Room temperature storage of mouse epididymal spermatozoa: exploration of factors affecting sperm survival

To explore optimal conditions for in vitro sperm survival, we examined the effects of several media used for murine egg culture and in vitro fertilization (IVF; including M16, M2, PB1, TYH, and CZB) on motility of murine spermatozoa stored at 22 degrees C under paraffin oil. Of media tested, M2 medium, that had been adjusted to pH 7.2 by adding N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), was found to be the best. Addition of various concentrations of HEPES to TYH did not improve sperm survival, suggesting that HEPES (and probably neutral pH) do not enhance survival of murine sperm. Since M16 has higher amounts of bicarbonate than M2 (25 mM versus 4.15 mM), four variations of M16 media containing 4.15, 8.30, 16.60, or 33.20 mM bicarbonate were prepared and tested. The modified M16 media with 4.15-16.60 mM bicarbonate yielded good sperm survival (comparable to M2 medium), while relatively high concentrations of bicarbonate (ranging from 16.60 to 33.20 mM) were deleterious to isolated sperm, suggesting the need for a minimum level of residual bicarbonate. However, the mechanism by which the lifespan of spermatozoa is extended remains unknown. The in vitro fertilizing abilities of spermatozoa left in M2 medium for 1, 3, and 5 days at 22 degrees C were 52.5, 21.8, and 7.0%, respectively, when the cleavage rate to the two-cell stage was examined. Transfer of two-cell embryos produced in vitro with spermatozoa stored for 1, 3, and 5 days at 22 degrees C resulted in production of fetuses with efficiencies of 42.5, 23.4, and 12.5%, respectively, which were lower than that of embryos derived from in vitro fertilization with fresh spermatozoa (68.1%). In conclusion, spermatozoa kept in M2 medium for up to 5 days at 22 degrees C can fertilize oocytes.     

Temporal synthesis of mitochondrial proteins from mouse epididymal spermatozoa

Mitochondria from ejaculated bovine spermatozoa contain a group of polypeptides ranging in molecular weights from 13,000 to 35,000 not found in other bovine or murine testicular mitochondria [Hecht and Bradley, 1981]. These proteins are present in the mitochondria isolated from both epididymal and ejaculated spermatozoa. To establish when during epididymal transport, spermiogenesis, and/or meiosis these proteins are synthesized, the synthesis intervals for the mitochondrial proteins from cauda epididymal spermatozoa were established following intratesticular injection of (³⁵S)methionine. Mice were killed every third day over a 33-day period and cauda epididymal spermatozoa were fractionated into mitochondrial and head components. Radioactivity in each fraction was monitored by liquid scintillation counting. Maximal incorporation was observed during spermiogenesis, although substantial amounts of protein were synthesized during meiosis. Analysis of the mitochondrial polypeptides by gel electrophoresis revealed that many polypeptides such as the cysteine-rich structural protein of the mitochondrial capsule were synthesized over prolonged intervals of spermiogenesis and meiosis rather than in a brief specific time period. These results suggest that spermatozoal mitochondria are produced by a sequential substitution of new proteins into the differentiating mitochondria rather than the abrupt appearance of a new class of mitochondria during spermatogenesis.     

Characterization of a decapacitation factor associated with epididymal mouse spermatozoa

Earlier studies demonstrated that epididymal mouse spermatozoa have a surface-associated factor which inhibits fertilizing ability in a reversible manner. The factor can be removed from uncapacitated spermatozoa by gentle centrifugation, resulting in immediately highly fertile gametes, and it can be added back to capacitated spermatozoa, resulting in poorly fertile cells in which the acrosome reaction has been blocked. Using such inhibition of in-vitro fertilizing ability as an assay, we have carried out experiments to characterize the factor. It appears to be an anionic polypeptide with Mr of approximately 40,000 (according to its behaviour on gel filtration). It is stable to heating at 100 degrees C for 15 min and is not destroyed by proteases at pH 8.0, yet inhibitory activity decreases during sperm incubation in capacitating conditions and is also destroyed in partially purified preparations by endogenous enzyme action during incubation at pH 5.0. Activity is not adsorbed to either concanavalin A-agarose or wheat-germ agglutinin-agarose, suggesting that terminal mannose and N-acetylglucosamine residues are not abundant. The factor causes rapid changes in the patterns of chlortetracycline fluorescence seen on sperm heads, a parameter used to assess the capacitated state. Removal of the factor from uncapacitated cells results in a shift to a predominance of capacitated patterns, while the addition of crude or partially purified factor to capacitated cells inhibits the acrosome reaction and causes a shift to the uncapacitated pattern in acrosome-intact spermatozoa. The factor therefore behaves as a decapacitation factor. However, it appears to differ from other characterized decapacitation factors in terms both of molecular size and of abundance of mannose and N-acetylglucosamine residues.     

Prolonged survival of mouse epididymal spermatozoa stored at room temperature

Summary: The viability and fertility of isolated mouse epididymal spermatozoa kept for up to 7 days at various temperatures (4°C, 22°C, and 37°C) were determined. Spermatozoa kept for 3 days at 22°C were still active, while those kept at 37°C or 4°C exhibited great reduction in motility within 2 days after isolation. In vitro fertilizing abilities of spermatozoa left for 0, 1, 2, and 3 days at 22°C were 69.2, 32.5, 9.5, and 4.9%, respectively, when the cleavage rate to two‐cell stage was examined. Transfer of two‐cell embryos produced in vitro with spermatozoa left for 1, 2, and 3 days at 22°C resulted in production of fetuses with efficiencies of respectively 30.2, 11.5, and 16.7%, which were lower (63.3%) than that of embryos derived from in vitro fertilization with fresh spermatozoa. These findings indicate that spermatozoa kept for up to 3 days at 22°C can fertilize oocytes, although at relatively low efficiency. genesis 31:147–155, 2001. © 2001 Wiley‐Liss, Inc.     

Calcium-dependent binding of mouse epididymal spermatozoa to the zona pellucida.

The minimal requirements and characteristics of epididymal sperm binding to the zona pellucida of the mouse egg were investigated using a new stop-fix centrifugation technique. This assay provided a precise physical definition of the association between the spermatozoon and the zona and permitted quantitation of the binding reaction at short time intervals. The results demonstrated that Ca²⁺ is an essential physiological component required for binding to occur. Sperm preincubated for 60 min in a simplified medium lacking Ca²⁺ did not acquire the ability to bind to eggs. In contrast, if sperm preincubation occurred in this medium supplemented with 1.7 mM Ca²⁺, binding was identical to that observed following sperm preincubation in the complete culture medium which supports both capacitation and fertilization in vitro. The Ca²⁺-dependent binding reaction was rapid, reversed by EGTA, specific for Ca²⁺, and did not require the transport of Ca²⁺ into the cell. Sperm bound to the zona surface following preincubation with Ca²⁺ were capable of fertilization in vitro when the eggs were subsequently transferred to the culture medium. It is proposed that this binding reaction represents a part of capacitation and not the acrosome reaction.     

Cyclic nucleotide metabolism in mouse epididymal spermatozoa during capacitation in vitro

The role of cyclic nucleotides in sperm capacitation is equivocal. Using conditions known to support mouse sperm capacitation after 120 min incubation in vitro, the cAMP and cGMP contents of epididymal spermatozoa were measured and the cGMP/cAMP ratio determined. The initial high cAMP content detected upon release of spermatozoa decreased within 30 min to a lower plateau, which was then maintained throughout incubation. With the cGMP content remaining approximately constant, the cGMP/cAMP ratio increased over 120 min. In the presence of 2 mM caffeine, an increased cAMP content was noted at 0 and 30 min before a fall to the plateau level. To investigate cyclic nucleotide metabolism, adenylate cyclase and phosphodiesterase activities were compared in two sperm populations, one essentially uncapacitated and the other incubated for 120 min. Adenylate cyclase activity, higher in the presence of 2 mM Mn²⁺ compared to Mg²⁺, showed increased activity at 120 min compared to 30 min incubation, while phosphodiesterase activity decreased during this period. The ability of spermatozoa to form adenosine and inosine from cAMP indicated endogenous 5′-nucleotidase and deaminase, as well as phosphodiesterase, activities. Although the endogenous cAMP content appeared to remain constant during the time that acrosome loss, hyperactivated motility and fertilizing ability can be demonstrated, activities of the enzymes responsible for cAMP metabolism indicate an increased potential for cAMP availability and turnover. The increased cGMP/cAMP ratio may also play a role during capacitation.     

The relationship between motility and the FITC-BSA binding properties of mouse epididymal spermatozoa

Mouse epididymal spermatozoa exposed to fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) immediately following dilution or after a 2-hour incubation period under fertilization conditions, were assessed by fluorescence microscopy for albumin adsorption. Motile spermatozoa exhibited light fluorescence in the midpiece and tail but not in the head. In contrast the majority of nonmotile spermatozoa displayed a strong and characteristic fluorescence in the post acrosomal region of the sperm head as well as the midpiece. Spermatozoa immobilised by short-term heat stress exhibited fluorescence in the post acrosomal region and midpiece as before but also in the acrosomal cap. The equatorial region failed to fluoresce. The significance of these observations on the involvement of albumin in capacitation is discussed.     

Morphological changes of mouse spermatozoa during in vitro storage

In addition to normal spermatozoa, spermatozoa with bent and coiled tails, as well fragments of spermatozoa, such as single heads and tails and complexes of heads and tails, were found in a suspension of epididymal spermatozoa obtained from mice kept in the state of sexual deprivation for a long time. The dynamics of these elements was traced in the suspensions maintained at 10 and 35°C. At 37°C, the numbers of normal spermatozoa and spermatozoa with bent tails significantly decreased, while that of spermatozoa with coiled tails remained unchanged. At 10°C, the number of only spermatozoa with bent tails decreased. Based on the published data and our results, we propose that spermatozoa unclaimed for fertilization not only disintegrate into characteristic fragments, but also undergo a sequence of forms characteristic for the haploid phase of multicellular organism life cycle.     

Freezing of epididymal spermatozoa from dogs after cool storage for 2 or 4 days

An experiment was conducted to investigate the freezing ability of canine epididymal spermatozoa after cool storage at 5 degrees C for 2 or 4 days. Spermatozoa were collected from the caudae epididymidis from 16 dogs. Total motility, plasma membrane integrity and acrosome integrity were evaluated immediately on harvesting, and after 2 and 4 days of storage at 5 degrees C, and at 0 and 2 h post-thaw at 37 degrees C. Sperm motility decreased significantly during cold storage, compared to freshly harvested spermatozoa (P < 0.001). Although there was no significant effect of pre-freeze storage time on post-thaw motility, there was a tendency towards decreased motility in spermatozoa that had been stored for 4 days, compared to spermatozoa that were frozen immediately after collection (P = 0.09). The number of post-thaw spermatozoa with an intact plasma membrane was decreased in spermatozoa cold-stored for 4 days (P < 0.001). There was no significant effect of pre-freeze storage time on the acrosomal status of post-thaw spermatozoa. In conclusion, canine epididymal spermatozoa were stored at 5 degrees C for up to 4 days without a clear detrimental effect on post-thaw motility and acrosome integrity, but storage may have decreased post-thaw motility. Results were, however, generally low.     

Cohesive properties of mammalian epididymal spermatozoa

Changing spermatozoan associations were observed in the epididymides of several mammals. These associations ranged from closely interwoven cylindrical bodies, found in the proximal part of the epididymis, to disorganized masses of spermatozoa, found in the distal part of the duct. It is suggested that changes in the cohesive properties of epididymal spermatozoa resulted in the formation and fragmentation of cylindrical bodies. These bodies, differeing in pattern and complexity according to the species, were found in all investigated mammals, including man. Cohesiveness appeared first in the upper part of the epididymidis, where it was confined to the spermatozoan tails. In general, there was a diminution of cohesive forces as the spermatozoa passed down the epididymal duct; consequently, the cylindrical bodies turned into disorganized masses of spermatozoa. There are indications that changes in the cohesive properties of spermatozoa may represent one aspect of spermatozoan maturation.     

Silver-staining patterns of mammalian epididymal spermatozoa

Epididymal spermatozoa from 12 species of mammals were stained using silver nitrate and examined with the light microscope. Silver nitrate differentiates many of the gross morphological features of spermatozoa, including the acrosome, subacrosomal region, perforatorium, postacrosomal sheath, neck, dense outer fibers of the core of the midpiece, annulus, principal piece, and end piece. Silver-staining patterns of spermatozoa reveal both species-specific and strain-specific differences, particularly of the sperm head. The biochemical basis of silver staining may be due in part to the presence of sulfhydryl- and disulfide-rich proteins; however, it cannot be explained entirely by the presence of these moieties. The detail obtained using silver nitrate staining, coupled with the ease and rapidity of the procedure, should be useful to workers in many areas of biological and medical research.     

Improvements and short-term viability of mouse epididymal spermatozoa recovered through the Spermprep filtration method

This study was designed to determine the effects of Sephadex filtration (Spermprep(trade mark)I method) on the separation of motile, morphologically normal, mouse epididymal spermatozoa and to study the viability of the recovered spermatozoa over a 3-h incubation period. Spermatozoa were harvested from the caudae epididymie (5 animals per run or replication; n=10) following bilateral testicular excision, after which they were incubated in 2-ml of Test-Yolk buffer (TYB) at 37 degrees C for 15-min. The specimens were then split into 2 1-ml aliquots, with Aliquot 1 as the control and Aliquot 2 as the filtered sample. The Spermprep(trade mark)I column was employed according to the manufacturer's specifications using TYB. During filtration (10-min), 2 different fractions were obtained: first 5-min (Sample 1) and second 5-min (Sample 2). The 2 fractions were evaluated and incubated at 37 degrees C and assessed for percentage of motility and grade of motility (0 to 4) every 30-min for 3-h. Filtration resulted in a significant improvement in the percentage and grade of motility (91.5% and 3.0 vs 76.5% and 2.5, respectively). The results indicate that filtration with the Spermprep(trade mark)I method improved the percentage and grade of motility (P<0.05) but not the percentage of normal morphology of the spermatozoa. In addition, the Spermprep(trade mark)I method enabled the recovery of 45% (8.3 x 10(6) spermatozoa recovered) of the total number of spermatozoa processed in the control aliquot (18.4 x 10(6) spermatozoa), which is consistent with previous observations. Most importantly, filtered spermatozoa incubated for 3-h showed a greater percentage and grade of motility than the control spermatozoa (63% and 1.66 vs 39% and 0.82, respectively. The Spermprep(trade mark)I filtration method selected a higher proportion of quality spermatozoa, which also displayed significant long-term motility (longevity) during in vitro incubation.     

Undiluted or extended storage of ram epididymal spermatozoa as alternatives to refrigerating the whole epididymes

The effect of storage procedure at 5°C on the quality of ram spermatozoa from the cauda epididymis was analyzed. Two strategies were tested at 0, 24, 48 and 72h post-mortem: (1) spermatozoa held in the epididymal fluid and stored either in the cauda epididymis (In-EPID) or in vitro (Ex-EPID), (2) epididymal spermatozoa extended in three media at 320, 370 and 420 mOsm/kg (D320, D370, D420). Analyzed parameters were: osmolality, pH, motility, acrosomal status and viability. In experiment 1, osmolality of the In-EPID samples, but not in Ex-EPID, increased with post-mortem time. Motility of In-EPID spermatozoa in samples, after 24h post-mortem, was higher compared to the Ex-EPID samples, although differences decreased at 48 and 72h. In experiment 2, total (TM) and progressive motility (PM) were not significantly affected by storage time for D320 and In-EPID samples. However, the motility of D370 and D420 samples significantly decreased with time. TM and PM of D320 were significantly higher than D370 and D420 at 72h. At 24h, sperm viability was higher for In-EPID (80.7±3.4%) than for the extended samples (44.8±2.9%, 37.7±3.9% and 48.6±6.0% for D320, D370 and D420, respectively), which also decreased faster with time. At 24h, the percentage of damaged acrosomes was low and similar for the four methods of storage, but damaged acrosomes increased with time for D320 and D370. Storing the spermatozoa in the epididymis is a good strategy for maintaining sperm quality in ram, at least for 48h. The D320 extender preserve motility of epididymal spermatozoa but does not protect the status of the acrosome.     

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.