Sperm Motility and Function: A Closer Look
Regina M Turner
Department of Clinical Studies, New Bolton Center
University of Pennsylvania School of Veterinary Medicine, Kennett Square, Pennsylvania USA
With the advent of modern molecular and cellular biological techniques, many fields of medicine, including reproductive biology, are moving toward molecular diagnoses and, in some cases, gene therapy for specific diseases. However, at this point in time, little is known about the molecular aspects of male factor infertility and in particular, sperm motility and function. Not until we have a more complete molecular understanding of the processes which contribute to normal sperm function can we hope to address this problem more efficiently in the clinic. This presentation will summarize the current knowledge of the molecular mechanisms which underlie normal mammalian sperm motility and sperm function and will introduce some of the initial findings on the molecular composition of the stallion sperm flagellum.
The Components of the Flagellum
The mammalian flagellum is structurally divided into four major parts: the connecting piece, the midpiece, the principal piece, and the end piece (Fawcett 1975). The flagellum is composed of a number of cytoskeletal elements whose proper assembly is critical for sperm motility. These include (1) the axoneme which runs throughout the length of the flagellum; (2) the outer dense fibers (ODFs) which surround the axoneme in the midpiece and principal piece; (3) the mitochondrial sheath (MS), which is located in the midpiece and (4) the fibrous sheath (FS) which is located in the principal piece. The presence and structure of the axoneme is highly conserved in all ciliated and flagellated eukaryotic cells. However, only mammalian sperm flagella contain additional accessory structures: the MS, the ODFs, and the FS. Understanding the proteins which make up each of these flagellar structures, and how these proteins interact to produce the normal flagellar beat, will be critical to understanding the molecular genetic causes of reduced sperm motility in infertile animals.
Proteins of the Flagellum
Although flagellar morphology has been well described, the molecular components that make up the various flagellar structures have been explored only recently and our understanding of the role of these structures in flagellar motility continues to evolve. Several key proteins will be highlighted and their functions summarized.
In mammals, the axoneme extends throughout the flagellum and generates the flagellum’s motive force. It is a complex structure composed of a characteristic 9+2 array of microtubules (composed primarily of a and b tubulins) and associated proteins (Fawcett 1975, Clermont et al. 1990). In spite of their potential relevance to sperm motility, very few of these axoneme-associated proteins have been well characterized.
Dyneins are the "motor" proteins located in the arms of the outer microtubular doublets and are members of a multi-gene family of proteins (Porter and Johnson 1989, Holzbaur and Vallee 1994, Milisav 1998). Activation of the axonemal dynein ATPase results in sliding of adjacent outer doublet microtubules and it has been proposed that this sliding results in flagellar bending (Gibbons and Rowe 1965, Tash and Means 1982).
The Mitochondrial Sheath
The sperm mitochondria are located only in the MS of the midpiece. Sperm mitochondria produce ATP for the cell through glycolysis and aerobic respiration. While these functions are similar to somatic mitochondria, sperm mitochondria possess several proteins or protein isoforms that are apparently unique and are not found in the mitochondria of somatic cells.
The Outer Dense Fibers
Historically, it has been suggested that the role of the ODFs is to provide passive elasticity to the motile flagellum (Fawcett 1975). In recent years, several ODF proteins have been cloned and characterized primarily in the mouse, rat, and human. Most of the cloned proteins appear to be cytoskeletal or structural. Other than its putative structural role, the role of the ODFs in sperm motility remains largely speculative.
The Fibrous Sheath
The FS is another sperm cytoskeletal structure which traditionally was thought to play a mechanical role in sperm motility by providing a rigid support for the flagellum and determining its planar beat (Lindemann et al. 1992). While this may be true to some extent, more recent evidence suggests a much more active role for the FS in sperm motility.
Functional Aspects of Flagellar Proteins
It has been shown that the cAMP-dependent phosphorylation of flagellar proteins is required for the initiation and maintenance of sperm motility (Tash and Means 1982, Tash and Means 1983, San Augustin and Witman 1994, Visconti et al. 1997). Currently, it is believed that the primary downstream target of cAMP in sperm is PK-A, making it likely that this kinase is responsible for these phosphorylation events (Visconti et al. 1997). The ubiquitous PK-A holoenzyme is composed of a regulatory (R) subunit dimer and two catalytic (C) subunits. When bound by the R subunit dimer, the C subunits are held inactive. In response to cAMP binding to the R dimer, the C subunit is released and becomes free to phosphorylate downstream target proteins. Multiple isoforms of PK-A exist that are defined by their R subunits (Taylor et al. 1990, Francis and Corbin 1994). Several different R isoforms (including isoforms RI and RII) are expressed during different stages of spermatogenesis and the different R subunits each have distinct intracellular localizations. Since the flagellum provides the motive force for the sperm cell, it is likely that those isoforms of PK-A which localize to the flagellum, particularly in the vicinity of the axoneme, will prove to be involved in the regulation of sperm motility. Unfortunately, few of the protein targets for PK-A phosphorylation in sperm have been identified. It has been shown that all of the components of PK-A signaling (RII, C and cAMP) are present in the FS (Horowitz et al. 1984, Macleod et al. 1994, Horowitz et al. 1989, Pariset and Weinman 1994). When flagella beat, the FS slides over the axoneme. FS sliding also has been shown to be dependent on cAMP.
AKAP82 and PK-A
A major protein of the FS in all mammalian sperm studied thus far is AKAP82 (Carrera et al. 1994). AKAPs (A-Kinase Anchor Proteins) are a class of proteins which anchor PK-A to various subcellular organelles, thus, directing the activity of the kinase to specific regions within the cell (Faux and Scott 1996, Rubin 1994). The identification of AKAP82 provides a mechanism by which the typically soluble PK-A localizes to the flagellum, a region relatively devoid of cytoplasm. In this way, AKAP82 may be involved in the molecular mechanisms that govern mammalian sperm motility.
The current model is that when cAMP binds to the anchored RII dimer, the active C subunits are released in proximity to specific flagellar target proteins, such as axonemal dyneins (Tash 1989). Thus, AKAPs localize the activity of the kinase to specific regions within the cell and, in so doing, increase its specificity.
Homologues of AKAP82 have now been reported in the human (hAKAP82) (Turner et al. 1998), rat (FS75) (El-Alfy et al. 1999), bovine (bAKAP82) (Moss et al. 1999) and equine (this report) and the amino acid sequence is highly conserved (approximately 79% identical and 91% conserved across all species) suggesting that the protein plays a crucial role in the FS. Additionally, the human homologue of AKAP82, and probably the equine homologue as well, are phosphorylated on tyrosine residues during sperm capacitation, raising the possibility that AKAP82 is somehow involved in this maturational process (Carrera et al. 1996)
The need to clarify the role of RII/AKAP interactions in sperm motility has become even more important in light of recent conflicting reports. In bovine sperm, it has been shown that disrupting RII-AKAP anchoring in sperm arrests sperm motility (Vijayaraghavan et al. 1997). In contrast, deletion of the RII subunit of PK-A in mice results in loss of PK-A anchoring but causes no obvious effects on sperm motility or fertility (Burton et al. 1999).
It was hypothesized that a cyclic nucleotide-gated ion channel in sperm or cAMP-mediated guanine nucleotide exchange factors in testes might be alternative pathways utilized by cAMP to cause flagellar motility instead of or in addition to the PK-A mediated pathway (Burton et al. 1999).
Phosphorylation of axonemal dynein appears to be a critical regulatory point in the initiation of flagellar motility (Tash 1989). Following phosphorylation, the dynein ATPase is activated and microtubule sliding occurs. Dephosphorylation of dynein by the calmodulin-dependent protein phosphatase calcineurin then reverses this process. This requires that phosphorylation/dephosphorylation occurs in an asynchronous manner along the length of the axoneme. This could be achieved, for example, by the AKAP-mediated differential localization of the R subunit in relation to the microtubule doublets of the axoneme (Tash 1989). It also has been shown that some members of the AKAP family bind phosphatases such as calcineurin and protein phosphatase-1, in addition to PK-A (Coghlan et al. 1995, Klauck et al. 1996). Therefore, another potential role for AKAPs in the FS may be to organize the activity of kinases and phosphatases that activate and inactivate axonemal proteins.
Tyrosine phosphorylation of a specific group of sperm proteins is closely associated with capacitation in the mouse, human and bull. This phosphorylation is cAMP-dependant and involves PK-A (Visconti et al. 1995, Leclerc et al. 1996, Carrera et al. 1996, Galantino-Homer et al. 1997). Similar results have been found in the stallion (H Galantino-Homer and . Noiles, personal communication, Rosenberger et al. 1998). Additionally, it has been shown in both the bull and the human that a significant percentage of the proteins which become tyrosine phosphorylated during capacitation are located in the principal piece, raising the possibility that anchoring of PK-A to the FS via AKAPs may prove to be involved in normal capacitation (Galantino-Homer et al. 1997, Carrera et al. 1996, Turner et al. 1999). Interestingly, in the stallion it also has been shown that the same pattern of tyrosine phosphorylated proteins appears following cryopreservation (S Meyers, personal communication, Bedford et al. in press). This is consistent with the hypothesis that cryopreservation results in premature capacitation and a resultant reduced lifespan of post-thaw sperm (Bailey et al. 2000)
Interestingly, in human sperm, it has been shown that the two major proteins which become tyrosine phosphorylated in a capacitation-dependent manner are hAKAP82 and its precursor, pro-hAKAP82 (Carrera et al. 1996, Turner et al. 1999). Early work from this laboratory suggests that equine AKAP82 and its precursor also become tyrosine phosphorylated during capacitation. Additionally, in human sperm, it has been shown that tyrosine dephosphorylation of these proteins occurs in the presence of calcium in a calmodulin-dependent fashion, suggesting that calcineurin is involved. This again raises the possibility that, by organizing both phosphatases and kinases in the FS, sperm AKAPs may act as scaffolds for cross talk between phosphorylation and dephosphorylation signaling pathways which regulate the activities of proteins central to capacitation.
Since capacitation and hyperactivation have been closely associated in several species (Boatman and Robbins 1991, Stauss et al. 1995, Llanos and Meizel 1983, Neill and Olds-Clarke 1987, Olds-Clarke 1989), and since pro-hAKAP82 and hAKAP82 (and possibly pro-eAKAP82 and eAKAP82) have been shown to be tyrosine phosphorylated in association with capacitation, another potential role for AKAPs in the FS might include the regulation of hyperactivation. In this regard, a recent study implicated the tyrosine phosphorylation and dephosphorylation of a single 80 kDa FS protein, likely to be the hamster homologue of AKAP82, as a key mediator of the onset and end (respectively) of hyperactivated motility in hamster sperm (Si and Okuno 1999, Si 1999). It has been suggested that tyrosine phosphorylation of FS proteins reduces the stiffness of the FS and thus permits the flagellum to bend in a larger arch, resulting in hyperactivated motility (Si and Okuno 1999)
Glycolytic Enzymes and Energy Production
Mitochondria are found only in the midpiece of sperm. However, large amounts of ATP are required along the full length of the motile flagellum. Flagellar PK-A needs ATP to phosphorylate its downstream targets and the dynein ATPases, the motors of the axoneme, also require ATP as an energy source. Mathematical models based on the diffusion constant of ATP and a morphometric estimate of the volume of the mouse sperm flagellum (Du et al. 1994) predict that ATP produced by the midpiece mitochondria would not be able to diffuse sufficiently along the length of the FS to supply the entire flagellum with enough energy to support the axonemal dynein ATPase (B.T. Storey, personal communication).
It has been shown that all of the enzymes of glycolysis may be present in the FS (Storey and Kayne 1975, Bradley et al. 1996, Westhoff and Kamp 1997, Bunch et al. 1998, Mori et al. 1998, Travis et al. 1998). This raises the possibility that mammals may have solved the problem of ATP diffusion by developing a system in which energy is produced by glycolysis within the FS, thus serving as a source of ATP to PK-A and dynein ATPases in the motile flagellum. Consistent with this hypothesis, it has been shown that mammalian sperm produce lactate from glucose under aerobic conditions (Storey and Kayne 1975).
The Genetics of Sperm Motility
Single gene defects typically result in severe but rare phenotypes. In the short term, studies focusing on these isolated genes result in significant benefits to a limited number of individuals. The full value of understanding these genes is realized when one uses the new information to refine or redefine the current understanding of basic physiology. The information also serves as a well-defined starting point from which to examine and identify other genes linked to multifactorial or multigenic diseases (i.e., genes that are responsible for the gradients of common abnormalities seen in clinical practice).
An understanding of the genetics of sperm motility has become even more critical with the advent of assisted reproduction technologies such as Intracytoplasmic Sperm Injection (ICSI). In the past, oligospermic males or males with severely impaired sperm motility have been essentially sterile; however techniques such as ICSI and Gamete Intrafallopian Transfer (GIFT) now allow us to minimize or even bypass the requirement for sperm motility. In particular, equine pregnancies have resulted from both ICSI and GIFT. The very real likelihood that genetic defects may be the underlying causes of some cases of severe aberrations of sperm numbers or sperm function in stallions raises the concern that these defects now will be passed on to future generations through the use of assisted reproductive technologies. Studies on the molecular composition of normal stallion sperm will provide new information on the underlying causes of genetic disorders of germ cells. This information will allow the theriogenologist to be more informed about potential genetic defects and may result in diagnostic tests to identify abnormal genes.
Some Questions for the Future
1. Can inhibition/restoration of RII/AKAP82 binding be used as an “off-on” switch for sperm motility?
2. Are single gene defects responsible for some of the less common and more severe cases of infertility seen in the clinic?
3. Are multiple genes involved in the gradations of sperm motility and functionality that are seen so commonly in the clinic?
4. Can we develop genetic screens for alleles that create more motile/functional vs. less motile/functional sperm?
5. Does aging (e.g. senile testicular degeneration) change the expression of key proteins that are required for normal spermatogenesis and sperm function?
1. Bailey JL, Bilodeau JF, Cormier N 2000. Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. Journal of Andrology 21: 1-7.
2. Bedford SJ, Meyers SA,Varner DD in press. Acrosomal status of fresh, cooled and cryopreserved stallion spermatozoa. Journal of Reproduction and Fertility, Suppl 56.
3. Boatman DE, Robbins RS 1991. Bicarbonate:carbon-dioxide regulation of sperm capacitation, hyperactivated motility, and acrosome reactions. Biology of Reproduction 44: 806-813.
4. Bradley MP, Geelan A, Leitch V, Goldberg E 1996. Cloning, sequencing, and characterization of LDH-C4 from a fox testis cDNA library. Molecular Reproduction and Development 44: 452-459.
5. Bunch DO, Welch JE, Magyar PL, Eddy EM, O'Brien DA 1998. Glyceraldehyde 3-phosphate dehydrogenase-S protein distribution during mouse spermatogenesis. Biology of Reproduction 58: 834-841.
6. Burton KA, Treash-Osio B, Muller CH, Dunphy EL, McKnight GS 1999. Deletion of type IIalpha regulatory subunit delocalizes protein kinase A in mouse sperm without affecting motility or fertilization. Journal of Biological Chemistry 274: 24131-24136.
7. Carrera A, Gerton GL, Moss SB 1994. Structural and functional similarities to the A-kinase anchoring proteins. Developmental Biology 165: 272-284.
8. Carrera A, Moos J, Ning X, Gerton G, Tesarik J, Kopf G, Moss S 1996. Regulation of Protein Tyrosine Phosphorylation in Human Sperm by a Calcium/Calmodulin-Dependent Mechanism: Identification of A Kinase Anchor Proteins as Major Substrates for Tyrosine Phosphorylation. Developmental Biology 180:284-296.
9. Clermont Y, Oko R, Hermo L 1990. Immunocytochemical localization of proteins utilized in the formation of outer dense fibers and fibrous sheath in rat spermatids: an electron microscope study. Anatomical Record 227: 447-457.
10. Coghlan VM, Perrino BA, Howard M, Langeberg LK, Hicks JB, Gallatin WM, Scott JD 1995. Association of protein kinase A and protein phosphatase 2B with a common anchoring protein. Science 267: 108-111.
11. Du J, Tao J, Kleinhans FW, Mazur P, Critser, J K 1994. Water volume and osmotic behaviour of mouse spermatozoa determined by electron paramagnetic resonance. Journal of Reproduction and Fertility 101: 37-42.
12. El-Alfy M, Moshonas D, Morales CR, Oko R 1999. Molecular cloning and developmental expression of the major fibrous sheath protein (FS 75) of rat sperm. Journal of Andrology 20: 307-318.
13. Faux MC, Scott JD 1996. Molecular glue: kinase anchoring and scaffold proteins. Cell 85: 9-12.
14. Fawcett DW 1975. The mammalian spermatozoon. Developmental Biology 44: 394-436.
15. Francis SH, Corbin JD 1994. The mammalian spermatozoon. Annual Reviews of Physiology 56: 237-272
16. Galantino-Homer HL, Visconti PE, Kopf GS 1997. Regulation of protein tyrosine phosphorylation during bovine sperm capacitation by a cyclic adenosine 3'5'-monophosphate-dependent pathway. Biology of Reproduction 56: 707-719.
17. Gibbons IR, Rowe A 1965. Dynein: a protein with adenosine triphosphate activity from cilia. Science 149: 424.
18. Holzbaur EL, Vallee RB 1994. DYNEINS: molecular structure and cellular function. Annual Reviews of Cell and Developmental Biology 10: 339-372.
19. Horowitz JA, Toeg H, Orr GA 1984. Characterization and localization of cAMP-dependent protein kinases in rat caudal epididymal sperm. Journal of Biological Chemistry 25: 832-838.
20. Horowitz JA, Voulala, P, Wasco W, MacLeod J, Paupard MC, Orr GA 1989. Biochemical and immunological characterization of the flagellar-associated regulatory subunit of a type II cyclic adenosine 5'-monophosphate-dependent protein kinase. Archives of Biochemistry and Biophysics 270: 411-418.
21. Klauck TM, Faux MC, Labudda K, Langeberg LK, Jaken S, Scott JD 1996. Coordination of three signaling enzymes by AKAP79, a mammalian scaffold protein. Science 27: 1589-1591.
22. Leclerc P, de Lamirande E, Gagnon C 1996. Cyclic adenosine 3',5'monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility. Biology of Reproduction 55: 684-692.
23. Lindemann CB, Orlando A, Kanous KS 1992. The flagellar beat of rat sperm is organized by the interaction of two functionally distinct populations of dynein bridges with a stable central axonemal partition. Journal of Cell Science 102: 249-260.
24. Llano MN, Meizel S 1983. Phospholipid methylation increases during capacitation of golden hamster sperm in vitro. Biology of Reproduction 28: 1043-1051.
25. Macleod J, Mei X, Erlichman J, Orr GA 1994. Association of the regulatory subunit of a type II cAMP-dependent protein kinase and its binding proteins with the fibrous sheath of rat sperm flagellum. European Journal of Biochemistry 225: 107-114.
26. Milisav I 1998. Dynein and dynein-related genes. Cell Motility and the Cytoskeleton 39: 261-272.
27. Mori C, Nakamura N, Welch JE, Gotoh H, Goulding EH Fujioka, Eddy EM 1998. Mouse spermatogenic cell-specific type 1 hexokinase (mHk1-s) transcripts are expressed by alternative splicing from the mHk1 gene and the HK1-S protein is localized mainly in the sperm tail. Molecular Reproduction and Development 49: 374-385.
28. Moss SB, Turner RM, Burkert KL, VanScoy Butt H, Gerton GL 1999. Conservation and function of a bovine sperm A-kinase anchor protein homologous to mouse AKAP82. Biology of Reproduction 61: 335-342.
29. Neill JM, Olds-Clarke P 1987. A computer-assisted assay for mouse sperm hyperactivation demonstrates that bicarbonate but not bovine serum albumin is required. Gamete Research 18: 121-140.
30. Olds-Clarke, P 1989. Sperm from tw32/+ mice: capacitation is normal, but hyperactivation is premature and nonhyperactivated sperm are slow. Developmental Biology 131: 475-482.
31. Pariset C, Weinman S 1994. Differential localization of two isoforms of the regulatory subunit RII alpha of cAMP-dependent protein kinase in human sperm: biochemical and cytochemical study. Molecular and Reproductive Development 39: 415-422.
32. Porter ME, Johnson KE 1989. Differential localization of two isoforms of the regulatory subunit RII alpha of cAMP-dependent protein kinase in human sperm: biochemical and cytochemical study. Annual Reviews of Cell and Developmental Biology 5: 119-151.
33. Rosenberger A, Meyer SA, Galantino-Homer H 1998. Tyrosine phosphorylation of stallion sperm during in vitro capacitation. Biology of Reproduction 58: 76-77.
34. Rubin CS 1994. A kinase anchor proteins and the intracellular targeting of signals carried by AMP. Biochem Biophys Acta 1224: 407-479.
35. San Augustin JT, Witman GB 1994. Role of cAMP in the reactivation of demembranated ram spermatozoa. Cell Motility and the Cytoskeleton 27: 206 - 218.
36. Si Y 1999. Hyperactivation of hamster sperm motility by temperature-dependent tyrosine phosphorylation of an 80-kDa protein. Biology of Reproduction 61: 247-252.
37. Si Y, Okuno M 1999. Role of tyrosine phosphorylation of flagellar proteins in hamster serm hperactivation. Biology of Reproduction 61: 240-246.
38. Stauss CR, Votta TJ, Suarez SS 1995. Sperm motility hyperactivation facilitates penetration of the hamster zona pellucida. Biology of Reproduction 53: 1280-1285.
39. Storey BT, Kayne FJ 1975. Energy metabolism of spermatozoa. V. The Embden-Myerhof pathway of glycolysis: activities of pathway enzymes in hypotonically treated rabbit epididymal spermatozoa. Fertility and Sterility 26: 1257-1265.
40. Tash JS 1989. Protein phosphorylation: the second messenger signal transducer of flagellar motility. Cell Motility and the Cytoskeleton 14: 332-339.
41. Tash JS, Means AR 1982. Regulation of protein phosphorylation and motility of sperm by cyclic adenosine monophosphate and calcium. Biology of Reproduction 26: 745-763.
42. Tash JS, Means AR 1983. . Cyclic adenosine 3',5' monophosphate, calcium and protein phosphorylation in flagellar motility. Biology of Reproduction 28: 75-104.
43. Taylor SS, Buechler JA, Yonemoto W 1990. cAMP-dependent protein kinase: Framework for a diverse family of regulatory enzymes. Annual Reviews of Biochemistry 59: 971-1005.
44. Travis AJ, Foster JA, Rosenbaum NA, Visconti PE, Gerton GL, Kopf GS, Moss SB 1998. Targeting of a germ cell-specific type 1 hexokinase lacking a porin- binding domain to the mitochondria as well as to the head and fibrous sheath of murine spermatozoa. Molecular Biology of the Cell 9: 263-276.
45. Turner RM, Eriksson RL, Gerton GL, Moss SB 1999. Relationship between sperm motility and the processing and tyrosine phosphorylation of two human sperm fibrous sheath proteins, pro-hAKAP82 and hAKAP82. Molecular Human Reproduction 5: 816-824.
46. Turner RM, Johnson LJ, Haig-Ladewig L, Gerton GL, Moss SB 1998. An X-linked gene encodes a major human sperm fibrous sheath protein, hAKAP82. Genomic organization, protein kinase A-RII binding, and distribution of the precursor in the sperm tail. Journal of Biological Chemistry 273: 32135-32141.
47. Vijayaraghavan S, Goueli SA, Davey MP, Carr DW 1997. A-anchoring inhibitor peptides arrest mammalian sperm motility. Journal of Biological Chemistry 272: 4747-4752.
48. Visconti P, Johnson L, Oyaski M, Fornes M, Moss S, Gerton G, Kopf G 1997. Regulation, localization and anchoring of protein kinase A subunits during mouse sperm capacitation. Developmental Biology 192: 351-363.
49. Visconti PE, Moore, GD, Bailey JL, Leclerc P, Connors SA, Pan D, Olds-Clarke P, Kopf GS 1995. Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Development 121: 1139-1150.
50. Westhoff D, Kamp G 1997. Glyceraldehyde 3-phosphate dehydrogenase is bound to the fibrous sheath of mammalian spermatozoa. Journal of Cell Science 110: 1821-1829.
Thanks to Dr. George Gerton and Dr. Stuart Moss for their advice and critical reading of this abstract. Thanks to Dr. Hannah Galantino-Homer, Dr. Stuart Meyers and Dr. Esther Noiles for allowing me to comment on some of their unpublished work.