Jyrki Suominen

Distribution of Glycoconjugates in Human Testis A Histochemical Study Using Fluorescein- and Rhodamine-conjugated Lectins

Summary: Differentiation in the seminiferous epithelium involves the orderly transformation of germ cells into spermatozoa. We have employed ten fluorescein‐ and rhodamine‐labeled lectins to visualize distinctive changes in the distribution of carbohydrate containing compounds during spermatogenesis and noticed the increase in RCA I, PNA, SB A and HP A binding sites during germ cell differentiation, suggesting the appearance of certain galactose and N‐acetylgalactosamine containing glycoconjugates. Besides, in the cytoplasm of all germ cell types the positive reactions with Con A, LCA, WGA, LPA and UE AI indicate the presence of mannose, N‐acetylglucosamine, sialic acid and fucose containing glycosubstances. Developing acrosomes demonstrated binding sites for most lectins, and particular HPA binding glycoconjugates were expressed in the equatorial segment region of late spermatids and testicular spermatozoa. In addition, the characteristic staining patterns of other testicular compartments are described.

Silent Infection in Male Accessory Genital Organs and Male Infertility

Stumme Infektion der Adnexe des Mannes und Infertilität

Glycoconjugates of human sperm surface. A study with fluorescent lectin conjugates and lens culinaris agglutinin affinity chromatography

Distribution of glycocompounds in human spermatozoa was studied by using fluorescent lectin-conjugates. Con A bound predominantly to acrosomal and posterior head regions whereas RCA I bound to the acrosomal region of intact spermatozoa, stained in suspension. Other lectins used (LCA, WGA, SBA, PNA) stained the the entire sperm surface. In airdried sperm smears binding of both Con A and RCA I were identical with the staining pattern obtained with living cells whereas LCA, WGA, SBA and PNA now bound heavily into acrosomal region. As a similar staining pattern was obtained with permeabilized sperm cells, this staining is apparently due to binding to intracellular structures. The efficiency of Lens culinaris agglutinin affinity chromatography in purification of human sperm glycoproteins was tested after their external radiolabelling with the neuraminidase/galactose oxidase/sodium borohydride method. 22% of applicated radioactivity could be eluted from the column with the specific inhibitory saccharide, and most of the radiolabelled surface glycoproteins of the whole sperm lysate, were also present in the LCA affinity column eluate. LCA affinity chromatography seems thus be an effective method to enrich membrane glycoproteins of human spermatozoa.

A comparison of eight techniques for the evaluation of the auto-immune response to spermatozoa after vasectomy

The tray agglutination tests (TAT), gelatin agglutination test (GAT), side agglutination test (SAT), tube-slide agglutination test (TSAT), sperm immobilization test (SIT), ATP-release cytotoxicity test (ARCT), indirect immunofluorescence technique (IFT) on methanol-fixed, intact spermatozoa, and a lymphocyte transformation test (LTT) were compared using a maximum of 329 blood samples taken from 47 men before and after vasectomy. The TAT, GAT, TSAT, SIT and ARCT discriminated between the pre- and post-vasectomy samples, and the sensitivity for sperm antibodies decreased in that order. The activity in the IFT and the LTT did not change significantly after vasectomy. In the TAT the mode of agglutination varied with serum dilution; the results for the 1:4 dilution showed the best agreement with the SAT results. Almost all TAT activity was detected by a combination of GAT and TSAT. Sperm agglutinins were present in all serum samples positive in the two complement-dependent tests, SIT and ARCT. If improved in sensitivity, the ARCT, which lacks the subjective elements of microscopy, might be suitable for the screening of male sera in clinical work. For the present, we recommend the TAT.

A New Method for Measurement of Cytotoxic Antibodies to Human Spermatozoa

The energy producing reactions of spermatozoa will cease after the spermatozoa have been damaged by a complement-mediated antibody reaction and, consequently, their ATP content falls rapidly. The finding was applied and used for determination of the cytotoxic reaction caused by sperm antibodies. This reaction could be recorded by comparing the difference in ATP content between damaged and control spermatozoa after 2 hr incubation. A serum dilution that still reduced the ATP content by 50% or more was regarded to be cytotoxic. Only those sera, collected from infertile patients, with an agglutinating titer of at lease 1:64 and an immobilizing titer of 1:4 were cytotoxic to spermatozoa. The method proved to be both simple and more sensitive than the supraviatal staining technique as a cytotoxicity test.

Glucose Content as a Parameter of Semen Quality

There are minute and varying amounts of glucose in seminal plasma. Findings on vasectomized men suggest that it is derived from accessory sex glands. The glucose content was shown to correlate negatively to the number of spermatozoa and the time period elapsed from ejaculation, indicating that seminal glucose was utilized by spermatozoa even in the presence of a 50-fold concentration of fructose. Large interindividual variations in the glucose utilization as reflected in the 14CO2 production from 14C-glucose by spermatozoa could not be explained by the parameters examined in routine semen analysis. Fructose and glucose maintained equally well the ATP content and motility of spermatozoa at the concentrations found normally in semen. Fructose may thus supplement and substitute for glucose as an energy source, and the addition of glucose into the semen or the measurement of its content would not give any advantage for the analysis of semen samples.

Prolactin in azoospermic men and its relation to testicular morphology, serum testosterone and gonadotrophin levels.

Prolaktin bei Azoospermie‐Patienten und seine Beziehung zur Hodenmorphologie, zum Serum‐Testosteron und zu den Gonadotropin‐Werten

Expression of Sox9 and type IIA procollagen during ocular development and aging in transgenic Del1 mice with a mutation in the type II collagen gene.

Purpose To study the expression and distribution of transcription factor Sox9 and type IIA procollagen in the developing and aging eyes of normal and transgenic Del1 mice carrying proa1(II) collagen transgenes with a short deletion mutation, which cause ocular abnormalities in this mouse line. Methods The eyes of Del1 mice were studied on embryonic days E14.5, E16.5 and E18.5, and at the ages of 4 and nine months, using their nontransgenic littermates as controls. Sox9 and proa1(IIA) collagen were detected by RNase protection assay and immunohistochemistry. Results RNase protection assay revealed Sox9 transcripts in the eyes of Del1 and control mice during development and aging. The mRNA for type IIA procollagen had a similar temporal expression pattern. On embryonic days E14.5, E16.5 and E18.5, Sox9 was located by immunohistochemistry in the nuclei and type IIA procollagen in the extracellular space of the developing retina. During growth and aging, the ocular expression of Sox9 mRNA and the immunohistochemical reaction for Sox9 antibody diminished, concomitant with the reduction in type II procollagen mRNA. However, at the age of nine months, levels of Sox9 and type IIA procollagen mRNAs were higher in the degenerating eyes of Del1 and control mice. Conclusions The similarities in the temporo-spatial distribution of Sox9 and type IIA procollagen suggest that this transcription factor is involved in the activation of type II collagen expression in the eye, as has been demonstrated in prechondrogenic mesenchyme and immature cartilage. The increased production of Sox9 and type IIA procollagen in the aging retina and vitreous is analogous to degenerating articular cartilage where attempted tissue repair has also been observed.

Ultrastructural Study of Separated Cell and Acrosomal Membranes from Bull Spermatozoa

Ejaculated bull spermatozoa were washed and incubated with a cationic detergent, Hyamine 2389, and the material removed was centrifuged on a three-step sucrose gradient. Five different bands were obtained, three of which contained cell and acrosomal membranes and also acrosomal material. The first of these, band III, was composed of two parts. The lower part contained U-shaped membrane profiles originating from the outer acrosomal membrane, acrosomal material attached to them, and vesicles of different sizes. The upper part, in addition to various vesicles (probably formed from cell membrane), contained acrosomal vesicles arranged in the form of aggregates or caps. The latter vesicles were formed by fusion and vesiculation of the cell and outer acrosomal membranes with tightly bound acrosomal material. Band IV was uniform and comprised various round vesicles with a smooth membrane, which had a typical unit-membrane structure. There were obviously formed from the cell membrane. The fifth band, in the saline portion with material unsedimented in the sucrose, included small vesicles and membrane fragments, the origin of which was obscure. The solubilized material was also left in this fraction.

Solubilization of Human Sperm Antigens

Various methods of solubilization of human sperm antigens were compared by testing the sperm extracts against rabbit antihuman spermatozoa immune serums with double immunodiffusion and rocket immunoelectrophoresis. Marked differences were found in the protein contents and the SDS-PAGE protein patterns of the extracts. Lithium diiodosalicylate was the most efficient in solubilizing the sperm proteins and in preserving their antigenicity. Nonionic detergents also gave, either alone or combined to sonification and 1.0 M KCl, 24-28 clearly discernible protein bands and several different immunoprecipitates. Ionic detergents deoxycholate and cetylpyridinium chloride solubilized a high yield of proteins, but their antigenity was poor.