Acrosome reaction in Octopus tankahkeei induced by calcium ionophore A23187 and a possible role of the acrosomal screw

The octopod sperm is unique especially in two aspects: the screw-shaped acrosome and its inner layered substructure (striation). The present study aims to investigate morphological changes of Octopus tankahkeei spermatozoa during the acrosome reaction (AR) and to pursue functions of the internal substructures revealed by inducing AR with the calcium ionophore A23187. Gradual changes of the spermatozoa were traced using fluorescence and electron microscopy. The AR process included the bulging, vesiculation, and dehiscence of the plasma membrane around the acrosome and the nucleus, as well as the vesiculation of the mitochondrial sheath. Membrane vesiculation outside the nucleus has never been reported in the order Octopoda. The rigid screw and the inner striation of the acrosome remained intact surmounting the nucleus, suggesting that these two structures have potential functions during fertilization. In addition, the detachment of the sperm head and the tail was commonly observed in this study, both in intact and acrosome-reacted sperm. Fluorescence microscopy revealed that the detached mitochondrial sheath usually gave weaker and more dispersive signals than the joint ones. This phenomenon implied that the intense energy release might promote the detachment of the mitochondrial sheath.     

The effects of different cryoprotectants and the temperature of addition on the survival of red deer epididymal spermatozoa

With the aim of finding an ideal cryoprotectant in a suitable concentration for red deer epididymal spermatozoa conservation, we evaluated the effects of four most commonly used cryoprotectants (CPAs), Glycerol (G), Ethylene glycol (EG), Propylene glycol (PG), and Dimethyl sulfoxide (DMSO), on the sperm survival. Besides, the effects of two temperatures of CPA addition--22 degrees C (ambient temperature) and 5 degrees C--on sperm quality were also tested. For each temperature tested, sperm samples were evaluated after 0, 15, 30 and 60 min of spermatozoa exposition to CPAs. Thus, sperm quality was in vitro judged by microscopic assessments of individual sperm motility (SMI), and of plasma membrane (Viability) and acrosome (NAR) integrities. Overall, DMSO showed the highest toxicity for red deer epididymal spermatozoa, and glycerol the lowest. Thus, at 60 min of incubation SMI results showed that the toxicity to red deer epididymal spermatozoa of the four CPAs are in the following sequence: G approximately = EG approximately = PG < DMSO ('less than' symbol means P < 0.05, and approximate symbol means P = 0.08). Furthermore, our results also showed a differential response of acrosome membrane to temperature of CPAs addition. Regardless of the CPA used, statistically significant variations (P < 0.05) were found between the two temperatures of addition of CPAs for acrosome integrity, the best being 22 degrees C (NAR = 83.8% vs. 69.8%). These data indicate that sperm quality of red deer epididymal spermatozoa, in addition to be affected by the cryoprotectant, can also be influenced by the temperature at which CPAs are added prior to freezing.     

Ultrastructure of the spermatozoa of Cicadella viridis (Linnaeus) and its bearing on the phylogeny of Auchenorrhyncha

The ultrastructure of mature spermatozoa of the leafhopper Cicadella viridis (Linnaeus) was investigated using light and transmission electron microscopy. The spermatozoon is composed of a head containing an acrosome and an elongated nucleus, and a long tail, which consists of a flagellum. The acrosome is conical and invaginated to form a subacrosomal space, and the acrosomal contents are filled with electron-dense tubular substructures. The nucleus is linear and filled with homogeneously condensed chromatin. The centriolar adjunct is parallel to the nucleus and connects the nucleus with the mid-piece/flagellum. The flagellum is formed by a 9+9+2 axoneme, two mitochondrial derivatives and two accessory bodies. The mitochondrial derivatives with an orderly array of peripheral cristae are symmetrical. The accessory bodies are small and slightly elliptical. The end of the axoneme shows progressive loss of microtubules. Comparison of sperm ultrastructure of C. viridis with those of other Auchenorrhyncha families supports the major relationships within Cicadomorpha as (Membracoidea (Cicadoidea, Cercopoidea)).     

Effect of the extender supplement Equex-STM on cryopreserved semen in the Assaf sheep

This study was designed to evaluate the effect of adding the detergent Equex-STM to the extender used to dilute semen for cryopreservation on several indicators of sperm preservation. Two consecutive ejaculates per day were obtained from 5 Assaf sheep on two days out of every week over three alternate months. The freezing protocol involved diluting the semen in Fiser's extender, to which 0.7 percent Equex-STM was added or omitted before cryopreserving the semen in straws by exposure to nitrogen vapor. Equex-STM supplementation gave rise to significantly (p=0.05) improved sperm quality variables after different periods of freezing (0 hours, 1 week and 1 month). The variables examined were: individual motility, viability, acrosome integrity, plasma membrane integrity (HOS test) and morphological anomalies. This improvement was independent of the ram and month of testing. In a second experiment in which we incubated the semen (0 and 6 hours) at 37 degree C after thawing, Equex-STM also showed a beneficial effect on sperm quality.     

Comparative ultrastructural study of spermatozoa in some oyster species from the Asian-Pacific Coast

Sperm organization in the oysters Crassostrea gigas, Crassostrea nippona, Crassostrea cf. rivularis and Saccostrea cf. mordax inhabiting Asian Pacific coast was studied. The spermatozoa of all studied species had a number of common morphological characters such as a cup-like acrosome with heterogeneous matrix on its top, an axial rod in the subacrosomal space, a barrel-shaped nucleus, four mitochondria in the midpiece, pericentriolar complexes, and a 9+2-organized flagellum. The spermatozoa of C. cf. rivularis differed from the other species by having cytoplasm processes in the midpiece region. Such structures have never been described in the Ostreidae. Additionally, each species could be identified by the shape and size of sperm compartments (acrosome, nucleus, anterior nuclear fossa). The most significant interspecific difference was found in the size of an anterior nuclear fossa. The smallest anterior nuclear fossa was found in C. cf. rivularis (about 0.24 μm in length reaching about 22% of the nuclear length) while the biggest in C. gigas from the Sea of Japan (about 0.53 μm in length reaching about 46% of the nuclear length). The spermatozoa of C. gigas collected from the Sea of Japan and Taiwan Strait differed significantly in almost all the studied parameters. Since sperm morphology has been successfully used for species differentiation, this suggests the existence of two species rather than two populations. The data obtained indicate the species-specific difference in the sperm ultrastructure within the Ostreidae, which may be identified both ultrastructurally and morphometrically.     

Ultrastructure of the mature spermatozoon of the bivalve Scapharca broughtoni (Mollusca: Bivalvia: Arcidae)

The ultrastructure of mature spermatozoa of the giant clam bivalve Scapharca broughtoni was investigated by transmission electron microscopy for the first time. The mature spermatozoon consists of a head which is composed of a cone-shaped acrosome, a round nucleus, and a tail region. A subacrosomal space contains an axial rod and a basal plate, the latter lying between the acrosome and the nucleus. Although the nucleus lacks an anterior invagination, an inverted shallow V-shaped posterior invagination is present within the nucleus. Within the middle portion of the spermatozoon lie five spherical mitochondria while the long whip-like end portion is composed of an axoneme with the typical 9 + 2 structure. Our conclusion is that the spermatozoon of S. broughtoni is of the type I anacrosomal “aquasperm”, and the morphology of acrosome and nucleus are an adaptation to external fertilization.     

Evaluation of damage in Pacific oyster (Crassostrea gigas) spermatozoa before and after cryopreservation using comet assay

We examined the applicability of the comet assay (single cell gel electrophoresis assay) to estimate the quality of frozen-thawed Pacific oyster (Crassostrea gigas) spermatozoa. Comet assay was performed on semen before and after cryopreservation followed by fluorescent staining with propidium iodide to assess DNA integrity. After cryopreservation, the percentage of spermatozoa with damaged DNA significantly increased, while only about half of the cells displayed intact DNA, even when protected with 10 percent DMSO. All the considered parameters (head length, head area, head intensity, total length, total area, total intensity, tail length percent, tail area percent, and tail intensity percent) were higher than the oyster sperm protected with 10 percent DMSO-artificial sea water after freezing and thawing. Only tail length percent, tail area percent, and tail intensity percent were increased significantly after cryopreservation. The tail length percent was found to be the most sensitive indicator of the cryopreservation-induced DNA damage. Our freeze-thawing procedure significantly affected oyster sperm DNA, as indicated by the reduced fertilization rate when frozen-thawed oyster sperm are used. Irreversible alteration of the genome may prevent fertilization or alter normal embryonic development. This study is the first to demonstrate that the comet assay is an inexpensive, rapid and sensitive method for determining DNA damage in Pacific oyster sperm quality assessments.     

High-pressure freezing and freeze-substitution fixation reveal the ultrastructure of immature and mature spermatozoa of the plant-parasitic nematode Trichodorus similis (Nematoda; Triplonchida; Trichodoridae)

The spermatozoa from testis and spermatheca of the plant-parasitic nematode Trichodorus similis Seinhorst, 1963 (Nematoda; Triplonchida; Trichodoridae) were studied with transmission electron microscopy (TEM), being the first study on spermatogenesis of a representative of the order Triplonchida and important to unravel nematode sperm evolution. Comprehensive results could only be obtained using high-pressure freezing (HPF) and freeze-substitution instead of chemical fixation, demonstrating the importance of cryo-fixation for nematode ultrastructural research. The spermatozoa from the testis (immature spermatozoa) are unpolarized cells covered by numerous filopodia. They contain a centrally-located nucleus without a nuclear envelope, surrounded by mitochondria. Specific fibrous bodies (FB) as long parallel bundles of filaments occupy the peripheral cytoplasm. No structures resembling membranous organelles (MO), as found in the sperm of many other nematodes, were observed in immature spermatozoa of T. similis. The spermatozoa from the uterus (mature or activated spermatozoa) are bipolar cells with an anterior pseudopod and posterior main cell body (MCB), which include a nucleus, mitochondria and MO appearing as large vesicles with finger-like invaginations of the outer cell membrane, or as large vesicles connected to the inner cell membrane. The peripheral MO open to the exterior via pores. In the mature sperm, neither FBs nor filopodia were observed. An important feature of T. similis spermatozoa is the late formation of MO; they first appear in mature spermatozoa. This pattern of MO formation is known for several other orders of the nematode class Enoplea: Enoplida, Mermithida, Dioctophymatida, Trichinellida but has never been observed in the class Chromadorea.     

Characterization of porcine oviductal epithelial cells, cumulus cells and granulosa cells-conditioned media and their ability to induce acrosome reaction on frozen-thawed bovine spermatozoa

The mammalian female reproductive tract cells have been wildely used in in vitro fertilization. We studied the secretory proteins of porcine oviductal epithelial cells (POEC), and cumulus cells (CC) co-cultured with granulosa cells (GC) in conditioned media (CM), and their ability in inducing acrosome reaction (AR) on frozen-thawed bovine spermatozoa. POEC and CC+GC were cultured in M 199 medium for 48, 96 and 144h prior to investigation of protein secretion. The results from SDS-PAGE showed secretory proteins sizes of about 17, 22 and >220kDa in both CM from POEC and CC+GC. To test the ability of CM from POEC and CC+GC in inducing AR, the CM was frozen for 1-3 months before incubating with frozen-thawed bovine spermatozoa. Percentages of the spermatozoa with AR were determined under an inverted microscope and it was found that the tested group 1 (incubated with fresh and frozen CM from POEC for 1-3 months) were 78.44+/-7.25, 75.78+/-4.41, 65.22+/-5.59 and 50.56+/-6.25, respectively. The tested group 2 (incubated with fresh and frozen CM from CC+GC for 1-3 months) had the percentages of the spermatozoa with AR at 88.67+/-4.03, 82.22+/-3.46, 71.00+/-3.16, and 58.56+/-4.69, respectively. Statistical analysis of all group percentages indicated that they were significantly different (P<0.05). Transmission electron microscope studies demonstrated that there were morphological changes on the sperm heads which resulted in the leakage of acrosin and subsequent AR. We concluded that the CM from both POEC and CC+GC efficiently increased in vitro AR on frozen-thawed bovine spermatozoa. Identification and functions of these secretory proteins are currently under investigation which may lead us to a better understanding of other beneficial effects in the utilization of both CM conditions.     

Cellular organisation of the mature testes and stages of spermiogenesis in Danio rerio (Cyprinidae; Teleostei)--structural and ultrastructural studies

The male gonads of Danio rerio occupy a position typical of the Teleostei species. The structure of the testes corresponds to the anastomosing tubular type with unrestricted spermatogonia and represents a cystic type of spermatogenesis. The results of this study indicate that four distinct stages of cell differentiation can be identified during spermiogenesis. These stages are characterised by chromatin condensation, the development of flagellum, nuclear rotation, the formation of nuclear fossa and the elimination of excess cytoplasm. A round head and the absence of an acrosome characterise the differentiated sperm. The midpiece is short and large, and C-shaped mitochondria form a ring surrounding the initial region of the flagellum. The axoneme shows a 9 + 2 pattern. In the D. rerio spermatozoa the flagellar axis is at an angle of 110° to the nucleus diameter running through the centriole.     

Fine structure of the spermatozoon of Perinereis macropus (Claparède, 1870) (polychaeta,Nereidae)

The mature spermatozoa of Perinereis macropus were investigated by transmission electron microscopy. The spermatozoon is composed with a large anterior part (head), a short middle piece and a long flagellum. The head contains a large acrosomal complex with a convex acrosomal vesicle, a subacrosomal space, a fibrillar crown and an acrosomal rod which penetrates into the nucleus invagination. The later is U shaped (in longitudinal section). The short middle piece contains about nine to eleven mitochondria and a centriole associated with the flagellum. This centriole, slightly eccentric to the sperm axis, anchors to the plasma membrane by nine satellite rays of the pericentriolar complex. The axoneme has a “9 + 2″ arrangement of microtubules. In cross section, the flagellar membrane extends in two lateral protuberances, aligned with the axis formed by the two central microtubules of the axoneme. The spermatozoon of P. macropus conforms to the primitive type with an acrosomal extension. Nevertheless, the acrosome complex ultrastructure shows noticeable modifications from the basic form. This finding agrees with the previously observed reproductive pattern (broadcast spawning – free-swimming larvae), and may be helpful to classify the sperm type of P. macropus.     

Effect of rapid freezing–thawing techniques on the sperm parameters and ultrastructure of Chinese Taihang black goat spermatozoa

Supercooling sperm in liquid nitrogen vapour is a feasible and economic technique for the practical production. The study aimed to reveal the negative effects of this rapid freezing and thawing processes on Taihang black goat spermatozoa and to find out the changing of spermatozoa motility and ultrastructure by using CASA and TEM. Qualified semen samples, which collected from twenty Chinese Taihang black goats using artificial vagina were pooled and investigated the kinematics parameters and ultrastructural morphology. The results showed that freezing–thawing caused a significant reduction in the spermatozoon total motility (P < 0.001), in rapid and medium cell numbers (P < 0.001) and motility parameters (VAP, VSL, VCL, ALH and BCF) (P < 0.01). Immotile spermatozoa number was increased significantly after freezing–thawing (P < 0.001). In the ultrastructural analysis, the shape with a sperm nucleus characterized by ruptures, bend and deformity was observed. The plasma membranes were broken, and nucleoplasm erupted. The mitochondria in the middle piece were disturbed by partial absence or additional accumulations. Swelling, coiling, vacuolization and structural disorganization of mitochondria were also observed. In conclusion, Freezing–thawing procedure has a detrimental effect on motility, membrane integrity and mitochondria of goat spermatozoa. Transmission electron microscopy provides an intuitive observation to investigate deformity spermatozoa.     

Morphology of the male reproductive system and spermiogenesis in Hypanthidium foveolatum (Alfken, 1930) (Hymenoptera: Apidae: Megachilinae)

The morphological aspects of male reproductive tract, spermiogenesis and spermatozoa are typical for each species and reflect its evolution, establishing a unique source of characters, which has been used to help solve phylogenetic problems. In Hypanthidium foveolatum the reproductive tract is composed of the testes comprising 28 testicular tubules, deferent ducts, seminal vesicles, accessory glands and an ejaculatory duct. The differentiation of spermatids occurs within cysts of up to 128 germ line cells each one. During the early spermatid phase, the nucleus resembles that of somatic cells. There follows a gradual chromatin condensation with an increase in nuclear electron density. In the spermatozoon, the nucleus contains heterogeneous chromatin with a loose appearance. The acrosome, shaped with the active participation of the Golgi complex, shows an electron-dense perforatorium involved by four electron-lucent acrosomal vesicle projections. The sperm tail presents an axoneme with a 9 + 9 + 2 microtubule pattern and two mitochondrial derivatives, which appear with different sizes. A dense crystalloid is formed initially in the mitochondrial matrix of the large derivative. The mitochondrial derivatives’ differentiation occurs concomitantly with an axoneme outgrowth. The centriolar adjunct is observed near the axoneme, anterior to the smaller mithocondrial derivative and exhibits an approximately triangular shape in cross-sections. Microtubules were observed around the head region and flagellar components during spermiogenesis.     

Spermiogenesis and sperm ultrastructure of Machilontus sp (Insecta: Archaeognatha) with phylogenetic consideration

The sperm structure of the jumping bristletail Machilontus sp has been described. The species shares several sperm characteristics with other genera of the same order Archaeognatha. During late spermiogenesis the spermatid bends at half of its length with the two limbs closely apposed within the same plasma membrane. The sperm has a helicoidal bi-layered acrosome with a filamentous perforatorium and a long nucleus. The elongated flagellum consists of an axoneme with 9 accessory microtubules external to the 9+2, two rows of conventional mitochondria and two accessory bodies. The accessory bodies are located lateral to the axoneme and are probably responsible for the shifting of the accessory tubules in two opposite groups of 5 and 4 tubules, respectively. These sperm characteristics represent common traits of all Archaeognatha.     

Ultrastructure and heteromorphism of spermatozoa in five species of bugs (Pentatomidae: Heteroptera)

Pentatomidae is one of the largest Heteroptera families, comprising about 10% of the species estimated for the suborder. In spite of existing studies, doubts remain regarding the systematics of Pentatomomorpha. In this study, five species of Pentatomidae spermatozoa were examined to achieve characteristics that enable inferences in the phylogeny of the group and in behavioral issues associated with the presence of polymorphisms. Spermatozoa polymorphisms, characterized by two classes of sizes, are found in Podisus nigrispinus, Podisus distinctus, and Brontocoris tabidus, whereas Thynacanta marginata (Dallas) and Supputius cincticeps have single-size spermatozoa. The head region consists of an acrosome, a nucleus, and part of the centriolar adjunct. In the more anterior region, the nucleus is parallel to the centriolar adjunct. In the nucleus-flagellum transition region, the nucleus overlaps the anterior region of the mitochondrial derivatives, just above the axoneme. The mitochondrial derivatives and the axoneme run the entire extent of the flagellum. In species in which the spermatozoa are polymorphic, the larger spermatozoa have derivatives mitochondrial approximately 3-fold larger than the smaller spermatozoa. Characteristics derived from the morphology of spermatozoa indicate synapomorphies and are promising for systematic studies.     

Does membrane lipid profile explain chilling sensitivity and membrane lipid phase transition of spermatozoa and oocytes?

Ram, fowl and bee spermatozoa, and oocytes of cows and zebrafish were used to study lipid membrane profiles, chilling sensitivity and lipid-phase transitions. The integrity of the membranes was determined by carboxyfluorescein diacetate (CFDA) staining following exposure for 15 minutes to low temperatures. Ram and fowl spermatozoa showed different degrees of loss of membrane integrity. Surprisingly, bee spermatozoa did not show any sensitivity to chilling, and their membranes remained intact down to 0 degree C. In bovine oocytes (at the GV stage) chilling injury was very severe at 16 degree C (membrane integrity decreased by 50%). Lipid phase transition (LPT) and membrane fluidity, which were evaluated by Fourier transform infrared (FTIR) microscopy, and fluorescence polarisation, showed phase transitions at the same temperatures as caused damage (between 30 and 12 degree C). The membrane lipid profiles showed high concentrations of polyunsaturated fatty acids (PUFA) in cold-sensitive ram spermatozoa and zebrafish oocytes, but the ratio between PUFA and saturated fatty acids was highest in cold-resistant bee spermatozoa and lowest in cold-sensitive bovine oocytes. These results suggest a close relationship among cold susceptibility, lipid phase transition and lipids profile in animal gametes.     

Cryopreservation of sea urchin (Evechinus chloroticus) sperm

A method was developed for cryopreserving sperm of the sea urchin, Evechinus chloroticus. Sperm fertilisation ability, mitochondrial function and membrane integrity were assessed before and after cryopreservation. Highest post-thaw fertilisation ability was achieved with lower concentrations (2.5%-7.5%) of dimethyl sulphoxide (DMSO). In contrast, post-thaw mitochondrial function and membrane integrity were higher for sperm frozen in intermediate and high DMSO concentrations (5%-15%). Surprisingly, some sperm frozen in seawater only, without DMSO, were able to survive post-thawing, although the fertilisation ability (10(6) sperm/ml; approximately 50% fertilisation), mitochondrial function and membrane integrity of these sperm were notably lower than of sperm frozen with DMSO (10(6) sperm cells/ml; 2.5%-7.5% DMSO; >85% fertilisation) at the concentrations tested. Amongst sperm from individual males, fertilisation ability varied before and after cryopreservation for both males frozen with and without cryoprotectant. Specific differences among males also varied. Sperm mitochondrial function and membrane integrity was similar among males before cryopreservation but differed considerably after cryopreservation. Cryopreserved sperm were able to fertilise eggs and develop to pluteus stage larvae. This study has practical applications and will provide benefits such as reduced broodstock conditioning costs, control of parental input and opportunities for hybridisation studies.