Occlusive lung arterial lesions in endothelial-targeted, fas-induced apoptosis transgenic mice.

Pulmonary arterial hypertension (PAH) is a lethal disease that is characterized by functional and structural abnormalities involving distal pulmonary arterioles that result in increased pulmonary vascular resistance and ultimately right heart failure. In experimental models of pulmonary hypertension, endothelial cell (EC) apoptosis is a necessary trigger for the development of obliterative lung arteriopathy, inducing the emergence of hyperproliferative and apoptosis-resistant vascular cells. However, it has not been established whether EC apoptosis is sufficient for the induction of complex lung arteriolar lesions. We generated a conditional transgenic system in mice to test the hypothesis that lung endothelial cell apoptosis is sufficient to induce a PAH phenotype. The Fas-induced apoptosis (FIA) construct was expressed under the control of endothelial-specific Tie2 promoter (i.e., EFIA mice), and administration of a small molecule dimerizing agent, AP20187, resulted in modest pulmonary hypertension, which was associated with obliterative vascular lesions localized to distal lung arterioles in a proportion of transgenic mice. These lesions were characterized by proliferating cells, predominantly CD68 macrophages. Although endothelial cell apoptosis was also seen in the kidney, evidence of subsequent arteriopathy was seen only in the lung. This model provides direct evidence that lung endothelial cell apoptosis acts as a trigger to initiate a PAH phenotype and provides initial insight into the potential mechanisms that underlie a lung-specific arterial response to endothelial injury.

Mammalian hyaluronidase induces ovarian granulosa cell apoptosis and is involved in follicular atresia.

During ovarian folliculogenesis, the vast majority of follicles will undergo atresia by apoptosis, allowing a few dominant follicles to mature. Mammalian hyaluronidases comprise a family of six to seven enzymes sharing the same catalytic domain responsible for hyaluronan hydrolysis. Interestingly, some of these enzymes have been shown to induce apoptosis. In the ovary, expression of three hyaluronidases (Hyal-1, Hyal-2, and Hyal-3) has been documented. However, their precise cellular localization and role in ovarian regulation have not yet been defined. We herein investigated the possible involvement of these enzymes in ovarian atresia. First, we established a mouse model for ovarian atresia (gonadotropin withdrawal by anti-equine chorionic gonadotropin treatment) and showed that the mRNA levels of Hyal-1, Hyal-2, and Hyal-3 were significantly increased in apoptotic granulosa cells as well as in atretic follicles. Second, using ovaries of normally cycling mice, we demonstrated the correlation of Hyal-1 mRNA and protein expression with cleavage of caspase-3. In addition, we showed that expression of all three hyaluronidases induced apoptosis in transfected granulosa cells. Significantly, the induction of apoptosis by hyaluronidases was independent of catalytic activity, because enzymatically inactive Hyal-1 mutant (D157A/E159A) was as efficient as the wild-type enzyme in apoptosis induction. The activation of the extrinsic apoptotic signaling pathway was involved in this induction, because increased levels of cleaved caspase-8, caspase-3, and poly-ADP-ribose polymerase (PARP) were observed upon hyaluronidase ectopic expression. Our present findings provide a better understanding of the role of hyaluronidases in ovarian functions, showing for the first time their involvement in follicular atresia.

Activation of endoproteolytic processing of insulin-like growth factor-II in fetal, early postnatal, and pregnant rats and persistence of circulating levels in postnatal life.

The process of posttranslational modifications of IGF-II likely has important physiological consequences. In addition to mature IGF-II, glycosylated proIGF-II(156-amino acid peptide) and two glycosylated big IGF-II forms, IGF-II(1-104) and IGF-II(1-87), have been identified in the human circulation. Due to lack of an appropriate methodology, different IGF-II isoforms have not been demonstrated and characterized in the rat circulation, thus preventing a better understanding of the physiological and pathological roles of IGF-II. In the present study, we characterized each IGF-II form and assessed its content in the rat circulation throughout life time by using a highly sensitive Western blot analysis, which is void of the IGF binding protein interference and distinguished all IGF-II forms. For the first time, we demonstrated the presence of IGF-II variants, including proIGF-II, IGF-II(1-87), and mature IGF-II, in the rat circulation during postnatal life, challenging the current impression that IGF-II is absent from sera of adult rats. ProIGF-II is glycosylated and is the predominant form in the rat circulation. Endoproteolytic processing of proIGF-II was clearly activated in fetal, neonatal, and pregnant rats, likely reflecting its involvement in fetal development through the generation of specific forms of IGF-II (e.g. mature IGF-II) that are required for their distinct biological functions. Taken together, our data also suggest that serum IGF-II profiles may reflect underlying physiological conditions.

Role and regulation of nodal/activin receptor-like kinase 7 signaling pathway in the control of ovarian follicular atresia.

Although the role of the TGF beta superfamily members in the regulation of ovarian folliculogenesis has been extensively studied, their involvement in follicular atresia is not well understood. In the present study, we have demonstrated for the first time that Nodal, a member of the TGF beta superfamily, is involved in promoting follicular atresia as evidenced by the following: 1) colocalization of Nodal and its type I receptor Activin receptor-like kinase 7 (ALK7) proteins in the granulosa cells was only observed in atretic antral follicles, whereas they were present in theca cells and granulosa cells of healthy follicles, respectively; 2) addition of recombinant Nodal or overexpression of Nodal by adenoviral infection induced apoptosis of otherwise healthy granulosa cells; 3) constitutively active ALK7 (ALK7-ca) overexpression mimicked the function of Nodal in the induction of granulosa cell apoptosis. Furthermore, overexpression of Nodal or ALK7-ca increased phosphorylation and nuclear translocation of Smad2, decreased X-linked inhibitor of apoptotic proteins (Xiap) expression at both mRNA and protein level and phospho-Akt content, as well as triggered mitochondrial release of death proteins Smac/DIABLO, Omi/HtrA2, and cytochrome c in the granulosa cells. Dominant-negative Smad2 significantly attenuated ALK7-ca-induced down-regulation of Xiap and thus rescued granulosa cells from undergoing apoptosis. In addition, whereas up-regulation of Xiap significantly attenuated ALK7-ca-induced apoptosis, down-regulation of Xiap sensitized granulosa cells to ALK7-ca-induced apoptosis. Furthermore, ALK7-ca-induced apoptosis was significantly attenuated by forced expression of activated Akt, and Akt rescued granulosa cells from undergoing apoptosis via proteasome-mediated ALK7 degradation. Taken together, Nodal plays an atretogenic role in the ovary where it induces granulosa cell apoptosis through activation of Smad2, down-regulation of the key survival molecules Xiap and phospho-Akt, as well as the activation of mitochondrial death pathway.

Growth differentiation factor 9 is antiapoptotic during follicular development from preantral to early antral stage.

Ovarian follicular atresia represents a selection process that ensures the release of only healthy and viable oocytes during ovulation. The transition from preantral to early antral stage is the penultimate stage of development in terms of gonadotropin dependence and follicle destiny (survival/growth vs. atresia). We have examined whether and how oocyte-derived growth differentiation factor 9 (GDF-9) and FSH regulate follicular development and atresia during the preantral to early antral transition, by a novel combination of in vitro gene manipulation (i.e. intraoocyte injection of GDF-9 antisense oligos) and preantral follicle culture. Injection of GDF-9 antisense suppressed basal and FSH-induced preantral follicle growth in vitro, whereas addition of GDF-9 enhanced basal and FSH-induced follicular development. GDF-9 antisense activated caspase-3 and induced apoptosis in cultured preantral follicles, a response attenuated by exogenous GDF-9. GDF-9 increased phospho-Akt content in granulosa cells of early antral follicles. Although granulosa cell apoptosis induced by ceramide was attenuated by the presence of GDF-9, this protective effect of GDF-9 was prevented by the phosphatidylinositol 3-kinase inhibitor LY294002 and a dominant negative form of Akt. Injection of GDF-9 antisense decreased FSH receptor mRNA levels in cultured follicles, a response preventable by the presence of exogenous GDF-9. The data suggest that GDF-9 is antiapoptotic in preantral follicles and protects granulosa cells from undergoing apoptosis via activation of the phosphatidylinositol 3-kinase/Akt pathway. An adequate level of GDF-9 is required for follicular FSH receptor mRNA expression. GDF-9 promotes follicular survival and growth during the preantral to early antral transition by suppressing granulosa cell apoptosis and follicular atresia.

Morphological patterns of angiogenesis in ovarian follicle capillary networks. A scanning electron microscopy study of corrosion cast.

To describe the morphological characteristics of the ovarian follicle (F) capillary neoformation and regression, the angiogenic figures were studied by means of scanning electron microscopy of corrosion casts in developing and mature F of rabbit, pig, and cow. Developing F showed gradual neoformation of thecal capillaries characterized by budding and then sprouting, likely from preexisting interstitial vessels. Postcapillary venules frequently showed vasoconstriction rings (sphincters). Vasodilation followed capillary elongation. Mature F, in addition to vessel elongation and dilation, also presented infolding of dilated capillary walls, followed by capillary duplication and sinusoidalization. Periovulatory F mainly showed functional changes, such as capillary dilation, signs of iperpermeabilization, and ischemia, the latter being limited to the apical follicular area. Vessel regression was characterized by thinning of capillaries and presence of avascular areas within the atretic F wall at any stage. This study showed two main types of angiogenic patterns. (a) longitudinal elongation (in series, sprouting angiogenesis) characterizing the initial phase of F development and (b) parallel duplication (in parallel, infolding or intussusceptive angiogenesis), ending in capillary lateral replication or splitting, secondary to functional microvascular changes. Indirect evidence of the establishment of postcapillary resistances contributing to capillary remodeling, was also shown. It is concluded that the sequence of capillary neoformation in mammalian ovarian F occurs in six steps: (1) budding, (2) sprouting (and elongation), (3) dilation, (4) infolding (intussusception), (5) duplication (splitting and elongation), and (6) sinusoidalization. Capillary regression hits F at any stage and characterizes F atresia.

Regulation of cell death and cell survival gene expression during ovarian follicular development and atresia.

Mammalian ovarian follicular development and atresia is closely regulated by the cross talk of cell death and cell survival signals, which include endocrine hormones (gonadotropins) and intra-ovarian regulators (gonadal steroids, cytokines and growth factors). The fate of the follicle is dependent on a delicate balance in the expression and actions of factors promoting follicular cell proliferation, growth and differentiation and of those inducing programmed cell death (apoptosis). As an important endocrine hormone, FSH binds to its granulosa cell receptors and promotes ovarian follicle survival and growth not only by stimulating proliferation and estradiol secretion of these cells, but also inhibiting the apoptosis by up-regulating the expression of intracellular anti-apoptotic proteins, such as XIAP and FLIP. In addition, intra-ovarian regulators, such as TGF-alpha and TNF-alpha, also play an important role in the control of follicular development and atresia. In response to FSH, Estradiol-17 beta synthesized from the granulosa cells stimulates thecal expression of TGF-alpha, which in turn increases granulosa cell XIAP expression and proliferation. The death receptor and ligand, Fas and Fas ligand, are expressed in granulosa cells following gonadotropin withdrawal, culminating in caspase-mediated apoptosis and follicular atresia. In contrast, TNF-alpha has both survival and pro-apoptotic function in the follicle, depending on the receptor subtype activated, but has been shown to promote granulosa cell survival by increasing XIAP and FLIP expression via the IkappaB-NFkappaB pathway. The pro-apoptotic action of TNF-alpha is mediated through the activation of caspases, via its receptor- (i.e. Caspases-8 and -3) and mitochrondria- (i.e. Caspase-9 and -3) death pathways. In the present manuscript, we have reviewed the actions and interactions of gonadotropins and intra-ovarian regulators in the control of granulosa cell fate and ultimately follicular destiny. We have highlighted the role and regulation of granulosa cell XIAP and FLIP expression, as well as their interactions with the death signaling pathways in the maintenance of granulosa cell survival during follicular development. We have provided strong evidence for these intracellular survival factors as key determinants for ovarian follicular destiny (growth versus atresia), the expression of which is regulated by a highly integrated endocrine, paracrine and autocrine mechanism. Further studies in these aspects will lead to a better understanding of the molecular and cellular regulation of follicular development and atresia, and provide invaluable insight into novel strategies in assisted reproduction in human infertility as well as in increasing reproductive efficiency in livestock industries.

Mural granulosa cell gene expression associated with oocyte developmental competence.

BACKGROUND: Ovarian follicle development is a complex process. Paracrine interactions between somatic and germ cells are critical for normal follicular development and oocyte maturation. Studies have suggested that the health and function of the granulosa and cumulus cells may be reflective of the health status of the enclosed oocyte. The objective of the present study is to assess, using an in vivo immature rat model, gene expression profile in granulosa cells, which may be linked to the developmental competence of the oocyte. We hypothesized that expression of specific genes in granulosa cells may be correlated with the developmental competence of the oocyte. METHODS: Immature rats were injected with eCG and 24 h thereafter with anti-eCG antibody to induce follicular atresia or with pre-immune serum to stimulate follicle development. A high percentage (30-50%, normal developmental competence, NDC) of oocytes from eCG/pre-immune serum group developed to term after embryo transfer compared to those from eCG/anti-eCG (0%, poor developmental competence, PDC). Gene expression profiles of mural granulosa cells from the above oocyte-collected follicles were assessed by Affymetrix rat whole genome array. RESULTS: The result showed that twelve genes were up-regulated, while one gene was down-regulated more than 1.5 folds in the NDC group compared with those in the PDC group. Gene ontology classification showed that the up-regulated genes included lysyl oxidase (Lox) and nerve growth factor receptor associated protein 1 (Ngfrap1), which are important in the regulation of protein-lysine 6-oxidase activity, and in apoptosis induction, respectively. The down-regulated genes included glycoprotein-4-beta galactosyltransferase 2 (Ggbt2), which is involved in the regulation of extracellular matrix organization and biogenesis. CONCLUSIONS: The data in the present study demonstrate a close association between specific gene expression in mural granulosa cells and the developmental competence of oocytes. This finding suggests that the most differentially expressed gene, lysyl oxidase, may be a candidate biomarker of oocyte health and useful for the selection of good quality oocytes for assisted reproduction.

Growth differentiation factor 9 promotes rat preantral follicle growth by up-regulating follicular androgen biosynthesis.

The transition from preantral to early antral stage is the penultimate stage of ovarian follicular development in terms of gonadotropin dependence and follicle destiny. Although oocyte-somatic cell communication is important in early follicular development, our knowledge of the precise role of the oocyte-derived growth differentiation factor (GDF)-9 during preantral follicle growth is incomplete. We examined whether and by what means oocyte-derived GDF-9 controls follicular development and steroidogenesis during the preantral to early antral transition, by a combination of in vitro gene manipulation (i.e. intraoocyte injection of GDF-9 antisense oligos) and preantral follicle culture. Intraoocyte injection of GDF-9 antisense suppressed rat preantral follicle growth in vitro, whereas GDF-9 enhanced follicular development. GDF-9 augmented testosterone production in preantral follicles. GDF-9 antisense suppressed androgen production and CYP17A1 mRNA expression in cultured follicles, a response attenuated by exogenous GDF-9. The nonaromatizable androgen 5alpha-dihydrotestosterone rescued the follicular growth arrest caused by GDF-9 down-regulation. The specific androgen receptor antagonist flutamide suppressed GDF-9-induced preantral follicle growth in vitro. The data suggest that GDF-9 plays an important role in promoting preantral follicle growth by up-regulating follicular androgen biosynthesis. GDF-9 is essential for CYP17A1 expression during follicular development from the preantral to the early antral stage.

Kit ligand 2 promotes murine oocyte growth in vitro.

Oocyte-granulosa cell communication, mediated by paracrine factors, is essential for oocyte development. Kit ligand (KITL) is expressed in granulosa cells as soluble (KITL1) or membrane-associated (KITL2) proteins. However, the relative biopotency of each isoform during oocyte development is unknown. Our initial results showed that Kitl2 was down-regulated in cultured granulosa cells. To determine the effect of the two isoforms of KITL on oocyte growth, Kitl-deficient fibroblasts were transfected with constructs expressing either KITL1 or KITL2, and growing oocytes were isolated from 12-day-old mice and cultured on the transfected fibroblasts for 2 days. At the end of culture, oocyte diameters were measured, the incidence of spontaneous germinal vesicle breakdown (GVBD) was noted, and oocytes were analyzed for KIT receptor expression. Oocyte growth occurred only in the presence of the KITL2-producing fibroblasts, and suppression of KITL2 expression impaired oocyte growth. Up-regulation of KIT expression occurred in the presence of KITL2 but not KITL1. The presence of KITL2 inhibited spontaneous GVBD. Meiosis inhibitors did not attenuate the GVBD that occurred in the absence of KITL2, suggesting that this process reflects oocyte degeneration rather than meiotic progression. These results indicate that KITL2 is the principal KITL isoform required for oocyte growth and survival in vitro.

Thyroxine treatment stimulated ovarian follicular angiogenesis in immature hypothyroid rats.

The development of mature ovarian follicles is greatly dependent on healthy thecal angiogenesis. Recent experimental evidence showed that thyroxine (T4) treatment promoted ovarian follicle development in immature hypothyroid (rdw) rats. However, an involvement of thyroid hormone in ovarian follicular angiogenesis has not yet been demonstrated. By morphological and molecular approaches, the present studies demonstrated that antral follicles in untreated, T4- or equine chorionic gonadotropin (eCG)-treated rdw rats were mainly small and/or atretic, and presented a poorly developed thecal microvasculature with ultrastructural evidence of diffuse quiescent or degenerative thin capillaries. However, T4 together with eCG increased the number of large antral and mature follicles with numerous activated capillaries and ultra-structural evidence of rich and diffuse angiogenesis in the theca layer. While T4 alone significantly increased mRNA expression of vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNFalpha), it decreased that of fetal liver kinase compared with those in the untreated group. Combined treatment of T4 and eCG markedly increased mRNA abundance of not only VEGF and TNFalpha, but also basic fibroblast growth factor. These data suggest that T4 may promote ovarian follicular angiogenesis in rdw rats by up-regulating mRNA expression of major angiogenic factors.

Acceleration of follicular development by administration of vascular endothelial growth factor in cycling female rats.

To address the role of follicular angiogenesis in the determination of ovulatory follicles and the effects of different vascular endothelial growth factor (VEGF) isoforms on follicular angiogenesis and development, mature female rats were treated with an angiogenic inhibitor (TNP-470), and also with VEGF 120 or 164 at different dosages (0.4, 0.8, 4.0 or 8.0 microg/kg body weight) for 3 days during the estrous cycle. Ovarian follicular angiogenesis, the population of large follicles and ovulation were examined. VEGF 120 (0.8 microg/kg) and 164 (8.0 microg/kg) treatments stimulated follicular angiogenesis in the theca interna layer, while TNP-470 treatment showed severe depression of follicular angiogenesis, and completely inhibited ovulation. After administration of VEGF 120 or 164, the number of healthy preovulatory follicles and ovulated oocytes increased significantly, concomitantly with a decrease in the number of atretic preovulatory follicles. The oocytes ovulated had normal fertilizability and developed to term with the same litter size as in the control rats. Our findings suggest that follicular angiogenesis may be a determinant of follicular development during the periovulatory phase, and that VEGF isoforms may play different important roles in regulating follicular angiogenesis.

Optimal conditions for successful in vitro fertilization and subsequent embryonic development in Sprague-Dawley rats.

The present study was conducted to determine the optimal conditions for successful in vitro fertilization (IVF) in Sprague-Dawley (SD) rats. The IVF of oocytes from SD and Wistar rats was compared in different fertilization media (mR1ECM, IVF-20, and modified Krebs-Ringer bicarbonate solution [mKRB]), and IVF conditions were then optimized for oocytes of the SD strain. Results showed that in mR1ECM medium, fertilization rates were markedly lower in SD rats (15%) than in the Wistar strain (73%), although this response was significantly improved by increasing the NaCl concentration. In addition, fertilization rates in SD rats were higher in modified IVF-20 (73%) than in IVF-20 (18%) and mKRB (53%). In contrast, fertilization rates in Wistar rats were higher in IVF-20 and modified IVF-20 than in mKRB (78%, 74%, and 36%, respectively). Further investigation concerning the effects of the NaCl supplementation (10- 40 mM) in IVF-20 on the fertilization of oocytes in the SD strain indicated that significantly higher percentages of oocytes were fertilized in IVF-20 supplemented with 30 mM NaCl (66%) and developed to the blastocyst stage (47%) in vitro. After transfer, embryos derived from this IVF system developed to term at a percentage comparable to that of in vivo-fertilized controls. In conclusion, differences exist in optimal IVF conditions between rat strains, and a modified culture medium has been successfully developed for assessment of the developmental competence of oocytes in SD rats.

Distribution and gene expression of cytoskeletal proteins in two-cell rat embryos and developmental arrest.

Previous observations in rat two-cell embryos suggested that distribution of microfilaments and microtubules are involved in developmental arrest. Therefore, we examined the distribution of cytoskeletal proteins, actin binding proteins, and microtubule-associated proteins in rat two-cell embryos. We also examined gene expression of beta-actin, alpha-tubulin, and cytoskeletal proteins that showed changes in their distributions. Distribution of cytoskeletal proteins was examined by immunocytochemistry. Although distributions of alpha-actinin, MAP1A, MAP1B/MAP5, and MAP2 were disturbed in arrested embryos, these abnormal distributions occurred following the initiations of developmental arrest and marked damage of microfilaments and microtubules. Gene expression of cytoskeletal proteins was examined by RT-PCR. Beta-actin and alpha-actinin mRNA was detected in normal late two-cell stage but not in arrested embryos. The difference occurred after zygotic gene activation. Expression of alpha-tubulin was detected in neither normal late two-cell stage nor arrested embryos. No MAP1A, MAP1B/MAP5, or MAP2 expression was detected in embryos during the two-cell stage. In conclusion, both distributions of microfilaments and microtubules are closely involved in rat developmental arrest, but other distributions of cytoskeletal proteins, actin binding proteins, and microtubule-associated proteins do not appear to have major roles in two-cell arrest. Furthermore, mRNA expression patterns are different between microfilaments and microtubules. Both distribution and mRNA transcription of microfilaments are involved in rat developmental arrest, whereas only distribution of maternal microtubules is disturbed in arrested embryos.

Changes of messenger RNA expression of angiogenic factors and related receptors during follicular development in gilts.

The interaction between angiogenic factors and related receptors is closely associated with follicular angiogenesis. The present study was performed to determine the relationships between the capillary network and mRNA expression of several angiogenic factors and related receptors during porcine follicular development. Ovaries in gilts were collected 72 h after eCG (1250 IU) treatment for histological observation. Granulosa cells and thecal tissues in small (diameter, <4 mm), medium (diameter, 4-5 mm), or large (diameter, >5 mm) individual follicles were collected for detection of mRNA expression of vascular endothelial growth factor (VEGF) 120, VEGF 164, basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) in granulosa cells and fms-like tyrosine kinase (Flt-1), fetal liver kinase (Flk-1) or the murine homologue of kinase domain region (KDR), bFGF receptor (bFGF-R), and EGF receptor (EGF-R) in thecal tissue by semiquantitative reverse transcription-polymerase chain reaction. The eCG treatment resulted in the emergence of healthy preovulatory follicles (diameter, >6.0 mm) that possessed more capillaries in the thecal cell layer and a significant increase in the percentage of atretic follicles of 1.0-2.9 mm in diameter. The number of capillaries in the thecal cell layer increased significantly in healthy follicles larger than 3 mm in diameter in the eCG group compared with those in controls. The expression of VEGF 120, VEGF 164, and bFGF mRNAs increased in granulosa cells of medium and large follicles from ovaries of prepubertal gilts after eCG treatment. The Flt-1, Flk-1/KDR, and bFGF-R mRNA expression increased in theca cells of medium and large follicles after eCG treatment. The expression of EGF mRNA increased in granulosa cells of small, medium, and large follicles from ovaries after eCG treatment, but the mRNA expression of EGF-R in thecal tissue did not change. These data indicate that preovulatory follicles possessed a larger capillary network and expressed more mRNAs of angiogenic factors in granulosa cells and related receptors in thecal tissue. We concluded that VEGF 120, VEGF 164, bFGF, and EGF may be greatly involved in the angiogenic process of follicular development in prepubertal gilts with eCG treatment.

Increased ovarian follicular angiogenesis and dynamic changes of follicular vascular plexuses induced by equine chorionic gonadotropin in the gilt.

Follicular angiogenesis and capillary degeneration are crucial ovarian processes in folliculogenesis. The present study was conducted to assess the changes in population of follicular vascular plexuses with different capillary status in prepubertal gilts 72 h after equine chorionic gonadotropin (eCG) (1,250 IU) treatment, using combined vascular corrosion casting and scanning electron microscopy. Follicular fluid concentrations of estradiol, progesterone and vascular endothelial growth factor (VEGF) were determined to confirm the follicular status. Based on the proliferative or degenerative characteristics of their capillaries, follicles were classified into three categories: active angiogenesis, low angiogenesis and degeneration. Irrespective of exogenous gonadotropin treatment in vivo, small follicular vascular plexuses (<4 mm in diameter) exhibited all three conditions in casted ovaries, while medium (4-5 mm) and large (>5 mm) plexuses showed only active angiogenesis or degeneration. eCG treatment significantly increased the population of large, but decreased that of small follicular plexuses. Most large follicular vascular plexuses showed active angiogenesis with higher follicular fluid estradiol: progesterone ratios and VEGF concentration. eCG also increased the percentage of medium follicular plexuses with active angiogenesis. The populations of small follicular plexuses with active angiogenesis were higher in controls, but decreased after eCG treatment. However, treatment of gilts with the gonadotropin increased the percentage of small plexuses (<1.0 mm) with low angiogenesis and those (1-3.9 mm) with extensive capillary degeneration. These findings are consistent with the hypothesis that angiogenesis is involved in selection and growth of small follicles in gilts under the regulation of gonadotropin.

Parthenogenetic activation and subsequent development of rat oocytes in vitro.

Studies were undertaken to determine whether electrical stimulation, or ethanol treatment alone or in combination with 6-dimethylaminopurine (6-DMAP) influenced the rate of parthenogenetic activation of rat oocytes. The percentages of activated oocytes with pronuclei (89-91%) and those developed to the two-cell stage (68-72%) were significantly higher after electrical stimulation with direct current (DC) at 100 V/mm, 99 microsec once or twice, than when other DC voltages (75, 150, and 200) were applied or when ethanol or 6-DMAP treatment was given alone. However, none of the activated oocytes developed beyond the four-cell stage. The percentages of activated oocytes with pronuclei (100%) that developed to the two-cell (100%), eight-cell (89%) and blastocyst stages (50%) were significantly higher when electrical stimulation was followed by treatment with 2 mM 6-DMAP for 4 hr than when other combined procedures were applied. In conclusion, the results of the present study clearly showed that combined treatment of electrical stimulation or ethanol with 6-DMAP induces parthenogenetic activation and subsequent development of rat oocytes in vitro.

Determination of optimal conditions for parthenogenetic activation and subsequent development of rat oocytes in vitro.

The present study was undertaken to determine optimal conditions for parthenogenetic activation and subsequent development of rat oocytes. Oocytes from immature Wistar-Imamichi (WI) and Sprague Dawley (SD) rats were activated by electrical stimulation in combination with 6-dimethylaminopurine (6-DMAP) to assess whether different rat strains display different responses to activation treatment. Since the cleavage rates of activated oocytes were significantly higher in WI than SD strain rats, WI rats were used for the subsequent experiments to determine the effects of post-hCG time, culture duration, different activation protocols (electrical stimulation with 6-DMAP or ionomycin with 6-DMAP) and osmolarity of the activation medium on the activation and subsequent development of WI rat oocytes. For oocytes activated by electrical stimulation combined with 6-DMAP, the percentages of oocytes that were activated and that developed to blastocysts were higher when oocytes were collected at 18-20 h than at any other time points after hCG injection (16, 22-24 h). Culturing for 2-6 h before activation treatment markedly decreased the percentage of activated oocytes that developed to beyond the four-cell stage. There were no differences in the percentages of oocytes with pronuclear formation and subsequent development to the two-cell and blastocyst stages between oocytes that were activated by electrical stimulation or ionomycin, both followed by 6-DMAP treatment. Activation of oocytes by ionomycin and 6-DMAP, both in low osmolarity media (246 mOsM), markedly increased the cleavage rates and percentages of high quality blastocysts (71%). The optimal conditions determined in the present study with simplified activation protocols and high efficiency of activation and subsequent development of WI rat oocytes will be helpful for further research involving nuclear transfer in the rat.

Induction of follicular development by direct single injection of vascular endothelial growth factor gene fragments into the ovary of miniature gilts.

Perifollicular angiogenesis is closely associated with ovarian follicular development. To investigate whether additional induction of perifollicular angiogenesis would support subsequent follicular development, we directly injected vascular endothelial growth factor (VEGF) gene fragments into the ovaries of miniature gilts, followed by gonadotroph treatment to stimulate follicle growth. In addition, to confirm extraexpression of the VEGF gene after injection, we assessed the expression of two isoforms of VEGF (VEGF 120 and VEGF 164) in granulosa cells and expression of fms-like tyrosine kinase (Flt-1), expression of fetal liver kinase (Flk-1), and density of capillary networks in theca cells. Direct injection of VEGF gene fragments into the ovaries was performed 7 days before eCG treatment. The ovaries in miniature gilts were removed 72 h after eCG treatment for histological examination. Granulosa cells and thecal tissues in the antral follicles (diameter, >4 mm) were collected to detect the mRNA expression of VEGF isoforms in the granulosa cells and of Flt-1 and Flk-1 in the thecal tissues by semiquantitative reverse transcription-polymerase chain reaction. The VEGF levels were measured in the follicular fluid by enzyme immunoassay. Injection of VEGF gene fragments increased the level of mRNA expression of VEGF 120 and 164 isoforms in the granulosa cells and VEGF protein contents in the follicular fluid. The number of preovulatory follicles and the capillary density in the theca interna increased significantly in the ovaries injected with VEGF gene fragments compared with those treated with eCG alone. The Flt-1, but not the Flk-1, mRNA expression show a tendency toward increasing in the thecal tissues of antral follicles in the ovaries injected with VEGF gene fragments. These results demonstrate that Flt-1 may be predominantly involved in the regulation of the capillary network in the theca interna during follicular development. Our data suggest that the regulation of perifollicular angiogenesis during follicular development is a very important factor in the development of ovulatory follicles. Our findings may offer an innovative technique for enhanced induction of follicular development in the ovary through gene and hormonal treatment, which may lead to prevention of infertility caused by ovarian dysfunction.