Identification of Candidate Mitochondrial Inheritance Determinants Using the Mammalian Cell-Free System

Reviewer #1 (Public Review):

In this manuscript, the authors used an unbiased method to identify proteins from porcine oocyte extracts associated with permeabilised boar spermatozoa in vitro. The identification of the proteins is done by mass spectrometry. A previous publication of this lab validated the cell-free extract purification methods as recapitulating early events after sperm entry in the oocyte. This novel method with mammalian gametes has the advantage that it can be done with many spermatozoa at the time and allows the identification of proteins associated with many permeabilised boar spermatozoa at the time. This allowed the authors to establish a list of proteins either enriched or depleted after incubation with the oocytes extract or even only associated with spermatozoa after incubation for 4h or 24h. The total number of proteins identified in their test is around 2 hundred and with very few present in the sample only when spermatozoa were incubated with the extracts.

The list of proteins identified using this approach and these criteria provide a list of proteins likely associated with spermatozoa remnants after their entry and either removed or recruited for the transformation of spermatozoa-derived structures.

Using WB and histochemistry labelling of spermatozoa and early embryos using specific antibodies the authors confirmed the association/dissociation of 6 proteins suspected to be involved in autophagy.

While this unique approach provides a list of potential proteins involved in sperm mitochondria clearance it's (only) a starting point for many future studies and does not provide the demonstration that any of these proteins has indeed a role in the processes leading to sperm mitochondria clearance since the protein identified may also be involved in other processes going-on in the oocyte at this time of early development.

Concerning the localisation of the 6 proteins further analysed, the authors must add how much the presented picture represents the observed patterns. They must include the details on the fraction of spermatozoa and embryos displaying the presented pattern.

Reviewer #2 (Public Review):

Mitochondria are essential cellular organelles that generate ATPs as the energy source for maintaining regular cellular functions. However, the degradation of sperm-borne mitochondria after fertilization is a conserved event known as mitophagy to ensure the exclusively maternal inheritance of the mitochondrial DNA genome. Defects on post-fertilization sperm mitophagy will lead to fatal consequences in patients. Therefore, understanding the cellular and molecular regulation of the post-fertilization sperm mitophagy process is critically important. In this study, Zuidema et. al applied mass spectrometry in conjunction with a porcine cell-free system to identify potential autophagic cofactors involved in post-fertilization sperm mitophagy. They identified a list of 185 proteins that might be candidates for mitophagy determinants (or their co-factors). Despite the fact that 6 (out of 185) proteins were further studied, based on their known functions, using a porcine cell-free system in conjunction with immunocytochemistry and Western blotting, to characterize the localization and modification changes these proteins, no further functional validation experiments were performed. Nevertheless, the data presented in the current study is of great interest and could be important for future studies in this field.

Reviewer #3 (Public Review):

In this manuscript, a cytosolic extract of porcine oocytes is prepared. To this end, the authors have aspirated follicles from ovaries obtained from by first maturing oocytes to meiose 2 metaphase stage (one polar body) from the slaughterhouse. Cumulus cells (hyaluronidase treatment) and the zona pellucida (pronase treatment) were removed and the resulting naked mature oocytes (1000 per portion) were extracted in a buffer containing divalent cation chelator, beta-mercaptoethanol, protease inhibitors, and a creatine kinase phosphocreatine cocktail for energy regeneration which was subsequently triple frozen/thawed in liquid nitrogen and crushed by 16 kG centrifugation. The supernatant (1.5 mL) was harvested and 10 microliters of it (used for interaction with 10,000 permeabilized boar sperm per 10 microliter extract (which thus represents the cytosol fraction of 6.67 oocytes).

The sperm were in this assay treated with DTT and lysoPC to prime the sperm's mitochondrial sheath.

After incubation and washing these preps were used for Western blot (see point 2) for Fluorescence microscopy and for proteomic identification of proteins.

Points for consideration:

1. The treatment of sperm cells with DTT and lysoPC will permeabilize sperm cells but will also cause the liberation of soluble proteins as well as proteins that may interact with sperm structures via oxidized cysteine groups (disulfide bridges between proteins that will be reduced by DTT).

2. Figure 3: Did the authors really make Western blots with the amount of sperm cells and oocyte extracts as the description in the figures is not clear? This point relates to point 1. The proteins should also be detected in the following preparations (1) for the oocyte extract only (done) (2) for unextracted nude oocytes to see what is lost by the extraction procedure in proteins that may be relevant (not done) (3) for the permeabilized (LPC and DTT treated and washed) sperm only (not done) (4) For sperm that were intact (done) (5) After the assay was 10,000 permeabilized sperm and the equivalent of 6.67 oocyte extracts were incubated and were washed 3 times (or higher amounts after this incubation; not done). Note that the amount of sperm from one assay (10,000) likely will give insufficient protein for proper Western blotting and or Coomassie staining. In the materials and methods, I cannot find how after incubation material was subjected to western blotting the permeabilized sperm. I only see how 50 oocyte extracts and 100 million sperm were processed separately for Western blot.

3. Figures 4, 5, 6, 7, and 8 see point 2. I do miss beyond these conditions also condition 1 despite the fact that the imaged ooplasm does show positive staining.

4. These points 1-3 are all required for understanding what is lost in the sperm and oocyte treatments prior to the incubation step as well as the putative origin of proteins that were shown to interact with the mitochondrial sheath of the oocyte extract incubated permeabilized sperm cells after triple washing. Is the origin from sperm only (Figs 5-8) or also from the oocyte? Is the sperm treatment prior to incubation losing factors of interest (denaturation by DTT or dissolving of interacting proteins pre-incubation Figs 3-8)?

5. Mass spectrometry of the permeabilized sperm incubated with oocyte extracts and subsequent washing has been chosen to identify proteins involved in the autophagy (or cofactors thereof). The interaction of a number of such factors with the mitochondrial sheath of sperm has been shown in some cases from sperm and others for an oocyte origin. Therefore, it is surprising that the authors have not sub-fractionated the sperm after this incubation to work with a mitochondrial-enriched subfraction.

I am very positive about the porcine cell-free assay approach and the results presented here. However, I feel that the shortcomings of the assay are not well discussed (see points 1-5) and some of these points could easily be experimentally implemented in a revised version of this manuscript while others should at least be discussed.