bluuebunny/crossref_metadata_2025 huggingface dataset:
Migration sedimentation technique as a predictive test for the fertilizing capacity of spermatozoa in an in‐vitro fertilization programme
Authors: HAUSER R. , HOMONNAI Z. T. , PAZ G. F. , YAVETZ H. , AMIT A. , LESSING J. B. , PEYSER M. R. , YOGEV L.
Published: May 2008

The migration‐sedimentation technique (MST) has been proposed as a means of separating high quality motile spermatozoa. The present study was conducted in order to evaluate whether sperm performance following separation by MST predicts their fertilizing capacity in an in‐vitro fertilization (IVF) programme. Ninety semen specimens were analysed for use in an IVF‐embryo transfer (ET) programme. Each specimen was divided into two parts: one was processed in the IVF programme and was used after sperm swim‐up separation for insemination of human ova. The other aliquot (0.2 ml) was separated by MST, and the sperm then characterized by their concentration, motility, degree of motility and morphology. Sperm characteristics after separation by MST were then correlated with the results of the IVF‐fertilization rates. In 79 of 90 IVF‐ET cycles, at least one oocyte was fertilized. All post‐MST sperm characteristics were significantly higher in cycles with fertilizations compared to IVF cycles without fertilization. A larger percentage of the total motile spermatozoa were recovered after MST in semen specimens with fertilization, compared to semen specimens without fertilization (39.9 ± 3.6 and 20.6 ± 6.6%, respectively; P<0.05). This value was correlated with the percentage of fertilized oocytes (r= 0.24; P<0.02). More IVF cycles with fertilizations were recorded in cases in which the recovery of motile sperm was >25%(P<0.005), or when more than 1.5 times 106 motile spermatozoa were recovered after MST (P <0.0001). As sperm characteristics after MST correlated significantly with their fertilizing capacity, the MST test could be used in evaluation of the fertilizing capacity of spermatozoa.
Enhanced detection of reactive oxygen species produced by human spermatozoa with 7‐dimethyl amino‐naphthalin–1, 2‐dicarbonic acid hydrazide
Authors: AITKEN R. J. , BUCKINGHAM D.
Published: May 2008

A new chemiluminescence technique has been assessed for the detection of reactive oxygen species generated by purified populations of human sperm. This revised protocol involves the use of horse‐radish peroxidase (HRP) in combination with a luminol analogue, 7‐dimethyl aminornaphthalin‐1,2‐dicarbonic acid hydrazide (DNDH), that exhibits two‐three times the quantal efficiency of luminol itself. The chemiluminescent signal generated with these reagents was significantly (P < 0.001) greater than that obtained with the conventional luminol‐based methodology for both the steady‐state situation and following stimulation of the sperm with PMA and A23187. Dose‐response analyses indicated that the DNDH/HRP chemi‐luminescence system could give linear standard curves with hydrogen peroxide concentrations into the nmol I‐1 range. In contrast, the exponential rise in chemiluminescence recorded with luminol was not observed until hydrogen peroxide concentrations exceeded 10 umol I‐1. It is concluded that the enhanced sensitivity of the DNDH/HRP system to low levels of hydrogen peroxide should facilitate the application of chemiluminescent techniques to the diagnosis of oxidative stress in cases of male infertility.
Effect of zinc on human sperm motility and the acrosome reaction
Authors: RIFFO M. , LEIVA S. , ASTUDILLO J.
Published: May 2008

This study has assessed the effect of zinc on human sperm motility and the acrosome reaction in vitro. Progressively motile human sperm were selected by swim‐up and by glass bead columns and then incubated in a medium in which capacitation happened in an asynchronous way. Different doses of zinc (1, 10, 100 and 1000 μM) were added for periods of 2, 4 or 6 h. Other samples were incubated with zinc (1000 μM), and after 1 h incubation, the zinc was removed. Aliquots of each culture were used to evaluate progressive motility and the acrosome reaction using a triple‐stain technique. Sperm motility was reduced when the amount of zinc added was ≥ 100 μM, and these doses also caused a significant reduction in the % of sperm undergoing the acrosome reaction. After removal of zinc and further incubation in zinc‐free medium for 1 h, an increase in the percentage of motile and acrosome‐reacted sperm was observed. However, the increase in acrosome reaction did not reach the values observed in controls. Results suggest that extracellular zinc acts as an inhibitor of human sperm motility and the acrosome reaction (and/or capacitation and the acrosome reaction). This inhibitory effect is reversible and occurs in a dose‐dependent fashion. The probable mechanisms involved are discussed.
Effects of induced peripheral anosmia on gonadal maturation in prepubertal male rabbits
Authors: ADAMOPOULOS D. A. , KONTOGEORGOS L. , VASSILOPOULOS P. , KAPOLLA N. , STATHOPOULOS E. , NICOPOULOU S.
Published: May 2008

The effect of induced peripheral anosmia on gonadal development and maturation was investigated in sexually immature male rabbits. Furthermore, the effect of agents promoting gonadal maturation (LHRH, hMG or methyl testosterone) on testicular development was examined in anosmic rabbits. Peripheral anosmia was induced by spraying the olfactory mucosa with 5% ZnSO4 solution; its effects were evaluated after a 45‐day period, corresponding to the duration of spermatogenesis. Evaluation was based on measurement of body‐weight, testicular size, testicular biopsy score count (TBSC) and a standard LHRH test (0 and 30 min) involving measurement of the blood levels of FSH, LH and testosterone before and at the end of the test. Markedly lower final and incremental values were noted in anosmic, compared to intact, animals for body‐weight (P > 0.001), TBSC (P > 0.001), FSH (P > 0.01) and LH (P > 0.05). On the other hand, treatment of the anosmic rabbits with 0.9% saline, resulted in lower FSH, TBSC and testicular size increments than in rabbits treated with LHRH, hMG or testosterone, while testosterone levels and body‐weight increments were similar in all groups. These findings indicate that induced peripheral anosmia is probably responsible for the inadequate gonadal maturation in prepubertal anosmic male rabbits. This relationship was confirmed by the observed stimulatory effect of administration of agents activating pituitary gonadotrophin secretion or gonadal function in anosmic animals.
Relationship between sperm quality and chromatin condensation measured by sperm DNA fluorescence using flow cytometry
Authors: ENGH E. , CLAUSEN O. P. F. , SCHOLBERG A. , TOLLEFSRUD A. , PURVIS K.
Published: May 2008

DNA flow cytometry of sperm from 100 randomly chosen men undergoing fertility investigation revealed a general association between reduced sperm quality, as judged by conventional parameters, and the appearance of sperm with lower degrees of chromatin condensation in the ejaculate as measured by DNA fluorescence intensity. Chromatin hypocondensation, as measured by increased fluorescence, was manifested to different degrees in different samples. In many cases of more extreme sperm pathology, such as oligoasthenoteratozoospermia (OAT), the whole population of spermatozoa appeared to be affected. Significant numbers of hypercondensed spermatozoa were present in both normozoospermic men and men with different degrees of disturbance in sperm quality. All of the different parameters of sperm quality could be correlated significantly with certain of the flow parameters, although not one in particular could be used to predict deviations from the normal flow profile. In several asthenoteratozoospermic men and a small proportion of men with OAT, the DNA profiles were normal, implying that in these cases the disturbance may not be so fundamental. The presence of leucocytes in the ejaculate was associated with a general increase in the preponderance of hypocondensed subpopulations of spermatozoa in men with OAT as well as in normozoospermic subjects, emphasizing the effect of inflammatory conditions in the reproductive tract on sperm quality.
The influence of sperm density on the motility characteristics of washed human spermatozoa
Authors: Wetzels Alexm. M. , Janssen Herman J. G. , Goverde Henny J. M. , Bastiaans Bart A. , Takahashi Kentaro , Rolland Rune
Published: May 2008

To study the effects of sperm density on the results of computer‐assisted semen analysis (CASA), 10 washed semen samples were diluted and measured with the CellTrak/S CASA system in a concentration range of 10–180×106 spermatozoa/ ml. All sperm motility parameters were influenced to some extent by sperm density. The motility percentage was influenced significantly in 5 samples (P<0.005), the straight line velocity in all samples (P<0.0005 in 7 samples), the curvilinear velocity in 3 samples (P< 0.005), the linearity in 9 samples (P<0.0005 in 6 samples) and the lateral head displacement in 9 samples (P< 0.005 in 6 samples). In general, the CellTrak/S data are influenced significantly if sperm density exceeds 50 × 106 spermatozoa/ml.

The influence of sperm density on the motility parameters can be explained both by the accuracy of the CASA system and by actual changes in the motility of the spermatozoa. In the light of other published studies, it is concluded that sperm motility measurements with CASA systems should be assessed using 25–50 × 106 spermatozoa/ml, especially in studies concerning lateral head displacement and the linearity, as in sperm hyperactivation studies.
Prostate‐specific antigen and prostate gland size in men receiving exogenous testosterone for male contraception
Authors: Wallace E. M. , Pye S. D. , Wild S. R. , Wu F. C. W.
Published: May 2008

Steroid regimens containing androgens are being evaluated currently as hormonal contraceptives for men. The possible non‐reproductive effects of such treatment were assessed during an efficacy trial using a prototype regime of 200 mg testosterone enanthate i.m. weekly. Prostatic function and size were monitored by regular rectal examination, blood levels of prostate‐specific androgen (PSA) were measured in 30 men and prostatic size was measured by trans‐rectal ultrasound imaging in a representative subgroup of five subjects for 12 months and for a further 6 months after discontinuation. Despite the sustained rise in serum levels of testosterone, oestradiol and dihydrotestosterone during treatment, there was no detectable increase in prostatic size on rectal examination or any significant change in blood concentrations of PSA. A small but significant increase (14.3 ± 2.0%) in maximal prostate transverse area was observed in four men while the remaining one showed no change. Our preliminary data demonstrate the feasibility and importance of monitoring prostatic function in the development of androgen‐containing male hormonal contraceptives.
Tissue‐specific gene expression as an indicator of epididymis‐specific functional status in the boar, bull and stallion
Authors: Uhlenbruck F. , Sinowatz F. , Amselgruber W. , Kirchhoff C. , Ivell R.
Published: May 2008

cDNA probes derived from genes expressed specifically in the human epididymis were used to examine gene expression in the epididymides of boar, bull and stallion by Northern hybridization. Two probes for the HE1 and HE4 gene products were found to recognize tissue‐specific transcripts in all three species, with a regionally differential distribution within the epididymis. Additionally, antibodies recognizing the HE4 protein were shown to react specifically in the epididymis of the boar and bull. An extensive study of the boar showed that, whereas mRNA for the HEl‐homologue was up‐regulated markedly only at puberty, the HE4‐homologue was already present at moderate levels prepubertally. The distribution of the HEl‐homologue changed at sexual maturity from a maximum in the cauda epididymis in the 3–4‐week‐old pig, to a maximum in the corpus/caput region in the adult, while the shift was in the opposite direction for the HE4‐homologue. Evidently, gene expression is not fixed regionally through epididymal development in this species. The abdominal epididymis of a hemicryptorchid pig also showed a differential change in expression for the two gene products by comparison with the scrotal testis from the same animal. The results suggest that the HE1 and HE4 gene homologues may be sensitive markers for physiological changes within the mammalian epididymis, and that the boar could prove a useful model to examine the regulation of these human epididymal transcripts.
Modification of human sperm function by human follicular fluid: a review
Authors: HONG C. Y. , CHAO H. T. , LEE S. L. , WEI Y. H.
Published: May 2008

This review assesses whether human follicular fluid (hFF) is able to modify human sperm function in vitro. Addition of hFF has been found to stimulate the motility of washed human spermatozoa, to increase the percentage of hyper‐activated spermatozoa, to induce the acrosome reaction, to attract spermatozoa to the site of fertilization and to facilitate penetration of the oocyte by spermatozoa. It is possible that hFF could provide a favourable environment around the oocyte for fertilization by spermatozoa. Inclusion of hFF in gamete transfer medium may also improve the success rate of assisted reproduction technology. Purification of individual components in hFF which modify different aspects of sperm function awaits further investigation.
Platelet‐activating factor acetylhydrolase in the male reproductive tract: origin and properties
Authors: JARVI K. , LANGLAIS J. , GAGNON C. , ROBERTS K. D.
Published: May 2008

The properties and origin of platelet‐activating factor acetylhydrolase‐ (PAF‐AH)‐like activity in the male reproductive tract were investigated. Seminal plasma (SP) and serum were obtained from normal donors and infertile patients while prostatic fluid (PF), seminal vesicle fluid (SVF) and vas deferens fluid were collected at autopsy. PAF‐AH‐like activity was found in all fluids tested. The specific activity of the enzyme in SP and PF was twice that of PAF‐AH in serum and 15–fold higher than that in SVF and vas deferens fluid. In SP, PAF‐AH‐like activity was Ca++‐independent, acid and heat labile, stable to freezing, not inhibited by phosphatidylcholine, but was inhibited by 10 mM disopropylfluoro‐phosphate (DFP) and 13 mM phenylmethylsulfonylfluoride (PMSF). These data indicate that the properties of the enzyme in SP are similar to those reported for PAF‐AH in serum. The variation in specific activity of PAF‐AH in reproductive tract fluids suggest that there are either activators of PAF‐AH in SP or inhibitors in one or several of the other reproductive tract fluids.
A search for circulating immunoglobulins blocking follicle‐stimulating hormone action in male idiopathic infertility
Authors: SIMONI M. , PASCHKE R. , NIESCHLAG E.
Published: May 2008

In this study, patients with idiopathic infertility were investigated for the presence of circulating antibodies which interfered with follicle‐stimulating hormone (FSH) activity. A retrospective search for autoantibodies was undertaken using single plasma samples obtained from 29 infertile men with azoo/oligo/asthenoteratozoospermia and eight controls with normozoospermia and normal blood FSH levels. Plasma levels of immunoactive FSH, bioactive FSH, immunoactive luteinizing hormone and testosterone were measured in the individual samples. Plasma immunoglobulin G (IgG) was isolated using protein‐A chromatography and tested at different dose levels for its ability to inhibit and/or stimulate basal and FSH‐induced aromatase activity in immature rat Sertoli cells in vitro. In the first set of trials, IgGs isolated from three infertile patients showed apparent inhibition of FSH‐stimulated aromatase activity, two showed further stimulation and five showed irregular fluctuations beyond the normal range of stimulation. When reanalysed at different doses, none of the IgG fractions exhibited abnormal interference with FSH action. It is concluded that, unlike many other endocrine disorders characterized by autoimmunity, the occurrence of autoantibodies of the IgG class blocking FSH action in male infertility is improbable.
Developmental expression and androgen regulation of 24 kDa secretory proteins by the murine epididymis
Authors: LEFRANÇOIS A. M. , JIMENEZ C. , DUFAURE J. P.
Published: May 2008

Two peptides with a molecular weight of 24 kDa and a Pi of 8.4–8.8 were found to be synthesized and secreted specifically by the caput epididymis of adult male mice under androgen control. The peptides can interact with spermatozoa. In the present study, the developmental pattern of [35S]‐methionine‐labelled proteins synthesized by the murine caput epididymis at 10, 20, 30 and 40 days of age were studied using two‐dimensional polyacrylamide gel electrophoresis (2D PAGE) and autoradiography. Active synthesis of the 24 kDa proteins was detected in the epididymis from 20 days of age, but secretion of the two peptides was only observed from 30 days of age onwards. To determine whether androgens influenced the active expression of 24 kDa proteins in the developing epididymis, their effect on [35S]‐methionine incorporation into proteins was assessed using 2D PAGE. Mice were either castrated, castrated then testosterone injected or simply testosterone injected at 10, 20, 30 or 40 days of age. Androgen control of 24 kDa protein expression was also studies in vitvo in epididymal organ culture over a 10‐day period, with or without testosterone. Androgens were not involved in the initiation of synthesis of the 24 kDa proteins from days 10 to 20, as shown by in‐vivo and in‐vitro experiments. However, androgens appeared to be essential for maintaining synthesis and secretion of the proteins from 20 days of age onwards. Administration of excessive testosterone was only able to increase secretion of the 24 kDa proteins in intact male mice aged 40 days.
Interleukin‐1 bioactivity and DNA synthesis in X‐irradiated rattestes
Authors: HAKOVIRTA H. , PENTTILÄ T. L. , PÖLLÄNEN P. , FRÖYSA B. , SÖDER O. , PARVINEN M.
Published: May 2008

Stage‐specific DNA synthesis and interleukin‐1 (IL‐1) bioactivity were measured in the rat testis at 2, 6, 12 and 25 days after local irradiation with 3 Gy to investigate whether there was any correlation between germ cell DNA synthesis during repopulation and IL‐1‐like bioactivity in the seminiferous epithelium. DNA synthesis by intermediate and type‐B spermatogonia was reduced significantly at 2, 6 and 12 days after irradiation in seminiferous tubules at stages II‐III and IV‐V. IL‐1 bioactivity was increased significantly at 6 and 25 days after irradiation at stages II‐VI during repopulation of spermatogonia. At 2, 6 and 25 days after irradiation at stages VIIa‐c a significant increase in IL‐1 bioactivity was observed that correlated with repair synthesis of DNA. Increased IL‐1 bioactivity was also observed at stages IX‐XII at 6 days post‐irradiation. These observations support the concept that IL‐1 is a spermatogonial growth factor which might also stimulate repair synthesis of DNA.
Intratesticular hormone levels and the route of secretion of hormones from the testis of the rat, guinea pig, monkey and human
Authors: Maddocks S. , Hargreave T. B. , Reddie K. , Fraser H. M. , Kerr J. B. , Sharpe R. M.
Published: May 2008

Blood samples were obtained from the testes of rats, guinea pigs and Macaque monkeys and from normal men undergoing vasectomy reversal, in order to assess the comparative dynamics of hormone secretion. In each species, blood was sampled from a vein on the surface of the testis (testicular venous blood, TV), from a vein in the spermatic cord above the pampiniform plexus (spermatic venous blood, SV) and from a vein elsewhere in the body (peripheral venous blood, PV). Plasma concentrations of testosterone and inhibin were then determined by radioimmunoassay. In all species, testosterone secretion profiles were comparable, with concentrations being greatest in TV blood. SV concentrations were reduced by 40–60% compared with TV levels, with a significantly greater reduction in PV levels. Inhibin secretion varied significantly between species, with the rat being the only animal to show significant increases in inhibin concentrations from TV to SV blood. Inhibin secretion in the guinea pig was most comparable with that of the rat, although the increased SV levels fell short of being significant. Macaque and human profiles contrasted with those of the rat and guinea pig, with the greatest inhibin concentrations being found in TV blood. Levels in SV blood were reduced by some 40%, and PV levels were reduced significantly further. These differences may be due to the different position of the rete testis, and its relationship to the testicular vasculature, in these species. The sampling procedure described provides a defined set of testicular blood samples that could contribute important information of relevance to physiological and clinical studies of testicular function. Hormone concentrations in TV blood samples provide the most accurate indication of intratesticular levels, and the present study demonstrates that TV blood can be sampled with relative ease from a major vein on the surface of the human testis. The observation that testosterone and inhibin concentrations in TV blood were 10 or 250 times greater, respectively, than in PV blood in men, suggests that greater clinical use of this sampling procedure may be warranted when surgical intervention is taking place for other reasons, as this could give insight into the pathophysiology of the human testis.
In‐vitro influence of germ cells on Sertoli cel l‐secreted proteins: a two‐dimensional gel electrophoresis analysis
Authors: Gearard Nadine , Jegou Bernard
Published: May 2008

Two‐dimensional polyacrylamide gel electrophoresis (2D PAGE) was used to analyse [35S]‐methionine‐labelled proteins secreted in vitro by Sertoli cells when cultured in the presence or absence of enriched preparations of pachytene spermatocytes (SPC), early spermatids (SPT) or residual bodies/cytoplasts from elongated spermatids (RB/CES). The presence of germ cells modified the pattern of Sertoli cell secreted proteins in co‐culture. Out of 21 Sertoli cell secreted polypeptide families visualized by 2D PAGE, one (referred to as number 12) was stimulated, whereas the secretion of polypeptides 1 and 3 was inhibited by all of the germ cell populations tested. Early spermatids and RB/CES both enhanced the secretion of protein number 10 and inhibited the production of protein 11. The RB/CES fraction specifically inhibited secretion of polypeptide 13. Of particular note was the finding that co‐culture with early spermatids or RB/CES induced the secretion of a novel polypeptide, termed GIP (germ cell‐induced protein), with an apparent molecular weight of 72 kDa and an isoelectric point of 5.9. Under the present experimental conditions, media conditioned by the different germ cell fractions inhibited the secretion of polypeptide 2 but enhanced the secretion of polypeptides 10 and 18; of note also was the finding that media conditioned by early spermatids or RB/CES induced the appearance of GIP. This study confirms and extends the concept that germ cells influence Sertoli cell function and that the effects observed differ according to the stage of development of the germ cells. However, the sensitivity of the 2D gel electrophoresis technique, and to some extent its reproducibility, limit its use for studying the paracrine control of Sertoli cells in culture.
Prevalence of antisperm antibodies by SpermMARtest in subjects undergoing a routine sperm analysis for infertility
Authors: SINISI A. A. , FINIZIO B. DI , PASQUALI D. , SCURINI C. , D'APUZZO A. , BELLASTELLA A.
Published: May 2008

To evaluate the prevalence of antisperm antibodies (ASA) attached to the sperm plasma membrane in male partners of infertile couples, the binding of latex particles to spermatozoa was investigated using SpermMARtest, included routinely in semen analysis. A total of 860 men were examined, who were referred consecutively for semen analysis. Of these, 750 men were referred because of infertility (0.6–10 years in duration) whereas 110 were volunteers with a history of previous fertility. Samples were assessed by the SpermMARtest kit using latex particles sensitized with human IgG. Sperm‐latex binding was read after 3 min and samples scored as negative, positive or highly positive when < 10, > 10–40, or >40% binding occurred, respectively. Of the samples 132 (17.3%) were excluded because of azoo‐ or severe oligo‐asthenozoospermia. IgG attached to spermatozoa were detected in nearly 13% of semen samples from the infertile population and in one of 110 fertile men (0.90/,). From the infertile group, 6.2% of samples showed > 40% binding, and 6.7% intermediate binding, with an overall ASA prevalence of 12.9% in subjects undergoing semen analysis for infertility.
Separation of human spermatozoa with hyaluronic acid induces, and Percoll® inhibits, the acrosome reaction
Authors: SLOTTE H. , ÅKERLÖF E. , POUSETTE Å.
Published: May 2008

The effect on the acrosome reaction of human spermatozoa of two widely used sperm separation media, hyaluronic acid (Sperm Select®) and Percoll®, was studied. Viable and highly motile fractions of human spermatozoa were separated from seminal plasma using self‐migration on a Percoll® gradient. After translocation of separated spermatozoa from the Percoll® solution to a culture medium, serum, Percoll® or hyaluronic acid (Sperm Select®) was added to aliquots of the spermatozoa containing culture medium. At increasing time intervals, the influx of 45Ca2+ into spermatozoa was measured and the concentration of viable spermatozoa that had undergone the acrosome reaction was analysed using the triple stain technique. Serum was found to be necessary to support sperm motility and viability. Compared to culture medium with serum only, addition of hyaluronic acid induced influx of 45Ca2+ and the acrosome reaction, whilst Percoll® inhibited both of these actions. Hyaluronic acid (Sperm Select®) added to spermatozoa separated by a ‘swim‐up’ method induced, and the addition of Percoll® inhibited, influx of 45Ca2+ when compared to the addition of culture medium with serum only. This study demonstrates that both hyaluronic acid (Sperm Select®) and Percoll® affect the acrosome reaction and the prerequisite for Ca2+ influx in human spermatozoa. These effects should be taken into consideration when using these media for preparation of spermatozoa for insemination or for fertilization in vitro.
Effect of age on seminiferous tubule protein secretion and the adverse effects of testicular toxicants in the rat
Authors: MCLAREN T. T. , FOSTER P. M. D. , SHARPE R. M.
Published: May 2008

This study has assessed the effect of age on protein secretion by seminiferous tubules (ST) isolated from rats and their response to Sertoli cell toxicants. ST were isolated from immature (aged 28 days), late pubertal (aged 45 days) and young adult (aged 70 days) rats and cultured in vitro for 24 h with 35S‐methionine in the presence or absence of FSH (1 mg/ml), m‐dinitrobenzene (m‐DNB) or nitrobenzene (NB) (both at 10‐4M). Incorporation of 35S‐methionine into newly synthesized proteins in the culture medium (secreted proteins) was assessed and the pattern of protein secretion evaluated using two‐dimensional sodium dodecylsulphate polyacrylamide gel electrophoresis (2‐D SDS‐PAGE). These data were compared with those obtained using cultures of immature rat Sertoli cells + germ cells (SC+GC). Addition of FSH, m‐DNB or NB in vitro either had no effect (NB) or had a small stimulatory effect (FSH and m‐DNB) on the incorporation of 35S‐methionine into overall secreted proteins by ST isolated from immature rats. At the same doses, addition of either FSH, m‐DNB or NB to SC+GC co‐cultures resulted in increased incorporation of radiolabel into secreted proteins in all instances. In contrast, the same additions to ST isolated from adult rats resulted in a 20–34% decrease in the overall incorporation of 35S‐methionine into secreted proteins. ST isolated from late pubertal rats showed a similar response to adult rats except that the decreases in incorporation induced by FSH, m‐DNB and NB were smaller. Analysis by 2‐D SDS‐PAGE revealed considerable age‐dependent differences in the proteins secreted by ST from immature and adult rats, of which 13 were identified as being of potential importance. Most of these proteins were prominent secretory products of ST from adult rats, but were minor or non‐detectable products of cultures of ST or SC+GC from immature rats. Most of these proteins disappeared or decreased in abundance after culture of ST with m‐DNB or NB. Two proteins showed the reverse pattern, being more prominent secretory products in immature than mature rats, and their secretion was unaffected or was increased by toxicant exposure. These results demonstrate that there are major age‐dependent differences in the secretion of total and specific proteins by isolated ST and that these are probably related to changes in the germ cell complement with age. The susceptibility of many of these proteins to perturbation by m‐DNB and NB may explain the adverse effects of these compounds on spermatogenesis. Finally, the present results suggest strongly that studies of the regulation of spermatogenesis should not use cells/tissue isolated from immature rats.
The influence of pentoxifylline on motility and viability of spermatozoa from normozoospermic semen samples
Authors: TOURNAYE HERMAN , JANSSENS RONNY , DEVROEY PAUL , VAN STEIRTEGHEM ANDRK
Published: May 2008

In order to evaluate the effects of pentoxifylline on sperm motility and longevity, a controlled in‐vitro study was conducted on normozoospermic donor semen samples using the Cellsoft automated system for sperm motility analysis. After incubation and selection, pentoxifylline was found to improve the recovery of spermatozoa and to increase their velocity. In the subgroup of progressively motile spermatozoa, curvilinear velocity was also enhanced. It is concluded that pentoxifylline has an effect on the vigour, but not on the pattern, of sperm motion. Pentoxifylline did not improve the motility characteristics of senescent spermatozoa in normozoospermic sperm samples. Sperm survival, as shown by supra‐vital staining, and motility longevity both decreased with time after pentoxifylline treatment.
Adenosine triphosphate and motility characteristics of fresh and cryopreserved human spermatozoa
Authors: McLAUGHLIN E. A. , FORD W. C. L. , HULL M. G. R.
Published: May 2008

The concentration of ATP and the motility of human spermatozoa was measured in fresh and cryopreserved cells from the same 15 ejaculates. No coherent picture of the relationship between motility and ATP concentration emerged in whole semen or in spermatozoa washed by repeated centrifugation and resuspension in Biggers Whitten and Whttingham medium. This may have been due to the presence of dead spermatozoa and contaminating cells. After preparation on a Percoll gradient, the ATP concentration in fresh and cryopreserved spermatozoa was the same (6±0.7 nmol/108 spermatozoa) but 85±2.5% of the fresh spermatozoa were progressively motile with an average path velocity of 55±3.5 pm/s compared to corresponding values of 33±5.3% and 44±3.4 μm/s in frozen/thawed spermatozoa. This suggests that the poor motility of cryopreserved spermatozoa does not result from deficient ATP production. No relationship was found between ATP concentration and the ability of motile spermatozoa in the ejaculate to survive freezing.
The value of quantitative DNA flow cytometry of testicular fine‐needle aspirates in assessment of spermatogenesis: a study of 137 previously maldescended human testes
Authors: GIWERCMAN A. , CLAUSEN O. P. F. , BRUUN E. , FRIMODT‐MOLLER C. , SKAKKEBAEK N. E.
Published: May 2008

In order to assess the suitability of DNA flow cytometry of fine‐needle aspirates for quantiftring spermatogenesis, the results from DNA flow cytometry were compared to histological evaluation of testicular biopsies taken concomitantly from 171 previously maldescended testes. In 137 of 171 cases, sufficient material for flow cytometric as well as histological evaluation was obtained.

Histological analysis of surgical biopsy specimens revealed spermatogenesis including the spermatid stage in 117 of the 137 gonads. In six of the 117 gonads no haploid cells were found using flow cytometry. On the other hand, surgical biopsies failed to reveal spermatogenesis in five cases in which the corresponding aspirates contained haploid cells. Both methods therefore seem equally sensitive in detection of spermatogenesis.

Other types of histological patterns also corresponded to distinct DNA histograms.

Thus, in 11 of 12 cases with Sertoli‐cell‐only pattern in all tubules, at least 95% of the cells had a diploid DNA content. Furthermore, predominance of tubules with maturation arrest at the primary spermatocyte level corresponded to an increased proportion of tetraploid cells.

When compared to surgical biopsy, DNA flow cytometry of testiclar fine‐needle aspirates is a more objective, easy and rapid method, which is more convenient for the patient. This study has indicatedthat DNA flow cytometry is a suitable method of quantitative assessment of spermatogenesis. One of the first target groups might be men with azoospermia. In such men, DNA flow cytometric analysis of fine‐needle aspirates and surgical biopsy are apparently of equal sensitivity in detecting gonads with spermatogenesis. We conclude that DNA flow cytometry may become an alternative method for the quantification of spermatogenesis.
The hamster Sertoli cell in early testicular regression and early recrudescence: a stereological and endocrine study
Authors: RUSSELL LONNIE D. , CHANDRASHEKAR V. , BARTKE ANDRZEJ , HIKIM AMIYA F. SINHA
Published: May 2008

Utilizing stereological techniques, the hamster Sertoli cell at Stage VII of the spermatogenic cycle was examined for early signs of regressive changes after short‐term photoperiod‐induced testicular regression and for early signs of stimulation after short‐term photoperiod‐induced recrudescence. After 4–6 weeks of exposure to short photoperiod, germ cell degeneration was prominent but there were only minimal changes in Sertoli cell structure. These included a decline in volume of multivesicular bodies and in the surface area of mitochondrial membranes and adluminal plasma membrane, while no changes were noted in the other parameters measured. Of the endocrine changes, only plasma FSH levels had declined at this time and there were virtually no significant correlations between plasma FSH levels and the various structural parameters. The general lack of Sertoli cell changes in response to a short exposure to inhibitory photoperiod in the seasonally breeding hamster is similar to the early response to hypophysectomy in the none‐seasonal rat. During early photoperiod‐related recrudescence, testis weight, tubular lumen volume and interstitial parameters were increased. Significantly more preleptotene spermatocytes (about a 50% increase) were present in stage VII tubules, though adluminal germ cells did not increase in number. At this time some structural features of the Sertoli cells were enhanced in volume (cytoplasm, smooth and rough endoplasmic reticulum) and surface area (outer and inner mitochondrial membranes, smooth and rough endoplasmic reticulum and the basal compartment plasma membrane). Blood levels of FSH and testosterone were increased significantly but correlated only with rough endoplasmic reticulum, suggesting that initiation of the synthetic processes was imporant in reestablishment of spermatogenesis. Thus, similar to our findings in the Leydig cell, the structural responses of the Sertoli cell during regression and after recrudescence do not involve the same organelles. During re‐stimulation, we suggest that hormonal stimulation affects primarily synthetic processes in the nucleus and rough endoplasmic reticulum of the Sertoli cell, and that its functions can be re‐established only after these processes have been stimulated.
Improvement in quality of cryopreserved human spermatozoa by swim‐up before freezing
Authors: PÉREZ‐SÉNCHEZ F. , COOPER T. G. , YEUNG C. H. , NIESCHLAG E.
Published: May 2008

Selecting a population of spermatozoa by the swim‐up technique yields, after freezing and thawing, a population of cells that contains proportionally more spermatozoa which are morphologically normal, fewer spermatozoa with damaged tail membranes, and a greater percentage of progressively motile spermatozoa with greater velocities and amplitudes of head displacement than those obtained after freezing and thawing the same semen samples in the normal way. This pattern was found for the semen from 10 patients and five volunteers. However, the cells selected by swim‐up were as susceptible to the stresses caused by freezing and thawing as unselected spermatozoa in the original semen sample, and the improvement came from the better quality of the initial sample.
Peroxidase‐positive round cells and microorganisms in human semen together with antibiotic treatment adversely influence the outcome of in‐vitro fertilization and embryo transfer
Authors: DE GEYTER CH. , DE GEYTER M. , BEHRE H. M. , SCHNEIDER H. P. G. , NIESCHLAG E.
Published: May 2008

Human semen contains not only spermatozoa but also other cells routinely differentiated as being peroxidase‐positive (e.g. leucocytes) and peroxidase‐negative (eg. immature germ cells and lymphocytes) cells. Considerable uncertainty exists about their role in male fertility. To assess the clinical value of both parameters, and of microorganisms in semen, 391 treatments with in‐vitro fertilization and embryo transfer were analysed retrospectively, and the concentrations of both peroxidase‐positive and ‐negative cells, together with the presence of microorganisms in semen, were compared with both the fertilization and pregnancy rates. The data indicate that the results of treatment were affected only by excessively elevated concentrations of peroxidase‐positive cells (>6×106/ml) and only marginally by the presence of microorganisms in the semen. The pregnancy rate after in‐vitro fertilization and embryo transfer was not improved by antibiotic treatments preceding gamete recovery by several weeks. The increased presence of peroxidase‐negative cells (e.g. germ cells) in semen was not associated with a significant change in the pregnancy rate. However, the concentration of peroxidase‐negative cells in semen correlated significantly with sperm numbers (p<0.01), sperm concentration (p<0.01), and normal morphology rates (p<0.01). It is concluded that short‐term antibiotic treatment of asymptomatic patients before assisted reproduction should be handled with caution. The widespread view that peroxidase‐negative cells in semen are harmful is rejected.
Localization of mRNAs by in‐situ hybridization to the residual body at stages IX‐X of the cycle of the rat seminiferous epithelium: fact or artefact?
Authors: MILLAR M. R. , SHARPE R. M. , MAGUIKE S. M. , GAUGHAN J. , WEST A. P. , SAUNDERS P. T. K.
Published: May 2008

Several recent articles have reported localization of specific mRNAs in the rat testis to stage IX and X seminiferous tubules using in‐situ hybridization. In all cases the expression was located basally in the tubules and appeared as discrete round clusters of grains close to the lamina propria. The localization was interpreted as being in Sertoli cells or leptotene spermatocytes. In this study we demonstrate that this pattern is most probably due to artefactual binding of probes to the residual body (RB).

In the present study testicular tissue, perfusion‐fixed with Bouin's and embedded in paraffin, was used, as this resulted in excellent morphological preservation such that RBs within tubules at stages VIII—X were clearly distinguishable. RNA content of the FU3s was demonstrated at stages VIII—X using methyl green pyronin staining, and could be eliminated by pretreatment with RNAse or trichloroacetic acid. Localization of mRNAs for 11 seminiferous tubule proteins was assessed using 35S‐labelled and digoxigenin‐labelled riboprobes (activin receptor‐11, α‐inhibin, transferrin, androgen‐binding protein (ABP), cyclic protein‐2 (CP‐2), CREM, sulphated glycoproteins 1 and 2 (SGP‐1 and SGP‐2), transition protein 2 (TP‐2) and cystatin‐C), and digoxigenin‐labelled oligonucleotide probes (transition protein‐1 (TP‐l), TP‐2 and protamine‐1). All of these probes showed localization to the correct cell type(s) within the seminiferous epithelium. In addition, six antisense riboprobes (activin receptor‐II, CREM, SGP‐2, CP‐2, cystatin C and α‐inhibin) showed hybridization to basally located residual bodies in tubules at stages IX—X on one or more occasions, whereas residual bodies around the edge of the lumen (stage VIII) or in transit through the seminiferous epithelium showed no hybridization; sense probes showed no localization to residual bodies.

A common feature of the probes which localized to the basal RBs was that they had been prepared using cDNA cloned into Bluescript SK— vector such that the antisense strand was generated from the T7 polymerase promotor. A cRNA prepared using T7 polymerase and Bluescript vector alone and a GC‐rich 27mer oligonucleotide corresponding to the region of the multiple cloning site of Bluescript adjacent to the T7 site both localized uniquely to basal RB.

It is concluded that the hybridization seen within RBs is probably a subtle artefact unique to RBs undergoing dissolution following fusion with Sertoli cell lysosomes, and may reflect nonspecific hybridization to GC‐rich fragments of RNA.
Expression and localization of N‐ and E‐cadherin in the human testis and epididymis
Authors: ANDERSSON ANNA‐MARIA , EDVARDSEN KLAUS , SKAKKEBÆK NIELS E.
Published: May 2008

Cellular interactions in the testis and epididymis are an important prerequisite for spermatogenesis and sperm maturation, and involve a well‐developed complex of intercellular junctions. Cadherins are cell surface proteins which mediate intercellular Ca2+ ‐dependent adhesion and are believed to be fundamentally important for maintaining multicellular structures. In the present study we report the expression of a 135 kDa N‐cadherin polypeptide in the human seminiferous epithelium by immunoblotting. The presence of N‐cadherin was demonstrated by immunohistochemistry on the surface of spermatogonia and primary spermatocytes, and possibly also around some early spermatids, whereas late spermatids were always negative. Endothelial cells also stained for N‐cadherin, whereas peritubular cells and Leydig cells did not. No expression of E‐cadherin could be demonstrated in the human testis. In the human epididymis E‐cadherin, but not N‐cadherin, was expressed and localized to the surface of the principal epithelial cells as shown by immunohistochemistry. These observations indicate that cadherins play an important role in the organization of the seminiferous and epididymal epithelium.
Increased 17‐OH‐Progesterone levels following hCG stimulation in men with idiopathic oligozoospermia and raised FSH levels
Authors: ANAPLIOTOU M. L. G. , LIPARAKI M. , AMERICANOS N. , GOULANDRIS N. , PAPAIOANNOU D.
Published: May 2008

Leydig cell function was investigated in 71 men with idiopathic oligospermia and compared to 14 fertile controls by assessing the steroidogenic response to GnRH and the repetitive administration of hCG (1500 IU X3). The oligospermic men were divided into two groups according to their basal serum FSH values (FSH<8, n=35; FSH>8, n=36), this level being defined by the mean + 3 SD of the levels in normal men (3.71+4.08 mIU/ml). Oversecretion of LH was supported by the findings of: (a) higher basal LH levels (p<0.0001) in both oligospermic groups, although still within the normal range; (b) higher Dmax LH and area LH (p<0.0001) levels in the FSH>8 group; (c) a strong position correlation (p<0.001) of the above parameters with the respective levels of FSH. No difference in basal testosterone levels was observed between the three groups, whereas basal levels of 17‐OHP were sigdcantly higher (p<0.05) in the group with FSH>8. The testosterone/LH ratio was significantly (p<0.0001) lower in the FSH>8 group, and was correlated inversely to the basal blood levels of FSH <0.0001) and to the area LH (p<0.04). After the hCG test, there was no difference in the testosterone and oestradiol response between the groups, whereas the secretion of 17‐OHP and the ratio of 17‐OHP/testosterone was sigdcantly higher (p<0.0001) in the group with FSH>8 compared with the other two groups. Using multiple regression, the total production of 17‐OHP and the 17‐OHP/testosterone ratio were found to be correlated positively with FSH levels. These results support the view that in men with idiopathic oligozoospermia associated with severe Sertoli cell dysfunction there is parallel oversecretion of LH and compensated dysfunction of the Leydig cells, as indicated by oversecretion or accumulation of 17‐OHP after hCG administration, and also by the low testosterone/LH ratio. This is possibly due to alterations in intratesticular paracrine factors deriving from the Sertoli cells, as suggested by the positive correlation between the altered steroidogenic indices and blood FSH levels.
Immunohistochemical localization of proliferating cell nuclear antigen as a tool to study cell proliferation in rodent and primate testes
Authors: SCHLATT STEFAN , WEINBAUER GERHARD E.
Published: May 2008

The immunohistochemical localization of proliferating cell nuclear antigen (PCNA) has become a widely used method for the detection of proliferating cells in cancerous tissues. PCNA expression is maximal around the S phase of the cell cycle. This study has evaluated the applicability of PCNA localization for the analysis of germ cell proliferation in rats, Djungarian hamsters, rhesus and cynomolgus monkeys and men, using Bouin's‐fixed, paraplast‐embedded tissue. In addition, involuted testes from photoinhibited hamsters, testes from immature rhesus monkeys and from GnRH antagonist‐treated rats and cynomolgus monkeys were included. Monoclonal mouse anti‐PCNA antibody (clone: PC10) was used for detection of the antigen. Visualization was performed by immunogold‐silver staining or avidin‐biotin staining. PCNA labelling was confined to the nuclei of spermatogonia and early spermatocytes within the seminiferous epithelium of all species. The distribution of PCNA among the different types of A‐spermatogonia in primates is in good agreement with the previously described proliferation pattern of these cells. No staining was observed in resting A‐dark spermatogonia, while differentiating A‐pale spermatogonia were positive at distinct stages of the seminiferous epithelium cycle. In the rodent species the pattern of labelled A‐spermatogonia was stage‐specific, but agreed only partly with the previously described pattern of mitotic figures of A‐spermatogonia. Hormonal withdrawal induced a decrease in the number of PCNA‐positive cells in adult rats, hamsters and monkeys. In immature testes from rhesus monkeys positive staining was present in spermatogonia but also in some Sertoli cells, indicating proliferative activity of Sertoli cells in the prepubertal stage. It is concluded that PCNA localization can be used to assess the proliferative status of the renewing spermatogonia in the primate testis and to analyse the proliferative activity of the seminiferous epithelium under various endocrine conditions.