Chinese hamster spermatozoa during epididymal maturation were examined by thin sectioning, freeze-fracture, and surface replica. Membrane-limited vesicles and tubules (MVTs) attach to the plasma membrane over the acrosome of the spermatozoa in the distal caput through proximal cauda epididymidis. The origin of these MVTs is likely to be spermatozoa degenerating in the epididymal lumen. The attachment of MVTs to the plasma membrane seems to be mediated by a paste-like substance covering the plasma membrane. A parallel striation pattern of intramembranous particles (IMPs) is seen in the plasma membrane of almost the entire postacrosomal region of the epididymal spermatozoa. The patterned domain begins to appear in the proximal caput epididymidis. The number and density of IMPs in the plasma membrane of the postacrosomal region increases with the development of striated pattern of IMPs in this region. In the cauda epididymidis, the redundant nuclear envelope elongates to form a shirt-like membrane covering the mitochondrial sheath. The elongated portion of the nuclear envelope is devoid of nuclear pores and has few IMPs.

Acrosome↗

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Effects of a single oral administration of ethinyl estradiol on early pregnancy in the mouse.

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Animals↗

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Application of ICSI for cryptorchid therapy.

Recent advances are provided in assisted reproductive technology and application of ICSI as treatment for infertile patients with previous cryptorchidism. Fertility rates were 25-81% and 8-48% in patients with unilateral and bilateral cryptorchidism, respectively. With the advent of assisted reproductive technology, pregnancy was not possible without sperm. Research in humans has resulted in pregnancy and live births after the injection of haploid germ cells (round spermatids) into the oocytes. In the mouse tetraploid germ cells (primary spermatocytes), nuclei have been injected into oocytes, which undergo meiosis, forming an embryo and live young. In animal models, round spermatids from both cryptorchid and previously cryptorchid testes can initiate normal embryo development when injected into mature oocytes. These cells may be used as substitutes for mature sperm. However, cryptorchidism in humans is often associated with inherent defects of spermatogenesis. With successive breakthroughs in treatment of impaired fertility associated with cryptorchidism, there is an increased risk of genetic abnormalities in the progeny. It is incumbent on clinicians who are involved in reproductive medicine to proceed cautiously with the development and application of assisted reproductive techniques to avoid creating future generations of genetically abnormal individuals.

Cryptorchidism↗

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Phospholipids in guinea pig spermatozoa before and after capacitation in vitro.

Phospholipids of guinea pigs spermatozoa were examined using high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). Two types of spermatozoa were compared: fresh epididymal (uncapacitated) spermatozoa and epididymal spermatozoa preincubated in Ca2+-free medium for 17-18 hr (capacitated spermatozoa). Determination of lipid phosphorus revealed that the total phospholipid content of spermatozoa did not change significantly during capacitation of spermatozoa. HPLC analyses of choline and ethanolamine phosphatides, lysophosphatides, and sphingomyelin revealed that the relative amounts of these phospholipids remained unchanged during capacitation. In contrast to spermatozoa, phospholipids in the medium surrounding spermatozoa reduced by 20% during capacitation of spermatozoa, suggesting that spermatozoa may utilize extracellular phospholipids as possible energy sources during their capacitation.

Animals↗

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Ca2+ is essential for the motility of plasma membrane-intact, but not of demembranated, hamster spermatozoa.

Extracellular Ca2+ is essential for the flagellar motility of membrane-intact hamster spermatozoa. When suspended in a medium completely free of Ca2+, most spermatozoa quickly lost their motility, and remained motionless until they were transferred back to Ca2+-containing medium. The motility could not be restored after the spermatozoa had been in Ca2+-free medium for more than 2 hr. Unlike membrane-intact spermatozoa, demembranated spermatozoa (spermatozoa without plasma membranes) exhibited active movement in Ca2+-free medium, and their motility was inhibited by Ca2+. In view of these facts, we suggest that the "hyperactivated motility" which membrane-intact spermatozoa display upon capacitation may be due to the activation of a Ca2+-dependent adenylate cyclase (and the resultant increase in intracellular cAMP), rather than being a direct effect of a rise in the intracellular Ca2+ concentration.

Animals↗

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Fluorescence energy transfer shows that various physical and chemical treatments of human sperm induce unpacking of chromatin.

Fluorescence resonance energy transfer (FRET) was used to study the changes which human sperm chromatin went through after various physical and chemical treatments. This technique showed a dilatation of the spatial relationship among chromatin liner arrays, with UV and DNAse among the treatments that gave rise to the highest increase. FRET image analysis showed that the chromatin linear arrays after treatment reach a spatial disarrangement similar to that brought about by sperm decondensation. Comparison of these results with the ability of human treated sperm to form pronuclei after microinjection into hamster eggs, suggests that the highly condensed spatial organization of sperm chromatin arrays may not be a necessary prerequisite for pronucleus formation.

Animals↗

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Round spermatid transfer and embryo development.

With the advent of assisted reproductive technology, pregnancy has become possible without sperm. The nucleus of the round spermatid, which contains a complete haploid set of chromosomes, can be used as a substitute for spermatozoa in animals and humans. In the mouse, round spermatids from surgically induced cryptorchid and prepubertal testes can fertilize normally and develop into normal offspring. The spermatid nucleus develops into a large pronucleus only when the oocyte is activated by artificial means, such as electrostimulation and oscillogen injection. Nuclei of mouse primary spermatocyte have been injected into oocytes and found to undergo meiosis, form an embryo and produce live young. Intracytoplasmic injection of male immature germ cells may become available as a treatment for patients with azoospermia due to maturation arrest.

Animals↗

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Mouse embryonic stem (ES) cell lines established from neuronal cell-derived cloned blastocysts.

We have established mouse embryonic stem (ES) cell lines from blastocysts derived by transfer of nuclei of fetal neuronal cells. These neuronal cell-derived embryonic cell lines had properties that characterize them as ES cells, including typical cell markers and alkaline phosphatase activity. Moreover, the cells had a normal karyotype and were pluripotent, as they were capable of differentiating into all three germ layers. Although they were derived from neuronal donor nuclei, the cells no longer expressed neuronal markers; however, they were capable of differentiating into cells with neuronal characteristics. These results suggest that the clone-derived cells have fully acquired an ES cell character. Thus, ES cells can be derived from embryos resulting from nuclear transfer, which results in reprogramming of the genetic information and acquisition of pluripotency. ES cells established from somatic cell-derived blastocysts could be useful not only as research tools for studying reprogramming but also as models for cell-based transplantation therapy.

Alkaline Phosphatase↗