Production of functional sperm from in vitro-cultured premeiotic spermatogonia in a marine fish
摘要: 体外培养生殖干细胞产生功能性配子快速获得种质资源，可缩短育种间隔。在性成熟周期较长的养殖鱼类中，它有望作为一种遗传育种的潜在策略。但在养殖鱼类中体外培养精原细胞产生功能性精子仍然是一种挑战。在该研究中，利用Percoll密度梯度离心从中华乌塘鳢精巢中分离了雄性生殖细胞。经细胞形态和分子标记鉴定表明，得到的细胞主要为减数分裂前精原细胞。随后，通过三维（3D）添加激素（Hor）的培养方法，对分离的高纯度中华乌塘鳢精原细胞进行培养，诱导产生游动精子，其比例为培养细胞的9.4%。人工授精实验结果表明，这些体外产生的精子能与中华乌塘鳢成熟卵子受精并产生正常的后代。该研究还发现，在培养系统中添加褪黑素，可显著提高精子产生效率，达到约16%。进一步研究表明，褪黑激素可能通过ERK1/2信号通路激活细胞周期、精子发生和减数分裂相关信号通路，促进精原细胞分化成功能性精子。该鱼类精原细胞3D培养系统有望作为一种新的干细胞育种策略，加速养殖鱼类遗传育种和种质改良。Abstract: In vitro production of functional gametes can revolutionize reproduction by reducing generation intervals and accelerating genetic breeding in aquaculture, especially in fish with relatively long generations. Nevertheless, functional sperm production from in vitro-cultured spermatogonia remains a challenge in most aquaculture fish. In this study, we isolated and characterized premeiotic spermatogonia from marine four-eyed sleepers (Bostrychus sinensis), which are prone to ovotesticular or sterile testicular development, and induced the differentiation of the spermatogonia into flagellated sperm in a three-dimensional (3D) culture system. Artificial insemination indicated that the in vitro-derived sperm were capable of fertilizing mature oocytes to develop into normal larvae. Furthermore, melatonin significantly promoted spermatogonia proliferation and differentiation through the ERK1/2 signaling pathway, and thus increased the efficiency in functional sperm production. The 3D culture system and resulting functional sperm hold great promise for improving the genetic breeding of aquaculture fish.
Figure 1. Isolation and characterization of premeiotic spermatogonia by Percoll density gradient centrifugation
A–C: Structure of developing testis used for spermatogenic cell isolation. Whole testis (A); Immunofluorescence of Vasa in testes of 5-month-old males (B, C); Magnified image in panel B (C). Vasa and nucleus are shown in green and red, respectively. SG, spermatogonia; PSP, primary spermatocyte; SSP, secondary spermatocyte; SPD, spermatid; SPZ, spermatozoa. D–F: Representative images of testicular cell suspension (CS) before centrifugation (D) and whole cells in Percoll gradient after centrifugation (E) and in middle layer (F). G, H: Immunofluorescence of Vasa in CS (G) and middle-layer cells (H). I: Proportion of Vasa+ cell in CS and middle-layer cells. J: PCR amplification of different cell markers in CS and top-, middle-, and bottom-layer cells. Bars indicate mean of three biological replicates in at least three independent experiments. Scale bar: 10 μm.
Figure 2. In vitro spermatogenesis under different culture conditions
A–D: Representative images of middle-layer cells after 1, 2, 3, and 4 weeks of culture in 2D and 3D systems in absence (2D, A1–A4; 3D, B1–B4) and presence (2D+Hor, C1–C4; 3D+Hor, D1–D4) of sex hormones. Scale bar: 50 μm. E: PCR amplification of meiosis markers dmc1 and acrosin in cells under different culture conditions at 4 WAC. F, G: DNA content in cells under different culture systems at 4 WAC. β-actin was used as a reference gene. Bars indicate mean of three biological replicates. **: P<0.01.
Figure 3. Dynamic changes, expression patterns, and DNA content in spermatogenic cells in 3D+Hor culture system
A–D: Immunofluorescence of Vasa (green) in spermatogenic cells in 3D+Hor culture system at 1, 2, 3, and 4 WAC, respectively. E: Immunofluorescence of Vasa (red) and EdU (green) in spermatogenic cells in 3D+Hor culture system at 4 WAC. F: Immunofluorescence of Vasa (green) in mature testis. G: PCR analysis of meiosis markers in spermatogenic cells in 3D+Hor culture system at 0, 1, 2, 3, 4, and 5 WAC. H, I: DNA content in cells derived from fresh testis samples and spermatogenic cells in 3D+Hor culture system at 1, 2, 3, 4, and 5 WAC, respectively. J: Representative image of spermatogenic cells in 3D+Hor culture system at 4 WAC. Arrows indicate flagellated sperm. Scale bar: 50 μm (A–F) and 10 μm (J).
Figure 5. Melatonin promoted spermatogonia proliferation
A–L: Immunofluorescence of Vasa (red) and EdU (green) in spermatogenic cells in 3D+Hor culture system exposed to 0 (A–C), 0.1 (D–F), 1 (G–I), and 10 (J–L) μmol/L melatonin (Mel), respectively. Scale bar: 20 μm. M–P: Proportion of EdU+, Vasa+, EdU+/Vasa+, and EdU+/Vasa− cells in 3D+Hor culture system exposed to different doses of melatonin. Different letters indicate significant differences between groups. Bars indicate mean of three biological replicates in at least three independent experiments. Q: qPCR analysis of different cell markers in spermatogenic cells in 3D+Hor culture system in absence or presence of 1 μmol/L melatonin. β-actin was used as a reference gene. Bars indicate mean of three biological replicates. *: P<0.05; **: P<0.01; ns: No significant difference. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test (M-P) and Student’s t-test (Q), respectively.
Figure 6. Melatonin promoted spermatogonia proliferation via melatonin receptor 1 (MT1)
A–E: Immunofluorescence of Vasa (green) in cells in 3D+Hor culture system or after exposure to melatonin (3D+Hor+Mel), melatonin and luzindole (3D+Hor+Mel+Luz), melatonin and 4-P-PDOT (3D+Hor+Mel+4PP), and 2-Iodomelatonin (3D+Hor+2-Iod). Scale bar: 20 μm. F: Proportion of Vasa+ cells in 3D+Hor culture system exposed to different stimuli. Bars indicate mean of three biological replicates in at least three independent experiments. G: qPCR analysis of cdk2, cyclin E, and pcna in spermatogenic cells in 3D+Hor culture system after 1 week of exposure to different stimuli. β-actin was used as a reference gene. Bars indicate mean of three biological replicates. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. Different letters indicate significant differences between groups.
Figure 7. Melatonin promoted differentiation of spermatogonia into haploid cells
A–C: qPCR analysis of spermatogenesis-related markers (A) and meiosis markers (B) in spermatogenic cells in 3D+Hor culture system after 4 weeks of exposure to different stimuli, as well as their corresponding DNA content (C). β-actin was used as a reference gene. Bars indicate mean of three biological replicates. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. Different letters indicate significant differences between groups.
Figure 8. Activation of ERK1/2 signaling pathway is required for spermatogenesis in vitro
A: Western blot analysis of DMC1, ERK1/2, and pERK1/2 in cells in 3D culture system after 4 weeks of exposure to different stimuli. β-actin was used as an internal control. B–E: qPCR analysis of cdk2, cyclin E, and pcna (B) or meiosis markers (C) and apoptosis-related genes (D) in cells in 3D+Hor culture system after 4 weeks of exposure to different stimuli, as well as their corresponding DNA content (E). β-actin was used as a reference gene. Bars indicate mean of three biological replicates. RX indicates ERK-specific inhibitor ravoxertinib. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test, and different letters indicate significant differences between groups.
Table 1. Proportion of embryos at different stages generated from mature oocytes mixed with fresh sperm or cultured cells in 3D+Hor culture system.
Sperm or cultured cells Eggs (n=3) Cleavage (%) Somite (%) Hatching (%) Fresh sperm 1 573 84.72±6.65 78.48±7.75 55.09±5.91 1 WAC 741 0 0 0 2 WAC 692 0.66±0.65 0 0 3 WAC 659 9.77±4.33 5.05±1.16 3.50±0.36 4 WAC 693 48.93±8.23 31.23±3.24 26.88±4.13 5 WAC 746 51.68±8.21 25.25±1.22 19.65±2.31
Table 2. Proportion of embryos at different stages generated from mature oocytes mixed with fresh sperm or cultured cells in 3D+Hor culture system exposed to different stimuli
Sperm or cultured cells Eggs (n=3) Cleavage (%) Somite (%) Hatching (%) Fresh sperm 861 75.76±4.17 65.14±5.82 53.83±5.30 3D+Hor 773 40.15±2.77 30.19±3.18 26.00±3.70 3D+Hor+Mel 712 53.44±6.32 43.19±4.87 38.56±4.86 3D+Hor+Mel+Luz 582 42.51±4.29 34.57±3.62 25.26±3.91 3D+Hor+2-Iod 647 58.66±7.23 45.74±4.34 36.74±3.45
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