Calvin Simerly

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

Abstract 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.

Development: Ubiquitin tag for sperm mitochondria

Like other mammals, humans inherit mitochondria from the mother only, even though the sperm contributes nearly one hundred mitochondria to the fertilized egg. In support of the idea that this strictly maternal inheritance of mitochondrial DNA arises from the selective destruction of sperm mitochondria, we show here that sperm mitochondria inside fertilized cow and monkey eggs are tagged by the recycling marker protein ubiquitin. This imprint is a death sentence that is written during spermatogenesis and executed after the sperm mitochondria encounter the eggs cytoplasmic destruction machinery.

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. Mol. Reprod. Dev. 56:89–98, 2000.

Acetylated α-tubulin in microtubules during mouse fertilization and early development

Abstract α-Tubulin in the microtubules of mouse oocytes and embryos is acetylated in a specific spatial and temporal sequence. In the unfertilized oocyte, a monoclonal antibody to the acetylated form of α-tubulin is bound predominantly at the poles of the arrested metaphase meiotic spindle. The labeling intensity of the spindle microtubules is weaker as observed by immunofluorescence using oocytes double-labeled for total tubulin and acetylated α-tubulin, and as measured by immuno high-voltage electron microscopy (immunoHVEM) with colloidal gold; cytasters are not acetylated. At meiotic anaphase, the spindle becomes labeled, and by telophase and during second polar body formation only the meiotic midbody is acetylated. The sperm axoneme retains its acetylation after incorporation though the interphase microtubules are not detected. First mitosis follows a pattern similar to that observed at the second meiosis and during interphase only the mitotic midbodies are acetylated. After treatment with cold, colcemid, or griseofulvin, the remaining stable microtubules are acetylated, but immunoHVEM observations suggest that these fibers might not have been acetylated prior to microtubule disruption. Taxol stabilization does not alter acetylation patterns. Acetylated microtubules are not necessarily old microtubules since acetylated fibers are observed at 30 sec after cold recovery. These results show the presence of acetylated microtubules during meiosis and mitosis and demonstrate a cell-cycle-specific pattern of acetylation, with acetylated microtubules found at the centrosomes at metaphase, an increase in spindle labeling at anaphase, and the selective deacetylation of all but midbody microtubules at telophase.

Sperm Aster Formation and Pronuclear Decondensation During Rabbit Fertilization and Development of a Functional Assay for Human Sperm

Abstract 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. γ-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.

Centriole and centrin degeneration during mouse spermiogenesis.

Centrosome reduction during mouse spermiogenesis has been studied by immunofluorescent microscopy using anticentrin antibody (20H5) and TEM. Centrin is detected as two spots in round spermatids, corresponding to a pair of centrioles. In elongating spermatids, centrin spots colocalize with the centrioles in the neck region, while the perinuclear ring from which manchette microtubules arise, does not label with the antibody 20H5. The proximal centriole of the elongating spermatids develops a prominent adjunct, which assembles an aster of microtubules. TEM studies after immunogold labeling revealed that centrin is associated with the distal and the proximal centrioles, but not with the adjunct. Centrin labeling in the neck region diminishes after spermiation stage, although it is not completely lost from all testicular sperm. Mature epididymal sperm do not display centrin labeling. Mouse sperm lose both distal and proximal centrioles at maturity. Loss of centrin staining appears to correlate with the degeneration of centrioles during mouse spermiogenesis.

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. Mol. Reprod. Dev. 56:325–328, 2000.

Rhesus offspring produced by intracytoplasmic injection of testicular sperm and elongated spermatids

OBJECTIVE To establish pregnancies in rhesus monkeys using testicular sperm and elongated spermatids injected into oocytes. DESIGN Comparative animal study. SETTING Regional Primate Research Center. ANIMAL(S) Prime, fertile rhesus monkeys. INTERVENTION(S) Oocytes collected by laparoscopy from gonadotropin-stimulated female rhesus monkeys were injected with testicular sperm or elongated spermatids obtained from the testis of males. Cleavage stage embryos were transferred to surrogate females. MAIN OUTCOME MEASURE(S) Fertilization, embryo cleavage, and the establishment of pregnancies. Fertilization failures were fixed and processed for the detection of microtubules and chromatin configurations. RESULT(S) Fertilization, assessed by the presence of two pronuclei within 15 hours after injection, was 60% for intracytoplasmic sperm injection with testicular sperm and 47% for elongated spermatid injection. Fertilized zygotes co-cultured in Connaughts Medical Research Labs (CMRL) medium on a Buffalo Rat Liver cell monolayer resulted in hatched blastocysts after testicular sperm extraction-intracytoplasmic sperm injection and elongated spermatids. Embryos transferred at the 4- to 8-cell stage gave rise to three pregnancies: 2/3 from testicular sperm and 1/1 from an elongated spermatid. Three healthy infants were delivered by cesarean. Oocytes that failed to fertilize typically remained arrested in metaphase of meiosis. CONCLUSION(S) Testicular sperm and elongated spermatids can be used for fertilization in the rhesus monkey resulting in live births.

U3 snRNPs and nucleolar development during oocyte maturation, fertilization and early embryogenesis in the mouse: U3 snRNA and snRNPs are not regulated coordinate with other snRNAs and snRNPs

U3 small nuclear ribonucleic acids (snRNA) and U3 small nuclear ribonucleoprotein (snRNP), which are thought to be responsible for ribosomal RNA processing, are quantitated and localized during oocyte maturation, fertilization, and early embryogenesis in the mouse. On the basis of Northern blot and nuclease protection experiments, it is estimated that there are about 5 x 10(4) U3 snRNA molecules in an ovulated oocyte and in a two-cell embryo. This number then increases roughly 50-fold to 2.7 x 10(6) molecules per embryo by the blastocyst stage. At all stages of development U3 snRNP antigens colocalize with nucleoli, as defined by differential interference contrast microscopy and an antibody to a nucleolar epitope. The synthesis and distribution of U3 snRNA and U3 snRNP follow a pattern independent from other major U snRNPs and snRNAs.

Centrosome reduction during mammalian spermiogenesis

Publisher Summary Spermiogenesis is the terminal differentiation of postmeiotic germ cells into spermatozoa. The cellular and molecular pathways of centrosome reduction during mouse, rhesus, and human spermiogenesis are described. This chapter discusses the fundamental events and stages of centrosome reduction during mammalian spermiogenesis. The developmental stages of isolated mouse spermatids show remarkable events of centrosomal changes: round spermatid stage, elongating spermatid stage, and late elongating stage. The process of centrosome reduction extends throughout the entire testicular and epididymal stages of spermiogenesis and comprises four stages: (a) loss of microtubule nucleating function, (b) formation of transitional microtubule organizing centers (MTOCs), (c) loss of centrosomal proteins, and (d) centriole degeneration. The observation of a systematic degeneration of distal centriole and γ-tubulin in nonrodent mammalian sperm implies that centrosome reduction could be a ubiquitous phenomenon taking place in all mammalian sperm to varying degrees, the mouse sperm representing the highest state and exhibiting complete loss of both centrioles and centrosomal proteins. The complete absence of centrosomes in mouse sperm consolidates the hypothesis that murine oocytes can compensate for the lack of paternal centrosomal contribution. The lack of a standard distal centriole and γ-tubulin in rhesus and human sperm does not interfere with the formation of the zygotic centrosome during fertilization and bipolar spindles during cleavages.