Metaphase I arrest and spontaneous parthenogenetic activation of strain LTXBO oocytes: chimeric reaggregated ovaries establish primary lesion in oocytes

Oocytes of strain LT mice, and related strains such as LTXBO, exhibit a high incidence of arrest in the progression of meiosis at metaphase I (MI) and in spontaneous parthenogenetic activation. Activation of these oocytes within the ovary leads to the formation of ovarian teratomas. In this study, the role of the oocyte's companion granulosa cells, the cumulus cells, was investigated using fully grown oocytes matured in vitro after isolation from LTXBO mice. Results showed that the role of cumulus cells in MI arrest is dichotomous. Cumulus cells temporarily helped to sustain MI arrest, but they also promoted a delayed progression to metaphase II. Cumulus cells also promoted parthenogenetic activation that occurred in association with the delayed progression to metaphase II. Next, the question of whether the lesion(s) promoting MI arrest and spontaneous activation is due to defects in the somatic cells or is intrinsic to the oocyte was addressed using chimeric reaggregated ovaries. An improved method for completely exchanging the germ cell and the somatic cell compartments of ovaries from newborn mice is described. These chimeric reaggregated ovaries, grafted beneath the renal capsule of SCID mice, allowed the complete development of LTXBO oocytes to occur in association with somatic cells from control (B6SJLF(1)) ovaries and development of control oocytes in association with LTXBO somatic cells. Oocyte growth and follicular development appeared generally normal in reaggregated ovaries. High incidences of MI arrest and spontaneous activation of LTXBO oocytes occurred regardless of the genotype of the somatic cells. Moreover, there was a low incidence of MI arrest and spontaneous activation of control oocytes, even though they underwent complete development and maturation associated with LTXBO somatic cells. It is concluded that the phenotypes of MI arrest and parthenogenetic activation in LTXBO oocytes are defects caused by lesions intrinsic to the oocyte. Nevertheless, the oocyte's companion somatic cells play crucial roles in the expression of these lesions.     

Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation

The expansion, or mucification, of the mouse cumulus oophorus in vitro requires the presence of an enabling factor secreted by the oocyte as well as stimulation with follicle-stimulating hormone (FSH). This study focuses on (1) the ability of mouse oocytes to secrete the enabling factor at various times during oocyte growth and maturation, (2) the temporal relationships between the development of the capacity of the oocyte to undergo germinal vesicle breakdown, the ability of the oocyte to secrete cumulus expansion-enabling factor, and the capacity of the cumulus oophorus to undergo expansion, and (3) the role of the oocyte in the differentiation of granulosa cells as functional cumulus cells. Growing, meiotically incompetent oocytes did not produce detectable amounts of cumulus expansion-enabling factor, but fully grown meiosis-arrested oocytes, maturing oocytes, and metaphase II oocytes did. Detectable quantities of enabling factor were produced by zygotes, but not by two-cell stage to morula embryos. The ability of oocytes to secrete cumulus expansion enabling factor and the capacity of cumulus cells to respond to FSH and the enabling factor are temporally correlated with the acquisition of oocyte competence to undergo germinal vesicle breakdown. Mural granulosa cells of antral follicles do not expand in response to FSH even in the presence of cumulus expansion-enabling factor, showing that mural granulosa cells and cumulus cells are functionally distinct cell types. The perioocytic granulosa cells of preantral follicles isolated from 12-day-old mice differentiate into functional cumulus cells during a 7-day period in culture. Oocytectomized granulosa cell complexes grown in medium conditioned by either growing or fully grown oocytes were comparable in size to intact complexes and maintained their 3-dimensional integrity to a greater degree than oocytectomized complexes grown in unconditioned medium. After 7 days, the oocytectomized complexes were stimulated with FSH in the presence of enabling factor, but no expansion was observed whether or not the oocytectomized complexes grew in the presence of oocyte-conditioned medium. These results suggest that a factor(s) secreted by the oocyte affects granulosa cell proliferation and the structural organization of the follicle, but continual close association with the oocyte appears necessary for the differentiation of granulosa cells into functional cumulus cells, insofar as they are capable of undergoing expansion.     

Somatic cell-oocyte interactions in mouse oogenesis: stage-specific regulation of mouse oocyte protein phosphorylation by granulosa cells

The relative rate of synthesis of a number of proteins and the protein phosphorylation pattern of growing and fully grown oocytes were influenced by the presence of granulosa cells. In particular, a 74-kDa phosphorylated protein was detected only in granulosa cell-enclosed growing mouse oocytes. When reaggregated with granulosa cells, the growing oocyte displayed the phosphorylated form of the 74-kDa protein but when oocytes were cultured on Sertoli cell monolayers or in granulosa cell-conditioned medium the 74-kDa protein was not phosphorylated. We propose that (1) granulosa cells regulate protein phosphorylation in mouse oocytes; (2) a 74-kDa protein is phosphorylated only in growing oocytes when surrounded by granulosa cells; and (3) granulosa cells, but not Sertoli cells, are competent to send the appropriate "signal" to the growing oocyte.     

TAF4b, a TBP associated factor, is required for oocyte development and function

Development of a fertilizable oocyte is a complex process that relies on the precise temporal and spatial expression of specific genes in germ cells and in surrounding somatic cells. Since female mice null for Taf4b, a TBP associated factor, are sterile, we sought to determine when during follicular development this phenotype was first observed. At postnatal day 3, ovaries of Taf4b null females contained fewer (P < 0.01) oocytes than ovaries of wild type and heterozygous Taf4b mice. However, expression of only one somatic cell marker Foxl2 was reduced in ovaries at day 15. Despite the reduced number of follicles, many proceed to the antral stage, multiple genes associated with granulosa cell differentiation and oocyte maturation were expressed in a normal pattern, and immature Taf4b null females could be hormonally primed to ovulate and mate. However, the ovulated cumulus oocyte complexes from the Taf4b null mice had fewer (P < 0.01) cumulus cells, and the oocytes were functionally abnormal. GVBD and polar body extrusion were reduced significantly (P < 0.01). The few oocytes that were fertilized failed to progress beyond the two-cell stage of development. Thus, infertility in Taf4b null female mice is associated with defects in early follicle formation, oocyte maturation, and zygotic cleavage following ovulation and fertilization.     

Proliferating cell nuclear antigen (PCNA) regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries

Primordial follicles, providing all the oocytes available to a female throughout her reproductive life, assemble in perinatal ovaries with individual oocytes surrounded by granulosa cells. In mammals including the mouse, most oocytes die by apoptosis during primordial follicle assembly, but factors that regulate oocyte death remain largely unknown. Proliferating cell nuclear antigen (PCNA), a key regulator in many essential cellular processes, was shown to be differentially expressed during these processes in mouse ovaries using 2D-PAGE and MALDI-TOF/TOF methodology. A V-shaped expression pattern of PCNA in both oocytes and somatic cells was observed during the development of fetal and neonatal mouse ovaries, decreasing from 13.5 to 18.5 dpc and increasing from 18.5 dpc to 5 dpp. This was closely correlated with the meiotic prophase I progression from pre-leptotene to pachytene and from pachytene to diplotene when primordial follicles started to assemble. Inhibition of the increase of PCNA expression by RNA interference in cultured 18.5 dpc mouse ovaries strikingly reduced the apoptosis of oocytes, accompanied by down-regulation of known pro-apoptotic genes, e.g. Bax, caspase-3, and TNFα and TNFR2, and up-regulation of Bcl-2, a known anti-apoptotic gene. Moreover, reduced expression of PCNA was observed to significantly increase primordial follicle assembly, but these primordial follicles contained fewer granulosa cells. Similar results were obtained after down-regulation by RNA interference of Ing1b, a PCNA-binding protein in the UV-induced apoptosis regulation. Thus, our results demonstrate that PCNA regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries.     

Oocyte control of metabolic cooperativity between oocytes and companion granulosa cells: energy metabolism

Intercellular communication between oocytes and granulosa cells is essential for normal follicular differentiation and oocyte development. Subtraction hybridization was used to identify genes more highly expressed in cumulus cells than in mural granulosa cells of mouse antral follicles. This screen identified six genes involved in glycolysis: Eno1, Pkm2, Tpi, Aldoa, Ldh1, and Pfkp. When oocytes were microsurgically removed from cumulus cell-oocyte complexes, the isolated cumulus cells exhibited decreased expression levels of genes encoding glycolytic enzymes, glycolysis and activity of the tricarboxylic acid (TCA) cycle. These decreases were prevented by culturing the cumulus cells with paracrine factors secreted by fully grown oocytes. Paracrine factors from fully grown oocytes exhibited greater ability than those from growing oocytes to promote expression of genes encoding glycolytic enzymes and glycolysis in the granulosa cells of preantral follicles. However, neither fully grown nor growing oocytes secreted paracrine factors affecting activity of the TCA cycle. These results indicate that oocytes regulate glycolysis and the TCA cycle in granulosa cells in a manner specific to the population of granulosa cells and to the stage of growth and development of the oocyte. Oocytes control glycolysis in granulosa cells by regulating expression levels of genes encoding glycolytic enzymes. Therefore, mouse oocytes control the intercellular metabolic cooperativity between cumulus cells and oocytes needed for energy production by granulosa cells and required for oocyte and follicular development.     

Inactivation of Retinoblastoma Protein (Rb1) in the Oocyte: Evidence That Dysregulated Follicle Growth Drives Ovarian Teratoma Formation in Mice

The origin of most ovarian tumors is undefined. Here, we report development of a novel mouse model in which conditional inactivation of the tumor suppressor gene Rb1 in oocytes leads to the formation of ovarian teratomas (OTs). While parthenogenetically activated ooctyes are a known source of OT in some mutant mouse models, enhanced parthenogenetic propensity in vitro was not observed for Rb1-deficient oocytes. Further analyses revealed that follicle recruitment and growth is disrupted in ovaries of mice with conditional inactivation of Rb1, leading to abnormal accumulation of secondary/preantral follicles. These findings underpin the concept that miscues between the germ cell and somatic compartments cause premature oocyte activation and the formation of OTs. Furthermore, these results suggest that defects in folliculogenesis and a permissive genetic background are sufficient to drive OT development, even in the absence of enhanced parthenogenetic activation. Thus, we have discovered a novel role of Rb1 in regulating the entry of primordial oocytes into the pool of growing follicles and signaling between the oocyte and granulosa cells during the protracted process of oocyte growth. Our findings, coupled with data from studies of other OT models, suggest that defects in the coordinated regulation between growth of the oocyte and somatic components in follicles are an underlying cause of OT formation.     

Developmental and molecular aberrations associated with deterioration of oogenesis during complete or partial follicle-stimulating hormone receptor deficiency in mice

Targeted disruption of the mouse FSH receptor gene (FSH-R) that mediates the action of the FSH results in a gene dose-related ovarian phenotype in the developing as well as the adult animal. While null females (FORKO) are sterile, the haplo-insufficient mice experience early reproductive senescence. The purpose of this study was to first record changes in oocyte development in the null FORKO and haplo-insufficient mice. Oocyte growth is significantly retarded in the null mutants with thinner zona pellucida in preantral follicles, but thicker zona pellucida in secondary follicles. This morphometric change indicates developmental aberrations in coordination of the germ cell (oocyte) and the somatic granulosa cell (GC) compartments. Markers for primordial germ cell proliferation and oocyte growth, such as the c-Kit/Kit-ligand and bone morphogenetic protein-15 (BMP-15) were downregulated in both null and +/- ovaries, suggesting disrupted communication between oocyte and GCs. Extensive changes in the expression of other oocyte-specific gene products like the zona pellucida glycoproteins (zona pellucida A, B, and C) indicate major alteration in the extracellular matrix surrounding the germ cells. This led to leaky germ cells that allowed infiltration of somatic cells. These results show that the loss of FSH-R signaling alters the follicular environment, where oocyte-granulosa interactions are perturbed, creating an out-of-phase germ cell and somatic cell development. We believe that these data provide an experimental paradigm to explore the mechanisms responsible for preserving the structural integrity and quality of oocytes at different ages.     

Mouse oocytes regulate metabolic cooperativity between granulosa cells and oocytes: amino acid transport

A search for genes expressed more highly in mouse cumulus cells than mural granulosa cells by subtraction hybridization yielded Slc38a3. SLC38A3 is a sodium-coupled neutral amino acid transporter having substrate preference for l-glutamate, l-histidine, and l-alanine. Detectable levels of Slc38a3 mRNA were found by in situ hybridization in granulosa cells of large preantral follicles, but levels were higher in all granulosa cells of small antral follicles; expression became limited to cumulus cells of large antral follicles. Expression of Slc38a3 mRNA in granulosa cells was promoted by fully grown oocytes from antral follicles but not by growing oocytes from preantral follicles. Fully grown oocytes were dependent on cumulus cells for uptake of l-alanine and l-histidine but not l-leucine. Fully grown but not growing oocytes secreted one or more paracrine factors that promoted cumulus cell uptake of all three amino acids but of l-alanine and l-histidine to a much greater extent than l-leucine. Uptake of l-leucine appeared dependent primarily on contact-mediated signals from fully grown oocytes. Fully grown oocytes also promoted elevated levels of Slc38a3 mRNA and l-alanine transport by preantral granulosa cells, but growing oocytes did not. Therefore, fully grown oocytes secrete one or more paracrine factors that promote cumulus cell uptake of amino acids that oocytes themselves transport poorly. These amino acids are likely transferred to oocytes via gap junctions. Thus, oocytes use paracrine signals to promote their own development via metabolic cooperativity with cumulus cells. The ability of oocytes to mediate this cooperativity is developmentally regulated and acquired only in later stages of oocyte development.     

Mouse antral oocytes regulate preantral granulosa cell ability to stimulate oocyte growth in vitro

In this study we evaluated whether mouse oocytes derived from early antral or preovulatory follicles could affect the ability of preantral granulosa cells to sustain oocyte growth in vitro. We found that early antral oocytes with a diameter > or =75 microm did not grow any further during 3 days of culture on preantral granulosa cell monolayers in vitro, while most of the oocytes with a smaller diameter increased significantly in size. Similarly, about 65% of growing oocytes isolated from preantral follicles grew when cultured on preantral granulosa cells. By coculturing with growing oocytes fully grown early antral or preovulatory oocytes, a small proportion (about 10%) of growing oocytes increased in diameter, and changes in granulosa cell morphology were observed. Such effects occurred as a function of the fully grown oocyte number seeded and were not associated with a decrease in coupling index values. By avoiding physical contact between antral oocytes and granulosa cells, the proportion of growing oocytes undergoing a significant increase in diameter was about 36%. These results indicate that fully grown mouse oocytes can control preantral granulosa cell growth-promoting activity through the production of a soluble factor(s) and the maintenance of functional communications with surrounding granulosa cells.     

Characterization of integrin expression in the mouse ovary

Integrin alpha:beta heterodimers mediate cell contacts to the extracellular matrix and initiate intracellular signaling cascades in response to a variety of factors. Integrins interact with many determinants of cellular phenotypes and play roles in controlling the development, structural integrity, and function of every type of tissue. Despite their importance, little is known about the regulation of integrin subunits in the mammalian ovary and how they function in folliculogenesis. To determine their relevance to ovarian physiology, we have studied the expression of integrin subunit mRNAs by Northern blot analysis and in situ hybridization in ovaries of wild-type, growth differentiation factor 9 (Gdf 9) knockout, FSHbeta (Fshb) knockout, and inhibin alpha (Inha) knockout mice. Integrin alpha6 mRNA is expressed in oocytes and granulosa cells of single-layer follicles and in oocytes and theca cells of multilayer follicles. Integrin alpha6 is highly expressed in Gdf 9 knockout ovaries, which are enriched in oocytes and primary (single layer) follicles because of a block at this stage of follicular development. Integrin alpha(v) mRNA is most highly expressed in the granulosa cells of multilayer growing follicles, and therefore only low levels of expression are detectable in the Gdf 9 knockout ovaries. Integrin beta1 mRNA exhibits a broad expression pattern in ovaries, including oocytes, granulosa cells, theca cells, and corpora lutea. Integrin beta3 mRNA is expressed in theca and interstitial cells and is upregulated in corpora lutea. It is nearly undetectable in ovaries of Fshb knockout mice, which develop preantral follicles but have no luteal cells. Integrin beta5 mRNA is predominantly expressed in granulosa cells of multilayer follicles. It is expressed at high levels in the Fshb knockout mice and in a compartmentalized manner in the granulosa cell/Sertoli cell tumors that develop in the Inha knockout mice. Specific integrins are associated with ovarian cellular phenotypes in mice, which raises intriguing possibilities as to integrin functions in oocyte competence, follicular development, luteinization, and granulosa cell proliferation.     

The expression of c-kit protein during oogenesis and early embryonic development.

The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor and was shown to be allelic with the white-spotting locus (W) of the mouse. Mutations at the W locus have pleiotropic effects on the development of hematopoietic stem cells, melanoblasts, and primordial germ cells. In order to elucidate the role of c-kit protein in gametogenesis and oocyte maturation, we have examined immunohistochemically the expression of c-kit in the ovaries of mice at late fetal and postnatal stages, and in early embryos. By the avidin-biotin-peroxidase (ABC) method using rat anti-mouse c-kit monoclonal antibody, the c-kit protein was detected in ovaries after the time of birth, but not before. The expression of c-kit was observed mainly on the surface of oocytes, but not in granulosa cells nor in interstitial regions. Oocytes of primordial to fully grown Graafian follicles showed the c-kit protein. When ovulation was induced by hCG, the expression of c-kit in ovulated unfertilized oocytes was weaker than in oocytes of Graafian follicles. In 1-cell embryos the c-kit protein was still observed, but with cell division its expression further decreased, and it was not detected in embryos of 4-cell, 8-cell, and morula stages. In summary, the highest expression of c-kit was observed on the surface of oocytes arrested in the diplotene stage of meiotic prophase. With ovulation and the resumption of meiotic maturation, its expression declined. These results suggest that the c-kit protein may play some role in meiotic arrest, oocyte growth, and oocyte maturation.     

Production of buffalo embryos using oocytes from in vitro grown preantral follicles

The present study examines the use of buffalo preantral follicles as a source of oocytes for in vitro embryo production. Preantral follicles were isolated from abattoir-derived buffalo ovaries and were grown for 100 days in five different culture systems: (1) minimum essential medium (MEM); (2) coconut water; (3) MEM + ovarian mesenchymal cell (OMC) co-culture; (4) MEM + granulosa cell (GC) co-culture; or (5) MEM + cumulus cell (CC) co-culture. Low growth rates for the preantral follicles were observed when follicles were cultured in MEM or coconut water medium. Moderate growth rates were seen for OMC and GC co-cultures, and high rates of growth were observed when follicles were grown in CC co-culture. The survival of preantral follicles was low in the MEM culture (<25%), but was over 75% in the other culture systems. Oocytes were not recovered from the MEM group, while an oocyte recovery rate of 80-100% was observed when the follicles were cultured with coconut water/somatic cells. Transferable embryos could be produced only with the oocytes obtained from preantral follicles grown in the OMC and CC co-culture systems. This study demonstrates, for the first time, that it is possible to produce buffalo embryos by in vitro fertilization of oocytes derived from in vitro grown preantral follicles.     

Mouse granulosa cells contribute more to the mRNA synthesis of mZP2 than oocyte does

In order to elucidate the biogenesis of mouse zona pellucida 2 (mZP2) protein, RT-PCR, and in situ hybridization were carried out to localize the expression of mouse ZP2 mRNA. Cumulus cells of the OCC (Oocyte-Cumulus cell Complex) were isolated from the oocytes after superovulation for the RNA extraction. The frozen sections of ovaries from adolescent and aged mice were prepared to hybridize with RNA probe of mouse ZP2. mRNA of ZP2 was detected in isolated cumulus cells by RT-PCR. Results of in situ hybridization showed that the mRNA of ZP2 was synthesized in both oocyte and granulosa cells at different folliculogenesis stages; and the expression of ZP2 mRNA in granulosa cells was stronger than that in oocyte; much weaker expression of mZP2 was detected in the follicles of aged mouse. These suggest that the entire amount of ZP2 mRNA generated in the granulosa cells layer should be much more than that in oocyte. Therefore, we think that the granulosa cells contribute more to the mZP2 mRNA synthesis than oocyte does.     

TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary

Although it is well established that both follicular assembly and the initiation of follicle growth in the mammalian ovary occur independently of pituitary hormone support, the factors controlling these processes remain poorly understood. We now report that neurotrophins (NTs) signaling via TrkB receptors are required for the growth of newly formed follicles. Both neurotrophin-4/5 (NT-4) and brain-derived neurotrophic factor (BDNF), the preferred TrkB ligands, are expressed in the infantile mouse ovary. Initially, they are present in oocytes, but this site of expression switches to granulosa cells after the newly assembled primordial follicles develop into growing primary follicles. Full-length kinase domain-containing TrkB receptors are expressed at low and seemingly unchanging levels in the oocytes and granulosa cells of both primordial and growing follicles. In contrast, a truncated TrkB isoform lacking the intracellular domain of the receptor is selectively expressed in oocytes, where it is targeted to the cell membrane as primary follicles initiate growth. Using gene-targeted mice lacking all TrkB isoforms, we show that the ovaries of these mice or those lacking both NT-4 and BDNF suffer a stage-selective deficiency in early follicular development that compromises the ability of follicles to grow beyond the primary stage. Proliferation of granulosa cells-required for this transition-and expression of FSH receptors (FSHR), which reflects the degree of biochemical differentiation of growing follicles, are reduced in trkB-null mice. Ovaries from these animals grafted under the kidney capsule of wild-type mice fail to sustain follicular growth and show a striking loss of follicular organization, preceded by massive oocyte death. These results indicate that TrkB receptors are required for the early growth of ovarian follicles and that they exert this function by primarily supporting oocyte development as well as providing granulosa cells with a proliferative signal that requires oocyte-somatic cell bidirectional communication. The predominance of truncated TrkB receptors in oocytes and their developmental pattern of subcellular expression suggest that a significant number of NT-4/BDNF actions in the developing mammalian ovary are mediated by these receptors.     

Consequences of fetal irradiation on follicle histogenesis and early follicle development in rat ovaries

Follicle histogenesis, in which follicles arise from fragmenting ovigerous cords, is a poorly understood mechanism that is strictly dependent upon the presence of germ cells. Our previous studies have shown that severely germ cell-depleted rat ovaries after fetal gamma-irradiation display modifications of follicular endowment and dynamics during the immature period. The primordial follicle stock was absent and the follicles with primary appearance remained quiescent longer than in control ovaries during the neonatal period. The aim of the present work was to analyze the initial steps of follicle histogenesis, and to investigate the etiology of the alterations observed in the development of irradiated ovaries. Just after birth, we observed, in addition to sterile ovigerous cords, the emergence of the first follicles which exhibited several abnormal features as compared to those of control ovaries. Most of the follicles appeared as primary follicles, as they were composed of a layer of cuboidal-shaped granulosa cells surrounding an enlarged oocyte. Interestingly, the granulosa cells of these primary-like follicles did not proliferate and did not express the genes for anti-Müllerian hormone (Amh) or bone morphogenetic protein receptor type II (Bmpr2), both of which are normally expressed from the primary stage onwards. In contrast, the oocytes strongly expressed the gene for growth and differentiation factor 9 (Gdf9), which is normally upregulated from the primary follicle stage onwards, which suggests an uncoupling of granulosa cell development from oocyte development. In addition, irradiated ovaries displayed a higher frequency of follicles that contained 2 or 3 oocytes, which are also referred to as multi-oocyte follicles (MOFs). Examination at the time of follicle histogenesis indicated that MOFs arise from incomplete ovigerous cord breakdown. Taken together, the results of this study indicate that severe perturbations of follicular histogenesis take place following irradiation and massive germ cell depletion during fetal life. In addition to the classically described sterile cords, we have pointed out the differentiation of MOFs and primary-like quiescent follicles, which finally evolve into growing follicles and participate in ovarian function. We propose that these phenotypes are closely correlated to the proportion of granulosa cells to oocytes at the time of neonatal follicle histogenesis.