Effect of endometrial cell-conditioned medium and platelet-rich plasma on the developmental competence of mouse preantral follicles: An in vitro study / 대한생식의학회지

Objective@#The aim of this study was to evaluate the impacts of platelet-rich plasma (PRP) and conditioned medium (CM) derived from endometrial stromal cells on mouse preantral follicle culture in a two-dimensional system to produce competent mature oocytes for fertilization. @*Methods@#In total, 240 preantral follicles were isolated from female mouse ovarian tissue and divided into four groups. The preantral follicles were isolated three times for each group and then cultured, respectively, in the presence of alpha minimum essential medium (control), PRP, CM, and PRP+CM. The in vitro growth, in vitro maturation, and cleavage percentage of the preantral follicles were investigated. Immunocytochemistry (IHC) was also conducted to monitor the meiotic progression of the oocytes. Additionally, the mRNA expression levels of the two folliculogenesis-related genes (Gdf9 and Bmp15) and two apoptosis-related genes (Bcl2 and Bax) were investigated using real-time polymerase chain reaction. @*Results@#In the PRP, CM, and PRP+CM groups, the preantral follicle maturation (evaluated by identifying polar bodies) were greater than the control group. The cleavage rate in the CM, and PRP+CM groups were also greater than the control group. IHC analysis demonstrated that in each treatment group, meiotic spindle was normal. In the PRP+CM group, the gene expression levels of Bmp15, Gdf9, and Bcl2 were greater than in the other groups. The Bax gene was more strongly expressed in the PRP and control groups than in the other groups. @*Conclusion@#Overall, the present study suggests that the combination of CM and PRP can effectively increase the growth and cleavage rate of mouse preantral follicles in vitro.

Effects of different doses and time-dependency of busulfan on testes parameters and spermatogenesis in a rat model: a quantitative stereological study

Objectives: The current study aimed at evaluating testis parameters and spermatogenesis changes in male rats administrated by different busulfan doses and time to construct a subfertile animal model by stereological methods

Materials and Methods: In the present study, 150 male Wistar rats randomly divided into 5 groups. All experimental groups were treated by different concentrations of busulfan [0.0, 2.5, 5, 10, and 15 mg/kg]. Rats were sacrificed 1, 15, and 30 days after busulfan treatment. The tissue processing was done for stereological study and the results were analyzed by the one-way ANOVA followed by the Duncan test

Results: The most stereological parameters such as testes weight and volume, tubules volume density, interstitial tissue [P<0.05], and germinal epithelium [P<0.01] were significantly reduced by busulfan treatment. Also, at different busulfan doses, the number of spermatogenic cells including spermatogonia [P<0.05], spermatocyte, round and elongated spermatid, and the Sertoli and Leydig cells [P<0.01] significantly decreased, compared with those of the control group. The decline was more obvious in higher busulfan doses and time [from the day 15 to 30] [P<0.05]

Conclusion: Most of testicular stereological parameters reduced during 15 days onwards after busulfan treatment in a dose-dependent manner

Hepatogenic Differentiation Capacity of Human Wharton’s Jelly Mesenchymal Stem Cell in a Co-culturing System with Endothelial Cells in Matrigel/collagen Scaffold in the Presence of Fetal Liver Extract

BACKGROUND: Human Wharton’s jelly mesenchymal stem cells (HWJMSCs) isolated from medical waste product can be considered as an accessible source of cells in regenerative medicine. Stem cell-derived hepatocytes have poor function and need appropriate niche to reconstruct the liver structure. Therefore, we attempted to find a novel approach in differentiating HWJMSCs into functional hepatic cells using 3D culture conditions and liver extract that recapitulates vital stage in liver development. MATERIALS AND METHODS: HWJMSCs were extracted from human Wharton’s jelly, characterized by flow cytometry, and differentiated towards osteogenic and adipogenic lineages. HWJMSCs were co-cultured with HUVECs in 3D matrigel/collagen scaffolds in the presence of fetal liver extract for 14 days. The expression of specific liver genes were evaluated by lectins, PAS and immunocytochemistry. RESULTS: According to flow cytometry data, isolated cells from HWJMSCs were shown to express MSC markers. HWJMSCs co-cultured with HUVECs in matrigel/collagen scaffold with extract expressed albumin, lectins UEA and PNA. Immunohistochemistry of the cells in matrigel/collagen scaffold with or without extract exhibited a positive reaction for CK19. CONCLUSIONS: Co-culturing of the HWJMSC/HUVEC in 3D matrigel/collagen scaffold is bimimicary of in vivo cell condition. The results showed that administration of the liver extract in 3D matrigel/collagen culture of HWJMSC/HUVEC can induce hepatocyte marker expression.

Comparison of the expression of hepatic genes by human Wharton's jelly mesenchymal stem cells cultured in 2D and 3D collagen culture systems

Background: Human Wharton's jelly mesenchymal stem cells [HWJMSCs] express liver-specific markers such as albumin, alpha-fetoprotein, cytokeratin-19, cytokeratin-18, and glucose-6-phosphatase. Therefore, they can be considered as a good source for cell replacement therapy for liver diseases. This study aimed to evaluate the effects of various culture systems on the hepatocyte-specific gene expression pattern of naive HWJMSCs

Methods: HWJMSCs were characterized as MSCs by detecting the surface CD markers and capability to differentiate toward osteoblast and adipocyte. HWJMSCs were cultured in 2D collagen films and 3D collagen scaffolds for 21 days and were compared to control cultures. Real time RT-PCR was used to evaluate the expression of liver-specific genes

Results: The HWJMSCs which were grown on non-coated culture plates expressed cytokeratin-18 and -19, alpha-fetoprotein, albumin, glucose-6-phosphatase, and claudin. The expression of the hepatic nuclear factor 4 [HNF4] was very low. The cells showed a significant increase in caludin expression when they cultured in 3D collagen scaffolds compared to the conventional monolayer culture and 2D collagen scaffold

Conclusion: Various culture systems did not influence on hepatocyte specific marker expression by HWJMSCs, except for claudin. The expression of claudin showed that 3D collagen scaffold provided the extracellular matrix for induction of the cells to interconnect with each other

Effect of follicular fluid and platelet-activating factor on lactate dehydrogenase C expression in human asthenozoospermic samples

Background: Application of follicular fluid [FF] and plateletactivating factor [PAF] in artificial insemination improves sperm motility. Lactate dehydrogenase C [LDH-C] is a key enzyme for sperm motility. In this study, the effects of FF and PAF on the sperm motility index and LDH-C expression were investigated. Moreover, LDH-C expression was compared between asthenozoospermic and normozoospermic samples

Methods: The expression of LDH-C was examined by quantitative real-time polymerase chain reaction [q-RT PCR] and western blotting after it was treated with optimized concentrations of FF and PAF in twenty asthenozoospermic samples. Also, LDH-C expression was evaluated in five normozoospermic samples

Results: Samples with 75% FF and 100 nM of PAF had an increase in their percentages of progressive and slowly motile sperms and a decrease in their percentages of non-progressive and non-motile sperms. Moreover, LDH-C mRNA transcripts were not changed following PAF and FF treatment, and LDH-C protein was detected in highly progressive motile specimens treated with FF in the asthenozoospermic samples. Furthermore, LDH-C expression was more detectable in the normal sperms

Conclusion: Our results indicated that PAF had more beneficial effects than FF on sperm motility in the asthenozoospermic samples [P=0.0001], although the LDH-C expressions of the sperms were not changed significantly in both groups. We found no association between LDH-C expression and sperm motility after FF and PAF actions. This finding, however, requires further investigation. The fact that LDH-C protein was detected in the normozoospermic, but not asthenozoospermic, samples could be cited as a reason for the infertility in these patients

comparison of the multiple oocyte maturation gene expression patterns between the newborn and adult mouse ovary

The interaction between follicular cells and oocyte leads to a change in gene expression involved in oocyte maturation processes. The purpose of this study was to quantify the expression of more common genes involved in follicular growth and oocyte developmental competence. In this experimental study, the expression of genes was evaluated with qRT-PCR assay in female BALB/c mice pups at 3-day of pre-pubertal and 8 week old virgin adult ovaries. The tissue was prepared by H and E staining for normal morphological appearance. The data were calculated with the 2-delta Ct formula and assessed using non-parametric two-tailed Mann-Whitney test. The p<0.05 was considered as significant. The data showed a significant increase in the level of Stra8 and GDF9 in adult compared with newborn mice ovaries [p=0.049]. In contrast, a significant decrease in the level of Mvh, REC8, SCP1, SCP3, and ZP2 was observed in adult mice ovaries compared to those in the newborn mice ovaries [all p=0.049 except SCP1: p=0.046]. There was no significant difference in the level of OCT4 and Cx37 expression between adult and newborn mice ovaries. The modifications in gene expression patterns coordinate the follicular developmental processes. Furthermore, the findings showed higher expression level of premeiotic gene [Stra8] and lower level of meiotic entry markers [SCP1, SCP3, and REC8] in juvenile than newborn mouse ovaries

Effects of Carthamus tinctorius on Semen Quality and Gonadal Hormone Levels in Partially Sterile Male Rats

PURPOSE: Traditional herbal medicine is just one of the many different approaches using plants in the remedy of diseases. Carthamus tinctorius (CT) or safflower is a popular plant that is used for coloring and flavoring in food industries. The effect of CT on spermatogenesis and sperm parameters has been reported in traditional medicine but has not yet been confirmed scientifically. Therefore, this study was designed to determine the effects of CT on spermatogenesis and the male reproductive system in an animal model. MATERIALS AND METHODS: Sixty male rats were divided into five groups. Four groups were injected with 5 mg/kg of busulfan as a model of partial infertility. Then, the experimental groups were treated with 10 mg/kg, 25 mg/kg, or 50 mg/kg of CT extract for 35 days. The control was treated with busulfan (infertile control) or distilled water only. After this period, the animals were sacrificed and blood samples were taken for hormonal assay. The semen was collected from the epididymis and the reproductive organs were assessed. Sperm count and motility were measured and smears were prepared for assessment of the other parameters. RESULTS: The results indicated that the percentage of sperm with good morphology, motility, and count increased significantly in the group treated with 10 mg/kg CT (p=0.002, p=0.03, and p=0.00001, respectively). The effects on hormonal changes and genital organ weights were also positive. CONCLUSIONS: It is probable that the CT extract affects spermatogenesis and as a result sperm quality. Further studies are needed.

Effects of sera taken from women with recurrent spontaneous abortion on sperm motility and apoptosis

Recurrent spontaneous abortion impacts almost 1% of couples. The sera from women with unexplained recurrent spontaneous abortion [URSA] have toxic effects on embryos that grow in the uterus. Therefore, the abnormal condition of the uterus may also affect sperm qualities. The objectives of this study were to search if these sera could induce DNA denaturation in sperm nuclei and also it could reduce sperm motility. Sera of 20 women with URSA history and sera from 20 women with at least two healthy children were added to the sperms samples from 20 healthy men for 2 hours. The sperm motility was assessed after incubation with sera. The samples were stained with Tdt mediated dUTP nick end labeling [TUNEL] assay for DNA fragmentation. The samples were analyzed with flow cytometry and the percentage of the TUNEL positive sperms were calculated. The data were analyzed by t-test. The incubation of the sperm samples in sera with URSA lead to a decrease in the percentage of the motile sperm from 55% in control to 41% in the treated group, significantly [p=0.038]. The percentage of the sperm with abnormal fragmented DNA increased after incubation with URSA [26.6%] compare to the control [21.2%]; however, it was not significant. It seems that sera from URSA patients could not induce a significant increase in the percentage of the sperms with nuclei contain DNA fragmentation. However, the sera of women with URSA could affect the fertility rate by reduction of the sperm motility