Barry Bean

Leptin indirectly affects estrous cycles by increasing metabolic fuel oxidation.

In previous experiments, lean Syrian hamsters fasted on days 1 and 2 of the estrous cycle failed to show sex behavior and ovulation normally expected to occur on the evening of day 4. The first goal of the present experiment was to determine whether systemic treatment with the ob (obese) protein leptin could reverse the effects of fasting on estrous cyclicity, social behaviors, and ovulation rate. Fasting-induced anestrus was reversed and normal sex and social behavior and ovulation rate were restored in hamsters injected intraperitoneally with 5 mg/kg leptin every 12 h during fasting on days 1 and 2 of the estrous cycle. A second goal was to test whether the effects of leptin could be prevented by treatment with pharmacological agents that block the oxidation of metabolic fuels. Glucose oxidation was blocked by treatment with 2-deoxy-d-glucose (2DG) and fatty acid oxidation was blocked by treatment with methyl palmoxirate (MP). 2DG (1000 mg/kg) or MP (20 mg/kg) was administered at doses that did not induce anestrus in hamsters fed ad libitum. As in the first experiment, fasting-induced anestrus was reversed by leptin treatment. However, when each injection of leptin was preceded by an injection of 2DG or MP, leptin treatment did not reverse fasting-induced anestrus. In summary, estrous cyclicity was not restored when oxidation of metabolic fuels was blocked, despite high endogenous levels of leptin. These results are consistent with the hypothesis that leptin acts indirectly on the reproductive system by increasing fuel oxidation.

Purification and Characterization of Plasma Membrane-Associated Human Sperm α-l-Fucosidase

Abstract Detergent and salt extraction studies, as well as cytochemical localization with fluorescein isothiocyanate-bovine serum albumin-l-fucose, have provided further evidence for the plasma membrane association of a novel human sperm, α-l-fucosidase. This α-l-fucosidase has been solubilized and purified 8600-fold to high specific activity (35 000 U/mg protein) by affinity chromatography on agarose-C24-fucosylamine. To our knowledge, this is the first report concerning the purification and characterization of a mammalian plasma membrane-associated α-l-fucosidase. Both SDS-PAGE and Western blot analysis indicated the α-l-fucosidase is highly purified and contains a single subunit with a molecular mass of 51 kDa. N-glycanase studies indicated the subunit contains N-glycans, and lectin blot analysis detected the presence of mannose, but no terminal galactose or sialic acid residues. Isoelectric focusing indicated the presence of two major α-l-fucosidase isoforms (pIs 6.5 and 6.7) and a possible minor isoform (pI 6.3). Treatment of α-l-fucosidase with neuraminidase did not change its isoform profile, providing further evidence for the enzymes lack of sialic acid residues. Kinetic analysis with 4-methylumbelliferyl α-l-fucopyranoside indicated that sperm α-l-fucosidase has a pH optimum near 7, an apparent Km of 0.08 mM, and a Vmax of 6.8 μmol/min/mg protein. The unusual properties of human sperm α-l-fucosidase argue in support of a potentially important, but presently unknown, role for this enzyme in human reproduction.

Hamster Sperm-Associated Alpha-L-Fucosidase Functions During Fertilization

Abstract Sperm-associated alpha-l-fucosidases have been identified in diverse organisms. Their wide phylogenetic distribution and known properties support the likelihood that l-fucose and alpha-l-fucosidase have fundamental function(s) during gamete interaction. This is consistent with the substantial evidence in the literature documenting the importance of carbohydrate moieties during fertilization. Direct enzyme assays were employed to evaluate the functional distribution of alpha-l-fucosidase in preparations of hamster sperm. In vitro fertilization was performed using Syrian hamster sperm and eggs to identify the functional role of hamster sperm-associated alpha-l-fucosidase during zona pellucida binding/penetration, sperm-egg membrane fusion, and postfusion events. Results reported here document the presence of hamster sperm-associated alpha-l-fucosidase and demonstrate that it functions during fertilization at the stage of sperm-oocyte membrane interaction and/or postfusion events within the zygote. Understanding the role of alpha-l-fucosidase during human fertilization could lead to development of improved infertility treatments.

Roles of mouse sperm-associated alpha-L-fucosidases in fertilization

Sperm‐associated α‐L‐fucosidases have been implicated in fertilization in many species. Previously, we documented the existence of α‐L‐fucosidase in mouse cauda epididymal contents, and showed that sperm‐associated α‐L‐fucosidase is cryptically stored within the acrosome and reappears within the sperm equatorial segment after the acrosome reaction. The enrichment of sperm membrane‐associated α‐L‐fucosidase within the equatorial segment of acrosome‐reacted cells implicates its roles during fertilization. Here, we document the absence of α‐L‐fucosidase in mouse oocytes and early embryos, and define roles of sperm associated α‐L‐fucosidase in fertilization using specific inhibitors and competitors. Mouse sperm were pretreated with deoxyfuconojirimycin (DFJ, an inhibitor of α‐L‐fucosidase) or with anti‐fucosidase antibody; alternatively, mouse oocytes were pretreated with purified human liver α‐L‐fucosidase. Five‐millimolar DFJ did not inhibit sperm–zona pellucida (ZP) binding, membrane binding, or fusion and penetration, but anti‐fucosidase antibody and purified human liver α‐L‐fucosidase significantly decreased the frequency of these events. To evaluate sperm‐associated α‐L‐fucosidase enzyme activity in post‐fusion events, DFJ‐pretreated sperm were microinjected into oocytes, and 2‐pronuclear (2‐PN) embryos were treated with 5 mM DFJ with no significant effects, suggesting that α‐L‐fucosidase enzyme activity does not play a role in post‐fusion events and/or early embryo development in mice. The recognition and binding of mouse sperm to the ZP and oolemma involves the glycoprotein structure of α‐L‐fucosidase, but not its catalytic action. These observations suggest that deficits in fucosidase protein and/or the presence of anti‐fucosidase antibody may be responsible for some types of infertility. Mol. Reprod. Dev. 80: 273–285, 2013.

Distribution, crypticity, stability, and localization of α‐L‐fucosidase of mouse cauda epididymal sperm

Sperm‐associated and semen‐specific isoforms of α‐L‐fucosidase are thought to function in fertilization in numerous organisms. Here, we report the localization, distribution, crypticity, and stability of this enzyme in mouse cauda epididymal sperm and cauda fluid. Western analysis revealed that the sperm‐associated α‐L‐fucosidase is present as two isoforms (Mr ∼49 and 56 kDa), whereas the cauda fluid α‐L‐fucosidase shows a single band at 50 kDa. α‐L‐Fucosidase activity was detected using the fluorogenic substrate 4‐MU‐FUC. Of the total α‐L‐fucosidase activity recovered in the cauda epididymal contents, 74% was found in the cell‐free cauda fluid and about 7% was found in sperm cells. During capacitation or permeabilization, cryptic intracellular stores of soluble enzyme were released to the supernatant, while leaving bound enzyme concentrated within the small volume of sperm. Moreover, membrane‐associated enzyme activity was still detectable in acrosome‐reacted cells. Immunofluorescence studies support the presence of α‐L‐fucosidase (originally localizing at the acrosomal area) at the equatorial segment after the acrosome reaction. α‐L‐Fucosidase activity of both cauda fluid and sperm at 37°C, 5% CO2 was relatively stable and detectable up to 72 hr. The stability and appearance of mouse sperm‐associated α‐L‐fucosidase in the equatorial segment after the acrosome reaction suggest that α‐L‐fucosidase may be involved in sperm–egg interaction. Mol. Reprod. Dev. 79: 208–217, 2012.

Human sperm CRISP2 is released from the acrosome during the acrosome reaction and re-associates at the equatorial segment.

Sperm CRISP2 has been proposed to be involved in sperm–egg fusion. After the acrosome reaction, it appears at the equatorial segment (EqS) of human sperm; the mechanism underlying the appearance of CRISP2 at the EqS remains unknown, though. Here, we provide evidence showing the re‐association of sperm acrosomal CRISP2 at the EqS during the acrosome reaction. Results showed that F‐actin is not involved in the relocalization of CRISP2. We found that basic, but not acidic, conditions can solubilize CRISP2 from sperm cells, suggesting that CRISP2 is a component of the acrosome and that it is released from the acrosome during the acrosome reaction. Purified, biotinylated human sperm acrosomal CRISP2 binds to the EqS of acrosome‐reacted sperm in a dose‐dependent manner, revealing that CRISP2 detected at the EqS of acrosome‐reacted sperm comes from the population stored in the acrosome. The association of CRISP2 at the EqS is very strong, and does not depend on ionic interactions or intermolecular disulfide bonds. Interestingly, the restriction of CRISP2 at the EqS was diminished when EGTA was present in the media, indicating that Ca2+ is required for maintaining CRISP2 at the EqS. This study supports the possibility that CRISP2 may help modify the EqS membrane to make this domain fusion‐competent. Mol. Reprod. Dev. 80: 488–502, 2013.

Stabilization of membrane‐associated α‐l‐fucosidase by the human sperm equatorial segment

Previous reports from this laboratory documented the existence of two novel isoforms of alpha-L-fucosidase in human semen and showed that membrane-associated alpha-L-fucosidase is cryptically held within the acrosomal compartment and enriched within the sperm equatorial segment. The occurrence of these novel isoforms is provocative. Sperm proteins potentially involved in sperm-egg interactions must maintain their functional integrity as they travel through the female reproductive tract. The goal of this project was to investigate the stability of membrane-associated alpha-L-fucosidase in human sperm. Whole seminal plasma and Percoll purified sperm cell populations were incubated for 72 h at 37 degrees C, with 5% CO(2) or ambient air. At various times during prolonged incubation, sperm cells were permeabilized with 0.1% Triton X-100 and enzyme assays using the fluorogenic substrate 4-MU-fuc were performed to evaluate the stability of both the seminal plasma and membrane-associated alpha-L-fucosidase. Here, we report seminal plasma alpha-L-fucosidase activity rapidly decreased within 24 h. Conversely, alpha-L-fucosidase activity from Percoll purified sperm cell populations persisted up to 72 h. Data from these experiments support the notions that (i) membrane-associated alpha-L-fucosidase is stable for extended periods of time, consistent with a possible role in sperm-egg interaction and (ii) membrane domains and compartmentalization within the human sperm are key to preserving protein integrity.

A Comparison of Human Semen from Healthy, Subfertile, and Post‐Vasectomy Donors by 31P NMR Spectroscopy

Preliminary characterizations of the phosphorylated compounds present in human semen were obtained using a JEOL FX90-Q Multinuclear NMR with a variable temperature accessory. Semen samples of 2.0 ml containing 12.5% D20 in a 10.0 mm sample tube were maintained at approximately 28.5 degrees Celsius. The spectra were recorded with a 250 msec pulse delay 1 sec acquisition time 0.95 sec acquisition delay and over the range of -16 ppm to 11 ppm. 3 distinguishable peaks were identified as glycerylphosphorycholine (GPC) at 0.00 ppm (this point was used as an internal standard) inorganic phosphate (Pi) at -2.45 ppm to -2.00 ppm and phosphorycholine (PC) at -3.4 ppm in accord with Arrata et al. A temporal study showed that in the hour immediately following ejaculation the original ratio of PC to total phosphate (P) decreased by 60% while the original ratio of GPC to total P did not change by a significant amount. These data contrast with those of arrata et al. who reported that PC hydrolyzed to Pi within 30 minutes and that GPC was stable. Data reported below were collected after a minimum of 60 minutes postejaculation following relative stabilization of GPC content. Semen specimens were evaluated either within 5 hours of collection (fresh) or quickly frozen without cryoprotectant and stored at -76 degrees Celsius (frozen). Ratios of GPC to total P differed with sample type. The authors observed the following ratios for GPC to total P: (0.17 0.15 0.16 0.19 0.099 0.16 0.15) N=7 with a mean of 0.15 +or- 0.028 for fresh healthy semen (0.085 0.070 0.086) N=3 with a mean of 0.080 +or- 0.008 for frozen health semen (0.074 0.074) N=2 with a mean of 0.074 for fresh subfertile semen (fig. 2) (0.11 0.040 0.060 0.00 0.060 0.00) N=6 with a mean of 0.045 +or- 0.042 for frozen subfertile semen and a ratio of (0.00 0.00 0.00 0.00) N=4 with a mean of 0.00 for postvasectomy specimens. While the data are generally consistent with those of Arrata et al. the authors detect a difference between fresh and frozen samples. This discrepancy may reflect partial biological hydrolysis of GPC and PC to Pi. The simple reliable quantification of GPC is important for further investigation of its biological significance. The authors suggest that 31P NMR analyses of human semen will play a valuable role in understanding detection and treatment of male infertility. (full text)

Fucosidases of Sperm and Milt in Darters (Percidae: Etheostomatini)

Darters (Percidae: Etheostomatini) offer attractive opportunities for analysis of the evolution of reproductive barriers and the molecular basis of fertilization. Here we report some basic characteristics of the sperm of darters, and, to our knowledge, the first report of sperm associated � -L-fucosidase in fishes. Fluorometric enzyme assays using 4- methylumbelliferyl-alpha-L-fucopyranoside (4-MU-fuc) as a substrate provided direct quantification of enzyme activity in both the soluble and cellular fractions of milt from all six species tested. Further analysis using SDS-PAGE and Western blotting revealed that fish sperm and milt contain � -L-fucosidase isoforms with molecular weights similar to those reported for human semen. Fertilization of darter ova by conspecific sperm, as assessed by development of the embryo to the 4 cell stage, was inhibited by pretreatment of sperm with the fucosidase-specific competitive inhibitor DFJ, implicating the sperm fucosidase in fertilization and/or early embryogenesis. Identification of sperm associated � -L- fucosidase in darters is consistent with the importance of carbohydrates during fertilization.

Rapid staining for the sperm penetration assay using a bisbenzimide dye**Supported in part by the Advanced Technology Center, Ben Franklin Partnership of the Commonwealth of Pennsylvania under grant no. 6.7, Bethlehem, Pennsylvania.

Our rapid staining method, accomplished by adding the vital fluorochrome bisbenzimide (Hoechst 33342) directly into the sperm droplet before fertilization, eases visual interpretation of sperm penetration and decondensation. For both within- and between-experiment comparisons, inclusion of bisbenzimide did not influence the overall SPA score or the progress of sperm-egg interaction. These results suggest that bisbenzimide can be used routinely to facilitate scoring of the SPA.