Stem Cell Factor Affects Fate Determination of Human Gonocytes in Vitro

REPRODUCTIONRESEARCH

Stem cell factor affects fate determination of human gonocytes in vitro

Jiongjiong Tu1,2, Liqing Fan1,2,KeTao2, Wenbing Zhu2, Jianjun Li3 and Guangxiu Lu1,2 1National Center of Human Stem Cell Research and Engineering, 2Institute of Human Reproduction and Stem Cell Engineering, Central South University, Changsha, Hunan 410078, China and 3Changsha Maternity and Child Health Hospital, Changsha, Hunan 410007, China Correspondence should be addressed to G Lu; Email: [email protected]

J Tu and L Fan contributed equally to this work

Abstract

The stem cell factor (SCF), binding its tyrosine kinase receptor c-Kit, has been shown to play essential roles in the proliferation, differentiation, and survival of germline cells. However, few reports are available about the effect of SCF on the development of human gonocytes within the fetal testis. The objective of this study was to investigate whether SCF affects the biological behaviors of human gonocytes before or after they enter the mitotic arrest stage. Employing an organ culture system, we observed that addition of exogenous SCF could inﬂuence the morphology of human gonocytes in vitro. Moreover, SCF was able to trigger the colony formation of round gonocytes, which were characterized positive for alkaline phosphatase activity, Oct-4, SSEA-4, and c-Kit as well. We found that SCF exerted actions in a dose- and age-dependent manner, although the stimulatory effect lasted no more than 14 days. We also showed that SCF played a role in suppressing the apoptosis of human gonocytes. Blocking of SCF signaling with either phosphatidylinositol 3-kinase or mitogen-activated protein kinase inhibitor resulted in similar apoptotic features as well as the SCF-withdrawal cultures. Taken together, we report that SCF acts as a potent regulator in the fate determination of human gonocytes. Our studies should form the basis for in vitro studies and facilitate investigation of the molecular mechanisms underlying this unique stage. Reproduction (2007) 134 757–765

Introduction Preliminary studies have reported that mutation of either SCF (Steel locus) or its receptor c-Kit (W locus) results in Male germline development is a highly ordered and reduced numbers of PGCs (McCoshen & McCallion complex process. In early gametogenesis, human 1975, Brannan et al. 1992, Buehr et al. 1993), and the gonocytes arise when their precursors, primordial germ interactions of SCF/c-Kit increase the proliferation and cells (PGCs), migrate into the embryonic genital ridge suppress apoptosis of PGCs and differentiated sperma- and become enclosed in the primitive seminiferous cords by 7 weeks of gestation (Wartenberg 1981). The togonia cells as well (Dolci et al. 1991, 2001, Godin gonocytes gradually reduce their mitotic cycles and et al. 1991, Pesce et al. 1993, Yan et al. 2000). Further enter the mitotic arrest stage by 16–18 weeks of gestation studies have revealed that phosphatidylinositol 3-kinase (Goto et al. 1999). Thereafter, a large number of (PI-3K) and MEK are involved in the intracellular gonocytes are committed to undergo apoptosis and the signaling of SCF in male germline development (Dolci survivors differentiate to spermatogonial stem cells, et al. 2001, De Miguel et al. 2002). which will initiate the wave of spermatogenesis in In humans, SCF has also been found expressed in the puberty (Helal et al. 2002). However, the precise surrounding tissues of PGCs, and c-Kit is conﬁned to mechanisms underlying these complex events are poorly PGCs as well (Sandlow et al. 1997, Hoyer et al. 2005). understood. When PGCs migrate into the embryonic gonad and In mouse embryos, stem cell factor (SCF) is expressed differentiate to gonocytes, the Sertoli cells within the in somatic tissues in a gradient pattern along the pathway primitive seminiferous cords could compensate for the of PGC migration, with the highest levels in the genital secretion of SCF as well as the tissues along PGCs ridges (Keshet et al. 1991, Matsui et al. 1991), whereas migration pathway (Strohmeyer et al. 1995). However, c-Kit is expressed on PGCs through their migratory phase the expression of c-Kit is reduced with the developmental and until shortly after they cease proliferation in the age consistent with the low mitotic activity of human embryonic gonads (Manova & Bachvarova 1991). gonocytes in vivo (Gaskell et al. 2004, Pauls et al. 2006).

q 2007 Society for Reproduction and Fertility DOI: 10.1530/REP-07-0161 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 10/02/2021 10:37:26PM via free access 758 J Tu, L Fan and others

Based on this evidence, we proposed that SCF signaling markers as well as embryonic germ cells (EGCs) that were might play an undefined role in the development of cultured from PGCs (Shamblott et al.1998, Turnpenny human gonocytes within the fetal testis. et al.2003, Liu et al. 2004, Park et al.2004, Pan et al. 2005). In the present paper, we developed an organ culture The result also showed that the cultured gonocyte colonies system to study human gonocytes in vitro. We tested were only dotted stained with c-Kit, suggesting that the different doses of SCF on the morphological change and c-Kit might be down-regulated in the present culture proliferation activity of human gonocytes derived from system. Nevertheless, the results indicated that these 12- to 14-week, 16- to 18-week, and 20- to 22-week gonocyte colonies have an undifferentiated phenotype. fetal testes. The SCF-induced gonocyte colonies were characterized by detection of alkaline phosphatase (AP) activity and pluripotent markers. In addition, PI-3K and SCF promotes formation of gonocyte colonies in a MEK inhibitors were used to examine the intracellular dose- and age-dependent manner signaling of SCF in human gonocytes. To examine the dose effect of SCF on the proliferation activity of human gonocytes, the colony number and size was evaluated at days 7 and 14 in the SCF-treated cultures Results as well as the controls. As shown in Fig. 4, few colonies SCF affects the morphology of human gonocytes in vitro were observed in the SCF-absent controls. The colony number was increased at 10 ng/ml SCF but mainly peaked The dissociated seminiferous cords, which were mainly at O2–4 cells. In contrast, there was a dramatic increase in composed of gonocytes and primitive Sertoli cells, were the colony number and size at 100 or 500 ng/ml SCF in cultured in gelatin-coated four-well tissue culture plates three tested age groups. The maximum dose of 500 ng/ml with or without SCF. In this organ culture system, resulted in no further enhancement, suggesting that different doses of SCF (10 ng/ml, 100 ng/ml and 100 ng/ml may be the saturated dose to enhance the 500 ng/ml) were tested on human gonocytes derived proliferation of gonocytes. The statistical data showed from 12- to 14-week, 16- to 18-week, and 20- to significant differences in response to SCF among 12–14 22-week fetal testes. After 2 days of culture, fibroblast- week, 16–18 week, and 20–22 week cultures. The like Sertoli cells began to firmly attach to the bottom of increased colonies, especially those with O8–16 cells the plates, while the gonocytes were exposed and and O16 cells, were obviously confined to the early anchored on the somatic monolayer. In the SCF-absent cultures. In addition, the colonies were significantly controls, most gonocytes developed cytoplasmic exten- reduced after day 14 when compared with those at day sions and converted to spindly shape (Fig. 1A). In 7, suggesting that SCF could not support the consistent contrast, many gonocytes remained in round shape growth of gonocytes in the present culture system. with the addition of SCF (Fig. 1B). In contrast to somatic We further determined the proliferation activity of cells, both the spindly and round gonocytes were positive for AP activity (Fig. 1C and D), which can be human gonocytes using BrdU incorporation tests and easily scored. The statistical data showed that the index 100 ng/ml SCF was added to stimulate colony formation. of round gonocytes was significantly higher in the Consistent with the colony distribution profile, the SCF-treated cultures, whereas treatments with 10, 100, proliferation index was significantly higher in the SCF- and 500 ng/ml SCF resulted in no significant differences. treated cultures, and the early cultures showed a The result also showed a less profound effect of SCF on relatively higher incorporation index as well. In contrast the late gonocytes than on the early gonocytes (Fig. 1E). to day 7 cultures, the proliferation index was dramatically reduced at day 14 in three tested age groups, suggesting that the proliferation activity of human gonocytes is Identification of the gonocyte-derived colonies with the impaired during the long-term cultures (Fig. 5). stimulation of SCF

After 3 days of stimulation with SCF, colonies could be SCF suppresses apoptosis of human gonocytes in vitro induced from round gonocytes, and they displayed a broad size distribution of O2–4 cells, O4–8 cells, O8–16 cells, We next examined the anti-apoptotic effect of SCF in vitro. and O16 cells by AP staining (Fig. 2). In contrast, most The gonocyte colonies were induced with the addition of spindly gonocytes remained in single or pair pattern and 100 ng/ml. At day 7, SCF was removed and the cells were few colonies were observed, suggesting that the round cultured without SCF for 24 h. As shown in Fig. 6A, the gonocytes retain stronger clonogenic capacity than the colonies began to detach from the somatic monolayer and spindly ones. The phenotypic characteristics of these dissociated into single cells with typical apoptotic features gonocyte-derived colonies were examined by immuno- such as shrunken membranes. These dissociated cells were ﬂuorescence staining. As shown in Fig. 3, the gonocyte conﬁrmed to be apoptotic by TUNEL staining. In contrast, colonieswere stained positive for Oct-4, SSEA-4, and c-Kit, the gonocyte colonies showed intact and integrated suggesting that these colonies share similar pluripotent appearances in the SCF-present controls. To block the

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Figure 1 Morphology of human gonocytes after 2 days of culture in vitro. (A and B) Representation of spindly gonocytes in the absence of SCF (A, arrows) and round gonocytes in the presence of SCF (B, arrowheads). (C and D) Representation of AP activity of spindly gonocytes (C, arrows) and round gonocytes (D, arrowheads). (E) Quantifi- cation of the round gonocytes with untreated controls versus stimulation with 10, 100, and 500 ng/ml SCF from 12–14 week, 16–18 week, and 20–22 week age groups. MeansGS.E.M. for three independent experiments are shown. Note that there was no significant difference within the treatments of 10, 100, and 500 ng/ml SCF and less profound effect of SCF on late gonocytes. ***P!0.001 when compared with controls. Scale barZ50 mm. effect of SCF, PI-3K inhibitor (LY294002) or MEK inhibitor formation in a dose- and age-dependent manner. SCF (U0126) was added with SCF in the culture medium. After also acted as an anti-apoptotic factor in the cultures. To 24 h incubation, the gonocyte colonies showed similar our knowledge, this is the first report describing the apoptotic features as well as the SCF-withdrawal con- regulatory mechanism underlying the development of ditions (Fig. 6A), suggesting that these two kinases are both human gonocytes. involved in downstream SCF signaling in human gono- The relative number of spindly versus round gonocytes cytes. The statistical data showed that the apoptotic index is difficult to assess in vivo, but in vitro studies found that was dramatically increased in the SCF-withdrawal and there were two obvious distinct morphological inhibitor-treated cultures in contrast to the SCF-present categories with the influence of exogenous SCF. The controls in three tested age groups (Fig. 6B). minimum dose of SCF (10 ng/ml) seems to be sufficient to rescue the round gonocytes, whereas no such effect was observed in the controls, although Sertoli cells Discussion produce endogenous SCF in a paracrine manner The study of human gonocytes and their niches is of (Strohmeyer et al. 1995). It is possible that these two paramount importance to understand the mechanisms of subpopulations are analogous to the migrating and self-renewal and differentiation of these unique germ degenerating gonocytes similar to previous observations stem cells. In the present study, we report that SCF in rodents (Orth & Boehm 1990, Hasthorpe et al. 1999). maintained round gonocytes and promoted their colony Orwig et al. (2002) have reported that it is essential for www.reproduction-online.org Reproduction (2007) 134 757–765

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Figure 2 AP staining of the gonocyte-derived colonies ranged from O2 to 4 cells (A), O4to8 cells (B), O8 to 16 cells (C), and O16 cells (D) after 3 days of stimulation with SCF. Scale barZ20 mm. gonocytes to change their appearance to acquire expressed pluripotent markers similar to EGCs, which migration ability, whereas the rest would undergo show a well-known pluripotency for the embryonic body apoptosis. In the present study, addition of exogenous formation and differentiation to other cell lineages (Turn- SCF may prevent this transformation and keep gonocytes penny et al. 2006). Thus, to some degree, SCF contributes in a relatively stationary status, and the low expression of to the maintenance of the pluripotency of human endogenous SCF by Sertoli cells may account for their gonocytes rather than triggering differentiation as reported morphological change in vitro. However, the effect of in late spermatogonia cells (Feng et al. 2002), suggesting exogenous SCF was less profound on late gonocytes, as that SCF plays different roles in speciﬁc stages. In addition, we have shown. A possible explanation could be that there are two potential explanations for the reduced c-Kit most late gonocytes have already been committed to on the cultured gonocytes. First, the organ culture system migration fate prior to culture, and SCF merely works on that mimics physiology niches may recapitulate the the residual round ones. programmed down-regulation of c-Kit as reported in vivo Interestingly, the rescued round gonocytes, rather than (Gaskell et al.2004, Pauls et al. 2006). Secondly,a negative the spindly ones, formed colonies with the stimulation of feedback loop model may account for this in that Cbl SCF. We found that these gonocyte-derived colonies proteins, a newly established family as components of the

Figure 3 Phenotype of the gonocyte-derived colonies induced by SCF. Immunoﬂuorescence staining showed that Oct-4 and SSEA-4 were strongly positive whereas c-Kit was dotted stained on the membrane of human gonocytes (arrowheads). Scale barZ50 mm.

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Figure 4 Effect of SCF on the formation of gonocyte colonies in vitro. Quantification of the colonies with untreated controls versus stimulation with 10, 100, and 500 ng/ml SCF from 12–14 week, 16–18 week, and 20–22 week age groups. Cumulative colony numbers were evaluated from 100 randomly selected colonies from at least three independent experiments and scored as four size groups of O2–4 cells, O4–8 cells, O8–16 cells, and O16 cells. Colonies with 1–2 cells were considered to be quiescent and excluded from the figure. MeansGS.E.M. for three independent experiments are shown. **P!0.01, ***P!0.001 when compared with controls. ###P!0.001 when compared with 10 ng/ml groups. There was no significant difference between 100 and 500 ng/ml groups. Note that the maximal effect of SCF was achieved on early gonocytes, and the colony number was reduced after 14 days of culture in vitro. ubiquitin ligation machinery, could induce phosphoryl- could explain the maximum effect of SCF on the early ation by SCF and, in turn, lead to the degradation of c-Kit cultures due to the activation of SCF signaling via SCF/c-Kit receptors (Joazeiro et al. 1999, Zeng et al.2005). complexes. Consistent with this idea, studies in mice have On the other hand, we found that SCF was able to shown that late gonocytes are almost c-Kit negative, which activate the proliferation of the less active or quiescent leads to the redundant effect of SCF on their colony gonocytes beyond 16–18 week of gestation (Goto et al. formation (Yoshinaga et al.1991, Vincent et al. 1998, Ohta 1999), suggesting that SCF is a potent mitogen in mediating et al. 2000, Kanatsu- Shinohara et al. 2003, 2004). the mitotic cycles of human gonocytes. The fact that SCF Although SCF acts as a potent mitogen in the first few functions in a dose-dependent manner indicates that the days, the overcrowded Sertoli cells associated with low expression of endogenous SCF in the local niches, as gonocytes may release negative factors and mask the effect well as the effect on morphological determination of of SCF during long-term cultures. It has been reported that human gonocytes, may also be responsible for the Sertoli cells not only favor the survival of gonocytes, but occurrence of mitotic arrest in vivo. According to the also inhibit their colony formation in vitro (Griswold 1995, immunohistochemistry analysis, the early fetal testis Hasthorpe et al. 1999). The strict control of germ cell houses more c-Kit-positive gonocytes when compared numbers in the fetal testis may be a protection mechanism with the late fetal testis (Gaskell et al.2004, Pauls et al. to prevent the formation of seminomas derived from the 2006), which agrees well with their age-dependent growth unlimited duplication of germ stem cells, which could be in response to SCF. Thus, the higher expression of c-Kit induced by the c-Kit mutations (Kemmer et al.2004). www.reproduction-online.org Reproduction (2007) 134 757–765

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Figure 5 Quantification of BrdU-positive gonocytes at 7 and 14 days with the stimulation of 100 ng/ml SCF. MeansGS.E.M. for three independent experiments are shown. In the left panels, ***P!0.001 when compared with controls. In the right panels, no significant differences were observed. Note that the higher incorporation index was confined to early gonocytes and the index of BrdU-positive gonocytes was reduced after 14 days culture in vitro.

Our results also showed that SCF acts as a survival However, the present culture conditions are not optimal factor in the culture of human gonocytes. Instead of for long-term maintenance, and many improvements undergoing apoptosis, the round gonocytes can survive need to be addressed. Although we demonstrate here and proliferate with the addition of SCF, suggesting that that SCF acts as both mitogen and survival factor for SCF may have a role in mediating the programmed death human gonocytes, much remains to be studied on of human gonocytes within the developing testis (Helal the intrinsic regulation of SCF in the fetal testis, which et al. 2002). It has been reported that SCF signaling would favor our further understanding of early gameto- up-regulates the expression of Bcl-2, a prosurvival genesis in humans. protein, thereby preventing apoptosis in a number of cell types (Carson et al. 1994, Jin et al. 1998, Zeuner et al. 2003, Dhandapani et al. 2005, Kimura et al. 2005). Materials and Methods In this study, we noted that PI-3 and MEK kinase pathway Testis collection are also involved in the anti-apoptotic mechanism of SCF signaling, which shares a relatively conserved Human fetal testes were obtained as a result of therapeutic termination of pregnancy using a protocol approved by the ethics pathway as well as the previous observations in mouse committee at Central South University of China, and the patients germline cells (Dolci et al. 1991, 2001, Pesce et al. gave their written informed consent according to the national 1993, Yan et al. 2000). guidelines (Medical Research Council 2001). The testes were In conclusion, in this paper, we report that SCF is a collected from fetuses of 12–14 weeks (nZ9), 16–18 weeks potent factor that affects the fate determination of human (nZ7), and 20–22 weeks (nZ7) of gestation. All terminations gonocytes, which provides a new insight into the were performed in the Gynecology and Obstetric Clinics, regulatory mechanism underlying this speciﬁc stage. Changsha Maternity & Child Health Hospital. The developmental

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Figure 6 Effect of SCF on suppressing apoptosis of human gonocytes in vitro. (A) Representation of apoptotic features of gonocytes with the treatments of SCF (100 ng/ml), SCF-withdrawal, SCF plus LY294002, and SCF plus U0126 for 24 h. Note that the single cells dissociated from the colonies were stained with TUNEL-positive signals (arrowheads). (B) Quantification of TUNEL signals showed significant apoptosis in the SCF-withdrawal, SCF plus LY294002, and SCF plus U0126 cultures. MeansGS.E.M. for three independent experiments are shown. ***P!0.001 when compared with SCF-present controls. Scale barZ50 mm. age of the fetuses was determined by the date of the last menstrual 0–500 ng/ml in the culture medium. For blocking experiments, bleeding. Weight and length measurements evaluated at the PI-3K and MEK inhibitors (Cell Signaling Technology, Beverly, autopsy were used to assure proper gestational development. MA, USA) were used at conventional concentration of 10 mM as reported (Dolci et al. 2001). All cultures were maintained at 37 8C in a humidified 5% CO2 atmosphere. Organ culture The fetal testes were decapsulated under a stereomicroscope in AP staining and colony counting Hank’s balanced salt solution (HBSS, Gibco) and mechanically dissociated with fine scissors. The spliced tissues were exposed To examine endogenous AP activity, cultured cells were fixed to 1 mg/ml collagenase (type IV, Sigma) and 1 mg/ml DNase I with 4% paraformaldehyde and stained with Fast Red Substrate (Sigma) at 37 8C for 20 min, followed by two washes in Pack (Zymed) by following the manufacturer’s instructions. Dulbecco’s PBS (DPBS, Gibco) at 40 g centrifugation for 5 min According to the AP staining, the gonocyte-derived colonies to remove redundant interstitial cells. The fragmented semi- were counted and grouped as O2–4 cells, O4–8 cells, O8–16 niferous cords were collected at 300 g centrifugation for 5 min cells, and O16 cells. Single or paired gonocytes were excluded and resuspended in culture medium. The resulting seminifer- for being considered quiescent. ous cords were aliquoted into four-well tissue culture plates (Nunclon; Nunc, Roskide, Denmark) pre-coated with 0.1% gelatin. The culture medium was changed every other day in all Immunofluorescence staining experiments. Basic culture medium used in this study was Cultured cells were fixed in 4% paraformaldehyde in PBS at room Dulbecco’s modified Eagle’s medium (D-MEM) supplemented temperature for 20 min and blocked with PBS containing 0.1% with 10% fetal bovine serum (FBS), 1!non-essential amino Triton X-100, 4% normal goat serum, and 1% BSA at room acids (NEAA), 50 mM 2-mercaptoethanol, 2 mM glutamine, temperature for 1 h. The cells were incubated with primary and 1 mM sodium pyruvate (all from Gibco). To investigate the antibodies at 4 8C overnight. The primary antibodies against dose effect of SCF, human recombinant stem cell factor (SCF; R human Oct-4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) & D Systems, Minneapolis, MN, USA) was added at and SSEA-4 (Chemicon International, Temecula, CA, USA) were www.reproduction-online.org Reproduction (2007) 134 757–765

Downloaded from Bioscientifica.com at 10/02/2021 10:37:26PM via free access 764 J Tu, L Fan and others diluted at 1:100, and antibody against human c-Kit (DAKO Foundation of China (No. 30170480 and No. 30470884). The Corporation, Carpinteria, CA, USA) was diluted at 1:300. After authors declare that there is no conflict of interest that would washing thrice with PBS, fluorescein isothiocyanate (FITC)- or prejudice the impartiality of this scientific work. rhodamine-conjugated secondary antibody (both from Santa Cruz Biotechnology) diluted at 1:100 was added and incubated at 37 8C for 1 h. Negative controls incubated with PBS instead of primary antibodies revealed no positive staining. 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