bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of antioxidants on human sperm preparationtechniques
Authors: GRIVEAU J. E. , LANNOU D. LE
Published: May 2008

The effect of two different sperm preparation techniques, Percoll gradient centrifugation and swim‐up from a washed pellet were tested on the functional competence of the selected spermatozoa. Percoll gradient centrifugation brought about an improvement in sperm motility parameters such as curvilinear velocity and straight‐line velocity, an increase in the rates of hyperactivation and the acrosome reaction and an increase in the percentage of motile spermatozoa after 24 h of incubation compared to the centrifugation, swim‐up procedure. The effects of antioxidants such as dithiothreitol (DTT) or reduced glutathione (GSH), and reactive oxygen species‐scavenging enzymes such as catalase or superoxide dismutase (SOD) during the stage of centrifugation before the swim‐up procedure were also studied. Though all of these agents prevented the fall in sperm motility after 24 h incubation, only DTT and SOD improved the rates of both hyperactivation and the acrosome reaction. GSH also improved the acrosome reaction, whereas catalase was without significant effect on the rates of hyperactivation or the acrosome reaction. These results indicate that Percoll gradient centrifugation selects spermatozoa with better functional competence than does centrifugation swim‐up. The damage caused by the centrifugation can be prevented by the addition of antioxidants, suggesting that the differences noted with the Percoll gradient method was due to better protection against peroxidative damage due to the centrifugation of unselected spermatozoa. However, the use of DTT is limited by virtue of the fact that this sulphydryl reducing agent leads to destabilization of the sperm chromatin. In contrast, GSH and SOD could have therapeutic potential.
Induction of spermatogenesis with gonadotrophins in Chinese men with hypogonadotrophic hypogonadism
Authors: KUNG A. W. C. , ZHONG Y Y. , LAM K. S. L. , WANG C.
Published: May 2008

The effects of gonadotrophin administration to 17 Chinese patients with hypogonadotrophic hypogonadsm (HH) on testicular volume and induction of spermatogenesis were studied. Ten subjects had isolated HH and seven had hypopituitarism. Twelve of the subjects had prepubertal onset of HH and five of them had been treated previously with hCG for induction of puberty. None had a history of cryptorchidism. During hCG treatment for induction of spermatogenesis, all subjects had an increase in serum levels of testosterone into the normal adult male range and their testes increased in size from 3 (1–20) ml to 11.6 (5–20) ml [median(range), p<0.02]. Six subjects required treatment with hCG alone. However, the remaining 11 subjects, after at least 6 months treatment with hCG, required the addition of human menopausal gonadotrophin (hMG) to induce spermatogenesis. Two subjects remained azoospermic. One had a history of mumps orchitis and the other had isolated elevation of blood FSH levels, suggestive of primary testicular failure in addition to HH. Excluding one subject with fertile eunuch syndrome, the mean duration for first appearance of spermatozoa was 13 (4–52) months. Twelve subjects became fertile and pregnancy was achieved in their partners after 20 (4–78) months. The weekly doses for hCG and hMG were 4000 (3000–10000) IU and 225 (225–450) IU, respectively. Patients who responded to hCG alone had a significantly larger pretreatment testicular volume, suggesting that they had only partial gonadotrophin deficiency. Prepubertal onset of hypogonadism was not a determining factor for requirement of hMG treatment. Pretreatment testicular volume correlated positively with the final testicular volume and negatively with the time to achieve spermatogenesis, but not with the final sperm concentration. Previous use of hCG for induction of puberty with resultant testicular growth, favoured a subsequent positive response to hCG. These subjects also required a lower dose of hCG for normalization of serum testosterone levels. In conclusion, gonadotrophins are very effective in stimulating testicular growth and spermatogenesis in subjects with HH, and the positive response to gonadotrophins can be determined by the pretreatment testicular volume.
Morphometric analysis of human sperm morphology
Authors: PÉREZ‐SÁNCHEZ F. , DE MONSERRAT J. J. , SOLER C.
Published: May 2008

Fourteen morphological forms of human spermatozoa were analysed morphometrically using semi‐automated image analysis techniques. Five basic (area, perimeter, length, width and mass) and five derived (ratio, length minus width, ellipticity, form and total mass) parameters were considered. Statistical analysis showed differences among all 14 types of human sperm heads. Basic parameters describing the size and shape were enough to distinguish most of the categories, whereas derived parameters as well as parameters dependent on stain intensity, were demonstrated to be useful for the discrimination of some morphological categories. The fact that statistical analysis showed differences among all 14 sperm types provides evidence for the reliability of our morphological classification. These results show that morphometry can be used for the fine study of sperm morphology and may serve as a database for future work dealing with sperm classification. As this is a pilot study to assess methodology, further studies will be required to validate the method in terms of its application and usefulness in assessing the fertilizing potential of human spermatozoa.
The routine assessment of sperm motility at room temperature and 37°C
Authors: BIRKS A. G. , IZZARD H. , MORROLL D. R. , PRIOR J. R. , TROUP S. A. , LIEBERMAN B. A. , MATSON I. L.
Published: May 2008

The World Health Organization (WHO) laboratory manual (1992) states that assessment of sperm motility can be performed at either 37OC or room temperature (20–24OC). The motility of spermatozoa in 44 semen samples (22 fresh samples and 22 frozen‐thawed samples) was assessed at both of these temperatures and a significant difference in the motility profiles was noted, specifically an increase at 37OC in the percentage (expressed here as median and ranges) of spermatozoa with excellent progressive motility and an overall increase in the percentage with total progressive motility. With fresh samples the excellent progressive motility increased from 41 (19–53) to 54 (30–66) and the overall motility from 58.5 (39–74) to 65.0 (40–79). With the frozen—thawed samples the excellent motility increased from 14 (1–33) to 25 (6–45) and the overall motility from 30.5 (14–51) to 33.0 (16–52). As the WHO laboratory manual was published. ‘In response to a growing need for the standardisation of procedures for the examination of human spermatozoa’ it is proposed that only one temperature for routine analysis should be used, namely 37OC, which may have more physiological relevance and eliminate effects of fluctuations in ambient laboratory temperature.
Spermatogenic arrest and ‘Sertoli cell‐only’ syndrome—common alcohol‐induced disorders of the human testis
Authors: PAJARINEN J. T. , KARHUNEN P. J.
Published: May 2008

The effect of alcohol on spermatogenesis and morphometric analysis of the human testis was investigated in a prospective autopsy study, with detailed alcohol‐use reports from relatives or friends of the deceased. The autopsy series comprised non‐alcoholic controls (daily intake <10 g; n=32) and heavy‐drinkers (n=44) with an intake of >80 g per day. Of the controls, 26 (81.3%) men showed normal spermatogenesis, and six (18.7%) partial spermatogenic arrest. Of the heavy drinkers, only 16 (36.4%) had normal spermatogenesis (p<0.00l), while 23 (52.3%) showed partial or complete spermatogenic arrest (p<0.00l) and five had Sertoli cell‐only (SCO) syndrome (pC0.05). The mean testicular weight of heavy‐drinkers was slightly (p<0.05) lower than in the controls. Compared to men with normal spermatogenesis, testicular weight was slightly lower both in controls and heavy‐drinkers with spermatogenic arrest, and was significantly (p<0.01) lower in heavy‐drinking men with SCO syndrome. Spermatogenic arrest was not correlated with fatty liver or cirrhosis of the liver, whereas four of the five men with SCO syndrome exhibited a fatty liver. Thus, our results suggest that the most common alcohol‐related pathological change in the testes is probably reversible arrest of spermatogenesis. Furthermore, we suggest that many heavy social drinkers may suffer from self‐inacted infertility, as one tenth of heavy‐drinkers in our study had SCO syndrome.
Regional variation of specific gene expression in the dog epididymis as revealed by in‐situ transcript hybridization
Authors: PERA I. , IVELL R. , KIRCHHOFF C.
Published: May 2008

In‐situ transcript hybridization was used to characterize the regional distribution of three marker gene transcripts which are expressed abundantly in the canine epididymis. The gene products CE1, CE4 and CE5, which are the canine equivalents of the human homologues HE1, HE4 and HE5, are shown to be expressed in a tissue‐specific and regionally characteristic pattern in the epicfidymal epithelium. CE1 mRNA was expressed very weakly in the efferent ducts but was expressed at a high level throughout the caput and corpus regions of the epididymis, decreasing somewhat in the distal cauda region. CE4 mRNA was not detectable in the efferent ducts and was only expressed moderately in the remainder of the epididymis, with greatest levels in the caput and proximal cauda regions, and decreasing in the distal cauda. CE5 mRNA showed the most marked regional variation in levels with little or no mRNA detectable in the caput and proximal corpus regions, but increasing dramatically in the distal corpus and cauda. In the transition region of the central corpus, the CE5 mRNA appeared to be expressed intermittently, giving a mottled signal appearance over the epididymal epithelium. The patterns of mRNA distribution for the three marker genes in the dog epididymis were, therefore, essentially similar to those for the equivalent human homologues, providing further support for the suitability of the dog epididymis as a model for the human.
Proteins in fluid from the proximal vas deferens of normal fertile and vasectomized men
Authors: FENG H.‐M. , WEN R.‐Q. , LI S.‐Q. , WANG C. , WANG Q.‐H. , JIANG Y.‐J. , LI Q.‐K.
Published: May 2008

Fluids were collected from the proximal vas deferens of 18 normal fertile men and 32 vasectomized men during vasectomy or vasovasostomy, respectively, and the protein concentration and pattern of proteins were then analysed by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS‐PAGE). In normal fertile men and vasectomized men there were no significant differences in total protein concentrations between fluids from the left and right vas deferens. The total protein concentration of proximal vas fluid from vasectomized men (82.8 ±7.5 mg/ml; mean ± SEM) was significantly higher than in normal fertile men (36.8 ± 4.5 mg/ml). In vasectomized men there was no significant correlation between the total protein concentration in vas fluid and the duration of vasectomy. The patterns of protein bands in proximal vas fluid differed from those in seminal plasma. There was no relationship between the pattern of protein bands in vas fluid and the duration of vasectomy. Five major groups of proteins from proximal vas fluid were identified and no new major proteins were found in proximal vas fluid after vasectomy. The percentage of 80K and 67K protein bands in vasectomized men was higher than that in normal fertile men. It is suggested that, after vasectomy, the physiological environment of the proximal vas deferens may be changed. One of these changes may be the higher total protein concentration (especially the 80K and 67K proteins) in vas fluid.
In vitro fertilization and the effect of progesterone and 17α‐hydroxyprogesterone on acrosome reaction of mouse epididymal spermatozoa
Authors: KHOLKUTE S. D. , RODRIQUEZ J. , DUKELOW W. R.
Published: May 2008

This study assessed the effect of progesterone and 17α‐hydroxyprogesterone (17α‐OH‐progesterone) at concentrations of 0.01–10 μg/ml, on the acrosome reaction and in vitro fertilizing ability of mouse epididymal spermatozoa. Cumulus masses containing oocytes were cultured in Brinster's medium, to which were added capacitated epididymal spermatozoa which had been incubated in medium with various concentrations of progesterone or 17α‐OH‐progesterone for 90 min. IVF success rate was assessed 20–24 h following insemination. Progesterone was found to increase the fertilization rate at the 1 μg/ml and 10 μg/ml concentrations while lower concentrations had no effect. However, 17α‐OH‐progesterone failed to show any effect on fertilizing ability. Incubation of epididymal spermatozoa in medium containing 1 μg/ml and 10μg/ml progesterone significantly increased the acrosome reaction as monitored by a chlortetracycline fluorescence assay. 17α‐OH‐progesterone, however, failed to show any effect on the acrosome reaction. The results suggest direct effects of progesterone, but not of 17α‐OH‐progesterone, on fertilization and the acrosome reaction of mouse spermatozoa.
Functional and structural characteristics of human epididymal spermatozoa retrieved by transcutaneous aspiration
Authors: FORESTA C. , ZORZI M. , GALEAZZI C. , ROSSATO M.
Published: May 2008

The present paper describes the development of a simple, repeatable technique for transcutaneous aspiration of epididymal spermatozoa in patients with inoperable excretory azoospermia. Some functional and structural parameters of the retrieved spermatozoa were also evaluated. Epididymal spermatozoa from patients and ejaculated spermatozoa from fertile donors showed similar percentages of acrosome‐reacted cells after incubation for 6 or 15 h in capacitating medium. Penetration of zona‐free hamster oocytes, expressed as the percentage of penetrated ova, was significally lower (p < 0.01) for spermatozoa from patients than for ejaculated spermatozoa from fertile donors. The percentage of epididymal spermatozoa with stable nuclei in sodium dodecyl sulphate was lower (p < 0.001) than for ejaculated spermatozoa from fertile donors. A positive linear correlation (r= 0.74, p < 0.005) was evident between the oocyte penetration rate and the percentage of spermatozoa with stable nuclei. In conclusion the percutaneous retrieval of epididymal spermatozoa in excretory azoospermia allows collection of motile spermatozoa, which can be evaluated for their structural and functional characteristics before repetition of the technique for retrieval of spermatozoa for assisted fertilization techniques.
Characterization of development‐specific, cell type‐specific mouse testicular antigens using testis‐specific polyclonal antibodies
Authors: TSUCHIDA J. , NISHINA Y. , AKAMATSU T. , NISHIMUNE Y.
Published: May 2008

Antisera were raised by immunizing rabbits with mouse testicular germ cells, and absorbed in vivo by injection into castrated male whole mice to obtain a specific antiserum which reacted with mouse germ cells. The expression of mouse testisspecific antigenic macromolecules was then studied immunochemically with the antiserum. Approximately 20 antigenic macromolecules with molecular weights ranging from 26 to 110 kD were detected in the normal adult testis. At least 12 of these were differentiation‐specific antigens appearing during development of germ cells, while others were expressed in testicular germ and/or somatic cells or detected only in mature spermatozoa. This technique for raising testis‐specific antisera could be useful for isolation of cDNA clones encoding their antigens, as well as for investigation of the physiological roles of these molecules in germ cell differentiation at the molecular level.
Influence of batches of bovine oocytes on the outcome of an intact zona pellucida binding assay and in vitro fertilization
Authors: ZHANG B. R. , LARSSON B. , RODRIGUEZ‐MARTINEZ H.
Published: May 2008

A series of experiments was performed in vitro to determine the variation between batches of abattoir‐derived bovine oocytes to bind and to be fertilized by frozen–thawed spermatozoa provided by a single ejaculate from a bull with high field fertility after artificial insemination. Fourteen batches of oocytes, collected over a 90‐day period, were divided randomly into two equal groups at each sampling period. One group of oocytes was subjected to in‐vitro maturation and subsequently in‐vitro fertilization (IVF) while the other group was used as intact oocytes in a zona binding assay (ZBA). Fertilized oocytes were evaluated for penetration and pronuclear formation and, in the ZBA, the number of spermatozoa bound to each oocyte was registered. Neither the results of the ZBA nor the IVF results varied significantly (p=0.45) between batches. Furthermore, when a sperm concentration of 0.5 × 106/ml was used, the number of spermatozoa bound to the zona pellucida correlated significantly with the frequency of oocytes penetrated after IVF (r= 0.76). The results suggest that variation between the batches of oocytes has no detrimental significance for the application of intact zona pellucida binding or IVF for assaying frozen–thawed bull semen, provided that a large number of oocytes/batch (> 15) are assayed.
Functional motion changes during sperm transit to the site of fertilization and in‐vitro applications: a review
Authors: MBIZVO M. T.
Published: May 2008

Continued research to define the parameters of sperm function should aid the evaluation of various approaches in infertility management as well as the efficacy of contraceptives for men which do not necessarily achieve azoospermia.

Many treatment forms have been advocated for male factor infertility but have yielded little effect. These include, for example, gonadotrophins, clomiphene citrate, the weakly androgenic steroid, mesterolone. Often, improvements in oligoasthenozoospermia that are not related to genital infection, do not attain normozoospermic levels.

Owing to lack of success with the various treatment modalities, assisted reproductive technology encompassing artificial insemination by husband or donor following in vitro enhancement of sperm function have assumed an important role in male infertility. Agents that have been shown to induce and support sperm capacitation processes such as hyperactivation, could serve an important role. These include human follicular fluid (HFF), maternal serum, fetal cord serum and methyl xanthine derivatives.
Sperm binding to the human zona pellucida after migration through human cervical mucus
Authors: VIGIL P. , RIQUELME R. , MORALES P.
Published: May 2008

During lactational amenorrhea a special type of cervical mucus, similar to that found during the luteal phase, is produced. This mucus, however, is able to support sperm migration. In the study described, the ability of spermatozoa to bind to the human zona pellucida (hZP) after migration through periovulatory and post‐partum mucus was studied. Mucus was obtained from exclusively breastfeeding women in amenorrhea at 30, 60, 120 and 180 days post‐partum. Periovulatory mucus samples from normally cycling women were used as a control. Flat capillary tubes were filled with BWW culture medium at the top and cervical mucus at the bottom. The tubes were immersed in a semen reservoir and the spermatozoa allowed to migrate through the mucus for 3 h into the culture media. Then the spermatozoa were coincubated with 3–4 hZP for 30 min and the number of bound spermatozoa per zona was counted. Periovulatory cervical mucus had an average Insler score of 14 ± 0.5 as compared to 4.6 ± 0.4 for post‐partum mucus. Spermatozoa recovered from periovulatory mucus were always able to bind to the hZP. Spermatozoa recovered from post‐partum mucus, however, were able to bind to the hZP in only 68 ± 7% of the cases. Moreover, spermatozoa recovered from post‐partum mucus bound to the ZP in lower numbers than did spermatozoa recovered from periovulatory mucus (p<0.03). These results suggest a greater ability of sperm‐hZP binding after migration through periovulatory mucus and they also indicate that sperm binding to the ZP is possible even after sperm migration through a low quality mucus.
Assessment of the vitality and acrosomal status of human spermatozoa using fluorescent probes
Authors: KINGER S. , RAJALAKSHMI M.
Published: May 2008

The acrosomal status and vitality of human spermatozoa are generally assessed simultaneously using the lectins Pisum sativum agglutinin (PSA), Peanut agglutinin (PNA) and Concanavalin A (Con A) in conjunction with either Hoechst 33258 (H258; a fluorescent DNA‐binding supravital stain with limited membrane permeability) or a hypo‐osmotic swelling (HOS) test. In the present study, sperm vitality was assessed using H258 under different staining conditions and compared with that estimated using eosin‐nigrosin (E‐N) under different staining conditions and the HOS test. The sensitivity of PNA‐ and Con A‐labelling methods were also compared by evaluating the acrosomal status of motile human spermatozoa after capacitation (1 and 4 h) in vitro followed by exposure to dimethylsulphoxide (DMSO) and a calcium ionophore. The E‐N method employing eosin‐staining for 15 sec, rather than for 30 sec, provided a more reliable estimate of sperm vitality. H258, as used in the H258/Con A‐labelling method (with and without ethanol fixation) rather than under the staining conditions equivalent to H258/PNA‐labelling, was as good for vitality assessment as the E‐N method employing eosin‐staining for 15 sec. However, the HOS test overestimated the proportion of dead spermatozoa compared to those obtained using different H258 and E‐N methods. Further, the Con A‐labelling method consistently scored a significantly lower percentage of spermatozoa undergoing the acrosome reaction compared to those estimated by the PNA‐labelling method. It is concluded that the different sensitivity of these methods can be attributed to the different binding specificity of the lectins. Since PNA and Con A specifically label the outer and inner acrosomal membrane, respectively, spermatozoa in the early stages of the acrosome reaction, undergoing modification of the outer acrosomal membrane but retaining their acrosomal contents, would be scored as acrosome‐reacted by the PNA‐labelling method but as acrosome‐intact by the Con A‐labelling method. The lower sensitivity of the Con A‐labelling method could also be due to the use of aldehyde‐fixed spermatozoa for Con A‐labelling. Also, the PNA‐labelling requires less time to complete than does the Con A‐labelling.
A simple method to detect sperm chromatin abnormalities: cytochemical mechanism and possible value in predicting semen quality in assisted reproductive procedures
Authors: ANDREETTA A. M. , STOCKERT J. C. , BARRERA C.
Published: May 2008

Protamine crosslinking by disulphide (‐SS‐) bonds is the main factor responsible for the stability of chromatin structure in mammalian spermatozoa. Sperm chromatin containing arginine/cysteine‐rich protamines shows a deeply modified cytochemical reactivity (e.g. basophilia) when compared with somatic chromatin. After methanol or ethanol‐acetic acid fixation and toluidine blue (TB) staining, most sperm heads in semen smears from human fertile donors exhibited a pale blue (orthochromatic) colour, while a few sperm heads exhibited violet‐blue or violet (metachromatic) staining. Smears from infertile patients showed an increased amount of metachromatic sperm nuclei. After reduction of ‐SS‐ bonds by dithiothreitol, sperm heads from all smears were metachromatic, suggesting that DNA phosphates then become available for TB stacking. Micro‐ and macro‐spectrophotometric studies confirmed the microscopic colour reaction of sperm nuclei. The ortho‐/metachromatic ratio seems a useful parameter for evaluation of altered chromatin structure in spermatozoa cells. Taking into account the current interest in complementary staining tests for evaluation of semen quality, the metachromatic TB reaction represents a simple cytochemical approach for detecting sperm chromatin abnormalities based on differences in ‐SS‐ crosslinking.
Intravasal injection of formed‐in‐place medical grade silicone rubber for vas occlusion
Authors: SOEBADI D. M. , GARDJITO W. , MENSINK H. J. A.
Published: May 2008

This paper describes two consecutive studies: a volume study and an efficacy study. The volume study determined the appropriate volume of Medical Grade Silicone Rubber (MSR) needed to achieve complete occlusion of the vas deferens. This was done by in‐vitro testing of 130 human vas specimens containing plugs of MSR formed in vivo. The volume of MSR needed to occlude the vas was 0.1531 ± 0.0059 ml injected by five to six turns of the applicator handwheel. There was a correlation between MSR volume, weight, and the number of turns of the applicator handwheel. An influence of body height upon the volume and weight of the MSR was also observed. An oval shaped clamp (15 mm long) was more effective in producing secure vas occlusion than was a round clamp (10 mm long). The mean outer diameter of the vas specimens was 1.80 ± 0.15 mm, and the mean maximum dilated inner diameter was 0.93 ± 0.11 mm.

The efficacy study was a clinical trial to compare MSR vas occlusion (using an oval 15 mm clamp and the appropriate volume derived from the volume study, n=58) with no‐scalpel vasectomy as the standard procedure (n=64). The azoospermia rate following MSR vas occlusion was not significantly different from that following no‐scalpel vasectomy, and was achieved in 3–6 months.
Insulin‐like growth factor‐I and transforming growth factor‐α stimulate differentiation of type A spermatogonia in organ culture of adult mouse cryptorchid testes
Authors: TAJIMA Y. , WATANABE D. , KOSHIMIZU U. , MATSUZAWA T. , NISHIMUNE Y.
Published: May 2008

This study assessed the effect of growth factors on testicular germ cell differentiation in vitro. Testicular fragments of experimentally prepared cryptorchid testes of adult mice were cultured for 9 days in serum‐free media containing various concentrations of IGF‐I, TGF‐α, FGF, and PDGF. Their histology was then examined under a light microscope. Each type of germ cell and mitotic cell in the seminiferous tubules was counted per 1000 Sertoli cells. IGF‐I at a concentration of 10 ng/ml induced maximal differentiation of type A spermatogonia. TGF‐α at concentrations ranging from 1 to 10 ng/ml also stimulated differentiation, whereas FGF and PDGF did not show any stimulation of spermatogonial differentiation in this experimental system.
High and unchanged sperm counts of Finnish men
Authors: VIERULA M. , NIEMI M. , KEISKI A. , SAARANEN M. , SAARIKOSKI S. , SUOMINEN J.
Published: May 2008

Several recent reports have suggested that the sperm counts of normal men are declining in most countries. In this study the sperm counts of Finnish men, and their possible changes during the past 28 years, were investigated. The material consisted of semen samples from 238 normal healthy men of unknown fertility and 5481 men from infertile couples. The means (medians) of semen volume, sperm density and total sperm count in normal men were 3.3 (3.0) ml, 133.9 (94.0) × 106/ml and 396.6 (309.0) × 106, respectively. These parameters and the relative frequency distribution of the sperm density were similar to those reported elsewhere in the 1940s. Multiple linear regression analysis revealed a significant decrease in semen volume, whereas sperm density and total sperm count of infertile men had not changed significantly during the past 28 years. In addition, no change in sperm counts was associated with the year of men's birth.
Chromomycin A3‐staining as an indicator of protamine deficiency and fertilization
Authors: LOLIS D. , GEORGIOU I. , SYRROU M. , ZIKOPOULOS K. , KONSTANTELLI M. , MESSINIS I.
Published: May 2008

Mature mammalian spermatozoa have a compact and stable nuclear structure conferred by protamines instead of histones, which are present in all other cellular types. Chromomycin A3 (CMA3) is a useful tool for the detection of protamine deficiency in sperm chromatin. The purpose of this study was to correlate the percentage of spermatozoa staining positively for CMA3 with sperm parameters and in‐vitro fertilization of human oocytes. Spermatozoa were collected from 56 fertile and 18 infertile men, and washed twice in PBS, fixed in two changes of methanol: acetic acid (3: 1 v: v) spread on rinsed slides treated with APES and dried. Twenty‐four of the semen samples were subjected to both Percoll and swim‐up, and were stained subsequently with CMA3. CMA3‐stained spermatozoa were expressed as a percentage in a count of 200 spermatozoa. A substantial variation in the percentage of CMA3‐stained cells was observed in ejaculated human spermatozoa, varying between 8% and 77%. A strong negative correlation (r= ‐0.64, p < 0.001) was found between sperm count and the percentage of CMA3‐stained spermatozoa. No correlation was found between CMA3‐stained spermatozoa and their motility, while excessive sperm morphological abnormalities were related positively to CMA3‐staining. Spermatozoa in samples exhibiting low (8–62%) CMA3‐staining had significantly higher fertilizing rates in vitro than did samples exhibiting high (49–77%) CMA3‐staining. The mean percentage of CMA3‐stained spermatozoa after swim‐up or Percoll preparation (26% vs 31%) did not differ significantly. These results demonstrate a close relationship between CMA3‐staining, fertilization and sperm count, and suggest potential application of this marker for the prediction of sperm quality and fertilizing capacity.
Testicular germ cell populations in the adult rat after continuous in‐vivo testicular infusion of inhibin‐A and activin‐A
Authors: ADEEKO A. O. , DUNNE E. , MATHER J. , MOORE A. , MORRIS I. D.
Published: May 2008

This study investigated the in‐vivo effects of inhibin‐A and activin‐A on germ cell subpopulations in the adult rat testis. Each animal received a total of 2 μg (dose 5 ng/0.5 μl/h) of activin, inhibin or 0.1% bovine serum albumin (vehicle control) delivered intratesticularly into the left testis via a cannula connected to an abdominal minipump implant. To establish whether the hormones exerted any effect on procarbazine‐induced germ cell depletion, a single intraperitoneal dose of this agent was given to a group of the rats 24 h after the start of testicular infusion of activin or inhibin. Rats were killed 14 days later. Inhibin treatment caused a reduction in the number of round spermatids without altering serum FSH levels. In procarbazine‐treated rats, infusion of inhibin reduced the number of pachytene spermatocytes and reduced blood FSH levels simultaneously. Infusion of activin had no significant effect on the numbers of germ cells, but reduced the number of dead cells in the seminiferous tubules of procarbazine‐treated rats. The data would therefore suggest that the effects of inhibin and activin on the seminiferous epithelium are influenced by the testicular microenvironment, and that they are capable of influencing the growth and survival of germ cells by both FSH‐ and non FSH‐mediated mechanisms. Further experiments are needed to identify the physiological role(s) of inhibin and activin in spermatogenesis.
Clinical and genetic variability in XX sex‐reversed patients
Authors: MANIERI C. , TAGLIABUE M. , NOVI R. F. , BISCEGLIE C. DI , ARDUINO C. , FORNENGO R. , BRANCALEONI V. , MARTINA V.
Published: May 2008

Three 46, XX hypogonadal subjects are described who exhibited different clinical and genetic characteristics. Two patients, with complete sex‐reversal, are sterile males with hypogonadal features; the third patient, with partial sex‐reversal, presented with a eunuchoid appearance and with ambiguous genitalia. Polymerase chain reaction (PCR) amplification of DNA from these patients showed the presence of a translocation of the sex‐determining region of the Y chromosome (Sry) only in the first two patients described.
Superoxide anion scavenging capacity of human seminal plasma
Authors: GAVELLA M. , LIPOVAC V. , VUČIĆ M. , ROČIĆ B.
Published: May 2008

This study has investigated the antioxidant capacity of human seminal plasma due to the presence of both high and low molecular weight antioxidant factors. Methods for the measurement of superoxide dismutase‐like activity (SOD‐like) and total antioxidant status (TAS) were automated, and had a within‐run coefficient of variation of 7.3% for SOD‐like activity and 4.8% for TAS. In 69 semen samples from unselected infertile men, SOD‐like activity in seminal plasma ranged from 2 to 16 U/ml, with a mean of 6.9 ± 2.8 U/ml. As SOD‐like activity was correlated positively with levels of citric acid (p<0.0001), zinc (p<0.0002) and acid phosphatase activity (p<0.0005), and there was no correlation with fructose levels, our results suggest that prostatic secretions are an important source of superoxide anion scavengers. Evaluation of SOD‐like activity in infertile men with accessory sex gland infections (n= 12) showed significantly lower activity (p<0.003) compared to values found in 12 infertile men without signs of infection. The values obtained for total antioxidant status (equivalent to the antioxidant capacity of alpha‐tocopherol analogue) ranged from 1.7 to 2.3 mmol/L, with a mean of 2.1 ± 0.1 (n= 40), reflecting the protective activity of ascorbate, urate and albumin, and to a very low extent of glutathione and taurine. The data obtained by TAS assay correlated with fructose, a major marker of vesicular secretion (p<0.005), suggesting that low molecular weight components with antioxidant capacity derive partly from the seminal vesicles. The results indicate that the relative contribution of antioxidant defence systems capable of counteracting the deleterious action of superoxide anions, depends on the secretory activity of accessory sex glands and is independent of excessive ROS production due to increased oxidative stress.
Are the characteristics of spermatozoa in the insemination medium useful for predicting in‐vitro fertilization results?
Authors: PARINAUD J. , RICHOILLEY G. , MOUTAFFIAN H. , VIEITEZ G. , MIEUSSET R.
Published: May 2008

To determine whether the characteristics of Percoll‐selected spermatozoa are more predictive of in‐vitro fertilization (IVF) results than are those of native semen, 118 semen samples from patients undergoing an IVF attempt were studied. Motility, using computer‐assisted sperm analysis, and morphology were recorded before and after sperm selection on a Percoll gradient. Percoll selection increased the percentage of morphologically normal spermatozoa (58.2 ± 19.9% versus 47.9 ± 17.9%; p <0.0001). This increase concerned almost all abnormalities and especially abnormalities of the midpiece (cytoplasmic droplets and bent tails). However, morphology of spermatozoa in the Percoll fraction had the same predictive value for IVF as did that for whole semen. Concerning motility, all parameters, except linearity, were improved by Percoll preparation, and their predictive value for IVF results was significantly higher in the Percoll fraction than in native semen. Therefore, it is concluded that, even if sperm characteristics are improved dramatically after Percoll selection, only motility analysis then gave more practical information than did analysis of sperm characteristics in native semen. This suggests that impairment of spermiogenesis, which presumably induces sperm abnormalities, is more important than are the actual characteristics of the spermatozoa.
Maturation of sperm motility in the epididymis of the common marmoset (Callithrix jacchus) and the cynomolgus monkey (Macaca fascicularis)
Authors: YEUNG C. H. , MORRELL J. M. , COOPER T. G. , WEINBAUER G. F. , HODGES J. K. , NIESCHLAG E.
Published: May 2008

The acquisition by spermatozoa of the capacity for movement upon liberation from the epididymal duct into buffer is one of the obvious changes associated with sperm maturation and, hence, studies of sperm kinematics might provide good indices for sperm maturation. Profiles of development in the movement pattern of spermatozoa taken from six regions of the epididymis were established in marmoset and cynomolgus monkeys using computer‐assisted sperm analysis. Motion of spermatozoa from initial regions was sluggish and irregular, then developed in the more distal region into erratic movement effected by an increase in the vigour of flagellation as reflected by curvilinear velocity. The tendency of immature spermatozoa to display circular motion was more common in the marmoset than in the cynomolgus monkey. In the marmoset the most drastic changes in percentage motility and kinematic parameters of spermatozoa occurred between the distal caput and the proximal corpus; maximal values for these parameters were found around the distal corpus epididymis. For the cynomolgus spermatozoa the sharpest gain in percentage motility was also achieved between the caput and proximal corpus epididymis, but with full kinematic development in the proximal cauda epididymis. These maturational changes were not synchronous among spermatozoa from the same site as they moved along the epididymis, as reflected in the heterogeneity of each sample in the kinematic properties of individual spermatozoa, especially those from the proximal corpus epididymis. The maturational profiles in the monkeys are comparable to the situation in the human, for which they may provide a useful model.
The effect of bradykinin and the bradykinin antagonist Hoe 140 on kinematic parameters of human spermatozoa
Authors: YEUNG C. H. , SPIER B. , COOPER T. G. , NACKE P. , NIESCHLAG E.
Published: May 2008

Bradykinin (BK) has been suggested to be an active substance in the disputed therapeutic use of kallikrein to improve semen quality. The effects of exogenous BK and its antagonist Hoe 140, which acts on one of the bradykinin receptors (BK2), were examined in two groups of patients attending the fertility clinic: those with asthenozoospermia (group I) and normozoospermia (group II). Bradykinin (10 nM–1 μM) added to washed human spermatozoa had no effect on most kinematic parameters and caused only a marginal increase (7%) in curvilinear velocity at 50 nM in group I patients; however, this increase was not suppressed by concomitant addition of the BK antagonist. The bradykinin antagonist itself had no effect on the percentage motility or kinematic motility parameters of washed human spermatozoa in either group of patients. The motility of spermatozoa in semen was also unaffected by the presence of the bradykinin antagonist. It is concluded that bradykinin does not act exogenously on washed spermatozoa nor endogenously on spermatozoa in semen to stimulate motility via BK2 receptors, regardless of the initial quality of the sperm motility.
Transplantation of the testis; from the past to the present
Authors: BARTEN E. J. , NEWLING D. W. W.
Published: May 2008

Since the development of surgery, the possibility of testis transplantation has fascinated man for centuries. Hunter and Berthold are considered to be the most important investigators in this field and, in addition, to be the founders of modern endocrinology. The association of testis transplantation with rejuvenation led to widespread popularity for this treatment in the first three decades of the twentieth century. At the same time, controversies concerning the aim of the treatment have coloured the early years of endocrinology. In the 1960s renewed interest in the subject arose for experimental reasons, leading to the development of microsurgical techniques for autotransplantation of high‐lying undescended testes in children. Homologous testis transplantation has never become a subject of great interest, probably for ethical reasons. This possible treatment for hypogonadism, however, has been developed experimentally and has been performed in man only in Russia and China, evidently with success. The details of these studies and their outcomes are discussed.
Scanning electron microscopy shows inhibited gubernacular development in relation to undescended testes in oestrogen‐treated mice
Authors: SHONO T. , HUTSON J. M. , WATTS L. , GOH D. W. , MOMOSE Y. , MIDDLESWORTH B. , ZHOU B. , RAMM‐ANDERSON S.
Published: May 2008

The morphological relationship between transabdominal testicular descent and the `swelling reaction' of the gubernaculum was investigated in oestrogen‐treated fetal mice by using scanning electron microscopy (scanning EM). In addition, flutamide was also administered to pregnant mice to determine whether androgens cause gubernacular growth and transabdominal testicular descent in offspring. In oestrogen‐treated fetal mice, scanning EM showed that both the gubernacular ‘swelling reaction’ and transabdominal testicular descent were inhibited, in addition to inhibition of Müllerian duct regression. The gubernaculum showed a flat, thin bulb (widest diameter 0.25 ± 0.04 mm) and an elongated cord (1.28 ± 0.41 mm) after oestrogen treatment in utero, which was significantly different in appearance from that in normal control mice (width 0.44 mm ± 0.06 mm, p < 0.001; length 0.27 ± 0.19 mm, p < 0.0001). However, flutamide‐treated mice showed much more normal gubernacular enlargement and transabdominal testicular descent. The width of the gubernacular bulb after flutamide exposure was 0.44 ± 0.05 mm, which was comparable to that in control animals; the length of the intra‐abdominal gubernaculum (0.44 ± 0.15 mm) was slightly longer than in controls (p < 0.02).

These results suggest that both the swelling reaction of the gubernaculum and transabdominal testicular migration are blocked by prenatal exposure to oestrogen. However, oestrogen exposure of the fetus does not block the swelling reaction of the gubernaculum by acting as an antiandrogen.
Viability of ram spermatozoa in relation to the abstinence period and successive ejaculations
Authors: OLLERO M. , MUIÑO‐BLANCO T. , LÓPEZ‐PÉREZ M. J. , CEBRIÁN‐PÉREZ J. A.
Published: May 2008

For successful fertilization, a functionally constituted sperm plasma membrane is necessary, and this is clearly dependent on the sperm maturation process. The latter involves a series of complex changes which result from a sequence of events occurring at different points within the epididymis. The transit time through the epididymis can be influenced by external factors such as sexual stimulus and ejaculatory frequency. The present work was undertaken to determine changes in ram sperm viability and other sperm quality characteristics in relation to ejaculatory frequency.

Three successive ejaculates were collected from rams during three different abstinence periods (collected every day, every 2 days and every 3 days). Cell viability (membrane integrity determined by fluorescence staining), progressive individual motility, and other in vitro parameters of sperm quality were evaluated. Second ejaculates showed the highest cell viability of the three periods studied, and increased as the abstinence period lengthened. The maximum proportion of viable cells (average 60%) was obtained in the second ejaculate after an abstinence period of 3 days. Likewise, overall and progressive individual motilities were higher in second ejaculates, the maximum value being 70% after 3 days of abstinence. The percentage of damaged or acrosome‐reacted spermatozoa was greater after 1 day of abstinence than after the other periods analysed, whereas the first ejaculate showed the highest value in all periods.

Differences in ejaculate volume were correlated strongly with both variables considered (abstinence period and ejaculate number). In the third ejaculate, about 27% more volume was obtained after 3 days of abstinence than after abstinence for 1 or 2 days. Sperm concentration increased significantly as the abstinence period lengthened, and also decreased significantly with ejaculate number in all cases. Therefore, the total number of spermatozoa in the ejaculate was clearly dependent on the abstinence period and the ejaculate number.

In conclusion, the results obtained suggest that using the second and/or a mixture of second and third ejaculates would improve the results in artificial insemination and in fertility studies. In addition, the use of better quality semen would facilitate progress in semen cryopreservation studies.
Immunodetection of cell adhesion molecules and extracellular matrix proteins in rat Leydig cell cultures
Authors: DENDUCHIS B. , SCHTEINGART H. , CIGORRAGA S. , VIANELLO S. E. , CASANOVA M. B. , LUSTIG L.
Published: May 2008

Cell adhesion molecules (CAMs) as well as extracellular matrix (ECM) proteins were identified in Leydig cell cultures using immunohistochemical and Western blot analysis. Leydig cells were isolated from 60‐day‐old rats and cultured for 4 days. For immunofluorescence and immunoperoxidase techniques, Leydig cells were incubated with antisera to ECM proteins (antibodies to laminin, type IV collagen and fibronectin); antisera to integrins (antibodies to β1, α3, α5 and α6 integrin subunits) and antisera to cell‐to‐cell adhesion molecules (antibodies to N‐CAM and N‐cadherin). Results of the two immunohistochemical techniques were similar. Laminin and type IV collagen were detected in the perinuclear area of Leydig cell cytoplasm and cell processes as bright granular immunofluorescence or as a brown reaction product using the immunoperoxidase technique. Leydig cells expressed α3 and α6 integrin subunits (mainly laminin receptors), while no reaction was detected with antibodies to the α5 integrin subunit (fibronectin receptor). Leydig cells also expressed cell‐to‐cell adhesion molecules such as N‐CAM and N‐cadherin.

Using Western blot analysis, Leydig cell extracts incubated with antibodies to laminin revealed two bands of around 200 kDa, which is characteristic of laminin 1 light chains. A band with electrophoretic mobility similar to that of the α2 (IV) collagen chain from EHS sarcoma and a band of around 230 kDa similar to fibronectin were also detected in Leydig cell extracts using specific antisera.

Leydig cells incubated with antibodies to the α3 integrin subunit revealed two bands below 120 kDa. Finally, Western blot results showed that Leydig cells expressed N‐CAM as two faint bands of around 140 kDa and N‐cadherin as a 120 kDa band. The present data suggest that Leydig cells in culture are able to synthesize ECM proteins and express ECM receptors (integrins), as well as cell‐to‐cell adhesion molecules such as N‐CAM and N‐cadherin.
Extensive polymorphism and geographical variation at a positively selected MHC class II B gene of the lesser kestrel (Falco naumanni)
Authors: ALCAIDE MIGUEL , EDWARDS SCOTT V. , NEGRO JUAN J. , SERRANO DAVID , TELLA JOSÉ L.
Published: May 2008

Understanding the selective forces that shape genetic variation in natural populations remains a high priority in evolutionary biology. Genes at the major histocompatibility complex (MHC) have become excellent models for the investigation of adaptive variation and natural selection because of their crucial role in fighting off pathogens. Here we present one of the first data sets examining patterns of MHC variation in wild populations of a bird of prey, the lesser kestrel, Falco naumanni. We report extensive polymorphism at the second exon of a putatively functional MHC class II gene, Fana‐DAB*1. Overall, 103 alleles were isolated from 121 individuals sampled from Spain to Kazakhstan. Bayesian inference of diversifying selection suggests that several amino acid sites may have experienced strong positive selection (ω = 4.02 per codon). The analysis also suggests a prominent role of recombination in generating and maintaining MHC diversity (ρ = 4Nc = 0.389 per codon, θ = 0.017 per codon). Both the Fana‐DAB*1 locus and a set of eight polymorphic microsatellite markers revealed an isolation‐by‐distance pattern across the Western Palaearctic (r = 0.67; P = 0.01 and r = 0.50; P = 0.04, respectively). Nonetheless, geographical variation at the MHC contrasts with relatively uniform distributions in the frequencies of microsatellite alleles. In addition, we found lower fixation rates in the MHC than those predicted by genetic drift after controlling for neutral mitochondrial sequences. Our results therefore underscore the role of balancing selection as well as spatial variations in parasite‐mediated selection regimes in shaping MHC diversity when gene flow is limited.