Methotrexate (MTX) an antifolate drug and leucovorin its antidote, are used in the treatment of both neoplastic and non-neoplastic diseases in young women. We hypothesize that MTX treatment might comprise a deleterious effect on fast proliferating reproductive cells, an unavoidable and unwanted side effect. MTX given dose dependently to rats for 20 days prevented vaginal cyclicity and caused a reduction in serum progesterone and estradiol. External morphology of reproductive tract displayed thinning of organs and reduction in their weights. To reveal mechanism of MTX action, we examined the histology of ovary, oviduct, uterus, cervix and vagina. Results suggested that in a dose-dependent fashion MTX restrained preantral and antral follicular growth in ovary. Epithelium and stroma of oviduct, uterus, cervix and vagina were disrupted and lost their normal structures. Such alterations in ovarian function raised serum follicle stimulating hormone, luteinizing hormonal profiles. Expression of steroidogenic acute regulatory protein and P450 cholesterol side chain cleavage gene, which are both essential for steroidogenesis, markedly decreased in ovary upon MTX treatment. Total RNA, DNA and protein concentrations, glucose 6 phosphate dehydrogenase, lactate dehydrogenase and alkaline phosphatase enzyme activities in ovary were distinctly altered. Leucovorin supplementation and withdrawal of the treatment, improved MTX caused effects partially. These results for the first time indicate that the malfunction of female reproductive organs by MTX treatment in young women is not only correlated to the disrupted circulating levels of hormones and histoarchitecture of tissues but also discrepancies in steroidogenic genes and hormone regulated enzyme activities in ovary.

Neurosteroids are steroids synthesised by brain cells. The molecular mechanism of neurosteroidogenesis from cholesterol has not yet been revealed. We studied the potential role of the steroidogenic acute regulatory (StAR) protein in neurosterodogenesis by using rat brain astrocytes. The novelty of the study is that regulation of StAR is described in primary cultures from embryonic mesencephalon and cerebellum regions of the brain. Dibutyryl cyclic AMP (dbcAMP) treatment increased StAR protein expression in astrocyte cultures. This was observed in immunoblots of mitochondrial fractions and by immunocytochemistry. Dual-labelling showed that the cyclic AMP-induced increase in StAR immunofluorescence was localised to mitochondria. In addition, mitochondrial cytochrome P450-side chain cleavage enzyme was demonstrated with a specific antibody, indicating the potential for pregnenolone production in these cells. Radioimmunoassay on ether-extracted conditioned media of control and dbcAMP treated cells demonstrated pregnenolone production by mesencephalic and cerebellar astrocyte cultures. Furthermore, 24-h pregnenolone levels, in the presence of inhibitors of further pregnenolone metabolism, were significantly increased by dbcAMP exposure. A murine StAR promoter-luciferase fusion plasmid was activated by dbcAMP in transiently transfected mesencephalic and cerebellar astrocytes. These novel results indicate that cyclic AMP signalling can regulate StAR expression and pregnenolone production in brain astrocytes, and provide additional insight into the role of StAR in neurosteroidogenesis.

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Background: Complete genome annotation will likely be achieved through a combination of computer-based analysis of available genome sequences combined with direct experimental characterization of expressed regions of individual genomes. We have utilized a comparative genomics approach involving the sequencing of randomly selected hamster testis cDNAs to begin to identify genes not previously annotated on the human, mouse, rat and Fugu (pufferfish) genomes.

Background.Exposure to methotrexate (MTX) causes infertility in young women treated for non-neoplastic diseases. Leucovorin an antidote to MTX is used to prevent its side effects. Reproductive tract cells are fast proliferating like cancer cells. Hence, MTX may cause deleterious effect on these cells.Aim. Explore MTX effect on biochemical markers and the protective role of leucovorin in the accessory reproductive organs viz; oviduct, uterus, cervix and vagina.Animals and Methods. Rats with regular oestrous cycle were randomly divided into five groups (n=6) as follows: Control, MTX LD (low dose), MTX HD (high dose), MTXHD+LCN (leucovorin, LCN), and MTXHD+WD (withdrawal): 20 days withdrawal. Animals were treated once per day intramuscularly (im) for 20 days. Rats were sacrificed on day 21. MTXHD treatment was withdrawn for additional 20 days and animals sacrificed on day 41. Oviducts, uterus, cervix and vagina were used for biochemical markers analysis.Results. MTX treatment decreased RNA, DNA and protein concentrations. Such similar effects were also observed in G6PD, LDH and ALP activities. Leucovorin and withdrawal of treatment partially recovered MTX effects. Uniquely, this study signifies MTX action on nucleic acids and protein levels and also G6PD, LDH and ALP activities in oviducts, uterus, cervix and vagina.