Jorge A. Blaquier

Isolation and characterization of specific rat epididymal proteins.

The partial purification and characterization of specific rat epididymal proteins (SEP) is reported. Starting from the cytosol fraction obtained from epididymal homogenates, protein C was purified 15-fold and proteins D--E were purified 19-fold. The molecular weight, determined by molecular sieving, of protein C was 22,400 while that of D--E was 37,000. These proteins stained as glycoproteins with periodic acid--Schiff reagent. The isoelectric point of protein D was 5.13 while that of protein E was 4.95. Protein C separated into 3 bands during isoelectric focussing. The major component focussed at 5.56 and the two minor components at pH 5.38 And 5.79. Using a specific antiserum we could confirm the organ specificity of SEP and their androgen-dependence.

Selective uptake and metabolism of androgens by rat epididymis. The presence of a cytoplasmic receptor

Abstract Epididymis actively concentrated androgens with a predominant intranuclear localization. Spermatozoa did not concentrate androgens. Macromolecular complexes were formed in both nuclei and cytoplasm. Density gradient centrifugation showed two binding zones in the cytosol. One had a sedimentation coefficient of 8.5± 0.3 and seemed to be highly specific for DHT. The second, approximately 3.5S, corresponded with the bulk of protein applied to the gradient. Testosterone was ineffective as a competitor of the formation of the 8.5S-DHT complex. Cyproterone acetate was inhibitory when administered in vivo , but less effective in vitro . Blockade of the SH groups prevented the formation of the 8.5S-DHT complex, which could be destroyed by digestion with Pronase.

Partial characterization of epididymal 5α reductase in the rat

Abstract Epididymal 5α reductase activity was found distributed in the crude nuclear fraction (44%) and microsomal fraction (41%). Spermatozoa contaminating the nuclear preparation accounted for only 3% of its activity. There were no regional differences in the distribution of total 5α reductase activity. However, the nuclear enzyme was more active in caput than in other regions. Maximal activity was found at pH 6.2 and at 32°C. Both enzymes had an absolute requirement of reduced dinucleotides. The microsomal preparation could only use NADPH while the nuclear enzyme could use NADPH and NADH. The apparent Km for the microsomal preparation was 0.62 ± 0.05 × 10−6M and Vmax was 555 ± 38 pmoles/mg protein/hour. The nuclear enzyme presented similar values. The reaction was not inhibited by accumulation of product in the medium, but other steroids such as progesterone, epitestosterone(17α-hydroxy-4-androsten-3-one) and 3-oxo-4-androstene-17β-carboxylic acid were potent competitive inhibitors. The reaction was strongly inhibited by Hg, Zn and Cu. The properties of the epididymal reductase are similar to those of the prostatic enzyme.

Quantitative determination of specific proteins in rat epididymis.

Abstract This paper describes the development of a radioimmunoassay for the measurement of specific proteins (SEP) in rat epididymis. Using [125I]-SEP the assay is useful within the range 0.6–30 ng SEP. With this method we confirmed the organ specificity of SEP which could neither be detected in the cytosol of several other organs in the rat nor in rat blood plasma. Furthermore, SEP appear to be species specific since the epididymal cytosol from man, stallion, bull, dog and rabbit did not react with serum anti rat SEP. Castration decreased the SEP content of rat epididymis. Preincubation of anti-SEP with cauda epididymis spermatozoa sharply decreased the binding capacity of the antiserum. The same treatment did not alter the binding capacity of control anti-LH serum. Immunofluorescence experiments show that SEP are attached to spermatozoa obtained from the cauda epididymis.

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.

Beneficial effect of autologous endometrial cell coculture in patients with repeated implantation failure

OBJECTIVE To confirm the beneficial effect of endometrial coculture in patients with repeated failures with assisted reproductive techniques (ART). DESIGN Patients with previous failures were offered a repetition of ART in conjunction with autologous endometrial coculture. SETTING Private fertility center. PATIENT(S) Sixty-eight couples who had attempted 92 cycles of IVF or intracytoplasmic sperm injection without obtaining an evolutive pregnancy. INTERVENTION(S) Patients repeated one cycle of ART with concomitant endometrial coculture of their embryos. MAIN OUTCOME MEASURES(S) Comparative pregnancy and delivery rates in conventional ART cycles vs. cycles with autologous endometrial coculture. RESULT(S) In the previous 92 cycles (146 ETs, fresh plus frozen) only 8 pregnancies were initiated, and all ended in spontaneous abortion. Upon repeating 68 cycles (76 ETs) using coculture, 39 pregnancies were obtained, of which 19 resulted in live births, 10 are ongoing evolutive pregnancies, and 10 ended in spontaneous abortions. CONCLUSION(S) These results confirm the usefulness of autologous endometrial coculture for the treatment of patients with repeated implantation failure.

Tissue androgens and androphilic proteins in rat epididymis during sexual development

Abstract The appearance of the epididymal 8s cytoplasmic receptor in the rat during sexual development was followed and correlated with the endogenous concentrations of three biologically active androgens in the epididymis, testosterone (T), dihydrotestosterone (DHT)and 5α-androstane-3α, 17β-diol (Diol). The results indicate that receptors could not be evidenced in the 10-day-old animal in which androgens were undetectable. The 8s receptor was first detected in the 20 day-old rat, coincidently with a rise in the concentration of DHT (0,24 ng/ epididymis) while T and Diol remained too low to be measured. At the 26th day of life a peak of androgen binding activity, sedimenting at approximately 4S, was seen and probably corresponds to ABP. This pattern of two binding peaks (8 and 4S) remained constant through adulthood. The association constant (Ka) of the 8s receptor from 35-, 45- and 60-day-old rats was found to be similar. T became detectable in the epididymides of the 35 day-old rat (0.03 ng/ep.) and increased with sexual maturation. However, DHT concentrations were higher than those of T at all ages studied and Diol could not be detected at any age. These results raise the possibility that the synthesis or availability of receptors in the developing animals is under androgenic control.

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.

Testosterone, Dihydrotestosterone, and Zinc Concentrations in Human Testis and Epididymis

Tissue testosterone, dihydrotestosterone, and zinc concentrations have been determined in testis and epididymis of 13 patients with carcinoma of the prostate, 1 patient with carcinoma of the penis, and 3 patients with carcinoma of the prostate on estrogens. The 13 patients not receiving estrogens had the following testicular levels: testosterone, of 529 +/- 63.1 ng/g (mean +/- SE); dihydrotestosterone, 23.7 +/- 2.58 ng/g; and zinc 62.2 +/- 7.6 micrograms/g. The epididymal levels were as follows: testosterone, 40.6 +/- 3.4 ng/g; dihydrotestosterone, 20.5 +/- 2.36 ng/g; and zinc, 67.2 +/- 11.1 micrograms/g. Patients on estrogen therapy showed much lower androgen values in the two organs but zinc was not changed significantly. Concentrations of androgens were not significantly different in the epididymal fractions of caput, corpus, and cauda. In testis, there was a positive correlation between zinc and androgens contents, while the opposite was suggested by the data in the epididymis. Even though these patients were not normal, there was no evidence of testicular or epididymal disturbances.

Human fertilization: epididymal hCRISP1 mediates sperm–zona pellucida binding through its interaction with ZP3

Human epididymal CRISP1 (hCRISP1) associates with sperm during maturation and participates in gamete fusion through egg complementary sites. Its homology with both rodent epididymal CRISP1 and CRISP4 reported to participate in the previous stage of sperm binding to the zona pellucida (ZP), led us to further investigate the functional role of hCRISP1 by studying its involvement in human sperm-ZP interaction. Human hemizona (HZ) were inseminated with human capacitated sperm in the presence of either anti-hCRISP1 polyclonal antibody to inhibit sperm hCRISP1, or bacterially-expressed hCRISP1 (rec-hCRISP1) to block putative hCRISP1 binding sites in the ZP. Results revealed that both anti-hCRISP1 and rec-hCRISP1 produced a significant inhibition in the number of sperm bound per HZ compared with the corresponding controls. The finding that neither anti-hCRISP1 nor rec-hCRISP1 affected capacitation-associated events (i.e. sperm motility, protein tyrosine phosphorylation or acrosome reaction) supports a specific inhibition at the sperm-egg interaction level. Moreover, immunofluorescence experiments using human ZP-intact eggs revealed the presence of complementary sites for hCRISP1 in the ZP. To identify the ligand of hCRISP1 in the ZP, human recombinant proteins ZP2, ZP3 and ZP4 expressed in insect cells were co-incubated with hCRISP1 and protein-protein interaction was analyzed by ELISA. Results revealed that rec-hCRISP1 mainly interacted with ZP3 in a dose-dependent and saturable manner, supporting the specificity of this interaction. Altogether, these results indicate that hCRISP1 is a multifunctional protein involved not only in sperm-egg fusion but also in the previous stage of sperm-ZP binding through its specific interaction with human ZP3.