Nuclear pore flow rate of ribosomal RNA and chain growth rate of its precursor during oogenesis of Xenopus laevis

The number of ribosomal RNA molecules which are transferred through an average nuclear pore complex per minute into the cytoplasm (nuclear pore flow rate, NPFR) during oocyte growth of Xenopus laevis is estimated. The NPFR calculations are based on determinations of the increase of cytoplasmic rRNA content during defined time intervals and of the total number of pore complexes in the respective oogenesis stages. In the mid-lampbrush stage (500–700 μm oocyte diameter) the NPFR is maximal with 2.62 rRNA molecules/pore/minute. Then it decreases to zero at the end of oogenesis. The nucleocytoplasmic RNA flow rates determined are compared with corresponding values of other cell types. The molecular weight of the rRNA precursor transcribed in the extrachromosomal nucleoli of Xenopus lampbrush stage oocytes is determined by acrylamide gel electrophoresis to be 2.5 × 10⁶ daltons. From the temporal increase of cytoplasmic rRNA (3.8 μg per oocyte in 38 days) and the known number of simultaneously growing precursor molecules in the nucleus the chain growth rate of the 40 S precursor RNA is estimated to be 34 nucleotides per second.     

Quantitation of the accumulation of histone messenger RNA during oogenesis in Xenopus leavis

The accumulation of messenger RNA coding for histone H3 in oogenesis of Xenopus laevis was studied by quantitative hybridization techniques, using a cloned genomic DNA fragment as a probe. This probe was isolated from cloned Xenopus histone DNA and contains most of the H3 coding sequences. Histone H3 mRNA accumulation was found to be completed before the maximum lampbrush stage. Hybridization of RNA blots with DNA probes containing genes for histones H2A, H2B, and H4 suggests the same accumulation pattern for the mRNAs coding for these histones as for histone H3 mRNA. The amount of H3 mRNA in the mature oocyte was established to be 130 ± 68 pg, i.e., about 5 × 10⁸ copies.     

Mitochondrial development during Drosophila oogenesis: distribution,density and in situ RNA hybridizations

The changes in distribution and density of mitochondria and the level of mitochondrial RNA during Drosophila oogenesis were studied simultaneously in the 3 cell types ie follicle cells, nurse cells and oocyte, making up the egg chamber. Up to stage 6, mitochondrial density (mitochondrial and cellular areas ratio) was elevated and increased similarly in both follicle and nurse cells. Thereafter the mitochondrial density of follicle cells continued to increase and that of the nurse cells declined markedly while the nurse cell mitochondria assembled in dense groups and decreased in size. This can be related to a transfer of nurse cell cytoplasm, including mitochondria, to the oocyte. In the oocyte from stage 4 to stage 7 we observed a significant decrease of the mitochondrial density due to the absence of mitochondrial biogenesis. Then the cytoplasm transfer caused mitochondrial density to increase up to the level found in the nurse cells at the end of oogenesis. The mature oocyte contains enough mitochondria to supply 15 000 somatic cells. Our results strongly suggest that the variations in size, distribution and density of mitochondria relate to the particular energetic requirements of the different cell types during the first half of oogenesis. Later they relate to the developmental requirements of the nurse cells and the oocyte, in particular the storage of mitochondria in the oocyte. The level of mitochondrial RNA was studied through in situ hybridization. Throughout oogenesis the follicle and nurse cell RNA evolved similarly. Up to stage 9, there was no change in RNA densities in these cells, suggesting a correlation with the cell volume and/or the nuclear DNA content. Thereafter the cellular RNA concentration declined rapidly. In the oocyte the RNA concentration evolved differently especially from stage 10 to the end, the RNA density being stabilized. This can be related to the injection of nurse cell mitochondria, followed by their assignment to reserve status. Our results suggest that the mt RNA density is under extramitochondrial control mechanisms.     

Ultrastructural observation of oogenesis in the crustacea amphipoda Orchestia gammarellus (Pallas)

The oogenesis of the Crustacea Amphipoda Orchestia gammarellus can be divided in five stages taking into consideration both the oocyte ultrastructure and the physiology of the ovary. The primary oogonium (12 μm in diameter) is lodged within the germinative zone: after division, the daughter cell (or secondary oogonium) leaves this area and enters meiotic prophase. Stage I is represented by the oocyte with visible chromosomes (12–18 μm in diameter) the cytoplasmic ultrastructure of which is comparable to that of the oogonium. Stage II or previtellogenesis is characterized by a considerable growth of the oocyte (18–80 μm in diameter) which becomes enriched in ribosomes and vesicles of the rough endoplasmic reticulum; the oocyte does not yet contain any vitelline reserve (proteinaceous and lipid). Stage III or primary vitello-genesis (80–160 μm in diameter) is typified by the synthetic activity of the rough endoplasmic reticulum, corresponding to an endogenous accumulation of proteinaceous yolk. Stage IV or secondary vitellogenesis (160–800 μm in diameter) only appears during the period of reproduction; by means of endocytosis the oocyte accumulates yolk spheres in addition to lipid droplets, the origin of which is uncertain; towards the end of vitellogenesis, cortical granules become a feature that is noted for the first time in Crustacea. The last stage or maturation (800 μm in diameter) starts right before or immediately after the exuviation of the female and ends with fertilization.     

Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals

Oogenesis in the anuran Xenopus laevis can be divided into six stages based on the anatomy of the developing oocyte. Stage I consists of small (50 to 100 μ) colorless oocytes whose cytoplasm is transparent. Their large nuclei and mitochondrial masses are clearly visible in the intact oocyte. Stage II oocytes range up to 450 μ in diameter, and appear white and opaque. Stage I and II are both previtellogenic. Pigment synthesis and yolk accumulation (vitellogenesis) begins during Stage III. Vitellogenesis continues through Stage IV (600 to 1000 μ), the oocytes grow rapidly, and the animal and vegetal hemispheres become differentiated. By Stage V (1000 to 1200 μ) the oocytes have nearly reached their maximum size and yolk accumulation gradually ceases. Stage VI oocytes are characterized by the appearance of an essentially unpigmented equatorial band. They range in size from 1200 to 1300 μ, are postivtellogenic and ready for ovulation. These stages of oocyte development have been correlated with physiological and biochemical data related to oogenesis in Xenopus.     

Intracellular pH plays a role in regulating protein synthesis in Xenopus oocytes

Full-grown Xenopus oocytes undergo meiotic maturation in response to progesterone stimulation. Using [¹⁴C]dimethyloxazolidine dione (DMO), we have measured a cytoplasmic alkalization in these oocytes starting at pH 7.14 ± 0.17 during the germinal vesicle (GV) stage, and increasing to 7.56 ± 0.14 at the time of germinal vesicle breakdown (GVBD). During this period, the rate of protein synthesis increases 2-fold from 18.9 ± 3.1 to 37.7 ± 8.8 ng/hr/oocyte. Artificial alkalization of GV stage oocytes to pH_i 7.68 ± 0.16, by exposure to the weak bases trimethylamine, methylamine, procaine, or imidazole, led to a 1.8-fold increase in the synthetic rate. Intracellular acidification from 7.5 back to 7.0 had no apparent effect on the elevated rate of protein synthesis following GVBD. Therefore, a cytoplasmic alkalization in the range of 7.5 to 7.6 seems to be one of the events that is necessary for initiating the increase in protein synthesis in maturing Xenopus oocytes; however, it does not appear that an elevated pH_i is necessary to maintain the increased synthetic rate following GVBD.     

Mitochondrial DNA content of mature spermatozoa and oocytes in the genetic model Drosophila

Although crucial to the success of fertilization and embryogenesis, little is known about the mitochondrial DNA (mtDNA) content of mature spermatozoa and oocytes across taxa and across different fertilization systems. Oocytes are assumed to hold a large population of mtDNAs that populate emerging cells during early embryogenesis, whereas spermatozoa harbor only a limited pool of mtDNAs that is believed to sustain functionality but fails to contribute paternal mtDNA to the zygote. Recent work suggests that mature sperm of the genetic model Drosophila melanogaster lack mtDNA, questioning the significance of zygotic mechanisms for the selective elimination of paternal mtDNA and their necessity for fertilization success. This finding further contradicts previous observations of the inheritance of paternal mtDNA in drosophilids. Using quantitative polymerase chain reaction, we estimate the mtDNA content of several laboratory strains of D. melanogaster and D. simulans to shed light on this discrepancy and to describe the mitochondrial/mtDNA load of gametes within this system. These measurements led to an average estimate of 22.91±4.61 mtDNA molecules/copies per spermatozoon across both species and to 1.07E+07±2.71E+06 molecules/copies per oocyte for D. simulans. As a consequence, the ratio of paternal and maternal mtDNA in the zygote was estimated at 1:4.65E+05.     

Follicle-stimulating hormone stimulated differentiation and progesterone production of bovine granulosa cells in culture

Progesterone production of granulosa cells cultured in vitro is stimulated and cell differentiation increased, by follicle-stimulating hormone (FSH). This study examined whether the increased progesterone production observed when bovine granulosa cells are cultured occurs because (1) progesterone production by undifferentiated and/or differentiated cells is increased or (2) the differentiation of granulosa cells is stimulated. Viable bovine granulosa cells (2−3×10⁵) from follicles 5–8 mm in diameter were cultured in the presence of 0, 1, 10 and 100 μu FSH (1 μu ≡ 1 μg NIH-FSH-S1) for 6 days at 37°C in a humidified atmosphere of 5% CO₂ in air in 1 ml of a 1:1 mixture of Dulbecco's modified Eagle medium: Ham's F10 medium supplemented with 365 μg ml⁻¹ l-glutamine, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. Progesterone production, total DNA and protein, and cell diameter were determined sequentially over the culture period. The increases in progesterone production (ng μg⁻¹ DNA per 24 h), cytoplasmic:nuclear ratio (μg protein μg⁻¹ DNA) and cell diameter (μm) over 6 days culture indicated that granulosa cells underwent differentiation in the presence of FSH. Progesterone production of undifferentiated granulosa cells (diameter 14 μm or less) was stimulated by FSH (P < 0.01) in a dose dependent manner (1.0±0.2, 2.9±0.3, 3.7±0.3 and 4.9±0.4 ng μg⁻¹ DNA per 24 h for 0, 1, 10 and 100 μu ml⁻¹ FSH respectively) but remained constant within dose (P > 0.05) during a 6 day culture period. FSH stimulated (P < 0.05) the rate of granulosa cell differentiation (10±3%, 53±13%, 74±21% and 82±10% differentiating cells per well for 0 μu, 1 μu, 10 μu and 100 μu ml⁻¹ FSH respectively) but did not stimulate (P > 0.05) progesterone production by differentiating granulosa cells (8.7±0.5 ng μg⁻¹ DNA per 24 h). In conclusion, the increase in progesterone production of FSH-stimulated granulosa cells cultured in vitro appears to be mainly due to an increase in the number of differentiating cells with a constant rather than an increasing progesterone production per cell.     

Oogenesis of microlecithal oocytes in the viviparous teleost Heterandria formosa

Viviparous teleosts exhibit two patterns of embryonic nutrition: lecithotrophy (when nutrients are derived from yolk that is deposited in the oocyte during oogenesis) and matrotrophy (when nutrients are derived from the maternal blood stream during gestation). Nutrients contained in oocytes of matrotrophic species are not sufficient to support embryonic development until term. The smallest oocytes formed among the viviparous poeciliid fish occur in the least killifish, Heterandria formosa, these having diameters of only 400 μm. Accordingly, H. formosa presents the highest level of matrotrophy among poeciliids. This study provides histological details occurring during development of its microlecithal oocytes. Five stages occur during oogenesis: oogonial proliferation, chromatin nucleolus, primary growth (previtellogenesis), secondary growth (vitellogenesis), and oocyte maturation. H. formosa, as in all viviparous poeciliids, has intrafollicular fertilization and gestation. Therefore, there is no ovulation stage. The full‐grown oocyte of H. formosa contains a large oil globule, which occupies most of the cell volume. The oocyte periphery contains the germinal vesicle, and ooplasm that includes cortical alveoli, small oil droplets and only a few yolk globules. The follicular cell layer is initially composed of a single layer of squamous cells during early previtellogenesis, but these become columnar during early vitellogenesis. They are pseudostratified during late vitellogenesis and reduce their height becoming almost squamous in full‐grown oocytes. The microlecithal oocytes of H. formosa represent an extreme in fish oogenesis typified by scarce yolk deposition, a characteristic directly related to matrotrophy. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

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 development during Drosophila oogenesis: distribution, density and in situ RNA hybridizations.

The changes in distribution and density of mitochondria and the level of mitochondrial RNA during Drosophila oogenesis were studied simultaneously in the 3 cell types ie follicle cells, nurse cells and oocyte, making up the egg chamber. Up to stage 6, mitochondrial density (mitochondrial and cellular areas ratio) was elevated and increased similarly in both follicle and nurse cells. Thereafter the mitochondrial density of follicle cells continued to increase and that of the nurse cells declined markedly while the nurse cell mitochondria assembled in dense groups and decreased in size. This can be related to a transfer of nurse cell cytoplasm, including mitochondria, to the oocyte. In the oocyte from stage 4 to stage 7 we observed a significant decrease of the mitochondrial density due to the absence of mitochondrial biogenesis. Then the cytoplasm transfer caused mitochondrial density to increase up to the level found in the nurse cells at the end of oogenesis. The mature oocyte contains enough mitochondria to supply 15,000 somatic cells. Our results strongly suggest that the variations in size, distribution and density of mitochondria relate to the particular energetic requirements of the different cell types during the first half of oogenesis. Later they relate to the developmental requirements of the nurse cells and the oocyte, in particular the storage of mitochondria in the oocyte. The level of mitochondrial RNA was studied through in situ hybridization. Throughout oogenesis the follicle and nurse cell RNA evolved similarly. Up to stage 9, there was no change in RNA densities in these cells, suggesting a correlation with the cell volume and/or the nuclear DNA content. Thereafter the cellular RNA concentration declined rapidly. In the oocyte the RNA concentration evolved differently especially from stage 10 to the end, the RNA density being stabilized. This can be related to the injection of nurse cell mitochondria, followed by their assignment to reserve status. Our results suggest that the mt RNA density is under extramitochondrial control mechanisms.     

Site and timing of synthesis of tubulin and other proteins during oogenesis in Drosophila melanogaster

Protein synthetic patterns during oogenesis in Drosophila melanogaster were examined; in particular the site, time, and rate of tubulin synthesis and accumulation during oogenesis were determined. Ovarian proteins were labeled with [³⁵S]methionine in vivo or in organ culure in vitro, and the proteins synthesized in egg chambers of specific developmental stages displayed by two-dimensional gel electrophoresis. A dissection technique was devised to examine proteins synthesized in each of the three cell types present in stage 10B egg chambers. The majority of proteins which were resolved by two-dimensional gel electrophoresis, including tubulin and actin, were synthesized throughout oogenesis and, at least to some extent, in each of the stage 10B cell types. Protein synthesis specific to developmental stage and/or cell type was also observed; for example, two nonchorion proteins were synthesized only in follicle cells and primarily at stage 10. A sensitive and specific radioimmune assay was developed in order to quantitate tubulin accumulation. Synthesis of several α-tubulin subunits and one β-tubulin subunit was observed. The tubulin content per egg chamber increased from 3 ng in stage 9 to 17 ng in stage 14, a period of about 13 hr. An accumulation rate of 1 ng/hr suggests that tubulin mRNA can account for about 4% of the total, nonmitochondrial, poly(A)⁺ RNA of the egg. Analysis of separated cell types at stage 10B revealed that both the follicle and nurse cells synthesize and accumulate appreciable amounts of tubulin. The stage 10B oocyte contains relatively little tubulin but actively synthesizes it. These two complementary analyses demonstrate that the tubulin present in the egg is synthesized within the oocyte-nurse cell syncytium, first in the nurse cells and later in the oocyte.     

Dioptrics of the facet lenses in the dorsal rim area of the cricket Gryllus bimaculatus

1. The optics of the corneal facet lenses from the dorsal rim area (DRA) and from the dorso-lateral areas (DA) of the compound eye of the cricket Gryllus bimaculatus were studied.
2. The DRA of the cricket eye contains quite normally shaped facet lenses. The diameter of the facet lens in the DA is 2-fold larger compared to that in the DRA. The radius of curvature of the front surface is distinctly less in the DA facet lenses, as the surface of the facet lenses in the DRA are virtually flat.
3. The averaged axial refractive index of the facet lenses of Gryllus bimaculatus, measured by interference microscopy, was 1.496 ± 0.008 (n = 42) in the DRA and 1.469 ± 0.004 (n = 39) in the DA. The geometrical thickness of the lenses was calculated to be 77 ± 3 μm (n = 42) in the DRA and 56 ± 1 μm (n = 39) in the DA.
4. Analysis of the diffraction pattern obtained with a point light source revealed distinct focusing properties of both the DRA and the DA facet lenses; striking Airy-like diffraction patterns were obtained in both cases.
5. Focal distances measured directly at the backfocal plane were 40 ± 8 μm (n = 84) in the DRA of all the animals studied, and 60–90 μm (n = 62) in DA depending on the animal. Analysis of the diffraction of the point light source yielded very similar focal distances: 40 ± 5 μm (n = 10) in DRA and 81 ± 8 μm (n = 11) in DA. In the DRA, focal distance of the facet lenses was smaller than the cone length, 58 ± 3 μm (n = 9) while in the DA the focal distance matched the effective cone length, 71 ± 5 μm (n = 16).

     

Amphibian oocytes and sphere organelles: are the U snRNA genes amplified?

The sphere organelles (spheres) ofXenopus and other amphibian oocytes are known to contain small nuclear ribonucleoprotein particles (snRNPs) and have been suggested to play a role in snRNP complex assembly. Coupled with the similarities that exist between spheres and nucleoli and the quantitative and kinetic aspects of snRNA synthesis in theXenopus oocyte, we have investigated whether or not the U snRNA encoding genes are amplified inXenopus oogenesis, the spheres being possible sites for the location of such extrachromosomal gene copies. By applying a number of quantitative nucleic acid hybridization procedures to both total and fractionated oocyte and somatic DNA, employing both homologous and heterologous U snRNA gene probes and suitable amplification and non-amplification control probes, we show that the U snRNA genes do not undergo any major amplification inXenopus oogenesis. Therefore, the analogy between the sphere organelles and nucleoli appears to be limited. The role of the spheres and their relationship to other snRNP containing structures, specifically B snurposomes, and the sphere organizer loci remains obscure.by A. Spradling     

Gonads of males and females of the mangrove tree crab Aratus pisonii (Grapsidae: Brachyura: Decapoda): a histological and histochemical view

Nicolau, C.F., Nascimento, A.A., Machado‐Santos, C., Sales, A. and Oshiro, L.M.Y. 2011. Gonads of males and females of the mangrove tree crab Aratus pisonii (Grapsidae: Brachyura: Decapoda): a histological and histochemical view. —Acta Zoologica (Stockholm) 00 :1–9. This study describes the microscopic anatomy of the male and female gonads and the spermatogenesis and oogenesis of the mangrove tree crab Aratus pisonii. Males and females were captured in a mangrove marsh in Guaratiba (23°04′S, 44°10′W), Rio de Janeiro State, Brazil. The testes are composed of spermatogonia I (7.82 ± 0.84 μm), spermatogonia II (6.12 ± 0.72 μm), spermatocytes I (5.62 ± 0.71 μm), spermatocytes II (5.00 ± 0.42 μm), spermatids (4.01 ± 0.33 μm), and spermatozoa (2.58 ± 0.18 μm). The spermatozoids are sent to the vas deferens, which is divided into three regions: anterior vas deferens, middle vas deferens and posterior vas deferens. There are no indications of development as the production of male gametes was continuous throughout the study period. In the females, there are four ovary development stages: previtellogenesis, early‐stage vitellogenesis, mature vitellogenesis, and postspawning. Five types of cells were found in the gonads: oogonia (5.23 ± 1.31 μm), oocytes in early development (19.84 ± 5.16 μm), previtellogenic oocytes (49.49 ± 6.87 μm), vitellogenic oocytes (87.51 ± 10.23 μm), and mature oocytes (174.78 ± 29.46 μm). The findings of this study indicate that A. pisonii females lay eggs on multiple occasions throughout the study period.     

The strength and timing of the mitochondrial bottleneck in salmon suggests a conserved mechanism in vertebrates

In most species mitochondrial DNA (mtDNA) is inherited maternally in an apparently clonal fashion, although how this is achieved remains uncertain. Population genetic studies show not only that individuals can harbor more than one type of mtDNA (heteroplasmy) but that heteroplasmy is common and widespread across a diversity of taxa. Females harboring a mixture of mtDNAs may transmit varying proportions of each mtDNA type (haplotype) to their offspring. However, mtDNA variants are also observed to segregate rapidly between generations despite the high mtDNA copy number in the oocyte, which suggests a genetic bottleneck acts during mtDNA transmission. Understanding the size and timing of this bottleneck is important for interpreting population genetic relationships and for predicting the inheritance of mtDNA based disease, but despite its importance the underlying mechanisms remain unclear. Empirical studies, restricted to mice, have shown that the mtDNA bottleneck could act either at embryogenesis, oogenesis or both. To investigate whether the size and timing of the mitochondrial bottleneck is conserved between distant vertebrates, we measured the genetic variance in mtDNA heteroplasmy at three developmental stages (female, ova and fry) in chinook salmon and applied a new mathematical model to estimate the number of segregating units (N(e)) of the mitochondrial bottleneck between each stage. Using these data we estimate values for mtDNA Ne of 88.3 for oogenesis, and 80.3 for embryogenesis. Our results confirm the presence of a mitochondrial bottleneck in fish, and show that segregation of mtDNA variation is effectively complete by the end of oogenesis. Considering the extensive differences in reproductive physiology between fish and mammals, our results suggest the mechanism underlying the mtDNA bottleneck is conserved in these distant vertebrates both in terms of it magnitude and timing. This finding may lead to improvements in our understanding of mitochondrial disorders and population interpretations using mtDNA data.     

Isolation and characterization of DNA from the entomopathogenBeauveria bassiana

Beauveria bassiana chromosomal (chr) and mitochondrial (mt) DNA were isolated, purified, and characterized. Modification of a recent rapid method of total DNA extraction was followed by DNA purification on CsCl-bisbenzimide gradients. Cytosine methylation at 5′-CpG-3′ doublets was undetectable in the total DNA as demonstrated byMspI andHpaII restriction enzyme digests. The chrDNA had a bouyant density of 1.7130 g/ml (G + C ratio of 53.4%) and a T_m of 92.6°C (G + C ratio of 56.9%). Mitochondrial DNA was isolated from total DNA or purified mitochondria and had a buoyant density of 1.7120 g/ml (G + C ratio of 52.3%) and a biphasic DNA melting curve. A minor melt at 76.2°C (16.8% G + C ratio) yielded a 10% increase in absorbance while the major melt at 93.1°C (58% G + C ratio) yielded a 30% increase in absorbance. The mtDNA was estimated by gel electrophoresis to be 17.6 ± 0.6 MDa and determined to be 28.5 kb by restriction enzyme analysis. Electron microscopy of the mtDNA revealed circular molecules having an average contour length of 8.84 ± 0.51 μm. A physical map of the mtDNA was generated by double restriction enzyme digestion using the restriction enzymesClaI,EcoRI,SalI,BstEII, andXhoI.     

Effect of developmental stage on bovine oocyte plasma membrane water and cryoprotectant permeability characteristics

Knowledge of bovine oocyte plasma membrane permeability characteristics at different developmental stages in the presence of cryoprotective agents (CPAs) is limited. The objective of this study was to determine the oolema hydraulic conductivity (L_p), cryoprotectant permeability (P_CPA), and reflection coefficient (σ) for immature (germinal vesicle stage, GV) and in vitro–matured (metaphase II, MII) bovine oocytes. Two commonly used cryoprotective agents, dimethyl sulfoxide (DMSO) and ethylene glycol (EG), were studied. Osmometric studies were performed using a micromanipulator connected to an inverted microscope at 22 ± 2°C. Each oocyte was immobilized via a holding pipette, and osmotically induced volume changes over time (dv/dt) were recorded. The L_p values for GV and MII oocytes in DMSO (L_p^DMSO) were 0.70 ± 0.06 and 1.14 ± 0.07 μm/min/atm (mean ± SEM) and in EG (L_p^EG) were 0.50 ± 0.06 and 0.83 ± 0.07 μm/min/atm, respectively. Estimates of P_DMSO for GV and MII oocytes were 0.36 ± 0.03 and 0.48 ± 0.03 μm/sec, and P_EG values for GV and MII oocytes were 0.22 ± 0.03, 0.37 ± 0.03 μm/sec, respectively. The σ values for GV and MII oocytes in DMSO (σ_DMSO) were 0.86 ± 0.03 and 0.90 ± 0.04 and in EG (σ_EG) were 0.94 ± 0.03 and 0.76 ± 0.04, respectively. These data demonstrate that bovine oolema permeability coefficients to water and cryoprotectants change after in vitro maturation. Furthermore, the bovine oocyte P_DMSO is higher than the P_EG. These results may provide a biophysical basis for developing criteria for choosing optimal CPAs and for minimizing damage during addition and removal of the CPAs. Additionally, these data support the hypothesis that different procedures may be required for optimal cryopreservation of different oocyte developmental stages. Mol. Reprod. Dev. 49:408–415, 1998. © 1998 Wiley-Liss, Inc.