Unistellate spermatozoa of decapods: comparative evaluation and evolution of the morphology

Decapod unistellate spermatozoa are primarily characterized by the presence of a single appendage (spike) extending from the acrosome. Among decapods, this type of spermatozoon is found only in shrimps of the families Sicyoniidae, Penaeidae, and Solenoceridae (suborder Dendrobranchiata) and of the infraorder Caridea (suborder Pleocyemata). This review comparatively discusses the morphological diversity of unistellate spermatozoal ultrastructure among these decapods, as well as the role of the primary structures involved in the fertilization and spermatozoal capacitation. Furthermore, the use of the unistellate spermatozoal ultrastructure to support phylogenetic relationships and of the current phylogenetic evidences to investigate the evolution of spermatozoa of decapods is discussed. Morphologically, the main differences between caridean and dendrobranchiate unistellate spermatozoa are the shape of the main body (inverted cup-shaped, and spherical, bulged or elongate, respectively) and complexity of the acrosomal region. The latter is directly related to the type of fertilization. For example, dendrobranchiates have more complex acrosomal regions than that carideans, and fertilization involves a visible acrosome reaction, which is not observed in carideans. Ultrastructural changes of spermatozoa throughout capacitation are unknown in carideans, but for dendrobranchiates generally occur in the acrosomal vesicle and subacrosomal region throughout attachment of the spermatophore to the thelycum, enabling fertilization by the spermatozoa. Comparative evaluation of spermatozoal morphology and current phylogenetic evidences corroborates the hypothesis that the spermatozoal spike of carideans and dendrobranchiates is the result of convergent evolution.     

Effect of cholesterol and phosphatidylcholine of different chain length on acrosome breakdown and fertilizing capacity of amphibian spermatozoa

The effect of different lipids on the fertilizing capacity of Bufo arenarum spermatozoa and on acrosome breakdown of Leptodactylus chaquensis spermatozoa was studied. Sonicated vesicles of egg yolk phosphatidylcholine (1 mM) were as effective as vesicles of egg yolk phosphatidylcholine:cholesterol (molar ratio 1:0.9) in inhibiting the fertilizing capacity of Bufo arenarum spermatozoa. This suggests that cholesterol depletion from the spermatozoa was not the cause of the fertility loss. Bufo arenarum spermatozoa were incubated with phosphatidylcholines with even chain length from 6 to 18 carbons. At a concentration of 0.01 mM, didecanoyl-phosphatidylcholine reduced fertilizing capacity to 10% in a few minutes and to 0% within 60 minutes. Didodecanoyl-phosphatidylcholine required 2 hours to reduce fertility to 10% and 4 hours to cause a 100% loss of fertilizing capacity. A concentration of didecanoyl-phosphatidylcholine as low as 5 × 10^?4 mM caused a more than 95% fertility loss in less than five minutes. At a concentration of 0.1 mM, didecanoyl-phosphatidylcholine induced complete acrosome breakdown in Leptodactylus chaquensis spermatozoa in 15 minutes, whereas didodecyl-phospatidylcholine required 2 hours. At a concentration 100-fold lower didecanoyl-phosphatidylcholine induced complete acrosome breakdown in 2 hours. Electron microscopic observations in both species showed loss of acrosome caused by the action of the didecanoyl-phosphatidylcholine. Longer chain phosphatidylcholines exerted an inhibitory effect on Bufo arenarum spermatozoa fertilizing capacity at a higher concentration when in a vesicular form.     

Induction of acrosome reaction of spermatozoa in <Emphasis Type="Italic">Eriocheir sinensis</Emphasis> by low temperature

The effects of temperatures, durations of treatment, and derivations from spermatophores or spermaries on in vitro acrosome reaction of the spermatozoa in the Chinese mitten crab Eriocheir sinensis were investigated. The results showed that the different temperatures resulted in extremely significant differences (p < 0.01) in the time of beginning acrosome reaction, the time of the maximum percentage of acrosome reaction, and the maximum percentage of acrosome reaction of the spermatozoa from spermatophores; and the low temperature (−20, −80 °C and liquid nitrogen) induced acrosome reaction of more than 90% spermatozoa while 15 and 4 °C didn’t. Similar results occur in the spermatozoa, treated with −80 °C for 15 min, from spermaries but the time of beginning acrosome reaction and the time of the maximum percentage of acrosome reaction were obviously longer than those from spermatophores. In conclusion, low temperature can induce acrosome reaction, which is a novel and efficient operating method of inducing acrosome reaction; the spermatozoa might be affected physiologically to capacitate with chilling. The study may be beneficial to new understandings of mechanism of acrosome reaction and provide the foundational material for artificial fertilization and breeding of this crab and other commercial aquatic crustaceans.     

Fluorescent localization of thiols and disulfides in marsupial spermatozoa by bromobimane labelling

The acrosome of marsupial spermatozoa is a robust structure which, unlike its placental counterpart, resists disruption by detergent or freeze/thawing and does not undergo a calcium ionophore induced acrosome reaction. In this study specific fluorescent thiol labels, bromobimanes, were used to detect reactive thiols in the intact marsupial spermatozoon and examine whether disulfides play a role in the stability of the acrosome. Ejaculated brushtail possum (Trichosurus vulpecula) and tammar wallaby (Macropus eugenii) spermatozoa were washed by swim up and incubated with or without dithiothreitol (DTT) in order to reduce disulfides to reactive thiols. Spermatozoa were then washed by centrifugation and treated with monobromobimane (mBBr), a membranepermeable bromobimane, or with monobromotrimethylammoniobimane (qBBr), a membrane-impermeable bromobimane. Labelled spermatozoa were examined by fluorescence microscopy and sperm proteins (whole sperm proteins and basic nuclear proteins) were analysed by gel electrophoresis. The membrane-permeable agent mBBr lightly labelled the perimeter of the acrosome of non-DTT-treated possum and wallaby spermatozoa, indicating the presence of peri-acrosomal thiol groups. After reduction of sperm disulfides by DTT, mBBr labelled the entire acrosome of both species. The membrane-impermeable agent qBBr did not label any part of the acrosome in non-DTT or DTT-treated wallaby or possum spermatozoa. Thiols and disulfides are thus associated with the marsupial acrosome. They are not found on the overlying plasma membrane but are either in the acrosomal membranes and/or matrix. The sperm midpiece and tail were labelled by mBBr, with increased fluorescence observed in DTT-treated spermatozoa. The nucleus was not labelled in non-DTT or DTT-treated spermatozoa. Electrophoretic analysis confirmed the microscopic observations: Basic nuclear protein (protamines) lacked thiols or disulfide groups. Based on these findings, the stability of the marsupial acrosome may be due in part to disulfide stabilization of the acrosomal membranes and/or acrosomal matrix. In common with placental mammals, thiol and disulfide containing proteins appear to play a role in the stability of sperm tail structures. It was confirmed that the fragile marsupial sperm nucleus lacked thiols and disulfides. © 1994 Wiley-Liss, Inc.     

Ultrastructure of the spermatozoon of Myrmecocichla formicivora (Vieillot, 1881) and Philetairus socius (Latham, 1790) (Aves; Passeriformes), with a new interpretation of the passeridan acrosome

Passerine spermatozoa exhibit apomorphies that distinguish them from non‐passerine neognaths and palaeognaths. The acrosome is longer than the nucleus (excepting the suboscines, most Corvida, and a few Passerida). A perforatorium and endonuclear canals are absent. The proximal centriole is absent (except in the suboscines). The distal centriole is secondarily short, contrasting with its elongate condition in palaeognaths and Galloanserae. In the Passerida a single mitochondrial strand winds extensively along the axoneme (restricted to the anterior axoneme in suboscines and Corvida). A fibrous, or amorphous, periaxonemal sheath, seen in palaeognaths and many non‐passerines, respectively, is absent. The acrosome in Myrmecocichla formicivora and Philetairus socius is bipartite: an acrosome core is surmounted by an acrosome crest; the core is ensheathed by a layer which is a posterior extension of the crest. The acrosome helix is a lateral extension of the crest and the crest layer with (Myrmecocichla) or without (Philetairus) protrusion of material of the acrosome core into it. In M. formicivora, as in other muscicapoids, a fibrous helix is intertwined with at least the more proximal region of the mitochondrial helix. The fibrous helix is absent at maturity in Philetairus and other described passeroid spermatozoa with the possible exception of Passer italiae. In Philetairus a granular helix precedes the mitochondrial helix.     

The acrosome in ascidians. I. Pleurogona

Summary

A vesicle which contains moderately electron-dense material has been found at the apex of mature spermatozoa in all representatives of three pleurogonan families: in Styela clava, Cnemidocarpa finmarkiensis and Botryllus schlosseri (family Styelidae), in Boltenia villosa and Herdmania momus (family Pyuridae), and in Molgula manhattensis (family Molgulidae). The vesicle described here resembles the acrosome of Ciona intestinalis spermatozoa. The Ciona acrosome shows structural changes at fertilization (Fukumoto, M., J. Ultrastruct. Res., 87 (1984) 252–262). This suggests that pleurogonan spermatozoa also have an acrosome. Some speculations are presented on ascidian fertilization.     

Acrosome staining and motility characteristics of sterlet spermatozoa after cryopreservation with use of methanol and DMSO

In this study we describe acrosome staining and motility characteristics of fresh and cryopreserved sterlet (Acipenser ruthenus L.) spermatozoa using soybean trypsin inhibitor-Alexa conjugate fluorescent staining and computer-aided sperm analysis (CASA), respectively. Methanol or dimethylsulfoxide (DMSO) were used as cryoprotectants. After cryopreservation a decline in sperm motility characteristics occurred, but no differential effect between cryoprotectant was observed. Cryopreservation caused a significant increase in the percentage of spermatozoa with acrosome stained by SBTI-Alexa for samples cryopreserved using DMSO compared to methanol. These data suggest that the low usefulness of DMSO for cryopreservation of sturgeon spermatozoa is related to its harmful specific effect towards the acrosome, probably by causing its precocious triggering, much before any egg contact.     

RELATION BETWEEN THE ACROSOME REACTION AND FERTILIZATION IN THE SEA URCHIN. II. FERTILIZATION IN ACIDIFIED SEA WATER WITH EGG-WATER-TREATED SPERMATOZOA*

Fertilization of sea urchin eggs fails to occur at a pH lower than 6.5. Analytical studies on this problem were made with Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudocentrotus depressus. If the spermatozoa have been pretreated with egg water, eggs can be fertilized even at pH 6.5 and 6.0. The acrosome reaction is inhibited at a pH lower than 6.5. Intact spermatozoa fail to adhere to the fixed eggs in acidified sea water, whereas egg-water-treated spermatozoa adhere even at pH 6.5 and 6.0. From these results we infer that the failure of fertilization at pH 6.5–6.0 is caused by non-occurrence of the acrosome reaction, and that fertilization reactions other than the acrosome reaction, such as the binding and fusion of the gametes, are not inhibited in this range of pH. At pH 5.5, the spermatozoa become inert and fertilization is inhibited or suppressed, even though egg-water-treated spermatozoa are employed.     

Ultrastructure of the Geogarypus nigrimanus Spermatozoon (Arachnida,Pseudoscorpionida)

The ultrastructure of the spermatozoon of Geogarypus nigrimanus (Arachnida, Pseudoscorpionida) is described. The spermatozoon is composed of a small elliptic nucleus, a short flagellum and a very long and complex acrosome. In the male genital ducts, as in other studied species of pseudoscorpions, the sperm components are rolled up to form a globular structure enclosed in a cyst wall. The Geogarypus spermatozoon with a reduced flagellum and a giant acrosome seems to be evolutionary more advanced than spermatozoa from other pseudoscorpions.     

Spermatozoon Ultrastructure of Two Holothurian Species of the Genus Cucumaria (Dendrochirotida,Holothuroidea) from the Sea of Japan

The ultrastructure of spermatozoa of Cucumaria japonica and a congeneric morphologically similar deep-sea species was studied. The spermatozoa of both C. japonica and C. conicospermium are similar to those of other holothurians: the acrosome is composed of an acrosomal granule and periacrosomal material; the centrioles lie at an acute angle to one another; and the proximal centriole is connected to the nuclear envelope by a flagellar rootlet. The spermatozoa of C. japonica differ from those of C. conicospermium in the shape of the head and the dimensions and position of the acrosome. In C. japonica, the acrosome is completely embedded in the nuclear fossa and measures 0.7 m. In C. conicospermium, only one-third of the acrosome is embedded in the nuclear fossa; this acrosome measures 1.3 m. A correlation between the structure of the sperm acrosome and that of the egg envelope is discussed.     

A comparative study on the fine structure of developing spermatozoa in the isopod,Oniscus asellus,and the amphipod,Orchestoidea sp.

Summary Developing spermatids and mature spermatozoa from the isopod, Oniscus asellus and the amphipod, Orchestoidea sp. have been examined with the light microscope and the electron microscope and have been found to have similar morphologies. As spermiogenesis proceeds the nucleus migrates to one pole of the spermatid at which point an acrosome, contiguous rod, and cross-striated tail develop. The acrosomal vesicle elongates to a cone-shaped, mature acrosome lying at the apex of a cross-striated tail and nucleus which are situated at approximate forty-five degrees to each other. The cross-striated tail originates as an evagination of the spermatid plasma membrane near the acrosomal vesicle. The tail eventually grows to lengths of four to five hundred microns. The mature, tail-like appendage is cross-striated at major 750 to 800 Å, and minor 125 to 150 Å, periodicities. When observed in vitro, mature sperm of both species appear non-motile.Possible homologies of this unusual spermatozoon with other types of spermatozoa are made and it is concluded that: 1) isopod and amphipod spermatozoa should be classified as non-flagellate; 2) the cross-striated tail, previously thought to be a flagellum, is a non-motile structure associated in development and possible function with the acrosome; and 3) the rodlike structure contiguous with the acrosome is similar to perforatoria described in some vertebrate sperm.Supported by U.S.P.H.S. Grant No. NB-06285 and Training Grant No. 5-Tl-GM-202. — The author wishes to express his grateful appreciation for the technical assistance given by Miss Ann Barnett during the course of this investigation.     

Quantitative changes ofRicinus communis agglutinin I andHelix pomatia lectin binding sites in the acrosome of rat spermatozoa during epididymal transit

Summary During passage through the epididymis, spermatozoa undergo a number of changes which result in their acquisition of fertility and motility. Some of the changes that occur include loss of the cytoplasmic droplet and changes in sperm morphology, metabolism and properties of the nucleus and plasma membrane. Changes have also been reported in the acrosomic system of mammalian spermatozoa during their transit through the epididymis. In the present study, the quantitative changes of the glycoconjugate content in the acrosome of rat spermatozoa were examined during their passage through the epididymis using lectin-colloidal gold cytochemistry. Various regions of the epididymis (initial segment, caput, corpus and cauda epididymidis) were fixed by perfusion with 1% or 2% glutaraldehyde buffered in sodium cacodylate (0.1M), dehydrated in ethanol and embedded without osmication in Lowicryl K4M. Lectin-colloidal gold labeling was performed on thin sections usingRicinus communis agglutinin I (RCA I) orHelix pomatia lectin (HPL) to detectd-galactose-andN-acetyl-d-galactosamine-containing glycoconjugates, respectively. The labeling density over the acrosome of the acrosomic system was evaluated as the number of gold particles per m² of profile area using a Zeiss MOP-3 image analyzer. The overall mean labeling densities over the acrosome of spermatozoa for each lectin was estimated from 4 rats and over the four distinct epididymal regions. The mean labeling density of the acrosome with RCA I and HPL showed a similar pattern along the epididymis, although RCA I revealed approximately twice as many gold particles per epididymal region. In either case, there was a significant decrease in the labeling density of the acrosome of spermatozoa between the initial segment or caput epididymidis and cauda epididymidis (p<0.01). A similar decrease was also noted between the initial segment and corpus epididymidis (p<0.01). No change was found between the initial segment and caput epididymidis. Controls showed a virtual absence of labeling. These results suggest that in addition to a multitude of changes occurring to spermatozoa during epididymal transit, there are also significant quantitative changes in the glycoconjugate content within the acrosome.     

Lysophosphatidylcholine disrupts the acrosome of tammar wallaby (macropus eugenii) spermatozoa

The acrosomal status of wallaby spermatozoa was evaluated by light and electron microscopy after incubation in 1–100 μM lysophosphatidylcholine (LPC) for up to 120 min. Treatment with 1 and 10 μM LPC for 120 min did not lead to acrosomal loss, or detectable alteration to the acrosome, as detected by Bryan's staining and light microscopy. Incubation with 25 μM LPC had little effect on acrosomal loss, however statistically significant changes (P < 0.05) in the acrosomal matrix (altered) were detected after 10-min incubation by light microscopy. Around 50% of acrosomes were altered after 20-min incubation in 50 μM LPC (P < 0.001), and 40% of spermatozoa had lost their acrosome after 60-min incubation (P < 0.001). Treatment with 75 and 100 μM LPC led to rapid acrosomal loss from around 50% of spermatozoa within 10 min (P < 0.001), and by 60 min acrosomal loss was 70–80%. LPC, like the diacylglycerol DiC₈ (1,2-di-octanoyl-sn-glycerol), is thus an effective agent to induce loss of the relatively stable wallaby sperm acrosome, and it also induces changes within the acrosomal matrix. Ultrastructure of the LPC-treated spermatozoa revealed that the plasma membrane and the acrosomal membranes were disrupted in a manner similar to that seen after detergent treatment (Triton X-100). There was no evidence of point fusion between the plasma membrane overlying the acrosome and the outer acrosomal membrane. The plasma membrane was the first structure to disappear from the spermatozoa. The acrosomal membranes and matrix showed increasing disruption with time and LPC concentration. Wallaby spermatozoa incubated with LPC at concentrations that induced significant acrosomal loss also underwent a rapid decline in motility that suggested that acrosomal loss may be due to cell damage, rather than a physiological AR. This study concluded that LPC-induced acrosomal loss from tammar wallaby spermatozoa is due to its action as a natural detergent and not as a phosphoinositide pathway intermediate. The study further demonstrates the unusual stability of the marsupial acrosomal membranes. © 1993 Wiley-Liss, Inc.     

Inhibition of the human sperm acrosome reaction by proteinase inhibitors

The analogue of the second messenger cAMP, dibutyryl cAMP (dbcAMP), was shown to induce the human sperm acrosome reaction to the same extent as calcium ionophore A23187, providing preliminary evidence for the involvement of the adenylate cydase system in the acrosome reaction (AR) of human spermatozoa. Using the human synchronous acrosome reaction system, proteinase inhibitors were tested for their effect on the dbcAMP-induced human sperm acrosome reaction. The proteinase inhibitor 4′-acctamidophenyl4-guanidinoben-zoate (AGB), an inhibitor of proacrosin activation and of acrosin, when added at either the onset of incubation or to capacitated spermatozoa, 5 min prior to stimulation by dbcAMP, significantly (P < 0.01) inhibited the acrosome reaction at final concentrations of 1 × 10^?4 M to 1 × 10^?6 M in comparison to dbcAMP treatment alone. At concentrations less than 1 × 10^?6 M, no significant inhibitory effect was seen. Similarly, para-aminobenzamidine (pAB), also an inhibitor of proacrosin activation and of acrosin, significantly (P < 0.01) inhibited the dbcAMP-induced acrosome reaction at final concentrations of 1 × 10-4 M to I × 10-6 M when added at either the onset of incubation or to capacitated spermatozoa, 5 min prior to stimulation by dbcAMP, in comparison to stimulation by dbcAMP alone. However, at concentrations less than 1 × 10^?6 M, no significant (P > 0.05) inhibitory effect was seen. These results indicate that a serine proteinase, most likely acrosin, has a role in the human sperm acrosome reaction and suggest that the enzyme functions after the involvement of the adenylate cyclase system.     

Ultrastructure of spermatozoa of members of Calappidae,Aethridae and Menippidae and discussion of their phylogenetic placement

The families Aethridae and Calappidae were originally considered as part of the same family; however, their morphology and molecular biology separate them into two families. In this context, we describe the ultrastructure of spermatozoa of species of the Calappidae, Aethridae and Menippidae to elucidate the relationships among taxa. The vasa deferentia were submitted to routine protocols for transmission electron microscopy. Our results indicate that the morphology of the spermatozoa of Hepatus pudibundussupports its exclusion from the Superfamily Calappoidea due to the presence of the apical striated layer. The spermatozoa of Menippe nodifrons is very similar to H. pudibundus and corroborates the recent phylogenetic analysis using sequence data of nuclear genes. Moreover, our results evidence two morphological patterns of spermatozoa within Calappidae. Calappa ocellata and C. cinerea show spermatozoa with a wide acrosome vesicle, a thick operculum shaped as a shallow “W” and a large thickened ring. Calappa gallusand C. hepatica show spermatozoa with a longer acrosome vesicle, a pointed operculum and a slender thickened ring. Our ultrastructure results conform with previous molecular proposal and show that spermatozoa ultrastructure can be an effective tool to adjust phylogenetic relationship when used in association with molecular data.     

Ultrastructure of spermatozoa and spermatophores of old world freshwater crabs (Brachyura: Potamoidea: Gecarcinucidae,Potamidae, and Potamonautidae)

We investigated the ultrastructure of spermatozoa and spermatophores of 19 palaeotropical freshwater crab species [12 species of the Gecarcinucidae, 6 of the Potamidae (Potamiscinae), and 1 species of the Potamonautidae (Deckeniinae: Hydrothelphusini)]. The investigated Potamiscinae have densely packed coenospermic spermatophores with the exception of Thaiphusa sirikit and Johora singaporensis that exhibit cleistospermia. In contrast, in the Gecarcinucidae the spermatozoa are loosely embedded in a mucous matrix. The gecarcinucid and potamiscine sperm differ, furthermore, in acrosomal structure and size. The acrosome in the Gecarcinucidae is much smaller and spherical, while the larger acrosome in the Potamiscinae has the tendency to be depressed. In the Potamiscinae, an additional middle acrosomal zone evolved between the acrosome ray zone and the outer acrosomal zone. Within the Gecarcinucidae, a differentiation into two groups (Gecarcinucinae and Parathelphusinae) is not supported by the present spermatological data. The sperm morphology of Hydrothelphusa aff. madagascariensis (Potamonautidae: Deckeniinae) differs from Potamonautes sidneyi (Potamonautidae: Potamonautinae) in acrosomal size and shape, and in the absence of a periopercular rim. A closer relationship of Deckeniinae and Gecarcinucidae cannot be confirmed by spermatology. J. Morphol., 2009. © 2008 Wiley‐Liss, Inc.     

Ram spermatozoa cocultured with epithelial cell monolayers: An in vitro model for the study of capacitation and the acrosome reaction

The effects of different epithelial cells, namely, hamster oviduct, sheep oviduct, and pig kidney epithelial cells (IBRS-2), on the viability, percentage of progressive motility (PPM), and acrosome reactions of ejaculated ram spermatozoa were investigated. Sperm aliquots were cultured on cells, cell-conditioned medium 199, or control medium 199. The PPM of unattached spermatozoa was estimated after 0, 3, 6, 9, 12, and 24 hr of incubation at 37°C under 5% CO₂ in air. Viability and the occurrence of true acrosome reactions were assessed using a triple-stain technique. Spermatozoa started to attach within 1 hr of coculture with the hamster or sheep oviductal epithelial cell (OEC) monolayers, and these spermatozoa showed vigorous tail motion. No spermatozoa were found to attach to the IBRS-2 monolayer. The PPM of unattached spermatozoa cocultured with the various types of epithelial cell monolayers for 12 hr was significantly higher than that of spermatozoa incubated in conditioned media or medium 199 alone (54% in hamster OEC vs. 40% in conditioned; 68% in sheep OEC vs. 38% in conditioned; 36% in control medium). On the other hand, after 24 hr of incubation, there were no differences in the PPM of spermatozoa cocultured with epithelial cells or incubated in conditioned media. The percentages of cells undergoing a true acrosome reaction reached maximum values (P < 0.05) in spermatozoa incubated for 9 hr in the presence of hamster OEC (22.5%) or for 12 hr on sheep OEC (20.5%) monolayers. IBRS-2, a commercial nonreproductive cell type, had a positive influence on both PPM and sperm viability but no effect on the occurrence of the acrosome reaction. Interactions leading to the acrosome reaction were thus observed only when spermatozoa were cocultured with OEC monolayers. The values of PPM in unattached sperm cells seen after 12 hr of coculture with OEC or IBRS-2 were still at a high level (52–67%) for in vitro fertilization. The coculture with OECs provides an “in vitro” model to study the capacitation processes in a situation that may resemble that occurring in vivo. Moreover, the coculture with hamster OECs may provide a convenient and standardized in vitro system to study mechanisms underlying capacitation and the acrosome reaction. © 1993 Wiley-Liss, Inc.     

Viability, acrosome morphology and fertilizing capacity of boar spermatozoa treated with strontium chloride

The positive effect of strontium ions (Sr2+) on sperm motility, capacitation and acrosome reaction has been demonstrated in the mouse, human, guinea pig and hamster. In the present study, we have evaluated the effect of Sr2+ on the viability and acrosome morphology of boar spermatozoa, and on the fertilization and development after the microinjection of Sr(2+)-treated spermatozoa into porcine oocytes. Before incubation, 79% of spermatozoa were classified as propidium iodide (PI)-negative (live) using the LIVE/DEAD Sperm Viability Kit. After incubation with strontium chloride (SrCl2), 39% (0 mM; no divalent cations), 25% (1.9 mM) and 24% (7.5 mM) of them were classified as PI-negative. The proportion of spermatozoa that had initiated the acrosome reaction was higher in Sr(2+)-containing medium than in Sr(2+)-free medium, when assessed by electron microscopy. There was no significant difference in percentage of spermatozoa initiating the acrosome reaction between Sr2+-treated groups (1.9 mM: 22%, 7.5 mM: 33%, p>0.05). After the microinjection of spermatozoa incubated with SrCl2, 67% (1.9 mM) and 61% (7.5 mM) of injected oocytes were successfully fertilized, and then 43% (1.9 mM) and 41% (7.5 mM) contained a fully decondensed sperm head. Sham-injected oocytes were significantly activated at a lower rate than Sr(2+)-treated groups (27%, p<0.05). Next, after microinjection of spermatozoa incubated with 1.9 mM SrCl2 (n=51), 45% of injected oocytes cleaved on day 2, and 18% developed to blastocysts on day 7 (sham-injection, n=48: 15% to cleavage and 0% to blastocyst). These results demonstrate that Sr2+ is likely to positively affect the fertilizing capacity of spermatozoa in the pig.     

Chlortetracycline analysis of boar spermatozoa after incubation,flow cytometric sorting,cooling, or cryopreservation

In this study, the effects of staining procedure with chlortetracycline (CTC) and method of analysis of boar spermatozoa after staining were examined. The hypothesis that incubation, flow cytometric sorting, cooling, and cryopreservation cause changes to boar sperm membranes which resemble capacitation and the acrosome reaction was also tested. Membrane status was evaluated by flow cytometry and by fluorescence microscopy after staining with CTC, and acrosome integrity was checked by flow cytometry after staining with FITC-pisum sativum agglutenin and propidium iodide (PI). Flow cytometry was also used to assess viability (percentages of live and dead cells) of boar sperm after staining with SYBR-14 and PI. Staining of spermatozoa with CTC alone and in combination with PI and/or Hoechst 33342 had no effect on the proportion of spermatozoa allocated to the F (uncapacitated), B (capacitated), or AR (acrosome-reacted) CTC fluorescent staining categories. The mean percentages of acrosome-intact and acrosome-reacted cells were 88.4 and 6.8 or 0.8 and 96.5 in semen treated with 0 or 100 μg/ml lysophosphatidylchloine (LPC), respectively (P < 0.001). Most spermatozoa were also in the AR CTC-stained category after treatment with LPC compared with a small proportion in the controls. Using flow cytometry to examine sperm suspensions stained with CTC, a gated population of spermatozoa with low fluorescence (population 1) comprised predominantly F-pattern cells (F-pattern: population 1 vs. population 2, 80.5 vs. 14.4%; P < 0.001), whereas population 2 (high fluorescence) comprised mainly B-pattern cells (B-pattern: population 1 vs. population 2, 8.5 vs. 62.3%; P < 0.001). Incubation (38°C, 4 hr), flow sorting, cooling (to 15 or 5°C) and freezing reduced the proportion of F-pattern and live spermatozoa, and increased the proportion of B-, AR-pattern, and dead spermatozoa, in comparison with fresh semen. There were more membrane changes in spermatozoa cooled to 5°C (30.4, 48.5, 21.1%) than in those cooled to 15°C (56.1, 32.6, 11.5% F-, B-, and AR-pattern spermatozoa, respectively). Mol. Reprod. Dev. 46:408–418, 1997. © 1997 Wiley-Liss, Inc.