Cloning in action: can embryo splitting,induced pluripotency and somatic cell nuclear transfer contribute to endangered species conservation?

The term ‘cloning’ refers to the production of genetically identical individuals but has meant different things throughout the history of science: a natural means of reproduction in bacteria, a routine procedure in horticulture, and an ever-evolving gamut of molecular technologies in vertebrates. Mammalian cloning can be achieved through embryo splitting, somatic cell nuclear transfer, and most recently, by the use of induced pluripotent stem cells. Several emerging biotechnologies also facilitate the propagation of genomes from one generation to the next whilst bypassing the conventional reproductive processes. In this review, we examine the state of the art of available cloning technologies and their progress in species other than humans and rodent models, in order to provide a critical overview of their readiness and relevance for application in endangered animal conservation.     

Production,characterization, and immunocytochemical applications of monoclonal antibodies to human sperm protamines

Three monoclonal antibodies against human protamines were obtained by immunization with total human basic nuclear proteins or purified protamine HP3. The specificity of antibodies was assessed by enzyme-linked immunosorbent assay (ELISA) and Western blot. They recognized three distinct epitopes: One was specific for the protamine P1 family, another was specific for the protamine P2 family and the third was common to both families. All were specific for the human species. Antibodies were used to detect protamines in germ cells by indirect immunofluorescence and by immunoelectron microscopy. Protamines appeared in spermtid nuclei at steps 4–5 of spermiogenesis, i.e., during the chromatin condensation process, and were not accumulated in the cytoplasm before entering the nucleus. © 1993 Wiley-Liss, Inc.     

Strontium supports capacitation and the acrosome reaction in mouse sperm and rapidly activates mouse eggs

Extracellular Ca²⁺ is required for capacitation and fertilization in the mouse, but very little is known about the ability of other divalent cations to substitute for Ca²⁺. In this study, Sr²⁺, Ba²⁺, and Mg²⁺ were evaluated for their ability to support capacitation, the acrosome reaction, hyperactivated motility, and fertilization. Ba²⁺ proved to be ineffective, but Mg²⁺-containing medium was able to support capacitation to a greater extent than unsupplemented Ca²⁺-deficient media; despite this, Ca²⁺ was required for fertilization. In contrast, Sr²⁺ proved capable of substituting for Ca²⁺ in all events. Furthermore, Sr²⁺-induced responses were indistinguishable from the corresponding Ca²⁺-induced ones: Sperm capacitated at the same rate and underwent the acrosome reaction to the same extent. However, demonstration of sperm:egg fusion in Sr²⁺ required the use of zona-free eggs. This was due not to the inability of the sperm to penetrate the zona but to the very rapid activation and cortical granule release by eggs in response to Sr²⁺. When zona-intact eggs were used, the block to polyspermy had been mounted by the time sperm had penetrated the zona. A 15 min exposure to Sr²⁺ was sufficient to block sperm fusion, but a longer exposure was required to ensure the resumption of meiosis in eggs; such a response was surprising in that the eggs were freshly ovulated and not susceptible to activation by many different treatments. Thus Sr²⁺ can profoundly affect both gametes in the mouse: It substitutes completely for Ca²⁺ in sperm responses and rapidly activates eggs, possibly by displacing Ca²⁺ from intracellular stores into the cytoplasm, where the Ca²⁺ can then trigger the various events of activation.     

One Hundred and One Years of Chemotaxis. Pfeffer,Pheromones, and Fertilization

Chemotaxis of flagellated unicellular gametes was discovered by Wilhelm Pfeffer. In most known systems the lure is a metabolic waste product ritualized for sexual attraction. A special case are the female gametes of brown algae. They secrete polyunsaturated C-8 or C-11 hydrocarbons mostly with straight chain or unsubstituted tri-, penta- or heptacyclic structures. Their biogenetic origin stems from polyunsaturated fatty acids. The reactions leading to the different structures comprise lipoxygenase-type radical formations and dehydrogenating decarboxylations. — Most gametes produce a variety of such hydrocarbons; however, only one of them is the active principle. Biological activity is highly stereospecific; but frequently the bouquets contain lures for other species. Although cross-fertilization is excluded, they may be used to defend the habitat against overgrowth by other seaweeds. Besides chemoattractive action, some secretions also synchronize hatching of the spermatozoids. One of the compounds responsible is characterized by an epoxy substitution. — For most of the structures, stereospecific and versatile syntheses are available, which were used to investigate the binding site. This forms a template for the signal molecule with specifically situated polarizing groups that interact with the π-electrons of the double bonds. The message is transformed into changes of flagellar beat patterns which ensure meeting of the partners followed by fertilization.     

The point of no return: Up to what point should we be allowed to withdraw consent to the storage and use of embryos and gametes?

This article discusses when it is ethically acceptable to withdraw consent for the storage and use of embryos and gametes. Currently, the law in the UK states that consent to use of a gamete or embryo can be withdrawn up to the point of the embryo's transfer to the recipient's uterus or when the gamete is used in providing treatment services; that is, the ‘point of no return’. In this article, we will consider other points of no return and argue that having a single point of no return, a one size fits all form of consent can, in some cases, lead to restrictions on individuals’ autonomy and cause particular types of harm. Therefore, having different points of no return that fit different circumstances could extend autonomy and allow people to enter into agreements that are tailored more to their own particular needs and circumstances.     

First observations on egg release in the oviparous sponge Chondrilla nucula (Demospongiae, Chondrosida, Chondrillidae) in the Mediterranean Sea

Abstract. The reproduction of the demosponge Chondrilla nucula in Portofino (Ligurian Sea, Italy) was studied during August 2001. Eighteen individuals were sampled and examined with light microscopy for the presence of gametes, and 5 individuals carrying oocytes were found. In addition to microscopic observations, reproductive individuals could be easily identified as female even at the macroscopic level because of the presence of a grayish layer in the mesohyl where oocytes were concentrated. Oogenesis resulted in modifications of the external sponge morphology and of the aquiferous system. Approximately one‐third of the sponge body was filled with oocytes with the consequent disappearance of choanocyte chambers in the reproductive portion of the sponge. Under laboratory conditions, we obtained fertilized eggs from females and observed the first stages of embryonic development. Our observations suggest that fertilization in specimens of Chondrilla nucula occurs internally and not in the water. During the 2 years following these observations, no reproductive specimens were found among the same population during the reported reproductive period.     

Contrasting breeding periodicity of nearby populations of the lugworm,Arenicola marina (Annelida,Polychaeta)

Summary

The breeding cycles of lugworm taken from three locations on the east coast of Ireland have been investigated through examination of coelomic fluid samples which contain the gametes, taken at monthly intervals. These reveal major differences in the timing and duration of breeding between the populations despite their geographic propinquity. The differences in breeding cycle cannot be ascribed to varietal or species differences and may arise from the different habitats occupied by the lugworm populations studied.