Acrosome reaction in spermatozoa from hagfish (Agnatha) Eptatretus burgeri and Eptatretus stouti: acrosomal exocytosis and identification of filamentous actin

Spermatozoa of the hagfishes Eptatretus burgeri and Eptatretus stouti, caught in the sea near Japan and North America, respectively, were found to undergo the acrosome reaction, which resulted in the formation of an acrosomal process with a filamentous core. The acrosomal region of spermatozoa of E. stouti exhibited immunofluorescent labeling using an actin antibody. The midpiece also labeled with the antibody. The acrosomal region showed a similar labeling pattern when sperm were probed with tetramethylrhodamine isothyocyanate (TRITC)-phalloidin; the midpiece did not label. Following induction of the acrosome reaction with the calcium (Ca2+) ionophore ionomycin, TRITC-phalloidin labeling was more intense in the acrosomal region, suggesting that the polymerization of actin occurs during formation of the acrosomal process, as seen in many invertebrates. The potential for sperm to undergo acrosomal exocytosis was already acquired by late spermatids. During acrosomal exocytosis, the outer acrosomal membrane and the overlying plasma membrane disappeared and were replaced by an array of vesicles; these resembled an early stage of the acrosome reaction in spermatozoa of higher vertebrates in which no formation of an acrosomal process occurs. It is phylogenetically interesting that such phenomena occur in spermatozoa of hagfish, a primitive vertebrate positioning between invertebrates and high vertebrates.     

Sperm ultrastructure in Myxinidocotyle and Acanthocotyle (Platyhelminthes, Monogenea, Acanthocotylidae)

Spermatozoa of Myxinidocotyle californica from the hagfish Eptatretus stoutii and of Acanthocotyle lobianchi from the skate Raja clavata show a similar ultrastructure: two axoncmes of the 9 + 1 type in parallel with the nucleus-and one mitochondrion. In the released Acanthocotyle spermatozoa nucleus and mitochondrion both have a triangular cross-section. No cortical microtubules are present. The ultrastructure of these two acanthocotylid spermatozoa thus corresponds to sperm pattern 2 according to Justine et al. (1985). This pattern is derived from the more primitive pattern 1, which in the Monogenea is found only in the Oligonchoinea Bychowsky, 1937 or the Polyopisthocotylea sensu Justine et al.     

Spermiogenesis in the Hagfish Eptatretus burgeri (Agnatha)

The fine structure of spermatid differentiation in a primitive vertebrate, the hagfish, whose spermatozoa bear acrosomes, was investigated. In early round spermatids, the acrosomal vesicles were spherical and located in a shallow nuclear indentation, flanked by the plasma and the nuclear membranes. The vesicle underwent a transition through lens-shaped and cap-shaped stages until it attained the shape of a bell in mature spermatozoa. Electron-dense acrosomal material that appeared as deposits in three portions of the vesicle finally joined in the center region at a late stage. Condensation of chromatin occurred in the anterior region of the nucleus. During transformation of the spermatids, many regularly spaced microtubules appeared beneath the plasma membrane except in the anteriormost region of the cell. The microtubules in a single alignment lay parallel to one another and encased the nucleus diagonally. During an early stage, the centrioles changed their orientation from perpendicular to longitudinal and rotated to become parallel to the long axis of the nucleus. Thus, the flagellum lay nearly straight along the cell axis. A cytoplasmic canal appeared transiently during the early stage. A droplet of cytoplasm was eliminated after descending along the flagella. The features of spermiogenesis in hagfish, which lies between invertebrates and vertebrates, are compared with those of other animals.     

The Complete Mitochondrial Genome of the Hagfish Myxine glutinosa: Unique Features of the Control Region

The complete sequence of the mitochondrial DNA of the hagfish Myxine glutinosa has been determined. The hagfish mtDNA (18,909 bp) is the longest vertebrate mtDNA determined so far. The gene arrangement conforms to the consensus vertebrate type and differs from that of lampreys. The exceptionally long (3628-bp) control region of the hagfish contains the typical conserved elements found in other vertebrate mtDNAs but is characterized by a large number of putative hairpins, which can potentially fold into a highly compact secondary structure that appears to be unique to hagfish. The comparison of the mtDNAs of two M. glutinosa specimens, excluding the control region, shows a 0.6% divergence at the nucleotide level as a sample of intraspecies polymorphism. Received: 21 August 2000 / Accepted: 2 March 2001     

Genome biology of the cyclostomes and insights into the evolutionary biology of vertebrate genomes

The jawless vertebrates (lamprey and hagfish) are the closest extant outgroups to all jawed vertebrates (gnathostomes) and can therefore provide critical insight into the evolution and basic biology of vertebrate genomes. As such, it is notable that the genomes of lamprey and hagfish possess a capacity for rearrangement that is beyond anything known from the gnathostomes. Like the jawed vertebrates, lamprey and hagfish undergo rearrangement of adaptive immune receptors. However, the receptors and the mechanisms for rearrangement that are utilized by jawless vertebrates clearly evolved independently of the gnathostome system. Unlike the jawed vertebrates, lamprey and hagfish also undergo extensive programmed rearrangements of the genome during embryonic development. By considering these fascinating genome biologies in the context of proposed (albeit contentious) phylogenetic relationships among lamprey, hagfish, and gnathostomes, we can begin to understand the evolutionary history of the vertebrate genome. Specifically, the deep shared ancestry and rapid divergence of lampreys, hagfish and gnathostomes is considered evidence that the two versions of programmed rearrangement present in lamprey and hagfish (embryonic and immune receptor) were present in an ancestral lineage that existed more than 400 million years ago and perhaps included the ancestor of the jawed vertebrates. Validating this premise will require better characterization of the genome sequence and mechanisms of rearrangement in lamprey and hagfish.     

Vertebrate features revealed in the rudimentary eye of the Pacific hagfish (Eptatretus stoutii)

Hagfish eyes are markedly basic compared to the eyes of other vertebrates, lacking a pigmented epithelium, a lens and a retinal architecture built of three cell layers: the photoreceptors, interneurons and ganglion cells. Concomitant with hagfish belonging to the earliest-branching vertebrate group (the jawless Agnathans), this lack of derived characters has prompted competing interpretations that hagfish eyes represent either a transitional form in the early evolution of vertebrate vision, or a regression from a previously elaborate organ. Here, we show the hagfish retina is not extensively degenerating during its ontogeny, but instead grows throughout life via a recognizable PAX6+ ciliary marginal zone. The retina has a distinct layer of photoreceptor cells that appear to homogeneously express a single opsin of the RH1 rod opsin class. The epithelium that encompasses these photoreceptors is striking because it lacks the melanin pigment that is universally associated with animal vision; notwithstanding, we suggest this epithelium is a homologue of gnathosome retinal pigment epithelium (RPE) based on its robust expression of RPE65 and its engulfment of photoreceptor outer segments. We infer that the hagfish retina is not entirely rudimentary in its wiring, despite lacking a morphologically distinct layer of interneurons: multiple populations of cells exist in the hagfish inner retina and subsets of these express markers of vertebrate retinal interneurons. Overall, these data clarify Agnathan retinal homologies, reveal characters that now appear to be ubiquitous across the eyes of vertebrates, and refine interpretations of early vertebrate visual system evolution.     

Progesterone treatment of boar spermatozoa improves male pronuclear formation after intracytoplasmic sperm injection into porcine oocytes

Boar spermatozoa were prepared for intracytoplasmic sperm injection (ICSI) by two different treatments to facilitate sperm chromatin decondensation and improve fertilisation rates after ICSI in pigs: spermatozoa were either frozen and thawed without cryoprotectants, or treated with progesterone. Morphological changes of the sperm heads after the treatments were examined and then the activation of oocytes and the transformation of the sperm nucleus following ICSI were assessed. After freezing and thawing, the plasma membrane and acrosomal contents over the apical region of sperm head were lost in all the spermatozoa. Following treatment with 1 mg/ml progesterone, the acrosome reaction was induced in 61% of spermatozoa. After injection of three types of spermatozoa, non-treated spermatozoa and progesterone-treated (i.e. acrosome-reacted) spermatozoa induced oocyte activation, but frozen-thawed spermatozoa induced oocyte activation at a significantly lower rate. Sixty-two per cent of sperm heads remained orcein-negative for 6 h, however, resulting in delayed sperm chromatin decondensation and low male pronuclear formation in the oocytes injected with a non-treated spermatazoon. Since the treatments of freezing and thawing and progesterone for spermatozoa accelerated the initial change in sperm chromatin and the latter treatment induced oocyte activation earlier, it is considered that the delay in oocyte activation and decondensation of sperm chromatin after injection of non-treated spermatozoa is caused by the existence of the sperm plasma membrane. These results show that progesterone treatment efficiently induces the acrosome reaction in boar spermatozoa without destroying their potency for oocyte activation, and the induction of the acrosome reaction results in the promotion of male pronuclear formation after ICSI.     

Hagfish (cyclostomata, vertebrata): searching for the ancestral developmental plan of vertebrates

The phylogenetic position of the hagfish remains enigmatic. In contrast to molecular data that suggest monophyly of the cyclostomes, several morphological features imply a more ancestral state of this animal compared with the lampreys. To resolve this question requires an understanding of the embryology of the hagfish, especially of the neural crest. The early development of the hagfish has long remained a mystery. We collected a shallow-water-dwelling hagfish, Eptatretus burgeri, set up an aquarium tank designed to resemble its habitat, and successfully obtained several embryos. By observing the histology and expression of genes known to play fundamental roles in the neural crest, we found that the hagfish crest develops as delaminating migratory cells, as in other vertebrates. We conclude that the delaminating neural crest is a vertebrate synapomorphy that seems to have appeared from the beginning of their evolutionary history, before the splitting away of the hagfish lineage.     

Functional and morphological diversity of sperm in Drosophila

Unlike mammals, where the males produce huge quantities of tiny spermatozoa, insects, and Drosophila in particular, exhibit a wide range of reproductive strategies. Sperm gigantism in Drosophila deviates from the rules that normally govern anisogamy, i.e. differences in the size and quantity of male and female gametes. Sperm gigantism has driven anatomical, physiological and cytological adaptations that affect the correlated evolution of the male and female reproductive systems, and has led to the evolution of a new structure, the roller, located between the testis and the seminal vesicle, and to sperm coiling to form pellets. The diversification of sperm strategy is investigated in the light of sexual selection processes that occur in the female genital tract after copulation. These processes, which bias paternity, result from interactions either between spermatozoa from different males, or between the spermatozoa and the environment within the female reproductive tract. In Drosophila, increased sperm size does not confer any reproductive advantage on the male. The evolution of sperm gigantism does not seem to be attributable to competition between spermatozoa from different males, as has been shown to occur in some vertebrate species. Alternative mechanisms, such as interactions between spermatozoa and the female reproductive system, are therefore currently viewed as being more likely explanations. In particular, the impact of sperm size on female reproductive physiology is being investigated to find out whether having large spermatozoa increases the likelihood of male reproductive success. Correlated adaptations of the spermatozoa and female storage organs also seem to be a major factor in determining sperm success, and their role in male-female conflicts is discussed briefly.     

Conservation of engrailed-like homeobox sequences during vertebrate evolution

The Drosophila melanogaster developmental gene engrailed (en) is a member of a distinct subfamily of homeobox genes with a wide phylogenetic distribution. Here we report the use of reduced stringency polymerase chain reaction (PCR) to amplify and clone 8 genes related to en from 5 vertebrate species, including representatives of the most ancient vertebrate lineages. Nucleotide and deduced amino acid sequence comparisons between mouse, toad, zebrafish, lamprey and hagfish genes reveal extensive evolutionary conservation, and suggests that 2 en-like genes have been retained in most vertebrate lineages.     

Proteoglycan from bovine follicular fluid enhances an acrosome reaction in bovine spermatozoa

Bovine epididymal and ejaculated spermatozoa were incubated for 22 and 9.5 h respectively, in a chemically defined medium. The percentages of sperm exhibiting an acrosome reaction were determined morphologically after fixing and staining specimens. Addition of bovine follicular proteoglycan or chondroitin sulfates ABC significantly increased the incidence of acrosome reaction. The stimulatory effects of the proteoglycan or chondroitin sulfates were negated by exposure to the enzyme chondroitinase ABC. Viability of spermatozoa was not affected by the various experimental treatments. Transmission electron microscopy of spermatozoa showed that vesiculation had occurred between the plasma and outer acrosomal membrane. These results suggest that proteoglycan present in follicular fluid at the time of ovulation may promote the acrosome reaction which precedes the ability of sperm to fertilize an ovum.     

Preferential attachment of cock spermatozoa to the perivitelline layer directly over the germinal disc of the hen's ovum.

In vitro incubation of cock spermatozoa with perivitelline layer (PL) from recently ovulated ova of the hen resulted in binding of spermatozoa to the PL and activation of the acrosome reaction. A simple quantitative technique was developed for assessing these events. Following incubation of the PL (0.5 cm2 sections) with spermatozoa, the PL section was rinsed and stained with Schiff's reagent. Microscopic examination revealed holes in the PL that were assumed to be sites of spermatozoa penetration. Utilizing this technique, a correlation was demonstrated between sperm concentration and the number of spermatozoa attaching to the PL and undergoing an acrosome reaction. Pre-treatment of spermatozoa with solubilized PL inhibited spermatozoa binding to pieces of intact PL. The PL overlying the germinal disc and a similarly sized section of PL from another area of the ovum were removed and incubated separately with spermatozoa (1 x 10(5) sperm/100 microliters). Spermatozoa showed preferential attachment and digestion of the PL from the germinal disc area (809 sperm/mm2) as compared to PL from other areas of the ovum (608 sperm/mm2). Spermatozoa attached to the PL in a circular, doughnut-shaped fashion in the area directly over the germinal disc.     

Effects of angiotensin II on the acrosome reaction in equine spermatozoa

Angiotensin II is a hormone with a wide array of physiological effects that exerts its effect via interaction with two major subtypes of receptor. The results of this study show that angiotensin II (from 1 to 100 nmol l(-1)) initiates acrosomal exocytosis in equine spermatozoa that have undergone capacitation in vitro in a TALP-TEST (Tyrode's albumin lactate pyruvate; 188.7 mmol TES l(-1), 84.8 mmol Tris l(-1)) buffer with cAMP. The acrosome reaction and sperm viability were assessed with fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA) and Hoechst 33258, respectively. The initiation of the acrosome reaction by angiotensin II was strongly inhibited by losartan, a specific angiotensin II type 1 receptor antagonist. Although angiotensin II as well as progesterone both initiated the acrosome reaction in equine spermatozoa, there was no synergistic effect when both agonists were added simultaneously. Initiation of acrosomal exocytosis by angiotensin II was accompanied by a rapid and transient calcium influx that was assessed in capacitated spermatozoa loaded with Fura-2AM. In addition, the angiotensin II-mediated calcium influx was inhibited when spermatozoa were preincubated with losartan. Western blotting with an antibody against angiotensin II type 1 receptor detected a major sperm protein of 60 kDa. Indirect immunofluorescence of non-capacitated spermatozoa with the angiotensin II type 1 receptor antibody revealed labelling in the midpiece and tail. In capacitated spermatozoa, the angiotensin II type 1 receptor was localized mainly over the anterior region of the sperm head, the equatorial segment and occasionally on the postacrosomal region in addition to the sperm tail. In conclusion, this study demonstrated the ability of angiotensin II to stimulate the acrosome reaction in capacitated equine spermatozoa. This effect is mediated via the angiotensin II type 1 receptor and is accompanied by an increase in intracellular calcium.     

Different functional states of ram spermatozoa analysed by partition in an aqueous two-phase system

The surface of spermatozoa plays a critical role in many stages involved in fertilisation. The plasma membrane undergoes important alterations in the male and female reproductive tract, which result in the ability of spermatozoa to fertilise eggs. One of these membrane modifications is sperm capacitation, a process by which sperm interacts with the zona pellucida receptors leading to the acrosome reaction. It has been proposed that the freezing process induces capacitation-like changes to spermatozoa, and that this premature capacitation could explain the reduction in longevity and fertilising capacity of cryopreserved mammalian spermatozoa. Our research focused on the relationship between membrane alterations occurring throughout freezing-thawing and the processes of capacitation and acrosome reaction. We used centrifugal countercurrent distribution (CCCD) analysis to compare the partition behaviour of ram spermatozoa that was either subjected to cold-shock or frozen-thawed with capacitated and acrosome reacted samples. In addition, the effect of the induced acrosome reaction on membrane integrity of ram spermatozoa was studied using biochemical markers and electron microscopy scanning. The CCCD analysis revealed important similarities between the surface characteristics of capacitated and cold-shocked sperm as well as between acrosome-reacted and frozen-thawed sperm. Cold-shocked and capacitated sperm showed an increased cell affinity for the lower dextran-rich phase as well as a decreased heterogeneity. Likewise, the induction of the acrosome reaction resulted in a loss of viability and an important decrease in cell surface heterogeneity compared to the untreated-control sample. Similar surface changes were found when semen samples were frozen with either Fiser or milk-yolk extender. These results confirm those obtained for membrane integrity by fluorescence markers. Thus, the high cell viability value found in the control sample (74.5%) was greatly decreased after cold-shock (22.2%), cryopreservation (26.38% Fiser medium, 24.8% milk-yolk medium) and acrosome reaction (6.6%), although it was preserved after inducing capacitation (46.7%). The study using electron microscopy scanning revealed dramatic structural alterations provoked by the induction of the acrosome reaction.     

L-arginine promotes capacitation and acrosome reaction in cryopreserved bovine spermatozoa

Sperm capacitation and acrosome reaction are essential for fertilization and they are considered as part of an oxidative process involving superoxide and hydrogen peroxide. In human spermatozoa, the amino acid L-arginine is a substrate for the nitric oxide synthase (NOS) producing nitric oxide (NO*), a reactive molecule that participates in capacitation as well as in acrosome reaction. L-arginine plays an important role in the physiology of spermatozoa and has been shown to enhance their metabolism and maintain their motility. Moreover, L-arginine has a protective effect on spermatozoa against the sperm plasma membrane lipid peroxidation. In this paper, we have presented, for the first time, the effect of L-arginine on cryopreserved bovine sperm capacitation and acrosome reaction and the possible participation of NOS in both processes. Frozen-thawed bovine spermatozoa have been incubated in TALP medium with different concentrations of L-arginine and the percentages of capacitated and acrosome reacted spermatozoa have been determined. L-arginine induced both capacitation and acrosome reaction. NO* produced by L-arginine has been inhibited or inactivated using NOS inhibitors or NO* scavengers in the incubation medium, respectively. Thus, the effect of NOS inhibitors and NO* scavengers in capacitated and non-capacitated spermatozoa treated with L-arginine has also been monitored. The data presented suggest the participation of NO*, produced by a sperm NOS, in cryopreseved bovine sperm capacitation and acrosome reaction.     

Sperm binding, in vitro fertilization, and in vitro embryonic development of bovine oocytes fertilized with spermatozoa incubated with norepinephrine

The final stages of sperm maturation, fertilization, and early embryonic development occur within the oviduct and are essential for successful reproduction in mammals. Norepinephrine was previously identified in native bovine oviductal fluid and its in vitro effects on bull sperm capacitation and the acrosome reaction have been determined. It was unknown how physiological concentrations of norepinephrine influence sperm binding, fertilization, and embryo development. Therefore, the objective of this study was to determine if pre-incubating bovine spermatozoa with physiological concentrations of norepinephrine prior to insemination of bovine oocytes would improve sperm-oocyte binding, fertilization, and embryonic development in vitro. Norepinephrine, in concentrations representing those measured in bovine oviductal fluid, was used to treat bovine spermatozoa prior to insemination. Spermatozoa incubated in norepinephrine were used to inseminate bovine oocytes matured in vitro, and oocytes were evaluated for sperm binding and fertilization. Additional experiments were conducted to evaluate how early in the co-incubation period oocytes were fertilized by spermatozoa pre-incubated with norepinephrine, and to test the developmental competence of those oocytes fertilized with norepinephrine-treated sperm. Sperm binding to the zona pellucida was reduced by pre-incubation with norepinephrine. Rates of fertilization and embryo development did not increase as a result of pre-incubating spermatozoa with norepinephrine, but as early as 4h after insemination, spermatozoa treated with 20 ng/ml norepinephrine fertilized more oocytes than spermatozoa incubated in medium alone. Interestingly, this concentration of norepinephrine was found to capacitate spermatozoa in previous studies. These data suggest that oocytes fertilized by spermatozoa incubated in 20 ng/ml norepinephrine fertilize earlier in vitro than sperm pre-incubated in medium alone, and provide additional support for the role of norepinephrine in sperm capacitation and the acrosome reaction.     

Mu opioid receptor-like sequences are present throughout vertebrate evolution

The sequence of the mu opioid receptor is highly conserved among human, rat, and mouse. In order to gain insights into the evolution of the mu opioid receptor, polymerase chain reaction (PCR) was used to screen genomic DNA from a number of different species using degenerate oligonucleotides which recognize a highly conserved region. DNA was assayed from representative species of both the protostome and deuterostome branches of the metazoan phylogenetic tree. Mu opioid receptor-like sequences were found in all vertebrate species that were analyzed. These species included bovine, chicken, bullfrog, striped bass, thresher shark, and Pacific hagfish. However, no mu opioid receptor-like sequences were detected from protostomes or from any invertebrates. The PCR results demonstrate that the region of the mu opioid receptor gene between the first intracellular loop and the third transmembrane domain (TM3) has been highly conserved during evolution and that mu opioid receptor-like sequences are present in the earliest stages of vertebrate evolution. Additional opioid receptor-like sequence was obtained from mRNA isolated from Pacific hagfish brain using rapid amplification of cDNA ends (RACE). The sequence of the Pacific hagfish was most homologous with the human mu opioid receptor (72% at the amino acid level between intracellular loop 1 and transmembrane domain 6) although over the same region high homology was also observed with the delta opioid receptor (69%), the kappa receptor (63%), and opioid receptor-like (ORL1) (59%). The hagfish sequence showed low conservation with the mammalian opioid receptors in the first and second extracellular loops but high conservation in the transmembrane and intracellular domains. Received: 5 January 1996 / Accepted: 7 March 1996     

Effects of the purified IGF-I complex on the capacitation and acrosome reaction of rabbit spermatozoa

A protein complex containing IGF-I, purified from rabbit seminal plasma, was used to investigate its effects on the capacitation and acrosome reaction of rabbit spermatozoa. Uncapacitated sperm (Pattern F), capacitated sperm (Pattern B), and acrosome-reacted sperm (Pattern AR) were determined by CTC staining, and the results were validated by PSA-staining. The addition of the IGF-I complex to the capacitative medium directed the spermatozoa to spontaneous acrosome reaction. On the other hand, IGF-I complex, added to capacitated spermatozoa, acted as inducer of the acrosome reaction. Results of IVF experiments showed high rates of fertilization with capacitated spermatozoa, acrosome-reacted by either A23187 or IGF I complex, whereas significantly lower rates were obtained with spermatozoa capacitated in the presence of IGF-I complex.