The journal missed an author request to correct their funding statement before indexing of this article.
The funding statement has now been updated from
\"The author(s) declared that financial support was received for this work and/or its publication. This work was supported by UTK startup fund awarded to BYBL, UTK Faculty Research Assistants Funding awarded to BYBL to support SF and LD, and UTK Advanced Undergraduate Research Activity awarded to SF, LD and AC. Funding for open access to this research was provided by University of Tennessee's Open Publishing Support Fund.\"
to
\"The author(s) declared that financial support was received for this work and/or its publication. This work was supported by UTK startup fund awarded to BYBL, UTK Faculty Research Assistants Funding awarded to BYBL to support SF and LD, and UTK Advanced Undergraduate Research Activity awarded to SF, LD and AC. Funding for open access to this research was provided by University of Tennessee's Open Publishing Support Fund. Funding for open access to this research was provided by University of Tennessee's Open Publishing Support Fund.\"
","date":"Mon Sep 28 2026","correctionType":"erratum"}],"versions":[{"id":"28bd900d-5a63-4fd3-a4e4-4bd760a4d12d","decisionLetter":"Dear Billy Lau,
Your article \"Pubertal development is unaffected in female Mecp2-heterozygous mice.\" has been accepted, with minor modifications.
Review
In this report, Fortich et al show that Mecp2-het female mice, model of Rett syndrome, do not display overt alterations of pubertal onset or oestral ciclicity. The study replicates recent findings by Martín-Sánchez et al., showing the same result in a Mecp2-mutant model derived to CD1 background.
The report is well written, methods are correct, and it is important to publish replication studies. (1) The only minor comment I have is that the authors should comment that in Martín-Sánchez et al., Mepc2-KO male mice, which are symptomatic, do show pubertal delay, supporting the hypothesis that neuroendocrine alterations might arise with aging and symptom onset in Mecp2-Het females. (2) Also, there is a typo in the figure, in the cytology image it reads \"neurtral\" instead than \"neutral\"
We kindly ask you to address each point and summarize your changes in line with the reviewer's response in the 'Comments to Editor' section on the platform. In order to expedite the processing of your revised manuscript, please be as specific as possible in your responses.
The microPublication Editorial Team
","decision":"revise","submitted":true,"abstract":"Rett syndrome (RTT) affects predominantly females and is characterized by regression after an initial period of typical development. Previously, we showed that while adolescent female Mecp2-heterozygous mice (Het, mouse model of RTT) perform pup retrieval comparably to wild-type littermate controls (WT), adult Het regress and become inefficient. Here, we analyzed the pubertal trajectory of naïve nulliparous WT and Het to test whether abnormal pubertal development contributes to this regression phenotype. We found no genotypic differences in pubertal onset nor estrous cyclicity during adolescence and adulthood. These findings further support the role of Mecp2 in experience-dependent plasticity within sensory cortices.
","acknowledgements":"We thank Drs. Kalynn Schulz and Marcia Chavez for training us in vaginal lavage and estrous phase staging. We also thank graduate student Aly Webb and undergraduate students Mariam Ceesay, Kiran Hussaini, Roshni Rameshkumar, Faith Webb and Samantha Webber for their contributions to data collection.
","authors":[{"affiliations":["University of Tennessee, Knoxville, TN, United States"],"credit":["formalAnalysis","methodology","writing_reviewEditing","writing_originalDraft","dataCuration","validation"],"email":"sfortich@vols.utk.edu","firstName":"Skylar","lastName":"Fortich","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Tennessee, Knoxville, TN, United States"],"credit":["formalAnalysis","methodology","writing_originalDraft","writing_reviewEditing","dataCuration","visualization"],"email":"ldemilio@vols.utk.edu","firstName":"Lily","lastName":"Demilio","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Tennessee, Knoxville, TN, United States"],"credit":["formalAnalysis","methodology","dataCuration"],"email":"anabelleclower26@gmail.com","firstName":"Anabelle","lastName":"Clower","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Tennessee, Knoxville, TN, United States"],"credit":["conceptualization","fundingAcquisition","methodology","project","supervision","visualization","writing_reviewEditing","writing_originalDraft","validation"],"email":"blau@utk.edu","firstName":"Billy","lastName":"Lau","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-2380-5341"}],"comments":"","dataTable":{"name":null,"url":null},"disclaimer":true,"funding":"The author(s) declared that financial support was received for this work and/or its publication. This work was supported by UTK startup fund awarded to BYBL, UTK Advanced Undergraduate Research Activity awarded to SF, LD and AC, and UTK Faculty Research Assistants Funding awarded to SF and LD.
","image":{"name":"Figure 1.jpg","url":"https://portal.micropublication.org/uploads/64a7e65faa15c1e76d17a6a4303bd8f4.jpg"},"imageCaption":"A. Schema of experimental design. Upon vaginal opening, animals were lavaged daily up to postnatal day 55 (P55), and lavaged again from P84-98. Cytology and imaging were performed to determine estrus phase. B. Example bright field images showing proestrus, estrus, metestrus and diestrus. Black arrows = nucleated epithelial cells, black arrowheads = cornified epithelial cells, white arrows = leukocytes. C-D. Vaginal opening (puberty onset; C) and first estrus (D) were not significantly different between WT (n=28, 9, respectively) and Het (n=35, 9, respectively). E-H. There were no significant differences between WT and Het in average cycle length during adolescence (E) and adulthood (G) nor in % time spent in the 4 different estrus phases during adolescence (F) and adulthood (H). E-F: WT (n=8) and Het (n=8). G-H: WT (n=10) and Het (n=9). C-H: Scattered plots: each dot represents an animal, lines are mean ± S.E.M. Mann-Whitney test: p>0.05, n.s. = no significance.
","imageTitle":"Pubertal onset and estrous cycling are not affected by Mecp2 mutations
","laboratory":{"name":"","WBId":""},"methods":"Methods
Animals
All experiments were performed in female mice (4-16 weeks old), maintained on a 12/12 h light/dark cycle (lights on 7 A.M.) and received food ad libitum. Genotypes used were Mecp2-heterozygote (Het; B6.129P2(C)-Mecp2tm1.1Bird/J, JAX # 003890) and Mecp2-WT siblings. All procedures were conducted in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and approved by the University of Tennessee-Knoxville Institutional Animal Care and Use Committee.
Pubertal onset and estrous cyclicity
Pubertal onset was evaluated by daily visual inspection for vaginal opening (VO), starting at postnatal day 21 (P21). On the day of VO, mice were daily vaginal lavaged for at least 2 weeks (adolescent age) to determine estrous cycle length and their relative duration in each cycle phase. The first detected estrus was used as the cycle-start marker. To assess estrous cyclicity in adulthood, 12-week-old mice (P84) underwent daily vaginal lavage for 2 weeks.
Cytology, Imaging and Phase Classification
Vaginal cells were collected by gently flushing the vaginal canal with 50–100 µL Milli-Q water using a glass eyedropper. The fluid was expelled and recollected in a single lavage, and this procedure was not repeated more than twice per session to avoid inducing pseudopregnancy. Samples were placed onto slides and air-dried on a hot plate (~50°C) for 2-3 minutes. Slides were then stained for 2-3 minutes with 0.1% Neutral Red (Sigma-Aldrich, #72210) dissolved in Milli-Q water, gently rinsed with distilled water and air-dried before imaging. Samples were imaged as brightfield montages using a Keyence microscope at 20x magnification. Cyclic phases - proestrus, estrus, metestrus and diestrus - were visually determined from the images by at least three human observers blinded to genotype. Phases were identified based on their characteristic cellular compositions, including nucleated epithelial cells, cornified epithelial cells and leukocyte. The human inter-rater agreement averaged 88.98% across 433 total phase counts, which is consistent with field standards (Caligioni, 2009).
Statistical Analysis
All statistical analyses and graphs were performed in GraphPad Prism 11. Mann-Whitney test was used to compare between WT and Het. Statistical significance was defined at p < 0.05. Data are presented as mean ± standard error mean. The figure was generated in Adobe Illustrator 2025.
","reagents":"","patternDescription":"Description
RTT is a rare neurodevelopmental disorder commonly affects girls, with a prevalence of 1 in 10,000 live female births. It is caused by mutations in the X-linked gene, methyl-CpG-binding protein 2 (MECP2) (Amir et al., 1999). RTT patients exhibit a typical development until 6-18 months of age, when they experience developmental stagnation followed by a period of regression. Regression is characterized by the loss of previous acquired skills, such as purposeful hand movement, communication skills and motor function, that persists throughout life (Neul et al., 2010; Halbach et al., 2013). Pubertal abnormalities have also been reported in RTT patients. For instance, Killian et al. (2014) sampled 802 RTT patients and reported that 25% exhibited premature thelarche, 28% premature adrenarche and 19% delayed menarche. A subset of RTT patients with milder mutations had less severe phenotype and earlier menarche. Another example was presented in a case study by Holm (1985), where the RTT patient exhibited precocious puberty at the age of 6, and subsequent case studies documented similar findings (Bas et al., 2013; Yang et al., 2021; Canton et al., 2023). Pubertal dysfunction is RTT is further underscored by the high prevalence of catamenial seizures (Humphrey et al., 2021). Despite substantial evidence of linking RTT with abnormal pubertal and reproductive phenotypes, whether these abnormalities contribute to regression remains unclear.
Previously, we identified a regression phenotype in a mouse model of RTT, female Mecp2-heterozygous mice (Het; Guy et al., 2001). At 6 weeks old (adolescent), Het perform as efficiently as wild-type littermate controls (WT) in an alloparental pup retrieval behavior (Mykins et al., 2023). By 12 weeks old, (adult), pre-symptomatic Het regress and perform the same task less efficiently than WT (Krishnan et al., 2017; Stevenson et al., 2021). One possible explanation for the Het regression phenotype is the abnormal pubertal development caused by Mecp2 mutations. Here, we tested this hypothesis by examining pubertal onset and estrus cyclicity in adolescent and adult mice. Pubertal onset was determined by the day of vaginal opening, and estrous cyclicity was determined by daily vaginal lavage followed by cytological analysis (Figure 1A). The mouse estrous cycle is divided into 4 phases distinguished by their distinct cellular composition (McLean et al., 2012): proestrus = mostly nucleated epithelial cells, with some cornified epithelial cells; estrous = mainly cornified epithelial cells; metestrus = mostly leukocytes, stringy tissue may be present; and diestrus = mostly leukocytes, with some cornified and nucleated epithelial cells (Figure 1B). We found that puberty onset was similar between WT and Het (Figure 1C; WT: 32.1 ± 0.7 days, n = 28 animals; Het: 33.7 ± 0.6 days, n = 35 animals; Mann-Whitney: p>0.05). In a subset of those mice, we determined the day of first observed estrus, an indirect indicator of reproductive maturation, and we also found no difference between WT and Het (Figure 1D; WT: 37.1 ± 1.0 days, n = 9 animals; Het: 35.44 ± 1.1 days, n = 9 animals; Mann-Whitney: p>0.05). To determine the phases of estrous cycle, cytological images were scored by 3 experimenters who were blinded to the genotypes. The human inter-rater agreement was 88.98%, consistent with field standards (Caligioni, 2009). In adolescence, there were no significant differences between the genotypes for their average estrus cycle length (Figure 1E) (WT: 4.7 ± 0.3 days, n = 8 animals; Het: 4.3 ± 0.3 days, n = 8 animals; Mann-Whitney test: p=0.42) nor their percentage of time spent at the 4 different phases (Figure 1F) (WT: proestrus - 2.0% ± 1.0, estrus - 44.5% ± 2.9, metestrus - 36.9% ± 6.7, diestrus - 16.6% ± 5.0, n = 8 animals; Het: proestrus - 5.3% ± 2.2, estrus – 50.9% ± 4.0, metestrus – 29.5% ± 5.8, diestrus: 14.3% ± 2.4 , n = 8 animals; Mann-Whitney test: p>0.05). In adults, we also did not find significant differences between WT and Het in their average estrus cycle length (Figure 1G) (WT: 4.2 ± 0.2 days, n = 10 animals; Het: 4.6 ± 0.2 days, n = 9 animals; Mann-Whitney test: p=0.41) nor percentage of time spent at the different phases (Figure 1H) (WT: proestrus - 8.0% ± 2.6, estrus – 41.0% ± 5.0, metestrus – 31.6% ± 4.2, diestrus – 19.3% ± 3.5, n = 10 animals; Het: proestrus – 3.3% ± 2.2, estrus – 49.3% ± 3.5, metestrus – 28.7% ± 4.9, diestrus: 18.7% ± 3.0, n = 9 animals; Mann-Whitney test: p>0.05).
Taken together, our results indicate that our female mouse model of RTT, female Mecp2-heterozygous mice (B6.129P2(C)-Mecp2tm1.1Bird/J), exhibits normal pubertal development and maturation of the endocrine system during the pre-symptomatic age. Our findings are consistent with those reported in another female mouse model of RTT (Martin-Sanchez et al., 2026). Our data further support the idea that the Het regression phenotype in pup retrieval behavior is unlikely to result from gross systemic endocrine disruption and is instead more consistent with age-dependent abnormalities in cellular and circuit plasticity within sensory cortices (Krishnan et al., 2017; Lau et al., 2020a; Lau et al., 2020b). Nevertheless, the absence of overt abnormalities in pubertal timing or estrous cyclicity does not exclude the possibility that more subtle neuroendocrine mechanisms contribute to age-dependent regression in Het. Future studies will be needed to determine whether Mecp2 mutations alter hormone-dependent modulation of maternal behavior and experience-dependent plasticity at the level of specific neural circuits.
","references":[{"reference":"Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. 1999. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nature Genetics 23: 185-188.
","pubmedId":"","doi":"10.1038/13810"},{"reference":"Baş VN, Çetinkaya S, Ağladıoğlu SYl, Aksoy Ae, Gülpınar Ba, Aycan Z. 2013. Report of the first case of precocious puberty in Rett syndrome. jpem 26: 937-939.
","pubmedId":"","doi":" 10.1515/jpem-2012-0418"},{"reference":"Caligioni CS. 2009. Assessing Reproductive Status/Stages in Mice. Current Protocols in Neuroscience 48: 10.1002/0471142301.nsa04is48.
","pubmedId":"","doi":"10.1002/0471142301.nsa04is48"},{"reference":"Canton APM, Tinano FvR, Guasti L, Montenegro LR, Ryan F, Shears D, et al., Latronico. 2023. Rare variants in the MECP2 gene in girls with central precocious puberty: a translational cohort study. The Lancet Diabetes & Endocrinology 11: 545-554.
","pubmedId":"","doi":"10.1016/S2213-8587(23)00131-6"},{"reference":"Guy J, Hendrich B, Holmes M, Martin JE, Bird A. 2001. A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome. Nature Genetics 27: 322-326.
","pubmedId":"","doi":"10.1038/85899"},{"reference":"Halbach N, Smeets E, Steinbusch C, Maaskant M, van Waardenburg D, Curfs L. 2012. Aging in Rett syndrome: a longitudinal study. Clinical Genetics 84: 223-229.
","pubmedId":"","doi":"10.1111/cge.12063"},{"reference":"Holm VA. 1985. Rett's syndrome: a progressive developmental disability in girls. J Dev Behav Pediatr 6(1):32-36. PMID:3973029.
","pubmedId":"","doi":""},{"reference":"Humphrey KN, Horn PS, Olshavsky L, Reebals L, Standridge SM. 2021. Features of Menstruation and Menstruation Management in Individuals with Rett Syndrome. Journal of Pediatric and Adolescent Gynecology 34: 144-153.
","pubmedId":"","doi":" 10.1016/j.jpag.2020.11.002"},{"reference":"Killian JT, Lane JB, Cutter GR, Skinner SA, Kaufmann WE, Tarquinio DC, et al., Percy. 2014. Pubertal Development in Rett Syndrome Deviates From Typical Females. Pediatric Neurology 51: 769-775.
","pubmedId":"","doi":"10.1016/j.pediatrneurol.2014.08.013"},{"reference":"Krishnan K, Lau BYB, Ewall G, Huang ZJ, Shea SD. 2017. MECP2 regulates cortical plasticity underlying a learned behaviour in adult female mice. Nature Communications 8: 10.1038/ncomms14077.
","pubmedId":"","doi":"10.1038/ncomms14077"},{"reference":"Lau BYB, Krishnan K, Huang ZJ, Shea SD. 2020a. Maternal Experience-Dependent Cortical Plasticity in Mice Is Circuit- and Stimulus-Specific and Requires MECP2. The Journal of Neuroscience 40: 1514-1526.
","pubmedId":"","doi":" 10.1523/JNEUROSCI.1964-19.2019"},{"reference":"Lau BYB, Layo DE, Emery B, Everett M, Kumar A, Stevenson P, et al., Krishnan. 2020b. Lateralized Expression of Cortical Perineuronal Nets during Maternal Experience is Dependent on MECP2. eneuro 7: ENEURO.0500-19.2020.
","pubmedId":"","doi":": 10.1523/ENEURO.0500-19.2020"},{"reference":"Martín‐Sánchez A, Jiménez‐Díaz D, Esteve‐Pérez R, Vasile‐Tudorache A, Read JE, Howard SR, Agustín‐Pavón C. 2026. Pubertal development and hypothalamic–pituitary–gonadal axis are altered in male mice lacking\n Mecp2. Journal of Neuroendocrinology 38: 10.1111/jne.70221.
","pubmedId":"","doi":"10.1111/jne.70221"},{"reference":"McLean AC, Valenzuela N, Fai S, Bennett SAL. 2012. Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification. Journal of Visualized Experiments : 10.3791/4389.
","pubmedId":"","doi":" 10.3791/4389"},{"reference":"Mykins M, Layo‐Carris D, Dunn LR, Skinner DW, McBryar AH, Perez S, et al., Krishnan. 2023. Wild‐type\n
Neul JL, Kaufmann WE, Glaze DG, Christodoulou J, Clarke AJ, Bahi-Buisson N, et al, Percy AK; RettSearch Consortium. 2010. Rett syndrome: Revised diagnostic criteria and nomenclature. Annals of Neurology 68: 944-950.
","pubmedId":"","doi":" 10.1002/ana.22124"},{"reference":"Stevenson PK, Casenhiser DM, Lau BYB, Krishnan K. 2021. Systematic analysis of goal‐related movement sequences during maternal behaviour in a female mouse model for Rett syndrome. European Journal of Neuroscience 54: 4528-4549.
","pubmedId":"","doi":"10.1111/ejn.15327"},{"reference":"Yang L, Jiang M, Yu R, Hu R, Xiong F, Li J. 2021. A case report of precocious puberty related to Rett syndrome and a literature review. Pharmazie 76(11):559-561.
","pubmedId":"","doi":" 10.1691/ph.2021.1747"}],"suggestedReviewer":{"name":"Carmen Agustín‐Pavón, Department of Cell Biology and Functional Biology, Faculty of Biological Sciences, Universitat de Valencia, València, Spain. Email: m.carmen.agustin@uv.es
","WBId":""},"title":"Pubertal development is unaffected in female Mecp2-heterozygous mice.
","reviews":[{"reviewer":{"displayName":"Carmen Agustín-Pavón"},"openAcknowledgement":false,"status":{"submitted":true}}]},{"id":"574f5249-9c8d-4064-8de4-48df7fe26b37","decisionLetter":"\n\n Dear Billy Lau,\n
\n\n We are happy to let you know that your article has been accepted for publication. Congratulations!\n
\n\n Please take a careful look at the production proof of your article: proof download.\n
\n\n Please make sure there are no typos, errors, or omissions in your article, including your title, author names, affiliations, \n reagents, etc. in addition to your reported results. These are little things that, if wrong, will still require a separate \n corrigendum article if they need correction after publication.\n
\n\n Please make any changes or approve the current version by following this link. Please be advised that, at this stage, only minor typographical and stylistic changes can be made, anything more substantial may need to undergo additional review.\n
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\n\n The microPublication Editorial Team\n
\n ","decision":"accept","submitted":true,"abstract":"Rett syndrome (RTT) affects predominantly females and is characterized by regression after an initial period of typical development. Previously, we showed that while adolescent female Mecp2-heterozygous mice (Het, mouse model of RTT) perform pup retrieval comparably to wild-type littermate controls (WT), adult Het regress and become inefficient. Here, we analyzed the pubertal trajectory of naïve nulliparous WT and Het to test whether abnormal pubertal development contributes to this regression phenotype. We found no genotypic differences in pubertal onset nor estrous cyclicity during adolescence and adulthood. These findings further support the role of Mecp2 in experience-dependent plasticity within sensory cortices.
","acknowledgements":"We thank Drs. Kalynn Schulz and Marcia Chavez for training us in vaginal lavage and estrous phase staging. We also thank graduate student Aly Webb and undergraduate students Kiran Hussaini, Roshni Rameshkumar, Faith Webb and Samantha Webber for their contributions to data collection.
","authors":[{"affiliations":["University of Tennessee, Knoxville, TN, United States"],"credit":["formalAnalysis","methodology","writing_reviewEditing","writing_originalDraft","dataCuration","validation"],"email":"sfortich@vols.utk.edu","firstName":"Skylar","lastName":"Fortich","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Tennessee, Knoxville, TN, United States"],"credit":["formalAnalysis","methodology","writing_originalDraft","writing_reviewEditing","dataCuration","visualization"],"email":"ldemilio@vols.utk.edu","firstName":"Lily","lastName":"Demilio","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Tennessee, Knoxville, TN, United States"],"credit":["dataCuration","methodology","validation"],"email":"mceesay@vols.utk.edu","firstName":"Mariam","lastName":"Ceesay","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Tennessee, Knoxville, TN, United States"],"credit":["formalAnalysis","methodology","dataCuration"],"email":"anabelleclower26@gmail.com","firstName":"Anabelle","lastName":"Clower","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Tennessee, Knoxville, TN, United States"],"credit":["conceptualization","fundingAcquisition","methodology","project","supervision","visualization","writing_reviewEditing","writing_originalDraft","validation"],"email":"blau@utk.edu","firstName":"Billy","lastName":"Lau","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-2380-5341"}],"comments":"We thank the Editor Team for accepting our manuscript!
We thank the reviewer's positive comments and suggestions. We have made the following changes:
1. Added Mariam Ceesay to the authorship list and removed her from the Acknowledgment section.
2. Updated the figure with the correct spelling of 'neutral', per the request of the reviewer.
3. Added the following sentence immediately after Martín-Sánchez et al. reference, per the request of the reviewer:
'The same study showed that symptomatic Mecp2-knock out male mice exhibit delayed pubertal onset, supporting the hypothesis that neuroendocrine alterations may emerge as female Mecp2-heterozygous mice age and become symptomatic.'
4. Added a reference (Mykins et al., 2024) to this sentence: By 12 weeks old, (adult), pre-symptomatic Het regress and perform the same task less efficiently than WT (Krishnan et al., 2017; Stevenson et al., 2021; Mykins et al., 2024).
","dataTable":{"name":null,"url":null},"disclaimer":true,"funding":"The author(s) declared that financial support was received for this work and/or its publication. This work was supported by UTK startup fund awarded to BYBL, UTK Advanced Undergraduate Research Activity awarded to SF, LD and AC, and UTK Faculty Research Assistants Funding awarded to SF and LD.
","image":{"name":"Figure 1.jpg","url":"https://portal.micropublication.org/uploads/e9a26341e519b0b5f075b0e79784a852.jpg"},"imageCaption":"A. Schema of experimental design. Upon vaginal opening, animals were lavaged daily up to postnatal day 55 (P55), and lavaged again from P84-98. Cytology and imaging were performed to determine estrus phase. B. Example bright field images showing proestrus, estrus, metestrus and diestrus. Black arrows = nucleated epithelial cells, black arrowheads = cornified epithelial cells, white arrows = leukocytes. C-D. Vaginal opening (puberty onset; C) and first estrus (D) were not significantly different between WT (n=28, 9, respectively) and Het (n=35, 9, respectively). E-H. There were no significant differences between WT and Het in average cycle length during adolescence (E) and adulthood (G) nor in % time spent in the 4 different estrus phases during adolescence (F) and adulthood (H). E-F: WT (n=8) and Het (n=8). G-H: WT (n=10) and Het (n=9). C-H: Scattered plots: each dot represents an animal, lines are mean ± S.E.M. Mann-Whitney test: p>0.05, n.s. = no significance.
","imageTitle":"Pubertal onset and estrous cycling are not affected by Mecp2 mutations
","laboratory":{"name":"","WBId":""},"methods":"Methods
Animals
All experiments were performed in female mice (4-16 weeks old), maintained on a 12/12 h light/dark cycle (lights on 7 A.M.) and received food ad libitum. Genotypes used were Mecp2-heterozygote (Het; B6.129P2(C)-Mecp2tm1.1Bird/J, JAX # 003890) and Mecp2-WT siblings. All procedures were conducted in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and approved by the University of Tennessee-Knoxville Institutional Animal Care and Use Committee.
Pubertal onset and estrous cyclicity
Pubertal onset was evaluated by daily visual inspection for vaginal opening (VO), starting at postnatal day 21 (P21). On the day of VO, mice were daily vaginal lavaged for at least 2 weeks (adolescent age) to determine estrous cycle length and their relative duration in each cycle phase. The first detected estrus was used as the cycle-start marker. To assess estrous cyclicity in adulthood, 12-week-old mice (P84) underwent daily vaginal lavage for 2 weeks.
Cytology, Imaging and Phase Classification
Vaginal cells were collected by gently flushing the vaginal canal with 50–100 µL Milli-Q water using a glass eyedropper. The fluid was expelled and recollected in a single lavage, and this procedure was not repeated more than twice per session to avoid inducing pseudopregnancy. Samples were placed onto slides and air-dried on a hot plate (~50°C) for 2-3 minutes. Slides were then stained for 2-3 minutes with 0.1% Neutral Red (Sigma-Aldrich, #72210) dissolved in Milli-Q water, gently rinsed with distilled water and air-dried before imaging. Samples were imaged as brightfield montages using a Keyence microscope at 20x magnification. Cyclic phases - proestrus, estrus, metestrus and diestrus - were visually determined from the images by at least three human observers blinded to genotype. Phases were identified based on their characteristic cellular compositions, including nucleated epithelial cells, cornified epithelial cells and leukocyte. The human inter-rater agreement averaged 88.98% across 433 total phase counts, which is consistent with field standards (Caligioni, 2009).
Statistical Analysis
All statistical analyses and graphs were performed in GraphPad Prism 11. Mann-Whitney test was used to compare between WT and Het. Statistical significance was defined at p < 0.05. Data are presented as mean ± standard error mean. The figure was generated in Adobe Illustrator 2025.
","reagents":"","patternDescription":"Description
RTT is a rare neurodevelopmental disorder commonly affects girls, with a prevalence of 1 in 10,000 live female births. It is caused by mutations in the X-linked gene, methyl-CpG-binding protein 2 (MECP2) (Amir et al., 1999). RTT patients exhibit a typical development until 6-18 months of age, when they experience developmental stagnation followed by a period of regression. Regression is characterized by the loss of previous acquired skills, such as purposeful hand movement, communication skills and motor function, that persists throughout life (Neul et al., 2010; Halbach et al., 2013). Pubertal abnormalities have also been reported in RTT patients. For instance, Killian et al. (2014) sampled 802 RTT patients and reported that 25% exhibited premature thelarche, 28% premature adrenarche and 19% delayed menarche. A subset of RTT patients with milder mutations had less severe phenotype and earlier menarche. Another example was presented in a case study by Holm (1985), where the RTT patient exhibited precocious puberty at the age of 6, and subsequent case studies documented similar findings (Bas et al., 2013; Yang et al., 2021; Canton et al., 2023). Pubertal dysfunction is RTT is further underscored by the high prevalence of catamenial seizures (Humphrey et al., 2021). Despite substantial evidence of linking RTT with abnormal pubertal and reproductive phenotypes, whether these abnormalities contribute to regression remains unclear.
Previously, we identified a regression phenotype in a mouse model of RTT, female Mecp2-heterozygous mice (Het; Guy et al., 2001). At 6 weeks old (adolescent), Het perform as efficiently as wild-type littermate controls (WT) in an alloparental pup retrieval behavior (Mykins et al., 2023). By 12 weeks old, (adult), pre-symptomatic Het regress and perform the same task less efficiently than WT (Krishnan et al., 2017; Stevenson et al., 2021; Mykins et al., 2024). One possible explanation for the Het regression phenotype is the abnormal pubertal development caused by Mecp2 mutations. Here, we tested this hypothesis by examining pubertal onset and estrus cyclicity in adolescent and adult mice. Pubertal onset was determined by the day of vaginal opening, and estrous cyclicity was determined by daily vaginal lavage followed by cytological analysis (Figure 1A). The mouse estrous cycle is divided into 4 phases distinguished by their distinct cellular composition (McLean et al., 2012): proestrus = mostly nucleated epithelial cells, with some cornified epithelial cells; estrous = mainly cornified epithelial cells; metestrus = mostly leukocytes, stringy tissue may be present; and diestrus = mostly leukocytes, with some cornified and nucleated epithelial cells (Figure 1B). We found that puberty onset was similar between WT and Het (Figure 1C; WT: 32.1 ± 0.7 days, n = 28 animals; Het: 33.7 ± 0.6 days, n = 35 animals; Mann-Whitney: p>0.05). In a subset of those mice, we determined the day of first observed estrus, an indirect indicator of reproductive maturation, and we also found no difference between WT and Het (Figure 1D; WT: 37.1 ± 1.0 days, n = 9 animals; Het: 35.44 ± 1.1 days, n = 9 animals; Mann-Whitney: p>0.05). To determine the phases of estrous cycle, cytological images were scored by 3 experimenters who were blinded to the genotypes. The human inter-rater agreement was 88.98%, consistent with field standards (Caligioni, 2009). In adolescence, there were no significant differences between the genotypes for their average estrus cycle length (Figure 1E) (WT: 4.7 ± 0.3 days, n = 8 animals; Het: 4.3 ± 0.3 days, n = 8 animals; Mann-Whitney test: p=0.42) nor their percentage of time spent at the 4 different phases (Figure 1F) (WT: proestrus - 2.0% ± 1.0, estrus - 44.5% ± 2.9, metestrus - 36.9% ± 6.7, diestrus - 16.6% ± 5.0, n = 8 animals; Het: proestrus - 5.3% ± 2.2, estrus – 50.9% ± 4.0, metestrus – 29.5% ± 5.8, diestrus: 14.3% ± 2.4 , n = 8 animals; Mann-Whitney test: p>0.05). In adults, we also did not find significant differences between WT and Het in their average estrus cycle length (Figure 1G) (WT: 4.2 ± 0.2 days, n = 10 animals; Het: 4.6 ± 0.2 days, n = 9 animals; Mann-Whitney test: p=0.41) nor percentage of time spent at the different phases (Figure 1H) (WT: proestrus - 8.0% ± 2.6, estrus – 41.0% ± 5.0, metestrus – 31.6% ± 4.2, diestrus – 19.3% ± 3.5, n = 10 animals; Het: proestrus – 3.3% ± 2.2, estrus – 49.3% ± 3.5, metestrus – 28.7% ± 4.9, diestrus: 18.7% ± 3.0, n = 9 animals; Mann-Whitney test: p>0.05).
Taken together, our results indicate that our female mouse model of RTT, female Mecp2-heterozygous mice (B6.129P2(C)-Mecp2tm1.1Bird/J), exhibits normal pubertal development and maturation of the endocrine system during the pre-symptomatic age. Our findings are consistent with those reported in another female mouse model of RTT (Martin-Sanchez et al., 2026). The same study showed that symptomatic Mecp2-knock out male mice exhibit delayed pubertal onset, supporting the hypothesis that neuroendocrine alterations may emerge as female Mecp2-heterozygous mice age and become symptomatic. Our data further support the idea that the Het regression phenotype in pup retrieval behavior is unlikely to result from gross systemic endocrine disruption and is instead more consistent with age-dependent abnormalities in cellular and circuit plasticity within sensory cortices (Krishnan et al., 2017; Lau et al., 2020a; Lau et al., 2020b). Nevertheless, the absence of overt abnormalities in pubertal timing or estrous cyclicity does not exclude the possibility that more subtle neuroendocrine mechanisms contribute to age-dependent regression in Het. Future studies will be needed to determine whether Mecp2 mutations alter hormone-dependent modulation of maternal behavior and experience-dependent plasticity at the level of specific neural circuits.
","references":[{"reference":"Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. 1999. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nature Genetics 23: 185-188.
","pubmedId":"","doi":"10.1038/13810"},{"reference":"Baş VN, Çetinkaya S, Ağladıoğlu SYl, Aksoy Ae, Gülpınar Ba, Aycan Z. 2013. Report of the first case of precocious puberty in Rett syndrome. jpem 26: 937-939.
","pubmedId":"","doi":" 10.1515/jpem-2012-0418"},{"reference":"Caligioni CS. 2009. Assessing Reproductive Status/Stages in Mice. Current Protocols in Neuroscience 48: 10.1002/0471142301.nsa04is48.
","pubmedId":"","doi":"10.1002/0471142301.nsa04is48"},{"reference":"Canton APM, Tinano FvR, Guasti L, Montenegro LR, Ryan F, Shears D, et al., Latronico. 2023. Rare variants in the MECP2 gene in girls with central precocious puberty: a translational cohort study. The Lancet Diabetes & Endocrinology 11: 545-554.
","pubmedId":"","doi":"10.1016/S2213-8587(23)00131-6"},{"reference":"Guy J, Hendrich B, Holmes M, Martin JE, Bird A. 2001. A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome. Nature Genetics 27: 322-326.
","pubmedId":"","doi":"10.1038/85899"},{"reference":"Halbach N, Smeets E, Steinbusch C, Maaskant M, van Waardenburg D, Curfs L. 2012. Aging in Rett syndrome: a longitudinal study. Clinical Genetics 84: 223-229.
","pubmedId":"","doi":"10.1111/cge.12063"},{"reference":"Holm VA. 1985. Rett's syndrome: a progressive developmental disability in girls. J Dev Behav Pediatr 6(1):32-36. PMID:3973029.
","pubmedId":"","doi":""},{"reference":"Humphrey KN, Horn PS, Olshavsky L, Reebals L, Standridge SM. 2021. Features of Menstruation and Menstruation Management in Individuals with Rett Syndrome. Journal of Pediatric and Adolescent Gynecology 34: 144-153.
","pubmedId":"","doi":" 10.1016/j.jpag.2020.11.002"},{"reference":"Killian JT, Lane JB, Cutter GR, Skinner SA, Kaufmann WE, Tarquinio DC, et al., Percy. 2014. Pubertal Development in Rett Syndrome Deviates From Typical Females. Pediatric Neurology 51: 769-775.
","pubmedId":"","doi":"10.1016/j.pediatrneurol.2014.08.013"},{"reference":"Krishnan K, Lau BYB, Ewall G, Huang ZJ, Shea SD. 2017. MECP2 regulates cortical plasticity underlying a learned behaviour in adult female mice. Nature Communications 8: 10.1038/ncomms14077.
","pubmedId":"","doi":"10.1038/ncomms14077"},{"reference":"Lau BYB, Krishnan K, Huang ZJ, Shea SD. 2020a. Maternal Experience-Dependent Cortical Plasticity in Mice Is Circuit- and Stimulus-Specific and Requires MECP2. The Journal of Neuroscience 40: 1514-1526.
","pubmedId":"","doi":" 10.1523/JNEUROSCI.1964-19.2019"},{"reference":"Lau BYB, Layo DE, Emery B, Everett M, Kumar A, Stevenson P, et al., Krishnan. 2020b. Lateralized Expression of Cortical Perineuronal Nets during Maternal Experience is Dependent on MECP2. eneuro 7: ENEURO.0500-19.2020.
","pubmedId":"","doi":": 10.1523/ENEURO.0500-19.2020"},{"reference":"Martín‐Sánchez A, Jiménez‐Díaz D, Esteve‐Pérez R, Vasile‐Tudorache A, Read JE, Howard SR, Agustín‐Pavón C. 2026. Pubertal development and hypothalamic–pituitary–gonadal axis are altered in male mice lacking\n Mecp2. Journal of Neuroendocrinology 38: 10.1111/jne.70221.
","pubmedId":"","doi":"10.1111/jne.70221"},{"reference":"McLean AC, Valenzuela N, Fai S, Bennett SAL. 2012. Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification. Journal of Visualized Experiments : 10.3791/4389.
","pubmedId":"","doi":" 10.3791/4389"},{"reference":"Mykins M, Layo‐Carris D, Dunn LR, Skinner DW, McBryar AH, Perez S, et al., Krishnan. 2023. Wild‐type\n
Mykins M, Bridges B, Jo A, Krishnan K. 2024. Multidimensional analysis of a social behavior identifies regression and phenotypic heterogeneity in a female mouse model for Rett syndrome. The Journal of Neuroscience : e1078232023.
","pubmedId":"","doi":"10.1523/JNEUROSCI.1078-23.2023"},{"reference":"Neul JL, Kaufmann WE, Glaze DG, Christodoulou J, Clarke AJ, Bahi-Buisson N, et al, Percy AK; RettSearch Consortium. 2010. Rett syndrome: Revised diagnostic criteria and nomenclature. Annals of Neurology 68: 944-950.
","pubmedId":"","doi":" 10.1002/ana.22124"},{"reference":"Stevenson PK, Casenhiser DM, Lau BYB, Krishnan K. 2021. Systematic analysis of goal‐related movement sequences during maternal behaviour in a female mouse model for Rett syndrome. European Journal of Neuroscience 54: 4528-4549.
","pubmedId":"","doi":"10.1111/ejn.15327"},{"reference":"Yang L, Jiang M, Yu R, Hu R, Xiong F, Li J. 2021. A case report of precocious puberty related to Rett syndrome and a literature review. Pharmazie 76(11):559-561.
","pubmedId":"","doi":" 10.1691/ph.2021.1747"}],"suggestedReviewer":{"name":"Carmen Agustín‐Pavón, Department of Cell Biology and Functional Biology, Faculty of Biological Sciences, Universitat de Valencia, València, Spain. Email: m.carmen.agustin@uv.es
","WBId":""},"title":"Pubertal development is unaffected in female Mecp2-heterozygous mice.
","reviews":[{"reviewer":{"displayName":"Carmen Agustín-Pavón"},"openAcknowledgement":false,"status":{"submitted":true}}]},{"id":"2ce7351e-c554-4ce3-b0ac-71101adeed06","decisionLetter":"\n\n Dear Authors,\n
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\n \"Fortich S, Demilio L, Ceesay M, Clower A, Lau BYB. 2026. Pubertal development is unaffected in female Mecp2-heterozygous mice.. microPublication Biology. 10.17912/micropub.biology.002400.\"\n
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\n ","decision":"publish","submitted":true,"abstract":"Rett syndrome (RTT) affects predominantly females and is characterized by regression after an initial period of typical development. Previously, we showed that while adolescent female Mecp2-heterozygous mice (Het, mouse model of RTT) perform pup retrieval comparably to wild-type littermate controls (WT), adult Het regress and become inefficient. Here, we analyzed the pubertal trajectory of naïve nulliparous WT and Het to test whether abnormal pubertal development contributes to this regression phenotype. We found no genotypic differences in pubertal onset nor estrous cyclicity during adolescence and adulthood. These findings further support the role of Mecp2 in experience-dependent plasticity within sensory cortices.
","acknowledgements":"We thank Drs. Kalynn Schulz and Marcia Chavez for training us in vaginal lavage and estrous phase staging. We also thank graduate student Aly Webb and undergraduate students Kiran Hussaini, Roshni Rameshkumar, Faith Webb and Samantha Webber for their contributions to data collection.
","authors":[{"affiliations":["University of Tennessee, Knoxville, TN, United States"],"credit":["formalAnalysis","methodology","writing_reviewEditing","writing_originalDraft","dataCuration","validation"],"email":"sfortich@vols.utk.edu","firstName":"Skylar","lastName":"Fortich","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Tennessee, Knoxville, TN, United States"],"credit":["formalAnalysis","methodology","writing_originalDraft","writing_reviewEditing","dataCuration","visualization"],"email":"ldemilio@vols.utk.edu","firstName":"Lily","lastName":"Demilio","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Tennessee, Knoxville, TN, United States"],"credit":["dataCuration","methodology","validation"],"email":"mceesay@vols.utk.edu","firstName":"Mariam","lastName":"Ceesay","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Tennessee, Knoxville, TN, United States"],"credit":["formalAnalysis","methodology","dataCuration"],"email":"anabelleclower26@gmail.com","firstName":"Anabelle","lastName":"Clower","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Tennessee, Knoxville, TN, United States"],"credit":["conceptualization","fundingAcquisition","methodology","project","supervision","visualization","writing_reviewEditing","writing_originalDraft","validation"],"email":"blau@utk.edu","firstName":"Billy You Bun","lastName":"Lau","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-2380-5341"}],"comments":"We made minor text edits for grammar, spelling and clarity. Please let me know if you would like a list of all the changes made. I'm happy to see another proof if needed. I am waiting on my university's decision on whether they can support this publication fee. Thank you!
","dataTable":{"name":null,"url":null},"disclaimer":true,"funding":"The author(s) declared that financial support was received for this work and/or its publication. This work was supported by UTK startup fund awarded to BYBL, UTK Faculty Research Assistants Funding awarded to BYBL to support SF and LD, and UTK Advanced Undergraduate Research Activity awarded to SF, LD and AC.
","image":{"name":"Figure 1.jpg","url":"https://portal.micropublication.org/uploads/e9a26341e519b0b5f075b0e79784a852.jpg"},"imageCaption":"A. Schema of experimental design. Upon vaginal opening, animals were lavaged daily up to postnatal day 55 (P55), and lavaged again from P84-98. Cytology and imaging were performed to determine estrous phase. B. Example bright field images showing proestrus, estrus, metestrus and diestrus. Black arrows = nucleated epithelial cells, black arrowheads = cornified epithelial cells, white arrows = leukocytes. C-D. Vaginal opening (puberty onset; C) and first estrus (D) were not significantly different between WT (n=28, 9, respectively) and Het (n=35, 9, respectively). E-H. There were no significant differences between WT and Het in average cycle length during adolescence (E) and adulthood (G) nor in % time spent in the 4 different estrous phases during adolescence (F) and adulthood (H). E-F: WT (n=8) and Het (n=8). G-H: WT (n=10) and Het (n=9). C-H: Scatter plots: each dot represents an animal, lines are mean ± S.E.M. Mann-Whitney test: p>0.05, n.s. = no significance.
","imageTitle":"Pubertal onset and estrous cycling are not affected by Mecp2 mutations
","laboratory":{"name":"","WBId":""},"methods":"Animals
All experiments were performed in female mice (4-16 weeks old), maintained on a 12/12 h light/dark cycle (lights on 7 A.M.) and received food ad libitum. Genotypes used were Mecp2-heterozygote (Het; B6.129P2(C)-Mecp2tm1.1Bird/J, JAX # 003890) and Mecp2-WT siblings. All procedures were conducted in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and approved by the University of Tennessee-Knoxville Institutional Animal Care and Use Committee.
Pubertal onset and estrous cyclicity
Pubertal onset was evaluated by daily visual inspection for vaginal opening (VO), starting at postnatal day 21 (P21). Beginning on the day of VO, mice were lavaged daily for at least 2 weeks (adolescent age) to determine estrous cycle length and their relative duration in each cycle phase. The first detected estrus was used as the cycle-start marker. To assess estrous cyclicity in adulthood, 12-week-old mice (P84) underwent daily vaginal lavage for 2 weeks.
Cytology, Imaging and Phase Classification
Vaginal cells were collected by gently flushing the vaginal canal with 50–100 µL Milli-Q water using a glass eyedropper. The fluid was expelled and recollected in a single lavage, and this procedure was not repeated more than twice per session to avoid inducing pseudopregnancy. Samples were placed onto slides and air-dried on a hot plate (~50°C) for 2-3 minutes. Slides were then stained for 2-3 minutes with 0.1% Neutral Red (Sigma-Aldrich, #72210) dissolved in Milli-Q water, gently rinsed with distilled water and air-dried before imaging. Samples were imaged as brightfield montages using a Keyence microscope at 20x magnification. Cyclic phases - proestrus, estrus, metestrus and diestrus - were visually determined from the images by at least three human observers blinded to genotype. Phases were identified based on their characteristic cellular compositions, including nucleated epithelial cells, cornified epithelial cells and leukocytes. The human inter-rater agreement averaged 88.98% across 433 total phase counts, which is consistent with field standards (Caligioni, 2009).
Statistical Analysis
All statistical analyses and graphs were performed in GraphPad Prism 11. Mann-Whitney test was used to compare WT and Het. Statistical significance was defined at p < 0.05. Data are presented as mean ± standard error mean (S.E.M.). The figure was generated in Adobe Illustrator 2025.
","reagents":"","patternDescription":"RTT is a rare neurodevelopmental disorder that predominantly affects girls, with a prevalence of 1 in 10,000 live female births. It is caused by mutations in the X-linked gene, methyl-CpG-binding protein 2 (MECP2) (Amir et al., 1999). RTT patients exhibit typical development until 6-18 months of age, when they experience developmental stagnation followed by a period of regression. Regression is characterized by the loss of previously acquired skills, such as purposeful hand movement, communication skills and motor function, that persists throughout life (Neul et al., 2010; Halbach et al., 2013). Pubertal abnormalities have also been reported in RTT patients. For instance, Killian et al. (2014) sampled 802 RTT patients and reported that 25% exhibited premature thelarche, 28% premature adrenarche and 19% delayed menarche. A subset of RTT patients with milder mutations had less severe phenotype and earlier menarche. Another example was presented in a case study by Holm (1985), where the RTT patient exhibited precocious puberty at the age of 6, and subsequent case studies documented similar findings (Bas et al., 2013; Yang et al., 2021; Canton et al., 2023). Pubertal dysfunction in RTT is further underscored by the high prevalence of catamenial seizures (Humphrey et al., 2021). Despite substantial evidence of linking RTT with abnormal pubertal and reproductive phenotypes, whether these abnormalities contribute to regression remains unclear.
Previously, we identified a regression phenotype in a mouse model of RTT, female Mecp2-heterozygous mice (Het; Guy et al., 2001). At 6 weeks old (adolescent), Het perform as efficiently as wild-type littermate controls (WT) in an alloparental pup retrieval behavior (Mykins et al., 2023). By 12 weeks old, (adult), pre-symptomatic Het regress and perform the same task less efficiently than WT (Krishnan et al., 2017; Stevenson et al., 2021; Mykins et al., 2024). One possible explanation for the Het regression phenotype is the abnormal pubertal development caused by Mecp2 mutations. Here, we tested this hypothesis by examining pubertal onset and estrous cyclicity in adolescent and adult mice. Pubertal onset was determined by the day of vaginal opening, and estrous cyclicity was determined by daily vaginal lavage followed by cytological analysis (Figure 1A). The mouse estrous cycle is divided into 4 phases distinguished by their distinct cellular composition (McLean et al., 2012): proestrus = mostly nucleated epithelial cells, with some cornified epithelial cells; estrus = mainly cornified epithelial cells; metestrus = mostly leukocytes, stringy tissue may be present; and diestrus = mostly leukocytes, with some cornified and nucleated epithelial cells (Figure 1B). We found that puberty onset was similar between WT and Het (Figure 1C; WT: 32.1 ± 0.7 days, n = 28 animals; Het: 33.7 ± 0.6 days, n = 35 animals; Mann-Whitney: p>0.05). In a subset of those mice, we determined the day of first observed estrus, an indirect indicator of reproductive maturation, and we also found no difference between WT and Het (Figure 1D; WT: 37.1 ± 1.0 days, n = 9 animals; Het: 35.44 ± 1.1 days, n = 9 animals; Mann-Whitney: p>0.05). To determine the phases of estrous cycle, cytological images were scored by 3 experimenters who were blinded to the genotypes. The human inter-rater agreement was 88.98%, consistent with field standards (Caligioni, 2009). In adolescence, there were no significant differences between the genotypes for their average estrous cycle length (Figure 1E) (WT: 4.7 ± 0.3 days, n = 8 animals; Het: 4.3 ± 0.3 days, n = 8 animals; Mann-Whitney test: p=0.42) nor their percentage of time spent at the 4 different phases (Figure 1F) (WT: proestrus - 2.0% ± 1.0, estrus - 44.5% ± 2.9, metestrus - 36.9% ± 6.7, diestrus - 16.6% ± 5.0, n = 8 animals; Het: proestrus - 5.3% ± 2.2, estrus – 50.9% ± 4.0, metestrus – 29.5% ± 5.8, diestrus: 14.3% ± 2.4 , n = 8 animals; Mann-Whitney test: p>0.05). In adults, we also did not find significant differences between WT and Het in their average estrous cycle length (Figure 1G) (WT: 4.2 ± 0.2 days, n = 10 animals; Het: 4.6 ± 0.2 days, n = 9 animals; Mann-Whitney test: p=0.41) nor percentage of time spent at the different phases (Figure 1H) (WT: proestrus - 8.0% ± 2.6, estrus – 41.0% ± 5.0, metestrus – 31.6% ± 4.2, diestrus – 19.3% ± 3.5, n = 10 animals; Het: proestrus – 3.3% ± 2.2, estrus – 49.3% ± 3.5, metestrus – 28.7% ± 4.9, diestrus: 18.7% ± 3.0, n = 9 animals; Mann-Whitney test: p>0.05).
Taken together, our results indicate that our female mouse model of RTT, female Mecp2-heterozygous mice (B6.129P2(C)-Mecp2tm1.1Bird/J), exhibits normal pubertal development and maturation of the endocrine system during the pre-symptomatic age. Our findings are consistent with those reported in another female mouse model of RTT (Martin-Sanchez et al., 2026). The same study showed that symptomatic Mecp2-knock out male mice exhibit delayed pubertal onset, supporting the hypothesis that neuroendocrine alterations may emerge as female Mecp2-heterozygous mice age and become symptomatic. Our data further support the idea that the Het regression phenotype in pup retrieval behavior is unlikely to result from gross systemic endocrine disruption and is instead more consistent with age-dependent abnormalities in cellular and circuit plasticity within sensory cortices (Krishnan et al., 2017; Lau et al., 2020a; Lau et al., 2020b). Nevertheless, the absence of overt abnormalities in pubertal timing or estrous cyclicity does not exclude the possibility that more subtle neuroendocrine mechanisms contribute to age-dependent regression in Het. Future studies will be needed to determine whether Mecp2 mutations alter hormone-dependent modulation of maternal behavior and experience-dependent plasticity at the level of specific neural circuits.
","references":[{"reference":"Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. 1999. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nature Genetics 23: 185-188.
","pubmedId":"","doi":"10.1038/13810"},{"reference":"Baş VN, Çetinkaya S, Ağladıoğlu SYl, Aksoy Ae, Gülpınar Ba, Aycan Z. 2013. Report of the first case of precocious puberty in Rett syndrome. jpem 26: 937-939.
","pubmedId":"","doi":" 10.1515/jpem-2012-0418"},{"reference":"Caligioni CS. 2009. Assessing Reproductive Status/Stages in Mice. Current Protocols in Neuroscience 48: 10.1002/0471142301.nsa04is48.
","pubmedId":"","doi":"10.1002/0471142301.nsa04is48"},{"reference":"Canton APM, Tinano FvR, Guasti L, Montenegro LR, Ryan F, Shears D, et al., Latronico. 2023. Rare variants in the MECP2 gene in girls with central precocious puberty: a translational cohort study. The Lancet Diabetes & Endocrinology 11: 545-554.
","pubmedId":"","doi":"10.1016/S2213-8587(23)00131-6"},{"reference":"Guy J, Hendrich B, Holmes M, Martin JE, Bird A. 2001. A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome. Nature Genetics 27: 322-326.
","pubmedId":"","doi":"10.1038/85899"},{"reference":"Halbach N, Smeets E, Steinbusch C, Maaskant M, van Waardenburg D, Curfs L. 2012. Aging in Rett syndrome: a longitudinal study. Clinical Genetics 84: 223-229.
","pubmedId":"","doi":"10.1111/cge.12063"},{"reference":"Holm VA. 1985. Rett's syndrome: a progressive developmental disability in girls. J Dev Behav Pediatr 6(1):32-36. PMID:3973029.
","pubmedId":"","doi":""},{"reference":"Humphrey KN, Horn PS, Olshavsky L, Reebals L, Standridge SM. 2021. Features of Menstruation and Menstruation Management in Individuals with Rett Syndrome. Journal of Pediatric and Adolescent Gynecology 34: 144-153.
","pubmedId":"","doi":" 10.1016/j.jpag.2020.11.002"},{"reference":"Killian JT, Lane JB, Cutter GR, Skinner SA, Kaufmann WE, Tarquinio DC, et al., Percy. 2014. Pubertal Development in Rett Syndrome Deviates From Typical Females. Pediatric Neurology 51: 769-775.
","pubmedId":"","doi":"10.1016/j.pediatrneurol.2014.08.013"},{"reference":"Krishnan K, Lau BYB, Ewall G, Huang ZJ, Shea SD. 2017. MECP2 regulates cortical plasticity underlying a learned behaviour in adult female mice. Nature Communications 8: 10.1038/ncomms14077.
","pubmedId":"","doi":"10.1038/ncomms14077"},{"reference":"Lau BYB, Krishnan K, Huang ZJ, Shea SD. 2020a. Maternal Experience-Dependent Cortical Plasticity in Mice Is Circuit- and Stimulus-Specific and Requires MECP2. The Journal of Neuroscience 40: 1514-1526.
","pubmedId":"","doi":" 10.1523/JNEUROSCI.1964-19.2019"},{"reference":"Lau BYB, Layo DE, Emery B, Everett M, Kumar A, Stevenson P, et al., Krishnan. 2020b. Lateralized Expression of Cortical Perineuronal Nets during Maternal Experience is Dependent on MECP2. eneuro 7: ENEURO.0500-19.2020.
","pubmedId":"","doi":": 10.1523/ENEURO.0500-19.2020"},{"reference":"Martín‐Sánchez A, Jiménez‐Díaz D, Esteve‐Pérez R, Vasile‐Tudorache A, Read JE, Howard SR, Agustín‐Pavón C. 2026. Pubertal development and hypothalamic–pituitary–gonadal axis are altered in male mice lacking\n Mecp2. Journal of Neuroendocrinology 38: 10.1111/jne.70221.
","pubmedId":"","doi":"10.1111/jne.70221"},{"reference":"McLean AC, Valenzuela N, Fai S, Bennett SAL. 2012. Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification. Journal of Visualized Experiments : 10.3791/4389.
","pubmedId":"","doi":" 10.3791/4389"},{"reference":"Mykins M, Layo‐Carris D, Dunn LR, Skinner DW, McBryar AH, Perez S, et al., Krishnan. 2023. Wild‐type\n
Mykins M, Bridges B, Jo A, Krishnan K. 2024. Multidimensional analysis of a social behavior identifies regression and phenotypic heterogeneity in a female mouse model for Rett syndrome. The Journal of Neuroscience : e1078232023.
","pubmedId":"","doi":"10.1523/JNEUROSCI.1078-23.2023"},{"reference":"Neul JL, Kaufmann WE, Glaze DG, Christodoulou J, Clarke AJ, Bahi-Buisson N, et al, Percy AK; RettSearch Consortium. 2010. Rett syndrome: Revised diagnostic criteria and nomenclature. Annals of Neurology 68: 944-950.
","pubmedId":"","doi":" 10.1002/ana.22124"},{"reference":"Stevenson PK, Casenhiser DM, Lau BYB, Krishnan K. 2021. Systematic analysis of goal‐related movement sequences during maternal behaviour in a female mouse model for Rett syndrome. European Journal of Neuroscience 54: 4528-4549.
","pubmedId":"","doi":"10.1111/ejn.15327"},{"reference":"Yang L, Jiang M, Yu R, Hu R, Xiong F, Li J. 2021. A case report of precocious puberty related to Rett syndrome and a literature review. Pharmazie 76(11):559-561.
","pubmedId":"","doi":" 10.1691/ph.2021.1747"}],"suggestedReviewer":{"name":"Carmen Agustín‐Pavón, Department of Cell Biology and Functional Biology, Faculty of Biological Sciences, Universitat de Valencia, València, Spain. Email: m.carmen.agustin@uv.es
","WBId":""},"title":"Pubertal development is unaffected in female Mecp2-heterozygous mice.
","reviews":[]},{"id":"36f95cdd-d376-4967-ac62-bf45817fc856","decisionLetter":"\n\n Dear Authors,\n
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\n \"Fortich S, Demilio L, Ceesay M, Clower A, Lau BYB. 2026. Pubertal development is unaffected in female Mecp2-heterozygous mice.. microPublication Biology. 10.17912/micropub.biology.002400.\"\n
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\n ","decision":"publish","submitted":true,"abstract":"Rett syndrome (RTT) affects predominantly females and is characterized by regression after an initial period of typical development. Previously, we showed that while adolescent female Mecp2-heterozygous mice (Het, mouse model of RTT) perform pup retrieval comparably to wild-type littermate controls (WT), adult Het regress and become inefficient. Here, we analyzed the pubertal trajectory of naïve nulliparous WT and Het to test whether abnormal pubertal development contributes to this regression phenotype. We found no genotypic differences in pubertal onset nor estrous cyclicity during adolescence and adulthood. These findings further support the role of Mecp2 in experience-dependent plasticity within sensory cortices.
","acknowledgements":"We thank Drs. Kalynn Schulz and Marcia Chavez for training us in vaginal lavage and estrous phase staging. We also thank graduate student Aly Webb and undergraduate students Kiran Hussaini, Roshni Rameshkumar, Faith Webb and Samantha Webber for their contributions to data collection.
","authors":[{"affiliations":["University of Tennessee, Knoxville, TN, United States"],"credit":["formalAnalysis","methodology","writing_reviewEditing","writing_originalDraft","dataCuration","validation"],"email":"sfortich@vols.utk.edu","firstName":"Skylar","lastName":"Fortich","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Tennessee, Knoxville, TN, United States"],"credit":["formalAnalysis","methodology","writing_originalDraft","writing_reviewEditing","dataCuration","visualization"],"email":"ldemilio@vols.utk.edu","firstName":"Lily","lastName":"Demilio","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Tennessee, Knoxville, TN, United States"],"credit":["dataCuration","methodology","validation"],"email":"mceesay@vols.utk.edu","firstName":"Mariam","lastName":"Ceesay","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Tennessee, Knoxville, TN, United States"],"credit":["formalAnalysis","methodology","dataCuration"],"email":"anabelleclower26@gmail.com","firstName":"Anabelle","lastName":"Clower","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Tennessee, Knoxville, TN, United States"],"credit":["conceptualization","fundingAcquisition","methodology","project","supervision","visualization","writing_reviewEditing","writing_originalDraft","validation"],"email":"blau@utk.edu","firstName":"Billy You Bun","lastName":"Lau","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-2380-5341"}],"comments":"We made minor text edits for grammar, spelling and clarity. Please let me know if you would like a list of all the changes made. I'm happy to see another proof if needed. I am waiting on my university's decision on whether they can support this publication fee. Thank you!
","dataTable":{"name":null,"url":null},"disclaimer":true,"funding":"The author(s) declared that financial support was received for this work and/or its publication. This work was supported by UTK startup fund awarded to BYBL, UTK Faculty Research Assistants Funding awarded to BYBL to support SF and LD, and UTK Advanced Undergraduate Research Activity awarded to SF, LD and AC. Funding for open access to this research was provided by University of Tennessee's Open Publishing Support Fund.
","image":{"name":"Figure 1.jpg","url":"https://portal.micropublication.org/uploads/e9a26341e519b0b5f075b0e79784a852.jpg"},"imageCaption":"A. Schema of experimental design. Upon vaginal opening, animals were lavaged daily up to postnatal day 55 (P55), and lavaged again from P84-98. Cytology and imaging were performed to determine estrous phase. B. Example bright field images showing proestrus, estrus, metestrus and diestrus. Black arrows = nucleated epithelial cells, black arrowheads = cornified epithelial cells, white arrows = leukocytes. C-D. Vaginal opening (puberty onset; C) and first estrus (D) were not significantly different between WT (n=28, 9, respectively) and Het (n=35, 9, respectively). E-H. There were no significant differences between WT and Het in average cycle length during adolescence (E) and adulthood (G) nor in % time spent in the 4 different estrous phases during adolescence (F) and adulthood (H). E-F: WT (n=8) and Het (n=8). G-H: WT (n=10) and Het (n=9). C-H: Scatter plots: each dot represents an animal, lines are mean ± S.E.M. Mann-Whitney test: p>0.05, n.s. = no significance.
","imageTitle":"Pubertal onset and estrous cycling are not affected by Mecp2 mutations
","laboratory":{"name":"","WBId":""},"methods":"Animals
All experiments were performed in female mice (4-16 weeks old), maintained on a 12/12 h light/dark cycle (lights on 7 A.M.) and received food ad libitum. Genotypes used were Mecp2-heterozygote (Het; B6.129P2(C)-Mecp2tm1.1Bird/J, JAX # 003890) and Mecp2-WT siblings. All procedures were conducted in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and approved by the University of Tennessee-Knoxville Institutional Animal Care and Use Committee.
Pubertal onset and estrous cyclicity
Pubertal onset was evaluated by daily visual inspection for vaginal opening (VO), starting at postnatal day 21 (P21). Beginning on the day of VO, mice were lavaged daily for at least 2 weeks (adolescent age) to determine estrous cycle length and their relative duration in each cycle phase. The first detected estrus was used as the cycle-start marker. To assess estrous cyclicity in adulthood, 12-week-old mice (P84) underwent daily vaginal lavage for 2 weeks.
Cytology, Imaging and Phase Classification
Vaginal cells were collected by gently flushing the vaginal canal with 50–100 µL Milli-Q water using a glass eyedropper. The fluid was expelled and recollected in a single lavage, and this procedure was not repeated more than twice per session to avoid inducing pseudopregnancy. Samples were placed onto slides and air-dried on a hot plate (~50°C) for 2-3 minutes. Slides were then stained for 2-3 minutes with 0.1% Neutral Red (Sigma-Aldrich, #72210) dissolved in Milli-Q water, gently rinsed with distilled water and air-dried before imaging. Samples were imaged as brightfield montages using a Keyence microscope at 20x magnification. Cyclic phases - proestrus, estrus, metestrus and diestrus - were visually determined from the images by at least three human observers blinded to genotype. Phases were identified based on their characteristic cellular compositions, including nucleated epithelial cells, cornified epithelial cells and leukocytes. The human inter-rater agreement averaged 88.98% across 433 total phase counts, which is consistent with field standards (Caligioni, 2009).
Statistical Analysis
All statistical analyses and graphs were performed in GraphPad Prism 11. Mann-Whitney test was used to compare WT and Het. Statistical significance was defined at p < 0.05. Data are presented as mean ± standard error mean (S.E.M.). The figure was generated in Adobe Illustrator 2025.
","reagents":"","patternDescription":"RTT is a rare neurodevelopmental disorder that predominantly affects girls, with a prevalence of 1 in 10,000 live female births. It is caused by mutations in the X-linked gene, methyl-CpG-binding protein 2 (MECP2) (Amir et al., 1999). RTT patients exhibit typical development until 6-18 months of age, when they experience developmental stagnation followed by a period of regression. Regression is characterized by the loss of previously acquired skills, such as purposeful hand movement, communication skills and motor function, that persists throughout life (Neul et al., 2010; Halbach et al., 2013). Pubertal abnormalities have also been reported in RTT patients. For instance, Killian et al. (2014) sampled 802 RTT patients and reported that 25% exhibited premature thelarche, 28% premature adrenarche and 19% delayed menarche. A subset of RTT patients with milder mutations had less severe phenotype and earlier menarche. Another example was presented in a case study by Holm (1985), where the RTT patient exhibited precocious puberty at the age of 6, and subsequent case studies documented similar findings (Bas et al., 2013; Yang et al., 2021; Canton et al., 2023). Pubertal dysfunction in RTT is further underscored by the high prevalence of catamenial seizures (Humphrey et al., 2021). Despite substantial evidence of linking RTT with abnormal pubertal and reproductive phenotypes, whether these abnormalities contribute to regression remains unclear.
Previously, we identified a regression phenotype in a mouse model of RTT, female Mecp2-heterozygous mice (Het; Guy et al., 2001). At 6 weeks old (adolescent), Het perform as efficiently as wild-type littermate controls (WT) in an alloparental pup retrieval behavior (Mykins et al., 2023). By 12 weeks old, (adult), pre-symptomatic Het regress and perform the same task less efficiently than WT (Krishnan et al., 2017; Stevenson et al., 2021; Mykins et al., 2024). One possible explanation for the Het regression phenotype is the abnormal pubertal development caused by Mecp2 mutations. Here, we tested this hypothesis by examining pubertal onset and estrous cyclicity in adolescent and adult mice. Pubertal onset was determined by the day of vaginal opening, and estrous cyclicity was determined by daily vaginal lavage followed by cytological analysis (Figure 1A). The mouse estrous cycle is divided into 4 phases distinguished by their distinct cellular composition (McLean et al., 2012): proestrus = mostly nucleated epithelial cells, with some cornified epithelial cells; estrus = mainly cornified epithelial cells; metestrus = mostly leukocytes, stringy tissue may be present; and diestrus = mostly leukocytes, with some cornified and nucleated epithelial cells (Figure 1B). We found that puberty onset was similar between WT and Het (Figure 1C; WT: 32.1 ± 0.7 days, n = 28 animals; Het: 33.7 ± 0.6 days, n = 35 animals; Mann-Whitney: p>0.05). In a subset of those mice, we determined the day of first observed estrus, an indirect indicator of reproductive maturation, and we also found no difference between WT and Het (Figure 1D; WT: 37.1 ± 1.0 days, n = 9 animals; Het: 35.44 ± 1.1 days, n = 9 animals; Mann-Whitney: p>0.05). To determine the phases of estrous cycle, cytological images were scored by 3 experimenters who were blinded to the genotypes. The human inter-rater agreement was 88.98%, consistent with field standards (Caligioni, 2009). In adolescence, there were no significant differences between the genotypes for their average estrous cycle length (Figure 1E) (WT: 4.7 ± 0.3 days, n = 8 animals; Het: 4.3 ± 0.3 days, n = 8 animals; Mann-Whitney test: p=0.42) nor their percentage of time spent at the 4 different phases (Figure 1F) (WT: proestrus - 2.0% ± 1.0, estrus - 44.5% ± 2.9, metestrus - 36.9% ± 6.7, diestrus - 16.6% ± 5.0, n = 8 animals; Het: proestrus - 5.3% ± 2.2, estrus – 50.9% ± 4.0, metestrus – 29.5% ± 5.8, diestrus: 14.3% ± 2.4 , n = 8 animals; Mann-Whitney test: p>0.05). In adults, we also did not find significant differences between WT and Het in their average estrous cycle length (Figure 1G) (WT: 4.2 ± 0.2 days, n = 10 animals; Het: 4.6 ± 0.2 days, n = 9 animals; Mann-Whitney test: p=0.41) nor percentage of time spent at the different phases (Figure 1H) (WT: proestrus - 8.0% ± 2.6, estrus – 41.0% ± 5.0, metestrus – 31.6% ± 4.2, diestrus – 19.3% ± 3.5, n = 10 animals; Het: proestrus – 3.3% ± 2.2, estrus – 49.3% ± 3.5, metestrus – 28.7% ± 4.9, diestrus: 18.7% ± 3.0, n = 9 animals; Mann-Whitney test: p>0.05).
Taken together, our results indicate that our female mouse model of RTT, female Mecp2-heterozygous mice (B6.129P2(C)-Mecp2tm1.1Bird/J), exhibits normal pubertal development and maturation of the endocrine system during the pre-symptomatic age. Our findings are consistent with those reported in another female mouse model of RTT (Martin-Sanchez et al., 2026). The same study showed that symptomatic Mecp2-knock out male mice exhibit delayed pubertal onset, supporting the hypothesis that neuroendocrine alterations may emerge as female Mecp2-heterozygous mice age and become symptomatic. Our data further support the idea that the Het regression phenotype in pup retrieval behavior is unlikely to result from gross systemic endocrine disruption and is instead more consistent with age-dependent abnormalities in cellular and circuit plasticity within sensory cortices (Krishnan et al., 2017; Lau et al., 2020a; Lau et al., 2020b). Nevertheless, the absence of overt abnormalities in pubertal timing or estrous cyclicity does not exclude the possibility that more subtle neuroendocrine mechanisms contribute to age-dependent regression in Het. Future studies will be needed to determine whether Mecp2 mutations alter hormone-dependent modulation of maternal behavior and experience-dependent plasticity at the level of specific neural circuits.
","references":[{"reference":"Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. 1999. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nature Genetics 23: 185-188.
","pubmedId":"","doi":"10.1038/13810"},{"reference":"Baş VN, Çetinkaya S, Ağladıoğlu SYl, Aksoy Ae, Gülpınar Ba, Aycan Z. 2013. Report of the first case of precocious puberty in Rett syndrome. jpem 26: 937-939.
","pubmedId":"","doi":" 10.1515/jpem-2012-0418"},{"reference":"Caligioni CS. 2009. Assessing Reproductive Status/Stages in Mice. Current Protocols in Neuroscience 48: 10.1002/0471142301.nsa04is48.
","pubmedId":"","doi":"10.1002/0471142301.nsa04is48"},{"reference":"Canton APM, Tinano FvR, Guasti L, Montenegro LR, Ryan F, Shears D, et al., Latronico. 2023. Rare variants in the MECP2 gene in girls with central precocious puberty: a translational cohort study. The Lancet Diabetes & Endocrinology 11: 545-554.
","pubmedId":"","doi":"10.1016/S2213-8587(23)00131-6"},{"reference":"Guy J, Hendrich B, Holmes M, Martin JE, Bird A. 2001. A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome. Nature Genetics 27: 322-326.
","pubmedId":"","doi":"10.1038/85899"},{"reference":"Halbach N, Smeets E, Steinbusch C, Maaskant M, van Waardenburg D, Curfs L. 2012. Aging in Rett syndrome: a longitudinal study. Clinical Genetics 84: 223-229.
","pubmedId":"","doi":"10.1111/cge.12063"},{"reference":"Holm VA. 1985. Rett's syndrome: a progressive developmental disability in girls. J Dev Behav Pediatr 6(1):32-36. PMID:3973029.
","pubmedId":"","doi":""},{"reference":"Humphrey KN, Horn PS, Olshavsky L, Reebals L, Standridge SM. 2021. Features of Menstruation and Menstruation Management in Individuals with Rett Syndrome. Journal of Pediatric and Adolescent Gynecology 34: 144-153.
","pubmedId":"","doi":" 10.1016/j.jpag.2020.11.002"},{"reference":"Killian JT, Lane JB, Cutter GR, Skinner SA, Kaufmann WE, Tarquinio DC, et al., Percy. 2014. Pubertal Development in Rett Syndrome Deviates From Typical Females. Pediatric Neurology 51: 769-775.
","pubmedId":"","doi":"10.1016/j.pediatrneurol.2014.08.013"},{"reference":"Krishnan K, Lau BYB, Ewall G, Huang ZJ, Shea SD. 2017. MECP2 regulates cortical plasticity underlying a learned behaviour in adult female mice. Nature Communications 8: 10.1038/ncomms14077.
","pubmedId":"","doi":"10.1038/ncomms14077"},{"reference":"Lau BYB, Krishnan K, Huang ZJ, Shea SD. 2020a. Maternal Experience-Dependent Cortical Plasticity in Mice Is Circuit- and Stimulus-Specific and Requires MECP2. The Journal of Neuroscience 40: 1514-1526.
","pubmedId":"","doi":" 10.1523/JNEUROSCI.1964-19.2019"},{"reference":"Lau BYB, Layo DE, Emery B, Everett M, Kumar A, Stevenson P, et al., Krishnan. 2020b. Lateralized Expression of Cortical Perineuronal Nets during Maternal Experience is Dependent on MECP2. eneuro 7: ENEURO.0500-19.2020.
","pubmedId":"","doi":": 10.1523/ENEURO.0500-19.2020"},{"reference":"Martín‐Sánchez A, Jiménez‐Díaz D, Esteve‐Pérez R, Vasile‐Tudorache A, Read JE, Howard SR, Agustín‐Pavón C. 2026. Pubertal development and hypothalamic–pituitary–gonadal axis are altered in male mice lacking\n Mecp2. Journal of Neuroendocrinology 38: 10.1111/jne.70221.
","pubmedId":"","doi":"10.1111/jne.70221"},{"reference":"McLean AC, Valenzuela N, Fai S, Bennett SAL. 2012. Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification. Journal of Visualized Experiments : 10.3791/4389.
","pubmedId":"","doi":" 10.3791/4389"},{"reference":"Mykins M, Layo‐Carris D, Dunn LR, Skinner DW, McBryar AH, Perez S, et al., Krishnan. 2023. Wild‐type\n
Mykins M, Bridges B, Jo A, Krishnan K. 2024. Multidimensional analysis of a social behavior identifies regression and phenotypic heterogeneity in a female mouse model for Rett syndrome. The Journal of Neuroscience : e1078232023.
","pubmedId":"","doi":"10.1523/JNEUROSCI.1078-23.2023"},{"reference":"Neul JL, Kaufmann WE, Glaze DG, Christodoulou J, Clarke AJ, Bahi-Buisson N, et al, Percy AK; RettSearch Consortium. 2010. Rett syndrome: Revised diagnostic criteria and nomenclature. Annals of Neurology 68: 944-950.
","pubmedId":"","doi":" 10.1002/ana.22124"},{"reference":"Stevenson PK, Casenhiser DM, Lau BYB, Krishnan K. 2021. Systematic analysis of goal‐related movement sequences during maternal behaviour in a female mouse model for Rett syndrome. European Journal of Neuroscience 54: 4528-4549.
","pubmedId":"","doi":"10.1111/ejn.15327"},{"reference":"Yang L, Jiang M, Yu R, Hu R, Xiong F, Li J. 2021. A case report of precocious puberty related to Rett syndrome and a literature review. Pharmazie 76(11):559-561.
","pubmedId":"","doi":" 10.1691/ph.2021.1747"}],"suggestedReviewer":{"name":"Carmen Agustín‐Pavón, Department of Cell Biology and Functional Biology, Faculty of Biological Sciences, Universitat de Valencia, València, Spain. Email: m.carmen.agustin@uv.es
","WBId":""},"title":"Pubertal development is unaffected in female Mecp2-heterozygous mice.
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