Spermatogenesis is a highly complex cell differentiation process that is governed by unique transcriptional regulation and massive chromatin alterations, which are required for meiosis and postmeiotic maturation. The underlying mechanisms involve alterations to the epigenetic layer, including histone modifications and incorporation of testis-specific nuclear proteins, such as histone variants and protamines. Histones can undergo methylation, acetylation, and phosphorylation among other modifications at their N-terminus, and these modifications can signal changes in chromatin structure. We have identified the temporal and spatial distributions of histone H3 mono-, di-, and trimethylation at lysine 4 (K4), and the lysine-specific histone demethylase AOF2 (amine oxidase flavin-containing domain 2, previously known as LSD1) during mammalian spermatogenesis. Our results reveal tightly regulated distributions of H3-K4 methylation and AOF2, and that H3-K4 methylation is very similar between the mouse and the marmoset. The AOF2 protein levels were found to be higher in the testes than in the somatic tissues. The distribution of AOF2 matched the cell- and stage-specific patterns of H3-K4 methylation. Interaction studies revealed unique epigenetic regulatory complexes associated with H3-K4 methylation in the testis, including the association of AOF2 and methyl-CpG-binding domain protein 2 (MBD2a/b) in a complex with histone deacetylase 1 (HDAC1). These studies enhance our understanding of epigenetic modifications and their roles in chromatin organization during male germ cell differentiation in both normal and pathologic states.

The determination for early cleavage-stage embryos of noninvasive morphologic and metabolic criteria that are predictive of blastocyst development and/or full-term viability remains an important research target. We describe the derivation of a logistic regression model that predicts the probability of porcine blastocyst formation in vitro. Pig zygotes, derived by in vitro maturation and fertilization of slaughterhouse oocytes, were cultured in NCSU-23 medium that was supplemented with a mixture of 20 amino acids (NCSU-23_aa). On Day 1, at 21, 23, 25, 27, 29 and 31 h postinsemination, cleaving embryos were evaluated morphologically in terms of the: i) number of blastomeres, ii) evenness of division, and iii) degree of fragmentation. These embryos were then placed in 1.5-μl drops of NCSU-23_aa for 24 h, after which time the three morphologic criteria were re-evaluated and 1.2 μl of spent medium were removed for analysis by HPLC, in order to determine the net rates of amino acid depletion and appearance. Embryos were then cultured singly in NCSU-23_aa by placing them between the filaments of a woven polyester mesh until Day 6, in order to permit the identification of individual embryos. Of 256 cleaved embryos, 28.7 ± 6.2% (n = 5 replicates) developed into blastocysts. Discriminant analysis was used to select a subset of amino acids (threonine, valine, lysine, and phenylalanine) that discriminated optimally between embryos that became blastocysts or degenerated. These discriminant scores were entered into the logistic regression. Significant univariate relationships were established between the probability of blastocyst development and amino acid score (odds ratio [OR] 0.53, 95% confidence interval [CI] 0.40–0.69, P < 0.001), cleavage time (OR 0.79, 95% CI 0.71–0.87, P < 0.001), degree of fragmentation on Day 1 (OR 0.55, 95% CI 0.35–0.84, P = 0.009) and Day 2 (OR 0.53, 95% CI 0.35–0.78, P = 0.002), evenness of division on Day 2 (OR 0.66, 95% CI 0.46–0.96, P = 0.028), and categorical values of blastomere number on Day 2 (all P < 0.02), although no single variate could accurately predict blastocyst formation. However, multivariate analysis of the cell numbers on Day 1 and Day 2 correctly classified 51.9% of the predicted blastocysts. The inclusion of cleavage time in the regression analysis raised this rate to 63.5%, which was increased to 66.2% by the addition of evenness of division and degree of fragmentation. Finally, the full logistic regression model, which incorporated amino acid score together with all the other morphologic and kinetic variables, correctly classified 80.8% of the predicted blastocysts. This represented 51.2% of the observed blastocysts. Our data are novel in that they not only define in a quantitative manner the influence of previously undescribed predictors of porcine blastocyst formation, but they also provide a simple model of preimplantation development with reasonable predictive accuracy. The present study also provides a basic model for the examination and incorporation of additional early morphologic and metabolic correlates of developmental competence and could potentially be applied to the selection of human embryos for transfer in clinical IVF.

The 26S proteasome, which is a multi-subunit protease with specificity for substrate proteins that are postranslationally modified by ubiquitination, has been implicated in acrosomal function and sperm-zona pellucida (ZP) penetration during mammalian fertilization. Ubiquitin C-terminal hydrolases (UCHs) are responsible for the removal of polyubiquitin chains during substrate priming for proteasomal proteolysis. The inhibition of deubiquitination increases the rate of proteasomal proteolysis. Consequently, we have hypothesized that inhibition of sperm acrosome-borne UCHs increases the rate of sperm-ZP penetration and polyspermy during porcine in vitro fertilization (IVF). Ubiquitin aldehyde (UA), which is a specific nonpermeating UCH inhibitor, significantly (P < 0.05) increased polyspermy during porcine IVF and reduced (P < 0.05) UCH enzymatic activity measured in motile boar spermatozoa using a specific fluorometric UCH substrate, ubiquitin-AMC. Antibodies against two closely related UCHs, UCHL1 and UCHL3, detected these UCHs in the oocyte cortex and on the sperm acrosome, respectively, and increased the rate of polyspermy during IVF, consistent with the UA-induced polyspermy surge. In the oocyte, UCHL3 was primarily associated with the meiotic spindle. Sperm-borne UCHL3 was localized to the acrosomal surface and coimmunoprecipitated with a peripheral acrosomal membrane protein, spermadhesin AQN1. Recombinant UCHs, UCHL3, and isopeptidase T reduced polyspermy when added to the fertilization medium. UCHL1 was detected in the oocyte cortex but not on the sperm surface, and was partially degraded 6–8 h after fertilization. Enucleated oocyte-somatic cell electrofusion caused polarized redistribution of cortical UCHL1. We conclude that sperm-acrosomal UCHs are involved in sperm-ZP interactions and antipolyspermy defense. Modulation of UCH activity could facilitate the management of polyspermy during IVF and provide insights into male infertility.

The angiotensin-converting enzyme (ACE) plays a crucial role in male fertilization and is a key regulator of blood pressure. Testicular ACE (tACE), the germinal specific isozyme expressed on different promoters, exclusively carries out the role of ACE in fertility, although the site and mode of action are not well known. To investigate the contribution of tACE in fertilization, we produced transgenic mouse lines carrying a dipeptidase-inactivated mutant. Although the transgenic mice showed normal blood pressure, kidney morphology, and fertility, reduced fertilization was observed after in vitro fertilization (IVF). The sperm-zona pellucida (ZP) binding was exclusively impaired in these lines in a manner similar to that observed in an Ace knockout mouse. The dipeptidase activity was reduced in epididymal ingredients but not in the testis. Furthermore, direct application of mutant protein did not suppress sperm-ZP binding of intact sperm during IVF, implying that the dipeptidase-inactivated mutant affects sperm modification in the epididymis for ZP binding. Our results indicate that the dipeptidase-inactivated tACE acts in vivo, suggesting that tACE contributes to fertilization as a dipeptidase at least in the epididymis.

Creating transgenic mammals is currently a very inefficient process. In addition to problems with transgene integration and unpredictable expression patterns of the inserted gene, embryo loss occurs at various developmental stages. In the present study, we demonstrate that this loss is due to chromosomal damage. We examined the integrity of chromosomes in embryos produced by microinjection of pronuclei, intracytoplasmic sperm injection (ICSI), and in vitro fertilization (IVF)-mediated transgenesis, and correlated these findings with the abilities of embryos to develop in vitro and yield transgenic morulas/blastocysts. Chromosomal analysis was performed after microinjection of the pronuclei in zygotes, as well as in parthenogenetic and androgenetic embryos. In all the pronuclei injection groups, significant oocyte arrest and increased incidence of chromosome breaks were observed after both transgenic DNA injection and sham injection. This indicates that the DNA damage is a transgene-independent effect. In ICSI-mediated transgenesis, there was no significant oocyte arrest. The observed chromosomal damage was lower than that after pronuclei microinjection in zygotes and was dependent upon the presence of exogenous DNA. The occurrence of DNA breaks, as measured by comet assay performed on the sperm prior to ICSI, showed that DNA damage was present in the sperm before fertilization. Embryonic development in vitro and transgene expression at the morula/blastocyst stage were higher in ICSI-mediated transgenesis than after microinjection of pronuclei into zygotes. Sperm-mediated gene transfer via IVF did not affect chromosome integrity, allowed good embryo development, but did not yield any transgenic embryos. The present study demonstrates that DNA damage occurs after both the microinjection of pronuclei and ICSI-mediated transgenesis, albeit through different mechanisms.

Paf (1-o-alkyl-2-acetyl-sn-gylcero-3-phosphocholine) is a putative autocrine survival factor for the preimplantation embryo. It acts to induce receptor-mediated calcium transients in the early embryo. Inhibitors of 1-o-phosphatidylinositol-3-kinase (PI3kinase), such as wortmannin and LY 294002, blocked these calcium transients, implicating the generation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in autocrine signal transduction in the early embryo. Perfusion of the embryo cytoplasm with a blocking antibody to PIP3 inhibited paf-induced calcium transients and hyperpolarization of the membrane potential. Furthermore, direct infusion of PIP3 into the embryo induced a nifedipine (10 μmol/L)- and diltiazem (10 μmol/L)-sensitive calcium current in the 2-cell embryo. PIP3 acts as a docking site on membranes for proteins that contain pleckstrin homology domains, such as the thymoma viral proto-oncogene protein (AKT) and phospholipase C gamma. The 2-cell embryo expressed three genes for AKT (Akt 1–3) and two genes for phospholipase C gamma (Plcg1 and Plcg2), and we confirmed the expression of both AKT and phospholipase C gamma 1 by immunolocalization. Paf induced increased accumulation of serine 473-phosphorylated AKT in the region of the plasma membrane, consistent with its recruitment to membrane PIP3. Inhibitors of PI3kinase, such as LY294002, and of AKT, e.g., deguelin and AKT-inhibitor, reduced zygote development in a dose-dependent manner, and this inhibition was partially reversed by the addition of paf to the culture medium. These results provide the first direct evidence that PIP3 and its responsive signaling pathways act in the 2-cell embryo. Since signal transduction via PI3kinase has important roles in governing the cell survival pathways, these results support the hypothesis that autocrine embryotropins, such as paf, act as survival factors.

Using two species of teleost fish, Japanese huchen (Hucho perryi) and common carp (Cyprinus carpio), we investigated whether sex steroids are involved in early oogenesis in vitro. Ovarian fragments were cultured to examine the effects of a progestin, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP), and an estrogen, estradiol-17 beta (E2). DHP and E2 significantly promoted DNA synthesis in ovarian germ cells, as judged by 5-bromo-2-deoxyuridine (BrdU) incorporation into these cells. Furthermore, to detect the initiation of the first meiotic division of early oogenesis, we assessed ultrastructurally the occurrence of synaptonemal complexes (SCs) and analyzed by immunohistochemistry the expression of a meiosis-specific marker, Spo11. In huchen, a higher percentage of oocytes with SC was seen in DHP-treated ovarian fragments than in control or E2-treated ovarian fragments. Spo11 was expressed in germ cells after DHP treatment of carp ovarian explants. These data suggest that the progression of germ cells through early oogenesis involves two sex steroids: E2, which acts directly on oogonial proliferation, and DHP, which acts directly on the initiation of the first meiotic division of oogenesis.

Nuclear receptors of the peroxisome proliferator-activated receptor (PPAR) family are implicated in implantation and early placental formation. In carnivores, the trophoblast invades to develop intimate contact with the endothelial cells of the maternal circulation, resulting in an endothelio-chorial form of placentation. Spatio-temporal investigation demonstrated that peroxisome proliferator-activated receptor gamma (PPARG) was strongly and specifically expressed in the mink trophoblast at the time of formation of the syncytiotrophoblast during early implantation, and in trophoblast of the placental labyrinth. The retinoid-X-receptor alpha (RXRA), the heterodimeric partner of PPARG in transcriptional regulation, is, with very few exceptions, co-expressed with PPARG in mink trophoblast. We used mink trophoblast cell lines together with a natural (15-deoxy-delta^12,14-prostaglandin J₂ ) or a synthetic (troglitazone) PPARG ligand to demonstrate that PPARG is an authentic regulator of gene expression in this tissue. Ligand-activated PPARG stimulated transcription of the PPRE-luc reporter gene transfected into these cell lines. The prostaglandin-induced morphologic changes were accompanied by attenuation in cell proliferation, an increase in PPARG mRNA and protein levels, and the appearance of enlarged and multinuclear cells. Furthermore, 15-deoxy-delta^12,14-prostaglandin J₂ stimulated the expression of invasion-related genes in trophoblast cells, namely, adipophilin and osteopontin. The results demonstrate that PPARG ligands attenuate proliferation and induce differentiation of mink trophoblast cells to the multlinuclear phenotype. The upregulation of differentiation-specific genes in the placenta under the influence of PPARG ligands provides a mechanism by which blastocyst and endometrial prostanoids regulate implantation, as well as the formation and maintenance of the placenta.

Fungi of the Fusarium species can infect food and feed commodities and produce the mycotoxins zearalenone (ZEA) and deoxynivalenol (DON). Since both toxins have been reported to reduce fertility, the mechanisms of ZEA and DON on inhibition of oocyte maturation were examined. Pig oocytes were matured in the presence of ZEA (a mycotoxin with estrogenlike activity), 17beta-estradiol, and DON (all 3.12 μmol/L). Zearalenone, 17beta-estradiol, and DON inhibited oocyte maturation and caused approximately 34% of the oocytes to form an aberrant spindle. Different ratios of ZEA:DON did not lead to a more severe inhibition of oocyte maturation. Both mycotoxins caused abnormal formation of the meiotic spindle. The developmental competence of oocytes matured in the presence of mycotoxins was further investigated after in vitro fertilization. Presence of ZEA (3.12 μmol/L) during maturation reduced the percentages of oocytes that cleaved and formed a blastocyst to about 12%, compared with 25% of control oocytes. Maturation in the presence of equimolar concentrations of DON was not compatible with development. The ploidy of blastomeres from blastocysts derived from mycotoxin-exposed oocytes was analyzed with fluorescent in situ hybridization. All blastocysts, even those from the control group, contained at least one blastomere with abnormal ploidy, but the variation in the percentages of aneuploid blastomeres was significantly larger in embryos from oocytes exposed to mycotoxins. It is concluded that ZEA and DON can lead to abnormal spindle formation, leading to less fertile oocytes and embryos with abnormal ploidy, and that the effects of ZEA and DON are not synergistic.

Epididymal protein CRISP1 participates in rat and mouse gamete fusion through its interaction with complementary sites on the egg surface. Based on in vivo observations, in the present study we investigated the possibility that CRISP1 plays an additional role in the sperm-zona pellucida (ZP) interaction that precedes gamete fusion. In vitro fertilization experiments using zona-intact rat and mouse eggs indicated that the presence of either an antibody against rat CRISP1 (anti-CRISP1) or rat native CRISP1 (rCRISP1) during gamete co-incubation produced a significant decrease in the percentage of fertilized eggs. However, differently to that expected for a protein involved in gamete fusion, no accumulation of perivitelline sperm was observed, suggesting that the inhibitions occurred at the sperm-ZP interaction level. Bacterially expressed recombinant CRISP1 (recCRISP1) also significantly inhibited egg fertilization. In this case, however, an increase in the number of perivitelline sperm was observed. Subsequent experiments evaluating the effect of anti-CRISP1 or rCRISP1 on the number of sperm bound per egg indicated that the protein is involved in the initial step of sperm-ZP binding. In agreement with these functional studies, indirect immunofluorescence experiments revealed that although rCRISP1 is capable of binding to both the ZP and the oolema, recCRISP1 only binds to the egg surface. The finding that deglycosylated rCRISP1 behaves as the untreated protein, whereas the heat-denatured rCRISP1 associated only with the oolema, indicates that the protein ZP-binding ability resides in the conformation rather than in the glycosydic portion of the molecule. The interaction between rCRISP1 and the ZP reproduces the sperm-ZP-binding behavior, as judged by the failure of the protein to interact with the ZP of fertilized eggs. Together, these results support the idea that CRISP1 participates not only in sperm-egg fusion but also in the prior stage of sperm-ZP interaction.

We investigated the function and expression of voltage-gated Na channels (VGSC) in the uteri of nonpregnant rats using organ bath techniques, intracellular [Ca² ] fluorescence measurements, and RT-PCR. In longitudinally arranged whole-tissue uterine strips, veratridine, a VGSC activator, caused the rapid appearance of phasic contractions of irregular frequency and amplitude. After 50–60 min in the continuous presence of veratridine, rhythmic contractions of very regular frequency and slightly increasing amplitude occurred and were sustained for up to 12 h. Both the early and late components of the contractile response to veratridine were inhibited in a concentration-dependent manner by tetrodotoxin (TTX). In small strips dissected from the uterine longitudinal smooth muscle layer and loaded with Fura-2, veratridine also caused rhythmic contractions, accompanied by transient increases in [Ca² ]_i, which were abolished by treatment with 0.1 μM TTX. Using end-point and real-time quantitative RT-PCR, we detected the presence of the VGSC alpha subunits Scn2a1, Scn3a, Scn5a, and Scn8a in the cDNA from longitudinal muscle. The mRNAs of the auxiliary beta subunits Scbn1b, Scbn2b, Scbn4b, and traces of Scn3b were also present. These data show for the first time that Scn2a1, Scn3a, Scn5a, and Scn8a, as well as all VGSC beta subunits are expressed in the longitudinal smooth muscle layer of the rat myometrium. In addition, our data show that TTX-sensitive VGSC are able to mediate phasic contractions maintained over long periods of time in the uteri of nonpregnant rats.

The SPAG16 gene encodes two major transcripts, one for the 71-kDa SPAG16L, which is the orthologue of the Chlamydomonas rheinhardtii central apparatus protein PF20, and a smaller transcript, which codes for the 35-kDa SPAG16S nuclear protein that represents the C-terminus (exons 11–16) of SPAG16L. We have previously reported that a targeted mutation in exon 11 of the Spag16 gene impairs spermatogenesis and prevents transmission of the mutant allele in chimeric mice. In the present report, we describe a heterozygous mutation in exon 13 of the SPAG16 gene, which causes a frame shift and premature stop codon, affording the opportunity to compare mutations with similar impacts on SPAG16L and SPAG16S for male reproductive function in mice and men. We studied two male heterozygotes for the SPAG16 mutation, both of which were fertile. Freezing-boiling of isolated sperm from both affected males resulted in the loss of the SPAG16L protein, SPAG6, another central apparatus protein that interacts with SPAG16L, and the 28-kDa fragment of SPAG17, which associates with SPAG6. These proteins were also lost after freezing-boiling cycles of sperm extracts from mice that were heterozygous for an inactivating mutation (exons 2 and 3) in Spag16. Our findings suggest that a heterozygous mutation that affects both SPAG16L and SPAG16S does not cause male infertility in man, but is associated with reduced stability of the interacting proteins of the central apparatus in response to a thermal challenge, a phenotype shared by the sperm of mice heterozygous for a mutation that affects SPAG16L.

Continuous exposure of follicles/oocytes to elevated levels of insulin compromises embryonic developmental competence, although the underlying cellular mechanisms are unknown. The objectives of the present study were to determine whether mouse oocytes have insulin receptors and a functional insulin signaling cascade, and whether insulin exposure during oocyte growth or maturation influences meiotic progression and chromatin remodeling. Immunoblot and immunocytochemical analyses of germinal vesicle-intact (GVI) oocytes demonstrated the presence of insulin receptor-β. Insulin receptor expression in oocytes was increased by gonadotropin stimulation, and remained elevated throughout meiotic maturation. Fully grown GVI oocytes contained 3-phosphoinositide-dependent protein kinase-1 (PDPK1), thymoma viral proto-oncogene 1 (AKT1), and glycogen synthase kinase 3 (GSK3). In vitro maturation of GVI oocytes in 5 μg/ml insulin had no influence on meiotic progression or the incidence of normal metaphase II (MII) chromosome condensation. Treatment of oocytes during maturation had no effect on GSK3A/B protein expression or phosphorylation of S21/9. However, the culturing of preantral follicles for 10 days with 5 μg/ml insulin increased the phosphorylation of oocyte GSK3B, indicating GSK3 inactivation. The rates of development to metaphase I (MI) were similar for oocytes obtained from insulin-treated follicles and controls, whereas the incidence of abnormal MI chromatin condensation was significantly higher in oocytes obtained from follicles cultured with insulin compared to those cultured without insulin. These results demonstrate that oocytes contain a functional insulin signaling pathway, and that insulin exposure during oocyte growth results in chromatin remodeling aberrations. These findings begin to elucidate the mechanisms by which chronic elevated insulin influences oocyte meiosis, chromatin remodeling, and embryonic developmental competence.

The adaptive growth of the uterus during pregnancy is a critical event that involves increased synthesis of extracellular matrix (ECM) proteins and dynamic remodeling of smooth muscle cell (SMC)-ECM interactions. We have previously found a dramatic increase in the expression of the mRNAs that encode fibronectin (FN) and its alpha5-integrin receptor (ITGA5) in pregnant rat myometrium near to term. Since the myometrium at term is exposed to considerable mechanical stretching of the uterine wall by the growing fetus(es), the objective of the present study was to examine its role in the regulation of FN and ITGA5 expression at late gestation and during labor. Using myometrial tissues from unilaterally pregnant rats, we investigated the temporal changes in Itga5 gene expression in gravid and empty uterine horns by Northern blotting and real-time PCR, in combination with immunoblotting and immunofluorescence analyses of the temporal/spatial distributions of the FN and ITGA5 proteins. In addition, we studied the effects of early progesterone (P4) withdrawal on Itga5 mRNA levels and ITGA5 protein detection. At all time-points examined, the Itga5 mRNA levels were increased in the gravid uterine horn, compared to the empty horn (P < 0.05). Immunoblot analysis confirmed higher ITGA5 and FN protein levels in the myometrium, associated with gravidity (P < 0.05). Immunodetection of ITGA5 was consistently high in the longitudinal muscle layer, increased with gestational age in the circular muscle layer of the gravid horn, and remained low in the empty horn. ITGA5 and FN immunostaining in the gravid horn exhibited a continuous layer of variable thickness associated directly with the surfaces of individual SMCs. In contrast to the effects of stretch, P4 does not appear to regulate ITGA5 expression. We speculate that the reinforcement of the FN-ITGA5 interaction: 1) contributes to myometrial hypertrophy and remodeling during late pregnancy; and 2) facilitates force transduction during the contractions of labor by anchoring hypertrophied SMCs to the uterine ECM.

Perturbations of the development of preimplantation embryos may have long-term consequences for the health of progeny. There are no standardized methods for assessing such risks. The OECD/OCDE 416 Guideline for Testing of Chemicals (Two-Generation Reproduction Toxicity Study) is a standardized assay for detecting potential toxic effects of chemicals. The present study assessed the utility of this guideline for identifying long-term consequences of perturbing preimplantation development. Extended culturing of mammalian zygotes commonly results in retarded preimplantation development. Mouse zygotes were cultured in vitro for 96 h until the blastocyst stage (cultured blastocysts) or blastocysts were collected from the Day-3.5 uterus (in vivo blastocysts). The resulting blastocysts were transferred to the uteri of pseudopregnant recipients (P generation). Progeny from both treatments were mated for a further two generations (F1 and F2 generations). There was no effect of treatment group on gross fertility across the generations tested. Progeny of the cultured blastocysts had lower body weights to the time of weaning compared to in vivo blastocysts in the P and F1 generations, but not in the F2 generation. At maturity, there was no effect of treatment group on body weight, although thyroid weight was higher in the in vivo blastocyst group in the P generation, while the brain, pituitary, and kidneys were larger in the progeny of the cultured blastocysts of the F1 generation. The OECD/OCDE 416 assessment may have a role as a standardized test for the assessment of the biological consequences of perturbing the growth environment of the preimplantation embryo. Embryo culture influenced the somatometric parameters of the resulting progeny, some of which were maintained across a generation.

Spermatogonial stem cells (SSCs) are responsible for life-long, daily production of male gametes and for the transmission of genetic information to the next generation. Unequivocal detection of SSCs has relied on spermatogonial transplantation, in which functional SSCs are analyzed qualitatively and quantitatively based on their regenerative capacity. However, this technique has some significant limitations. For example, it is a time-consuming procedure, as data acquisition requires at least 8 weeks after transplantation. It is also laborious, requiring microinjection of target cells into the seminiferous tubules of individual testes. Donor-recipient immunocompatibility for successful transplantation and large variations in data obtained represent further limitations of this technique. In the present study, we provide evidence that a recently developed SSC culture system can be employed as a reliable, short-term in vitro assay for SSCs. In this system, donor cells generate three-dimensional structures of aggregated germ cells (clusters) in vitro within 6 days. We show that each cluster originates from a single cell. Thus, by counting the clusters, cluster-forming cells can be quantified. We observed a strong linear correlation between the numbers of clusters and SSCs over extended culture periods. Therefore, cluster numbers faithfully reflect SSC numbers. These results indicate that by simply counting the number of clusters, functional SSCs can be readily detected within 1 week in a semi-quantitative manner. The faithfulness of this in vitro assay to the transplantation assay was further confirmed under two experimental situations. This in vitro cluster formation assay provides a reliable short-term technique to detect SSCs.