Trine B. Haugen

World Health Organization reference values for human semen characteristics

BACKGROUND Semen quality is taken as a surrogate measure of male fecundity in clinical andrology, male fertility, reproductive toxicology, epidemiology and pregnancy risk assessments. Reference intervals for values of semen parameters from a fertile population could provide data from which prognosis of fertility or diagnosis of infertility can be extrapolated. METHODS Semen samples from over 4500 men in 14 countries on four continents were obtained from retrospective and prospective analyses on fertile men, men of unknown fertility status and men selected as normozoospermic. Men whose partners had a time-to-pregnancy (TTP) of < or =12 months were chosen as individuals to provide reference distributions for semen parameters. Distributions were also generated for a population assumed to represent the general population. RESULTS The following one-sided lower reference limits, the fifth centiles (with 95th percent confidence intervals), were generated from men whose partners had TTP < or = 12 months: semen volume, 1.5 ml (1.4-1.7); total sperm number, 39 million per ejaculate (33-46); sperm concentration, 15 million per ml (12-16); vitality, 58% live (55-63); progressive motility, 32% (31-34); total (progressive + non-progressive) motility, 40% (38-42); morphologically normal forms, 4.0% (3.0-4.0). Semen quality of the reference population was superior to that of the men from the general population and normozoospermic men. CONCLUSIONS The data represent sound reference distributions of semen characteristics of fertile men in a number of countries. They provide an appropriate tool in conjunction with clinical data to evaluate a patients semen quality and prospects for fertility.

The mature form of interleukin-1α is constitutively expressed in immature male germ cells from rat

In the present study we show that immature germ cells from rat testis contain both interleukin-1 alpha (IL-1 alpha) mRNA and immunoreactive proteins. In contrast, in primary cultures of Sertoli cells and peritubular cells. IL-1 alpha mRNA and immunoreactive protein were below the levels of detection. Immunoblots of lysates from these germ cells showed the presence of strong 17 kDa bands (mature forms of IL-1 alpha) and a much weaker 33 kDa band (precursor form). The finding of cell-associated mature forms of IL-1 alpha in germ cells indicates that immature male germ cells are able to process IL-1 alpha independent of its secretion. Data from isolated cell fractions, as well as from whole testis tissue from rats of various ages, indicate that IL-1 alpha expression takes place in late pachytene spermatocytes and early round spermatids. Whether IL-1 alpha plays a role intracellularly in germ cells or exerts its effects on neighboring Sertoli cells remains to be shown.

Differential distribution of splice variants of estrogen receptor beta in human testicular cells suggests specific functions in spermatogenesis.

A growing number of estrogen receptor beta (ER beta) splice variants are reported. Several of these have been discovered in testis, but with few exceptions little is known about their cellular localization. The aim of this study was to identify and elucidate the mRNA expression pattern of the different ER beta splice variants in human testicular cells. Northern analysis was performed on whole testis and fractions enriched in germ cells from untreated men and from estrogen-treated men undergoing sex change surgery. Probes were constructed in order to systematically screen for and identify various ER beta splice variants. Several ER beta bands were observed in the human testis, in which splice variants constituted the major part of total ER beta transcripts. Interestingly, only two ER beta wild-type transcripts were detected. These seem to be virtually absent from the haploid germ cells and are probably mainly located in somatic cells and/or primary spermatocytes. Several novel ER beta deletion variants were found in high levels in the haploid germ cell fractions and were nearly absent in testicular cells from the estrogen-treated men. The cell-dependent distribution raises the question whether splice variants may have specific functions in spermatogenesis, and whether the differential splicing of ER beta is regulated in a cell-specific manner.

Expression and Regulation of Δ5-Desaturase, Δ6-Desaturase, Stearoyl-Coenzyme A (CoA) Desaturase 1, and Stearoyl-CoA Desaturase 2 in Rat Testis

Abstract In mammalian cells, essential polyunsaturated fatty acids (PUFAs) are converted to longer PUFAs by alternating steps of elongation and desaturation. In contrast to other PUFA-rich tissues, the testis is continuously drained of these fatty acids as spermatozoa are transported to the epididymis. Alteration of the germ cell lipid profile from spermatogonia to condensing spermatids and mature spermatozoa has been described, but the male gonadal gene expression of the desaturases, responsible for the PUFA-metabolism, is still not established. The focus of this study was to characterize the expression and regulation of stearoyl-CoA desaturase 1 (SCD1), stearoyl-CoA desaturase 2 (SCD2), and Δ5- and Δ6-desaturase in rat testis. Desaturase gene expression was detected in testis, epididymis, and separated cells from seminiferous tubulus using Northern blot analysis. For the first time, SCD1 and SCD2 expression is demonstrated in rat testis and epididymis, both SCDs are expressed in epididymis, while testis mainly contains SCD2. Examination of the testicular distribution of Δ5- and Δ6-desaturase and SCD1 and SCD2 shows that all four desaturases seem to be localized in the Sertoli cells, with far lower expression in germ cells. In light of earlier published results showing that germ cells are richer in PUFAs than Sertoli cells, this strengthens the hypothesis of a lipid transport from the Sertoli cells to the germ cells. As opposed to what is shown in liver, Δ5- and Δ6-desaturase mRNA levels in Sertoli cells are up-regulated by dexamethasone. Furthermore, dexamethasone induces SCD2 mRNA. Insulin also up-regulates these three genes in the Sertoli cell, while SCD1 mRNA is down-regulated by both insulin and dexamethasone. Δ5- and Δ6-desaturase, SCD1, and SCD2 are all up-regulated by FSH. A similar up-regulation of the desaturases is observed when treating Sertoli cells with (Bu)2cAMP, indicating that the desaturase up-regulation observed with FSH treatment results from elevated levels of cAMP. Finally, testosterone has no influence on the desaturase gene expression. Thus, FSH seems to be a key regulator of the desaturase expression in the Sertoli cell.

Fish model for assessing the in vivo estrogenic potency of the mycotoxin zearalenone and its metabolites.

The in vivo estrogenic potency of zearalenone (ZEA), a mycotoxin produced by different strains of Fusarium fungi, and its metabolites (alpha- and beta-zearalenol), have been studied in fish. Estrogenicity was evaluated using an in vitro competitive receptor binding assay and in vivo induction of vitellogenesis and zonagenesis, two estrogen receptor (ER)-mediated responses that are integral aspects of fish oogenesis. The ER binding affinities of alpha-zearalenol and ZEA in rainbow trout (Oncorhynchus mykiss) were approximately 1/150 and 1/300 to that of estradiol, respectively. Juvenile salmon (Salmo salar) were exposed to a single intraperitoneal injection of ZEA, alpha-zearalenol and beta-zearalenol (each at 1 and 10 mg/kg) and compared to fish injected with estradiol-17 beta (E2; 5 mg/kg) and controls. Using indirect enzyme-linked immunosorbent assay (ELISA) with homologous antibodies, a dose-dependent induction of vitellogenin (Vtg) and eggshell zona radiata proteins (Zr-proteins) were observed 7 days after exposure to ZEA and alpha-zearalenol. beta-Zearalenol did not elevate plasma Vtg levels, but a non-significant elevation of plasma Zr-proteins levels was observed at the highest dose (10 mg/kg). Generally, alpha-zearalenol and ZEA possess estrogenic potencies that are approximately 50% compared to that of E2, and their order of estrogenic potency (in both in vitro receptor competitive binding and in vivo induction of Vtg and Zr-proteins levels) is: alpha-zearalenol > ZEA > beta-zearalenol. Our results show that blood plasma analysis of Vtg and Zr-proteins levels provides a suitable in vivo fish model for assessing the estrogenic potencies of ZEA and its metabolites.

Elevated Expression of Lanosterol 14α-Demethylase (CYP51) and the Synthesis of Oocyte Meiosis-Activating Sterols in Postmeiotic Germ Cells of Male Rats.

Mammalian CYP51 encodes lanosterol 14α-demethylase (P45014DM) that is involved in the postsqualene part of cholesterol biosynthesis. This enzyme removes the 14α-methyl group from lanosterol and 24,25-dihydrolanosterol producing intermediates in cholesterol biosynthesis, the oocyte meiosis-activating sterols FF-MAS and MAS-412. Human and rat CYP51 messenger RNAs (mRNAs) are expressed in all tissues, with highest levels in the testis due to the presence of an additional shorter CYP51 transcript in this tissue. In situ hybridization shows the highest CYP51 mRNA levels in seminiferous tubules, with only background levels in Leydig cells. The rat testis-specific CYP51 mRNA arises from the use of an upstream polyadenylation site and is restricted to germ cells, being most abundant in elongating spermatids in stages VII–XIV, whereas somatic CYP51 transcripts are present in all cells. In contrast, the mRNA levels of squalene synthase are maximal in round spermatids, and no germ cell-specific transcript is observe...

Alcohol and male reproductive health: a cross-sectional study of 8344 healthy men from Europe and the USA

STUDY QUESTION Is there an association between alcohol intake and semen quality and serum reproductive hormones among healthy men from the USA and Europe? SUMMARY ANSWER Moderate alcohol intake is not adversely associated with semen quality in healthy men, whereas it was associated with higher serum testosterone levels. WHAT IS KNOWN ALREADY High alcohol intake has been associated with a wide range of diseases. However, few studies have examined the correlation between alcohol and reproductive function and most have been conducted in selected populations of infertile men or have a small sample size and the results have been contradictory. STUDY DESIGN, SIZE, DURATION A coordinated international cross-sectional study among 8344 healthy men. A total of 1872 fertile men aged 18-45 years (with pregnant partners) from four European cities and four US states, and 6472 young men (most with unknown fertility) aged 18-28 years from the general population in six European countries were recruited. PARTICIPANTS/MATERIALS, SETTING, METHODS The men were recruited using standardized protocols. A semen analysis was performed and men completed a questionnaire on health and lifestyle, including their intake of beer, wine and liquor during the week prior to their visit. Semen quality (semen volume, sperm concentration, percentage motile and morphologically normal sperm) and serum reproductive hormones (FSH, LH, testosterone, sex hormone-binding globulin, and inhibin B and free testosterone) were examined. MAIN RESULTS AND THE ROLE OF CHANCE The participation rate for our populations was 20-30%. We found no consistent association between any semen variable and alcohol consumption, which was low/moderate in this group (median weekly intake 8 units), either for total consumption or consumption by type of alcohol. However, we found a linear association between total alcohol consumption and total or free testosterone in both groups of men. Young and fertile men who consumed >20 units of alcohol per week had, respectively, 24.6 pmol/l (95% confidence interval 16.3-32.9) and 19.7 pmol/l (7.1-32.2) higher free testosterone than men with a weekly intake between 1 and 10 units. Alcohol intake was not significantly associated with serum inhibin B, FSH or LH levels in either group of men. The study is the largest of its kind and has sufficient power to detect changes in semen quality and reproductive hormones. LIMITATIONS, REASONS FOR CAUTION The participation rate was low, but higher than in most previous semen quality studies. In addition, the study was cross-sectional and the men were asked to recall their alcohol intake in the previous week, which was used as a marker of intake up to 3 months before. If consumption in that week differed from the typical weekly intake and the intake 3 months earlier, misclassification of exposure may have occurred. However, the men were unaware of their semen quality when they responded to the questions about alcohol intake. Furthermore, we cannot exclude that our findings are due to unmeasured confounders, including diet, exercise, stress, occupation and risk-taking behavior. WIDER IMPLICATIONS OF THE FINDINGS Our study suggests that moderate alcohol intake is not adversely associated with semen quality in healthy men, whereas it was associated with higher serum testosterone levels which may be due to a changed metabolism of testosterone in the liver. Healthy men may therefore be advised that occasional moderate alcohol intake may not harm their reproductive health; we cannot address the risk of high alcohol consumption of longer duration or binge drinking on semen quality and male reproductive hormones. STUDY FUNDING/COMPETING INTERESTS All funding sources were non-profitable and sponsors of this study played no role in the study design, in data collection, analysis, or interpretation, or in the writing of the article. The authors have no conflicts of interest.

A Novel Isoform of Human Cyclic 3′,5′-Adenosine Monophosphate-Dependent Protein Kinase, Cα-s, Localizes to Sperm Midpiece

Abstract Using rapid amplification of cDNA ends, a cDNA encoding a novel splice variant of the human Cα catalytic subunit of cAMP-dependent protein kinase (PKA) was identified. The novel isoform differed only in the N-terminal part of the deduced amino acid sequence, corresponding to the part encoded by exon 1 in the previously characterized murine Cα gene. Sequence comparison revealed similarity to an ovine Cα variant characterized by protein purification and micropeptide sequencing, Cα-s, identifying the cloned human cDNA as the Cα-s isoform. The Cα-s mRNA was expressed exclusively in human testis and expression in isolated human pachytene spermatocytes was demonstrated. The Cα-s protein was present in ejaculated human sperm, and immunofluorescent labeling with a Cα-s-specific antibody indicated that Cα-s was localized in the midpiece region of the spermatozoon. The majority of Cα-s was particulate and could not be released from the sperm midpiece by cAMP treatment alone. Furthermore, detergent extraction solubilized approximately two-thirds of the Cα-s pool, indicating interaction both with detergent-resistant cytoskeletal and membrane structures. In addition, we recently identified the regulatory subunit isoforms RIα, RIIα, and an A-kinase anchoring protein, hAKAP220 in this region in sperm that could target Cα-s. This novel Cα-s splice variant appeared to have an independent anchor in the human sperm midpiece as it could not be completely solubilized even in the presence of both detergent and cAMP.

The pathway from arachidonic to docosapentaenoic acid (20:4n-6 to 22:5n-6) and from eicosapentaenoic to docosahexaenoic acid (20:5n-3 to 22:6n-3) studied in testicular cells from immature rats.

The concentration-dependent metabolism of 1-(14)C-labelled precursors of 22:5n-6 and 22:6n-3 was compared in rat testis cells. The amounts of [(14)C]22- and 24-carbon metabolites were measured by HPLC. The conversion of [1-(14)C]20:5n-3 to [3-(14)C]22:6n-3 was more efficient than that of [1-(14)C]20:4n-6 to [3-(14)C]22:5n-6. At low substrate concentration (4 microM) it was 3.4 times more efficient, reduced to 2.3 times at high substrate concentration (40 microM). The conversion of [1-(14)C]22:5n-3 to [1-(14)C]22:6n-3 was 1.7 times more efficient than that of [1-(14)C]22:4n-6 to [1-(14)C]22:5n-6 using a low, but almost equally efficient using a high substrate concentration. When unlabelled 20:5n-3 was added to a cell suspension incubated with [1-(14)C]20:4n-6 or unlabelled 22:5n-3 to a cell suspension incubated with [1-(14)C]22:4n-6, the unlabelled n-3 fatty acids strongly inhibited the conversion of [1-(14)C]20:4n-6 or [1-(14)C]22:4n-6 to [(14)C]22:5n-6. In the reciprocal experiment, unlabelled 20:4n-6 and 22:4n-6 only weakly inhibited the conversion of [1-(14)C]20:5n-3 and [1-(14)C]22:5n-3 to [(14)C]22:6n-3. The results indicate that if both n-6 and n-3 fatty acids are present, the n-3 fatty acids are preferred over the n-6 fatty acids in the elongation from 20- to 22- and from 22- to 24-carbon atom fatty acids. In vivo the demand for 22-carbon fatty acids for spermatogenesis in the rat may exceed the supply of n-3 precursors and thus facilitate the formation of 22:5n-6 from the more abundant n-6 precursors.

Residual bodies and IL-1α stimulate expression of mRNA for IL-1α and IL-1 receptor type I in cultured rat Sertoli cells1

Abstract The cytokine interleukin (IL)-1α may be produced both by Sertoli cells and immature male germ cells from rat and is thought to play a role in autocrine and/or paracrine regulation of the spermatogenesis. The localization of IL-1 receptors in seminiferous tubules is unknown. In this study we found a constitutive expression of IL-1 receptor type I (IL-1 RI) mRNA in cultured Sertoli cells and peritubular cells from rat, whereas no such transcripts were observed in immature germ cells (pachytene spermatocytes and round spermatids). An autostimulation of IL-1α mRNA synthesis has previously been described in other cell types. Stimulation of Sertoli cells with recombinant IL-1α for 0–7 h resulted in a rapid increase in both IL-1α and IL-1 RI mRNA. When Sertoli cells were cultured with residual bodies for 0–48 h, mRNA levels for both IL-1α and IL-1 RI were increased in a biphasic manner. We suggest that phagocytosis of residual bodies triggers an autocrine IL-1α loop in Sertoli cells where both IL-1α and one of its receptors are stimulated.