EMBO reports - Peer Review Process File - EMBO-2015-41060
Manuscript EMBO-2015-41060
Sororin loads to the synaptonemal complex central region
independently of meiotic cohesin complexes
Natalia Felipe-Medina, Miguel Ruiz-Torres, Ines Berenguer, Alberto Viera, Sara Perez, Miguel
Ruiz-Torres, Jose Luis Barbero, Elena Llano, Tomoyuki Fukuda, Manfred Alsheimer, Alberto
Pendas, Ana Losada, José Suja, Rocio Gómez Lencero
Corresponding author: Rocio Gómez Lencero, Universidad Autonoma de Madrid
Review timeline:
Submission date:
Editorial Decision:
Revision received:
Editorial Decision:
Revision received:
Accepted:
22 July 2015
09 September 2015
11 December 2015
13 January 2016
03 February 2016
05 February 2016
Editor: Achim Breiling/Martina Rembold
Transaction Report:
(Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity,
letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this
compilation.)
1st Editorial Decision
09 September 2015
Thank you for the submission of your research manuscript to our journal. We have now received the
full set of referee reports, which can be found at the end of this e-mail.
As you will see, the referees acknowledge that the findings are potentially interesting. However, all
three referees also point out several technical concerns and have a number of suggestions for how
the study should be strengthened, and I think that all of them should be addressed. As the reports are
below, I will not detail them here. However, addressing the dependence of Sororin on cohesion at
later stages of meiosis (referee 2, point 4) and its dependence on double-strand breaks (referee 3),
additional co-stainings suggested by referee 1 and referee 3 to substantiate the findings and the
avoidance of over-interpretation of data would be necessary. In particular, both referee 2 (point 2)
and referee 3 are concerned about the specificity of the Sororin antibodies used in the study, which
has to be addressed.
Overall, most concerns seem important, reasonable and should be addressed. An exception is point 3
from referee 2. Given that no data has been published on the meiotic phenotype of Pds5b-deficient
mice, it would be out of the scope of the study.
Given these constructive comments, we would like to invite you to revise your manuscript with the
understanding that the referee concerns must be fully addressed and their suggestions taken on
board. Please address all referee concerns in a complete point-by-point response. Acceptance of the
manuscript will depend on a positive outcome of a second round of review. It is EMBO reports
© European Molecular Biology Organization
1
EMBO reports - Peer Review Process File - EMBO-2015-41060
policy to allow a single round of revision only and acceptance or rejection of the manuscript will
therefore depend on the completeness of your responses included in the next, final version of the
manuscript.
Revised manuscripts should be submitted within three months of a request for revision; they will
otherwise be treated as new submissions. Please contact us if a 3-months time frame is not sufficient
for the revisions so that we can discuss the revisions further.
You can either publish the study as a short report or as a full article. I suggest the short reports
format. For short reports, the revised manuscript should not exceed 35,000 characters (including
spaces and references) and 5 main plus 5 expanded view figures. The results and discussion section
must further be combined, which will help to shorten the manuscript text by eliminating some
redundancy that is inevitable when discussing the same experiments twice. Commonly used
materials and methods can further be moved to the supplementary information, however, please note
that materials and methods essential for the understanding of the experiments described in the main
text must remain in the main manuscript file. The legend for expanded view figures has to be
included at the end of the text file. For a normal article there are no length limitations, but the results
and discussion section must be separate and the entire materials and methods included in the main
manuscript file.
Please also include scale bars in all microscopy images.
We now strongly encourage the publication of original source data with the aim of making primary
data more accessible and transparent to the reader. The source data will be published in a separate
source data file online along with the accepted manuscript and will be linked to the relevant figure.
If you would like to use this opportunity, please submit the source data (for example scans of entire
gels or blots, data points of graphs in an excel sheet, additional images, etc.) of your key
experiments together with the revised manuscript. Please include size markers for scans of entire
gels, label the scans with figure and panel number, and send one PDF file per figure or per figure
panel.
As part of the EMBO publication's Transparent Editorial Process, EMBO reports publishes online a
Review Process File to accompany accepted manuscripts. This File will be published in conjunction
with your paper and will include the referee reports, your point-by-point response and all pertinent
correspondence relating to the manuscript.
You are able to opt out of this by letting the editorial office know ([email protected]). If you
do opt out, the Review Process File link will point to the following statement: "No Review Process
File is available with this article, as the authors have chosen not to make the review process public
in this case."
I look forward to seeing a revised version of your manuscript when it is ready. Please let me know if
you have questions or comments regarding the revision.
REFEREE REPORTS
Referee #1:
The manuscript by Gomez et al studies the localization of Sororin, a regulator of cohesin, in mouse
spermatocytes. By using their technique of spermatocyte spreading, they find that Sororin localizes
at the central regions (CRs) of the synaptonemal complex during meiosis. This localization pattern is
different from that of cohesin complexes, which are known to localize at axial elements. Their
analyses of spermatocytes from Rec8-/- mice and Smc1b-/- mice show that the localization of
Sororin at CRs is independent of cohesin. Based on these data, they speculate that Sororin has a role
at CRs independent of cohesin.
A novel point of this manuscript is cohesin-independent localization of Sororin. This conclusion is
robustly supported by their careful analysis that compares the localization of Sororin and cohesins
and by their analysis of cohesin-KO mice. Although the function of Sororin independent of cohesin
© European Molecular Biology Organization
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EMBO reports - Peer Review Process File - EMBO-2015-41060
is unclear from the data shown in the manuscript, the description of cohesin-independent
localization of Sororin would be a significant contribution to the field and interest researchers in the
specific area. The data are of high quality and the images are impressively beautiful. Sororin's
potential roles independent of cohesin are appropriately discussed. I believe the current manuscript
is suitable for publication in EMBO reports.
I have only one minor suggestion that would further improve the manuscript.
- It would be helpful to further confirm their conclusion if they could include a data for double
staining of Rec8-myc and Sororin at early zygotene in the manuscript.
Referee #2:
In this study, the authors have analyzed the localization of Sororin, a protein required for sister
chromatid cohesion in somatic cells, by immunofluorescence during male mouse meiosis. In
somatic cells, Sororin contributes to sister chromatid cohesion by preventing the release of cohesin
complexes from DNA. Sororin is thought to achieve this by binding to cohesin and by inhibiting
Wapl, a protein that can release cohesin from DNA. There are conflicting reports about how Sororin
interacts with cohesin, both the cohesin subunits Pds5A and Pds5B (Nishiyama et al., Cell 2010)
and SA2 have been reported as Sororin binding partners (Zhang and Pati, Cell Cycle 2015). Specific
isoforms of cohesin complexes also exist in meiotic cells, where they are essential for proper
homologous pairing, chromosome cohesion and chromosome segregation, but it is not known if
Sororin is expressed in meiotic cells and has a function in sister chromatid cohesion in meiosis.
In this study, the authors describe staining patterns obtained with Sororin antibodies in surfacespread mouse spermatocytes and show that this pattern changes in interesting ways during meiotic
progression. From zygotene to diplotene Sororin antibodies stain the central region of the
synaptonemal complex (SC), where the two homologous chromosomes are synapsed and where the
protein SYCP1 is located. Surprisingly, during these stages of prophase I, Sororin staining does not
co-localize with cohesin, its binding partner in somatic cells. Cohesin is known to be present at
axial/lateral elements, where also Wapl and Pds5B have previously been detected. Moreover, the
authors provide evidence that Sororin localization to the central region of the SC does not depend on
the meiotic-specific cohesin subunits Rec8 and Smc1b. Based on these results the authors suggest
that Sororin might be involved in chromosome pairing and synapsis and that its functions during
early stages of prophase I might be independent of cohesin complexes. From late diplotene to
anaphase II, Sororin staining is detected at centromeres, where cohesin is known to exist, and its
localization there depends on Sugoshin2 (Sgo2). Based on this observation the authors suggest that
Sororin might work in combination with Sgo2 in order to protect centromeric cohesion until
anaphase II onset. In summary, the authors propose that Sororin might have two distinct and
independent roles during male mouse meiosis, a cohesin independent role in chromosome pairing
and synapsis, and a cohesin dependent role in protecting centromeric cohesion.
This manuscript addresses an interesting topic, as previous work has revealed the importance of
Sororin for sister chromatid cohesion in somatic cells, but nothing is known about the existence and
possible functions of Sororin in meiotic cells. The presented microscopic data are of high quality,
and the observed staining patterns very interesting. However, this study has several limitations. One
is the lack of loss-of-function data. But one can argue that this is beyond the scope of this study, and
since we know nothing about Sororin in meiosis and the observed staining patterns are interesting
and suggestive, I would argue that it is fine to publish this study without functional data, as long as it
is clear that the manuscript does not overstate any conclusions (see for example comment 1 on the
title of the manuscript below). The second limitation is more serious. How do we know that the
observed staining patterns are representing the location of Sororin? More controls will be needed to
address this central point (see comment 2 below with some suggestions for what could be done).
This is particularly important as in somatic cells Sororin is thought to be recruited to chromatin only
via cohesin, so can the authors exclude that the central element staining observed with Sororin
antibodies is not caused by a cross-reacting antigen? The third limitation is whether the authors can
really exclude that the observed Sororin staining pattern is cohesin independent. It would be
technically possible to address this more rigorously and I would recommend doing this (see points 3
and 4 below). If these points can be addressed I would support publication of this interesting study.
© European Molecular Biology Organization
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Specific points:
1. The authors do not provide functional data supporting the role of Sororin in chromosome pairing
and centromere dynamics in male mouse meiosis. To properly test the role of Sororin in these
processes would have been necessary to analyze chromosome pairing and centromere dynamics in
the absence of Sororin. I would suggest that the authors change the title in a way that it reflects what
the manuscript actually shows, for example by saying "Sororin localizes to the central elements
during mouse male meiosis independently of meiotic cohesin complexes".
2. As already mentioned, more work is required to control the specificity of the Sororin antibodies
used. For example: Do the two Sororin antibodies mentioned in the Methods give comparable
staining patterns? This should be shown. How does the entire Western blot in Figure S1 look like,
are there no other cross-reacting bands? Do these antibodies also cross-react with Sororin in somatic
cells? This could be analyzed on the same Western side by side. If the answer is yes, it would be
really helpful to try to deplete Sororin in somatic cells by RNA interference to see if the recognized
bands do indeed represent Sororin. Likewise, if the Sororin antibodies stained somatic cells in
immunofluorescence experiments and these signals were reduced by Sororin specific siRNAs, this
would also ad confidence to the interpretation. Alternatively, it could be tested in coimmunoprecipitation experiments from somatic cell lysates if the proteins detected by these
antibodies are associated with cohesin. I realize that it is difficult to be absolutely sure about the
specificity of antibodies, but since literally all conclusions in this manuscript depend on this
specificity the authors need to provide more evidence for this. For an example of how RNAi can be
used to control the specificity of Sororin antibodies see Carretero et al., 2013.
3. In Figure 3, the authors show that Sororin localization at the central region of the SC depends on
Sycp1, but not on Rec8 and Smc1β. In mitosis, Sororin binding to DNA depends on Pds5b and
Pds5a (Nishiyama et al., 2010; Carretero et. al. 2013). Moreover, in mouse spermatocytes, Pds5b
signal is depleted from the chromosome axis when homologues undergo desynapsis at diplotene
stage, at the same time Sororin is removed from the central region of the SC. Additionally, Pds5b
signal persists in Smc1β-/- spermatocytes (Fukuda and Hoog. 2010). This poses the possibility that
Sororin localization in male spermatocytes might still depend on some cohesin complexes
(presumably the ones that contain Smc1α and Rad21L) and on Pds5b. For this reason, it would be
interesting to analyze Sororin localization in Pds5b-/- spermatocytes.
4. In Figures 4 and 5, the authors show that Sororin localizes to centromeres from late diplotene
through anaphase II. They present a very similar pattern of localization to Shugoshin2 (Sgo2)
(Gomez et al, 2007). Sgo2 is required to protect centromeric cohesion and its levels are reduced in
cohesin mutants during female mouse meiosis (Lister et al., 2010). If Sororin and Sgo2 participate in
the same pathway to protect centromeric cohesin, one would expect that the centromeric levels of
Sororin are also reduced in cohesin mutants. The authors showed that in early stages of prophase I,
Sororin localization does not depend on the meiotic cohesin subunits Smc1β and Rec8. However in
these later stages of meiosis, if Sororin is indeed protecting centromeric cohesin, one would expect
that the centromeric localization of Sororin depends on cohesin. Therefore, it would be important to
test whether this centromeric localization is cohesin dependent. The authors could use the cohesin
mutants they used in Figure 3 and force spermatocytes in metaphase I using okadaic acid as
described in Llano et al., 2014 and assay for Sororin localization.
Minor points:
Page 3, Ciosk et al. 2000 is not the correct reference for the cohesin loading complex in vertebrates.
I personally believe the readability of this manuscript for a general audience could be increased by
using fewer abbreviations (AE, LE, TF, CE, SC), even though it would increase the length a little
bit.
Referee #3:
Review of Gomez et al., "Sororin participates in ..."
© European Molecular Biology Organization
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Cohesin regulators are key to cohesin function, which is essential for proper gametogenesis. Thus,
for basic germ cell biology and for considerations of reproductive health it is imperative to
understand the role of the pro- and anti-cohesion factors in meiosis. So far no animal models
deficient in specific regulators were published that would shed light on these roles and even a
thorough description of the spatiotemporal appearance of many regulators is elusive. The mansucript
by Gomez et al fills one of these gaps as it describes the status of sororin, a pro-cohesion factor, in
spermatocytes.
The scope of the study is obviously limited to describing rather than including manipulation. Still,
this study provides important insights into the fate of this central regulator of cohesin not only in
wild-type mice but also in cohesin, cohesin protector, and synaptonemal complex mutants.
Interestingly, sororin localizes to the central element of the SC where it interacts with SYCP1,
indicating either a cohesin-independent role - a hypothesis the authors favor - or that cohesin
complexes somehow reach into this region, which is low in nucleic acids and supposed to be low in
cohesin.
However, we know little about cohesin's precise arrangement on the AEs and the SC. We alos know
nothing about the structure and associations of the cohesin complexes on meiotic chromosomes. It is
unclear whether the cohesins form single rings, handcuffed double-rings, multimers or whatever
structure and it is by no means certain that cohesins cannot reach into the central element region. As
sororin interacts with the N/C head domain interface of cohesin this would imply that the head
domains and the associated kleisins/SA proteins are more oriented towards the central elements than
assumed. Thus, I suggest that the authors consider this as well, besides postulating a quite nebulous
cohesin-independent role. Even in absence of REC8 and SMC1b there is considerable cohesin on
prophase I chromosomes, with which sororin may associate.
The role of sororin in supporting (I would rather not say "protecting" since that term is associated
with shugoshin) centromeric cohesion appears more in line with previous considerations.
Considering the proposed role of sororin in pairing it would be very informative to add some data sororin/sycp3/sycp1 staining - of Spo11-deficient spermatocytes where non-homologous pairing
occurs. This would answer two questions: does sororin loading depend on DSBs and is it specific for
homologously paired chromosomes? I am sure that colleagues like Dr Keeney would provide some
slides to stain.
The images are generally very nice and conclusive.
p. 8: it is very important to take the nature of the antibodies into account when interpreting the IF
images, since it may matter a lot which region of SMC3 is recognized by this antibody.
Also, the wouldn't it be possible to stain sororin and SMC3 simultaneously using directly labeled
antibody?
Since the claim of distinct localization of SMC3 and sororin is very important to this paper, one
would like to see confirmation by use of another independent antibody, at least for SMC3.
The authors propose co-localization of REC8 and sororin at the SC. This may suggest that the SMC
head domains point towards the CR. However, the localization of REC8 in the CR should be more
thoroughly demonstrated as it is not entirely clear from Fig. 2.
Figure S1: it is good to show the specificity of the anti sororin antibodies by Western blot, but then
please show the entire gel lane and not only a small area of the gel. Otherwise this figure does not
serve its purpose. There may be other bands appearing at other mol mass positions.
There is no sororin at centromeres in Sgo2-/- cells, but is this an effects of absence of Shugoshin or
of cohesin removal? The authors may want to co-stain the Sgo2-/- centromeres for SMC3.
The authors suggest association of sororin with SGO2. Do they have data such as IP data to
substantiate this proposal?
Minor points:
© European Molecular Biology Organization
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EMBO reports - Peer Review Process File - EMBO-2015-41060
p.7: why is it "surprising" that sororin appears at centromeres? The supportive role for centromeric
cohesion is quite along expectations.
p. 8, line 167: as noted above, the high-resolution localization of cohesins at the SC is not yet
known.
p. 8: typo "SYC3"
p. 8, line 176: reference for the Rec8-myc transgenics is missing (Nobuaki Kudo et al).
p.13, lines 280 & 285: ...independent of...
p.14, line 304: typo
p.15, line 317: not "that", but "as"
There are more typos and there is incorrect English - please have it checked.
1st Revision - authors' response
11 December 2015
Response to the Editor
Editor´s comments:
Thank you for the submission of your research manuscript to our journal. We have now received the
full set of referee reports, which can be found at the end of this e-mail.
As you will see, the referees acknowledge that the findings are potentially interesting. However, all
three referees also point out several technical concerns and have a number of suggestions for how
the study should be strengthened, and I think that all of them should be addressed. As the reports are
below, I will not detail them here. However, addressing the dependence of Sororin on cohesion at
later stages of meiosis (referee 2, point 4) and its dependence on double-strand breaks (referee 3),
additional co-stainings suggested by referee 1 and referee 3 to substantiate the findings and the
avoidance of over-interpretation of data would be necessary. In particular, both referee 2 (point 2)
and referee 3 are concerned about the specificity of the Sororin antibodies used in the study, which
has to be addressed.
Author response:
The explanation regarding the new experiments done can be found below in the point-by point
response to the corresponding referees.
Overall, most concerns seem important, reasonable and should be addressed. An exception is point 3
from referee 2. Given that no data has been published on the meiotic phenotype of Pds5b-deficient
mice, it would be out of the scope of the study.
Author Response:
We want to inform that our group, in collaboration with the group led by Dr. Ana Losada (CNIO,
Madrid) is currently studying the male meiotic phenotype of conditional knockouts for PDS5A,
PDS5B, and double PDS5A/B mice. We agree with you that these new data are out of the scope of
the present study.
Given these constructive comments, we would like to invite you to revise your manuscript with the
understanding that the referee concerns must be fully addressed and their suggestions taken on
board. Please address all referee concerns in a complete point-by-point response. Acceptance of the
manuscript will depend on a positive outcome of a second round of review. It is EMBO reports
policy to allow a single round of revision only and acceptance or rejection of the manuscript will
© European Molecular Biology Organization
6
EMBO reports - Peer Review Process File - EMBO-2015-41060
therefore depend on the completeness of your responses included in the next, final version of the
manuscript.
Revised manuscripts should be submitted within three months of a request for revision; they will
otherwise be treated as new submissions. Please contact us if a 3-months time frame is not sufficient
for the revisions so that we can discuss the revisions further.
You can either publish the study as a short report or as a full article. I suggest the short reports
format. For short reports, the revised manuscript should not exceed 35,000 characters (including
spaces and references) and 5 main plus 5 expanded view figures. The results and discussion section
must further be combined, which will help to shorten the manuscript text by eliminating some
redundancy that is inevitable when discussing the same experiments twice. Commonly used
materials and methods can further be moved to the supplementary information, however, please note
that materials and methods essential for the understanding of the experiments described in the main
text must remain in the main manuscript file. The legend for expanded view figures has to be
included at the end of the text file. For a normal article there are no length limitations, but the results
and discussion section must be separate and the entire materials and methods included in the main
manuscript file.
Author response:
As you will read below in the point-by-point response to the referees, we have now performed all
the new experiments that the referees requested. Accordingly, we have changed the extension of
previous figures (new panel E in Figure 3, and new Supplementary Figure 1), we have added new
figures (new Figure 7, and new Supplementary Figures 2 and 3), and we have corrected another one
(panel E in Figure 2). This leads to the incorporation of new information in the Material and
Methods, Results, and Discussion sections, new Figure Legends, and the inclusion and deletion of
some References.
After considering the editor recommendation, we evaluated the possibility of reorganizing our
manuscript into the short report format. However, we believe that with that option important
information could be excluded while reducing number of figures or text length. We also considered
combining figures, but as they are quite rich in content, the combination would reduce too much the
size of the cells shown, and therefore, it could lead to a reduced quality or significantly reduced
resolution of the visualized structures. For this, we will very much appreciate if we could keep the
full article format.
Please also include scale bars in all microscopy images.
Author response:
All microscopy figures now include a scale bar, we are sorry for the mistake.
We now strongly encourage the publication of original source data with the aim of making primary
data more accessible and transparent to the reader. The source data will be published in a separate
source data file online along with the accepted manuscript and will be linked to the relevant figure.
If you would like to use this opportunity, please submit the source data (for example scans of entire
gels or blots, data points of graphs in an excel sheet, additional images, etc.) of your key
experiments together with the revised manuscript. Please include size markers for scans of entire
gels, label the scans with figure and panel number, and send one PDF file per figure or per figure
panel.
Author response:
We thank the journal for offering us this opportunity, however, we consider that all the important
information is included in the manuscript, and we believe that there is no need to include any
additional data separately.
As part of the EMBO publication's Transparent Editorial Process, EMBO reports publishes online a
Review Process File to accompany accepted manuscripts. This File will be published in conjunction
© European Molecular Biology Organization
7
EMBO reports - Peer Review Process File - EMBO-2015-41060
with your paper and will include the referee reports, your point-by-point response and all pertinent
correspondence relating to the manuscript.
You are able to opt out of this by letting the editorial office know ([email protected]). If you
do opt out, the Review Process File link will point to the following statement: "No Review Process
File is available with this article, as the authors have chosen not to make the review process public
in this case."
Author response:
We agree in making public the review process.
Response to the Referee #1
Referee´s comments:
The manuscript by Gomez et al studies the localization of Sororin, a regulator of cohesin, in mouse
spermatocytes. By using their technique of spermatocyte spreading, they find that Sororin localizes
at the central regions (CRs) of the synaptonemal complex during meiosis. This localization pattern is
different from that of cohesin complexes, which are known to localize at axial elements. Their
analyses of spermatocytes from Rec8-/- mice and Smc1b-/- mice show that the localization of
Sororin at CRs is independent of cohesin. Based on these data, they speculate that Sororin has a role
at CRs independent of cohesin.
A novel point of this manuscript is cohesin-independent localization of Sororin. This conclusion is
robustly supported by their careful analysis that compares the localization of Sororin and cohesins
and by their analysis of cohesin-KO mice. Although the function of Sororin independent of cohesin
is unclear from the data shown in the manuscript, the description of cohesin-independent
localization of Sororin would be a significant contribution to the field and interest researchers in the
specific area. The data are of high quality and the images are impressively beautiful. Sororin's
potential roles independent of cohesin are appropriately discussed. I believe the current manuscript
is suitable for publication in EMBO reports.
I have only one minor suggestion that would further improve the manuscript.
- It would be helpful to further confirm their conclusion if they could include a data for double
staining of Rec8-myc and Sororin at early zygotene in the manuscript.
Author response:
We are very grateful with referee #1 for his/her kind and positive comments on our work.
Following the referee recommendation, we have changed panel E of previous Figure 2 showing the
colabeling of Sororin and REC8-myc in a pachytene spermatocyte by a new panel E showing in this
case a zygotene one. Thus, all panels in this Figure show zygotene spermatocytes. The text now
reflects this change in page 7 lines 177-178, and in the corresponding figure legend.
Response to the Referee #2
Referee´s comments:
In this study, the authors have analyzed the localization of Sororin, a protein required for sister
chromatid cohesion in somatic cells, by immunofluorescence during male mouse meiosis. In
somatic cells, Sororin contributes to sister chromatid cohesion by preventing the release of cohesin
complexes from DNA. Sororin is thought to achieve this by binding to cohesin and by inhibiting
Wapl, a protein that can release cohesin from DNA. There are conflicting reports about how Sororin
interacts with cohesin, both the cohesin subunits Pds5A and Pds5B (Nishiyama et al., Cell 2010)
and SA2 have been reported as Sororin binding partners (Zhang and Pati, Cell Cycle 2015). Specific
isoforms of cohesin complexes also exist in meiotic cells, where they are essential for proper
© European Molecular Biology Organization
8
EMBO reports - Peer Review Process File - EMBO-2015-41060
homologous pairing, chromosome cohesion and chromosome segregation, but it is not known if
Sororin is expressed in meiotic cells and has a function in sister chromatid cohesion in meiosis.
In this study, the authors describe staining patterns obtained with Sororin antibodies in surfacespread mouse spermatocytes and show that this pattern changes in interesting ways during meiotic
progression. From zygotene to diplotene Sororin antibodies stain the central region of the
synaptonemal complex (SC), where the two homologous chromosomes are synapsed and where the
protein SYCP1 is located. Surprisingly, during these stages of prophase I, Sororin staining does not
co-localize with cohesin, its binding partner in somatic cells. Cohesin is known to be present at
axial/lateral elements, where also Wapl and Pds5B have previously been detected. Moreover, the
authors provide evidence that Sororin localization to the central region of the SC does not depend on
the meiotic-specific cohesin subunits Rec8 and Smc1b. Based on these results the authors suggest
that Sororin might be involved in chromosome pairing and synapsis and that its functions during
early stages of prophase I might be independent of cohesin complexes. From late diplotene to
anaphase II, Sororin staining is detected at centromeres, where cohesin is known to exist, and its
localization there depends on Shugoshin2 (Sgo2). Based on this observation the authors suggest that
Sororin might work in combination with Sgo2 in order to protect centromeric cohesion until
anaphase II onset. In summary, the authors propose that Sororin might have two distinct and
independent roles during male mouse meiosis, a cohesin independent role in chromosome pairing
and synapsis, and a cohesin dependent role in protecting centromeric cohesion.
This manuscript addresses an interesting topic, as previous work has revealed the importance of
Sororin for sister chromatid cohesion in somatic cells, but nothing is known about the existence and
possible functions of Sororin in meiotic cells. The presented microscopic data are of high quality,
and the observed staining patterns very interesting. However, this study has several limitations. One
is the lack of loss-of-function data. But one can argue that this is beyond the scope of this study, and
since we know nothing about Sororin in meiosis and the observed staining patterns are interesting
and suggestive, I would argue that it is fine to publish this study without functional data, as long as it
is clear that the manuscript does not overstate any conclusions (see for example comment 1 on the
title of the manuscript below). The second limitation is more serious. How do we know that the
observed staining patterns are representing the location of Sororin? More controls will be needed to
address this central point (see comment 2 below with some suggestions for what could be done).
This is particularly important as in somatic cells Sororin is thought to be recruited to chromatin only
via cohesin, so can the authors exclude that the central element staining observed with Sororin
antibodies is not caused by a cross-reacting antigen? The third limitation is whether the authors can
really exclude that the observed Sororin staining pattern is cohesin independent. It would be
technically possible to address this more rigorously and I would recommend doing this (see points 3
and 4 below). If these points can be addressed I would support publication of this interesting study.
Author response:
We are very grateful for your suggestions, which are very constructive and helped to improve this
manuscript. We are aware that this work might be lacking functional data, but we agree that as this
is the first study that shows Sororin in mouse meiosis, our work could be of high interest for the
scientific community. We have addressed all recommendations. The experiments done and the
changes introduced in the text are detailed below in a point-by-point response.
Specific points:
1. The authors do not provide functional data supporting the role of Sororin in chromosome pairing
and centromere dynamics in male mouse meiosis. To properly test the role of Sororin in these
processes would have been necessary to analyze chromosome pairing and centromere dynamics in
the absence of Sororin. I would suggest that the authors change the title in a way that it reflects what
the manuscript actually shows, for example by saying "Sororin localizes to the central elements
during mouse male meiosis independently of meiotic cohesin complexes".
Author response:
To avoid overstatements and therefore offer a more fair and accurate title of our work, we have
followed this recommendation. Now, the title of our manuscript, taking into account the restrictions
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in the number of characters, is similar to the title suggested by the referee and reads as follows:
“Sororin loads to the synaptonemal complex central region independently of meiotic cohesin
complexes”.
2. As already mentioned, more work is required to control the specificity of the Sororin antibodies
used. For example: Do the two Sororin antibodies mentioned in the Methods give comparable
staining patterns? This should be shown.
Author response:
We agree with the referee that it is important to emphasize that both antibodies used for this study
give comparable staining patterns. In the previous version of the manuscript, we wrote in the
Material and Methods section (page 14 lines 386-387 of the first submission) “The two antibodies
gave also similar results in immunofluorescence, being the serum C-106 the one that has been used
for the images presented in this work”. As that sentence reads, we found very similar
immunofluorescence results with both antibodies: anti-Sor provided by one of the coauthors (Dr.
Losada, CNIO), and C-106 provided by another coauthor (Dr. Barbero (CIB/CSIC). The anti-Sor
antibody has been previously described and validated (Remeseiro et al., 2012; Carretero et al.,
2013). However, we agree with the referee that this needs to be even better clarified in our
manuscript. Now the sentence states (page 15 lines 417-420 of the revised manuscript): “The two
antibodies used in this study (anti-Sor and C-106) offered highly similar results in
immunofluorescence and therefore present comparable staining patterns of Sororin during
prophase I (Fig EV3). As the serum C-106 provided slightly cleaner results, it is the one used for the
images presented in this work”.
The new Supplementary Figure 3, (Fig EV3) shows the similar staining pattern of both antibodies in
wild-type zygotene spermatocytes. It is cited in page 6 lines 155-156 as follows: “This pattern of
localization of Sororin at the SC central region was consistent with both antibodies used for this
study (Fig EV3A and B)”. In addition, and given that one of the most interesting results that our
manuscript describes is the presence of Sororin at the SC central region independently of meiotic
cohesin complexes, we also show the staining of Sororin with both antibodies on Smc1β-/pachytene-like spermatocytes (new Supplementary Fig. 3 C,D, Fig EV3C and D). This additional
information is also cited in page 8 lines 230-232 as follows: “This pattern of localization of Sororin
at the SC central region in Smc1β-/- spermatocytes was consistently found when employing both
antibodies used for this study (Fig EV3C and D)”. The legend of this new figure appears in page 26
line 772.
How does the entire Western blot in Figure S1 look like, are there no other cross-reacting bands? Do
these antibodies also cross-react with Sororin in somatic cells? This could be analyzed on the same
Western side by side. If the answer is yes, it would be really helpful to try to deplete Sororin in
somatic cells by RNA interference to see if the recognized bands do indeed represent Sororin.
Likewise, if the Sororin antibodies stained somatic cells in immunofluorescence experiments and
these signals were reduced by Sororin specific siRNAs, this would also ad confidence to the
interpretation. Alternatively, it could be tested in co-immunoprecipitation experiments from somatic
cell lysates if the proteins detected by these antibodies are associated with cohesin. I realize that it is
difficult to be absolutely sure about the specificity of antibodies, but since literally all conclusions in
this manuscript depend on this specificity the authors need to provide more evidence for this. For an
example of how RNAi can be used to control the specificity of Sororin antibodies see Carretero et
al., 2013.
Author response:
As the referee suggests we now show in the new Supplementary Figure 1 (Fig EV1A) the entire
Western blots obtained with both anti-Sororin antibodies on testes extracts.
To determine the specificity of the antibodies used in this study, and demonstrate that they are
indeed recognizing Sororin, we have performed 2 different experiments:
1. We have knocked-down Sororin in mouse somatic cells as suggested by the referee (please see
modified Supplementary Figure 1B and C, Fig EV1B and C). As recommended by the referee, we
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took into consideration the specificity experiments of Sororin antibodies described by Carretero et
al. (2013). This publication already describes the specificity of the antibody generated by Dr. Losada
(anti-Sor) that we used in this work. However, we have tested it again in comparison with the
antibody generated by Dr. Barbero (C-106), which has been validated for the first time in this work.
The siRNA experiments were done using mouse C2C12 cells using Sororin siRNA (Thermo
Scientific Dharmacon) or control siRNA (Scrambled). Immunoblot analyses of whole cell extracts
prepared 48 h after transfection with both antibodies show a significant decrease in the expected
band of endogenous Sororin (around 36 kDa). This demonstrates that both antibodies used in this
work recognize Sororin in mouse cells.
2. To further support the specificity of the antibodies, we have also overexpressed Sororin in human
somatic cells (please see new Supplementary Figure 2, Fig EV2). HEK 293T cells were transfected
with pEGFP-N1-Sororin or empty pEGFP vector as a control. After the verification of the
transfection (see new Supplementary Figure 2D, Fig EV2D), we carried out immunoblot
experiments, using both anti-Sor and C-106 antibodies (Fig EV2A and B), and anti GFP (Fig
EV2C). Both anti-Sororin antibodies detect a strong band at around 72 kDa that corresponds with
the expected mass of GFP-Sororin (fused protein 36 kDa + 36 kDa). By immunofluorescence, the
two antibodies show clear nuclear signals that correlate with the level of overexpression of GFPSororin (Fig EV2E). We believe that this experiment reinforces the fact that we are using antibodies
that specifically detect Sororin.
Both siRNA and overexpression experiments are shown in the modified Supplementary Figure 1
(Fig EV1) and the new Supplementary Figure 2 (Fig EV2). The methodology is included in the
Material and Methods section (page 16 lines 434-443), and the citations to these figures are properly
included in the corresponding place of the Results section (page 6 lines 137-139). The legends of
these new Supplementary Figures 1 and 2 have also been included in the text.
We believe that the experiments recommended by the referee significantly improve our manuscript
and enhance its credibility, and therefore we are grateful for this suggestion.
3. In Figure 3, the authors show that Sororin localization at the central region of the SC depends on
Sycp1, but not on Rec8 and Smc1β. In mitosis, Sororin binding to DNA depends on Pds5b and
Pds5a (Nishiyama et al., 2010; Carretero et. al. 2013). Moreover, in mouse spermatocytes, Pds5b
signal is depleted from the chromosome axis when homologues undergo desynapsis at diplotene
stage, at the same time Sororin is removed from the central region of the SC. Additionally, Pds5b
signal persists in Smc1β-/- spermatocytes (Fukuda and Hoog, 2010). This poses the possibility that
Sororin localization in male spermatocytes might still depend on some cohesin complexes
(presumably the ones that contain Smc1α and Rad21L) and on Pds5b. For this reason, it would be
interesting to analyze Sororin localization in Pds5b-/- spermatocytes.
Author response:
The referee is right. For this study we have worked with mice deficient in the meiotic cohesins
REC8 and SMC1β. Thus, we can only conclude that Sororin localization at the SC central region is
independent on meiotic cohesin complexes with these subunits. Due to the existence of multiple
cohesin complexes during mouse meiosis we cannot exclude the presence of other “mitotic” cohesin
complexes with RAD21, or other “meiotic” complexes with RAD21L in these mutants with which
Sororin could interact. In any case, we have now included a sentence mentioning the possibilities
suggested by the referee in the Discussion section (page 12 lines 326-328).
On the other hand, we are currently studying the male meiotic phenotype of mice deficient for PDS5
proteins in collaboration with Dr. Ana Losada (CNIO). We will for sure address the relevance of
PDS5 in loading Sororin. However, in our opinion these results are out of the scope of the present
manuscript.
4. In Figures 4 and 5, the authors show that Sororin localizes to centromeres from late diplotene
through anaphase II. They present a very similar pattern of localization to Shugoshin2 (Sgo2)
(Gomez et al, 2007). Sgo2 is required to protect centromeric cohesion and its levels are reduced in
cohesin mutants during female mouse meiosis (Lister et al., 2010). If Sororin and Sgo2 participate in
the same pathway to protect centromeric cohesin, one would expect that the centromeric levels of
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Sororin are also reduced in cohesin mutants. The authors showed that in early stages of prophase I,
Sororin localization does not depend on the meiotic cohesin subunits Smc1β and Rec8. However in
these later stages of meiosis, if Sororin is indeed protecting centromeric cohesin, one would expect
that the centromeric localization of Sororin depends on cohesin. Therefore, it would be important to
test whether this centromeric localization is cohesin dependent. The authors could use the cohesin
mutants they used in Figure 3 and force spermatocytes in metaphase I using okadaic acid as
described in Llano et al., 2014 and assay for Sororin localization.
Author response:
This is indeed a good question. Since spermatocytes lacking cohesin subunits SMC1β and REC8
arrest in prophase I, we followed the referee’s suggestion to use okadaic acid to force entry into
metaphase I.
We proceeded with the okadaic acid experiment using Smc1β-/- spermatocytes (generously provided
again by Dr. Rolf Jessberger, Technische Universität Dresden, Germany), in parallel with wild-type
spermatocytes as control. In cultured wild-type spermatocytes, Sororin labelling could be detected at
the SC central region in prophase I stages, but not at the centromeres of metaphase I chromosomes
(new Fig 7A and B). Thus, we cannot use this protocol to address the importance of cohesin for
Sororin recruitment to centromeres. However, we can propose that the phosphatase activity of the
SGO2 partner PP2A is likely to be important for Sororin presence at centromeres in metaphase I.
We cannot discard that other dynamic processes are also affected. For example, the okadaic acid
treated metaphase I chromosomes showed an atypical accumulation of SYCP3 at the interchromatid
domain along the arms, and little accumulation of SYCP3 at their centromeres (new Fig 7C and D).
We describe these new results in the main text (page 10 lines 288-307) and discuss them in the
Discussion section (page 13 lines 371-375).
As mentioned above, this new results are included in the new Figure 7. If the referees and editor
agree, we propose to include this figure in the main text. Although this experiment does not offer
additional information about the recruitment of Sororin in the absence of cohesins (in this case
SMC1β), it does offer additional and interesting information about the okadaic acid methodology
and the possible interactions among PP2A-SGO2-Sororin, which are worth to mention. However,
we will be open to changes if the editor or reviewers consider that this figure should be better placed
in the Supplementary material.
Minor points:
Page 3, Ciosk et al. 2000 is not the correct reference for the cohesin loading complex in vertebrates.
Author response:
This reference has been deleted and changed by Gillespie and Hirano (2004) in the text and the
Reference section.
I personally believe the readability of this manuscript for a general audience could be increased by
using fewer abbreviations (AE, LE, TF, CE, SC), even though it would increase the length a little
bit.
Author response:
As suggested by the referee, we have deleted the abbreviations AE, LE, TF, and CE, and also CR
and ACA, to increase the readability of the manuscript. Now the corresponding full names appear in
the manuscript. However, we decided to keep the SC (synaptonemal complex) abbreviation.
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Response to Referee #3
Referee´s comments:
Review of Gomez et al., "Sororin participates in ..."
Cohesin regulators are key to cohesin function, which is essential for proper gametogenesis. Thus,
for basic germ cell biology and for considerations of reproductive health it is imperative to
understand the role of the pro- and anti-cohesion factors in meiosis. So far no animal models
deficient in specific regulators were published that would shed light on these roles and even a
thorough description of the spatiotemporal appearance of many regulators is elusive. The manuscript
by Gomez et al fills one of these gaps as it describes the status of sororin, a pro-cohesion factor, in
spermatocytes.
The scope of the study is obviously limited to describing rather than including manipulation. Still,
this study provides important insights into the fate of this central regulator of cohesin not only in
wild-type mice but also in cohesin, cohesin protector, and synaptonemal complex mutants.
Interestingly, sororin localizes to the central element of the SC where it interacts with SYCP1,
indicating either a cohesin-independent role - a hypothesis the authors favor - or that cohesin
complexes somehow reach into this region, which is low in nucleic acids and supposed to be low in
cohesin. However, we know little about cohesin's precise arrangement on the AEs and the SC. We
also know nothing about the structure and associations of the cohesin complexes on meiotic
chromosomes. It is unclear whether the cohesins form single rings, handcuffed double-rings,
multimers or whatever structure and it is by no means certain that cohesins cannot reach into the
central element region. As sororin interacts with the N/C head domain interface of cohesin this
would imply that the head domains and the associated kleisins/SA proteins are more oriented
towards the central elements than assumed. Thus, I suggest that the authors consider this as well,
besides postulating a quite nebulous cohesin-independent role. Even in absence of REC8 and
SMC1b there is considerable cohesin on prophase I chromosomes, with which sororin may
associate. The role of sororin in supporting (I would rather not say "protecting" since that term is
associated with shugoshin) centromeric cohesion appears more in line with previous considerations.
Considering the proposed role of sororin in pairing it would be very informative to add some data sororin/sycp3/sycp1 staining - of Spo11-deficient spermatocytes where non-homologous pairing
occurs. This would answer two questions: does sororin loading depend on DSBs and is it specific for
homologously paired chromosomes? I am sure that colleagues like Dr Keeney would provide some
slides to stain.
Author response:
We found this suggestion very interesting and we thank the referee for transmitting it to us. We
contacted Dr. Scott Keeney (Sloan Kettering Institute, New York) and Dr. Ignasi Roig (UAB,
Barcelona), who also possess Spo11-/- mutant mice. They both were very kind and we received slides
with spermatocyte spreads from Spo11-/- mice (Baudat et al., 2000). Our results, now included in
Figure 3E, showed that Sororin was present at non-homologous paired regions. Consequently, the
loading of Sororin to the SC central region is not specific for homologously paired chromosomes. In
addition, our results also suggest that Sororin loading into the central region does not depend on the
occurrence of double strand breaks.
The origin of the Spo11-/- mice is included now in the Material and Methods section (page 15 line
390), and new data appear in Figure 3E and have been included in the Results section (page 9 lines
236-245), and accordingly discussed (page 12 lines 338-341).
The images are generally very nice and conclusive.
Author response:
We are grateful for the kind referee comment, which is highly appreciated.
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p. 8: it is very important to take the nature of the antibodies into account when interpreting the IF
images, since it may matter a lot which region of SMC3 is recognized by this antibody. Also, the
wouldn't it be possible to stain sororin and SMC3 simultaneously using directly labeled antibody?
Since the claim of distinct localization of SMC3 and sororin is very important to this paper, one
would like to see confirmation by use of another independent antibody, at least for SMC3.
Author response:
We thank the referee suggestion, but we have to take into account that both antibodies (anti-SMC3
and anti-Sororin) were raised in rabbit, so even using directly labeled antibodies we believe that the
interpretation of a double immunolocalization would be risky. We bought two commercial antiSMC3 antibodies from Santa Cruz (a goat polyclonal against rat SMC3, sc-8198; and a mouse
monoclonal against rat SMC3, sc-365540) in order to detect simultaneously Sororin and SMC3.
Unfortunately, neither of these anti-SMC3 antibodies worked in immunofluorescence. This is the
reason by which we preferred to detect Sororin and SMC3 separately.
The K987 rabbit anti-SMC3 antibody that we used was developed by Dr. Barbero against aa. 9781217 of mouse SMC3. We have also employed a rabbit anti-SMC3 antibody from Abcam (ab-9263)
which recognizes the last 100 aa from human SMC3. Both antibodies recognized the axial/lateral
elements of the synaptonemal complex, as it has been published using other different antibodies
(Eijpe et al., 2000 - J. Cell Sci. 113, 673-682; Kudo et al., 2006 - Cell 126, 135-146). Thus, we are
confident that the SMC3 staining pattern along axial/lateral elements is accurate.
The authors propose co-localization of REC8 and sororin at the SC. This may suggest that the SMC
head domains point towards the CR. However, the localization of REC8 in the CR should be more
thoroughly demonstrated as it is not entirely clear from Fig. 2.
Author response:
We do not suggest that REC8 and Sororin colocalize during prophase I. On the contrary, we suggest
that Sororin is located at the central region of the synaptonemal complex, whereas it is known, and
we confirm, that REC8 is located along the axial/lateral elements. We are sorry if this was not
sufficiently well explained in our manuscript.
As we understand that showing a pachytene with a double immunolocalization of REC8 and Sororin
could generate misunderstandings, we have substituted that pachytene with a zygotene spermatocyte
where both REC8 and Sororin are detected (as also suggested referee #1). This new panel in Figure
2E confirms that REC8 and Sororin have different localizations. This clarification appears in the text
in page 7 lines 175-180.
Figure S1: it is good to show the specificity of the anti sororin antibodies by Western blot, but then
please show the entire gel lane and not only a small area of the gel. Otherwise this figure does not
serve its purpose. There may be other bands appearing at other mol mass positions.
Author response:
This was also a concern of referee #2. Please see our response above.
There is no sororin at centromeres in Sgo2-/- cells, but is this an effect of absence of Shugoshin or
of cohesin removal? The authors may want to co-stain the Sgo2-/- centromeres for SMC3.
Author response:
We thank the referee for this question, which is a very important one. The localization of SMC3 at
metaphase I centromeres in Sgo2-/- spermatocytes has been previously reported by our group in
collaboration with Dr. Pendás group (see Supplementary Fig S6 in Llano et al, 2008). In this work,
we described that SMC3 localized as a small signal at the inner centromeric domain, as does the
cohesin subunit REC8, in metaphase I Sgo2-/- spermatocytes. However, in the absence of SGO2
cohesins disappear from centromeres in anaphase I. Thus, in these Sgo2-/- spermatocytes cohesins
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are present at metaphase I centromeres. We now mention this study in the main text (page 10 lines
284-287).
The authors suggest association of sororin with SGO2. Do they have data such as IP data to
substantiate this proposal?
Author response:
We unfortunately do not have any IP data that demonstrates the association of SGO2 and Sororin.
Our work can only suggest that Sororin is not recruited to the centromeres in the absence of SGO2
(experiments done with the Sgo2-/- mice, Figure 6). We could potentially suggest an interaction, but
not a formal association. In our manuscript the affirmation of an “association between Shugoshin
and Sororin” does not appear per se. Nevertheless, in order to avoid overstatements and too
speculative hypotheses, we have verified that the manuscript does not propose the formal
association of Sororin and SGO2.
Minor points:
p.7: why is it "surprising" that sororin appears at centromeres? The supportive role for centromeric
cohesion is quite along expectations.
Author response:
We agree with the referee that it was expected to localize Sororin at the centromeres taking into
account previous publications. Hence, the word “surprising” has been deleted in the text.
p. 8, line 167: as noted above, the high-resolution localization of cohesins at the SC is not yet
known.
Author response:
We agree with the referee that there is much to be described regarding the precise high-resolution
localization of cohesins at the SC. Accordingly, we have deleted that cohesins underlie the
axial/lateral elements.
p. 8: typo "SYC3". Corrected
p. 8, line 176: reference for the Rec8-myc transgenics is missing (Nobuaki Kudo et al).
Author response:
This reference has been included. We thank the referee for noticing this unfortunate mistake.
p.13, lines 280 & 285: ...independent of.... Sentences corrected.
p.14, line 304: typo. The title’s section has been changed to Sororin at the centromeres. In addition,
the sentence “Thus, Sororin adds to these proteins that load to ther centromeres from CRs” has been
changed to this one: “Our results indicate that Sororin is another protein that shows a dynamic
relocalization from the SC central regions to the centromeres” (page 13 lines 353-354).
p.15, line 317: not "that", but "as" . Corrected
There are more typos and there is incorrect English - please have it checked.
Author response:
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All the text has been checked and these specific mistakes corrected. English has been revised in the
entire document.
2nd Editorial Decision
13 January 2016
Thank you for the submission of your revised manuscript to our editorial offices. We have now
received the enclosed reports on it. As you will see, both referees find the manuscript suitable for
publication in EMBO reports. Nevertheless, referee 2 has raised some points that should be
addressed. Given these evaluations, I would like to give you the opportunity to revise your
manuscript, with the understanding that the concerns of referee 2 must be fully addressed.
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REFEREE REPORTS
Referee #1:
In the revised manuscript, the authors have appropriately addressed my minor comment and
provided data that further support their conclusion. I fully support publication of the manuscript at
EMBO Reports.
Referee #2:
The revised version of this study describes the localization of Sororin during mouse
spermatogenesis. The authors show a dynamic Sororin localization pattern during prophase I. From
zygotene to diplotene, Sororin co-localizes with SYCP1 at the central region of the synaptonemal
complex (SC), where the two homologous chromosomes are synapsed. Surprisingly, during these
stages of prophase I, Sororin does not co-localize with cohesin, and also not with Wapl and Pds5b,
which have been reported to be located at the lateral elements of the SC. Moreover, Sororin
localization to the central region of the SC does not depend on the meiotic-specific cohesin subunits
Rec8 and Smc1b. Based on these results the authors suggest that Sororin might play a role in
chromosome pairing and synapsis independently of meiotic cohesin complexes. From late diplotene
to anaphase II, Sororin re-localizes to centromeres and its centromeric localization depends on
Sugoshin2 (Sgo2). This suggests that Sororin might work in combination with Sgo2 in order to
protect centromeric cohesion until anaphase II onset. Together, these results raise the interesting
possibility that Sororin might have two distinct and independent roles during male mouse meiosis,
one cohesin-independent and one cohesin dependent function.
In this revised version of the manuscript the authors clarified most of the concerns and criticisms
raised in the initial review.
Regarding point 1, the authors changed the title to a more adequate one. The new title, "Sororin
loads to the synaptonemal complex central region independently of meiotic cohesin complexes",
describes the main finding of the manuscript without overstatements about Sororin function during
mammalian meiosis.
Concerning point 2, the authors now provided IF images using a second Sororin antibody, and the
results obtained confirm the results previously obtained with another antibody. Moreover, the
authors have characterized the specificity of the Sororin antibodies Further. The authors now show
that Sororin depletion by siRNA in mouse C2C12 cells leads decreases endogenous Sororin level in
immunoblot experiments. Unfortunately, the blot presented for the C-106 antibody contains a lot of
"background" signal that makes it more difficult to interpret the data. It would help if this could be
improved. The authors also overexpressed GFP-mouse-Sororin in human HEK 293T cells. Both
anti-Sororin antibodies detected a band at around 72 kDa which corresponds to the expected mass of
the GFP-Sororin fusion protein. The authors also showed that the two anti-Sororin antibodies show
clear nuclear signals that correlate with the level of overexpression of GFP-Sororin.
Concerning point 3, the authors commented on the possibility that other cohesin complexes, namely
Rad21, Rad21L and Smc1a-containing complexes might interact with Sororin and be required for
Sororin localization during prophase I.
Concerning point 4, the authors made an effort to test whether the centromeric localization of
Sororin depends on cohesin by forcing prophase I arrested Smc1b-/- spermatocytes into metaphase I
using okadaic acid. However, Sororin labeling could not be detected at the centromeres of
metaphase I chromosomes upon okadaic acid treatment in wild type oocytes. Therefore, the authors
could not conclude whether the centromeric localization of Sororin in metaphase I depends on
cohesin. Instead, their result suggests that the activity of PP2A, which is recruited to centromeres
together with Sgo2, is required for the centromeric localization of Sororin during metaphase I. This
is an interesting result complementary to the finding that Sororin localization at centromeres in
metaphase I depends on Sgo2. This result should therefore be mentioned and discussed in the main
text of this manuscript.
As suggested by reviewer 1, the authors show co-stained images of Sororin and Rec-myc, which
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indicate more clearly that Sororin and cohesin complexes do not co-localize in early stages of
prophase I.
Following a suggestion by reviewer 3, the authors show that in the absence of Spo11 Sororin
localizes normally to the central region of the SC. This result indicates that Sororin loading does not
depend on double strand breaks formation and that Sororin is present in non-homologous paired
regions. This result does, however, not support the hypothesis that Sororin might play a role in
homologous pairing, synapsis and recombination. The authors should discuss this more explicitly in
the discussion section and take it into account when they speculate on the role of Sororin in early
prophase I.
After minor revision to address the suggestions above I would support publication of this manuscript
in EMBO Reports.
2nd Revision - authors' response
03 February 2016
Response to the Referee #1
Referee´s comments:
In the revised manuscript, the authors have appropriately addressed my minor comment and
provided data that further support their conclusion. I fully support publication of the manuscript at
EMBO Reports.
Author response:
We are very grateful with referee #1 for his/her kind and positive comments on our work.
Response to the Referee #2
Referee´s comments:
The revised version of this study describes the localization of Sororin during mouse
spermatogenesis. The authors show a dynamic Sororin localization pattern during prophase I. From
zygotene to diplotene, Sororin co-localizes with SYCP1 at the central region of the synaptonemal
complex (SC), where the two homologous chromosomes are synapsed. Surprisingly, during these
stages of prophase I, Sororin does not co-localize with cohesin, and also not with Wapl and Pds5b,
which have been reported to be located at the lateral elements of the SC. Moreover, Sororin
localization to the central region of the SC does not depend on the meiotic-specific cohesin subunits
Rec8 and Smc1b. Based on these results the authors suggest that Sororin might play a role in
chromosome pairing and synapsis independently of meiotic cohesin complexes. From late diplotene
to anaphase II, Sororin re-localizes to centromeres and its centromeric localization depends on
Sugoshin2 (Sgo2). This suggests that Sororin might work in combination with Sgo2 in order to
protect centromeric cohesion until anaphase II onset. Together, these results raise the interesting
possibility that Sororin might have two distinct and independent roles during male mouse meiosis,
one cohesin-independent and one cohesin dependent function.
In this revised version of the manuscript the authors clarified most of the concerns and criticisms
raised in the initial review.
Regarding point 1, the authors changed the title to a more adequate one. The new title, "Sororin
loads to the synaptonemal complex central region independently of meiotic cohesin complexes",
describes the main finding of the manuscript without overstatements about Sororin function during
mammalian meiosis.
Concerning point 2, the authors now provided IF images using a second Sororin antibody, and the
results obtained confirm the results previously obtained with another antibody. Moreover, the
authors have characterized the specificity of the Sororin antibodies Further. The authors now show
that Sororin depletion by siRNA in mouse C2C12 cells leads decreases endogenous Sororin level in
© European Molecular Biology Organization
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EMBO reports - Peer Review Process File - EMBO-2015-41060
immunoblot experiments. Unfortunately, the blot presented for the C-106 antibody contains a lot of
"background" signal that makes it more difficult to interpret the data. It would help if this could be
improved.
Author response:
In order to improve the western blot in Figure EV1B, we have incubated the same membrane again
with the C-106 antibody after a high speed centrifugation to separate possible aggregates. We have
also increased the number and length of the washes in TBST. Although there is still background, the
image is cleaner and it is now easier to see the clear disappearance of the band corresponding to
Sororin after siRNA.
The authors also overexpressed GFP-mouse-Sororin in human HEK 293T cells. Both anti-Sororin
antibodies detected a band at around 72 kDa which corresponds to the expected mass of the GFPSororin fusion protein. The authors also showed that the two anti-Sororin antibodies show clear
nuclear signals that correlate with the level of overexpression of GFP-Sororin.
Concerning point 3, the authors commented on the possibility that other cohesin complexes, namely
Rad21, Rad21L and Smc1a-containing complexes might interact with Sororin and be required for
Sororin localization during prophase I.
Concerning point 4, the authors made an effort to test whether the centromeric localization of
Sororin depends on cohesin by forcing prophase I arrested Smc1b-/- spermatocytes into metaphase I
using okadaic acid. However, Sororin labeling could not be detected at the centromeres of
metaphase I chromosomes upon okadaic acid treatment in wild type oocytes. Therefore, the authors
could not conclude whether the centromeric localization of Sororin in metaphase I depends on
cohesin. Instead, their result suggests that the activity of PP2A, which is recruited to centromeres
together with Sgo2, is required for the centromeric localization of Sororin during metaphase I. This
is an interesting result complementary to the finding that Sororin localization at centromeres in
metaphase I depends on Sgo2. This result should therefore be mentioned and discussed in the main
text of this manuscript.
Author response:
We agree with the referee that this is an interesting finding, and following his/her suggestion, we
have now further discussed it in the manuscript. In order to better address this, we have
immunodetected SGO2 in cultured spermatocytes treated with okadaic acid, as well as in control
cultured spermatocytes. This has allowed us to have a better understanding of our previous results.
To present these data and be able to discuss them in the manuscript, we have prepared a small new
supplementary Figure EV4.
We have included the details for the SGO2 antibody in the material and methods section in page 19
line 450. These new data have been included in the results section in pages 10-11 lines 296-309.
According to this, the title of this section of the results has been changed to “The centromere
loading of Sororin is independent on SGO2” (page 10). These results are now discussed in page 13
lines 375-387 and in page 14 lines 393-398. The summarized last phrase of the discussion has also
been slightly modified so that it presents all the conclusions (page 14 lines 404-409). The abstract
has also been slightly modified according to these new findings.
As suggested by reviewer 1, the authors show co-stained images of Sororin and Rec-myc, which
indicate more clearly that Sororin and cohesin complexes do not co-localize in early stages of
prophase I.
Following a suggestion by reviewer 3, the authors show that in the absence of Spo11 Sororin
localizes normally to the central region of the SC. This result indicates that Sororin loading does not
depend on double strand breaks formation and that Sororin is present in non-homologous paired
regions. This result does, however, not support the hypothesis that Sororin might play a role in
homologous pairing, synapsis and recombination. The authors should discuss this more explicitly in
the discussion section and take it into account when they speculate on the role of Sororin in early
prophase I.
© European Molecular Biology Organization
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EMBO reports - Peer Review Process File - EMBO-2015-41060
Author response:
Following the referee suggestion, we have now included a sentence that clearly indicates that our
results using the Spo11-/- model support that the loading of Sororin to the SC central region is not
specific for homologously paired chromosomes, and therefore Sororin is not directly implicated in
homologous pairing during prophase I. As our results indeed suggest that Sororin loading into the
central region does not depend on the occurrence of double strand breaks, we have kept this
statement in the manuscript.
These changes now appear in the results section in page 9 lines 243-245. According to this, the title
of the section has been changed to “The loading of Sororin at the central region of the
synaptonemal complex is independent on double strand breaks and homologous pairing” (page 9).
The changes in the discussion appear in pages 12-13 lines 352-357. In addition, all previous
sentences indicating that Sororin could be implicated in homologous paring have now been
deleted/modified in the manuscript. As an example the sentence on page 12 line 351 now reads
“Sororin….could be involved in …processes such as chromosome pairing”, where the words
“homologous pairing” have been changed for “chromosome pairing”.
After minor revision to address the suggestions above I would support publication of this manuscript
in EMBO Reports.
Author response:
We are grateful with Referee 2 as all his/her comments have help us to improve our manuscript. We
hope that he/she find these last changes satisfactory and now finds the manuscript suitable for
publication.
3rd Editorial Decision
05 February 2016
I am very pleased to accept your manuscript for publication in the next available issue of EMBO
reports. Thank you for your contribution to our journal.
© European Molecular Biology Organization
20
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Manuscript Number: EMBOR-­‐2015-­‐41060V2
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1.a. How was the sample size chosen to ensure adequate power to detect a pre-­‐specified effect size?
At least 3 mice were analysed for every detection and protein pattern of distribution.
1.b. For animal studies, include a statement about sample size estimate even if no statistical methods were used.
At least 3 mice were analysed for every detection and protein pattern of distribution. All animals were handled according to the Ethical Committee.
2. Describe inclusion/exclusion criteria if samples or animals were excluded from the analysis. Were the criteria pre-­‐established?
A parallel control immunolocalization was always done when analysing the proteins studied in our work.
3. Were any steps taken to minimize the effects of subjective bias when allocating animals/samples to treatment (e.g. randomization procedure)? If yes, please describe. No
For animal studies, include a statement about randomization even if no randomization was used.
We declare that all animals were treated and examined under the same conditions. All of them were of similar age and showed perfect health conditions. Samples were made always following the same protocols and experiments were repeated at least trice.
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We declare that control experiments were always done for every procedure. For example, control cultured seminiferous tubules were studied at the same time than treated (with Okadaic Acid) ones.
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There are no stadistical studies in our work.
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There are no stadistical studies in our work.
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There are no stadistical studies in our work.
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7. Identify the source of cell lines and report if they were recently authenticated (e.g., by STR profiling) and Cell lines used in this study (C2C12 an HEK 293T) are daily used in Dr. Pendás laboratory, co-­‐author tested for mycoplasma contamination.
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D-­‐ Animal Models
8. Report species, strain, gender, age of animals and genetic modification status where applicable. Please Mice used in this study were as follows: Wild-­‐type (C57BL/6), and knockouts for Syce3-­‐/-­‐ [1], Sycp1-­‐/-­‐ detail housing and husbandry conditions and the source of animals.
[2], Rec8-­‐/-­‐ [3], Smc1β-­‐/-­‐ [4] , Spo11-­‐/-­‐ [5], Sgo2-­‐/-­‐ [6], and REC8-­‐myc transgenic mice [7]. Animals were handled according with regulatory standards and experiments were approved by the UAM Ethics Committee.
1. Schramm S, Fraune J, Naumann R, Hernandez-­‐Hernandez A, Hoog C, Cooke HJ, Alsheimer M, Benavente R (2011) A novel mouse synaptonemal complex protein is essential for loading of central element proteins, recombination, and fertility. PLoS Genet 7: e1002088
2. de Vries FA, de Boer E, van den Bosch M, Baarends WM, Ooms M, Yuan L, Liu JG, van Zeeland AA, Heyting C, Pastink A (2005) Mouse Sycp1 functions in synaptonemal complex assembly, meiotic recombination, and XY body formation. Genes Dev 19: 1376-­‐89
3. Bannister LA, Reinholdt LG, Munroe RJ, Schimenti JC (2004) Positional cloning and characterization of mouse mei8, a disrupted allelle of the meiotic cohesin Rec8. Genesis 40: 184-­‐94
4. Revenkova E, Eijpe M, Heyting C, Hodges CA, Hunt PA, Liebe B, Scherthan H, Jessberger R (2004) Cohesin SMC1 beta is required for meiotic chromosome dynamics, sister chromatid cohesion and DNA recombination. Nat Cell Biol 6: 555-­‐562
5. Baudat F, Manova K, Yuen JP, Jasin M, Keeney S (2000) Chromosome synapsis defects and sexually dimorphic meiotic progression in mice lacking Spo11. Mol Cell 6: 989-­‐998
6. Llano E, Gomez R, Gutierrez-­‐Caballero C, Herran Y, Sanchez-­‐Martin M, Vazquez-­‐Quinones L, Hernandez T, de Alava E, Cuadrado A, Barbero JL, et al. (2008) Shugoshin-­‐2 is essential for the completion of meiosis but not for mitotic cell division in mice. Genes Dev 22: 2400-­‐13
9. For experiments involving live vertebrates, include a statement of compliance with ethical regulations and identify the committee(s) approving the experiments.
This work was supported by grants SAF2011-­‐28842-­‐C02-­‐01 and BFU2014-­‐53681-­‐P to JAS, who has approved permission from the Ethical Committee of the Universidad Autónoma de Madrid, who´s president is D. Rafael Garesse Alarcón ([email protected]). The responsible of animal wealth is Dr. David Muñoz Valverde ([email protected]). Who can be contacted at any time in case you have any questions. 10. We recommend consulting the ARRIVE guidelines (see link list at top right) (PLoS Biol. 8(6), e1000412, We confirm compliance of the ARRIVE guidelines.
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Primary Data
Wetmore KM, Deutschbauer AM, Price MN, Arkin AP (2012). Comparison of gene expression and mutant fitness in Shewanella oneidensis MR-­‐1. Gene Expression Omnibus GSE39462
Referenced Data
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AP-­‐MS analysis of human histone deacetylase interactions in CEM-­‐T cells (2013). PRIDE PXD000208
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