Fig 1.
Immunohistochemical labeling of PGR and kisspeptin in neurons from the AVPV, and cells from the ovary and uterus of 2-3-month-old WT (left), Kiss1-PgrKO (middle), and global-PgrKO (right).
Black arrows indicate PGR positive nuclei, black arrowheads indicate cytoplasmic kisspeptin, open arrowheads indicate nuclei that do not express PGR. Bar in AVPV = 20 μm; bar in uterus and ovary = 25 μm.
Fig 2.
Loss of Pgr in kisspeptin neurons leads to altered cyclicity, progressively impaired fertility, decreased serum LH, and absence of corpora lutea formation.
A. Two representative estrous-cycle cycling patterns from 1.5- and 6-month-old WT and Kiss1-PgrKO mice. B. Fertility assay results for 3.5- and 5-month-old WT and Kiss1-PgrKO mice (asterisk represents p = 0.03). C. Box plot data of diestrus serum LH concentration of 2-3-month-old WT (n = 5) and Kiss1-PgrKO (n = 10) mice and 6-7-month-old WT (n = 6) and Kiss1-PgrKO (n = 4) mice; diamonds represent the mean. a represents difference between 6–7 moth old WT and Kiss1-PgrKO mice (p = 0.024). D. Representative images of 6-month-old WT and Kiss1-PgrKO ovaries (n = 5); CL–corpus luteum, AF–antral follicle, PA–preantral follicle, bar = 100 μm. Tabular data presentation for quantitative analysis of corpora lutea numbers from 2- and 6-month-old WT and Kiss1-PgrKO mice; a represents a difference between 2- and 6-months old WT mice, p = 0.024; b represents a difference between 6-month-old WT and Kiss1-PgrKO mice, p = 0.026. Data represent means ± SD.
Fig 3.
Kiss1-PgrKO mice have impaired kisspeptin-mediated GnRH release.
A. Serum LH before and after stimulation of kisspeptin neurons by exogenous administration of estradiol in 6-8-month-old ovariectomized WT and Kiss1-PgrKO mice (n = 3); a represent statistical difference between WT, and b represents statistical difference between Kiss1-PgrKO mice (p = 0.037 and p = 0.01, respectively). B. Serum LH concentration 10 min after GnRH stimulation in 5-month-old WT (n = 3) and Kiss1-PgrKO (n = 6) mice (p = 0.47). C. Gross morphology, histology, oocyte morphology, and box plot data representation of oocytes from 5-month-old WT (n = 3) and Kiss1-PgrKO (n = 6) mice after ovarian hyperstimulation with gonadotropins; grossly corpora lutea are small, round, tan, nodular structures that can be seen bulging from the ovarian surface (black arrows), and histologically corpora lutea are characterized as multiple large, round, nodular structures that expand the ovarian cortex (CL–corpora lutea). Gross image scale bar = 1 mm, histology scale bar = 100 μm, oocyte scale bar = 250 μm.
Fig 4.
PGR and kisspeptin are co-localized with ERα in kisspeptin neurons but are not expressed in AVPV kisspeptin neurons of ERαKO mice.
A. Double immunohistochemistry of PGR and kisspeptin in WT and ERαKO mice. Black arrows indicate dark brown nuclear PGR staining. Black arrowheads indicate brown red cytoplasmic kisspeptin staining. Open arrowhead indicates hematoxylin-stained nucleus of a kisspeptin positive and PGR negative neuron. Bar = 25 μm. B. Co-localization of PGR, ERα, and kisspeptin in kisspeptin neuron in the hypothalamic AVPV nucleus from a 2-month-old female C57BL/6 mouse in the estrus stage. From left to right: PGR (blue), kisspeptin (red), ERα (green), and merged image. Yellow arrows indicate nuclear PGR, ERα, and merged PGR/ERα. Green arrows indicate cytoplasmic kisspeptin. White arrows indicate ERα positive nucleus.
Fig 5.
Quantification of kisspeptin positive immunofluorescent cells in the AVPV of WT and Kiss1-PgrKO mice.
A. Immunofluorescence of kisspeptin in the AVPV of 5-month-old WT and Kiss1-PgrKO; arrows indicate kisspeptin positive cells (bar = 25 μ). B. Quantification of the raw cell number of kisspeptin positive cells from the AVPV of 2-3- and 5-month-old WT and Kiss1-PgrKO mice; the raw cell number was calculated from 3 sections per animal. Data represent means ± SD.
Fig 6.
PGR does not regulate Kiss1 gene expression.
Kiss1 mRNA expression level in 6-month-old WT (n = 9) and Kiss1-PgrKO (n = 4) mice.