Distribution of I-particles NIRFIP-180 in testis injected via the rete testis route.

(A) A solution containing 0.05% Fast Green and 1% fluorescent i-particles NIRFiP-180 was prepared, 10 µL was injected into the seminiferous tubules of adult males, through the rete testes and its efferent channels. Injection was performed at constant pressure under a binocular microscope. The progression of filling of the seminiferous tubules was monitored thanks to the Fast Green (B). The testes were only filled to 2/3 capacity in order to prevent damage to the tissue. (C) Representative distribution of fluorescent i-particles NIRFiP-180 in a whole cross-section of an injected testis. Nuclei were counterstained with DAPI (blue emission) to reveal tubules. (D) Enlargement of a seminiferous tubule showing particles localized inside the lumens of the tubules. Scales bars: 1 mm and 500 mm.

In vivo injection and electroporation do not alter the morphological structure of the testes, seminiferous tubules, or sperm cells.

(A, B) Testicular morphology was not affected by in vivo injection and electroporation of EEV-GFP (A) or GFP-mRNA (B). Controls corresponds to contralateral testes injected/electroporated with control solution (PBS, 0.05% FG). (A1, B1) comparison of the testicular morphology of adult testes injected with nucleic acid vectors or control solutions. (A2, B2) Comparison of testicular weight and (A3, B3) testicular length on day 7 after injection/electroporation. Data are represented as a box plot median (n=4 for each condition). A Wilcoxon matched pairs test was used to assess the significance of any differences in testis weights and lengths, and p values of ≤0.05 were considered statistically significant.

(C) Intact testicular structure after in vivo injection and electroporation with EEV-GFP and GFP-mRNA. Comparison of testicular cross section structures. Testes paraffin sections were stained with eosin/hematoxylin and observed by light microscopy (20X magnification). (C1) Control, (C2) EEV-GFP injected and (C3) GFP-mRNA injected. Scales bars: 1000 µm.

(D) Seminiferous tubule structures are not affected by in vivo injection and electroporation with EEV-GFP and GFP-mRNA. Enlargement of cross sections showing the fine structure of a seminiferous tubule for control (D1), EEV-GFP (D2) and GFP-mRNA (D3). In each tubule the different layers of spermatogenic cells are indicated, Sertoli cells (S), Spermatogonia (Sg), Spermatocytes (Scytes), rounds Spermatids (Stids) and sperm cells (Spz), Leydig cells (L). Scales bars: 500 and 20 µm.

(E) The area of seminiferous tubules is not affected by in vivo injection and electroporation with EEV-GFP and GFP-mRNA. Comparison of the seminiferous tubule diameter after injection of nucleic acid vectors or control solutions. Data are represented as a box plot median. The areas of seminiferous tubules (μm2) were measured for round cross sections of n > 35 tubules per testis section (n= 5 testis sections per condition). Statistical significance was verified using a Student t-test.

(F) Injection/electroporation do not impact epidydimal sperm cells. Representative sperm observed by light microscopy on day 7 after injection/electroporation with Control solution, EEV-GFP, or GFP-mRNA. Scale bars: 10 μm.

Kinetics of EEV-GFP expression following in vivo injection/electroporation: whole testicular expression

(A1-H1) Whole-mount testes on days 0, 1, 7, 14, 21, 28, 35 and 42 after in vivo injection/ electroporation with EEV-GFP. (A2-H2) Under fluorescence observation, GFP expression was detectable in transfected testes from 12-week-old B6D2 mice. (C3-H3) Insets show the absence of autofluorescence in non-transfected control testes. (4X magnification). The GFP expression presented a punctiform pattern in seminiferous tubules and was detected from 1 day to 42 days. Scales bars: 1 mm and 100 μm.

Kinetics of GFP-mRNA expression following in vivo injection/electroporation: whole testicular expression

(A1-F1) Whole-mount testes on days 0, 1, 7, 14, 21, and 28 after in vivo injection/ electroporation with GFP-mRNA. (A2-F2) Under fluorescence observation, GFP expression was detectable in transfected testes from 12-week-old B6D2 mice. (A3-F3) Insets show the absence of autofluorescence in non-transfected control testes. (4X magnification). The GFP expression presented a continuous pattern in seminiferous tubules and was detected from 1 day to 21 days. Scale bars: 1 mm and 100 μm.

(G) Comparison of number of injected mice exhibiting reporter gene expression. Mice injected with GFP-mRNA exhibited GFP expression from day 1 to day 21. Mice injected with EEV-GFP exhibited GFP expression from day 1 to 49 days. (for EEV-GFP n=11 on day 1; n=13 on day 2; n=10 on day 3; n=14 on day 7; n= 5 on day 10; n= 12 on day 15; n=11 on day 21; n= 12 on day 28; n=15 on day 35; n=17 on day 42 and n=9 on day 49) ; ( for GFP-mRNA n=3 on day 1; n=4 on day 3; n=15 on day 7; n= 21 on day 15; n=15 on day 21 and n= 5 on day 28).

Kinetics of EEV and mRNA expression by in vivo bioluminescence imaging.

(A) In vivo bioluminescence imaging quantification of luciferase expression over time following injection/electroporation of EEV-GFP-luc. (A1): EEV-GFP-Luc was injected into the testes and electroporated on day 0. Bioluminescence signal was quantified at several time points. Results are expressed as the percentage of the maximal signal (mean ± SEM; n=5 mice up to D2; n=4 from D3 to D28; n=3 from D35 to D98 and n=3 from D105 to D119). (A2) In vivo bioluminescence images of a representative mouse at several time points after administering EEV-GFP-LUC or PBS, and ex vivo bioluminescence images of testes after 119 days.

(B) In vivo bioluminescence imaging quantification of luciferase expression over time induced by injection/electroporation of LUC-mRNA. (B1) LUC-mRNA was injected into the testes and electroporated on day 0. Bioluminescence signal was quantified in the whole testis at several time points. Results are expressed as the percentage of the maximal signal (mean ± SEM; n = 5 mice). (B2): In vivo bioluminescence images of a representative mouse at several time points after administering LUC-mRNA or PBS, and ex vivo bioluminescence images of caput, testes, and cauda after 28 days.

(C) Decay over time of the number of mice expressing reporter genes. Mice were injected on day 0 with LUC-mRNA or EEV-GFP-LUC and the number of mice showing bioluminescence in the testis was counted at different time points, from day 1 to day 119. For EEV-GFP: n=13 at D1; n=13 at D2; n=4 from D3 to D28; n=3 from D35 to D98 and n=3 from D105 to D119. For mRNA-Luc: n = 5 mice for all time points.

Testicular and cellular GFP-mRNA expression measured on optically cleared testis after 3D image reconstructions from light sheet microscopy acquisitions.

Testes were injected/electroporated with GFP-mRNA on day 0. On day 1, whole testes were fixed and subjected to optical clearing. (A) Testes were observed before and after optical clearing on a binocular microscope. The right image shows the transparency of the testis after complete clearing, revealing the blue mesh throughout the organ. (B) The internal structure of a cleared testis was 3D reconstructed after lightsheet microscopy image acquisition. The reconstruction was possible only for a half testis due to light penetration constraints. Two opposing faces of the same testis are presented, allowing the distribution of GFP fluorescence throughout the seminiferous tubules to be measured. Pink fluorescence corresponds to the autofluorescence of interstitial cells located around the seminiferous tubules. Scale bars A: 1 mm and B:500 mm.

Cellular expression of EEV-GFP following in vivo injection/ electroporation.

Testes were injected/electroporated with EEV-GFP on day 0. On day 1 and on day 7, whole testes were fixed and subjected to optical clearing. Cleared tested were observed by fluorescence microscopy. (A1-A3) On day 1, transfected seminiferous tubules showed dotted green fluorescence at low magnification (X10/0.45). Nuclei were counterstained with DAPI (blue staining) to reveal the structure of the seminiferous tubules. At the cellular level, fluorescence was detectable (B1-B3) in germ cells including Spermatogonia (Sg), Spermatocytes (Scytes) and round Spermatids (RStides), as well as (C1-C3) in Sertoli cells (SC). (D1-D3) On day 7, the GFP signal was lower at low magnification (X10/0.45) and detectable (E1-E3) only in Sertoli cells (40x/1.15 WI) (n=3). E4 is an enlargement of the red square in E3, allowing the cell type to be identified. Scale bars: 100 μm, 15 μm and 3 μm.

Cellular expression of GFP-mRNA following in vivo injection/electroporation.

Testes were injected/electroporated with GFP-mRNA on day 0. On day 1 and day 7, whole testes were fixed and subjected to optical clearing. Cleared testes were observed by fluorescence microscopy. (A1-A3) On day 1, transfected seminiferous tubules showed strong broad-ranging green fluorescence at low magnification (X10/0.45). Nuclei were counterstained with DAPI (blue staining) to reveal the structure of the seminiferous tubule. At the cellular level, fluorescence was detectable in germ cells (B1-B3) including Spermatogonia (Sg), Spermatocytes (Scytes) and round Spermatids (RStides), Sperm cells (Spz) and Sertoli cells (SC). B4 is an enlargement of the red square in B3, allowing the cell types to be identified. (D1-D3) On day 7, the GFP signal remained strong at low magnification (X10/0.45) and (E1-E3) was still detectable in all germ cell types and Sertoli cells (40x/1.15 WI) (n=3). E4 is an enlargement of the red square in E3, showing that testicular sperm were also stained. Scale bars: 100 μm, 15 μm and 3 μm.

ARMC2 localization in dissociated testicular cells observed by immunofluorescence.

Cells from WT and Armc2 KO mice were stained with antibodies against ARMC2 (red signal) and tubulin (green signal). In WT mice, ARMC2 is located in the flagellum of spermatids. In KO mice, no ARMC2 signal (red fluorescence) was observed in any cells.

In vivo co-injection of Armc2-mRNA and eGFP-mRNA followed by electroporation do not affect testes morphology and weight.

Adult WT mouse testes were injected with a solution containing Armc2-mRNA and eGFP-mRNA. After injection, the testes were electroporated and mice were euthanized two weeks later. (A): Whole testis under white and blue lights on a fluorescence microscope (A1): control testes not injected or electroporated (A2): testes injected with Armc2-mRNA and eGFP-mRNA. eGFP-mRNA was co-injected to validate the transfection efficiency. (B): Ratio of injected/electroporated testis weights to control testis weights at several time points post-injection (3-, 6-, 10-, 15-, 21-, 28- and 35-days post-surgery). n= 1 mouse per time.

Sperm motility is restored in Armc2 KO mice at 21 and 35 days after injection and electroporation of Armc2-mRNA.

Adult Armc2 KO mouse testes were injected with a solution containing Armc2-mRNA. After injection, the testes were electroporated. At different times (3-, 6-, 10-, 15-, 21-, 28-, and 35-days post-injection), sperm were extracted from the cauda epididymis of the injected testis, and the sample was then examined with a CASA system to identify the percentage of motile spermatozoa. n= 2 for 15 days, n= 3 for 3, 6 and 21 days, n= 4 for 10 days, and n= 5 for 28 and 35 days.

B) Morphology of sperm cells in Armc2 KO mice injected or not with Armc2-mRNA. (B1-2): microscopic observation of epididymal sperm cells from a mature WT mouse. (B3-4): epididymal sperm cells from a mature Armc2 KO mouse 35 days after injection/electroporation with Armc2-mRNA. (B5-6): epididymal sperm cells from a control Armc2 KO male. Normal sperm cells were observed in the injected condition with Armc2-mRNA. (White arow) Scale bars:10 µm.

Damaged tubules observed by optical microscopy following overstimulation Adult mouse testes were in vivo injected and over electroporated using 10 square electric pulses to induce damage. (A) Control testis (no injection/electroporation). (B1-B2) Over electroporated testes showing damaged tubules as pearly white striations. Scale bars: 1 mm.

EEV and mRNA maps (A) EEV-plasmid map. The EEV-plasmid contains GFP, EVB ori, GFP, Luciferase, and EBNA sequences under the control of the GAGs promotor. (B) The mCherry plasmid contains the mCherry gene under the control of a T7 promotor. (C) The EEV-Armc2 plasmid contains GFP, oriP, EBNA and Armc2 sequences under the control of the CMV and T7 promotors. (D) mcherry-mRNA was synthetized as described in Material and Methods. It was validated by agarose gel electrophoresis: Lane 1: DNA size marker ladder (100 bp), lane 2: capped mcherry-mRNA (IVT product), lane 3: mcherry-mRNA after DNAse treatment. Capped and poly A tailed mcherry-mRNA migrated to the expected size of 876 bp. (E) Validation of Armc2-mRNA. HEK cells were transfected with Armc2-mRNA or EEV-Armc2. ARMC2 protein was detected by Western blot with an anti-HA primary antibody. The expected size for the ARMC2 protein is 98 kDa.

Testicular expression of mcherry-mRNA following in vivo electroporation (A1, B1, C1, D1, E1 and F1) Whole-mount testes on days 0, 1, 7, 14, 21, 28 after in vivo injection/ electroporation. (A2, B2, C2, D2, E2 and, F2) Using fluorescence microscopy, transfected testes from 12-week-old B6D2 mice express red mCherry fluorescence. mCherry was detected in a diffuse pattern throughout the seminiferous tubules from day 1 to day 15. (A3, B3, C3, D3, E3 and F3) images showing the absence of autofluorescence in non-transfected Control testes. (4X magnification). Scales bars: 1 mm and 100 μm.

Cellular expression of mcherry-mRNA following in vivo injection/ electroporation.

Cross sections (20 µm) of mouse testes on day 1 (AB) and day 7 (CD) after in vivo injection and electroporation with mcherry-mRNA, observed under fluorescence microscopy. Red signals correspond to successfully transfected testicular tubular cells; nuclei were counterstained with DAPI (blue). At the cellular level, mCherry fluorescence was detectable in Sertoli cells (SC); Spermatogonia (SG); Spermatocytes (Scytes); round Spermatids (RStides), and Sperm cells (Spz); on day 1 and day 7 (Plan-Apochromat 63x/1,4 Oil, WD 190µm) (n=3). Scale bars: 10 µm and 5 mm.

Decay over time of the number of mice exhibiting reporter gene expression following injection/electroporation of the three different mRNAs. Mice were injected on day 0 with LUC-mRNA, GFP-mRNA or mcherry-mRNA and the number of mice showing bioluminescence or fluorescence in the testis was counted at different time points. For LUC-mRNA n= 5 mice at each time point. For mcherry-mRNA n=3 on day 1; n=4 on day3; n=15 on day 7; n= 21 on day 15; n=15 on day 21; n= 5 on day 28; n=5 on day 35; and for GFP-mRNA n=3 on day 12; n=7 on day 2; n=7 on day 3; n=12 on day 7; n= 13 on day 15; n=10 on day 21 ; n= 9 on day 28; n=17 on day 35 and n=5 on day 42).