Gonadotrophs have a dual origin, with most derived from early postnatal pituitary stem cells

Abstract

Gonadotrophs are the essential pituitary endocrine cells for reproduction. They produce both luteinizing (LH) and follicle-stimulating (FSH) hormones that act on the gonads to promote germ cell maturation and steroidogenesis. Their secretion is controlled by the hypothalamic gonadotrophin-releasing hormone (GnRH), and gonadal steroid feedback. Gonadotrophs first appear in the embryonic pituitary, along with other endocrine cell types, and all expand after birth. While gonadotrophs may display heterogeneity in their response to GnRH, they appear, at least transcriptionally, as a homogenous population. The pituitary also contains a population of stem cells (SCs), whose contribution to postnatal growth is unclear, in part because endocrine cells maintain the ability to proliferate. Here we show an unsuspected dual origin of the murine adult gonadotroph population, with most gonadotrophs originating from postnatal pituitary stem cells starting early postnatally and up to puberty, while embryonic gonadotrophs are maintained. We further demonstrate that postnatal gonadotroph differentiation happens independently of gonadal signals and is not affected by impairment of GnRH signalling. The division of gonadotrophs based on separate origins has implications for our understanding of the establishment and regulation of reproductive functions, both in health and in disease.

Fig. 1. Cell type-specific expansion in the postnatal pituitary.

A Percentages of endocrine cells from dissociated pituitaries stained for each endocrine hormone (GH for somatotrophs, PRL for lactotrophs, TSH for thyrotrophs, LH for gonadotrophs and POMC for both corticotrophs and melanotrophs) from P5 to one-year-old males and females (n > =3 pituitaries/age/sex, Supplementary Table S1). B Estimated numbers of endocrine cells per pituitary. Total cell numbers were independently counted (Supplementary Fig. 1, Supplementary Table 2) and percentages assessed in A used to estimate the number of cells for each endocrine population. C Evolution of each population: the percentage at each age is shown in relation to the average percentage at P5. The size of dots relates to the proportion of each population in the gland (Supplementary Table S1). D Percentage of EdU-positive cells/marker following a one-hour EdU pulse. Each dot represents one pituitary (n = 3 pituitaries/age/sex, Supplementary Table S3). Source data are provided as a Source Data file.

Fig. 2. Single cell analysis of early postnatal stem cells and their immediate progeny.

A UMAP clustering of integrated P3 male and female SOX9iresGFP^+ve datasets. B Two-sided pair-wise comparison for proportion test with Benjamini Hocberg correction for multiple testing performed to compare male and female cells. Significance is exclusively shown for clusters where distribution was different from all other clusters. This shows that the proportion of Ednrb^+ve (cleft) SCs is superior in females (p.adj = 1.53.10^-2 to 5.28.10^-60) while the proportion of differentiating gonadotrophs is higher in males (p.adj = 1.29.10^-3 to 5.83.10⁻⁵⁰, source data are provided as a Source Data file). C UMAP representation of the pseudotime gonadotroph trajectory from Ednrb^+ve SCs to gonadotrophs. Extra-pituitary cells (Pixt1^-ve, Supplementary Fig. 2) were filtered out. D Heatmap showing genes whose expression pattern correlate with the gonadotroph pseudotime trajectory. E Heatmap displaying regulons according to the gonadotroph trajectory pseudotime. F Double immunostaining for LEF1 and SOX2 from P4 to P21 in male pituitaries. At P4, LEF1 is mostly co-expressed with SOX2 (arrow), with a small number of SOX2^-ve;LEF1^+ve cells nearby (indicated by <). At P14, LEF1 remains co-expressed with SOX2. By P21, LEF1 no longer colocalises with SOX2. G, H At P4, LEF1 is co-expressed in some FOXL2^+ve (G) and POU1F1^+ve (H) progenitors. This experiment was performed at least three times at each timepoint with independent samples. Scale bars represent 30 μm in all panels.

Fig. 3. SOX2^+ve SC and LEF1^+ve progenitors give rise to most adult gonadotrophs from early postnatal stages up to puberty.

A Timeline of postnatal lineage tracing induction in Sox2^2A-rtTA/+;TetO-Cre;Rosa26^ReYFP/+ and harvest timepoints for quantification. B Immunofluorescence for lineage-traced eYFP^+ve SCs and their progeny with each pituitary hormone. SC progeny contributes to all endocrine lineages, as exemplified by eYFP co-localisation with each hormone in a 7-week-old male. C Quantification of SC contribution to each cell type in both sexes in 7-week-old mice, as measured by the percentage of eYFP;hormone double^+ve cells in the total hormone^+ve population counted in dissociated pituitaries (n > =3pituitaries /age/sex Supplementary Table 4). D Time course of postnatal SC contribution to the gonadotroph population from P5 to 1 year of age in both sexes (n > =3 pituitaries/age/sex). There is no difference between sexes in SC contribution to any cell type (P > 0.05, multiple two-tailed unpaired t-test with Benjamini-Hochberg post hoc test, Supplementary Table 5). E Immunofluorescence for lineage-traced eYFP^+ve SCs and gonadotroph markers FOXL2 and LH. SCs rapidly commit to the gonadotroph lineage postnatally, with numerous eYFP;FOXL2 double^+ve;LH^-ve cells present at P3 (top), ( < ). From P7 onwards (bottom), most eYFP;FOXL2 double^+ve cells are also LH^+ve, (arrow). F Timeline of lineage tracing induction in Lef1^CreERT2/+; Rosa26^ReYFP mice, with tissue counts performed at P12 (n = 3 pituitaries). G LEF1^+ve cell progeny contributes to all endocrine cell types, as evidenced by the co-localisation of eYFP with all hormones in a P12 male. Scale bar = 20 µm for all panels. Graphs show individual data points (each dot represents one animal) and group median. AL anterior lobe, IL intermediate lobe. Source data are provided as a Source Data file.

Fig. 4. Postnatal and embryonic gonadotrophs occupy different domains in the pituitary.

A Immunofluorescent staining for LH and eYFP showing non-lineage traced, eYFP^-ve;LH^+ve, ventral gonadotrophs at P7 (upper panel) in Sox2rtTA;eYFP pituitaries induced at birth. At P21 (lower panel), SC-derived lineage traced eYFP^+ve;LH^+ve gonadotrophs are enriched dorsally. B Quantification of stem cell-derived gonadotrophs (eYFP;LH^+ve) in ventral (gold dots) and dorsal (blue dots) pituitary regions at P7 (n = 4 pituitaries) and P21 (n = 3 pituitaries). Pituitaries were sectioned transversely, with the first 50% designated ventral and the rest dorsal. Immunofluorescence cell counts were performed to calculate the percentage of eYFP;LH^+ve /LH^+ve per region. Each dot represents one animal, and the diamond indicates the median value. Data from a minimum of N = 3 female pituitaries with at least 700 gonadotrophs counted per animal/pituitary. Similar patterns were observed in males (unpaired two-tailed t-test after angular transformation, p = 0.0204 for P7 and p = 0.0166 for P21). Source data are provided as a Source Data file. C, D Whole-mount eYFP and FSH double immunofluorescence on a Sox2rtTA;eYFP pituitary induced at birth (C) and a Foxl2CreERT2;eYFP pituitary induced in utero (D), with the induction timing indicated above. The experiment was performed on at least three independent samples, in both sexes, with a similar result obtained. The scale bars in A represents 50 μm and the scale bar in C represents 200 μm for both C and D.

Fig. 5. Regulation of neonatal SCs differentiation.

A Differentiation was induced in pituispheres from P5 (top) or 7-week-(bottom) old male pituitaries. The presence of differentiated cells within spheres was evaluated by immunofluorescence using LH or collectively GH, PRL and TSH antibodies for POU1F1 lineage cell detection. SOX2 staining confirms the presence of pituisphere forming-cells. B The proportion of spheres containing LH or POU1F1 lineages cells were compared between ages in both sexes. Multiple two-tailed unpaired-tests with Holm-Šídák post hoc test, cultures from N = 3 animals per sex and per age (for females, adj.p = 0.0002 at P5 and 0.0097 in adults; for males adj.p = 0.07 at P5 and 0.004 in adults). C Timeline of GnRH antagonist (cetrorelix) administration and lineage tracing induction (Dox) in Sox2rtTa;eYFP pups. D Immunofluorescence for eYFP and LH in pituitaries of P33 cetrorelix treated male and control mice showing no apparent differences in SC-derived gonadotroph emergence. E,F Quantification in both sexes at P33 shows no effect of cetrorelix on either the proportion of LH^+ve/DAPI^+ve cells (E) or the proportion of eYFP; LH double^+ve/LH^+ve cells (F). G Timeline of gonadectomy and lineage tracing induction (Dox) in Sox2rtTa;eYFP pups. H) Immunofluorescence for eYFP and LH in pituitaries of P21 gonadectomised male and sham-operated control showing no apparent differences in SC-derived gonadotroph emergence. I–J Quantification in both sexes at P21 shows no effect of gonadectomies on either the proportion of LH^+ve/DAPI^+ve cells (I) or eYFP; LH double^+ve/LH^+ve cells (J). K Timeline of androgen antagonist (flutamide) treatment and lineage tracing induction (Dox) in Sox2rtTa;eYFP pups. Created in BioRender. Rizzoti, K. (2025) https://BioRender.com/ uge0o92. L,M Quantification in males at P21 shows no effect of flutamide on either the proportion of LH^+ve/DAPI^+ve cells (L) or the proportion of eYFP; LH double^+ve/LH^+ve cells (M). Scale bar = 80 μm for all images. On graphs, each dot represents one animal with median values indicated. Source data are provided as a Source Data file.