Transferring isolated mitochondria into tissue culture cells

We have developed a new method for introducing large numbers of isolated mitochondria into tissue culture cells. Direct microinjection of mitochondria into typical mammalian cells has been found to be impractical due to the large size of mitochondria relative to microinjection needles. To circumvent this problem, we inject isolated mitochondria through appropriately sized microinjection needles into rodent oocytes or single-cell embryos, which are much larger than tissue culture cells, and then withdraw a ‘mitocytoplast’ cell fragment containing the injected mitochondria using a modified holding needle. These mitocytoplasts are then fused to recipient cells through viral-mediated membrane fusion and the injected mitochondria are transferred into the cytoplasm of the tissue culture cell. Since mouse oocytes contain large numbers of mouse mitochondria that repopulate recipient mouse cells along with the injected mitochondria, we used either gerbil single-cell embryos or rat oocytes to package injected mouse mitochondria. We found that the gerbil mitochondrial DNA (mtDNA) is not maintained in recipient rho0 mouse cells and that rat mtDNA initially replicated but was soon completely replaced by the injected mouse mtDNA, and so with both procedures mouse cells homoplasmic for the mouse mtDNA in the injected mitochondria were obtained.     

Mitochondrial function in the human oocyte and embryo and their role in developmental competence

The role of mitochondria as a nexus of developmental regulation in mammalian oogenesis and early embryogenesis is emerging from basic research in model species and from clinical studies in infertility treatments that require in vitro fertilization and embryo culture. Here, mitochondrial bioenergetic activities and roles in calcium homeostasis, regulation of cytoplasmic redox state, and signal transduction are discussed with respect to outcome in general, and as possible etiologies of chromosomal defects, maturation and fertilization failure in human oocytes, and as causative factors in early human embryo demise. At present, the ability of mitochondria to balance ATP supply and demand is considered the most critical factor with respect to fertilization competence for the oocyte and developmental competence for the embryo. mtDNA copy number, the timing of mtDNA replication during oocyte maturation, and the numerical size of the mitochondrial complement in the oocyte are evaluated with respect to their relative contribution to the establishment of developmental competence. Rather than net cytoplasmic bioenergetic capacity, the notion of functional compartmentalization of mitochondria is presented as a means by which ATP may be differentially supplied and localized within the cytoplasm by virtue of stage-specific changes in mitochondrial density and potential (ΔΨm). Abnormal patterns of calcium release and sequestration detected at fertilization in the human appear to have coincident effects on levels of mitochondrial ATP generation. These aberrations are not uncommon in oocytes obtained after ovarian hyperstimulation for in vitro fertilization. The possibility that defects in mitochondrial calcium regulation or bioenergetic homeostasis could have negative downstream development consequences, including imprinting disorders, is discussed in the context of signaling pathways and cytoplasmic redox state.     

The importance of mitochondrial metabolic activity and mitochondrial DNA replication during oocyte maturation in vitro on oocyte quality and subsequent embryo developmental competence

Mitochondrial metabolic capacity and DNA replication have both been shown to affect oocyte quality, but it is unclear which one is more critical. In this study, immature oocytes were treated with FCCP or ddC to independently inhibit the respective mitochondrial metabolic capacity or DNA replication of oocytes during in vitro maturation. To differentiate their roles, we evaluated various parameters related to oocyte maturation (germinal vesicle break down and nuclear maturation), quality (spindle formation, chromosome alignment, and mitochondrial distribution pattern), fertilization capability, and subsequent embryo developmental competence (blastocyst formation and cell number of blastocyst). Inhibition of mitochondrial metabolic capacity with FCCP resulted in a reduced percent of oocytes with nuclear maturation; normal spindle formation and chromosome alignment; evenly distributed mitochondria; and an ability to form blastocysts. Inhibition of mtDNA replication with ddC has no detectable effect on oocyte maturation and mitochondrial distribution, although high-dose ddC increased the percent of oocytes showing abnormal spindle formation and chromosome alignment. ddC did, however, reduce blastocyst formation significantly. Neither FCCP nor ddC exposure had an effect on the rate of fertilization. These findings suggest that the effects associated with lower mitochondrial DNA copy number do not coincide with the effects seen with reduced mitochondrial metabolic activity in oocytes. Inhibiting mitochondrial metabolic activity during oocyte maturation has a negative impact on oocyte maturation and subsequent embryo developmental competence. A reduction in mitochondrial DNA copy number, on the other hand, mainly affects embryonic development potential, but has little effect on oocyte maturation and in vitro fertilization.     

Mitogenomes of Polar Bodies and Corresponding Oocytes

The objective of the present study was to develop an approach that could assess the chromosomal status and the mitochondrial DNA (mtDNA) content of oocytes and their corresponding polar bodies (PBs) with the goal of obtaining a comparative picture of the segregation process both for nuclear and mtDNA. After Whole Genome Amplification (WGA), sequencing of the whole mitochondrial genome was attempted to analyze the segregation of mutant and wild-type mtDNA during human meiosis. Three triads, composed of oocyte and corresponding PBs, were analyzed and their chromosome status was successfully assessed. The complete mitochondrial genome (mitogenome) was almost entirely sequenced in the oocytes (95.99% compared to 98.43% in blood), while the percentage of sequences obtained in the corresponding PB1 and PB2 was lower (69.70% and 69.04% respectively). The comparison with the mtDNA sequence in blood revealed no changes in the D-loop region for any of the cells of each triad. In the coding region of blood mtDNA and oocyte mtDNA sequences showed full correspondence, whereas all PBs had at least one change with respect to the blood-oocyte pairs. In all, 9 changes were found, either in PB1 or PB2: 4 in MT-ND5, 2 in MT-RNR2, and 1 each in MT-ATP8, MT-ND4, MT-CYTB. The full concordance between oocyte and blood in the 3 triads, and the relegation of changes to PBs, revealed the unexpected coexistence of different variants, giving a refined estimation of mitochondrial heteroplasmy. Should these findings be confirmed by additional data, an active mechanism could be postulated in the oocyte to preserve a condition of ‘normality’.     

In vitro embryo production efficiency in cattle and its association with oocyte adenosine triphosphate content, quantity of mitochondrial DNA, and mitochondrial DNA haplogroup

Mitochondria have a broad range of functions that affect reproduction, and structural as well as quantitative variation in mtDNA has been associated with gamete quality and reproductive success. To investigate the mitochondria effect on in vitro embryo production, we collected oocytes by ultrasound-guided follicular aspiration from donor cows known to differ in the developmental capacity, measured by the blastocyst formation rate, of their oocytes. To evaluate the potential effects of mtDNA and mitochondrial function on oocyte quality, the donor cows' mtDNA control region was sequenced and, after pairwise comparisons of polymorphisms, animals were grouped into two major haplogroups. The number of mtDNA molecules per oocyte was quantified by real-time PCR, and the adenosine triphosphate (ATP) content was measured in each oocyte to identify variations between haplogroups. Overall, ATP stocks in oocytes of the two haplogroups differed significantly (P < 0.05; means +/- SEM) both at the germinal vesicle and metaphase II stages (2.8 +/- 0.06 pmol vs. 2.6 +/- 0.07 pmol and 2.9 +/- 0.1 pmol vs. 2.3 +/- 0.06 pmol, respectively). The proportion of development to blastocyst was significantly different between haplogroups (22.3 +/- 2.1 % vs. 36.7 +/- 2.9 %). The number of mtDNA molecules per oocyte was highly variable (377 327 +/- 14 104, ranging from 2.0 x 10(3) to 1.2 x 10(6)) but not significantly different between the two haplogroups; significant differences were observed between animals without any apparent relationship to blastocyst production. These data suggest that mitochondria and mtDNA haplogroup affect the developmental capacity of bovine oocytes in vitro.     

Embryo quality,and not chromosome nondiploidy,affects mitochondrial DNA content in mouse blastocysts

It has been shown recently that there is premature mitochondria biosynthesis in blastocysts from older women whose egg or embryo quality is poor and that aneuploid blastocysts also have a high number of mitochondrial DNA (mtDNA) copies. Whether nondiploidy/aneuploidy or reduced egg or embryo quality causes premature mitochondrial biosynthesis is not known. This study constructed haploid, diploid, triploid, and tetraploid blastocysts by parthenogenetic activation, intracytoplasmic sperm injection with one or two sperm heads, blastomere electrofusion, respectively, and generated reduced cytoplasm quality embryos from diabetic mouse and in vitro fertilization of aged oocytes, and examined whether nondiploidy or reduced cytoplasm quality causes premature mitochondrial biosynthesis. MtDNA numbers of each blastocyst from different models were tested by absolute quantitative real-time polymerase chain reaction. It was found that mtDNA content in preimplantation embryos was not associated with their chromosome ploidy, while mtDNA copy numbers in embryos with suboptimal quality were increased. Therefore, it might be the reduced cytoplasmic quality, and not chromosome nondiploidy, that causes premature mitochondria biosynthesis in blastocysts.     

Segregation of mitochondria in the cytoplasm of Xenopus vitellogenic oocytes

In actively growing vitellogenic oocytes of Xenopus laevis mitochondria segregate into 2 populations. One stays around the nucleus, actively replicates mitochondrial DNA (mtDNA), and builds up most of the stock of the mitochondria in the full-grown oocyte. The other moves toward the vegetal pole and stops replicating mtDNA early in vitellogenesis. Organelles of this population are components of the germ plasm of the cell.     

Polar body mutation load analysis in a patient with A3243G tRNALeu(UUR) point mutation

Diseases associated with point mutations in the mitochondrial DNA (mtDNA) are maternally inherited. We evaluated whether pre-implantation genetic diagnosis, based on polar body mutation load detection could be used to distinguish healthy from affected oocytes. Restriction Fragment Length Polymorphism (RFLP) analysis was used and validated, to determine A3243G tRNA(Leu(UUR)) mutation load in metaphase II oocytes and their respective first polar bodies. The results of this study show for the first time that the mutation load measured in the polar bodies correlates well with the mutation load in the respective oocytes. Therefore, human polar body analysis can be used as diagnostic tool to prevent transmission of mitochondrial disorders.     

Mitochondrial DNA synthesis in oocytes of Xenopus laevis

Mitochondria isolated from stage 3 (about half-grown) oocytes of Xenopus laevis exhibit a DNA synthetic rate in vitro of 2.35 ± 0.28 pg/oocyte/h. Similarly, stage 6 (full-grown) oocyte mitochondria synthesize DNA (mtDNA) at 0.28 ± 0.02 pg/oocyte/h. By comparison, the rate of mtDNA synthesis by intact stage 6 oocytes following microinjection of [³H]-dTTP was calculated to be 0.43 ± 0.08 pg/oocyte/h, indicating that the observed in vitro rates may represent minimum values. Measurements of DNA polymerase activity associated with mitochondria isolated from stage 3 oocytes are almost three times those recorded with stage 6 oocyte mitochondria. It appears that active replication of complete mtDNA molecules, which accompanies accumulation of mitochondria by the egg, is terminated midway through oogenesis.     

Mitochondrial dysfunction in reproduction

The mitochondrial genome passes from one generation to the next by way of the egg's cytoplasm, so ordinarily an individual's mitochondrial DNA (mtDNA) is entirely derived from his or her mother. A potential mother has a finite number of eggs, or oocytes, all of which were formed when she herself was still a fetus, many years before she can conceive. The eggs are progressively depleted through childhood and her reproductive years at a much faster rate than is accounted for by ovulation. Up to a decade before the ultimate depletion of ovarian follicles (and hence oocytes) at or soon after menopause, cytoplasmic senility of the remaining eggs leads to physiological sterility; a phenomenon that is suspected of being mitochondrially based and has been termed the oopause. When ovulation and conception occur, oxidative phosphorylation and other mitochondrial functions of the fertilized oocyte are thought to be essential to the early embryo well before it implants in the uterus. The competition between follicles to deliver the oocyte that will be fertilized and which will found a new generation could also be mitochondrially based, but the mechanism remains to be elucidated. Increasing experience with the culture of human embryos in vitro is highlighting the importance of mitochondrial metabolism generally, and the avoidance of excessive generation of reactive oxygen species in particular. Paradoxes abound in the experimental data, however. Although natural selection operates on mitochondria only in females (and in extreme cases through the survival of their offspring), reproductive disturbance from mitochondrial mutations is most obvious in males, who typically have reduced sperm motility. mtDNA point mutations such as T8993G, which is serious enough to cause the death of infants from Leigh disease in the first few years of life, can carry through the female germ line apparently unhindered; yet mtDNA deletions that cause a less severe phenotype, and which typically manifest at a later age, are effectively blocked from transmission to offspring--a phenomenon in accord with early experimental observations that deleted mtDNA species are less common in cleaving embryos than in unselected preovulatory oocytes. A mitochondrial basis for ooplasmic aging has not been convincingly established, but the novel IVF-based practice of micro-aspiration and transfer of ooplasm from younger eggs to older eggs, which includes the transfer of mitochondria, appears in preliminary studies to have some clinical efficacy in rejuvenating fertility in older women.     

Transmitochondrial differences and varying levels of heteroplasmy in nuclear transfer cloned cattle

To assess the extent of cytoplasmic genetic variability in cloned cattle produced by nuclear transplantation procedures, we investigated 29 individuals of seven male cattle clones (sizes 2–6) from two different commercial sources. Restriction enzyme and direct sequence analysis of mitochondrial DNA (mtDNA) detected a total of 12 different haplotypes. Transmitochondrial individuals (i.e., animals which share identical nuclei but have different mitochondrial DNA) were detected in all but one of the clones, demonstrating that mtDNA variation among cloned cattle is a very common phenomenon which prevents true genetic identity. The analyses also showed that the cytoplasmic genetic status of some investigated individuals and clones is further complicated by heteroplasmy (more than one mtDNA type in an individual). The relative proportions of different mtDNA‐types in two animals with mild heteroplasmy were estimated at 2:98% and 4:96% in DNA samples derived from blood. This is in agreement with values expected from karyoplast‐cytoplast volume ratios. In contrast, the mtDNA haplotype proportions observed in six other heteroplasmic animals of two different clones ranged from 21:79% to 57:43%, reflecting a marked increase in donor blastomere mtDNA contributions. These results suggest that mtDNA type of donor embryos and recipient oocytes used in nuclear transfer cattle cloning should be controlled to obtain true clones with identical nuclear and cytoplasmic genomes. Mol. Reprod. Dev. 54:24–31, 1999. © 1999 Wiley‐Liss, Inc.     

Effects of acetyl-L-carnitine on lamb oocyte blastocyst rate,ultrastructure, and mitochondrial DNA copy number

Viable lambs can be produced after transfer of in vitro–derived embryos from oocytes harvested from prepubertal lambs. However, this occurs at a much lower efficiency than from adult ewe oocyte donors. The reduced competence of prepubertal oocytes is believed to be due, at least in part, to deficiencies in cytoplasmic maturation. Differences in the cytoplasmic ultrastructure between prepubertal and adult oocytes have been described in the sheep, pig, and cow. Prepubertal lamb oocytes have been shown to have a different distribution of mitochondria and lipid droplets, and less mitochondria and storage vesicles than their adult counterparts. L-carnitine plays a role in supplying energy to the cell by transporting long-chain fatty acids into mitochondria for β-oxidation to produce ATP. Both L-carnitine and its derivative acetyl-L-carnitine have been reported to increase the blastocyst rate of oocytes from mice, cows, and pigs, treated during IVM. L-carnitine has also been shown to increase mitochondrial biogenesis in adipose cells. Therefore, the aims of this study were to determine if treatment of oocytes from prepubertal lambs with acetyl-L-carnitine during IVM could increase the blastocyst rate and alter mitochondria, vesicle, or lipid droplet number, volume, or distribution. The blastocyst rate was doubled in prepubertal lamb oocytes treated with acetyl-L-carnitine when compared to untreated oocytes (10.0% and 4.6%, respectively; P = 0.028). Light microscopy, scanning electron microscopy, and stereology techniques were used to quantify organelles in untreated and acetyl-L-carnitine–treated lamb oocytes, and quantitative polymerase chain reaction methods were used to measure the mitochondrial DNA copy number. There were no differences in mitochondrial volume, number, or mitochondrial DNA copy number. Acetyl-L-carnitine treatment increased the cytoplasmic volume (P = 0.015) of the oocytes, and there were trends toward an increase in the vesicle volume (P = 0.089) and an altered distribution of lipid droplets (P = 0.076). In conclusion, acetyl-L-carnitine can be used to increase the in vitro blastocyst rate of juvenile oocytes and therefore to improve juvenile in vitro embryo transfer methods. These methods can be used for livestock improvement by increasing the rate of genetic gain. Further work is required to identify the contents of the vesicles and confirm the mode of action of acetyl-L-carnitine in improving oocyte competence.     

Mitochondria: Participation to infertility as source of energy and cause of senescence

Mitochondria is a powerhouse organelle involved in ATP synthesis, calcium signaling, reactive oxygen species (ROS) by oxidative stress production, cell cycle arrest via apoptosis and sex steroid hormones biosynthesis. Improvement of sperm parameters such as motility, capacitation, acrosome reaction, and oocyte interaction, involve regulation of ROS levels by the mitochondria. In human, the relation between the quantitative level of mitochondrial DNA (mtDNA), oocyte cytoplasm maturation and fertilization potential, is not clear. It has been hypothesized that oocytes without sufficient wild type mtDNA and therefore able to generate ATP, would not normally be ovulated. This is reflected in the low numbers of mtDNA observed in degenerate oocytes obtained through super ovulation protocols during assisted reproductive technology programs. Different theories place mitochondria in a central role of oxidative damage to cells and tissues related to infertility declining and aging. Mitochondria-dependent apoptosis seems to be responsible for the pre and post-natal decline in germ cells, embryo development, implantation failure, and miscarriages.     

Der Einfluß von Li- und SCN-Ionen auf die Differenzierungsleistungen desAmbystoma-Ektoderms und ihre Veränderung bei kombinierter Einwirkung beider Ionen

Summary The development of the mouse oocyte during the primordial, primary and secondary follicular growth stages was studied by means of the electron microscope.During the early stages of oocyte maturation, mitochondrial multiplication takes place along with an apparent temporary transition from round to oval shape. The internal structure of many of the mitochondria is altered by separation of membranes of a crista to form a vacuole. This enlarges to pear-shaped configurations and gradually it forms so large a structure as to result in compression of adjacent cristae, thereby altering the entire appearance of the organelle.Dense round bodies encapsulated by a single membrane are found in the cytoplasm of oocytes of primary follicles near the periphery. The Golgi complex appears in primary follicle oocytes as an aggregation of vesicles. Gradually the number of lamellae in the complexes increase and these organelles become more peripherally located. The Balbiani yolk nuclei apparently is represented by a conglomeration of Golgi complexes and are present only in primordial and young primary follicle oocytes.The endoplasmic reticulum appears in the early stages only as rough-surfaced vesicles. At later stages individual cisternae become prominent. Apparently, a modified form of E. R. appears during maturation of the secondary follicle oocyte.Multivesicular complexes, each consisting of two components, small vesicles and larger vesicles enclosing microvesicles (multivesicular bodies), were commonly found during all stages of oocyte growth. The secondary follicle oocytes frequently contain multilamellar bodies. These are commonly found in juxtaposition to the multivesicular complexes and also near the egg periphery and occasionally near the nuclear envelope.This investigation was supported by a Public Health Service Research Career Program Award (5-K3-HD-5356-07) from the National Institute of Child Health and Human Development.     

Effect of oocyte mitochondrial DNA haplotype on bovine somatic cell nuclear transfer efficiency

The development capability of reconstructed bovine embryos via ovum pick-up (OPU)-somatic cell nuclear transfer (SCNT) technique has been influenced by the maternal lineage of oocyte cytoplasm, but the underlying mechanism remains unclear. Since mitochondria are the richest maternal-inherited organelle, in this study, we intended to clarify the effect of mtDNA haplotypes on cloning efficiency. By PCR-RFLP method, we identified mtDNA haplotypes A and B, differing in six restriction sites. Reconstructed embryos with haplotype A cytoplast achieved better fusion and blastocyst formation rate (64.6% and 39.4%), as compared with haplotype B (53.6% and 26.3%; P < 0.05). To further evaluate the role of mitochondria, the quantity of mtDNA, ATP content, and mRNA level of mtDNA-encoded COXI, COXIII in both oocytes were measured. Our data indicated that mtDNA copy number in haplotype A oocyte was significantly higher than that in haplotype B oocyte, both at the GV (10(5.03 +/- 0.69) vs. 10(4.81 +/- 0.86) copies/oocyte) and MII stages (10(5.31 +/- 0.71) vs. 10(5.13 +/- 0.63) copies/oocyte; logarithmically transformed values; P < 0.05). ATP content in type A oocyte was also greater at the GV (1.67 +/- 0.09 vs. 1.27 +/- 0.1 pmol) and MII stages (5.18 +/- 0.07 vs. 2.68 +/- 0.03 pmol; P < 0.05). Similarly, the mRNA expression level of mtDNA-encoded COXI and COXIII in haplotype A oocyte was significantly higher comparing to haplotype B oocyte (3.3 +/- 2.0 x 10(3) vs. 0.68 +/- 0.45 x 10(3); 24.9 +/- 10.5 x 10(3) vs. 9.4 +/- 3.3 x 10(3), respectively; P < 0.05). The data suggest that mitochondrial structure, quantity, and function may significantly affect the developmental competence of reconstructed embryos.     

小鼠单个GV期卵母细胞中ATP8基因的表达分析

目的:探讨小鼠GV期卵母细胞线粒体中ATP8(ATP合酶亚基8)基因的表达情况。方法:应用挤压法从卵巢中分离获得生发泡期(germinal vesicle,GV)卵母细胞;用RT-PCR检测GV期单个卵母细胞中ATP8基因的表达:其中cDNA的合成分两种方法进行:一是将GV期单个卵母细胞直接进行RT合成cDNA,二是先用DNA酶加EcoRⅠ酶祛除mtDNA和核DNA后再进行RT;回收产物构建克隆质粒并测序。结果:1.5%琼脂糖电泳显示、测序结果均表明ATP8基因在GV期卵母细胞中有表达。结论:小鼠GV期卵母细胞特异表达的ATP8基因可能与卵母细胞的正常发育成熟相关。     

Polarization of mitochondria in the unfertilized mouse oocyte

Maturation of an immature oocyte into one capable of being fertilized involves tightly choreographed movements of chromosomes and organelles. The localizaton of mitochondria during maturation was studied in live mouse oocytes by confocal laser scanning microscopy (CLSM). Mitochondria were labeled with rhodamine 123 or Mitotracker (Molecular Probes, Eugene, OR) both of which are cell permeant and accumulate in mitochondria; acridine orange was used to mark chromatin. Prior to maturation, oocytes appeared to be radially symmetrical with no evident polarity; fully mature oocytes exhibited obvious polarity marked by the position of the metaphase II spindle in the cortex. CLSM revealed several interesting features of mitochondrial distribution: (1) A cortical clump of mitochondria was seen approximately 30-45to one side of the metaphase II spindle and marked the region of polar body I extrusion. (2) Large foci of mitochondria (7–14μM) were frequently found around the central region of the mature oocyte, while the central region often exhibited markedly fewer mitochondria. (3) Small mitochondrial foci (3μM) in the cortex and near the GV characterized several oocytes which failed to mature. (4) Non-spindle-associated mitochondria were not uniformly distributed in the mature oocyte but were concentrated in the hemisphere containing the metaphase II spindle. (5) The distal margins of this mitochondrial hemisphere were sharply demarcated at the cortex. These findings should help us understand organelle localization during mammalian oocyte maturation, and may give insights into possible causes of infertility and into early events of preimplantation development. © 1995 Wiley-Liss, Inc.     

A Balbiani body and the fusome mediate mitochondrial inheritance during Drosophila oogenesis

Maternally inherited mitochondria and other cytoplasmic organelles play essential roles supporting the development of early embryos and their germ cells. Using methods that resolve individual organelles, we studied the origin of oocyte and germ plasm-associated mitochondria during Drosophila oogenesis. Mitochondria partition equally on the spindle during germline stem cell and cystocyte divisions. Subsequently, a fraction of cyst mitochondria and Golgi vesicles associates with the fusome, moves through the ring canals, and enters the oocyte in a large mass that resembles the Balbiani bodies of Xenopus, humans and diverse other species. Some mRNAs, including oskar RNA, specifically associate with the oocyte fusome and a region of the Balbiani body prior to becoming localized. Balbiani body development requires an intact fusome and microtubule cytoskeleton as it is blocked by mutations in hu-li tai shao, while egalitarian mutant follicles accumulate a large mitochondrial aggregate in all 16 cyst cells. Initially, the Balbiani body supplies virtually all the mitochondria of the oocyte, including those used to form germ plasm, because the oocyte ring canals specifically block inward mitochondrial transport until the time of nurse cell dumping. Our findings reveal new similarities between oogenesis in Drosophila and vertebrates, and support our hypothesis that developing oocytes contain specific mechanisms to ensure that germ plasm is endowed with highly functional organelles.     

Maternal inheritance of the mouse mitochondrial genome is not mediated by a loss or gross alteration of the paternal mitochondrial DNA or by methylation of the oocyte mitochondrial DNA

To evaluate whether the absence or modification of paternal mitochondrial DNA or methylation of the oocyte mitochondrial DNA could be the molecular basis for maternal inheritance of mitochondria in mammals, the mitochondrial genome has been analyzed in four meiotic and postmeiotic testicular cell types, and in oocytes from the mouse. All four testicular cell types including spermatozoa contain mitochondrial DNA. Between meiosis and the end of spermatogenesis the number of mitochondrial genomes per haploid genome decreases 8- to 10-fold with spermatozoa containing approximately one copy of the mitochondrial genome per mitochondrion. Restriction enzyme digestions with six different enzymes indicate no gross differences in DNA sequence in the testicular mitochondrial DNA from meiotic cells, early haploid cells, late haploid cells, and spermatozoa. By the criterion of differential digestion with the isoschizomers, MspI and HpaII, the mitochondrial DNA is not differentially methylated during spermatogenesis. No methylation differences were detected in mitochondrial DNA from sperm and oocytes following digestion with seven methylation-sensitive restriction enzymes.