Grégoire Harichaux

Proteomic characterization and cross species comparison of mammalian seminal plasma

UNLABELLED Seminal plasma contains a large protein component which has been implicated in the function, transit and survival of spermatozoa within the female reproductive tract. However, the identity of the majority of these proteins remains unknown and a direct comparison between the major domestic mammalian species has yet to be made. As such, the present study characterized and compared the seminal plasma proteomes of cattle, horse, sheep, pig, goat, camel and alpaca. GeLC-MS/MS and shotgun proteomic analysis by 2D-LC-MS/MS identified a total of 302 proteins in the seminal plasma of the chosen mammalian species. Nucleobindin 1 and RSVP14, a member of the BSP (binder of sperm protein) family, were identified in all species. Beta nerve growth factor (bNGF), previously identified as an ovulation inducing factor in alpacas and llamas, was identified in this study in alpaca and camel (induced ovulators), cattle, sheep and horse (spontaneous ovulators) seminal plasma. These findings indicate that while the mammalian species studied have common ancestry as ungulates, their seminal plasma is divergent in protein composition, which may explain variation in reproductive capacity and function. The identification of major specific proteins within seminal plasma facilitates future investigation of the role of each protein in mammalian reproduction. BIOLOGICAL SIGNIFICANCE This proteomic study is the first study to compare the protein composition of seminal plasma from seven mammalian species including two camelid species. Beta nerve growth factor, previously described as the ovulation inducing factor in camelids is shown to be the major protein in alpaca and camel seminal plasma and also present in small amounts in bull, ram, and horse seminal plasma.

Quantitative proteomics and bioinformatic analysis provide new insight into protein function during avian eggshell biomineralization

UNLABELLED Gallus gallus eggshell is a bioceramic composed of 95% calcium carbonate in calcitic form and 3.5% extracellular organic matrix. The calcification process occurs in the uterine fluid where biomineralization follows a temporal sequence corresponding to the initiation, growth and termination stages of crystal growth. Eggshell texture and its ultrastructure are regulated by organic matrix proteins, which control mineralization process and influence the eggshell biomechanical properties. We performed proteomic qualitative analyses and identified 308 uterine fluid proteins. Quantitative analysis showed differential abundances at the three stages of shell biomineralization for 64 of them. Cluster analysis revealed a first group of proteins related to mineralization and mainly present at the onset of calcification including OVOT, OVAL, OC-17, and two novel calcium binding proteins (EDIL3, MFGE8). A second group of proteins mainly present at the initiation and termination of shell formation was potentially involved in the regulation of the activity of the uterine fluid proteins (e.g. molecular chaperones, folding proteins, proteases and protease inhibitors). OCX21, a protein highly concentrated in the fluid and the shell, belongs to this group. A third group equally represented at all stages of shell mineralization corresponded to antibacterial proteins that could protect the forming egg against microbial invasion. BIOLOGICAL SIGNIFICANCE The calcitic avian eggshell protects the developing embryo and, moreover, ensures that the nutritious table egg remains free of pathogens. The eggshell is formed by nucleation upon a fibrous scaffold (the eggshell membranes) followed by an interaction between the growing mineral crystals and the shell organic matrix. This interaction leads to a highly ordered shell microstructure and texture which contribute to its exceptional mechanical properties. Shell mineralization occurs in three distinct phases of calcification (initiation, growth and termination), which are associated with distinct populations of matrix proteins that are secreted into the acellular uterine fluid as modulators of the process. The recent development of high-throughput methods has led to the identification of many proteins in the shell, but little is known concerning their role in shell formation. In order to determine precisely the importance of particular proteins relative to eggshell mineralization, this project used qualitative and quantitative proteomics of the uterine fluid constituents, coupled with bioinformatic analysis, to predict the functional role of proteins secreted at each of the three main stages of shell calcification. Besides its relevance to food production and to hen reproduction, eggshell calcification is furthermore a relevant model for studying calcium carbonate biomineralization on a two-dimensional membrane support. Better understanding of this process will provide insight into the fabrication of ceramics at ambient pressure and temperature.

Ram seminal plasma proteome and its impact on liquid preservation of spermatozoa

UNLABELLED Seminal plasma is composed of secretions from the epididymis and the accessory sex glands and plays a critical role in the fertilising ability of spermatozoa. In rams, analysis of seminal plasma by GeLC-MS/MS has allowed the identification of more than 700 proteins, including a high abundance of Binder of Sperm family proteins (BSP1, BSP5, SPADH1, SPADH2), the spermadhesin family (bodhesin2), lactoferrin and newly identified proteins like UPF0762 (C6orf58 gene). When spermatogenesis was stopped by scrotal insulation, changes in the proteome profile revealed the sperm origin of 40 seminal proteins, such as glycolysis pathway enzymes, the chaperonin containing TCP1 (CCT) complex and the 26S proteasome complex. Sperm mobility after liquid preservation (24h in milk at 15°C) is male dependent and can be correlated to differences in the seminal plasma proteome, detected by spectral counting. The negative association of zinc alpha-2 glycoprotein (ZAG) with semen preservation was confirmed by the use of recombinant human ZAG, which induced an increase in mobility of fresh sperm, but then decreased sperm mobility after 24h of incubation. Several sperm membrane proteins interacting with the cytoskeleton, glycolysis enzymes and sperm-associated proteins involved in capacitation correlated with better liquid storage and can be considered as seminal biomarkers of sperm preservation. BIOLOGICAL SIGNIFICANCE Extensive analysis of the ram seminal plasma proteome reveals a complex and diverse protein composition. This composition varies between males with different sperm preservation abilities. Several proteins were shown to originate from the spermatozoa and positively correlate with sperm liquid preservation, indicating that these proteins can be traced as sperm biomarkers within the seminal plasma. The zinc alpha-2 glycoprotein (ZAG) was found to have a biphasic effect on sperm mobility, with a short-term stimulation followed by a long-term exhaustion of sperm mobility after a 24h preservation period.

The Highly Prolific Phenotype of Lacaune Sheep Is Associated with an Ectopic Expression of the B4GALNT2 Gene within the Ovary

Prolific sheep have proven to be a valuable model to identify genes and mutations implicated in female fertility. In the Lacaune sheep breed, large variation in litter size is genetically determined by the segregation of a fecundity major gene influencing ovulation rate, named FecL and its prolific allele FecLL. Our previous work localized FecL on sheep chromosome 11 within a locus of 1.1 Mb encompassing 20 genes. With the aim to identify the FecL gene, we developed a high throughput sequencing strategy of long-range PCR fragments spanning the locus of FecLL carrier and non-carrier ewes. Resulting informative markers defined a new 194.6 kb minimal interval. The reduced FecL locus contained only two genes, insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) and beta-1,4-N-acetyl-galactosaminyl transferase 2 (B4GALNT2), and we identified two SNP in complete linkage disequilibrium with FecLL. B4GALNT2 appeared as the best positional and expressional candidate for FecL, since it showed an ectopic expression in the ovarian follicles of FecLL/FecLL ewes at mRNA and protein levels. In FecLL carrier ewes only, B4GALNT2 transferase activity was localized in granulosa cells and specifically glycosylated proteins were detected in granulosa cell extracts and follicular fluids. The identification of these glycoproteins by mass spectrometry revealed at least 10 proteins, including inhibin alpha and betaA subunits, as potential targets of B4GALNT2 activity. Specific ovarian protein glycosylation by B4GALNT2 is proposed as a new mechanism of ovulation rate regulation in sheep, and could contribute to open new fields of investigation to understand female infertility pathogenesis.

Proteomes of the Female Genital Tract During the Oestrous Cycle

The female genital tract includes several anatomical regions whose luminal fluids successively interact with gametes and embryos and are involved in the fertilisation and development processes. The luminal fluids from the inner cervix, the uterus and the oviduct were collected along the oestrous cycle at oestrus (Day 0 of the cycle) and during the luteal phase (Day 10) from adult cyclic ewes. The proteomes were assessed by GeLC-MS/MS and quantified by spectral counting. A set of 940 proteins were identified including 291 proteins differentially present along the cycle in one or several regions. The global analysis of the fluid proteomes revealed a general pattern of endocrine regulation of the tract, with the cervix and the oviduct showing an increased differential proteins abundance mainly at oestrus while the uterus showed an increased abundance mainly during the luteal phase. The proteins more abundant at oestrus included several families such as the heat shock proteins (HSP), the mucins, the complement cascade proteins and several redox enzymes. Other proteins known for their interaction with gametes such as oviductin (OVGP), osteopontin, HSPA8, and the spermadhesin AWN were also overexpressed at oestrus. The proteins more abundant during the luteal phase were associated with the immune system such as ceruloplasmin, lactoferrin, DMBT1, or PIGR, and also with tissue remodeling such as galectin 3 binding protein, alkaline phosphatase, CD9, or fibulin. Several proteins differentially abundant between estrus and the luteal phase, such as myosin 9 and fibronectin, were also validated by immunohistochemistry. The potential roles in sperm transit and uterine receptivity of the proteins differentially regulated along the cycle in the female genital tract are discussed.

Quantitative proteomics provides new insights into chicken eggshell matrix protein functions during the primary events of mineralisation and the active calcification phase.

Eggshell is a bioceramic composed of 95% calcium carbonate mineral and 3.5% organic matrix. Its structural organisation is controlled by its organic matrix. We have used quantitative proteomics to study four key stages of shell mineralisation: 1) widespread deposition of amorphous calcium carbonate (ACC), 2) ACC transformation into crystalline calcite aggregates, 3) formation of larger calcite crystal units and 4) development of a columnar structure with preferential calcite crystal orientation. This approach explored the distribution of 216 shell matrix proteins found at the four stages. Variations in abundance according to these calcification events were observed for 175 proteins. A putative function related to the mineralisation process was predicted by bioinformatics for 77 of them and was further characterised. We confirmed the important role of lysozyme, ovotransferrin, ovocleidin-17 and ovocleidin-116 for shell calcification process, characterised major calcium binding proteins (EDIL3, ALB, MFGE8, NUCB2), and described novel proteoglycans core proteins (GPC4, HAPLN3). We suggest that OVAL and OC-17 play a role in the stabilisation of ACC. Finally, we report proteins involved in the regulation of proteins driving the mineralisation. They correspond to numerous molecular chaperones including CLU, PPIB and OCX21, protease and protease inhibitors including OVM and CST3, and regulators of phosphorylation.

Proteomic analysis of egg white heparin-binding proteins: towards the identification of natural antibacterial molecules.

The chicken egg resists most environmental microbes suggesting that it potentially contains efficient antimicrobial molecules. Considering that some heparin-binding proteins in mammals are antibacterial, we investigated the presence and the antimicrobial activity of heparin-binding proteins from chicken egg white. Mass spectrometry analysis of the proteins recovered after heparin-affinity chromatography, revealed 20 proteins, including known antimicrobial proteins (avidin, lysozyme, TENP, ovalbumin-related protein X and avian bêta-defensin 11). The antibacterial activity of three new egg candidates (vitelline membrane outer layer protein 1, beta-microseminoprotein-like (LOC101750704) and pleiotrophin) was demonstrated against Listeria monocytogenes and/or Salmonella enterica Enteritidis. We showed that all these molecules share the property to inhibit bacterial growth through their heparin-binding domains. However, vitelline membrane outer layer 1 has additional specific structural features that can contribute to its antimicrobial potential. Moreover, we identified potential supplementary effectors of innate immunity including mucin 5B, E-selectin ligand 1, whey acidic protein 3, peptidyl prolyl isomerase B and retinoic acid receptor responder protein 2. These data support the concept of using heparin affinity combined to mass spectrometry to obtain an overview of the various effectors of innate immunity composing biological milieus, and to identify novel antimicrobial candidates of interest in the race for alternatives to antibiotics.

Regulation of the bovine oviductal fluid proteome

Our objective was to investigate the regulation of the proteome in the bovine oviductal fluid according to the stage of the oestrous cycle, to the side relative to ovulation and to local concentrations of steroid hormones. Luminal fluid samples from both oviducts were collected at four stages of the oestrous cycle: pre-ovulatory (Pre-ov), post-ovulatory (Post-ov), and mid- and late luteal phases from adult cyclic cows (18-25 cows/stage). The proteomes were assessed by nanoLC-MS/MS and quantified by label-free method. Totally, 482 proteins were identified including a limited number of proteins specific to one stage or one side. Proportions of differentially abundant proteins fluctuated from 10 to 24% between sides at one stage and from 4 to 20% among stages in a given side of ovulation. In oviductal fluids ipsilateral to ovulation, Annexin A1 was the most abundant protein at Pre-ov compared with Post-ov while numerous heat shock proteins were more abundant at Post-ov compared with Pre-ov. Among differentially abundant proteins, seven tended to be correlated with intra-oviductal concentrations of progesterone. A wide range of biological processes was evidenced for differentially abundant proteins, of which metabolic and cellular processes were predominant. This work identifies numerous new candidate proteins potentially interacting with the oocyte, spermatozoa and embryo to modulate fertilization and early embryo development.

The Family Secrets of Avian Egg-Specific Ovalbumin and Its Related Proteins Y and X

ABSTRACT The ovalbumin gene family in Gallus gallus is composed of three homologous genes located within a 46 kb locus on chromosome 2: ovalbumin, ovalbumin-related protein Y (OVAY), and ovalbumin-related protein X (OVAX) genes. The expression of these genes in hen oviduct is under estrogen control, but their relative hormonal responsiveness and subsequent protein concentration in egg, is distinctive. Interestingly, all three proteins lack the classical signal peptide for secretion. Ovalbumin, OVAX, and OVAY belong to the serine protease inhibitor (serpin) family whose members share a common tertiary structure. Ovalbumin and OVAX are one of the few members of this family that do not express any protease inhibition activity whereas OVAY has been predicted to be inhibitory, by comparison with the consensus sequence for inhibitory serpins. In contrast to ovalbumin and OVAY, OVAX interacts with heparin, a negatively charged glycosaminoglycan, via a positively charged domain exposed at the surface of the molecule. Ovalbumin is the major egg white protein and might be a source of amino acids for the developing embryo. The physiological function of OVAY is not known, but recent data have revealed a possible role of this protein in early embryonic development. Considering the antibacterial activities of OVAX, this protein might play a role in egg defense. This review sheds light on the expression, biochemistry, and structural specificities of these three highly similar paralogs. It gives new clues in favor of diverging functions, which are likely to have arisen by duplication events from a common ancestral gene.

Data for chicken semen proteome and label free quantitative analyses displaying sperm quality biomarkers

Understanding of biology of the avian male gamete is essential to improve the conservation of genetic resources and performances in farming. In this study, the semen proteome of the main domestic avian species (Gallus gallus) and evaluation of the molecular phenotype related to sperm quality were investigated using GeLC–MS/MS approach and label-free quantitative proteomic based on Spectral Counting (SC) and extracted ion chromatograms (XIC) methods. Here we describe in details the peptide/protein inventory of chicken ejaculated spermatozoa (SPZ) and seminal plasma (SP). We also show differential analyses of chicken semen (SPZ and corresponding SP) from 11 males demonstrating different levels of fertilizing capacity and sperm motility. The interpretation and description of these data can be found in a research article published by Labas and colleagues in the Journal of Proteomics in 2014 [1]. This is a new resource for exploring the molecular mechanisms involved in fertilizing capacity and to reveal new sets of fertility biomarkers.