Decrease in bicarbonate transport activities during epididymal maturation of porcine sperm

Kinetic studies of [14C]HCO3- and [35S]SO4(2-) transport show the existence of a common transport system for the two anions on the plasma membranes of porcine epididymal sperm. Km value for bicarbonate (8.3mM) is within the range of physiological concentration of bicarbonate. The anion transport activity is dependent on the temperature but independent of co-existence of Na+. Its pH dependency and sensitivity to the several anion transport inhibitors are similar to those observed in erythrocyte. Anion transport activity decreases during sperm maturation in epididymis.     

Suppression of endogenous estrogen during development affects porcine epididymal sperm maturation

Estrogen plays an important role in male reproduction, critical for sustained fertility in some species. Reducing estrogen's interaction with its receptor(s) in monkey and mouse models is associated with reduced sperm motility and, in some cases, documented elimination of sperm fertilizing ability, suggesting that normal epididymal function may be estrogen dependent. The objective of these experiments was to evaluate the effects of reduced endogenous estrogen on development of epididymal function in the pig, a species in which males have very high levels of endogenous estrogen. Letrozole, a potent inhibitor of estrogen synthesis, was administered to neonatal boars from 1 week of age and markedly suppressed estrogen production. Epididymal function assessed as acquisition of sperm fertilizing ability (in vitro fertilization of zona-free oocytes) was reduced in Letrozole-treated animals at 24 and 28 weeks of age (23% and 30% fertilization, respectively compared with 37% and 54% in vehicle controls) but had recovered by 32 weeks of age. Cauda epididymal sperm numbers were reduced in treated animals (35% of control values at 20 weeks of age) but appeared to be recovering at 32 weeks of age. Reduction of endogenous estrogen had no effect on other aspects of epididymal function (percentage of motile sperm, sperm motion parameters, sperm head morphometrics, or ability of sperm to undergo an acrosome reaction). Reducing endogenous estrogen during postnatal development appears to have transient effects on porcine epididymal function. These transient effects suggest that the pig, with its high endogenous estrogen, may respond differently than other species to reduced estrogen synthesis.     

alpha-Tocopherol modifies tyrosine phosphorylation and capacitation-like state of cryopreserved porcine sperm

Sperm cryopreservation is associated with the production of reactive oxygen species (ROS) leading to membrane destabilization, which induces capacitation-like changes, increases protein tyrosine phosphorylation, and decreases their fertilizing ability. alpha-Tocopherol, a lipid peroxidation inhibitor, preserves the functionality of cryopreserved porcine sperm. Our aim was to evaluate the effect of alpha-tocopherol on sperm quality parameters as well as capacitation-like changes and modifications in protein tyrosine phosphorylation. Boar sperm frozen with or without 200 microg/mL of alpha-tocopherol were thawed and maintained at 37 degrees C for 10 min in BTS. Routine parameters of semen quality were evaluated by optical microscopy and membrane changes were determined by the epifluorescence chlortetracycline technique. Changes in protein tyrosine phosphorylation were examined using a specific anti-phosphotyrosine monoclonal antibody. Motility was higher (18%, P<0.05) in semen with alpha-tocopherol. Viability did not differ (P>0.05) between treatments. However, there was less (P<0.05) capacitation-like changes in semen with alpha-tocopherol compared to control samples. A MW 32 kDa tyrosine-phosphorylated protein was detected in extracts of cryopreserved sperm; the intensity of immunostaining was lower in semen containing alpha-tocopherol compared to the control (0.211+/-0.030 versus 0.441+/-0.034 arbitrary units). Additionally, this band was not detected in fresh sperm. The addition of alpha-tocopherol to the extender prior to cryopreservation of boar semen protected sperm membranes against oxidative damage and reduced both tyrosine phosphorylation and the capacitation-like state.     

Cryopreservation of epididymal stallion sperm

Any event that makes semen collection or mating impossible, such as death, castration, or injury, may terminate a stallion’s breeding career. Fortunately, stallion sperm which are capable of fertilization can be harvested from the epididymis, and frozen for future use. However, the fertility of frozen–thawed epididymal sperm has been found to be lower than that of ejaculated sperm. Therefore, this study aimed to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of different cryoprotectants and freezing protocols on sperm quality. Dimethylformamide was tested alone or combination with pasteurized egg yolk as substitute of fresh egg yolk. In addition, the effect of the pre-freeze stabilization on sperm quality was analyzed. Heterospermic samples obtained from stallion epididymis were collected and cryopreserved in lactose–egg-yolk extender or in the same extender with varying content of cryoprotectant and content of egg yolk, stabilized and no-stabilized. Sperm motility, viability, hypoosmotic swelling test (HOST) and acrosome integrity were evaluated post-thawing. No improvement was observed on the replacement of fresh yolk by pasteurized egg yolk, whereas the results suggest that dimethylformamide is a cryoprotectant suitable for cryopreservation of equine epididymal semen, even better than glycerol. In addition, we found that the stabilization before freezing on epididymal stallion sperm, can improve sperm quality parameters.     

Low-temperature preincubation enhances survival and regeneration of cryopreserved Saussurea involucrata callus

Saussurea involucrata Kar. et Kir. is one of the most well-known Chinese medicinal plants, and it is utilized for a variety of medical conditions. Due to the overexploitation of this endangered species, it is crucial to develop methods for both conservation and propagation. To address this issue, we have developed and optimized a simple and effective vitrification process for the cryopreservation of S. involucrata callus tissue. The optimized method consisted of a 3-d incubation period on medium containing 0.3 M sucrose, transfer to a plant vitrification solution (PVS2) containing 30% (v/v) glycerol, 15% (v/v) ethylene glycol, 15% (v/v) dimethylsulfoxide, and 0.4 M sucrose first at 60% PVS2 for 40 min, then at 100% PVS2 for 60 min, followed by immediate immersion and storage in liquid nitrogen. To thaw the tissue, tissues were rewarmed at 40°C for 2 min. This method resulted in a survival rate of approximately 56% and a regrowth rate of approximately 40%. Survival and regrowth were significantly improved by the addition of a low-temperature preincubation step. Incubating the calli at 4°C for 12 d prior to initiating the optimized cryopreservation protocol increased the survival rate of the tissue to 75%, increased the regrowth rate to 60%, and more than doubled the number of regenerated shoots per explant. Following cryopreservation, greater than 90% of the regenerated shoots formed complete plantlets, and 81% of the regenerated plantlets survived and grew vigorously under greenhouse conditions.     

Freezing of stallion epididymal sperm

Inseminations with frozen-thawed epididymal sperm have resulted in low-pregnancy rates of mares. If fertility of epididymal sperm could be improved, it would help to preserve genetic material from stallions that have suffered severe injuries, been castrated or have died. The aim of the present study was to investigate the effect of different extenders and pre-freezing addition of capacitation media on freezability of epididymal sperm and on storage at 5 degrees C for 24h. In experiment 1, epididymal sperm samples were diluted and subsequently frozen with three different extenders: Botu-Crio, EDTA-Lactose and INRA-82. Motility analysis using computer assisted sperm analyzer (CASA) demonstrated better motility for sperm in Botu-Crio than in the other extenders; EDTA-Lactose yielded better motility than INRA-82 on most evaluated parameters. There was no difference in membrane integrity among the studied extenders. From 18 inseminated mares, 12 (66%) were pregnant 15 days after AI with frozen-thawed epididymal sperm showing that Botu-Crio was able to maintain the fertility potential. In experiment 2, the effect of incubation of epididymal sperm before freezing in three capacitation media (Fert Talp, Sperm Talp, Talp+Progesterone), seminal plasma, or control was tested. Based on post-thaw motility evaluation by CASA, samples incubated in Sperm Talp showed better motility values. There were no differences in plasma or acrosomal membranes or in mitochondrial potential among groups. We concluded that Botu-Crio was better than the other extenders in the ability to preserve epididymal sperm and that pre-freeze addition of Sperm Talp was also beneficial.     

Lipolytic activity of porcine adipose tissue after preincubation

1. The stimulated lipolytic rate in porcine adipose tissue slices was markedly increased after preincubation with or without isoproterenol. 2. Preincubation with and without insulin, dibutyryl-cAMP or theophylline suggested that the activation of the stimulated rate resulted from inhibition or inactivation of cAMP-phosphodiesterase activity during preincubation. 3. Preincubation with isoproterenol also caused activation of the basal (no exogenous hormone) lipolytic activity. 4. However, much of this activation appeared to result from carry-over of isoproterenol into the incubation medium because it could be lowered by additional washing of the tissue, by lower isoproterenol concentration or by propranolol.     

Zona pellucida protein binding ability of porcine sperm during epididymal maturation and the acrosome reaction

In many mammals, the first interaction between gametes during fertilization occurs when sperm contact the zona pellucida surrounding the egg. Although porcine sperm first contact the zona pellucida via their plasma membrane, the regions of the sperm surface that display zona receptors have not been determined. We have used the Alexa 488 fluorophore conjugated to solubilized porcine zona pellucida proteins to observe zona receptors on live boar sperm. Zona proteins bound live, acrosome-intact sperm on the anterior portion of the sperm head, concentrated in a thin band over the acrosomal ridge. When sperm membranes were permeabilized by fixation or acrosome reactions induced by the ionophore A23187, zona binding was extended to a broad area covering the entire acrosomal region. Zona binding proteins were present in the acrosomes of sperm from all regions of the epididymis. In contrast, zona binding sites were found on the plasma membrane of most sperm from the corpus and cauda epididymis, but on only 6% of caput epididymal sperm. In conclusion, acrosome-intact boar sperm exhibit concentrated zona protein binding over the acrosomal ridge and acquire this binding in the corpus region of the epididymis, correlating with the developmental stage at which sperm gain the ability to fertilize oocytes.     

Cryopreservation of epididymal dog sperm.

A method of cryopreservation was developed for sperm salvaged from the cauda epididymis and vas deferens of domestic dog testes. Four modifications of the glycerol concentration of a buffer used for cryopreservation of dog ejaculates and two freezing rates were assessed for their effect upon post-thaw spermatozoal motility and morphology. There was no statistical difference between the four glycerol concentrations or the two freezing rates and the buffer containing 6% glycerol and the freezing rate provided by 0.5 ml straws was chosen for further study. This method resulted in a significant reduction in the percentage of live spermatozoa detected with Hoechst staining and a reduction in the percentage of capacitated spermatozoa after freeze-thawing. However, there was no difference in the ability of frozen-thawed spermatozoa to penetrate homologous oocytes.This study demonstrates that cryopreservation of epididymal canine sperm can be performed using methods similar to those established for ejaculates of the same species, and that despite some damage, spermatozoa retain their functional ability.     

Rat sperm enzymes during epididymal transit

The activities of cAMP and cGMP phosphodiesterases (EC 3.1.4.1), adenylate cyclase (EC 4.6.1.1) and protein carboxyl-methylase (EC 2.1.1.24) were measured in the particulate and soluble (105 000 g supernatant) fractions of washed spermatozoa isolated from five segments of the adult rat epididymis. The activities of both phosphodiesterases decreased during epididymal transit, whereas adenylate cyclase and protein carboxyl-methylase underwent a progressive increase, the latter showing the most marked alteration. Both cAMP and cGMP phosphodiesterases as well as the adenylate cyclase were all associated primarily with the particulate fraction, and the extent to which these enzymes were associated with the membranes increased as the spermatozoa passed through the epididymis. Sperm protein carboxyl-methylase activity was, on the other hand, predominantly soluble in all segments of the epididymis. Adenylate cyclase, cAMP phosphodiesterase and protein carboxyl-methylase activities were found predominantly in the sperm tails, whereas cGMP phosphodiesterase was equally distributed between heads and tails. These observations imply that the acknowledged increase in intracellular cAMP levels which occurs in spermatozoa during epididymal transit may be a consequence of both increased synthesis (adenylate cyclase) and reduced hydrolysis (phosphodiesterase).     

Effect of storage method and extender osmolality in the quality of cryopreserved epididymal ram spermatozoa

Post-mortem sperm recovery and cryopreservation could be a complement to germplasm banking in sheep, especially for endangered breeds. This study is an attempt to identify factors for improving the success of cryopreserving ram epididymal spermatozoa, considering the decrease of sperm quality with post-mortem time. Epididymal spermatozoa from 9 rams were kept at 5°C using three storage methods: within the epididymes, undiluted sperm mass, and diluted in extenders of different osmolality (TES-Tris-fructose at 320, 370 or 420 mOsm/kg, 20% egg yolk, 8% glycerol). At 0, 24, 48 and 72h, spermatozoa were cryopreserved using each extender. Samples were analyzed before and after cryopreservation by CASA (motility) and flow cytometry (viability and acrosomal status). Post-mortem time decreased pre-freezing and post-thawing sperm quality. Some storage x extender combinations improved the effect of post-mortem time on sperm quality. Both epididymis storage combined with the 420 extender, and storing the spermatozoa diluted in the 320 extender improved post-thawing quality, especially at long post-mortem times. Storing the spermatozoa diluted in the 370 extender was detrimental for the acrosomal status. These findings have practical applications. The simplest storage method (within the epididymes) seems to be adequate if hyperosmotic extenders were used for freezing. An alternative method could be storing the spermatozoa diluted in a hypoosmotic extender. These recommendations are limited to the osmolalities tested in this study (420 mOsm/kg and 320 mOsm/kg); other osmolalities should be tested.     

Direct binding of boar ejaculate and epididymal spermatozoa to porcine epididymal epithelial cells is also needed to maintain sperm survival in in vitro co-culture

The aim of the present study was to compare the influence of cultured epididymal epithelial cells (EEC) from corpus, caput or cauda, oviductal epithelial cells (OEC) and non-reproductive epithelial cells (LLC-PK1) on function and survival of epididymal and ejaculated spermatozoa, in the latter case to determine whether such influence differed between morphologically normal and abnormal spermatozoa. For this purpose, either spermatozoa were directly co-cultured with EEC from caput, corpus, or cauda, OEC and LLC-PK1 cells (experiment 1) or a membrane-diffusible insert was included in these co-cultures (experiment 2). EEC cultured from the three epididymal regions did not differently affect the sperm parameters. Morphologically normal spermatozoa presented a higher ability to bind EEC, OEC, and LLC-PK1 than abnormal spermatozoa with cytoplasmic droplets or with tail/head malformations. Epididymal spermatozoa were more able to bind EEC during the first 24 h of co-culture, while ejaculated spermatozoa presented a higher capacity to bind OEC between 30 min and 3 h of co-incubation. In all cases, the ability to bind to epithelial cells was higher when they were co-cultured with EEC and OEC than with LLC-PK1. After 2 h of co-culture, the viability of epididymal spermatozoa was better maintained when they bound EEC than when they bound OEC. Conversely, the viability of ejaculated spermatozoa was better maintained when bound OEC than when bound EEC after 24 and 48 h of co-culture. Our work, apart from corroborating the involvement of morphologically normal spermatozoa in the formation of sperm reservoir, highlights the importance of direct contact spermatozoa-EEC in maintaining the sperm survival in in vitro co-culture, and also suggests that a specific binding between EEC and epididymal spermatozoa exists.     

Morphological abnormalities in zebrafish cryopreserved sperm

The aim of the present study was to evaluate the effect of cryopreservation on the morphology of zebrafish sperm (Danio rerio). Sperm from 30 males were collected and divided in two treatments: fresh and cryopreserved semen. The following were measured sperm morphology, motility and membrane integrity. Cryopreservation reduced motility, the number of normal cells and the membrane integrity, as well as increased the percentage of sperm abnormalities. The most frequent types of morphological changes found in cryopreserved semen were macrocephaly, loose head, degenerated head, proximal gout, curled tail and short tail. This study opens the way for further investigations on morphological changes and for a new classification of these changes in fish semen due to cryopreservation.     

Effect of external cryoprotectants as membrane stabilizers on cryopreserved rainbow trout sperm

The process of freezing and thawing induces certain cellular damage in rainbow trout (Oncorhynchus mykiss) spermatozoa. We have previously demonstrated that after freezing and thawing decreased fertility in rainbow trout (Oncorhynchus mykiss) spermatozoa, is related to sublethal damage to the plasma membrane. External cryoprotectants are known to stabilize the sperm cell membrane against such damage. In the current study, we used a basic freezing extender containing #6 Erdahl and Graham and 7% DMSO and added egg yolk, BSA, and a soybean-protein complex (DanPro S760) singly and in various combinations. To assess the effect of these cryoprotectants we evaluated the percentage of cells with progressive motility, permeability of cells to propidium iodide (viability) after exposure for 30 sec, 2, 5, 10 and 15 min. to hypo- and isoosmotic solutions of 10 and 300 mOsm, and the in vitro fertility rate. Fertility trials were performed using 1.87 x 10(7) spermatozoa/egg. Some of the tested stabilizers increased motility, increased viability, or reduced cell fragility after freezing and thawing. Nevertheless these quality improvements demonstrated by the "in vitro" tests do not always correlate with high fertility. The best membrane protection in terms of resistance to hypoosmotic shock was achieved when BSA and egg yolk were added to the extender. The highest fertility rates were obtained with DanPro S760 alone or in combination with BSA; the use of BSA with egg yolk did not improve this parameter. Our results demonstrated that some external cryoprotectants effectively increased membrane resistance during freezing and thawing, but some of the tested mixtures interfered with fertilization. Soybean protein concentrate provided good protection and increased fertility rates in cryopreserved trout spermatozoa.     

Localization of a maturation-dependent epididymal sperm surface antigen recognized by a monoclonal antibody raised against a 135-kilodalton protein in porcine epididymal fluid.

A specific 135-kDa protein was purified from porcine cauda epididymal fluid. Analysis of its N-terminal amino acid sequence revealed it to be a new protein. Stable clones of hybridomas that produced monoclonal antibodies against the purified 135-kDa protein were established. A clone, B-11, reacting both with epididymal fluid and with sperm plasma membranes was selected and used in this study. Immunoblotting analysis showed that B-11 reacted only with a 135-kDa protein among epididymal fluid proteins. In contrast, B-11 did not recognize a similar 135-kDa sperm protein but did strongly react with a 27-kDa protein among sperm membrane proteins, extracted by NP-40 in the presence of protease inhibitors. B-11 also reacted only with a 27-kDa protein fragment among trypsin digests of the 135-kDa epididymal protein. The 135-kDa protein was first detected, by ELISA or immunoblotting analysis, at the beginning of the corpus epididymis. Maximal levels were reached in the distal corpus and levels were slightly decreased in the cauda epididymis. On the other hand, the surface of caput sperm were found to contain small amounts of antigen(s), the concentration of which gradually increased during epididymal transit. In immunocytochemical studies, the antigen was detectable in the epithelial cells from the initial segment to the corpus of the epididymis but not in the caudal cells. In the lumen, the presence of the 135 kDa protein was apparent in the corpus (at a maximum in the middle and distal corpus) and to a lesser degree in the caudal lumen. The 27-kDa protein was distributed all over the equatorial region of the acrosome of less than 10% of caput epididymal sperm. As sperm passed through the corpus epididymis, the percentage of immunoreactive cells increased and the protein was restricted to specific domains of the sperm head. Thus, on the mature sperm, antigen was localized in a crescent-shaped area of the equatorial segment just behind the anterior part of the acrosome and on the apical rim of the sperm head. This is the first observation of a sperm surface antigen derived from an epididymal protein as a proteolytic fragment that interacts with specific regions of the sperm membrane during the process of spermatozoa maturation.     

DNA integrity of Polyodon spathula cryopreserved sperm

Comet assay was used to detect DNA integrity of paddlefish (Polyodon spathula) sperm following cryopreservation. At the same time, sperm velocities prior to freezing and post‐thawing were also assessed by the computer‐assisted sperm analysis (CASA) system. Significant differences (P < 0.05) were detected in the degree of DNA damage in cryopreserved sperm using different extenders. According to osmolality of the extenders, DNA damages of Sb (20 mm Tris, 75 mm sucrose, 0.5 mm KCl, pH 8.5) sperm was the least, which showed that the percentage of tail DNA of Sb (17.87–35.28%) was lower than those of Sa (20 mm Tris, 50 mm sucrose, 0.5 mm KCl, pH 8.5) and Sc (20 mm Tris, 100 mm sucrose, 0.5 mm KCl, pH 8.5). Moreover, A and B class sperm cells provided most of the Sb sperm (>50%). However, in light of the concentration of methanol, DNA damages of M8 (8% methanol concentration) sperm were the least, including a lower percentage of the tail DNA (21.56–30.86%), and C and D class sperm cells (<30%), regardless of the osmolality of the extenders. In conclusion, when the dilution was 20 mm Tris, 75 mm sucrose, 0.5 mm KCl, pH 8.5 and the concentration of methanol was 8%, the extenders were the best for cryopreservation of paddlefish sperm. In addition, the results indicated that the extent of damage to sperm motility caused by freeze‐thawing (VCL, VSL) was correlated with DNA breakage (|r| > 0.8). This implied that cryopreservation could damage sperm DNA of paddlefish and affect the sperm velocities when the osmolality and the concentrations of the cryoprotectants of the extender were inappropriate.     

Changes in the nature of calcium transport systems on the porcine sperm plasma membrane during epididymal maturation.

Comparative studies of 45Ca(2+)-transport across the plasma membrane were performed using porcine caput, corpus and cauda epididymal sperm. The Ca(2+)-uptake is dependent on the presence of the substrates for respiration and is sensitive to verapamil. The Ca(2+)-efflux is mediated by both Na(+)-dependent and -independent systems. In the immature sperm in caput epididymis, Na(+)-independent efflux is predominant, but it is gradually replaced by Na(+)-dependent efflux during the epididymal transit. The net activity of Ca2+ accumulation into sperm increases with the epididymal maturation.     

Effect of human oviductal epithelial cell cultural medium on cryopreserved human sperm survival

The human oviduct is known as a functional site for gamete transportation, retention, fertilization and zygote development. Previous studies have shown that human oviductal epithelial cell cultural medium (OECCM) has a positive effect on prolongation of sperm motility for some cryopreserved human sperm without cryodamage. However, for most cryopreserved sperm, OECCM could not improve their survival prolongation. In this study, we assessed the influence of human OECCM on the motility longevity of cryopreserved human sperm with an in vitro incubation method.     

Apoptosis in fresh and cryopreserved buffalo sperm

The objectives of present study were (a) validation of annexin V/PI assay for estimation of sperm apoptosis in buffalo (Experiment 1) and (b) determining the effect of stages of cryopreservation on sperm apoptosis and its correlation with sperm motility and plasma membrane integrity (Experiment 2). In Experiment 1, different levels of apoptosis were artificially induced in buffalo semen (100 × 10⁶ sperm/aliquot) through graded doses of camptothecin (5, 10 and 20 μM/aliquot). Higher concentrations of camptothecin (10 and 20 μM) successfully (P < 0.05) induced apoptosis as compared to the lower (5 μM) dose and/or control. In Experiment 2, semen samples (n = 9, three pooled semen samples from each of the three buffalo bulls separately) were cryopreserved using vapor freezing. The mean percentage of apoptotic, necrotic and viable sperm did not differ between fresh and before freezing stages. However, freezing and thawing increased (P < 0.05) the percentage of apoptotic sperm (25.4 ± 0.6 vs. 36.5 ± 1.9) while decreased (P < 0.05) the necrotic (35.1 ± 1.2 vs. 29.7 ± 0.7) and viable sperm (37.2 ± 1.3 vs. 32.8 ± 1.9, (P < 0.07). Likewise, the mean percent motility and plasma membrane integrity decreased (P < 0.05) (64 ± 2.1 vs. 49.4 ± 1.3) and (79.6 ± 0.5 vs. 38.7 ± 0.3) respectively, at post thaw compared to other stages. Coefficient of correlation, combined at all stages for each variable revealed that sperm apoptosis was inversely correlated with sperm motility and plasma membrane integrity. It is concluded that (a) the annexin V/PI assay can be used as a tool to determine the buffalo semen apoptosis and (b) freezing and thawing induces apoptosis in buffalo sperm.