Contraceptive and Infertility Target DataBase: a contraceptive drug development tool for targeting and analysis of human reproductive specific tissues†.

The long and challenging drug development process begins with discovery biology for the selection of an appropriate target for a specific indication. Target is a broad term that can be applied to a range of biological entities such as proteins, genes, and ribonucleic acids (RNAs). Although there are numerous databases available for mining biological entities, publicly available searchable, downloadable databases to aid in target selection for a specific disease or indication (e.g., developing contraceptives and infertility treatments) are limited. We report the development of the Contraceptive and Infertility Target DataBase (https://www.citdbase.org), which provides investigators an interface to mine existing transcriptomic and proteomic resources to identify high-quality contraceptive/infertility targets. The development of similar databases is applicable to the identification of targets for other diseases and conditions.

Octogenarians and aortic valve surgery: surgical outcomes in the geriatric population.

BACKGROUND: The era of percutaneous aortic valve intervention has challenged the continuing indication for surgical aortic valve replacement (SAVR). AIM: The aim of this study is to evaluate clinical outcomes of the elderly patients who underwent surgical aortic valve replacement via median sternotomy, in order to assess the impact of surgery on patient outcomes and discharge destination. METHODS: The study involves a retrospective observational analysis in a single centre, including all octogenarian patients who underwent aortic valve surgery between January of 2011 and July of 2016. The study assessed pre-operative co-morbidities and post-operative outcomes, including long-term mortality and discharge destination following on from surgery. RESULTS: The mean age of patients was 82.7 years (± 2.9), 67% of whom were male. The mean EuroSCORE II was 8.1 (± 7.6). The most common pre-operative co-morbidities were dyslipidaemia (82%), hypertension (80%), and ischaemic heart disease (78.8%). The median length of stay was 10 days (± 6.9 days). Discharge home occurred in 71.8% of patients, with 21.2% of patients requiring transfer to a rehabilitation facility, and 1.2% of patients required placement into an aged care facility. There were five peri-operative deaths, equating to 5.9% of the cohort. CONCLUSION: Despite high EuroSCORE II values for the majority of our patients, our data adds to overall suggestions that the octogenarian population can be considered eligible for SAVR and should not be excluded due to age alone. The use of the EuroSCORE II index more accurately predicts adequacy for treatment however does not entirely predict overall course of events, and proceduralist discretion should still be used.

A systematic review and meta-analysis of the clinical outcomes of TAVI versus SAVR in the octogenarian population.

BACKGROUND: Surgical aortic valve replacement (SAVR) has shown safe, robust results in elderly populations, and up until recently, was the gold standard for management of severe aortic stenosis. The approach to severe aortic stenosis in high-risk populations, such as octogenarians, has been challenged with the development of transcatheter-based strategies. We sought to systematically analyse outcomes between surgical and transcatheter aortic valve replacement (TAVI) in octogenarians. METHOD: Electronic databases were searched from their inception until November 2018 for studies comparing SAVR to TAVI in octogenarians, according to a predefined search criterion. The primary end point was mortality, and secondary end points included post-procedural complications. RESULTS: The review yielded four observational studies. The total number of patients included was 1221 including 395 who underwent TAVI and 826 SAVR. On average, patients from both subgroups carried a high number of cardiac risk factors, and STS-PROM scoring yielded mean values equating to high-risk population groups, with significantly higher values for TAVI patients across the board. The presence of post-procedural moderate aortic regurgitation was noted only in the TAVI population (OR = 8.88; 95% CI (1.47-53.64), χ2 = 1.22; p = 0.02; I 2 = 0%). Otherwise, there were no significant differences when accounting for mortality (OR = 0.68; 95% CI (0.44-1.05), χ2 = 1.88; p = 0.60; I 2 = 0%), permanent pacemaker implantation groups (OR = 0.45; 95% CI (0.44-1.49), χ2 = 0.11; p = 0.19; I 2 = 0%), and neurological events (OR = 0.72; 95% CI (0.42-1.23), χ2 = 2.57; p = 0.23; I 2 = 22%). DISCUSSION: The analysed data on TAVI versus SAVR in the octogenarian population show that TAVI shows similar outcomes with relation to mortality and inpatient admission times, in a population with significantly higher risk profiles than their SAVR counterparts. TAVI has higher occurrences of post-procedural AR. TAVI still does not have robust long-term data to ensure its efficacy and rate of complications, but is showing promising results nonetheless.

Outcomes of patients who undergo percutaneous coronary intervention with covered stents for coronary perforation: A systematic review and pooled analysis of data.

OBJECTIVES: This review aims to evaluate the adverse outcomes for patients after treatment with covered stents. BACKGROUND: Coronary perforation is a potentially fatal complication of percutaneous coronary revascularization which may be treated using covered stents. Studies have evaluated long-term outcomes among patients who received these devices, but hitherto no literature review has taken place. METHODS: We conducted a systematic review of adverse outcomes for patients after treatment with covered stents. Data from studies were pooled and outcomes were compared according to stent type. RESULTS: A total of 29 studies were analyzed with data from 725 patients who received covered stents. The proportion of patients with chronic total occlusions, vein graft percutaneous coronary intervention (PCI), intracoronary imaging and rotational atherectomy were 16.9, 11.5, 9.2, and 6.6%, respectively. The stents used were primarily polytetrafluoroethylene (PTFE) (70%) and Papyrus (20.6%). Mortality, major adverse cardiovascular events, pericardiocentesis/tamponade and emergency surgery were 17.2, 35.3, 27.1, and 5.3%, respectively. Stratified analysis by use of PTFE, Papyrus and pericardial stents, suggested no difference in mortality (p = .323), or target lesion revascularization (p = .484). Stent thrombosis, pericardiocentesis/tamponade and emergency coronary artery bypass surgery (CABG) occurred more frequently in patients with PTFE stent use (p = .011, p = .005, p = .012, respectively). In-stent restenosis was more common with pericardial stent use (<.001, pooled analysis for first- and second-generation pericardial stents). CONCLUSIONS: Cases of coronary perforation which require implantation of a covered stent are associated with a high rate of adverse outcomes. The use of PTFE covered stents appears to be associated with more stent thrombosis, pericardiocentesis/tamponade, and emergency CABG when compared to Papyrus or pericardial stents.

Complex disease management of pregnant young patient with familial hypercholesterolaemia complicated by coronary artery disease and cerebrovascular disease.

Inherited disorders of lipid metabolism may cause a heavy burden of cardiovascular disease early in life. Familial hypercholesterolaemia (FH) with abnormalities of LDL metabolism results in marked LDL elevations and accelerated, multivessel atherosclerosis presenting in teenage or young adulthood. We describe the case of a 33-year-old woman who presented with exertional angina in the setting of pregnancy who was found post-partum to have severe triple-vessel disease including left main disease on coronary angiography (Figs. 1 and 2). She was also noted to have a typical supravalvular "hourglass" [1] abnormality of the aortic root (Fig. 3), and heavy calcification of the proximal aorta precluding conventional aortic cross clamping and bypass surgery. After discussion with the multidisciplinary team, her disease was felt to be amenable to a beating-heart coronary bypass technique with an anaortic approach to minimise the possibility of cerebral embolism. Significant extracranial cerebrovascular disease, a major risk for cardiopulmonary bypass, reinforced the beating-heart technique. Her ongoing management has consisted of medical therapy with cessation of breast feeding, statins, ezetimibe, and introduction of PCSK9-inhibitor therapy. This case illustrates a number of the difficulties associated with management of widespread atherosclerotic disease associated with FH, in which an excellent outcome was achieved with the assistance of a multi-disciplinary team.

The sixth vital sign: what reproduction tells us about overall health. Proceedings from a NICHD/CDC workshop.

STUDY QUESTION: Does the fertility status of an individual act as a biomarker for their future health? SUMMARY ANSWER: Data support an association between reproductive health and overall health for men and women. WHAT IS ALREADY KNOWN: Various chronic conditions, such as diabetes, obesity and cancer, can compromise fertility, but there are limited data for the converse situation, in which fertility status can influence or act as a marker for future health. Data reveal an association between infertility and incident cardiovascular disease and cancer in both men and women. STUDY DESIGN SIZE AND DURATION: A National Institute of Child Health and Human Development-Centers for Disease Control and Prevention workshop in April 2016 was convened that brought together experts in both somatic diseases and conditions, and reproductive health. Goals of the workshop included obtaining information about the current state of the science linking fertility status and overall health, identifying potential gaps and barriers limiting progress in the field, and outlining the highest priorities to move the field forward. PARTICIPANTS/MATERIALS SETTING AND METHODS: Approximately 40 experts participated in the workshop. MAIN RESULTS AND THE ROLE OF CHANCE: While the etiology remains uncertain for infertility, there is evidence for an association between male and female infertility and later health. The current body of evidence suggests four main categories for considering biological explanations: genetic factors, hormonal factors, in utero factors, and lifestyle/health factors. These categories would be key to include in future studies to develop a comprehensive and possibly standardized look at fertility status and overall health. Several themes emerged from the group discussion including strategies for maximizing use of existing resources and databases, the need for additional epidemiologic studies and public health surveillance, development of strategies to frame research so results could ultimately influence clinical practice, and the identification of short and long-term goals and the best means to achieve them. LIMITATIONS REASONS FOR CAUTION: Further research may not indicate an association between fertility status and overall health. WIDER IMPLICATIONS OF THE FINDINGS: Currently medical care is compartmentalized. Reproductive medicine physicians treat patients for a short period of time before they transition to others for future care. Going forward, it is critical to take an interdisciplinary patient care approach that would involve experts in a broad range of medical specialties in order to more fully understand the complex interrelationships between fertility and overall health. If infertility is confirmed as an early marker of chronic disease then screening practices could be adjusted, as they are for patients with a family history of malignancy. STUDY FUNDING/COMPETING INTERESTS: Funding for the workshop was provided by the Fertility and Infertility Branch, National Institute of Child Health and Human Development, National Institutes of Health and the Division of Reproductive Health, National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control. There are no conflicts of interest to declare. The findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention or the National Institutes of Health. TRIAL REGISTRATION NUMBER: Not applicable.

Transducin-like enhancer of split-6 (TLE6) is a substrate of protein kinase A activity during mouse oocyte maturation.

Fully grown oocytes in the ovary are arrested at prophase of meiosis I because of high levels of intraoocyte cAMP that maintain increased levels of cAMP-dependent protein kinase (PKA) activity. Following the luteinizing hormone surge at the time of ovulation, cAMP levels drop, resulting in a reduction in PKA activity that triggers meiotic resumption. Although much is known about the molecular mechanisms of how PKA activity fluctuations initiate the oocyte's reentry into meiosis, significantly less is known about the requirement for PKA activity in the oocyte after exit from the prophase I arrest. Here we show that although PKA activity decreases in the oocyte upon meiotic resumption, it increases throughout meiotic progression from the time of germinal vesicle breakdown (GVBD) until the metaphase II (MII) arrest. Blocking this meiotic maturation-associated increase in PKA activity using the pharmacological inhibitor H89 resulted in altered kinetics of GVBD, defects in chromatin and spindle dynamics, and decreased ability of oocytes to reach MII. These effects appear to be largely PKA specific because inhibitors targeting other kinases did not have the same outcomes. To determine potential proteins that may require PKA phosphorylation during meiosis, we separated oocyte protein extracts on an SDS-PAGE gel, extracted regions of the gel that had corresponding immune reactivity towards an anti-PKA substrate antibody, and performed mass spectrometry and microsequencing. Using this approach, we identified transducin-like enhancer of split-6 (TLE6)-a maternal effect gene that is part of the subcortical maternal complex-as a putative PKA substrate. TLE6 localized to the oocyte cortex throughout meiosis in a manner that is spatially and temporally consistent with the localization of critical PKA subunits. Moreover, we demonstrated that TLE6 becomes phosphorylated in a narrow window following meiotic resumption, and H89 treatment can completely block this phosphorylation when added prior to GVBD but not after. Taken together, these results highlight the importance of oocyte-intrinsic PKA in regulating meiotic progression after the prophase I arrest and offer new insights into downstream targets of its activity.

Extracellular adenosine 5'-triphosphate alters motility and improves the fertilizing capability of mouse sperm.

Extracellular adenosine 5'-triphosphate (ATPe) treatment of human sperm has been implicated in improving in vitro fertilization (IVF) results. We used the mouse model to investigate mechanisms of action of ATPe on sperm. ATPe treatment significantly enhanced IVF success as indicated by both rate of pronuclear formation and percentage cleavage to the 2-cell stage. However, ATPe did not increase the percentage of sperm undergoing spontaneous acrosomal exocytosis nor change the pattern of protein tyrosine phosphorylation normally observed in capacitated sperm. ATPe altered sperm motility parameters; in particular, both noncapacitated and capacitated sperm swam faster and straighter. The percentage of hyperactivated sperm did not increase in capacitated ATPe-treated sperm compared to control sperm. ATPe induced a rapid increase in the level of intracellular calcium that was inhibited by two distinct P2 purinergic receptor inhibitors, confirming that these receptors have an ionotropic role in sperm function. The observed motility changes likely explain, in part, the improved fertilizing capability when ATPe-treated sperm were used in IVF procedures and suggest a mechanism by which ATPe treatment may be beneficial for artificial reproductive techniques.

Recombinant mouse sperm ZP3-binding protein (ZP3R/sp56) forms a high order oligomer that binds eggs and inhibits mouse fertilization in vitro.

Many candidates have been proposed as zona pellucida-binding proteins. Without precluding a role for any of those candidates, we focused on mouse sperm protein ZP3R/sp56, which is localized in the acrosomal matrix. The objective of this study was to analyze the role of ZP3R/sp56 in mouse fertilization. We expressed recombinant ZP3R/sp56 as a secreted protein in HEK293 cells and purified it from serum-free, conditioned medium. In the presence of reducing agents, the recombinant ZP3R/sp56 exhibited a molecular weight similar to that observed for the native ZP3R/sp56. Reminiscent of the native protein, recombinant ZP3R/sp56 formed a high molecular weight, disulfide cross-linked oligomer consisting of six or more monomers under non-reducing conditions. Recombinant ZP3R/sp56 bound to the zona pellucida of unfertilized eggs but not to 2-cell embryos, indicating that the changes that take place in the zona pellucida at fertilization affected the interaction of this protein with the zona pellucida. The extent of in vitro fertilization was reduced in a dose-dependent manner when unfertilized eggs were preincubated with recombinant ZP3R/sp56 (74% drop at the maximum concentrations assayed). Eggs incubated with the recombinant protein showed an absence of or very few sperm in the perivitelline space, suggesting that the reduction in the fertilization rate is caused by the inhibition of sperm binding and/or penetration through the zona pellucida. These results indicate that sperm ZP3R/sp56 is important for sperm-zona interactions during fertilization and support the concept that the acrosomal matrix plays an essential role in mediating the binding of sperm to the zona pellucida.

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.

Effects of extracellular adenosine 5'-triphosphate on human sperm motility.

Extracellular adenosine 5'-triphosphate (ATP) previously has been shown to increase the fertilization percentage in human in vitro fertilization (IVF) performed for male factor infertility. The objective of this study is to determine the effects of extracellular adenosine 5'-triphosphate (ATPe) on human sperm function by examining its effects on end points of sperm capacitation. Sperm obtained from healthy volunteers with normal semen parameters, asthenozoospermic men, and cryopreserved samples were incubated in medium with or without 2.5 mM ATPe. The effects of ATPe on acrosomal exocytosis, protein tyrosine phosphorylation, and sperm motility parameters were quantified. Although ATPe did not affect acrosomal exocytosis or protein tyrosine phosphorylation in sperm from healthy donors, it significantly altered several motility parameters, with the largest effects manifested in increased curvilinear velocity and percentage hyperactivation. ATPe similarly affected sperm selected for poor motility and thawed cryopreserved sperm but to a lesser extent than its effects on sperm with normal motility. ATPe increased straight-line velocity and linearity of sperm obtained from asthenozoospermic men. Human sperm motility characteristics are altered by ATPe; this finding may explain its previously reported beneficial effect on human IVF. These results suggest that ATPe could constitute a new therapeutic modality in the treatment of male infertility.

Knockdown of the cAMP-dependent protein kinase (PKA) Type Ialpha regulatory subunit in mouse oocytes disrupts meiotic arrest and results in meiotic spindle defects.

In mammalian oocytes, cyclic AMP-dependent protein kinase (PKA) is responsible for maintaining meiotic arrest. We examined the role of the predominant regulatory subunit, RIalpha in regulating PKA activity during mouse oocyte maturation by knocking down the protein levels using an RNA interference approach. In oocytes in which RIalpha protein was reduced to non-detectable levels, compensatory decreases were also observed in the RIIalpha and catalytic (Calpha) subunit levels. These oocytes resumed meiosis, despite culture under conditions that maintain elevated intracellular cAMP levels, suggesting that the remaining Calpha was not sufficient to maintain meiotic arrest. The resulting eggs, however, displayed meiotic spindle abnormalities and abnormal cleavage planes leading to extrusion of large polar bodies. These results demonstrate that RIalpha is required for regulating PKA activity in maturing oocytes and that compensatory upregulation of RII does not occur. Furthermore, we implicate PKA as a modulator of spindle morphology and function during meiosis.

Aberrant distribution of ADAM3 in sperm from both angiotensin-converting enzyme (Ace)- and calmegin (Clgn)-deficient mice.

Male mice deficient for the calmegin (Clgn) or the angiotensin-converting enzyme (Ace) gene show impaired sperm migration into the oviduct and loss of sperm-zona pellucida binding ability in vitro. Since CLGN is a molecular chaperone for membrane transport of target proteins and ACE is a membrane protein, we looked for ACE on the sperm membranes from Clgn-/- mice. ACE was present and showed normal activity, indicating that CLGN is not involved in transporting ACE to the sperm membranes. The ablation of the Adam2 and Adam3 genes generated animals whose sperm did not bind the zona pellucida, which led us to examine the presence of ADAM2 and ADAM3 in Clgn-/- and Ace-/- sperm. ADAM3 was absent from Clgn-/- sperm. In the Ace-/- mice, while ADAM2 was found normally in the sperm, ADAM3 disappeared from the Triton X-114 detergent-enriched phase after phase separation, which suggests that ACE is involved in distributing ADAM3 to a location where it can participate in sperm-zona pellucida binding. This diminished amount of ADAM3 in the Triton X-114 detergent-enriched phase may explain the inability of Clgn-/- and Ace-/- sperm to bind to the zona pellucida.

Adenylate kinases 1 and 2 are part of the accessory structures in the mouse sperm flagellum.

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.

Protein domains govern the intracellular distribution of mouse sperm AKAP4.

A-kinase anchor proteins (AKAPs) spatially restrict cAMP-dependent protein kinase by tethering it to various cellular structures. In the polarized sperm cell, various compartmentalized functions, such as motility generated by the flagellum, are modulated by cAMP-dependent protein kinase. This important regulatory enzyme is associated with AKAP4, the principal component of the fibrous sheath; AKAP4 is synthesized as a precursor, pro-AKAP4, which is cleaved into mature AKAP4 during fibrous sheath assembly. To define the domains responsible for the intracellular distribution and assembly of AKAP4 into a macromolecular complex, various AKAP4-green fluorescent protein (GFP) constructs were introduced into somatic cell lines. The presence of the pro domain, either alone or as part of pro-AKAP4, resulted in a diffuse cytoplasmic localization of the GFP fusion protein, suggesting that, the pro domain keeps the AKAP4 precursor unassembled in vivo until it is transported to the developing tail structure and incorporated into the fibrous sheath. When the mature AKAP4-GFP fusion protein was expressed, it localized in a punctate cytoplasmic pattern. Two domains critical for this punctate localization, T2a and T2b, are homologous to the T2-tethering domain of rat AKAP5 that is important for binding to the actin cytoskeleton in transfected HEK293 cells. In contrast to AKAP5, the distribution of AKAP4 was dependent on the microtubular cytoskeleton. The interaction of AKAP4 with the microtubular network provides evidence that the longitudinal columns of the fibrous sheath, which contain AKAP4, may interact directly with the outer microtubular doublets of the sperm axoneme.

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.

Deficiency of SPAG16L causes male infertility associated with impaired sperm motility.

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 M(r) approximately 71 x 10(3) (SPAG16L) and M(r) approximately 35 x 10(3) (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.