Journal: bioRxiv

Article Title: Actl7b -deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis

doi: 10.1101/2022.12.19.520998

Figure Lengend Snippet: (A) Western blots on the protein input (whole WT testis), the non-bound fraction of the beads only control (n.b. c.), the IP eluate of the beads only control (IP c.), non-bound fraction of the anti-ACTL7B-coupled beads (n.b.), the IP eluate of the anti-ACTL7B-coupled beads (IP). Anti-DYNLL2, anti-DYNLL1 and anti-ACTL7B were used. (B) Western blots on the protein input (whole WT testis), the IP eluate of the anti-DYNLL1-coupled beads (IP D1), the IP eluate of the anti-DYNLL2-coupled beads (IP D2), the IP eluate of the beads only control (IP c.). Anti-ACTL7B and anti-DYNLL2 were used for western blots. (C) Western blots on protein extractions from whole Actl7b+/+, Actl7b+/- and Actl7b-/- testis. Anti-ACTL7B, anti-DYNLL2, anti-HOOK1, anti-ACTL7A and anti-DYNLL2 were used. α-tubulin was used as loading control. (D) Graphical depiction of DYNLL1 and DYNLL2 immunolocalization during spermiogenesis based on literature . (E) DYNLL1 staining in Actl7b+/+, Actl7b+/- and Actl7b-/- elongating spermatids. Dapi was used as counterstain. Scale: 20 µm. (F) DYNLL2 staining in Actl7b+/+, Actl7b+/- and Actl7b-/- elongating spermatids. Dapi was used as counterstain. Scale: 20 µm.

Article Snippet: Sections stained with anti-DYNLL1, anti-DYNLL2 and anti-ACTIN were processed using the VectaFluor™ Horse Anti-Rabbit IgG, DyLight® 488 Antibody Kit (Vector Laboratories, Burlingame, CA, USA; DI-1788), sections stained against Ezrin were processed with the VectaFluor™ Anti-Mouse IgG, DyLight® 594 Kit (Vector Laboratories, DI-2794), sections stained against PRM2 and ODF2 were processed with the VectaFluor™ Duet Immunofluorescence Double Labeling Kit, DyLight® 594 Anti-Rabbit, DyLight® 488 Anti-Mouse (Vector Laboratories; DK-8828) and sections stained with anti-ACTL7B were processed using the Vectastain® ABC-AP Kit (Vector Laboratories; AK-5001) and ImmPACT® Vector® Red alkaline phosphatase substrate (Vector Laboratories; SK-5105).

Techniques: Western Blot, Staining

Journal: bioRxiv

Article Title: Actl7b -deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis

doi: 10.1101/2022.12.19.520998

Figure Lengend Snippet: (A) Graphical representation of CRISPR-Cas9-mediated gene editing of the Actl7b locus using two guide RNAs (indicated by black arrow heads) targeting the intron-less Actl7b coding sequence. 473 bp were deleted causing a frameshift leading to a premature stop. (B) Agarose gel of genotyping polymerase chain reaction of Actl7b+/+, Actl7b+/- and Actl7b-/- mice (WT band: 607 bp; KO band:134 bp). bp= base pairs (C) Graphical representation of Actl7b expression and ACTL7B immunolocalization during spermiogenesis based on literature (Hisano et al . 2003b, Tanaka et al . 2003 , Guo et al . 2018 ). (D) Immunohistochemical staining against ACTL7B on Bouin-fixed, paraffin-embedded Actl7b+/+, Actl7b+/- and Actl7b-/- testis sections counterstained with hematoxylin. Scale: 20 μm

Article Snippet: Sections stained with anti-DYNLL1, anti-DYNLL2 and anti-ACTIN were processed using the VectaFluor™ Horse Anti-Rabbit IgG, DyLight® 488 Antibody Kit (Vector Laboratories, Burlingame, CA, USA; DI-1788), sections stained against Ezrin were processed with the VectaFluor™ Anti-Mouse IgG, DyLight® 594 Kit (Vector Laboratories, DI-2794), sections stained against PRM2 and ODF2 were processed with the VectaFluor™ Duet Immunofluorescence Double Labeling Kit, DyLight® 594 Anti-Rabbit, DyLight® 488 Anti-Mouse (Vector Laboratories; DK-8828) and sections stained with anti-ACTL7B were processed using the Vectastain® ABC-AP Kit (Vector Laboratories; AK-5001) and ImmPACT® Vector® Red alkaline phosphatase substrate (Vector Laboratories; SK-5105).

Techniques: CRISPR, Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Expressing, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Actl7b -deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis

doi: 10.1101/2022.12.19.520998

Figure Lengend Snippet: (A) Pregnancy rate of Actl7b+/+, Actl7b+/- and Actl7b-/- males mated with female WT C57BL/6J mice (n = number of males). (B) Average litter sizes monitored after mating of Actl7b+/+, Actl7b+/- males with female WT C57BL/6J mice (n = number of males). 5 plugs per male were monitored. (C) Mean testis weight of Actl7b+/+, Actl7b+/- and Actl7b-/- males (n = number of males). (D) Testis to body weight ratio of Actl7b+/+, Actl7b+/- and Actl7b-/- males (n = number of males). (E) Representative picture of testis dissected from Actl7b+/+, Actl7b+/- and Actl7b-/- littermates with similar body weight. (F) Hematoxylin-Eosin staining of testis (IV-VI of the epithelial cycle), caput epididymis and cauda epididymis of Actl7b+/+, Actl7b+/- and Actl7b-/- males. Scale: 50 μm

Article Snippet: Sections stained with anti-DYNLL1, anti-DYNLL2 and anti-ACTIN were processed using the VectaFluor™ Horse Anti-Rabbit IgG, DyLight® 488 Antibody Kit (Vector Laboratories, Burlingame, CA, USA; DI-1788), sections stained against Ezrin were processed with the VectaFluor™ Anti-Mouse IgG, DyLight® 594 Kit (Vector Laboratories, DI-2794), sections stained against PRM2 and ODF2 were processed with the VectaFluor™ Duet Immunofluorescence Double Labeling Kit, DyLight® 594 Anti-Rabbit, DyLight® 488 Anti-Mouse (Vector Laboratories; DK-8828) and sections stained with anti-ACTL7B were processed using the Vectastain® ABC-AP Kit (Vector Laboratories; AK-5001) and ImmPACT® Vector® Red alkaline phosphatase substrate (Vector Laboratories; SK-5105).

Techniques: Staining

Journal: bioRxiv

Article Title: Actl7b -deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis

doi: 10.1101/2022.12.19.520998

Figure Lengend Snippet: (A) Hematoxylin-Eosin staining of Bouin-fixed paraffin-embedded testis sections of Actl7b-/- mice. Immature apoptotic germ cells can be seen to be released into the lumen (marked by green arrows). In late stage VIII elongated spermatids with an abnormal morphology, which were not spermiated, were seen (marked by green arrow heads) and round spermatids blocked in development with dark cytoplasm were found (marked by vermillion arrows). Vacuolation of seminiferous tubules was detected (marked by vermillion arrow heads). Scale: 50 µm (B) Representative transmission electron micrograph of a disorganized Actl7b-/- seminiferous tubule. vermillion arrow heads = vacuoles, encircled in vermillion = flagellar cross sections, s2 = secondary spermatocyte, 1 = step 1 spermatid with centrally positioned nucleus and chromatoid body, 2-3 = step 2-3 spermatid with centrally positioned nucleus and acrosomal vesicle not yet connected to nucleus, 9 = step 9 spermatid with approaching centriole, 12-13 = step 12-13 spermatid with not yet fully condensed chromatin and manchette, 13-15 = abnormal step 13-15 spermatids, eP = early pachytene spermatocytes, SC = Sertoli cell nucleus, white arrow heads = basal lamina, pM = peritubular myoid cell, Scale: 10 µm (C-D) Transmission electron micrographs of vesicles filled with degrading spermatids detected in Actl7b-/- seminiferous tubules. N = condensed nuclei, dN = degraded nucleus, G = granular material, vermillion arrow heads = acrosomal structures, green arrow heads = flagellar cross sections, green arrows = mitochondria. Scales: 5 µm, 2 µm

Article Snippet: Sections stained with anti-DYNLL1, anti-DYNLL2 and anti-ACTIN were processed using the VectaFluor™ Horse Anti-Rabbit IgG, DyLight® 488 Antibody Kit (Vector Laboratories, Burlingame, CA, USA; DI-1788), sections stained against Ezrin were processed with the VectaFluor™ Anti-Mouse IgG, DyLight® 594 Kit (Vector Laboratories, DI-2794), sections stained against PRM2 and ODF2 were processed with the VectaFluor™ Duet Immunofluorescence Double Labeling Kit, DyLight® 594 Anti-Rabbit, DyLight® 488 Anti-Mouse (Vector Laboratories; DK-8828) and sections stained with anti-ACTL7B were processed using the Vectastain® ABC-AP Kit (Vector Laboratories; AK-5001) and ImmPACT® Vector® Red alkaline phosphatase substrate (Vector Laboratories; SK-5105).

Techniques: Staining, Transmission Assay

Journal: bioRxiv

Article Title: Actl7b -deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis

doi: 10.1101/2022.12.19.520998

Figure Lengend Snippet: (A) Periodic acid Schiff staining of testis of Actl7b+/+, Actl7b+/- and Actl7b-/- males. Acrosomal structures are indicated by vermillion arrow heads. Scale: 50 μm (B) IHC staining against ODF2 (red fluorescence) and PRM2 (green fluorescence) of Actl7b+/+, Actl7b+/- and Actl7b-/- testis tissue sections. DAPI (in grey) was used as the counterstain. Scale: 50 μm (C) Transmission electron micrographs of testicular sperm presented with condensed chromatin from Actl7b+/+ and Actl7b-/- males. Scale: 2 µm

Article Snippet: Sections stained with anti-DYNLL1, anti-DYNLL2 and anti-ACTIN were processed using the VectaFluor™ Horse Anti-Rabbit IgG, DyLight® 488 Antibody Kit (Vector Laboratories, Burlingame, CA, USA; DI-1788), sections stained against Ezrin were processed with the VectaFluor™ Anti-Mouse IgG, DyLight® 594 Kit (Vector Laboratories, DI-2794), sections stained against PRM2 and ODF2 were processed with the VectaFluor™ Duet Immunofluorescence Double Labeling Kit, DyLight® 594 Anti-Rabbit, DyLight® 488 Anti-Mouse (Vector Laboratories; DK-8828) and sections stained with anti-ACTL7B were processed using the Vectastain® ABC-AP Kit (Vector Laboratories; AK-5001) and ImmPACT® Vector® Red alkaline phosphatase substrate (Vector Laboratories; SK-5105).

Techniques: Staining, Immunohistochemistry, Fluorescence, Transmission Assay

Journal: bioRxiv

Article Title: Actl7b -deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis

doi: 10.1101/2022.12.19.520998

Figure Lengend Snippet: (A) Transmission micrograph of a lumen of an Actl7b-/- seminiferous tubule. (B) Transmission micrograph of a lumen of an Actl7b+/+ seminiferous tubule. (C-F) Representative images of Actl7b-/- spermatids with condensed nuclei. Scales A-B: 5 µm, C-F: 2µm

Article Snippet: Sections stained with anti-DYNLL1, anti-DYNLL2 and anti-ACTIN were processed using the VectaFluor™ Horse Anti-Rabbit IgG, DyLight® 488 Antibody Kit (Vector Laboratories, Burlingame, CA, USA; DI-1788), sections stained against Ezrin were processed with the VectaFluor™ Anti-Mouse IgG, DyLight® 594 Kit (Vector Laboratories, DI-2794), sections stained against PRM2 and ODF2 were processed with the VectaFluor™ Duet Immunofluorescence Double Labeling Kit, DyLight® 594 Anti-Rabbit, DyLight® 488 Anti-Mouse (Vector Laboratories; DK-8828) and sections stained with anti-ACTL7B were processed using the Vectastain® ABC-AP Kit (Vector Laboratories; AK-5001) and ImmPACT® Vector® Red alkaline phosphatase substrate (Vector Laboratories; SK-5105).

Techniques: Transmission Assay

Journal: bioRxiv

Article Title: Actl7b -deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis

doi: 10.1101/2022.12.19.520998

Figure Lengend Snippet: (A) Western blots on the protein input (whole WT testis), the non-bound fraction of the beads only control (n.b. c.), the IP eluate of the beads only control (IP c.), non-bound fraction of the anti-ACTL7B-coupled beads (n.b.), the IP eluate of the anti-ACTL7B-coupled beads (IP). Anti-DYNLL2, anti-DYNLL1 and anti-ACTL7B were used. (B) Western blots on the protein input (whole WT testis), the IP eluate of the anti-DYNLL1-coupled beads (IP D1), the IP eluate of the anti-DYNLL2-coupled beads (IP D2), the IP eluate of the beads only control (IP c.). Anti-ACTL7B and anti-DYNLL2 were used for western blots. (C) Western blots on protein extractions from whole Actl7b+/+, Actl7b+/- and Actl7b-/- testis. Anti-ACTL7B, anti-DYNLL2, anti-HOOK1, anti-ACTL7A and anti-DYNLL2 were used. α-tubulin was used as loading control. (D) Graphical depiction of DYNLL1 and DYNLL2 immunolocalization during spermiogenesis based on literature . (E) DYNLL1 staining in Actl7b+/+, Actl7b+/- and Actl7b-/- elongating spermatids. Dapi was used as counterstain. Scale: 20 µm. (F) DYNLL2 staining in Actl7b+/+, Actl7b+/- and Actl7b-/- elongating spermatids. Dapi was used as counterstain. Scale: 20 µm.

Article Snippet: Sections stained with anti-DYNLL1, anti-DYNLL2 and anti-ACTIN were processed using the VectaFluor™ Horse Anti-Rabbit IgG, DyLight® 488 Antibody Kit (Vector Laboratories, Burlingame, CA, USA; DI-1788), sections stained against Ezrin were processed with the VectaFluor™ Anti-Mouse IgG, DyLight® 594 Kit (Vector Laboratories, DI-2794), sections stained against PRM2 and ODF2 were processed with the VectaFluor™ Duet Immunofluorescence Double Labeling Kit, DyLight® 594 Anti-Rabbit, DyLight® 488 Anti-Mouse (Vector Laboratories; DK-8828) and sections stained with anti-ACTL7B were processed using the Vectastain® ABC-AP Kit (Vector Laboratories; AK-5001) and ImmPACT® Vector® Red alkaline phosphatase substrate (Vector Laboratories; SK-5105).

Techniques: Western Blot, Staining

Journal: bioRxiv

Article Title: Actl7b -deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis

doi: 10.1101/2022.12.19.520998

Figure Lengend Snippet: (A-C) Volcano plots showing differential abundance (DA) of proteins in Actl7b+/- compared to Actl7b+/+ (A) , Actl7b-/- compared to Actl7b+/- (B) and Actl7b-/- compared to Actl7b+/+ (C) testis. Proteins showing a significant DA are indicated in teal (lower abundant) and yellow (higher abundant) (adjusted p-value > 0.05). Top DA proteins are labeled with their corresponding gene symbol. (D-E) Venn diagrams showing the overlap of significantly higher (D) and lower (E) abundant proteins in the comparisons of Actl7b-/- vs. Actl7b+/+ and Actl7b-/- vs. Actl7b+/+ testis (adjusted p-value ≤ 0.05, LFC ≤ 1). (F-G) Dot plots of the top twenty gene ontology (GO)-biological process terms significantly enriched in the higher abundant (F) and lower abundant (G) proteins in Actl7b-/- vs. Actl7b+/+ samples (p ≤ 0.05, LFC ≤ 0.05). Dot sizes represent the number of proteins contributing to the GO-term.

Article Snippet: Sections stained with anti-DYNLL1, anti-DYNLL2 and anti-ACTIN were processed using the VectaFluor™ Horse Anti-Rabbit IgG, DyLight® 488 Antibody Kit (Vector Laboratories, Burlingame, CA, USA; DI-1788), sections stained against Ezrin were processed with the VectaFluor™ Anti-Mouse IgG, DyLight® 594 Kit (Vector Laboratories, DI-2794), sections stained against PRM2 and ODF2 were processed with the VectaFluor™ Duet Immunofluorescence Double Labeling Kit, DyLight® 594 Anti-Rabbit, DyLight® 488 Anti-Mouse (Vector Laboratories; DK-8828) and sections stained with anti-ACTL7B were processed using the Vectastain® ABC-AP Kit (Vector Laboratories; AK-5001) and ImmPACT® Vector® Red alkaline phosphatase substrate (Vector Laboratories; SK-5105).

Techniques: Labeling