Sperm acrosome status before and during fertilization in the Chinese hamster (Cricetulus griseus), and observation of oviductal vesicles and globules.

The present study was conducted to determine exact location where the acrosome reaction of fertilizing spermatozoa begins in the oviduct of the Chinese hamster. Unlike spermatozoa of other rodent species, Chinese hamster spermatozoa did not spontaneously undergo the acrosome reaction in fertilization-supporting media. In naturally mated females, spermatozoa in the uterus had intact acrosomes, whereas those in the lower oviductal isthmus had visibly thin acrosomal caps. The acrosomal cap was lost when spermatozoa passed through the cumulus oophorus. Thus, Chinese hamster spermatozoa begin the acrosome reaction in the lower isthmus and complete it in the cumulus oophorus. The mucosal epithelium of the oviductal isthmus released many "transparent" vesicles into the lumen, was very fragile and readily sloughed off by rough handling or rapid flushing with medium. Globular materials that oozed out of the dissected oviduct were most likely mucosa cells destroyed by rough handling. Although the oviducts of Chinese hamsters may be exceptionally delicate, this observation nevertheless warns us to cautiously handle the oviducts of any species when studying oviduct secretions that could be involved in inducing capacitation and the acrosome reaction of spermatozoa within the female genital tract.

Sperm competition risk affects ejaculate strategy in terms of sperm number but not sperm size in squid.

In polygamous species, the mode of sperm storage in females influences evolution of sperm quantitative and qualitative traits because it provides the arena for sperm competition, cryptic female choice and fertilization processes. In this study, we compared ejaculate traits of two squid species, Heterololigo bleekeri and Loligo reynaudii. Both species show dimorphic sperm traits associated with alternative reproductive tactics where consort and sneaker males transfer sperm to different storage sites within a female (on the oviduct and near the mouth, respectively). Due to differences in reproductive behaviours and sperm placement, sperm competition risk is expected to be higher in sneakers than in consorts of both species and higher overall in L. reynaudii. Our results demonstrate that the instantaneous number of released sperm is adjusted to the expected sperm competition risk via an elaborate sperm package. Consort sperm are similar in size; however, sneaker sperm have a significantly longer flagellum in H. bleekeri than in L. reynaudii, most likely due to intra-tactic conflicts associated with sperm storage conditions. From consideration of the different mating tactics, we suggest that while levels of sperm competition determine quantitative traits, sperm quality traits are determined more by the mode of sperm storage and fertilization.

Sperm acrosome reaction: its site and role in fertilization.

Manner and roles of sperm acrosome reaction in a variety of animals were compared.

Mouse sperm begin to undergo acrosomal exocytosis in the upper isthmus of the oviduct.

Recent evidence demonstrated that most fertilizing mouse sperm undergo acrosomal exocytosis (AE) before binding to the zona pellucida of the eggs. However, the sites where fertilizing sperm could initiate AE and what stimuli trigger it remain unknown. Therefore, the aim of this study was to determine physiological sites of AE by using double transgenic mouse sperm, which carried EGFP in the acrosome and DsRed2 fluorescence in mitochondria. Using live imaging of sperm during in vitro fertilization of cumulus-oocyte complexes, it was observed that most sperm did not undergo AE. Thus, the occurrence of AE within the female reproductive tract was evaluated in the physiological context where this process occurs. Most sperm in the lower segments of the oviduct were acrosome-intact; however, a significant number of sperm that reached the upper isthmus had undergone AE. In the ampulla, only 5% of the sperm were acrosome-intact. These results support our previous observations that most of mouse sperm do not initiate AE close to or on the ZP, and further demonstrate that a significant proportion of sperm initiate AE in the upper segments of the oviductal isthmus.

Sneaker Male Squid Produce Long-lived Spermatozoa by Modulating Their Energy Metabolism.

Spermatozoa released by males should remain viable until fertilization. Hence, sperm longevity is governed by intrinsic and environmental factors in accordance with the male mating strategy. However, whether intraspecific variation of insemination modes can impact sperm longevity remains to be elucidated. In the squid Heterololigo bleekeri, male dimorphism (consort and sneaker) is linked to two discontinuous insemination modes that differ in place and time. Notably, only sneaker male spermatozoa inseminated long before egg spawning can be stored in the seminal receptacle. We found that sneaker spermatozoa exhibited greater persistence in fertilization competence and flagellar motility than consort ones because of a larger amount of flagellar glycogen. Sneaker spermatozoa also showed higher capacities in glucose uptake and lactate efflux. Lactic acidosis was considered to stabilize CO2-triggered self-clustering of sneaker spermatozoa, thus establishing hypoxia-induced metabolic changes and sperm survival. These results, together with comparative omics analyses, suggest that postcopulatory reproductive contexts define sperm longevity by modulating the inherent energy levels and metabolic pathways.

Chemical and physical guidance of fish spermatozoa into the egg through the micropyle†,‡.

Eggs of teleost fish, unlike those of many other animals, allow sperm entry only at a single site, a narrow canal in the egg's chorion called the micropyle. In some fish (e.g., flounder, herring, and Alaska pollock), the micropyle is a narrow channel in the chorion, with or without a shallow depression around the outer opening of micropyle. In some other fish (e.g., salmon, pufferfish, cod, and medaka), the micropyle is like a funnel with a conical opening. Eggs of all the above fish have a glycoprotein tightly bound to the chorion surface around the micropyle. This glycoprotein directs spermatozoa into the micropylar canal in a Ca2+-dependent manner. This substance, called the micropylar sperm attractant or MISA, increases fertilization efficiency and is essential in herring. In flounder, salmon, and perhaps medaka, fertilization is possible without MISA, but its absence makes fertilization inefficient because most spermatozoa swim over the micropyle without entering it. The mechanism underlying sperm-MISA interactions is yet to be determined, but at least in herring the involvement of Ca2+ and K+ channel proteins, as well as CatSper and adenylyl cyclase, is very likely. In some other fish (e.g., zebrafish, loach, and goldfish), the chorion around the micropyle is deeply indented (e.g., zebrafish and loach) or it has radially or spirally arranged grooves around the outer opening of the micropyle (e.g., goldfish). MISA is absent from the eggs of these fish and sperm entry into micropylar canal seems to be purely physical.

An intact acrosome is required for the chemotactic response to progesterone in mouse spermatozoa.

Mammalian sperm become fertilization-competent in the oviduct, during a process known as capacitation that involves the acquisition of the ability to exocytose the acrosome but also the chemotactic responses-both of which contribute to successful fertilization. Chemotaxis is used by spermatozoa to orient and to locate the egg; the acrosome reaction facilitates sperm binding to and fusing with the egg membrane. Mammalian spermatozoa are able to sense picomolar concentrations of progesterone, which drives chemotactic behavior. The state of the acrosome during the chemotactic response, however, is unknown. Genetically modified mouse spermatozoa were employed in a chemotaxis assay under fluorescence microscopy to evaluate their acrosome status while swimming, allowing us to elucidate the acrosome integrity of sperm responding to progesterone-induced chemotaxis. We first showed that wild-type mouse spermatozoa chemotactically respond to a gradient of progesterone, and that the genetic modifications employed do not affect the chemotactic behavior of sperm to progesterone. Next, we found that acrosome-intact, but not acrosome-reacted, spermatozoa orient and respond to picomolar concentrations of progesterone and that chemotaxis normally occurs prior to the acrosome reaction. Our results suggest that premature commitment to acrosome exocytosis leads to navigation failure, so proper control and timing of the acrosome reaction is required for fertilization success and male fertility.

Site of Mammalian Sperm Acrosome Reaction.

Until recently, no special attention has been paid to the question of the site of mammalian sperm acrosome reaction (AR) in the female reproductive tract. Because AR is an essential process that enables the spermatozoon to fertilize, it is generally believed that it occurs at a specific step during sperm-egg interaction. It is generally thought that "the site of action coincides with the site of commitment." Thus, understanding the roles of AR and acrosomal substances is needed to gain insight into the site of the sperm commitment to undergo AR.

Redistribution of the intra-acrosomal EGFP before acrosomal exocytosis in mouse spermatozoa.

Mammalian spermatozoa must undergo complex physiological and morphological alterations within the female reproductive tract before they become fertilization competent. Two important alterations are capacitation and the acrosome reaction (AR), by which spermatozoa become capable of penetrating the zona pellucida (ZP) of the oocyte. Although various biochemical stimulants have been reported to induce the AR, the true physiological inducer in vivo remains to be identified. Previously, it has been reported that most fertilizing spermatozoa undergo the AR before contacting the ZP and that only a small fraction of in vitro-capacitated spermatozoa can penetrate the ZP. Therefore, it is important to identify which capacitating spermatozoa undergo the AR in response to potential AR inducers such as progesterone. Here we show that spermatozoa undergo a dynamic rearrangement of the acrosome during in vitro capacitation. This involves the rapid movement of an artificially introduced soluble component of the acrosome, enhanced green fluorescent protein (EGFP), from the acrosomal cap region to the equatorial segment (EQ) of the sperm head. Spermatozoa exhibiting the EQ pattern were more sensitive to progesterone than were those without it. We suggest that spermatozoa that are ready to undergo acrosomal exocytosis can be detected by real-time EGFP imaging. This offers a promising new method for identifying where spermatozoa undergo the AR in the female reproductive tract in vivo.

Integrative omics analysis reveals differentially distributed proteins in dimorphic euspermatozoa of the squid, Loligo bleekeri.

In the coastal squid Loligo bleekeri, each male produces one of two types of fertilization-competent spermatozoa (eusperm) that exhibit morphological and behavioral differences. Large "consort" males produce short-tailed spermatozoa that display free-swimming behavior when ejaculated into seawater. Small "sneaker" males, on the other hand, produce long-tailed spermatozoa that exhibit a self-swarming trait after ejaculation. To understand the molecular basis for adaptive traits employed by alternative male mating tactics, we performed the transcriptome deep sequencing (RNA-seq) and proteome analyses to search for differences in testicular mRNAs and sperm proteins, respectively. From mature male testes we identified a total of 236,455 contigs (FPKM ≧1) where 3789 and 2789 were preferentially (≧10-fold) expressed in consort and sneaker testes, respectively. A proteomic analysis detected 4302 proteins in the mature sperm as post-translational products. A strongly biased (≧10-fold) distribution occurred in 55 consort proteins and 61 sneaker proteins. There was no clear mRNA-protein correlation, making a ballpark estimate impossible for not only overall protein abundance but also the degree of biased sperm type expressed in the spermatozoa. A family encoding dynein heavy chain gene, however, was found to be biased towards sneakers, whereas many enzymes involving energy metabolism were heavily biased towards consort spermatozoa. The difference in flagellar length matched exactly the different amount of tubulins. From these results we hypothesize that discrete differential traits in dimorphic eusperm arose from a series of innovative alterations in the intracellular components of spermatozoa.

Unresolved questions concerning mammalian sperm acrosomal exocytosis.

In recent years, the study of mammalian acrosomal exocytosis has produced some major advances that challenge the long-held, general paradigms in the field. Principally, the idea that sperm must be acrosome-intact to bind to the zona pellucida of unfertilized eggs, based largely on in vitro fertilization studies of mouse oocytes denuded of the cumulus oophorus, has been overturned by experiments using state-of-the-art imaging of cumulus-intact oocytes and fertilization experiments where eggs were reinseminated by acrosome-reacted sperm recovered from the perivitelline space of zygotes. In light of these results, this minireview highlights a number of unresolved questions and emphasizes the fact that there is still much work to be done in this exciting field. Future experiments using recently advanced technologies should lead to a more complete and accurate understanding of the molecular mechanisms governing the fertilization process in mammals.

Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization.

To fuse with oocytes, spermatozoa of eutherian mammals must pass through extracellular coats, the cumulus cell layer, and the zona pellucida (ZP). It is generally believed that the acrosome reaction (AR) of spermatozoa, essential for zona penetration and fusion with oocytes, is triggered by sperm contact with the zona pellucida. Therefore, in most previous studies of sperm-oocyte interactions in the mouse, the cumulus has been removed before insemination to facilitate the examination of sperm-zona interactions. We used transgenic mouse spermatozoa, which enabled us to detect the onset of the acrosome reaction using fluorescence microscopy. We found that the spermatozoa that began the acrosome reaction before reaching the zona were able to penetrate the zona and fused with the oocyte's plasma membrane. In fact, most fertilizing spermatozoa underwent the acrosome reaction before reaching the zona pellucida of cumulus-enclosed oocytes, at least under the experimental conditions we used. The incidence of in vitro fertilization of cumulus-free oocytes was increased by coincubating oocytes with cumulus cells, suggesting an important role for cumulus cells and their matrix in natural fertilization.

Males conditionally inseminate at three female body locations according to female mating history and female maturity status in a squid.

In some squids, such as those in the family Loliginidae, upon copulation, females receive and store male-delivered sperm capsules, spermatangia, at two different body locations: the buccal membrane and the distal end of the oviduct. This insemination site dimorphism is associated with alternative reproductive strategies. However, in Loliolus sumatrensis, a species of Loliginidae, the females possess three insemination sites: buccal membrane (BM), basal left IV arm (ARM) and lateral head behind the left eye (EYE), therefore we studied such the unusual phenomena. We developed microsatellite markers and genotyped the paternity of each spermatangium on three sites. We found multiple paternity at every single site and simultaneous usage of all three sites by a few males. The seasonal dynamics of a population in the Seto Inland Sea revealed a set priority for the initial use of insemination sites as BM, followed by ARM and then EYE, whereas the maximum number of stored spermatangia was greater in EYE > ARM > BM. Female maturity status was correlated with the usage pattern of insemination sites but not with the number of stored spermatangia at any insemination site. These results suggest that a male squid inseminates at different locations according to female mating history and female maturity status.

The macroscopic structure of RADA16 peptide hydrogel stimulates monocyte/macrophage differentiation in HL60 cells via cholesterol synthesis.

Cells can sense physical properties of surrounding 3-dimensional (3D) culture substrata; however, the physiological influences of such sensing are not fully understood. Here, we studied the physiological characteristics and activities of the macroscopic structure of a routinely used 3D culture substrata, the RADA16 self-assembling peptide scaffold. We found that RADA16 exhibited three distinct assembly patterns depending on its concentration, and one of these assemblies, formed with 0.01% (w/v) RADA16, was capable of inducing differentiation of human myelocytic leukemia HL-60 cells into monocytes/macrophages. This activity was largely reduced by destroying the 3D structure of the assembly, suggesting that the assembly intrinsically retained the ability to induce HL-60 differentiation. When cultured in the RADA16 scaffold, HL-60 cells accumulated intracellular cholesterol about 10 times more than normally cultured cells. Both the RADA16 culture and cholesterol loading brought about similar gene expression profiles. These results showed that HL-60 cells can sense the physical properties of the RADA16 scaffold through a mechanism that may involve intracellular pathways of cholesterol synthesis and/or transport.

Arrest at metaphase of meiosis I in starfish oocytes in the ovary is maintained by high CO2 and low O2 concentrations in extracellular fluid.

During the spawning process in starfish, oocytes are arrested at metaphase of meiosis I (MI) within the ovary, and reinitiate meiosis only after they have been released into the seawater. However, this arrest does not occur if the ovary is removed from the animal. As the pH of the coelomic fluid is buffered by CO2/H(+)/HCO3(-), we investigated the involvement of gas concentrations in MI arrest. In vivo, the CO2 level in the coelomic fluid was high (∼1.5% vs. 0.04% in air) and the O2 level was low (0.1-1.0% vs. ∼20% in air). When these gas conditions were reproduced in isolated coelomic fluid or seawater, ovarian oocytes arrested at MI, just as in vivo. Isolated oocytes from the ovary required the similar high CO2 and low O2 level to remain arrested in MI and had an intracellular pH of ∼6.9. Intracellular pH increased to ∼7.3 when oocytes were transferred to seawater equilibrated with air, a condition that mimics that of spawning. We used ammonium acetate to clamp intracellular pH at different levels and found that MI arrest occurred when intracellular pH was ∼6.9. Our results support the idea that high CO2 and low O2 in the ovarian environment lead to low intracellular pH and MI arrest, while spawning into the seawater with low CO2 and high O2 results in high intracellular pH and release from MI arrest. The biological significance of MI arrest is that oocytes are spawned into seawater at the optimal physiological state of MI when the least polyspermy occurs.

Egg Development After In Vitro Insemination in Japanese Quail (Coturnix japonica).

In vitro fertilization has been widely used to produce offspring in several mammalian species. We previously successfully produced Japanese quail chicks using intracytoplasmic sperm injection (ICSI), whereas in vitro insemination was not successful. This may be due to the difficulties associated with mimicking the sperm-egg fusion process and subsequent events in physiological polyspermic fertilization in vitro. In the present study, we observed egg development after in vitro insemination and investigated the inactivation of metaphase-promoting factor (MPF) and cytostatic factor (CSF), which are downstream of the Ca2+ signaling pathway in the egg, due to fertilizing sperm. We found a sperm number-dependent increase in hole formation caused by sperm penetration of the perivitelline membrane, the extracellular coat surrounding the egg. Egg development was observed following in vitro insemination; however, the developmental rate and stages after 24-h culture were inferior to those of ICSI eggs, even when insemination was performed with a high number of sperm (2 × 104). We also noted the downregulation of inositol 1,4,5-trisphosphate receptor-1, ryanodine receptor-3, cyclin B1, and c-MOS, which are important regulatory components of MPF and CSF in the egg, which was dependent on the number of sperm used for insemination. However, the decreases observed in these components did not reach the levels observed in the ICSI eggs. Collectively, the present results suggest that a sperm number higher than 2 × 104 is required for the progression of the Ca2+ signaling pathway, which initiates subsequent egg development in Japanese quail.

Regulation of intracellular pH by p90Rsk-dependent activation of an Na(+)/H(+) exchanger in starfish oocytes.

Starfish oocytes arrest at metaphase of the first meiotic division (MI arrest) in the ovary and resume meiosis after spawning into seawater. MI arrest is maintained by lower intracellular pH (pH(i)) and release from arrest by cellular alkalization. To elucidate pH(i) regulation in oocytes, we cloned the starfish (Asterina pectinifera) Na(+)/H(+) exchanger 3 (ApNHE3) expressed in the plasma membrane of oocytes. The cytoplasmic domain of ApNHE3 contains p90 ribosomal S6 kinase (p90Rsk) phosphorylation sites, and injection of a constitutively active p90Rsk and the upstream regulator Mos to immature oocytes, stimulated an increase in pH(i). This increase was blocked by 5-(N-ethyl-N-isopropyl)-amiloride, a NHE inhibitor, and SL0101, a specific Rsk inhibitor. The MAPK kinase (MEK) inhibitor U0126 blocked the Mos-induced, but not the p90Rsk-induced, pH(i) increase, suggesting that the Mos-MEK-MAPK-p90Rsk pathway promotes ApNHE3 activation. In a cell-free extract, the Mos-MEK-MAPK-p90Rsk pathway phosphorylates ApNHE3 at Ser-590, -606, and -673. When p90Rsk-dependent ApNHE3 phosphorylation was blocked by a dominant-negative C-terminal fragment, or neutralizing antibody, the p90Rsk-induced pH(i) increase was suppressed in immature oocytes. However, ApNHE3 is up-regulated via the upstream phosphatidylinositol 3-kinase pathway before MAPK activation and the active state is maintained until spawning, suggesting that the p90Rsk-dependent ApNHE3 phosphorylation is unlikely to be the primary regulatory mechanism involved in MI arrest exit. After meiosis is completed, unfertilized eggs maintain their elevated pH(i) ( approximately 7.4) until the onset of apoptosis. We suggest that the p90Rsk/ApNHE3-dependent elevation of pH(i) increases fertilization success by delaying apoptosis initiation.

Why small males have big sperm: dimorphic squid sperm linked to alternative mating behaviours.

BACKGROUND: Sperm cells are the target of strong sexual selection that may drive changes in sperm structure and function to maximize fertilisation success. Sperm evolution is regarded to be one of the major consequences of sperm competition in polyandrous species, however it can also be driven by adaptation to the environmental conditions at the site of fertilization. Strong stabilizing selection limits intra-specific variation, and therefore polymorphism, among fertile sperm (eusperm). Here we analyzed reproductive morphology differences among males employing characteristic alternative mating behaviours, and so potentially different conditions of sperm competition and fertilization environment, in the squid Loligo bleekeri. RESULTS: Large consort males transfer smaller (average total length = 73 µm) sperm to a female's internal sperm storage location, inside the oviduct; whereas small sneaker males transfer larger (99 µm) sperm to an external location around the seminal receptacle near the mouth. No significant difference in swimming speed was observed between consort and sneaker sperm. Furthermore, sperm precedence in the seminal receptacle was not biased toward longer sperm, suggesting no evidence for large sperm being favoured in competition for space in the sperm storage organ among sneaker males. CONCLUSIONS: Here we report the first case, in the squid Loligo bleekeri, where distinctly dimorphic eusperm are produced by different sized males that employ alternative mating behaviours. Our results found no evidence that the distinct sperm dimorphism was driven by between- and within-tactic sperm competition. We propose that presence of alternative fertilization environments with distinct characteristics (i.e. internal or external), whether or not in combination with the effects of sperm competition, can drive the disruptive evolution of sperm size.

Effect of sperm surface oligosaccharides in sperm passage into sperm storage tubules in Japanese quail (Coturnix japonica).

In birds, the ejaculated spermatozoa do not directly pass to the site of fertilization but rather are stored initially in specialized structures, referred to as sperm storage tubules (SSTs), located in the utero-vaginal junction (UVJ) of the oviduct. The fertilizing capacity of spermatozoa in the SSTs is maintained for an extended period (i.e., several days to months). Although many studies have been conducted to ascertain the mechanisms involved in sperm storage, the understanding of the phenomenon is limited. In this study, there was investigation of the effects of sperm surface oligosaccharides in sperm passage into SSTs in Japanese quail. Results from lectin staining of ejaculated spermatozoa indicated galactose/N-Acetylgalactosamine (Gal/GalNAc), N-Acetylglucosamine (GlcNAc) or mannose/glucose (Man/Glc) moieties were present on the sperm surface, indicating the presence of glycoproteins/glycolipids containing these oligosaccharides. When ejaculated spermatozoa were co-incubated with UVJ explants, the lectins derived from Agaricus bisporus and Canavalia ensiformis had marked inhibitory effects on sperm passage into SSTs. Preincubation of UVJ explants with these lectins, however, had no effect indicating there were no effects of UVJ oligosaccharides in this process. Furthermore, none of these lectin had effects on values of sperm motility variables. These results indicate that O-glycans with terminal ß-Gal or GalNAc and N-glycans with terminal α-D-Man or α-D-Glc may have functions in the process of sperm passage into SSTs.

Longer and faster sperm exhibit better fertilization success in Japanese quail.

In birds, sperm storage tubules (SST) located in the utero-vaginal junction are thought to be a site of sperm selection; however, the exact mechanism of sperm selection is poorly understood. Here, we investigated sperm entry into the SST and subsequent fertilization success under a competitive situation created by artificial insemination of a sperm mixture obtained from 2 males. We employed 2 quail strains, a wild-type and a dominant black (DB) type, as this allows easy assessment of paternity by feather coloration. We found paternity of embryos was biased toward DB males when a sperm mix with similar sperm numbers from the 2 males strains was artificially inseminated into females. Our novel sperm staining method with 2 different fluorescent dyes showed that the DB-biased fertilization was because of the better ability of DB sperm to enter the SST. Moreover, we found that DB sperm had a longer flagellum and midpiece. These characteristics probably allow sperm to swim faster in a high viscosity medium, which may be a similar environment to the lumen of the female reproductive tract. Our results indicated that sperm competition occurs to win a place in the SST and that filling the SST with their own spermatozoa is a critical step to achieve better fertilization success for the male Japanese quail.