Platelet-rich plasma promotes the development of isolated human primordial and primary follicles to the preantral stage.

This study aimed to assess the effects of platelet-rich plasma (PRP) on growth and survival of isolated early human follicles in a three-dimensional culture system. After fresh and vitrified-warmed ovarian tissue was digested, isolated early preantral follicles and ovarian cells were separately encapsulated in 1% alginate (w/v). The encapsulated follicles and ovarian cells were cultured together in a medium supplemented with foetal bovine serum (FBS), PRP, PRP + FBS, or human serum albumin (HSA) for 10 days. Growth and survival of the follicles were assessed by measurement of diameter and staining with trypan blue. Follicular integrity was assessed by histological analysis. After culturing, all follicles increased in size, but growth rate was greater in follicles isolated from fresh samples than those from vitrified-warmed ones (P < 0.001). Similarly, follicular viability of fresh samples after culturing was higher than that of vitrified-warmed ones. The growth and survival rates of follicles from both fresh and vitrified groups cultured in PRP supplemented media were significantly higher than those of other groups (growth P < 0.001 and survival P < 0.05, in both groups). In conclusion, media supplementation with PRP can better support viability and growth of isolated human early preantral follicles in vitro.

Development of sheep primordial follicles encapsulated in alginate or in ovarian tissue in fresh and vitrified samples.

In vitro follicle growth is a promising strategy for female fertility preservation. This study was conducted to compare the development of ovine follicles either isolated or in the context of ovarian cortical pieces after short term (8 days) three-dimensional culture in fresh and vitrified samples. Four different experiments were conducted; I) culture of ovarian cortical pieces encapsulated in 0.5% and 1% alginate and without alginate encapsulation (CP-0.5%, CP-1% and CP, respectively), II) culture of isolated primordial and primary follicles encapsulated in 1% and 2% alginate (IF-1% and IF-2%, respectively), III) culture of fresh and vitrified-warmed cortical pieces (F-CP and Vit-CP, respectively), and IV) culture of fresh and vitrified-warmed encapsulated isolated follicles (F-IF and Vit-IF, respectively). The number of secondary follicles after culture was negatively influenced by encapsulation of ovarian cortical pieces (6.3 ± 3.3 and 10.6 ± 0.9 vs 21.5 ± 2.3 in CP-0.5% and CP-1% vs CP, respectively). The diameter of follicles in IF-2% was higher than IF-1% (54.06 ± 2 vs 41.9 ± 1.5) and no significant difference in follicular viability was observed between the two groups. The proportions of different follicular types and their viability after culture in vitrified-warmed cortical pieces were comparable with fresh ones. The viability of vitrified-warmed isolated follicles was lower than fresh counterparts. The growth rate of fresh follicles was higher than vitrified-warmed follicles after culture (47.9 ± 1 vs 44.6 ± 1). In conclusion, while encapsulation of ovarian cortical pieces decreased the follicles' development, it could better support the growth of isolated follicles. Moreover, the viability and growth rate of isolated-encapsulated follicles was decreased by vitrification.

Morphologic and proliferative characteristics of goat type A spermatogonia in the presence of different sets of growth factors.

PURPOSE: The present study by using different growth factors was aimed to develop the best practical culture condition for purification of goat undifferentiated SSCs and their colonization under in vitro and in vivo conditions. METHODS: The enzymatically isolated SSCs obtained from one month old goat testes were cultured in DMEM plus FCS supplemented with different sets of growth factors (GDNF, LIF, bFGF, and EGF) for 2 weeks. At the end of each week, the morphological characteristics of cells and colonies alongside with purification rate of undifferentiated type A spermatogonia were evaluated by immunocytochemical staining and flow cytometry. RESULTS: The number and size of colonies in treatment groups were significantly (P < 0.01) higher than corresponding values in control group. In immunocytochemical evaluation, the proportion of KIT and PGP9.5 positive cells were significantly (P < 0.001) higher in control and treatment groups, respectively. CONCLUSIONS: The culture medium comprising all four growth factors, especially the one supplemented with the higher concentration of GDNF, was superior to the other groups with respect to the population of undifferentiated type A spermatogonia and its propagation in culture system. Additionally, goat SSCs could colonize within the mouse testis following xenotransplantation.

In-utero transfer of decidualized endometrial stromal cells increases the frequency of regulatory T cells and normalizes the abortion rate in the CBA/J × DBA/2 abortion model.

Introduction: Failure to adequate decidualization leads to adverse pregnancy outcomes including pregnancy loss. Although there are plenty of reports underscoring immune dysfunction as the main cause of abortion in CBA/J females mated with DBA/2 males (CBA/J × DBA/2), little is known about the potential role of impaired endometrial decidualization. Methods: Endometrial stromal cells (ESCs) from CBA/J mice were in-vitro decidualized, and the proteome profile of the secretome was investigated by membrane-based array. CBA/J mice were perfused In-utero with either decidualized ESCs (C×D/D), undecidualized ESCs (C×D/ND), or PBS (C×D/P) 12 days before mating with DBA/2 males. Control mice were not manipulated and were mated with male DBA/2 (C×D) or Balb/c (C×B) mice. On day 13.5 of pregnancy, reproductive parameters were measured. In-vivo tracking of EdU-labeled ESCs was performed using fluorescence microscopy. The frequency of regulatory T cells (Tregs) in paraaortic/renal and inguinal lymph nodes was measured by flow cytometry. The proliferation of pregnant CBA/J splenocytes in response to stimulation with DBA/2 splenocytes was assessed by 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) flow cytometry. Results: In C×D/D mice, the resorption rate was reduced to match that seen in the C×B group. Intrauterine perfused ESCs appeared in uterine stroma after 2 days, which remained there for at least 12 days. There was no difference in the number of implantation sites and embryo weight across all groups. The frequency of Tregs in the inguinal lymph nodes was similar across all groups, but it increased in the paraaortic/renal lymph nodes of C×D/D mice to the level found in C×B mice. No significant changes were observed in the proliferation of splenocytes from pregnant C×D/D compared to those of the C×D group in response to stimulation with DBA/2 splenocytes. Decidualization of ESCs was associated with a profound alteration in ESC secretome exemplified by alteration in proteins involved in extracellular matrix (ECM) remodeling, response to inflammation, senescence, and immune cell trafficking. Discussion: Our results showed that the deficiency of Tregs is not the primary driver of abortion in the CBA/J × DBA/2 model and provided evidence that impaired endometrial decidualization probably triggers endometrial immune dysfunction and abortion in this model.

Isolation, identification, and culture of goat spermatogonial stem cells using c-kit and PGP9.5 markers.

INTRODUCTION: Presently the techniques for making transgenic animals are cumbersome, required costly instruments and trained man-power. The ability of spermatogonial stem cells (SSCs) to integrate foreign genes has provided the opportunity for developing alternate methods for generation of transgenic animals. One of the big challenges in this field is development of the methods to identify and purify donor SSCs by antibody mediated cell sorting. PURPOSE: The present study was aimed to identify goat subpopulations of SSCs using polyclonal antibodies against PGP9.5 and c-kit molecular markers as well as the growth characteristics of SSCs during short term culture. METHODS: One month old goats' testicular samples were subjected for immunohistochemical and immunocytochemical evaluations. The enzymatically isolated SSCs were cultured in DMEM plus FCS supplemented with (treatment) or without (control) growth factors (GDNF, LIF, FGF, and EGF) for 2 weeks. At the end of culture the morphological characteristics of SSCs colonies and immunocytochemical staining were evaluated. RESULTS: The number and size of colonies in treatment groups were significantly (P < 0.01) higher than corresponding values in controls. The presence of PGP 9.5 and c-kit antigens was confirmed in immunocytochemical evaluation. In immunocytochemical evaluation, the proportion of c-kit and PGP9.5 positive cells were significantly (P < 0.001) higher in control and treatment groups, respectively. CONCLUSIONS: The presence of PGP9.5 and c-kit antigens was confirmed in goat SSCs. Moreover, culture medium supplementation with growth factors could effectively retain the undifferentiation status of SSCs, reflected as a higher population of PGP9.5 positive cells, after short term culture.

Bioactive Materials Derived from Menstrual Blood Stem Cells Enhance the Quality of In Vitro Bovine Embryos.

Backgrounds: The aim of this study was to determine whether the addition of bioactive materials derived from Menstrual Blood Stem Cells (MenSCs) to the oocyte maturation medium may improve the quality of bovine embryos in vitro. Methods: MenSCs were collected from 6 healthy women (between 26 and 36 years old) and after 3 days of culture, their bioactive materials were frozen. The bovine Cumulus-Oocyte-Complexes (COCs) were aspirated from ovarian slaughterhouse and the oocytes with more than three layers of cumulus cells were cultured in vitro in media supplemented with (treatment) and without (control) 10% MenSCs' bioactive materials. After IVM/IVF, the presumptive zygotes were cultured for 8 days. Results: The blastocyst rate on day 8 in treatment group was higher than control (40.2±1.9 vs. 23±4.2.3, p=0.001). The ratio of Trophectoderm (TE) and Inner Cell Mass (ICM) (ICM/TE) cells was also greater in treatment group compared to control (30.3±2 vs. 14.9±1; p=0.001). The re-expansion of vitrified blastocysts, 24 hours after warming, in treatment group was higher than control (93.3±2.5 vs. 66.2±8.8; p=0.01). The expression of some genes related to pluripotency and implantation (OCT4, CDX2, and IFNT) were increased in treatment group compared to control (p<0/05). Conclusion: In conclusion, the addition of MenSCs' bioactive materials during in vitro maturation of bovine oocytes could improve the quantity and quality of bovine IVP embryos. Also, the expression of some genes associated with pluripotency and implantation in the blastocyst would be increased.

Assessing Pain Behavioral Responses and Neurotrophic Factors in the Dorsal Root Ganglion, Serum and Peritoneal Fluid in Rat Models of Endometriosis.

Objective: Pain is the most frequently reported symptom involving in endometriosis. The alterations of neurotrophic factors and certain neuropeptides in the dorsal root ganglion (DRG), as well as serum and peritoneal fluid (PF), were evaluated in rat models of endometriosis. Materials and methods: Twenty-four Sprague Dawley female rats were selected and maintained in a standard condition with 12 hours' dark-light cycles. All the rats were randomly assigned to 3 groups: Control (intact rats); Sham (the operation was conducted without endometriosis induction); and Endometriosis (endometriosis induction was performed). The formalin test was performed for all groups on the first and the 21st day of the study. The assessments of Brain-Derived Neurotrophic Factor (BDNF), Nerve Growth Factor (NGF), Calcitonin Gene-Related Peptide (CGRP), and Substance P levels were carried out by enzyme-linked immunosorbent assay (Elisa). The data were analyzed by One-Way ANOVA. The Tukey's test was used as post-hoc. Results: Endometriosis induction significantly increased the mean pain scores in the endometriosis group in all three phases of the formalin test. The concentrations of DRG-CGRP (p=0.035), BDNF (p<0.001), and NGF (p=0.006) in the endometriosis group were significantly higher than that of the other groups while serum-BDNF (p<0.001), Substance P (p=0.009), and NGF (p=0.015) were significantly lower in endometriosis group compared to other groups. The concentrations of PF-BDNF (p=0.025) and Substance P (p=0.009) were significantly lower than those of other groups. Conclusion: The present results delineate that endometriosis induction could lead to hyperalgesia. This may be related to the significant increases in the BDNF, NGF, and CGRP in DRG.

Histone Modifications of H3K4me3, H3K9me3 and Lineage Gene Expressions in Chimeric Mouse Embryo.

OBJECTIVE: Chimeric animal exhibits less viability and more fetal and placental abnormalities than normal animal. This study was aimed to determine the impact of mouse embryonic stem cells (mESCs) injection into the mouse embryos on H3K9me3 and H3K4me3 and cell lineage gene expressions in chimeric blastocysts. MATERIALS AND METHODS: In our experiment, at the first step, incorporation of the GFP positive mESCs (GFP-mESCs) 129/Sv into the inner cell mass (ICM) of pre-compacted and compacted morula stage embryos was compared. At the second and third steps, H3K4me3 and H3K9me3 status as well as the expression of Oct4, Nanog, Tead4, and Cdx2 genes were determined in the following groups: i. In vitro blastocyst derived from In vivo morula subjected to mESCs injection (blast/chimeric), ii. In vivo derived blastocyst (blast/In vivo), iii. In vitro blastocyst derived from culture of morula In vivo (blast/morula), and iv. In vitro blastocyst derived from morula In vivo subjected to sham injection (blast/sham). RESULTS: Subzonal injection of GFP-mESCs at the pre-compacted embryos produced more chimeric blastocysts than compacted embryos (P<0.05). The number of trophectoderm (TE), ICM, ICM/TE and total cells in chimeric blastocysts were less than the corresponding numbers in blastocysts derived from other groups (P<0.05). In ICM and TE of chimeric blastocysts, the levels of H3K4me3 and H3K9me3 were respectively decreased and increased compared to the blastocysts of the other groups (P<0.05). Expressions of Oct4, Nanog and Tead4 were decreased in chimeric blastocysts compared to the blastocysts of the other groups (P<0.05), while this was not observed for Cdx2. CONCLUSION: In the present study, embryo compaction significantly reduced the rate of incorporation of injected mESCs into the ICM. Moreover, in chimeric blastocysts, the levels of H3K9me3 and H3K4me3 were altered. In addition, the expressions of pluripotency and cell fate genes were decreased compared to blastocysts of the other groups.

Effects of silymarin, cabergoline and letrozole on rat model of endometriosis.

OBJECTIVE: Silymarin as an herbal drug has potent antioxidant effects that could make it a good choice for endometriosis therapy. The aim of the current study was to determine the effects of silymarin as an herbal drug on induced endometrial lesion in rat model of endometriosis. MATERIALS AND METHODS: A total of 32 mature, female Sprague-Dawley rats were allocated into 4 experimental groups. The duration of study was about 6 months. Endometriosis implants were surgically prepared and autografted into 32 rats. Three weeks after endometriosis induction, animals were randomly allocated into four groups: Group 1 received cabergoline (CAB group); Group 2 received letrozole (LET group); Group 3 received silymarin (SIL group) and Group 4 received no medication (CONT group). Experimental groups were treated for 3 weeks and then were sacrificed for volume and histopathological evaluation of implants and biochemical assessment. Serum and peritoneal levels of vascular endothelial growth factor (VEGF), total antioxidant activity (TAC) and tumor necrosis (TNF)-α were measured. RESULTS: Mean volume of the implants decreased significantly in silymarin (p < 0.001), letrozole (p < 0.001) and cabergoline (p < 0.001) groups compared to the control. Histopathologic score was significantly lower in silymarin (p: 0.039), letrozole (p: 0.017) and cabergoline (p < 0.001) groups compared to the control. Those receiving silymarin had significantly higher serum TAC compared to control after 21 days of therapy (p < 0.001). CONCLUSION: Silymarin, Letrozole, and Cabergoline administration resulted in decreased size and histopathologic grade of the induced endometrial lesions in a rat model. Silymarin appears to be a virtual novel therapeutic agent for treatment of endometriosis.

Reconstruction of mammalian oocytes by germinal vesicle transfer: A systematic review.

Nuclear transfer procedures have been recently applied for clinical and research targets as a novel assisted reproductive technique and were used for increasing the oocyte activity during its growth and maturation. In this review, we summarized the nuclear transfer technique for germinal vesicle stage oocytes to reconstruct the maturation of them. Our study covered publications between 1966 and August 2017. In result utilized germinal vesicle transfer techniques, fusion, and fertilization survival rate on five different mammalian species are discussed, regarding their potential clinical application. It seems that with a study on this method, there is real hope for effective treatments of old oocytes or oocytes containing mitochondrial problems in the near future.

The Effect of Angiotensin on the Quality of In Vitro Produced (IVP) Sheep Embryos and Expression of Na(+)/K(+)/ATPase.

BACKGROUND: The presence of rennin-angiotensin components in mammalian ovaries and their involvement in ovarian physiology have been established. In the present study, effects of angiotensin II (Ang II) on sodium-potassium adenosine triphosphatase (Na(+)/K(+)/ATPase) expression and development of sheep embryos was evaluated. METHODS: The abattoir-derived Cumulus Oocyte Complexes (COC) were randomly allocated into three experimental groups; group I) in vitro Maturation (IVM) of oocytes in the presence of Ang II followed by in vitro fertilization (IVF)/in vitro Culture (IVC) (IVM group), group II) IVM/IVF of oocytes followed by IVC wherein the embryos were exposed to Ang II on day 4 of IVC (D4 group), and group III) IVM/IVF and IVC of oocytes without any angiotensin (Control). The blastocyst and hatching rates were recorded on days 6 to 8. Day 8 embryos were immunostained with primary and secondary antibodies against Na(+)/K(+)/ATPase α1 and ß1 subunits. RESULTS: Addition of Ang II during IVM and IVC significantly increased the hatching rate of blastocysts on day 8 compared to the control. The trophectoderm and total blastocyst cells' numbers were significantly increased by addition of Ang II to the IVM and IVC media, though the expression of Na(+)/K(+)/ATPase α1 and ß1 subunits were positively influenced by the addition of Ang II on day 4 (D4 group). CONCLUSION: In conclusion, it seems Ang II through positive effects on embryos, expressed as the greater hatching rate and blastocyst cell number, could increase the sheep embryo developmental rate. These improvements might be partly related to the greater expression of Na(+)/K(+)/ATPase α1 and ß1 subunits when Ang II was added during IVC.

The Effect of Media Supplementation with Angiotensin on Developmental Competence of Ovine Embryos Derived from Vitrified-warmed Oocytes.

BACKGROUND: This study was aimed to assess the effects of angiotensin II (Ang II) supplementation to the In Vitro Maturation (IVM) and In Vitro Culture (IVC) media of vitrified-warmed ovine oocytes on their developmental competence and expression of Na(+)/K(+)/ATPase in resulting embryos. METHODS: The slaughterhouse-derived immature oocytes (n=1069) were randomly distributed into four experimental groups: groups I and II) IVM/IVF and IVC of fresh and vitrified oocytes without angiotensin supplementation (Control-Fresh and Control-Vit groups, respectively); group III) IVM of vitrified oocytes in the presence of Ang II followed by IVF/IVC (Vit-IVM group); and group IV) IVM/IVF of vitrified oocytes followed by IVC wherein the embryos were exposed to Ang II on day 4 of IVC (Vit-D4 group). The embryos were immunostained with primary antibodies against Na(+)/K(+)/ATPase α1 and ß1 subunits. RESULTS: In Vit-IVM and Vit-D4 groups, the rates of expanded and total blastocysts on day 7 as well as the proportion of blastocysts on day 8 were increased. The expression of Na(+)/K(+)/ATPase α1 and ß1 subunits were positively influenced by the addition of Ang II on day 4 (Vit-D4 group). CONCLUSION: The addition of Ang II to the IVM and IVC media could improve blastocysts formation in vitrified sheep oocytes. This improvement might be related to the greater expression of Na(+)/K(+)/ATPase α1 and ß1 subunits when Ang II was added during IVC.

Comparative evaluation of in vivo biocompatibility and biodegradability of regenerated silk scaffolds reinforced with/without natural silk fibers.

Nowadays, exceptional advantages of silk fibroin over synthetic and natural polymers have impelled the scientists to application of this biomaterial for tissue engineering purposes. Recently, we showed that embedding natural degummed silk fibers in regenerated Bombyx mori silk-based scaffold significantly increases the mechanical stiffness, while the porosity of the scaffolds remains the same. In the present study, we evaluated degradation rate, biocompatibility and regenerative properties of the regenerated 2% and 4% wt silk-based composite scaffolds with or without embedded natural degummed silk fibers within 90 days in both athymic nude and wild-type C57BL/6 mice through subcutaneous implantation. In all scaffolds, a suitable interconnected porous structure for cell penetration was seen under scanning electron microscopy. Compressive tests revealed a functional relationship between fiber reinforcement and compressive modulus. In addition, the fiber/fibroin composite scaffolds support cell attachment and proliferation. On days 30 to 90 after subcutaneous implantation, the retrieved tissues were examined via gross morphology, histopathology, immunofluorescence staining and reverse transcription-polymerase chain reaction as shown in Figure 1. Results showed that embedding the silk fibers within the matrix enhances the biodegradability of the matrix resulting in replacement of the composite scaffolds with the fresh connective tissue. Fortification of the composites with degummed fibers not only regulates the degradation profile but also increases the mechanical performance of the scaffolds. This report also confirmed that pore size and structure play an important role in the degradation rate. In conclusion, the findings of the present study narrate key role of additional surface area in improving in vitro and in vivo biological properties of the scaffolds and suggest the potential ability of these fabricated composite scaffolds for connective tissue regeneration. spjba;30/6/793/FIG10885328215601925F1fig1-0885328215601925Figure 1.Illustrative summary of the main methods and findings.RS: regenerated silk; RSF: regenerated fibroin/ silk fiber composite scaffolds; H&E: Hematoxylin and eosin; COX-1: Cyclooxygenase.

White matter hyperintensities and chronicity of depression.

OBJECTIVE: White matter hyperintensities (WMHs) on T(2)-weighted magnetic resonance imaging (MRI) of the brain are associated with advanced age and late-life depression. Most investigations predominantly found these lesions in frontal lobe and basal ganglia supporting the hypothesis of a fronto-striatal dysfunction in depression. A prospective study was undertaken to investigate the association between extent of WMHs and clinical outcome in elderly depressed patients. METHODS: Thirty-one non-demented depressed subjects underwent a 1.5 T cranial MRI scan. The MRI scans were analysed in consensus by two experienced radiologists. Each MRI scan was assessed for presence and extent of WMHs, which are differentiated in periventricular hyperintensities (PVHs) and deep white matter hyperintensities (DWMHs). A total of 21 patients of the original cohort of 31 patients were re-assessed 5 years after baseline assessment. We ascertained the severity of depressive symptoms, the longitudinal course of depression, the cognitive decline and the global assessment of functioning at follow-up visit. RESULTS: (1) Subjects with greater extent of WMHs had a significant higher Hamilton Depression Rating Scale (HAM-D) score, (2) had more severe longitudinal courses of depression (3) and had a lower Mini-Mental State Examination (MMSE) score. CONCLUSIONS: WMHs on MRI are associated with poorer outcome in elderly depressed subjects. Further studies are needed to evaluate WHMs as prognostic factor for an appropriate treatment decision-making.

Comparative evaluation of differentiation potential of menstrual blood- versus bone marrow-derived stem cells into hepatocyte-like cells.

Menstrual blood has been introduced as an easily accessible and refreshing stem cell source with no ethical consideration. Although recent works have shown that menstrual blood stem cells (MenSCs) possess multi lineage differentiation capacity, their efficiency of hepatic differentiation in comparison to other stem cell resources has not been addressed so far. The aim of this study was to investigate hepatic differentiation capacity of MenSCs compared to bone marrow-derived stem cells (BMSCs) under protocols developed by different concentrations of hepatocyte growth factor (HGF) and oncostatin M (OSM) in combination with other components in serum supplemented or serum-free culture media. Such comparison was made after assessment of immunophenotye, trans-differentiation potential, immunogenicity and tumorigeicity of these cell types. The differential expression of mature hepatocyte markers such as albumin (ALB), cytokeratin 18 (CK-18), tyrosine aminotransferase and cholesterol 7 alpha-hydroxylase activities (CYP7A1) at both mRNA and protein levels in differentiating MenSCs was significantly higher in upper concentration of HGF and OSM (P1) compared to lower concentration of these factors (P2). Moreover, omission of serum during differentiation process (P3) caused typical improvement in functions assigned to hepatocytes in differentiated MenSCs. While up-regulation level of ALB and CYP7A1 was higher in differentiated MenSCs compared to driven BMSCs, expression level of CK-18, detected level of produced ALB and glycogen accumulation were lower or not significantly different. Therefore, based on the overall comparable hepatic differentiation ability of MenSCs with BMSCs, and also accessibility, refreshing nature and lack of ethical issues of MenSCs, these cells could be suggested as an apt and safe alternative to BMSCs for future stem cell therapy of chronic liver diseases.

Enrichment of undifferentiated type a spermatogonia from goat testis using discontinuous percoll density gradient and differential plating.

BACKGROUND: The well documented source for adult multipotent stem cells is Spermatogonial Stem Cells (SSCs). They are the foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. The aim of this study was to assess the effect of percoll density gradient and differential plating on enrichment of undifferentiated type A spermatogonia in dissociated cellular suspension of goat testes. Additionally, we evaluated the separated fractions of the gradients in percoll and samples in differential plating at different times for cell number, viability and purification rate of goat SSCs in culture. METHODS: Testicular cells were successfully isolated from one month old goat testis using two-step enzymatic digestion and followed by two purification protocols, differential plating with different times of culture (3, 4, 5, and 6 hr) and discontinuous percoll density with different gradients (20, 28, 30, and 32%). The difference of percentage of undifferentiated SSCs (PGP9.5 positive) in each method was compared using ANOVA and comparison between the highest percentage of corresponding value between two methods was carried out by t-test using Sigma Stat (ver. 3.5). RESULTS: The highest PGP9.5 (94.6±0.4) and the lowest c-Kit positive (25.1±0.7) in Percoll method was significantly (p ≤ 0.001) achieved in 32% percoll gradient. While the corresponding rates in differential plating method for the highest PGP9.5 positive cells (81.3±1.1) and lowest c-Kit (17.1±1.4) was achieved after 5 hr culturing (p < 0.001). The enrichment of undifferentiated type A spermatogonia using Percoll was more efficient than differential plating method (p < 0.001). CONCLUSION: Percoll density gradient and differential plating were efficient and fast methods for enrichment of type A spermatogonial stem cells from goat testes.

A technique for facile and precise transfer of mouse embryos.

BACKGROUND: Successful Embryo Transfer (ET) technique is a fateful step of all efforts to achieve live births from in vitro produced embryos in assisted reproductive techniques or in knockout, transgenic or cloned animal projects. Small reproductive tract of mice and limitation of current techniques may not well satisfy the requirements for mass production of genetically modified mice. Genetic abnormalities of embryos, receptivity and uterine contractions, expulsion of embryos, blood, mucus or bacterial contamination on the transfer pipette tip, technical problems and even animal strain may affect embryo transfer outcome. METHODS: In this study, two techniques of embryo transfer in mice were compared. In conventional technique the oviduct wall was punctured with a 30-gauge needle and the loaded Pasteur pipette with embryos and medium was inserted into the hole. In new technique, embryos that were loaded in modified micropipette with minimal medium were transferred directly to the oviduct by manual piston micro-pump easily. Embryo viability was evaluated considering the percentage of live healthy newborns. RESULTS: Results of the two techniques were compared by t-test within the NPAR1WAY procedure of SAS software (ver. 9.2). The average live birth rates in the novel methods was significantly higher (42.4%) than the conventional method (21.7%, p<0.05). CONCLUSION: In conclusion, using new embryo transfer technique improved birth rate by preventing embryos expulsion from the oviduct, saving time and easy transfer of embryos with minimum volume of medium.

Comparison of proliferative and multilineage differentiation potential of sheep mesenchymal stem cells derived from bone marrow, liver, and adipose tissue.

BACKGROUND: Despite major progress in our general knowledge related to the application of adult stem cells, finding alternative sources for bone marrow Mesenchymal Stem Cells (MSCs) has remained to be challenged. In this study successful isolation, multilineage differentiation, and proliferation potentials of sheep MSCs derived from bone marrow, adipose tissue, and liver were widely investigated. METHODS: The primary cell cultures were prepared form tissue samples obtained from sheep 30-35 day fetus. Passage-3 cells were plated either at varying cell densities or different serum concentrations for a week. The Population Doubling Time (PDT), growth curves, and Colony Forming Unit (CFU) of MSCs was determined. The stemness and trilineage differentiation potential of MSCs were analyzed by using molecullar and cytochemical staining approaches. The data was analyzed through one way ANOVA using SigmaStat (ver. 2). RESULTS: The highest PDT and lowest CFU were observed in adipose tissue group compared with other groups (p<0.001). Comparing different serum concentrations (5, 10, 15, and 20%), irrespective of cell sources, the highest proliferation rate was achieved in the presence of 20% serum (p<0.001). Additionally, there was an inverse relation between cell seeding density at culture initiation and proliferation rate, except for L-MSC at 300 cell seeding density. CONCLUSION: All three sources of fetal sheep MSCs had the identical trilineage differentiation potential. The proliferative capacity of liver and bone marrow derived MSCs were similar at different cell seeding densities except for the higher fold increase in B-MSCs at 2700 cells/cm (2) density. Moreover, the adipose tissue derived MSCs had the lowest proliferative indices.

Culture of Ovine IVM/IVF Zygotes in Isolated Mouse Oviduct: Effect of Basal Medium.

BACKGROUND: The basal medium that supports Isolated Mouse Oviduct (IMO) is important for supporting embryo development and quality. METHODS: The culture of ovine IVM/IVF zygotes was done in IMO using SOFaaciBSA and SOFaaBSA as basal medium of IMO and in SOFaaBSA alone as control. For preparation of IMO mature inbred strain C57BL/6 female mice were synchronized and mated with vasectomized males. The females with vaginal plug were sacrificed and the zygotes were transferred in to the isolated oviduct at 20 hpi. The oviducts were cultured with SOFaaciBSA and SOFaaBSA for 6 days. Another group of zygotes were cultured in SOFaaBSA alone as control. RESULTS: Culture of zygotes in the IMO with SOFaaciBSA and SOFaaBSA, did not significantly affect the development and quality of embryos (p > 0.05). The hatching rate, total and trophectoderm cells number in IMO groups' blastocysts were significantly higher than SOFaaBSA alone. The morphological appearance of IMO blastocysts was superior to SOFaaBSA alone. When the quality of oocytes was poor, IMO could better support ovine embryo development either with SOFaaBSA or SOFaaciBSA than SOFaaBSA alone and there was a significant difference in blastocyst formation at day 6 with SOFaaBSA alone. CONCLUSION: The culture of ovine IVM/IVF zygotes in IMO using two highly efficient ruminant embryo culture media not only could support development of ovine embryos similar to the level in non IMO culture system (SOFaaBSA alone) but also could improve the quality of resulting embryos. Additionally, IMO could better support the development of ovine embryos derived from poor quality oocytes compared to the SOFaaBSA alone.

Regulations and ethical considerations in animal experiments: international laws and islamic perspectives.

Growing usage of animals in the research projects has drawn more attention to their welfare and ethics surrounding this practice. Dissemination of information about the existing ethical consideration and alternatives in animal experiments has two important functions; first, it increases the researcher's awareness of the possible methods of using animals in the experiment, and second, to ensure that potential users are aware of the established alternatives. For example, legislations enacted in many countries during the 1980s state that laboratory animal applications should be reduced, refined and replaced wherever possible according to principles of the 3Rs. Thus, scientists around the world tried to apply the 3Rs in their biomedical researches regarding welfare of the laboratory animals. However, the Qur'an, the holy book of Muslims, and also Hadiths contain the obligatory ways to keep and treat animals since their revelations. According to Islamic viewpoint, animals represent Allah's ability and wisdom, and humans must pay attention to their health and living conditions. Several Islamic manuscripts state that animals have their own position in the creation hierarchy and humans are responsible for supplying minimal facilities and their welfare. This paper has tried to review ethical consideration in animal experiments and regarding Islamic resources in this case to encourage providing comprehensive ethical regulations in animal experiments which its establishment could be beneficial for animal ethics committees or research institutes.