Stuart B. Moss

Functional Relationships between Capacitation-dependent Cell Signaling and Compartmentalized Metabolic Pathways in Murine Spermatozoa

Spermatozoa are highly polarized cells with specific metabolic pathways compartmentalized in different regions. Previously, we hypothesized that glycolysis is organized in the fibrous sheath of the flagellum to provide ATP to dynein ATPases that generate motility and to protein kinases that regulate motility. Although a recent report suggested that glucose is not essential for murine sperm capacitation, we demonstrated that glucose (but not lactate or pyruvate) was necessary and sufficient to support the protein tyrosine phosphorylation events associated with capacitation. The effect of glucose on this signaling pathway was downstream of cAMP, and appeared to arise indirectly as a consequence of metabolism as opposed to a direct signaling effect. Moreover, the phosphorylation events were not affected by uncouplers of oxidative respiration, inhibitors of electron transfer, or by a lack of substrates for oxidative respiration in the medium. Further experiments aimed at identifying potential regulators of sperm glycolysis focused on a germ cell-specific isoform of hexokinase, HK1-SC, which localizes to the fibrous sheath. HK1-SC activity and biochemical localization did not change during sperm capacitation, suggesting that glycolysis in sperm is regulated either at the level of substrate availability or by downstream enzymes. These data support the hypothesis that ATP specifically produced by a compartmentalized glycolytic pathway in the principal piece of the flagellum, as opposed to ATP generated by mitochondria in the mid-piece, is strictly required for protein tyrosine phosphorylation events that take place during sperm capacitation. The relationship between these pathways suggests that spermatozoa offer a model system for the study of integration of compartmentalized metabolic and signaling pathways.

Proteomic Profiling of Accessory Structures from the Mouse Sperm Flagellum

The flagellum of a mammalian spermatozoon consists of an axoneme surrounded in distinct regions by accessory structures known as the fibrous sheath, outer dense fibers, and the mitochondrial sheath. Although the characterization of individual proteins has provided clues about the roles of these accessory structures, a more complete understanding of flagellar function requires the identification of all the polypeptides in these assemblies. Epididymal mouse sperm were treated with SDS to dislodge sperm heads and to extract the axoneme and membranous elements. The remaining flagellar accessory structures were purified by sucrose gradient centrifugation. Analysis of proteins from these structures by two-dimensional gel electrophoresis and colloidal Coomassie Blue staining showed a highly reproducible pattern of >200 spots. Individual spots were picked, digested with trypsin, and identified by mass spectrometry and peptide microsequencing. Approximately 50 individual proteins were identified that could be assigned to five general categories: 1) proteins previously reported to localize to the accessory structures, e.g. ODF2 in the outer dense fibers, the sperm-specific glyceraldehyde-3-phosphate dehydrogenase in the fibrous sheath, and glutathione peroxidase in the mitochondrial sheath, validating this proteomic approach; 2) proteins that had not been shown to localize to any accessory structure but would be predicted to be present, e.g. glycolytic enzymes; 3) proteins known to be part of the flagellum but not localized to a specific site, e.g. adenylate kinase; 4) proteins not expected to be part of the accessory structures based on their previously reported locations, e.g. tektins; and 5) unknown proteins for which no information is available to make a determination as to location. The unexpected presence of the tektins in the accessory structures of the flagellum was confirmed by both immunoblot and immunofluorescence analysis. This proteomic analysis identified a number of unexpected and novel proteins in the accessory structures of the mammalian flagellum.

The circadian clock protein BMAL1 is necessary for fertility and proper testosterone production in mice.

Although it is well established that the circadian clock regulates mammalian reproductive physiology, the molecular mechanisms by which this regulation occurs are not clear. The authors investigated the reproductive capacity of mice lacking Bmal1 (Arntl, Mop3), one of the central circadian clock genes. They found that both male and female Bmal1 knockout (KO) mice are infertile. Gross and microscopic inspection of the reproductive anatomy of both sexes suggested deficiencies in steroidogenesis. Male Bmal1 KO mice had low testosterone and high luteinizing hormone serum concentrations, suggesting a defect in testicular Leydig cells. Importantly, Leydig cells rhythmically express BMAL1 protein, suggesting peripheral control of testosterone production by this clock protein. Expression of steroidogenic genes was reduced in testes and other steroidogenic tissues of Bmal1 KO mice. In particular, expression of the steroidogenic acute regulatory protein (StAR) gene and protein, which regulates the rate-limiting step of steroidogenesis, was decreased in testes from Bmal1 KO mice. A direct effect of BMAL1 on StAR expression in Leydig cells was indicated by in vitro experiments showing enhancement of StAR transcription by BMAL1. Other hormonal defects in male Bmal1 KO mice suggest that BMAL1 also has functions in reproductive physiology outside of the testis. These results enhance understanding of how the circadian clock regulates reproduction.

Deficiency of SPAG16L Causes Male Infertility Associated with Impaired Sperm Motility

Abstract The axonemes of cilia and flagella contain a “9+2” structure of microtubules and associated proteins. Proteins associated with the central doublet pair have been identified in Chlamydomonas that result in motility defects when mutated. The murine orthologue of the Chlamydomonas PF20 gene, sperm-associated antigen 16 (Spag16), encodes two proteins of Mr ∼71 × 103 (SPAG16L) and Mr ∼35 × 103 (SPAG16S). In sperm, SPAG16L is found in the central apparatus of the axoneme. To determine the function of SPAG16L, gene targeting was used to generate mice lacking this protein but still expressing SPAG16S. Mutant animals were viable and showed no evidence of hydrocephalus, lateralization defects, sinusitis, bronchial infection, or cystic kidneys—symptoms typically associated with ciliary defects. However, males were infertile with a lower than normal sperm count. The sperm had marked motility defects, even though ultrastructural abnormalities of the axoneme were not evident. In addition, the testes of some nullizygous animals showed a spermatogenetic defect, which consisted of degenerated germ cells in the seminiferous tubules. We conclude that SPAG16L is essential for sperm flagellar function. The sperm defect is consistent with the motility phenotype of the Pf20 mutants of Chlamydomonas, but morphologically different in that the mutant algal axoneme lacks the central apparatus.

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

Mammalian sperm motility is regulated by a cascade of cAMP-dependent protein phosphorylation events mediated by protein kinase A. A-kinaseanchor proteins (AKAPs) direct protein kinase A activity by tethering the enzyme near its physiological substrates. We have characterized a major human sperm fibrous sheath AKAP, hAKAP82, and its precursor, pro-hAKAP82, the homologues of the mouse fibrous sheath proteins mAKAP82 and pro-mAKAP82. The cDNA sequence of pro-hAKAP82 was highly homologous to the mouse sequence, and the functional domains of the pro-hAKAP82 protein, the protein kinase A binding, and the pro-hAKAP82/hAKAP82 cleavage sites were identical to those of the mouse protein. The genomic organization of mousepro-AKAP82 was determined. Alternative splicing occurred in both the mouse and human pro-AKAP82 genes that resulted in at least two distinct transcripts and possibly two different proteins. Compared with pro-mAKAP82, considerably less pro-hAKAP82 was processed to hAKAP82 in human sperm. Although pro-mAKAP82 localizes only to the proximal portion of the principal piece of the flagellum, pro-hAKAP82 localized to the entire length of the principal piece. The pro-hAKAP82 gene mapped to human chromosome Xp11.2, indicating that defects in this gene are maternally inherited. These studies suggest several roles for hAKAP82 in sperm motility, including the regulation of signal transduction pathways.

Dissecting the Axoneme Interactome The Mammalian Orthologue of Chlamydomonas PF6 Interacts with Sperm-Associated Antigen 6, The Mammalian Orthologue of Chlamydomonas PF16

The axoneme central apparatus is thought to control flagellar/ciliary waveform and maintain the structural integrity of the axoneme, but proteins involved in these processes have not been fully elucidated. Moreover the network of interactions among them that allows these events to take place in a compact space has not been defined. PF6, a component of the Chlamydomonas central apparatus, is localized to the 1a projection of the C1 microtubule. Mutations in the Chlamydomonas PF6 gene result in flagellar paralysis. We characterized human and murine orthologues of PF6. The murine Pf6 gene is expressed in a pattern consistent with a role in flagella and cilia, and the PF6 protein is indeed localized to the central apparatus of the sperm flagellar axoneme. We discovered that a portion of PF6 associates with the mammalian orthologue of Chlamydomonas PF16 (sperm-associated antigen 6 (SPAG6)), another central apparatus protein that is localized to the C1 microtubule in algae. A fragment of PF6 corresponding to the PF6 domain that interacts with SPAG6 in yeast two-hybrid assays and colocalizes with SPAG6 in transfected cells was missing from epididymal sperm of SPAG6-deficient mice. SPAG6 binds to the mammalian orthologue of PF20, which in Chlamydomonas is located in bridges connecting the C2 and C1 microtubules. Thus, PF6, SPAG6, and PF20 form a newly identified network that links together components of the axoneme central apparatus and presumably participates in its dynamic regulation of ciliary and flagellar beat.

Adenylate Kinases 1 and 2 Are Part of the Accessory Structures in the Mouse Sperm Flagellum

Abstract Proper sperm function depends on adequate ATP levels. In the mammalian flagellum, ATP is generated in the midpiece by oxidative respiration and in the principal piece by glycolysis. In locations where ATP is rapidly utilized or produced, adenylate kinases (AKs) maintain a constant adenylate energy charge by interconverting stoichiometric amounts of ATP and AMP with two ADP molecules. We previously identified adenylate kinase 1 and 2 (AK1 and AK2) by mass spectrometry as part of a mouse SDS-insoluble flagellar preparation containing the accessory structures (fibrous sheath, outer dense fibers, and mitochondrial sheath). A germ cell-specific cDNA encoding AK1 was characterized and found to contain a truncated 3′ UTR and a different 5′ UTR compared to the somatic Ak1 mRNA; however, it encoded an identical protein. Ak1 mRNA was upregulated during late spermiogenesis, a time when the flagellum is being assembled. AK1 was first seen in condensing spermatids and was associated with the outer microtubular doublets and outer dense fibers of sperm. This localization would allow the interconversion of ATP and ADP between the fibrous sheath where ATP is produced by glycolysis and the axonemal dynein ATPases where ATP is consumed. Ak2 mRNA was expressed at relatively low levels throughout spermatogenesis, and the protein was localized to the mitochondrial sheath in the sperm midpiece. AK1 and AK2 in the flagellar accessory structures provide a mechanism to buffer the adenylate energy charge for sperm motility.

AKAP7γ is a nuclear RI-binding AKAP ☆

Spatial regulation of protein kinase A (PKA) is accomplished by its sequestration via A-kinase anchor proteins (AKAPs). PKA activity is critical for mammalian oocyte development, suggesting that PKA must be appropriately positioned in these large cells. A screen for AKAPs in oocytes identified AKAP7γ, an AKAP originally found in pancreas. Yeast two-hybrid analysis and co-immunoprecipitation studies showed that AKAP7γ bound the type I PKA regulatory subunit (RI) and that the RI-binding domain overlapped the previously identified type II PKA regulatory subunit (RII) binding domain. Overexpressed AKAP7γ localized to the nuclei of HEK 293 cells via a nuclear localization signal. In addition, endogenous AKAP7γ protein was found in both the nucleus and cytoplasm of oocytes. This work identifies AKAP7γ as the first nuclear AKAP to bind RI and suggests that AKAP7γ may be responsible for positioning PKA via RI and/or RII to regulate PKA-mediated gene transcription in both somatic cells and oocytes.

Dissecting the axoneme interactome: The mammalian orthologue of chlamydomonas PF6 interacts with SPAG6, the mammalian orthologue of chlamydomonas PF16

The axoneme central apparatus is thought to control flagellar/ciliary waveform and maintain the structural integrity of the axoneme, but proteins involved in these processes have not been fully elucidated. Moreover the network of interactions among them that allows these events to take place in a compact space has not been defined. PF6, a component of the Chlamydomonas central apparatus, is localized to the 1a projection of the C1 microtubule. Mutations in the Chlamydomonas PF6 gene result in flagellar paralysis. We characterized human and murine orthologues of PF6. The murine Pf6 gene is expressed in a pattern consistent with a role in flagella and cilia, and the PF6 protein is indeed localized to the central apparatus of the sperm flagellar axoneme. We discovered that a portion of PF6 associates with the mammalian orthologue of Chlamydomonas PF16 (sperm-associated antigen 6 (SPAG6)), another central apparatus protein that is localized to the C1 microtubule in algae. A fragment of PF6 corresponding to the PF6 domain that interacts with SPAG6 in yeast two-hybrid assays and colocalizes with SPAG6 in transfected cells was missing from epididymal sperm of SPAG6-deficient mice. SPAG6 binds to the mammalian orthologue of PF20, which in Chlamydomonas is located in bridges connecting the C2 and C1 microtubules. Thus, PF6, SPAG6, and PF20 form a newly identified network that links together components of the axoneme central apparatus and presumably participates in its dynamic regulation of ciliary and flagellar beat.

Properties and localization of a tyrosine phosphorylated form of hexokinase in mouse sperm

Mouse sperm possess a phosphotyrosine‐containing hexokinase type 1 (HK1) that is associated with the plasma membrane fraction of these cells (Kalab et al., 1994; J. Biol Chem 269:3810–3817.). This apparent plasma membrane association appears unique, since somatic HK1 is normally cytoplasmic or bound to the outer mitochondrial membrane via contact sites with a voltage‐dependent anion channel (porin) through a porin‐binding domain. In male germ cells, three cDNA clones have been described that encode unique HK1 isoforms (HK1‐sa, HK1‐sb, HK1‐sc) that do not contain porin binding domains (Mori et al., 1993: Biol Reprod 49:191–203). This suggests that these proteins might not be localized to the outer mitochondrial membrane and could have alternative functions in germ cells and/or sperm. We demonstrate in the mouse that male germ cells and sperm could potentially express four HK1 isoforms (HK1‐sa, HK1‐sb, HK1‐sc, and the somatic HK1). At the protein level, at least one of the HK1 isoforms becomes phosphorylated on tyrosine residues during spermatogenesis. Treatment of sperm membrane fractions to dissociate the phosphotyrosine‐containing HK1 (pY‐mHK1) yields results demonstrating that pY‐mHK1 has properties of an integral membrane protein. Indirect immunofluorescence using a monoclonal antibody to HK1 demonstrates specific staining both in the head and tail regions of sperm. Surface biotinylation of intact sperm followed by precipitation with either polyclonal HK1 antiserum or with avidin‐Sepharose suggests that pY‐mHK1 possesses an extracellular domain. These results suggest that mouse sperm contain at least one HK1 isoform that is present on the sperm head, has an extracellular domain, and behaves as an integral membrane protein.