Factors affecting cellular outgrowth from porcine inner cell masses in vitro

During early embryonic development, endodermal cells leave the inner cell mass (ICM) and migrate over an extracellular matrix located on the blastocoelic side of the trophectoderm to form extraembryonic endoderm. Two experiments were conducted to evaluate factors supporting porcine endodermal cell migration in vitro. In Exp. 1, porcine ICM were cultured on matrices of collagen IV, fibronectin, or laminin. Percentages of ICM generating cellular outgrowth on fibronectin (5/11; 45%) and laminin (4/10; 40%) were similar (P > 0.10); however, collagen IV (0/10; 0%) failed (P < 0.05) to support cellular outgrowth. Inner cell mass and outgrowth areas and numbers of cells in outgrowths were similar (P > 0.10) for fibronectin and laminin, and increased (P < 0.05) with time in culture. In Exp. 2, ICM were cultured on fibronectin or laminin in medium containing 0 or 500 microg/mL of the inhibitory tripeptide, arg-gly-asp (RGD), or on laminin in medium containing 0 or 10 microg/mL recombinant human tissue inhibitor of matrix metalloproteinases-2 (rhTIMP-2). Inner cell mass and outgrowth areas and numbers of cells in the outgrowths for ICM cultured on fibronectin did not differ (P > 0.10) due to the presence of RGD. Inner cell masses cultured on laminin in medium containing 500 microg/mL RGD had fewer cells in the outgrowths and slower rates of cell migration compared with 0 microg/mL (P < 0.05). No differences (P > 0.10) in ICM and outgrowth areas and numbers of cells in the outgrowths were observed for ICM cultured on laminin in medium containing 0 or 10 microg/mL rhTIMP-2. Both fibronectin and laminin supported porcine ICM outgrowth in vitro; however, because outgrowth on fibronectin was not inhibited by RGD, endodermal cells must express an integrin that recognizes an alternative sequence in fibronectin. Cell migration on laminin was inhibited by RGD, suggesting either RGD competes with laminin for binding sites on endodermal cells or binding RGD alters endodermal cell migration on laminin. Because rhTIMP-2 had no effect on cell outgrowth, porcine ICM do not appear to be responsive to the proliferative effects of rhTIMP-2.     

The effects of canine bone marrow stromal cells on neuritogenesis from dorsal root ganglion neurons in vitro

The present in vitro study was designed to evaluate whether canine bone marrow stromal cells (BMSCs) promote neurite outgrowth from dorsal root ganglion (DRG) neurons. Bone marrow aspirates were collected from iliac crests of three young adult dogs. DRG neurons were cultured on BMSCs, fibroblasts, or laminin substrates. DRG neurons were also cultured in BMSC- or fibroblast-conditioned media. DRG neurons grown on BMSCs extended longer neurites and developed a much more elaborate conformation of branching neurites compared to those on fibroblasts or laminin. Quantitative analysis revealed that these effects were associated with the emergence of increased numbers of primary and branching neurites. The effect appears to be dependent upon cell-cell interactions rather than by elaboration of diffusible molecules. With more extensive investigations into the basic biology of canine BMSCs, their ability for promoting neurite outgrowth may be translated into a novel therapeutic strategy for dogs with a variety of neurological disorders.     

Role of beta1 integrins in adhesion of canine mastocytoma cells to extracellular matrix proteins

The interactions of tumor cells with the extracellular matrix (ECM) are a crucial step in invasion and metastasis. Integrins are adhesive molecules forming heterodimers with alpha and beta subunits that play a definitive role in these interactions. In this study, mastocytoma (mast cell tumor: MCT) cell-ECM interaction was investigated using 3 canine MCT cell lines: CM-MC (originating from cutaneous MCT), VI-MC (originating from intestinal MCT), and CoMS (originating from oral MCT). Flow cytometric analysis showed that all cells highly expressed the integrin beta1 and alpha1 through alpha5 subunits and that they moderately expressed the alpha6 subunit. In adhesion studies, CoMS weakly but spontaneously adhered to fibronectin (FN), which was enhanced by phorbol ester (TPA), while CM-MC and VI-MC required cell activation by TPA to adhere to FN. Anti-beta1 and alpha5 integrin antibodies strongly inhibited cell adhesion to FN in CM-MC and CoMS and moderately inhibited cell adhesion in VI-MC. Only VI-MC adhered to laminin (LN) under activation by TPA. Anti-beta1 integrin antibodies strongly inhibited cell adhesion to LN, but all anti-alpha integrin antibodies failed to inhibit cell adhesion to LN. No cells adhered to collagen types I and IV. Canine MCT cells from different origins expressed similar integrin patterns; however, there were some differences in adhesive behavior in response to various ECM proteins and activating stimuli.     

Immunohistochemical characterization of the extracellular matrix in normal mitral valves and in chronic valve disease (endocardiosis) in dogs

This study aimed to characterize the composition and distribution of the extracellular matrix (ECM) components in normal canine mitral valves (MV) and in chronic heart valve disease (CVD).MV of 50 dogs (normal (n = 9), mild (n = 13), moderate (n = 17), severe (n = 11) CVD) were investigated macroscopically, histologically (H.-E., picrosirius red) and immunohistochemically (collagen I, III, IV, V, VI, elastin, laminin, fibronectin, heparan sulphate).In normal MV, ECM components were expressed in a typical layered pattern. In mild CVD, basement membrane components (laminin, collagen IV, fibronectin) were increased. Advanced CVD was characterized by myxomatous nodular lesions displaying a marginal and a central region comprised mainly of collagen I, VI and fibronectin in the former and collagen I and III in the latter. Collagen IV and laminin appeared multifocally in marked CVD.In conclusion, not only an accumulation of proteoglycans, but also a distinctly altered expression of basement membrane components, and collagens characterizes CVD.     

Localization of extracellular matrix receptors in ICGN mice, a strain of mice with hereditary nephrotic syndrome.

Fibrotic degeneration was examined in the kidneys of ICR-derived glomerulonephritis (ICGN) mice, a novel inbred mouse line with a hereditary nephrotic syndrome of unknown etiology considered to be a good model of human idiopathic nephrotic syndrome. In the present study, we histochemically revealed changes in accumulation of extracellular matrix (ECM) components and in localization of integrins, cellular receptors for ECM, in the kidneys of ICGN mice with the progression of renal failure. Excessive accumulation of basement membrane (laminin and collagen IV) and interstitial (type III collagen) ECM components were demonstrated in the glomeruli and tubulointerstitum of ICGN mice. Marked deposition of type I collagen and tenascin was seen only in the glomeruli of ICGN mice but not in those of ICR mice as normal controls. Increased expression of integrin alpha1-, alpha2-, alpha5- and beta1-subunits in glomeruli with fibrotic degeneration and abnormal distribution of alpha6-subunit were noted in the kidneys of ICGN mice. Excessive laminin, a ligand of alpha6beta1-integrin, was demonstrated on the tubular basement membrane, but alpha6-subunit diffusely disappeared on the basal side of the tubular epithelial cells. We presumed that abnormal integrin expression in renal tubules causes epithelial cell detachment, and consequently tubular nephropathy, and results in disorder of ECM metabolism causing excessive accumulation of ECM components in the kidneys of ICGN mice.     

Angiogenesis in the Bovine Corpus Luteum: An Immunocytochemical and Ultrastructural Study*

Immediately after ovulation a neovascular response occurs at the level of the theca interna. Pericytes and endothelial cells of post-capillary venules locally remodel the surrounding stroma, elongate and migrate into the avascular granulosa folds of the ruptured follicle. In order to examine the composition of the extracellular matrix as well as the growth characteristics of these newly formed vessels, we used immunohistochemical and electron microscopic methods. Initial sprouts were characterized by the appearance of a fibrillary network of fibronectin along the main axis of the sprout. Type IV collagen stained weakly and extracellular deposits of laminin were amorphous and patchy around immature capillary sprouts. In advanced maturational stages of the sprouts the capillaries were surrounded by increased deposits of fibronectin, whereas laminin and type IV collagen displayed a distinct and well-developed line around endothelial cells and pericytes. These observations indicate that the microvascular extracellular matrix undergoes a series of quantitative rather than qualitative changes during capillary development before achieving final maturation. Ultrastructural analyses showed that early capillary sprouts in the bovine corpus luteum were usually preceded by pericytes migrating at the tips of the sprouts. Endothelial cells comigrated in cohesive cylindrical projections, forming immediately a slit-like lumen which satisfies the criteria of the intercellular canalization type. Pericytes at the tips of endothelial sprouts exhibited a slender, bipolar morphology and were regularly surrounded by fragmented basal lamina, which is well-developed around pericytes in a more proximal position of the sprout. The regular association of pericytes with the leading front of the capillary sprouts suggests that these cell types may serve as guiding structures aiding outgrowth of endothelial cells in the bovine corpus luteum.     

The dynamic expression of extracellular matrix in the bovine endometrium at implantation

Remodeling of uterine endometrial extracellular matrix (ECM) is pivotal to successful implantation and placentation, and has been well described in the rodents and humans. However, bovine endometrial ECM remodeling is still vaguely defined, especially at the time of implantation. Therefore, this study investigated the distribution of four ECMs namely, types I and IV collagen, laminin and fibronectin, from days 0 to 30 of gestation in bovine endometrium by immunofluorescence microscopy. A change in the distribution pattern of ECMs was evident by day 14 of gestation as features at this stage were clearly different from those of day 14 of the estrous cycle. The immunoreactivity of type I collagen, fibronectin and laminin decreased from day 14 of gestation and was obscured by day 24 of gestation. The type I collagen fibers formed were of thinner consistency than those of the estrous cycle and showed a coarser meshwork within the epithelium sites during the implantation period. In addition, the type IV collagen and laminin immunoreactivities of epithelial basement membrane also remarkably declined at exactly the same time. By day 30 of gestation, the four ECMs had regenerated with the formation of the placentome. In conclusion, this study reveals that remodeling of ECM is essential for the successful establishment of pregnancy in the bovine.     

Expression and adhesive ability of gicerin, a cell adhesion molecule, in the pock lesions of chorioallantoic membranes infected with an avian poxvirus.

The expression and adhesive activities of gicerin, a cell adhesion protein, in the pock lesions on chicken chorioallantoic membranes (CAM) infected with an avian poxvirus were studied. In normal CAMs, gicerin was found on the flattened epithelial cells, and neurite outgrowth factor (NOF) was in the basement membrane. However, in the pock lesions on infected CAMs, gicerin was overexpressed on the cell membranes of hyperplastic epithelial cells forming thick epithelial layers. Neurite outgrowth factor was also found mainly in the basement membrane, but occasionally showed aberrant expression among hyperplastic cells. In vitro analyses, using the dissociated cells from pock lesions, demonstrated that an anti-gicerin polyclonal antibody inhibit cell aggregation activity and cell adhesion to NOF. These results suggest that gicerin might promote the cell-cell and cell-extracellular matrix protein bindings of the hyperplastic epithelial cells by its homophilic and heterophilic adhesive activities, and contribute to pock formation on the infected CAMs.     

Expression of fibronectin and its integrin receptor α5β1 in canine mammary tumours

Fibronectin and its integrin receptor α5β1 were studied by immunohistochemical methods in five normal canine mammary glands, four dysplastic glands and 18 mammary tumours. The aim of the study was to evaluate the possible changes in the α5β1 integrin receptor and its ligand fibronectin in relation to the metastatic capacity of canine mammary neoplasms. The immunostaining of α5β1 was very uniform in the hyperplastic glands but uneven in the mammary tumours. The expression of α5 and β1 was diminished in metastatic tumours but there were some α5-positive cells with pronounced features of malignancy and immaturity. Stromal fibronectin was increased in most cases and cytoplasmic staining of fibronectin was observed in epithelial and myoepithelial cells in mammary neoplasms but not in normal or dysplastic mammary tissue. There was no relationship between the content of α5β1 and the expression of fibronectin in canine mammary tumours.     

激活素A促进鸡胚背根神经节神经突起生长的作用机制

采用半定量PCR检测激活素II型受体（ActRII）、钙基因相关肽（cGRP）、血管活性肠肽（VIP）mRNA表达,液相一质谱联用仪检测5-羟色胺释放水平。结果显示,5μg／L激活素A能明显促进鸡胚背根神经节神经突起生长,同时可见激活素A促进ActRIIA、CGRPmRNA表达及神经递质5-羟色胺释放,但对ActRIIB及VIPmRNA表达无影响。结果表明,ActivinA可能通过ActivinA—ActRIIA途径发挥作用,其促进鸡胚背根神经节神经突起生长与其上调CGRP表达及5-HT释放有关。     

Avian pathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> bind fibronectin and laminin

Avian colisepticemia frequently occurs after respiratory tract damage, the primary site for infection allows bacteria to encounter an exposed basement membrane, where laminin and fibronectin are important components. We investigated the ability of an isolate of avian pathogenic Escherichia coli to bind fibronectin and laminin. Using Far-western dot blot analysis, we demonstrated the ability of this microorganism to bind basement membrane proteins fibronectin and laminin. Results from an ELISA-based approach indicate that the binding to these membrane proteins was bacterial-dose dependent. Furthermore, two specific E. coli polypeptides, of 32 kDa and 130 kDa, reacted with laminin and fibronectin, respectively. Further evaluation of these potential bacterial adhesins may provide insights into the pathogenesis of colibacillosis.     

Meat Science and Muscle Biology Symposium: the influence of extracellular matrix on intramuscular and extramuscular adipogenesis

The extracellular matrix (ECM) and specific ECM components can have a major influence on cell growth, development, and phenotype. The influence of the ECM and ECM components on adipogenesis in vivo and in vitro will be reviewed in this paper. Engelbreth-Holm-Swarm substratum and laminin per se markedly increased attachment, spreading, and hypertrophy of preadipocytes in serum-free primary cultures of porcine adipose tissue stromal-vascular cells. Furthermore, primary cultures of stromal-vascular cells showed that preadipocytes express ECM components after preadipocyte recruitment. Staining for plant lectins, type IV collagen, and laminin in fetal pig adipose tissue demonstrates that adipocyte reactivity for laminin was strong throughout fetal development and was similar for developing adipocytes and vasculature. However, lectin binding and type IV collagen reactivity of blood vessels preceded that for adipocytes. Therefore, these studies indicated that the ECM and in particular laminin may play a critical role in morphological aspects of preadipocyte development. Specific inhibitors and modulators of collagen synthesis have been used to evaluate the role of collagens in the differentiation of bovine intramuscular preadipocytes (BIP) and other preadipocyte cell lines. Triglyceride accretion of BIP cells was inhibited by a general inhibitor of collagen biosynthesis, whereas specific inhibitors or modulators of type IV collagen inhibited 3T3-L1 cell differentiation. Further study revealed that compared with collagens types I to IV, type V and VI collagens have an important and active role in BIP adipogenesis. The growth of intramuscular bovine adipose tissue may be dependent on collagen newly synthesized and organized by the adipocytes per se. The role of extracellular or ECM proteolysis in regulating adipogenesis also will be reviewed in this paper. Many members of the matrix metalloproteinase (MMP) family are expressed by adipocytes, and specific inhibition of MMP-9 greatly reduces adipogenesis in vitro. Possibly, MMP and other proteases regulate turnover of key adipocyte ECM proteins that are involved in the regulation of preadipocyte proliferation and differentiation.     

The effects of estrogen and progesterone on prostaglandins and integrin beta 3 (beta3) subunit expression in primary cultures of bovine endometrial cells

In cattle, endometrial expression of integrin alphavbeta3 is reduced on day 16 of the estrous cycle, coinciding with the critical period during which the decision is made to initiate luteolysis or continue with pregnancy. The objective of these experiments was to examine the relationship between estrogen and progesterone treatments, endometrial integrin alphavbeta3 expression, and prostaglandin F2alpha (PGF2alpha) and E2 (PGE2) production. Epithelial and stromal cells from intercaruncular (ICAR) and caruncular (CAR) bovine endometrium were treated with 17beta-estradiol (0.1 and 1.0 nM) and/or progesterone (1.0 and 10 nM) in a manner designed to mimic the steroid fluctuations of the estrous cycle. All cell types expressed estrogen receptor and progesterone receptor mRNA and protein. Intercaruncular stromal cells were the most responsive to steroidal regulation. Estrogen suppressed expression of integrin subunit beta3 mRNA in ICAR stromal cells (P< or =0.05). Progesterone and estrogen + progesterone treated cells did not differ in beta3 expression from controls (P> or =0.05). Steroid treatment did not affect PGF2alpha production in any cell type (P> or =0.05), however, estrogen decreased PGE2 production in all cells except CAR stroma (P< or =0.05). The results indicate that in bovine endometrium expression of integrin alphavbeta3 and production of PGE2 is influenced by estrogen.     

Adherence of Pasteurella multocida to fibronectin

For many pathogens, adherence and/or invasion involve association with host extracellular matrix molecules, such as fibronectin (Fn). Pasteurella multocida was found to bind significantly to Fn and collagen type IX but not to laminin and collagen types IV and X. The binding of P. multocida to Fn was dose-dependent and was inhibited by heparin (Hep). Removal of polysaccharide capsule enhanced the binding capacity of the bacterium to Fn and inhibition by Hep. Protease treatment of bacteria decreased binding, implicating surface protein(s) as adhesive components. Investigation of the binding domain(s) of P. multocida on the Fn molecule revealed preferential binding to the N-terminal Hep-binding domain of Fn but not to the carboxyl-terminal Hep-binding domain. Furthermore, Fn, and anti-Fn antibodies inhibited P. multocida adherence to Madin-Darby bovine kidney cells, suggesting the involvement of Fn in the bacterium adherence to host cells. Ligand blotting, batch affinity purification and MALDI-TOF mass spectrometry implicated several proteins as putative adhesins of P. multocida in the Fn-mediated adherence. Taken together, the data suggest that P. multocida-Fn interaction may play a role in the bacterium adherence to host cells, and this may be mediated by bacterial surface proteins with preferential affinity for the Hep-1 binding domain of Fn.     

Buffalo (Bubalus bubalis) ES Cell–Like Cells are Capable of In Vitro Skeletal Myogenic Differentiation

When buffalo embryonic stem (ES) cell–like cells that expressed surface markers SSEA‐4, TRA‐1‐60, TRA‐1‐81, CD9 and CD90 and intracellular markers OCT4, SOX2 and FOXD3, as shown by immunofluorescence, and that expressed REX‐1 and NUCLEOSTEMIN as confirmed by RT‐PCR, were subjected to suspension culture in hanging drops in absence of LIF and buffalo foetal fibroblast feeder layer support, they differentiated to form three‐dimensional embryoid bodies (EBs). Of 231 EBs examined on Day 3 of suspension culture, 141 (61.3 ± 3.09%) were of compact type, whereas 90 (38.4 ± 3.12%) were of cystic type. The cells obtained from EBs were found to express NF‐68 and NESTIN (ectodermal lineage), BMP‐4 and α‐skeletal actin (mesodermal lineage), and α‐fetoprotein, GATA‐4 and HNF‐4 (endodermal lineage). When these EBs were cultured on gelatin‐coated dishes, they spontaneously differentiated to several cell types such as epithelial‐ and neuron‐like cells. When EBs were cultured in the presence of 1 or 2% DMSO or 10^?8 m or 10^?7 m retinoic acid for 25 days, ES cells could be directed to form muscle cell–like cells, the identity of which was confirmed by expression of α‐actinin by immunofluorescence and of MYF‐5, MYOD and MYOGENIN genes by RT‐PCR. MYOD was first detected on Day 10 in both treatment groups and on Day 15 in controls, whereas MYOGENIN was first detected on Day 10, Day 15 and Day 25 in the presence of retinoic acid, in the presence of DMSO and in controls, respectively. The present study demonstrates the ability of buffalo ES cell–like cells to undergo directed differentiation to cells of skeletal myogenic lineage.     

Three-dimensional culture of keratinocytes and the formation of basement membrane for canine footpad substitute

A pad equivalent for a dog was prepared as a substitute for the loss of footpad. In addition to the time course of formation on epidermal morphogenesis, we investigated expressions of alpha(6) integrin subunit as adhesive molecule, and laminin and type IV and VII collagens as extracellular matrices of basement membrane components. Epithelium of the pad equivalent was thick enough to be easily confirmed at 5 days at the air-liquid interface, but many creases appeared on it at 7 days, and it shrank at 10 and 14 days. Keratinocytes were increased in 4 to 5 cell layers at 1 day at the air-liquid interface, differentiating into basal cell layer. Granular and corneal cell layers were confirmed until 5 days, and maintained their shape at least until 14 days. Alpha 6 integrin was expressed at almost the same fluorescent intensity as native pad tissue at 1 day at the dermal-epidermal junction. Laminin and type IV collagen were intermittently expressed at 5 and 10 days, respectively, at the dermal-epidermal junction, and at 14 days the fluorescence showed almost the same intensity as native pad tissue. The expression of type VII collagen was discontinuous at 2 days at the dermal-epidermal junction, but remained as it was at 14 days. The present findings suggested that although the formation of anchoring fibrils in basement membrane was incomplete, the pad equivalent in the dog was reconstructed similar to a native pad by epidermal morphogenesis.     

Primary cell culture and morphological characterization of canine dermal papilla cells and dermal fibroblasts

Skin biopsies were taken from female dogs, the primary hair follicles isolated and the dermal papilla dissected. After incubation in supplemented Amniomax complete C100 medium in 24-well culture plates, the dermal papilla cells (DPC) grew to confluence within 3 weeks. Thereafter, they were subcultivated every 7 days. Dermal fibroblast (DFB) cultures were established by explant culture of interfollicular dermis in serum-free medium, where they reached confluence in 10 days. They were subcultivated every 5 days. For immunohistochemistry, cells were grown on cover slips for 24 h, fixed and stained with antibodies against collagen IV and laminin. DPC showed an aggregative growth pattern and formation of pseudopapillae. Intensive staining for collagen IV and laminin could be observed until the sixth passage. DFB grew as branching, parallel lines and showed only weak staining for collagen IV and laminin.     

Serum markers of lamellar basement membrane degradation and lamellar histopathological changes in horses affected with laminitis

In order better to evaluate the extent to which degradation of the lamellar basement membrane (LBM) by matrix metalloproteinases (MMP) occurs in equine laminitis, we determined the concentration of type IV collagen and laminin in normal and laminitic horses, using specific immunoassays. Blood samples were obtained from both the jugular and the cephalic veins of horses (n = 10) before and after the induction of acute alimentary laminitis by carbohydrate overload. Jugular and cephalic venous blood samples were also obtained from horses affected with naturally occurring laminitis (n = 16) and nonlaminitic controls (n = 8). The serum collagen IV concentration was not changed following the induction of laminitis in the experimental group. Serum collagen IV concentration was increased in jugular venous blood obtained from cases of naturally occurring laminitis (mean +/- s.e. 218.04 +/- 18.59 ng/ml) compared with nonlaminitic controls (157.50 +/- 10.93 ng/ml) (P<0.05). Serum collagen IV concentration was also increased in jugular venous blood obtained from severely laminitic horses (219.50 +/- 18.18 ng/ml) compared with nonlaminitic controls (157.50 +/- 10.93 ng/ml) (P<0.05). A difference in serum concentration of collagen IV was not identified based on chronicity of naturally occurring laminitis. Serum laminin concentration did not differ between laminitic and nonlaminitic horses. Differences in serum laminin concentration were not identified based on sampling location (jugular or cephalic vein), severity of laminitic pain, or chronicity of spontaneous laminitis. In conclusion, the circulating concentration of collagen IV was increased in horses affected with naturally occurring laminitis. The potential role for serum collagen IV assay for characterisation of equine laminitis warrants further investigation.     

Laminin‐111 Inhibits Bovine Fertilization but Improves Embryonic Development in vitro,and Receptor Integrin‐β1 is Involved in Sperm–Oocyte Binding

This study detected the distribution of laminin during embryonic formation by immunofluorescence. To determine the possible function of laminin on developmental ability of in vitro fertilized embryos, the presumptive zygotes were divided and transferred to CR1aa medium supplemented with different concentrations (0 μg/ml, 5 μg/ml, 10 μg/ml and 20 μg/ml) of laminin. To explore the association with sperm–oocyte fusion, oocytes and/or sperm were pre‐incubated with laminin or anti‐β1 antibody before insemination. Laminin was absent in mature oocytes and could be detected first at the 8‐cell stage and then displayed an increasing tendency. Adding 10 μg/ml laminin to the culture medium improved embryonic development including cleavage rate, blastocyst rate, total cell numbers in the blastocyst and cell numbers in the inner cell mass. Laminin inhibited sperm–oocyte fusion when incubated with oocytes and/or sperm before in vitro fertilization, and only integrin‐β1 of sperm was involved in sperm–oocyte binding. Inhibition may be caused by blocking β1, but why laminin inhibits fertilization is still unknown. The results suggest that laminin plays an important role during embryonic formation and has a negative function in sperm–oocyte fusion, but improves embryonic development. However, only integrin‐β1 is involved in sperm–oocyte binding.