Genetic Analysis of Kit Ligand Functions During Mouse Spermatogenesis

Kit ligand (KITL), also known as mast cell growth factor (MGF), stem cell factor (SCF), and Steel factor,1 and its receptor, KIT, are essential for embryonic and postnatal development of both male and female germ cells. While germ cells express KIT at multiple stages in their development, somatic cells that support the growth and differentiation of germ cells express KITL. Numerous studies have revealed that KITL promotes proliferation and suppresses apoptosis of differentiated spermatogonia and has a role in either initiating or maintaining meiosis. However, many details of the molecular mechanisms by which KITL signaling affects proliferation, apoptosis, and differentiation of postnatal male germ cells are not known. This review summarizes the structure, organization, and expression of the Kitl gene in mice. In addition, the functions of KITL and its receptor during spermatogenesis are reviewed, with an emphasis on information gained through the analysis of mouse mutants. In mice, KITL and KIT are encoded by the Steel (Sl) locus and the Dominant White Spotting (W) locus, respectively, which are two classical mutant loci. Long before the Sl and W gene products were identified, numerous studies revealed that they are essential for spermatogenesis. Readers are encouraged to read the earlier literature; however, in this review we summarize research findings that were published since 1990, the year in which KITL was shown to be the product of the Sl locus. Although a great deal of information has been revealed about KITL functions during spermatogenesis, we raise several important questions that remain to be addressed. Kitl is located on chromosome 10 of mice and its coding region comprises 9 exons. Of these 9 exons, only exon 6 is known to be alternatively spliced. In testes, Sertoli cells, but not germ cells, were shown to express Kitl messenger RNA (mRNA) as well as biologically active KIT ligand (KITL) (Motro et al, 1991; Rossi et al, 1991; Tajima et al, 1991). Although Kitl expression in Sertoli cells is under developmental, tissue-specific, stage-specific, and hormonal regulation (Manova et al, 1993; Rossi et al, 1993; Tajima et al, 1993; Hakovirta et al, 1999; Yan et al, 1999), little is known of its transcriptional and posttranscriptional regulation. In studies using cultured Sertoli cells from mice, rats, and humans, sequences in the adjacent 5′-flanking region of Kitl have been identified that mediate transcriptional activation in response to stimulation by follicle-stimulating hormone (FSH) or cyclic adenosine monophosphate (cAMP) analogues (Taylor et al, 1996; Jiang et al, 1997; Grimaldi et al, 2003). However, it is not clear whether these cultured cells reflect the transcriptional state of the gene in vivo. For example, DNA sequences from a 10-kilobase (kb) region upstream of the Kitl transcriptional start site were not sufficient to drive lacZ reporter gene expression in the gonads of transgenic mice (Yoshida et al, 1996). This absence of expression in the gonads occurred even though this same fragment, or an even smaller fragment containing only 2 kb of sequence upstream to the start site, was able to direct reporter gene expression to neural tissue and skin in a pattern identical to that of the endogenous gene. Furthermore, chromosomal rearrangements located up to 200 kb away from the Kitl transcription start site exert tissuespecific effects on Kitl mRNA expression (Bedell et al, 1995), indicating that transcriptional regulation of this gene is complex and may require sequences located a large distance from the gene. A potentially important aspect of Kitl mRNA expression in the adult testis is that it is stage-specific, and as such, may reflect requirements for KITL function during different aspects of spermatogenesis. The first indication of this came from a study that used in situ hybridization of testes sections from mice at different ages (Manova et al, 1993). In this study, all tubules from testes of mice younger than postnatal day 9 (P9) had high levels of Kitl mRNA, but by P13, Kitl mRNA levels were greatly reduced in about half the tubules. It is interesting that Kitl mRNA expression in P13 testes was maximal in tubules that contain proliferating spermatogonia but lack meiotic cells, and was greatly reduced in tubules that contained meiotic cells but no proliferating spermatogonia. Stage-specific expression of Kitl mRNA was confirmed by analysis of rat testis in which maximal expression was again observed in tubules (specifically stages II-VI) that have proliferating spermatogonia (Hakovirta et al, 1999; Yan et al, 1999). Addition of FSH to of tubules from rat testes in a in Kitl mRNA with maximal in stages et al, 1999). In this same study, the in Kitl mRNA was shown to from both transcription and mRNA mechanisms may a for Kitl mRNA levels to be in response to during spermatogenesis. In support of the that regulation of Kitl mRNA expression may to the of the of KITL to of tubules from rat testes in a in DNA (Hakovirta et al, 1999). KITL is as either a or as a and these two from RNA and et al, 1991; et al, Kitl that by the or absence of exon 6 and also and et al, are in a in the testis (Manova et al, 1993; Rossi et al, 1993; et al, 1999). The expression of these alternatively products and the of their expression are in the Kitl are that contain important and by et al, and et al, a an a and a The sequence of is during and is to that of many for the cell The the of KITL and sequences that to KIT with high A of functions as the of KITL and is of The of is and two studies revealed that and of KITL require sequences in its et al, and 1999). Although it was that the KITL has no with a study has revealed a in this that KITL and growth to the of cells and For KITL in the testes, may have important The Kitl mRNA a that is at the site in exon 6 to a of as growth of KITL not require sequences in the and though be an important for KITL little information about KITL the only known to KITL are a on mast cells et al, and which is in et al, The alternatively Kitl mRNA, Kitl mRNA, the site and a KITL of It is clear from many in studies that both and are biologically these studies were with germ cells by and and spermatogonia et al, 1991; Rossi et al, 1993; et al, Yan et al, as well as with cells that require KITL as cells, and However, studies with cultured cells have shown that has in and promotes a activation of KIT than et al, et al, Furthermore, studies with mice that the role in many in the While both and et al, 1991; et al, 1997; Tajima et al, and are at both and et al, et al, the of and in KITL functions are not known. studies revealed that KITL is a of the of et al, et al, a that in sequence et al, The of these are in the that are in an with A and and and et al, studies have been only with and it be of to whether the of and have the same the of its or may the of the of KITL. in the of and to their In postnatal testes, KITL is by Sertoli cells, KIT is by germ cells and by cells by et al, 1993). of the expression of Kitl mRNA and of KITL in Sertoli cells has important to the functions of KITL during spermatogenesis. of mast cells with Sertoli cells as an for KITL Tajima et that mast cell proliferation occurred only was direct the 2 cell This for that the biologically active of KITL from these Sertoli cells, which were from mice, was studies revealed that the of the 2 alternatively Kitl as well as of Kitl mRNA, are at stages of the testis (Manova et al, 1993; et al, 1993; et al, 1999). findings that is a for either or at different stages of spermatogenesis. of exon 6 is by that the Kitl mRNA, which is in an at a than Kitl mRNA et al, 1999). by et it is that the of the Sertoli cell KITL function by the of Kitl mRNA in of the Kitl on in spermatogenesis observed with a mouse mutant that it has been that is important than in the postnatal However, as it that an that is in sequence expression or Furthermore, the functions of during stages of spermatogenesis be by the and earlier functions of has been shown to both proliferation et al, 1993; et al, and meiotic et al, et al, of cultured spermatogonia. The of KITL in Sertoli cells also that KITL functions may at different stages of spermatogenesis. to KITL is in the of Sertoli cells but is in the region of Sertoli cells in testes and in stages of tubules of testes (Manova et al, 1993). Furthermore, a pattern for KITL in these cells was observed in stages and of testes, with from the to the et al, studies that the of KITL be in response to a during different stages of spermatogenesis KITL be for proliferation, KITL be for the of important for cells and for KITL and KIT function is the et al, 1991). This is for an in the of KIT and signaling of KITL by its to and that of mice a of A spermatogonia but not of spermatogonia and or or et al, 1991). The of was to expression of KIT in these cell in all spermatogonia spermatogonia are for and are for studies have confirmed that KIT is on but not spermatogonia and that promotes the proliferation and suppresses apoptosis of cultured spermatogonia et al, et al, 1999; Yan et al, The and differentiated spermatogonia is important the are to be stem cells. and a for stem cell that of testes cells testes et al, In study, was used to of spermatogonia from adult testes, and the that only and not have stem cell et al, It is interesting that the of either or for has an on the of testis cells. The testes of mice at to were to a for than were testes from adult mice at of et al, A for this is that about of the Kitl mRNA in the testes is Kitl mRNA, the of this mRNA to Kitl mRNA is greatly in mice (Manova et al, 1993). be in the testis than in the testis however, that to be it was that spermatogonia from spermatogonia from mice have adult et al, 2003). KIT expression is a of it be of to whether the of spermatogonia a of at of these cells to an spermatogonia may be stem cells from of adult stem cells, or KIT may have in the adult In to the role in has been that KITL signaling is in initiating maintaining or However, have been whether KIT is on meiotic and cells. may on the of in situ hybridization and the of used for or on the of germ cells testes sections cell For example, studies used in situ hybridization (Manova et al, and with et al, to Kit mRNA and KIT respectively, in mouse testis are in with the levels in spermatogonia and In both KIT expression was not in or this lack of KIT expression in meiotic and cells, with cultured spermatogonia have that KITL and KIT have in meiosis. of and of testis cell the of both Kit mRNA and KIT in was et al, In addition, of germ cells through in be using an Sertoli cell and this meiotic was shown to be on KITL and KIT et al, important in spermatogenesis has from the of a cell of A spermatogonia that was by of the of et al, that the of both meiotic and germ cells from the spermatogonia. Although both studies that KITL is a that the cells or the used in these in may not the in KITL a role in in an important to be is whether or not KIT is in meiotic cells. that KIT is in meiotic and germ cells in testis sections has been by two that used of that were different from used of a and KIT expression was on A and et al, 1999). This of in cells that were shown to lack that the KIT by may be or not on meiotic and cells. The that KIT is at high levels on these cells in has been by et used mice in which of the Kit gene was by a of a lacZ reporter gene. expression of the reporter was under all of the in their for Kit transcriptional regulation and the pattern of endogenous Kit In the adult reporter expression was observed in A spermatogonia as well as in and Furthermore, of the testes of these mice, which have reduced KIT function they are for the Kit by of the lacZ revealed a in et al, this a in or a in of to be all that KITL and KIT are for proliferation and of spermatogonia and for either or of in the The is to the molecular for these KITL and KIT have different functions in and are these functions through in KIT signaling are in these cells or in somatic cells of the testes that the of KITL and of these questions be through a of the signaling and of the and function of these The summarizes the information on the signaling with an emphasis on this signaling in spermatogonia. KITL signaling is for the development of germ cells, cells, mast cells, and Although KITL is a KIT is a receptor that has sequence and to the growth factor receptor, of et al, 1993; et al, KITL KIT and activation of its KIT is on and or to a of signaling A of signaling have been identified in cell et al, et al, et al, and the the also known as the 2 the and the the and on the of these signaling it has been to which or are to functions in and to whether of KIT are the same or different in different cell However, important information is from in and in studies and many have been that be for these complex that different signaling have been to complex signaling in a of cultured cells. This was used in two of KITL signaling in of mouse spermatogonia et al, et al, In both of to in stimulation of DNA Furthermore, both studies that the response of these cells to was by of that signaling through and that a is of a of the was shown in both studies to be in response to in response to the of and in cell in response to KITL are by both their are in the two In et that a response in spermatogonia from mice but not spermatogonia from mice. This is with about the expression of KIT in germ cells at these ages (Manova et al, et al, spermatogonia are and be and not to spermatogonia are differentiated and be and to et used spermatogonia from for their study, and these cells not be to be to KITL they were all that a response to KITL its with KIT, it is that the spermatogonia used by et were in In support of this the proliferation of spermatogonia from mice not younger was shown to be on KIT et al, as the of mice the of and the the of and cells in these testis cell from mice. In studies of KITL it be to potentially are for cells or the of and cells are The studies by et also that of apoptosis in spermatogonia not through or Although the was not revealed in this study, the that KITL signaling in spermatogonia may through different to apoptosis than to cell is known to be a aspect of spermatogenesis and its is to a of and by and Although a of known of apoptosis have been shown to function during spermatogenesis, little is known of the molecular from KITL signaling to apoptosis in testis cells. of to of rat tubules was shown to expression of known as and known as which have and to expression of which has functions et al, In addition, that the apoptosis that in the testes of mice with Kit is on et al, 1999), a known as and et al, a receptor known as factor receptor However, using of and that of germ cells from testes is of function et al, 2003). Although these studies for be to a of the mechanisms by which KITL affects the or of apoptosis in the interesting aspect of KITL signaling is that the used to proliferation of may be different from that used to proliferation of spermatogonia. though both and spermatogonia are on for their response to not to a role in in et al, as it in spermatogonia and in cell et that stimulation of with was by an and not by a Furthermore, proliferation of was with a of and not with an of the in studies of proliferation in and spermatogonia are with in studies and it is clear that signaling through is important for this in spermatogonia but not The of to signaling in cultured germ cells to be a in studies as in the studies to be in studies of KITL signaling are the or used to apoptosis of and the in germ cell development in which the response to KITL from to and the of that to the of and spermatogonia to KITL. for in signaling and spermatogonia has from studies of mice in the Kit gene. The is of only a Kit or Kitl that in of both et that spermatogenesis in even though the mice are with only about the of the reduced of is by development, the of the on may reflect in signaling and spermatogonia. The in the have not been identified to but important about KIT signaling in these cell studies that used mice in KIT that is an aspect of the signaling in spermatogenesis et al, et al, mice in which in the KIT was to This to KIT but for signaling mice that KIT and have reduced cell reduced skin mast cells, and reduced of and by et al, 1993; et al, the mice have skin mast cells, and While of mutant and were the mutant were development in the was up which proliferation and apoptosis of spermatogonia were observed and no meiotic or cells were these studies that signaling through is for KIT function in proliferation, and differentiation of spermatogonia in vivo. In addition, KITL signaling through may the of cells but not the or of these cells, and this may have a on spermatogenesis et al, 2003). Although it is to that signaling is not for KIT functions in cells, mast cells, or the of or that the of in these cells be The from the analysis of mice the lacZ in the and the of the of a in complex Although the the lacZ in Kit and the were through using gene in embryonic stem cells. In to a large of mouse in KIT and KITL were identified through to as classical In the we and summarize the information gained about spermatogenesis through analysis of Kitl mutant mice. In mice, KITL is encoded by the Steel (Sl) locus, and KIT is encoded by the Dominant White Spotting (W) locus by et al, 1993; et al, the classical have been used to and at both loci. information on and many mouse may be at The and at Although these were revealed on the of and development of several cell The of a or mutant is with in either or both mast cell and reduced as well as in the of are to in development of germ cells, cells stem cells, cells, and mast and and the of in cell are the products of the Sl and W were identified, expression studies in mice revealed that both are in many than be from the mutant it is known that several cell are in and mice, et al, et al, and and In addition, these mutant mice have been to have and (Motro et al, 1996). The molecular and of a for KITL are than different mutant all of which exert This in which a than that gene is to KITL important aspect of that for their in studies is that different that are with to are for of gene functions that may be or may be at DNA sequence have been identified in many mutant et al, for that have of the Kitl coding in the Kitl coding and that of the Kitl coding with coding or that in the absence of KITL have that as the Kitl of the and and an or with et al, et al, 2003). In addition, for have in development and in but they are not for studies of postnatal germ cell development they to the first the but have effects on et al, and effects on et al, and in and both of which contain coding that of KITL and are have been used in a of studies to the role of KITL during spermatogenesis. The an of kb from DNA that the and and potentially a but et al, 1991; et al, 1991). Although mice are and their is than that by et al, This that the a KITL In studies have revealed that the or are biologically active et al, 1991; et al, 1991). However, little is known about the in expression levels or of encoded the of different may be of which all lack the and and two contain sequences at the The of the in gene is in exon and two that by the or absence of exon as in be that express two It is that the absence of the or the of the sequences in greatly reduced expression of both of these at the cell or and the the cell at the site to a sequence is identical to that of However, the site and the of is for and and it is not clear whether this even be by the in vivo. The a that affects et al, that an and a with are encoded but the not the site for the of Although the is not for of the in the with its et al, In addition, is that the is a the sequences for of the mutant to and It is interesting that and that of the that the cell of it to the than to the of cells. that also in Sertoli cells, and KITL be at the for to a a for spermatogenesis in A for reduced KITL function in these is that the KITL may or Sertoli cells. and mice are with greatly reduced of germ cells to in development of by germ cells are in these at it is to direct effects of these on spermatogenesis from effects on this that from germ cell of the testes of these has revealed important information about KITL function during spermatogenesis. In all aspects of spermatogenesis, the are in than in mutants. This that be KITL function in the mice. the of germ cells, tubules in the testes contain germ cells that of spermatogenesis et al, of spermatogenesis by of in the that spermatogenesis is in mice with adult mice. studies have that and adult testes with to of spermatogonia and the in which these cells and In and at about or and respectively, of the tubules contain spermatogonia but no meiotic or cells et al, the from the of the that KITL and KIT are essential to the and proliferation of spermatogonia. However, in the of the of the spermatogonia were shown to be indicating that they are spermatogonia. This is with a that in all spermatogonia were at the which is to be the of spermatogonia and It is interesting that proliferation of spermatogonia in the is at a than in mice and et al, but the of these cells in the not of a high of apoptosis and growth cell factor is also by Sertoli cells and proliferation et al, It has been that the proliferation of spermatogonia in is to the mutant testes express a greatly of mRNA and the proliferation of these cells in the mutant testes was by of an et al, of germ cells tubules has been a for and molecular of stem cells. using and mice of different with to and this has of information about the functions of KITL and KIT during spermatogenesis. of spermatogonia from the testes of in spermatogenesis and of the germ cells et al, et al, The same occurred Kitl were testes by et al, and et al, et al, indicating that the of spermatogenesis in the mutant testes is not an to the that stem cells are in testes, and their differentiation is as as KITL is However, revealed the that testes have stem cells than even for the reduced of et al, This the interesting that KITL function is to stem cells in as in the mutant testes, in which the of these stem cells are a not the large of that stem cells are the cells are in a of molecular and are in germ testes, of which in either signaling or of germ cells to The in which cells from testes were mutant has also important an was with and mutant the spermatogonia and in et al, no differentiation of the cells was observed in these mutant testes, important that KITL function is for However, the of proliferating spermatogonia in the to the that endogenous spermatogonia in these mutant testes but not their of apoptosis and this it the the of endogenous spermatogonia in the mutant testes the of spermatogonia in the mutant testes Although many remain to be about KITL functions during spermatogenesis, it is clear that KITL has a role in the proliferation, and differentiation of spermatogonia and that this may be or by the of germ cells. the sequence in 10 mutant of which have little or no for mouse or development of cells, 2 of the and are for these et al, 2003). In the a in exon was identified that a in et al, a mutant product of only of be by of both Kitl and Kitl messenger In addition, of exon was observed and is to a of only the first of with to that this is to be et al, and it is that the is the only biologically active by the The from a that an in the and of the identified in mouse KITL While KITL from tissue cells is known to be at et al, et al, the that the absence of this has on KITL function in is not known. studies have revealed that the and exert effects on the of germ cells in the that in reduced of germ cells in the postnatal testes and in It is interesting that the testes of these with to the of in spermatogenesis and in of all mice for the or a in spermatogenesis to the of germ cells. However, analysis of younger mice to revealed that the and have identical but both have effects on spermatogenesis that are than that of In the many tubules lack germ cells, but the of tubules with germ cells is in this mutant than in or mutants. the and tubules from the contain all stages of and cells and in The of the on the testis from which it may be to aspects of KITL function in the It is interesting that the effects of the in the testis are different from their effects on cells and in For example, the has a on cells than the the has a on spermatogenesis than the Although the for these effects on different cells are not these that in the and either the cells the mutant cells Sertoli with to their expression or of KITL or that cells and spermatogonia in different to the mutant KITL Although it is clear that the functions of KITL in spermatogenesis are in differentiated spermatogonia and in several interesting questions remain to be about these are the molecular mechanisms that the KITL proliferation and of KITL in stem KITL not at all in these cells, or is it only under as in mice or in germ KITL and of are the molecular that its functions in meiotic germ the signaling of KIT different in male germ cells from cells, as cells or are and molecular have on the response of the of KITL function using or mouse with in either KITL or KIT, with in and testis cell of these and aspects of KITL The and two that the