From the list on the left, make sure you are in UI Settings. 6B).

Acrosome reaction rates of spermatozoa treated with DMSO and A23187 were 19.7 ± 10.8% (n = 4) and 83.3 ± 5.5% (n = 4), respectively. When Sof1 mutant spermatozoa were used for in vitro fertilization (IVF) with cumulus-intact oocytes, Sof1 KO spermatozoa could penetrate the ZP, but they accumulated in the perivitelline space (Fig. I am having a similar problem. Thus, the function of FIMP remains to be studied in more detail.

However, Spaca6 KO spermatozoa were rarely found to fuse with the oocyte plasma membrane (Fig. In the present study, we focused on three testis-enriched genes (sperm−oocyte fusion required 1 [Sof1], transmembrane protein 95 [Tmem95], and sperm acrosome membrane-associated protein 6 [Spaca6]), which are predicted as membrane proteins by in silico analyses. As mice lacking 1,024 bp and 1,039 bp delete the SOF1 ORF, these mice were used for subsequent analyses. S1) were used for this assay.

As depicted by RT-PCR analyses (using primers as shown in SI Appendix, Table S1), Tmem95 was exclusively expressed in mouse testis, and testis expression was first observed on day 21 postpartum, during which spermiogenesis initiates (Fig. Render Job 1 failed as the current clip could not be processed. (B) Detection of Spaca6 mRNA in mouse testes at various postnatal days by RT-PCR. Transgenic mice which expressed mouse Sof1, Tmem95, and Spaca6 rescued the sterility of Sof1, Tmem95, and Spaca6 KO males, respectively.

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In this S6D). (D) Detection of SOF1 in A23187-treated spermatozoa. In conclusion, we discovered three genes, Sof1, Tmem95, and Spaca6, that encode sperm proteins required for fusion with oocytes.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1922650117/-/DCSupplemental. Such adhesion is believed to originate from the interaction between IZUMO1 overexpressed on the plasma membrane of HEK293T cells and the receptor of IZUMO1, JUNO, on the oocyte membrane. IZUMO1 was found to interact with SOF1 (SI Appendix, Fig. Spermatozoa were incubated with the ZP-free oocytes prestained with Hoechst 33342 for 30 min.

We established three mutant lines carrying 1-base pair (bp) (endonuclease-mediated mutation [em] 1), 1,024-bp (em2), or 1,039-bp (em3) deletions (SI Appendix, Fig. (E) Oocyte observation of females mated with Sof1 del/del males having the Sof1-PA Tg insertion. S4 B and C and Table S2). Izumo1 KO spermatozoa can penetrate the zona pellucida (ZP), but spermatozoa accumulate in the perivitelline space due to a fusion defect (9). IVF was performed as described previously (45). However, in vitro studies implied that IZUMO1 may be responsible for sperm−oocyte membrane adhesion instead of fusion (16, 17).

S8D), and normal sperm motility (SI Appendix, Fig. Click Relink Media. Further, when Sof1 del/del males were caged with wt/wt females for 2 mo, the males failed to sire offspring (Fig. TMEM95 may not act as a fusogenic protein that directly mediates the membrane fusion between spermatozoa and oocytes. The site may not work properly if you don't, If you do not update your browser, we suggest you visit, Press J to jump to the feed. Proteins of cauda epididymal spermatozoa were extracted with sample buffer containing β-mercaptoethanol (Nacalai Tesque) as described previously (38). S7A). S2G, S6A, and S9A). ; Takeda Science Foundation grants to Y.F.

I went in and deleted it's media input and put it back. Render job 1 failed as the current clip could not be processed. S12). 2B). The litter sizes of Sof1 del/del and Sof1-PA Tg rescue males were 0 and 10.2 ± 0.5, respectively. In 2014, by using oligomerized IZUMO1 ectodomains, Bianchi et al.

The tagged SPACA6 was localized to the acrosome cap before the acrosome reaction and translocated to the equatorial segment after the acrosome reaction (Fig. Hey, after a few weeks I reopened a project, changed a few minor things in grading and wanted to render it out again.

Sucks but it works, New comments cannot be posted and votes cannot be cast, More posts from the blackmagicdesign community, Looks like you're using new Reddit on an old browser. S11D). The clip 2020-02-28 22.55.25.MOV could not be decoded correctly.

Izumo1, Sof1, Tmem95, Spaca6, and Fimp ORFs were cloned from mouse testis cDNA, a Kozak sequence was added upstream of the ATG start codon, and a sequence containing an epitope tag was added in-frame prior to the stop codon, using RT-PCR. The wt/wt males were examined, in the meantime, as a positive control.

While about 97% of the control sperm-inseminated oocytes formed 2 PN, KO spermatozoa could not fuse with ZP-free oocytes.

A search for gRNA and off-target sequences was performed using CRISPRdirect software (https://crispr.dbcls.jp/) (40) and Benchling (https://www.benchling.com/academic/). After 10 and 120 min of incubation in TYH medium, the localization of IZUMO1 in control and KO spermatozoa was visualized by immunocytochemistry.

5E).

The video clips were rendered with a digitization rate of 25 frames per second, and the stereo soundtracks were digitized at 44.1 kHz with 16-bit resolution. Spermatozoa were incubated with Hoechst 33342-preloaded oocytes for 30 min, and the membrane fusion was determined by the transfer of Hoechst 33342 fluorescence signal to fused sperm heads (yellow arrows).

3F).

Testis appearance, size, and weight in del/del males were normal compared to their del/wt littermates (SI Appendix, Fig.

Based on public databases such as UniProt and Simple Modular Architecture Research Tool (SMART), SPACA6 displays similar domain structures compared with IZUMO1.

In this particular case, the error occurred at the very end of rendering and resulting video seemed to be fine. To examine the physiological function of SOF1, we generated Sof1 mutant mice by introducing a guide RNA (gRNA)/Cas9 expression vector into oocytes (for indel) and embryonic stem (ES) cells (for deletion) (SI Appendix, Fig.

A monoclonal antibody against β-Actin (ACTB) was purchased from Abcam (ab6276). Sof1 is originally registered as 1700034O15Rik in Mouse Genome Informatics and recently changed to Llcfc1 because it conserves an “LLLL and CFNLAS” motif, but its physiological function remains to be studied.

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