Developmental changes in expression of myeloid cell leukemia-1 in human germ cells during oogenesis and early folliculogenesis.

The regulation of germ cell number in the developing ovary is central to female reproduction. Members of the Bcl-2 family of proapoptotic and antiapoptotic proteins have been implicated in this process in rodents. We investigated the expression of Mcl-1, Bcl-2, Bax, and BAD at 13-21 gestational wk in the human fetal ovary and of Mcl-1 in the adult ovary. mRNA expression of Mcl-1 and its short form Mcl-1s, Bcl-2, Bax, and BAD was demonstrated in fetal ovary by RT-PCR. Hybridization array analysis suggested a selective increase in Mcl-1 expression between 14 and 18 wk gestation, which was confirmed by quantitative PCR. There was a corresponding change in the expression of Mcl-1 protein, detected by immunohistochemistry, from germ cells at the periphery of the ovary at 14-16 wk to the largest germ cells, including oocytes within newly formed primordial follicles, at 21 wk. Mcl-1 was also expressed by oocytes of primordial and preantral follicles in the adult. Bax and BAD immunostaining was detected in both somatic and germ cells in the fetal ovary, whereas Bcl-2 was restricted to somatic cells: no changes in expression were observed. Apoptotic cells, detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, were observed in all fetal ovaries but were infrequent. These results confirm that Bcl-2 family members are differentially expressed in several cell types within the developing human ovary. Increased mRNA expression and the changing distribution of Mcl-1 in germ cells as they develop into primordial follicles as well as persistence in the growing oocyte in the adult may indicate an important role for this survival/antiapoptotic factor throughout germ cell development and maturation.

Germ cell specific expression of c-kit in the human fetal gonad.

The proto-oncogene receptor, c-kit, and its ligand have been demonstrated to be essential to the processes of germ cell migration, proliferation and survival in the rodent. The aim of the present study was to investigate the expression of c-kit mRNA and protein in human fetal ovary and testis across the gestational period 13-21 weeks. In the ovary, this crucial period of development spans the transition from oogonial replication by mitosis to primordial follicle formation. In the testis, germ cells (gonocytes) are mitotically active. Expression of c-kit mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) in both ovary and testis at all gestational ages examined. Testicular germ cell specific expression of c-kit mRNA was confirmed by RT-PCR using specific cell types recovered by laser capture microscopy. The expression of c-kit protein by both male and female germ cells was demonstrated by immunohistochemistry at all gestational ages examined, and was confirmed by immunoblotting. In both, c-kit was localized to the cell membrane except in oocytes within primordial follicles where it was localized to the cytoplasm. These data demonstrate that the expression of c-kit mRNA and protein is germ cell specific in human fetal gonads and are consistent with an important role for the c-kit/kit ligand signalling system in germ cell proliferation and survival in the developing human gonad.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in human fetal testis and ovary.

Matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) are major regulators of tissue remodelling of the extracellular matrix (ECM) and may also be involved in the control of growth factor availability. We have investigated their production and localization in the developing human gonad during mid-gestation using zymographic techniques and immunohistochemistry. The secretion of MMP-2, MMP-9 and all four TIMP was demonstrated from both testis and ovary, with the predominant gelatinase produced by both being MMP-2. In the testis, MMP-1, MMP-2, MMP-9 and all TIMP family members were localized to the interstitium and to varying degrees within the tubules. MMP-9 and TIMP-4 were abundant in both Sertoli cells and gonocytes and MMP-1 and TIMP-1 were localized in particular to Sertoli cells. In the ovary, all TIMP and MMP-1, MMP-2 and MMP-9 were localized to the oogonium/oocyte cytoplasm with varying intensities and MMP-1, TIMP-2 and TIMP-3 were also detected in the ovarian stroma. This study demonstrates that MMP-1, MMP-2, MMP-9 and all TIMP family members are secreted by the developing ovary and testis and are localized to specific cell and tissue sites. MMP and TIMP are likely to play a role in ECM remodelling during gonadal development and also in the cell and matrix interactions that control a range of cellular functions.

Can protocol-specified doses of chemotherapy and radiotherapy be used as a measure of treatment actually received? A CCG/NIH study on long-term survivors of acute lymphocytic leukemia.

In a cohort of 593 long-term survivors of acute lymphocytic leukemia identified through the Children's Cancer Group (CCG), treatment abstracts were obtained and compared to protocol information on radiation therapy and intravenous chemotherapy. This was done in order to evaluate the actual compliance to protocol-specified treatment, and assess if protocol-specified doses can be used in studies of late effects of treatment. The compliance to protocol-specified type of treatment ranged between 95.3% (intrathecal methotrexate) and 98.6% (adriamycin) for chemotherapy, and between 94.1% (cranial radiation) and 97.0% (extended field radiation) for radiation. Concordance with the protocol-specified chemotherapy dose (+/- 25%) was 57.5% for adriamycin, 91.3% for daunomycin, and 48.5% for cyclophosphamide. When concordance was low, most patients received doses that were lower than expected. Concordance with chemotherapy was significantly lower for high-dose regimens than for low-dose regimens. Concordance with protocol-specified radiation dose (+/- 10%) was 87.4% for cranial radiation, 87.8% for spinal radiation, and 85.7% for extended field radiation. Concordance with treatment did not differ by gender, relapse status, or age at diagnosis. In this cohort of leukemia survivors, the validity of type of treatment was greater than the validity of dosage. Great care should be used when drawing conclusions about effects of treatment dosage. Although costly and time consuming, it appears that chart reviews are the most appropriate way to collect information about dose-related effects of therapy.

An exploratory analysis of risk factors for childhood malignant germ-cell tumors: report from the Childrens Cancer Group (Canada, United States).

A study of 105 patients with childhood malignant germ-cell tumors (MGCT) and 639 community controls was conducted utilizing a large epidemiologic database collected by the Childrens Cancer Group from 25 member institutions in the United States and Canada. This study was designed to explore the risk factors of this malignancy whose etiology remains poorly understood. A structured, self-administered questionnaire was used to collect exposure information, and data were analyzed using an unconditional logistic regression model with adjustment for relevant confounders. Consistent with the findings from studies of adult MGCT, gestational age was associated inversely with risk of MGCT, with a 70 to 75 percent reduction in risk for children born at term compared with those born pre-term. Parental, particularly maternal, self-reported exposure to chemicals or solvents (odds ratio [OR] = 4.6, 95 percent confidence interval [CI] = 1.9-11.3) and OR = 2.2, CI = 1.1-4.7 for maternal and paternal exposure, respectively) and plastic or resin fumes (OR = 12.0, CI = 1.9-75.0 [maternal] and OR = 2.5, CI = 1.0-6.5 [paternal]) were associated with elevated risk of MGCT. New findings, not reported previously, include a positive relationship of MGCT risk with birthweight and prolonged breastfeeding, an inverse association between MGCT risk and number of cigarettes smoked by the mother during pregnancy, and a 3.1-fold increased risk (CI = 1.5-6.6) associated with maternal urinary infections during index pregnancy. Although these findings need confirmation from future studies, they suggest a potential influence of in utero exposure to maternal endogenous hormones, parental environmental exposures, and maternal diseases during pregnancy in the development of childhood MGCT.