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Liu, Y.-X.

Publications and source records attributed to Liu, Y.-X..

16 recordsLinked to original sources

A thalamostriatal brake counteracts cortical recruitment of striatal ensembles in levodopa-induced dyskinesia

Levodopa-induced dyskinesia (LID) is a disabling complication of Parkinsons disease therapy, yet how upstream circuits recruit and restrain dyskinesia-linked striatal ensembles remains unclear. Using FosTRAP-based ensemble access in a unilateral 6-hydroxydopamine mouse model, we identified secondary motor cortex (M2) and parafascicular thalamus (PF) as dominant afferents with opposing functions. Projection-wide M2 activation promoted dyskinesia, whereas PF activation suppressed ongoing dyskinesia and shifted behavior toward non-dyskinetic states. Chronic levodopa reduced overall presynaptic terminal abundance while preserving putative contacts onto ensemble neurons, thereby increasing effective pathway-to-ensemble coupling. This remodeling followed distinct pathway rules: M2 contacts became spatially dispersed and biased toward NMDAR-mediated excitation, whereas PF inputs recruited stronger polysynaptic inhibition. Dyskinesia preferentially re-engaged ensemble-projecting M2 neurons, but reactivated PF neurons were topographically segregated from PF neurons directly innervating the ensemble. Accordingly, selective M2-to-ensemble stimulation promoted dyskinesia, whereas selective PF-to-ensemble stimulation was ineffective. Finally, ensemble-restricted Grin1 knockdown reduced peak dyskinesia and weakened M2-driven dyskinesia. These findings define LID as a targetable imbalance between cortical ensemble recruitment and thalamostriatal restraint.

neuroscience↗

Microglial pruning of extinction-ensemble synapses preserves fear memory

Fear extinction suppresses learned fear without erasing the original memory, yet how competing fear and extinction ensembles are selectively updated remains unknown. Here, we show that microglia preserve fear memory by selectively editing extinction-ensemble synapses during retrieval. In medial prefrontal cortex, microglial processes, but not somata, expand, ramify, and tighten their engagement with extinction-ensemble dendrites and spines, where they preferentially engulf excitatory postsynaptic material and bias spine remodeling toward elimination. This selectivity is instructed by local find-me, eat-me, and dont-eat-me cues: purinergic signaling recruits microglial processes, phosphatidylserine exposure licenses engulfment of extinction-ensemble synapses, and CD47-SIRP protects fear ensembles from removal. Weakening microglial recruitment or engulfment, or removing this protection, accelerates extinction without impairing fear acquisition. These findings identify microglial processes as active gatekeepers of ensemble competition and reveal a neuroimmune mechanism that preserves fear memory by limiting extinction. In BriefDuring memory updating, microglial processes selectively engage extinction-ensemble synapses rather than globally pruning active circuits. Local recruitment, engulfment, and protection cues determine this choice, allowing microglia to preserve fear memory by limiting extinction. HighlightsO_LIExtinction retrieval selectively recruits microglial processes to extinction ensembles. C_LIO_LIMicroglia preferentially engulf excitatory postsynaptic material from extinction ensembles. C_LIO_LILocal "find-me," "eat-me," and "dont-eat-me" cues determine synapse selection. C_LIO_LIDisrupting microglial pruning facilitates extinction without impairing fear learning. C_LI

neuroscience↗

abcFISH enables multiplexed, single-molecule visualization of circular RNA spatial heterogeneity

Functional RNAs often exhibit distinct subcellular localization, but studying circular RNA (circRNA) localization has been challenging due to their extensive sequence overlap with linear mRNAs. We developed amplicon-based circular RNA fluorescence in situ hybridization (abcFISH), a method that employs an optimized rolling circle amplification (RCA) strategy targeting back-splicing junction (BSJ) sites for robust, high-specificity, single-molecule circRNA imaging. abcFISH enables quantitative, multiplexed imaging in cells and tissues, allowing us to reveal alternative circularization patterns from the single locus; uncover cell-type-specific circPOLR2A(9,10) expression related to a combinatorial effect of RNA-binding proteins; map the distinct spatial distribution patterns of multiple circRNAs in neurons and brain tissues; and show the functional interplay of circRNA Cdr1as and lncRNA Cyrano co-localization. Furthermore, we applied abcFISH to validate the efficacy of therapeutic double-stranded circRNA aptamers (ds-cRNAs), simultaneously visualizing AAV-delivered ds-cRNAs and a consequent reduction in astrocyte infiltration around transduced cells. Collectively, abcFISH establishes a robust and user-friendly toolkit for deciphering circRNA localization and function in vivo.

molecular biology↗

Genome-Wide Investigation of Transcription Factor Occupancy and Dynamics Using cFOOT-seq

Gene regulation relies on the precise binding of transcription factors (TFs) at regulatory elements, but simultaneously detecting hundreds of TFs on chromatin is challenging. We developed cFOOT-seq, a cytosine deaminase-based TF footprinting assay, for high-resolution, quantitative genome-wide assessment of TF binding in both open and closed chromatin, even with small cell numbers. By utilizing the dsDNA deaminase SsdAtox, cFOOT-seq converts accessible cytosines to uracil while preserving genomic integrity, making it compatible with techniques like ATAC-seq for sensitive and cost-effective detection of TF occupancy at single-molecule and single-cell level. Our approach enables the delineation of TF footprints, quantification of occupancy, and examination of chromatin influences. Notably, cFOOT-seq, combined with FootTrack analysis, predicts TF occupancy dynamics. We demonstrate its application in capturing cell type-specific TFs, analyzing TF dynamics during reprogramming, and revealing TF dependencies on chromatin remodelers. Overall, cFOOT-seq represents a robust approach for investigating the genome-wide dynamics of TF occupancy and elucidating the cis-regulatory architecture underlying gene regulation.

molecular biology↗

An ultra-short-acting benzodiazepine in thalamic nucleus reuniens undermines fear extinction via intermediation of hippocamposeptal circuits

Benzodiazepines, commonly used for anxiolytics, hinder conditioned fear extinction, and the underlying circuit mechanisms are unclear. Utilizing remimazolam, an ultra-short-acting benzodiazepine, we reveal its impact on the thalamic nucleus reuniens (RE) and interconnected hippocamposeptal circuits during fear extinction. Systemic or RE-specific administration of remimazolam impedes fear extinction by reducing RE activation through A type GABA receptors. Remimazolam enhances long-range GABAergic inhibition from lateral septum (LS) to RE, underlying the compromised fear extinction. RE projects to ventral hippocampus (vHPC), which in turn sends projections characterized by feed-forward inhibition to the GABAergic neurons of the LS. This is coupled with long-range GABAergic projections from the LS to RE, collectively constituting an overall positive feedback circuit construct that promotes fear extinction. RE-specific remimazolam negates the facilitation of fear extinction by disrupting this circuit. Thus, remimazolam in RE disrupts fear extinction caused by hippocamposeptal intermediation, offering mechanistic insights for the dilemma of combining anxiolytics with extinction-based exposure therapy.

neuroscience↗

Transcription factor AhR regulates glutathione S-transferases (GSTs) conferring resistance to lambda-cyhalothrin in Cydia pomonella

Transcription factor aryl hydrocarbon receptor (AhR) can enhance insect resistance to insecticides by regulating the detoxification metabolic network. Our previous studies have confirmed that overexpression of cytochrome P450 monooxygenases (P450s) and glutathione S-transferases (GSTs) are both involved in lambda-cyhalothrin resistance in Cydia pomonella. In this study, we report that AhR regulates GSTs thus conferring lambda-cyhalothrin resistance in C. pomonella. Spatiotemporal expression patterns indicated that AhR gene of C. pomonella (CpAhR) was highly expressed in the Malpighian tubules of larvae. Moreover, the expression of CpAhR was induced by lambda-cyhalothrin exposure and was up-regulated in a lambda-cyhalothrin-resistant population. RNA interference (RNAi) of the expression of CpAhR could effectively decrease the relative expression level of CpGSTe3 and enzyme activity of GSTs, but not P450s, further reducing the tolerance of larvae to lambda-cyhalothrin. Furthermore, {beta}-naphthoflavone (BNF), a novel agonist of AhR, can effectively increase the expression of CpAhR and the activity of the GSTs enzyme, resulting in the enhancement of larvae tolerance to lambda-cyhalothrin. These results demonstrate that lambda-cyhalothrin exposure can effectively activate the expression of CpAhR and increase GSTs enzyme thus leading to the development of resistance to lambda-cyhalothrin, which enriches the theory of insecticide resistance regulation in C. pomonella.

molecular biology↗

Unraveling the Intricate Cargo-BBSome Coupling Mechanism at the Ciliary Tip

Certain ciliary transmembrane and membrane-tethered signaling proteins migrate from the ciliary tip to base via retrograde intraflagellar transport (IFT), essential for maintaining their ciliary dynamics to enable cells to sense and transduce extracellular stimuli inside the cell. During this process, the BBSome functions as an adaptor between retrograde IFT trains and these signaling protein cargoes. The Arf-like 13 (ARL13) small GTPase resembles ARL6/BBS3 in facilitating these signaling cargoes to couple with the BBSome at the ciliary tip prior to loading onto retrograde IFT trains for transporting towards the ciliary base, while the molecular basis for how this intricate coupling event happens remains elusive. Here, we report that Chlamydomonas ARL13 only in a GTP-bound form (ARL13GTP) anchors to the membrane for diffusing into cilia. Upon entering cilia, ARL13 undergoes GTPase cycle for shuttling between the ciliary membrane (ARL13GTP) and matrix (ARL13GDP). To achieve this goal, the ciliary membrane-anchored BBS3GTP binds and activates the ciliary matrix-residing ARL13GDP as an ARL13 guanine nucleotide exchange factor. At the ciliary tip, ARL13GTP binds and recruits the ciliary matrix-residing and post-remodeled BBSome as an ARL13 effector to anchor to the ciliary membrane. This makes the BBSome spatiotemporally become available for the ciliary membrane-tethered phospholipase D (PLD) to couple with. Afterward, ARL13GTP hydrolyzes GTP for releasing the PLD-laden BBSome to load onto retrograde IFT trains. According to this model, hedgehog signaling defects associated with ARL13b and BBS3 mutations in humans could be satisfactorily explained, providing us a mechanistic understanding behind BBSome-cargo coupling required for proper ciliary signaling. Significance statementCertain signaling proteins export in a BBSome-dependent manner from cilia via retrograde IFT. As IFT cargoes, their coupling with the BBSome is mediated by ARL13 and BBS3. Here, we report that ARL13 in a GTP-bound state binds the membrane for diffusing into cilia followed by running GTPase cycle with BBS3 acting as an ARL13-specific guanine nucleotide exchange factor. ARL13GTP recruits the IFT-shed BBSome as its effector to anchor to the membrane of the ciliary tip, making it spatiotemporally available for the ciliary membrane-tethered PLD to bind. Afterward, ARL13 GTP hydrolysis releases the PLD-laden BBSome for retrograde IFT train loading. ARL13 and BBS3 facilitate the cargo-BBSome coupling in such a way providing a regulatory mechanism for signaling protein export from cilia.

cell biology↗

Metabolic functional redundancy of the CYP9A subfamily members leads to P450-mediated lambda-cyhalothrin resistance in Cydia pomonella

BACKGROUNDThe evolution of insect resistance to pesticides poses a continuing threat to sustainable pest management. While much is known about the molecular mechanisms that confer resistance in model insects and few agricultural pests, far less is known about fruit pests. RESULTSHere we found that functional redundancy and preference of metabolism by cytochrome P450 monooxygenases (P450s) genes in the CYP9A subfamily confer resistance to lambda-cyhalothrin in Cydia pomonella, a major invasive pest of pome fruit. A total of four CYP9A genes, including CYP9A61, CYP9A120, CYP9A121, and CYP9A122, were identified from C. pomonella. Among these, CYP9A120, CYP9A121, and CYP9A122 were predominantly expressed in the midgut of larvae. The expression levels of these P450 genes were significantly induced by LD10 of lambda-cyhalothrin and were overexpressed in a field-evolved lambda-cyhalothrin resistant population. Knockdown of CYP9A120 and CYP9A121 by RNA-mediated interference (RNAi) increased the susceptibility of larvae to lambda-cyhalothrin. In vitro assays demonstrated that recombinant P450s expressed in Sf9 cells can metabolize lambda-cyhalothrin, but with functional redundancy and divergence through regioselectivity of metabolism. CYP9A121 preferred to convert lambda-cyhalothrin to 2'-hydroxy-lambda-cyhalothrin, whereas CYP9A122 only generated 4'-hydroxy metabolite of lambda-cyhalothrin. Although possesses a relatively low metabolic capability, CYP9A120 balanced catalytic competence to generate both 2'- and 4'-metabolites. CONCLUSIONCollectively, these results reveal that metabolic functional redundancy of three members of the CYP9A subfamily leads to P450-mediated lambda-cyhalothrin resistance in C. pomonella, thus representing a potential adaptive evolutionary strategy during its worldwide expansion.

molecular biology↗

Sublethal and transgenerational effects of lambda-cyhalothrin and abamectin on the development and reproduction of Cydia pomonella

The codling moth Cydia pomonella (Lepidoptera: Tortricidae) is a major invasive pest of pome fruits and walnuts worldwide. Lambda-cyhalothrin (LCT) and abamectin (AM) have been frequently used in C. pomonella control, but control of this pest is very difficult because shortly after hatching, larvae of this insect bore tunnels and hide inside host plant fruit. In this study, a simulated field spray bioassay method was developed against neonate larvae of C. pomonella and concentration-response bioassays were conducted to evaluate the susceptibility of the neonate larvae to LCT and AM. Exposure of neonate larvae to sublethal concentrations (LC30) of LCT or AM significantly reduced the survival rate of larvae (4th and 5th instars), lowered the mean weight of larvae and pupae, and decreased the daily maximal number of eggs laid and the total number of eggs laid (fecundity) per female. The sublethal effects, including reduced body mass, mean fecundity and net reproductive rate, extended mean generation time, and shortened oviposition period, were also found in transgenerational offspring. Furthermore, the transgenerational maternal effects were more obvious for AM than LCT, in comparison to the control. Additionally, the estimated population size was decreased by exposure to LC30 of LCT and AM, and the observed reduction of fecundity and population size within and across generations was likely the result of the downregulation of the reproduction-related vitellogenin gene (CpVg) after exposure to LC30 of LCT and AM. These results provide a better understanding of the overall effects of LCT and AM on C. pomonella and the transgenerational effects which should be taken into consideration when using insecticides in order to control C. pomonella.

pharmacology and toxicology↗

RABL2 Regulates Ciliation via Controlling IFT-B1 Basal Body Recruitment and ARL3-mediated BBSome Ciliary Retrieval

Highly conserved intraflagellar transport (IFT) trains and certain small GTPases coordinate to direct ciliation and to maintain the ciliary dynamics of signaling molecules via the IFT cargo adaptor BBSome. Unlike murine Rab-like 2 (RABL2) GTPase that enters cilia to drive outward transition zone (TZ) passage of the BBSome, human orthologue fails to enter cilia but resides at the ciliary base, indispensable for ciliation. However, mechanisms underlying how RABL2 regulates ciliation and BBSome barrier passage remain elusive. Here, we show that Chlamydomonas RABL2 regulates basal body targeting of the IFT-B1 subcomplex component of IFT trains as a RABL2-specific effector, mediating ciliation via controlling IFT-B1 basal body amount available for assembling anterograde IFT trains. RABL2GTP binds IFT-B1 to perform IFT; sheds from retrograde IFT trains at the proximal ciliary region right above the TZ; and converts to RABL2GDP rapidly. Next, RABL2GDP activates the ciliary membrane anchored Arf-like 3 (ARL3) GTPase (ARL3GDP) as a ARL3-specific guanine nucleotide exchange factor. Upon detaching from the ciliary membrane, the active ARL3GTP recruits its BBSome effector, autonomous of retrograde IFT train association, to move cross the TZ for ciliary retrieval. This ensures proper BBSome ciliary turnover for maintaining phototactic response of Chlamydomonas cells. For finishing RABL2 ciliary cycle, RABL2GDP passes the TZ for ciliary retrieval by loading onto the ARL3GTP/BBSome as a BBSome cargo. Our data thus propose that RABL2 mediates ciliation and BBSome ciliary retrieval simultaneously but via distinct molecular pathways. Significance statementIntraflagellar transport (IFT) and its cargo adaptor BBSome are indispensable for ciliation and ciliary singling. Rab-like 2 (RABL2) GTPase mediates ciliation and outward transition zone (TZ) passage of BBSomes with mechanisms yet to be determined. Here, we report that RABL2 decides ciliation by controlling the basal body amount of its effector IFT-B1 available for the assembly of anterograde IFT trains. RABL2GTP cycles through cilia as an IFT-B1 cargo; sheds from IFT at the ciliary base; and undergoes nucleotide exchange for activating ARL3 as an ARL3-specfic guanine nucleotide exchange factor. ARLGTP recruits IFT-shed BBSomes to pass the TZ for ciliary retrieval. RABL2GDP exists cilia via ARL3GTP/BBSome as a BBSome cargo. Therefore, RABL2 functions both outside and inside cilia for initiating IFT and BBSome ciliary retrieval, respectively.

cell biology↗

Sequence-based Functional Metagenomics Reveals Novel Natural Diversity of Functioning CopA in Environmental Microbiomes

Exploring natural diversity of functional genes/proteins from environmental DNA (eDNA) in a high-throughput fashion remains challenging. In this study, we developed a sequence-based functional metagenomics procedure for mining the diversity of copper resistance gene copA in global microbiomes, by combining the metagenomic assembly technology, local BLAST, evolutionary trace analysis (ETA), chemical synthesis, and conventional functional genomics. In total, 88 metagenomes were collected from a public database and subjected to copA detection, resulting in 93,899 hits. Manual curation of 1214 hits of high-confidence led to the retrieval of 517 unique CopA candidates, which were further subjected to ETA. Eventually 175 novel copA sequences of high-quality were discovered. Phylogenetic analysis showed that almost all of these putative CopA proteins are distantly related to known CopA proteins, with 55 sequences from totally unknown species. Ten novel and three known copA genes were chemically synthesized for further functional genomic tests using the Cu-sensitive Escherichia coli ({Delta}copA). Growth test and Cu uptake determination showed that five novel clones had positive effects on host Cu resistance and uptake. One recombinant harboring copA-like 15 (copAL15) successfully restored Cu resistance of host with a substantially enhanced Cu uptake. Two novel copA genes were fused with the gfp gene and expressed in E. coli for microscopic observation. Imaging results showed that they were successfully expressed and their proteins were localized to the membrane. The results here greatly expand the diversity of known CopA proteins, and the sequence-based procedure developed overcomes biases in length, screening methods, and abundance of conventional functional metagenomics.

microbiology↗

Chlamydomonas RABL4/IFT27 Mediates Phototaxis via Promoting BBSome-dependent Ciliary Export of Phospholipase D

Phospholipase D (PLD) interacts with the BBSome for loading onto retrograde intraflagellar transport (IFT) trains to exit cilia in Chlamydomonas reinhardtii. PLD ciliary retention and depletion of Rab-like 4 (RABL4) GTPase IFT27 cause the same non-phototactic phenotype but not impair IFT and ciliation. Here, we show that the IFT-B1 subunit IFT27 binds its partner IFT25 to form the heterodimeric IFT25/27 in an IFT27 nucleotide state-independent manner. IFT25/27, IFT-A, and IFT-B are irrelevant for maintaining the stability of one another. GTP-loading onto IFT27 enhances the IFT25/27 affinity for IFT-B1 in cytoplasm, while GDP-loaded IFT27 does not prevent IFT25/27 from entering and cycling through cilia by integrating into IFT-B1. Upon at the ciliary tip, IFT25/27 cycles on and off IFT-B1 and this process is irrelevant with the nucleotide state of IFT27. During BBSome remodeling at the ciliary tip, IFT25/27 promotes BBSome reassembly independent of IFT27 nucleotide state, making post-remodeled BBSomes available for PLD to interact with. Therefore, IFT25/27 facilitates BBSome-dependent PLD export from cilia via controlling availability of intact BBSomes at the ciliary tip, providing a regulatory mechanism for IFT27 to mediate phototaxis in C. reinhardtii.

cell biology↗

ARL3 Mediates BBSome Ciliary Turnover by Promoting Its Outward Diffusion through the Transition Zone

Ciliary receptors and their certain downstream signaling components undergo intraflagellar transport (IFT) as BBSome cargoes to maintain their ciliary dynamics for sensing and transducing extracellular stimuli inside the cell. Cargo laden BBSomes shed from retrograde IFT at the proximal ciliary region above the transition zone (TZ) followed by diffusing through the TZ for ciliary retrieval, while how the BBSome barrier passage is controlled remains elusive. Here, we show that the BBSome is a major effector of the Arf-like 3 (ARL3) GTPase in Chlamydomonas. Under physiological condition, ARL3GDP binds the membrane for diffusing into and residing in cilia. Following a nucleotide conversion, ARL3GTP dissociates with the ciliary membrane and binds and recruits the IFT-detached and cargo (phospholipase D, PLD)-laden BBSome at the proximal ciliary region to diffuse through the TZ and out of cilia. ARL3 deficiency impairs ciliary signaling, e.g. phototaxis of Chlamydomonas cells, by disrupting BBSome ciliary retrieval, providing a mechanistic understanding behind BBSome ciliary turnover required for ciliary signaling.

cell biology↗

Schisandrin B for treatment of male infertility

The decline of male fertility and its consequences on human populations are important public-health issues. However, there are limited choices for treatment of male infertility. In an attempt to identify a compound that could promote male fertility, we identified and characterized a library of small molecules from an ancient formulation Wuzi Yanzong-Pill, which was used as a folk medicine since the Tang dynasty of China. We found that SB enabled evident repairs in oligoasthenospermia-associated testicular tissue abnormality and in spermatogenesis disruption, resulting in significant improvements of sperm count, mobility, and reproductive ability in oligoasthenospermia mice. Furthermore, SB could alter substantial testicular genes (2033), among which, upregulation of Fst while downregulation of Inhba involved in reproductive signaling pathway could explain its role in enhancing spermatogenesis. The encouraging preclinical data with pharmacokinetics warranted a rapid development of this new class of therapeutic agent. Our finding provides a strong potent drug for treatment of male infertility.

pharmacology and toxicology↗

Small RNA expression pattern in multiply inbred lines and their hybrids of maize embryo

Heterosis, also known as hybrid vigor or transgression, is the phenomenon wherein an F1 hybrid produced from crossing two cultivars of the same or different species shows superior phenotypes. Heterosis in maize has been found and applied in breeding for more than one hundred years. However, its underlying molecular mechanisms are still poorly understood. To investigate whether small RNAs (sRNAs) participate in the regulation of heterosis, we profiled the sRNA expression patterns in the germ seeds of five inbred lines and theirs three F1 hybrids using high-throughput sequencing technology. The sequencing result show maize sRNAs are enriched in 22-nt length. Nearly 90% of 22-nt small RNA dominated clusters (SRCs) are in repeat regions, which mainly originated from Gypsy and Copia in retrotransposon. About 25% differentially expressed SRCs exist between parents, and hybrid gain almost all differentially expressed 22-nt SRCs. Twenty-four-nt sRNA also enriched in maize, which showed great sequence diversity and overall low expression across the genome. More than half of 24-nt SRCs originate from repeat, and 80% of them come from DNA transposon. Nearly 30% of 24-nt SRCs located in genes or their flanking regions, especially in flanking regions of \"lipid metabolic process\" and \"macromolecule modification\" related genes. Several thousands 24-nt SRCs are paternal or maternal specific expressed, and hybrids gain only half of differentially expressed 24-nt SRCs. Hundreds of 24-nt SRCs show high parent or above high parent expression pattern in different hybrids, and them mainly from Tourist, haT, and CACTA in DNA transposon and enrich nearby \"tRNA aminoacylation for protein translation\" related genes. Also, some 21-nt SRCs show conserved expression pattern in low parent. They were enriched in anti-sense region of some genes, which functions are about oxidative phosphorylation and translation. miRNAs have a global downregulated tendency in hybrids. zma-miR408-5p, zma-miR1432-5p and zma-miR528-5p are significant downregulated in each hybrid, and this phenomenon may cause their target genes more stable and contribute to hybrid vigor. Taken together, our results illustrated that sRNAs may contribute to heterosis at the very early stage of seed germination through repressing of retrotransposon activity, regulation gene activity at gene and genic flanking regions, and promotion some gene expressions by downregulated miRNAs.

plant biology↗

Retinoic acid promotes in vitro development of haploid germ cells from pre-pubertal porcine spermatogenic cells

Spermatogonial stem cells (SSCs) self-renew and contribute genetic information to the next generation. Inducing directional differentiation of porcine SSCs may be an important strategy in exploring the mechanisms of spermatogenesis and developing better treatment methods for male sterility. Here, we established an in vitro culture model for porcine small seminiferous tubule segments, to induce SSCs to differentiate into single-tail haploid spermatozoa. The culture model subsequently enabled spermatozoa to express the sperm-specific protein acrosin, and oocytes to develop to blastocyst stage after round spermatid injection. The addition of retinoic acid (RA) to the differentiation media promoted the efficiency of haploid differentiation. RT-PCR analysis indicated that RA stimulated the expression of Stra8 but reduced the expression of NANOS2 in spermatogonia. Genes involved in post-meiotic development, Prm1 and Tnp1, were up-regulated in the presence of RA. The addition of RAR inhibitor, BMS439, showed that RA enhanced the expression of cAMP responsive-element binding protein through RAR, and promoted the formation of round spermatids.

developmental biology↗