Neuroscience

Prolactin-mediates a lactation-induced suppression of arcuate kisspeptin neuronal activity necessary for lactational infertility in mice

Eleni CR Hackwell
Sharon R Ladyman
Jenny Clarkson
H James McQuillan
Ulrich Boehm
Allan E Herbison
Rosemary SE Brown
David R Grattan author has email address

Centre for Neuroendocrinology, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand
Department of Anatomy, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand
Maurice Wilkins Centre for Molecular Biodiscovery, Auckland, New Zealand
Department of Physiology, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand
Saarland University School of Medicine, Centre for Molecular Signalling (PZMS), Experimental Pharmacology, Homburg, Germany
Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom

https://doi.org/10.7554/eLife.94570.2

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Figures and data

Prlr^lox/lox/Camk2a^Cre mice do not undergo the normal period of lactational infertility and the lactation-induced suppression of kisspeptin immunoreactivity is absent.
(A) Kisspeptin immunoreactivity shown in representative photomicrographs from diestrus, nulligravid, non-lactating (NL; left) and lactating (right) Prlr^lox/lox control and Prlr^lox/lox/Camk2a^Cre mice (from anteroventral periventricular nucleus (AVPV) region of RP3V). (B) Representative photomicrographs showing mid arcuate nucleus (mARC) of lactating Prlr^lox/lox (top) and lactating Prlr^lox/lox/Camk2a^Cre mice (bottom). (C) Total kisspeptin cell number for the RP3V (NL Prlr^lox/lox (n = 6) versus lactating Prlr^lox/lox control (n = 8) p = 0.0100, NL Prlr^lox/lox/Camk2a^Cre (n = 5) versus lactating Prlr^lox/lox/Camk2a^Cre (n = 8) p = 0.6409). Two-way ANOVA followed by Tukey’s multiple comparisons test. (D) Quantification of kisspeptin fibre density in the arcuate nucleus (Fiji software, measured in percentage voxels per region of interest), showing total kisspeptin fibre density in the arcuate nucleus (lactating Prlr^lox/lox control n = 8, lactating Prlr^lox/lox/Camk2a^Cre n = 7, p = 0.0020, unpaired two-tailed t test). (E) Lactating Prlr^lox/lox/Camk2a^Cre mice (blue, n = 8) resume estrous cycles significantly earlier (100% within 6-10 days of lactation) than lactating Prlr^lox/lox controls (grey, n = 10) (p = <0.0001, Log-rank (Mantel-Cox) test). Scale bar image and insert = 50μm. Values are shown as mean ± SEM.

Prolactin action in the brain during lactation is necessary for the suppression of pulsatile LH secretion.
Examples of pulsatile LH levels in the blood from lactating Prlr^lox/lox control and lactating Prlr^lox/lox/Camk2a^Cre mice that have either been treated with vehicle (sesame oil, s.c., grey, veh) or 4mg/kg mifepristone (in sesame oil, s.c., blue, mif). Graphs show LH pulse frequency (B; interaction p = 0.2807, genotype p = 0.0024, state p = 0.8558), and mean LH levels (C; interaction p = 0.8697, genotype p = 0.0031, state p = 0.8586). Lactating vehicle-treated Prlr^lox/lox (n = 8), lactating mifepristone-treated Prlr^lox/lox (n = 8), lactating vehicle-treated Prlr^lox/lox/Camk2a^Cre (n = 8), lactating mifepristone-treated Prlr^lox/lox/Camk2a^Cre (n = 7). Asterisks indicate LH pulse peaks as detected by PULSAR Otago analysis. Two-way ANOVA followed by Tukey’s multiple comparisons test. Values are shown as mean ± SEM.

Longitudinal recordings of arcuate kisspeptin neuron GCaMP population activity throughout different reproductive states in the same Kiss1^Cre mice.
Representative neuronal activity from three Kiss1^Cre mice throughout the NL (diestrus, nulligravid, non-lactating), pregnant, lactating, and post-weaning states. The time points monitored in order were: diestrus NL, day 4 pregnancy, day 14 pregnancy, day 18/19 pregnancy (overnight), day 7 lactation, day 14 lactation, day 18 lactation, 24 hours after weaning (day 22 postpartum), return to normal cycling following weaning (return to estrous cycles), and 10 days post ovariectomy (OVX). Asterisks indicate SEs. Note: change in y axes scale on all 3 OVX timepoints and mouse shown in dataset (B) from day 4 of pregnancy onwards.

Synchronised Ca²⁺ events (SE) are consistently correlated with pulsatile LH secretion across different reproductive states in Kiss1^Cre mice.
(A) When fibre photometry was paired with serial blood sampling for pulsatile LH secretion, the relationship between SEs and LH pulses was examined. Each time an SE was seen during a recording with blood sampling, pulsatile secretion of LH was also observed, with 100% correlation (p = <0.0001, chi-squared test; 73 out of 73 SEs observed led to an LH pulse). Representative examples of paired photometry and blood sampling are shown from the NL (diestrus, nulligravid, non-lactating) state, from day 7 lactation, and from day 14 lactation. (B) Quantitative analysis of SE frequency per hour across different reproductive states in Kiss1^Cre mice (p = 0.0012, mixed effect analysis (fixed type III) with Tukey’s multiple comparisons tests). (C) Quantitative analysis of relative SE amplitude of normalised ΔF/F across different reproductive states (p = 0.0118, mixed effect analysis (fixed type III) with Tukey’s multiple comparisons tests). Black asterisks indicate SEs, red asterisks indicate LH pulse peaks as detected by PULSAR Otago analysis. Values shown as mean ± SEM.

Activity of arcuate kisspeptin neurons on day 18 and 19 of pregnancy in Kiss1^Cre mice.
Fibre photometry recordings of mice on the evening of day 18 of pregnancy (1800 hours) to the morning of day 19 of pregnancy (0800 hours) show low amplitude SEs. 3-hour section blown up for ease of viewing. (C) No difference was seen between frequency of SEs (per 60 minutes) in the NL (diestrus, nulligravid, non-lactating) versus day 18/19 pregnancy (p = 0.4063, paired two-tailed t test), however a significant decrease in relative SE amplitude is seen (D; p = 0.0141, paired two-tailed t test). Asterisks indicate SEs. Dotted line in insert of (A) and (B) indicates 3 standard deviations (3SD). Grey shaded region = lights off.

Prlr^lox/lox/Kiss1^Cre mice do not undergo the normal period of lactational infertility and show early reactivation of arcuate kisspeptin neurons prior to estrus in lactation.
(A) Prlr^lox/lox/Kiss1^Cre mice resume estrous cycles significantly earlier (63% by day 10 of lactation, n = 32, blue) than Prlr^lox/lox controls (4% by day 19 of lactation, n = 30, grey) (p = <0.0001, Log-rank (Mantel-Cox) test). (B) Representative fibre photometry traces from day 5 of lactation from either a Kiss1^Cre control mouse or Prlr^lox/lox/Kiss1^Cre mice. Mice with Prlr knocked out of arcuate kisspeptin neurons (Prlr^lox/lox/Kiss1^Cre) show SEs early in lactation. Asterisks indicate SEs.

Proportion of kisspeptin neurons showing Prlr deletion using RNAscope in either Prlr^lox/lox/Camk2a^Cre or Prlr^lox/lox/Kiss1^Cre mice.
Representative photomicrographs showing RNAscope labelling for Prlr (blue) and Kiss1 (red) in the rostral periventricular region of the third ventricle (RP3V, A) or arcuate nucleus (ARC, C, E), in either intact (Prlr^lox/lox control n = 5, Prlr^lox/lox/Camk2a^Cre, n = 5, A) or ovariectomised (OVX; Prlr^lox/lox control n = 4, Prlr^lox/lox/Camk2a^Cre n = 5, C; Prlr^lox/lox control n = 6, Prlr^lox/lox/Kiss1^Cre n = 7, E) mice. Compared to Prlr^lox/lox control mice, Prlr^lox/lox/Camk2a^Cre mice show a significant decrease in the percentage of Kiss1-expressing cells co-expressing Prlr in both the RP3V (B; p = <0.0001) and arcuate nucleus (D; p = 0.0009) (unpaired two-tailed t tests). (F) A significant decrease in the percentage of Kiss1-expressing cells co-expressing Prlr was seen in Prlr^lox/lox/Kiss1^Cre compared to Prlr^l^ox/lox controls (p = <0.0001, unpaired two-tailed t test). (G) No correlation was found between percentage of Kiss1 cells co-expressing with Prlr and the day of estrus return during lactation (p = 0.1912, simple linear regression). Solid black arrows = doubled labelled cells expressing both Kiss1 and Prlr; black outlined arrows = Kiss1 cells with sparse or no co-labelling for Prlr. Scale bar = 150μm, insert = 60μm. Values are shown as mean ± SEM.

Mifepristone dose response and effect on litter weight gain.
(A) Mifepristone dose response trial showing dose of 4mg/kg was sufficient to terminate pregnancy in all mice (p = 0.072, chi-squared test, n = 6 both groups). (B) Mifepristone or vehicle treatment had no effect on litter weight gain from day 3 to day 5 of lactation (interaction of time x genotype & treatment p = 0.5322; time p = <0.0001; genotype and treatment p = 0.8811; subject p = <0.0001; two-way ANOVA). Values are shown as mean ± SEM.

Pulsatile LH secretion profiles of Prlr^lox/lox/Camk2a^Cre mice and Prlr^lox/lox controls following vehicle or mifepristone treatment.
Individual LH pulse data from lactating Prlr^lox/lox control and Prlr^lox/lox/Camk2a^Cre mice treated with either vehicle (sesame oil, s.c., grey) or 4mg/kg mifepristone (in sesame oil, s.c., blue). Lactating vehicle-treated Prlr^lox/lox (n = 8), lactating mifepristone-treated Prlr^lox/lox (n = 8), lactating vehicle-treated Prlr^lox/lox/Camk2a^Cre (n = 8), lactating mifepristone-treated Prlr^lox/lox/Camk2a^Cre (n = 7). Asterisks indicate LH pulse peaks as detected by PULSAR Otago analysis.

Gestational and maternal phenotyping of Prlr^lox/lox/Camk2a^Cre and Prlr^lox/lox/Kiss1^Cre mice and their respective Prlr^lox/lox controls.
(A-H) Data show; gestational weight gain (A; Prlr^lox/lox n = 11, Prlr^lox/lox/Camk2a^Cre n = 11; E; Prlr^lox/lox n = 31, Prlr^lox/lox/Kiss1^Cre n =27), gestation length (B; Prlr^lox/lox n = 11, Prlr^lox/lox/Camk2a^Cre n = 11; F; Prlr^lox/lox n = 31, Prlr^lox/lox/Kiss1^Cre n =27), number of live pups on day 3 of lactation (C; Prlr^lox/lox n = 11; Prlr^lox/lox/Camk2a^Cre n = 11; G; Prlr^lox/lox n = 31, Prlr^lox/lox/Kiss1^Cre n =27), and litter weight gain between day 3-8 of lactation (D; Prlr^lox/lox n = 8; Prlr^lox/lox/Camk2a^Cre n = 8) or day 3-20 of lactation (H; Prlr^lox/lox n = 22, Prlr^lox/lox/Kiss1^Cre n = 20). There were no differences in any of these parameters (A, C, E, G, unpaired two-tailed t test; B, F, Mann Whitney test; D, H, repeated measures mixed effect analysis, fixed effects (type III) with Šídák’s multiple comparisons test. Grey = Prlr^lox/lox; blue in top row A-D = Prlr^lox/lox/Camk2a^Cre, blue in bottom row E-H = Prlr^lox/lox/Kiss1^Cre. N numbers of Prlr^lox/lox/Kiss1^Cre mice and respective Prlr^lox/lox controls changed due to: Prlr^lox/lox (n = 5), and Prlr^lox/lox/Kiss1^Cre (n = 5) mice euthanised prior to day 20 lactation due to COVID-19 lockdown; Prlr^lox/lox/Kiss1^Cre (n = 2) showed estrus and therefore underwent blood sampling (with respective Prlr^lox/lox controls (n =2)). All these mice were euthanised 2 hours following blood sampling; a Prlr^lox/lox dams’ (n =1) litter losing weight and being excluded from subsequent analysis. Values are shown as mean ± SEM.

Miniature synchronised event (SE)-like activity on day 14 of pregnancy in Kiss1^Cre mice does not result in pulsatile LH secretion.
(A) Paired fibre photometry and blood sampling from representative mouse on day 14 of pregnancy showing miniature SE-like activity have no significant effect on pulsatile LH secretion (red).

Statistics table. Abbreviations for tables below: DF = degrees of freedom; mc = multiple comparison; CI = 95% confidence interval; MW U = Mann Whitney U; MEA = Mixed effect analysis (fixed effects (type III)), RM = repeated measures. Supp = supplementary data figure.

Statistics table. Abbreviations for tables below: DF = degrees of freedom; mc = multiple comparison; CI = 95% confidence interval; MW U = Mann Whitney U; MEA = Mixed effect analysis (fixed effects (type III)), RM = repeated measures. Supp = supplementary data figure.

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