Nathalie Melaine

Emmanuelle Com

Pascale Bellaud

Laëtitia Guillot

Mélanie Lagarrigue

Nick A Morrice

Blandine Guével

Regis Lavigne

Juan-Felipe Velez

La Calle

Jörg Dojahn

Laetitia Guillot

Régis Lavigne

Juan-Felipe Velez De La Calle

Charles Pineau

Deciphering the Dark Proteome Use of the Testis and Characterization of Two Dark Proteins

Keywords: human proteome project, testis, missing proteins, uPE1, dark proteins, immunohistochemistry, data mining PrYUSRqp

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The primary function of the testis is the production of male gametes, also known as spermatozoa. In mammals, this organ can be divided in two compartments: the seminiferous tubules and the interstitium. The interstitium is a rich connective tissue matrix that mostly hosts Leydig cells, of which the primary function is the production of testosterone in the adult testis.

The seminiferous tubules, which account for 60 to 80% of the total testis volume in mammals 1 , are the site of spermatogenesis, a complex, intricate, tightly controlled, and specialized process [START_REF] Eddy | Male Germ Cell Gene Expression[END_REF][START_REF] Jégou | Paracrine Control of Testis Function[END_REF] . Correct operation of the communication network between nurturing somatic cells and germ cells depends on the continuous production of nearly 1,000 gametes/s in an adult man [START_REF] Baker | Proteomic insights into the maturation and capacitation of mammalian spermatozoa[END_REF] . The effectiveness of this communication network is such that dysfunction of any one of the cellular types which contribute to it has an inevitable cascading effect on other cell types. Thus, the structural and biochemical organization of the human testis makes it one of the most complex organs in the body.

Phase 1 of the Chromosome-Centric Human Proteome Project (C-HPP) aims to catalogue proteins as gene products encoded by the human genome, in a chromosome-centric manner [START_REF] Legrain | The human proteome project: Current state and future direction[END_REF] , and confirm their existence at the protein level by mass spectrometry. Since the C-HPP was launched in 2012, with the help of coordinated efforts worldwide, neXtProt, the reference protein knowledge-base for C-HPP [START_REF] Gaudet | The neXtProt knowledgebase on human proteins: 2017 update[END_REF] , now contains 19,656 entries (release 2018-01-17). The number of experimentally validated proteins (PE1) has reached 17,470 (89%), whereas the actual count of missing proteins (MPs) stands at 2,186. Nevertheless, 1,260 of those "PE1" have unknown functions and are referred to as "uPE1" (neXtProt 2018-01-17 release). As part of the next phase of the project, the C-HPP consortium has launched the "neXt-CP50 Challenge" (C-HPP 4 newsletter n°7, may 2018), which aims to functionally characterize the PE1 proteins with completely unknown functions. An experimental workflow has been proposed to fulfill this goal 7 , which includes immunological detection, transcriptomic analysis and in vitro and in vivo functional studies. Additionally, the C-HPP also focuses on "dark proteins", which include not only uPE1 but also missing proteins (PE 2-4), smORFs and proteins from lncRNAs or any uncharacterized transcripts.

It was suggested that the production of proteins that have been systematically overlooked since the launch of the C-HPP may be restricted to unusual organs or cell types and, particularly, the testis. [START_REF] Lane | Metrics for the Human Proteome Project 2013-2014 and Strategies for Finding Missing Proteins[END_REF] It was shown over a decade ago that the testis expresses the highest number of tissuespecific genes. [START_REF] Son | Database of mRNA gene expression profiles of multiple human organs[END_REF] More recently, two very elegant large-scale studies by Uhlén and collaborators 10, 11 confirmed that the testis is probably the most promising organ to search for missing proteins. Interestingly, the selective pressure on most genes involved in spermatogenesis implies a very high degree of germ-cell specific expression. Thus, approximately half of testisspecific genes are expressed in meiotic and post-meiotic germ cells, whereas a small number was identified in the premeiotic germ cell lineage or somatic testicular cells (i.e., Sertoli and Leydig cells). [START_REF] Djureinovic | The human testis-specific proteome defined by transcriptomics and antibody-based profiling[END_REF] Whole testis extracts have been largely used to search for elements of the missing proteome 13, 14 , whereas the Franco-Swiss contribution to the C-HPP initiative successfully combined the search for missing proteins that lack conclusive mass spectrometric evidence with an extensive examination of the sperm proteome, unambiguously validating over 300 MPs.

The study presented in this paper originates from members of the French C-HPP initiative and aims to characterize dark proteins in the human testis. First, we analyzed the mRNA expression of 520 selected "uncharacterized" proteins extracted from the Human Protein Atlas RNA database and demonstrated that most display higher expression in the testis than in the other human tissues analyzed. We then performed trans-chromosome-based data analysis to catalogue all uncharacterized proteins in a total protein extract from human testis in which cytoplasmic and membrane proteins were preferentially extracted and peptides separated by pH fractionation. A high-quality mass spectrometry dataset was collected on a high-resolution 6600 TripleTOF® system (Sciex). A total of 5,578 proteins were unambiguously identified with at least two peptides, 30 of which were annotated as "uncharacterized". Two uncharacterized proteins were further selected based on their testis specificity. Combining publicly available expression data and immunohistochemistry, we showed that A0AOU1RQG5 is a putative cancer/testis antigen, specifically expressed in the testis, where it accumulates in the cytoplasm of elongated spermatids. We also demonstrate that PNMA6E, which has been referenced as a uPE1 since the beginning of our study, is enriched in the testis, where it is found in the germ cell nuclei during spermatogenesis. Finally, we looked for the gene and protein expression of other dark proteins, uPE1 and missing proteins (PE2-4), in a series of human tissues. Most of the uPE1 and missing proteins are highly expressed in the testis at both transcript and protein levels. Thus, the testis may be a relevant organ to study the dark proteome, as the function of most dark proteins may be related to spermatogenesis and germ cell differentiation.

MATERIALS AND METHODS

Ethics, donor consent, and sample collection and preparation

6

Human testes were obtained from a patient with a significant family history of cancer and diagnosed with seminoma. Voluntary ablation of the gonads, followed by testosterone supplementation, was performed at the IVF unit of the Pasteur Clinic (Brest, France) and samples were considered as biological waste. Written informed consent was obtained from the donor for their use for research. The testes were washed in PBS, frozen in liquid nitrogen, and stored at -80°C until protein extraction.

Protein extraction and digestion

A piece of frozen human testis was resuspended in extraction buffer (20 mM Hepes pH 7.4) supplemented with a protease inhibitor mix (1 mM EDTA, 0.5 mM dithiothreitol (DTT), 1 mM 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), and 10 µM L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E64)). The tissue suspension was sonicated on ice. The resulting lysate was then centrifuged 30 min at 15,000 × g at 4°C. The supernatant, containing the soluble proteins, was kept on ice and the pellets retrieved in 100 mM Na 2 CO 3 and sonicated. This suspension was centrifuged at 105, 000 × g at 4°C for 45 min and the supernatant, enriched for membrane proteins, pooled with the first supernatant containing the soluble proteins. The pellet, containing the membrane fraction, was re-suspended in 8 M urea, 30 mM

Tris, and 4% CHAPS supplemented with the same protease inhibitor mix as above, sonicated, frozen and thawed, and finally centrifuged at 105,000 x g at 4°C for 1 h. The supernatant, enriched for membrane proteins, was pooled with the first two supernatants. The protein concentration was determined using a Bradford colorimetric assay and the protein extract stored at -80°C until use.

The protein extract (407 µg in 25 µL) was diluted in 275 µL 8 M Urea, 0.1 M ammonium bicarbonate, pH 7.8. The proteins were reduced and alkylated by adding 5.3 µL 700 mM DTT, 7 incubating at 37°C for 30 min, and then adding 17.7 µL 135 mM iodoacetamide and incubating an additional 30 min in the dark at room temperature. Finally, 10.2 µL of a 0.4 µg/µL trypsin solution and 677 µL 100 mM ammonium bicarbonate were added to the sample, to adjust the final volume to 1 mL, and incubated overnight at 37°C. The tryptic digested sample was then desalted using 100 mg Sep-Pak tC18 reverse phase cartridges. After elution, the tryptic peptides were stored in 50-100 µL 0.1% formic acid at -80°C.

High pH fractionation of the testis tryptic digest

A human testis peptide sample (407 µg at 8 mg/mL) was injected onto a 250 x 4.6 mm Durashell RP 5 µm column (Bonna-Agela) connected to a Shimadzu Nexera HPLC system fitted with a UV detector. The column was equilibrated at 1 mL/min with buffer A (2 mM ammonium hydroxide /2% acetonitrile in water) and the peptides eluted using a discontinuous gradient of buffer A and buffer B (2 mM ammonium hydroxide /90% acetonitrile /10% water) as follows: 0 min (4% B), 6 min (4% B), 42 min (28% B), 50 min (50% B), 50.1 min (80% B), and 55 min (80% B). Fractions were collected every 2 min from 6 to 52 min and dried under vacuum.

Fractions were reconstituted in 40 µL 5% acetonitrile/0.1% FA in water plus 1 µL 10x HRM peptides (Biognosys) and analyzed by microflow LC-MS as described below.

Microflow LC-MS analysis

LC-MS analysis was performed on the 6600 TripleTOF system (Sciex) connected to an NanoLC TM 425 system (Sciex). The 6600 TripleTOF was operated with a Duospray TM ion source fitted with a 50-mm microflow electrode in positive polarity at 5,500 V, with the GS1 at 10 and curtain gas at 25. The nanoLC TM 425 system was run with a low microflow module (1-10 mL/min) at 5 mL/min with buffer A (0.1% formic acid in water) and buffer B (0.1% formic 8 acid in acetonitrile). The LC system was run in the trap/elute mode with a 5 x 0.5 mm Triart C 18 FDR of 1% at the protein level using a FDR calculation methodology previously described.

16

The number of target and decoy values at PSM, peptide and protein levels together with respective FDR values were reported in supplementary Table 3. All validated Mascot search results obtained from each analyzed fraction were merged and a protein inference list created.

Proteins identified with the exact same set of peptides or with a subset of the same peptides were grouped in a protein set. This protein set was represented by a typical protein corresponding to the protein identified with the best score or, for the same protein set, with the SwissProt accession number, if applicable.

The identified peptides from proteins of interest were analyzed with the neXtProt peptide uniqueness checker [START_REF] Schaeffer | The neXtProt peptide uniqueness checker: a tool for the proteomics community[END_REF] to verify that they were unique to the inferred protein.

RNA-Seq analysis

The RNA-Seq data of isolated testicular cells (Leydig cells, peritubular cells, Sertoli cells, spermatocytes, and round spermatids) and total testis, were obtained from the SRA (https://www.ncbi.nlm.nih.gov/sra) with identification SRX1426397 to SRX1426408. [START_REF] Jégou | Meiotic Genes Are Enriched in Regions of Reduced Archaic Ancestry[END_REF] The fastq format collection of paired reads was obtained using a Galaxy (https://galaxyproject.org/) tool wrapper based on the fastq-dump utility of the SRA Toolkit. [START_REF] Leinonen | The Sequence Read Archive[END_REF] The transcript abundance of ENSG00000277535 and ENSG00000214897 was estimated using Kallisto tool wrapper on the Galaxy interface [START_REF] Bray | Near-optimal probabilistic RNA-seq quantification[END_REF] by alignment on the human genome (Ensembl release 92, GRCh38) and 10 expressed in "Transcripts Per Million" (TPM). The cut-off was set to 1 TPM. The TPM average is given as a histogram for each cell duplicate.

Datamining

Protein evidence (PE1-4) was retrieved from the neXtProt database (Release 2018-01-17). The uPE1 list was retrieved using the SPARLQ query of NXQ-00022 selecting only the validated PE1 proteins. Ensembl and UniProt IDs were retrieved using Retrieve/ID mapping tools on the UniProt website (http://www.uniprot.org/).

Our data set was compared to the neXtProt uPE1 list using a Venn diagram (J Ven V tool Data on the levels of 13,205 proteins in human tissues, based on immunohistochemistry using tissue microarrays, were obtained from the HPA (Normal tissue data). The data are based on The Human Protein Atlas version 18 and Ensembl version 88.38. For each organ, the highest staining found in a cell type was assigned to the whole organ and global staining was then re-calculated per organ and expressed as 0 for not detected, 1 for low, 2 for medium, and 3 for high.

Heatmaps were generated using the Heatmapper tool [START_REF] Babicki | Heatmapper: web-enabled heat mapping for all[END_REF] (http://www1.heatmapper.ca/). Row clustering was performed using the "complete linkage" method and the Euclidean method was used for distance measurement.

Polyclonal antibody production 11

Two specific peptides (Figs. 3 and5) were designed for A0A0U1RQG5 and A0A0J9XYQ4.

Protein sequences were first blasted against the human protein database to determine the homology region. Peptides were then chosen based on several criteria: no sequence homology, no post-translational modification, and low hydrophobicity. Peptide synthesis and production of rabbit polyclonal antibodies were performed by Biotem (Apprieu, France) using the "42 days"

protocol. The antibodies were purified by Protein-A affinity chromatography.

Immunohistochemistry

The testicular expression of the two proteins of interest was confirmed by immunohistochemistry on human testes fixed in 4% paraformaldehyde and embedded in paraffin, as previously described. [START_REF] Jumeau | Human Spermatozoa as a Model for Detecting Missing Proteins in the Context of the Chromosome-Centric Human Proteome Project[END_REF] Briefly, paraffin-embedded tissues were cut into 4 µm thick slices, mounted on slides, and dried at 58°C for 60 min. Immunohistochemical staining was performed on a Discovery Automated IHC stainer using the Ventana DABMap and OMNIMap detection kit (Ventana Medical Systems, Tucson, USA). Antigen retrieval was performed using the proprietary Ventana Tris-based buffer solution, CC1, at 95 to 100°C for 48 min. Tissue sections were then saturated for 1 h with 5% BSA in TBS and endogenous peroxidase was blocked with Inhibitor-D, 3% H 2 O 2 (Ventana) for 8 min at 37°C. After rinsing in TBS, slides were incubated at 37°C for 60 min with polyclonal rabbit antibodies specific for the selected peptide diluted in TBS containing 0.2% Tween 20 (v/v) and 3% BSA (TBST-BSA). Nonimmune

Data Availability

The mass spectrometry proteomics data, including raw files and identification files, form a complete submission with the ProteomeXchange Consortium. [START_REF] Vizcaíno | ProteomeXchange 39 provides globally coordinated proteomics data submission and dissemination[END_REF] Data were submitted via the PRIDE partner repository under the dataset identifiers PXD009598 and 10.6019/PXD009598.

RESULTS AND DISCUSSION

Identification of "uncharacterized" dark proteins in the human testis

The aim of the present work was to study dark proteins in the testis with priority given to uncharacterized proteins. We assessed whether the testis is a relevant organ for identifying uncharacterized proteins by first analyzing the tissue expression of 520 mRNAs, corresponding to referenced uncharacterized proteins. Data were extracted for the 19,613 expressed genes in the HPA RNA database (Fig. 1). Most of these uncharacterized genes/proteins were more highly expressed in the testis than in the 36 other human tissues analyzed. Fallopian tubes and, to a lesser extent, the cerebral cortex, also appear to be relevant tissues for studying uncharacterized proteins (Fig. 1). This observation strengthened our choice to look for uncharacterized proteins in the human testis.

Page 12 of 42 We generated a high-quality and high-resolution mass spectrometry dataset from a total human testis and were able to identify 6,897 protein groups with a 1% FDR threshold at the PSM and protein levels and at least one peptide with a minimal length of nine amino acids. The dataset was filtered by the number of peptides and UniProt description (Table 1 and Supplemental Table S1). Among the 5,578 protein groups we identified with at least two peptides, 30 were annotated as uncharacterized based on information from the 2016-07 UniProt release. Examination of the mRNA expression profile corresponding to these 30 uncharacterized dark proteins in the HPA database (Table 1) showed 15 to have a testis-enriched profile. Two candidates were selected for According to UniProt release 2016-07, 30 identified proteins were described as uncharacterized proteins. Two of these were chosen according to their expression pattern in reproductive organs:

A0A0U1RQG5 and PNMA6E (A0A0J9YXQ4) which are testis-enriched proteins.

A0A0U1RQG5 is testis-specific, whereas PNMA6E is also expressed at low level in the ovary and Fallopian tube.

further investigation: i) A0A0U1RQG5, for which the mRNA displayed a clear testis-specific expression profile and ii) A0A0J9XYQ4, for which the mRNA displayed an expression profile with a potential link to reproductive tissues. Indeed, the gene corresponding to A0A0J9XYQ4 was highly expressed in the testis, but also showed low expression in the female genital tract.

The status of the 30 uncharacterized proteins was updated in the last neXtProt release (2018-01-17). Eighteen were shown to exist at the protein level (PE1), of which 14 have an unknown function (uPE1) and are thus awaiting further characterization. Twenty-four are still annotated as "uncharacterized" (Table 1). A0A0U1RQG5 was not reviewed in neXtProt, whereas A0A0J9XYQ4 is evidenced at the protein level, known as PNMA6E, but with an unknown function. We further characterized these two dark proteins by designing immunogenic peptides for the production of specific polyclonal antibodies for immunohistochemistry studies of human testis sections. In our study, A0A0U1RQG5 was identified by two unique non-nested peptides with a length of 13 and 10 aa (Fig. 2C). According to the Expression Atlas data from several laboratories (Fig. 3A), the AL772284.2 gene transcript is only expressed in the adult testis. Data on the level of mRNA expression of the AL772284.2 gene was obtained from recently published RNA-Seq data on isolated human testicular cells, expressed as transcripts per million (TPM; Fig. 3B).

AL772284.2 is expressed in spermatocytes and round spermatids. We produced a polyclonal antibody using two specific peptides for A0A0U1RQG5 (Fig. 2C). The peptides were blasted against human entries (UniProtKB Human database) to ensure that they did not share homology with other human proteins. The purified polyclonal antibody was used for immunohistochemistry studies of human testis sections (Fig. 3C). There was moderate immunoreactivity in the seminiferous epithelium at all stages of spermatogenesis, whereas the cytoplasmic lobes of elongated spermatids showed strong staining. In the interstitium, there was moderate staining of

Leydig cells, a classical feature

Page 16 of 42 and 10 aa (C). The two immunogenic peptides used for antibody production were designed in regions that share 44% (peptide 1) and 63% (peptide 2) homology with CT47B and CT47A; 2 proteins which present the highest homology with A0A0U1RQG5 (C).

of the immunohistochemistry of testis sections, often corresponding to non-specific staining.

Our results show that expression of the AL772284.2 gene is testis-specific and the corresponding protein, A0A0U1RQG5, is germ cell-specific. A0A0U1RQG5 contains CT47 domains and is probably a novel cancer testis antigen (CTA). CTAs are overexpressed in tumors but restricted to immune privileged sites in normal tissues. CTA are classed into three groups: 1) specific to the

N A N A N A N A N A 0 N A N A N A N A N A N A 0 N A 0 N A 0 N A N A N A N A N A N A N A N A N A N A N A N A N A N A N A N A N A N A N A 1 6 N A Illumina Body Map 0 0 N A N A 0 N A N A 0 N A N A N A N A 0 N A 0 0 0 0 N A N A 0 N A N A N A N A N A 0 N A N A 0 N A N A 0 N A N A N A 1 2 0
GTEx N A 0 0 N A N A 0 0 N A N A N A N A 0 N A 0 N A N A 0 0 0 0 0 N A N A N A 0 0 0 0 N A 0 N A 0 0 N A 0 0

1 7 0 ENCODE (M. Snyder lab) 0 0 N A N A 0 N A N A N A N A N A N A N A 0 N A 0 N A 0 0 0 N A N A 0 N A N A 0 N A N A N A N A N A N A N A 0 N A N A N A 9 N A 32
Uhlen's Lab 0 0 N A 0 N A N A 0 0 0 0 0 N A 0 N A 0 N A 0 0 0 N A 0 0 0 N A 0 0 0 0 0 N A 0 0 0 0 N A N A 19 testis, 2) testis-and brain-specific, and 3) testis-specific and expressed at low levels in no more than two organs. [START_REF] Hofmann | Genome-wide analysis of cancer/testis gene expression[END_REF] Our candidate, A0A0U1RQG5, is testis-specific, with a gene located on the X chromosome, similarly to half of all CTAs, such as MAGEs, leading to their being named CT-X genes. CT-Xs are more testis-restricted than other CTAs. [START_REF] Hofmann | Genome-wide analysis of cancer/testis gene expression[END_REF] CTAs have been widely studied for their potential to target cancer cells in personalized immunotherapy (for review see 27, 28 ).

Recently, Babatunde and collaborators [START_REF] Babatunde | Cancer/Testis genes in relation to sperm biology and function[END_REF] reviewed the potential roles of CTAs in testis function.

Most of the functions of CTAs functions remain unclear, but some appear to be involved in gametogenesis, sperm metabolism, or motility.

29

PNMA6E is enriched in the human testis and is expressed during spermatogenesis

The second selected protein is paraneoplastic Ma antigen 6E (PNMA6E), which is testisenriched (Table 1). The PNMA6E gene is located on chromosome Xq28 (position 153,395,640 to 153,401,420) on the reverse strand and has two splice variants: PNMA6E-201 and PNMA6E-202 (Fig. 4A andB). PNMA6E-201 was identified in our dataset by 11 non-nested peptides with a minimal length of nine aa. Nine are unique to the PNMA6E gene (two are shared with PNMA6F) and four are specific to the PNMA6E-201 isoform (Fig. 4C). The transcript is 2,192 bp in length and contains PNMA domains (Fig. 4A).

Page 19 of 42

21

The resulting protein, PNMA6E, has a length of 647 aa and a sequence of 322 aa in the C terminal part (325-647) shares 100% identity with PNMA6E-202 (Fig. 4C). Public expression data from the Expression Atlas shows that PNMA6E is highly expressed in the human testis (Fig. 5A) and present at low levels in the ovary and Fallopian tubes. The PNMA family is composed of 15 members, which are predominantly expressed in the brain (Fig. 6), whereas PNMA6E is only expressed in testis and, at a low level, in female organs (ovary and Fallopian tubes; Figs. 5,6). We further investigated the expression of the PNMA6E gene using RNA-Seq data from isolated testicular cells. Its expression is restricted to germ cells (Fig. 5B). Furthermore, we performed immunohistochemistry experiments on human testis sections using a polyclonal antibody directed against two specific peptides of the PNMA6E protein (see Material and Methods section; Fig. 4C). PNMA6E showed nuclear staining (Fig. 5) in both cells of the interstitium and the seminiferous tubules. In the seminiferous epithelium, germ cells increasingly expressed this protein from premeiotic germ cells (i.e., spermatogonia and primary spermatocytes) to pachytene spermatocytes and round spermatids. However, PNMA6E was no longer found in elongated spermatids. Leydig cells do not express the PNMA6E mRNA (Fig. 5B). Thus, the moderate immunostaining observed in Leydig cells probably corresponds to nonspecific staining.

PNMA family members share several domains, with high sequence homology, and are quite conserved among species 30 . Particularly, PNMA6E shares 64% homology with its mouse orthologue. Although PNMA members share conserved domains, their expression patterns differ. [START_REF] Pang | PNMA family: Protein interaction network and cell signalling pathways implicated in cancer and apoptosis[END_REF] In contrast to other members of the PNMA family, PNMA6E expression is clearly restricted to the testis and, to a lower extent, to the female genital tract (Figs. 5A and6).

Page 21 of 42 stages IV to VI of the seminiferous epithelium [START_REF] Clermont | The cycle of the seminiferous epithelium in man[END_REF] using rabbit polyclonal antiserum designed against two specific peptides. Non-immune serum was used as a negative control (data not shown). In the interstitium, dense immunoreactivity was observed in the nucleus of Leydig cells.

A significant number of peritubular cell nuclei also displayed a moderate signal.

Immunoreactivity was more intense in germ cell nuclei, in which the signal increased from premeiotic germ cells to pachytene spermatocytes (arrowhead) and round spermatids (arrows), whereas there was no staining of elongated spermatids.

GTEx N A 0 N A N A 0 N A N A 0 N A N A N A 0 N A N A 0 0 0 0 0 N A N A N A 0 0 0 0 N A 0 N A 1 2 N A 0 0 2 6 0
32 Uhlen's Lab 0 0 0 N A 0 0 0 N A 0 0 0 N A 0 N A 0 0 0 N A 0 0 0 N A 0 0 PNMAs also differ in their association with disease (for review see [START_REF] Pang | PNMA family: Protein interaction network and cell signalling pathways implicated in cancer and apoptosis[END_REF] ): aberrant expression of PNMA1, 2, and 3 are correlated with paraneoplastic disorders or cancer [START_REF] Sahashi | Anti-Ma2 antibody related paraneoplastic limbic/brain stem encephalitis associated with breast cancer expressing Ma1, Ma2, and Ma3 mRNAs[END_REF] , whereas MOAPS mediates apoptotic signaling. [START_REF] Pang | PNMA family: Protein interaction network and cell signalling pathways implicated in cancer and apoptosis[END_REF] Recently, mouse pnma5 was shown to be involved in meiotic progression in oocytes by controlling the phosphorylation of two important kinases, PDK1 and Gsk3beta. [START_REF] Zhang | Pnma5 is essential to the progression of meiosis in mouse oocytes through a chain of phosphorylation[END_REF] The authors also demonstrated that pnma5 knockdown mice exhibit abnormal oocyte fertilization.

0 0 0 N A 0 0 . 9 5 0 N A N A 2 8 0 Illumina Body Map 0 0 N A 0 N A 0 N A N A N A N A 0 N A 0 0 0 0 N A N A 0 N A N A N A N A N A 0 N A N A 0 N A N A 0 . 9 N A N A N A 1 6 0 Mammalian Kaessmann N A N A N A N A N A N A N A N A N A N A 0 N A 0 N A 0 N A N A N A N A N A N A N A N A N A N A N A N A N A N A N A N A N A N A N A 3 1 N A ENCODE (M. Snyder lab) 0 0 N A 0 N A N A N A N A N A N A 0 N A 0 N A 0 0 0 N A N A 0 N A N A 0 N A N A N A N A N A N A N A 0 N A N A N A 1 2 N A Low Medium Hight
In conclusion, PNMA6E expression is restricted to the genital tract. The protein is highly enriched in the testis, particularly in germ cells up to meiosis. The expression of PNMA6E in only the human testis, and to a lower extent in the ovary and Fallopian tubes, favors a role in gametogenesis, similar to PNMA5, which is considered to be a female fertility factor [START_REF] Zhang | Pnma5 is essential to the progression of meiosis in mouse oocytes through a chain of phosphorylation[END_REF] .However, this awaits further investigation. tissues, including that from the female and male reproductive tracts. All PNMA genes, except PNMA6E, are expressed in brain tissues. PNMA1, PNMA5, and PNMA6E display a testisenriched pattern, whereas PNMA6E is exclusively expressed in the testis and, to a lower extent, in the ovary and Fallopian tube.

uPE1 and MPs are highly expressed in the testis

In the last part of our study, we investigated the expression pattern of other dark proteins:

uPE1 and missing proteins (PE2-4) (Figs. 7 and8). We examined the mRNA expression of the 19,613 genes and the level of the 13,205 proteins from the HPA database. The status of the evidence for the existence of the protein was assigned to each entry (neXtProt release 2018-01-17). Among the 19,613 genes, 1,229 correspond to uPE1 and 1,822 correspond to MPs (PE2-4), others are PE1, PE5 or unreferenced in neXtProt. Among the 13,205 proteins found in HPA, 754 are considered to be uPE1 whereas 424 are MPs. We found that uPE1 genes were highly expressed in the testis using a clustering method (Fig. 7A). At the protein level, many uPE1 also showed high expression levels in the testis (Fig. 7B). Unsurprisingly, most were found at high levels in seminiferous tubules, suggesting that uPE1 are mainly expressed in the germ lineage.

Two other significant enriched expression clusters could be observed in the cerebral cortex and Fallopian tubes. Finally, we sought uPE1 identified in our testis dataset (Fig. 7C). Among the 1,260 uPE1 referenced in the latest neXtProt release, 238 were identified in the present study, corresponding to approximately 19% of the current uPE1 in neXtProt.

We used a clustering method to also compare the 1,822 MP genes found in the HPA database (Figs. 8A, B andC). Most genes corresponding to MPs were testis-enriched, similar to uPE1 genes. At the protein level, the 424 MPs found in the HPA database also displayed a testisenriched pattern and were, to a large extent, highly expressed in seminiferous tubules, similar to uPE1, and thus most likely expressed in germ cells. Some MPs were also enriched in the cerebral cortex, placenta, and epididymis.

Page 25 of 42 Our results clearly show the great potential of the human testis, not only to search for additional missing proteins, but also to characterize the function of uPE1 in a biological context.

The dark proteins studied here are, to a large extent, testis-enriched and their expression could be traced to the seminiferous tubules. This confirms previous studies showing that most testisenriched genes are expressed by germ cells. [START_REF] Djureinovic | The human testis-specific proteome defined by transcriptomics and antibody-based profiling[END_REF] Although seminiferous tubules account for 90% of the total testis mass, approximately two-thirds of the cells in these tubules are haploid spermatids at various stages of development. This suggests that testis-enriched uPE1 and MPs are primarily expressed in germ cells and thus play a role in spermatogenesis. Other interesting clusters for characterization of dark proteins are indeed the cerebral cortex, the Fallopian tubes, the placenta, and the epididymis (Figs. 7 and8), suggesting their involvement in biological mechanisms specific to these organs. In Page 28 of 42 29 a testis-enriched cluster (1). These MPs were enriched in the testis and most were also found at high levels in the seminiferous tubules. Other significantly enriched clusters were observed in the cerebral cortex (2), placenta (3), and epididymis (4). such a context, future studies in our lab will involve collecting information about the proteins and genes from the clusters highlighted in this study to associate them with organ-related functions.

CONCLUSION

Here, we investigated the global expression of dark proteins in the human testis, focusing on uncharacterized proteins, as well as uPE1 and MPs. We were unable to find evidence of new missing proteins for validation in our dataset according to the latest C-HPP Guidelines. Only nine missing proteins were potentially identified by single peptide that pass through the automatic validation, among which 5 MPs were evidenced with one unique peptide (supplementary Table 2), suggesting that the dynamic range in a total testis extract is still too large to access low-copy number proteins by conventional pre-fractionation and LC-MS/MS analysis. Although, we have clearly reached the limit of detection of shotgun mass spectrometry, our results confirm that the testis is a promising organ to search for additional MPs, as previously suggested 8, 10, 11 . Monitoring the expression of genes corresponding to missing proteins in the whole testis is indeed a relevant prerequisite to search for MPs. However, such an approach could be misleading, because even though MP genes may be highly expressed in the testis, expression of the corresponding proteins could vary or they could be expressed in particular subcellular locations that render them difficult to access and identify. In such a context, the use of a targeted-MS approach was shown to be efficient in the search for selected testis-enriched MPs, as carried out by Carapito and collaborators on ejaculated spermatozoa [START_REF] Carapito | Validating Missing Proteins in Human 38 Sperm Cells by Targeted Mass-Spectrometry-and Antibody-based Methods[END_REF] . Equalization of .

These authors have recently created the Dark Proteome Database DPD (http://darkproteome.ws)

to better understand and characterize the Dark Proteome. The database presently provides a general map for the dark proteome but needs to be further implemented with additional annotation and prediction sources. Undoubtedly, such a database will be important for aiding the characterization of dark proteins, regardless of the definition used.

Determining the biological functions of dark proteins in the human testis is a very long-term project. Although genome editing may open new perspectives in the coming years [START_REF] Vassena | Genome engineering through CRISPR/Cas9 technology in the human germline and pluripotent stem cells[END_REF] , the CRISPR/Cas9 system is far from being routine and allowing large-scale functional studies of spermatogenesis. [START_REF] Kherraf | Creation of knock out and knock in mice by CRISPR/Cas9 to validate candidate genes for human male infertility, interest, difficulties and feasibility[END_REF] A more realistic approach will be to preferentially select candidate proteins based on their expression pattern. Thus, priority should be given to dark proteins specifically expressed during meiosis and spermiogenesis, two unique processes of the testis. As an example, the PNMA6E expression pattern observed here suggests that the protein may play an important role during meiosis up to early spermiogenesis. Late spermatid-and spermatozoa-specific proteins may also play a role in sperm biology (e.g., mobility, interaction with the oocyte). In both cases, gene invalidation studies could be considered. The selection criteria will be the existence of an orthologous gene in the mouse, with no paralogs, and coordination with the International Mouse Phenotyping Consortium (http://www.mousephenotype.org) to accelerate the production of knockout mice on the orthologous gene of interest. However, demonstration of a link between a defect in the expression of a specific protein and/or a defect with its posttranslational modification and human infertility will take several years. Coordination with clinicians of Assisted Reproductive Technologies will also be necessary to determine the most relevant sperm pathologies, based on observed phenotypes, and then the collection of the cohort of samples. Coutton et al. [START_REF] Coutton | Mutations in CFAP43 and CFAP44 cause male infertility and flagellum defects in Trypanosoma and human[END_REF] succeeded in using Trypanosoma brucei gene invalidation to rapidly study the function of proteins suspected to be important for sperm flagella. The authors demonstrated that the use of such an original model, which shares an extremely conserved flagellar structure with mammals, for gene invalidation could induce severe flagella defects.

Indeed, a comparable approach could be envisioned for a selected set of testicular dark proteins.

Here, we demonstrated that uPE1, MPs, and other uncharacterized proteins are, to a large extent, highly expressed in the testis and, more precisely, within the seminiferous tubules for uPE1 and MPs, even if we could only identify 30 uncharacterized proteins. Nevertheless, we identified 238 uPE1, corresponding to 19% of the uPE1 referenced in neXtProt. Our results clearly show that the testis is an organ of choice, not only for the identification of MPs, but also for the study and characterization of uPE1 and other uncharacterized proteins. Interestingly, in addition to somatic protein isoforms and due to the presence of the germ cell lineage, the testis is 32 also suspected to express an array of germinal isoforms, a significant number of which being produced through alternative splicing. It might be wise to also explore that domain. Only a few laboratories worldwide work on testicular function and this is probably why many dark proteins, expressed in the testis, are still uncharacterized or missing. In addition, the testis is a complex organ with an array of very specific functions and unique phenomena (e.g., chromatin remodeling, repackaging, and transcriptional reprogramming in haploid germ cells) which involve specific genes and proteins 9, 10 . We hope that the presented data will significantly aid the further characterization of dark proteins. Finally, thorough characterization of dark proteins in the testis will contribute to a better understanding of the molecular mechanisms underlying spermatogenesis and should yield striking discoveries in the context of male infertility.

  rabbit serum (1:1,000) was used as a negative control. After several washes in TBS, sections were incubated for 16 min with a biotinylated goat anti-rabbit antibody (Roche) at a final dilution of 1:500 in TBST-BSA. The signal was enhanced using the Ventana DABMap or OMNIMap kit. Sections were then counterstained for 16 min with hematoxylin (commercial 12 solution, Microm) and 4 min with bluing reagent (commercial solution, Microm), before rinsing with Milli-Q water. After removal from the instrument, slides were manually dehydrated and mounted in Eukitt (Labnord, Villeneuve d'Ascq, France). Finally, immunohistology images were obtained using NDP.Scan acquisition software (v2.5, Hamamatsu) and visualized with NDP.View2 software (Hamamatsu). Representative images are shown in figures 3 and 5.

Figure 1 :

 1 Figure 1: Tissue expression of uncharacterized genes in 37 human tissues. mRNA expression of 19,613 genes were obtained from the HPA database. Gene were filtered by names reviewed as uncharacterized. This heat map represents the clustering of gene expression of uncharacterized genes in 37 different human tissues. Interestingly, most of these uncharacterized genes are testis-enriched.

Page 13 of 42

 42

Figure 2 :

 2 Figure 2: Structure of the AL772284.2 gene and sequence of the corresponding

Figure 3 :

 3 Figure 3: Expression of AL772284.2 gene in human tissues. (A) mRNA expression pattern

Figure 4 :

 4 Figure 4: Structure of the PNMA6E gene and sequence of the corresponding protein. The

Figure 5 :

 5 Figure 5: Expression of PNMA6E in human tissues. (A) mRNA expression pattern in various human tissues. According to Expression Atlas, PNMA6E mRNA expression is only found at a high level in the testis, whereas the ovaries and fallopian tubes also display positive, but low, expression. (B) mRNA levels in different cell types of the testis. The mRNA level is expressed in TPM and the cut-off is set at 1 TPM (green dotted line). In the testis, mRNA was detected only in male germ cells. (C) Antibody staining validation of the PNMA6E protein in adult human testis. The corresponding protein was detected in transverse testis sections at

Figure 6 :

 6 Figure 6: Human tissue expression of the PNMA family gene members. mRNA expression

Figure 7 :

 7 Figure 7: Testicular enrichment of uPE1 proteins in humans. Data on the (A) mRNA

Figure 8 :

 8 Figure 8: Updated status of missing proteins in the human testis. Data on the (A) mRNA expression levels of 19,613 genes and (B) staining pattern of 13,205 proteins were obtained from the HPA database. The protein evidence status was assigned to each entry (neXtProt release 2018-01-17). (A) Heat maps representing the clustering of gene expression corresponding to missing proteins (PE2-4) in 37 human tissues. Many of the MP genes are testis-enriched. (B) Among the 13,205 proteins found in the HPA database, 424 are MPs (PE2-4). Clustering showed

MS/MS data analysis/protein identification and validation

	Each search result was thus validated with a peptide rank of 1, a minimal peptide length of 9
	amino acids, a FDR of 1% on mascot e-value at the peptide spectrum match (PSM) level, and a
	3 mm trap column (YMC) and a 150 x 0.3 mm Triart C 18 3 mm trap column. Samples were
	injected onto the trap column at 10 mL/min with the loading pump and then eluted with a linear
	gradient of acetonitrile at 5 mL/min.
	Ten microliters of each fraction from the high pH fractionation were analyzed in data-
	dependent acquisition using a linear acetonitrile gradient (0-45 min (2-40% B), 50 min (80% B),
	and 55 min (80% B) with the following acquisition parameters: survey scan m/z 400-1250 (250
	ms) followed by Top 30 precursors (2-5+) with the intensity >150 cps. The TOF MS/MS was
	acquired for 50 ms in high sensitivity mode from m/z 100-1500 using rolling collision energy
	(CE spread 5) and precursors were excluded for 15 s after one occurrence.
	ProteinPilot	TM 5.0 (Sciex) was used to perform automatic mass recalibration of MS and
	MS/MS spectra and export the recalibrated peak lists as mgf files. The Mascot server v2.2.07
	(http://www.matrixscience.com) database search engine was used for peptide and protein
	identification, using its automatic decoy database search to calculate the false discovery rate
	(FDR). MS/MS spectra were compared to the UniProt Homo Sapiens reference proteome
	database (UP000005640, release 2016-07, 92,578 protein sequences which contains canonical
	and variant isoforms). Peptide and MS/MS tolerances were set at 0.05 and 0.1 Da, respectively.
	The enzyme selectivity was set to full trypsin with one mis-cleavage allowed. Protein
	modifications were fixed carbamidomethylation of cysteines and variable oxidation of
	methionine. Mascot dat files obtained from each query were then imported into Proline Studio
	1.3 software, used for the validation of identification results (http://proline.profiproteomics.fr/).
		9

Table 1 : List of the uncharacterized proteins identified in the human testis.

 1 Dark proteins annotated as uncharacterized in the UniProt release 2016-07 are provided together with the corresponding gene name. For each entry, the protein updated description in the UniProt release

	2018-03, evidence level (neXtProt release 2018-01-17, N/A not applicable), domain/family
	(UniProt), and mRNA level expression in human testis (Expression Atlas) were provided.

novel cancer/testis antigen, is testis-specific and expressed in the germ cell lineage A0A0U1RQG5

  is a transcript of the AL772284.2 gene (Fig.2A and B; Ensembl version 92) composed of three exons and located on chromosome Xq21.1 (position 119,073,226 to 119,076,373) on the forward strand. The transcript is 1,147 bps in length and encodes a protein of 324 aa (A0AOU1RQG5). No splice variant is referenced in the Ensembl database. The annotated transcript contains two "cancer/testis antigen 47" domains, suggesting that the A0A0U1RQG5 protein may be a cancer/testis antigen belonging to family 47. Furthermore, A0A0U1RQG5 shares 61.4% homology with CT47B and 60.3% with CT47A (data not show).

	Page 15 of 42
	A0A0U1RQG5, a

  Page 29 of 42 protein abundance using the Proteominer approach may also be relevant for the identification of low expressed MPs[START_REF] Li | Digging More Missing Proteins Using an Enrichment Approach with ProteoMiner[END_REF] , particularly for the identification of membrane or nuclear MPs. The use of RNA-Seq information on MP gene expression will be an important asset to design proteotypic peptides for selected protein candidates prior to targeted-MS analyses. The search for MPs in isolated testicular cell extracts and/or subcellular compartments has indeed been envisioned but is still technically challenging due to limited routine access to human testes.The C-HPP consortium also focuses on the characterization of uPE1 and, more widely, dark proteins. According to the Protein Structure group, the dark proteome consists of proteins for which the experimental structure has never been determined and which are inaccessible to homology modeling. More than half of dark proteins are not intrinsic disorder proteins, have no transmembrane domain, and have a low compositional bias, as shown by Perdigão et al.

	35, 36

ACKNOWLEDGMENTS

We are grateful to Gauthier Husson (Université de Strasbourg, CNRS, IPHC UMR 7178, LSMBO, Strasbourg, France) for recalibration of the raw MS/MS data. We also thank the GenOuest bioinformatics Core Facility (UMR6074 IRISA CNRS/INRIA/Université de Rennes1) for access to the Galaxy web platform at galaxy.genouest.org used for the RNA-Seq data analysis.

Funding Sources

This work was partially funded through the Fondation pour la Recherche Médicale (FRM Grant DBS20140930778). This work was also supported by grants from Biogenouest and the Conseil Régional de Bretagne, awarded to CP.

PRIDE Archive (http://www.ebi.ac.uk/pride/archive/) via the PRIDE partner repository with the data set identifier PXD009598 and 10.6019/PXD009598.

Acc

Uniprot

Notes

The authors declare no competing financial interest.


Y Pascal

D Labrousse

M Petit

S Lefebvre

F Costa

Experimental investigation of the reliability of Printed Circuit Board (PCB)-embedded power dies with pressed contact made of metal foam

A Printed Circuit Board (PCB)-embedding process using pressed metal foam to connect the top-side pads of power dies is considered. The manufacturing process, simple and highly cost-effective, is described in detail; samples are manufactured and their reliability and robustness are characterised. It is shown that thermally cycled prototypes exhibit reliability close to that of Direct Bounded Copper (DBC) substrates. Samples submitted to 150 A-surges have highly scattered reliability. SiC MOSFETs submitted to destructive current limiting tests and repetitive short-circuit tests performed similarly to dies reported in TO247 packages. A discussion on the development of reliability-assessment-methods, especially suited for PCB-embedding processes, is proposed.

Introduction

Although wire-bonding processes are mature and fully mastered, they have limited performances. For instance, wire bonds can account for more than half of the conduction loss of a packaged low-voltage transistor while adding an inductance in the order of ten nanohenries. Furthermore, wire-bonded dies can only be cooled from their bottom-sides. Last, wire bonds have limited reliability and robustness [START_REF] Hamidi | Reliability and lifetime evaluation of different wire bonding technologies for high power IGBT modules[END_REF].

These limitations call for a new way to connect the top-sides of dies. Many academic and industrial researchers [START_REF]Current Developments in 3D Power Packaging with Focus on Embedded Substrate Technologies[END_REF][START_REF] Wang | Review of state-of-the-art integration technologies in power electronic systems[END_REF][START_REF] Ménager | Industrial and lab-scale power module technologies: A review[END_REF] tackle this issue, using copper [START_REF] Rittner | Robust top side contact technology on power semiconductors -results from the public funded project 'propower[END_REF] or aluminium [START_REF] Milke | High temperature behaviour and reliability of Al-Ribbon for automotive applications[END_REF] ribbons or a flexible Printed Circuit Board (PCB) [START_REF] Stockmeier | SKiN: double side sintering technology for new packages[END_REF] instead of aluminium wires, using pressed contacts made with spring-like metal wool [START_REF] Zhu | A Solution to Press-Pack Packaging of SiC MOSFETS[END_REF] or made of plain metal [START_REF] Vagnon | Electrical characterization of a pressed contact between a power chip and a metal electrode[END_REF][START_REF] Vagnon | A bus-bar-like power module based on three-dimensional power-chip-on-chip hybrid integration[END_REF].

Embedding the dies within the PCB-substrate itself is another approach to wire-bond-free components and modules [START_REF] Gottwald | P2 Pack -the paradigm shift in interconnect technology[END_REF][START_REF] Kearney | PCB-embedded semiconductors for low-voltage power electronic applications[END_REF][START_REF] Regnat | Optimized power modules for silicon carbide MOSFET[END_REF][START_REF] Manessis | Development of advanced power modules for electric vehicle applications[END_REF][START_REF] Wyss | analysis of pcb embedded power semiconductors for a 30 kw boost pfc converter[END_REF][START_REF] Sharma | PCB embedded power package with reinforced top-side chip contacts[END_REF]. As a matter of fact, in such device, the top-side pads of the dies are often connected by a layer of copper electrodeposited on the top of the dies. This process, however, is rather complex and costly since it involves sputtering seed layers on the top-side metallisation layers of the dies, which is seldom compatible with today's PCB factories. Some publications tackled the reliability of these assemblies. In [START_REF] Manessis | Technical Understanding of Resin-Coated-Copper (RCC) Lamination Processes for Realization of Reliable Chip Embedding Technologies[END_REF], it is reported that all samples passed 1000 thermal cycles between -55 °C and +125 °C without showing any delamination but no electrical measurement was made. In [START_REF] Kearney | PCB-embedded semiconductors for low-voltage power electronic applications[END_REF], a reduced temperature swing was used (Δ𝑇 𝑗 < 90 K) and none of the aged sampled reached the failure criteria, defined as a 10 % increase in IGBT saturation voltage, after 400 k cycles. In [START_REF] Pascal | Using Laminated Metal Foam as the Top-Side Contact of a PCB-Embedded Power Die[END_REF], we proposed a new technique to connect the top-side of a die, using a pressed contact made of metal foam. This paper deals with the reliability and the robustness of this process. Various sources of stress were experimentally investigated: samples were thermally cycled, submitted to current surges and submitted to short-circuit tests. These tests were intended, depending upon the nature of the stress applied, either to assess the reliability and robustness of the connection itself, or to ensure that the proposed packaging process does not degrade the reliability and robustness of the die. Few samples were submitted to each test, hence the limited statistical significance.

Additionally, a discussion on the relevance of defining new reliability indicators, suited to PCBembedded power electronics, is proposed.

Packaging process

The manufacturing process is quite straightforward; it consists of only three steps. First, the bottom side of the die is soldered on a "bottom PCB" (Fig. 1.a). Then, the bottom PCB (and the die), layers of prepreg, a polyimide mask (with one hole placed above each top-side pad of the die), a piece of metal foam for each top-side pad, and the top PCB are stacked (Fig. 1.b). This is then laminated, as specified by the prepreg manufacturer (Fig. 1.c).

The mask allows using pieces of foam wider than the pads and roughly positioning them above it. As a matter of fact, provided that the polyimide mask is precisely positioned above the die (i.e. that the holes in the mask are aligned with the pads), it will prevent the foam, slightly distorted during the lamination process, from short-circuiting adjacent pads (cf. Fig.

1.c).

The foam is neither soldered, nor sintered, nor glued to neither the copper nor the die; it is solely pressed between the top-PCB and the die and kept in place by the epoxy resin.

Nickel foam Ni-5763 was used [START_REF] Recemat | Data sheet Nickel Foam[END_REF]. It is mainly composed of nickel (99.5 %), alloyed with iron (0.2 %), copper and zinc (0.1 % each). The foam thickness and porosity before lamination were, respectively, 1.4 mm and 96 % whereas they drop to about 300 µm and 70 %, respectively, afterward.

In spite of its rather high porosity (see Fig. 2) and thanks to its three-dimensional structure (Fig. 3), the foam is able to carry current between the die and the top PCB and thereby electrically connect them.

3

Aging with high-intensity current pulses

Motivation

Fig. 2 and Fig. 4 show that the contact between the foam and the die top-side metallisation are highly localised. This will induce local high current densities that, in conjunction with the contact resistance, might induce hot-spots at these points. These might be highly stressful to both the die and the package.

Method

To investigate this potential failure mechanism, current pulses with amplitude 150 A and duration 200 µs (Fig. 5) were forced through diodes embedded using the process under study. The diodes were 60 V-18 A -rated and the die size was 4.2 mm × 6.5 mm × 250 µm. The pulse amplitude and duration were chosen low enough so that the mean die temperature would remain close to the ambient temperature whereas the energy of each pulse would be high enough to induce metallurgical changes at the interface between the foam and the die.

The peak mean current density in the die was close to 700 A/cm². The pulses frequency was 12.5 Hz and each pulse delivered 30 mJ to the device; the mean dissipated power was thus 0.38 W. The die mean temperature was therefore about 4 K above room temperature. Most of the energy was dissipated within the diode junction. Assuming that, throughout the pulse, the heating of the die was homogenous and adiabatic, it can be estimated that the temperature of the active region of the die increased by only a few kelvins during a pulse.

In each sample, so as to further intensify the stress on the interface between the foam and the die without increasing the stress on the die itself, a piece of foam much smaller than the die was used: its surface (2 mm × 4 mm, in Fig. 6: 𝑤 𝑓 = 2 mm) was only 30 % that of the die (4.2 mm × 6.5 mm, in Fig. 6: 𝑤 𝑑 = 4.2 mm).

After every 1000 to 10,000 pulses, the static characteristic of the Device Under Test was measured between 0 and 150 A using a Tektronix 371A High Power Programmable Curve Tracer (Fig. 6). Each characteristic was modelled by a resistance seriesconnected with a voltage-source. This resistance, defined as the derivative of the (𝑉 𝑓 , 𝐼 𝑓 ) characteristic at a given current 𝐼 𝑓 0 , included not only the connection using pressed metal foam, but also the die and the solder.

Results

One of our samples failed as a short circuit after only 7 k pulses, one failed as an open circuit after 1 M pulses, one has not shown any sign of aging after 13 M pulses. At this point, a wire-bonded die (used as reference) sustained 10 M pulses without showing any degradation.

The evolutions of the elements of the model of a sample are given in Fig. 7. The shape of the resistance curve compares with that observed during a bond wire lift-off [START_REF] Hamidi | Reliability and lifetime evaluation of different wire bonding technologies for high power IGBT modules[END_REF].

Optical observations of metallurgical cuts of failed devices did not reveal any sign of degradation. In particular, no fracture in the resin surrounding the foam could be spotted.

Conclusions on the aging with current pulses

The foam being a highly non-homogenous medium might explain the dispersion of the results; this would therefore be a significant limitation to the proposed connecting technic. On the other hand, the dispersion might be due to the process lack of maturity.

Optically studying failed sampled proved to be insufficient to determine the causes of failures. These might be linked to hot-spots on the die or in the foam, or to a degradation of the interfaces between the foam and either the copper of the top-PCB or the aluminium of the die.

4

Passive thermal cycling

Motivation

As in any compact assembly involving materials with different Coefficients of Thermal Expansion (CTE), thermal cycling can be highly stressful for laminated assemblies. In particular, the high CTE mismatch between standard epoxy-based PCB substrate (usually CTE≈60 ppm/K [START_REF] Perrin | 2 MHz high-density integrated power supply for gate driver in high-temperature applications[END_REF]) and the die (CTE=2.6 ppm/K for silicon, 23 ppm/K for aluminium) can induce cracks or delamination [START_REF] Perrin | 2 MHz high-density integrated power supply for gate driver in high-temperature applications[END_REF][START_REF] Ciappa | Selected failure mechanisms of modern power modules[END_REF].

Furthermore, since the contact between the die topside metallisation and the foam is only pressed, small relative movements are possible at the interface. This might result in a local matting or abrasion of the top of the die. On the other hand, this could increase the reliability of the assembly by removing the strain otherwise appearing at the feet of bond wires and responsible for plastic deformations [START_REF] Ciappa | Selected failure mechanisms of modern power modules[END_REF].

The reliability of eight dies embedded using our process was therefore assessed through passive thermal cycling.

Method

Eight diodes with die size 4.2 mm × 6.5 mm were embedded using the proposed process. They were then placed in a climatic chamber and thermally cycled between -40 °C and 150 °C. Each cycle lasted 70 min, including 15 min-long dwell times. The temperature slope was less than 8 K/min.

Results

Before and after 220 cycles, the resistances of the samples were measured (at 𝐼 𝑓 0 = 10 A) and compared (cf. Fig. 8). The resistance of all the diodes increased, by 5 % to 108 %; the mean increase was 54 %. The blocking capabilities of the components, on the other hand, were not affected by the test. This likely indicates that the dies were not damaged and that the pressed contacts themselves evolved during the aging.

Metallurgical cuts were made on aged assemblies and studied using an optical microscope but no sign of degradation, such as delamination (like those reported in [START_REF] Perrin | 2 MHz high-density integrated power supply for gate driver in high-temperature applications[END_REF]), or resin fracture, could be found in any sample.

Most power modules use Direct Bounded Copper (DBC) as substrate, the reliability of which has been widely studied. In [START_REF] Bouarroudj | Temperature-level effect on solder lifetime during thermal cycling of power modules[END_REF], delaminations in the solder between the DBC and the base plate appeared after only 25 cycles between -40 °C and 120 °C. In [START_REF] Giinther | Failure Mechanisms Of Direct Copper Bonding Substrates (DCB)[END_REF], catastrophic failures due to cracks in the ceramic were observed after 200 to 300 -40 °C/150 °C cycles. Similar results were reported in [START_REF] Hung | Investigation of solder crack behavior and fatigue life of the power module on different thermal cycling period[END_REF][START_REF] Devoto | Thermal performance and reliability of bonded interfaces for power electronics packaging applications[END_REF].

Conclusion on thermal cycling

This shows that DBCs submitted to the same harsh test conditions as our prototypes would have likely catastrophically failed whereas the physical integrity of our samples was not impaired. Nevertheless, the very high resistance increase denotes a severe degradation of the assembly, possibly within the foam or at the interface between the foam and either the die or the top-PCB. Such resistance increase is a strong limiting factor for reliability aspects.

Metallurgical cuts were not sufficient to determine failure mechanisms; a more thorough failure analysis will be required to draw definite conclusions.

5

Robustness in Current Limitation Mode

Motivation

Dies packaged using bond wire have their top-side pads connected in only as many points as the number of bond wires usedusually 2 or 3 in a discrete package such as TO247. On the other hand, in the proposed packaging process, the contact between the foam and the die is disseminated over the entire die Fig. 8. Resistance of diodes packaged using our process before and after thermal cycling; 𝐼 𝑓 0 = 10 A surface. This is an improvement that should result in smaller surface current, and therefore reduced resistive voltage drop, in the top-side metallisation.

As a result of it, the potential of the source pad may be expected to be more homogenous and the current more evenly distributed over the cells, thus limiting the risk of hot-spot. This could result in an improved robustness, in particular to short-circuit tests.

Method

Transistors were tested in current limiting mode using the set-up shown in Fig. 9: a high-voltage power supply was applied across the Device Under Test (DUT), which was then turn on until failure. A circuit breaker was used to disconnect the power supply after failure, when the current reaches a threshold value 𝐼 ̂ and prevent the DUT from exploding.

The current through the DUT was measured using a coaxial shunt (400 MHz, 𝑅 𝑠ℎ𝑢𝑛𝑡 = 10 mΩ, from T&M), the voltage across it was measured using a PHV1000-RO passive voltage probe (400 MHz, 1:100, from PMK), the gate current was measured with a CT2 current probe (200 MHz, 1mV/mA, from Tektronix) and all was monitored using a HDO8108 oscilloscope (12 bits, 1 GHz, 2.5 GS/s, from Teledyne-LeCroy).

Bare dies SiC MOSFETs with reference CPM2-1200-0025b (rating: 1200 V -98 A -25 mΩ, with die size 6.44 mm × 4.04 mm), from Cree were used.

So as to be able to easily perform an optical study of the dies after failure, a custom set-up able to mimic the properties of our process without requiring lamination was designed. Fig. 10 shows a crosssectional view of the set-up. A stack composed of: -a bottom PCBon which the die was soldered; -a polyimide mask; -pieces of foam, positioned by a single layer of prepreg (which shall not be cured); -a top PCB; was placed between two plates of aluminium (required due to the high flexibility of PCB) that were maintained by four bolts and springs. The set-up acted as a press-pack package: the springs applied a pressure of about 3 MPa (close to that otherwise applied during the curing of the prepreg) on the foam and maintained it in contact with the die.

Three prototypes were tested using this set-up whereas a fourth had been fully embedded, laminated.

Results

Measured waveforms

The gate currents and voltages measured on our samples during a current-limiting short-circuit are given in Fig. 11. Gate leakage current appeared a few µs after the beginning of the test resulting, in conjunction with the 51 Ω-gate resistance, in a decrease in the gate voltage. This current migh denote a gate oxide failure [START_REF] Boige | Gate leakage-current analysis and modelling of planar and trench power SiC MOSFET devices in extreme short-circuit operation[END_REF]. The shape of the drain current in a representative sample (Fig. 11) was as expected. At first, the rate of the current increase is limited by the test voltage and the loop inductance; subsequent evolutions of the saturation current can be explained by the temperature dependences of the threshold voltage and majority carrier mobility in the channel [START_REF] Castellazzi | Short-circuit tests on SiC power MOSFETs[END_REF]. From about 3.5 µs after the start of the test, the saturation current starts decreasing due to the drop in majority carrier mobility, later accentuated by the drop in gate voltage due to the gate leakage current.

The peak saturation current was 430 A (i.e. 1.6 kA/cm²), that is 4.4 times the nominal value. This ratio is comparable to those reported in the literature for similar devices [START_REF] Boige | Gate leakage-current analysis and modelling of planar and trench power SiC MOSFET devices in extreme short-circuit operation[END_REF][START_REF] Chen | Robustness in short-circuit Mode of SiC MOSFETs[END_REF][START_REF] Wang | Temperature-Dependent Short-Circuit Capability of Silicon Carbide Power MOSFETs[END_REF][START_REF] Sun | Comparison and analysis of short circuit capability of 1200V single-chip SiC MOSFET and Si IGBT[END_REF][START_REF] Awwad | Short-circuit evaluation and overcurrent protection for SiC power MOSFETs[END_REF]. The drain current, gate voltage, drain-source voltage and gate current are highly similar for all devices, the difference being the time to failure, which varies from 9.3 µs to 12 µs.

5.3.2

Dissipated energy leading to failure The dissipated energy until failure is defined as the amount of energy dissipated in a component until its failure as a short-circuit. The value of this parameter, normalised by the die area, was calculated for four prototypes and is given in Fig. 12. In spite of the low representativeness of statistics calculated over such a low number of samples, this shows that the results are quite repeatable in so far as the measured values are within ±1.6 % of the mean critical energy for nonlaminated samples (i.e. using the press-pack like setup). Furthermore, Fig. 12 shows that using the presspack-like package shown in Fig. 10 gives comparable results as laminated devices whilst allowing a more thorough study of failed devices (cf. next section).

Test on dies from the same generation (2 nd ) of the same manufacturer (Cree), in a TO247 packaged have been reported in the literature. For instance, [START_REF] Boige | Gate leakage-current analysis and modelling of planar and trench power SiC MOSFET devices in extreme short-circuit operation[END_REF][START_REF] Chen | Robustness in short-circuit Mode of SiC MOSFETs[END_REF][START_REF] Wang | Temperature-Dependent Short-Circuit Capability of Silicon Carbide Power MOSFETs[END_REF][START_REF] Sun | Comparison and analysis of short circuit capability of 1200V single-chip SiC MOSFET and Si IGBT[END_REF][START_REF] Awwad | Short-circuit evaluation and overcurrent protection for SiC power MOSFETs[END_REF] investigated the dissipated energy leading to failure of a C2M0080120 (80 mΩ -1200 V) transistor. [START_REF] Chen | Robustness in short-circuit Mode of SiC MOSFETs[END_REF] found it to be 6.9 J/cm² (at 𝑉 𝑑𝑠 = 600 V, 𝑅 𝑔 = 47 Ω, 𝑉 𝑑𝑟𝑖𝑣𝑒𝑟 = 18 V), [START_REF] Boige | Gate leakage-current analysis and modelling of planar and trench power SiC MOSFET devices in extreme short-circuit operation[END_REF] reported a value only 5 % greater (at 𝑉 𝑑𝑠 = 600 V, 𝑅 𝑔 = 47 Ω, 𝑉 𝑑𝑟𝑖𝑣𝑒𝑟 = 21 V) and analogous results were reported in other papers.

These values are close to those found in the case of dies embedded using the proposed process, suggesting that using a contact made of pressed metal foam or bond wires contributes similarly to a die current limiting robustness.

5.3.3

Study of failed transistors After failure, all sampled had their drains, sources and gates short-circuited. An optical inspection of the dies showed that they were all burned (cf. Fig. 13) and fractured. The passivation of the dies was greatly distorted, showing undulations with characteristic length in the order of a hundred of µmcomparable observations were made in [START_REF] Eni | Short-Circuit Degradation of 10-kV 10-A SiC MOSFET[END_REF]. In some spots, the passivation had even been ejected away from the die (Fig. 13).

Simulations [START_REF] Wang | Temperature-Dependent Short-Circuit Capability of Silicon Carbide Power MOSFETs[END_REF][START_REF] März | Explaining the short-circuit capability of SiC MOSFETs by using a simple thermal transmission-line model[END_REF][START_REF] Romano | MOSFETs, A Comprehensive Study of Short-Circuit Ruggedness of Silicon Carbide Power[END_REF][START_REF] Sadik | Short-Circuit Protection Circuits for Silicon-Carbide Power Transistors[END_REF] have shown that during a short-circuit test, the temperature of the surface of the die can rise well above the melting point of aluminium (660 °C). The optical study of our samples showed that the top-side metallisation layers of all dies had melted (Fig. 14). The cells clearly stand Fig. 12. Energy density leading to failure of CPM2-1200-0025b dies. out from the surface of a new die but cannot be distinguished in a failed one. Small waves, demonstration of a flow of liquid aluminium could be spotted, mostly in the regions of the metallisation that were not in contact with the foam. This flow of liquid metal could result, not only from the pressure applied on the die, but also from the Lorentz force due to the interaction between the magnetic field created by the high current density and the electrical charges themselves.

From the moment the component failed and during the time it took the circuit breaker to open the circuit, the current rises rapidly, at a rate mainly limited by the loop inductance. In our set-up, the peak current reached about 4.2 kA and lasted 3.5 µs. There is therefore no way of telling to what extent the damages observed correlates to the device failure or were a consequence of this current spike.

Conclusion on curent limitation mode tests

These tests show that dies packaged using the proposed process or conventional techniques have similar current limitation mode robustness, which may be explained by the fact that the weakest element of the assembly is, in this test, not the connection but rather the die itself. Still, this shows that the proposed connection technique does not degrade this aspect of the robustness of the die, nor does it seem to increase its dispersion.

Secondly, these tests suggest that our press-pack like set-up (Fig. 10) mimics quite accurately the performances of the complete process.

6

Short-circuit Aging

Method

The same test bench as in the previous section was used to submit prototypes to repetitive, nondestructive, Short-Circuit (SC) tests. The bus voltage was set to 600 V, the gate voltage was -5/+18 V and the gate resistor was 51 Ω. Two devices were tested in the press-pack-like mechanical set-up shown in Fig. 10 and aged. One was submitted to SCs long enough for a gate leakage current to appear whereas the other was submitted to shorter SCs. Every few shortcircuits, the on-state resistance of the DUT, including the die, foam, contacts and solder resistances, was measured. The short-circuit repetition period was 3 s.

Results -4.5 µs-long SCs

A prototype was submitted to 4.5 µs-long repetitive short-circuits. The dissipated energy was about 890 mJ (that is 3.4 J/cm²), per SC, i.e. 40 % of the energy leading to failure. Fig. 15 shows that during the first 140 SC, the saturation current remained quite constant (the decrease was only 1 %); so did the gate voltage, proof that the leakage current was negligible (≪ 1 mA). During subsequent SC, however, a DC gate leakage current appeared, reaching and stabilizing itself around 80 mA after 400 SC. Similar results were found on C2M0080120, which are TO247-packaged MOSFETs from the same generation of the same manufacturer [START_REF] Chen | Studies of SiC power devices potential in power electronics for avionic applications[END_REF] as ours. The conjunction of the gate voltage reduction, a shift of the threshold voltage, and an increase in the on-state resistance of the component (due to the restructuration of the top-side metallisation layer) might explain the drop in saturation current [START_REF] Pietranico | Study of ageing of the metallization layer of power semiconductor devices[END_REF]. An optical study of the device after the experiment showed that the top-side metallisation had undergone severe reconstruction, the surface having lost its shine and having become rough. However, the on-state resistance of the DUT remained constant throughout the entire experiment; evidence that using a contact disseminated over the entire surface of the die is beneficial in term in metallisation layer aging.

Results -3 µs-long SCs

A sample was submitted to 3 µm-long SC, i.e. short enough so that no gate leakage current appeared. The dissipated energy was about 530 mJ per SC, i.e. 2 J/cm² or 25 % of the energy leading to failure.

After 10 kSC, the decrease of saturation current was negligible (-2 %). Furthermore all changes in onstate resistance could be correlated with ambient temperature variations or explained by the lack of repeatability of the connection of the device to the test-bench.

A TO247-packaged component from the same generation of the same manufacturer as ours was aged in [START_REF] Chen | Robustness in short-circuit Mode of SiC MOSFETs[END_REF][START_REF] Chen | Studies of SiC power devices potential in power electronics for avionic applications[END_REF][START_REF] Othman | Robustness of 1.2 kV SiC MOSFET devices[END_REF]. The test conditions were alike: same bus voltage, same SC duration, comparable ratio between the dissipated energy and the energy leading to failure. It was reported that the resistance increased quite faster: +3.7 % after 3.000 SC.

Conclusion on short-circuit aging

Notwithstanding their low statistical significance, these tests show that the reliability of SiC-MOSFETs packaged using the proposed process is comparable to that of wire-bonded chips. It thus appears that the proposed connection method does not degrade the performances of the die.

Discussion

Repeatability

We showed in [START_REF] Pascal | PCB-Embedding of Power Dies using Pressed Metal Foam[END_REF] that the process appeared to be quite repeatable since 54 of our 59 embedded diodes had resistances between 3 and 5 mΩ, with a standard deviation of 470 µΩ. Yet, this study shows a great variability among the reliability of the samples.

This apparent paradox might be due to the fact that the resistance due to the packaging is negligible vs. the resistance of the die itself whereas the reliability of the component isat least for the high-current pulse and thermal cycling tests -primarily defined by the packaging.

Furthermore, the tests were performed on few prototypes and should be conducted on larger sets of samples in order to gain statistical significance.

Hypotheses could explain the high dispersion of the test results. Although most of the steps of the process are simple and mastered, to this point, samples have been manufactured using unautomated laboratory equipment; the repeatability of the process might therefore not be optimal. Additionally, foam is a random medium, which leads to a random distribution of contacts over the die metallization, different from one prototype to another. One could expect pores to be small enough compared to the die area for the contact to be homogenized, but this is yet to be verified.

A more extended study is yet to be conducted so as to improve and assess the process reproducibility. It will then become possible to more thoroughly evaluate the process reliability.

Reliability indicators for PCB-embedding processes

The structure of assemblies with PCB-embedded dies is fundamentally different from that of standard power modules: no bond-wire is used, not only laminated but also electrodeposited copper layers are used, DBC are typically replaced by organic substrate (potentially anisotropic), the assemblies are more symmetrical: dies can be flipped and the top-side connections are similar to the bottom-side ones. The failure mechanisms of such assemblies might therefore differ from those of standard modules. Without diminishing the relevance of common, standardised, aging test, these structural changes might call for the development of new reliabilityassessing-methods.

For instance, aging by current overloads, as in section 3 of this paper, might be a relevant performance indicator.

On the opposite, a 5 % increase of the resistance is a common failure criterion for wire bonded power modules. It is based on the fact that this increase induces a 5 % conduction loss increase and that it is an evidence that most bonding wires are already lifted-off (or on the verge to be so) and that the module is close to a catastrophic failure. Then, this criterion may have limited relevance in the case of PCBembedded electronics, which is characterised by different failure mechanisms.

Conclusion

A PCB-embedding process using pressed metal foam to connect the top-side of PCB-embedded power dies has been considered. Results of an experimental DOI: https://doi.org/10.1016/j.microrel.2018.06.064 9/10 study of the reliability and robustness of this process have been presented: embedded dies were submitted to passive thermal cycling, current surges, destructive short-circuit tests, or repetitive short-circuit tests. Although the reliability and robustness has been shown to vary greatly from one sample to the other, some performed as well as state-of-the-art DBCs and wire-bonded packages. Finally, the development of new reliability-assessment-methods, suited to PCBembedding processes, has been discussed.

Fig. 1 .

 1 Fig. 1. Embedding of a MOSFET using pressed metal foam. (a): the die is soldered on the bottom PCB; (b): prepreg layers, a polyimide mask & pieces of metal foam (one for each top-side pad) are positioned; (c): the stack is laminated, the epoxy resin polymerizes and floods the foam.

Fig. 4 .

 4 Fig. 4. Cross-sectional view of a contact between the foam and the die.

Fig. 2 .

 2 Fig. 2. Partial cross-sectional view of an embedded diode. The die is 250 µm-thick and 6.5 mm-wide.

Fig. 3 .

 3 Fig. 3. Photography of the foam before lamination.

Fig. 5 .

 5 Fig. 5. Current and dissipated power during a current surge.

Fig. 6 .

 6 Fig. 6. Kelvin-connection of an embedded diode to the curve tracer: current source and voltage sensing, connected on the right-and left-hand side in the schematic, respectively.

Fig. 7 .

 7 Fig. 7. Inset: measured characteristic (blue) and model (red) of a diode, for a given current 𝐼 𝑓 0 . Main plot: evolution of the resistance 𝑅 𝑠 and voltage source 𝑉 𝑓 0 modeling a diode packaged using our process until failureaging by current surges. 𝐼 𝑓 0 = 20 A.

Fig. 10 .

 10 Fig. 10. Schematic of the press-pack-like mechanical set-up used for the tests.

Fig. 9 .

 9 Fig. 9. Schematic of the set-up used in current-limiting tests.

Fig. 13 .

 13 Fig. 13. Photography of a die before and after test.

Fig. 11 .

 11 Fig. 11. Waveforms acquired during the tests on a sample: drain current and cumulative dissipated energy (top); gate to source voltage and gate current (bottom).

Fig. 15 .

 15 Fig. 15. Peak saturation current (blue) and on-state gate to source voltage (red) vs. number of short-circuits Fig. 14. Melted aluminium above the passivation protecting a gate finger (the dendrite of aluminium is roughly 25 µm-high; left image) and below the piece of foam (right image).

Acknowledgements

This work was supported by a grant overseen by the French National Research Agency (ANR-15-CE05-0010).