_94947; Yamanaka et al. 2011) for five nights at 4ºC. Z-series stacks were photographed through the SON and PVN at 1 µm intervals using a 60 × objective on a Nikon A1R confocal microscope and three-dimensional images were reconstructed using Nikon NIS Elements AR. The percentage of synaptophysin on kisspeptin fibers in the regions of interest were calculated as a percentage of co-localization using IMARIS. There was no remaining SON tissue from G7 mice to include in this part of the study.

Caspase-induced cell death was targeted to kisspeptin neurons in Kiss-Cre mice by stereotaxic injection into the APVP/PeN at least five days (range: 5–14 days) before mating. This timeline was chosen to allow full recovery from the effects of surgery before mating and because pilot experiments (data not shown) revealed that between two and four weeks was required for maximal kisspeptin neuron ablation. Kiss-Cre⁺ and Kiss-Cre^‒ mice were anesthetized with 4.5% isoflurane, placed in a stereotaxic apparatus and anesthesia was maintained at 2% isoflurane. Mice were given simultaneous bilateral injections (using 1 µL Hamilton syringes, Hamilton Company, NV, USA) of Cre-dependent caspase-3 delivered through the AAV5-flex-taCasp3-TEVp virus or AAV5-flex-C3-Tp (Vector Core, University of North Carolina; 1 µL/side of brain) system into the AVPV/PeN (0.7 mm anterior, 0.3 mm lateral and 5.7 mm ventral to bregma (Paxinos and Watson 2007)). The syringes were left in situ for 3 min before and 10 min after the injections. Mice were then allowed to recover for at least five days post injection. Estrous cycles were monitored daily from the day of injection. For those mice that displayed a first proestrus within the 5 days post injection recovery period, cycle length was determined by the number of days between the first two proestrous days following injection. Timed mating with a male occurred on the first proestrus, after the recovery period, to determine the duration of pregnancy in each mouse. Female mice were checked for the presence of a vaginal plug, which denoted day 1 of gestation. Mice were smeared for 14 days post-mating and pregnancy was confirmed by the presence of continual diestrous smears.

On G14, Kiss-Cre⁺ and Kiss-Cre^‒ mice were moved to a separate room dedicated to video recording for acclimatization to the new environment. Two cameras (Sony Chip Wired Bird Box Camera, Green Feathers, Bristol, UK) with infra-red night vision were installed on either side of each cage to record parturition. Cameras were connected to a desktop computer enabled with the software, iSpy 64-bit CCTV Recorder, which recorded continuously with tracked time and date. Dams were monitored continuously from G17 until PP4. The dams were randomized, coded and underwent blind analysis, with the experimenter being unaware of the groups. The precise timing of birth was determined from the video recording by the appearance and complete delivery of the first pup, and gestation length (days) was calculated. After analysis of all the data, the results were decoded, grouped, and expressed as mean ± SEM. Total parturition duration (min) was determined as the time between the complete delivery of the first pup to the complete delivery of the last pup. Mean time (min) of birth between each pup was determined as the time between the complete delivery of consecutive pups. Total number of live pups were counted at the end of parturition and each day thereafter. Four days post-partum (or 28 days post-caspase injection in mice that did not get pregnant), mice were anesthetized with sodium pentobarbitone (10 mg kg⁻¹, IP), transcardially perfused and the brains removed, frozen and sliced on a freezing microtome, as described above.

Free-floating chromogen immunohistochemistry for kisspeptin was undertaken as previously reported (Clarkson and Herbison 2006; Hellier et al. 2018) to evaluate and confirm the presence or absence of kisspeptin immunoreactivity in the AVPV/PeN after caspase-3 ablation in Kiss-Cre mice. Sections were incubated in polyclonal rabbit anti-kisspeptin-10 antibody (1:25,000; AC 564, A. Caraty, INRA, France, RRID:AB_2622231; Franceschini et al. 2006) for 48 h at 4 °C. Sections were then incubated in biotinylated goat anti-rabbit IgG (1:200; ZA0520, Vector Laboratories, Burlingame, CA, USA) for 90 min at room temperature, followed by incubation in Vectastain Elite ABC (Vector Laboratories, Burlingame, CA, USA). Sections were incubated in 3,3’-diaminobenzidine (DAB Peroxidase (HRP) Substrate Kit, Vector Laboratories) with nickel enhancement that resulted in a black precipitate and then mounted onto gelatin-coated slides and cover-slipped with Fluoromount-G™ mounting medium (00-4958-02, Invitrogen, USA).

To determine whether the number of kisspeptin-positive neurons in the AVPV/PeN differed between the Kiss-Cre⁺ pregnant and non-pregnant mice and Kiss-Cre^‒ groups, sections were visualized under an Olympus BX61 light microscope and photographed at 10 × objective using QImaging Micropublisher 5.0 RTV camera (QImaging, Canada). Numbers of kisspeptin-positive soma were counted in three sections per mouse and the mean value for each group calculated. Data were analyzed as mean number of kisspeptin neurons ± SEM.

Single-label fluorescent immunohistochemistry for kisspeptin in the SON, PVN and ARC sections was carried out as previously described (Seymour et al. 2017). Briefly, every second or third section from the SON, PVN or ARC of each mouse brain was immunolabeled with anti-kisspeptin-10 antibody (1:25,000; AC 564, A. Caraty, INRA, France, RRID:AB_2622231; Franceschini et al. 2006) and secondary antibody Alexa Fluor goat anti-rabbit 568 (1:500, A11036, Molecular Probes, USA), respectively.

To determine whether the density of kisspeptin-positive fibers differed between the Kiss-Cre⁺ pregnant and non-pregnant mice and Kiss-Cre^‒ groups, images from the SON, PVN and ARC were acquired using an inverted confocal microscope (Nikon A1R Inverted Confocal Laser Scanning Microscope, Nikon Instruments Inc, Tokyo, Japan) in Z-series stacks. A Z-series of 15–17 stacks was captured using excitation at 488 nm and 561 nm, 20 × objective, a pinhole of 4 µm and a slice interval of 1.65 µm at dimensions 1024 μm × 1024 μm. Fiber density was measured as previously described (Patisaul et al. 2008). Using Image J (NIH), a sub stack of images was created to control for section thickness throughout the samples. Images were then binarized to a threshold to allow best signal-to-noise ratio and skeletonized to make the fibers one pixel thick for fiber thickness normalization. The voxel counter plugin (NIH) was then used to count the number of voxels. The fraction of signal-containing voxels to total voxels in each region was calculated, and the mean value for each group was calculated. Data were analyzed as mean fiber density in % ± SEM of three sections per mouse.

All data were analyzed using GraphPad Prism (Version 8.00). Values are expressed as mean ± SEM unless stated otherwise. Two-tailed unpaired Student’s t-tests were used to compare between two groups and one-way ANOVA was used to compare between three or more groups where n ≥ 5; where the F-ratio was significant, all-pairwise Dunnett’s post hoc tests were applied. Where n < 5, non-parametric Mann–Whitney tests were used to compare between two groups and Kruskal–Wallis ANOVA on ranks was used to compare between three or more groups; where the ANOVA on ranks was significant, all-pairwise Dunn’s post hoc tests were applied. P ≤ 0.05 was considered significant.

Kisspeptin-positive fibers were observed among oxytocin-positive neurons throughout the rostral extent of the SON in NP, G7, G14, G19 and PP7 C57/B6J mice (Fig. 1B–F) and PVN (Fig. 2B–F). Some fibers were also evident in the perinuclear zone (PNZ), surrounding the SON, in all groups (Fig. 1B–F). Kisspeptin fibers bundled around oxytocin fibers and cell bodies in the SON (Fig. 1G) and PVN (Fig. 2G, H). The volume fraction of kisspeptin labeling was similar in the SON of all groups (Fig. 1H; F_4,34 = 0.69, P = 0.60, one-way ANOVA). In the PVN, the volume fraction of kisspeptin labeling was different between groups (Fig. 2I, F4,51 = 4.68, P = 0.003, one-way ANOVA), with higher expression in G7 mice than in NP mice (P = 0.02, NP vs G7, Dunnett’s post hoc test).

Kisspeptin fibers made few appositions with oxytocin neurons in the SON (Fig. 3A, B) and PVN of (Fig. 3E–F) C57/B6J mice. There were a similar number of appositions with oxytocin cell bodies (H₍₂₎ = 7.96, P = 0.09) and proximal dendrites (H₍₂₎ = 6.10, P = 0.10) in the SON in NP, G7, G14, G19 and PP7 mice (Fig. 3C, D). While there was a difference in the number of appositions with oxytocin cell bodies in the PVN (Fig. 3G, F_4,48 = 4.04, P = 0.007), there was no difference between NP and any of the other groups, rather the difference was between G7 and G14 compared to G19 (Dunnett’s post hoc tests). There was a higher number of appositions on the proximal dendrites of oxytocin neurons in the PVN of G14 mice than in NP mice (Fig. 3H, F_4,44 = 3.46, P = 0.015, one-way ANOVA, P = 0.007, Dunnett’s post hoc test).

There was a similar level of synaptophysin co-localization in kisspeptin fibers in both the SON (H₍₂₎ = 2.58, P = 0.29) and PVN (F_2,14 = 0.17, P = 0.84) in NP, G19 and PP7 C57/B6J mice (Fig. 4).

Bilateral injections of Caspase-3 effectively ablated kisspeptin cell bodies in the AVPV/PeN in Kiss-Cre⁺ female mice but not Kiss-Cre^‒ female mice (Fig. 5A–C). Fourteen Kiss-Cre⁺ mice had successful ablation of kisspeptin neurons on both sides of the AVPV/PeN. There was a difference in the number of kisspeptin neurons in the AVPV/PeN (Fig. 5D) between the groups (F_2,17 = 39.37, P = 0.039, one-way ANOVA). The mean maximal number of kisspeptin neurons per section in Kiss-Cre⁻ mice on PP4 was higher than in Kiss-Cre⁺ PP4 mice (P < 0.001, Dunnetts post hoc test) and in Kiss-Cre⁺ NP mice (P < 0.001, Dunnetts post hoc test).

Kisspeptin-positive fibers were observed in the SON, PVN and ARC in all groups of Kiss-Cre^± mice but a marked reduction was seen in both groups of Kiss-Cre⁺ mice after kisspeptin cell ablation in the AVPV/PeN. In the SON (Fig. 5E–G) the mean density of kisspeptin fibers (%) was different between the groups (H₍₂₎ = 7.19, P = 0.02) with Kiss-Cre⁺ PP4 mice having less compared to Kiss-Cre⁻ PP4 mice (P = 0.04, Dunn’s post hoc test). There were also less kisspeptin fibers in the Kiss-Cre⁺ NP mice than in the Kiss-Cre⁻ PP4 mice (P = 0.02, Dunn’s post hoc test, Fig. 5H).

The fiber density (%) in the PVN (Fig. 5I–K) was different between the groups (H₍₂₎ = 9.82, P = 0.003) with less fibers in the Kiss-Cre⁺ PP4 mice than in Kiss-Cre⁻ mice (P = 0.01, Dunn’s post hoc test). There were also less kisspeptin fibers in the Kiss-Cre⁺ NP mice than in the Kiss-Cre⁻ PP4 mice (P = 0.01, Dunn’s post hoc test, Fig. 5L).

In the ARC (Fig. 5M–P) the mean density of kisspeptin fibers (%) was not different between the groups (H₍₂₎, P = 0.24, Fig. 5P).

Ablation of the AVPV/PeN kisspeptin neurons in female Kiss-Cre⁺ mice disrupted the estrous cycle. Kiss-Cre⁺ mice had longer estrous cycles (Fig. 6A, P = 0.003, Student’s t-test) and less days in proestrus compared with Kiss-Cre⁻ mice (Fig. 6B, P = 0.003, Student’s t-test). Kiss-Cre⁺ mice that fell pregnant spent longer in proestrus than Kiss-Cre⁺ mice that did not fall pregnant (Fig. 6D–E). There was a large proportion of Kiss-Cre⁺ females (nine out of 14) that failed to get pregnant after bilateral injection with AAV5-flex-C3-Tp, while only one female (one out of ten) Kiss-Cre^‒ failed to get pregnant after the stereotaxic injection (Fig. 6F, P < 0.01, Chi-square test). Hence, the ablation of the AVPV/PeN kisspeptin neurons in female Kiss-Cre⁺ mice also reduced their ability to get pregnant. There was no difference in the duration of gestation between Kiss-Cre⁺ and Kiss-Cre⁻ females (Fig. 6G, P = 0.8, Student’s t-test). There was no difference in the duration of parturition (min) between Kiss-Cre⁺ and Kiss-Cre⁻ dams (Fig. 6H, P = 0.8, Student’s t-test). There was also no difference in the time between delivery of each pup between the two groups (Fig. 6I, P = 0.87, Student’s t-test). There was no difference in the mean litter size on PP1 between Kiss-Cre⁺ and Kiss-Cre⁻ dams (Fig. 6J, P = 0.55, Student’s t-test). Caspase-3 ablation of AVPV/PeN kisspeptin neurons did not affect the number of pups surviving in each Kiss-Cre⁺ litter when compared to Kiss-Cre⁻ litters (Fig. 6K). No difference was observed in the percent of pup survival between Kiss-Cre⁺ mice, and Kiss-Cre⁻ mice (Fig. 6L, P = 0.9, Student’s t-test). One Kiss-Cre⁺ dam lost all its pups after PP2 (0% survival), whereas no Kiss-Cre⁻ dams lost more than 50% of pups post-partum. The mean weight of pups born to the Kiss-Cre⁺ mice was higher on PP4 than the pups born to Kiss-Cre⁻ mice (Fig. 6M, P = 0.04, Mann–Whitney test).