(1/353) A novel trans-complementation assay suggests full mammalian oocyte activation is coordinately initiated by multiple, submembrane sperm components.
To initiate normal embryonic development, an egg must receive a signal to become activated at fertilization. We here report that the ability of demembranated sperm heads to activate is abolished after incubation over the range 20-44 degreesC and is sensitive to reducing agents. On the basis of this observation, we have developed a microinjection-based, trans-complementation assay in order to dissect the heat-inactivated sperm-borne oocyte-activating factor(s) (SOAF). We demonstrate that the failure of heat-inactivated sperm heads to activate an egg is rescued by coinjection with dithiothreitol-solubilized SOAF from demembranated sperm heads. The solubilized SOAF (SOAFs) is trypsin sensitive and is liberated from demembranated heads in a temperature-dependent manner that inversely correlates with the ability of sperm heads to activate. This argues that SOAFs is a proteinaceous molecular species required to initiate activation. Injection of oocytes with mouse or hamster sperm cytosolic factors, but not SOAFs alone, induced resumption of meiosis, further suggesting that these cytosolic factors and SOAF are distinct. Collectively, these data strongly suggest that full mammalian oocyte activation is initiated by the coordinated action of one or more heat-sensitive protein constituents of the perinuclear matrix and at least one heat-stable submembrane component. (+info)
(2/353) Destruction of protamine in human sperm inhibits sperm binding and penetration in the zona-free hamster penetration test but increases sperm head decondensation and male pronuclear formation in the hamster-ICSI assay.
PURPOSE: Our purpose was to investigate the fertilizing ability of human protamine-damaged sperm in a heterologous system using hamster oocytes. METHODS: The protamine of the sperm were damaged by exposure to dithiothreitol, a disulfide-reducing agent. Their ability to penetrate and form male pronuclei were investigated using the zona-free hamster penetration test and the hamster-intracytoplasmic sperm injection assay, respectively. RESULTS: The zona-free hamster penetration test revealed that protamine-damaged sperm are unable to bind and penetrate the hamster oocyte. On the other hand, hamster-intracytoplasmic sperm injection assay results showed that 56.9% and 39.2% of the injected oocytes developed male pronuclei in protamine-damaged and live-intact sperm groups, respectively, with a significant difference in these rates (P < 0.01). CONCLUSIONS: This study shows that protamine-damaged sperm are able to undergo sperm head decondensation and male pronuclear formation only when injected into the ooplasm, although they cannot bind and penetrate through the zona and enter the ooplasm. (+info)
(3/353) Gender preselection in cattle with intracytoplasmically injected, flow cytometrically sorted sperm heads.
We investigated the development to the blastocyst and subsequent live-offspring stages of in vitro-matured bovine oocytes intracytoplasmically injected with flow cytometrically sorted bull sperm heads. Bull sperm heads, prepared by ultrasound sonication, were distinguished and sorted on the basis of their relative DNA contents using a flow cytometer/cell sorter modified for sorting sperm. By fluorescence in situ hybridization, the proportion of sperm confirmed as having Y specific DNA in the fraction sorted for the Y sperm was 82%. Injection with single sorted sperm heads of in vitro-matured oocytes (cultured for 24 h) resulted in 46.6% cleavage and 6.9% blastocyst development rates. Embryo transfer of 48 blastocysts (Days 7-8) to recipients (one per recipient) resulted in 20.8% pregnancy and 20.8% normal live offspring production rates. The birth of 8 male and 2 female calves represents an 80% sex preselection accuracy rate. (+info)
(4/353) Fertility of ejaculated and testicular megalohead spermatozoa with intracytoplasmic sperm injection.
In this study the fertility and outcome of intracytoplasmic sperm injection (ICSI) using megalohead spermatozoa from the ejaculates and testicles was evaluated. Seventeen males with megalohead and pinhead sperm forms in their ejaculate were studied in 22 cycles. A high number of sperm heads without tails and abundant round spermatid forms were commonly observed. Round-headed spermatozoa were seldom accompanied by these severely abnormal spermatozoa. The majority of megalohead spermatozoa were observed to have multiple tails, were predominant in the sample, and were used for ICSI. Ejaculated megalohead spermatozoa were used for ICSI in 15 cycles, while testicular spermatozoa were used in seven cycles where there were no vital spermatozoa or spermatozoa of low vitality in the ejaculate. The same abnormal morphology was observed in the testicles as in the ejaculated spermatozoa in the same males. Mean (+/- SD) low motility 4.7 +/- 5.6% and sperm count (3.8 +/- 4.19 x 10(6)) were common findings in these severely teratozoospermic patients. A low fertilization rate (43.2%) was achieved by using megalohead sperm forms (group I, n = 17) in comparison with the control group (60.2%) which had zero normal sperm morphology according to strict criteria (group II, n = 30) (P <0.01). Furthermore, a low pregnancy rate (9.1%) was obtained in the megalohead sperm group in comparison with the control group (40%) (P <0.05). Low fertilization and pregnancy rates may be due to a high incidence of chromosomal abnormalities from severely defective spermatozoa in the ejaculate. Couples should be counselled and warned about possible low fertilization and pregnancy rates with ICSI when only pinhead and megalohead forms with a high number of sperm heads without tails are present in the ejaculate. (+info)
(5/353) Mammalian transgenesis by intracytoplasmic sperm injection.
Coinjection of unfertilized mouse oocytes with sperm heads and exogenous DNA encoding either a green fluorescent protein (GFP) or beta-galactosidase reporter produced 64 to 94 percent transgene-expressing embryos, reflecting DNA-sperm head association before coinjection. Nonselective transfer to surrogate mothers of embryos in the GFP series generated about 20 percent offspring expressing the integrated transgene. These data indicate that exogenous DNA can reproducibly be delivered into an oocyte by microinjected spermatozoa and suggest an adaptable method of transgenesis. (+info)
(6/353) Self-organization of microtubule asters induced in Xenopus egg extracts by GTP-bound Ran.
The nucleotide exchange activity of RCC1, the only known nucleotide exchange factor for Ran, a Ras-like small guanosine triphosphatase, was required for microtubule aster formation with or without demembranated sperm in Xenopus egg extracts arrested in meiosis II. Consistently, in the RCC1-depleted egg extracts, Ran guanosine triphosphate (RanGTP), but not Ran guanosine diphosphate (RanGDP), induced self-organization of microtubule asters, and the process required the activity of dynein. Thus, Ran was shown to regulate formation of the microtubule network. (+info)
(7/353) Stimulation of microtubule aster formation and spindle assembly by the small GTPase Ran.
Ran, a small guanosine triphosphatase, is suggested to have additional functions beyond its well-characterized role in nuclear trafficking. Guanosine triphosphate-bound Ran, but not guanosine diphosphate-bound Ran, stimulated polymerization of astral microtubules from centrosomes assembled on Xenopus sperm. Moreover, a Ran allele with a mutation in the effector domain (RanL43E) induced the formation of microtubule asters and spindle assembly, in the absence of sperm nuclei, in a gammaTuRC (gamma-tubulin ring complex)- and XMAP215 (Xenopus microtubule associated protein)-dependent manner. Therefore, Ran could be a key signaling molecule regulating microtubule polymerization during mitosis. (+info)
(8/353) Ran-GTP stabilises microtubule asters and inhibits nuclear assembly in Xenopus egg extracts.
Ran is an abundant GTPase of the Ras superfamily that is highly conserved in eukaryotes. In interphase cells, Ran is mainly nuclear and thought to be predominantly GTP-bound, but it is also present in the cytoplasm, probably GDP-bound. This asymmetric distribution plays an important role in directing nucleocytoplasmic transport. Ran has also been implicated in cell cycle control, including the transition from mitosis to interphase when the compartmentalisation of the nucleus is established. Here, we have examined the role of Ran in this transition using a cell-free system of Xenopus egg extracts supplemented with sperm heads that provides a model for microtubule aster formation and post-M phase nuclear assembly. Ran-GTP, added as wild-type protein, a mutant defective in GTPase activity (Q69L), or generated by addition of the specific nucleotide exchange factor RCC1, stabilises large microtubule asters nucleated at the sperm centrosome, prevents the redistribution of NuMA from the aster to the nucleus and blocks chromatin decondensation. In contrast, Ran GDP does not stabilise microtubules or inhibit nuclear assembly. RanT24N and RanBP1, which oppose the generation of Ran-GTP by RCC1, arrest nuclear growth after disappearance of the aster. Ran associates with microtubule asters in egg extracts and with mitotic spindles in somatic Xenopus cells, suggesting that it may affect microtubule stability directly. These results show that Ran has a novel function in the control of microtubule stability that is clearly distinct from nucleocytoplasmic transport. The Ran GDP/GTP switch may play a role in co-ordinating changes in the structure of microtubules and the assembly of the nucleus associated with the transition from mitosis to interphase. (+info)