The fibrous sheath is a cytoskeletal structure located in the principal piece of mammalian sperm flagella. Previous studies showed that glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS), a germ cell-specific glycolytic isozyme that is required for sperm motility, is tightly bound to the fibrous sheath. To determine if other glycolytic enzymes are also bound to this cytoskeletal structure, we isolated highly purified fibrous sheath preparations from mouse epididymal sperm using a sequential extraction procedure. The isolated fibrous sheaths retain typical ultrastructural features and exhibit little contamination by axonemal or outer dense fiber proteins in Western blot analyses. Proteomic analysis using peptide-mass fingerprinting and MS/MS peptide fragment ion matching identified GAPDHS and two additional glycolytic enzyme subunits, the A isoform of aldolase 1 (ALDOA) and lactate dehydrogenase A (LDHA), in isolated fibrous sheaths. The presence of glycolytic enzymes in the fibrous sheath was also examined by Western blotting. In addition to GAPDHS, ALDOA, and LDHA, this method determined that pyruvate kinase is also tightly bound to the fibrous sheath. These data support a role for the fibrous sheath as a scaffold for anchoring multiple glycolytic enzymes along the length of the flagellum to provide a localized source of ATP that is essential for sperm motility.
This investigation was conducted to determine the initial period of gonadal sensitivity to estrogen in genetically male rainbow trout (Oncorhynchus mykiss). Fish were immersed in approximately 250 μg estradiol‐17β (E2)/l for two 2 hr periods during different stages of embryonic development beginning 30 days postfertilization (DPF) and continuing until 68 DPF. Histological analysis of gonad samples indicated a significant proportion of E2‐treated fish had intersex gonads; these gonads were primarily comprised of testicular tissue with one or more oocytes scattered throughout. The most sensitive period for altering normal testicular development was found to occur between 44 and 51 DPF (63% intersex), while the labile period in general was determined to span 24 days (from 30 to 54 DPF). Additionally, quantitative RT‐PCR was used to measure estrogen receptor (ER) mRNA expression in whole individual untreated embryos at six weekly time points throughout the period of E2‐exposure. Although the intersex condition was not observed throughout the entire E2‐exposure period, ER mRNA was detected at each time point assayed. Mol. Reprod. Dev. 56:495–501, 2000. © 2000 Wiley‐Liss, Inc.
Signaling by cAMP-dependent protein kinase (PKA) plays an important role in the regulation of mammalian sperm motility. However, it has not been determined how PKA signaling leads to changes in motility, and specific proteins responsible for these changes have not yet been identified as PKA substrates. Anti-phospho-(Ser/Thr) PKA substrate antibodies detected a sperm protein with a relative molecular weight of 270,000 (p270), which was phosphorylated within 1 min after incubation in a medium supporting capacitation. Phosphorylation of p270 was induced by bicarbonate or a cAMP analog, but was blocked by the PKA inhibitor H-89, indicating that p270 is likely a PKA substrate in sperm. In addition, phosphorylation of p270 was inhibited by stearated peptide st-Ht31, suggesting that p270 is phosphorylated by PKA associated with an A-kinase anchoring protein (AKAP). AKAP4 is the major fibrous sheath protein of mammalian sperm and tethers regulatory subunits of PKA to localize phosphorylation events. Phosphorylation of p270 occurred in sperm lacking AKAP4, suggesting that AKAP4 is not involved directly in the phosphorylation event. Phosphorylated p270 was enriched in fractionated sperm tails and appeared to be present in multiple compartments including a detergent-resistant membrane fraction. PKA phosphorylation of p270 within 1 min of incubation under capacitation conditions suggests that this protein may have an important role in the initial signaling events that lead to the activation and subsequent hyperactivation of sperm motility.
This study developed a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method to measure estrogen receptor-(ER ) mRNA in the rainbow trout (Oncorhynchus mykiss). Using RT-PCR, and primers based on the known ER DNA sequence in this species, cDNA sequences representing most of the protein coding region were obtained from ovary poly A(+) RNA. Using these DNA sequences as probes in Northern blot hybridizations confirmed that a single transcript of 4·2 kilobases in poly A(+) RNA could be detected in liver and ovary RNA. For the quantitative RT-PCR assay an internal standard RNA molecule was produced to control for inherent inter-tube differences in amplification efficiency and permit accurate quantification of ER mRNAs. The quantitative RT-PCR assay proved to be highly specific for ER mRNA with a detection limit of 6·9 fg, which corresponds to 273 fg ER mRNA/µg total RNA. The quantitative RT-PCR assay was used to measure the levels of ER mRNA in ovaries of rainbow trout at different stages of reproductive development. Ovarian ER mRNA expression was found during two distinct periods of reproductive development, in pre-vitellogenic ovaries of fish with ovarian follicle diameters (OFDs) c100 µm and in mid-vitellogenic ovaries with OFDs >1000 µm. ER mRNA could not be detected in the ovaries of fish with OFDs >100 µm but c1000 µm. The highest levels of ER mRNA were found in late vitellogenic ovaries of fish with OFDs >2000 µm.
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