Androgen-Controlled Synthesis of Specific Proteins in the Rat Epididymis*

alpha-L-Fucosidase (alpha-fucosidase) is the most active of a number of glycosidases measured in rat epididymal sperm through use of artificial 4-methylumbelliferyl substrates. In addition, enzyme activity can be detected in purified populations of testicular germ cells; in comparison to round spermatids, caput epididymal sperm show at least a 1.8-fold increase in alpha-fucosidase activity per cell. Metabolic labeling of cultured testicular germ cells, followed by immunoprecipitation and SDS-PAGE, reveals synthesis of alpha-fucosidase as a polypeptide of 54 kDa in pachytene spermatocytes and round spermatids but no synthesis in condensing spermatids. This polypeptide is N-glycosylated but does not undergo further processing as determined by pulse/chase labeling and treatment with endoglycosidase H. In contrast, the alpha-fucosidase synthesized by cultured clone 9 cells (a rat liver-derived cell line) undergoes processing from a 54-kDa precursor to a slightly larger intermediate centered at 56 kDa on SDS-PAGE (via carbohydrate modification) and finally to a 52-kDa mature polypeptide. Immunoblotting confirms the presence of the 54-kDa form of alpha-fucosidase in testicular germ cells and shows the existence of a 52-kDa mature polypeptide in epididymal sperm. In addition, immunoreactive polypeptide is more prominent in caput epididymal sperm preparations; this is consistent with the increase in enzyme activity. In the absence of alpha-fucosidase synthesis beyond the round spermatid stage of development, it appears that alpha-fucosidase may be acquired from the luminal fluid by sperm during transit through the excurrent duct system. This hypothesis is supported by evidence that metabolically labeled cultured epididymal epithelial cells synthesize and secrete into culture medium an immunoprecipitable 58-kDa alpha-fucosidase polypeptide. Analysis of sperm isolated from the first part of the caput epididymis indicates that both acquisition and processing of sperm-associated alpha-fucosidase takes place prior to or concomitant with arrival of sperm in the epididymis.

Section: Discussionmentioning

confidence: 99%

Synthesis and Processing of Rat Sperm-Associated α-L-Fucosidase1

Hancock¹,

Raab²,

Aronson³

1993

“…Subsequent analysis of purified CRISP1 by mass spectrometry using an ESI-ion trap LCQ-Duo mass spectrometer (Thermo Fisher Scientific) revealed that all tryptic peptides within the sample corresponded to CRISP1. Polyclonal antibody anti-CRISP1 (8 mg/ml) was produced by immunization of rabbits with purified CRISP1 as previously described [37].…”

Section: Collection Of Epididymal Fluidsmentioning

confidence: 99%

Evidence for the Involvement of Zinc in the Association of CRISP1 with Rat Sperm During Epididymal Maturation

Maldera

Vasen

Ernesto

et al. 2011

Rat epididymal protein CRISP1 (cysteine-rich secretory protein 1) associates with sperm during maturation and participates in fertilization. Evidence indicates the existence of two populations of CRISP1 in sperm: one loosely bound and released during capacitation, and one strongly bound that remains after this process. However, the mechanisms underlying CRISP1 binding to sperm remain mostly unknown. Considering the high concentrations of Zn(2+) present in the epididymis, we investigated the potential involvement of this cation in the association of CRISP1 with sperm. Caput sperm were coincubated with epididymal fluid in the presence or absence of Zn(2+), and binding of CRISP1 to sperm was examined by Western blot analysis. An increase in CRISP1 was detected in sperm exposed to Zn(2+), but not if the cation was added with ethylenediaminetetra-acetic acid (EDTA). The same results were obtained using purified CRISP1. Association of CRISP1 with sperm was dependent on epididymal fluid and Zn(2+) concentrations and incubation time. Treatment with NaCl (0.6 M) removed the in vitro-bound CRISP1, indicating that it corresponds to the loosely bound population. Flow cytometry of caput sperm exposed to biotinylated CRISP1/avidin-fluorescein isothiocyanate revealed that only the cells incubated with Zn(2+) exhibited an increase in fluorescence. When these sperm were examined by epifluorescence microscopy, a clear staining in the tail, accompanied by a weaker labeling in the head, was observed. Detection of changes in the tryptophan fluorescence emission spectra of CRISP1 when exposed to Zn(2+) supported a direct interaction between CRISP1 and Zn(2+). Incubation of either cauda epididymal fluid or purified CRISP1 with Zn(2+), followed by native-PAGE and Western blot analysis, revealed the presence of high-molecular-weight CRISP1 complexes not detected in fluids treated with EDTA. Altogether, these results support the involvement of CRISP1-Zn(2+) complexes in the association of the loosely bound population of CRISP1 with sperm during epididymal maturation.

“…For experiments involving the effect of a polyclonal antibody against rCRISP1 (anti-CRISP1) on fertilization, sperm were incubated for 30 min in the presence or absence of the antibody (7.2 mg/ml) [26], or in the presence of normal rabbit serum or normal rabbit immunoglobulin (IgG; Sigma) prior to the addition of the eggs to the IVF drops. For those cases in which the effect of rCRISP1 on fertilization was examined, eggs were incubated for 30 min in medium alone or in medium containing 6 lM purified rCRISP1 [5,6]; ovalbumin (OA; Sigma); recCRISP1, expressed as a fusion protein coupled to maltose-binding protein (MBP) [14]; or MBP prior to insemination.…”

Section: In Vitro Fertilization (Ivf)mentioning

confidence: 99%

A Novel Function for CRISP1 in Rodent Fertilization: Involvement in Sperm-Zona Pellucida Interaction1

Busso

Cohen

Maldera

et al. 2007

Epididymal protein CRISP1 participates in rat and mouse gamete fusion through its interaction with complementary sites on the egg surface. Based on in vivo observations, in the present study we investigated the possibility that CRISP1 plays an additional role in the sperm-zona pellucida (ZP) interaction that precedes gamete fusion. In vitro fertilization experiments using zona-intact rat and mouse eggs indicated that the presence of either an antibody against rat CRISP1 (anti-CRISP1) or rat native CRISP1 (rCRISP1) during gamete co-incubation produced a significant decrease in the percentage of fertilized eggs. However, differently to that expected for a protein involved in gamete fusion, no accumulation of perivitelline sperm was observed, suggesting that the inhibitions occurred at the sperm-ZP interaction level. Bacterially expressed recombinant CRISP1 (recCRISP1) also significantly inhibited egg fertilization. In this case, however, an increase in the number of perivitelline sperm was observed. Subsequent experiments evaluating the effect of anti-CRISP1 or rCRISP1 on the number of sperm bound per egg indicated that the protein is involved in the initial step of sperm-ZP binding. In agreement with these functional studies, indirect immunofluorescence experiments revealed that although rCRISP1 is capable of binding to both the ZP and the oolema, recCRISP1 only binds to the egg surface. The finding that deglycosylated rCRISP1 behaves as the untreated protein, whereas the heat-denatured rCRISP1 associated only with the oolema, indicates that the protein ZP-binding ability resides in the conformation rather than in the glycosydic portion of the molecule. The interaction between rCRISP1 and the ZP reproduces the sperm-ZP-binding behavior, as judged by the failure of the protein to interact with the ZP of fertilized eggs. Together, these results support the idea that CRISP1 participates not only in sperm-egg fusion but also in the prior stage of sperm-ZP interaction.