Meiotic Oocytes Research Articles

Sex Differentiation and Early Gonadogenesis in Sebastes inermis Cuvier

Early gonadal development and sexual differentiation of dark-banded rockfish (Sebastes inermis Cuvier) were followed from parturition to 400 days post parturition (dpp). During this period, average total length (TL) increased from 0.57 to 13.18 cm. Primordial germ cells (PGCs) were first detected at 0.68 cm TL (10 dpp). When fish reached 1.52 cm TL (50 dpp), initial stages of ovarian differentiation were identified by the presence of PGCs containing condensed chromatin and their transformation into meiotic oocytes. At 10.23 cm TL (300 dpp), the ovaries gradually developed into oocytes in the primary yolk stages. Ovary growth was rapid after sex differentiation, but testis tissue continued to multiply without growing until fish reached 6.97 cm TL (200 dpp), after which the production of spermatocytes, spermatogonia, and cyst cells was apparent. Histological analysis of gonadal structure suggested a gonochoristic sexual development pathway. Our analysis of the sex ratio at 400 dpp showed a significantly higher proportion of males.

Actin-driven chromosomal motility leads to symmetry breaking in mammalian meiotic oocytes

Movement of meiosis I (MI) chromosomes from the oocyte centre to a subcortical location is the first step in the establishment of cortical polarity. This is required for two consecutive rounds of asymmetric meiotic cell divisions, which generate a mature egg and two polar bodies. Here we use live-cell imaging and genetic and pharmacological manipulations to determine the force-generating mechanism underlying this chromosome movement. Chromosomes were observed to move toward the cortex in a pulsatile manner along a meandering path. This movement is not propelled by myosin-II-driven cortical flow but is associated with a cloud of dynamic actin filaments trailing behind the chromosomes/spindle. Formation of these filaments depends on the actin nucleation activity of Fmn2, a formin-family protein that concentrates around chromosomes through its amino-terminal region. Symmetry breaking of the actin cloud relative to chromosomes, and net chromosome translocation toward the cortex require actin turnover.

Sex-specific crossover patterns in Zebrafish (Danio rerio)

Some species display intersex variation in their rate of meiotic recombination, where recombination is usually suppressed in the heterogametic sex. Although no heteromorphic sex chromosomes have been detected in zebrafish (Danio rerio), genetic analysis has indicated a lower frequency of recombination in males relative to females. Our study of the meiotic recombination pattern in female zebrafish indicates that adult females have only a few meiotic oocytes that are found in groups in the ventral zone of the ovarian surface. We used antibody staining of human mutL homolog 1 (MLH1) protein to mark the sites of putative chiasmata to seek a physical basis for the pattern of recombination and its relative frequency in both sexes. We report that MLH1 foci are found mostly in distal regions of the synaptonemal complexes (SCs) in males, but tend to be more evenly distributed in females. Our cytological analysis yields a ratio of MLH1 foci per chromosome in males versus females of 1:1.55. This lower level of recombination in males is in general agreement with previously published results from linkage map analysis. However, the similar ratio of MLH1 foci per unit length of SCs in both sexes demonstrates a correlation between SC length and the frequency of recombination rather than a mechanism that suppresses recombination in males. Thus, chiasma interference seems to provide similar expression in males and females in agreement with the situation in humans, where oocytes with longer SCs display a higher level of recombination that is not a consequence of more closely spaced crossovers along the SCs.

Assembly of ovarian follicles in the caeciliansIchthyophis tricolorandGegeneophis ramaswamii:light and transmission electron microscopic study

Though much is known about various aspects of reproductive biology of amphibia, there is little information on the cellular and mechanistic basis of assembly of ovarian follicles in this group. This is especially true of the caecilians. Therefore, taking advantage of the abundant distribution of caecilians in the Western Ghats of India, two species of caecilians, Ichthyophis tricolor and Gegeneophis ramaswamii, were subjected to light and transmission electron microscopic analysis to trace the sequential changes during the assembly of ovarian follicles. The paired ovaries of these caecilians are elongated sac-like structures each including numerous vitellogenic follicles. The follicles are connected by a connective tissue stroma. This stroma contains nests of oogonia, primary oocytes and pregranulosa cells as spatially separated nests. During assembly of follicles the oocytes increase in size and enter the meiotic prophase when the number of nucleoli in the nucleus increases. The mitochondrial cloud or Balbiani vitelline body, initially localized at one pole of the nucleus, disperses through out the cytoplasm subsequently. Synaptonemal complexes are prominent in the pachytene stage oocytes. The pregranulosa cells migrate through the connective tissue fibrils of the stroma and arrive at the vicinity of the meiotic prophase oocytes. On contacting the oocyte, the pregranulosa cells become cuboidal in shape, wrap the diplotene stage oocyte as a discontinuous layer and increase the content of cytoplasmic organelles and inclusions. The oocytes increase in size and are arrested in diplotene when the granulosa cells become flat and form a continuous layer. Soon a perivitelline space appears between the oolemma and granulosa cells, completing the process of assembly of follicles. Thus, the events in the establishment of follicles in the caecilian ovary are described.

Developmental reprogramming after chromosome transfer into mitotic mouse zygotes

Until now, animal cloning and the production of embryonic stem cell lines by somatic cell nuclear transfer have relied on introducing nuclei into meiotic oocytes. In contrast, attempts at somatic cell nuclear transfer into fertilized interphase zygotes have failed. As a result, it has generally been assumed that unfertilized human oocytes will be required for the generation of tailored human embryonic stem cell lines from patients by somatic cell nuclear transfer. Here we report, however, that, unlike interphase zygotes, mouse zygotes temporarily arrested in mitosis can support somatic cell reprogramming, the production of embryonic stem cell lines and the full-term development of cloned animals. Thus, human zygotes and perhaps human embryonic blastomeres may be useful supplements to human oocytes for the creation of patient-derived human embryonic stem cells.

Histone acetylation and subcellular localization of chromosomal protein BRD4 during mouse oocyte meiosis and mitosis†

Most specific and general transcription factors (TFs) become dissociated from hypoacetylated mitotic chromosomes, which may contribute to transcriptional silencing during mitosis. Only some chromosomal proteins, such as bromodomain containing protein 4 (BRD4), have a potential to associate with mitotic chromosomes in a histone acetylation-dependent manner. It remains to be fully demonstrated whether similar displacement of nuclear factors takes place in meiotic oocytes whose chromosomes become globally deacetylated. To address this, we here examined the subcellular localization of BRD4 in conjunction with the acetylation status of histones in mouse oocytes. Immunofluorescence studies revealed that BRD4 preferentially localized to mitotic chromosomes in early embryos. In contrast, not only endogenous BRD4 but also exogenous BRD4 overexpressed by mRNA microinjection were displaced from meiotic chromosomes whose histones H3 and H4 were deacetylated. Treatment with trichostatin A (TSA), an inhibitor of histone deacetylases, induced histone hyperacetylation of meiotic chromosomes from which endogenous BRD4, however, remained dissociated. Finally, meiotic chromosomal localization of BRD4 could be achieved by BRD4 overexpression together with TSA-induced histone hyperacetylation. These results indicate that, unlike mitosis, histone acetylation is necessary but not sufficient for chromosomal localization of BRD4 during meiosis, suggesting that meiotic oocytes may have additional mechanism(s) for displacement of chromosomal proteins and TFs.

Gonadogenesis in early developmental stages of Acipenser naccarii and influence of estrogen immersion on feminization

Gonad development processes and the effects of a single 8-hour immersion treatment with 17β-estradiol (E2, 400 μg L−1) on sex differentation in the Adriatic sturgeon, Acipenser naccarii, were investigated. After migration of germ cells, gonadal ridges appeared in 16- to 18-day old larvae and undifferentiated gonads in 55- to 60-day old larvae. Putative ovaries with notches in the germinal epithelium and presumed testes with smooth germinal epithelium appeared in 180–185-day old juveniles. Ovaries with proliferating oogonia and early meiotic oocytes clusters were observed in 292-day old juveniles. Testes did not exhibit germ cell mitosis until 430 days of age. Developmental stages in E2-treated animals closely followed those of controls up to 430 days. The treatment significantly increased the percentage of ovaries when administered to embryos about 1.5 day before hatching, while did not significantly altered the normal 1/1 sex ratio when administered to 1.5-day old pre-larvae and 10-day old larvae. It is likely that in A. naccarii exogenous E2 administration may act through a feedback mechanism of self-supporting steroid production and that steroids are the physiological inducers of sex differentiation, as in most teleosts. The E2-immersion treatment, easier than time-consuming administration through food, could be a good approach to control sex differentiation and caviar production.

Regulated expression of TATA-binding protein-related factor 3 (TRF3) during early embryogenesis

RNA polymerase (Pol) II transcription persists in TATA-box-binding protein (TBP)(-/-) mutant mouse embryos, indicating TBP-independent mechanisms for Pol II transcription in early development. TBP-related factor 3 (TRF3) has been proposed to substitute for TBP in TBP(-/-) mouse embryos. We examined the expression of TRF3 in maturing oocytes and early embryos and found that TRF3 was co-expressed with TBP in the meiotic oocytes and early embryos from the late one-cell stage onward. The amounts of TBP and TRF3 changed dynamically and correlated well with transcriptional activity. Chromatin immunoprecipitation (ChIP) assay revealed that different gene promoters in mouse embryonic stem (ES) cells recruited TRF3 and TBP selectively. Comparative analyses of TRF3 and TBP during cell cycle showed that both factors proceeded through cell cycle in a similar pace, except that TRF3 was slightly delayed than TBP in entering the nucleus when cells were exiting the M-phase. Data from expression and biochemical analyses therefore support the hypothesis that TRF3 plays a role in early mouse development. In addition, results from co-localization study suggest that TRF3 may be also involved in Pol I transcription.

Centrosome changes during meiosis in horse oocytes and first embryonic cell cycle organization following parthenogenesis, fertilization and nuclear transfer

Various types of cell cycle organization occur in mammals. In this study, centrosome changes during meiosis in horse oocytes, and first cell cycle organization following fertilization, parthenogenesis and nuclear transfer, were monitored. Cumulus oocyte complexes harvested from horse ovaries obtained from slaughtered mares were cultured in vitro. Meiotic oocytes of germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I and II (MI and MII) stages were selected at various set times during in vitro maturation. Embryos at the first cell cycle stage were generated by subjecting MII stage oocytes to fertilization by intracytoplasmic sperm injection (ICSI), parthenogenetic treatment or nuclear transfer. Centrosome changes during meiosis and the first cell cycle organization were detected by indirect immunofluorescent staining, using a mouse anti-alpha-tubulin antibody for microtubules and a rabbit anti-gamma-tubulin antibody for centrosomes. These examinations showed that the centrosomes of the horse oocyte reorganize themselves from the beginning of GV stage to leave only PCM of gamma-tubulin surrounding both poles of the MI and MII stage spindles. These MII oocytes can organize the separation of metaphase chromosomes during the first embryonic cell cycle by parthenogenetic treatment. When the MII oocytes were subjected to ICSI or nuclear transfer, one or two red-stained centrosomes of gamma-tubulin were introduced by the fertilising spermatozoon or the donor cell which associated with the sperm chromatin in the fertilized embryos and with the donor cell chromatin and microtubules in the cloned embryos. This finding suggests that centrosomes are not an essential component in the formation of the metaphase spindle during meiotic maturation of horse oocytes, but they can be introduced from the spermatozoon or donor cell and are necessary for the organization of normal embryonic development.

Interactions of the meiotic spindle with mitotic chromosomes in GV mouse oocytes

During mitosis, a spindle checkpoint detects chromosome misalignment and halts the cell cycle progression. In meiosis of female germ cells, however, it is debatable whether such a checkpoint is present. This research employed a unique model in the mouse, mitotic chromosomes transferred to meiotic cytoplasts to investigate whether a meiotic oocyte's microtubule apparatus can effectively separate mitotic metaphase chromosomes, and whether a spindle checkpoint exists during its division. The intact germinal vesicle (GV) oocytes, enucleated GV cytoplasts, and enucleated GV cytoplasts at 15 hin-vitro maturation were transferred with a metaphase fibroblast cell. When mitotic chromosomes were transferred into enucleated or intact mouse GV oocytes, the first bipolar meiotic spindles were established and the reconstructed oocytes were able to extrude polar bodies. However, none of the reconstructed oocytes showed complete and accurate alignment of chromosomes, except the enucleated GV cytoplasts reconstructed after maturation. The spindle formation and polar body extrusion suggest that the first meiotic spindle was functional, and the chromosome misalignment did not prevent the onset of anaphase. The data indicate that a spindle checkpoint, providing surveillance of misaligned chromosomes, was overridden or compromised by the incompatibility between somatic chromosomes and meiotic spindles during the first meiotic division.

Gonadal morphogenesis and sex differentiation in intraovarian embryos of the viviparous fishZoarces viviparus (Teleostei, Perciformes, Zoarcidae): A histological and ultrastructural study

It is essential to know the timing and process of normal gonadal differentiation and development in the specific species being investigated in order to evaluate the effect of exposure to endocrine-disrupting chemicals on these processes. In the present study gonadal sex differentiation and development were investigated in embryos of a viviparous species of marine fish, the eelpout, Zoarces viviparus, during their intraovarian development (early September to January) using light and electron microscopy. In both sexes of the embryos at the time of hatching (September 20) the initially undifferentiated paired bilobed gonad contains primordial germ cells. In the female embryos, ovarian differentiation, initiated 14 days posthatch (dph), is characterized by the initial formation of the endoovarian cavity of the single ovary as well as by the presence of some early meiotic oocytes in a chromatin-nucleolus stage. By 30 dph, the endoovarian cavity has formed. By 44 dph and onward, the ovary and the oocytes grow in size and at 134 dph, just prior to birth, the majority of the oocytes are at the perinucleolar stage of primary growth and definitive follicles have formed. In the presumptive bilobed testis of the male embryos, the germ cells (spermatogonia), in contrast to the germ cells of the ovary, remain quiescent and do not enter meiosis during intraovarian development. However, other structural (somatic) changes, such as the initial formation of the sperm duct (30 dph), the presence of blood vessels in the stromal areas of the testis (30 dph), and the appearance of developing testicular lobules (102 dph), indicate testicular differentiation. Ultrastructually, the features of the primordial germ cells, oogonia, and spermatogonia are similar, including nuage, mitochondria, endoplasmic reticulum, and Golgi complexes.

Spindle imaging in human oocytes: the impact of the meiotic cell cycle

Recent studies using polarized light microscopy revealed a correlation between the presence of a spindle in human metaphase II meiotic oocytes and the fertilization rate following intracytoplasmic sperm injection (ICSI). Using a new spindle imaging system, it was possible to visualize the spindle image and the conventional light microscopic view of the oocyte simultaneously. Using this system, time-lapse studies of the meiotic cycle of human oocytes were performed. The video sequences showed that during the transition from metaphase I to metaphase II, the spindle completely disappears for approximately 40–60 min. These data support the idea that at least in some oocytes, the absence of the spindle is more likely an indicator for physiological progression through an important developmental stage of meiosis rather than a cellular disturbance. In view of the low fertilization rates of oocytes with absence of spindles as reported in the literature, the underlying problem could simply be the incorrect timing of ICSI. The time-lapse sequence itself may be viewed online at www.rbmonline.com/Article/2126 [pay-per-view or by subscription].

Strontium promotes calcium oscillations in mouse meiotic oocytes and early embryos through InsP3 receptors, and requires activation of phospholipase and the synergistic action of InsP3

Sr2+ is the most efficient agent for mouse oocyte activation and functions by inducing Ca2+ oscillations. However, its specific mechanism of action remains unknown. Here we investigated the specificity and possible mechanism of Sr2+-induced Ca2+ oscillations in mouse oocytes and early embryos. Ca2+ oscillations in oocytes and embryos were measured by ratiometric fluorescence imaging using fura-2AM. The role of phospholipase C (PLC) and inositol trisphosphate (InsP3) receptors in Sr2+-induced Ca2+ oscillations was examined by selective inhibitors. Sr2+ can induce Ca2+ oscillations in both immature and mature oocytes, and in early embryos. A cell cycle stage-dependent phenomenon to Sr2+ stimulation was observed in 1-cell embryos. By using a low molecular weight heparin to antagonize the function of InsP3 receptors, we were able to show that InsP3 receptors are essential for Sr2+-induced Ca2+ oscillations. Treating metaphase II (MII) oocytes with the PLC inhibitor, U73122, abolished Sr2+-induced increases in Ca2+. This inhibitory effect of U73122 could be rescued by microinjection of InsP3, indicating that Sr2+-induced Ca2+ oscillations require the synergistic action of InsP3. Sr2+-induced calcium oscillations in mouse oocytes and early embryos are mediated through InsP3 receptors, and require PLC activation and the synergistic action of InsP3.

The use ofXenopus oocytes and embryos as a route towards cell replacement

When nuclei of somatic cells are transplanted to enucleated eggs of Xenopus, a complete reprogramming of nuclear function can take place. To identify mechanisms of nuclear reprogramming, somatic nuclei can be transplanted to growing meiotic oocytes of Xenopus, and stem cell genes activated without DNA replication. The combination of somatic cell nuclear transfer with morphogen signalling and the community effect may lead towards the possibility of cell replacement therapy. When mechanisms of nuclear reprogramming are understood, it may eventually be possible to directly reprogramme human somatic cell nuclei without the use of eggs.

Ultrastructure of oogenesis in the bluefin tuna,Thunnus thynnus

Ovarian ultrastructure of the Atlantic bluefin tuna (Thunnus thynnus) was investigated during the reproductive season with the aim of improving our understanding of the reproductive biology in this species. The bluefin, like the other tunas, has an asynchronous mode of ovarian development; therefore, all developmental stages of the oocyte can be found in mature ovaries. The process of oocyte development can be divided into five distinct stages (formation of oocytes from oogonia, primary growth, lipid stage, vitellogenesis, and maturation). Although histological and ultrastructural features of most these stages are similar among all studied teleosts, the transitional period between primary growth and vitellogenesis exhibits interspecific morphological differences that depend on the egg physiology. Although the most remarkable feature of this stage in many teleosts is the occurrence of cortical alveoli, in the bluefin tuna, as is common in marine fishes, the predominant cytoplasmic inclusions are lipid droplets. Nests of early meiotic oocytes derive from the germinal epithelium that borders the ovarian lumen. Each oocyte in the nest becomes surrounded by extensions of prefollicle cells derived from somatic epithelial cells and these form the follicle that is located in the stromal tissue. The primary growth stage is characterized by intense RNA synthesis and the differentiation of the vitelline envelope. Secondary growth commences with the accumulation of lipid droplets in the oocyte cytoplasm (lipid stage), which is then followed by massive uptake and processing of proteins into yolk platelets (vitellogenic stage). During the maturation stage the lipid inclusions coalesce into a single oil droplet, and hydrolysis of the yolk platelets leads to the formation of a homogeneous mass of fluid yolk in mature eggs.

MAP Kinase is Activated dring the Maturation of Porcine Oocytes

In an attempt to evaluate the function of MAP kinase in porcine oocytes and to develop a method of the assessment of its activity, myelin basic protein (MBP) was used as a substrate to detect the MAP kinase activity of porcine oocytes which had undergone maturation in vitro. The existence of MAP kinase and MAP kinase kinase (MAPKK) was verified in immature porcine germinal vesicle (GV) oocytes at 0 h culture via Western blotting. Porcine oocytes exhibited a low level of MAP kinase activity during the first 20 h of culture, which increased at 25 h, during which time a breakdown in the nuclear membrane occurred. Significantly higher increases (p<0.05) of MAP kinase activity were detected at 30 h of culture. Using the gel phosphorylation method, MBP was phosphorylated at two positions corresponding to mammalian MAP kinase-extracellular signal-regulated kinase (ERK 1) (44 kDa) and ERK 2 (42 kDa). The absolute levels of those proteins did not increase during 40 h of culture, suggesting that the detected increase in MAP kinase activity was the result of phosphorylation rather than changes in the total amount of protein. MAPKK and MAP kinase were dephosphorylated in first-stage (MI) meiotic oocytes by the addition of cycloheximide, a protein synthesis inhibitor. These results of this study indicate that the MAP kinase cascade does exists in porcine oocytes and that its activation leads to oocyte maturation. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 8 : 1069-1075)

Sex differentiation and hormonal sex reversal in the bagrid catfish Pseudobagrus fulvidraco (Richardson)

Sex differentiation of the bagrid catfish Pseudobagrus fulvidraco (Richardson) is described from hatching to the 100th day post-hatch (water temperature 25 °C). Primordial germ cells (PGCs) were observed on the 3rd day post-hatch (0.78 cm total length (TL), 0.006 g body weight (BW)), and began to protrude into the peritoneal cavity from the 8th day post-hatch (1.17 cm TL, 0.02 g BW). On the 12th day post-hatch (1.46 cm TL, 0.06 g BW), initial ovarian differentiation was identified by the PGCs with condensed chromatin, and their transformation into meiotic oocytes. Oocytes were in the secondary and tertiary yolk stages on the 100th day post-hatch (10.04 cm TL, 11.38 g BW). While ovaries gradually grew after sex were differentiated, testes continued to multiply from the 20th day post-hatch (2.30 cm TL, 0.24 g BW) when testicular differentiation was first identified. On the 100th day post-hatch (9.84 cm TL, 12.79 g BW), testes contained spermatocytes exhibiting meiotic activity, spermatozoa and cyst cells. Therefore, the sex differentiation type of the bagrid catfish is identified as gonochoristic. Effect of oral administration of either estradiol-17β (5–40 ppm) or tamoxifen (25–200 ppm) for 20 days (from 3rd to 20th day post-hatch) on mortality and growth of the bagrid catfish was studied. Estradiol-17β treatment at the concentration of 400 ppm successfully produced 100% females; however, estradiol-17β treatment induced mortality seemingly related to dose-dependent. Estradiol fashion did not affect growth of the bagrid catfish at doses of 0–20 ppm ( P>0.05), but significant growth depression occurred at 40 ppm ( P<0.05). Tamoxifen treatment at 200 ppm produced 90% males and mortality appeared to be dose-dependent. All groups of tamoxifen-treated fish exhibited significant growth acceleration comparing to the control group, but no significant difference was observed among the treated groups ( P>0.05). The combined results of oral administration with estradiol-17β and tamoxifen suggest that these hormones be effective for sex reversal in the bagrid catfish and the labile period of sex differentiation fall between the 8th and 30th day post-hatch.

CHANGES IN PERICENTRIN LOCALIZATION DURING MEIOSIS

SUMMARY The centrosome in oocytes, an acentriolar aggregate of centrosomal material, is regulated in a dynamic manner throughout the process of meiotic maturation. Recently, it has been demonstrated that meiotic spindle assembly is likely regulated by chromosomal and microtubule/microtubule associated modulators. Given the importance of centrosomal role in spindle assembly, we aimed to assess the distribution of the eintegral centrosomal protein, pericentrin, during the course of meiotic maturation. Distribution of pericentrin during meiotic progression was examined by the serial confocal images in z-axis. It was also evaluated after exposure of oocytes to some pharmacological agents (i.e. Taxol and Methylbenzimidazole Carbamate) that perturb spindle and/or centrosomal integrity. We examined pericentrin in various shapes and locations during different meiotic stages (i.e. arcs, rings, fractures etc.). After taxol exposure, pericentrin incorporation into both spindle poles and cytoplasmic centrosomes was increased. Treatment of oocytes with MBC, a potent drug that has been previously shown to disrupt spindle poles in meiotic oocytes, influenced early events such as chromosome capture and spindle assembly, and altered the number and distribution of cytoplasmic centrosomes. In conclusion, the dynamic reorganization of pericentrin and changes in centrosome microtubule nucleation capacity are involved in critical cell cycle transitions during meiotic maturation.

Sex-specific gene expression during meiotic prophase I: Xlr (X linked, lymphocyte regulated), not its male homologue Xmr (Xlr related, meiosis regulated), is expressed in mouse oocytes.

The Xmr (Xlr related, meiosis regulated) gene product is abundantly expressed in primary spermatocytes and is notably associated with nonrecombining segments of sex chromosomes in the XY body. Here we determined whether Xmr was expressed in meiotic oocytes. This was done by reverse transcription-polymerase chain reaction and cDNA sequencing, Western blot analysis, and immunocytochemistry. Unexpectedly, no Xmr message was detected in mouse fetal oocytes. Instead, Xlr (X linked, lymphocyte regulated), a closely related gene expressed in fetal thymus cells at the time of antigen-receptor gene rearrangement, was expressed in oocytes throughout meiotic prophase I. These findings indicate a sex-specific expression of two closely related members of the Xlr gene family during meiotic prophase I. The XLR protein may provide a useful marker for studies on chromatin condensation or DNA recombination in oocytes. In addition, because of the localization of the Xlr sequence family on the mouse X chromosome, the human equivalent of Xlr is a candidate gene for premature ovarian failure.

Calcium-binding proteins and calcium-release channels in human maturing oocytes, pronuclear zygotes and early preimplantation embryos.

The study aim was to investigate the presence and localization of Ca2+-binding proteins and Ca2+-release receptor channels in human maturing oocytes, pronuclear zygotes and preimplantation embryos. Immunocytochemical analysis, using specific antibodies against the proteins being studied, followed with confocal laser microscopy, was performed on human oocytes and embryos. Calreticulin and calsequestrin (the two major calcium storage proteins of somatic cells), two types of calcium release receptors, the inositol trisphosphate and ryanodine receptors (InsP(3)R-2, RyRs-1,2,3), and the molecular chaperone, calnexin, were identified in all investigated cell types. Calreticulin was predominant in the cell cortex and in the nuclear envelope, while calsequestrin was distributed throughout the entire cytoplasm. Generally, localization of the InsP(3)R-2 and RyRs was similar to that of calreticulin and calsequestrin respectively. Both types of receptor were enriched in the subplasmalemmal region of meiotic oocytes. In addition, the InsP(3)R was detected in the nuclear structures of oocytes and blastomeres. Calnexin distribution overlapped with that of calreticulin but appeared to be present in distinct subcompartments. Human oocytes and embryos express the calcium sequestration and release proteins in highly organized and developmentally regulated patterns. Fine-tuning of these proteins may play a crucial role in regulation of Ca2+ transience during oocyte maturation, fertilization and early embryo development.