Cauda Epididymidis Research Articles

Prolonged deleterious effects of neonatal handling on reproductive parameters of pubertal male rats

The purpose of the present study was to investigate the long-lasting effects of neonatal handling on reproductive parameters of male rats. Neonatal handling (pups separated from their mothers, kept isolated at environmental temperature for 20 min and submitted to 1 min of tactile stimulation) was applied from post partum Days 1 to 14 (a stress-hyporesponsive period, SHRP) and the animals were killed at puberty (61 days of age). The number of mature spermatids and the daily sperm production were estimated in homogenates from the right testes and cauda epididymidis. Histometric parameters (diameter of seminiferous tubule, germinal epithelium thickness and number of Sertoli cells) were evaluated in paraplast sections of the left testes. The association of the slightly aversive stimuli applied during the SHRP proved to have lasting deleterious effects on male reproduction, causing lower testicular weight and reduced values of seminiferous tubule diameter and germinal epithelium thickness at puberty, which resulted in a 25% reduction in the daily sperm production and in the number of mature spermatids. Similarly, the number of Sertoli cells per tubular cross section was 20% smaller and the weight and number of spermatozoa were reduced more than 40% in the cauda epididymidis of animals handled.

15 EFFECT OF BULL EPIDIDYMIS STORAGE CONDITIONS ON CRYOPRESERVED EPIDIDYMAL SPERM IN VITRO FERTILITY AND LIPID PEROXIDATION STATUS

Although cryopreservation of epididymal sperm has been studied extensively in several species, some factors that could negatively influence its quality are still unknown, such as the storage conditions of the epididymides prior to sperm collection. Studies indicate that the lower the storage temperature, the better the sperm quality after collection (Kaabi et al. 2003 Theriogenology 60, 1249–1259). An additional factor is lipid peroxidation in which sperm membrane resistance against reactive oxygen species (ROS) attacks is an important component. The objective of this experiment was to study whether the epididymis storage temperature following slaughter, as well as the intrinsic resistance against oxidative stress, affects the fertilizing capacity of cryopreserved epididymal bull sperm in vitro. Twelve epididymides (6 bulls) were collected after slaughter and divided into 2 groups, (stored at either 4 or 37�C for 2), after which semen was collected from the caudae epididymides and cryopreserved. Subsequently, one aliquot of the frozen–thawed semen samples was subjected to induced lipid peroxidation with ferrous sulfate and ascorbate (37�C; 2 h), after which tiobarbituric acid-reactive substances (TBARS), as an index of lipid peroxidation, were measured according to a method previously described (Beorlegui et al. 1997 Andrologia 29, 37–42). A second aliquot was used for in vitro fertilization in a routine IVF–IVC setup in duplicate (24-h maturation, SOF culture medium in 5% CO2, 5% O2, and 90% N2). In vitro embryo production results at Day 7 and TBARS levels were statistically analyzed using SAS (SAS Institute, Inc., Cary, NC, USA). No influence of storage temperature was observed at either TBARS level (4�C: 943.6 � 173.4; 37�C: 751.4 � 136.2 ng of TBARS/108 spermatozoa; P = 0.3) or on blastocyst rates (4�C: 23.0 � 2.8; 37�C: 18.7 � 3.6% of blastocysts; P = 0.2). However, the percentage of hatched blastocysts tend to be higher for epididymides stored at 4�C when compared to those stored at 37�C (6.4 � 1.8 and 2.3 � 0.9, respectively; P = 0.06). In addition, a negative correlation was found between TBARS concentrations and blastocyst rates (R = –0.57; P < 0.05). Compared to fresh samples collected from epididymides under the same conditions (unpublished data), levels of TBARS were two- to threefold higher for the cryopreserved sperm, indicating that lipid peroxidation appears to play a role in the decrease in quality of cryopreserved epididymal sperm. In conclusion, temperature during the epididymides short-term storage prior to sperm cryopreservation did not seem to influence the sperms' in vitro fertilizing capacity. On the other hand, an alternative to improve cryopreserved epididymal sperm in vitro fertility (or fertilizing capacity) could be the addition of antioxidants to semen extenders. Further studies are necessary to confirm this hypothesis.

Inhibitory Effect of Ethylene Glycol Monoethyl Ether on Rat Sperm Motion

Ethylene glycol monoethyl ether (EGEE) is known to have testicular toxicity. To elucidate whether EGEE has any effect on sperm motion, especially in the case of short time exposure, we conducted a series of in vivo experiments with rats, as well as an in vitro study with rat sperm. Sperm from cauda epididymides and spermaducts was analyzed for the change in motion with a Hamilton-Thorne Sperm analyzer. Administration of EGEE at 600 mg/kg/d for five weeks significantly decreased total and progressive motility of sperm to 15-30% of controls, in both the cauda epididymis and the spermaduct. The time-course experiment using a single dose of 1,000 mg/kg showed that damage to sperm motion was evident at 12-24 h after EGEE administration. Addition of EGEE to the medium of sperm had no effect on its motion, but the metabolite ethoxyacetic acid showed a significant inhibitory effect. These results suggest that besides its toxicity to spermatogenesis, the metabolite of EGEE may also directly affect the motion of mature sperm.

17 WHITETAIL DEER OFFSPRING FROM ARTIFICIAL INSEMINATION WITH EPIDIDYMAL SPERM HARVESTED FROM A HUNTER-KILLED BUCK

The ability to cryopreserve epididymal sperm from mature postmortem bucks has long been of interest to both wildlife conservationists and deer ranchers. At present, there is loss of valuable genetics from the hunter harvest of trophy males. Increasing adult body weight and antler size of adult males would be of substantial economic value to the deer hunting industry. In this preliminary trial, 6 yearling pen-raised Whitetail does (47 to 58 kg), in good body condition, were isolated from all bucks prior to the onset of the fall breeding season. Females were synchronized for AI with a 14-day caprine CIDR and 200 IU of eCG (IM) at the time of CIDR removal. Does were timed AI 60 to 63 h after eCG with one 0.5-mL straw of frozen–thawed Whitetail sperm. All sperm used for AI were harvested from a single mature Whitetail buck that was hunter harvested during the previous hunting season. Within 3 h after death, the testes with scrotum were removed, enclosed in a plastic Ziploc bag, and then placed in a Styrofoam ice chest containing frozen cold packs. The ice chest was transported to the laboratory where sperm were extracted at 4�C in the late evening (<12 h postmortem) by flushing the cauda epididymides with Triladyl� one-step extender (Minitube, Verona, WI, USA) in a retrograde flow from a small incision made in the cauda. The sperm–Triladyl mixture was flushed from the cauda incision into a sterile 50-mL tube using a 10-mL plastic syringe modified by heating and then stretching the tip until small enough to thread into the vas deferens. The sperm plus extender was then held at 4 to 10�C for 12 h and frozen at a concentration of <50 million/straw using a commercial bull freezing protocol (Genex Custom Collection Center, Baton Rouge, LA, USA). A random sample of straws was then thawed, resulting in an overall post-thaw motility of 60%. The remaining straws were left frozen in liquid nitrogen until the next breeding season. On the first of December, does (n = 6) were given 0.1 mL of Domosedan� (Pfizer Animal Health, Groton, CT, USA) IV and inseminated transcervically using a modified caprine speculum. All does were handled in a custom-built deer barn, and AI was performed by one technician in a drop-bottom deer chute (Deer Handler; Delclayna, Alberta, Canada). At 48 days after AI, 3 of the 6 does (50%) were diagnosed pregnant by transrectal ultrasonography. All pregnant females gave birth, producing 5 offspring (1 male and 1 female singletons and a set of mixed sex triplets) that ranged from 1.9 to 4.3 kg and had a mean gestation length of 196 days (range = 190–203 days). In summary, results indicate that live offspring can be produced from epididymal sperm harvested from mature hunter-harvested Whitetail bucks. Further experiments are needed to optimize techniques and protocols for the harvesting and usage of these gametes.

217 QUALITY CHANGES IN POST-THAW SPRINGBOK (ANTIDORCAS MARSUPIALIS) EPIDIDYMAL SPERM MAINTAINED IN FERTILIZATION MEDIUM

The availability of gametes from the cropping of excess wildlife species provides the opportunity for the advancement of knowledge into assisted reproductive technology for possible future conservation measures. Little is known about the longevity of springbok (Antidorcas marsupialis) spermatozoa maintained in fertilization medium. The aim of this project was to determine the quality changes of post-thawed springbok spermatozoa incubated in fertilization medium by measuring plasma membrane integrity over time. Testes (n = 12) were obtained from two geographically distinct free-ranging springbok populations in South Africa. Spermatozoa were flushed from the cauda epididymides within three hours of the animals' death. Samples from an individual male were pooled, diluted to 400 × 106 sperm/mL with Biladyl (Minitüb, Tiefenbach, Germany) fraction A (no glycerol) and equilibrated in a water bath for 6 h at 4°C. An equal volume of Biladyl fraction B (containing 12% glycerol) was added to the sample to make a final concentration of 200 × 106 sperm/mL. Samples were loaded into 0.25-mL straws and frozen in liquid nitrogen vapor (5 cm above the liquid nitrogen level) for 20 min after which they were plunged into liquid nitrogen. Straws from each sample were thawed for 20 s at 36°C in a water bath. Thawed spermatozoa (100 μL) was added to 1 mL IVF-TALP medium containing heparin and PHE (Vajta et al. 1996 Theriogenology 45, 683–689) in 2-mL Nunc tubes (AEC, Amersham, South Africa) and incubated at 38.7°C, in humidified 5% CO2 balance air for 30 h. Aliquots were extracted from the incubating spermatozoa to determine plasma membrane integrity at 6-h intervals. Propidium iodide (Sigma, South Africa) at 50 ng/mL (10 min at RT) was used to evaluate membrane integrity under fluorescence microscopy at ×400, with a 450-nm excitation filter, a 510-nm dicroic beam splitter, and a 520-nm barrier filter. Cells with damaged plasma membrane have nuclei that fluorescence red. Eosin/nigrosin was also used to evaluate membrane integrity under ×400 bright-field microscopy. Cells with damaged plasma membrane stain purple-red, whereas the balance of cells remain translucent. The average post-thaw motility of spermatozoa in populations A and B was 69% (n = 6) and 68% (n = 6), respectively. Plasma membrane integrity of post-thawed springbok spermatozoa deceased steadily in IVF-TALP medium over the 30-h period (Table 1). Cryopreserved epididymal sperm derived from free-ranging springbok populations survive in IVF-TALP media and may be useful in future conservation activities where an isolated gene pool requires genetic supplementation through one or more assisted reproduction techniques such as IVF or AI. Further research is required to confirm and extend these findings. Table 1. Percentage plasma membrane integrity of post-thawed springbok sperm over time

95 EFFECT OF CRYOPROTECTANT ADDITION AND REMOVAL AT 4°C ON MOTILITY AND PLASMA MEMBRANE INTEGRITY OF BOVINE CAUDAL EPDIDYMAL SPERMATOZOA

Cryopreservation of spermatozoa harvested from the epididymides would be a means of salvaging germplasm from genetically valuable males that die unexpectedly from injury, disease, or poaching. It is well known that the addition of cryoprotective agents (CPAs) is essential for sperm survival following the freezing process. However, CPAs can cause loss in sperm viability due to osmotic damage or chemical toxicity. The objective of this study was to determine the effects of single-step addition and/or removal of glycerol (GLY) or ethylene glycol (EG) on motility and plasma membrane integrity of bovine epididymal spermatozoa. Paired testes were obtained from mature bulls (n = 10) at a local abattoir and transported to the laboratory at 25–28°C within 4–6 h post-mortem. Epididymal spermatozoa were harvested by multiple incisions from the caudae epididymides of each bull, pooled, and washed in Brackett-Oliphant medium by centrifugation for 5 min at 500g. Pellets were resuspended in egg yolk-Tris-glucose-citric acid monohydrate medium (EYT-GC) at a concentration of 120 × 106 cells/mL and cooled to 4°C at a rate of 0.1°C/min. Specimens were allocated to each of five treatment groups: control (no CPA), 7% GLY, and 14% GLY, 7% EG, 14% EG. Then, replicate samples were diluted 1:1 in EYT-GC medium containing twice the final desired concentration of CPA. After being exposed for 10 min, each sample was diluted directly into EYT-GC at 4°C. Motility was assessed by means of a computer assisted semen analysis system and plasma membrane integrity was determined by SYBR 14 and propidium iodide staining followed by fluorescence microscopy. Differences among treatments were analyzed using one way ANOVA (P < 0.05). The results (Table 1) show that maximum survival, as assessed by measurements of motility and membrane integrity, was achieved with spermatozoa exposed to 7% EG. Almost identical intermediate levels of survival were observed with spermatozoa exposed to 7% GLY or 14% EG. The lowest survival was observed for spermatozoa exposed to 14% GLY. The results indicate that the use of EG as a cryoprotectant may minimize toxicity and osmotic damage to fresh bovine epididymal spermatozoa. Its efficacy as a CPA is currently being determined. Table 1. Sperm motility and membrane integrity (mean ± SEM) after addition of CPA to epididymal sperm

Superoxide dismutase, catalase, and glutathione peroxidase during epididymal maturation and prolonged storage of spermatozoa in the Mexican big-eared bat (Corynorhinus mexicanus)

We studied the activities of reactive oxygen species (ROS) scavenging enzymes during epididymal spermatozoon maturation and storage in Corynorhinus mexicanus (G.M. Allen, 1916), a vespertilionid bat that stores spermatozoa in the epididymides for several months after regression of the testes. Depending on the phase of the epididymal reproductive cycle, two different patterns of antioxidant enzyme activities were observed in C. mexicanus. Catalase activity is clearly present in both caput and cauda epididymides throughout the entire annual reproductive cycle, being particularly high during the post-testicular phase of epididymal function. Superoxide dismutase (SOD) activity, present during the testicular phase of epididymal transport and maturation of spermatozoa, is almost completely absent or inhibited in both epididymal segments during the post-testicular epididymal storage period. GPx activity is low during the testicular phase of epididymal spermatozoon maturation and is high in both epididymal segments during the storage phase of epididymal function. From our results, we postulate that (i) the pattern of epididymal antioxidant enzyme activities in C. mexicanus is significantly different from the pattern that is proposed to be unique for mammals; (ii) epididymal function in these species of bats can be clearly divided into two phases, a testicular-dependent phase that is related to the spermatozoon maturation function of the epididymides and a testicular-independent phase that is related to the long-term spermatozoon storage function observed in these mammals; (iii) the study of the regulation of the redox potential of the microenvironment, associated with mammalian spermatozoa as they transit through the epididymides, must be particularly focused on the anatomical region where ROS generation scavenging and spermatozoon maturation storage processes take place.

Changes in spermatozoal chromatin packaging and susceptibility to oxidative challenge during aging

Our goal was to test the hypothesis that spermatozoal chromatin packaging changes with age and that aging affects the susceptibility of spermatozoal DNA to oxidative damage. Laboratory study. Academic facility. Young (4 months) and old (21 months) Brown Norway rats. Spermatozoa were collected from the cauda epididymidis and were incubated in saline or H2O2. Thiols levels, chromatin condensation, DNA susceptibility to acid-induced DNA denaturation, and DNA damage were evaluated using monobromobimane, chromomycin A3 (CMA3), acridine orange, and polymerase chain reaction, respectively. Spermatozoa from old rats had 25% fewer disulfides but similar levels of free thiols as compared with young. The CMA3 staining was decreased by 13% with age. Levels of chromatin denaturation and DNA damage were similar in control groups. After exposure to oxidant, free thiols became oxidized by about 20% irrespective of age, but CMA3 staining changed little. The acridine orange assay, however, showed a trend for greater chromatin denaturation in spermatozoa from old rats after oxidant treatment. Furthermore, the DNA from spermatozoa of old rats was significantly more susceptible to developing DNA breaks and modification after oxidative challenge. Spermatozoal chromatin packaging changes with aging and vulnerability to oxidative damage increases.

A comprehensive evaluation of the testicular toxicity of dichlorvos in Wistar rats

Assessments of the reproductive toxicity of organophosphorus insecticides are important public health issues. This study aimed at defining the testicular toxicity of dichlorvos (DDVP) since this toxicity was suspected by our previous survey on pesticide sprayers and in some earlier publications during the 1970s. Ten-week-old Wistar rats were divided into four groups ( n = 8 or 9) and were injected subcutaneously with DDVP (0, 1, 2 or 4 mg/kg) 6 days a week for 9 weeks. After that period, erythrocyte cholinesterase (ChE) activities decreased dose-dependently, showing 44–55% inhibition among the treated groups. No significant difference was observed in the reproductive organ weights in any treated groups compared with the control group. Sperm motility decreased slightly but significantly in the 1 and 4 mg/kg groups, and significant regressions were observed between sperm motility and both blood ChE activity and urinary concentration of dimethyl phosphate (DMP), a urine metabolite of DDVP. However, sperm counts and sperm morphology in the cauda epididymidis, plasma testosterone concentrations, and histopathology in the testes in all the treated groups were not significantly different from those of the control group. Since only the sperm motility deteriorated by DDVP exposure at doses inducing marked inhibition of cholinesterase activities in the rats, it was suggested that the risk of testicular dysfunction posed to occupationally exposed humans would be small in terms of the effect of DDVP exposure alone. This conclusion was also supported by an estimate of the decrease in human sperm motility based on the urinary DMP concentrations observed in actual occupational settings.

Effect of isolated fractions of Barleria prionitis root methanolic extract on reproductive function of male rats: preliminary study

Male rats treated with 100 mg/kg for 60 days of isolated fractions of the Barleria prionitis root methanolic extract (Fr. I and Fr. II) showed a significant reduction on spermatogenesis without affecting general body metabolism. Sperm motility as well density in cauda epididymides was reduced significantly. The fertility was decreased by 33.4% in Fr. I and 100% in Fr. II treated rats. The blood parameters were within the normal range. Total protein, glycogen and sialic acid contents of testes were reduced after the plant fractions treatment. Seminal vesicular fructose was decreased significantly after the treatment. The population of various spermatogenic cells such as primary spermatocytes, secondary spermatocytes and round spermatids were declined significantly in Fr. II treatment groups whereas in Fr. I treated animals preleptotene spermatocyte and spermatid number was decreased. There was no significant change in the number of Sertoli cells and spermatogonia in any of the treatment group.

Role of exosomes in sperm maturation during the transit along the male reproductive tract

Even tough differentiated spermatozoa are unable of transcriptional or translational activity; the sperm surface undergoes major modifications in macromolecules composition during the transit along the male reproductive tract. This is the result of sequential, well orchestrated interactions between the male reproductive tract secretions and the transiting male gamete. This is particularly true when spermatozoa transit along the epididymis. The epididymis is a long convoluted tubules in which the spermatozoa leaving the testis have to transit. The unraveled epididymal tubule can be as long as 80 m in stallion, and the transit time of spermatozoa is of 3–12 days depending on the species. The epididymis is usually divided in three segments: the caput (proximal part), the corpus, and cauda. While the cauda epididymides acts as a sperm reservoir, the caput and corpus are responsible for sperm maturation. This means that, under androgen control, the epididymal epithelium secretes proteins that will interact sequentially with sperm surface. Some of the sperm proteins acquired during maturation along the excurrent duct behave as integral membrane proteins. In fact, some epididymal originating proteins are glycosylphosphatidylinositol (GPI)-anchored to the sperm plasma membrane. Our laboratory has shown that some of these proteins are secreted in an apocrine manner by the epididymal epithelium and are associated to exosomes, called epididymosomes. Epididymosomes are rich in sphingomyelin and are characterized by a high cholesterol/phospholipids ratio. Many proteins are associated to epididymosomes, some of which are selectively transferred to spermatozoa during the epididymal transit. We have identified some of these exosomes associated proteins transferred to the maturing spermatozoa. These include two enzymes involved in the polyol pathway: an aldose reductase and a sorbitol dehydrogenase. A cytokine named MIF (macrophage migration inhibitory factor) is another protein associated to exosomes who is transferred to spermatozoa during the epididymal transit. We hypothesized that both the polyol pathway and MIF secreted in an apocrine fashion by the epididymal epithelium modulate sperm motility during the transit along the male reproductive tract. Finally, P25b, belonging to a family of sperm surface proteins (P26h/P34H) necessary for the binding to the surface of the egg, is also acquired through the interaction between epididymosomes and the male gamete. In vitro studies have defined the conditions of protein transfer when epididymal spermatozoa are co-incubated with epididymosomes. The transfer of selected proteins to specific membrane domains of spermatozoa is saturable, temperature and pH-dependent, being optimal at pH 6.5. The presence of zinc in the incubation medium, but not of calcium neither magnesium, significantly increases the efficiency of protein transfer. These results show that exosomes play a role in sperm epididymal maturation which is an essential event to produce male gametes with optimal fertilizing ability.

Transport of IgG across the Blood-Luminal Barrier of the Male Reproductive Tract of the Rat and the Effect of Estradiol Administration on Reabsorption of Fluid and IgG by the Epididymal Ducts1

In rats immunized systemically with tetanus toxoid the concentration of specific anti-tetanus-toxoid-specific IgG in fluid from the rete testis and cauda epididymidis were respectively 0.6% and 1.4% the concentration in blood serum. The extratesticular duct system reabsorbed 97% of the IgG and 99% of the fluid leaving the rete, but estradiol administration affected the site of reabsorption. In untreated rats, the ductuli efferentes reabsorbed 94% of the IgG and 96% of the fluid leaving the rete, whereas estradiol-treated rats reabsorbed 83% of the IgG and 86% of the fluid, and the ductus epididymidis fully compensated for these different effects of estradiol on the ductuli efferentes. The concentrations of IgG in secretions of the seminal vesicles and prostate gland were lower (0.1% and 0.3% respectively of the titers in blood serum) than in fluids from the extratesticular ducts, and were not affected by the administration of estradiol. RT-PCR showed that Fcgrt (neonatal Fc receptor, also known as FcRn) is expressed in the reproductive ducts, where IgG is probably transported across epithelium, being particularly strong in the ductuli efferentes (where most IgG was reabsorbed) and distal caput epididymidis. It is concluded that IgG enters the rete testis and is concentrated only 2.5-fold along the extratesticular duct system, unlike spermatozoa, which are concentrated 95-fold. Further, the ductus epididymidis can recognize and compensate for changes in function of the ductuli efferentes.

Synaptotagmin VI and VIII and Syntaxin 2 Are Essential for the Mouse Sperm Acrosome Reaction

The sperm acrosome is a large secretory granule that undergoes calcium-stimulated exocytosis by a mechanism analogous to neuronal secretion. In neurons the core SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, composed of syntaxin (Stx), SNAP-25, and VAMP2, mediates vesicle fusion, whereas calcium regulation is thought to be accomplished by the synaptotagmin (Syt) family, some of which exhibit calcium-dependent binding to syntaxin and SNAP-25. Sperm express Syt VI and VIII and Stx2, which are co-localized to the acrosomal compartment where they might mediate exocytosis in response to calcium influx. Therefore, we examined the calcium dependence and isoform-specific interaction of Syt and Stx. We found that Stx2 binds to Syt I, VI, and VIII in a calcium-dependent manner with EC(50) values of 175, 233, and 96 mum calcium, respectively. We also determined that the EC(50) for calcium of the acrosome reaction in streptolysin O-permeabilized sperm is 87 mum, which closely coincides with the calcium sensitivity of Stx2 and Syt VIII interaction. Consistent with this is the greater potency of recombinant Syt VIII, VI, and Stx2 compared with other isoforms in inhibiting the acrosome reaction in streptolysin O-permeabilized sperm. Similarly, introduction of Syt VIII-specific antibodies was equally effective in inhibiting the acrosome fusion. Taken together, our data suggest a critical role for Syt VIII and Stx2 in membrane fusion and acrosome reaction in the sperm.

Identification of non-mitochondrial NADPH oxidase and the spatio-temporal organization of its components in mouse spermatozoa

Though the spermatozoa are known to produce superoxide anion radicals, the enzyme system(s) that produce superoxide in these cells are not yet identified. Using Western blot assays and confocal laser scan microscopy, we detected gp91 phox and p67 phox associated with spermatozoa from testis and epididymis. We could not detect p22 phox in any of the sperm samples analyzed. While the expression of gp91 phox p67 phox appeared to be constitutive, p47 phox was detectable only in spermatozoa from testis and vas deferens. Importantly, p40 phox could be seen in very high quantities in testicular spermatozoa, which also showed the highest levels of NADPH-oxidase activity. Spermatozoa from cauda epididymidis and vas deferens also showed the presence of p40 phox, though the amount was low when compared with that of testicular spermatozoa. The absence of p22 phox and the striking correlation between the presence of p40 phox and the NADPH-oxidase activity suggest that the NADPH oxidase associated with spermatozoa is p22 phox-independent and that its activity is positively modulated by p40 phox. Further, since the confocal imaging detected that the subunits of the NADPH oxidase are located significantly on the head domains, the spermatozoa appear to present a case with dominant non-mitochondrial superoxide anion producing capabilities.

Ubiquitin C-Terminal Hydrolase L-1 Is Essential for the Early Apoptotic Wave of Germinal Cells and for Sperm Quality Control During Spermatogenesis1

Ubiquitination is required throughout all developmental stages of mammalian spermatogenesis. Ubiquitin C-terminal hydrolase (UCH) L1 is thought to associate with monoubiquitin to control ubiquitin levels. Previously, we found that UCHL1-deficient testes of gad mice have reduced ubiquitin levels and are resistant to cryptorchid stress-related injury. Here, we analyzed the function of UCHL1 during the first round of spermatogenesis and during sperm maturation, both of which are known to require ubiquitin-mediated proteolysis. Testicular germ cells in the immature testes of gad mice were resistant to the early apoptotic wave that occurs during the first round of spermatogenesis. TUNEL staining and cell quantitation demonstrated decreased germ cell apoptosis and increased numbers of premeiotic germ cells in gad mice between Postnatal Days 7 and 14. Expression of the apoptotic proteins TRP53, Bax, and caspase-3 was also significantly lower in the immature testes of gad mice. In adult gad mice, cauda epididymidis weight, sperm number in the epididymis, and sperm motility were reduced. Moreover, the number of defective spermatozoa was significantly increased; however, complete infertility was not detected. These data indicate that UCHL1 is required for normal spermatogenesis and sperm quality control and demonstrate the importance of UCHL1-dependent apoptosis in spermatogonial cell and sperm maturation.

Glutathione peroxidase activity in cell cultures from different regions of human epididymis

To study the secretory activity and androgen regulation of glutathione peroxidase (GPx) in epithelial cell cultures from human epididymis. Tissue was obtained from patients undergoing therapeutic orchidectomy for prostatic cancer. Epithelial cell cultures were obtained from the caput, corpus and cauda epididymides. Enzymatic activity was measured in conditioned media by colorimetric methods in absence or presence of 1, 10 or 100 nmol/L testosterone. The effect of 1 micromol/L flutamide was also evaluated. GPx activity was higher in cultures from corpus and cauda than caput epididymidis. The presence of different concentrations of testosterone increase enzyme activity in cell cultures from all epididymal regions. Addition of flutamide reverses the androgen dependent increase of GPx activity. GPx activity is secreted from human epididymal cells in a region dependent manner and is regulated by androgens.

The effect of prolonged cold storage of eland ( Taurotragus oryx) cauda epididymides on the spermatozoa: possible implications for the conservation of biodiversity

The objectives of this study were to investigate the effects of prolonged storage of cauda epididymides at 4 °C on spermatozoa, and to determine the practicality of utilising epididymal sperm, harvested from testes collected during routine culling of game animals, in assisted reproductive technologies. Testes from eland ( Taurotragus oryx) were collected and epididymides removed and maintained at 4 °C. Sperm motility, viability, morphology and membrane integrity were examined at 12 h intervals for 108 h. Sperm motility and viability were significantly lower at the end of the experiment than at the start ( P < 0.05) and there was individual variation in the rate at which motility and viability declined. The total number of normal sperm decreased significantly with prolonged storage at 4 °C. Midpiece defects were the most common and head and tail abnormalities were rare. A significant decrease in acrosomal and nuclear membrane integrity was observed with prolonged cold storage but there was no significant change in cell membrane integrity. However, about 30% of epididymal sperm survived for 3 days at 4 °C and more than 10% survived for 4 days, and it should be possible to use sperm from culled animals in some assisted reproductive technologies.

Repeated in utero and lactational 2,3,7,8-tetrachlorodibenzo- p-dioxin exposure affects male gonads in offspring, leading to sex ratio changes in F 2 progeny

The effects of in utero and lactational 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on the reproductive system of male rat offspring (F 1) and the sex ratio of the subsequent generation (F 2) were examined. Female Holtzman rats were gavaged with an initial loading dose of 400 ng/kg TCDD prior to mating, followed by weekly maintenance doses of 80 ng/kg during mating, pregnancy, and the lactation period. Maternal exposure to TCDD had no significant effects on fetus/pup (F 1) mortality, litter size, or sex ratio on gestation day (GD) 20 or postnatal day (PND) 2. The TCDD concentration in maternal livers and adipose tissue on GD20 was 1.21 and 1.81 ng/kg, respectively, and decreased at weaning to 0.72 in the liver and 0.84 in the adipose tissue. In contrast, the TCDD concentration in pup livers was 1.32 ng/kg on PND2 and increased to 1.80 ng/kg at weaning. Ventral prostate weight of male offspring was significantly decreased by TCDD exposure on PND28 and 120 compared with that of controls. Weight of the testes, cauda epididymides, and seminal vesicle, and sperm number in the cauda epididymis were not changed by TCDD exposure at PND120. TCDD- or vehicle-exposed male offspring were mated with unexposed females. The sex ratio (percentage of male pups) of F 2 offspring was significantly reduced in the TCDD-exposed group compared with controls. These results suggest that in utero and lactational TCDD exposures affect the development of male gonads in offspring (F 1), leading to changes in the sex ratio of the subsequent generation (F 2).

Radiological quiz

Marsupial spermatozoa tolerate cold shock well, but differ in cryopreservation tolerance. In an attempt to explain these phenomena, the fatty acid composition of the sperm membrane from caput and cauda epididymides of the Eastern grey kangaroo, koala, and common wombat was measured and membrane sterol levels were measured in cauda epididymidal spermatozoa. While species-related differences in the levels of linolenic acid (18:3, n-6) and arachidonic acid (20:4, n-6) were observed in caput epididymal spermatozoa, these differences failed to significantly alter the ratio of unsaturated/saturated membrane fatty acids. However in cauda epididymidal spermatozoa, the ratio of unsaturated/saturated membrane fatty acids in koala and kangaroo spermatozoa was approximately 7.6 and 5.2, respectively; substantially higher than any other mammalian species so far described. Koala spermatozoal membranes had a higher ratio of unsaturated/saturated membrane fatty acids than that of wombat spermatozoa (t = 3.81; df = 4; p ⩽ 0.02); however, there was no significant difference between wombat and kangaroo spermatozoa. The highest proportions of DHA (22:6, n-3), the predominant membrane fatty acid in cauda epididymidal spermatozoa, were found in wombat and koala spermatozoa. While species-related differences in membrane sterol levels (cholesterol and desmosterol) were observed in cauda epididymidal spermatozoa, marsupial membrane sterol levels are very low. Marsupial spermatozoal membrane analyses do not support the hypothesis that a high ratio of saturated/unsaturated membrane fatty acids and low membrane sterol levels predisposes spermatozoa to cold shock damage. Instead, cryogenic tolerance appears related to DHA levels.

Viable rat offspring derived from oocytes intracytoplasmically injected with freeze-dried sperm heads

A 2 x 3 factorial designed experiment was conducted in order to examine whether freeze-dried rat spermatozoa can participate in full-term development following intracytoplasmic sperm injection (ICSI). A sperm suspension from cauda epididymides of Sprague-Dawley (SD) rats was prepared with or without ultrasonic treatment. The sonicated and non-sonicated sperm suspensions were processed for freeze-thawing (FT groups; 100 microl sample was cooled in liquid nitrogen vapor, stored for 1 day at--196 degrees C, and thawed in a 25 degrees C water bath) and freeze-drying (FD groups; 100 microl sample was frozen in liquid nitrogen for 20 s, lyophilized for 6 h, stored at 4 degrees C for 2 days, and rehydrated with 100 microl ultrapure water), or were subjected to immediate use for ICSI (fresh control groups). The sperm heads were microinjected into denuded SD oocytes using a piezo-driven micropipette 2-4 microm in diameter. The presumptive zygotes were transferred into oviducts of pseudopregnant Wistar female rats. Viable rat offspring were produced from all six experimental groups. Ultrasonic treatment of rat spermatozoa was effective in increasing the offspring rate (23.3% vs 6.7% in fresh control groups, 35.0% vs 7.6% in FT groups, 9.2% vs 2.5% in FD groups). The acrosomal region appeared to be intact even after ultrasonic FT and FD treatments as well as in the fresh controls, while the lateral dorsal region of the sperm membrane was more or less damaged in the sonicated, FT and FD samples. Thus, the successful participation of freeze-dried spermatozoa in full-term development was demonstrated by applying ICSI in the rat.