Biology of Reproduction最新文献

{"title":"Disruption of oocyte SUMOylation impacts critical regulatory processes during folliculogenesis in mice.","authors":"Zian Liao, Tessa E Steenwinkel, Bruno Moscoso, Ernesto Salas, Bethany K Patton, Amanda Rodriguez, Anna Malovannaya, Stephanie A Pangas","doi":"10.1093/biolre/ioaf035","DOIUrl":"10.1093/biolre/ioaf035","url":null,"abstract":"<p><p>The conjugation of small ubiquitin-like modifiers (SUMO) to target proteins, known as SUMOylation, plays a crucial role in regulating protein homeostasis, activity, interaction with other proteins, and subcellular localization. Loss of SUMOylation in non-growing oocytes by conditional deletion of the E2 sumo conjugating enzyme, Ube2i, at the primordial follicle stage leads to female sterility due to complex changes in oocyte development, including altered folliculogenesis, defective meiotic progression, and premature loss of the ovarian reserve. In this study, proteomics was used to compare control and Ube2i conditional knockout ovaries during the first wave of folliculogenesis to identify key differences that may drive the premature follicle loss phenotype. Data are available via ProteomeXchange with identifier PXD055913. Label-free mass spectrometry results showed that 238 proteins were significantly altered more than 2-fold (P < 0.05). Proteins upregulated in the Ube2i conditional knockout ovaries included those involved in mRNA splicing and WNT signaling, while those downregulated were related to metabolism, mitochondria, and the maternal effect proteins NLRP2 and NLRP9B. The majority of differentially expressed proteins showed no change by transcriptome analysis, indicating protein level regulation and revealing potential SUMOylation targets with necessary roles in oocyte and follicle development.</p>","PeriodicalId":8965,"journal":{"name":"Biology of Reproduction","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143466525","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Age-Related Integrative Transcriptomic Profiling of Human Granulosa Cells Reveals mRNA-microRNA Regulatory Network Associated with Key Ovulation Dynamics.","authors":"Heather M Rogers, Ahmed Gad, Gentry K Cork, Nico G Menjivar, William B Schoolcraft, Dawit Tesfaye, Ye Yuan","doi":"10.1093/biolre/ioaf034","DOIUrl":"https://doi.org/10.1093/biolre/ioaf034","url":null,"abstract":"<p><p>Advanced maternal age (AMA) patients experience decreased success from assisted reproductive technologies (ART), attributed to the quantity and quality of oocytes, which is significantly influenced by the intrafollicular granulosa cells (GCs). In this study, we compared the mRNA and microRNA (miRNA) transcriptomes between young (< 32 y.o.) and AMA (> 38 y.o.) patients' GCs to identify potential ovarian aging-related molecular signatures. We identified 293 and 21 differentially expressed genes (DEGs) and miRNAs (DE miRNAs), respectively, between young and aged GCs. Highly expressed mitochondrial-encoded genes, MT-ND3, MT-ND6, and MT-CYB, were downregulated in aged GCs, indicating potential mitochondrial insufficiency. Additionally, pathway analysis indicates DEGs are involved in inflammation, cytokine signaling, extracellular matrix (ECM) remodeling, and angiogenesis. Key DEGs related to these processes include CXCL8, IL1B, NLRP3, SIGIRR, ANGPT2, ADAM8, and ADAMTS14. Additionally, target gene prediction and pathway analysis of DE miRNAs indicates their potential post-transcriptional regulation of genes associated with cell signaling, mitochondrial function, oxidative stress, apoptosis, and senescence pathways in addition to cytokine signaling, angiogenesis, and ECM remodeling. To investigate regulatory mechanisms further, we looked at the DEGs' convergence with the DE miRNAs predicted target genes and we identified miR-483-3p, miR-1268a, miR-4497, miR-7704, miR-135a-5p, miR-1261, and miR-4791 as potential crucial regulators of genes involved in pathways associated with inflammation, ECM, and angiogenesis. This data suggests that aged GCs have an impaired ability to elicit the same pro-inflammatory response combined with dysregulation of angiogenesis and ECM remodeling compared to young GCs, and miRNA may play a role in regulating key ovulatory processes. While this study identifies potential regulatory relationships between DE miRNAs and DEGs, experimental validation is necessary to confirm the relationships and biological relevance.</p>","PeriodicalId":8965,"journal":{"name":"Biology of Reproduction","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143466397","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unveiling the role of FOXL2 in female differentiation and disease: a comprehensive review.","authors":"Jia He, Zican Wang, Lici Yang, Yongjian Jiang, Ge Yan, Yongwei Pan, Fei Gao, Jinxiang Yuan, Yang Gao","doi":"10.1093/biolre/ioaf013","DOIUrl":"https://doi.org/10.1093/biolre/ioaf013","url":null,"abstract":"<p><p>Ovarian differentiation relies on the accurate and orderly expression of numerous related genes. Forkhead box protein L2 (FOXL2) is one of the earliest ovarian differentiation markers and transcription factors. In sex determination, FOXL2 maintains the differentiation of the female pathway by inhibiting male differentiation genes, including SOX9 and SF1. In addition, FOXL2 promotes the synthesis of follicle-stimulating hormone and anti-Müllerian hormone to support follicle development. Mutations in FOXL2 are associated with numerous female reproductive diseases. A comprehensive and in-depth study of FOXL2 provides novel strategies for the diagnosis and treatment of such diseases. This review discusses the mechanism of FOXL2 in female sex differentiation and maintenance, hormone synthesis, and disease occurrence and reveals the role of FOXL2 as a central factor in female sex development and fertility maintenance. This review will serve as a reference for identifying novel targets of other regulatory factors interacting with FOXL2 in female sex determination and follicle development and for the diagnosis and treatment of female reproductive diseases.</p>","PeriodicalId":8965,"journal":{"name":"Biology of Reproduction","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143456650","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"SATINN v2: automated image analysis for mouse testis histology with multi-laboratory data integration.","authors":"Ran Yang, Fritzie T Celino-Brady, Jessica E M Dunleavy, Katinka A Vigh-Conrad, Georgia Rae Atkins, Rachel L Hvasta, Christopher R X Pombar, Alexander N Yatsenko, Kyle E Orwig, Moira K O'Bryan, Ana C Lima, Donald F Conrad","doi":"10.1093/biolre/ioaf033","DOIUrl":"10.1093/biolre/ioaf033","url":null,"abstract":"<p><p>Analysis of testis histology is fundamental to the study of male fertility, but it is a slow task with a high skill threshold. Here, we describe new neural network models for the automated classification of cell types and tubule stages from whole-slide brightfield images of mouse testis. The cell type classifier recognizes 14 cell types, including multiple steps of meiosis I prophase, with an external validation accuracy of 96%. The tubule stage classifier distinguishes all 12 canonical tubule stages with external validation accuracy of 63%, which increases to 96% when allowing for ±1 stage tolerance. We addressed generalizability of SATINN, through extensive training diversification and testing on external (non-training population) wildtype and mutant datasets. This allowed us to use SATINN to successfully process data generated in multiple laboratories. We used SATINN to analyze testis images from 8 different mutant lines, generated from 3 different labs with a range of tissue processing protocols. Finally, we show that it is possible to use SATINN output to cluster histology images in latent space, which, when applied to the 8 mutant lines, reveals known relationships in their pathology. This work represents significant progress towards a tool for robust, automated testis histopathology that can be used by multiple labs.</p>","PeriodicalId":8965,"journal":{"name":"Biology of Reproduction","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143439663","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Developmental programming: preconceptional and gestational exposure of sheep to biosolids on offspring ovarian dynamics†.","authors":"Katherine M Halloran, Yiran Zhou, Michelle Bellingham, Richard G Lea, Neil P Evans, Kevin D Sinclair, Peter Smith, Vasantha Padmanabhan","doi":"10.1093/biolre/ioae166","DOIUrl":"10.1093/biolre/ioae166","url":null,"abstract":"<p><p>Developmental exposure to environmental chemicals perturbs establishment and maintenance of the ovarian reserve across the reproductive lifetime, leading to premature follicle depletion and ovarian aging. Considering humans are exposed to a complex mixture of environmental chemicals, real-life models assessing their cumulative impact on the ovarian reserve are needed. Biosolids are a source of a real-life mixture of environmental chemicals. While earlier studies demonstrated that grazing pregnant sheep on biosolids-treated pastures did not influence establishment of the ovarian reserve in fetal life, its impact on subsequent depletion of ovarian reserve during reproductive life of offspring is unknown. We hypothesized that developmental exposure to biosolids accelerates depletion of ovarian reserve. Ovaries were collected from F1 juveniles (9.5 weeks) and adults (2.5 years) born to F0 ewes grazed on control inorganic fertilizer pastures or biosolids-treated pastures from before conception and throughout gestation. The impact on follicular density, activation rate, and anti-Müllerian hormone (mediator of activation) expression by immunohistochemistry was determined. Activation rate was increased in F1 biosolids-treated pastures juveniles with a corresponding reduction in primordial follicle density. In contrast, activation rate and ovarian reserve were similar between control and F1 biosolids-treated pastures adults. The density of anti-Müllerian hormone-positive antral follicles was lower in biosolids-treated pastures juveniles, whereas anti-Müllerian hormone expression tended to be higher in antral follicles of biosolids-treated pastures adults, consistent with the changes in the ovarian reserve. These findings of detrimental effects of developmental exposure to biosolids during juvenile life that normalizes in adults is supportive of a shift in activation rate likely related to peripubertal hormonal changes.</p>","PeriodicalId":8965,"journal":{"name":"Biology of Reproduction","volume":" ","pages":"331-345"},"PeriodicalIF":3.1,"publicationDate":"2025-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11833488/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142675034","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"SCGB1D4 downregulation links to fibrosis in intrauterine adhesion patients and rat models†.","authors":"Jing Zhao, Yuanhui Wang, Chanchan Ma, Yifan Feng, Yunmeng Wang, Shiying Sun","doi":"10.1093/biolre/ioae172","DOIUrl":"10.1093/biolre/ioae172","url":null,"abstract":"<p><p>Intrauterine adhesions (IUA) represent a prevalent uterine endometrial disorder frequently correlated with menstrual irregularities and infertility. Some members of the secretoglobin(SCGB) family have demonstrated anti-fibrotic effects, however, the specific role of SCGB1D4, one of the family members, in anti-fibrosis remains unclear. This study aimed to investigate the expression of SCGB1D4 in IUA tissues, validate the role of SCGB1D4 in endometrial fibrosis, and assess its potential therapeutic significance by analyzing clinical features and constructing rat and cell models. Clinical characteristics of patients with intrauterine adhesions (IUA) were compared and analyzed against control subjects. Additionally, a rat uterine adhesion model was successfully established using a combination of mechanical injury and infection. The expression levels of SCGB1D4 in patient tissues and animal models were detected through immunohistochemistry, Western blot, and real-time fluorescence quantitative PCR, and the changes in fibrosis markers COL1A1 and α-SMA were also evaluated. Furthermore, human endometrial stromal cell lines (HESCs) induced by transforming growth factor-β-1 conversion were differentiated into myofibroblasts to establish cell models of intrauterine adhesion. We detected the expression of SCGB1D4 and fibrosis-related factors by real-time fluorescence quantitative PCR and Western blot. Cell proliferation and cell cycle changes were assessed using flow cytometry and CCK8. IUA patients showed increased miscarriage rates and decreased endometrial thickness. Clinical tissue specimens revealed significantly lower expression of SCGB1D4 in the endometrial tissues of IUA patients, accompanied by a notable increase in COL1A1 and α-SMA. The established rat model of intrauterine adhesion exhibited decreased expression of SCGB1D4 and a significant increase in fibrosis. After overexpression of SCGB1D4 on the IUA cell model, SCGB1D4 expression was elevated, while COL1A1 and α-SMA expression was significantly reduced. Cell proliferation was inhibited and cell cycle distribution was altered. This study has confirmed the low expression of SCGB1D4 in patients with IUA, as well as in animal and cell models. Furthermore, the overexpression of SCGB1D4 in a cell model of IUA demonstrates that it may play a key role in inhibiting fibrosis. SCGB1D4 holds promise as a potential therapeutic target for IUA, providing a new avenue for overcoming fertility issues caused by IUA.</p>","PeriodicalId":8965,"journal":{"name":"Biology of Reproduction","volume":" ","pages":"273-285"},"PeriodicalIF":3.1,"publicationDate":"2025-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142715286","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Preliminary urinary profiles of prolactin during gestation and the synergistic effects of embryonic diapause hormones in the giant panda (Ailuropoda melanoleuca)†.","authors":"Wang Shenfei, Zhang Mengshi, Li Feiping, Hu Xianbiao, Li Yan, Wang Juan, Liu Xiangyu, Hou Rong, Liu Yuliang, Cai Kailai","doi":"10.1093/biolre/ioae178","DOIUrl":"10.1093/biolre/ioae178","url":null,"abstract":"<p><p>The embryonic diapause of the giant panda (Ailuropoda melanoleuca) has caused great difficulties in monitoring pregnancy in this vulnerable species. The secretion of prolactin (PRL) from anterior pituitary glandular lactotropic cells is an important signal for the termination of embryonic dormancy. Currently, the mechanism by which PRL affects embryonic diapause in giant pandas and methods for detecting PRL in this species is poorly understood. In this study, the first sandwich enzyme immunoassay for detecting PRL in giant panda urine was established by using two antigiant panda PRL antibodies prepared as coating and labeling antibodies, and PRL recombinant proteins prepared via the prokaryotic system as standards. The established method was used to detect the levels of PRL in the urine of giant pandas during pregnancy. At the same time, the changes in PRL levels in giant pandas and the relationship between PRL and progestagen levels were analyzed during the luteal phase. The results showed that in female giant pandas, PRL levels significantly increased before the progestagen peak, and during the luteal phase, the PRL level was significantly greater in giant pandas that gave birth than in those that did not give birth and those in the nonestrus group. To the best of our knowledge, this is the first study to preliminarily explore the mode of action of PRL in the gestation period of giant pandas and lays a foundation for further study of the regulatory mechanisms of endocrine hormones in the giant panda.</p>","PeriodicalId":8965,"journal":{"name":"Biology of Reproduction","volume":" ","pages":"319-330"},"PeriodicalIF":3.1,"publicationDate":"2025-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142827190","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The effects of bisphenol A and its analogs on steroidogenesis in MA-10 Leydig cells and KGN granulosa cells†.","authors":"Lama Iskandarani, Sabrina Romanelli, Barbara F Hales, Bernard Robaire","doi":"10.1093/biolre/ioae165","DOIUrl":"10.1093/biolre/ioae165","url":null,"abstract":"<p><p>Bisphenols are a family of chemicals used in the manufacture of consumer products containing polycarbonate plastics and epoxy resins. Studies have shown that exposure to bisphenol A (BPA) may disrupt steroidogenesis and induce adverse effects on male and female reproduction, but little is known about BPA replacements. We determined the effects of six bisphenols on the steroidogenic function of MA-10 Leydig cells and KGN granulosa cells by measuring the levels of progesterone and estradiol produced by these cells as well as the expression of transcripts involved in steroid and cholesterol biosynthesis. MA-10 and KGN cells were exposed for 48 h to one of six bisphenols (0.01-50 μM): BPA, bisphenol F, bisphenol S, bisphenol AF, bisphenol M, or bisphenol TMC, under both basal and dibutyryl cAMP (Bu2cAMP)-stimulated conditions. In MA-10 cells, most bisphenols increased the Bu2cAMP-stimulated production of progesterone. In KGN cells, there was a general decrease in progesterone production, while estradiol levels were increased following exposure to many bisphenols. Quantitative real-time polymerase chain reaction analyses revealed that all six bisphenols (≥1 μM) upregulated the expression of STAR, a cholesterol transporter, in both cell lines after stimulation. Key transcripts directly involved in steroid and cholesterol biosynthesis were significantly altered in a cell line, chemical, and concentration-dependent manner. Thus, BPA and five of its analogs can disrupt steroid production in two steroidogenic cell lines and alter the levels of transcripts involved in this process. Importantly, BPA replacements do not appear to have fewer effects than BPA.</p>","PeriodicalId":8965,"journal":{"name":"Biology of Reproduction","volume":" ","pages":"399-414"},"PeriodicalIF":3.1,"publicationDate":"2025-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11833478/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142614336","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Anti-Müllerian hormone as a predictor of oocyte yield following controlled ovarian stimulation in the rhesus macaque†.","authors":"Jared V Jensen, Philberta Y Leung, Emily C Mishler, Fernanda C Burch, Nadine Piekarski, Cecily V Bishop, Carol B Hanna","doi":"10.1093/biolre/ioae159","DOIUrl":"10.1093/biolre/ioae159","url":null,"abstract":"<p><p>Anti-Müllerian hormone (AMH) is widely used in the clinic as a biomarker for ovarian reserve and to predict ovarian response to gonadotropin stimulation. Patients with higher AMH levels tend to yield more oocytes and have better outcomes from assisted reproductive technology procedures. The goal of this study is to determine if AMH can be used to predict the outcome of controlled ovarian stimulation in rhesus macaques, which are commonly used in biomedical research, to refine animal use while maximizing oocyte yield. We hypothesized that pre-stimulation AMH values can be used to predict oocyte yield and quality. Regularly cycling adult macaques underwent controlled ovarian stimulation and baseline (pre-stimulation) plasma AMH levels were determined using an AMH-specific enzyme-linked immunoassay. Oocytes were collected by laparoscopic or ultrasound-guided aspiration, then counted and evaluated for quality and stage of meiosis. Sperm from established fertile males were used to inseminate the oocytes in vitro with fertilization success checked 14-16 h later. Females were grouped by oocyte yield: low ≤17; mid = 18-41; high ≥42. We found that high and mid yielders had significantly higher AMH than low yielders (p < 0.0001) and the percent of mature oocytes was greater in the high and mid yielders. There were no significant differences in oocyte quality or ova fertilization rate. These data suggest that AMH is a useful measure for controlled ovarian stimulation success in rhesus macaques and can be used to identify suitable animals for oocyte donation before entering them into a stimulation protocol.</p>","PeriodicalId":8965,"journal":{"name":"Biology of Reproduction","volume":" ","pages":"392-398"},"PeriodicalIF":3.1,"publicationDate":"2025-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142614306","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Two-directional trafficking of the IFT25 protein in the developing mouse sperm flagella.","authors":"Wei Li, Changmin Niu, Yi Tian Yap, Tao Li, Cheng Zheng, Mariska Goswami, Sanjana Kandiraju, Opeyemi Dhikhirullahi, Jie Xu, Jifeng Zhang, Christopher V Kelly, Zhibing Zhang","doi":"10.1093/biolre/ioae171","DOIUrl":"10.1093/biolre/ioae171","url":null,"abstract":"<p><p>Intraflagellar transport 25 is a component of the intraflagellar transport 25-B complex. In mice, even though this intraflagellar transport component is not required for cilia formation in somatic cells, it is essential for sperm formation. However, the intracellular localization of this protein in male germ cells is not known given no reliable antibodies are available for histologic studies, and the dynamic trafficking in the developing sperm flagella is not clear. To examine localization of the protein in male germ cells and further investigate the mechanism of intraflagellar transport in sperm formation, particularly to look into the dynamic trafficking of the protein, we generated a mouse intraflagellar transport 25-green fluorescent protein knock-in mouse model using the clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats associated protein 9 system, with the mouse intraflagellar transport 25 protein fused with a green fluorescent protein tag in the C-terminus. Three independent lines were analyzed. Western blotting using both anti-intraflagellar transport 25 and anti-green fluorescent protein antibodies showed that the intraflagellar transport 25-green fluorescent protein fusion protein was highly abundant only in the testis, which is consistent with the endogenous intraflagellar transport 25 protein. Examination of localization of the intraflagellar transport 25-green fluorescent protein in isolated germ cells revealed that the fusion protein was present in the cytoplasm of spermatocytes and round spermatids and a strong signal was present in the developing sperm flagellar. The homozygous knock-in mice had normal spermatogenesis, fertility and sperm parameters. Diffusion analysis of intraflagellar transport 25 within the developing flagellar revealed the presence of both mobile and immobile fractions as revealed by fluorescence recovery after photobleaching. Kymograph and fluorescence recovery after photobleaching analyses demonstrate the transport of intraflagellar transport 25-green fluorescent protein within the developing tail demonstrate no apparent preference for trafficking toward and away from the cell body. The speed of trafficking depends on the stage of sperm development, ranging from highly mobile unrestricted diffusion initially, mobile punctate structures in developing sperm, and immobile punctate structures in mature sperm. Our studies demonstrate that mouse intraflagellar transport 25 travels along the developing sperm flagella in two directions that might be essential for functional sperm formation.</p>","PeriodicalId":8965,"journal":{"name":"Biology of Reproduction","volume":" ","pages":"309-318"},"PeriodicalIF":3.1,"publicationDate":"2025-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142675050","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}