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A molecular cell biology toolkit for the study of meiosis in the silkworm Bombyx mori - PubMed

  • ️Sun Jan 01 2023

A molecular cell biology toolkit for the study of meiosis in the silkworm Bombyx mori

Youbin Xiang et al. G3 (Bethesda). 2023.

Abstract

Meiosis is usually described as 4 essential and sequential processes: (1) homolog pairing; (2) synapsis, mediated by the synaptonemal complex; (3) crossing over; and (4) segregation. In this canonical model, the maturation of crossovers into chiasmata plays a vital role in holding homologs together and ensuring their segregation at the first meiotic division. However, Lepidoptera (moths and butterflies) undergo 3 distinct meiotic processes, only one of which is canonical. Lepidoptera males utilize 2 meiotic processes: canonical meiosis that produces nucleated fertile sperm, and a noncanonical meiosis that produces anucleated nonfertile sperm which are nonetheless essential for reproduction. Lepidoptera females, which carry heteromorphic sex chromosomes, undergo a completely achiasmate (lacking crossovers) meiosis, thereby requiring an alternative mechanism to ensure proper homolog segregation. Here, we report that the development of a molecular cell biology toolkit designed to properly analyze features of meiosis, including the synaptonemal complex structure and function, in the silkworm Bombyx mori. In addition to standard homology searches to identify Bombyx orthologs of known synaptonemal complex encoding genes, we developed an ortholog discovery app (Shinyapp) to identify Bombyx orthologs of proteins involved in several meiotic processes. We used this information to clone genes expressed in the testes and then created antibodies against their protein products. We used the antibodies to confirm the localization of these proteins in normal male spermatocytes, as well as using in vitro assays to confirm orthologous interactions. The development of this toolkit will facilitate further study of the unique meiotic processes that characterize meiosis in Lepidoptera.

Keywords: Bombyx mori; HOP1; Lepidoptera; PCH2; SYCP1; SYCP2; SYCP3; meiosis; synaptonemal complex.

© The Author(s) 2023. Published by Oxford University Press on behalf of the Genetics Society of America.

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Conflict of interest statement

Conflicts of interest The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

EM images of SC from B. mori spermatocytes. Two representative images of the SC in spermatocytes. In the left image, the 2 LEs that attach to the chromatin of the homologous chromosomes are labeled and shown by the bracket. The LEs, which contain axial element components and cohesin complexes, are attached to each other along their length by TF proteins. The overlapping TFs form an electron dense region in the middle called the central element (CE). Scale bar, 100 nm.

Fig. 2.
Fig. 2.

a) The CM1 of SYCP1 in silkworm, mouse, human, and cattle (Fraune et al. 2012; Xiang et al. 2014). Left blue arrowhead corresponds to aa position 16 for silkworm, aa 96 for mouse, aa 99 for human, and aa 100 for cattle. Full sequence alignment can be found in Supplementary Fig. 4. The conservation threshold for colored shading of the residues was set to 50%. Identical residues are shown below the alignment. (*) indicates residues important for in vitro self-assembly in human SYCP1 (Dunce et al. 2018). b) CC prediction of silkworm and mouse SYCP1. Only first 500 aa residues were used for both species in the analysis.

Fig. 3.
Fig. 3.

a) Schematic depiction of SYCP2 from silkworm (1,800 aa) and mouse (1,500 aa) showing the location of the Armadillo repeat (rainbow) and the pleckstrin homology domain that make up the SYCP2 domain, the closure motif (pink), and the CC region (turquoise) [reviewed in Ur and Corbett (2021)]. The sequence of these domains for silkworm is shown in Supplementary Fig. 5. b) The secondary structure prediction of the SYCP2 domain in silkworm and mouse. The secondary structure of SYCP2 from silkworm and mouse was generated using Phyre2 web portal (

http://www.sbg.bio.ic.ac.uk/∼phyre2/html/page.cgi?id=index

). For silkworm SYCP2, Phyre2 identified with 98.1% confidence the protein database molecule SYCP2 domain of 295 residues, and silkworm SYCP2 identified a SYCP2 domain of 371 residues with 100% confidence [shown in rainbow colors which correspond to the same colors in the schematic in (a)]. c) CC prediction of silkworm and mouse SYCP2. The last 500 aa residues were used for both species in the analysis. The location of the CC domain is shown in the schematic in (a).

Fig. 4.
Fig. 4.

a) Protein sequence alignment of SYCP3 from silkworm and mouse. Default of 30% was used for colored shading of the conservation for residues in JalView. Comparison of silkworm and mouse SYCP3 proteins show 25% identity and 51% positive substitutions. b) Schematic depiction of the location of the Cor1 superfamily domain in SYCP3 of silkworm and mouse. Cor1 superfamily domain for each protein was identified by NCBI blastp search. Cor1 domain in silkworm spans residues 56–170 and in mouse 101–224. c) Bombyx mori SYCP3 has a predicted CC domain.

Fig. 5.
Fig. 5.

a) Protein sequence alignment of HOP1 from silkworm and HORMAD1 mouse. The HORMA domain sequence is underlined in black. Comparison of silkworm and mouse proteins show 31% identity and 51% positive substitutions. Default of 30% was used for colored shading of the conservation for residues in JalView. b) Schematic depiction of the location of the HORMA domain which spans residues 28–234. The PHD superfamily domain identified by NCBI is just downstream of the HORMA domain at residues 279–325 (orange box).

Fig. 6.
Fig. 6.

a) Protein sequence alignment of PCH2 from silkworm and mouse. Comparison of PCH2 from silkworm and TRIP13/PCH2 from mouse show 43% identity and 65% positive substitutions. Default of 30% was used for colored shading of the conservation for residues in JalView. Black bar under the sequence is the AAA protein-family signature domain that is a characteristic of AAA + ATPases. Red and green bars under sequence are the canonical Walker A and B motifs, respectively. b) Schematic depiction of the location of the AAA protein-family signature domain and spans residues 280–299 in silkworm and 292–312 in mouse.

Fig. 7.
Fig. 7.

Meiotic chromosome spermatocyte spreads of pachytene nuclei. a) Immunostaining of pachytene nuclei stained with DAPI (blue), SMC3 (green), and SMC1 (red) in the merge with individual panels shown in white. Cohesin complex containing SMC1/3 localizes along the chromatin axes. b) Immunostaining of pachytene nuclei stained with HOP1 (blue), SYCP1 (green), and SYCP2 (red) in the merge with individual panels shown in white. Axial/LE components HOP1 and SYCP2 coassociate with the SC TF component SYCP1. c) Immunostaining of pachytene nuclei stained with SYCP1 (blue), SYCP3 (green), and SYCP2 (red) in the merge with individual panels shown in white. SYCP2 and SYCP3 both coassociate with SYCP1 on the SC. All image panels are projections. Scale bars, 15 μm.

Fig. 8.
Fig. 8.

Meiotic chromosome spermatocyte spreads of zygotene, pachytene, and late pachytene nuclei. Staging of nuclei was determined based on SC morphology within each nucleus and shown is only the denoted antibody (SMC1, SMC3, HOP1, SYCP2, SYCP3, and SYCP1) for each image. Scale bars, 10 μm.

Fig. 9.
Fig. 9.

Interaction of B. mori proteins using yeast two-hybrid assay. a) Only residues 190–1,800 of SYCP2 interacts with SYCP3 on the selection plate SD-HIS. SYCP2 N-terminal and C-terminal residues were 190–1,469 and 1,470–1,800, respectively. Note that when full length SYCP2 (1–1,800) was tested, no interaction was found. This could be due to structural constraints of the SYCP2 domain near the GAL4-AD of the vector or unknown constraints using the yeast two-hybrid systems. b) HOP1 and SYCP2, as well as (c) HOP1 and PCH2 interact based on growth on the selection plate SD-HIS. HOP1 does not interact with empty prey vector [shown in (b) and (c) for independent experiments].

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