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Abbreviations used: Apaf-1 -apoptosis protease activating factor-1; Bcl -B-cell lymphoma family; BH -Bcl-2 homology; tBid -truncated Bid; BIR -baculoviral IAP repeat; BRUCE -BIR repeat-containing ubiquitin-conjugating enzyme; CARDcaspase recruitment domain; CDR -cysteine-rich extracellular domain; DISC -deathinducing signaling complex; DD -death domain; DED -death effector domain; DRdeath-inducing receptor; ER -endoplasmic reticulum; FADD -Fas-associated death domain protein; FLIP -FADD-like-ICE-inhibitory protein or FLICE inhibitory protein; G1 -GAP1; IAP -inhibitor of apoptosis; IL -interleukin; MOMP -mitochondrial outer membrane permeabilization; PCD -programmed cell death; PUMA -p53-up-regulated modulator of apoptosis; PS -phosphatidylserine; PTP -permeability transition pore; RIP -receptor interacting protein; TACE -TNF alpha-converting enzyme; TGF-β -transforming growth factor-β; TNF-α -tumor necrosis factor α; TNFR-1 -tumor necrosis factor receptor-1; TRAIL -TNF-related apoptosis-inducing ligand; TRADD -TNF-receptor associated protein with death domain; TUNEL -terminal deoxynucleotidyl transferase-mediated (TdT-mediated) dUTP-digoxigenin nick end labeling; VDAC -voltage-dependent anion channel Abstract: Apoptosis, or programmed cell death (PCD), involves a complex network of biochemical pathways that normally ensure a homeostatic balance between cellular proliferation and turnover in nearly all tissues. Apoptosis is essential for the body, as its deregulation can lead to several diseases. It plays a major role in a variety of physiological events, including embryonic development, tissue renewal, hormone-induced tissue atrophy, removal of inflammatory cells, and the evolution of granulation tissue into scar tissue. It also has an essential role in wound repair. The various cellular and biochemical mechanisms involved in apoptosis are not fully understood. However, there are two major pathways, the extrinsic pathway (receptor-mediated apoptotic CURRENT CONCEPTS IN APOPTOSIS: THE PHYSIOLOGICAL SUICIDE PROGRAM REVISITED

The obese gene (ob) product, leptin, has recently been shown to be produced by adipocytes and to circulate in the plasma acting as a hormone to modulate appetite and metabolism. Intriguingly, the ob/ob mutant female mouse, which does not produce an active form of leptin due to a mutation of the ob gene, has been shown to be acyclic and sterile. This sterility can be reversed by treatment with recombinant leptin, but not by diet restriction – suggesting that leptin is required for normal reproductive function. The mechanism(s) whereby leptin modulates reproductive function are unknown; however, it is possible that leptin could directly regulate reproductive tissues. To determine whether endocrine and neuroendocrine tissues could be targets for leptin action, we examined whether these tissues express the leptin receptor mRNA by utilizing reverse-transcription polymerase chain reaction (RT-PCR) analysis in selected tissues from the male and female rat. The results revealed that the leptin receptor mRNA transcript is highly expressed in the ovary, uterus and testis, moderately expressed in the hypothalamus and anterior pituitary, with low to no expression in the adrenal. The RT-PCR results were confirmed by Northern analysis. Furthermore, immortalized GnRH (GT1-7 and NLT) neurons and ovarian granulosa cells were also demonstrated by RT-PCR analysis to express the leptin receptor, suggsting that GnRH neurons and steroid-producing cells of the ovary could be targets for leptin action. Immunohistochemical studies revealed dense immunolocalization of the leptin receptor in the choroid plexus, and interestingly, in the arcuate nucleus/median eminence of the female rat – a key sit in the control of feeding and reproduction. Finally, treatment of the ob/ob mouse with recombinant leptin (0.15 mg/kg/day × 2 weeks) was found to markedly upregulate side chain cleavage and 17α-hydroxylase mRNA levels in the ovary, demonstrating that leptin, acting either through a direct or indirect mechanism, can regulate gene expression in reproductive tissues.

The neurotransmitter glutamate plays an important role in the control of gonadotropin releasing hormone (GnRH) secretion. Recent evidence suggests that the novel transmitter nitric oxide may also play a role in controlling GnRH release and may be an important mediator of glutamate effects. To explore the role of nitric oxide in these events, the present study determined the distribution of the enzyme which catalyzes nitric oxide production, nitric oxide synthase (NOS) in the hypothalamus, its association with GnRH neurons, and whether NOS neurons contain NMDA receptors. NOS was localized by staining hypothalamic sections from female rats for NADPH-diaphorase activity. Specific antibodies for GnRH and the NMDA R₁receptor subunit were used for double-staining to determine NOS association with GnRH neurons and the presence of NMDA R₁ receptor subunits in hypothalamic NOS neurons. The studies showed intense NOS cell body and fiber staining in the organum vasculosum of the lamina terminalis (OVLT) where numerous GnRH cell bodies are located. Other major GnRH cell body sites such as the median preoptic nucleus (MPN) and medial preoptic area (MPOA) displayed moderate staining of NOS cell bodies and fibers. Intense NOS staining was also observed in the median eminence, ventromedial nucleus, paraventricular nucleus and supraoptic nucleus of the hypothalamus. While no GnRH neurons were found to double stain for NOS in the hypothalamus, GnRH neurons were frequently surrounded by NOS neurons in the OVLT, MPN and MPOA with potential contacts between NOS and GnRH neurons in these areas. In addition, there was significant overlap of GnRH and NOS fibers in the lateral portion of the internal zone of the median eminence where GnRH fibers and terminals converge. Double-staining studies for NADPH-diaphorase and NMDA R₁ receptor subunit showed that many NOS neurons in the OVLT, MPOA, ventromedial nucleus, paraventricular nucleus and supraoptic nucleus co-localize the NMDA R₁ receptor subunit. Localization of NMDA R₁ receptor subunit immunoreactivity in B-NOS neurons in the hypothalamus was further confirmed by using combined immunohistochemistry-in situ hybridization. Finally, the functional importance of this co-localization was shown by the finding that central administration of a nitric oxide synthase inhibitor blocked the ability of NMDA to induce LH secretion. Taken as a whole, these studies provide evidence which support a role for nitric oxide as an important regulator of GnRH neurons in the female. They also suggest that hypothalamic NOS neurons are targets for glutamate regulation as evidenced by co-localization of the NMDA Ri receptor subunit.

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