Development Of Secondary Follicles Research Articles

Melatonin promotes hair regeneration by modulating the Wnt/β-catenin signalling pathway.

Melatonin (MLT) is a circadian hormone that reportedly influences the development and cyclic growth of secondary hair follicles; however, the mechanism of regulation remains unknown. Here, we systematically investigated the role of MLT in hair regeneration using a hair depilation mouse model. We found that MLT supplementation significantly promoted hair regeneration in the hair depilation mouse model, whereas supplementation of MLT receptor antagonist luzindole significantly suppressed hair regeneration. By analysing gene expression dynamics between the MLT group and luzindole-treated groups, we revealed that MLT supplementation significantly up-regulated Wnt/β-catenin signalling pathway-related genes. In-depth analysis of the expression of key molecules in the Wnt/β-catenin signalling pathway revealed that MLT up-regulated the Wnt/β-catenin signalling pathway in dermal papillae (DP), whereas these effects were facilitated through mediating Wnt ligand expression levels in the hair follicle stem cells (HFSCs). Using a DP-HFSCs co-culture system, we verified that MLT activated Wnt/β-catenin signalling in DPs when co-cultured with HFSCs, whereas supplementation of DP cells with MLT alone failed to activate Wnt/β-catenin signalling. In summary, our work identified a critical role for MLT in promoting hair regeneration and will have potential implications for future hair loss treatment in humans.

Melatonin promotes proliferation of Inner Mongolia cashmere goat hair follicle papilla cells through Wnt10b

The study demonstrated that melatonin (MT) can induce the development of secondary hair follicles in Inner Mongolian cashmere goats through the Wnt10b gene, leading to secondary dehairing. However, the mechanisms underlying the expression and molecular function of Wnt10b in dermal papilla cells (DPC) remain unknown. This research aimed to investigate the impact of MT on DPC and the regulation of Wnt10b expression, function, and molecular mechanisms in DPC. The findings revealed that MT promotes DPC proliferation and enhances DPC activity. Co-culturing DPC with overexpressed Wnt10b and MT showed a significant growth promotion. Subsequent RNA sequencing (RNA-seq) of overexpressed Wnt10b and control groups unveiled the regulatory role of Wnt10b in DPC. Numerous genes and pathways, including developmental pathways such as Wnt and MAPK, as well as processes like hair follicle morphogenesis and hair cycle, were identified. These results suggest that Wnt10b promotes the growth of secondary hair follicles in Inner Mongolian cashmere goats by regulating crucial factors and pathways in DPC proliferation.

Neurotrophin-4 promotes in vitro development and maturation of human secondary follicles yielding metaphase II oocytes and successful blastocyst formation.

Does a matrix-free culture system supplemented with neurotrophic factor 4 (NT4) improve human in vitro follicular development and meiotic maturation, ultimately resulting in fertilizable oocytes? NT4 supplementation of in vitro culture significantly enhances the growth, steroid hormone production, and maturity potential of human secondary follicles derived from fresh ovarian medulla (from post- and pre-pubertal patients), thereby yielding fertilizable oocytes. Reconstituting folliculogenesis in vitro is of paramount importance in the realms of fertility preservation, reproductive biology research, and reproductive toxicity assessments. However, the efficiency of in vitro culture systems remains suboptimal, as the attainment of fertilizable oocytes from in vitro growth (IVG) of human follicles remains unachieved, with the data being particularly scant regarding follicles from prepubertal girls. We have previously found that mouse oocytes from secondary follicles derived from IVG are deficient in neuroendocrine regulation. NT4 and its corresponding receptor have been identified in human follicles. Significantly, the addition of NT4 during the IVG process markedly enhances both follicle growth and oocyte maturation rates in mice. Fresh medulla tissue obtained during tissue preparation for ovarian tissue cryopreservation (OTC) were collected from 10 patients aged from 6 to 21 years old, all of whom had undergone unilateral oophorectomy as a means of fertility preservation. Isolated secondary follicles were individually cultured in vitro with or without NT4 in a matrix-free system. Secondary follicles, extracted via enzymatic digestion and mechanical disruption from each patient, were randomly allocated to either a control group or an NT4-supplemented group (100 ng/ml), followed by individual culture on an ultra-low attachment plate. Follicle growth and viability were assessed by microscopy. Levels of anti-Müllerian hormone (AMH), estradiol, and progesterone in the medium were quantified. An oocyte-specific marker was identified using confocal fluorescence microscopy following DEAD box polypeptide 4 (DDX4) staining. The competence of individual oocytes for maturation and fertilization were assessed after IVM and ICSI with donated sperm samples. Overall, isolated follicles from both groups survived up to 6 weeks with increasing diameters over the duration (P < 0.05), reaching terminal diameters of almost 1 mm with confirmed steroidogenesis and expression of oocyte marker (DDX4), and producing morphologically normal MII oocytes. When compared with the control group, the NT4 group had a similar initial follicular diameter (206 ± 61.3 vs 184 ± 93.4 μm) but exhibited a significant increase in follicular diameter from the ninth day of culture onwards (P < 0.05). From Week 3, estradiol and progesterone production were significantly increased in the NT4 group, while no significant difference was observed in AMH production between groups. The proportion of 'fast-growth' follicles in the NT4 group was significantly higher than that in the control group (13/23 vs 6/24, P < 0.05). An increased efficiency of MII oocyte maturation per live follicle in the NT4 group was also observed (control group vs NT4 group, 4/24 vs 10/23, P < 0.05). It is noteworthy that an MII oocyte obtained from the control group exhibited abnormal fertilization after ICSI. In contrast, an MII oocyte acquired from the NT4 group progressed to the blastocyst stage and showed potential for transfer. N/A. The cohort examined in this study was all patients diagnosed with beta-thalassemia major. Whether this culture system is effective for patients with other diseases remains unknown. Since the chosen dose of NT4 was established based on dose finding in mice, the optimal dose for use in a human IVG system needs further confirmation. The oocytes and embryos procured from this study have not been quantified for ploidy status or epigenetic signatures. Fresh medulla tissue obtained during tissue preparation for OTC may serve as a precious source of fertilizable oocytes for female fertility preservation, even for pre-pubertal girls, without the threat of tumour reintroduction. After further characterization and optimization of the system, this culture system holds the potential to provide a powerful future research tool, for the comprehensive exploration of human follicular development mechanisms and for conducting reproductive toxicity evaluations. This work was supported by the National Key R&D Program of China (grant number 2022YFC2703000) and National Natural Science Foundation of China (grant numbers 82271651 and 81871214). The medium used in human follicle in vitro culture in this study has been applied for a national invention patent in China (No. 202211330660.7). The inventors of the patent, in order, are: Y.G., C.F., and X.L.

Genetic Knockin Approaches to Reconstructing DLBCL-Subtypes from Human Germinal Center B-Cells

Background and Significance: The clinical and genetic heterogeneity of DLBCL has led to the recent classification of the disease into five genetically distinct subtypes. The MCD/C5 subtype of DLBCL is defined by genetic alterations of CD79B at the first tyrosine residue of its ITAM motif (CD79B Y196H), crucial for signaling downstream of the BCR, and MYD88 (MYD88 L265P), a signaling adaptor protein essential for Toll Like receptor signaling. CD79B Y196H and MYD88 L265P mutations together define the MCD/C5 subtype and were found to be co-dependent for proliferation and oncogenic BCR-signaling in vitro. However, the mechanism of epistatic interaction between CD79B Y196H and MYD88 L265P is unknown. The MCD/C5 DLBCL subtype is associated with particularly poor clinical outcomes, however only a few human cell lines and a genetic mouse model are available for preclinical testing. Here, we developed a novel genetic approach to engineer the MCD/C5 subtype of DLBCL in primary human tonsillar germinal center B cells. To this end, we introduced the CD79B Y196H and MYD88 L265P mutations as genetic knockin alleles utilizing CRISPR-Cas9 homology directed repair templates (HDRT). In addition, we developed humanized NBSGW mice carrying a mouse Tslp-transgene to enable development of lymph nodes and secondary lymphoid organs to propagate genetically engineered GC B-cells carrying CD79B Y196H and MYD88 L265P knockin mutations for lymphoma development ( Figure 1). Results: Primary human germinal center B-cells were isolated from tonsils and grown on follicular dendritic feeder cells expressing CD40L and IL21. Single strand DNA templates were designed for CD79B, with and without Y196H mutation. Constructs were linked to fluorescent reporters by T2A sites and flanked by homology arms of ~280 bp targeting the guide RNA cut site. The target site was selected to disrupt the endogenous gene, while placing the construct under transcriptional control of endogenous promoter/enhancer elements. We achieved a knock-in efficiency of 5-8%, prior to single-cell cloning and genotyping to confirm the presence of heterozygous CD79B Y196H mutant CD79B Y196 wild-type mutations ( Figure 1). Preliminary biochemical studies show a delayed response and reduced intensity of signaling downstream of the BCR upon stimulation in CD79B Y196H mutant, compared to CD79B Y196 wild-type knockin germinal center B-cells. A similar approach was taken to design temples encoding Exon 5 of MYD88, with and without the L265P mutation, utilizing two guide RNAs to excise and replace Exon 5, linked to a mCherry reporter by a T2A site. Upon successfully introducing the MYD88 HDR templates into our CD79B wild-type and mutant clones to generate four distinct genotypes based on wildtype and CD79B Y196H and MYD88 L265P mutations alone or in combination. In addition, traditional NSG mice lack lymph nodes and secondary lymphoid organs. Lack of lymphoid follicles in NSG mice, owing to defective IL7Rα-signaling, contributed to lack of lymphoma formation observed in the previous model. In our model, we substituted the missing IL7Rα-signal with transgenic expression of murine Tslp, which enables development of embryonic lymphoid tissue inducer (LTi) cells that are required for the development of secondary lymphoid organs and germinal center follicles supporting initiation of human DLBCL development. Conclusion: Previous approaches to model DLBCL starting from germinal center B-cells were based on lentiviral overexpression of mutant oncogenes. Thereby, tonsillar germinal center B-cells with lentiviral BCL2 and BCL6 overexpression did not form lymphomas in NSG mice, however, were permissive to malignant transformation by MYC. Building on this previous work, we propose an improved model for preclinical testing for genetically defined DLBCL subtypes. Our two main improvements involve (i) genetic modeling based on knockin-strategies targeting endogenous loci and transcriptional control elements to phenocopy physiological gene expression levels and transcriptional regulation, and (ii) substitution of defective IL7Rα-signaling by transgenic expression of murine Tslp to rescue development of LTi cells. Transgenic expression of mTslp enables LTi-dependent formation of lymph nodes and secondary lymphoid organs supporting lymphoma development.

Paternal cadmium exposure affects estradiol synthesis by impairing intracellular cholesterol homeostasis and mitochondrial function in offspring female mice

Cadmium (Cd) is a toxic heavy metal commonly found in nature and an endocrine disrupting chemical (EDC). Previous studies found that Cd can damage several organs, including the kidneys, bones, cardiovascular system and reproductive system. However, the effect of paternal Cd exposure on the offspring is unclear. In this study, 1 mg/kg of cadmium chloride (CdCl2) was injected intraperitoneally every other day in 8-week-old C57BL/6 J male mice to study the effects on their female offspring. Our results showed an increase in body weight, water intake and food intake in F1 female mice from the Cd-exposed group. The development of secondary follicles and antral follicles in the ovaries of Cd-treated was inhibited. Serum estradiol (E2) was found to be decreased. Further analysis revealed significant downregulation of StAR, P450scc, 17β-HSD, CYP17A1 and CYP19A1, which are related to E2 synthesis. Serum total cholesterol was increased and free cholesterol was reduced. Total cholesterol in ovarian tissue was decreased. qRT-PCR and Western blot analysis revealed a decrease in the mRNA and protein expression of HMGCR, LDLR, and ABCA1, which are associated with cholesterol homeostasis. Oil red O staining indicated that lipid droplets (LDs) were accumulated in ovarian tissues, while the expression of ATGL and HSL proteins associated with lipid droplet degradation was significantly downregulated. In juvenile female mice, ultrastructural alterations of mitochondria in the ovaries were observed by transmission electron microscopy (TEM). In adult female mice, the expression of proteins associated with mitochondrial dynamics (DRP1 and MFN2) was significantly reduced in the ovaries. Overall, our study suggests that paternal Cd exposure inhibits follicular development, and affects serum E2 synthesis by impairing cholesterol homeostasis and affecting mitochondrial function.

Melatonin regulates the periodic growth of secondary hair follicles through the nuclear receptor RORα.

Cashmere is the fine bottom hair produced by the secondary hair follicles of the skin. This hair is economically important. Previous studies by our research group have shown that exogenous melatonin (MT) can regulate the periodic growth of secondary hair follicles, induce the secondary development of villi, and alter the expression of some genes related to hair follicle development. Few studies on the regulation of villus growth by MT binding receptors have been published. In this study, MT was implanted subcutaneously behind the ear of Inner Mongolia cashmere goats. RT-qPCR, in situ hybridization, Western blot analysis, immunofluorescence and RNAi techniques were used to investigate the receptors and functions of MT in regulating the development of secondary hair follicles in Inner Mongolia cashmere goats. The results showed that MT binds to the nuclear receptor RORα on dermal papilla stimulates hair follicle development and promotes villus growth. The RORα mRNA expression in the skin of Inner Mongolia cashmere goats was periodic and showed a trend of first increasing and then decreasing. The expression began to increase in February, peaked in April, and reached the lowest level in May. RORα significantly affected the mRNA expression of β-catenin gene, a key gene in hair follicle development, in the presence of MT. It will lay a solid molecular foundation for further research on the regulation mechanism between MT receptor and villus growth and development and to achieve artificial regulation of villus growth time and yield to improve the effect of villus production.

Effect of induced molting on ovarian function remodeling in laying hens

Induced molting (IM) can restore the laying rate of aged laying hens to the peak level of laying and rejuvenate ovarian function for the second laying cycle. To explore the mechanism of ovarian function remodeling during IM in laying hens, in this study, ninety 71-wk-old laying lady hens with 60% laying rate and uniform weight were selected for molting induction by fasting. Samples (serum and fresh ovarian tissue) were collected on the day before fasting (F0), the 3rd and 16th days of fasting (F3, F16), and the 6th, 15th, 32nd days of refeeding (R6, R15, and R32), and the number of follicles in each period was counted. Then, the reproductive hormone levels in serum and antioxidant levels in ovarian tissues were detected at different stages, and the gene expression of the KIT-PI3K-PTEN-AKT pathway and GDF-9 in ovaries was measured by qRT-PCR. The results showed that the laying rate increased rapidly after refeeding and returned to the prefasting level by R32. At F16 and R6, the number of mature follicles significantly decreased; the number of primary and secondary follicles significantly increased; the contents of follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and progesterone (P4) in serum decreased; the relative expression of KIT, PI3K, AKT, and GDF-9 significantly increased; and that of PTEN significantly decreased. At R15 and R32, except for GDF-9, which maintained a high expression state, other indicators showed opposing trends to those observed at F16 and R6. In conclusion, IM activated the KIT-PI3K-PTEN-AKT signaling pathway and promoted the activation of primordial follicles during the fasting period and early resumption of feeding; gonadotropin secretion increased gradually, which promoted the rapid development of primary and secondary follicles to mature follicles and ovulation. This study explained the mechanism of ovarian function remodeling in the process of IM and provided a theoretical basis for improving the ovarian function of laying hens and optimizing the IM program.

Melatonin promotes the development of the secondary hair follicles by regulating circMPP5

BackgroundThe quality and yield of cashmere fibre are closely related to the differentiation and development of secondary hair follicles in the skin of cashmere goats. The higher the density of secondary hair follicles, the higher the quality and yield of cashmere from the fleece. Development of secondary hair follicles commences in the embryonic stage of life and is completed 6 months after birth. Preliminary experimental results from our laboratory showed that melatonin (MT) treatment of goat kids after their birth could increase the density of secondary hair follicles and, thus, improve the subsequent yield and quality of cashmere. These changes in the secondary hair follicles resulted from increases in levels of antioxidant and expression of anti-apoptotic protein, and from a reduction in apoptosis. The present study was conducted to explore the molecular mechanism of MT-induced secondary hair follicle differentiation and development by using whole-genome analysis.ResultsMT had no adverse effect on the growth performance of cashmere kids but significantly improved the character of the secondary hair follicles and the quality of cashmere, and this dominant effect continued to the second year. Melatonin promotes the proliferation of secondary hair follicle cells at an early age. The formation of secondary hair follicles in the MT group was earlier than that in the control group in the second year. The genome-wide data results involved KEGG analysis of 1044 DEmRNAs, 91 DElncRNAs, 1054 DEcircRNAs, and 61 DEmiRNAs which revealed that the mitogen-activated protein kinase (MAPK) signaling pathway is involved in the development of secondary hair follicles, with key genes (FGF2, FGF21, FGFR3, MAPK3 (ERK1)) being up-regulated and expressed. We also found that the circMPP5 could sponged miR-211 and regulate the expression of MAPK3.ConclusionsWe conclude that MT achieves its effects by regulating the MAPK pathway through the circMPP5 sponged the miR-211, regulating the expression of MAPK3, to induce the differentiation and proliferation of secondary hair follicle cells.In addition there is up-regulation of expression of the anti-apoptotic protein causing reduced apoptosis of hair follicle cells. Collectively, these events increase the numbers of secondary hair follicles, thus improving the production of cashmere from these goats.

Characterization of Growth Secondary Hair in Min Pig Activated by Follicle Stem Cell Stimulated by Wnt and BMP Signaling Pathway

In China, the national-level protected pig, the Min pig, is characterized by the development of secondary hairs and hair follicles in winter. Factors that dominate the genotype in the growth of secondary hairs are not clear through the concrete cell signaling pathways. This study compared hair phenotypes based on morphological structure, transcriptomics, and potential targeting molecules in the breeds of Min, Berkshire, and Yorkshire pigs. The results indicated that Min pigs have specific characteristics for the growth of secondary hairs compared with the Berkshire and Yorkshire pigs. The transcriptome analyses and quantitative reverse transcription-polymerase chain reaction results revealed that secondary hair growth was activated by follicle stem cells. The specific inhibitors of Wnt and BMP were studied using respective signals. The density of follicles, activity of follicle stem cells, and relative gene expression results have shown that Wnt and BMP stimulate the activity of follicle stem cells, and the Wnt signaling molecule has a significantly better effect than the BMP signaling molecule on stem cells. Wnt and BMP can promote the growth of local secondary hair and gene expression. Therefore, this study was conducted to verify the development mechanisms of secondary hairs, which have potential applications in laboratory animals and comparative medicine.

Melatonin Promotes the Development of Secondary Hair Follicles in Adult Cashmere Goats by Activating the Keap1-Nrf2 Signaling Pathway and Inhibiting the Inflammatory Transcription Factors NFκB and AP-1.

Exogenous melatonin (MT) has been used to promote the growth of secondary hair follicles and improve cashmere fiber quality, but the specific cellular-level mechanisms involved are unclear. This study was carried out to investigate the effect of MT on the development of secondary hair follicles and on cashmere fiber quality in cashmere goats. The results showed that MT improved secondary follicle numbers and function as well as enhanced cashmere fiber quality and yield. The MT-treated goat groups had high secondary-to-primary ratios (S:P) for hair follicles, greater in the elderly group (p < 0.05). Antioxidant capacities of secondary hair follicles improved fiber quality and yield in comparison with control groups (p < 0.05/0.01). Levels of reactive oxygen and nitrogen species (ROS, RNS) and malondialdehyde (MDA) were lowered (p < 0.05/0.01) by MT. There was significant upregulation of antioxidant genes (for SOD-3; GPX-1; NFE2L2) and the protein of nuclear factor (Nrf2), and downregulation of the Keap1 protein. There were significant differences in the expression of genes for secretory senescence-associated phenotype (SASP) cytokines (IL-1β, IL-6, MMP-9, MMP-27, CCL-21, CXCL-12, CXCL-14, TIMP-1,2,3) plus their protein of key transcription factors, nuclear factor kappa B (NFκB) and activator protein-1 (AP-1), in comparison with the controls. We concluded that MT could enhance antioxidant capacity and reduce ROS and RNS levels of secondary hair follicles through the Keap1-Nrf2 pathway in adult cashmere goats. Furthermore, MT reduced the expression of the SASP cytokines genes by inhibiting the protein of NFκB and AP-1 in the secondary hair follicles in older cashmere goats, thus delaying skin aging, improving follicle survival, and increasing the number of secondary hair follicles. Collectively, these effects of exogenous MT enhanced the quality and yield of cashmere fibers, especially at 5-7 years old.

Construction and functional analysis of ceRNA regulatory network related to the development of secondary hair follicles in Inner Mongolia cashmere goats.

Cashmere goat hair follicles are divided into primary hair follicles and secondary hair follicles. The primary hair follicles produce coarse hair, and the secondary hair follicles produce cashmere. The development of hair follicles is affected by a variety of signaling molecules and pathways. Studies have shown that non-coding RNAs are widely involved in the development of hair follicles of the goat, including small RNAs (miRNAs), long non-coding RNAs (lncRNA), and circular RNAs (circRNAs). In recent years, circRNAs, as a new type of circular closed non-coding RNAs, have attracted great attention due to their high stability. However, its regulatory effect on cashmere goat hair follicles mainly focuses on the periodic regulation of secondary hair follicles, and there is no report on the development of cashmere goat hair follicles during the fetal period. Therefore, this study was based on the circRNA, miRNA, and mRNA expression profiles obtained by whole-transcriptional sequencing of the skin tissue of the Inner Mongolia cashmere goats in the fetal period (days 45, 55, 65, and 75) and screening out the morphological changes of hair follicles at different periods. A total of 113 circRNAs related to the development of secondary hair follicles were present. According to the principle of the ceRNA regulatory network, a ceRNA regulatory network composed of 13 circRNAs, 21 miRNAs, and 110 mRNAs related to the development of secondary hair follicles was constructed. Then, qRT-PCR and Sanger sequencing identified circRNA2034, circRNA5712, circRNA888, and circRNA9127 were circRNAs. Next, the dual-luciferase reporter gene verified the targeting relationship of circRNA5712-miR-27b-3p-Dll4. In conclusion, this study constructed a ceRNA regulatory network for the development of cashmere goat secondary hair follicles, laying a foundation for the analysis of circRNAs regulating the morphogenesis and development of cashmere goat secondary hair follicles through the ceRNA mechanism.

Novel circRNAs from cashmere goats: Discovery, integrated regulatory network, and their putative roles in the regeneration and growth of secondary hair follicles

Circular RNAs (circRNAs) were identified as noncoding RNAs with covalently closed structures differing from other types of RNA molecules. It was thought that circRNAs were implicated in the development of secondary hair follicles (SHFs) of cashmere goats. In this investigation, a total of 21 novel circRNAs were identified from the skin tissue of cashmere goats. Of them, nine circRNAs were found to be significantly higher in expression at anagen skin tissue than those at telogen of cashmere goats. Based on bioinformatics analysis, a complicated regulatory relationship was revealed among the nine upregulated circRNAs at anagen with related signalling pathways, such as Wnt signalling pathway, TGF-beta signalling pathway, mTOR signalling pathway, MAPK signalling pathway, and axon guidance. Further, the expression pattern analysis of the nine upregulated circRNAs at anagen along with their host genes in SHFs suggested their potential functional roles in the regeneration and growth of anagen SHFs of cashmere goats. Our results provided novel significant information for elucidating the molecular regulatory mechanisms understanding the regeneration and growth of SHFs in cashmere goats, which will be essential for artificially regulating the growth of cashmere to increase cashmere yield.

Identification of Genes Related to Hair Follicle Cycle Development in Inner Mongolia Cashmere Goat by WGCNA.

Cashmere goat from Inner Mongolia is an excellent local breed in China, and the related cashmere product is a kind of precious textile raw material with high price. Cashmere is generated from secondary hair follicles, which has obvious annual periodicity and includes three different stages: anagen, catagen, and telogen. Therefore, we investigated skin transcriptome data for 12 months using weighted gene co-expression network analysis (WGCNA) to explore essential modules, pathways, and genes responsible for the periodic growth and development of secondary hair follicles. A total of 17 co-expression modules were discovered by WGCNA, and there is a strong correlation between steelblue module and month (0.65, p = 3E−09), anagen (0.52, p = 1E−05), telogen (−0.6, p = 8E−08). Gene expression was generally high during late anagen to catagen (June to December), while expression was downregulated from telogen to early anagen (January–May), which is similar to the growth rule of hair follicle cycle. KEGG pathway enrichment analyses of the genes of steelblue module indicated that genes are mainly enriched in Cell cycle, Wnt signaling pathway, p53 signaling pathway and other important signal pathways. These genes were also significantly enriched in GO functional annotation of the cell cycle, microtubule movement, microtubule binding, tubulin binding, and so on. Ten genes (WIF1, WNT11, BAMBI, FZD10, NKD1, LEF1, CCND3, E2F3, CDC6, and CDC25A) were selected from these modules, and further identified as candidate biomarkers to regulate periodic development of hair follicles using qRT-PCR. The Wnt signaling pathway and Cell cycle play an important role in the periodic development of hair follicles. Ten genes were identified as essential functional molecules related to periodic development of hair follicle. These findings laid a foundation for understanding molecular mechanisms in biological functions such as hair follicle development and hair growth in cashmere goats.

Screening of microRNA and mRNA related to secondary hair follicle morphogenesis and development and functional analysis in cashmere goats

microRNA (miRNA) is a type of endogenous short-chain non-coding RNA with regulatory function found in eukaryotes, which is involved in the regulation of a variety of cellular and biological processes. However, the research on the development of cashmere goat secondary hair follicles is still relatively scarce. In this study, small RNA libraries and mRNA libraries of 45 days, 55 days, 65 days, and 75 days of fetal skin of cashmere goats were constructed, and the constructed libraries were sequenced using Illumina Hiseq4000, and the expression profiles of miRNA and mRNA in cashmere goat fetal skin were obtained. The differentially expressed miRNAs and mRNAs in six control groups were identified and the qRT-PCR experiment shows that the sequencing results are accurate. Sixty-six miRNAs related to secondary hair follicle development were screened, and used TargetScan and miRanda to predict 33 highly expressed miRNA target genes. At the same time, 664 mRNAs related to the development of secondary hair follicles were screened, and GO enrichment and KEGG pathway analysis were performed. It was found that some miRNA target genes were consistent with the screening results of mRNAs related to secondary hair follicle development and were enriched in Notch signaling pathway, TGF-β signaling pathway. Therefore, miR-145-5p-DLL4, miR-27b-3p-DLL4, miR-30e-5p-DLL4, miR-193b-3p-TGF-β1, miR-181b-5p-NOTCH2, and miR-103-3p-NOTCH2 regulatory network related to the development of secondary hair follicles were constructed and the results of dual-luciferase reporter gene assay indicated that there is a targeted relationship between chi-miR-30e-5p and DLL4, which will provide a basis for molecular mechanism of miRNA-mRNA in the development of the hair follicles in cashmere goats.

Development of sheep secondary follicles and preservation of aromatase and metalloproteinases 2 and 9 after vitrification and in vitro culture.

The cryopreservation of secondary follicles (SF) is a promising alternative to preserve the reproductive potential both in humans and animals in situations in which the transplantation of ovarian tissue is not possible. The objective of the present study was cryopreserved SF isolated sheep. Beyond follicular morphology, viability and development, we investigated proteins related to steroidogenic function and basement membrane remodeling [metalloproteinases 2 (MMP-2) and 9 (MMP-9)] in fresh SF (FSF) and vitrified SF (VSF) followed by in vitro culture for 6 (D6) or 12days (D12). The percentage of intact follicles, follicular and oocyte diameter of the VSF were lower than FSF on both days of culture (P < 0.05). The VSF viability was statistically reduced from D6 (95.5%) to D12 (77.3%) but did not differ from the FSF on both days (D6:96.2% to D12:86.5%). Antrum formation in the VSF (D6: 59.13%; D12: 79.56%) was significantly lower than the FSF (D6: 79.61%; D12: 92.23%). However, an increase in this percentage was observed from D6 to D12 in both groups. Aromatase showed stronger labeling on FSF D6 and VSF D12 compared to other treatments (P < 0.05). MMP-2 showed a similar pattern of labeling in FSF D6 and VSF D12, similarly to that observed in FSF D12 and VSF D6. MMP-9 was similar in FSF and VSF cultivated for 6 and 12days. In conclusion, VSF are able to grow and develop during 12days of in vitro culture and showed evidence of preservation of steroidogenic function and remodeling of the basement membrane.

Human dermal fibroblasts support the development of human primordial/primary follicles in a 3-dimensional alginate matrix culture system.

BackgroundAlginate matrix 3-dimensional culture offers the opportunity for the development and maturation of human secondary follicles in vitro. However, alginate may not be the most suitable culture system for human primordial/primary follicles in vitro. Thus, the innovation of alginate matrix 3-dimensional culture systems for human primordial/primary follicles could hold promise as an ideal approach to restoring fertility.MethodsWe extracted primordial/primary follicles from ovarian tissues collected from patients with non-ovarian benign gynecological conditions. Fibroblasts were isolated from dermal tissue from 1 male patient who had undergone posthectomy. The isolated human follicles were randomly divided into 2 groups and encapsulated within fibroblast-alginate-hydrogels or alginate hydrogels. The survival and growth of human primordial/primary follicles were measured after 21 days of in vitro culture.ResultsThe dermal fibroblasts in alginate hydrogel microcapsules were round in shape, and were distributed as uniform clouds on the surface and gaps of the alginate. After 21 days of culture, the survival rate of follicles in the fibroblast-alginate group was higher than that of the alginate group (P<0.05). The diameter of follicles in the fibroblast-alginate group and the alginate group after 21 days of culture was 152.80±13.64 and 129.14±9.95 μm, respectively (P<0.05). After 21-day culture, the mean cpm (log-converted) for 3H-thymidine incorporated by granulosa cells in the fibroblast-alginate and alginate groups was 6.87±0.24 and 4.63±0.38, respectively (P<0.05). After 21 days of culture, the messenger RNA expression levels of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) were significantly higher in oocytes in fibroblast-alginate hydrogels than in those in alginate hydrogels (P<0.05).ConclusionsHuman fibroblasts are beneficial to the development of human follicles in 3-dimensional culture alginate gel systems over a long period of time. More studies are required to investigate the molecular biological mechanisms of human fibroblasts that promote follicle growth in vitro.

Inhibition of prolactin promotes secondary skin follicle activation in cashmere goats.

The aim of this study was to investigate the involvement of prolactin (PRL) on development of secondary skin follicles in cashmere goats. Goats were randomly assigned to either a bromocriptine treatment or control group. Samples of cashmere fiber, blood, and skin were collected from all goats after 1 mo. The results indicated that the length, growth rate, and diameter of fibers were not influenced (P > 0.05) by the inhibition of PRL resulting from the treatment with bromocriptine. There was a tendency for increases in total follicle number, primary and secondary follicle numbers, and in the ratio of secondary to primary follicles following treatment with bromocriptine, but these differences were not significant (P > 0.05). The percentage of active secondary follicles in anagen was increased (P < 0.05) in the bromocriptine-treated goats, but there was no effect of treatment on the percentage of active primary follicles. Bromocriptine decreased (P < 0.05) circulating concentrations of PRL and Insulin-like growth factor 1 (IGF1) and increased (P < 0.05) those of melatonin (MT), but there was no effect of this treatment on the serum concentrations of cortisol, growth hormone, tetraiodothyronine, and triiodothyronine. In bromocriptine-treated goats, mRNA expressions of PRL and MT membrane receptor 1a (MTNR1a) were decreased (P < 0.05) and mRNA expression of MT nuclear receptor (RORα) was increased (P < 0.05), but there was no effect of the treatment on expression of long PRL receptor, short PRL receptor, MT membrane receptor 1b and IGF1. It is concluded that inhibition of PRL promotes secondary hair follicle development in the anagen phase, possibly by downregulating MTNR1a and up-regulating RORα gene expression in the skin.

Placentation in the African Elephant (Loxodonta africana).

The female elephant shows a 3-week "follicular phase" to commence her 16-week estrous cycle at the end of which a second surge in pituitary luteinizing hormone (LH) release matures and ovulates an ovarian follicle in association with estrous behavior and mating, whereas the first LH surge at the start of the follicular phase causes luteinization of 3-5 partially developed follicles. The prolonged pregnancy of 22months is supported by a zonary endotheliochorial placenta which secretes placental lactogen (ePL) from around 40days of gestation in association with replacement of the lumenal epithelium of the endometrium by trophoblast and the development of large corpora lutea (CLs) in the maternal ovaries from the previously formed luteinized follicles in response to the first LH peak early in the follicular phase. The zonary placenta develops above, rather than within, the endometrium. The elephant placenta secretes neither estrogens nor progestagens throughout gestation, as pregnancy maintenance relies on 5α-dihyroprogesterone and other 5α reduced progestagens secreted by secondary CLs stimulated by ePL and the stromal tissue of the fetal gonads, which become extremely enlarged during the second half of the 22-month pregnancy. In female fetuses, this ovarian enlargement includes the development and subsequent regression of multiple primary and secondary follicles with a consequent substantial decline in primary follicle numbers at birth. During the next 8-9years of pre-pubertal life, however, oocyte and primary follicle numbers recover to levels near those found in late gestation, which may be evidence of postnatal oogenesis occurring in the elephant.

Effects of dexamethasone on growth, viability and ultrastructure of bovine secondary follicles cultured in vitro.

This study aimed to evaluate the effects of dexamethasone on development, viability, antrum formation and ultrastructural integrity of bovine secondary follicles cultured in vitro for 18 days. Bovine ovaries were obtained from slaughterhouses and secondary follicles of ~150-200 µm diameter were isolated and cultured in the laboratory in TCM-199+ alone or supplemented with different concentrations of dexamethasone (1, 10, 100 and 1000 ng/ml). Follicle viability was evaluated after the culture period, using calcein-AM (viable) and ethidium homodimer (nonviable). Follicle diameters and antrum formation were evaluated at days 0, 6, 12 and 18. Before or after in vitro culture, follicles were fixed for histological and ultrastructural analysis. Follicle diameters were evaluated using analysis of variance and Kruskal-Wallis test, while chi-squared test was used to evaluate the percentage of viable follicles and antrum formation (P < 0.05). Follicles cultured for 6 days with all treatments increased their diameters significantly, but there was no significant difference between treatments at the end of the culture period. In vitro cultured follicles showed antral cavity formation at the end of the culture period, but no influence of dexamethasone was seen. Ultrastructural analysis showed that follicles cultured with dexamethasone (1, 10, 100 and 1000 ng/ml) had well preserved granulosa cells. However, oocytes from follicles cultured with 10, 100 or 1000 ng/ml dexamethasone showed signs of degeneration. It can be concluded that follicles cultured in vitro in the presence of dexamethasone demonstrated continuous in vitro growth, but oocytes from follicles cultured with 10, 100 or 1000 ng/ml dexamethasone had poor ultrastructure.

Effect of Nutritional Restriction on the Hair Follicles Development and Skin Transcriptome of Chinese Merino Sheep.

Simple SummaryThe morphogenesis hair follicles begins in the embryonic stage. The first follicles formed are the primary wool follicles (PF), followed by secondary wool follicles (SF), and then secondary-derived follicles that branch from the SF in Merino sheep. The morphogenesis and development of SF in sheep determine wool yield and quality. At present, the mechanism of SF development and branching is still unclear. In this study, hair follicle morphogenesis and development were inhibited through nutritional restrictions during pregnancy, and transcriptome sequencing was performed to screen for differentially expressed genes related to the SF development in the fetus. Our findings are helpful to understand the mechanisms of SF development and branching of Merino sheep.The high concentration of secondary branched wool follicles is a distinctive feature of the Merino sheep. At present, the molecular control of the development and branching of secondary wool follicles (SF) remains elusive. To reveal the potential genes associated with the development of hair follicles, we investigated the characteristics of prenatal and postnatal development of wool follicles, and the transcriptional expression profile in fetuses/lambs from dams under either maternal maintenance or sub-maintenance (75% maintenance) nutrition. The density of SF and the ratio of SF to primary wool follicles (PF) were reduced (p < 0.05) in fetuses from day 105 to 135 of gestation under sub-maintenance nutrition. Differentially expressed genes were enriched in the binding, single-organism process, cellular process, cell and cell part Gene Ontology (GO) functional categories and metabolism, apoptosis, and ribosome pathways. Four candidate genes, SFRP4, PITX1, BAMBI, and KRT16, which were involved in secondary wool follicles branching and development, were identified. Our results indicate that nutritional intervention imposed on pregnant ewes by short-term sub-maintenance nutrition could provide a strategy for the study of wool follicle development. Overall insight into the global gene expression associated with SF development can be used to investigate the underlying mechanisms of SF branching in Merino sheep.