Jorge G. Tezón

Participation of glycosylated residues in the human sperm acrosome reaction: Possible role of N-acetylglucosaminidase

Sperm binding to the egg zona pellucida is mediated by complementary protein-carbohydrate interaction. This binding results in the exocytosis of the sperm acrosome, or acrosome reaction (AR). We report the effect of different neoglycoproteins (sugar residues covalently bound to bovine serum albumin) on the human sperm AR. p-Aminophenyl-N-acetyl-beta-D-glucosaminide-BSA (BSA-GlcNAc) and p-aminophenyl-alpha-D-mannopyranoside-BSA (BSA-Man) at 1 micrograms/ml were capable of inducing the greatest percentages of AR (3-fold stimulation with respect to controls), while other NeoGPs had only a weak effect on this process. The BSA-GlcNAc-induced acrosome reaction was inhibited by N-acetylglucosamine (GlcNAc), p-nitrophenyl-GlcNAc, and purified soluble beta-N-acetylglucosaminidase (beta NAG). The induction of the AR with BSA-Man could be inhibited by mannose, while soluble alpha-mannosidase was only partially effective. These data suggest that binding sites for GlcNAc and mannose may be involved in the induction of the AR in human sperm. The characteristics of the BSA-GlcNAc induction suggest that the beta NAG molecule may be the mediator of this effect.

Calcium requirements for human sperm function in vitro

OBJECTIVE To determine extracellular calcium (Ca(2+)) requirements for the maintenance of human sperm function in vitro. DESIGN Prospective study. SETTING Basic research laboratory. PATIENT(S) Normozoospermic volunteers provided fresh semen samples; follicular fluid (human FF) and oocytes were collected from women undergoing IVF-ET. INTERVENTION(S) Spermatozoa were incubated for </=18 hours in media containing different CaCl(2) concentrations (maximum, 2.5 mM [control]). MAIN OUTCOME MEASURES Protein tyrosine phosphorylation patterns, development of hyperactivated motility, induction of the acrosome reaction (AR) in response to human FF, and sperm interaction with homologous zona pellucida (ZP). RESULT(S) Cells maintained for 18 hours in medium containing >/=0.1 mM of Ca(2+) were able to undergo the AR when exposed to human FF in the presence of 2.5 mM of Ca(2+). Calcium concentrations of >/=0.22 mM were sufficient to reach protein tyrosine phosphorylation levels and hyperactivated motility values similar to those of controls. Higher Ca(2+) concentrations (>/=0.58 mM) were required to produce maximum human FF-induced AR in previously capacitated cells and to obtain an adequate sperm-ZP binding. CONCLUSION(S) Different steps of the fertilization process have distinctive Ca(2+) requirements. Whereas 0.22 mM of Ca(2+) is sufficient for the development of some capacitation-related events, human FF-induced AR and sperm-ZP interaction require 0.58 mM of this cation.

The organ culture of human epididymal tubules and their response to androgens

Human epididymal tubules from 9 patients undergoing orchidectomy were cultured for periods of up to 8 days with preservation of the histological structure of the tissue. Addition of androgens (testosterone, 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol) significantly increased the epithelial height, the incorporation of [3H]amino acids and [3H]thymidine. The effect on protein synthesis was significant 48 h after the onset of treatment and was maximal after 3 days. The effect on DNA replication was maximal during the 3rd day of treatment. In both instances maximal activity was achieved at a concentration of 10(-7) M of testosterone. Cyproterone acetate (10(-5) M) was able to negate all effects of androgens.

Effect of incubating human sperm at room temperature on capacitation-related events

OBJECTIVE To determine the effect of human sperm incubation at room temperature (20 degrees C) upon capacitation-related events. DESIGN Prospective study. SETTING Basic research laboratory. PATIENT(S) Semen samples were obtained from normozoospermic volunteers. Human follicular fluid (hFF) was collected from women undergoing assisted reproductive treatment. INTERVENTION(S) Spermatozoa were incubated for up to 18 hours at 20 degrees C and/or 37 degrees C. MAIN OUTCOME MEASURE(S) Protein tyrosine phosphorylation patterns, development of hyperactivated motility, and induction of acrosome reaction (AR) in response to hFF. RESULT(S) Spermatozoa incubated for 18 hours at 20 degrees C showed an array of tyrosine phosphorylated proteins similar to noncapacitated cells. After incubation at 20 degrees C, the percentage of spermatozoa displaying hyperactivated motility and undergoing acrosomal loss in response to hFF was significantly lower when compared with cells kept the same time at 37 degrees C. Conversely, spermatozoa incubated overnight at 37 degrees C could respond to hFF, either at 37 degrees C or 20 degrees C. When preincubation at 20 degrees C was followed by sperm exposure to 37 degrees C, capacitation-related events could be activated. In capacitated cells (16 hours at 37 degrees C), 2-hour incubation at 20 degrees C led to a significant decrease in acrosome reaction inducibility, suggesting sperm decapacitation. CONCLUSION(S) Human sperm incubation at room temperature does not allow capacitation, although it does not affect hFF-induced acrosome reaction in capacitated cells. The blocking effect is overcome when spermatozoa are exposed to 37 degrees C.

Expression of Human Proacrosin in Escherichia coli and Binding to Zona Pellucida

Abstract Proacrosin is a multifunctional protein present in the sperm acrosome. This study characterizes the expression of human proacrosin in bacteria and assesses zona pellucida binding activity. The cDNA encoding human proacrosin was subcloned in pGEX-3X and pET-22b vectors. In the pGEX system, expression of the full-length fusion protein was not detected. In the pET system, an expression product with an apparent molecular size similar to that expected for the proenzyme (Rec-40, 42–44 kDa) was recognized by a monoclonal antibody to human acrosin, AcrC5F10. A 32–34-kDa protein (Rec-30), not recognized by AcrC5F10 on Western blots, was the major expression product. Proteins of 21 (Rec-20) and 18 (Rec-10) kDa were recovered as insoluble expression products as were Rec-40 and Rec-30, and truncated products from the C terminus were detected in the soluble fraction. Rec-40 and Rec-30 coexisted at any culture time tested. Immune serum raised against Rec-30 (AntiRec-30) stained the acrosomal region of permeabilized human spermatozoa and recognized the recombinant proteins and proacrosin from human sperm extracts. Amino acid sequence analysis indicated that Rec-30, Rec-20, and Rec-10 are N-terminal fragments of proacrosin. The recombinant proteins Rec-40, -30, -20, and -10 were found to interact with homologous 125I-zona pellucida glycoproteins.

Effect of Antisperm Antibodies Present in Human Follicular Fluid upon the Acrosome Reaction and Sperm–Zona pellucida Interaction

Problem: To determine the ability of IgGs isolated from follicular fluids (hFFIgGs) to induce the acrosome reaction (AR) in human spermatozoa and to inhibit sperm–zona pellucida (ZP) interaction.

Identification of human sperm proteins involved in the interaction with homologous zona pellucida

OBJECTIVE To identify human sperm proteins involved in homologous zona pellucida (ZP) interaction. DESIGN Prospective study. SETTINGS Basic research laboratory. PATIENT(S) Semen samples from normozoospermic donors, tissue sections from surgical pieces, and ZP from nonfertilized oocytes. INTERVENTION(S) Antibodies for sperm proteins (HSE; high salt extract) were developed (anti-HSE) and partially characterized. Participation of sperm proteins on ZP-interaction was tested with the hemizona assay (HZA). Antigens were immunolocalized in sperm and tissues. MAIN OUTCOME MEASURE(S) Sperm and tissue immunostaining; Western blotting; and number of sperm bound to the ZP. RESULT(S) Anti-HSE antibodies recognized several polypeptides in HSE (9 to 200 kd). Specific antibodies for 49 and 66 kd proteins (p49, p66) were obtained. Both (anti-p49 and anti-p66) stained the head of ejaculated and capacitated sperm. In the HZA, sperm preincubation with a mixture of anti-p49 and anti-p66 (100 micro g/mL) resulted in a decrease in the number of spermatozoa bound to the ZP. Presence of p66 (10 micro g/mL) inhibited sperm-ZP interaction. In contrast, p49 did not alter sperm binding to the ZP. Immunohistochemical analysis showed that p66 is present in the epididymis. No staining was observed in testicular sections. CONCLUSION(S) We found that p66 is an epididymal protein that participates in human sperm interaction with homologous ZP.

Transforming growth factor-β activities in‘in vivo’ lines of hormone-dependent and independent mammary adenocarcinomas induced by medroxyprogesterone acetate in BALB/c mice

SummaryWe have determined the presence of transforming growth factor-β (TGF-β)-like polypeptides in mammary adenocarcinomas induced by medroxyprogesterone acetate (MPA) in BALB/c mice. In hormone-dependent tumors (HD) from nontreated and MPA-treated mice a high molecular weight (43 kDa) transforming activity was purified by Bio-Gel P-60 chromatography. This TGF was able to confer the neoplastic phenotype on NRK-49F cells without the addition of epidermal growth factor (EGF), though its activity was potentiated by EGF. It did not compete for binding to the EGF receptor, had no mitogenic activity on monolayer cultures of NRK fibroblasts, and was a potent inhibitor of DNA synthesis induced in these cells by EGF and insulin. In HD and hormone-independent tumors (HI) another TGF with a Mr of 13 kDa was isolated. This transforming activity showed the same biological properties as 43 kDa TGF, with the exception that in the absence of EGF it did not stimulate soft agar growth of NRK-49F cells. The synthesis of both factors in ‘in vivo’ HD tumors seems to be under MPA control, since it is much lower in HD tumors from MPA-treated mice. Further purification of the 13 and 43 kDa TGFs by hydrophobic interaction HPLC demonstrated that each one eluted in a different position, and that their elution profile differed from the TGF-β from human platelets. The biological activity of the 13 and 43 kDa TGFs was not neutralized by a specific anti-TGF-β antibody.

Studies on the mechanism of the antiandrogenic effect of a putative 5α -reductase inhibitor

Abstract The mechanism of the antiandrogenic effect of 5,10-seco-19-norpregnane-4,5-diene-3,10,20-trione (secosteroid), reputedly an irreversible inhibitor of 5α -reductase, was investigated. Its addition (10 μM) to culture media effectively suppressed the synthesis of rat epididymal proteins specifically induced by 0.1 μM testosterone (T) or dihydrotestosterone (DHT). Under the same conditions, secosteroid did not change the rate at which labeled T was metabolized to 5α -reduced compounds. In a comparative study, secosteroid inhibited 5α -reductase in an isolated microsomal fraction while not affecting the enzyme activity in minced tissue. Secosteroid was shown to be a competitor of the binding of [3H]T and [3H]DHT (both at 4nM) to the epididymal cytosol androgen receptor, with ID50 of 1 μM for the former and 4μM for the latter, thus explaining the mechanism involved in its antiandrogenic properties.

HUMAN SPERM β‐GLUCURONIDASE IS POORLY EXTRACTABLE BY TRITON X‐100

A part of sperm glycosidase activities was detected as detergent‐insoluble after sequential extractions with Triton X‐100. Sixty per cent of total β‐glucuronidase activity was found in the detergent‐insoluble fraction. This portion of β‐glucuronidase was resistant to extractions in the presence of 1mKCl, chaotropic agents, colchicine or cytochalasine B, being only partially solubilized by 3mKCl or DNAse I treatment. Results demonstrate that β‐glucuronidase is tightly associated to the Triton X‐100 resistant fraction.