In addition to their contribution to the research on early human development, human embryonic stem (hES) cells may also be used for cell-based therapies. Traditionally, these cells have been cultured on mouse embryonic fibroblast feeder layers, which allow their continuous growth in an undifferentiated state. However, the use of hES cells in human therapy requires an animal-free culture system, in which exposure to mouse retroviruses is avoided. In this study we present a novel feeder layer-free culture system for hES cells, based on medium supplemented with 15% serum replacement, a combination of growth factors including transforming growth factor Î²1 (TGFÎ²1), leukemia inhibitory factor, basic fibroblast growth factor, and fibronectin matrix. Human ES cells grown in these conditions maintain all ES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of the three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. The culture system presented here has two major advantages: 1) application of a well-defined culture system for hES cells and 2) reduced exposure of hES cells to animal pathogens. The feeder layer-free culture system reported here aims at facilitating research practices and providing a safer alternative for future clinical applications of hES cells.
Belclare and Cambridge are prolific sheep breeds, the origins of which involved selecting ewes with exceptionally high litter size records from commercial flocks. The variation in ovulation rate in both breeds is consistent with segregation of a gene (or genes) with a large effect on this trait. Sterile ewes, due to a failure of normal ovarian follicle development, occur in both breeds. New naturally occurring mutations in genes for the oocyte-derived growth factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are described. These mutations are associated with increased ovulation rate in heterozygous carriers and sterility in homozygous carriers in both breeds. This is the first time that a mutation in the gene for GDF9 has been found that causes increased ovulation rate and infertility in a manner similar to inactivating mutations in BMP15, and shows that GDF9 is essential for normal folliculogenesis in sheep. Furthermore, it is shown, for the first time in any species, that individuals with mutations in both GDF9 and BMP15 have a greater ovulation rate than sheep with either of the mutations separately.
One of the most promising applications of microarrays is the study of changes in gene expression associated with the growth and development of mammalian tissues. The testis provides an excellent model to determine the ability of microarrays to effectively characterize the changes in gene expression as an organ develops from birth to adulthood. To this end, a developmental testis gene expression time course profiling the expression patterns of â¼36â000 transcripts on the Affymetrix MGU74v2 GeneChip platform at 11 distinct time points was created to gain a greater understanding of the molecular changes necessary for and elicited by the development of the testis. Additionally, gene expression profiles of isolated testicular cell types were created that can aid in the further characterization of the specific functional actions of each cell type in the testis. Statistical analysis of the data revealed 11â252 transcripts (9846 unique) expressed differentially in a significant manner. Subsequent cluster analysis produced five distinct expressional patterns within the time course. These patterns of expression are present at distinct chronological periods during testis development and often share similarities with cell-specific expression profiles. Analysis of cell-specific expression patterns produced unique and characteristic groups of transcripts that provide greater insight into the activities, biological and chronological, of testicular cell types during the progression of spermatogenesis. Further analysis of this time course can provide a distinct and more definitive view into the genes implicated, known and unknown, in the maturation, maintenance, and function of the testis and the integrated process of spermatogenesis.
The human endometrium regenerates from the lower basalis layer, a germinal compartment that persists after menstruation to give rise to the new upper functionalis layer. Because adult stem cells are present in tissues that undergo regeneration, we hypothesized that human endometrium contains small populations of epithelial and stromal stem cells responsible for cyclical regeneration of endometrial glands and stroma and that these cells would exhibit clonogenicity, a stem-cell property. The aims of this study were to determine 1) the clonogenic activity of human endometrial epithelial and stromal cells, 2) which growth factors support this clonogenic activity, and 3) determine the cellular phenotypes of the clones. Endometrial tissue was obtained from women undergoing hysterectomy. Purified single- cell suspensions of epithelial and stromal cells were cultured at cloning density (300â500/cm 2 ) in serum medium or in serum- free medium supplemented with one of eight growth factors. Small numbers of epithelial (0.22%) and stromal cells (1.25%) initiated colonies in serum-containing medium. The majority of colonies were small, containing large, loosely arranged cells, and 37% of epithelial and 1 in 60 of stromal colonies were classified as large, comprising small, densely packed cells. In serum-free medium, transforming growth factor-Î± (TGFÎ±), epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) strongly supported clonogenicity of epithelial cells, while leukemia-inhibitory factor (LIF), hepatocyte growth factor (HGF), stem-cell factor (SCF), insulin-like growth factor-I (IGF- I) were weakly supportive, and basic fibroblast growth factor (bFGF) was without effect. TGFÎ±, EGF, PDGF-BB, and bFGF supported stromal cell clonogenicity, while HGF, SCF, LIF, and IGF- I were without effect. Small epithelial colonies expressed three epithelial markers but not stromal markers; however, large epithelial colonies showed little reactivity for all markers except Î± 6 -integrin. All stromal colonies contained fibroblasts, expressing stromal markers, and in some colonies, myofibroblasts were also identified. This analysis of human endometrium has demonstrated the presence of rare clonogenic epithelial and stromal cells with high proliferative potential, providing the first evidence for the existence of putative endometrial epithelial and stromal stem cells.
The human endometrium regenerates from the lower basalis layer, a germinal compartment that persists after menstruation to give rise to the new upper functionalis layer. Because adult stem cells are present in tissues that undergo regeneration, we hypothesized that human endometrium contains small populations of epithelial and stromal stem cells responsible for cyclical regeneration of endometrial glands and stroma and that these cells would exhibit clonogenicity, a stem-cell property. The aims of this study were to determine 1) the clonogenic activity of human endometrial epithelial and stromal cells, 2) which growth factors support this clonogenic activity, and 3) determine the cellular phenotypes of the clones. Endometrial tissue was obtained from women undergoing hysterectomy. Purified single-cell suspensions of epithelial and stromal cells were cultured at cloning density (300-500/cm(2)) in serum medium or in serum-free medium supplemented with one of eight growth factors. Small numbers of epithelial (0.22%) and stromal cells (1.25%) initiated colonies in serum-containing medium. The majority of colonies were small, containing large, loosely arranged cells, and 37% of epithelial and 1 in 60 of stromal colonies were classified as large, comprising small, densely packed cells. In serum-free medium, transforming growth factor-alpha (TGFalpha), epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) strongly supported clonogenicity of epithelial cells, while leukemia-inhibitory factor (LIF), hepatocyte growth factor (HGF), stem-cell factor (SCF), insulin-like growth factor-I (IGF-I) were weakly supportive, and basic fibroblast growth factor (bFGF) was without effect. TGFalpha, EGF, PDGF-BB, and bFGF supported stromal cell clonogenicity, while HGF, SCF, LIF, and IGF-I were without effect. Small epithelial colonies expressed three epithelial markers but not stromal markers; however, large epithelial colonies showed little reactivity for all markers except alpha-integrin. All stromal colonies contained fibroblasts, expressing stromal markers, and in some colonies, myofibroblasts were also identified. This analysis of human endometrium has demonstrated the presence of rare clonogenic epithelial and stromal cells with high proliferative potential, providing the first evidence for the existence of putative endometrial epithelial and stromal stem cells.
Abstract The mammalian sperm must be highly motile for a long time to fertilize a egg. It has been supposed that ATP required for sperm flagellar movement depends predominantly on mitochondrial respiration. We assessed the contribution of mitochondrial respiration to mouse sperm motility. Mouse sperm maintained vigorous motility with high beat frequency in an appropriate solution including a substrate such as glucose. The active sperm contained a large amount of ATP. When carbonyl cyanide m-chlorophenylhydrazone (CCCP) was applied to suppress the oxidative phosphorylation in mitochondria, the vigorous motility was maintained and the amount of ATP was kept at the equivalent level to that without CCCP. When pyruvate or lactate was provided instead of glucose, both sperm motility and the amount of ATP were high. However, they were drastically decreased when oxidative phosphorylation was suppressed by addition of CCCP. We also found that sperm motility could not be maintained in the presence of respiratory su...
Cell fate determination between self-renewal or differentiation of spermatogonial stem cells (SSCs) in the testis is precisely regulated to maintain normal spermatogenesis. However, the mechanisms underlying the process remain elusive. To address the problem, we developed a model SSC culture system, first, by establishing techniques to obtain enriched populations of stem cells, and second, by establishing a serum-free culture medium. Flow cytometric cell sorting and the SSC transplantation assay demonstrated that Thy-1 is a unique surface marker of SSCs in neonatal, pup, and adult testes of the mouse. Although the surface phenotype of SSCs is major histocompatibility complex class I â Thy-1 + Î±6-integrin + Î±v-integrin â/dim throughout postnatal life, the most enriched population of SSCs was obtained from cryptorchid adult testes by cell-sorting techniques based on Thy-1 expression. This enriched population of SSCs was used to develop a culture system that consisted of serum-free defined medium and STO (SIM mouse embryo-derived thioguanine and ouabain resistant) feeders, which routinely maintained stem cell activity for 1 wk. Combining the culture system and the transplantation assay provided a mechanism to study the effect of single growth factors. A negative effect was demonstrated for several concentrations of basic fibroblast growth factor and leukemia inhibitory factor, whereas glial cell line-derived neurotrophic factor and stem cell factor appeared to have a positive effect on stem cell maintenance. The stem cell enrichment strategies and the culture methods described provide a reproducible and powerful assay system to establish the effect of various environmental factors on SSC survival and replication in vitro.
Active demethylation of cytosine residues in the sperm genome before forming a functional zygotic nucleus is thought to be an important function of the oocyte cytoplasm for subsequent embryonic development in the mouse. Conversely, this event does not occur in the sheep or rabbit zygote and occurs only partially in the cow. The aim of this study was to investigate the effect of limited methylation reprogramming in the normal sheep embryo on reprogramming somatic nuclei. Sheep fibroblast somatic nuclei were partially demethylated after electrofusion with recipient sheep oocytes and undergo a stepwise passive loss of DNA methylation during early development, as determined by 5-methylcytosine immunostaining on interphase embryonic nuclei. A similar decrease takes place with in vivo-derived sheep embryos up to the eight-cell stage, although nuclear transfer embryos exhibit a consistently higher level of methylation at each stage. Between the eight-cell and blastocyst stages, DNA methylation levels in nuclear transfer embryos are comparable with those derived in vivo, but the distribution of methylated DNA is abnormal in a high proportion. By correlating DNA methylation with developmental potential at individual stages, our results suggest that somatic nuclei that do not undergo rapid reorganization of their DNA before the first mitosis fail to develop within two to three cell cycles and that the observed methylation defects in early cleavage stages more likely occur as a direct consequence of failed nuclear reorganization than in failed demethylation capacity. However, because only embryos with reorganized chromatin appear to survive the 16-cell and morula stages, failure to demethylate the trophectoderm cells of the blastocyst is likely to directly impact on developmental potential by altering programmed patterns of gene expression in extraâembryonic tissues. Thus, both remodeling of DNA and epigenetic reprogramming appear critical for development of both fertilized and nuclear transfer embryos.
Abstract Cryopreservation induces many changes in sperm cells, including membrane disorders and cell death. We tested the hypothesis that apoptosis, a form of programmed cell death, can contribute to the fatal effect of cryopreservation on sperm cells. A multiparametric study of apoptosis on bovine sperm is proposed, using flow cytometry, including mitochondrial membrane potential (ΔΨm), caspase activation, membrane permeability, nucleus condensation, DNA fragmentation, and phosphatidylserine (PS) externalization. The relevance of each test was first validated on a human somatic cell line, U937. Cryopreservation and/or thawing induced significant changes in all apoptotic markers in living bull sperm cells except those concerning the nucleus. After cryopreservation, 44.9% ± 17% (vs. 11.3% ± 10.6% before cryopreservation) of sperm cells showed low ΔΨm, 12% ± 6.3% (vs. 2.2% ± 1.0% before) contained active caspases, and 10.8% ± 5.8% (vs. 1.4% ± 1.1% before) exhibited high membrane permeability. However, cryop...
Abstract In the human fetal testis, germ cells that have migrated to the genital ridges become enclosed within testicular cords by 8 wk of gestation. Most papers refer to all types of germ cells as being “gonocytes” or “prespermatogonia,” giving the impression that they are identical. Detailed morphological studies, however, have suggested a heterogeneous population. We have used single, double, and triple immunohistochemistry to evaluate the differentiation of cells within fetal testes recovered during the first (7–9 wk) and second (14–19 wk) trimesters. In the first trimester, differentiation of Sertoli cells preceded the formation of testicular cords and the differentiation of interstitial (Leydig, peritubular myoid) cells. Immunostaining for CHK2, C-KIT, placental alkaline phosphatase, PCTAIRE-1, and MAGE-A4 revealed that the proportion of germ cells expressing each of these proteins was correlated with gestational age. Expression of the pluripotency marker OCT4 was restricted to a population of small...
Oxygen concentrations used during in vitro embryo culture can influence embryo development, cell numbers, and gene expression. Here we propose that the preimplantation bovine embryo possesses a molecular mechanism for the detection of, and response to, oxygen, mediated by a family of basic helix-loop-helix transcription factors, the hypoxia-inducible factors (HIFs). Day 5 compacting bovine embryos were cultured under different oxygen tensions (2%, 7%, 20%) and the effect on the expression of oxygen-regulated genes, development, and cell number allocation and HIFÎ± protein localization were examined. Bovine in vitro-produced embryos responded to variations in oxygen concentration by altering gene expression. GLUT1 expression was higher following 2% oxygen culture compared with 7% and 20% cultured blastocysts. HIF mRNA expression (HIF1Î±, HIF2Î±) was unaltered by oxygen concentration. HIF2Î± protein was predominantly localized to the nucleus of blastocysts. In contrast, HIF1Î± protein was undetectable at any oxygen concentration or in the presence of the HIF protein stabilizer desferrioxamine (DFO), despite being detectable in cumulus cells following normal maturation conditions, acute anoxic culture, or in the presence of DFO. Oxygen concentration also significantly altered inner cell mass cell proportions at the blastocyst stage. These results suggest that oxygen can influence gene expression in the bovine embryo during postcompaction development and that these effects may be mediated by HIF2Î±.
Successful ovulation and implantation processes play a crucial role in female fertility. Adamts-1, a matrix metalloproteinase with disintegrin and thrombospondin motifs, has been suggested to be regulated by the progesterone receptor in the hormonal pathway leading to ovulation. With the primary aim of investigating the role of Adamts-1 in female fertility, we generated Adamts-1 null mice. Forty-five percent of the newborn Adamts-1 null mice die, with death most likely caused by a kidney malformation that becomes apparent at birth. Surviving female null mice were subfertile, whereas males reproduced normally. Ovulation in null females was impaired because of mature oocytes remaining trapped in ovarian follicles. No uterine phenotype was apparent in Adamts-1 null animals. Embryo implantation occurred normally, the uteri were capable of undergoing decidualization, and no morphological changes were observed. These results demonstrate that a functional Adamts-1 is required for normal ovulation to occur, and hence the Adamts-1 gene plays an important role in female fertility, primarily during the tissue remodeling process of ovulation.
We investigated the impact of cryopreservation and thawing on levels of caspases-3, -8, and -9 activity, intact mitochondrial membrane potential (DeltaPsi(m)), and DNA fragmentation in human spermatozoa. Eleven pools of cryopreserved and eight pools of fresh semen samples were examined. Mature and immature fractions were separated on a two-layer density gradient (47% and 90%) and further subdivided based on the externalization of phosphatidylserine and its binding to annexin Wabeled superparamagnetic microbeads (ANMB). Levels of activated caspases were assessed using fluorescein-labeled inhibitors of caspases (FLICA), DeltaPsi(m) using a lipophilic cationic dye, and DNA fragmentation by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Cryopreservation was significantly associated with activation of caspases-3, -8, and -9, as well as disruption of the mitochondrial membrane potential but no significant changes were observed in DNA fragmentation. In mature sperm, caspase activation was only detected in the ANMB(+) fraction, whereas in immature sperm, both ANMB(+) and ANMB(-) fractions showed activated caspase levels. In ANMB(+) immature sperm, apoptosis seemed to be triggered by a surface ligand-receptor mechanism as well as by disruption of mitochondria, whereas in ANMB(-) immature sperm, apoptosis was induced by activation of caspase-9 following loss of intact DeltaPsi(m). These results demonstrate that selection of annexin V-negative mature spermatozoa might be of clinical relevance for fertility preservation, as this sperm fraction shows no activated apoptosis during the cryopreservation process.
In the course of mammalian spermiogenesis, a unique chromatin remodeling process takes place within elongating and condensing spermatid nuclei. The histone-to-protamine exchange results in efficient packaging and increased stability of the paternal genome. Although not fully understood, this change in chromatin architecture must require a global but transient appearance of endogenous DNA strand breaks because most of the DNA supercoiling is eliminated in the mature sperm. To establish the extent of DNA strand breakage and the stage specificity at which these breaks are created and repaired, we performed a sensitive terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay to detect in situ DNA strand breaks on both mice and human testis cross sections. In the mouse, we established that DNA strand breaks are indeed detected in the whole population of elongating spermatids between stages IX and XI of the seminiferous epithelium cycle perfectly coincident with the chromatin remodeling as revealed by histone H4 hyperacetylation. Similarly, TUNEL analyses performed on human testis sections revealed an elevated and global increase in the levels of DNA strand breaks present in nuclei of round-shaped spermatids also coincident with chromatin remodeling. The demonstration of the global character of the transient DNA strand breaks in mammalian spermiogenesis suggests that deleterious consequences on genetic integrity of the male gamete may arise from any disturbance in the process. In addition, this investigation may shed some light on the origin of the low success rate that has been encountered so far with intracytoplasmic injection procedures making use of round spermatids in humans.
Testicular maturation and sperm production throughout the life of the male form the basis of male fertility. It is difficult to elucidate the intricate processes controlling testicular maturation and spermatogenesis in primates in vivo due to the long time span required for sexual maturation and also to the lack of accessible in vitro or in vivo models of primate spermatogenesis. Ectopic xenografting of neonatal testis tissue into mice provides an accessible model to study and manipulate the propagation and differentiation of male germ cells from immature donor animals. However, it was not clear whether this approach would be applicable to slowly maturing primates. Here we report that grafting of testis tissue from immature rhesus monkeys ( Macaca mulatta ) into host mice resulted in the acceleration of testicular maturation and production of fertilization-competent sperm in testis xenografts. The system reported here provides a powerful, practical approach to study timing and control of testicular maturation and regulation of primate spermatogenesis without the necessity for experimentation in primates. This approach could potentially be applied to produce fertile sperm from sexually immature individuals of rare or valuable primate species or from prepubertal boys undergoing sterilizing therapy for cancer.
Spermatogenesis is dependent on a small population of stem cells. Despite the biological significance of spermatogonial stem cells, their analysis has been hampered by their scarcity. However, spermatogonial stem cells can be enriched by selection with an antibody against cell-surface molecules. In this investigation, we searched for new antigens expressed on spermatogonial stem cells. Using the spermatogonial transplantation technique, we examined expression of the CD9 molecule, which is commonly expressed on stem cells of other tissues. Selection of both mouse and rat testis cells with anti-CD9 antibody resulted in 5- to 7-fold enrichment of spermatogonial stem cells from intact testis cells, indicating that CD9 is commonly expressed on spermatogonial stem cells of both species. Therefore, CD9 may be involved in the common machinery in stem cells of many self-renewing tissues, and the identification of a common surface antigen on spermatogonial stem cells of different species has important implications for the development of a technique to enrich stem cells from other mammalian species.
Equol (7-hydroxy-3[4â²hydroxyphenyl]-chroman) is the major metabolite of the phytoestrogen daidzein, one of the main isoflavones found abundantly in soybeans and soy foods. Equol may be an important biologically active molecule based on recent studies demonstrating that equol can modulate reproductive function. In this study, we examined the effects of equol on prostate growth and LH secretion and determined some of the mechanisms by which it might act. Administration of equol to intact male rats for 4â7 days reduced ventral prostate and epididymal weight and increased circulating LH levels. Using binding assays, we determined that equol specifically binds 5Î±-dihydrotestosterone (DHT), but not testosterone, dehydroepiandrosterone, or estrogen with high affinity. Equol does not bind the prostatic androgen receptor, and has a modest affinity for recombinant estrogen receptor (ER) Î², and no affinity for ERÎ±. In castrated male rats treated with DHT, concomitant treatment with equol blocked DHT's trophic effects on the ventral prostate gland growth and inhibitory feedback effects on plasma LH levels without changes in circulating DHT. Therefore, equol can bind circulating DHT and sequester it from the androgen receptor, thus altering growth and physiological hormone responses that are regulated by androgens. These data suggest a novel model to explain equol's biological properties. The significance of equol's ability to specifically bind and sequester DHT from the androgen receptor have important ramifications in health and disease and may indicate a broad and important usage for equol in the treatment of androgen-mediated pathologies.
In the growing follicle, communication between the oocyte and its surrounding follicular cells is essential for normal oocyte and follicular development. Maturation of the fully grown oocyte in vivo is associated with the loss of cumulus cell-oocyte gap junctional communication, preventing entry of meiotic-modulating factors such as cAMP into the oocyte. We have previously shown that oocyte and cumulus cell cAMP levels can be independently regulated using inhibitors of cell-specific phosphodiesterase (PDE) isoenzymes. The objectives of this study were to examine the effects of cell type-specific PDE inhibitors on the maintenance of cumulus cell-oocyte gap junction communication (GJC) and oocyte meiotic progression. Cumulus-oocyte complexes (COCs) were aspirated from antral follicles of abattoir-derived ovaries. Cumulus cell-oocyte GJC during oocyte maturation was quantified using the fluorescent dye, calcein-AM. COCs were cultured in the presence of specific PDE inhibitors, milrinone (an oocyte PDE3 inhibitor) or rolipram (a cumulus cell PDE4 inhibitor), and were pulsed with calcein-AM to allow dye transfer between the two cell types. Following cumulus cell removal, fluorescence in denuded oocytes was measured by microphotometry, and meiotic progression was assessed. In control COCs, dye transfer from cumulus cells to the oocyte fell progressively from 0 to 9 h, after which oocyte-cumulus cell GJC was completely lost. Loss of GJC was significantly attenuated (P < 0.05) during this time in response to treatment with milrinone and rolipram. Forskolin maintained GJC at the initial 0 h level until 3-4 h of culture, whereas treatment with milrinone and forskolin together actually increased the level of dye transfer above that in COCs treated with forskolin alone. Importantly, all treatments that prolonged GJC also delayed meiotic resumption, with meiosis generally resuming when fluorescence had fallen to similar to40% of initial levels. These results, together with our previous studies, demonstrate that treatments that maintain or elevate cAMP levels in cumulus cells, oocytes, or both result in prolonged oocyte-cumulus cell communication and delayed meiotic resumption.
The mouse is an excellent model for studying the genetic basis of placental development, but analyses are restricted by the lack of quantitative data describing normal murine placental structure. This study establishes a technique for generating such data, applies stereological techniques on systematic uniform random sections of placentas between E12.5âE18.5 of gestation (E1.0 = day of the vaginal plug), and considers the results in the context of development of the labyrinth zone. Half of each placenta was wax embedded and exhaustively sectioned to determine absolute volumes of the labyrinth zone (Lz), junctional zone (Jz), and decidua using the Cavalieri principle. The other half was resin embedded and 1-Î¼m sections were used to generate all volume, surface, and length densities within the Lz. Maximum placental volume is reached by E16.5, whereas the Lz volume fraction increases until E18.5 at the expense of the Jz and decidua. Within the Lz, the absolute volume and surface area of maternal blood spaces (MBS) expand rapidly between E14.5 and E16.5, with no increase thereafter. In contrast, fetal capillary development is linear and continues for longer than that of the MBS. The interhemal membrane separating maternal and fetal circulations undergoes thinning prior to expansion of maternal and fetal surface areas, achieving a harmonic mean thickness of 4.39 Î¼m by E18.5. The specific diffusion capacity for oxygen of the interhemal membrane is maximal by E16.5, which may be necessary to support rapid fetal growth until the end of gestation.