bluuebunny/crossref_metadata_2025 huggingface dataset:
‘Water‐test’: a simple method to assess sperm‐membrane integrity
Authors: LOMEO A. M. , GIAMBERSIO A. M.
Published: May 2008

The functional integrity of the membrane of sperm from semen samples collected from 10 fertile men (Group A) and from 50 infertile men (Group B) was assessed by studying the swelling reaction of sperm when suspended in a medium of distilled water (‘Water‐test’). The results were correlated with routine semen‐analysis and with the results of Eosin‐Y staining. The mean values for the ‘Water‐test’ were significantly different (P<0.01) between Groups A and B (84.8 ± 3.5 versus 70.9 ± 13.3, respectively). No significant correlations were observed in either Group A or B, between values for the ‘Water‐test’ and values for the sperm count, the percentage of sperm with normal morphology and the percentage of motile sperm. There was a good correlation in both Group A (r = 0.86, P<0.01) and Group B (r = 0.92, P<0.01) between values for the ‘Water‐test’ and those for the Eosin‐Y test. These results indicate that the ‘Water‐test’ is a simple and reliable test for evaluating sperm membrane integrity.
Effects of piperine on hamster sperm capacitation and fertilization in vitro
Authors: PIYACHATURAWAT P. , SRIWATTANA W. , DAMRONGPHOL P. , PHOLPRAMOOL C.
Published: May 2008

The effect of piperine on the fertilizing ability of hamster sperm was investigated in vitro. Sperm were incubated in a capacitation medium for 3 h prior to co‐incubation with hamster eggs in a fertilization medium for another 3 h. Addition of 0.18–1.05 mM piperine to the capacitation medium reduced both the percentage of eggs fertilized and the degree of polyspermia in a dose‐dependent manner. When piperine was added to the fertilization medium alone, a significant reduction in fertilization was observed only at high doses (0.70–1.05 mM). The presence of piperine in the capacitation medium inhibited the acrosome reaction in a dose‐dependent manner but had no effect on sperm motility, whether this was measured quantitatively or qualitatively. Piperine also inhibited the influx of calcium into sperm during capacitation. It is suggested that such an inhibition might be a major cause of a reduction of the acrosome reaction and the subsequent impairment of fertilizing ability of sperm.
Male fertility in long‐term survivors of childhood acute lymphoblastic leukaemia
Authors: WALLACE W. H. B. , SHALET S. M. , LENDON M. , MORRIS‐JONES P. H.
Published: May 2008

To study long‐term testicular function following the treatment of acute lymphoblastic leukaemia (ALL) in childhood, 37 young adult males were assessed at two separate time points. The initial assessment was made by a wedge testicular biopsy after completion of treatment (median 9.7 years; range 4.1–16.3 years) and the subsequent assessment (median 18.6 years; range 15.4–26.8 years) consisted of the clinical examination of pubertal stage, measurement of serum gonadotrophins and testosterone and, in 19 patients, semen analysis.

All 37 men completed pubertal development normally and had a testosterone concentration within the normal adult range. Six men showed evidence of severe damage to the seminiferous epithelium, five were azoospermic and one, who did not provide semen for analysis, had a reduced mean testicular volume (11 mls; normal ≥15 mls) and a raised basal FSH level (13 IU 1‐‐1; normal ≤6 IU I‐1). All six men with germ‐cell damage had received either cyclophosphamide or both cyclophosphamide and cytosine arabinoside as part of their chemotherapy regimen.

Approximately 10.7 years earlier all 37 men had undergone a testicular biopsy after completion of their chemotherapy. Morphological damage to the seminiferous epithelium had been calculated by estimating the tubular fertility index (TFI), which is the percentage of seminiferous tubules containing identifiable spermatogonia (age‐matched normal = 100%).

Of the 11 males who had a TFI >50%, consistent with severe germ‐cell damage, five had recovered normal germ‐cell function more than 10 years later. Twenty‐three of the 26 males, who had a TFI >50% at testicular biopsy, showed completely normal testicular function when reassessed subsequently, and in the remaining three men the results were inconclusive. It is concluded that the long‐term outlook for male fertility after chemotherapy for ALL in childhood improves with time after treatment, but remains poor for at least 10 years in the more severely affected.
Effect of pentoxifylline on superoxide anion production by human sperm
Authors: GAVELLA M. , LIPOVAC V. , MAROTTI T.
Published: May 2008

The in‐vitro effect of pentoxifylline, a methylxanthine derivative, on superoxide anion generation by sperm was studied in 27 men with superoxide anion production ranging from 0.57 to 13.8 nmoles 02 per 106 sperm. Superoxide anion release stimulated by phorbol myristate acetate, a potent activator of oxygen radical generation, was reduced by 29–72% following the addition of 10 mM pentoxifylline. The inhibitory effect of this millimolar concentration of the drug did not depend on the initial superoxide production in the presence of phorbol myristate acetate. These results point to a potential use for pentoxifylline in the treatment of male infertility in men with an increased superoxide anion generation capacity.
Evidence for the involvement of sperm angiotensin converting enzyme in fertilization
Authors: FORESTA C. , MIONI R. , ROSSATO M. , VAROTTO A. , ZORZI M.
Published: May 2008

Recently it has been observed that ejaculated human sperm possess high angiotensin converting enzyme (ACE) activity and that this enzyme is released during the process of capacitation. This observation raises the possibility that ACE may be involved in the fertilization process. To verify this hypothesis, we tested the effects of a potent ACE inhibitor, Captopril, on acrosome reaction induced by capacitating medium (3.5% HSA‐added BWW) and on ability of human capacitated spermatozoa to penetrate zona‐free hamster oocytes.

Addition of Captopril (100 nmol 1‐‐1) modified neither sperm motility nor viability at any time considered, but significantly reduced the acrosome reaction percentages of sperm incubated in capacitating medium. Furthermore, Captopril significantly reduced the percentage of penetrated oocytes. The mean penetration rates both in the absence and presence of Captopril were 65.5 ± 4.9% and 26.9 ± 2.3% (P<0.001) respectively. These findings provide evidence that sperm release of ACE during capacitation may have a physiological role in the regulation of the mechanisms that allow sperm acrosome reaction and thus fertilizability.
Combination of a GnRH agonist with an antiandrogen or bromocriptine in the treatment of prostatic cancer; slight potentiation of antigonadal effects
Authors: HUHTANIEMI I. , PARVINEN M. , VENHO P. , RANNIKKO S.
Published: May 2008

The effect of combined treatment with a GnRH agonist (buserelin depot, BUS, 6.6 mg every 2 months) with an antiandrogen (cyproterone acetate, CPA, 300 mg day‐1) or a prolactin‐suppressing agent (bromocriptine, BR, 20 mg day‐1) on pituitary‐testicular function were studied in patients with advanced prostatic carcinoma. The patients (n= 5–6 per group) were treated in this fashion for 6 months and thereafter orchidectomized. Serum testosterone and gonadotrophin responses were followed during treatment, and histology and certain endocrine parameters were studied using testicular tissue obtained at orchidectomy. Serum LH was suppressed in all treatment groups from mean levels of 4–6 IU 1‐‐1 to less than 0.1 1U 1‐‐1, whilst serum FSH levels decreased in all groups during the first month of therapy from 4.5–7 to 1–2 IU 1‐‐1, but recovered thereafter. Only minor increases in serum gonadotrophin levels were evident 3 months after castration. No differences in gonadotrophin responses were seen between the different treatment groups. Serum levels of testosterone were suppressed from 15–20 nmol 1‐‐1 to the castrate range (˜1 nmol 1‐‐1), in each of the treatment groups. Testicular weight decreased significantly more (P<0.05) in the BUS + CPA group, compared to the other treatments. No differences were found in the testicular concentration of testosterone, or LH and FSH receptors between the three treatment groups. On histological examination, spermatogenesis was found to be impaired severely in all groups, with the lowest Johnsen score in the BUS + BR group (2.16 ± 0.13, vs. 2.73± 0.25 with BUS alone; P<0.05). Seminiferous tubular diameters were reduced similarly in all treatment groups. In conclusion, the combination of CPA or BR with BUS in the treatment of prostatic carcinoma does not potentiate the suppression of gonadotrophin or testosterone secretion, evidently because the GnRH agonist exerts a maximal suppressing effect. However, other antigonadal effects were enhanced slightly, including suppressed testicular weights by CPA and further suppression of spermatogenesis by BR.
Evaluation of the acrosome reaction using monoclonal antibodies against different acrosomal antigens ‐ comparison with the triple stain technique
Authors: Braun J. , Hirsch T. , Krause W. , Ziegler A.
Published: May 2008

The acrosome reaction of human sperm was evaluated in vitro with the aid of there monoclonal antibodies TüS‐1, TüS‐19 and TüS‐20, which react with antigens in the anterior part of the acrosome. Data were compared with results obtained using the triple stain technique.

Seminal smears were performed prior to, and following incubation for 5 and 24 h in Hams‐F10 containing 3% human serum albumin. Using the triple stain technique, 15.2 ± 7.1% of sperm exhibited acrosome activation after 5 h and 16.8 ± 8.4% after incubation for 24 h. The corresponding values obtained using antibody TüS‐1 were 12.9 ± 5.8 and 13.2 ± 2.2%, with TüS‐19 10.1 ± 3.8 and 10.8 ± 1.4% and with TüS‐20, 9.0 ± 3.4 and 10.4 ± 2.9%. When the same sperm smears were stained firstly with rose Bengal, and secondly with TüS‐1 both methods stained the same cells and the same region of the cells, thus indicating that both methods stain the same substrate.

Staining of the acrosome with monoclonal antibodies gives clearer results than the triple stain technique, and could be used in preference in future studies dealing with diagnosis of the acrosome reaction in vitro.
Reappraisal of the hypo‐osmotic swelling test to improve assessment of seminal fertility status
Authors: SMITH R. , MADARIAGA M. , BUSTOS‐OBREGON E.
Published: May 2008

The hypo‐osmotic swelling (HOS) test has been proposed as a useful assay for evaluation of the functional competence of the human sperm membranes. To assess this further, the HOS‐test was evaluated in 187 semen samples collected from fertile men and from male patients consulting for infertility. These samples were classified as normal, oligo‐, astheno‐ or oligoasthenozoospermic on the basis of their standard semen variables. The percentage of total sperm tail swelling and of sperm exhibiting different tail swelling patterns was recorded. In the fertile men and in the group of patients with normal semen variables, significantly more (P0.001) HOS‐reactive sperm were observed after hypo‐osmotic treatment in comparison with those groups exhibiting abnormal semen parameters. Swelling of the sperm in a hypo‐osmotic medium was highly correlated with both progressive motility (r= 0.62, P < 0.001) and sperm viability (r= 0.65, P < 0.001). A weak positive correlation was also observed between sperm swelling and sperm morphological features (r= 0.31, P < 0.005) and between sperm swelling and sperm concentration (r= 0.31, P < 0.005). No significant correlation was observed between sperm swelling and in‐vitro sperm fertilizing capacity as assessed by the zona‐free hamster oocyte penetration assay. However the majority of the semen samples (87.3%) showing a normal penetration rate (≥ 10%) also exhibited a 60% (or higher) reaction in the HOS‐test. Finally, the results indicate that when the cut‐off values for the rate of total swollen sperm and tail tip (B pattern) swollen sperm were set at 45% and 20% respectively, rates were above the cut‐off value in all fertile and normozoospermic groups. These findings provide additional evidence of the worth of this assay in the evaluation of the subfertile men as a relevant part of a comprehensive male fertility assessment.
Comparative study on the use of human follicular fluid or egg yolk medium to enhance the performance of human sperm in the zona‐free hamster oocyte penetration assay
Authors: CHAN S. Y. W. , TUCKER M. J.
Published: May 2008

A prospective study was conducted on 61 unselected semen samples from infertile patients to compare the efficacy of human follicular fluid (hFF) or TEST‐egg yolk medium treatment in enhancing sperm performance in the zona‐free hamster oocyte penetration (HOP) test. Both hFF and TEST‐egg yolk enhanced penetration scores significantly in the HOP test in comparison with the control swim‐up sperm preparation. The TEST‐egg yolk medium treatment, however, exhibited the highest penetration scores, and there were higher percentages of preparations passing the 10 or 20|X% penetration rate cut‐off point with sperm exposed to the TEST‐egg yolk medium than those exposed to hFF. These results indicate that modification of the original HOP‐test protocol by incorporation of TEST‐egg yolk medium gives a greatly increased penetration rate when compared to the addition of hFF in such a system.
Separation of phospholipase A2 in the testis and cauda epididymis of the adult rat by chromatofocusing
Authors: RÖNKKÖ S.
Published: May 2008

Phospholipase A2 (PLA2) activity was measured in the reproductive organs of adult male rats. Phosphatidylethanolamine and phosphatidylcholine labelled with 14C linoleic (lino‐PE lino‐PC) and arachidonic acid (ara‐PE ara‐PC) at the 2‐position were used as substrates. Lino‐PE was hydrolysed most strongly by homogenates of the distal cauda epididymis but the testis vas deferens and caput and corpus epididymis also contained hydrolytic activity. Ara‐PC and lino‐PC were hydrolysed by homogenates of the cauda epididymis and testis. No hydrolysis of ara‐PE was detected using homogenates of reproductive tissues.

Chromatofocusing of testis homogenate resulted in the appearance of two active forms of PLA2 with different pl‐values (6.5 and 5.6) when lino‐PE was used as substrate. Maximum activities of both enzymes with 1 mM Ca2+ were observed at pH 9.5. These isoenzymes have marked differences in response to Cu2+ Nethylmaleimide and p‐bromophenacyl bromide (p‐BPB). Cu2+ and Nethylmaleimide had almost no effect on PLA2 activity with a pi value of 6.5 but inhibited the other isoenzyme strongly; the latter was almost more resistant to p‐bromophenacyl bromide. Both enzymes hydrolysed lino‐PE most strongly.

Chromatofocusing of an homogenate of cauda epididymis also revealed two isoenzymes of PLA2 with different pl‐values (6.0 and 5.0). The latter form was resistant to p‐bromophenacyl bromide but was more sensitive to Triton X‐100 and sodium deoxycholate than was other isoenzyme. The pH optimum of the isoenzyme with a pi value of 6.0 ranged from 6.25 to 8.75 whilst the other isoenzyme was most active at pH 8.0–8.75. Both isoenzymes hydrolysed lino‐PE most actively whilst lino‐PC and ara‐PC were deacylated at much lower rates.
Passive immunization against prostatic ‘inhibin’ peptide as a male contraceptive
Authors: VANAGE G. R. , GARDE S. V. , SHETH A. R. , GOPALKRISHNAN K.
Published: May 2008

Passive immunization of adult male hamsters for 12 weeks against peptide (As‐PIP), a sperm coating antigen, resulted in selective elevation of the blood levels of FSH, impairment of spermatogenesis, and complete infertility when males were mated with normal cycling females. Passive immunization of male marmosets with As‐PIP for 8 weeks was also effective and was reversible, without causing any obvious change in mating behaviour. These preliminary studies in hamsters and marmosets indicate that antibodies to a prostatic ‘inhibin’ peptide represent a promising new approach to male contraception.
Influence of rat testicular macrophages on Leydig cell function in vitro
Authors: LOMBARD‐VIGNON N. , GRIZARD G. , BOUCHER D.
Published: May 2008

The influence of co‐cultures of rat testicular macrophages and Leydig cells (LC) on LC morphology and steroidogenesis was investigated with and without macrophage stimulation by a bacterial lipopolysaccharide (LPS). LC showed an elongated form in the presence of stimulated testicular macrophages. In the presence of non‐stimulated testicular macrophages a significant inhibition of testosterone production was observed (decrease of 33%) from 48 h in co‐culture while an increase of 16% was obtained at the same culture time, after stimulation of macrophages by LPS. When LC were treated with testicular macrophage‐conditioned media (MCM) obtained from LPS‐treated macrophages, they became fusiform and there was stimulation (78%) of steroid production. After human FSH stimulation (1 ‐1000 mIU m1,‐1), MCM from testicular macrophages was no more effective in enhancing testosterone production by LC than was media from untreated LC. Similar experiments with LPS were conducted with macrophages of peritoneal origin. Peritoneal macrophages stimulated or not by LPS in co‐cultures with LC or peritoneal MCM did not significantly modify testosterone production. However, these cells were able to modify LC morphology when LPS‐MCM was added to LC‐culture medium. The present results suggest strongly that testicular macrophage‐LC interactions could be important in the control of LC steroidogenesis.
Effect of testosterone deprivation on expression of the androgen receptor in rat prostate, epididymis and testis
Authors: BLOK L. J. , BARTLETT J. M. S. , VRIES J. BOLT‐DE , THEMMEN A. P. N. , BRINKMANN A. O. , WEINBAUER G. F. , NIESCHLAG E. , GROOTEGOED J. A.
Published: May 2008

Adult rats were treated with ethane dimethane sulphonate (EDS) to eliminate the Leydig cells. This treatment resulted in very low levels of testosterone in the blood and in the testis. Furthermore, histological evaluation of spermatogenesis showed no marked differences between control and EDS‐treated animals.

In the ventral prostate, 5 days after EDS‐treatment, a 4.0 ± 0.3–fold up‐regulation of androgen receptor (AR) mRNA was observed, together with a 2.2 ± 0.2–fold increase in actin mRNA. In the epididymis, a 2.0 ± 0.5–fold increase in AR mRNA level was observed, without a change in actin mRNA level. In the testes of EDS‐treated rats, the AR mRNA level was not changed (1.02 ± 0.17–fold of controls), and there was also no change in actin mRNA level at 5 days after EDS‐treatment. These results indicate that AR mRNA expression in the ventral prostate and epididymis is regulated differentially by testosterone when compared to regulation in the testis.

Testicular androgen binding sites were assayed by Scatchard analysis of the binding of 3H‐R1881 to a nuclear fraction, that was isolated by a method which involved the use of liquid nitrogen and high sucrose buffer. The number of specific binding sites per testis in EDS‐treated rats with testosterone‐implants, remained unaltered compared to control rats (9.1 ± 1.4 pmol/testis). In these rats, 20% of the normal testicular testosterone level was sufficient to maintain the androgen receptor in a tight nuclear binding (transformed) form. In testes from EDS‐treated rats without testosterone‐implants, the AR did not fractionate into the nuclear fraction; however, the total testicular AR content in these animals was close to control levels, as measured by nuclear 3H‐R1881 binding after receptor transformation through injection of a high dose of testosterone (10 mg) 2 h before killing the rats (testosterone pulse). In the different experimental groups, FSH was not required to maintain the total testicular AR content (ligand binding).
Acidification of testicular and epididymal fluids in the rat after surgically‐induced varicocele
Authors: CAFLISCH C. R.
Published: May 2008

Experimental left varicocele (ELV) in adult rats produces a bilateral increase in testicular blood‐flow and temperature which may alter the intraluminal environment in which sperm mature. The purpose of the present study was to evaluate the effect of ELV on the in‐situ pH, PCO2, and bicarbonate concentration ([HCO3]) in seminiferous tubules (ST), initial segment (IS), proximal caput (PCP), middle caput (MCP), middle corpus (MCR), and proximal cauda epididymidis (PCD) of the rat employing pH and PCO2, microelectrodes. Adult male rats with ELV or sham‐surgeries (control) were studied after 30 days. Relative to controls, ELV significantly reduced the in‐situ PCO2, in the testicular artery, ST, IS, PCP, MCP, MCR and PCD. In spite of this reduction, all values remained significantly higher than the systemic arterial blood PCO2. Values for in‐situ pH in ST, IS, PCP, MCP, MCR, and PCD of control rats were significantly more acidic than systemic arterial blood. Furthermore, ELV decreased the pH in ST, IS, PCP, and MCP when compared to control values. The calculated [HCO‐3] in ST from control animals was less than half that in systemic arterial blood and was reduced further in IS, PCP, and MCP. Varicocele did not change the [HCO‐3] in systemic arterial blood but reduced markedly the values in ST, IS, PCP, and MCP. These alterations in the in‐situ pH, PCO2, and [HCO‐3] in structures of the rat testis and epididymis may play a role in the anti‐spermatic effect of ELV.
Pharmacokinetic and pharmacodynamic studies of the effect of ketoconazole on reproductive function in male rats
Authors: WANG J. M. , WU X. L. , YOU W. , LING L. X. , WU J. , ZHANG G. Y.
Published: May 2008

A single oral dose (300 mg kg‐1) of ketoconazole induced reversible immobilization of rat epididymal spermatozoa at 8–24 h after dosing. This occurred when the drug concentrations in cauda epididymal fluid and seminal plasma were at their peak (18.0 ± 7.3 and 13.5 ± 3.0 μg ml‐1, respectively), and which was preceded by a peak plasma concentration (Cmax) of 64.82 ± 2.47 μg ml‐1 at 5.15 ± 0.68 h (Tmax). In contrast, rete testis fluid collected from the same animals contained only minute amounts of ketoconazole (0.47 ± 0.34 pig ml‐1). Plasma testosterone concentration showed a sharp decline within 4 h of dosing, followed by a recovery from suppression, even after administration of a low dose (100 mg kg‐1) which did not affect sperm motility. These findings suggest that ketoconazole gains access to the post‐testicular sex organs and affects the mature spermatozoa therein much more readily than it affects testicular spermatogenesis. Synthesis and screening of compounds with a related molecular structure but which exhibit more pronounced spermicidal and less pronounced anti‐androgenic effects are thus suggested in the hope that rapidly acting and reversible male contraceptives might be identified and developed.
Immunohistochemical localization of calmodulin
Authors: TSUJI Y. , SANO M. , NAGAHAMA M. , TSUTSUI Y. , MIYAKE K.
Published: May 2008

The localization of calmodulin in the normal human testis was studied by the indirect immunoperoxidase method. Three different types of fixative were used: phosphate‐buffered formalin, Bouin's solution or Carnoy's solution. Immunoreac‐tivity specific for calmodulin was not detectable in the testis fixed in Carnoy's solution. The specimens fixed in phosphate‐buffered formalin and in Bouin's solution were stained. The immunostaining for calmodulin was observed in pachytene spermatocytes, secondary spermatocytes and round spermatids but not in spermatogonia, or in pre‐leptotene, leptotene and zygotene spermatocytes, elongated spermatids, spermatozoa or Leydig cells. Sertoli cells were not stained or were stained slightly. Among pachytene spermatocytes, the cells at the early stage were barely stained but those at the middle and late stages were slightly and intensely stained, respectively. The present report provides the first confirmation of the localization of calmodulin in the human testis.
Relative roles of testosterone and the germ cell complement in determining stageldependent changes in protein secretion by isolated rat seminiferous tubules
Authors: Sharpe Richard M. , Millar Mike , Mckinnell Chris
Published: May 2008

This study has compared the effect of withdrawal of testosterone (± replacement) with that of selective depletion of pachytene spermatocytes (PS) or round/elongating spermatids (RS), or both PS and RS, on the level of overall protein secretion by seminiferous tubules (ST) isolated at particular stage‐groups of the spermatogenic cycle. Testosterone withdrawal was induced by destroying the Leydig cells with a single injection of ethane‐dimethane sulphonate (EDS), with or without concomitant replacement of testosterone by injection; ST at stages II‐V, VI‐VIII or IX‐XII were then isolated from control and treated rats at 4 days after treatment. Methoxyaceticacid (MAA) was administered, in either one or two doses, to selectively destroy 80–100% of pachytene and later spermatocytes; ST at stages I‐V, VI‐VIII or IX‐XIV were then isolated at specific times after treatment such that ST were depleted selectively of either PS, RS or PS + RS. Isolated ST (5 cm) were then cultured for 22 h at 34°C with 35S‐methionine and its incorporation into secreted proteins then quantified.

Based on the incorporation of 35S‐methionine, ST at stages VI‐VIII showed a significantly higher level of protein secretion than did ST at earlier or later stages. This difference was abolished following testosterone withdrawal but was maintained by testosterone replacement. The normal increase in protein secretion by ST at stages VI‐VIII was also prevented if either PS or RS were depleted, whereas depletion of either PS or RS alone had no significant effect on protein secretion by ST at stages I‐V, and only the depletion of RS significantly reduced protein secretion by ST at stages IX‐XIV. Depletion of both PS + RS reduced protein secretion significantly by ST at all stages. In contrast to the data for total protein secretion, the levels of sulphated glycoprotein (SGP)‐1 and SGP‐2 secreted by ST at stages VI‐VIII showed that these two Sertoli cell proteins were unaffected by germ cell depletion except after co‐depletion of PS + RS when secretion of SGP‐1 was halved. It is concluded that: (i) the increase in overall protein secretion by ST at stages VI‐VIII in the normal spermatogenic cycle is both androgen‐ and germ cell‐dependent: (ii) the modulatory effect of germ cells on ST‐protein secretion varies according to the stage of the spermatogenic cycle and the germ cell type and does not affect all secreted proteins: (iii) the changing germ cell complement is probably the trigger for the stage‐dependent changes in overall protein secretion by ST.
Predictive value of morphologically normal sperm concentration in the medium for in‐vitro fertilization
Authors: ENGINSU M. E. , DUMOULIN J. C. M. , PIETERS M. H. E. C. , EVERS J. L. H. , GERAEDTS J. P. M.
Published: May 2008

Morphological evaluation of spermatozoa using strict criteria (MEUSC) and conventional sperm parameters were studied with respect to in‐vitro fertilization and pregnancy outcome before and after a swim‐up selection procedure. Recovered oocytes were inseminated with 50 000 progressively motile spermatozoa, and this study assess the influence of the total number of spermatozoa and of the percentage with strictly normal morphology in the insemination sample on the outcome of IVF. The results showed that the percentages of spermatozoa with normal morphology using strict criteria, both in native and in post‐swim‐up samples, were the best predictors of IVF outcome. Their respective cut‐off points were 5% and 8%. The number of morphologically normal spermatozoa inseminated also showed a good correlation with fertilization. However, it was not possible to find a proper cut‐off point for this parameter. The patients were categorized on the basis of their native and post‐swim‐up scores. Category 1, in which both parameters were below their respective cut‐off points, showed a 7% fertilization rate and a 0% pregnancy rate. Category 3, in which both parameters were above their cut‐off points, showed a 70% fertilization rate and a 23% pregnancy rate. This suggests that sperm morphology can be used as a criterion for patient selection for IVF as an aid to identification of possibly subfertile males.
Paternity in a patient with testicular seminoma and contralateral testicular intraepithelial neoplasia
Authors: DIECKMANN K.‐P. , LOY V.
Published: May 2008

A 32‐year‐old patient with unilateral βhCG‐positive seminoma and contra‐lateral testicular intraepithelial neoplasia (TIN; so‐called carcinoma‐in‐situ) with no metastases (clinical stage I) received one course of adjuvant carboplatin therapy. He refused further treatment of TIN in his remaining testis. His wife became pregnant by him 4 months later and delivered a healthy child at term. This case shows that patients with TIN in their remaining solitary testis are not necessarily infertile, and testes afflicted with TIN must also contain tubules that retain normal spermatogenic potential. Surveillance may be an treatment option for patients with TIN in their remaining testis in cases where there is a strong desire for paternity.
Seminal leucocyte subpopulations and sperm function in fertile and infertile Chinese men
Authors: KUNG A. W. C. , HO P. C. , WANG C.
Published: May 2008

The level of seminal leucocytes and the prevalence of leucocytospermia was determined in a group of fertile and infertile southern Chinese men in Hong Kong. Sixteen normal fertile semen donors and 49 men with male factor infertility were studied prospectively. None had antisperm antibodies and past or present evidence of genital tract infection. Seminal leucocytes and their subsets were analysed using monoclonal antibodies and an immunocytochemical alkaline phosphatase‐anti‐alkaline phosphatase conjugate technique. Seminal leucocytes were detectable in 94% and 86% of the fertile and infertile men respectively, with the predominant subset being granulocytes. Leucocytospermia (> 1 × 106 leucocytes/ml) was found in only one of the 49 (2%) infertile men without clinical evidence of genito‐urinary infection. Inverse correlations were observed between (1) the percentage of spermatozoa with normal morphology and the number of T‐helper/inducer cells, (2) the linearity of sperm movement and the number of T‐lymphocytes. In conclusion, the level of seminal leucocytes and the prevalence of leucocytospermia is low in infertile Chinese subjects. The effect of seminal leucocytes on sperm function in these subjects needs further evaluation.
Investigation in real time of the effect of gravitation on human spermatozoa and their tendency to swim‐up and swim‐down
Authors: Makler A. , Stoller J. , Blumenfeld Z. , Feigin P. D. , Brandes J. M.
Published: May 2008

To investigate in real time if and how natural gravity affects rates of swim‐up and swim‐down of human spermatozoa, samples of motile or immobilized spermatozoa in a sealed mini‐chamber were placed vertically on a 90° tilted microscope. The mode of their sedimentation, as well as the difference in the rate of their swimming up and down, were observed directly over 30 min and analysed from photomicrographs. Under the influence of natural gravity force, most immobilized spermatozoa turned their heads down in about 5 min and then sank slowly at an average speed of 0.2 μ/s. The number of motile spermatozoa that swam down was 5–6 times more than those swimming up. It can be implied that in spite of the mild force exerted by 1g on suspended spermatozoa in comparison to the high g force obtained by centrifugation, the overall effect of gravity on the rate of swimming up or down becomes dominant. Gravity causes the sperm heads to turn downward after which the oriented spermatozoa continue to move down by their own tail movements, causing accumulation of motile spermatozoa at the bottom. This may explain why in some recent studies swim‐down was superior to the swim‐up procedure during sperm separation by self‐migration.
Inverse relationship between the induction of human sperm capacitation and spontaneous acrosome reaction by various biological fluids and the superoxide scavenging capacity of these fluids
Authors: Lamirande E. de , Eiley D. , Gagnon C.
Published: May 2008

Capacitation of spermatozoa is essential for fertilization, and can be induced by various agents or biological fluids. Previous reports have shown that foetal cord serum (FCS) and the superoxide anion trigger human sperm hyperactivation and capacitation, and that superoxide dismutase (SOD) prevents these processes. We investigated: (1) the capacity of seminal plasma (SP) and follicular fluid (FF)(whole, or fractionated into high and low molecular weight components), in the presence or absence of SOD, to induce the spontaneous acrosome reaction(no stimulant needed, AR) and capacitation (as measured by the lysophosphatidyl‐choline‐induced AR, LPC‐AR); (2) a possible relationship between the levels of AR and capacitation obtained with these biological fluids and the superoxide scavenging capacity of the same fluids. The highest levels of LPC‐AR were obtained with FF ultrafiltrate (48 ± 6%), followed by SPultrafiltrate (31.9 ± 0.8%), FF (30 ± 5%), dialysed FF (27 ± 4%), and finally, by FCSultrafiltrate (23 ± 1%.), SP (21 ± 1%) and dialysed SP (18.9 ± 0.8%).A similar order of potency for the fluids existed when sperm AR was studied, the levels of AR observed ranging from 26 ± 2% to 5.3 ± 0.8% after incubation with FF ultrafiltrate and SP respectively. None of these treatments had detrimental effects on sperm motility. In the presence of SOD, there was always an important reduction (52–86%) of the AR and LPC‐AR observed. A highly significant inverse linear relationship was observed between the SOD‐like activity of the fluids tested and the AR (r= 0.86, p < 0 0.001) and LPC‐AR (r= 0.91, p < 0.001)observed in the presence of these fluids. The results suggest that biological fluids contain inducers of the AR and capacitation, and that these probably act by a common mechanism, possibly by direct or indirect induction of an NADPH oxidase in the sperm membrane. The data suggest strongly that the SOD‐like activity of a specific fluid is probablyone of the most important factors that will determine its capacity to induce the AR and capacitation.
Phenotypic characterization of lymphocytic cell infiltrates into the testes of rats undergoing autoimmune orchitis
Authors: Lustig L. , Lourtau L. , Perez R. , Doncel G. F.
Published: May 2008

Experimental autoimmune orchitis (EAO) was induced in adult Wistar rats by active immunization with a testicular homogenate (TH) and adjuvants. Fifty per cent of the immunized rats developed EAO. Testicular damage became evident at 50 days after the primary immunization and increased in severity at 80 days. Phenotypic characterization of T‐cell subsets (CD4+ and CD8+) and Ia+ cells was performed on cryostat sections of testis obtained from normal rats, rats immunized with adjuvant (control group) and rats immunized with TH and adjuvants (experimental group) at 50 and 80 days. Labelled cells were only detected in the interstitial area; no labelled cells were observed inside the seminiferous tubules with any of the monoclonal antibodies used (W3/25, OX‐8, OX‐6). A significant increase in the numbers of CD4+ and CD8+, as well as of Ia+ cells, were observed in the testis of rats with severe EAO at 80 days after the first immunization. Rats of the same experimental group without testicular damage showed no major differences compared to rats from the control group, with the exception of a lower number of CD8+ cells. Variations in the lymphocyte subsets in lymph nodes draining the site of immunization showed the opposite pattern to that observed in the testis. In conclusion, these data suggest the traffic of specifically sensitized lymphocytes from lymph nodes to the testis and an active role of CD4+, CD8+ and Ia+ cells in the pathogenesis of EAO in the rat.
Sperm function in Indonesian men treated with testosterone enanthate*
Authors: ARSYAD K. M.
Published: May 2008

This study was undertaken to assess the fertilizing capacity (sperm function) of residual spermatozoa produced by groups of men (n= 7) rendered oligozoospermic by treatment with weekly intramuscular injection of 50 or 100 mg testosterone enanthate (TE). The treatment was continued for 6 months and the effects compared with a control group (n= 6) in which men received weekly intramuscular injections of sesame oil (1 ml). Sperm function was assessed by tests of membrane integrity (supravital stain and hypoosmotic swelling [HOS] test) and sperm penetration (artificial cervical capillary tube test). The quality of sperm movement was also assessed. These parameters of sperm function were all reduced consistently in a dose‐dependent manner with maximal suppression occurring between weeks 15 and 25 of treatment, and full recovery to baseline levels by 12 months after cessation of treatment. The decrease in sperm function was correlated with the degree of suppression of sperm output with the proportion of morphologically normal spermatozoa and the degree of suppression of gonadotrophin levels. The weekly dose of 50 mg TE induced severe oligozoospermia (concentration < 5 million/ml) in four of seven men, whereas 100 mg TE induced azoospermia in all seven men by week 20. It is concluded that, unlike published observations on men of European background, the administration of TE at 100 mg per week may achieve adequate contraceptive efficacy in Indonesian men.
Effects of retinol on glycoprotein synthesis by Sertoli cells in culture: dolichyl phosphomannose synthase activation
Authors: GUMA FATIMA C. R. , BERNAFCD ELENA AIDA
Published: May 2008

Sertoli cells were isolated from Wistar rats aged 19 days and cultured for 48 h. The addition of retinol (10 μM) to the culture medium significantly stimulated the incorporation of [2–3H]mannose into lipid‐linked oligosaccharide and into cellular and secreted glycoproteins. Incorporation of [U‐ 14C] leucine into proteins and of [5, 6–3 H] uridine into RNA was unaffected by retinol treatment. Incubation of microsomal fractions of retinol‐treated cells showed an increase in mannose incorporation into dolichyl phosphomannose, into dolichyl pyrophosphoryl oligosaccharide and into proteins. Chromatographic analysis of the fraction soluble in chloroform/methanol (2: 1 v/v) did not show the presence of retinyl phosphomannose either in control or in retinol‐treated cells. When the formation of dolichyl phosphomannose was studied in microsomes isolated from control cells and from cells treated with 10 μM retinol for 48 h in the presence of exogenous dolichyl phosphate, the results showed that the retinol effect was due to stimulation of dolichyl phosphomannose synthase.
Outer dense fibres of human spermatozoa: partial characterization and possible physiological functions
Authors: HENKEL R. , STALF T. , MERTENS N. , MISKA W. , SCHILL W.‐B.
Published: May 2008

The outer dense fibres are accessory fibres in the spermatozoon. They represent up to 30% of the protein portion in human spermatozoa and are involved in sperm progressive motility. If outer dense fibres are missing or developed poorly, spermatozoa are only locally motile. For isolation of the outer dense fibres, human spermatozoa were sonicated at 25 kHz and the flagella were separated by density gradient centrifugation in Percoll. Thereafter, membranes and fibrous sheath were dissolved under reducing conditions in the cationic detergent cetyltrimethylammonium bromide for 30, 60 and 90 min, respectively. The isolation steps were monitored by phase contrast microscopy and electron microscopy. After SDS‐polyacrylamide gel electrophoresis and silver staining of isolated outer dense fibres, two protein bands at 55 and 67 kDa could be detected. By means of rhodamine B staining, no phosphorus could be detected in the outer dense fibre proteins.
Flow cytometric evaluation of the acrosome reaction of human spermatozoa: a new method using a photoactivated supravitaI stain
Authors: HENLEY N. , BARON C. , ROBERTS K. D.
Published: May 2008

A flow cytometric assay using a double‐stain method for the measurement of the acrosome reaction of human spermatozoa is described. The use of a stable photoactivated stain, ethidium monoazide, allowed evaluation of the viability of spermatozoa. This stain was more stable in fixed samples than propidium iodide, which is not bound covalently to DNA and is therefore removed readily during the washing procedure. The permeabilized acrosome was labelled with Pisum sativum agglutinin conjugated with fluoroisothiocyanate. Since this lectin binds to the acrosome and acrosomal contents, a decrease in the fluorescence intensity allows the cytometric evaluation of the acrosome reaction.

Microscopic analysis and flow cytometric analysis were well correlated and cell sorting was performed to ensure the homogeneity of each different subpopulation encountered.
Effects of male accessory sex glands on fertilization, polyspermy and morphometry of early embryos in the golden hamster
Authors: CHOW P. H. , DOCKERY P. , CHEUNG M. l. L. , CHEUNG W. S. O.
Published: May 2008

Preimplantation embryos sired by hamsters without accessory sex glands (ASG) were found to have a higher mortality rate and a slower cleavage rate than those sired by sham‐operated males at 72 h post coitum (P.c.). A time‐course study of fertilization in vivo was conducted to determine whether this effect was due to delayed fertilization. Ultrastructural morphometry of 48 h embryos was also undertaken to establish the earliest manifestation of developmental anomalies. Compared to sham‐operated controls (SH), ablation of all the ASG (TX), or just the ventral prostate (VPX) or ampullary gland (AGX) had no effect on the timing of sperm penetration, extrusion of the second polar body and pronuclear formation. Females mated with AGX males tended to have more polyspermic embryos (9.7%; p < 0.05). The volumes, volume fractions (Vv) of the blastomere nuclei, mitochondria and yolk material of the four‐cell embryos sired by these same groups of males were assessed using point counting techniques. No difference in the Vv of yolk and mitochondria could be observed between groups. However, the SH group did have a significantly larger proportion of the cell occupied by the nucleus (p < 0.05), and the TX group had a higher proportion of the nucleus occupied by nucleoli when compared with the SH group (p <0.01). Smaller nuclei and larger nucleoli in the TX group was interpreted as an early manifestation of a slower division rate of the blastomeres.
Clinical study of azoospermia
Authors: MATSUMIYA K. , NAMIKI M. , TAKAHARA S. , KONDOH N. , TAKADA S. , KIYOHARA H. , OKUYAMA A.
Published: May 2008

This study evaluated how many patients with azoospermia might have fertility potential using assisted conception techniques. A total of 102 male patients with aioospermia were included in the study. Thirteen patients had sex chromosomal abnormalities. Testicular biopsy performed in the other 89 patients showed incomplete spermatogenesis in 47 of them whereas 42 had complete spermatogenesis. In the latter 42 patients, distal vasography demonstrated bilateral obstruction of the excurrent ducts in 14 patients whereas no distal obstruction of the ducts was found in 28. The 89 patients were divided into three groups according to the findings of testicular biopsy and distal vasography. In the 14 patients with both complete spermatogenesis and distal obstruction of the excurrent ducts, surgical procedures are applicable. The pathogenesis of the 28 patients with complete spermatogenesis but without distal obstruction of the ducts should be clarified for further treatment.
Post‐thaw sperm motility, cAMP concentration and membrane lipid per oxidation after stimulation with pentoxifylline and platelet‐activating factor*
Authors: WANG R. , BELL M. , HELLSTROM W. J. G. , SIKKA S. C.
Published: May 2008

Earlier studies have demonstrated that pentoxifilline (PTX) and platelet‐activating factor (PAF) can significantly improve the motion parameters of post‐thaw human spermatozoa. This study has investigated the effects of PAF, PTX and their combination on cyclic adenosine monophosphate (CAMP) concentrations and membrane lipid peroxidation (LPO) in post‐thaw human spermatozoa. Washed spermatozoa from normal volunteers (n=10) were cryopreserved in Test‐yolk buffer using a standard protocol. After 2 weeks the sperm samples were thawed, washed and incubated with either 1 p˜ PAF, 3 m˜ PTX or 0.5 p˜ PAF plus 1.5 m˜ PTX. Video sequences were recorded at 0, 30, 60, and 120 min for analysis of sperm motion parameters using the Cell Track Sperm Analysis System. Concentrations of cAMP were assessed by radioim‐munoassay, and LPO levels were measured by malondialdehyde‐thiobarbituric acid reactivity. Our studies indicate a time‐course stimulatory effect with overall maximal stimulation observed in samples treated with the combination of PAF and PTX. The maximal stimulation of percentage motility compared to control was observed at 60 min in samples treated with PAR PTX, or PAF plus PTX. PAF plus PTX stimulated straight‐line velocity (VSL), curvilinear velocity (VCL) and lateral head displacement (ALH) after 30 min incubation. The primary effect of PAF was observed on VSL, while the main effect of PTX was on VCL. CAMP concentrations were 3‐fold higher than controls in samples treated with PTX or PAF plus PTX. CAMP concentrations in PAF‐treated samples did not differ significantly from controls. No significant differences were observed between any groups for LPO. This study demonstrates that both PAF and PTX can improve the motion parameters of post‐thaw human spermatozoa with the maximal effect observed by their combination. The stimulatory effect of PTX in these post‐thaw samples is related to increased CAMP levels, while another mechanism is likely to be operational for PAR Protection from lipid peroxidative membrane damage is not suggested to be the mechanism of action for PAF or PTX under these conditions.
Sperm motility inhibitor from human seminal plasma: presence of a precursor molecule in seminal vesicle fluid and its molecular processing after ejaculation
Authors: ROBERT MARTIN , GAGNON CLAUDE
Published: May 2008

Human seminal plasma contains a protein factor that has the capacity to inhibit the movement of demembranated and intact spermatozoa. This factor ‘seminal plasma motility inhibitor’ (SPMI) has been shown to originate exclusively from the seminal vesicles. The present results demonstrate that the biological activity of SPMI in semen decreases rapidly from 1000 U/d, immediately after ejaculation, to 220 U/ml 2 h later. Immunoblots of seminal plasma proteins probed with an antibody against human SPMI, revealed the rapid processing of a predominant 52 kDa SPMI antigen, present in the seminal vesicle secretions. This precursor was degraded initially into intermediate molecular mass fragments of 25–40 kDa, and subsequently into smaller fragments of 17–21 kDa. When seminal vesicle fluid was mixed with prostatic secretions (3: 1 v/v), proteases present in prostatic secretions were shown to be responsible for processing of the SPMI precursor. Addition of protease inhibitors such as phenylmethylsulphonyl fluoride (PMSF, 5 mM), benzamidine (100 mM) or ethylenediaminetetraacetic acid (EDTA, 5 mM) to the mixture of seminal vesicle and prostate secretions partially prevented the loss of activity of SPMI by 54%, 27% and 9%, respectively. However, the simultaneous addition of PMSF and benzamidine conferred almost total stability to the SPMI precursor activity. These results demonstrate that SPMI exists as a predominant 52 kDa precursor form in the seminal vesicles and is processed rapidly after ejaculation into less active, lower molecular mass forms by one or more serine proteases and/or metallo‐proteases of prostatic origin which are present in liquefied semen.