Post-Testicular Sperm Maturation: Centriole Pairs, Found in Upper Epididymis, are Destroyed Prior to Sperm's Release at Ejaculation.

The fertilizing sperm's lengthiest unchartered voyage is through the longest, least-investigated organ in a man's body - the Epididymis. Over six meters long in men, ~80 meters in stallions and over one-hundred times a mouse's body length, there are few functions known aside from sperm storage and nutrition. While spermatogenesis is completed in the testes, here we demonstrate sperm centriole reduction occurs within the epididymis. Investigations of GFP-CENTR mice and controls demonstrate both the presence of centriole pairs in the upper caput region of the epididymis and, the destruction, first, of the distal and, then, of the proximal centriole as the sperm transits to the cauda and vas deferens in preparation for its climactic release. These centrioles can neither recruit γ-tubulin nor nucleate microtubules when eggs are inseminated or microinjected, yet numerous maternally-nucleated cytasters are found. These sperm centrioles appear as vestigial basal bodies, destroyed in the mid-to-lower corpus. Post-testicular sperm maturation, in which sperm centrioles found in the caput are destroyed prior to ejaculation, is a newly discovered function for the epididymis.

Intracytoplasmic sperm injection: stiletto conception or a stab in the dark.

To describe the importance of molecular and cellular analyses in intracytoplasmic sperm injection (ICSI) the authors review the literature on biological challenges in ICSI and associated techniques. Several matters can be proposed in molecular and cellular challenges in ICSI for safety and efficacy: (1) a reliable and convenient animal model for understanding the molecular and cellular basis of human ICSI must be established, and molecular and cellular analysis of the first cell cycle of human fertilization should be better understood; (2) a proper assay for human sperm function that contributes to the indication for ICSI should be developed; and (3) de novo and transmitted genetic security in ICSI should be examined.

Arrest of cell cycle progression during first interphase in murine zygotes microinjected with anti-PCM-1 antibodies.

To investigate the function of the centrosome protein PCM-1, antibodies against PCM-1 were microinjected into either germinal vesicle stage meiotic oocytes or fertilized mouse eggs, and cell cycle progression events (i.e., microtubule assembly, chromosome and centrosome organization, meiotic maturation) were assayed. These studies determined that microinjected PCM-1 antibodies arrested cell cycle progression, with anti-PCM-1 arresting fertilized eggs at the pronucleate stage when injected during G1. Analysis of the injected eggs determined that centrosome disruption and microtubule cytaster disorganization accompanied the cell cycle arrest. Anti-PCM-1 blocked neither pronuclear centration, completion of mitosis when microinjected into zygotes at G2, nor meiotic maturation when microinjected into immature oocytes. These results identify a novel role for PCM- 1 in cell cycle regulation, and indicate that PCM-1 must fulfill an essential function for cells to complete interphase.

Fertilization imaged in 2-, 3- and four dimensions: molecular insights for treating infertility.

Fertilization in humans follows a complex series of events including binding of the sperm to the oocyte plasma membrane, oocyte activation, the completion of meiotic maturation of the oocyte with the extrusion of the second polar body, the decondensation of the sperm nucleus and the maternal chromosomes into male and female pronuclei and the restoration of the sperm centrosome. This duplicates and separates, forming two mitotic spindle poles upon which the parental genomes can intermix to complete fertilization. The use of intracytoplasmic sperm injection (ICSI) has been highly effective as a treatment for severe male infertility and thousands of ICSI babies have been born world-wide. Working with rhesus monkey gametes, we have developed a preclinical animal model for understanding the cell biological basis of ICSI. Typically, ICSI results in abnormal nuclear remodeling during sperm decondensation due to the presence of the sperm acrosome and perinuclear structures normally removed at the oolemma during in vitro fertilization. These unusual modifications raise concerns that the ICSI procedure itself might lead to the observed increase in chromosome anomalies reported for

Transgenic monkeys produced by retroviral gene transfer into mature oocytes.

Transgenic rhesus monkeys carrying the green fluorescent protein (GFP) gene were produced by injecting pseudotyped replication-defective retroviral vector into the perivitelline space of 224 mature rhesus oocytes, later fertilized by intracytoplasmic sperm injection. Of the three males born from 20 embryo transfers, one was transgenic when accessible tissues were assayed for transgene DNA and messenger RNA. All tissues that were studied from a fraternal set of twins, miscarried at 73 days, carried the transgene, as confirmed by Southern analyses, and the GFP transgene reporter was detected by both direct and indirect fluorescence imaging.

ICSI choreography: fate of sperm structures after monospermic rhesus ICSI and first cell cycle implications.

We have dissected the initial stages of fertilization by intracytoplasmic sperm injection of single spermatozoa into prime oocytes from fertile rhesus monkeys (Macaca mulatta). DNA decondensation was delayed at the apical portion of the sperm head. It is possible that this asynchronous male DNA decondensation could be related to the persistence of the sperm acrosome and perinuclear theca after injection. However, incomplete male pronuclear formation did not prevent sperm aster formation, microtubule nucleation and pronuclear apposition. In contrast, DNA synthesis was delayed in both pronuclei until the sperm chromatin fully decondensed, indicating that male pronuclear formation constitutes an important checkpoint during the first embryonic cell cycle.

Contractile apparatus of the normal and abortive cytokinetic cells during mouse male meiosis.

Mouse male meiotic cytokinesis was studied using immunofluorescent probes against various elements of cytokinetic apparatus and electron microscopy. In normal mice, some spermatocytes fail to undergo cytokinesis after meiotic I or II nuclear divisions, forming syncytial secondary spermatocytes and spermatids. Abnormal cytokinetic cells develop sparse and dispersed midzone spindles during the early stage. However, during late stages, single and compact midzone spindles are formed as in normal cells, but localize asymmetrically and attach to the cortex. Myosin and f-actin were observed in the midzone spindle and midbody regions of normally cleaving cells as well as in those cells that failed to develop a cytokinetic furrow, implying that cytokinetic failure is unlikely to be due to defect in myosin or actin assembly. Depolymerization of microtubules by nocodazole resulted in the loss of the midbody-associated f-actin and myosin. These observations suggest that actin-myosin localization in the midbody could be a microtubule-dependent process that may not play a direct role in cytokinetic furrowing. Anti-centrin antibody labels the putative centrioles while anti-(gamma)-tubulin antibody labels the minus-ends of the midzone spindles of late-stage normal and abnormal cytokinetic cells, suggesting that the centrosome and midzone spindle nucleation in abnormal cytokinetic cells is not different from those of normally cleaving cells. Possible use of mouse male meiotic cells as a model system to study cytokinesis has been discussed.

Ubiquitinated sperm mitochondria, selective proteolysis, and the regulation of mitochondrial inheritance in mammalian embryos.

The strictly maternal inheritance of mitochondria and mitochondrial DNA (mtDNA) in mammals is a developmental paradox promoted by an unknown mechanism responsible for the destruction of the sperm mitochondria shortly after fertilization. We have recently reported that the sperm mitochondria are ubiquitinated inside the oocyte cytoplasm and later subjected to proteolysis during preimplantation development (P. Sutovsky et al., Nature 1999; 402:371-372). Here, we provide further evidence for this process by showing that the proteolytic destruction of bull sperm mitochondria inside cow egg cytoplasm depends upon the activity of the universal proteolytic marker, ubiquitin, and the lysosomal apparatus of the egg. Binding of ubiquitin to sperm mitochondria was visualized by monospecific antibodies throughout pronuclear development and during the first embryonic divisions. The recognition and disposal of the ubiquitinated sperm mitochondria was prevented by the microinjection of anti-ubiquitin antibodies and by the treatment of the fertilized zygotes with lysosomotropic agent ammonium chloride. The postfecundal ubiquitination of sperm mitochondria and their destruction was not seen in the hybrid embryos created using cow eggs and sperm of wild cattle, gaur, thus supporting the hypothesis that sperm mitochondrion destruction is species specific. The initial ligation of ubiquitin molecules to sperm mitochondrial membrane proteins, one of which could be prohibitin, occurs during spermatogenesis. Even though the ubiquitin cross-reactivity was transiently lost from the sperm mitochondria during epididymal passage, likely as a result of disulfide bond cross-linking, it was restored and amplified after fertilization. Ubiquitination therefore may represent a mechanism for the elimination of paternal mitochondria during fertilization. Our data have important implications for anthropology, treatment of mitochondrial disorders, and for the new methods of assisted procreation, such as cloning, oocyte cytoplasm donation, and intracytoplasmic sperm injection.

SNAREs in mammalian sperm: possible implications for fertilization.

Soluble N-ethylmalameide-sensitive factor attachment protein receptor (SNARE) proteins are present in mammalian sperm and could be involved in critical membrane fusion events during fertilization, namely the acrosome reaction. Vesicle-associated membrane protein/synaptobrevin, a SNARE on the membrane of a vesicular carrier, and syntaxin 1, a SNARE on the target membrane, as well as the calcium sensor synaptotagmin I, are present in the acrosome of mammalian sperm (human, rhesus monkey, bull, hamster, mouse). Sperm SNAREs are sloughed off during the acrosome reaction, paralleling the release of sperm membrane vesicles and acrosomal contents, and SNARE antibodies inhibit both the acrosome reaction and fertilization, without inhibiting sperm-egg binding. In addition, sperm SNAREs may be responsible, together with other sperm components, for the asynchronous male DNA decondensation that occurs following intracytoplasmic sperm injection, an assisted reproduction technique that bypasses normal sperm-egg surface interactions. The results suggest the participation of sperm SNAREs during membrane fusion events at fertilization in mammals.

TransgenICSI reviewed: foreign DNA transmission by intracytoplasmic sperm injection in rhesus monkey.

This brief review considers the status of transgenesis by intracytoplasmic sperm injection (ICSI) with nonhuman primates. GFP expressing rhesus macaques embryos (mean = 34.6%; N = 81) were produced by ICSI using rhodamine-tagged DNA encoding the green fluorescence protein (GFP) gene bound on sperm. Rhodamine signal was lost at the egg surface during in vitro fertilization (IVF) but could be traced by dynamic imaging during ICSI within the egg cytoplasm. GFP gene was expressed as early as the 4-cell stage in ICSI embryos but not in embryos produced by in vitro fertilization (IVF). The percentage of GFP expressing blastomeres increased during embryogenesis to the blastocyst stage. Three offspring resulted from seven embryo transfers-a set of anatomically normal twins (a male and a female) stillborn 35 days premature, and a healthy male born at term. Although transgene was not detected in the offspring, the successful production of live primates using DNA bound sperm by ICSI suggests an alternative route to creating transgenic animals. It also raises concern regarding transmission of infectious material during ICSI.

Microfilament stabilization by jasplakinolide arrests oocyte maturation, cortical granule exocytosis, sperm incorporation cone resorption, and cell-cycle progression, but not DNA replication, during fertilization in mice.

Jasplakinolide (JAS), which induces microfilament polymerization and stabilization, inhibits microfilament-mediated events in murine oocyte maturation and fertilization in a fashion unlike the effects of cytochalasin B (CCB) and latranculin A (LAT A). JAS prevents egg polar body emission at a much lower concentration than either CCB or LAT A. Microfilament bundles were detected on the entire egg cortex after JAS exposure. Conversely, microfilament patterns did not change after exposure to CCB, and few microfilaments were observed after exposure to LAT A. Eggs that were allowed to recover from JAS were unable to recover normal microfilament organization. During oocyte maturation, JAS prevented both spindle migration to the oocyte cortex and first polar body emission. During in vitro fertilization, sperm head entered the eggs and formed pronuclei, but sperm tail entry, pronuclear centration, and second polar body emission were not detected. DNA synthesis occurs in these JAS-treated zygotes. JAS inhibited not only the formation, but also the disassembly, of incorporation cones. JAS was also found to prevent cortical granule exocytosis following artificial activation, and cortical granules were still beneath the plasma membrane even after activation. Finally, incorporation of microinjected nonmuscle actin into the microfilament network of mice eggs was delayed by JAS. We conclude that JAS acts as a microfilament inhibitor during maturation and fertilization and is more powerful than other inhibitors. Its mechanism differs in that it promotes assembly and stabilization of microfilaments. JAS is a novel cell permeable tool for the investigation of microfilament-dependent events in early mammalian development.

Cellular and molecular events after in vitro fertilization and intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) has heralded an era of tremendous improvements in treating male infertility leading to the births of thousands of babies. However, recent concerns over possible long-term effects of ICSI on offspring has prompted the development of a preclinical, nonhuman primate model to assess the safety of ICSI. Fluorescent imaging of rhesus macaque IVF zygotes revealed that this species shares many similarities with humans in terms of cytoskeletal and chromatin dynamics during fertilization. However, rhesus monkey zygotes fertilized by ICSI resulted in abnormal nuclear remodeling leading to asynchronous chromatin decondensation in the apical region of the sperm head, delaying the onset of DNA synthesis. The persistence of the acrosome and perinuclear theca on the apex of sperm introduced into the oocyte by ICSI may constrict the DNA in this region. Despite these differences, normal rhesus monkey ICSI embryos have been produced and have lead to several births after transfer. The irregularities described in this paper raise concerns that the ICSI procedure may result in chromatin damage during DNA decondensation and further highlight the need for devising improved pre-clinical assessment prior to global acceptance of this, and other, novel methods of assisted reproduction.

Sperm aster formation and pronuclear decondensation during rabbit fertilization and development of a functional assay for human sperm.

Microtubule organization and chromatin configurations in rabbit eggs after in vivo rabbit fertilization and after intracytoplasmic injection with human sperm were characterized. In unfertilized eggs, an anastral barrel-shaped meiotic spindle, oriented radially to the cortex, was observed. After rabbit sperm incorporation, microtubules were organized into a radial aster from the sperm head, and cytoplasmic microtubules were organized around the male and female pronuclei. The microtubules extending from the decondensed sperm head participated in pronuclear migration, and organization around the female pronucleus may also be important for pronuclear centration. Support for these observations was found in parthenogenetically activated eggs, in which microtubule arrays were organized around the single female pronucleus that formed after artificial activation. These observations support a biparental centrosomal contribution during rabbit fertilization as opposed to a strictly paternal inheritance pattern suggested from previous studies. In rabbit eggs that received injected human donor sperm, an astral array of microtubules radiated from the sperm neck and enlarged as the sperm head underwent pronuclear decondensation. gamma-Tubulin was observed in the center of the sperm aster. We conclude that the rabbit egg exhibits a blended centrosomal contribution necessary for completion of fertilization and that the rabbit egg may be a novel animal model for assessing centrosomal function in human sperm and spermatogenic cells following intracytoplasmic injection.

Highly degenerated distal centrioles in rhesus and human spermatozoa.

In humans and other mammals except rodents, the spermatozoa contribute the proximal centriole during fertilization. The inheritance of the distal centriole is not yet fully clear. In the present work, the distal centrioles of rhesus and human spermatozoa have been studied by transmission electron microscopy. The round and elongating rhesus spermatids possess both proximal and distal centrioles. The distal centriole extends posteriorly as an axoneme while the proximal centriole produces a microtubular adjunct. Ejaculated rhesus and human spermatozoa have intact proximal centrioles, but the distal centrioles have degenerated. The central pair of microtubules of the axoneme extends continuously into the distal centriolar region up to the sperm head. Serial transverse and longitudinal sections of the sperm neck region reveal few scattered microtubule duplexes or triplets in the distal centriolar region. The loss of the centriolar microtubules is more extensive on the ventral side of the neck region, the side where the proximal centriole resides. The distal centriole degenerates caudally from the rostral area. Immunogold electron microscopy with anti-beta-tubulin antibody showed that the distal centriolar regions possess 50% fewer gold particles than the proximal centrioles, indicating a significant loss of centriolar microtubules in the distal centriolar region. The A-tubules of the remaining triplets are filled with a dense material, as observed in the axoneme. Thus, rhesus and human spermatozoa introduce only proximal centrioles intact, whereas the distal centrioles are mostly disorganized in the mature spermatozoa.

Clonal propagation of primate offspring by embryo splitting.

Primates that are identical in both nuclear and cytoplasmic components have not been produced by current cloning strategies, yet such identicals represent the ideal model for investigations of human diseases. Here, genetically identical nonhuman embryos were produced as twin and larger sets by separation and reaggregation of blastomeres of cleavage-stage embryos. A total of 368 multiples were created by the splitting of 107 rhesus embryos with four pregnancies established after 13 embryo transfers (31% versus 53% in vitro fertilization controls). The birth of Tetra, a healthy female cloned from a quarter of an embryo, proves that this approach can result in live offspring.

Cytoskeletal aspects of assisted fertilization.

During mammalian fertilization, the zygotic centrosome organizes a large sperm aster, critical for uniting the male and female pronuclei prior to first mitosis. Fluorescent imaging of inseminated human oocytes has shown that centrosomal defects may result in abnormal microtubule nucleation preventing genomic union, suggesting a novel cause of fertilization failure. Working with rhesus monkey gametes, we have developed a preclinical model for understanding the cell biological basis of intracytoplasmic sperm injection (ICSI). Typically, ICSI results in abnormal nuclear remodeling during sperm decondensation due to the presence of the sperm acrosome and perinuclear theca, structures normally removed at the oolemma during IVF; this is turn causes a delay in the onset of DNA synthesis. These unusual modifications raise concerns that the ICSI procedure itself may result in chromatin damage during DNA decondensation and further highlight the need for a more rigorous assessment of methods of assisted reproduction prior to their global application.

Dynamic imaging of the metaphase II spindle and maternal chromosomesin bovine oocytes: implications for enucleation efficiency verification, avoidanceof parthenogenesis, and successful embryogenesis.

Manipulations of DNA and cellular structures are essential for the propagation of genetically identical animals by nuclear transfer. However, none of the steps have been optimized yet. This study reports a protocol that improves live dynamic imaging of the unfertilized bovine oocyte's meiotic spindle microtubules with microinjected polymerization-competent X-rhodamine-tubulin and/or with vital long-wavelength excited DNA fluorochrome Sybr14 so that the maternal chromosomes can be verifiably removed to make enucleated eggs the starting point for cloning. Suitability of the new fluorochromes was compared to the conventional UV excitable Hoechst 33342 fluorochrome. Enucleation removed the smallest amount of cytoplasm (4-7%) and was 100% efficient only when performed under continuous fluorescence, i.e., longer fluorescence exposure. This was in part due to the finding that the second metaphase spindle is frequently displaced (60.7 +/- 10%) from its previously assumed location subjacent to the first polar body. Removal of as much as 24 +/- 3% of the oocyte cytoplasm underneath the polar body, in the absence of fluorochromes, often resulted in enucleation failure (36 +/- 6%). When labeled oocytes were exposed to fluorescence and later activated, development to the blastocyst stage was lowest in the group labeled with Hoechst 33342 (3%), when compared to Sybr14 (19%), rhodamine-tubulin (23%), or unlabeled oocytes (37%). This suggests that longer wavelength fluorochromes can be employed for live visualization of metaphase spindle components, verification of their complete removal during enucleation, and avoidance of the confusion between artifactual parthenogenesis versus "cloning" success, without compromising the oocyte's developmental potential after activation.