Objective: The purpose of this study was to investigate the effect of controlled ovarian hyperstimulation (COH) on gap junction, and to induce the effect of an estrogen level overdose on gap junction in vitro by COH. Here, we mainly focus on connexion43 (Cx43), progesterone receptor (PR) and prolactin-related protein (PRP), and CyclinD3 genes expression, as well as the expression of Cx43 protein, were investigated. Materials and methods: Mature BDF-1 mice were divided into different COH, and the mouse uterus was isolated, Paraffin sections evaluate the effect of COH on mouse uterine endometrial morphology. The other part was used for the extraction of mouse uterine endometrial stromal cells (ESC), some related gene changes are detected. Human ESC were isolated from human endometrium by primary culture, the estrogen concentrations 10(-6) mol/L, 10(-7) mol/L were added, the changes of Cx43 gene and related proteins were detected, too. Results: (1) HE staining showed that in the ovulatory endometrium of mice in the high super ovulation group, uterine glands in the stromal layer were significantly increased, the relative vascular tissues was less abundant. (2) In three groups of COH mice, the expression of Cx43, PR, and PRP genes in ESC was significantly different (P < 0.05). (3) In vitro ESC in the COH group showed significant differences in Cx43, PR, and CyclinD3 gene expression (P < 0.05), and showed an obvious dose effect. In addition, Western blot analysis showed that the Cx43 protein and Cx43 gene expression were similar. Conclusions: (1) Animal experiments study showed that Cx43 gene expression in ESC was significantly decreased in hyper COH, in addition, the advance in gene expression was significantly earlier, suggesting decidualization appeared significantly earlier. (2) In vitro COH demonstrated when the estrogen concentration used was higher, the expression level of Cx43 gene and protein was lower. Combined with animal experiments, the endometrium decidualization was advanced in mice that were underwent hyper COH, which may reflect the endometrial receptivity. (C) 2019 Taiwan Association of Obstetrics & Gynecology. Publishing services by Elsevier B.V.

Objective: We studied the effects of interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) on the proliferation of porcine theca interna (TI) cells and further elucidated the roles of IL-6 and TNF-alpha in the pathogenesis of polycystic ovary syndrome.Materials and Methods: TI cells were treated with 10 pg/mL, 100 pg/mL, and 1000 pg/mL IL-6 or TNF-alpha. TI cell proliferation was then examined by carboxyfluorescein diacetate succinimidyl ester labeling and flow cytometry.Results: Cell proliferation was not significantly different in TI cells cultured in medium alone (control) or in the presence of IL-6. At 72 hours of treatment, the mean fluorescence intensity was significantly lower in TI cells treated with 100 pg/mL and 1000 pg/mL TNF-alpha than in the control (p < 0.05).Conclusion: TNF-alpha, but not IL-6, was able to promote TI cell proliferation. Our results suggest that TNF-alpha might play a role in hyperandrogenism, cortex thickness, and the increased ovary volume observed in polycystic ovaries. Copyright (C) 2016, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC.

Generating male germ cells in vitro from multipotent stem cells is still a challenge for stem cell biologists. The difficulty is caused by the lack of knowledge about spermatogenesis molecular-controlling mechanisms. In vivo, PGCs differentiate into male germ cells in a very complicated environment through many middle steps. In this study, we use the pluripotent p19 cells to test their responses to different retinoic acid (RA) concentrations by evaluating markers for stem cells (bmp4, egr3), primordial germ cells (ddx4), spermatogonia (c-kit), premeiotic cells (stra8), and male germ cells (dazl and plzf). We have found that cyp26b1, which will catalyze RA, increases dramatically in p19 cells 1 d after RA treatment. Bmp3, egr3, and stra8 are stimulated after 1 d of RA treatment and then recover to normal after 3 d of RA treatment. C-kit keeps being expressed when treated with 10 nM–4 μM RA. Dazl and plzf are gained after 3 d of stimulation. The morphology of RA (100 nM–4 μM)-treated cells changes distinctively, and cell colonies are formed. Typical neural cell-like and germ cell-like morphologies appear in the 100 nM and 4 μM RA groups, respectively. We conclude that 100–500 nM RA can cause responses in p19 cells, but a high concentration of RA (1–4 μM) can drive these pluripotent cells’ differentiation towards male germ cells. However, high concentrations of RA are also toxic. Some colonies that survived from 4 μM RA begin to express ddx4 and c-kit. Selection of the c-kit + , dazl + , and ddx4 + cells after RA stimulation and creating a special culture medium for their propagation might benefit successful spermatogenesis induction in vitro.

目的:比较玻璃化法与慢速程序法对人卵裂期胚胎冷冻复苏的效果.方法:将接受体外受精-胚胎移植患者的取卵周期移植后剩余的胚胎,分别进行玻璃化法或慢速程序法冷冻,比较两组胚胎解冻后的复苏率、胚胎完整率、胚胎种植率、临床妊娠率及观察非当日移植的复苏胚胎的发育潜能等指标.结果:玻璃化冷冻组的活产率均显著高于慢速程序化冷冻;玻璃化冷冻后的胚胎复苏率和胚胎完整率显著高于慢速程序化冷冻;复苏周期移植取消率显著低于慢速程序化冷冻;而临床妊娠率和种植率的差异无统计学意义.结论:玻璃化冷冻和慢速程序化冷冻相比有更高的复苏率和胚胎利用率.

Gene abnormality during spermatogenesis stages before mature spermatozoa can cause their inability to fertilize eggs. In order to solve this problem of male infertility, studying gene function and its regulation mechanism during spermatogenesis becomes more and more important. Tctex5 is a novel sperm motility-related gene discovered in mouse testis recently and encodes Tctex5 interacting with PP1 gamma, which is predominantly expressed in the testis. In this study, we have compared expression of Tctex5 in male germ cell lines representing different spermatogenic stages (spermatogenic stem cells, differentiating spermatogonia and differentiated spermatocytes, respectively) and the Sertoli cell line in mouse. Restricted to the nuclei, expression of Tctex5 decreases from highest in the spermatogenic stem cell stage to lowest in the differentiated spermatocyte stage. The estimated interacting protein of Tctex5, PP1 gamma, is co-expressed in the nuclei. PP1 gamma expression showed a similar pattern with that of Tctex5. When Tctex5 is knocked down by siRNA, expression of the interacting protein PP1 gamma is also reduced. Tctex5 expression is reduced when germ cell enters into meiosis indicating that Tctex5 may act as a regulating factor to prepare cell entering into meiosis. Its down expression caused PP1 gamma expression to be reduced. This may change the phosphorylation level of many proteins in germ cells which may further regulate meiosis, apoptosis or other cell events.

Induced multipotent stem (iMS) cells are originated from somatic cells and become multipotent by genetic and/or epigenetic modifications. Previous studies have shown that the fish oocytes extracts (FOE) can induce skin fibroblast cells into iMS cells. In this study, we aim to determine whether FOE can similarly induce mouse peripheral blood mononuclear cells (PBMCs) into the iMS state and if so, whether they can survive longer when they are transplanted into the irradiation female mice. PBMCs of GFP-transgenic male mice were cultured and transiently reprogrammed by FOE. They were deemed reaching the iMS state after detection of expression of stem cell markers. The iMS-like PBMCs were transplanted into female C57BL mice by tail vein injection. The spleen wet weights as well as numbers of colonies of the recipient mice were examined. The results showed the spleen wet weights and numbers of spleen colonies of FOE-induced group were all significantly higher than those of the non-induced group and negative control group. On day 90 after transplantation, FISH analysis detected the presence of Y chromosome in the induced group, but not of the other groups. The current findings demonstrate that FOE-induced PBMCs are able to survive longer in irradiated female mice.

It has been proven that c-kit is crucial for proliferation, migration, survival and maturation of spermatogenic cells. A periodic expression of c-kit is observed from primordial germ cells (PGCs) to spermatogenetic stem cells (SSCs), However, the expression profile of c-kit during the entire spermatogenesis process is still unclear. This study aims to reveal and compare c-kit expression profiles in the SSCs before and after the anticipated differentiation, as well as to examine its relationship with retinoic acid (RA) stimulation.

To observe the relative activity of sperm mitochondria and the proportion of ROS-positive sperm before and after capacitation and progesterone (Pg)-induced hyperactivation, and investigate the functional characteristics of sperm mitochondria.We collected 20 samples of normal human spermatozoa that met the criteria of WHO Laboratory Manual for the Examination and Processing of Human Semen (5th ed) and cultured them with the swim-up method in a CO2 incubator at 37 degrees C for 1 hour. We divided the sperm into a pre-capacitation and a capacitated group, and further incubated the capacitated sperm in an upright tube with (Pg-induced group) or without (control group) slow-releasing Pg at 37 degrees C for another hour. Then we determined the relative activity of mitochondria and the percentage of ROS-positive cells in the sperm samples using JC-1 and DCF staining.The relative activities of mitochondria were significantly increased in the capacitated, control and Pg-induced groups (6.23, 14.36 and 12.33) as compared with the pre-capacitation group (1.42) (P < 0.05), while the percentages of balanced mitochondria (mitochondria with equal amount of high and low electric potentials) remarkably reduced (4.27%, 5.03% and 8.57% vs 21.64%, P < 0.05). The percentages of ROS-positive sperm in the pre-capacitation, capacitated, control and Pg-induced groups were 2.89%, 0.70%, 4.25% and 1.90%, respectively, significantly lower in the capacitated than in the pre-capacitation group (P < 0.01), but dramatically increased in the control group after another hour of swim-up incubation and markedly higher than in the Pg-induced group (P < 0.01).Progesterone induction can hyperactive human sperm motility, inhibit the relative activity of mitochondria, keep mitochondria potential at a more balanced level, and reduce the production of ROS, which may help to raise the rate of in vitro fertilization and improve the quality of embryos.

It is important issue for assisted reproductive techniques(ART) to improve the pregnancy rate and security. As the most abundant organelles in oocytes,mitochondria not only take part in the energy supply for oocyte maturation, fertilization,transcription and translation during embryonic development,but also play roles in some key procedures such as the oocyte activation,calcium oscillation,and apoptosis. More importantly,all mitochondria in embryo were originated from oocyte,not the sperm. It has been demonstrated that mitochondria dysfunction is closely related to oocyte aging,low fertilization rate,poor embryo quality,and developmental arrest of embryo,etc. In this paper,the roles of mitochondrial function in oocyte maturation,fertilization and embryo development, as well as how to protect oocyte mitochondria and how to improve the function of oocyte mitochondria during ART, were reviewed.

Abstract The objective of this study was to investigate the effects of transplanted bone marrow mesenchymal stem cells (BMSCs) administered via internal jugular vein injection, carotid artery injection, or intraventricular transplantation for the treatment of cerebral infarction, which was modeled in rats. The neurological scores of the treated rats and the distribution of the transplanted cells (GFP-labeled) in the infarction area were evaluated. The cerebral infarction model was produced by inserting a modified Zea-longa suture, which generated middle cerebral artery occlusion (MCAO). The GFP-labeled BMSCs were transplanted through the jugular vein or the carotid artery or by stereotactic intraventricular delivery to the infarction models 1 week after the cerebral infarction was established. The ‘Nerve Function Score’ of the model rats was recorded before and after BMSC transplantation. Brain tissue sections were examined under a fluorescence microscope. We determined that the transplanted BMSCs rescued brain function, which was indicated by a decrease in the neurological scores (P<0·05) following BMSC transplantation. The effect of BMSC transplantation was reflected in decreases in the neurological score in the intraventricular transplantation group, the carotid artery transplantation group, and the jugular vein graft group*. The transplanted BMSCs were able to migrate to the brain injury area and the cortex and survived the infarction; thus, BMSCs may promote the recovery of nerve function.

"ocyte aging" is an important cause for IVF failure and poor embryo quality.At present,many women delay their pregnancy to the later part of their childbearing years which makes "ocyte aging" a more important etiology for infertility.Mitochondrial dysfunction is associated with somatic cells senescence.Because mitochondria are the most abundant organelles in oocytes/embryos,we speculate that mitochondrial dysfunction may have an important role in oocyte maturation,fertilization and embryo development too.In this article,we have reviewed the function of mitochondria during oocyte maturation,fertilization and the following embryo development.Techniques improving mitochondrial function in the oocytes could not only prevent the oocyte aging process,but also improve the quality of embryos from assisted reproductive technology(ART).

Treatment of male infertility started from the intrauterine insemination with husband's semen(IUH).In the early stage,assisted reproductive technology(ART) was mainly for female patients with infertility,specifically for the tubal factor infertility.The outcome of ART treatment of male factor infertility was not good as that of female factor infertility,because of the low in vitro fertilization rate due to the low spermatozoa motility.To get clinical pregnancies,Gordon and Talansly in 1988 successfully developed ′zona drilling(ZD)′,and Cohen developed ′partial zonal dissection(PZD)′.Although these techniques partially overcome the difficulties of fertilization,they were not widely applied due to the low monospermic fertilization rate(15%-25%),the high polyspermic fertilization rate,the relative high requirement for sperm number and motility,and the low clinical pregnancy rate(10%).In 1992,Palermo successfully developed the intracytoplasmic sperm injection(ICSI) technique for the creative treatment of male infertility.ART should never be the first choice for the treatment of male infertility,it is a important backup choice after routine treatment.The good treatment for male infertility should be evaluated overall factors,including male fertility function,fertility ability of female partner,selectable ART methods,safety and low-invasiveness and low cost.

Spermatogenesis is the process of production of male gametes from SSCs. The SSCs are the stem cells that differentiate into male gametes in the testis. in the mean time, the Spg are remarkable for their potential multiple trans-differentiations, which make them greatly invaluable for clinical applications. However, the molecular mechanism controlling differentiation of the Spg is still not clear. Among the discovered spermatogenesis-related genes, c-kit seems to be expressed first by the Spgs thus may play a central role in switching on the differentiation process. Expression of Kit and the activation of the Kit/Kitl pathway coincide with the start of differentiation of Spgs. Several genes have been discovered to be related to the Kit/Kitl pathway. in this review, we have summarized the recent discoveries of c-kit and the Kit/Kitl pathway-related genes in the spermatogenic cells during different stages of spermatogenesis.

Mammalian spermatozoa acquire the capacity for motility and fertilization during the transit through the epididymis under the control of different factors, such as CAMP, intracellular pH, intracellular calcium and phosphorylation of sperm proteins. As the acquisition of functional competence including gaining motility during epididymal transit occurs in the complete absence of contemporaneous gene transcription and translation on the part of the spermatozoa, it is widely accepted that post-translational modifications are the only means by which spermatozoa can acquire functionality. Serine-threonine protein phosphatase 1 (PP1) together with their testis/ sperm-specific interacting proteins might be involved in this regulatory mechanism. PP1 alpha, PP1 beta/delta, PP1 gamma 1 and PP1 gamma 2 are all expressed in the testis whereas PP1 gamma 2 is the only isoform expressed on spermatozoa. I2, I3, sds22, 14-3-3 and hsp90 are associated with PP1 gamma 2 in spermatozoa located on the sperm head and tail. Activity of PP1 gamma 2 and the binding pattern to these regulatory proteins changes in spermatozoa recruited from the Caput and those from the cauda part of the epididymis. In this review, we summarize the possible roles of PP1 on spermatozoa during spermatogenesis and flagellar motility control. We suggest that PP1 might take part in the inhibition of the sperm motility activation by interacting with AKAPs and CAMKII. A hypothesized signaling pathway of mammalian sperm motility activation and PP1's function has been proposed.

Intra-uterine insemination (IUI) is one of the most frequently used fertility treatments for couples with unexplained male subfertility. The objective of this study was to compare the pregnancy rate in humans after sperm washing by sperm wash medium (Conception Technology, CA) and SperMagic medium (Reprobiotech, NY) for IUI. This study will provide useful data for SpermMagic medium for use on human IUI, particularly for male factor infertility. SperMagic medium containing sperm stimulators was specially designed for male factor and/or poor quality sperm with lower motility. There were 640 patients (35±5 years old) with unexplained and male subfertility, they were undergoing 1240 treatment cycles by using standard stimulation protocol. Patients were randomly divided into two groups. Group I (630 cycles) was performed IUI by inseminators with a standard insemination dose (≥5-10 million of fresh spermatozoa), the sperm specimen was prepared by pre-warmed SperMagic medium + 10% SPS (Copper Surgical Sage, NJ) Group II (610 cycles) was performed similar procedure for IUI, however, the sperm specimen was prepared by pre-warmed sperm wash medium (Conception Technology, CA) + 10% SPS. The pregnancy rates were compared between two groups. The data suggests that the use of SperMagic medium resulted in significantly increased pregnancy rate in group I (114/63, 18.10% per cycle) as compared with Group II (68/610, 11.15% per cycle) (P<0.05). It is concluded that IUI could achieve a higher pregnancy rate in human after using SperMagic medium to prepare sperm. This data indicates SperMagic medium might be great beneficial for human IUI treatment, especially for male factor infertility.

Aim: To determine the possible roles of the t-complex testis expressed gene 5 (Tctex5) on sperm functions, the full-length sequence of mRNA was studied and compared in the testis between the normal wild-type and the sterile t-haplotype mutant mice. Methods: We applied rapid amplification of cDNA ends, Northern blot and reverse transcription polymerase chain reaction to analyze the full length of Tctex5 mRNAs isolated from testes of the wild-type and the t-haplotype mice. Reverse transcription polymerase chain reaction was used to semi-quantitatively compare expression of Tctex5 transcripts in the 16 tissues and 9.5 day stage embryos in the wild-type mice. E-translation was applied to estimate the amino acid sequences. Results: One long and one short transcript of Tctex5 mRNA were discovered in mouse testis of wild-type (Tctex5(long-+) and Tctex5(short-+)) and t-haplotype (Tctex5(long-t) and Tctex5(short-t)) mice, respectively. Being enhanced only in the testis, Tctex5(long-t) had 17 point mutations and one 15-bp-deletion in the exon 1 region, comparing with the Tctex5(long-+), whereas the Tctex5(short-t) was similar to the Tctex5(short-+). The short isoforms of Tctex5 mRNAs in the two models encoded exactly the same peptides, but the long isoforms did not. The estimated peptide encoded by Tctex5(long-t) had significant mutations on putative sites of phosphorylation and PP1 binding. Conclusion: We established that mutations that occur in the Tctex5 long transcript of the t-haplotype mice are important for normal sperm function, whereas the short transcript of Tctex5 might have a conserved function among different tissues.

目的探讨雄鼠生殖细胞中是否有Tctex5基因的表达。方法从三株体外培养的雄鼠生殖细胞CRL-1715、CRL-2053和CRL-2196细胞株中提取总RNA和蛋白,采用RT-PCR和Western blot分别从mRNA和蛋白质水平分析基因产物的表达。结果Tctex5基因在CRL-1715、CRL-2053、CRL-2196三株细胞中都有高表达。结论Tctex5基因作为一个未知功能的基因,在雄鼠生殖细胞中有丰富的表达,可能参与了精子发生的功能调节,作为男性正常生育的一个新的标志物,它可能在精子发生中扮演了一个重要的角色,该基因在雄性生殖细胞中的发现可能会对男性不育的诊断与治疗提供新的途径。

The Y chromosome evolves from an autochromosome and accumulates male-related genes including sex-determining region of Y-chromosome (SRY) and several spermatogenesis-related genes. The human Y chromosome (60 Mb long) is largely composed of repetitive sequences that give it a heterochromatic appearance, and it consists of pseudoautosomal, euchromatic, and heterochromatic regions. Located on the two extremities of the Y chromosome, pseudoautosomal regions 1 and 2 (PAR1 and PAR2, 2.6 Mb and 320 bp long, respectively) are homologs with the termini of the X chromosome. The euchromatic region and some of the repeat-rich heterochromatic parts of the Y chromosome are called "male-specific Y" (MSY), which occupy more than 95% of the whole Y chromosome. After evolution, the Y chromosome becomes the smallest in size with the least number of genes but with the most number of copies of genes that are mostly spermatogenesis-related. The Y chromosome is characterized by highly repetitive sequences (including direct repeats, inverted repeats, and palindromes) and high polymorphism. Several gene rearrangements on the Y chromosome occur during evolution owing to its specific gene structure. The consequences of such rearrangements are not only loss but also gain of specific genes. One hundred and fifty three haplotypes have been discovered in the human Y chromosome. The structure of the Y chromosome in the GenBank belongs to haplotype R1. There are 220 genes (104 coding genes, 111 pseudogenes, and 5 other uncategorized genes) according to the most recent count. The 104 coding genes encode a total of about 48 proteins/protein families (including putative proteins/protein families). Among them, 16 gene products have been discovered in the azoospermia factor region (AZF) and are related to spermatogenesis. It has been discovered that one subset of gene rearrangements on the Y chromosome, "micro-deletions", is a major cause of male infertility in some populations. However, controversies exist about different Y chromosome haplotypes. Six AZFs of the Y chromosome have been discovered including AZFa, AZFb, AZFc, and their combinations AZFbc, AZFabc, and partial AZFc called AZFc/gr/gr. Different deletions in AZF lead to different content spermatogenesis loss from teratozoospermia to infertility in different populations depending on their Y haplotypes. This article describes the structure of the human Y chromosome and investigates the causes of micro-deletions and their relationship with male infertility from the view of chromosome evolution. After analysis of the relationship between AZFc and male infertility, we concluded that spermatogenesis is controlled by a network of genes, which may locate on the Y chromosome, the autochromosomes, or even on the X chromosome. Further investigation of the molecular mechanisms underlying male fertility/infertility will facilitate our knowledge of functional genomics.

Expression profile of microRNA (miRNA) in mouse oocytes and preimplantation embryos has been revealed by a novel high throughput microarray assay. A total of 97 (43 “new” and 54 known) including mouse, human, and predicted miRNAs have been discovered in the preimplantation mouse embryos which can be classified into developmental stage‐dependent groups and non–stage‐dependent group according to the statistical analysis of the expression patterns. Potential gene targets of each group of miRNAs are estimated by TargetsScan system and significantly changed signaling pathways and biological processes underlying these gene targets are searched by PANTHER classification system between the stage‐dependent miRNAs and the non–stage‐dependent miRNAs. Expression of some miRNAs is confirmed by reverse transcriptase‐polymerase chain reaction. It is shown that dynamic synthesis and degradation of miRNAs coexists in the preimplantation development of mouse embryos. However, the overall quantity of miRNAs and percentage of the stage‐dependent miRNAs increase as the preimplantation embryos develop. Developmental Dynamics 237:2315–2327, 2008. © 2008 Wiley‐Liss, Inc.

The objective of this study was to identify and compare the expression profiles of progesterone receptor (PR) and estrogen receptor alpha (ERalpha) in the testes of men with obstructive azoospermia (OA), maturation arrest (MA), and Sertoli cell-only (SCO) histology. Testicular biopsies were obtained from 10 patients with OA, 10 patients with MA (either early or late arrest), and 8 patients with SCO who did not have hormonal abnormalities and varicoceles. Expression of PR and ERalpha was detected by immunofluorescence and Western blot. PR was expressed in the spermatogenic, Leydig, and Sertoli cells in the testes of OA patients. In the MA and SCO patients, the expression of PR was reduced in all cell types as compared with that in the OA patients. Western blot demonstrated that both the full-size (120 KDa) and the truncated (52 KDa) isoforms of the PR were expressed in the OA and MA testes. However, in the SCO testes, only the truncated isoform of PR (52 KDa) was expressed. ERalpha (66 KDa) was expressed principally in the spermatogenic and Leydig cells in the OA testes. By immunohistochemistry staining, expression of ERalpha was decreased in the spermatogenic and Leydig cells of the MA testes, whereas its expression was enhanced in the Leydig cells of the SCO testes. However, by Western blot, expression of ERalpha was significantly reduced in the SCO testes as compared with that in the OA and MA testes. We conclude that PR and ERalpha may play a role in the pathogenesis of the MA and SCO phenotype in patients with infertility.