Ultrastructural study of spermatogenesis and sperm in the African medicinal leech Hirudo troctina Johnson, 1816 (Annelida, Hirudinida).

This paper presents the process of spermatogenesis in the leech Hirudo troctina Johnson, 1816 using light, fluorescent and transmission electron microscopy. At the onset of spermatogenesis in testes, the pear-shaped spermatogonia divide mitotically without full cytokinesis and as a result isogenic groups are formed (clusters, clones) with 2, 4, 8, 16, 32, 64, 128 spermatogonia and, finally, 256 primary spermatocytes occur. The final meiotic divisions of spermatocytes give rise to clones with 1024 spermatids. There are hundreds of developing germ-line clones in each testis. In each clone, the male germ cells divide in full synchrony and they are in the same phase of spermatogenesis. During complex spermiogenesis each spermatid becomes a filiform spermatozoon with a helicoid nucleus, which is characterized by the presence of a long acrosome with two regions - anterior and posterior, which are followed by a helicoid nucleus, a midpiece with only one mitochondrion and a long flagellum. Our results were compared to those on other clitellate annelids that have been studied to date, especially to sperm formation in Hirudo medicinalis Linnaeus, 1785. Only minor differences were found in the length and the diameter of different organelles and the number of spermatids in germ-line clones.

Quercetin improves the postthaw characteristics of cryopreserved sex-sorted and nonsorted stallion sperm.

Excessive reactive oxygen species generation during sex sorting and cryopreservation of stallion sperm leads to DNA fragmentation, lipid peroxidation, and motility loss. In this study we investigated whether antioxidant supplementation during sex sorting and cryopreservation could ameliorate the effects of reactive oxygen species on stallion sperm. In experiment 1, the postthaw characteristics of stallion sperm (N = 9) cryopreserved in the presence or absence of catalase (200 U/mL), cysteine (0.2 mg/mL), or quercetin (0.15 mM) was examined. Motility and acrosome integrity were assessed at 0, 1, and 3 hours after thawing. The sperm chromatin structure assay (SCSA; detectable DNA fragmentation index [DFI], mean DFI, and DFI) was used to assess DNA integrity immediately after thawing. Quercetin increased the total postthaw motility (25.3% vs. 20.9%; P < 0.05), but there was no beneficial effect of catalase or cysteine. Based on these results, the effect of quercetin during cryopreservation on the postthaw zona binding ability of sperm was assessed using a heterologous (bovine) zona binding assay. Quercetin increased the number of sperm bound per oocyte (13.6 vs. 9.2; P < 0.05) compared with the control. In experiment 2, the effect of quercetin (0.15 mM) in the media used during semen storage and transport, Hoechst 33342 staining and cryopreservation of stallion sperm (N = 9) was investigated. Motility, acrosome integrity, and viability were assessed at 0, 1, and 3 hours after thawing and SCSA was performed at 0 hours after thawing. Quercetin supplementation during sex sorting and cryopreservation improved DNA integrity (SCSA; detectable DFI of 54.9% vs. 74.6%, P < 0.05; mean DFI of 270.2 vs. 288.1, P < 0.05; and DFI of 26.3% vs. 28.5%, P < 0.05) compared with control sex-sorted sperm. There was no beneficial effect of quercetin on the motility, acrosome integrity, or viability of sex-sorted sperm. In conclusion, quercetin significantly improved the motility and zona binding ability of cryopreserved stallion sperm, and reduced DNA fragmentation in sex-sorted, cryopreserved stallion sperm.

Effects of supplement therapy on sperm parameters, protamine content and acrosomal integrity of varicocelectomized subjects.

PURPOSE: Surgical repair of varicocele has long been a procedure to correct spermatogenesis. However, the outcome has been reported to be inadequate. We combined varicocelectomy with supplement therapy to evaluate the concurrent effect of these procedures. METHODS: A prospective randomized controlled study was undertaken to investigate the effects of zinc sulfate, folic acid and zinc sulfate/folic acid on sperm quality, protamine content and acrosomal integrity following surgical repair of varicocele. Male subjects with palpable varicocele were included in the study and randomized into four groups. Subjects received Zinc sulfate, Folic acid, Zinc sulfate/Folic acid or placebo for 6 months. A semen sample was obtained before surgery and 3 and 6 months after surgical repair. Semen samples were evaluated for sperm parameters as well as chromatin content and acrosomal integrity. RESULTS: Most of the evaluated parameters showed a mild improvement after varicocelectomy in the placebo group. Interestingly, co-administration of Zinc sulfate and folic acid improved most factors significantly. Folic acid administration but not zinc sulfate could increase sperm number. Hence, Zinc sulfate was better than folic acid when change in morphology was assessed, and none of them was significantly effective in sperm motility. In Zinc sulfate and Folic acid groups, protamine content and halo formation rate significantly improved. CONCLUSIONS: We may conclude that co-administration of zinc and folic acid significantly improved sperm parameters and increased varicocelectomy outcomes. So, medical treatment with compatible drugs after surgery might be advantageous for obtaining acceptable results.

Internal anatomy and ultrastructure of the male reproductive system of the Norway lobster Nephrops norvegicus (Decapoda: Astacidea).

The Norway lobster (Nephrops norvegicus) is economically important in Europe. However, apart from the female reproductive system, very little is known about its internal anatomy. This article focuses on studying the internal anatomy and ultrastructure of the male reproductive system. This system follows the general pattern found among decapod crustaceans, with several peculiarities. Testes are composed of lobular sperm ducts in which the spermatozoa are fully constituted. The spermatozoa present three lateral arms and a long acrosome, which gives a false appearance of flagellated spermatozoa. The two testes form a double H under the heart, and the vas deferens (VD) arise from each side at the posterior edge of the double H. The main characteristic of the VD is the presence of a sphincter in the enlarged area of the distal end of the middle VD. The MVD here shows an increase in musculature of the wall as compared to the VD, which regulates the passage of the sperm cord to the distal VD (DVD) and thence to the thelycum of the female. The wall of the spermatophore is formed in the distal part of the proximal VD, which surrounds the unique sperm cord present in the VD. Isolated spermatophores are not observed in the VD. The sperm cord is pinched off during copulation by the musculature of the DVD. Then, a portion of the sperm cord is transferred from each VD to form the isolated spermatophores. The wall of the spematophores and the spermatozoa that are observed inside the thelycum have the same morphology as those observed in the VD.

Deficiency in the omega-3 fatty acid pathway results in failure of acrosome biogenesis in mice.

An omega-3 fatty acid, docosahexaenoic acid (DHA), is enriched in testicular membrane phospholipids, but its function is not well understood. The Fads2 gene encodes an enzyme required for the endogenous synthesis of DHA. Using Fads2-null mice (Fads2-/-), we found in our preceding studies that DHA deficiency caused the arrest of spermiogenesis and male infertility, both of which were reversed by dietary DHA. In this study, we investigated a cellular mechanism underlying the DHA essentiality in spermiogenesis. Periodic acid-Schiff staining and acrosin immunohistochemistry revealed the absence of acrosomes in Fads2-/- round spermatids. Acrosin, an acrosomal marker, was scattered throughout the cytoplasm of the Fads2-/- spermatids, and electron microscopy showed that proacrosomal granules were formed on the trans-face of the Golgi. However, excessive endoplasmic reticulum and vesicles were present on the cis-face of the Golgi in Fads2-/- spermatids. The presence of proacrosomal vesicles but lack of a developed acrosome in Fads2-/- spermatids suggested failed vesicle fusion. Syntaxin 2, a protein involved in vesicle fusion, colocalized with acrosin in the acrosome of wild-type mice. In contrast, syntaxin 2 remained scattered in reticular structures and showed no extensive colocalization with acrosin in the Fads2-/- spermatids, suggesting failed fusion with acrosin-containing vesicles or failed transport and release of syntaxin 2 vesicles from Golgi. Dietary supplementation of DHA in Fads2-/- mice restored an intact acrosome. In conclusion, acrosome biogenesis under DHA deficiency is halted after release of proacrosomal granules. Misplaced syntaxin 2 suggests an essential role of DHA in proper delivery of membrane proteins required for proacrosomal vesicle fusion.

In vitro evaluation of liquid-stored buffalo semen at 5°C diluted in soya lecithin based extender (Bioxcell®), tris-citric egg yolk, skim milk and egg yolk-citrate extenders.

This study was designed to compare the quality of liquid-stored buffalo bull spermatozoa in soya lecithin based extender Bioxcell(®) (BIOX), milk (MILK), tris-citric egg yolk (TEY) and egg yolk-citrate (EYC) extender at 5°C. Semen was collected from five Nili-Ravi buffalo (Bubalus bubalis) bulls of 6-7 years of age with artificial vagina over a period of 3 weeks (two consecutive ejaculates once in a week). Semen ejaculates having more than 60% motility were pooled, split into four aliquots, diluted (37°C; 10 × 10(6) motile spermatozoa/ml), cooled from 37 to 5°C in 2 h (0.275°C/min) and stored for 5 days. Sperm motility, viability, plasma membrane integrity (PMI) and normal acrosomal ridge were studied at first, third and fifth day of storage. Higher values of progressive sperm motility (%), sperm viability (%), sperm PMI (%) and normal apical ridge (%) were observed in BIOX, MILK and TEY extenders at first, third and fifth day of storage than EYC extender. Progressive sperm motility, sperm viability and sperm PMI in BIOX(®) extender were not different from MILK and TEY extenders at 1st and third day storage period. However, at fifth day of storage, the values for these parameters remained significantly higher (p<0.05) in BIOX(®) compared with MILK, TEY and EYC extenders. At fifth day of storage, the semen quality parameters for Bioxcell(®) were comparable to those with MILK and TEY extenders at third day of storage. In conclusion, motility, viability and PMI of buffalo bull spermatozoa remained similar in Bioxcell(®) , milk and TEY extender at first and third days of storage at 5°C. Yet, the values for the aforementioned parameters in Bioxcell(®) were higher compared with milk, TEY and EYC extender at fifth day of storage at 5°C.

The cryoprotective effects of vitamin B12 supplementation on bovine semen quality.

The present study aimed to investigate the effects of vitamin B(12) supplementation on standard bovine semen quality parameters and anti-oxidative enzyme activities. Vitamin B(12) was supplemented at concentrations of 1.25, 2.5, 3.75 and 5.0 mg/ml to bovine semen cryoprotective medium. The results indicated that the motility and straight line velocity, curvilinear velocity, mean coefficient, velocity of the average path values of sperm supplemented with 2.50 mg/ml vitamin B(12) were significantly higher than that of other groups (p<0.05). No significant difference was observed for linearity index, lateral head displacement values and the percentage of grade A spermatozoa between the extenders containing 2.50 and 3.75 mg/ml vitamin B(12) (p>0.05). The percentages of acrosome-intact and plasma membrane-intact spermatozoa were significantly improved (p<0.05) by supplementing with 2.50 mg/ml vitamin B(12) . The results of biochemical assay revealed that vitamin B(12) supplementation did not cause significant changes in superoxide dismutase levels compared with control (p>0.05). However, the catalase levels were higher in the treatment supplemented with vitamin B(12) at 2.50 mg/ml, when compared with other groups (p<0.05). The extender supplemented with vitamin B(12) significantly decreased glutathione peroxidase activity compared with the control (p<0.05). The supplementation of 3.75 mg/ml vitamin B(12) caused the highest value of glutathione reductase activity, compared with other groups (p<0.05). In conclusion, the extender supplemented with vitamin B(12) could reduce the oxidative stress provoked by freezing-thawing and improve bovine semen quality. Further studies are required to obtain more concrete results on the determination of lipid peroxidation and antioxidant capacities of vitamin B(12) in cryopreserved bovine semen.

Selenium in blood, semen, seminal plasma and spermatozoa of stallions and its relationship to sperm quality.

The essential trace element selenium is indispensable for male fertility in mammals. Until now, little data existed regarding the relationship between selenium and sperm quality in the stallion. Selenium, or selenium-dependent glutathione peroxidase activity, was determined in red blood cells, semen, seminal plasma and spermatozoa, and the percentages of spermatozoa with progressive motility (PMS), intact membranes (PMI), altered (positive) acrosomal status (PAS) and detectable DNA damage, determined by the sperm chromatin structure assay, were evaluated in 41 healthy stallions (three samples each). The pregnancy rate per oestrus cycle (PRC) served as an estimation of fertility. An adverse effect on stallion fertility caused by low dietary selenium intake was excluded, as all stallions had sufficient selenium levels in their blood. Interestingly, no significant correlations (P > 0.05) between the selenium level in blood and the selenium level in seminal plasma or spermatozoa were found, suggesting that the selenium level in blood is no indicator of an adequate selenium supply for spermatogenesis. The selenium level in spermatozoa (nmol billion(-1)) was correlated with PMI, PMS and PAS (r = 0.40, r = 0.31 and r = -0.42, respectively; P

Effect of alpha-tocopherol supplementation during boar semen cryopreservation on sperm characteristics and expression of apoptosis related genes.

Boar semen is extremely vulnerable to cold shock and sensitive to peroxidative damage due to high content of unsaturated fatty acids in the phospholipids of the plasma membrane and the relatively low antioxidant capacity of seminal plasma. The present study evaluated the influence of alpha-tocopherol supplementation at various concentrations in the boar semen extender during cryopreservation on post-thawed sperm motility characteristics (total sperm motility, MOT; local motility, LCM; curvilinear velocity, VCL; straight linear velocity, VSL; and average path velocity, VAP), sperm qualities (viability, acrosomal integrity and apoptosis), expression of stress protein (HSP70), and the expression of pro-apoptotic (Bax and Bak) and anti-apoptotic (Bcl-2l and Bcl-xl) genes. Semen collected from 10 Duroc boars was cryopreserved in lactose-egg yolk buffer supplemented with various concentrations of alpha-tocopherol (0, 100, 200, 400, 600 and 800 microM) using the straw-freezing procedure and stored at -196 degrees C for a minimum period of one month. In frozen-thawed groups, sperm motility was significantly (P<0.05) lower than that of fresh sperm. In fresh sperm, HSP70 immunoreactivity expression was observed in the equatorial region, but in frozen-thawed groups, expressions were mostly observed in the sperm head. Higher apoptosis rates were observed in 600 and 800 microM alpha-tocopherol supplemented frozen-thawed groups. In alpha-tocopherol supplemented frozen-thawed groups immediately after thawing, the expression was similar to that of fresh group. But after incubation at 37 degrees C for 3h, the expression in 200 and 800 microM alpha-tocopherol supplemented groups was higher than that of others. Expression of pro-apoptotic genes was significantly higher and anti-apoptotic genes was significantly (P<0.01) lower in alpha-tocopherol supplemented frozen-thawed groups compared to fresh sperm group. In conclusion, alpha-tocopherol, supplemented at 200 microM concentration in boar semen extender during cryopreservation had a positive effect on post-thawed sperm survivability.

Morphological changes induced by Caesalpinia bonducella seed extract on rat sperm: scanning electron microscope study.

Morphological changes in the sperm of albino rats observed under scanning electron microscope illustrate the disturbance in the plasma membrane, as well as in the acrosomal membrane upon treatment with graded doses of an alcoholic seed extract of Caesalpinia bonducella. Considerable changes in the shape and size of the sperm head were observed, with the middle region of the sperm head being slightly constricted dorsoventrally. Most sperm appeared morphologically abnormal in the head region showing the distortion at the anterior region and bulging of the acrosomal membrane when compared with the control. The results of this study suggest that such effects might have resulted from general disturbance in proteins and alteration in the cauda epididymal milieu, probably due to an androgen deficiency consequent to the treatment with Caesalpinia bonducella.

Refrigerated storage of red deer epididymal spermatozoa in the epididymis, diluted and with vitamin C supplementation.

We have approached the problem of refrigerated storage of epididymal sperm samples from red deer by comparing three options: storing the genital (testicles within the scrotum), diluting the semen in extender or diluting the semen in extender supplemented with an anti-oxidant. Twenty-nine pairs of testes were collected. Spermatozoa from one of each of the pairs were immediately recovered, and diluted to 400 x 10(6) sperm/ml in Tris-citrate-fructose with 20% egg yolk. Control group was stored as such, and Anti-oxidant group was supplemented with 0.8 mm vitamin C. The remaining epididymides and the diluted samples were stored at 5 degrees C and spermatozoa were analysed at 0, 24, 96 and 192 h for: motility [computer-assisted semen analysis (CASA)], acrosomal integrity, sperm viability (eosine/nigrosine staining), normal tails and chromatin status [sperm chromatin structure assay (SCSA)]. In general, seminal quality decreased with storage time. Vitamin C supported progressive motility better at 24 h (median 42% vs 23% Control and 15% epididymis), reduced the incidence of tail abnormalities and protected chromatin. Storing the semen in the epididymis slowed down motility loss, but slightly increased the occurrence of tail abnormalities and viability was lower at 192 h. However, regarding chromatin status, sperm stored in the epididymis was protected similarly to those diluted in the medium supplemented with vitamin C. Although the differences between the three groups were small, there were some advantages in supplementing the extender with vitamin C. Besides, refrigerating the epididymis may be a good option when immediate processing is not available.

Rhodiola sacra aqueous extract (RSAE) improves biochemical and sperm characteristics in cryopreserved boar semen.

Although Rhodiola sacra aqueous extract (RSAE) has been used in many studies as an antioxidant, its effects on semen characteristics and its antioxidant properties during cryopreservation of boar sperm have never been evaluated. Semen was collected from five Duroc boars (2-4-year-old) twice weekly and frozen-thawed in extender with RSEA. Motion characteristics were assessed with a computer-aided semen analysis (CASA) system, whereas other sperm quality end points were assessed by routine methods. The effective concentration of RSEA in extender ranged from 4 to 8mg/L and the effect of RSEA on sperm quality was better in glycerol-free extender than extender containing glycerol (P<0.05). In frozen-thawed boar semen, there was a direct correlation (P<0.05) between RSEA concentration and glutathione (GSH) concentrations, mitochondrial activity, and hypoosmotic swelling test (HOST), and an inverse correlation (r=-0.982, P<0.05) between RSEA concentration and malondialdehyde (all end points were significantly higher at 6mg/L than in the control group). In summary: (i) the effective concentration of RSEA in extender ranged from 4 to 8mg/L; (ii) the effect of RSEA on sperm quality was better in extender without glycerol; and (iii) there was a significant correlation between RSEA concentrations and concentrations of GSH and MAD in frozen-thawed boar semen (antioxidant effects of RSEA were concentration-dependent). Further studies are needed to define the active ingredient in RSEA that protects boar sperm against ROS.

Effect of antioxidant supplementation in semen extenders on semen quality and reactive oxygen species of chilled canine spermatozoa.

The objective of this study was to evaluate quality of chilled dog semen processed with extenders containing various antioxidants. Single ejaculates from five dogs were always pooled and evaluated for concentration, sperm motility, progressive motility (RSF-movement), viability, acrosomal integrity and by the hypo-osmotic swelling (HOS)-test. Also, superoxide (O(2)(-)) production, hydroxyl radicals (OH) and total reactive oxygen species (tROS) were determined. Pooled semen was divided in seven aliquots (for control and test conditions), which were diluted to a final concentration of 67x10(6)spermatozoa/ml with TRIS-glucose-egg yolk extender with or without the following supplements: control (without antioxidants), vitamin C (0.5mM), N-acetyl-l-cysteine (NAC; 0.5mM), taurine (0.2mM), catalase (100u/ml), vitamin E (0.1mM) and 5-(4-dimethylamino-phenyl)-2-phenyl-penta-2,4-dienoic acid (B16; 0.1mM). The semen aliquots were chilled and preserved at 4 degrees C. Portions of chilled semen were removed at 24 and 72h, and semen quality was evaluated after rewarming. At 24h the mean (+/-S.E.M.) sperm motility was higher (p<0.001) when vitamin E, taurine and B16 were added in the extender, whereas more spermatozoa with RSF-movement were observed (p<0.001) in the vitamin E, catalase, B16 and taurine groups. Sperm viability was higher (p=0.040) in B16 and vitamin E groups and the percentage of swollen spermatozoa was higher (p=0.002) only in the B16 group. Acrosomal integrity and OH were not significantly influenced by any of the antioxidants tested. Superoxide production was significantly lower when vitamin C, B16 and vitamin E were added in semen extenders compared with the control (p=0.017). All antioxidant groups, except vitamin C and NAC, contained less tROS compared to the control group, but only the B16 group value differed significantly (p=0.05). At 72h sperm motility was higher (p<0.001) when vitamin E, catalase, B16, taurine and NAC were added in the extender. More spermatozoa with RSF-movement were observed (p<0.001) in the vitamin E, catalase, B16, taurine and NAC treatment groups. Sperm viability was higher (p=0.001) when vitamin E, B16, taurine and vitamin C were added in semen extenders. HOS-test percentages were higher (p=0.016) in the B16, vitamin E, catalase and NAC groups. Acrosomal integrity was not influenced in any case. Production of O(2)(-) was significantly higher using catalase compared to all the other groups (p=0.006), while OH was not significantly influenced by any of the antioxidants tested. The addition of vitamin E, catalase and B16 in semen extenders resulted in significantly lower tROS values compared with the controls (p<0.0005). The results suggest that vitamin E and B16 had the most pronounced effect in preserving semen quality of chilled dog spermatozoa.

[Protection of extract from Cuscuta japonica on human sperm acrosome and ultrastructure].

OBJECTIVE: To study the oxidation damage of active oxygen (ROS) to human sperm acrosome and ultrastructure, and study the function mechanism about Cuscuta japonica treating male's infertility and asthenoospermia. METHOD: By using the Percoll gradient centrifugation, the sperm with normal physiological function were selected for the normal sperm model, and the sperm suspension were divided into the normal group, the model group, the positive control group (Vitamin C group), and the lugh, the median and the low dose gvoups of C. japonica. The ROS made from hypoxanthine-xanzine xanzine(HX-XO) and different content (0.125, 0.25, 0.5 g x mL(-1)) of extract were incubated with sperm in the oxygen environment. The acrosomic integrity rate were calculated and the sperm acrosome and ultrastructure were observed. RESULT: The content (0.125, 0.5 g x mL(-1)) of extract had no obvious difference as compared with Vitamin C (0.25 mg x mL(-1)) in protecting the acrosome and ultrastructure, but the content (0.25 mg x mL(-1)) of extract was significantly better than Vit C (P < 0.001). CONCLUSION: The suitable content of extract from C. japonica can significantly protect the sperm membrane, the acosomic structure and the mitochondrion function from the damage caused by ROS.

Effect of graded doses of Momordica charantia seed extract on rat sperm: scanning electron microscope study.

Morphological changes in sperm of albino rats observed under scanning electron microscopy illustrate the disturbance in the plasma membrane as well as in the acrosomal membrane on treatment with effect of graded doses of alcohol seed extract from Momordica charantia. Considerable changes in the shape and size of the sperm head were observed, with the middle region of the sperm head being slightly constricted dorsoventrally. Most sperm appeared morphologically abnormal in the mid-region of the tail, with formation of a balloon-like cytoplasmic droplet. The results of this study suggest that such effects may have resulted from a general disturbance in proteins and an alteration in the cauda epididymal milieu, probably due to androgen deficiency consequent to the anti-androgenic property of Momordica charantia seeds.

Effects of p-nonylphenol and resveratrol on body and organ weight and in vivo fertility of outbred CD-1 mice.

The aim of this study was to analyse the multigenerational effects of para-nonylphenol (NP) and resveratrol (RES) on the body weight, organ weight and reproductive fitness of outbred CD-1 mice. The data indicate that in male mice, NP had an effect on the weight of selected reproductive organs and the kidneys in the parental (P) generation males. Effects on selected reproductive organs, the liver and kidneys in the F1-generation males were also seen. In females, effects of NP on body weight and kidney weight were seen in the P generation, but no effects on any measured parameter were seen in the F1 generation. RES had no effect on body weight but did have some effect on selected male and female reproductive organs in the P generation. RES altered the spleen and liver weights of P-generation males and the kidney weight of F1-generation males. Acrosomal integrity (using a monoclonal antibody against intra-acrosomal sperm proteins) was assessed for both generations of NP- and RES-treated mice. A significant reduction in acrosomal integrity was seen in both generations of NP-treated, but not in RES-treated, mice. Fewer offspring were observed in the second litter of the F2 generation of mice treated with NP; no similar effect was seen in RES-treated mice. The litter sex ratio was not different from controls. Unlike RES, NP had a negative effect on spermatogenesis and sperm quality with a resultant impact on in vivo fertility.

Superoxide dismutase affects the viability of thawed European mouflon (Ovis g. musimon) semen and the heterologous fertilization using both IVF and intracytoplasmatic sperm injection.

This study evaluated the effects of superoxide dismutase (SOD) on viability and acrosome integrity of European mouflon spermatozoa after cryopreservation and on the fertilization rates of sheep oocytes after i.v.f. or intracytoplasmatic sperm injection (i.c.s.i.). Frozen semen was thawed and washed with synthetic oviduct fluid supplemented with 0.6% bovine serum albumin. After centrifugation, the spermatozoa pellet was split into two culture systems: (i) without SOD; and (ii) in the presence of 1500 IU mL(-1) SOD. Sperm viability and acrosome integrity were evaluated simultaneously, immediately after thawing and after 3, 6 and 9 h of culture (5% CO2, 39 degrees C, 90% humidity), by incubating sperm with propidium iodide and fluorescein isothiocyanate-labelled Pisum sativum agglutinin. At the same time, sperm were assessed for motility using a standard scoring system (independent operators' observation of sperm) that graded degree of motility (i.e. 1 = immotile to 10 = maximum motility, as observed at the moment of thawing). For i.v.f., frozen-thawed semen derived from the two culture systems was placed in culture together with in vitro-matured sheep oocytes. For i.c.s.i., semen derived from the same culture systems as that for i.v.f. was used, and incubated for 1 h under standard conditions. The results showed a marked difference (P < 0.01) between the percentages of live spermatozoa in medium with SOD and those obtained in medium alone, after 3, 6 and 9 h of culture. The percentages of intact acrosome spermatozoa were higher in medium with SOD after 6 h (P = 0.05) of culture. Spermatozoa motility decreased significantly in SOD containing medium at 3 and 6 h of culture compared with motility in control medium. Fertilization rates were significantly lower in medium with SOD than in medium alone, whereas in the i.c.s.i. system fertilization rates were significantly higher in the presence of SOD. The results indicate that the addition of SOD to the culture media enhances the viability rates and the acrosome integrity of cryopreserved mouflon spermatozoa.

Insulin-dependent diabetes in men is associated with hypothalamo-pituitary derangement and with impairment in semen quality.

BACKGROUND: The objective of the study was to investigate the hypothalamo-pituitary-testicular axis and sperm structure at the transmission electron microscope (TEM) level in men affected by insulin-dependent diabetes. METHODS: Twenty-two diabetic men and 24 controls were recruited. GnRH (100 micro g) was administered and FSH- and LH-induced secretion was evaluated. Semen samples were collected and sperm concentration and motility were determined using a Makler chamber. Ejaculated sperm were fixed and observed with a TEM. RESULTS: The response of gonadotrophins to GnRH was significantly lower in diabetics than in control men. Sperm motility was also significantly lower. At the electron microscope level, sperm from diabetics exhibited a higher percentage of immaturity- and apoptosis-related defects than sperm from controls. CONCLUSIONS: The reduced response of gonadotrophins to GnRH in diabetic men may indicate a decreased acute releasable pool of pituitary gonadotrophins. The results of TEM examination showed that sperm from men with diabetes presented severe structural defects in comparison with sperm from controls. It is possible that the reproductive impairment recognized in men with diabetes could be the result of interference by the disease on the hypothalamo-pituitary-testicular axis at multiple levels, as indicated by the reduced gonadotrophin response to appropriate stimuli and by the abnormal ultrastructure of ejaculated sperm. The defective spermatogenesis may be the consequence of a direct testicular effect of the disease.

Effects of a purified fraction from Echeveria gibbiflora aqueous crude extract on guinea-pig spermatozoa.

Guinea-pig spermatozoa in the presence of a purified fraction from Echeveria gibbiflora aqueous crude extract suffer a hypotonic-like effect. The phenomena exhibited included a distension of the plasma membrane over the acrosome region, inducing the formation of a huge 'head-bubble'. The agglutination effect was so enhanced that instead of inducing sperm clusters, it produced cane-like 'stalk' structures. The immobilizing activity was induced instantaneously after the addition of the purified fraction. At electron microscope level it was possible to observe a heavy amount of electron dense material of the purified fraction embedded or intercalated along the plasma membrane. It was also possible to corroborate the dispersion of the acrosomal content and the disappearance of the external acrosome membrane. The purified fraction induced loosening of the plasma membrane all along the sperm cell, however, the distension of the membrane was only produced in the apical portion of the sperm head and not in the post equatorial region. The results suggest that the plant may yield a compound suitable for use as a vaginal barrier or male contraceptive agent.

Effect of Maytenus ilicifolia Mart.ex. Reiss on spermatogenesis.

The effect of the ethanolic extract of Maytenus ilicifolia Mart.ex. Reiss leaves on spermatogenesis was studied in Swiss mice by evaluating morphological characteristics by light and electron microscopy. The extract was administered at a dose of 200 mg/kg/day intraperitoneally for 20 days, and at a dose of 800 mg/kg/day orally for 30 days. Structural analysis of the germ epithelium showed that treated animals were not noticeably different from control animals. The alterations included some exfoliated immature germ cells, occasional germ cell death (recognized as pyknotic nuclei) and a few vacuolized seminiferous tubules. Ultrastructurally, enlarged lipid droplets were found in Sertoli cells and swollen acrosomes occurred in early spermatids of animals treated with the higher dose. Sperm production indicated that the ethanolic extract of M. ilicifolia leaves did not contain substances sufficient to arrest spermatogenesis.

PIP: Several plant extracts used to regulate female fertility have proved effective for the male reproductive system as well. Maytenus ilicifolia Mart.ex. Reiss, a plant native to parts of South America, has been used as a contraceptive, abortifacient, and emmenagogue by women in Paraguay and Argentina. The present study evaluated the effect of the ethanolic extract of Maytenus ilicifolia Mart.ex. Reiss leaves on spermatogenesis in Swiss mice. The extract was administered at doses of 200 mg/kg/day intraperitoneally for 20 days and 800 mg/kg/day orally for 30 days. Light microscopy revealed apparently normal spermatogenesis in the seminiferous tubules of treated animals. Although the spermatogenic process was not altered, ultrastructural alterations were observed, including some exfoliated immature germ cells, occasional germ cell death, and a few vacuolized seminiferous tubules. Enlarged lipid droplets were found in Sertoli cells and swollen acrosomes occurred in early spermatids of mice treated with the higher dose. However, sperm production indicated that the ethanolic extract did not contain substances sufficient to arrest spermatogenesis. Thus, the indigenous use of Maytenus ilicifolia as a medicinal plant probably does not cause a disturbance of spermatogenesis as a side effect.