Nuclear HKII-P-p53 (Ser15) Interaction is a Prognostic Biomarker for Chemoresponsiveness and Glycolytic Regulation in Epithelial Ovarian Cancer.

In epithelial ovarian cancer (EOC), carboplatin/cisplatin-induced chemoresistance is a major hurdle to successful treatment. Aerobic glycolysis is a common characteristic of cancer. However, the role of glycolytic metabolism in chemoresistance and its impact on clinical outcomes in EOC are not clear. Here, we show a functional interaction between the key glycolytic enzyme hexokinase II (HKII) and activated P-p53 (Ser15) in the regulation of bioenergetics and chemosensitivity. Using translational approaches with proximity ligation assessment in cancer cells and human EOC tumor sections, we showed that nuclear HKII-P-p53 (Ser15) interaction is increased after chemotherapy, and functions as a determinant of chemoresponsiveness as a prognostic biomarker. We also demonstrated that p53 is required for the intracellular nuclear HKII trafficking in the control of glycolysis in EOC, associated with chemosensitivity. Mechanistically, cisplatin-induced P-p53 (Ser15) recruits HKII and apoptosis-inducing factor (AIF) in chemosensitive EOC cells, enabling their translocation from the mitochondria to the nucleus, eliciting AIF-induced apoptosis. Conversely, in p53-defective chemoresistant EOC cells, HKII and AIF are strongly bound in the mitochondria and, therefore, apoptosis is suppressed. Collectively, our findings implicate nuclear HKII-P-p53(Ser15) interaction in chemosensitivity and could provide an effective clinical strategy as a promising biomarker during platinum-based therapy.

Maintenance of hPSCs under Xeno-Free and Chemically Defined Culture Conditions.

Previously, the majority of human embryonic stem cells and human induced pluripotent stem cells have been derived on feeder layers and chemically undefined medium. Those media components related to feeder cells, or animal products, often greatly affect the consistency of the cell culture. There are clear advantages of a defined, xeno-free, and feeder-free culture system for human pluripotent stem cells (hPSCs) cultures, since consistency in the formulations prevents lot-to-lot variability. Eliminating all non-human components reduces health risks for downstream applications, and those environments reduce potential immunological reactions from stem cells. Therefore, development of feeder-free hPSCs culture systems has been an important focus of hPSCs research. Recently, researchers have established a variety of culture systems in a defined combination, xeno-free matrix and medium that supports the growth and differentiation of hPSCs. Here we described detailed hPSCs culture methods under feeder-free and chemically defined conditions using vitronetin and TeSR-E8 medium including supplement bioactive lysophospholipid for promoting hPSCs proliferation and maintaining stemness.

The effects of soybean isoflavones and 17ß-estradiol in uterus and mammary glands of diabetic rat models.

The objective of this study was to evaluate the action of soy isoflavones and 17 beta estradiol on the extracellular matrix in the uterus and mammary gland of diabetic rats. Sixty adult female rats underwent ovariectomy, then randomized into seven groups of ten animals each: Non-diabetic: GI Sham control animals ovariectomized; and GII control ovariectomized that received propylene glycol vehicle. Diabetic: GIII Sham control diabetic animals ovariectomized; GIV ovariectomized diabetic animals receiving propylene glycol vehicle; GV diabetic ovariectomized animals treated with soy isoflavones (150 mg/kg by gavage); GVI ovariectomized diabetic rats treated with estrogen (17b-estradiol, 10 mg/kg, subcutaneously); GVII diabetic ovariectomized animals treated with soy isoflavones (150 mg/kg by gavage), and with estrogen (17b-estradiol, 10 mg/kg combination therapy). Treatments occurred during 30 consecutive days. After animals euthanasia, a portion of the uterus was immersed in liquid nitrogen for molecular biology analysis, the other portion of uterus and mammary glands were removed and processed for paraffin embedding. Soy isoflavones (GV) and 17b estradiol improved the production of compounds of extracellular matrix, such as small leucine-rich proteoglycans (SLRPs). The combination of both therapies had an additive effect in SLRPs expression. Soy isoflavones contribute to the uterine integrity of SLRPs of diabetic rats.

Regulation of androgen receptor signaling by ubiquitination during folliculogenesis and its possible dysregulation in polycystic ovarian syndrome.

Although chronic hyperandrogenism suppresses antral follicular development, a phenomenon often observed in polycystic ovarian syndrome (PCOS), whether and how deregulation of androgen receptor (AR) signaling is involved, is not well understood. In the present study, we examined the role of ring finger protein 6 (RNF6) in AR ubiquitination and the possible dysregulation in the expression and actions of growth differentiation factor 9 (GDF9) and kit-ligand (Kitlg) in a chronic androgenized PCOS rat model. 5α-dihydrotestosterone (DHT) treatment in vivo inhibited antral follicle growth, a response mediated through increased RNF6 content, suppressed K63- but increased K48-linked AR ubiquitination as well as the mRNA expression and content of soluble KIT-L (sKitlg) and content of GDF9. These androgenic responses were attenuated by gonadotropin treatment in vivo. Growth of antral follicles from DHT-treated rats in vitro was significantly slower when compared to those of control but was significantly enhanced by exogenous GDF9, suggesting the DHT-induced antral follicular growth arrest is in part the results of GDF9 suppression. Our findings indicate how hyperandrogenism modulates RNF6 content and subsequently AR ubiquitination, resulting in antral follicle growth arrest in a chronically androgenized PCOS rat model.

Ring Finger Protein 6 Mediates Androgen-Induced Granulosa Cell Proliferation and Follicle Growth via Modulation of Androgen Receptor Signaling.

The destiny of the ovarian follicle (growth or atresia) is tightly regulated by the actions and interactions of endocrine, paracrine, and autocrine factors. Although androgens are known to be important in the regulation of folliculogenesis, whether they facilitate or suppress follicular growth has been controversial, and the mechanisms involved are not fully understood. Moreover, the role and regulation of androgen receptor (AR) in mediating androgen signaling during follicular development is not clear. Here, we report that the active androgen dihydrotestosterone upregulates the expression of AR and its E3 ligase ring finger protein 6 (RNF6), increasing site-specific AR polyubiquitination and AR transcriptional activity for soluble Kit ligand (sKit-L) expression in preantral follicle growth. RNF6 silencing suppressed dihydrotestosterone-induced AR ubiquitination (lysine residue 63) and proliferation and suppressed apoptosis in preantral granulosa cells, with these responses being overcome by the presence of exogenous sKit-L. Taken together, our findings support the notion that RNF6 plays an important role in androgen-induced, follicle-stage-dependent follicle growth and that it acts by facilitating AR-mediated granulosa cell sKit-L expression and proliferation. Our findings offer insights into the regulatory mechanism of androgen action in ovarian follicular growth.

Usage and perceptions of phosphodiesterase type 5 inhibitors among the male partners of infertile couples.

OBJECTIVE: We aimed to investigate the prevalence of erectile dysfunction (ED) and the usage of phosphodiesterase type 5 (PDE5) inhibitors for ED treatment in infertile couples. METHODS: A total of 260 male partners in couples reporting infertility lasting at least 1 year were included in this study. In addition to an evaluation of infertility, all participants completed the International Index of Erectile Function (IIEF)-5 questionnaire to evaluate their sexual function. The participants were asked about their use of PDE5 inhibitors while trying to conceive during their partner's ovulatory period and about their concerns regarding the risks of PDE5 inhibitor use to any eventual pregnancy and/or the fetus. RESULTS: Based on the IIEF-5 questionnaire, 41.5% of the participants (108/260) were classified as having mild ED (an IIEF-5 score of 17-21), while 10.4% of the participants (27/260) had greater than mild ED (an IIEF-5 score of 16 or less). The majority (74.2%, 193/260) of male partners of infertile couples had a negative perception of the safety of using a PDE5 inhibitor while trying to conceive. Only 11.1% of men (15/135) with ED in infertile couples had used a PDE5 inhibitor when attempting conception. CONCLUSION: ED was found to be common in the male partners of infertile couples, but the use of PDE5 inhibitors among these men was found to be very low. The majority of male partners were concerned about the risks of using PDE5 inhibitors when attempting to conceive. Appropriate counseling about this topic and treatment when necessary would likely be beneficial to infertile couples in which the male partner has ED.

Spermatogonial stem cell enrichment using simple grafting of testis and in vitro cultivation.

Enrichment of spermatogonial stem cells (SSCs) from the mammalian adult testis faces several limitations owing to their relatively low numbers among many types of advanced germ cells and somatic cells. The aim of the present study was to improve the isolation efficiency of SSCs using a simple tissue grafting method to eliminate the existing advanced germ cells. Sliced testis parenchyma obtained from adult ICR or EGFP-expressing transgenic mice were grafted heterotropically under the dorsal skin of nude mice. The most advanced germ cells disappeared in the grafted tissues after 2-4 weeks. Grafted tissues were dissociated enzymatically and plated in culture dishes. During in vitro culture, significantly more SSCs were obtained from the grafted testes than from non-grafted controls, and the isolated SSCs had proliferative potential and were successfully maintained. Additionally, EGFP-expressing SSCs derived from graft parenchyma were transplanted into bulsufan-treated recipient mice testes. Finally, we obtained EGFP-expressing pups after in vitro fertilization using spermatozoa derived from transplanted SSCs. These results suggest that subcutaneous grafting of testis parenchyma and the subsequent culture methods provide a simple and efficient isolation method to enrich for SSCs in adult testis without specific cell sorting methods and may be useful tools for clinical applications.

Three-step method for proliferation and differentiation of human embryonic stem cell (hESC)-derived male germ cells.

The low efficiency of differentiation into male germ cell (GC)-like cells and haploid germ cells from human embryonic stem cells (hESCs) reflects the culture method employed in the two-dimensional (2D)-microenvironment. In this study, we applied a three-step media and calcium alginate-based 3D-culture system for enhancing the differentiation of hESCs into male germ stem cell (GSC)-like cells and haploid germ cells. In the first step, embryoid bodies (EBs) were derived from hESCs cultured in EB medium for 3 days and re-cultured for 4 additional days in EB medium with BMP4 and RA to specify GSC-like cells. In the second step, the resultant cells were cultured in GC-proliferation medium for 7 days. The GSC-like cells were then propagated after selection using GFR-α1 and were further cultured in GC-proliferation medium for 3 weeks. In the final step, a 3D-co-culture system using calcium alginate encapsulation and testicular somatic cells was applied to induce differentiation into haploid germ cells, and a culture containing approximately 3% male haploid germ cells was obtained after 2 weeks of culture. These results demonstrated that this culture system could be used to efficiently induce GSC-like cells in an EB population and to promote the differentiation of ESCs into haploid male germ cells.

DNA fragmentation of human sperm can be detected by ligation-mediated real-time polymerase chain reaction.

OBJECTIVE: To determine whether ligation-mediated real-time polymerase chain reaction (LM-RT-PCR), which combines LM-PCR, and RT-PCR, can detect sperm DNA fragmentation (DF) in human semen samples. DESIGN: Three-way comparison of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), sperm chromatin dispersion (SCD), and LM-RT-PCR for detecting sperm DNA fragmentation. SETTING: University hospital-based research laboratory. PATIENT(S): Twenty-five men presenting at an infertility clinic. INTERVENTION(S): Basic analysis of sperm concentration, motility, vitality, and morphology, with each semen sample equally divided into three aliquots that were evaluated for fragmentation using TUNEL, SCD, and LM-RT-PCR assays. MAIN OUTCOME MEASURE(S): In TUNEL and SCD assays, counts of the number of sperm with tetramethylrhodamine (TMR) red signals or no halo; in LM-RT-PCR results, evaluation of the threshold cycles (Ct) and relative fluorescence unit (RFU) values. RESULT(S): The median percentage of sperm with positive results for fragmentation in the TUNEL and SCD assays were 20.5% and 20.7%, respectively. To compare the accuracy of the TUNEL, SCD, and LM-RT-PCR assays, we divided the semen samples into two groups according to the TUNEL results: low and high percentage of sperm fragmentation. In the LM-RT-PCR results, the values of the cycles of threshold (Ct) and relative fluorescence unit (RFU) statistically significantly differed between the low and high percentage of sperm fragmentation groups. Comparisons among the TUNEL, SCD, and LM-RT-PCR assays revealed that the correlation patterns according to DNA fragmentation were similar in both the groups with high and low percentage of DNA fragmentation. Our morphologic analysis indicated that the fragmentation of sperm DNA did not appear to influence sperm morphology. CONCLUSION(S): These results indicate that the LM-RT-PCR technique is another useful tool for detecting DNA fragmentation, a parameter of sperm quality in human semen alone or combined with TUNEL or SCD assays.

Evaluation of sperm deoxyribonucleic acid (DNA) damage and effects on embryo development using a mouse cryptorchidism model.

OBJECTIVE: To investigate the effects of sperm deoxyribonucleic acid (DNA) damage on fertilization and embryo development using a mouse cryptorchidism model of sperm DNA damage induction. MATERIALS AND METHODS: Male ICR mice (aged 5-6 weeks) underwent cryptorchidism on their left testicles and sham operations on their right testicles. Spermatogenesis and sperm DNA fragmentation were assessed after 1, 2, and 4 weeks using hematoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assays. Intracytoplasmic sperm injection into the oocytes of BDF1 females (aged 4-6 weeks) was performed using DNA-damaged sperm and normal sperm, and the fertilization rates and embryonic development were compared. RESULTS: The testicular weight and size gradually decreased after induction of cryptorchidism, with progressive reduction of spermatogenesis and increased DNA damage after 1, 2, and 4 weeks. After intracytoplasmic sperm injection, the fertilization and blastocyst development rates were significantly lower in the cryptorchidism group; however, about one quarter of the embryos arising from DNA-damaged sperm continued to develop. CONCLUSION: This was an in vivo animal study to evaluate the effects of sperm DNA damage using a cryptorchidism model. Sperm DNA damage increased significantly over time after cryptorchidism. This model could be useful in investigating male factor infertility and evaluating the biologic effects of paternal DNA damage on fertilization and future embryonic development.

Reversible infertility associated with testosterone therapy for symptomatic hypogonadism in infertile couple.

PURPOSE: Androgen replacement therapy has been shown to be safe and effective for most patients with testosterone deficiency. Male partners of infertile couples often report significantly poorer sexual activity and complain androgen deficiency symptoms. We report herein an adverse effect on fertility caused by misusage of androgen replacement therapy in infertile men with hypogonadal symptoms. MATERIALS AND METHODS: The study population consisted of 8 male patients referred from a local clinic for azoospermia or severe oligozoospermia between January 2008 and July 2011. After detailed evaluation at our andrology clinic, all patients were diagnosed with iatrogenic hypogonadism associated with external androgen replacement. We evaluated changes in semen parameters and serum hormone level, and fertility status. RESULTS: All patients had received multiple testosterone undecanoate (NebidoR) injections at local clinic due to androgen deficiency symptoms combined with lower serum testosterone level. The median duration of androgen replacement therapy prior to the development of azoospermia was 8 months (range: 4-12 months). After withdrawal of androgen therapy, sperm concentration and serum follicle-stimulating hormone level returned to normal range at a median 8.5 months (range: 7-10 months). CONCLUSION: Misusage of external androgen replacement therapy in infertile men with poor sexual function can cause temporary spermatogenic dysfunction, thus aggravating infertility.

In vitro culture-induced pluripotency of human spermatogonial stem cells.

Unipotent spermatogonial stem cells (SSCs) can be transformed into ESC-like cells that exhibit pluripotency in vitro. However, except for mouse models, their characterization and their origins have remained controversies in other models including humans. This controversy has arisen primarily from the lack of the direct induction of ESC-like cells from well-characterized SSCs. Thus, the aim of the present study was to find and characterize pluripotent human SSCs in in vitro cultures of characterized SSCs. Human testicular tissues were dissociated and plated onto gelatin/laminin-coated dishes to isolate SSCs. In the presence of growth factors SSCs formed multicellular clumps after 2-4 weeks of culture. At passages 1 and 5, the clumps were dissociated and were then analyzed using markers of pluripotent cells. The number of SSEA-4-positive cells was extremely low but increased gradually up to ~ 10% in the SSC clumps during culture. Most of the SSEA-4-negative cells expressed markers for SSCs, and some cells coexpressed markers of both pluripotent and germ cells. The pluripotent cells formed embryoid bodies and teratomas that contained derivatives of the three germ layers in SCID mice. These results suggest that the pluripotent cells present within the clumps were derived directly from SSCs during in vitro culture.

Isolation and characterization of novel, highly proliferative human CD34/CD73-double-positive testis-derived stem cells for cell therapy.

Human adult stem cells are a readily available multipotent cell source that can be used in regenerative medicine. Despite many advantages, including low tumorigenicity, their rapid senescence and limited plasticity have curtailed their use in cell-based therapies. In this study, we isolated CD34/CD73-double-positive (CD34(+)/CD73(+)) testicular stromal cells (HTSCs) and found that the expression of CD34 was closely related to the cells' stemness and proliferation. The CD34(+)/CD73(+) cells grew in vitro for an extended period of time, yielding a multitude of cells (5.6×10(16) cells) without forming tumors in vivo. They also differentiated into all three germ layer lineages both in vitro and in vivo, produced cartilage more efficiently compared to bone marrow stem cells and, importantly, restored erectile function in a cavernous nerve crush injury rat model. Thus, these HTSCs may represent a promising new autologous cell source for clinical use.

Embryonic demise caused by targeted disruption of a cysteine protease Dub-2.

BACKGROUND: A plethora of biological metabolisms are regulated by the mechanisms of ubiquitination, wherein this process is balanced with the action of deubiquitination system. Dub-2 is an IL-2-inducible, immediate-early gene that encodes a deubiquitinating enzyme with growth regulatory activity. DUB-2 presumably removes ubiquitin from ubiquitin-conjugated target proteins regulating ubiquitin-mediated proteolysis, but its specific target proteins are unknown yet. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the functional role of Dub-2, we generated genetically modified mice by introducing neo cassette into the second exon of Dub-2 and then homologous recombination was done to completely abrogate the activity of DUB-2 proteins. We generated Dub-2+/- heterozygous mice showing a normal phenotype and are fertile, whereas new born mouse of Dub-2-/- homozygous alleles could not survive. In addition, Dub-2-/- embryo could not be seen between E6.5 and E12.5 stages. Furthermore, the number of embryos showing normal embryonic development for further stages is decreased in heterozygotes. Even embryonic stem cells from inner cell mass of Dub-2-/- embryos could not be established. CONCLUSIONS: Our study suggests that the targeted disruption of Dub-2 may cause embryonic lethality during early gestation, possibly due to the failure of cell proliferation during hatching process.

Natural course of idiopathic oligozoospermia: comparison of mild, moderate and severe forms.

OBJECTIVES: To investigate the natural courses of mild, moderate and severe idiopathic oligozoospermia, and which factors or semen variables were of utmost importance in predicting the courses. METHODS: A total of 208 men (age 29-47years) who were diagnosed with mild, moderate and severe idiopathic oligozoospermia in a 9-year-period between January 2000 and December 2008 were followed up for more than 6months. RESULTS: Overall, 16 (24.6%) of 65 patients with severe oligozoospermia developed azoospermia, whereas two (3.1%) patients with moderate oligozoospermia developed azoospermia and none of the patients with mild oligozoospermia developed azoospermia. Initial follicle stimulating hormone level and testicular volume between the subgroups were significantly different (P=0.0071 and 0.0039, respectively). The subgroup of patients who became azoospermic (n=18) showed statistically significant differences in terms of body mass index and the level of prolactin (PRL) from the subgroup that maintained the initial lingering sperm count (n=190; P=0.0086 and 0.0154, respectively). As the vitality of semen variables increased 1%, the risk of progression to azoospermia diminished by 0.892-fold, according to Cox's proportional hazards model analysis. A receiver operating characteristic curve analysis showed that the area under the curve was 0.755 and the sperm concentration value with the highest sensitivity and specificity was the reference value of 3-5 million/mL, with a sensitivity of 0.746 and specificity of 0.711 (P=0.01). CONCLUSIONS: Patients with severe oligozoospermia should be warned of the possibility of becoming azoospermic and hence sperm freezing should be encouraged as early as possible.

Natural course of severe oligozoospermia in infertile male: influence on future fertility potential.

Most couples with severe male factor infertility are treated with assisted reproduction technology and little has been known about the prognosis of severe male factor infertility itself. We investigated the prognosis of infertile male patients with severe oligozoospermia. Thirty-nine patients with severe nonobstructive oligozoospermia were followed more than 6 months without any medical or surgical intervention. Retrospective analyses of the natural sequence of the condition and influences on the future fertility potential of the study participants were conducted. Sperm concentration, motility, and morphology between first semen analysis and last semen analysis were not significantly different. However, during the follow-up period, 5 (12.8%) patients became azoospermic. In 7 (17.9%) patients, the sperm count declined to a severe level that could be detected only after centrifugation. Three patients underwent microdissection testicular sperm extraction (TESE) for sperm retrieval after confirmation of azoospermia. The sperm retrieval was successful only in 1 of the 3 patients. Therefore, male patients diagnosed with severe oligozoospermia should be informed about possible aggravation of their residual spermatogenesis function and the necessity of intermittent follow-up semen analyses. If follow-up semen tests show a declining tendency, sperm cryopreservation may be recommended for these patients. If azoospermia develops during the follow-up period, early TESE procedure should be considered to improve the chance of sperm retrieval.

Stem cell factor/c-Kit signaling in in vitro cultures supports early mouse embryonic development by accelerating proliferation via a mechanism involving Akt-downstream genes.

PURPOSE: stem cell factor (SCF)/c-Kit regulates the proliferation and survival of germ cells or stem cells; however, little is known about the role of SCF/c-Kit in pre-implantation embryo development. METHODS: using exogenous SCF supplementation and c-Kit siRNA injection, we investigated the role and mechanism of SCF/c-Kit in pre-implantation mouse embryos. RESULTS: addition of soluble SCF to the culture medium improved blastocyst formation. c-Kit gene silencing reduced the rate of blastocyst formation and delayed embryonic development. The number of proliferating cells in c-Kit gene-silenced blastocysts decreased, whereas the number of apoptotic cells in blastocysts obtained from both experimental and the control groups was not affected. RT-PCR, immunostaining and western blotting revealed that proliferation-related Akt downstream targets were substantially affected by c-Kit gene silencing. CONCLUSION: SCF/c-Kit signaling through Akt downstream targets is likely involved in mediating the cleavage and proliferation of blastomeres during mouse pre-implantation embryogenesis.

Expression profile of genes identified in human spermatogonial stem cell-like cells using suppression subtractive hybridization.

Spermatogenesis is the process by which testicular spermatogonial stem cells (SSCs) self-renew and differentiate into mature sperm in the testis. Maintaining healthy spermatogenesis requires proper proliferation of SSCs. In this study, we sought to identify factors that regulate the proliferation of SSCs. Human SSC (hSSC)-like cells were isolated from azoospermic patients by a modified culture method and propagated in vitro. After four to five passages, the SSC-like cells spontaneously ceased proliferating in vitro, so we collected proliferating (P)-hSSC-like cells at passage two and senescent (S)-hSSC-like cells at passage five. Suppression subtractive hybridization (SSH) was used to identify genes that were differentially expressed between the P-hSSC-like and S-hSSC-like cells. We selected positive clones up-regulated in P-hSSC-like cells using SSH and functionally characterized them by reference to public databases using NCBI BLAST tools. Expression levels of genes corresponding to subtracted clones were analyzed using RT-PCR. Finally, we confirmed the differential expression of 128 genes in positive clones of P-hSSC-like cells compared with S-hSSC-like cells and selected 23 known and 39 unknown clones for further study. Known genes were associated with diverse functions; 22% were related to metabolism. Fifteen of the known genes and two of the unknown genes were down-regulated after senescence of hSSC-like cells. A comparison with previous reports further suggests that known genes selected, SPP1, may be related to germ cell biogenesis and cellular proliferation. Our findings identify several potential novel candidate biomarkers of proliferating- and senescencet-hSSCs, and they provide potentially important insights into the function and characteristics of human SSCs.

Identification of an intermediate state as spermatogonial stem cells reprogram to multipotent cells.

The aim of this study was to understand the mechanisms that allow mSSC lines to be established from SSCs. Small, multilayer clumps of SSCs formed during two to four weeks of in vitro culture and were then transferred to MEF feeders. Small, round, monolayer colonies containing cells destined to convert to mSSCs, designated as intermediate state SSCs (iSSCs), first appeared after two to three passages. During an additional nine passages (47-54 days) under the same culture conditions, iSSCs slowly proliferated and maintained their morphology. Ultimately, a cell type with an ES-like morphology (mSSC) appeared from the iSSC colonies, and two mSSC cell lines were established. The mSSCs had a high proliferative potential in serum-free ES culture medium and have been successfully maintained since their first establishment (> 12 months). We also compared the specific characteristics of iSSCs with those of SSCs and mSSCs using immunocytochemistry, FACS, RT-PCR, DNA methylation, and miRNA analyses. The results suggest that iSSCs represent a morphologically distinct intermediate state with characteristic expression patterns of pluripotency-related genes and miRNAs that arise during the conversion of SSCs into mSSCs. Our results suggest that iSSCs could be a useful model for evaluating and understanding the initiation mechanisms of cell reprogramming.

Effect of liquid nitrogen vapor storage on the motility, viability, morphology, deoxyribonucleic acid integrity, and mitochondrial potential of frozen-thawed human spermatozoa.

OBJECTIVE: To evaluate the effectiveness of liquid nitrogen (LN(2)) vapor (indirect contact method) for the storage of human semen. DESIGN: Experimental study. SETTING: University hospital-based fertility center. PATIENT(S): We evaluated 150 patients with normal sperm parameters. INTERVENTION(S): Human semen (N = 120) was mixed with Semen Freezing Medium, divided into two groups, frozen, and stored in LN(2) or LN(2) vapor. Frozen semen from each group (n = 40) was thawed after storage for 1 week, 1 month, or 3 months and then analyzed. In the second experiment, semen (n = 30) was divided into four groups, frozen, and stored in LN(2) or stored 7 cm, 12 cm, or 17 cm above the surface of LN(2) for 1 week. MAIN OUTCOME MEASURE(S): The motility and viability of sperm were evaluated by basic analysis, the morphology was analyzed with use of staining, the DNA integrity was assessed with use of terminal deoxyuridine triphosphate nick end-labeling assays, and active mitochondria were detected with use of rhodamine 123 staining. RESULT(S): The LN(2) and LN(2) vapor groups did not differ with regard to sperm motility, viability, morphology, DNA integrity, or active mitochondria when the cryostorage periods were compared. Furthermore, the quality of sperm stored within 17 cm of the surface of LN(2) for 1 week did not change. CONCLUSION(S): The storage of human semen in LN(2) vapor, without direct contact with LN(2), may represent a useful alternative for the effective storage of human semen.