In 1996, the US Environmental Protection Agency was given a mandate by Congress to develop a screening program that would evaluate whether variously identified compounds could affect human health by mimicking or interfering with normal endocrine regulatory functions. Toward this end, the Agency chartered the Endocrine Disruptor Screening and Testing Advisory Committee in October of that year that would serve to recommend a series of in vitro and in vivo protocols designed to provide a comprehensive assessment of a chemical's potential endocrine-disrupting activity. A number of these protocols have undergone subsequent modification by EPA, and this review focuses specifically on the revised in vivo screening procedure recommended under the title Research Protocol for Assessment of Pubertal Development and Thyroid Function in Juvenile Female Rats. Background literature has been provided that summarizes what is currently known about pubertal development in the female rat and the influence of various forms of pharmaceutical and toxicological insult on this process and on thyroid activity. Finally, a section is included that discusses technical issues that should be considered if the specified pubertal endpoints are to be measured and successfully evaluated.

Tumor necrosis factor-alpha (TNF) is a cytokine produced not only by various cells of the immune system, but also by various cells in the reproductive system. We have demonstrated that oocytes are an important source of TNF and that the onset of oocytic TNF expression occurs around birth. TNF receptors are localized on oocytes, granulosa cells and interstitial cells allowing for the possibility of autocrine or paracrine actions of TNF. The late fetal/early neonatal period represents a time during which several key events occur, including formation of the primordial follicle and extensive oocyte apoptosis. We have utilized an ovary culture system to examine the involvement of TNF in early ovarian function. This culture system allows both primordial follicle function and apoptosis to occur in vitro. Our results show that TNF can decrease oocyte and primordial follicle number through stimulation of oocyte apoptosis in vitro. TNF thus may serve as an important intraovarian factor involved in the determination of the size of the primordial follicle pool.

Tumor necrosis factor alpha (TNFalpha) is an intraovarian cytokine that may play a role in ovarian development and function. Effects of TNFalpha are mediated by binding to at least one of two TNFalpha receptor subtypes (with molecular masses of approximately 60 and 80 kDa); therefore, the overall goal of this study was to determine whether rat ovaries have TNFalpha receptors during critical times in development. Two approaches were used: 1) demonstration of specific binding of radiolabeled TNFalpha to ovarian cells and 2) semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis for each of the two TNFalpha receptors. Ovarian cells were obtained on Embryonic Day 19, day of birth (Day 0), and Days 2, 5, 10, and 20. TNFalpha binding was present on all days, with significantly greater binding on Day 20. Messenger RNA for both receptor subtypes was detected on all days using RT-PCR analysis but was significantly greater for the 60-kDa receptor on Day 20. In conclusion, rat ovaries contained receptors capable of binding TNFalpha and mRNA for both receptor subtypes. Identification of ovarian TNFalpha receptors provides support for a role of TNFalpha in ovarian development and function.

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