Fumikazu Kotsuji

Oocyte-granulosa-theca cell interactions during preantral follicular development

The preantral-early antral follicle transition is the penultimate stage of follicular development in terms of gonadotropin dependence and follicle destiny (growth versus atresia). Follicular growth during this period is tightly regulated by oocyte-granulosa-theca cell interactions. Formation of the theca cell layer is a key event that occurs during this transitional stage. Granulosal factor(s) stimulates the recruitment of theca cells from cortical stromal cells, while oocyte-derived growth differentiation factor-9 (GDF-9) is involved in the differentiation of theca cells during this early stage of follicular development. The preantral to early antral transition is most susceptible to follicular atresia. GDF-9 promotes follicular survival and growth during transition from preantral stage to early antral stage by suppressing granulosa cell apoptosis and follicular atresia. GDF-9 also enhances preantral follicle growth by up-regulating theca cell androgen production. Thecal factor(s) promotes granulosa cell proliferation and suppress granulosa cell apoptosis. Understanding the intraovarian mechanisms in the regulation of follicular growth and atresia during this stage may be of clinical significance in the selection of the best quality germ cells for assisted reproduction. In addition, since certain ovarian dysfunctions, such as polycystic ovarian syndrome and gonadotropin poor-responsiveness, are consequences of dysregulated follicle growth at this transitional stage, understanding the molecular and cellular mechanisms in the control of follicular development during the preantral-early antral transition may provide important insight into the pathophysiology and rational treatment of these conditions.

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.

Granulosa Cells Promote Differentiation of Cortical Stromal Cells into Theca Cells in the Bovine Ovary

Abstract Formation of a theca cell (TC) layer is an important physiologic event that occurs during early follicular development. Nevertheless, little is known concerning the nature and regulation of the formation of the TC layer during follicular growth. Using an established coculture system in this study, we examined the hypothesis that stromal cells differentiate into TCs during early follicular development and that this process involves interaction with granulosa cells (GCs). Ovarian stromal cells from the bovine ovarian cortex (SC) and medulla (SM) were cultured with or without GCs from small antral follicles. The presence of GCs increased the number of lipid droplets and mitochondria, and it stimulated androstenedione production in SC and SM. However, luteinizing hormone/choriogonadotropin receptor (LHCGR) mRNA abundance and hCG-induced cAMP and androstenedione production were increased in SC but not in SM by the presence of GCs. The present results indicate that GCs are involved in the functional differentiation and the acquisition of LH responsiveness in stromal cells of the ovarian cortex. We suggest that GC-SC interaction is important in the formation of the TC layer during early follicular development, although the nature of this interaction remains to be determined.

Luteinizing hormone-induced Akt phosphorylation and androgen production are modulated by MAP Kinase in bovine theca cells

BackgroundTheca cells play an important role in controlling ovarian steroidogenesis by providing aromatizable androgens for granulosa cell estrogen biosynthesis. Although it is well established that the steroidogenic activity of theca cells is mainly regulated by LH, the intracellular signal transduction mechanisms that regulate thecal proliferation and/or steroidogenesis remain obscure. In this study, we examined whether and how LH controls the PI3K/Akt signaling pathway and androgen production in bovine theca cells. We also explored whether this LH-induced PI3K/Akt activation is modulated with other signaling pathways (i.e. PKA and MAPK).MethodsOvarian theca cells were isolated from bovine small antral follicles and were incubated with LH for various durations. Phospho-Akt and total-Akt content in the cultured theca cells were examined using Western blotting. Androstenedione levels in the spent media were determined using EIA. Semi-quantitative RT-PCR analyses were conducted to analyze the mRNA levels of CYP17A1 and StAR in the theca cells. To examine whether Akt activity is involved in theca cell androgen production, the PI3K inhibitors wortmannin and LY294002 were also added to the cells.ResultsAkt is constitutively expressed, but is gradually phosphorylated in cultured bovine theca cells through exposure to LH. LH significantly increased androstenedione production in bovine theca cells, whereas addition of the wortmannin and LY294002 significantly decreased LH-induced androstenedione production. LH significantly increased CYP17A1 mRNA level in theca cells, whereas addition of LY294002 significantly decreased LH-induced CYP17A1 expression. Neither LH nor PI3K inhibitors alter the mRNA levels of StAR in theca cells. Although H89 (a selective inhibitor of PKA) does not affect LH-mediated changes in Akt, U0126 (a potent MEK inhibitor) suppressed LH-induced Akt phosphorylation, CYP17A1 expression, and androgen production in theca cells.ConclusionThese results indicate that LH stimulates CYP17 mRNA expression and androgen production in theca cells via activation of the PI3K/Akt pathway. The LH-induced Akt phosphorylation and androgen production are modulated by the MAPK signaling in bovine theca cells.

Growth Differentiation Factor-9 Mediates Follicle-Stimulating Hormone-Thyroid Hormone Interaction in the Regulation of Rat Preantral Follicular Development

FSH regulates follicular growth in a stage-development fashion. Although preantral follicle stage is gonadotropin responsive, FSH is not required for preantral follicular growth. With the antrum, the follicles continue growing under the influence of FSH and become gonadotropin dependent. Although thyroid hormone is important for normal female reproductive function, its role and interaction with FSH in the regulation of preantral ovarian follicular growth is yet to be defined. In the present study, we have examined the action and interaction of FSH and T(3) in the regulation of the growth of preantral follicles, especially in their transition from preantral to early antral stage, using an established follicle culture system and evaluated the involvement of growth differentiation factor-9 (GDF-9) in this process in vitro. We have demonstrated that although T(3) alone had no effect on follicular development, it markedly enhanced FSH-induced preantral follicular growth. Although FSH alone significantly down-regulated FSH receptor (FSHR) mRNA abundance in the preantral follicles and T(3) alone was ineffective, expression of the message was significantly increased in the presence of both hormones. In addition, intra-oocyte injection of GDF-9 antisense oligonucleotides (GDF-9 morpholino) induced follicular cell apoptosis and suppressed follicular growth induced by FSH and T(3). These responses were attenuated by exogenous GDF-9. Our findings support the concept that thyroid hormone regulates ovarian follicular development through its direct action on the ovary and that promotes FSH-induced preantral follicular growth through up-regulation of FSHR, a mechanism dependent on the expression and action of oocyte-derived GDF-9.

Neonatal suppurative parotitis possibly associated with congenital cytomegalovirus infection and maternal methyldopa administration.

Correspondence: Hirokazu Tsukahara, Department of Pediatrics, Faculty of Medical Sciences, University of Fukui, Fukui 910-1193, Japan. Email: htsuka@fmsrsa.fukui-med.ac.jp Received 21 April 2004; accepted 28 February 2005. Suppurative parotitis is uncommon in neonates. 1,2 We have recently encountered a neonatal case of suppurative parotitis and speculate that its manifestation was associated with congenital cytomegalovirus (CMV) infection and maternal methyldopa administration. To our knowledge, no such cases have been documented in the previous literature.

An operative technique for conservative management of placenta accreta

BACKGROUND: Control of bleeding is the goal of management for placenta accreta, which usually necessitates hysterectomy. A Committee Opinion of The American College of Obstetricians and Gynecologists (ACOG) has addressed the difficulties of conservative treatments. CASES: Placentas of 2 primiparous women with placenta accreta were removed operatively from their uteri. One woman underwent a low transverse cesarean delivery, and the other had delivered vaginally. In each case, the anterior uterine wall was incised vertically between the lower segment and fundus before manual removal. After eversion of the uterus, the placenta was successfully detached from the uterine wall after intramyometrial administration of oxytocin. CONCLUSION: A vertical incision in the anterior uterine wall and subsequent eversion of the uterus may aid in avoiding hysterectomy with placenta accreta.

Massive Subchorionic Hematoma: Peculiar Prenatal Images and Review of the Literature

Massive subchorionic hematoma is a localized collection of blood or hematoma in the placenta, and can result in serious obstetrical complications. The condition can be diagnosed antenatally by ultrasound. However, no reports have previously described the same condition featuring an intraplacental fluid–fluid level on imaging studies. We report a case of massive subchorionic hematoma diagnosed prenatally, and propose an additional peculiar finding detectable on both the ultrasound and magnetic resonance images: the intraplacental fluid–fluid level. We also review previously reported cases that were detected by ultrasonography.

Assessment of fluorodeoxyglucose uptake by leiomyomas in relation to histopathologic subtype and the menstrual state.

Objective: The aim of this study was to assess fluorodeoxyglucose (FDG) uptake by leiomyomas in relation to histopathologic subtype and the menstrual state. Methods: We performed a retrospective study of 92 patients referred for uterine leiomyomas confirmed by histopathologic findings. On FDG-positron emission tomography, quantitative evaluation was performed using the mean values of the standardized uptake value (SUV) with reference to magnetic resonance imaging findings. Of the 92 patients, 56 had typical leiomyomas (41 premenopausal and 15 postmenopausal women), whereas 36 patients with subtype leiomyomas consisted of 21 premenopausal and 15 postmenopausal women. Results: Although no significant differences in SUV were observed between the typical and subtype leiomyomas, tumoral SUV was significantly higher in the premenopausal patients than in the postmenopausal patients. Uterine tumoral FDG uptake during the proliferative phase was also slightly lower than in the other menstrual phases. Conclusions: The FDG uptake by leiomyomas in premenopausal women was greater than that in postmenopausal women.

Micelles of Pulmonary Surfactant in Human Amniotic Fluid at Term

Studies using in vitro analysis have shown that the interaction between pulmonary surfactant and vernix caseosa could explain the appearance of amniotic fluid turbidity. That phenomenon is interpreted based on the “roll-up” hypothesis. We tested the roll-up hypothesis by examining the presence of micelles of pulmonary surfactant in human amniotic fluid at term. Amniotic fluid samples were collected from each of six healthy pregnant women at term and at 16 wk of gestation. These samples were stained negatively and analyzed using an electron microscope. Ultrastructures present in amniotic fluid were compared with the structure of micelles derived from suspended surfactant TA isolated from bovine lung. Surfactant TA formed spheroidal and rod-shaped micelles 10–70 nm in diameter above the critical micelle concentration. Identical micelle particles were described in human amniotic fluid at term. In addition, surfactant protein B was identified in the micelle fraction of amniotic fluid. However, no micelles were found in human amniotic fluid taken at 16 wk of gestation. Our results support the view that pulmonary surfactant could induce the detachment of vernix caseosa and increase the turbidity of the amniotic fluid.