It is known that mammalian primordial germ cells (PGCs), the precursors of oocytes and prospermatogonia, depend for survival and proliferation on specific growth factors and other undetermined compounds. Adhesion to neighboring somatic cells is also believed to be crucial for preventing PGC apoptosis occurring when they lose appropriate cell to cell contacts. This explains the current impossibility to maintain isolated mouse PGCs in culture for periods longer than a few hours in the absence of suitable cell feeder layers producing soluble factors and expressing surface molecules necessary for preventing PGTC apoptosis and stimulating their proliferation. In the present paper, we identified a cocktail of soluble growth factors, namely KL, LIF, BMP-4, SDF-1, bFGF and compounds (N-acetyl-L-cysteine, forskolin, retinoic acid) able to sustain the survival and self-renewal of mouse PGCs in the absence of somatic cell support. We show that under culture conditions allowing PGC adhesion to an acellular substrate, such growth factors and compounds were able to prevent the occurrence of significant levels of apoptosis in PGCs for two days, stimulate their proliferation and, when LIF was omitted from the cocktail, allow most of them to enter into and progress through meiotic prophase I. These results consent for the first time to establish culture conditions for purified mammalian PGCs in the absence of somatic cell support and should make easier the molecular dissection of the processes governing the development of such cells crucial for early gametogenesis.

To determine the properties of the cAMP-specific, rolipramsensitive phosphodiesterases (cAMP-PDEs) that are expressed in different organs, monoclonal and polyclonal antibodies were raised against different epitopes present in the cAMP-PDE sequences. Of the several antibodies generated against peptides and fusion proteins, one monoclonal and four polyclonal antibodies recognized both the native cAMP-PDEs as well as the denatured proteins on Western immunoblot analysis. An immunoprecipitation assay demonstrated that these antibodies recognized the recombinant rat PDE4A, PDE4B, and PDE4D proteins with different avidity. The polyclonal antibody K118 and the monoclonal M3S1 were most specific for rat PDE4B and PDE4D forms, respectively, whereas the AC55 antiserum displayed the highest affinity for PDE4A forms. This selectivity was confirmed by Western blot analysis using recombinant rat PDE4A, PDE4B, and PDE4D proteins expressed in a heterologous system. These antibodies were used to characterize the cAMP-PDEs expressed in the rat brain. An immunoblot of extract of cortex and cerebellum demonstrated that at least seven different polypeptides specifically cross-reacted with the different antibodies, indicating that multiple cAMP-PDEs are expressed in this tissue. On the basis of cross-reactivity with PDE4D but not PDE4A or PDE4B antibodies, 93-and 105-kDa PDE4D species were detected in the cortex and cerebellum extract. These forms are different from the 68-kDa PDE4D form expressed in endocrine cells after hormonal stimulation. Although the 93-kDa form was recovered in both the soluble and particulate fractions, the 105-kDa polypeptide was mostly particulate in the cortex and cerebellum extracts. PDE4B forms of 90 -87 kDa were recovered in both soluble and particulate compartments of the brain extract. These forms were different from the previously identified PDE4A variants of 110 and 75 kDa. These data demonstrate that the presence of multiple cAMP-PDE genes is translated into cAMP-PDE proteins of different sizes and distinct immunological properties and that multiple variants derived from these cAMP-PDE genes are expressed in different regions of the brain and different subcellular compartments. These immunological tools will be useful to identify different cAMP-PDE forms expressed in organs targeted for pharmacological intervention with PDE4 inhibitors.The high affinity, cAMP-specific phosphodiesterases [type 4 according to the nomenclature proposed by Beavo et al. (1994)] are a class of enzymes with similar kinetic properties that are inhibited by the antidepressant rolipram and structurally related compounds. Although the presence of these forms has long been recognized, their distinctive properties are becoming evident only recently (Conti et al., 1995b). Early attempts to purify these forms have been hampered by their low abundance and instability (Conti and Swinnen, 1990). The molecular mass attributed to this group of enzymes ranges between 29 and 89 kDa (Conti and Swinnen, 1990). The definition of th...

Strict control of cell proliferation and cell loss is essential for the coordinated functions of different cell populations in complex multicellular organisms. Oogenesis is characterized by a first phase occurring during embryo-fetal life and in common with spermatogenesis, during which mitotic proliferation of the germline stem cells, the primordial germ cells (PGC), prevails over germ cell death. The result is the formation of a relatively high number of germ cells depending on the species, ready to enter sex specific differentiation. In the female, PGC enter into meiosis and become oocytes, thereby ending their stem cell potential. After entering into meiosis in the fetal ovary, oocytes pass through leptotene, zygotene and pachytene stages before arresting in the last stage of meiotic prophase I, the diplotene or dictyate stage at about the time of birth. The most part of oocytes die during the fetal period or shortly after birth. It is widely accepted that in mammals a female is born with a fixed number of oocytes within the ovaries, which over the years progressively decreases without possibility for renewal. Once the oocyte reserve has been exhausted, ovarian senescence, driving what is referred to as the menopause in women, rapidly ensues. The fertile lifespan of a female depends by the size of the oocyte pool at birth and the rapidity of the oocyte pool depletion. Which mechanisms control PGC proliferation? Why do most of the oocytes die during fetal life and what are the mechanisms of such massive degeneration? Is it possible to prolong the lifespan of a female by reducing oocyte lost during the fetal life? This review reports some of the most recent results obtained in an attempt to answer these questions.

Apoptosis is the main cause of primordial germ cell and oocyte degeneration in the developing fetal ovary. In this study we examined by immunohistochemistry and immunoblotting the expression of the anti-and pro-apoptotic proteins Bcl-2 and Bax in primordial germ cells and fetal oocytes during pre natal oogenesis in the mouse embryo. While Bcl-2 and Bax were not detectable in primordial germ cells in vivo, both proteins were upregulated when they undergo apoptosis in culture. Treatment with the stem cell factor (SCF), a growth factor known to partially reduce primordial germ cell apoptosis, resulted in decreased Bax expression. Bcl-2 was barely detectable in oocytes entering into meiosis and its expression did not change during the stage of meiotic prophase I examined. On the contrary, high levels of Bax was expressed in degenerating oocytes while low levels of the protein was present in many apparently healthy oocytes between 15.5 days post coitum (d.p.c.) and birth, when Bax was downregulated. Oocytes isolated from 15.5 days post coitum (d.p.c.) ovaries that progress through prophase I and undergo a wave of apoptosis at the stage of pachytene/diplotene in vitro, showed a pattern of Bax expression similar to the in vivo condition. Although the addition of SCF to the culture medium reduced significantly apoptosis in oocytes at the pachytene/ diplotene stages, it was not possible to directly correlate this effect with the downregulation of Bax in the surviving oocytes. These findings indicate that whereas a balance between Bcl-2 and Bax might regulate apoptosis of proliferating primordial germ cells under a partial control by SCF, Bax-mediated apoptosis in meiotic oocytes may be due to intrinsic meiotic checkpoints which act to monitor aberrant DNA recombination rather than to a growth factor-dependent process. Elimination of supernumerary oocytes might be a subsequent apoptotic phenomenon controlled by the availability of growth factors such as SCF within the ovary.

In previous reports we have shown that FSH and beta-adrenergic agonists regulate the levels of mRNA and increase the activity of a high affinity cAMP phosphodiesterase (cAMP-PDE) in the immature rat Sertoli cell in culture. To identify and characterize the hormone-inducible form(s), the cAMP-PDE activity of the Sertoli cell was partially purified and its properties were determined using biochemical and immunological tools. The cAMP-PDE activity present in the 100,000g supernatant of Sertoli cell extracts was purified more than 2000-fold by four HPLC chromatographic steps. The major purified form of cAMP-PDE had a specific activity of 1-2 mumol/(min.mg of protein). Polyacrylamide gel electrophoresis and silver staining analysis showed that a 67-68 kDa polypeptide comigrated with the major peak of cAMP hydrolytic activity. The molecular weight of the crude or purified enzyme determined by gel filtration and sucrose density gradients was 150,000, suggesting that the native enzyme is an oligomeric structure. This PDE hydrolyzed cAMP with a Km of 1.97 +/- 0.26 microM. The hydrolysis of cAMP was neither inhibited nor stimulated by cGMP concentrations lower than 50 microM. Cyclic nucleotide catalysis required Mg2+, but was insensitive to Ca2+. The activity of this form was competitively inhibited by several inhibitors with the following potency: rolipram > RO 20-1724 > methylisobutylxanthine > cilostamide = milrinone. Because mRNAs derived from two distinct PDE4B and PDE4D genes are present in the Sertoli cell, selective and nonselective PDE antibodies were used to determine the origin of the inducible PDE protein.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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A novel solvable extension of the goldfish N-body problem is presented. Its Newtonian equations of motion read ζ̈n=2aζ̇nζn+2∑m=1,m≠nN(ζ̇n−aζn2)(ζ̇m−aζm2)∕(ζn−ζm), n=1,…,N, where a is an arbitrary (nonvanishing) constant and the rest of the notation is self-evident. The isochronous version of this model is characterized by the Newtonian equations of motion z̈n−3iωżn−2ω2zn=2a(żn−iωzn)zn+2∑m=1,m≠nN(żn−iωzn−azn2)(żm−iωzm−azm2)∕(zn−zm), n=1,…,N, where ω is an arbitrary positive constant and the points zn(t) move now necessarily in the complex z-plane. The generic solution of this second model is completely periodic with a period Tk=kT which is an integer multiple k (not larger than N!, indeed generally much smaller) of the basic period T=2π∕ω and which is independent of the initial data (for sufficiently small, but otherwise arbitrary, changes of such data). These many-body models have an intriguing variety of equilibrium configurations (genuine: with no two particles sitting at the same place), but only for small values of N (N=2,3,4 for the first model, N=2,3,4,5 for the second). Other versions of these models are also discussed. The study of the behavior of the second, isochronous model around its equilibrium configurations yields some amusing diophantine results.