Analysis of programmed cell death in mouse fetal oocytes.

We report a short-term culture system that allows to define novel characteristic of programmed cell death (PCD) in fetal oocytes and to underscore new aspects of this process. Mouse fetal oocytes cultured in conditions allowing meiotic prophase I progression underwent apoptotic degeneration waves as revealed by TUNEL staining. TEM observations revealed recurrent atypical apoptotic morphologies characterized by the absence of chromatin margination and nuclear fragmentation; oocytes with autophagic and necrotic features were also observed. Further characterization of oocyte death evidenced DNA ladder, Annexin V binding, PARP cleavage, and usually caspase activation (namely caspase-2). In the aim to modulate the oocyte death process, we found that the addition to the culture medium of the pan-caspase inhibitors Z-VAD or caspase-2-specific inhibitor Z-VDVAD resulted in a partial and transient prevention of this process. Oocyte death was significantly reduced by the antioxidant agent NAC and partly prevented by KL and IGF-I growth factors. Finally, oocyte apoptosis was reduced by calpain inhibitor I and increased by rapamycin after prolonged culture. These results support the notion that fetal oocytes undergo degeneration mostly by apoptosis. This process is, however, often morphologically atypical and encompasses other forms of cell death including caspase-independent apoptosis and autophagia. The observation that oocyte death occurs mainly at certain stages of meiosis and can only be attenuated by typical anti-apoptotic treatments favors the notion that it is controlled at least in part by stage-specific oocyte-autonomous meiotic checkpoints and when activated is little amenable to inhibition being the oocyte able to switch back and forth among different death pathways.

Experimental approaches to the study of primordial germ cell lineage and proliferation.

New information regarding primordial germ cell (PGC's) segregation and proliferation over the last decade is reviewed. Advances have been obtained in the mouse but current knowledge of human PGC's remains scant. Questions still fully or partially unresolved about the emergence of the germline in mammals are addressed. (i) When and where is the germ line set aside in the embryo? (ii) How is the germ line segregated from the somatic lineages? (iii) Which factors guide PGC's to the gonadal ridges? (iv) Which factors regulate PGC's proliferation? The main purpose of this review is to outline the information obtained using mainly in vitro culture systems about two aspects of these processes namely the segregation of PGC's and their proliferation.

Insulin receptor substrate (IRS) transduction system: distinct and overlapping signaling potential.

Insulin receptor substrate (IRS) proteins play a central role in maintaining basic cellular functions such as growth and metabolism. They act as an interface between multiple growth factor receptors possessing tyrosine kinase activity, such as the insulin receptor, and a complex network of intracellular signalling molecules containing Src homology 2 (SH2) domains. Four members (IRS-1, IRS-2, IRS-3, IRS-4) of this family have been identified which differ in their subcellular distribution and interaction with SH2 domain proteins. In addition, differential IRS tissue- and developmental-specific expression patterns may contribute to specificity in their signaling potential.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP-receptor type 1 expression in rat and human placenta.

Pituitary adenylate cyclase-activating polypeptide (PACAP), the new hypophysiotropic factor member of the vasoactive intestinal peptide (VIP)/secretin/glucagon/GHRH family of neuropeptides, exerts its biological action by interacting with both PACAP-selective type I receptors (PAC1) and type II receptors (VPAC1), which bind both PACAP and VIP. The placenta is a site of production of hypophysiotropic factors that participate in the control of local hormone production, as well as the respective hypothalamic-pituitary neurohormones. In the present study, we show the expression of PACAP gene and irPACAP distribution within rat and human placental tissues, by means of RT-PCR and immunohystochemical experiments. In both rat and human placenta, we evaluated the expression of PAC1 gene by Northern hybridization analysis performed with a 32P-labeled 706 nt complementary DNA probe, derived from the full-length coding region of the rPAC1 complementary DNA. The results of these experiments demonstrate the presence, in both human and rat placenta, of a 7.5-kb transcript similar in size to those detected in the ovary, brain, and hypothalamus. Alternative splicing of two exons occurs in human and rat PAC1 gene generating splice variants with variable tissue-specific expression. To ascertain which of the splice variants were expressed in placental tissue we performed RT-nested PCR using primers flanking the insertion sequence termed hip/hop cassette in rat or SV1/SV2 box in human gene. Electrophoretic analysis of the PCR products showed a different pattern of expression of messenger RNA splicing variants in human and rat placenta. In particular, the rat placenta expresses the short PAC1 receptor (PAC1short), the rPAC1-hip or hop (which are indistinguishable with the primers used), and the rPAC1-hip-hop, whereas the human placenta expresses only the PAC1SV1 (or SV2) variant, structurally homologous to the rat PAC1 hip (or hop). Sequence analysis of the human PCR-amplified PAC1 variant was therefore carried out and revealed that human placenta only expresses the PAC1SV2 isoform. The presence and characterization of PACAP binding sites was then investigated in human placenta by radioligand binding studies performed on crude membrane preparation using [125I]PACAP27 as tracer. Scatchard analysis of the binding results revealed the presence of two binding sites, one with high affinity and low capacity (Kd 0.33+/-0.04 nM; Bmax 36.9+/-12.1 fmol/mg protein) and one with low affinity and high capacity (Kd 24+/-6.9 nM, Bmax 9.3+/-0.19 pmol/mg protein). The relative potencies of PACAP-related peptides for inhibition ofradioligand binding were: PACAP27 > or = PACAP38 > VIP, whereas GHRH and other unrelated peptides, such as CRH and beta-endorphin, did not inhibit [125I]PACAP27 binding. In conclusion, in this study, we provide evidence for the expression of PACAP within rat and human placenta. We also demonstrate that both human and rat placenta express the PAC1 gene and that the human tissue has binding sites for PACAP. These findings may suggest a role for PACAP in the regulation of placental physiology through autocrine and/or paracrine mechanisms.

Thyroid hormone effects on mouse oocyte maturation and granulosa cell aromatase activity.

In the present study we evaluated the role of T3 on the in vitro processes of mouse cumulus cell-oocyte complex expansion, oocyte meiotic maturation, and granulosa cell aromatase activity. Results obtained from cumuli oophori isolated from immature and adult mice ovaries demonstrated that T3 at all concentrations tested (0.1-100 nM) did not affect basal or FSH-induced cumulus expansion or interfere with oocyte meiotic maturation up to metaphase II stage. On the contrary, T3 inhibited in a time- and dose-dependent manner FSH-induced aromatase activity in cultured granulosa cells obtained from either adult or immature female mice. The half-maximal dose (ED50) of T3 inhibition was 0.87 +/- 0.21 nM, which is in agreement with the reported dissociation constant of T3 nuclear receptor (Kd = 0.4-5 nM) in mammalian granulosa cells. Time-course experiments demonstrated higher sensitivity to T3 of adult granulosa cells with respect to immature granulosa cells in culture. Indeed, in immature granulosa cells T3 inhibition became significantly evident only after 6 days of hormonal treatment, whereas in adult granulosa cells the inhibitory effect was present after only 2 days of treatment. (Bu)2cAMP- or 3-isobutyl-1-methyl-xanthine-stimulated aromatase activity was also significantly decreased by T3, thus suggesting that the inhibition was downstream from cAMP formation. Lastly, analysis of aromatase messenger RNA (mRNA) levels by semiquantitative RT-PCR demonstrated the ability of FSH to increase aromatase mRNA level in cultured granulosa cells by 2.4 +/- 0.5-fold. In agreement with the effect on enzyme activity, the stimulatory effect of FSH on aromatase mRNA level was greatly reduced after T3 cotreatment. In conclusion, T3 inhibition of aromatase activity may be of physiological relevance in the complex multihormonal regulation of mammalian follicle development and may contribute to explaining the alteration in female reproductive functions after thyroid hormone hypo- or hypersecretion.