Introduction

In the mammalian testis, spermatogenesis is a tightly regulated developmental process by which undifferentiated germ cells continuously produce mature sperm throughout a lifetime. Through several mitotic divisions, type A spermatogonial stem cells either renew themselves or differentiate into later-stage spermatogonia to eventually become pachytene spermatocytes [1]. Pachytene spermatocytes undergo two meiotic divisions and become haploid round spermatids, which eventually transform into mature elongated spermatozoa. The rigorous balance between cellular proliferation and cell death via apoptotic pathways, as well as the drastic changes in cell morphology, suggests the presence of a highly organized network of cell cycle regulatory mechanisms during different stages of spermatogenesis. It is increasingly clear that cyclins play a key role in governing a well-defined sequence of mitotic and meiotic divisions that characterize mammalian spermatogenesis. Some regulatory mechanisms of meiosis involving cyclins are similar to their mitotic counterparts, and other mechanisms are probably unique to meiosis.

Cyclins are regarded as key components of the machinery that drives cells through the cell cycle, and have been identified in a variety of organisms ranging from yeasts to humans. Based on their amino acid similarity and the timing of their appearance during the cell cycle, at least nine families of cyclin have been identified in mammals; from cyclin A to I. Cyclin is a regulatory subunit that regulates the activation state of catalytic subunit cyclin-dependent serine/threonine kinase (Cdk) in the cyclin-Cdk complex, through the phosphorylation and dephosphorylation of certain cites in the Cdk subunit. Although the role of cyclins in mammalian spermatogenesis is only beginning to be investigated, observations have shown that various cyclins exhibit distinct patterns of expression in male germ cell lineage. Multiple members of the A-, B-, and D-type cyclin families are most undervalued in recent research (Fig. 1).

Fig. 1
figure 1

Periodic expression of different cyclins during spermatogenesis in mammals. Cyclin A1 is present in late pachytene to diplotene spermatocytes in the male germ line. Cyclin A2 protein is present only in spermatogonia (data not shown) and preleptotene spermatocytes, and no longer detected when germ cells enter the leptotene stage. Cyclin B1 is present in primary spermatogonia at all stages of meiotic prophase and its presence is strongest in zygotene. It is then destroyed at meiotic anaphase I, resynthesized (to a lower level), and destroyed again at second meiotic anaphase. The curve of cyclin B2 is similar to cyclin B1; however, it is not first detected until early pachytene. Cyclin B3 is expressed in leptotene and zygotene phases, and accumulates to its highest expression level in zygotene. All three kinds of cyclin D are expressed in spermatogonia. To date, no cyclin D1 expression has been found in spermatocytes and spermatids, while cyclin D2 and D3 are found in spermatocytes (data not shown)

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The A-type cyclins

There have been only two kinds of A-type cyclins discovered in mammals; cyclin A1 (encoded by Ccna1) and cyclin A2 (encoded by Ccna2) [2, 3]. Cyclin A has remained somewhat enigmatic, partly due to the lack of homologues that exist in yeast. Mammalian cyclin A1 is a highly tissue-specific A-type cyclin. In mice, cyclin A1 is prominently expressed in the male germ cells in normal testis, and at very low levels in the brain [4] and peripheral blood [5, 6]. Relatively high levels of cyclin A1 have been detected in several leukemic cell lines and in the peripheral blood cells of patients with certain hematological malignancies [5]. Cyclin A1 is essential for progression through the meiotic cell cycle in spermatocytes. In contrast, cyclin A2 is expressed in all tissues analyzed. In the mouse, cyclin A1 and A2 share 44% identity at the amino acid level and share 84% identity within the two highly conserved cyclin boxes [2, 3].

Cyclin A1

The cyclin A1 gene (Ccna1) is expressed during meiosis and is vital for spermatogenesis. It has a similar pattern of expression in mice and humans and is likely to have the same essential role during spermatogenesis in both species. In the adult mouse testis, Ccna1 mRNA and protein are present in late pachytene to diplotene spermatocytes but not at significant levels during the second division of meiosis. Male mice lacking cyclin A1 protein were generated through homologous recombination in embryonic stem (ES) cells, and the offspring were healthy, but homozygous males cannot enter the first meiotic division during spermatogenesis, thus leading to sterility [7]. The meiotic arrest found to be accompanied by a defect in MPF (M-phase promoting factor) activation. Treated with the protein phosphatase type-1 and -2A inhibitor okadaic acid, the arrested germ cells restored the MPF activity and induced entry into M phase. Conversely, inhibition of tyrosine phosphatases by vanadate suppressed the okadaic acid-induced metaphase induction. It revealed that cyclin A1 participates in the regulation of other protein kinases or phosphatases critical for the G2–M transition in normal meiotic male germ cells. It has been proposed that the role of cyclin A1/Cdk may be to participate in the regulation of the phosphorylation of Cdc25 phosphatase, which is required for the activation of MPF, and therefore critical for the G2/M-phase transition in normal meiotic male germ cells [8]. Further study of the regulation of cyclin A1 kinase complexes, at both mRNA transcriptional level and post-translational level, will better position its role within the full context of the cell cycle regulatory network at the meiotic G2–M junction. Besides the defect in MPF activation, meiosis arrest in Ccna1–/- spermatocytes is associated with apoptosis as evidenced by TUNEL-positive staining. Adult testicular tubules exhibit severe degeneration and some tubules in the older animals are almost devoid of germ cells at various stages of spermatogenesis [9]. An unusual clustering of centromeric heterochromatin is also identified in the cyclin A1-deficient spermatocytes. Phosphorylation of histone H3 at serine 10 in pericentromeric heterochromatin (which normally occurs in late diplotene), and the level of pericentromeric aurora B kinase (known to phosphorylate histone H3 during meiosis) are both partially reduced in spermatocytes from testes of heterozygous mice and further reduced in homozygous null spermatocytes. These data suggest a critical function for cyclin A1 in the pericentromeric region in the late diplotene stage of meiosis, perhaps in assembly of the passenger protein [10].

The molecular features and functions of cyclin A1 are still not well understood. It has been shown that methylation of the CpG island of the human cyclin A1 promoter, is correlated with nonexpression in cancer cell lines. However, expression in the testes of mice is independent of promoter methylation, and even strong promoter methylation did not suppress promoter activity. Appropriate tissue-specific repression of the cyclin A1 promoter may occur independently of CpG methylation [11]. Other research reveals that the fragment of mouse Ccna1 spanning −1.3 to +0.8 kb from the putative transcriptional start site contains sequences necessary for the stage-specific expression of Ccna1 in meiotic cell cycle. These sequences also appear to maintain repression of Ccna1 in the correct adult tissues and stages of spermatogenic differentiation. Further, sequences located between −4.8 and −1.3 kb appear to contribute to enhanced expression of Ccna1 [12]. Evidenced by TUNEL-positive staining in the testes during the first wave of spermatogenesis, it was recently demonstrated that the apoptotic response to the absence of cyclin A1 in cyclin A1-deficient spermatocytes involves upregulation of Bax and subsequent activation of caspase 3. Furthermore, mice lacking both cyclin A1 and p53 were generated, and the double-mutant testes revealed a decrease in the number of apoptotic cells. These findings suggest that p53 may be involved in removing the arrested germ cells from the seminiferous tubules [13]. Additionally, the apoptotic response was partially rescued in the presence of p53, although the triggers for this apoptotic response have not yet been determined. It was also indicated that cyclin A1 interacts with p53 and phosphorylated p53 in complex with Cdk2. Ccna1-deficient testes accumulate unrepaired DNA double-strand breaks as detected by immunohistochemistry for phosphorylated H2AX. This suggests that Ccna1-deficiency in spermatogenesis is associated with defects in DNA double-strand break repair, which is enhanced by the loss of p53 [14]. Dmrt7 is a novel gene expressed only in testes of adult mice. Expression of Ccna1 mRNA decreased significantly in Dmrt7-knockout mice (Dmrt7 −/−), and these null males were infertile. No sperm was detected in a histological analysis [15]. It suggests that Dmrt7 may be involved in the regulation of cyclin A1 at the pachytene stage. Diederichs et al. identified several novel proteins (INCA1, KARCA1, and PROCA1) as cyclin A1/CDK2 interaction partners in a yeast triple-hybrid approach. Interactions were confirmed by GST pull-down assays and co-immunoprecipitation. INCA1, the most frequently isolated unknown gene, was cloned and their expression pattern in testis development was studied [16]. The research provides evidence that the cyclin A1-CDK2 complex plays a role in several signaling pathways important for cell cycle control and meiosis and established a new basis for functional analyses of cyclin A1.

Cyclin A2

Cyclin A2 has different biological functions to cyclin A1. In cultured cells, it can bind and activate Cdk1 and Cdk2, functioning during both the G1-S and G2-M phases. The cyclin A2/Cdk complex phosphorylates histone H1 and the pocket protein family member p107 in vitro. Targeted mutagenesis of Ccna2 has revealed that it is essential for normal development. It has been shown that Ccna2-/- mice exhibit early embryonic lethality [17]. This embryonic lethality has obviated studies to determine the function of cyclin A2 in spermatogenesis. The use of conditional targeted mutagenesis, and perhaps RNAi, would be helpful in elucidating the role of cyclin A2 during spermatogenesis.

While cyclin A2 appears to be almost ubiquitously expressed in the mouse male germ line, cyclin A2 protein is present only in spermatogonia and preleptotene spermatocytes, earlier than those in which cyclin A1 is detected. Cyclin A2 is no longer detected as germ cells enter the leptotene stage. In immature animals in which spermatogenesis has not proceeded beyond early pachytene stages, the expression of cyclin A2 in preleptotene spermatocytes was highly synchronous in germ cells about to enter meiosis [18]. The specific and nonoverlapping (with cyclin A1) presence of cyclin A2 in the testis indicates unique conserved biological functions for these two distinct A-type cyclins in male germ cell development. Their differential expression patterns may correlate with their specific roles in spermatogenesis. Elevated levels of cyclin A2 and histone kinase activities of cyclin A2/Cdks have been detected in the majority of male germ cell tumors (GCTs) including carcinoma in situ (CIS), seminoma, and nonseminoma GCTs, and were correlated with the transformation and progression of the tumors, suggesting that aberrant expression of cyclin A2 and its Cdks is a significant factor in male germ cell tumorigenesis [19]. Thyroid hormone receptor-beta1 (TRbeta1) belongs to the ligand-inducible transcription factor superfamily. The expression of cyclin A2 upon serum stimulation in TRbeta1 expressing cells was investigated. There existed a strong downregulation of cyclin A2 mRNA, concomitant with low protein level. It reveals that TRbeta1 can inhibit of the transcriptional activation of cyclin A2 [20]. Wilms’ tumor 1-associating protein (WTAP) has been reported to be a ubiquitously expressed nuclear protein. WTAP-knockout mice exhibited proliferative failure and early embryonic lethality, an etiology almost identical to Ccna2-/- mice. Moreover, WTAP knockdown via small interfering RNA induced G2 accumulation, which can be partially rescued by adenoviral overexpression of cyclin A2 [21]. These findings establish WTAP as an essential factor for the stabilization of cyclin A2 mRNA, thereby regulating G2/M cell-cycle transition.

The B-type cyclins

In mammalian cells, M-phase promoting factor (MPF), also known as “maturation promoting factor, ” consists of Cdc2 kinase and a B-type cyclin. It is the most clearly defined Cdk-cyclin complex compared with other cyclin-Cdk complexes in the cell cycle. MPF triggers the G2/M-phase transition during the mitotic and meiotic cell cycles. Activation of MPF at the end of G2 is controlled by the accumulation and binding of cyclin B to Cdc2. During mitosis, cyclin B increases steadily during the interphase, peaks at the G2/M-phase transition, and is then destroyed. This destruction leads to the inactivation of the MPF and finally causes the cell to exit mitosis. To date, three B-type cyclins have been found in mammals. While expected to have crucial role in germ cell development, only a limited number of studies have focused on the role of cyclins in spermatogenesis. Cyclin B and its kinase are known to have an association with spermatogonial and early spermatocyte proliferation in the testis.

Cyclin B1 and B2

Cyclin B1 and B2 were the first two B-type cyclins identified in mammals, and both of them are expressed in tissues that contain dividing cells such as the gut, spleen, ovaries, and testis. In the mammalian testis, mRNA expression of two B-type cyclins correlates with second meiotic division and early round spermatid development, but they have different expression patterns in the mouse testis. Cyclin B1 is present in primary spermatogonia at all stages of meiotic prophase, and its presence is strongest in zygotene. It is destroyed at meiotic anaphase I, resynthesized (to a lower level), and destroyed again at second meiotic anaphase. In contrast, cyclin B2 is barely detectable until early pachytene, and disappears at anaphase like cyclin B1. Neither of them is detected in postmeiotic spermatids. These different patterns of expression may indicate that cyclin B1 can compensate for the loss of cyclin B2 in spermatogenesis. There are also some differences in subcellular localization between cyclin B1 and B2. While cyclin B2 is membrane associated, cyclin B1 can also be found in the cytoplasm [22].

Although cyclin B2-null mice of both sexes prove to be fertile, the litter sizes of homozygous mating pairs are consistently smaller than those of their heterozygous littermates. In contrast, homozygous B1-null mice exhibit early embryonic lethality [22]. Hsp70-2, the only member of HSP family expressed in spermatogenic cells at a significant level, is shown to play an essential role in cyclin B1/CDC2 assembly in pachytene spermatocytes as a molecular chaperone for the meiotic cell cycle in spermatogenesis when checked by immunoprecipitation-coupled western blot and in vitro reconstitution experiments. Furthermore, addition of HSP70-2 to testis from Hsp70-2(-/-) mice not only restored cyclin B1/Cdc2 complex formation but also reconstituted Cdc2 kinase activity in vitro [23]. These studies provide novel evidence for a link between an HSP70 molecular chaperone and Cdc2 kinase activity, which are essential for the meiotic cell cycle in spermatogenesis. Comparison of the crystal structure of human cyclin B1 to 2.9 A, with cyclin A and cyclin E, reveals remarkably similar N-terminal cyclin box motifs but significant differences among the C-terminal cyclin box lobes [24]. Examination of the structure provides insight into the molecular basis for differential affinities of protein-based cyclin/Cdk inhibitors such as p27, substrate recognition, and Cdk interaction. Selenium was recently reported to induce oxidative stress, significantly affect the expression of cyclin B1 in spermatogenic cells, and lead to apoptosis in germ cells and male infertility in male mice, which were fed a deficient diet supplemented with Se [25]. This effect of Se-induced oxidative stress on the cell cycle regulators and apoptotic activity of germ cells provides new dimensions to molecular mechanisms underlying male infertility. Deletion of the DNA methyltransferase genes DNMT1 and DNMT3b leads to an increase in transcription of cyclin B2. Furthermore, DNA methylation in vitro prevents transcriptional activation of the cyclin B2 promoter in G2/M. Analysis in vivo of the cyclin B2 core promoter revealed that the CDE/CHR (cell cycle-dependent element and cell cycle genes homology region) is partially methylated. However, CpG methylation is independent of the cell cycle indicated by quantitative in vivo analysis of the CpG-methylation level of the CDE during cell division [26]. Collectively, DNA methylation affects cell cycle-dependent transcription of cyclin B2 but that regulation through CDE/CHR is independent of cytosine methylation. Justin D et al. recently have applied a novel method for rapid identification of protein kinase substrates, thus identified more than 70 Cdk1-cyclin B substrates and phosphorylation sites. Cdk1 was engineered to accept an ATP analog that allows it to label its substrates with a bio-orthogonal phosphate analog tag. Using a highly specific, covalent capture-and-release methodology, tagged peptides derived from labeled substrate proteins can be rapidly purified [27]. This approach has the potential to expand our understanding of cyclin/Cdk and other connections in signaling networks.

Cyclin B3

Cyclin B3, a more distant B-type cyclin in mammals, was originally found to express in chickens, frogs, flies, and nematode worms but not in mammals. Cyclin B3 (Ccnb3) is conserved from Caenorhabditis elegans to Homo sapiens and expressed in both males and females. Cyclin B3 is an early meiotic cyclin that is expressed in leptotene and zygotene phases during spermatogenesis, accumulates to its highest expression levels in zygotene, and diminishes in pachytene. Its unique expression pattern indicates it may either regulate events during the leptotene and zygotene phases, such as recombination or synapsis, or its turnover may be important for proper exit of cells from zygotene and their progression into pachytene [28]. In transgenic mice, prolonged cyclin B3 expression to the end of meiosis leads to severe defects in spermatogenesis and a reduction in sperm counts. Transgenic testes reveal some tubules that are depleted of germ cells, some containing an abnormal cell mass in the lumen, and others with round spermatid defects. These distinct morphological defects were accompanied by increased apoptosis in the seminiferous tubules of transgenic mice [29]. It indicates that downregulation of cyclin B3 at the zygotene-pachytene transition is required to ensure normal spermatogenesis. The function of cyclin B3 in male germ line cells is still not clear. Further studies on the effect of deregulating cyclin B3 expression, and identification of cyclin B3-interacting proteins in the leptotene and zygotene cells, will provide increased understanding of cyclin B3 function.

The D-type cyclins

In mammals, three D-type cyclins have been identified: D1, D2, and D3. The genes are located on different chromosomes but share substantial homology and are expressed in a tissue-specific manner. D-type cyclins are proto-oncogenic components of the RB pathway, a G1/S regulatory mechanism centered on the retinoblastoma (Rb) family of proteins that control cell progress through the G1/S restriction point. Each of them is able to regulate G1/S transition in vitro and has been reported to form complexes with Cdk2, Cdk4, Cdk5, and Cdk6. Unlike other cyclins that fluctuate periodically during the cell cycle, the levels of D-cyclins are induced by and respond to extracellular growth factors [30], and exhibit only moderate oscillations during the cell cycle with peak levels achieved near G1-S. The three D-type cyclins are differentially expressed in a number of isolated cell types and cell lines, indicating distinct roles in cell cycle regulation in particular cell lineages. In the testis, the expression patterns of cyclins D1, D2, and D3 only partly overlap, which suggests different roles for these D-type cyclins during spermatogenesis. Generally speaking, cyclin D1 and D3 seem to be involved in cell cycle regulation, whereas cyclin D2 is likely to have a role in spermatogonial differentiation. The roles of the D-type cyclins during spermatogenesis are undefined in many respects and require further investigation.

Cyclin D1

Cyclin D1 was the first D-type cyclin found in mammals. Increased cyclin D1 levels have been found in two opposing cell cycle situations; cell proliferation and cell cycle arrest. Several factors such as retinoblastoma protein [31], p53 [32], and p21Cip1/WAF1 [33] involved in G1/S cell cycle control are able to regulate cyclin D1 synthesis. Using immunohistochemistry, the expression of the cyclin D1 was studied in the developing and adult mouse testis. Cyclin D1 is expressed only in spermatogonia throughout stages of the seminiferous epithelium, indicating a role for cyclin D1 in the regulation of spermatogonia proliferation during the G1/S phase transition in particular [34]. No cyclin D1 expression has been found in spermatocytes and spermatids to date. Sertoli cells are also found lacking cyclin D1 expression, while results of Ravnik and colleagues showed that Sertoli cells in the adult mouse testis express cyclin D1 mRNA detected by Northern and in situ hybridization in the testis [35]. This may suggest low translational activity or a high turnover of cyclin D1 protein in these cells. The subcellular localization of cyclin D1 and the molecular and biochemical pathways in proliferating spermatogonia remain unclear. Human apoptosis related protein (Apr3) was demonstrated to be obviously upregulated by All-trans-retinoic acid (ATRA) in many ATRA sensitive cells. FACS analysis showed that Apr3 overexpression could cause an obvious G1/S phase arrest. Furthermore, truncated Apr3 antagonized the negative role of Apr3 on cell cycle and cyclin D1 [36]. Taken together, these data suggest that Apr3 should play an important role in ATRA signal pathway and is vital for the negative regulation on cell cycle and cyclin D1.

Cyclin D2

Cyclin D2 is first expressed at the start of spermatogenesis when gonocytes produce A1 spermatogonia. In the adult testis, cyclin D2 is expressed in spermatogonia around stage VIII of the seminiferous epithelium when undifferentiated Aal spermatogonia differentiate into differentiating A1 spermatogonia. Cyclin D2 is also expressed in spermatocytes and spermatids. In spermatocytes, cyclin D2 was detected in the nuclei of pachytene, which at that time, are in the prophase stage of first meiotic division. Cyclin D2 mRNA is also found present in Sertoli cells during testicular development [37]. Frequently elevated levels of cyclin D2 were found in some invasive testicular tumors, including seminomas and embryonal carcinomas, and in cell lines derived from such tumors [38]. These findings extend our concepts of the biology of the cyclin D3 as well as oncopathology of the human adult testis. They also inspire further research into the emerging role of the cyclin D proteins in the establishment and/or maintenance of differentiated phenotypes. Cyclin D2-deficient male mice are fertile but display hypoplastic testes. It is recently reported that capillary morphogenesis gene (CMG)-1 may play a unique role in the transcriptional regulation of the cyclin D2 gene, through a reporter assay using a genomic region upstream of the mouse cyclin D2 gene. When CMG-1 expression was attenuated by a target-specific siRNA in a mouse spermatocyte-derived cell line, the morphology of the cells was changed and the expression of cyclin D2 was abrogated [39]. This novel upregulated germ line stem gene provides useful information for the future investigation of spermatogonial stem cells and the early phase of male germ cell differentiation. Tan reported a robust, stage-specific upregulation in cyclin D2 expression induced by the blockage of androgen action in Sertoli cells. Either treatment of adult rats with flutamide or Sertoli cell-selective ablation of the androgen receptor (AR) in mice using Cre/loxP technology resulted in stage-dependent cyclin D2 immunoexpression in Sertoli cells. Confocal microscopy revealed that immunoexpression of AR and cyclin D2 was mutually exclusive within individual seminiferous tubules in flutamide treated rats. These results pointed out that AR expression and androgen action upregulate the expression of cyclin D2 in mature Sertoli cells, which are involved in the regulation of spermatogenesis [40].

Cyclin D3

In the adult testis, cyclin D3 protein is found in some spermatogonia, spermatocytes, spermatids, Sertoli cells and Leydig cells, suggesting that cyclin D3 may control G1/S transition in spermatogonia but has a different role in Sertoli and Leydig cells. Cyclin D3 is highly expressed in spermatogonia in the early stages of spermatogenesis. As the germ cells enter into meiosis and progress to the leptotene stage, cyclin D3 is reduced to undetectable levels. However, at later stages of spermatogenesis, cyclin D3 mRNA is expressed in haploid, round spermatids. It is supposed that cyclin D3 might have dual functions during spermatogenesis; regulation of proliferation in spermatogonia, and a role in noncell cycle functions such as specialized morphogenetic differentiation and chromatin remodeling. Cyclin D3 forms complexes with both Cdk4 and p27 in the testis but not with Cdk2 or Cdk5 when detected by immunoprecipitation and immunoblotting. Cdk4 co-localized with cyclin D3 only in spermatogonia but not in elongating spermatids, which suggests that the role of cyclin D3 during spermatogenesis may be mediated by different partners in a cell type specific pattern. The kinase activity of cyclin D3 on histone H1 was also reported in the mouse testis, with the highest levels detected in day 7 postnatal testicular lysates and decrease during development [41]. Other research revealed that stem cell factor (SCF)/c-kit recruits the rapamycin-sensitive PI3-K/p70S6K signaling pathway to induce cyclin D3 expression and further phosphorylation of Rb leading to promoted spermatogonial cell proliferation [42]. These observations suggest unique roles for cyclin D3 in the regulation of cell proliferation and differentiation in the germ line, and the differential regulation of mitotic and meiotic cell cycles during spermatogenesis.

Other cyclins

Cyclin E is believed to control G1/S phase progression. It is associated with Cdk2 and activates kinase activity shortly before entry of cells into the S phase. Two mammalian E-type cyclins, E1 and E2, have been recognized and considered analogous to the two A-type cyclins. Both cyclin E1, and E2 expressions have been detected in adult mice testes, and the expression patterns of the two cyclins in the tissues of adult mice are not identical [43]. Cyclin E1- and E2-deficient mice show apparently normal development [44], however, cyclin E2-deficient male mice exhibit reduced fertility, with about one-half of the males being sterile. Testes of the mutant males exhibited reduced numbers of spermatogenic cells resulting in an overall atrophy of the testes. Therefore, cyclin E may also play a vital role during spermatogenesis in mammals.

Other types of cyclins have not been examined in detail during spermatogenesis. More investigation of other cyclins is needed to define their expression and function in male germ line cell proliferation and differentiation.

Perspectives

Our current understanding of the various types of cyclins and their Cdk counterparts during mammalian spermatogenesis is still not clear, and some types have been overlooked. While many cyclin-deficient and cyclin-overexpression transgenic mice have been produced, the clues provided are insufficient to solve the enigmatic role of cyclins in the regulation of spermatogonial multiplication and stem cell renewal. Little is known about multiple cyclin-dependant signaling pathways in the differentiation and self-renewal of male germ line cells in mammals. Take cyclin A1 for instance. The target molecules of cyclin A1/Cdk2 are unknown and the mechanisms of apoptosis associated with Ccna1-/- spermatocytes are still not well elucidated. Interestingly, some members of the Cdk family have not been found to bind with cyclin. Their association with cyclin and involvement in cell cycle regulation remain unclear and require further investigation.

A search for specific inhibitors of cyclin–Cdk complexes, use of conditional targeted mutagenesis, and use of the novel specific gene silencing method by RNAi, would facilitate further investigation of mammalian cyclins. The inhibitors of cyclin-Cdk complexes frequently trigger apoptosis and in some instances they appear to contribute toward differentiation. Certain inhibitors can induce apoptosis in dividing cancer cells, but protect normal cells from apoptosis. These properties of the inhibitors may provide opportunities in testicular tumor treatment. Understanding the genetic program controlling mitotic and meiotic divisions of the germ line [45–48] will provide an insight into understanding infertility and provide a new perspective on the treatment of male sterility and human testicular tumors.