Monitoring dimeric status of IZUMO1 during the acrosome reaction in living spermatozoon

The acrosome reaction (AR) is indispensable for successful spermatozoon-oocyte fusion. Recent studies have indicated that sperm IZUMO1 gradually gathers in the equatorial segment (EQ), which is the initiation site of sperm-egg fusion, after the AR. In addition, by examining the binding process of oocytes and Izumo1-expressing cultured cells to reconstitute the early steps of fertilization, we previously demonstrated that robust IZUMO1-dependent adhesion specifically occurs at the contact site along with the dimerization of IZUMO1. However, when IZUMO1 dimerizes after the AR in living spermatozoon is unknown. Here, we report dynamics of IZUMO1 dimerization during the AR in spermatozoa by combining transgenic mice and time-lapse imaging using a set of bimolecular fluorescence complementation (BiFC) probes. Surprisingly, dimeric IZUMO1 was already formed at the acrosomal cap region before the AR and redistributed into the EQ after the AR. We categorized the translocation of the dimer into two types: Type 1, the near-simultaneous appearance of BiFC signals with IZUMO1-mCherry; and Type 2, the delayed formation of dimer in the EQ. Those findings suggest that, before encountering oocytes, spermatozoa are prepared to boost their affinity with JUNO.     

Genetic studies of anther culture ability in rice (Oryza sativa)

Inheritance of three anther and culture characters, callus induction, green plant regeneration and culture efficiency was studied using incomplete diallel crosses with a gamete model. It was suggested that callus induction was mainly controlled by gametic additive effects and with less effect of the maternal effects. Green plant regeneration was mainly determined by maternal effects with less influence of gametic additive effects. Culture efficiency was controlled by gametic additive, maternal and cytoplasmic effects. Cultivar Lunhui 422 showed positive genetic effects for all three traits and was a very good parent for rice anther culture breeding. Significant positive heterosis was observed for callus induction. Both gametic additive and maternal correlations contributed to the significant genotypic and phenotypic correlations between callus induction and green plant regeneration suggesting these two traits to be linked.Abbreviations 2,4-d 2,4-dichlorphenoxyacetic acid - NAA -napthaleneacetic acid     

From the cellular to the molecular dimension: The actual challenge for human fertilization research

The form that rat spermatozoa assume when swelling in hyposmotic media depends on the position of the cytoplasmic droplet, previous exposure to hypertonic media, and the stiffness of the flagellum. Bending at the end of the midpiece occurs when the swelling droplet is situated at this site; this occurs in midcaput cells, but sperm from more proximal sites do not bend in this fashion. Stiffening of caput sperm stored in vitro reduces the incidence of such midpiece bending but looping at the tip of the tail still occurs, and previous exposure of caput spermatozoa to hypertonic media also prevents hairpin bend formation. Mature sperm from the cauda are too stiff to form hairpin loops when placed in hypotonic media unless first treated with a penetrating disulphydryl-reducing agent, after which swollen spherical vesicles can result from very flexible flagella confined within an intact membrane. Long-chain acylcarnitines are more potent lytic agents than acylcholines, but, for both, chain lengths of 16 carbon atoms is optimal for preventing the swelling of rat caput sperm.     

Induction of cross-fertilization between sea urchin eggs and starfish sperm by polyethylene glycol treatment

Cross-fertilization between sea urchin eggs (Strongylocentrotus nudus) and starfish sperm (Asterina pectinifera) was induced by treatment with polyethylene glycol (PEG). Without treatment with PEG, the denuded egg surface (jelly coat- and vitelline coat-free) engulfed the head of acrosome-reacted sperm; however, sperm penetration did not occur [Kyozuka and Osanai, 1988]. When these eggs were exposed briefly to PEG (molecular weight 3,000) in seawater, the sperm entered the egg by membrane fusion. Cortical granules were discharged, and embryogenesis began following sperm penetration. PEG did not induce parthenogenesis in Strongylocentrotus eggs. Egg activation is thus closely linked with gamete membrane fusion.     

Morphological observations on sperm–egg interactions during in vivo fertilization in the dasyurid marsupial Sminthopsis crassicaudata

The aim of the present study was to determine the morphological changes that take place in the male and female gametes during in vivo fertilization in the Australian marsupial, the fat-tailed dunnart, Sminthopsis crassicaudata. Plastic sections were cut of sperm and eggs recovered from the oviducts of recently mated individuals, and light microscopy of thick, and transmission EM of thin, sections was carried out. It was found that, before penetration of the zona, the spermatozoon came to lie along the outer surface with its rostral tip forming a depression in the zona substance. During penetration, zona material was packed tightly around the spermatozoon, and no large hole was formed. A spermatozoon within the perivitelline space had made contact with the oolemma by way of its apical tip. In a spermatozoon partly incorporated into the ooplasm, fusion appeared to have taken place between its plasma membrane and that of the oolemma. Mucoid coat material became deposited outside the zona at this time; its existence and/or the release of cortical granule content probably prevented polyspermy. Once inside the egg cytoplasm, the sperm head sometimes travelled a considerable distance before chromatin decondensation occurred. In addition, it appeared to rotate somewhat on its axis at this time. Finally, some membranous structures were found around two condensed sperm heads in the ooplasm, which may have been part of the pronuclear envelope. Thus this study on in vivo fertilization in the dunnart documents, for the first time, some aspects of fertilization in an Australian marsupial as seen with the transmission electron microscope; it indicates a few differences from those previously found for the American opossum.     

我国被子植物体外受精研究——十年回顾

简要回顾了十余年来我国被子植物体外受精领域的研究历程和主要成就.我国科学家在性细胞的分离, 尤其是生活胚囊和雌配子的分离方面走在国际前列. 在一定程度上我国科学家的开拓性工作引起了国际同行的广泛兴趣和重视, 推动了该领域的迅速发展, 为实现被子植物真正意义的体外受精做出了独特的贡献.十年来, 我国已建立了自己独特的被子植物离体受精实验技术系统并利用该系统与国际同行合作进行了一系列研究, 尤其在探讨配子相互作用、卵细胞激活、避免多精入卵的机制等方面做出了创新性工作, 为正确认识体外受精系统的优越性与局限性, 也为深入探讨受精机制提供了有价值的新资料.目前正以体外受精操作系统结合细胞生物学和分子生物学等多种手段对受精过程中的一些重要事态与机理做进一步探讨.     

An unexpectedly sophisticated, V-shaped spermatozoon in Demospongiae (Porifera): reproductive and evolutionary implications

The demosponge Crambe crambe shows a peculiar spermatogenesis, hard to be reconciled with the basal position of sponges in the animal phylogeny. Early spermatogenesis stages showed most of the simple features expected in sponges. However, spermiogenesis departed from the anticipated process. Spermatids lengthened remarkably, forming a deep cytoplasmic pit around the cilium insertion, with the proximal axoneme bending to produce a V-shaped spermatozoon surprisingly similar to that known in the phylum Phoronida. The cytology was unexpectedly complex, with a needle-like nucleus of helically condensed chromatin, a conical acrosome with a subacrosomal rod, and a mitochondrion connected to the basal body by striated rootlets. These findings establish that the spermatozoon of broad-casting demosponges occurs in two structural categories ('primitive' and 'modified' type). This dualistic condition must necessarily have pre-dated the evolutionary apparition of higher metazoans, if we are to keep regarding sponges as the most primitive animals. We hypothesize that internal fertilization in C. crambe – and incidentally other demosponges – may depart from the general model assumed for spermcasting sponges. The V-shape of this spermatozoon suggests a design to favour autonomous penetration through the dense mesohyl to reach the oocytes, rather than engulfment and transportation by carrier cells towards the oocyte.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 413–426.     

Sexual and Asexual Processes in Protoperidinium steidingerae Balech (Dinophyceae), with Observations on Life-History Stages of Protoperidinium depressum (Bailey) Balech (Dinophyceae)

ABSTRACT. A suite of morphological, histological, and molecular techniques was used to reveal for the first time division, sexuality, mandatory dormancy period of hypnozygotes, and identity of life-history stages of any Protoperidinium spp. In both Protoperidinium steidingerae and Protoperidinium depressum , asexual division occurred by eleutheroschisis within a temporary cyst, yielding two daughter cells. Daughter cells were initially round and one-half to two-thirds the size of parent cells then rapidly increased in size, forming horns before separating. Gamete production and fusion was constitutive in clonal and non-clonal cultures, indicating that both species may be homothallic. Gametes were isogamous, approximately half the size and lacking the pink pigmentation of the vegetative cells, and were never observed to feed. Gamete fusion resulted in a planozygote with two longitudinal flagella. Planozygotes of P. steidingerae formed hypnozygotes. The fate of planozygotes of P. depressum is unknown. Hypnozygotes of P. steidingerae had a mandatory dormancy period of ca. 70 days. Germination resulted in planomeiocytes with two longitudinal flagella. Nuclear cyclosis occurred in the planozygotes of P. depressum , but in the planomeiocytes of P. steidingerae . The plate tabulation and gross morphology of gametes of P. steidingerae and P. depressum differed markedly from those of vegetative cells. Thus, misidentification of morphologically distinct life-history stages and incomplete examination of thecal plate morphology in field specimens has likely led to taxonomic confusion of Protoperidinium spp. in previous studies.