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.

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.

The "soluble" adenylyl cyclase in sperm mediates multiple signaling events required for fertilization.

Mammalian fertilization is dependent upon a series of bicarbonate-induced, cAMP-dependent processes sperm undergo as they "capacitate," i.e., acquire the ability to fertilize eggs. Male mice lacking the bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC), the predominant source of cAMP in male germ cells, are infertile, as the sperm are immotile. Membrane-permeable cAMP analogs are reported to rescue the motility defect, but we now show that these "rescued" null sperm were not hyperactive, displayed flagellar angulation, and remained unable to fertilize eggs in vitro. These deficits uncover a requirement for sAC during spermatogenesis and/or epididymal maturation and reveal limitations inherent in studying sAC function using knockout mice. To circumvent this restriction, we identified a specific sAC inhibitor that allowed temporal control over sAC activity. This inhibitor revealed that capacitation is defined by separable events: induction of protein tyrosine phosphorylation and motility are sAC dependent while acrosomal exocytosis is not dependent on sAC.

Patterns of expression of sperm flagellar genes: early expression of genes encoding axonemal proteins during the spermatogenic cycle and shared features of promoters of genes encoding central apparatus proteins.

Sperm are motile cells. Thus, a significant component of the spermatogenic cycle is devoted to the formation of flagellum, a process that must be coordinated to insure proper construction. To document the temporal pattern of flagellar gene expression, we employed real-time PCR to assess changes in accumulation of a cohort of genes encoding axoneme, outer dense fibre (ODF) and fibrous sheath (FS) proteins during the first wave of spermatogenesis in the mouse. Axoneme genes were expressed first at the pachytene spermatocyte stage, followed by expression of transcripts encoding ODF and FS components. However, there were differences among these families with respect to the time of initial expression and the rate of mRNA accumulation. To gain understanding of factors that determine these patterns of expression, we cloned the promoters of three axoneme central apparatus genes (Pf6, Spag6 and Pf20). These promoters shared common features including the absence of a TATA box, and putative binding sites for several factors implicated in spermatogenesis (CREB/CREM, SOX17 and SPZ1) as well as ciliogenesis (FOXJ1). Collectively, our findings demonstrate a sequential pattern of expression of flagellar component genes, differential times of expression or rates of transcript accumulation within each class and shared promoter features within a class.

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.

PAR-3 defines a central subdomain of the cortical actin cap in mouse eggs.

The evolutionarily conserved partitioning defective (PAR) protein PAR-3 is pivotal for establishing and maintaining cell polarity. During mammalian oocyte maturation, the radially symmetric oocyte is transformed into a highly polarized metaphase II (MII)-arrested egg. We therefore examined several aspects of PAR-3 expression during oocyte maturation. We cloned two novel PAR-3 transcripts from an oocyte library that likely encode proteins of Mr = 73 K and 133 K that are phosphorylated during maturation. PAR-3, which is found throughout the GV-intact oocyte, becomes asymmetrically localized during meiosis. Following germinal vesicle breakdown, PAR-3 surrounds the condensing chromosomes and associates with the meiotic spindles. Prior to emission of the first and second polar bodies, PAR-3 is located within a central subdomain of the polarized actin cap, which overlies the spindle. This cortical PAR-3 localization depends on intact microfilaments. These results suggest a role for PAR-3 in establishing asymmetry in the egg and in defining the future site of polar body emission.

Differential RNA expression and polyribosome loading of alternative transcripts of the Akap4 gene in murine spermatids.

An X chromosome-linked gene, Akap4, is expressed only during spermiogenesis and encodes the major fibrous sheath protein of the mouse sperm flagellum. All sperm contain the AKAP4 protein even though only X chromosome-bearing spermatids express the gene, indicating that the Akap4 mRNA and/or protein must be shared among the conjoined spermatids via the intercellular bridges. There are two mouse Akap4 cDNA clones, Akap82 and Fsc1, which represent mRNAs that arise by alternative processing of a single gene. Although Akap82 and Fsc1 encode identical mature proteins, they differ in their 5' UTRs. We hypothesized that the expression pattern of these two mRNAs might be relevant to the issue of mRNA and/or protein transport into adjacent spermatids. Expression of both transcripts began in round spermatids, but the amount of the Akap82 transcript in condensing spermatids increased twofold relative to Fsc1. Significantly, only the Akap82 transcript was found on polyribosomes and translated in spermatids. These results indicate that the Akap82 transcript and/or its protein must be shared among the conjoined X and Y chromosome-bearing spermatids. Although Fsc1 was not polysomal, both the Akap82 and Fsc1 transcripts were deadenylated during spermiogenesis, suggesting that deadenylation is not always correlated with loading of mRNAs onto polyribosomes in germ cells. The distinct 5' UTR sequences in Akap82 and Fsc1 did not differ in their ability to regulate translation of reporter constructs either in vivo or in vitro. Antisense RNA transcripts complementary to both the Akap82 and Fsc1 mRNAs were present, suggesting that translatability may be regulated by these RNAs.

Haploinsufficiency for the murine orthologue of Chlamydomonas PF20 disrupts spermatogenesis.

PF20 was first identified in Chlamydomonas rheinhardtii as an essential component of the axoneme central apparatus. We discovered that the mouse Pf20 gene encodes two major transcripts (2.5 and 1.4 kb), which are expressed in different patterns during spermatogenesis, yielding proteins of 71 and 35 kDa, respectively. Both proteins contain contiguous WD repeats in their C termini. The meiotically expressed 71-kDa protein is incorporated into the central apparatus, whereas the 35-kDa protein, which accumulates in postmeiotic male germ cells, is abundant in the nucleus. We disrupted the Pf20 gene domains that encode the C-terminal WD repeats in embryonic stem cells. Highly chimeric mice carrying the mutant Pf20 allele had impaired spermatogenesis with a significant loss of germ cells at the round spermatid stage, in association with disorganization of sperm axoneme structure. The mutated Pf20 allele was never transmitted, indicating that Pf20 haploinsufficiency caused the defects in spermatogenesis. The 35-kDa PF20 protein was shown to bind to meiosis-expressed gene 1 (MEIG1), a chromosome/chromatin-binding protein initially expressed during meiosis but retained in the germ cell nucleus throughout later stages of spermatogenesis. Our findings reveal an essential role for Pf20 in mouse spermatogenesis, sustaining postmeiotic germ cell viability. The different patterns of expression of the two PF20 proteins suggest the possibility that the Pf20 gene has multiple functions during spermatogenesis.

AKAP7gamma 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 AKAP7gamma, an AKAP originally found in pancreas. Yeast two-hybrid analysis and co-immunoprecipitation studies showed that AKAP7gamma 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 AKAP7gamma localized to the nuclei of HEK 293 cells via a nuclear localization signal. In addition, endogenous AKAP7gamma protein was found in both the nucleus and cytoplasm of oocytes. This work identifies AKAP7gamma as the first nuclear AKAP to bind RI and suggests that AKAP7gamma may be responsible for positioning PKA via RI and/or RII to regulate PKA-mediated gene transcription in both somatic cells and oocytes.

Sperm from the calmegin-deficient mouse have normal abilities for binding and fusion to the egg plasma membrane.

Calmegin is a putative testis-specific molecular chaperone required for the heterodimerization of fertilin alpha/beta and the appearance of fertilin beta on the sperm surface. Calmegin-deficient mice are almost completely sterile. The cause of the sterility initially was considered to be impaired abilities in sperm/zona pellucida (ZP) and sperm/egg plasma membrane (EPM) binding, and in the ascension of sperm to the oviduct, phenotypes similar to those seen in sperm from fertilin beta-deficient animals. We have developed a new method in which eggs were prepared without any detectable ZP3 on their surfaces by using a piezo-driven micromanipulator. Using these eggs and sperm containing the green fluorescent protein in their acrosomes, which can distinguish acrosome-intact from acrosome-reacted sperm, the binding and fusing abilities of calmegin-deficient sperm were reexamined. Under these conditions, acrosome-reacted sperm retained their ability to bind to and fuse with the EPM. The reduction in EPM binding of sperm from the calmegin(-/-) animals was apparently due to the artifactual binding of large numbers of acrosome-intact sperm from calmegin(+/-) mice to ZP remnants remaining on the EPM prepared with acidic Tyrode's solution. Thus, the sperm defect in calmegin-null animals is not at the level of sperm-EPM binding but rather may involve either sperm-ZP binding and/or sperm transit to the oviduct. Because fertilin beta is absent from calmegin-deficient mice, these results also suggest that the role of fertilin beta in sperm-EPM interaction needs to be reevaluated.