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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 1, 2005 as doi:10.1096/fj.05-4626fje. |
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* Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Histology and Medical Embryology, University of Rome "La Sapienza," Rome, Italy;
Department of Public Health and Cell Biology, Section of Histology and Embryology, University of Rome, Tor Vergata, Rome, Italy;
Department of Surgery "P. Valdoni," University of Rome "La Sapienza," Rome, Italy; and
Department of Experimental Medicine, University of L’Aquila, L’Aquila, Italy
1Correspondence: Department of Histology and Medical Embryology, University of Rome "La Sapienza," 00161 Rome, Italy. E-mail: claudia.giampietri{at}uniroma1.it
SPECIFIC AIMS
To analyze the presence and role of Fas/FasL pathway in fetal gonocytes, we investigated the expression of the Fas receptor and of its inhibitor c-Flip in mouse fetal testes and studied their role in controlling gonocytes apoptosis.
PRINCIPAL FINDINGS
1. c-Flip is expressed in fetal male mouse gonads and its expression is developmentally regulated
c-Flip was investigated at mRNA level by reverse transcription polymerase chain reaction (RT-PCR). c-Flip long isoform (c-FlipL) band was undetectable in sex indifferent 11.5 days post coitum (dpc) gonadal ridges and in 12.5 dpc testes as well; it was weakly expressed in 13.5 and 14.5 dpc testes while it was present at significant levels in 16.5 and 18.5 dpc testes (Fig. 1
A). The expression and localization of c-FlipL protein were then investigated by immunohistochemistry. c-FlipL was found to be expressed only in fetal gonocytes, weakly at 16.5 dpc and strongly at 18.5 dpc (Fig. 2
A, B).
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All together these results indicate that c-Flip expression in testis is restricted to gonocytes starting around 13.5 dpc resulting in detectable c-FlipL protein accumulation after 16.5 dpc.
2. Fas is expressed in the fetal mouse gonad
Fas was investigated at mRNA level by RT-PCR. A weak Fas expression was found in 13.5, 16.5 and 18.5 dpc testes correlating at least in part with c-Flip expression (Fig. 1B
). Immunohystochemical analyses confirmed Fas expression at protein level.
3. Fas-dependent apoptosis in fetal testes is dependent on c-Flip levels
To demonstrate that c-FlipL might exert a functional role in protecting fetal gonocytes from Fas-mediated apoptosis, we investigated the frequency of apoptosis in gonocytes using an organ-culture system. The results showed that 18 dpc control testes were resistant to Fas-induced apoptosis caused by an anti-Fas agonist antibody, while high levels of apoptosis were found in testes where c-Flip synthesis was specifically inhibited by antisense c-Flip oligos.
4. Apoptosis in gonocytes cultures
Since c-Flip expression is expected to reduce cell sensitivity to Fas-mediated apoptotic stimulus, we performed in vitro culture experiments aimed to evaluate the susceptibility of purified male germ cells obtained from various developmental ages to undergo spontaneous apoptosis. To this aim we cultured purified germ cells obtained from 11.5 dpc gonadal ridges (strictly primordial germ cells, PGCs) or 12.5-16.5 dpc testes (gonocytes). TUNEL analyses showed that the frequency of apoptotic cells decreased as function of the developmental age being maximum in 11.5 dpc PGCs and minimum in 16.5 dpc gonocytes. Therefore there is an inverse relation between c-Flip expression level and gonocyte spontaneous apoptosis sensitivity.
5. Initiator caspase-10 is expressed in fetal male mouse gonad and its activity is developmentally regulated
c-FlipL is known to inhibit caspase-8 and -10 recruitment to Fas death receptor in different cell systems, by direct competition; we therefore investigated whether these caspases are expressed in fetal testes. According to c-FlipL expression, caspase-10 is expressed at 13.5, 16.5 and 18.5 dpc as a proenzyme. In addition caspase-10 active form band is expressed at higher levels at 13.5 dpc (a time point showing no c-FlipL expression) as compared with 16.5 and 18.5 dpc (when c-FlipL is present). Caspase activity was investigated by measuring DEVD-AFC cleavage. We found higher caspase activity in 13.5 dpc fetal gonad extracts if compared with 16.5 and 18.5 dpc extracts, thus matching with Western blot results.
Therefore, the activity of caspase-10 is inversely related to the expression of c-FlipL.
CONCLUSIONS AND SIGNIFICANCE
The continuous production of mammalian sperm is maintained by the proliferation and differentiation of germ stem cells or spermatogonia stem cells that originate from gonocytes. Although the time and mechanisms of gonocyte differentiation into spermatogonial stem cells remain to be determined, it is clear that gonocytes are subjected to severe selection through apoptotic degeneration. In rodents massive gonocyte degeneration occurs during the embryonic period coinciding with their enclosure into the seminiferous cords and the end of proliferation. Besides some pro-(p53, bax) and anti- (bcl-x) apopotic proteins, a few factors, such as the transcription factor Zfp148 and the mRNA binding protein Dazla, have been identified as important regulators of gonocyte survival. However, definite apoptotic pathways still need to be clarified. In this work we show that mouse gonocytes express the proapoptotic Fas receptor and c-FlipL, a protein able to inhibit the proapoptotic Fas-signaling, this latter present specifically in gonocytes and mainly at later stages of fetal testis development. This led us to hypothesize that in gonocytes apoptotis can occur throughout activation of Fas and that this process is prevented when c-FlipL is expressed. In the mouse embryo, massive gonocyte apoptosis occurs indeed mainly at early stages of testis development when we observed Fas but not c-FlipL expression in such cells. On the other hand, degeneration is minimal at late stages when we found expression of both proteins. Using in vitro culture of fetal testes, we were able to demonstrate that Fas activation causes gonocyte apoptosis only when c-Flip synthesis is inhibited. Interestingly a protective effect of c-FlipL against Fas-dependent apoptosis on adult male germ cells have been previously reported by us. Our results showed that in the presence of c-FlipL gonocytes are less susceptible to undergo apoptosis in vitro when apoptosis occurs without an extrinsic stimulation of Fas. These experiments suggest a possible role of this protein in interfering with spontaneous apoptosis in gonocyte culture. This process might occur as a consequence of activation of intrinsic apoptotic pathways or alternatively through a Fas-mediated pathway. Therefore our observation might indicate a novel role for c-Flip in regulating intrinsic pathways independent from Fas activation.
A further indication supporting a role of c-FlipL in preventing gonocyte apoptosis and allowing to identify a possible c-FlipL target comes from the observation that gonocytes expressing c-FlipL show lower caspase-10 activity in comparison to the high enzyme activity found in early germ cells not expressing this protein. Caspase-10 together with caspase-8 are generally involved in Fas intracellular pathways and are preferentially targets of the c-Flip inhibitory action.
Previous observation that gonadal somatic cells play an active role in inducing gonocyte apoptosis pointed to these cells as possible source of proapoptotic signals, which might include FasL. In rodents, the adult testis represents a source of constitutive FasL production. Besides the controversial immunoregolative function of FasL in the testis, the Fas system has been proposed as a key regulator of physiological male germ cell apoptosis. In this model, FasL expressed by Sertoli cells would trigger apoptosis of germ cells expressing Fas. Alternatively, we have previously proposed that FasL produced in rodents mainly by germ cells might play a role in self inducing apoptosis and /or in protecting sperm from immune reaction in the female genital tract. We demonstrate here that mouse gonocytes possess a functional Fas system and hypothesize that the activation of the Fas system is normally prevented in gonocytes at late stages of fetal testis development by the presence of c-FlipL and that this protein plays a general anti-apoptotic role in such cells.
In female germ cells, programmed cell death with features of apoptosis occurs in oocytes during the fetal life. However, this process seems to involve intrinsic apoptotic pathways activated by lack of growth factors and/or defects in meiotic processes. In contrast with male, transcripts for Fas were not detectable in fetal mouse oocytes (our unpublished observation). Moreover, in postnatal and adult ovary there are not clear evidence about an involvement of the Fas/FasL system in oocyte degeneration. In conclusion, our results identify c-FlipL as a crucial modulator of gonocyte apoptosis within the fetal testis. How the Fas/FasL and c-Flip systems are modulated and integrated with other apoptotic pathways, likely involved in gonocyte degeneration, remains to be further clarified.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4626fje;
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