Inner cell mass localization of NANOG precedes OCT3/4 in rhesus monkey blastocysts.

The mechanism by which the inner cell mass (ICM) and trophectoderm (TE) become specified is poorly understood. Considerable species variation is evident in the expression of lineage-specific and embryonic stem cell (ESC) regulatory markers. We sought to investigate localization patterns of these markers in rhesus macaque compact morulae and blastocysts. NANOG protein was restricted to the ICM of blastocysts. In contrast to a previous report, the expression of CDX2 was detected in the primate blastocyst, localized specifically to the TE. Unlike the mouse embryo, OCT4 protein was detected using two different antibodies in both the ICM and TE. The ubiquitous pattern of OCT4 expression is consistent with observations in human, cow, and pig embryos. Significantly, lack of restricted OCT4 protein, and ICM localization of NANOG in primate blastocysts, suggests that NANOG may determine inner cell mass fate more specifically during primate development or may be less susceptible to culture artifacts. These results contrast markedly with current mechanistic hypotheses, although other factors may lie upstream of NANOG to constitute a complex interactive network. This difference may also underlie observations that regulatory mechanisms in ESC differ between mice and primates.

Comparison of protocols for cryopreservation of rhesus monkey spermatozoa by post-thaw motility recovery and hyperactivation.

Cryopreservation of spermatozoa is useful for gene banking and for in vitro fertilisation (IVF). This study compared several published cryopreservation techniques to find the most efficient for rhesus macaques. Effectiveness was assessed by sperm longevity (post-thaw motility % and duration) and ability to hyperactivate in response to chemical activators (caffeine, dibutyryl cyclic AMP). Each ejaculate from three males was treated with four published cryopreservation protocols (Seier et al. 1993; Sanchez-Partida et al. 2000; Si et al. 2000; Isachenko et al. 2005). Upon thawing, each sub-sample was incubated either at 37 degrees C in 5% CO2 in air with or without activators or at approximately 22 degrees C in atmospheric air without activators for 0-24 h. Samples cryopreserved using one method showed zero motility and were not included in the 2 ;2 G-test statistical analysis. The other methods all demonstrated good immediate post-thaw motility rates (68%, 73% and 62% respectively) and underwent capacitation after exposure to activators. Sperm motility in each treatment decreased over time at both temperatures but overall, incubation at 22 degrees C preserved motility better in all three methods. In summary, cryopreservation of rhesus spermatozoa using the method published by Sanchez-Partida et al. or Seier et al. appeared best, potentially supporting gene banking as well as allowing for multiple IVF uses from the same sample.

Ovarian senescence in the rhesus monkey (Macaca mulatta).

BACKGROUND: A decline in fertility is evident in human females past their middle thirties. This 'reproductive senescence', marked by a sharp decline in pregnancy rates, may be attributed to reductions in numbers of available oocytes and their quality. Because Old World primates exhibit ovarian morphology and physiological control and timing of menstrual cycles closely resembling those of humans, the current study investigated the rhesus macaque as a potential model for human reproductive senescence. METHODS: Ovaries collected from females aged 1-25 years and divided into five age groups were analysed histologically. RESULTS: General ovarian morphology demonstrated significant changes as the females approached menopause. The proportions of primordial and primary follicles all demonstrated significant differences across age groups (primordial: 77.1, 79.9, 69.7, 62.9, 55.1%; primary: 21.5, 18.8, 28.5, 35.2, 43.1% for age groups 1 to 5 respectively; P<0.0001 for both). Samples from females approaching or undergoing the menopausal transition (aged 20-25 years) demonstrated evidence of ovarian senescence, having scattered and atretic follicles, low numbers of primordial follicles and reduced stromal tissue. CONCLUSION: This study supports the value of the rhesus monkey as a model for reproductive ageing because its ovary undergoes follicular reservoir depletion similar to that seen in humans.

Imaging mitochondrial organization in living primate oocytes and embryos using multiphoton microscopy.

We employed multiphoton laser scanning microscopy (MPLSM) to image changes in mitochondrial distribution in living rhesus monkey embryos. This method of imaging does not impair development; thus, the same specimen can be visualized multiple times at various developmental stages. Not only does this increase the amount of information that can be gathered on a single specimen but it permits the correlation of early events with subsequent development in the same specimen. Here we demonstrate the utility of MPLSM for determining changes in mitochondrial organization at various developmental stages and show that rhesus zygotes possess a distinct accumulation of mitochondria between the pronuclei prior to syngamy. We present evidence that suggests that this pronuclear accumulation may be positively correlated with development to the blastocyst stage-in the same embryo-thereby illustrating how MPLSM can be used to correlate cellular dynamics of primate oocytes and early embryos with their developmental potential. Understanding the relationship between mitochondrial distribution and the subsequent development of mammalian embryos, particularly primates, will increase our ability to improve embryo culture technologies, including those used for human assisted reproduction.

Recombinant human albumin supports hamster in-vitro fertilization.

BACKGROUND: Serum albumin is normally required to support sperm capacitation and IVF, but its mechanism of action is not well understood. Commercial serum albumin preparations are contaminated with a variety of other proteins and compounds, and their biological activity is variable. Recombinant human albumin (rHA) might replace serum albumin for IVF. METHODS: rHA was examined for its ability to capacitate hamster spermatozoa and to support fertilization in vitro. A standardized hamster IVF system was used to compare the capacitation-supporting activities of rHA and two commercial preparations of bovine serum albumin (BSA) in a chemically defined culture medium. Epididymal spermatozoa were incubated for 4 h at 37 degrees C under 5% CO(2) in air in either the basic medium containing rHA, one of the two BSA preparations or no protein, and then cultured in the same medium with ovulated oocytes for another 4 h. The experiment was replicated five times. RESULTS: Spermatozoa incubated in protein-free medium fertilized only one oocyte (2% of total), significantly less than any of the other three treatment conditions (P < 0.01); spermatozoa incubated in medium containing rHA or BSA fertilized 86-93% of oocytes. There were no differences between the three albumin-containing treatment groups. CONCLUSION: rHA is equivalent to commercial serum albumin preparations in its ability to support sperm capacitation and fertilization in this test system. This finding has considerable practical implications for human IVF and may also help efforts to elucidate the mechanism of sperm capacitation.

Development of in-vitro-derived bovine embryos in protein-free media: effects of amino acids, glucose, pyruvate, lactate, phosphate and osmotic pressure.

In experiment 1, the effects of a group of either 20 (i.e. glutamine + essential + non-essential) or 11 (i.e. hamster embryo culture medium (HECM)-6) amino acids were evaluated in modified potassium simplex optimised medium (mKSOM) or basic medium (BM)-3. In experiment 2, the effects of glucose, pyruvate, lactate, phosphate or all four substrates were evaluated in low- or high-osmotic pressure BM-3 (255 and 275 mOsmol respectively) containing 20 amino acids (BM-3-20aa). In experiment 1, mKSOM containing 20 amino acids (mKSOM-20aa) supported the highest frequency of total, expanded (Days 7, 8 and 9) and hatched blastocysts. In experiment 2, supplement type affected the frequency of development to at least the morula stage (Day 7), expanded (Day 8), hatched (Day 9) or total blastocysts and cell number per blastocyst. Osmotic pressure affected the frequency of expanded blastocysts (Day 7) and blastocyst cell number. Regardless of the osmotic pressure, BM-3-20aa containing glucose (0.2 mM) supported the highest frequency of blastocyst development. The interaction between supplement type and osmotic pressure was not significant; however, treatment mean differences were more marked in high- than in low-osmotic pressure medium. In conclusion, the beneficial effects of amino acids on in vitro embryo development are influenced by the base medium. Moreover, glucose-containing media supported a higher frequency of embryonic development than pyruvate- and/or phosphate-supplemented media, indicating that glucose plays more important roles in non-energy generating pathways.

How animal embryo research led to the first documented human IVF.

Research studies using animal gametes led to the first documented human IVF in 1969. A key contribution was the development of a reliable culture medium for IVF of hamster oocytes, which was then used successfully with human gametes in the laboratory. This article describes how this was accomplished. Hamster IVF was obtained using a bicarbonate-buffered culture medium based on Tyrode's solution. A dose-response experiment showed that IVF in this species was highly dependent on the pH of the culture medium. At pH 7.2 or less, very few oocytes were penetrated and spermatozoa did not undergo acrosome reactions. Over the pH range 7.3-7.5, 40% of oocytes were fertilized in vitro, while at pH 7.6 or higher, more than 75% of oocytes were fertilized and most spermatozoa exhibited acrosome reactions. There was no apparent effect of pH on sperm motility. These experiments showed that in the hamster, fertilization in vitro is highly pH dependent, most likely through pH effects on the sperm acrosome reaction and sperm:zona binding. The data led to formulation of a culture medium, Tyrode-B, that consistently supported high levels of IVF of hamster oocytes. When this medium was used for human gametes, IVF was observed and documented for the first time. Spermatozoa penetrating through the zona pellucida and sperm components within the ooplasm were detected, and some oocytes exhibited two pronuclei. These observations suggested that human IVF might be useful for alleviation of infertility.

Amino acid requirements for maturation of rhesus monkey (Macacca mulatta) oocytes in culture.

This study evaluated the effects of different amino acid formulations on supporting meiotic and cytoplasmic maturation of rhesus monkey (Macacca mulatta) oocytes in vitro. Five hundred and forty-six cumulus-oocyte complexes (COCs) aspirated from unstimulated adult monkey follicles (> or = 1000 microm in diameter) were cultured in either modified Connaught Medical Research Laboratories 1066 medium (mCMRL-1066) or in one of eight chemically defined media (modified basic medium 5 supplemented with 5.5 mmol glucose l(-1), 0.003 mmol pantothenic acid l(-1) and different amino acid formulations) as below: (1) modified basic medium 5 (mBM5) containing no amino acid; (2) mBM5 + 0.2 mmol glutamine l(-1); (3) mBM5 + 11 amino acids from hamster embryo culture medium 6 (HECM-6) (11 AA); (4) mBM5 + Eagle's non-essential amino acids (NEA); (5) mBM5 + NEA + 0.2 mmol glutamine l(-1); (6) mBM5 + Eagle's essential amino acids (EA) without glutamine; (7) mBM5 + EA + 0.2 mmol glutamine l(-1); (8) mBM5 + Eagle's 20 amino acids (20 AA) + 0.2 mmol glutamine l(-1); and (9) mCMRL-1066 (control). All media contained FSH, LH, oestradiol and progesterone. After maturation, mature oocytes were subjected to the same fertilization and embryo culture procedures. COCs matured in treatment 5 had greater potential to progress to metaphase II (66%; P < 0.05) than did those in treatments 1 (37.3%), 2 (48.3%), 3 (41%), 6 (41%) and 9 (43%). Oocytes matured in treatment 8 had the best morula (53%) and blastocyst (18%) developmental responses (P < 0.05). The lowest (P < 0.05) morula and blastocyst developmental responses were obtained from COCs matured in treatments 1 (0%) and 6 (8%). The other media supported intermediate embryonic development (range 11-38% of morula and blastocyst). These results indicate that the choice of amino acids affects the competence of oocyte maturation and that Eagle's 20 AA with 0.2 mmol glutamine l(-1) is more efficient than the other amino acid formulations for maturation of rhesus monkey oocytes.

Relationship between development, metabolism, and mitochondrial organization in 2-cell hamster embryos in the presence of low levels of phosphate.

The effect of low concentrations of inorganic phosphate (P(i)) on development, metabolic activity, and mitochondrial organization in the same cohorts of cultured hamster embryos was evaluated. Two-cell embryos were collected from eCG-stimulated golden hamsters and cultured in HECM-10 with 0.0 (control), 1.25, 2.5, or 5.0 microM KH(2)PO(4). Glucose utilization through the Embden-Meyerhof pathway (EMP) and tricarboxylic acid (TCA)-cycle activity were determined following 5 h of culture. Mitochondrial organization in living embryos was evaluated using multiphoton microscopy at 6 h of culture. Development was assessed at 27 h (on-time 8-cell stage) and 51 h (on-time blastocyst stage) of culture. Total cell numbers, as well as cell allocation to the trophectoderm and inner cell mass were determined for morula- and blastocyst-stage embryos. Culture with P(i) did not alter TCA-cycle activity. However, culture with > or 2.5 microM P(i) significantly increased (P < 0.01) EMP activity compared to control. Mitochondrial organization was significantly (P < 0.01) disrupted by P(i) in a dose-dependent manner. Development to the 8-cell, morula/blastocyst, and blastocyst stages was significantly reduced (P < 0.05) in the presence of > or =2.5 microM P(i) compared to both control and 1.25 microM P(i). This study clearly demonstrates that, for hamster embryos, inclusion of even exceptionally low concentrations of P(i) in culture medium dramatically alters embryo physiology. Additionally, although 2-cell embryos can tolerate some structural disruption without concomitant, detrimental effects on development or metabolic activity, metabolic disturbance is associated with decreased developmental competence.

Differences in intracellular pH regulation by Na(+)/H(+) antiporter among two-cell mouse embryos derived from females of different strains.

Regulation of intracellular pH (pH(i)) by two-cell-stage embryos derived from female mice of three different strains (CF-1, Balb/c, and BDF) was investigated. Embryos recovered at a slow rate from intracellular acidosis produced by a pulse of NH(4)Cl; the rate did not differ significantly among strains. Recovery was reversibly inhibited by amiloride or the absence of Na(+), implicating Na(+)/H(+) antiporter activity. The threshold pH(i) (setpoint) below which Na(+)/H(+) antiporter activity was elicited was approximately 7.15 for each strain. No recovery from induced acidosis occurred in the absence of external Na(+) in any strain, and thus embryos could be maintained in acidosis for an extended period. Upon reintroduction of Na(+), embryos derived from either CF-1 or BDF females recovered at a slow rate comparable to that measured in embryos not maintained for a period in Na(+)-free medium, but embryos derived from Balb/c females consistently recovered at a highly accelerated rate. This accelerated recovery appeared to be due, in part, to an activation of the Na(+)/H(+) antiporter in Balb/c-derived embryos, which did not occur in CF-1- or BDF-derived embryos. Thus, embryos derived from different strains of female mice differ in their control of mechanisms for pH(i) regulation.

Altering intracellular pH disrupts development and cellular organization in preimplantation hamster embryos.

In early cleavage stage hamster embryos, the inability to regulate intracellular pH (pHi) properly is associated with reduced developmental competence in vitro. The disruption of mitochondrial organization is also correlated with reduced development in vitro. To determine the relationship between pHi and the disruption of cytoplasmic organization, we examined the effects of altering pHi on hamster embryo development, mitochondrial distribution, and cytoskeletal organization. The weak base trimethylamine was used to increase pHi and was found to reduce embryo development and disrupt the perinuclear organization of mitochondria. The weak acid 5,5-dimethyl-2,4-oxazolinedione was used to decrease pH(i) and was also found to reduce development and disrupt the perinuclear organization of mitochondria. With either treatment, the microfilament organization was perturbed, but the microtubule cytoskeleton was not. However, the temporal progression of the disruption of mitochondrial distribution was more rapid in alkalinized embryos than acidified embryos, as revealed by two-photon imaging of living embryos. Additionally, the disruption of the microfilament network by the two treatments was not identical. The cytoplasmic disruptions observed were not due to acute toxicity of the compounds because embryos recovered developmentally when the treatment compounds were removed. These observations link ionic homeostasis, structural integrity and developmental competence in preimplantation hamster embryos.

Differential effect of hexoses on hamster embryo development in culture.

The effects of glucose, fructose, and galactose on hamster embryo development in the absence of phosphate were studied in culture. One- and two-cell embryos were cultured to the blastocyst stage in HECM-9 medium without hexose or in medium with increasing concentrations of hexoses. Embryo development, cell number, and cell allocation were assessed in blastocysts. Blastocyst viability was determined by transfer to pseudopregnant recipients. Although 0.25 mM fructose increased mean cell number, low glucose concentrations had no stimulatory effect on development to blastocyst. Both galactose and 5.0 mM glucose were detrimental to embryos. Addition of 0.5 mM glucose increased implantation and fetal viability as compared with controls. Compared with 0.5 mM glucose, treatment with 0.25 mM fructose gave similar implantation and fetal viability, whereas 5.0 mM glucose tended to decrease implantation and significantly decreased fetal development. These data demonstrate that morphology is a poor indicator of embryo viability and that exposure of preimplantation embryos to glucose or fructose is important for embryo viability post-transfer. Although no difference in blastocyst viability was detected between embryos cultured with 0.25 mM fructose and those cultured with 0.5 mM glucose, increased cell numbers obtained with fructose suggest that fructose may be more appropriate than glucose for inclusion in culture medium.

Effect of age and breeding season on the developmental capacity of oocytes from unstimulated and follicle-stimulating hormone-stimulated rhesus monkeys.

Effects of age and season on the developmental capacity of oocytes from unstimulated and FSH-stimulated rhesus monkeys were examined. Immature cumulus-oocyte complexes were matured in vitro in modified CMRL-1066 medium containing 20% bovine calf serum and subjected to in vitro fertilization followed by embryo culture. After fertilization, ova from unstimulated prepubertal monkeys displayed lower development to morula (4%) than those from unstimulated adult females (18% in breeding season and 22% in nonbreeding season). No developmental difference was found between ova from unstimulated adult monkeys in breeding and nonbreeding seasons. However, ova from FSH-primed prepubertal monkeys displayed greater development to blastocyst stage (54%) than those from adult monkeys in the breeding season (16%) and nonbreeding season (0%); and ova from FSH-primed adult females in the breeding season had significantly (P < 0.05) greater developmental competence than those obtained in the nonbreeding season (> or = morula stage, 54% vs. 3%; blastocyst stage, 16% vs. 0%). These data indicate that 1) rhesus monkey oocytes acquire developmental competence in a donor age-dependent manner, and 2) animal age and breeding season modulate the effect of FSH on oocyte developmental competence in the rhesus monkey.

Maturation of rhesus monkey oocytes in chemically defined culture media and their functional assessment by IVF and embryo development.

This study compared success of in-vitro maturation of rhesus monkey oocytes in protein-free versus serum-containing culture systems, assessed by embryo development subsequent to IVF. Four media were tested: (i) modified Connaught Medical Research Laboratories medium (mCMRL-1066); (ii) hamster embryo culture medium-10 (HECM-10); (iii) control: mCMRL-1066 + 20% bovine calf serum (BCS); (iv) HECM-10 + 20% BCS. Immature oocytes from FSH-stimulated rhesus monkeys were allocated among the media containing ovine FSH (5 microg/ml) and LH (10 microg/ml) and cultured for 36-40 h. Metaphase II ova were inseminated and putative zygotes were cultured in mCMRL-1066 + 20% BCS until development arrested. Ova matured in all four media had similar (P> 0.05) potential to initiate (66, 67, 82 and 69% respectively) and complete meiotic maturation (60, 50, 76 and 57% respectively). Inseminated ova in all groups had similar potential to be fertilized (86, 83, 84 and 90% respectively), cleave (71, 83, 76 and 90% respectively) and develop to the blastocyst stage (19, 17, 16 and 30% respectively). These results indicate for the first time that primate oocytes can be successfully matured in protein-free medium, with subsequent blastocyst development, comparable to responses in complex medium with serum. This finding will facilitate studies on mechanisms regulating primate oocyte maturation.

Energy substrate requirement for in vitro maturation of oocytes from unstimulated adult rhesus monkeys.

The energy substrates lactate, pyruvate, and glucose were evaluated for supporting in vitro cytoplasmic maturation of rhesus monkey oocytes. A total of 321 cumulus-oocyte complexes (COCs) aspirated from > or = 1000 microm diameter follicles of unstimulated adult monkeys were matured in one of six media with various individual or combinations of energy substrates: (1) mCMRL-1066 (control); (2) HECM-10 (containing 4.5 mM lactate); (3) HECM-10+0.2 mM pyruvate; (4) HECM-10 + 5.0 mM glucose; (5) HECM-10+ 0.2 mM pyruvate + 5.0 mM glucose; and (6) HECM-10 minus lactate + 5.0 mM glucose. All media contained gonadotropins, oestradiol, and progesterone. Following maturation, all mature oocytes were subjected to the same in vitro fertilization and embryo culture procedures. Oocytes matured in control medium or in treatment groups 4 and 6 had the best morulae+ blastocysts developmental responses (35, 36, and 32%, respectively, P < 0.05). HECM-10 + 0.2 mM pyruvate + 5.0 mM glucose for COC maturation supported intermediate embryonic development (16% morulae + blastocysts). The lowest (P < 0.05) morula + blastocyst developmental responses were obtained after maturation of COCs in HECM-t10 and HECM-10 + 0.2 mM pyruvate (4 and 6%, respectively). The COCs matured in glucose-containing medium showed greater levels of cumulus expansion than those in glucose-free medium. These results indicate that (a) glucose is both necessary and sufficient as the energy substrate for supporting optimal cytoplasmic maturation in vitro of oocytes from unstimulated rhesus monkeys; (b) pyruvate suppresses the stimulatory effect of glucose on oocyte maturation; (c) glucose is involved in cumulus expansion; (d) cumulus expansion is not a reliable indicator of primate oocyte competence.

In utero and in vitro proteinase activity during the Mesocricetus auratus embryo zona escape time window.

The goal of the present study was to investigate proteinase activity in uterine flushates collected during the zona loss time window (68-80 h post-egg activation) in both pregnant and pseudopregnant hamsters and in culture medium conditioned by hatching blastocysts. Several prominent enzyme activities appeared in all pregnant and pseudopregnant uterine flushates. However, only a 45, 43 x 10(-3) M:(r) doublet coincided with the zona loss time window; these bands were absent outside of this time window and were not found in conditioned medium. In medium conditioned by hatching blastocysts, enzyme activity was represented by a 70, 65 x 10(-3) M:(r) doublet identical to a doublet seen in all uterine flushates collected and in serum. There were 12 pregnant and 8 pseudopregnant uterine flushates that were capable of zona lytic activity in vitro (positive bioassays). Of these positive bioassays, five pregnant and four pseudopregnant uterine flushates exhibited the 45, 43 x 10(-3) M:(r) doublet (correlative positive bioassays). These data suggest that there is an important uterine contribution to blastocyst escape from the zona pellucida, consisting of proteinases secreted during a finite time window prior to blastocyst attachment that are different from the proteinases responsible for the zona lytic activity in vitro.

Effect of antibiotics on development in vitro of hamster pronucleate ova.

Antibiotics are commonly added to embryo culture media, but effects on embryo development have not been examined thoroughly. Hamster ova were used to investigate whether penicillin, streptomycin or gentamicin affect embryo development in vitro. Ova were collected 10 h post activation by spermatozoa in vivo and cultured in five treatments: 1) CONTROL: chemically-defined medium HECM-9 with no antibiotics; 2) HECM-9 with 100 IU/mL penicillin; 3) HECM-9 with 50 microg/mL streptomycin; 4) HECM-9 with 10 microg/mL gentamicin and 5) HECM-9 with both 100 IU/mL penicillin and 50 microg/mL streptomycin. Individually, penicillin, streptomycin and gentamicin did not affect embryo development to the 8-cell stage at 58 h post oocyte activation, or morula/blastocyst stages, or blastocysts alone at 82 h post activation. However, when penicillin and streptomycin were both present in the culture medium the percentages of 8-cell embryos at 58 h and blastocysts at 82 h were significantly lower than the control. No antibiotic treatment improved hamster embryo development in vitro. We caution against the use of penicillin and streptomycin together for hamster embryo culture, and show that it is not necessary to include any antibiotics in embryo culture media for up to 72 h if proper sterile technique is used with an oil overlay.

Development of zona-free hamster ova to blastocysts in vitro.

The zona pellucida (ZP) enclosing the mammalian ovum is important for its protection and for initial stages of fertilization, but the role of the ZP during embryo development is less clear. This study was designed to investigate if the hamster ZP is needed for embryo development from 1-cell to blastocyst in vitro, and to compare methods for removing the ZP. A total of 395 hamster pronucleate ova were collected 10 h post activation from superovulated, mated female hamsters. The ZP was removed from some ova using either 0.05% pronase, 0.05% trypsin or acid Tyrode's solution. To prevent ZP-free ova from sticking together, they were cultured singly in 30-50 microL drops of HECM-6 culture medium together with ZP-intact ova as controls. There was no significant difference among treatment groups in embryo development to blastocyst: 36/87 (42%) in the ZP intact group; 35/75 (47%) in the pronase-treated ZP-free group; 37/74 (50%) in the trypsin-treated ZP-free group; and 37/71 (52%) in the acid-treated ZP-free group. These results indicate that 1) the ZP is unnecessary for hamster embryo development in vitro from the pronucleate ovum stage to blastocyst; 2) none of the three ZP-removal methods was detrimental to embryo development; 3) embryos do not need to be cultured in groups during in vitro development from 1-cell to blastocyst.

Mitochondrial distribution and function in oocytes and early embryos.

Active mitochondria relocate during oocyte maturation or fertilization in several species. Detailed studies with hamster oocytes and early embryos reveal a pattern of active mitochondria migrating to surround the pronuclei to form a pattern that persists through the early cleavage stages. Although the functional significance of this relocation is unknown, it appears to be an important part of normal development in hamsters. Treatments that disrupt embryo development in vitro (such as the presence of inorganic phosphate or alteration of intracellular pH) also disrupt the normal pattern of mitochondrial distribution. Active mitochondria also reorganize during maturation in bovine oocytes and during fertilization in rhesus monkey oocytes. Examination of these changes in mitochondrial organization may provide insights into the regulation of normal embryo development and might serve as predictors of oocyte or embryo developmental competence.