Andrologia. 2019;51:e13305. wileyonlinelibrary.com/journal/and
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1 of 11 https://doi.org/10.1111/and.13305© 2019 Blackwell Verlag GmbH
1 | INTRODUCTION
Fusion between sperm and oocyte is mediated by multiple molecules in both gametes involving cell‐to‐cell and cell‐to‐matrix interactions. The superfamily of cell adhesion receptors is called integrins, and they primarily recognise extracellular matrix ligands on the oocyte surface. Disintegrins are thought to be integrin ligands, perturb‐ ing integrin‐mediated adhesion on the sperm cell surface (Gould, Koukoulis, & Virtanen, 1990; Sutovsky, 2018; Wright & Bianchi, 2016). Integrins serve as structural receptors enabling cell–cell and cell–matrix interaction, as well as signalling receptors that regulate Ca2+ ions, pH and inositol turnover, and protein phosphorylation
in the oocyte (McNamee, Liley, & Ingber, 1996; Schwartz, 1993). Integrins are heterodimeric proteins constituted by an extracellular and a cytoplasmic domains, formed by α and β subunits (Shrimali & Reddy, 2000). In several vertebrate cells, 18 different longer α‐sub‐ units (α1–11, αv, αllb, αE, αL, αM, αX, αD) and eight shorter β‐subunits (β1–8) have already been identified. Thus, α and β subunits are adhe‐ sion receptors and mediate cell attachment to the extracellular ma‐ trix and cell‐to‐cell interactions (Evans, 2001; Giancotti & Ruoslahti, 1999). Although integrin α and β subunits have been highly conserved during evolution, each integrin has a distinct distribution and binds to a special ligand (Huhtala, Heino, Casciari, de Luise, & Johnson, 2005). The β‐cytoplasmic tails of integrins are the target‐binding Received: 7 January 2019
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Revised: 4 March 2019|
Accepted: 25 March 2019DOI: 10.1111/and.13305
O R I G I N A L A R T I C L E
Expression dynamics of Integrin Subunit Beta 5 in bovine
gametes and embryos imply functions in male fertility and
early embryonic development
Ana Velho
1,2| Hongfeng Wang
1| Leslie Koenig
1| Kamilah E. Grant
1|
Erika S. Menezes
1| Abdullah Kaya
3| Arlindo Moura
2| Erdogan Memili
11Department of Animal and Dairy Sciences, Mississippi State University, Starkville, Mississippi
2Department of Animal Sciences, Federal University of Ceara, Fortaleza, Brazil 3Department of Reproduction and Artificial Insemination, Selcuk University, Konya, Turkey
Correspondence
Erdogan Memili, Department of Animal and Dairy Sciences, Mississippi State University, Starkville, MS.
Email: em149@ads.msstate.edu
Funding information
Mississippi Agricultural Forestry Experiment Station; Brazilian Research Council; Selcuk University; Improvement of Higher Education; Merial Veterinary Scholars Programme; College of Veterinary Medicine of Mississippi State University
Abstract
Integrins have been shown to act as signalling receptors, and they primarily recognise extracellular matrix ligands on the oocyte surface. However, their possible roles in oo‐ cyte activation and embryo development are not clearly understood. The objectives of this study were to evaluate expression of Integrin Subunit Beta 5 (ITGβ5) in bovine sperm, oocytes, and early embryos and to ascertain the evolutionary conservation of ITGβ5. To accomplish these objectives, we used western blotting to study expres‐ sion levels of ITGβ5 protein in sperm and RT‐qPCR to determine expression levels of ITGβ5 transcripts in oocytes and embryos. We have also used bioinformatic analy‐ sis to determine the evolutionary conservation of the ITGβ5 protein among various species. Western blotting showed that ITGβ5 protein was detectable in bull sperm. Moreover, results of RT‐qPCR showed that levels of ITGβ5 were significantly higher in the two‐cell embryos, followed by the 8–16‐cell embryos. However, no significant difference in expression levels were noted for the morula and blastocyst stages as compared to MII oocytes. Bioinformatic analysis revealed that ITGβ5 is conserved among various species. We conclude that expression of ITGβ5 in bovine gametes and embryos implies an important role in fertilisation and embryogenesis.
K E Y W O R D S
sites for many cytoskeletal and signalling proteins, some of which interact with specific α tails (Danen, 2006). Integrins mediate cell ad‐ hesion to vitronectin (Hynes, 1987), which can play a role in sperm– oocyte interaction (Fusi, Bernocchi, Ferrari, & Bronson, 1996).
Takada, Ye, and Simon (2007) suggested that phosphoryla‐ tion of the tyrosine in the NPxY/F motifs may be the mode of integrin interactions with other proteins at the cytoplasmic re‐ gion of the membrane. Integrins can mediate the downstream results of cell adhesion and are therefore prime targets for the development of therapeutic agents for treatment of many diseases (Humphries, 2000).
Integrin alpha4‐beta7 participates in adherence of lympho‐ cytes to fibronectin, a vascular cell adhesion molecule‐1 (VCAM‐1) and homotypic cell‐clustering lymphocyte (Ruegg et al., 1992). Both transcriptomic and proteomic approaches showed evidence that mouse oocytes express a group of integrins at their surface. These receptors recognise the amino acid tripeptide Arg‐Gly‐Asp (RGD) as a ligand recognition sequence (Ruoslahti & Pierschbacher, 1987), and the RGD‐containing peptides have been shown to in‐ hibit competitively sperm–oocyte adhesion, impairing sperm penetration (Bronson & Fusi, 1990). Bovine spermatozoa display integrin alpha 5 at their equatorial segment after acrosome re‐ action, and it participates in the process of spermatozoa–oocyte interaction in bovine species. Since integrin alpha 5 has been de‐ tected on oolemma and spermatozoa, it has been suggested an interaction between the endogenous fibronectin ligand and cor‐ responding receptors on both sperm cell and oolemma, initiating sperm–egg binding (Thys et al., 2009). Silva et al. (2015) detected ITGβ5 in the human testis and epididymal fluid. In addition, synon‐ ymous single nucleotide polymorphisms in ITGβ5 were associated with bull fertility (Feugang et al., 2009).
Although integrins have been shown to act as signalling recep‐ tors, their possible roles in male fertility, fertilisation, and embry‐ onic development are not clearly understood. This breach in the knowledge of the molecular mechanisms regulating these particu‐ lar proteins prevents full understanding of all crucial steps in early mammalian embryology development and advancement in the
assisted reproductive technologies. In this context, the objectives of the present study were to determine the expression of ITGβ5 in bovine sperm, oocytes, and early embryos and to ascertain the full spectrum of evolutionary diversification of ITGβ5.
2 | MATERIALS AND METHODS
2.1 | Determination of bull fertility
Frozen semen samples and bull fertility data from eight mature and progeny tested Holstein bulls that had satisfactory semen quality were provided by Alta Genetics. Sires were selected based on their fertility as previously described by Peddinti et al. (2008). Factors that influence fertility performance of sires (i.e., breeding event, en‐ vironmental factors and herd management factors) were adjusted using threshold models similar to described by Zwald, Weigel, Chang, Welper, and Clay (2004a); (Zwald, Weigel, Chang, Welper, & Clay, 2004b). Using Probit.F90 software (Chang, Gianola, Heringstad, & Klemetsdal, 2004), fertility prediction of each sire was calculated ac‐ cording to the average conception rates from more than 300 breed‐ ing outcomes along with their per cent deviation from the conception rates. The method to determine bull fertility used in our study is also similar to those from previous investigations about fertility biomark‐ ers in bulls, conducted by several authors in the last decades (de Oliveira et al., 2013; Killian, Chapman, & Rogowski, 1993; Moura, Chapman, & Killian, 2007; Peddinti et al., 2008; Velho et al., 2018). The calculation of fertility scores was based on the actual concep‐ tion rates confirmed by either veterinary palpation or ultrasound of cows inseminated with hundreds/thousands frozen‐thawed semen straws from each bull. Based on this calculation, per cent deviation of conception rates was used to categorise the fertility of bulls. For this marker discovery study, bulls were selected among the ones per‐ forming conception rates of two standard deviations above or below the average of the bull population in the database. Thus, bulls which had per cent difference of their conception rate above average were defined as high fertility (HF) with average of 3,174 breeding out‐ comes. Those bulls that had per cent difference of their conception rate below average were classified as low fertility (LF) having 1,662 breeding outcomes (Table 1).
2.2 | Determination of expression levels of ITGΒ5 in
bull sperm by Western Blotting
2.2.1 | Extraction and quantification of
sperm proteins
Sperm proteins were isolated according to established methods in our laboratory and as described by Escoffier et al. (2015), with modifications. Briefly, frozen semen from the eight mature Holstein bulls with different in vivo fertility were thawed in water bath at 37°C for 30 s. Spermatozoa were then pelleted by centrifugation (700 g, 4°C, 5 min) and washed twice with phosphate‐buffered sa‐ line (PBS) containing protease inhibitor cocktail (Roche Diagnostics
TA B L E 1 Fertility data of the Holstein bulls. The fertility score
of each bull was obtained using the Probit.F90 software, and it is expressed as the per cent deviation of its conception rates from the average conception rate of all bulls
Bull number ST DEV
Fertility score (% difference from average fertility)
1 1.71 3.7 2 1.22 2.6 3 1.16 2.5 4 −1.43 −3.1 5 −1.62 −3.5 6 −1.87 −4.0 7 −1.94 −4.2 8 −2.25 −4.9
Corporation) (700 g, 4°C, 5 min). Total sperm count was deter‐ mined using a hemacytometer. Approximately, 20 × 106 cells were resuspended in 100 µl of 1× Laemmli sample buffer (2% SDS, 10% glycerol, 0.0625 M Tris–HCl) without β‐mercaptoethanol, vortexed for 1 min, and heated at 98°C for 5 min. Samples were then cen‐ trifuged at 10,000 g at 4°C for 5 min. The supernatants contain‐ ing sperm membrane proteins were recovered and transferred to a new tube where 5% β‐mercaptoethanol was added. Then, the samples were heated one more time at 98°C for 5 min and frozen at −80°C until further use.
After thawing the samples, the protein concentration was deter‐ mined in triplicates using Quick Start™ Bradford Protein Assay Kit 2 Quick Start™ Kit (Bio‐Rad Laboratories). In addition, sperm proteins were precipitated with cold acetone at −20°C for 3 hr, followed by centrifugation (10,000 g, 4°C, 10 min). Supernatants were discarded, and pellets were resuspended in 30 µl of 1× Laemmli sample buffer (Bio‐Rad Laboratories) with 5% β‐mercaptoethanol, vortexed (10 s), boiled for 10 min, and stored at −80°C.
2.2.2 | Western blotting analysis of
membrane proteins
An aliquot of sperm membrane proteins (20 μg) was mixed and separated with a vertical polyacrylamide gel electrophoresis (4%– 20% SDS–PAGE; Mini‐Protean TGX™ gel, Bio‐Rad Laboratories). Protein bands were transferred from the gels to an Immobilon®‐P polyvinylidene difluoride (PVDF) membrane using HEP‐1 semidry electroblotting (Thermo Fisher Scientific) set at 46 mA for 2.5 hr. Binding sites were blocked with 5% bovine serum albumin (BSA) in PBS‐0.1% Tween 20 (PBS‐T) at room temperature under mild agitation for 1 hr, followed by incubation with primary antibod‐ ies against ITGβ5 (1:2,000; goat polyclonal IgG; sc‐5402; Santa Cruz Biotechnology) and tubulin beta 2C (TUBβ2C) (1:500, mouse monoclonal IgG; sc‐134230; Santa Cruz Biotechnology) with 1% of BSA in PBS‐T at room temperature for 1 hr. Tubulin beta was used as internal control as described previously by Ardon and Suarez (2013). Membranes were then washed three times with PBS‐T for 10 min each and incubated with secondary antibodies (1:10,000; rabbit anti‐goat IgG‐HRP; sc‐2768; Santa Cruz or Donkey anti‐ mouse IgG‐HRP; sc‐134230; Santa Cruz Biotechnology) and Precision Protein™ StrepTactin‐HRP Conjugate (1:10,000, Bio‐Rad Laboratories) with 1% of BSA in PBS‐T at room temperature for 1 hr. Membranes were washed three times with PBS‐T for 10 min each. Bands were detected using a chemiluminescence reagent (Clarity™ Western ECL Substrate, Bio‐Rad Laboratories, and Image Laboratory software (Bio‐Rad Laboratories) for 30 s. Analyses of ITGβ5 band intensities were performed using ImageJ software version 1.49 (National Institutes of Health). After the image was generated, bands were normalised and background‐reduced, and bands signal was quantified by the sum of the intensities measured in all the band pixels. Similar to described by Ardon and Suarez (2013), the relative quantity of ITGβ5 bands was normalised and calculated using TUBβ2C bands.
2.3 | In vitro maturation, fertilisation, and embryo
development
The procedures for in vitro maturation, fertilisation, and embryo de‐ velopment were performed as previously described (Kaya et al., 2017; Memili & First, 1999). Bovine cumulus–oocyte complexes were aspi‐ rated from antral follicles (2–8 mm in diameter) at local slaughterhouse and were then washed in TL (tyrode‐lactate)‐HEPES medium and matured in TCM‐199, 10% foetal calf serum (FCS), sodium pyruvate (0.2 mM), gentamycin (25 μg/ml), FSH (5 μg/ml, FSH‐P; Scherring‐ Plough Animal Health), and 1 μg/ml estradiol. Only oocytes with evenly granulated cytoplasm surrounded by multiple layers of com‐ pact cumulus cells were used in the experiments. Ten cumulus–oocyte complexes were matured per 50 μl drop of maturation medium under mineral oil at 39°C, 5% CO2 in a humidified atmosphere. Motile sperm were separated from cryopreserved sperm of a previously tested bull using percoll gradient and was diluted to 50 × 106 sperm cells/ ml to fertilise the matured oocytes as described previously (Parrish, Krogenaes, & Susko‐Parrish, 1995). Fertilisation drops were supple‐ mented with 2 µl of the diluted sperm, 2 µl of 5 µg/ml heparin, and 2 µl of PHE solution (20 µM penicillamine, 10 µM hypotaurine, 1 µM epinephrine) into the fertilisation drops. Following 16–18 hr co‐cul‐ ture of oocytes and sperm, cumulus cells were removed by vortexing the presumptive zygotes in a 1.5‐ml Eppendorf tube for 3 min. The cumulus‐free presumptive zygotes were washed three times in TL‐ HEPES. Twenty‐four hours post‐insemination (hpi), the embryos were mechanically stripped free, with a glass pipette, of cumulus cells and attached sperm, washed, and cultured in CR1‐aa medium (Rosenkrans, Zeng, MCNamara, Schoff, & First, 1993) under ambient conditions described above. Fertilisation time in the present study was consid‐ ered as 0 hr. Developmental data were evaluated for bovine two‐cell embryos, 8–16‐cell embryos, morulae, and blastocysts stage embryos.
2.4 | Real‐time PCR to determine levels of ITGβ5
transcripts in bovine oocytes and embryos
2.4.1 | Primer design
Primers were designed using Primer3Plus software (Untergasser et al., 2007) and ITGΒ5 mRNA reference sequence reported on NCBI (NM_174679). Primers were chosen based on the following criteria: number of base pairs, C‐G percentage, and melting temperature (Tm). Primers were tested using Primer Premier Software which looked at the potential for primer dimers, cross‐dimers, false priming, and for‐ mation of hairpins. Initially, two primers were tested. The first primer (181 ITGβ5) was 20‐base long and had Tm of 60°C, and the C‐G per‐ centage was 55%. The second chosen primer (230 ITGβ5) was 20‐ base long and had a Tm of 60°C, and the C‐G percentage was 45%.
Primer conditions were tested using cDNA from blastocysts and MII cells. Primers were set up with a temperature gradient from 56°C to 60°C at 1.5 MgCl2 and then at an optimal primer condition (181 ITGβ5: 1.25 MgCl2 at 58°C and 230 ITGβ5: 1.25 MgCl2 at 55.7°C) determined by previous tests. The concentration of cDNA blastocyst was 40 ng/
µl, and the concentration of cDNA MII was 55 ng/µl. Samples were placed in Eppendorf Master cycler with a PCR programme of 95°C for 3 min, 95°C for 1 min, 58°C for 1 min, 72°C for 1 min, and 72°C for 5 min. Temperature gradient was set up from 56°C to 60°C. Samples were then subjected to SDS–PAGE at 110V for 90 min. Results were visualised using a UV transilluminator (Bio‐Rad Laboratories).
2.4.2 | RNA extraction, quantification,
purification, and cDNA synthesis
Total RNA was isolated from bovine MII oocytes, two‐cell embryos, 8–16‐cell embryos, morulae, and blastocysts (Sagirkaya et al., 2006) using a RNeasy Micro Kit (Qiagen) according to the manufacturer's protocol. Quantity and purity of isolated RNA were determined using a NanoDrop spectrophotometer (NanoDrop Technologies). Quality of total isolated RNA was determined using the Agilent Bioanalyzer 2,100 RNA 6,000 picochip kit (Agilent Technologies). RNA from all groups was used for cDNA synthesis using SuperScript III Platinum Two Step qRT‐PCR kit according to the manufacturer's protocol. The samples were incubated at 25°C for 10 min, 42°C for 50 min, and 85°C for 5 min. Then 0.5 µl of Escherichia coli RNase H was added to each tube and incubated at 37°C for 20 min.
2.4.3 | Real‐Time PCR
Real‐time quantitative PCR was performed to assess transcript abundance of ITGΒ5 relative to the housekeeping gene GAPDH.
Quantitative assessment of RNA amplification was detected by SYBR® GreenER™ qPCR SuperMixes for iCycler (Invitrogen). A primer mix was prepared using 5 µl of the forward primer, 5 µl of the reverse primer, and 40 µl nuclease free water. The following kit components were combined in one tube for a total of 66 reactions: 495µl SYBR® GreenER™ qPCR SuperMix for iCycler®, 19.8µl primer mix, and 409.2 µl DEPC water. A 14‐µl aliquot of this mixture was placed into each well on the PCR plate and 1 µl cDNA (total volume of mixture: 15 µl) was added to all sample wells, while 1 µl of ddH2O was added to the negative control wells. The PCR plate was centri‐ fuged for 1 min at 1,000 g and then placed in the iCycler iQ Real‐Time PCR instrument. The RT‐qPCR cycling steps were as follows: 50°C for 2 min, 95°C for 8.5 min, 40 cycles of 10 s at 95°C, 40 cycles of 30 s at 60°C, 40 cycles of 30 s at 72°C, 95°C for 1 min, and 55°C for 1 min. A melting curve was established starting at 55°C with 0.5°C increases for 80 cycles of 10 s. Expression values were calculated using the relative standard curve method as described previously (Feugang et al., 2010).
2.5 | Statistical analysis
All experiments were carried out in triplicates, and expression values were calculated using the relative standard curve method. Standard curves were generated using 10‐fold serial dilutions for GAPDH and the target gene by measuring the cycle number at which ex‐ ponential amplification occurred. Results from the RT‐qPCRs were analysed using one‐way analysis of variance (ANOVA) by SAS 9.1.3
TA B L E 2 ITGB5 across species. Chromosome, gene ID, mRNA sequence, protein sequence, and amino acids (AA) lengths for 11 ITGB5
sequences are derived provided
Species Chromosome Gene ID mRNA Protein AA Other
Bovine [Bos Taurus] 1 282564 NM_174679.2 NP_777104 800 Human [Homo Sapiens] 3q21.2 3693 NM_002213.3 NP_002204 799 Mouse [Mus Musculus] 16 B3 16419 NM_010580.1 Q6PE70 799 Zebrafish [Danio rerio] 9 563556 NM_001082836.1 NP_001076305 823 Chicken [Gallus gallus] 7 395142 NM_204483.1 NP_989814 812 Rat [Rattus norvegicus] 11q22 257645 NM_147139.2 NP_671480 799 Chimpanzee [Pan troglodytes] 3 460646 XM_516706 XP_516706 899 Predicted Rhesus Monkey [Macaca mulatta] 2 715710 XM_001113909 XP_001113909 799 Predicted Frog
[Xenopus laevis] N/A 446423 NM_001093119 NP_001086588 802
Canine
[Canine lupus familiaris]
33 608977 XM_846152.1 XP_851245 819 Predicted
Opossum
[Didelphis virginiana]
(SAS Institute Inc.). The relative expression software tool (REST) was used to compare all samples. The mathematical model used by REST is based on the PCR efficiencies and the crossing point deviation between samples (Feugang et al., 2010).
2.6 | Bioinformatics analyses of the ITGβ5
2.6.1 | Comparative functional genomics of ITGβ5
Complete information regarding the chromosome, gene ID, mRNA sequence, protein sequence, and amino acid lengths for all 11 ITGβ5 sequences are summarised in Table 2. Note that chimpan‐ zee, rhesus monkey, canine, and opossum are all predictable se‐ quences. Opossum ITGβ5 sequence was predicted using N‐SCAN on the UCSC Genome Browser. When compared to the bovine ITGβ5 sequence, the predicted opossum sequence had two extra exons (#6 and #13) that showed very little conservation. Those exons were removed, and a new protein sequence was estab‐ lished using European Molecular Biology Open Software Suite (EMBOSS) Transeq.2.6.2 | Alignment of multiple amino acid
sequences of ITGβ5
The 17 ITGβ5 amino acid sequences from bull (Bos taurus), man (Homo sapiens), mouse (Mus musculus), baboon (Papio cynocephalus), monkey‐rhesus (Macaca mulata), dog (Canis lupus familiaris), sheep (Ovis aries), horse (Equus caballus), cat (Felis catus), gorilla (Gorilla gorilla), zebrafish (Danio rerio), guinea pig (Cavia porcellus), frog (Xenopus laevis), elephant (Loxodonta africana), turkey (Meleagris gal-lopavo), rooster (Gallus gallus), and pig (Sus scrofa domesticus) were
obtained from the Uniprot programme (http://www.unipr ot.org/). Subsequently, the alignment of these multiple ITGβ5 sequences was generated by the Clustal Omega software (http://www.ebi.ac.uk/ Tools/ msa/clust alo/), which is available online at the European Bioinformatics Institute (http://www.ebi.ac.uk/).
2.6.3 | Evaluation of per cent identity of amino
acid sequences of bovine ITGβ5 protein compared to
other species
The Clustal Omega was employed to determine the percentage of amino acid sequence identity of bovine ITGβ5 protein versus other species (man, mouse, baboon, rhesus monkey, dog, sheep, horse, cat, gorilla, zebrafish, guinea pig, frog, elephant, turkey, rooster, and pig).
2.6.4 | Dottup analysis of sequence similarities of
ITGβ5 among bovine, human, and mouse
Pairwise sequence comparisons of ITGβ5 protein sequences were done using an online dottup tool from the European Molecular Biology Open Software Suite (EMBOSS). Comparisons used were bovine versus human, bovine versus mouse, and human versus mouse.
2.6.5 | Phylogenetic trees of ITGΒ5
Phylogenetic trees with the 17 ITGβ5 amino acid sequences of man, mouse, baboon, rhesus monkey, dog, sheep, horse, cat, gorilla, ze‐ brafish, guinea pig, frog, elephant, turkey, rooster, and pig were ob‐ tained using MEGA 6 software.
F I G U R E 1 Expression levels of
ITGβ5 protein in bull sperm membrane. (a) Western blotting analysis of bulls (1–8) with varying fertility scores using antibodies against ITGB5 protein. (b) Relative bands intensities of ITGβ5 in bull sperm membrane (1–8) with differing fertility scores. Relative intensity was normalised and calculated using TUBβ2C bands. MW: molecular weight
2.6.6 | Conserved domains of ITGβ5
The ITGβ5 protein sequences were analysed using the Conserved Domain Database (CDD) from NCBI/BLAST (Basic Local Alignment Search Tool). Conserved domains from the multiple sequence align‐ ment were determined using in‐house software that is part of the MSAVIS package (Lindeman et al., 2007).
3 | RESULTS
3.1 | ITGβ5 protein is present in bull sperm
We performed western blotting analysis to evaluate the expression of ITGβ5 in sperm from bulls of different in vivo fertility. TUBβ2C ex‐ pression was used as loading controls. Our results showed that ITGβ5 protein is present in bull sperm. The levels of expression among bulls with different fertilities are depicted in Figure 1a. Relative intensi‐ ties of the ITGβ5 bands in bull sperm membrane ranged from 0.93 to 1.87 pixels among bulls with different fertilities (Figure 1b).
3.2 | Expression dynamics of ITGβ5 transcripts in
bovine oocytes and early embryos
Levels of ITGβ5 transcripts were determined in MII oocytes, two‐ cell embryos, 8–16‐cell embryos, morula, and blastocysts using RT‐qPCR (Figure 2a). The highest transcripts abundance of ITGΒ5 was detected in two‐cell embryos (p < 0.05), followed by 8–16‐cell embryos. No differences in relative levels of transcripts were noted for the morula and blastocyst stages as compared to the MII oocytes (p > 0.05). Quantity of ITGΒ5 transcript in MII oocytes was equiva‐ lent to that in 8–16‐cell embryos. (p > 0.05; Figure 2b).
3.3 | ITGβ5 evolutionary conservation
Using Clustal Omega we obtained the percentage of amino acid se‐ quence identity of bovine versus other species. The percentage of identity of bull versus sheep was 96.5%, bulls versus man was 90.55%, and bull versus mouse was 89.41%. Percentage of identity of bovine ITGβ5 protein compared to other species ranged from 96.5% for bull
FI G U R E 2 ITGβ5 transcripts in MII oocytes, two‐cell embryos, 8–16‐cell embryos, morula, and blastocysts. (a) Levels of transcripts and (b)
number of transcripts of ITGβ5 in MII oocytes, two‐cell embryos, 8–16‐cell embryos, morula and blastocysts. RT‐PCR was performed to assess transcript abundance of ITGβ5 relative to the housekeeping gene GAPDH. Transcripts in bovine oocytes and early embryos were statistically analysed using one‐way analysis of variance. Different letters correspond to significant differences among developmental stages (p < 0.05)
versus sheep to 68.69% for bull versus frog (Table 3). Dottup analy‐ sis of bovine, mouse, and human ITGβ5 protein sequences showed a significant structural conservation with few gaps between the three sequences (Figure 3). In addition, the phylogenetic tree revealed that ITGβ5 protein presents evolutionary conservation among the 17 spe‐ cies evaluated. The phylogenetic tree also showed a greater similarity among the amino acid sequence of ITGβ5 protein of bull and sheep than bull and the other species studied (Figure 4). The extraordinary degree of conservation of ITGΒ5 results in a very shallow tree indi‐ cating that this protein may serve a very important functional role across many species. The conserved domains were determined by analysing the 17 amino acid sequences of ITGβ5 protein in the NCBI/ BLAST Conserved Domain Database (CDD) database. The conserved domains that occurred in all species studied included Von Willebrand factor type A (vWFA) superfamily, integrin beta tail, and integrin beta cytoplasmic domain (Figure 5).
4 | DISCUSSION
Embryo biotechnologies are extremely important both for propa‐ gation of mammals including farm animals, endangered species, and humans around the world. Increasing numbers of embryos have been produced in vitro for production of offspring with higher genetic merit. Low male fertility, fertilisation, and developmental failures as well as abnormalities during pre‐and post‐implantation are currently hindering the continued success of embryonic bio‐ technologies such as IVF and somatic cell nuclear transfer (SCNT).
For this reason, there exists the urgent need to determine the gene products that improve successful fertilisation and embryonic developmental competency. A comprehensive understanding of bovine early developmental biology will enable determination of molecular networks in fertilisation and in developing embryos that are capable for viable development and improvement in multi‐cel‐ lular organisms (Moore & Hasler, 2017; Sirard, 2018; Wrenzycki, 2018).
Clarifying mechanisms of fertilisation and competence of embry‐ onic development are of great importance. Once the mechanisms of fertilisation and embryogenesis are known, they enable powerful new approaches to yield healthier and more efficient animals by producing developmentally competent sperm and embryos for improved animal production. In the present study, we evaluated the presence of ITGβ5 on sperm, determined ITGβ5 transcripts in oocytes, and early embryos, in an effort to identify molecular markers and networks responsible for fertility, and to identify conserved sequence, domains and motifs in ITGβ5 across mammalian species. Therefore, we showed, for the first time, that ITGβ5 is present in bull spermatozoa. In addition, we pro‐ vided evidence, for the first time, that the expression levels of ITGβ5 were significantly higher in the two‐cell embryos, followed by the 8–16‐cell embryos, but no significant difference in expression levels in the morula and blastocyst stages as compared to the MII oocytes. Furthermore, our bioinformatics study showed that ITGβ5 is con‐ served across several species and probably exists across many more.
Integrins have also been shown to mediate cellular adhesion to vitronectin and to influence on sperm–oocyte interaction (Fusi et al., 1996). In men, vitronectin has also been reported to be synthesised during spermatogenesis and subsequently released following capac‐ itation (Fusi et al., 1994). However, studies have shown that human α and β integrin receptors only appear following capacitation (Fusi et al., 1996). Our results showed that ITGβ5 is highly detectable in bull sperm and oocytes. This finding is in agreement with previous studies that ITGβ5 protein is present in ram seminal plasma (Rocha et al., 2015). Moreover, our findings are consistent with those pub‐ lished earlier by Finaz and Hammami‐Hamza (2000), which reported that ITGβ5 is involved in sperm–egg fusion and is located on the sur‐ face of both the mature human sperm and oocyte. The results of the present study also are in agreement with reports that integrin β1 protein is expressed on the surface of human sperm (Barraud‐ Lange et al., 2007; Feugang et al., 2009; Glander & Schaller, 1993; Reddy, Meherji, & Shahani, 1998). The significance of this binding is attributed to the adhesion/fusion process of sperm–oocyte during fertilisation, which stimulates a cascade of events. Feugang et al. (2009) reported a decrease in bovine oocyte's capability of being fertilised by sperm that were incubated with anti‐ITGβ5 antibodies, which suggested the presence of the ITGβ5 protein in the bovine sperm membrane. In the present study, using bioinformatics analy‐ ses, we showed that ITGβ5 contains vWFA domains which are con‐ served among various species and involved in cellular adhesion and protein–protein interaction.
The expression of integrins, tetraspanins, and ADAM families by both gametes has been implicated in sperm–egg interaction, a
TA B L E 3 Percentage of amino acid sequence identity of bovine
ITGβ5 protein versus 16 other species. The identity percentages were obtained using Clustal Omega software
Species
Percentage of identity of ITGβ5 protein of bull (Bos
taurus) versus other species
Sheep (Ovis aries) 96.50
Pig (Sus scrofa domesticus) 95.00
Horse (Equus caballus) 94.98
Baboon (Papio cynocephalus) 92.67
Elephant (Loxodonta africana) 92.02
Gorilla (Gorilla gorilla) 90.77
Cat (Felis catus) 90.75
Man (Homo sapiens) 90.55
Monkey‐rhesus (Macaca mulata) 90.30
Mouse (Mus musculus) 89.41
Guinea pig (Cavia porcellus) 88.53
Dog (Canis lupus familiaris) 86.04
Rooster (Gallus gallus) 76.54
Turkey (Meleagris gallopavo) 76.01
Zebrafish (Danio rerio) 70.62
complex molecular process and interaction leading to gamete fu‐ sion (Barraud‐Lange et al., 2007; Sutovsky, 2018; Wright & Bianchi, 2016). Thus, as large heterodimeric glycoproteins, integrins are cell
surface receptors for extracellular matrix that can participate in cell–cell adhesion (Whittaker & Hynes, 2002). Our results suggest that the expression of ITGβ5 in the sperm and oocyte membrane is
F I G U R E 3 Dottup analysis of ITGβ5. Sequence similarity among bovine, human, and mouse. A solid diagonal line indicates sequence
similarity, breaks in the line indicate low similarity, and a shift in the line indicates an insertion or deletion. The window size used was 10
Bovine vs. Human Human vs. Mouse Bovine vs. Mouse
Bovine ITGB5 Human ITGB5 Bovine ITGB5
F I G U R E 4 Phylogenetic tree of ITGβB5, evolutionary comparison across multiple species. Optimal tree configurations depict
diversification of ITGβ5. Branch lengths are drawn to scale to show common ancestry among species for ITGβ5 protein. The analysis involved 17 amino acid sequences. Evolutionary analyses were conducted in MEGA7
F I G U R E 5 Conserved domains in ITGB5 in 17 species. Conserved domains were found using NCBI's BLAST. Conserved domains were
important in the adhesion and fusion stages of these cells during the fertilisation process.
Fusion of male and female gametes initiates communication and triggers cellular cleavage (R. Bronson, Peresleni, Golightly, & Preissner, 2000) and such initial cleavage marks the first act of sign of cellular totipotency. Continuing development of the embryo re‐ sults in production of the pluripotent inner cellular mass and the trophectoderm (Mitalipov & Wolf, 2009). Thus, in a study carried out in mice by Miranda, Pericuesta, Ramirez, and Gutierrez‐Adan (2011), it was suggested that the expression of Nanog represents the onset of pluripotency and the decrease in prion protein gene (PRNP) via inhibition of ITGβ5 preventing expression of Nanog and thereby inhibiting pluripotency (Miranda et al., 2011). Thus, if toti‐ potency is ITGβ5 dependent, then its sperm‐borne low expression could prevent early embryonic development or its sustainability due to inability to cleave following initial fertilisation.
Integrins mediate communication between sperm and oo‐ cyte and initiate intracellular signalling pathways (Giancotti & Ruoslahti, 1999). Even more, these molecules influence early em‐ bryonic development (Taga & Suginami, 1998). In this context, previous studies have suggested that integrins play a role in the transfer of information into the cell to provide support, regula‐ tion of movement, and alteration of gene expression. Additionally, integrins also have been shown to play multiple roles in fertilisa‐ tion, embryogenesis, and implantation. These proteins facilitate sperm—oocyte binding and subsequently increase their expres‐ sion during implantation and early pregnancy (Sueoka et al., 1997). We have determined here that expression levels of ITGβ5 were significantly higher in the two‐cell embryos than those of the eight‐cell and succeeding embryonic developmental stages. The two‐cell embryonic stage immediately follows zygote formation, and this suggests the importance of ITGβ5 beyond sperm–egg binding. Increase in transcript expression at this level is indica‐ tive of possible ITGβ5‐dependent embryogenesis or transcription interaction. This could pinpoint the molecular interface of sperm– egg fusion necessary to initiate or sustain early embryonic devel‐ opment. Thus, our data suggest that there may be activation of an ITGβ5‐dependent early embryonic development at least until the eight‐cell stage followed by a significant decrease. Our findings support the notion that the ITGβ5 is indeed an important contrib‐ utor to the onset of early embryonic development. Moreover, the computational analyses showed that ITGβ5 is highly conserved among various species and that coupled with all our findings indi‐ cate that integrins can play multiple roles in the phases of fertilisa‐ tion, embryogenesis, and implantation. Because of the significant similarities between bovine and other mammals, the new data and knowledge generated in this study can be used to advance both fundamental science and technology of other species including humans and endangered species. Future studies aimed at deter‐ mining function(s) of ITGB5 in preimplantation development are expected to further the molecular and cellular mechanisms con‐ trolling early embryogenesis.
ACKNOWLEDGEMENTS
This study was funded in part by Mississippi Agricultural Forestry Experiment Station, Brazilian Research Council (CNPq), and by Selcuk University. A. Velho and E. Menezes were funded through scholarships by the Improvement of Higher Education (CAPES) in Brazil. L. Koenig was funded by the Merial Veterinary Scholars Programme and by the College of Veterinary Medicine of Mississippi State University.
ORCID
Erdogan Memili https://orcid.org/0000‐0002‐8335‐5645
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How to cite this article: Velho A, Wang H, Koenig L, et al.
Expression dynamics of Integrin Subunit Beta 5 in bovine gametes and embryos imply functions in male fertility and early embryonic development. Andrologia. 2019;51:e13305. https ://doi.org/10.1111/and.13305
