- Research article
- Open Access
Variational modularity at the cell level: insights from the sperm head of the house mouse
© Medarde et al.; licensee BioMed Central Ltd. 2013
Received: 1 March 2013
Accepted: 21 August 2013
Published: 3 September 2013
Modularity is an important feature in the evolvability of organisms, since it allows the occurrence of complex adaptations at every single level of biological systems. While at the cellular level the modular organization of molecular interactions has been analyzed in detail, the phenotypic modularity (or variational modularity) of cell shape remains unexplored. The mammalian spermatozoon constitutes one of the most complex and specialized cell types found in organisms. The structural heterogeneity found in the sperm head suggests an association between its inner composition, shape and specificity of function. However, little is known about the extent of the connections between these features. Taking advantage of the house mouse sperm morphology, we analyzed the variational modularity of the sperm head by testing several hypotheses related to its structural and functional organization. Because chromosomal rearrangements can affect the genotype-phenotype map of individuals and thus modify the patterns of covariation between traits, we also evaluate the effect of Robertsonian translocations on the modularity pattern of the sperm head.
The results indicated that the house mouse sperm head is divided into three variational modules (the acrosomal, post-acrosomal and ventral spur module), which correspond to the main regions of the cytoskeletal mesh beneath the plasma membrane, i.e., the perinuclear theca. Most of the covariation is concentrated between the ventral spur and the acrosomal and post-acrosomal modules. Although the Rb fusions did not alter the main modularity pattern, they did affect the percentages of covariation between pairs of modules.
The structural heterogeneity of the cytoskeleton is responsible for the modular organization of the sperm head shape, corroborating the role that this structure has in maintaining the cell shape. The reduction in percentages of shape covariation between pairs of modules in Rb sperms suggests that chromosomal rearrangements could induce changes in the genotype-phenotype map. Nevertheless, how these variations affect sperm fertilization success is yet to be elucidated.
Organisms are composed of elements that, although coordinated, show obvious signs of heterogeneity with respect to certain kinds of processes [1, 2]. These elements, called modules, are internally integrated but relatively independent of one another . Thus, modularity is considered a key feature of biological organization that allows the modification of certain parts of organisms with minor effects on other parts, thereby contributing to evolvability . Modularity occurs at every single level of biological organization, from molecular interactions to networks of ecological connections [1, 2]. Variational modularity (that is, groups of correlated characters) has long been recognized in morphological traits [1, 2] since it provides the evolutionary flexibility required to induce adaptive changes in certain regions of complex phenotypic structures. At the cell level, the structural and functional modularity of molecular networks have been studied in detail [4, 5], but to our knowledge, the variational modularity of cell morphology has not been examined to date. The relations between different kinds of modularity in biological organization are still not well understood, and their comparative study may provide insights into evolutionary processes . The male gametes of certain mammals may represent an ideal model for testing the connections between different kinds of modularity, as they are highly polarized cells with structurally and functionally differentiated regions that are morphologically recognizable .
Previous studies in mice from the Robertsonian system found in Barcelona (BRbS) revealed that chromosomal rearrangements affect the size and the shape of the sperm head . This Rb system represents a unique contact zone between standard (St) and Rb mice since there is no evidence of a Rb race in which a group of individuals from the same geographical area share a set of metacentrics in homozygous condition . Diploid numbers range from 27 to 40 chromosomes, and seven different metacentrics (Rb3.8, 4.14, 5.15, 6.10, 7.17, 9.11 and 12.13) have been described up to now [14–16]. The relative stability of its metacentric staggered structure  as well as the phenotypic differences associated with karyotype detected in animals from this area  suggest the presence of partial barriers to gene flow. In this scenario, the study of the factors involved in the establishment of reproductive barriers between individuals may take on special relevance. Thus, assuming that Rb fusions could induce variations in the genotype-phenotype map of the sperm head , and that changes in the variational modularity patterns may play an important role in the evolvability of the sperm features, a second aim of this study is to evaluate the effect, if any, of the Rb translocations on the pattern of variational modularity of the western house mouse sperm head.
Results and discussion
The Procrustes ANOVA performed on the replicated subsample showed highly significant differences between sperm heads (MS sperms = 0.000388, MS error = 0.000001, P < 0.0001). The mean squares for sperm head variation exceeded the mean squares for replicates by 388-fold, indicating low measurement error and consequently strong repeatability of the landmark location in the sperm head.
Fine morphological analysis of mouse sperm heads combining scanning electron microscopy and geometric morphometrics revealed significant allometry of cell shape (P < 0.001) with 11.8% and 8.6% of shape variation explained by changes in cell size in the St and Rb groups, respectively. Allometric shape changes affected all the landmarks to a similar degree and mainly involved a narrowing of the sperm head and a stretching of the hook. The existence of significant size-dependent shape changes is interesting because evidence of shape allometry at the cell level is very scarce. The precise mechanisms that underlie this association are unknown. However, recent studies have indicated a correlation between cell shape and growth [18, 19], and between cell size and the behaviour of the cytoskeletal machinery . Given that the cytoskeleton is mainly responsible for shaping the cell during growth, it is reasonable to suppose that these behavioural changes in the cytoskeletal machinery may be partly responsible for the association between size and shape of the cell. Because allometry represents a global integration factor, the residuals of the multivariate regression of the Procrustes coordinates onto log CS were used for further analyses.
Eigenvalues and percentages of variance and cumulative variance explained by the first ten principal components (out of 34) of the PCA obtained with the residuals from the multivariate regression analysis
Results of 2B-PLS analyses for standard (St) and Robertsonian (Rb) samples
%Total Cov PLS1
AC vs PA
AC vs VS
PA vs VS
AC vs PA
AC vs VS
PA vs VS
Our results reveal for first time the existence of variational modularity in a cellular structure such as the house mouse sperm head and highlight the important role of the cytoskeleton in maintaining the shape of the cell. The presence of Rb translocations did not affect the variational modularity pattern. However, the lower percentages of shape covariation between pairs of modules in Rb sperms heads suggest a certain influence of the Rb rearrangements. Understanding the mechanisms that alter covariation between phenotypic traits in the sperm head is an aspect of great importance given its possible effect on the evolvability of these specialized cells. However, the extent to which these changes affect sperm fertilization success is a subject for further studies.
Thirty-one live-trapped males in the BRbS were used for analyses. Karyotypes were obtained from a suspension of bone marrow cells, following Ford . Metaphase chromosome spreads were stained by a G-banding method . Chromosome identification was performed following the Committee on Standardized Genetic Nomenclature for mice . The left caudate epididyme from 13 St and 18 Rb house mice was dissected and disaggregated in 5 ml of phosphate buffer (PB) 0.1 M at room temperature. After homogenization, 1 ml of sperm solution was filtered through a nucleopore membrane (0.2 μm) and fixed in 2.5% glutaraldehyde, 2% paraformaldehyde and PB 0.1 M solution. Samples were then rinsed in PB 0.1 M, postfixed in 1% osmium tetraoxide, rinsed in PB 0.1 M, dehydrated in graded series of ethanol and dried by the critical-point method. Membranes were observed in an S-570 scanning electron microscope (SEM; Hitachi Ltd.) at an accelerating voltage of 15 kV. From each individual, an average of 20 sperm heads in a horizontal plane, with the hook orientated to the left side and without evident structural alterations were randomly captured (Figure 1).
To determine the form of the sperm heads, sixteen landmarks and three semilandmarks were digitized using the tpsDig2 software  (Figure 1). The criteria used for the landmark assignation were the following: (1) top of the hook, (2) point where the hook and the upper ventral spur overlap, (3) prominence in the axis of the upper ventral spur, (4 and 7) top of the upper and lower ventral spurs, (5 and 6) inner distance between the ventral spurs, (8–11) insertion edge of the sperm head with flagellum, (12 and 13) terminal edges of the post-acrosomal sheath, (14,15 and 19) basal and apical ridge of the equatorial crest. The semilandmarks (points 16–18) were digitized as equidistant points by the tpsDig2 ‘resample curve by length’ option. Measurement error is an important source of variation affecting morphometric data that can increase the likelihood of type II errors and lead to biased results [28, 29]. In order to evaluate the impact of measurement error in the current set of landmarks around the sperm head, in a subsample of 40 images all landmarks were digitized three times. Geometric morphometrics and modularity analyses were performed using the routines implemented by MorphoJ software . Shape variation in the landmark configurations was obtained by the full Procrustes fit and the orthogonal projection to the tangent space . Size was defined as centroid size (CS) . In the replicated subsample, a Procrustes ANOVA comparing variation among and within sperm heads was performed to obtain the measurement error associated with landmark location [33, 34]. Given that variation between sperm heads clearly exceeded that of measurement error (see Results) subsequent analyses were based on a single digitization of landmarks per head. Shape allometry, the scaling of shape with size, may conceal the patterns of modularity ; thus, the dependence of shape on size was calculated by means of a linear multivariate regression of the Procrustes coordinates onto the logarithm of CS . Statistical significance was obtained using a permutation test with 10,000 iterations under the null hypothesis of size and shape independence . The residuals obtained in the multivariate regression analyses were used for subsequent analyses . First, principal component analysis (PCA) was performed with the covariance matrix of the residuals. Then, the division of the sperm head into three different sets of morphological modules was tested (Figure 1). To measure the covariation between the hypothesized sets of landmarks, the RV coefficient or the multi-set RV coefficient was obtained . To test for modularity, this value was compared with the distribution of RV values of all the alternative partitions of spatially contiguous subsets of landmarks (adjacency graphs in Figure 1) containing the same number of landmarks as the hypothesized partitions and with 10,000 random partitions . Finally, we used a two-block partial least square (2B-PLS) to examine covariation between the detected modules [37, 38]. Because of differences in mice karyotypes, these analyses were performed separating the sample into two different chromosomal groups: i) St, sperms produced by animals with 40 chromosomes and ii) Rb, sperms from animals ranging from 30 to 39 chromosomes.
Permission to capture was granted by the Departament de Medi Ambient of the Generaltitat de Catalunya (Spain). Animals were handled in compliance with the guidelines and ethical approval by the Comissió d’Ètica en l’Experimentación Animal y Humana (CEEAH) of the Universitat Autònoma de Barcelona and by the Departament d’Agricultura, Ramaderia, Pesca, Alimentació i Medi Natural (Direcció General de Medi Natural i Biodiversitat) of the Generalitat de Catalunya (reference of the experimental procedure authorization: DAAM 6328).
We are indebted to J. Martínez and A. Sánchez-Chardi for their help in processing the samples by SEM and Michael Maudsley of the Serveis Lingüístics (University of Barcelona) for revising the English. This study was funded by the Spanish Ministerio de Economía y Competitividad (project number CGL2010-15243) and by a PIF grant from the Universitat Autònoma de Barcelona to NM.
- Wagner GP, Pavlicev M, Cheverud JM: The road to modularity. Nature Rev Genet. 2007, 8: 921-931.PubMedView ArticleGoogle Scholar
- Klingenberg CP: Morphological integration and developmental modularity. Annu Rev Ecol Evol Syst. 2008, 39: 115-32. 10.1146/annurev.ecolsys.37.091305.110054.View ArticleGoogle Scholar
- Schlosser G, Wagner GP: Introduction: the modularity concept in developmental and evolutionary biology. Modularity in development and evolution. Edited by: Schlosser G, Wagner GP. 2004, Chicago: The University of Chicago Press, 1-11.Google Scholar
- Hartwell LH, Hopfield JJ, Leibler S, Murray AW: From molecular to modular cell biology. Nature. 1999, 402: C47-C52. 10.1038/35011540.PubMedView ArticleGoogle Scholar
- Spirin V, Mirny LA: Protein complexes and functional modules in molecular networks. Proc Nat Acad Sci. 2003, 100: 12123-12128. 10.1073/pnas.2032324100.PubMed CentralPubMedView ArticleGoogle Scholar
- Klingenberg CP: Evolution and development of shape: integrating quantitative approaches. Nature Rev Genet. 2010, 11: 623-635.PubMedGoogle Scholar
- Breed WG: The spermatozoon of Eurasian murine rodents: its morphological diversity and evolution. J Morphol. 2004, 261: 52-69. 10.1002/jmor.10228.PubMedView ArticleGoogle Scholar
- Selvaraj V, et al: Segregation of micron-scale membrane sub-domains in live murine sperm. J Cell Physiol. 2006, 206: 636-646. 10.1002/jcp.20504.PubMedView ArticleGoogle Scholar
- Selvaraj V, Asano A, Buttke DE, Sengupta P, Weiss RS, Travis AJ: Mechanisms underlying the micron-scale segregation of sterols and GM1 in live mammalian sperm. J Cell Physiol. 2009, 218: 522-536. 10.1002/jcp.21624.PubMed CentralPubMedView ArticleGoogle Scholar
- Oko R, Clermont Y: Isolation, structure and protein composition of the perforatorium of rat spermatozoa. Biol Reprod. 1988, 39: 673-687. 10.1095/biolreprod39.3.673.PubMedView ArticleGoogle Scholar
- Oko R, Moussakova L, Clermont Y: Regional differences in composition of the perforatorium and outer periacrosomal layer of the rat spermatozoon as revealed by immunocytochemistry. Am J Anat. 1990, 188: 64-73. 10.1002/aja.1001880108.PubMedView ArticleGoogle Scholar
- Medarde N, Martínez J, Sánchez-Chardi A, López-Fuster MJ, Ventura J: Effect of Robertsonian translocations on sperm head form in the house mouse. Biol J Linn Soc. In pressGoogle Scholar
- Hausser J, Fedyk S, Fredga K, Searle JB, Volobouev V, Wójcik JM, Zima J: Definition and nomenclature of the chromosome races of Sorex araneus. Folia Zool. 1994, 43: 1-9.Google Scholar
- Gündüz I, López-Fuster MJ, Ventura J, Searle JB: Clinal analysis of a chromosomal hybrid zone in the house mouse. Genet Res. 2001, 77: 41-51. 10.1017/S0016672300004808.PubMedView ArticleGoogle Scholar
- Sans-Fuentes MA, Muñoz-Muñoz F, Ventura J, López-Fuster MJ: Rb(7.17), a rare Robertsonian fusion in wild populations of the house mouse. Genet Res. 2007, 89: 207-213.PubMedView ArticleGoogle Scholar
- Medarde N, López-Fuster MJ, Muñoz-Muñoz F, Ventura J: Spatio-temporal variation in the structure of a chromosomal polymorphism zone in the house mouse. Heredity. 2012, 109: 78-89. 10.1038/hdy.2012.16.PubMed CentralPubMedView ArticleGoogle Scholar
- Muñoz-Muñoz F, Sans-Fuentes MA, López-Fuster MJ, Ventura J: Evolutionary modularity of the mouse mandible: dissecting the effect of chromosomal reorganizations and isolation by distance in a Robertsonian system of Mus musculus domesticus. J Evol Biol. 2011, 24: 1763-1776. 10.1111/j.1420-9101.2011.02312.x.PubMedView ArticleGoogle Scholar
- Savriama Y, Neustupa J, Klingenberg CP: Geometric morphometrics of symmetry and allometry in Micrasterias rotata (Zygnemophyceae, Viridiplantae). Beih Nova Hedw. 2010, 136: 43-54.Google Scholar
- Needleman DJ: Cellular allometry: the spindle in development and inheritance. Curr Biol. 2009, 19: 846-847. 10.1016/j.cub.2009.08.028.View ArticleGoogle Scholar
- Korley R, Pouresmaeili F, Oko R: Analysis of the protein composition of the mouse sperm perinuclear theca and characterization of its major protein constituent. Biol Reprod. 1997, 57: 1426-32. 10.1095/biolreprod57.6.1426.PubMedView ArticleGoogle Scholar
- Toshimori K, Ito C: Formation and organization of the mammalian sperm head. Arch Hystol Cytol. 2003, 66: 383-396. 10.1679/aohc.66.383.View ArticleGoogle Scholar
- Alibert P, Auffray JC: Genomic coadaptation, outbreeding depression, and, developmental stability. Developmental stability. Causes and consequences. Edited by: Polak M. 2003, New York: Oxford University Press, 116-134.Google Scholar
- Sans-Fuentes MA, Ventura J, López-Fuster MJ, Corti M: Morphological variation in house mice from the Robertsonian polymorphism area of Barcelona. Biol J Lin Soc. 2009, 97: 555-570. 10.1111/j.1095-8312.2009.01237.x.View ArticleGoogle Scholar
- Ford CE: The use of chromosomes markers. Tissue Grafting and Radiation. Edited by: Micklem HS, Loutit JF. 1966, New York: Academic Press, 197-206.Google Scholar
- Mandahl N: Methods in solid tumor cytogenetics. Human Cytogenetics,a practical approach, Volume II. Edited by: Rooney DE, Czepulkowski BH. 1992, London: IRL Press, 155-187.Google Scholar
- Committee on Standardized Genetic Nomenclature for Mice: Standard karyotype of the mouse Mus musculus. J Hered. 1972, 63: 69-72.Google Scholar
- Rohlf FJ: TpsDig2, version 2.16. New York: State University of New York;. 2010, Available at http://life.bio.sunysb.edu/morph/Google Scholar
- Bailey RC, Byrnes J: A new, old method for assessing measurement error in both univariate and multivariate morphometric studies. Syst Zool. 1990, 39: 124-130. 10.2307/2992450.View ArticleGoogle Scholar
- Arnqvist G, Martensson T: Measurement error in geometric morphometrics: empirical strategies to assess and reduce its impact on measures of shape. Acta Zool Hung. 1998, 44: 73-96.Google Scholar
- Klingenberg CP: MorphoJ: an integrated software package for geometric morphometrics. Mol Ecol Resources. 2011, 11: 353-357. 10.1111/j.1755-0998.2010.02924.x.View ArticleGoogle Scholar
- Dryden IL, Mardia KV: Statistical shape analysis. 1998, Chichester: John WileyGoogle Scholar
- Bookstein FL: Morphometric tools for landmark data. 1991, Cambridge: Cambridge Univ Press, 1Google Scholar
- Klingenberg CP, McIntyre GS: Geometric morphometrics of developmental instability: analyzing patterns of fluctuating asymmetry with Procrustes methods. Evolution. 1998, 52: 1363-1375. 10.2307/2411306.View ArticleGoogle Scholar
- Klingenberg CP, Barluenga M, Meyer A: Shape analysis of symmetric structures: quantifying variation among individuals and asymmetry. Evolution. 2002, 56: 1909-1920.PubMedView ArticleGoogle Scholar
- Klingenberg CP: Morphometric integration and modularity in configurations of landmarks: tools for evaluating a priori hypotheses. Evol Dev. 2009, 11: 405-421. 10.1111/j.1525-142X.2009.00347.x.PubMed CentralPubMedView ArticleGoogle Scholar
- Good P: Permutation tests: a practical guide to resampling methods for testing hypotheses. 1994, New York: Springer-VerlagView ArticleGoogle Scholar
- Rohlf FJ, Corti M: The use of two-block partial least-squares to study covariation in shape. Syst Biol. 2000, 49: 740-753. 10.1080/106351500750049806.PubMedView ArticleGoogle Scholar
- Singh N, Harvati K, Hublin JJ, Klingenberg CP: Morphological evolution through integration: a quantitative study of cranial integration in Homo, Pan, Gorilla and Pongo. J Hum Evol. 2012, 62: 155-164. 10.1016/j.jhevol.2011.11.006.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.