DNAJB13 is a radial spoke protein of mouse '9+2' axoneme.

It has been shown that DNAJB13, a type II heat shock protein 40, is highly expressed in the testis and is an axonemal component of mouse mature spermatozoa. By multi-tissue reverse transcription polymerase chain reaction, we found that Dnajb13 gene was expressed not only in the testis but also in several other ciliated cell-containing tissues like brain, lung and oviduct. Immunohistochemistry on mouse trachea and oviduct sections shown that DNAJB13 was present in the motile cilia of those tissues. To define further its localization in the axoneme, immunoelectron microscopy of mouse sperm flagella was performed and shown that DNAJB13 was localized to radial spokes of the axoneme. Taken together, our data indicate that DNAJB13 is a radial spoke protein of the mouse '9+2' axoneme.

The physiological roles of the boar ejaculate.

During ejaculation in the boar, sperm cohorts emitted in epididymal cauda fluid are sequentially exposed and resuspended in different mixtures of accessory sex gland secretion. This paper reviews the relevance of such unevenly composed fractions of seminal plasma (SP) in vivo on sperm transport and sperm function and how this knowledge could benefit boar semen processing for artificial insemination (AI). The firstly ejaculated spermatozoa (first 10 ml of the sperm-rich fraction, SRF [P1]) remain mainly exposed to epididymal cauda fluid and its specific proteins i.e. various lipocalins, including the fertility-related prostaglandin D synthase; than to prostatic and initial vesicular gland secretions. P1-spermatozoa are hence exposed to less bicarbonate, zinc or fructose and mainly to PSP-I spermadhesin; than if they were in the rest of the SRF and the post-SRF (P2). Since the P1-SP is less destabilizing for sperm membrane and chromatin, P1-spermatozoa sustain most in vitro procedures, including cryopreservation, the best. Moreover, ejaculated firstly, the P1-spermatozoa seem also those deposited by the boar as a vanguard cohort, thus becoming overrepresented in the oviductal sperm reservoir (SR). This vanguard SR-entry occurs before the endometrial signalling of SP components (as PSP-I/PSP-II and cytokines) causes a massive influx of the innate defensive PMNs to cleanse the uterus from eventual pathogens, superfluous spermatozoa and the allogeneic SP. The SP also conditions the mucosal immunity of the female genital tract, to tolerate the SR-spermatozoa and the semi-allogeneic conceptus. These in vivo gathered data can be extrapolated into procedures for handling boar spermatozoa in vitro for AI and other biotechnologies, including simplified cryopreservation.

Evaluation of the one-step eosin-nigrosin staining technique for human sperm vitality assessment.

BACKGROUND: The one-step eosin-nigrosin staining technique for assessment of sperm vitality was developed in the 1950s for various mammalian species. Although commonly used on human sperm in semen, a validation for this use has not previously been published. METHODS: The technique was evaluated on 1235 consecutive semen samples. RESULTS: The one-step eosin-nigrosin staining technique gave valid results when evaluated with sperm motility data obtained according to World Health Organization standard (1992, 1999). The mean for the sums of stained (i.e. supposedly dead) and motile sperm using the one-step eosin-nigrosin technique was 91% (SD +/- 10%). The distribution of sums for percentage stained and percentage motile sperm was similar, regardless of whether the samples had many or few dead sperm. CONCLUSIONS: Standardization and quality control of basic semen analysis demands robust, reliable and simple techniques that are easy to learn, and easy to continue to perform in the same way. The one-step eosin-nigrosin technique does not need negative phase contrast optics but can be run with ordinary bright-field microscopy. Since it also includes fewer methodological steps to control, it seems preferable in terms of standardization and quality control management. It should therefore be recommended in the basic semen analysis when sperm vitality is to be assessed.

ESHRE basic semen analysis courses 1995-1999: immediate beneficial effects of standardized training.

BACKGROUND: Many reports have shown problems with the high variability in results of semen analyses. The Special Interest Group in Andrology (SIGA) of the European Society of Human Reproduction and Embryology (ESHRE) implemented a standardized training course which has been run in different regions of the world on more than 20 occasions since 1994. The aim of the present analysis was to investigate to what extent training resulted in any immediate effects on the variability of assessments made by different observers. METHODS: The variability in participants' results from the beginning to the end of each course was analysed in eight courses given between 1995 and 1999. RESULTS: For assessments of sperm concentration, motility, vitality and morphology, substantial improvement was seen over the duration of the course. CONCLUSIONS: A comprehensive, structured training course does lead to substantial reductions in inter-observer variability in semen analysis. This supports our contention that providing a thorough theoretical background and repeated practical training, combined with daily feedback of results, is highly effective in raising the technical skills of laboratory personnel performing semen analysis.

Percutaneous cutting needle biopsies for histopathological assessment and sperm retrieval in men with azoospermia.

BACKGROUND: Twenty-three men (45 testes) with azoospermia underwent percutaneous testicular biopsy under local anaesthesia. METHODS: In all but one of the 45 testes two biopsies were taken close to each other, one with a 16 gauge (n = 44) and another with a 14 gauge (n = 45) cutting needle, both with a 19 mm notch. Three quarters of the tissue was used for histopathological assessment and one quarter for direct microscopy. RESULTS: The histopathological findings were similar between the two needles. The observations with direct microscopy corresponded with the histopathological assessments concerning the presence of mature spermatids in 41 of 45 (91%) biopsies using the 14 gauge and in 40 of 44 (91%) biopsies using the 16 gauge needle. There were no post-operative complications except for minimal pain and minor local swelling. CONCLUSIONS: Percutaneous material retrieved using 16 gauge and 14 gauge needles is sufficient for histopathological assessment, and the two needles are equally reliable for testicular sperm retrieval. However, needle biopsy with one puncture may not be representative of the entire testis.

Specific localization of RBM1a in the nuclei of all cell types except elongated spermatids within seminiferous tubules of the human.

Recent studies have indicated that at least three regions (AZF a-c) on the long arm of the Y-chromosome code for factors are involved in spermatogenesis. One of the candidate genes in the AZFb region is RBM1a, coding for a protein with an RNA binding motif. In this study, poly clonal antibodies raised against a 15 amino acid peptide, corresponding to residues 263-304 of the deduced amino acid sequence of RBM1a, has been used to localize the RBM1a protein in the human testis. Immunohistochemistry on normal human testis using this RBM1a antibody, localized the antigen to the nuclei of spermatogonia, primary spermatocytes, and round spermatids but not to the nuclei of elongated spermatids. The antibody also specifically identified the nuclei of Sertoli cells, although the fluorescence was not as strong as in the germ cell nuclei it identified. No specific fluorescence was seen in the nuclei of either peritubular, endothelial or Leydig cells. Western blot of normal human testicular tissue using the anti-RBM1a antibody gave rise to a single specific band of approximately 55 kDa, corresponding to the expected size of RBM1a. In view of its expression in germ cells, and because RBM1a has an RNA binding domain, RBM1a may be involved in RNA processing, such as RNA splicing or RNA export which are events necessary for normal spermatogenesis.

Human fertility does not decline: evidence from Sweden.

OBJECTIVE: To assess changes in human fertility over time. DESIGN: Time-trend analyses and age-period-cohort modeling. SETTING: Sweden, 1983-1993. PATIENT(S): All primiparous women aged > or =20 years during the study period. There were 401,653 women who were identified through the nationwide Medical Birth Register. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Risk of subfertility, defined as > or =1 year of involuntary childlessness. RESULT(S): Subfertility problems decreased dramatically over successive maternal birth cohorts. Further, the risk of subfertility increased with age and decreased with increasing formal education. CONCLUSION(S): A decrease in male fertility cannot be ruled out on the basis of these results, but if present, it is minor and totally outweighed by other favorable developments. As the main explanation for our findings, we propose a decrease in the prevalence of secondary subfertility as a result of the eradication of gonorrhea.

A comparison between open and percutaneous needle biopsies in men with azoospermia.

Open testicular biopsy is a classic method of investigation in men with azoospermia. Recently, percutaneous needle biopsy of the testis has been used in attempts to obtain material for histopathological diagnosis in such cases and to retrieve spermatozoa for intracytoplasmic sperm injection (ICSI). To determine whether a 19 gauge (G) and a 21G butterfly needle could be used for percutaneous aspiration of testicular tissue to determine the presence of mature spermatids and assess spermatogenesis, 10 patients (16 testes) and 12 patients (17 testes) underwent 19G or 21G needle biopsy respectively, immediately followed by open testicular biopsy, with both procedures under local anaesthesia. Biopsy with each needle size was compared with open biopsy. With the 19G needle, in the 14 cases where material was obtained there was full agreement with open biopsy regarding the presence or absence of mature spermatozoa, whereas with the 21G needle only nine of the 13 biopsies yielding material were predictive in this respect. Each needle size correlated poorly with open biopsy regarding evaluation of spermatogenesis. We conclude that percutaneous biopsy with a 19G butterfly needle is a quick and reliable method for demonstrating spermatozoa for ICSI. But for a detailed histopathological diagnosis, however, the needle biopsies gave poor results, whereas the material from the open testicular biopsies was assessable.

Trisomy 5q12-->q13.3 in a patient with add(13q): characterization of an interchromosomal insertion by forward and reverse chromosome painting.

We report on a patient with azoospermia, mild mental retardation, and minor physical anomalies. Chromosome analysis demonstrated the presence of additional material on the long arm of one chromosome 13. Forward chromosome painting using chromosome-specific libraries showed an insertion of material from chromosome 5. Further characterization with flow sorting of the aberrant chromosome and amplification by DOP-PCR followed by reverse chromosome painting showed specific trisomy of 5q12-->q13.3.

Presence of synaptonemal complex protein 1 transversal filament-like protein in human primary spermatocytes.

The synaptonemal complex (SC) is involved in the pairing of chromosomes during meiosis. We found that antibodies raised against a protein component (P1) of the mouse synaptonemal complex, mouse SCP1, also identified the SC in human primary spermatocytes. Biopsies from 18 men presented with infertility were evaluated by light-field microscopy and grouped into five categories: normal spermatogenesis, Sertoli cell-only syndrome, meiotic disturbances, spermiogenic (i.e. differentiation) disturbances, and other combined disturbances. In all the normal subjects the SCP1 antibody distinctly stained the synaptonemal complexes of primary spermatocytes, whereas Sertoli cells, spermatogonia or spermatids were never stained. In three of the groups, which had germ cells but showed spermatogenic disturbances, the staining was similar to that seen in normal subjects. In sharp contrast to this, in sections from men with Sertoli cell-only syndrome no specific staining was seen. This study demonstrates that a SCP1-related protein is also conserved in the synaptonemal complex in meiotic cells from man. Further studies will reveal to what extent the absence or the non-functionality of SCP1 contributes to male infertility.

Rapid and slow hydroxylators of seminal E prostaglandins among men in barren unions.

E prostaglandins are formed in seminal vesicles and can be oxygenated by (omega-1)-hydroxylation catalysed by cytochrome P450 to 19(R)-hydroxy metabolites. The latter are not further metabolized. Prostaglandin E1 (PGE1), PGE2, 19-hydroxy-PGE1 and 19-hydroxy-PGE2 were measured in seminal fluid of 95 men, who attended the clinic for infertility. After extractive isolation, the E prostaglandins were converted to B prostaglandins by alkali treatment and analysed by high performance liquid chromatography on beta-cyclodextrin silica with 17-phenyl-PGE2 as an internal standard. The relative amount of 19-hydroxy E-prostaglandins varied between 26% and 97%. Most (86%) of the men were classified as rapid or normal hydroxylators with PGE/19-hydroxy PGE ratios below 0.75, while 14% were slow hydroxylators. The relative amount of 19-hydroxy E1 and 19-hydroxy E2 showed a 96% covariation, which supports that a common enzymatic mechanism is operating on both E1 and E2 prostaglandins and that this mechanism is the major determinant for formation of 19-hydroxy compounds. We conclude that the relative amounts of PGE1, PGE2, 19-hydroxy-PGE1 and 19-hydroxy-PGE2 in seminal fluid vary, possibly due to polymorphic expression of this enzymatic mechanism. Total output of 19-hydroxy-PGE compounds, but not the primary PGE compounds was correlated with the output of seminal fructose, supporting that the 19-hydroxy prostaglandins are the normal end products of the seminal vesicles. Low sperm concentration found among men with high output of E prostaglandins could here simply be explained by dilution of spermatozoa by a high output of seminal vesicular fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

Sperm chromatin stability and zinc binding properties in semen from men in barren unions.

Sperm chromatin stability and zinc binding properties were studied in semen samples from 115 men living in barren unions. Of these men, 26% had a high proportion of swelling sperm, i.e. less than 80% sperm with stable chromatin after exposure to the detergent sodium dodecyl sulphate. From 2-67% of seminal zinc was bound to high molecular weight ligands of vesicular origin (HMW). This shows that, among infertile men, liquefied seminal plasma has huge variations in zinc chelating properties. The relationship between prostatic palpatory status, the proportion of abnormal sperm, the percentage zinc bound to HMW (HMW-Zn), the time between ejaculation and analysis and chromatin stability were studied. Samples with low chromatin stability were found more frequently in men with low HMW-Zn levels in semen. The proportion of stable sperm decreased in samples with prolonged exposure to seminal plasma. Neither the proportion of stable sperm heads nor the percentage zinc bound to HMW could be used to predict the future chances of the infertile men fathering children when studied 15-180 min after ejaculation. To differentiate between initial zinc-dependent stability and superstability developed in seminal plasma, other more sensitive methods must be developed.

The fertility potential of male cystic fibrosis patients.

Men suffering from cystic fibrosis (CF) are considered to be infertile because of azoospermia. In testicular biopsies from two patients with CF normal spermatogenesis was found despite the absence of sperm in the ejaculate. Fructose and prostaglandin were not detectable in the semen whereas the levels of acid phosphatase and zinc were within normal limits, indicating normal prostatic function and absence of seminal vesicles. These findings may improve the possibility for male patients with CF to father a child.

Ejaculatory sequence in men with low sperm chromatin-zinc.

The composition of seminal plasma in the sperm-rich split ejaculate fraction was studied in a group of men with a low zinc content in their sperm chromatin, to evaluate the availability of zinc at ejaculation. Men with low-chromatin zinc had, in the sperm-rich split-ejaculate fraction, high amounts of seminal-vesicular fluid, a low zinc:fructose molar ratio, and a high percentage of zinc bound to high molecular weight ligands of seminal vesicular origin (HMW-Zn). This indicates premature admixture of vesicular fluid at ejaculation. It is suggested that the zinc:fructose molar ratio and HMW-Zn in the sperm-rich fractions could be used as a measure of the availability of zinc in seminal plasma.

Seminal fluid from men with agenesis of the Wolffian ducts: zinc-binding properties and effects on sperm chromatin stability.

Zinc-binding properties were studied in 'prostatic fluid', i.e. in seminal plasma from patients with agenesis of the Wolffian ducts, and in split-ejaculate fractions dominated by seminal vesicular fluid. The effect of seminal fluid, with different zinc-binding properties, on the stability of zinc-dependent sperm chromatin was assessed by exposing sperm to 1% sodium dodecyl sulphate (SDS) for 60 min. Citrate was the only zinc ligand in 'prostatic fluid', as revealed by gel chromatography. Zinc in this fluid enhanced the stability of sperm chromatin. In contrast, the stability of sperm chromatin was decreased in seminal plasma dominated by vesicular fluid. These results are in accordance with the concept that prostatic fluid ensures the appropriate zinc content and stability of sperm chromatin, whereas abundance of vesicular fluid may jeopardize chromatin stability by reducing chromatin zinc content.