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. Author manuscript; available in PMC: 2023 Apr 15.
Published in final edited form as: Mol Cell Endocrinol. 2022 Feb 2;546:111577. doi: 10.1016/j.mce.2022.111577

Deletion of the homeodomain gene Six3 from kisspeptin neurons causes subfertility in female mice

Shanna N Lavalle 1, Teresa Chou 1, Jacqueline Hernandez 1, Nay Chi P Naing 1, Michelle Y He 1, Karen J Tonsfeldt 1, Pamela L Mellon 1,*
PMCID: PMC8934285  NIHMSID: NIHMS1779602  PMID: 35121076

Abstract

The homeodomain transcription factor SIX3 is a known regulator of eye, nose, and forebrain development, and has recently been implicated in female reproduction. Germline heterozygosity of SIX3 is sufficient to cause subfertility, but the cell populations that mediate this role are unknown. The neuropeptide kisspeptin is a critical component of the reproductive axis and plays roles in sexual maturation, ovulation, and the maintenance of gonadotropin secretion. We used Cre-Lox technology to remove Six3 specifically from kisspeptin neurons in mice to test the hypothesis that SIX3 in kisspeptin neurons is required for reproduction. We found that loss of Six3 in kisspeptin neurons causes subfertility and estrous cycle irregularities in females, but no effect in males. Overall, we find that SIX3 expression in kisspeptin neurons is an important contributor to female fertility.

Keywords: Six3, Fertility, Kisspeptin, Cre/LoxP, Hypothalamus

1. Introduction

Activation and maintenance of the hypothalamic-pituitary-gonadal (HPG) axis is necessary for the transition from adolescence to adulthood and reproductive competence. Dysregulation along the HPG axis can lead to reduced or complete loss of fertility. Idiopathic hypogonadotropic hypogonadism (IHH) and its anosmic counterpart, Kallmann syndrome, are examples of reproductive disorders that lead to delayed or absent puberty. IHH is a genetic disorder characterized by insufficient production or secretion of gonadotropin-releasing hormone (GnRH), leading to reduced sex steroid production and hypogonadism (Au et al., 2011; Bianco and Kaiser, 2009; Topaloglu, 2017). In humans, approximately 5% of normosmic IHH cases are attributed to genetic disruptions of kisspeptin signaling (Bianco and Kaiser, 2009).

The neuropeptide kisspeptin has established roles in puberty, ovulation, and maintenance of pulsatile GnRH and resulting luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion (Gottsch et al., 2004; Kauffman, 2010; Messager et al., 2005). Kiss1 is differentially regulated in the arcuate nucleus (ARC) and the anteroventral periventricular nucleus (AVPV), leading to differing roles in reproduction (Smith et al., 2005a, 2005b). ARC kisspeptin neurons are considered important for the pulse generator and mediate sex steroid negative feedback, leading to reduced Kiss1 expression in the presence of androgens and estrogens (Herbison, 2018; McQuillan et al., 2019; Smith et al., 2005b). AVPV kisspeptin neurons are sexually dimorphic and primarily occur in females, where they mediate the circadian-gated, estradiol (E2)-induced LH surge and induce Kiss1 expression in the presence of E2 (Dror et al., 2013; Dungan et al., 2007; Smith et al., 2005a, 2006).

Recent studies in mice have implicated homeodomain transcription factors, such as VAX1 and SIX3, as potential IHH gene candidates (Hoffmann et al., 2014, 2019; Pandolfi et al., 2018). In mice, deletion of Vax1 from GnRH neurons results in infertility (Hoffmann et al., 2016), whereas deletion of Vax1 from kisspeptin neurons alters Kiss1 gene expression but does not impact fertility (Lavalle et al., 2021). Six3, which plays a critical role in eye, nose, and forebrain development (Conte et al., 2005; Lagutin et al., 2003), is also critical to reproduction in mice. The loss of a single allele results in strong reproductive defects, many of which recapitulate those seen in patients with IHH and Kallmann syndrome, including reduced GnRH neurons in the hypothalamus, altered estrous cyclicity, subfertility, and anosmia (Pandolfi et al., 2018). However, heterozygous Six3+/− mice maintain approximately 55% of GnRH neurons in the hypothalamus during adulthood, which is sufficient to maintain reproductive function in female mice (Herbison et al., 2008). Furthermore, conditional deletion of Six3 specifically from GnRH neurons did not recapitulate any of the phenotypes seen in the heterozygous Six3+/− mice, and there were no impairments to fertility, suggesting that SIX3 in reproduction occurs in cell populations outside of GnRH neurons (Pandolfi et al., 2018).

In our current study, we tested the hypothesis that SIX3 in kisspeptin neurons is necessary for fertility by specifically deleting Six3 from Kiss1-expressing cells. We found that SIX3 within kisspeptin neurons is necessary for female fertility, but not male, and may play a role in the timing of LH release in the presence of E2.

2. Materials and methods

2.1. Animals

All animal procedures were performed in accordance with the University of California San Diego, Institutional Animal Care and Use Committee regulations. To delete Six3 from Kiss1-expressing cells, we crossed Six3flox/flox mice (RRID:MGI:3693848) (Liu et al., 2006) with Kiss1Cre mice (RRID:IMSR_JAX:023,426) (Cravo et al., 2011) to generate Six3Flox/Flox:Kiss1Cre mice (Six3KissCre) and Six3flox/flox:Kiss1WT mice (Six3flox/flox). We used Ai14 Rosa-tdTomato mice (RRID: IMSR_JAX:007914) (Madisen et al., 2010) to create a Kiss1 reporter line by crossing them to Kiss1Cre mice to generate Kiss1Cre:tdTomato mice. All mice were on a mixed NMRI and C57/Bl6 background. Mice were group housed and maintained on a 12-h light, 12-h dark cycle with ad libitum chow and water.

Genotyping of the mice was performed by PCR with genomic DNA collected from tail tips. Six3 wildtype (WT) and Six3 flox alleles were detected using Six3-For: 5′-CCCCTAGCCTAACCCAAACAT-3′ and Six3-Rev: 5′-CGGCCCATGTACAACGCGTAT-3′ primers. Germline recombination was detected using Six3-For and Six3-Rec-Rev: 5′-TTCCCCTCTTTGACTCCTATG-3′ primers. Cre-For: 5′-GCATTACCGGTCGTAGCAACGAGTG-3′ and Cre-Rev: 5′-GAACGCTAGAGCCTGTTTTGCACGTTC-3′ primers were used to detect the Cre allele. tdTomato was detected using tdtF1: 5′-GGCATTAAAGCAGCGTATCC-3′, tdtR1 5′-CTGTTCCTGTACGGCATGG-3, tdtF2: 5′-CCGAAAATCTGTGGGAAGTC-3′, and tdtR2: 5′-AAGGGAGCTGCAGTGGAGTA-3. Mice that were positive for Six3 germline recombination in tail samples or exceeded 60 g in body weight were removed from the study.

2.2. Fluorescent in-situ hybridization

20 μm serial coronal sections were collected from fresh frozen brains, spanning the length of the AVPV, ARC, and reticular thalamic nucleus (Di Giorgio et al., 2013; Sunnen et al., 2014; Tonsfeldt et al., 2019). AVPV sections were collected from ovariectomized (OVX)+E2 treated females. ARC and reticular thalamic nucleus sections were collected from OVX females. Sections were mounted on Superfrost Plus Microscope Slides (Fisher Scientific) and stored at −80 °C until further processing. Slides were fixed in chilled 4% PFA, washed two times with 1X PBS, and dehydrated in ethanol. Probes to detect Kiss1 (500,141), cFos (316,921-C2), and Six3 (855,211-C3) were used with the highly sensitive RNAscope Multiplex Fluorescent v2 Assay (Advanced Cell Diagnostic, 323,100), according to manufacturer’s instructions. Sections were counterstained with DAPI and coverslipped with ProLong Gold (Invitrogen). All AVPV slides were processed in a single assay and all ARC and reticular thalamic nucleus slides were processed in a single assay.

2.3. Microscopy and analysis

Fluorescent microscopy was performed at the Nikon Imaging Core (UCSD) using a Nikon Eclipse Ti2-E microscope with Plan Apo objectives. Samples were excited by the Lumencor SpectraX and acquired with a DS-Qi2 CMOS camera using NIS-Elements software, or with a laser scanning confocal (A1R HD, Nikon), acquired with an iXon Ultra 897 EMCCD camera (Andor). All AVPV slides were imaged at the same time and under the same conditions. All ARC and thalamic reticular nucleus slides were imaged at the same time and under the same conditions. The number of Kiss1 cells that colocalized with Six3 or atypical signal was determined manually, using FIJI Cell Counter tool. NIS-Elements: General Analysis software was used to objectively quantify the intensity of Kiss1, cFos, and Six3 signals. A signal threshold of 2.5 standard deviations above background was used for defining positive signal of each gene. cFos and Six3 signals were quantified only in Kiss1-positive cells.

2.4. Fertility and plugging assessment

To assess fertility in females, 15–18-week-old virgin Six3flox/flox or Six3KissCre females were paired with a virgin wildtype male for 90 days. The number of litters and the number of pups in each litter were recorded. Fertility of the wildtype males of the infertile pairs were not further assessed due to the age at disbanding. To assess male mating behavior and fertility, 10–16-week-old virgin Six3flox/flox or Six3KissCre males were paired with a wildtype female for 60 days. For the first 10 days, females were checked for the presence of plugs. The number of pups produced from each litter were recorded.

2.5. Ovarian histology

Following the fertility assay, female mice were separated from males and provided time to birth their final litters, then ovaries were collected during diestrus. Upon dissection, ovaries were placed in a solution of 60% EtOH, 30% formaldehyde, and 10% glacial acetic acid. Fixed ovaries were paraffin embedded, serial sectioned at 12 μm on a microtome, and stained with hematoxylin and eosin (H&E; Sigma-Aldrich). The number of Graafian follicles, identified by the presence of large antral space surrounding the oocyte, and the number of corpora lutea within a single ovary was quantified independently by two individuals, blinded to genotype. Every fifth section was scored to ensure follicles and corpora lutea were not double counted.

2.6. Pubertal onset and estrous cyclicity

After weaning, mice were checked daily for vaginal opening in females and preputial separation in males to determine pubertal onset (Hoffmann, 2018). In females, vaginal smears were taken daily from pubertal onset until first estrus or when they reached 90 days of age. To assess estrous cyclicity, vaginal smears were taken for 16 consecutive days from 12-week-old females. Vaginal smears were stained with 0.1% methylene blue, and a single observer, blinded to genotype, determined stage of cell cycle based on the composition of cell types present (Byers et al., 2012).

2.7. Hormone analysis

For diestrus-staged female mice, blood was collected from tail vein via capillary tube (Drummond microcaps 40 μL), sealed with Critoseal, allowed to clot at room temperature for 1 h, centrifuged at 2000×g for 15 min, and then serum was collected and stored at −20 °C until assayed on Luminex Magpix. Serum LH and FSH were measured by Luminex assay according to manufacturer’s instructions (LH: lower detection limit, 4.92 pg/mL; intra-assay CV, <14.10%; inter-assay CV 7.28%; FSH: lower detection limit, 9.5 pg/mL; intra-assay CV, 12.13% inter-assay CV 7.22%). For OVX + E2-treated females and intact males, blood samples were collected at time of euthanasia, allowed to clot at room temperature for 1 h, centrifuged at 2000×g for 15 min, and then serum was collected and stored at −20 °C until assayed for LH and FSH by The University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core. LH was measured using LH RIA with a reportable range between 0.02 and 75.0 ng/mL (intra-assay CV = 5.5%, inter-assay CV = 8.4%). FSH was measured using FSH RIA with a reportable range between 3.0 and 75.0 ng/mL (intra-assay CV = 7.5%, inter-assay CV = 10.1%).

2.8. Luteinizing hormone (LH) surge

12–16-week-old virgin female mice weighing between 18 and 28 g were ovariectomized between zeitgeiber time (ZT) 2–5. A pellet containing 0.75 μg of 17-β estradiol dissolved in sesame oil was implanted subcutaneously to mimic proestrus levels of E2 (Dror et al., 2013). Two days after surgery, mice were sacrificed either in the morning (AM), between ZT 4–5, or at the time of lights off (PM), between ZT 12–13. Serum and brains were collected at sacrifice. An LH surge was conservatively defined as LH values that were three standard deviations above the AM average (Dungan et al., 2007). AM values plus 3 standard deviations amounted to: 0.65 for Six3Flox/Flox, 0.90 for Six3KissCre, and 0.92 when values for Six3Flox/Flox and Six3KissCre were combined. We used the most stringent value of 0.92 for defining a surge in all mice.

2.9. Cell culture

KTaR-1 (RRID:CVCL_VS93) and KTaV-3 (RRID:CVCL_VS94) immortalized kisspeptin cell lines (Jacobs et al., 2016) were kindly provided by Dr. Patrick Chappell (Oregon State University). Cells were maintained in complete media consisting of DMEM (Corning) with 10% fetal bovine serum (FBS) (Omega Scientific) and 1% penicillin-streptomycin (HyClone) and incubated at 37°C with 5% CO2.

2.10. Plasmids

The −1313/+26 human KISS1-pGL2-luciferase plasmid (hKiss-Luc) (Mueller et al., 2011) was kindly provided by Alejandro Lomniczi and Sergio Ojeda. The −4058/+455 mouse Kiss1-pGL4-luciferase plasmid (Atkin et al., 2013) was kindly provided by Dr. Steven Kliewer (UT Southwestern). We subcloned the −4058/+455 mouse Kiss1 promoter sequence from the pGL4 backbone into a pGL2 backbone using the KpnI-HF, XhoI, and SalI-HF restriction enzymes in 10X Cut Smart Buffer (New England BioLabs) and religated using Quick Ligase Kit (New England BioLabs). This resulted in the −4058/+455 mouse Kiss1-Luciferase-pGL2 plasmid (mKiss-Luc). The murine −1 kb cFos-pGL3-Luciferase (cFos-Luc) (Ely et al., 2011) and the cFos-Luc with cis-mutations at −59 (μ−59), −313 (μ−313), and at both −59 and −313 (μ−313x-59) bp were previously described (Hoffmann et al., 2018). The reporter plasmid containing β-galactosidase constitutively driven by the Herpes virus thymidine kinase promoter (TK-βgal) was used to control for transfection efficiency. We obtained the SIX3-pSG5 expression plasmid from Origene Technologies (Rockville, MD) and the murine cFOS-CMV expression plasmid (cFOS) was previously described (Glidewell-Kenney et al., 2013). Sequences of all plasmids were confirmed by Sanger Sequencing (Eton Bioscience).

2.11. Transient transfections and luciferase assays

One day prior to transient transfections, KTaR-1 or KTaV-3 cells were seeded at 30 × 104 cells per well in 12-well plates with complete media and allowed to grow overnight. Transient transfections were performed using Polyjet In Vitro DNA Transfection Reagent (SignaGen Laboratories), following manufacturer’s instructions. Cells were transfected with 500 ng of reporter plasmid (hKiss-Luc, mKiss-Luc, cFos-Luc, μ−313, μ−59, μ−313x-59) or backbone (pGL2 or pGL3), and co-transfected with 100 ng of TK-βgal and 20 ng SIX3 expression vector or pSG5-empty vector (EV) or 50 ng cFOS expression vector or CMV-empty vector (EV). Polyjet/DNA was removed 24 h after transfection treatment and replaced with complete medium. 48 h from start of transfection, cells were harvested for luciferase assay. To harvest cells, medium was aspirated from wells, cells were washed with 1X PBS, and then lysed with 0.1 M K-phosphate buffer, pH 7.8, containing 0.2% Triton X-100. Luciferase and β-galactosidase assays were performed as previously described. All conditions were performed in triplicate. Within each well, luciferase values were normalized to TK-βgal values, then triplicate luciferase/TK-βgal values were averaged. hKiss-Luc and mKiss-Luc values were normalized to pGL2 and cFos-Luc, μ−313, μ−59, and μ−313x-59 values were normalized to pGL3.

2.12. Statistical analysis

Differences between groups were detected by Student’s t-test, Welch’s t-test, one-way ANOVA, or two-way ANOVA using Prism 9 (Graphpad). For one-way and two-way ANOVA, significant effects were followed by Tukey’s Honest Significant Difference test. Residuals were checked for normality using Shapiro Wilk test (p < 0.05). When needed, data were log transformed and reanalyzed, where indicated.

3. Results

3.1. Loss of SIX3 from Kiss1-expressing cells reduces fecundity in female mice

To test the hypothesis that SIX3 within kisspeptin neurons regulates reproduction, we specifically deleted Six3 from Kiss1 cells by crossing Six3Flox/Flox mice to Kiss1Cre mice to generate Six3Flox/Flox:Kiss1Cre mice (Six3KissCre) and Six3Flox/Flox:Kiss1WT mice (Six3F/F). Exon 1 of the Six3 allele, which contains both the Six domain and the homeodomain, is flanked by LoxP sites, allowing this region to be excised from Kiss1 cells in Six3KissCre mice and remain intact in Six3Flox/Flox mice (Fig. 1A). We used fluorescent in situ hybridization to assess knockdown efficiency in the AVPV of Six3Flox/Flox and Six3KissCre mice using a Six3 probe that spans the 5’ UTR and Exon 1 of the Six3 gene (Fig. 1A and B). We found that 46.7% of AVPV kisspeptin neurons colocalized with Six3 in Six3Flox/Flox mice compared to 6.7% in Six3KissCre mice (Fig. 1C). We also found that Six3 is expressed in 20.7% of Kiss1-positive cells in the ARC of Six3Flox/Flox mice and is reduced to 2.8% in Six3KissCre mice (Fig. 1D and E). We observed distinct punctate Six3 signal in both Kiss1-negative and Kiss1-positive cells in Six3Flox/Flox mice (Fig. 1D and E). In Six3KissCre mice, 50.6% of AVPV Kiss1 cells and 43.5% of ARC Kiss1 cells displayed atypical, large, and circular fluorescent signal, potentially an artifact left from the excision by Kiss1-Cre (Fig. 1B, D). Six3KissCre mice maintained the clear punctate signal in Kiss1-negative cells in the AVPV, ARC, and reticular thalamic nucleus, a region reported to have high Six3 expression levels (Conte et al., 2005; Sunnen et al., 2014), demonstrating nearby cell populations retain intact Six3 and that the atypical signal is not representative of high Six3 expression (Fig. 1B, D, F). We did observe atypical signal in a subset of cells that did not express Kiss1 mRNA in the AVPV and ARC of Six3KissCre mice. This may be due to cells that previously expressed Kiss1 but were no longer expressing Kiss1 mRNA, as there are more cells labeled with tdTomato in a Kiss1-tdTomato reporter mouse than detectable Kiss1 mRNA in the AVPV of an OVX + E2 treated Six3Flox/Flox mouse (Fig. 1G), because tdTomato reporter records cells that have expressed Kiss-cre at any time throughout development or because Kiss1 levels are below detection in some of the cells while the tdTomato signal is more robust and not regulated by steroid hormones.

Fig. 1.

Fig. 1.

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Six3 is expressed in AVPV and ARC kisspeptin neurons in mice. A) Schematic of the Six3 gene in kisspeptin cells in Six3Flox/Flox (Six3F/F) and Six3KissCre mice. Triangles represent LoxP sites. Exon 1 (E1) and exon 2 (E2) are represented by the larger black rectangles. Dotted line indicates the region targeted by the Six3 probe. Representative images of fluorescent in situ hybridization to detect Kiss1 (green) and Six3 (magenta) in the B) AVPV of OVX + E2 treated and D) ARC of OVX females. Sections were counterstained with DAPI (blue) to visual nuclei. White arrowheads depict examples of Six3 and Kiss1 colocalization, yellow arrowheads depict Six3 expression in Kiss1-negative cells, and white arrows depict atypical Six3 signal in Kiss1-positive cells. Quantification of the percent of Six3 colocalized with Kiss1 in the C) AVPV and E) ARC. Data were analyzed by Student’s t-test. Significance indicated by ***p < 0.0005, ****p < 0.0001. N = 3–4. F) Fluorescent images of Six3 (magenta) in situ hybridization in the reticular thalamic nucleus of Six3Flox/Flox and Six3KissCre OVX females. G) Fluorescent images of tdTomato (red) immunostaining and Kiss1 (green) in situ hybridization in the AVPV of intact Kiss1Cre:tdTomato and OVX + E2 Six3Flox/Flox females, respectively.

To assess the role of kisspeptin cell specific SIX3 in reproduction, we paired Six3Flox/Flox and Six3KissCre females with a wildtype male for 90 days. We found that there were no significant differences in the number of days to produce their first litter (Fig. 2A), or in the total number of litters produced. However, a subset of Six3KissCre mice did not produce a single litter within the 90 days (Fig. 2A and B). We also examined litter size and found that the fertile Six3KissCre females had significantly fewer pups per litter compared to Six3Flox/Flox females (Fig. 2C). To determine if the reduced fecundity was due to an ovulatory defect, we assessed ovarian morphology (Fig. 2D). Six3KissCre females had a significant reduction in the number of Graafian follicles compared to Six3Flox/Flox females (Fig. 2E), and, while there appeared to be a reduction in the number of corpora lutea in Six3KissCre mice (Fig. 2F), the difference was not significant. In contrast to females, male Six3KissCre mice had no impairments to fertility as they were able to plug wildtype female mice in a similar time frame as Six3Flox/Flox males (Fig. 2G) and produced a similar number of pups per litter (Fig. 2H). Because the loss of Six3 from kisspeptin neurons had no effect on male fertility, we focused the remainder of the study on female Six3KissCre mice.

Fig. 2.

Fig. 2.

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Six3 within kisspeptin cells is necessary for female fecundity. A) Number of days between pairing and first litter in Six3F/F and Six3KissCre female mice. Dotted line indicates a 90-day cutoff. B) Number of litters produced in 90 days by female mice. C) Average number of pups born per litter in female mice. D) Representative images of ovaries from Six3F/F and Six3KissCre females collected after fertility assay. Arrows depict examples of Graafian follicles, CL depicts examples of corpora lutea. E) Quantification of the number of Graafian follicles per ovary. F) Quantification of the number of corpora lutea per ovary. G) Number of days for Six3F/F and Six3KissCre males to plug wildtype females. H) Average number of pups produced per litter by wildtype females when paired with male Six3F/F or Six3KissCre mice. All data were analyzed by Student’s t-test or Welch’s t-test. N = 5–10. Significance indicated by * p < 0.05, **p < 0.005. Purple squares represent the infertile Six3KissCre female mice.

3.2. Loss of SIX3 in kisspeptin cells disrupts female estrous cycles

We next determined whether sexual maturation was affected by the loss of SIX3 from kisspeptin neurons in female mice. We found that the loss of Six3 did not alter the time to pubertal onset, as indicated by vaginal opening (Fig. 3A) or the weight at vaginal opening (Fig. 3B). The time to reach first estrus, another marker of sexual maturation, was also unaffected (Fig. 3C). Female mice have a four to five day estrous cycle in which they transition from diestrus to proestrus and then estrus (Byers et al., 2012), as shown in representative images of our Six3Flox/Flox females (Fig. 3D). Six3KissCre females, however, had impaired estrous cycles (Fig. 3E). Six3KissCre females spent significantly more time in diestrus and a significantly less time in estrus (Fig. 3F) and took approximately twice as long to complete a full cycle (diestrus to diestrus; Fig. 3G) compared to Six3Flox/Flox females.

Fig. 3.

Fig. 3.

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Loss of Six3 from kisspeptin neurons disrupts estrous cyclicity. A) Time to pubertal onset determined by vaginal opening. N = 13–26. B) Weight of mice the day of vaginal opening. N = 13–26. C) Time to first estrus stage. N = 11–22. Representative cycles from: D) two Six3F/F females and E) two Six3KissCre females. D indicates diestrus, P indicates proestrus, and E indicates estrus. F) Percent of time spent in each stage of the estrous cycle. White circles represent Six3F/F mice and grey squares represent Six3KissCre mice. N = 8–9. G) Average time to complete one estrous cycle, measured from diestrus to diestrus. N = 8–9. Data were analyzed by two-way ANOVA or Student’s t-test. Significance is indicated by: *p < 0.05, **p < 0.005. Purple squares represent the infertile Six3KissCre female mice.

3.3. Six3KissCre females have elevated LH levels during the morning of induced LH surge

Circulating FSH and LH were similar between diestrus Six3Flox/Flox and Six3KissCre females when collected between ZT 4–6 (Fig. 4A and B). Because some of the Six3KissCre mice did not produce any litters during the fertility assay, we performed an induced LH surge paradigm (OVX + E2) and found that 7 of 9 Six3KissCre females were able to induce an LH surge, as well as 5 of 5 Six3Flox/Flox mice (Fig. 4C). Interestingly, AM Six3KissCre females had significantly elevated levels of LH compared to AM Six3Flox/Flox mice (Fig. 4C); however, 7 of 9 Six3KissCre mice still had PM LH levels more than three times the standard deviation of the AM Six3KissCre levels.

Fig. 4.

Fig. 4.

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Serum gonadotropins in female mice. Circulating serum levels of A) FSH and B) LH in diestrus-staged female mice collected between ZT 4–6. Data were analyzed by Student’s t-test. N = 11–13. C) Serum LH levels in OVX + E2 treated mice collected at ZT 4–5 (AM) or ZT 12–13 (PM). Dotted line indicates surge threshold (combined AM average + 3 SD = 0.9 ng/mL). Data were log transformed prior to analysis by two-way ANOVA. N = 5–7. Significance of p < 0.05 indicated by different letters.

To understand what might be driving the elevated AM LH levels in Six3KissCre mice, we collected brains from both AM and PM Six3Flox/Flox and Six3KissCre OVX + E2-treated females and visualized AVPV Kiss1 and cFos expression by in situ hybridization (Fig. 5A). The intensity of Kiss1 was significantly higher in the PM groups compared to the AM groups (Two-way ANOVA, p < 0.0001). The loss of SIX3 from Six3KissCre mice did not alter the number of Kiss1-expressing cells (Fig. 5B) or the intensity of the Kiss1 signal in the AVPV of AM or PM females (Fig. 5C). There were no differences in ARC Kiss1 intensity between Six3Flox/Flox and Six3KissCre OVX + E2-treated females at either the AM or PM time points (AM Six3Flox/Flox = 0.235 ± 0.054, AM Six3KissCre = 0.192 ± 0.049, PM Six3Flox/Flox = 0.145 ± 0.066, PM Six3KissCre = 0.135 ± 0.012, N = 3–4). The percent of Kiss1 cells colocalized with cFos was significantly higher in the PM group compared to the AM group (Two-way ANOVA, p < 0.05) and there was a significant reduction in the percent colocalization in AM Six3KissCre mice compared to AM Six3Flox/Flox mice (Fig. 5D). Consistent with our LH findings, the intensity of cFos was significantly increased in AM Six3KissCre mice compared to AM Six3Flox/Flox mice; however, no difference was observed in cFos intensity between PM Six3Flox/Flox and PM Six3KissCre mice (Fig. 5E).

Fig. 5.

Fig. 5.

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The loss of SIX3 from kisspeptin neurons reduces the percent of Kiss1 cells colocalized with cFos. A) Representative images of fluorescent in situ hybridization to detect Kiss1 (green), cFos (magenta) in the AVPV of OVX + E2 treated females collected at ZT 4–5 (AM) or ZT 12–13 (PM). Kiss1 and cFos colocalization are visualized in white. Arrows depict examples of Kiss1-cFos colocalization. Sections were counterstained with DAPI (blue) for visualization of the nuclei. B) The number of Kiss1 expressing cells. C) Mean intensity of Kiss1 in arbitrary units (A.U.). D) Percent of Kiss1-cells colocalized with cFos. E) Mean intensity of cFos within Kiss1-positive cells. Student’s t-test between indicated pairs. N = 3–4. Significance indicated by * p < 0.05 and **p < 0.05.

3.4. Six3 expression in AVPV kisspeptin neurons is regulated by time of day

Because we only observed changes in cFos colocalization and expression in OVX + E2-treated mice in the AM, we investigated whether Six3 expression levels were affected by time of day. We used fluorescent in situ hybridization to assess Six3 expression patterns in the AVPV of Six3Flox/Flox OVX + E2-treated females (Fig. 6A). We found that there was no significant difference in the percentage of Kiss1 cells that colocalized with Six3 between the AM and PM timepoints (Fig. 6B). However, we did observe that Six3 intensity was significantly lower in the evening (PM), when the LH surge is expected to occur, than in the morning (AM) (Fig. 6C), indicating that Six3 expression in AVPV kisspeptin neurons is regulated by time of day.

Fig. 6. Six3 intensity is regulated by time of day in OVX + E2-treated Six3 WT mice.

Fig. 6.

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A) Representative images of fluorescent in situ hybridization to detect Kiss1 (green) and Six3 (magenta) in the AVPV of OVX + E2 treated females collected at ZT 4–5 (AM) or ZT 12–13 (PM). Sections were counterstained with DAPI (blue) to visual nuclei. White triangles depict examples of Kiss1 and Six3 colocalization. B) Quantification of the percent of Kiss1 cells colocalized with Six3. (C) Quantification of Six3 mean intensity within Kiss1-positive cells. Data were analyzed by student’s t-test. N = 3. Significance indicated by **p < 0.05.

3.5. SIX3 represses cFos-Luc transcription in vitro

While the loss of SIX3 from AVPV kisspeptin cells did not alter Kiss1 mRNA in vivo, we next tested whether overexpression of SIX3 could modulate Kiss1 transcription in vitro. We used immortalized kisspeptin cell lines derived from the AVPV (KTaV-3) or ARC (KTaR-1) of a female mouse hypothalamus (Jacobs et al., 2016) and cotransfected a human Kiss1 luciferase (hKiss-Luc) or mouse Kiss1 luciferase (mKiss-Luc) reporter plasmid with a SIX3 expression vector. We found that overexpression of SIX3 significantly repressed hKiss-Luc transcription in both KTaV-3 (Fig. 7A) and KTaR-1 (Fig. 7B) cells but did not alter mKiss-Luc transcription in either cell line (Fig. 7C and D). Because we observed altered cFos expression in AVPV kisspeptin cells in the morning of an induced LH surge, we determined whether SIX3 could regulate transcription of cFos, which has one complete ATTA and a partial (ATTxA) SIX3 binding site located −313 and −59 base pairs upstream of the proximal promoter, respectively (Fig. 6E). Using a murine −1 Kb cFos-luciferase (cFos-Luc), we demonstrate that SIX3 can act as a repressor of cFos-Luc transcription in KTaV-3 cells (Fig. 7F). We then tested whether the repression by SIX3 was being mediated through either of the 2 sites. We found that loss of a single ATTA or ATTxA site was not sufficient to prevent SIX3 induced repression of the cFos promoters. However, SIX3 could no longer repress cFos-Luc transcription when both sites were mutated (Fig. 7G). We were also interested in whether cFOS could act as a transcription factor to regulate the Kiss1 promoter which contains an AP-1 consensus sequence, a potential binding site for cFOS. We found that overexpression of cFOS significantly induced mKiss-Luc transcription (Fig. 7H).

Fig. 7. SIX3 represses cFos-Luc transcription in vitro.

Fig. 7.

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hKiss-Luc was cotransfected with 20 ng SIX3 or empty vector (EV) in A) KTaV-3 and B) KTaR-1 cells. mKiss-Luc was co-transfected with 20 ng SIX3 or EV in C) KTaV-3 and D) KTaR-1 cells. E) Schematic of SIX3 binding sites on the murine −1 Kb cFos promoter. F) cFos-Luc was co-transfected into KTaV-3 cells with 20 ng SIX3 or EV. G) cFos-Luc or cFos-luc reporters containing cis-mutations at −313 (μ −313), −59 (μ −59), or both (μ −313 and −59) base pairs were co-transfected with 20 ng SIX3 or EV. Data are represented as fold change of SIX3/EV. H) mKiss-luc was co-transfected into KTaV-3 cells with 50 ng cFOS or EV. For all experiments, bars represent means ± SEM, N = 3–5. Data were analyzed using Student’s t-test or One-way ANOVA. Significance indicated by * p < 0.05, **p < 0.005, ***p < 0.0005, or different letters p < 0.05.

4. Discussion

Heterozygous loss of the homeodomain transcription factor, Six3, has previously been shown to cause subfertility in mice (Hoffmann et al., 2019; Pandolfi et al., 2018); while Six3 is expressed in several areas of the adult brain and pituitary, the specific cell types that mediate this effect are unknown. Six3 heterozygous mice have functional pituitaries, normal ovarian and testicular morphology, and the homozygous loss of Six3 from GnRH neurons produced no reproductive impairments (Pandolfi et al., 2018). We therefore decided to examine the role of Six3 in kisspeptin neurons, which act upstream of GnRH neurons. We found that the loss of Six3 in kisspeptin neurons shared some features of subfertility and disrupted estrous cyclicity seen in the Six3 heterozygous mice, indicating that the heterozygous phenotype is mediated at least in part by loss of Six3 in kisspeptin neurons.

We confirm that Six3 is expressed in approximately half of AVPV and 20% of ARC kisspeptin neurons in adult Six3Flox/Flox mice, and that kisspeptin-specific Six3 expression is disrupted in Six3KissCre mice, leading to reduced fertility. Although we observed an atypical fluorescent signal in Kiss1-positive cells in Six3KissCre mice, we do not believe this signal represents functional Six3 mRNA that is being translated to SIX3 protein. If a truncated protein were made containing exon 2, it would not contain the functional Six domain or homeodomain and would not be detected by the Six3 probe that targets exon 1. It is likely an artifact produced by the recombination as we do not see this atypical signal in regions that express Six3 but do not express Kiss1-Cre, such as the reticular thalamic nucleus. Conditional deletion of Six3 using the Six3-flox mouse has been used by others to demonstrate developmental, reproductive, and circadian phenotypes in mice (Hoffmann et al., 2021; Liu et al., 2006; Pandolfi et al., 2018). The Kiss1Cre mouse is well characterized and has been used by many in the field to efficiently delete genes specifically in kisspeptin neurons (Cravo et al., 2011; Ho et al., 2021; Stephens et al., 2015). If Six3 remained functional, we would not see the reproductive phenotypes that were observed in the Six3KissCre mice.

We found that the loss of Six3 from kisspeptin neurons resulted in a subset of Six3KissCre females being unable to produce a single litter within a 90-day fertility assay. Furthermore, the Six3KissCre females that were able to produce litters had reduced fecundity compared to Six3Flox/Flox females, which was a more robust reproductive effect compared to Six3 heterozygous females who had a delay to first litter, but normal fecundity (Pandolfi et al., 2018). The decreased fecundity is likely due to an impairment in follicular maturation or survival as there were significantly fewer Graafian follicles in Six3KissCre mice. There was no correlation between the number of Graafian follicles or corpora lutea in the infertile Six3KissCre mice compared to the subfertile Six3KissCre mice, suggesting the source of complete infertility is not driven by alterations in follicular development. While there is some Kiss1-Cre expression in the ovary (Ho et al., 2021), Six3 is not detected in the adult ovary (Hsu et al., 2014), so we speculate that the reduction in Graafian follicles is driven by the loss of SIX3 in kisspeptin neurons. These phenotypes may be caused by disruption in sex steroid feedback, which is mediated by kisspeptin neurons and was disrupted on the morning of proestrus (Smith, 2008, 2013).

We also found that the increased cycle length in Six3KissCre females was comparable to Six3 heterozygous females, suggesting that SIX3 in kisspeptin neurons underlies this phenotype (Pandolfi et al., 2018). The infertile Six3KissCre mice had more severe cycling impairments compared to the other Six3KissCre mice, which may contribute to their inability to produce litters. Future studies of gonadotropin release would be needed to determine a more precise mechanism.

Successful ovulation is prompted by the circadian-gated LH surge that occurs on the evening of proestrus (de la Iglesia and Schwartz, 2006; Robertson et al., 2009). We found that Six3KissCre females were able to generate an induced LH surge and that the loss of SIX3 did not alter Kiss1 levels, cFos levels, or Kiss1-cFos colocalization at the time of the LH surge. Therefore, these mice can respond appropriately to the cues needed to induce ovulation in the presence of exogenous estrogen. Surprisingly, Six3KissCre females had elevated LH serum levels in the morning of an E2-induced LH surge, when AVPV kisspeptin neuron activity and LH levels are expected to remain low (Marraudino et al., 2017; Wang et al., 2016). This finding suggests an impairment in negative feedback, which is regulated by ARC kisspeptin neurons. However, there were no detectable differences in ARC Kiss1 mRNA on the morning of the induced LH surge, and diestrus female Six3KissCre mice had normal circulating LH and FSH levels.

We next explored whether the cause of the elevated morning LH could be increased or mis-timed kisspeptin secretion from AVPV kisspeptin neurons. We found that AVPV Kiss1 gene expression during the morning of the induced LH surge was not higher in Six3KissCre mice. We observed a modest reduction in the percent of Kiss1 cells colocalized with cFos in AM Six3KissCre mice, but almost a doubling of cFos expression, leading us to postulate that SIX3 could indirectly mediate kisspeptin secretion through repression of cFos (Bullitt, 1990; Hoffman et al., 1993; Kovacs, 2008). In addition to being a marker of neuronal activity and immediate early gene (IEG), cFOS is a pleiotropic transcription factor that can form heterodimers with other IEGs to form the AP-1 transcription factor complex, which can then regulate gene expression (Coss et al., 2004; Karin et al., 1997). The murine cFos proximal promoter contains both a complete (−313 bp) and partial (−59 bp) SIX3 consensus sequence (Hoffmann et al., 2018) and we show here that SIX3 can act through either of these sites to repress cFos-Luc transcription in AVPV kisspeptin neurons in vitro. Other studies have indicated a role of cFOS as a potential regulator Kiss1 gene expression, as cFOS-null mice have an 80% reduction in AVPV Kiss1 mRNA per cell (Xie et al., 2015); however, a direct relationship between the two has not been established. We demonstrate that cFOS induces the mKiss1 promoter and can be suppressed by SIX3 overexpression in vitro. We postulate that, in our model, the absence of SIX3 from the kisspeptin cells in the Six3KissCre mice drives the increase in cFos observed in the Kiss1 neurons in the AM of the induced surge. While we do not observe a corresponding increase in Kiss1 in vivo, only 30% of Kiss1 cells expressed cFos in the AM, which could make it difficult to detect cFos-induced changes in total Kiss1 mRNA levels.

We found that SIX3 differentially regulates the human and mouse kisspeptin promoters in vitro, by acting as a repressor of hKiss-Luc transcription and having no effect on mKiss-Luc transcription in either ARC or AVPV kisspeptin cell lines. SIX3 is highly conserved with the amino acid sequence being identical between mouse and human (Kawakami et al., 2000), suggesting that the contrast is due to differences in the human and mouse promoters. SIX3 regulates transcription through binding of ATTA and ATTA-like motifs (Manavathi et al., 2007; Zhu et al., 2002). While both the human and mouse KISS1/Kiss1 promoters contain several ATTA sites, the positions of the motifs are not conserved (http://genome.ucsc.edu/) (Kent et al., 2002).

Alternatively, the effects of SIX3 on cFos expression may be unrelated to increased LH levels, and SIX3 is instead directly regulating neuronal excitability. AVPV kisspeptin neurons integrate signals from the suprachiasmatic nucleus (SCN) to regulate the timing of the LH surge (Piet et al., 2015; Tonsfeldt et al., 2022). A recent study, which deleted Six3 from mature neurons using Synapsin-Cre, revealed that SIX3 in mature neurons is important for proper alignment of circadian rhythms and fertility (Hoffmann et al., 2021). In the present study, we found that Six3 expression in AVPV kisspeptin neurons is regulated by time of day and may be involved in gating of neuronal activity prior to the surge.

Overall, our study establishes the importance of kisspeptin-specific SIX3 in regulating estrous cycles and fertility in female mice, which contributes to the reproductive phenotype of Six3 heterozygous mice. We also propose that Six3 is a novel regulator of cFos. Further, we demonstrate that Six3 is regulated by time of day in AVPV kisspeptin neurons and may play a role in the circadian control of the LH surge, through repression of cFos within kisspeptin neurons.

Acknowledgements

We thank Alejandro Lomniczi and Sergio Ojeda for providing the hKiss-Luc plasmid, Steven Kliewer for providing the mKiss-Luc plasmid, and Patrick E. Chappell for providing the KTaR-1 and KTaV-3 cell lines. We thank Jessica Cassin for obtaining the single cell germ cell RNA expression data. We also thank Ichiko Saotome and Austin Chin for technical assistance and Daphne Bindels and the Nikon Imaging Core for developing the analysis pipeline to quantify our RNAscope images.

Funding

This work was supported by NIH grants R01 HD082567, R01 HD100580, and R01 HD072754 (to P.L.M.) and by P50 HD012303 as part of the National Centers for Translational Research in Reproduction and Infertility (P.L.M.). P.L.M. was partially supported by P30 DK063491, P42 ES010337, and P30 CA023100, all of which also supported the UCSD Transgenic Mouse and Embryonic Stem Cell Core. S.N. L. was partially supported by P42 ES010337, T32 GM008666, and a supplement to P50 HD012303. T.C. was partially supported by the Ledell Family Research Undergraduate Research Scholarship Award for Science and Engineering. J.H. was partially supported by The Endocrine Society Summer Research Fellowship Award, the BrightSpinnaker Fellowship Award, and the McNair Research Program. N.C.P.N. was partially supported by the McNair Research Program. M.Y.H. was partially supported by Doris A. Howell Research Scholarship for Women’s Health and the Julia Brown Research Scholarship for Health and Medical Professions, or Medical Research. K.J.T. was partially supported by T32 HD007203, F32 HD090837, and P42 ES010337. The University of Virginia Ligand Assay Core was supported by NIH P50 HD028934.

Footnotes

Declaration of competing interest

Authors have no conflict of interest.

CRediT authorship contribution statement

Shanna N. Lavalle: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization. Teresa Chou: Validation, Formal analysis, Investigation, Writing – review & editing. Jacqueline Hernandez: Validation, Investigation, Writing – original draft, Writing – review & editing. Nay Chi P. Naing: Validation, Investigation, Writing – review & editing. Michelle Y. He: Validation, Investigation, Writing – review & editing. Karen J. Tonsfeldt: Methodology, Validation, Data curation, Investigation, Writing – review & editing, Visualization. Pamela L. Mellon: Conceptualization, Methodology, Validation, Formal analysis, Resources, Data curation, Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition.

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