In vitro culture and morphological characterization of prepubertal buffalo (Bubalus bubalis) putative spermatogonial stem cell

Purpose

Spermatogonial stem cells (SSCs) have the unique ability both to self-renew and to produce progeny that undergo differentiation to spermatozoa. The present study has been carried out to develop a method to purify and enrich the pure populations of spermatogonial stem cell like cells in buffalo.

Methods

The spermatogonial cells were isolated from testes of 3–7 month old buffalo calves and disaggregated by double enzymatic digestion. Mixed population of isolated cells were then plated on Datura stramonium agglutinin (DSA) lectin coated dishes for attachment of Sertoli cells. The desired cells were obtained from suspension medium after 18 h of incubation and then loaded on discontinuous density gradient using percoll (20–65 %) and different types of spermatogonia cells were obtained at interface of each layer. These cells were cultured in vitro.

Results

Spermatogonial cells isolated have spherical outline and two or three eccentrically placed nucleoli, created a colony after proliferation during first week or immediately after passage. After 7–10 days of culture, the resulted developed colonies of spermatogonial cells expressed the spermatogonial specific genes like Plzf and VASA; and other pluripotency related markers viz. alkaline phosphtase, DBA, CD9, CD90, SSEA-1, OCT-4, NANOG and REX-1.

Conclusion

Our results show that the isolated putative spermatogonial stem cells exhibit the expression of pluripotency related and spermatogonial specific genes. This study may help to establish a long term culture system for buffalo spermatogonia.     

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.     

In vitro culture and characterization of spermatogonial stem cells on Sertoli cell feeder layer in goat (Capra hircus)

Purpose

To develop an efficient protocol for isolation, purification and long-term culture of spermatogonial stem cell (SSC) in goat.

Methods

The isolation of SSC was performed by testicular disaggregation by enzymatic digestion using collagenase IV, trypsin and DNase I. Further SSCs were enriched using Percoll density gradient centrifugation. The purity of SSCs was assessed by immunocytochemistry (ICC) using α6 integrin. The SSCs were co-cultured on Sertoli cell feeder layer. The SSC colonies were characterized by studying the expression of SSC specific markers (viz., α6 integrin and PLZF) using ICC. The abundance of mRNAs encoding the markers of SSC (viz., β1 integrin and Oct-4) and Sertoli cells (viz., vimentin) was also assayed using quantitative real-time PCR (qPCR).

Results

The viability of isolated testicular cells was > 90 % and the Percoll density gradient method resulted in 3.65 folds enrichment with a purity of 82.5 %. Co-culturing of SSCs with Sertoli cell feeder layer allowed the maintenance of stable SSC colonies even after one and half months of culture. The results of ICC analysis showed the expression of α6 integrin and PLZF in almost all the SSC colonies. qPCR analysis revealed a differential expression of mRNAs encoding β1 integrin, Oct-4 and vimentin markers.

Conclusion

Results of this study demonstrate a simple enzymatic digestion and Percoll density gradient method for isolation and enrichment of SSCs, and suitability of Sertoli cell feeder layer for long term in vitro culture of SSC in goats. Results also suggest the possible application of non-caprine antibodies against SSC specific markers for the identification and subsequent assessment of SSCs in goats.     

Co-culture of mouse spermatogonial stem cells with sertoli cell as a feeder layer,stimulates the proliferation and spermatogonial stemness profile

Objectives

Sertoli cells effect the fate map of spermatogonial stem cells (SSCs) to self-renew via providing the special microenvironments. Maintenance of proliferation and self-renewal activity of SSCs may be usable as a therapeutic strategy, leads to increase the recovery of male fertility. This research was aimed to evaluate the effect of mouse sertoli cells on spermatogonia stem cells proliferation and the expression pattern of stemness markers.

Does prepubertal testicular tissue vitrification influence spermatogonial stem cells (SSCs) viability?

Purpose

Testicular cryopreservation prior to chemotherapy or radiotherapy in children with cancer is one of the ways to preserve fertility. However, cryopreservation may cause damage to the testicular parenchyma cells. The objective of this study was to investigate effects of vitrification on the intracellular LDH leakage, cell cycle/apoptotic responses and apoptosis-related gene expression patterns in the spermatogonial stem cells (SSCs) obtained from the vitrified testis.

Methods

The testes of the mice pups (6-day-old, BALB/c) both vitrified and fresh groups were digested with enzymes (collagenase, DNaseΙ, trypsin-EDTA) to disperse the cells. The SSCs, type A, were isolated from the rest of testicular cells by MACS. The amount of damage to the SSCs immediately was evaluated by Cytotoxicity assay, Flow cytometry assay and Real-time PCR.

Results

The intracellular LDH leakage in the SSCs,harvested from the vitrified testes, was less reported compared with the fresh ones. Moreover, the percentage of apoptotic and necrotic SSCs obtained from the vitrified testes was lower than that of yielded from the fresh samples. Also, the apoptosis-related genes of the SSCs,collected from the vitrified testes, changed their expression profile as increasing P53 and BCL-2 expression levels and decreasing Bax and Fas expression levels.

Conclusions

The study indicates that vitrification of prepubertal testicular tissue does not increase the expression profile of apoptosis-related genes such as Bax and Fas in the testicular SSCs consistent with diminished cell apoptotic/necrotic responses and no increasing intracellular LDH leakage.     

Molecular and ultrastuctural changes of rat pre-implantation embryos during two-cell developmental arrest

Background

Rat pre-implantation embryos often suffer 2-cell stage developmental arrest and fail to progress further under in-vitro conditions.

Objective

In order to understand underlying mechanism leading to 2-cell arrest, we investigated the molecular changes, culture conditions and subcellular changes.

Methods

Gene expression in in-vivo developed 2-cell embryos (in-vivo), in- vitro developed 2-cell embryos (in-vitro), and in-vitro 2-cell arrested embryos (arrested) were investigated using microarrays and real-time PCR. Ultra-structural changes were determined using electron microscopy.

Results

Gene expression was similar between in-vivo and in-vitro embryos. Over 2400 genes changed in arrested embryos compared to in-vivo and in-vitro embryos. The mRNAs encoding proteins involved in translation were elevated in arrested embryos. In-vivo and in-vitro embryos highly expressed genes that were involved in cell cycle, and protein catabolic process compared to arrested embryos. Gene expression data suggested subcellular changes associated with 2-cell block. Transmission electron microscopy showed that in-vivo embryos had healthy subcellular structure, whereas arrested embryos did not have a nuclear membrane, contained small mitochondria and autophagic vacuoles. Furthermore, gene expression data was used for the optimization of culture media conditions to obtain better in-vitro embryonic development. Comparison of five and 20 % oxygen in culture resulted in two times more blastocyst formation with 5 % oxygen.

Conclusions

These results showed that although all experimental groups appeared morphologically similar, arrested embryos had ultra-structural and molecular changes associated with oxidative stress and apoptosis. In-vitro culture under low oxygen and media additives reduced 2-cell block in rat embryos.     

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

PurposeThe 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.MethodsThe 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.ResultsThe 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.ConclusionsThe 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.     

Ovarian stem cells are always accompanied by very small embryonic-like stem cells in adult mammalian ovary

Background

Existing dogma that a female is born with fixed number of eggs was challenged by the detection of stem cells in adult mammalian ovary. Data has accumulated in support of ovarian stem cells (OSCs) proliferation, maintenance in culture, formation of germ cell nests and differentiation into oocytes and primordial follicle assembly using different strategies.

Results

Flow cytometry analysis identified >8 μm OSCs which are DDX1 positive and are considered equivalent to spermatogonial stem cells (SSCs) in testis. Analysis of both ovarian and testicular smears obtained after enzymatic digestion has led to the identification of an additional stem cell population termed very small embryonic-like stem cells (VSELs). VSELs and OSCs/SSCs differ from each other in their size and OCT-4 expression. VSELs express pluripotent markers including nuclear OCT-4 whereas OSCs/SSCs express cytoplasmic OCT-4 suggesting a differentiated state. VSELs can be studied by flow cytometry as small sized cells which are LIN-/CD45-/Sca-1+. We have reported 0.02?±?0.008, 0.03?±?0.017 and 0.08?±?0.03 % of total cells as VSELs in normal, chemoablated and after FSH treatment to chemoablated mouse ovary.

Conclusions

VSELs have remained poorly studied till now because of their very small size and rare occurrence. Spinning cells obtained after enzymatic digestion of ovarian tissue at a speed of 1000G (rather than 1200 rpm) throughout processing allows reliable detection of the VSELs by flow cytometry. VSELs exist in aged, chemoablated and non-functional ovary and providing a healthy niche to support their function offers an interesting strategy to manage infertility.

     

bFGF and VEGF improve the quality of vitrified-thawed human ovarian tissues after xenotransplantation to SCID mice

Purpose

The aim of this research is to study whether basic fibroblast growth factor (bFGF) alone or in combination with vascular endothelial growth factor (VEGF) could improve the quality of vitrified-thawed human ovarian tissue xenotransplanted to severe combined immune deficiency (SCID) mice.

Methods

After collection and cryopreservation, thawed human ovarian tissue were cultured in vitro for 2 days and then xenografted to severe combined immune deficiency (SCID) mice for 7 days. The in vitro culture medium was separated into six groups, including (A) the blank control group, (B) the human recombinant bFGF (150 ng/ml) group, (C) the bFGF (150 ng/ml)+human recombinant VEGF (25 ng/ml) group, (D) bFGF (150 ng/ml)+VEGF (50 ng/ml) group, (E) bFGF (150 ng/ml)+ VEGF (75 ng/ml) group and (F) bFGF (150 ng/ml) + VEGF (100 ng/ml) group. In addition, eight pieces of thawed ovarian tissue were transplanted without in vitro culture, which serve as the fresh control group. The effect of transplantation was assessed by histological analysis, immunohistochemical staining for CD34, Ki-67, and AC-3 expression, and microvessel density (MVD).

Results

There was no significant difference between the fresh and blank control group. Compared to the blank control group, the number of follicles, MVD, and rate of Ki-67-positive cells increased significantly in groups B, C, D, E, and F, while apoptosis decreased significantly. Compared to the bFGF treatment group, no significant difference appeared in group C, D, E, and F.

Conclusions

The administration of bFGF alone or in combination with VEGF improved the quality of postgraft human ovarian tissue, though VEGF, regardless of different concentrations, did not influence effect of bFGF.

     

Short-term in-vitro culture of goat enriched spermatogonial stem cells using different serum concentrations

Purpose  

To investigate the effect of serum supplementing on short-term culture, fate determination and gene expression of goat spermatogonial stem cells (SSCs).     

Recent advances of in vitro culture systems for spermatogonial stem cells in mammals

Background

Spermatogonial stem cells (SSCs) in the mammalian testis are unipotent stem cells for spermatozoa. They show unique cell characteristics as stem cells and germ cells after being isolated from the testis and cultured in vitro. This review introduces recent progress in the development of culture systems for the establishment of SSC lines in mammalian species, including humans.

Methods

Based on the published reports, the isolation and purification of SSCs, identification and characteristics of SSCs, and culture system for mice, humans, and domestic animals have been summarized.

Results

In mice, cell lines from SSCs are established and can be reprogrammed to show pluripotent stem cell potency that is similar to embryonic stem cells. However, it is difficult to establish cell lines for animals other than mice because of the dearth of understanding about species‐specific requirements for growth factors and mechanisms supporting the self‐renewal of cultured SSCs. Among the factors that are associated with the development of culture systems, the enrichment of SSCs that are isolated from the testis and the combination of growth factors are essential.

Conclusion

Providing an example of SSC culture in cattle, a rational consideration was made about how it can be possible to establish cell lines from neonatal and immature testes.     

T-cadherin expression in uterine leiomyoma

Objective

T-cadherin is a tumor-suppressor with low expression in many malignant tumors, but with high expression in endothelial cells and so on. In this study we investigated whether T-cadherin was expressed and if together with bFGF play a role in the occurrence and development of uterine leiomyoma.

Method

Uterine leiomyoma, the adjacent normal myometrium, control normal myometrium without uterine leiomyoma and vascular features of myoma were collected. Immunohistochemistry, western blot and relative quantitative real time PCR were used to evaluate bFGF and T-cadherin on the three specimens. Data were statistically analysed.

Results

T-cadherin was observed on the leiomyoma cellular layers but not in the endochylema, extracellular matrix and leiomyoma vascular endothelial cell, bFGF in the leiomyoma endochylema but not observed in the extracellular matrix and leiomyoma vascular endothelial cell. The protein and mRNA expression of bFGF and T-cadherin in uterine leiomyoma were significantly with higher expression than that in adjacent normal myometrium and control normal myometrium. In addition, T-cadherin correlated well with bFGF. There was relationship between T-cadherin and color Doppler flow imaging (CDFI).

Conclusion

bFGF and T-cadherin have high expressions in uterine leiomyoma, and T-cadherin is associated with CDFI, indicating that a cross talk between bFGF and T-cadherin plays an important role in the occurrence and development of uterine leiomyoma or even malignant tumors.     

Clinical significance of amphiregulin and epidermal growth factor in colostrum

Background

Colostrum contains a wide variety of crucial nutritional elements including growth factors for newborn infants to adapt to the extrauterine environment.

Objective

To investigate the clinical significance of epidermal growth factor receptor ligands in milk during the first month of lactation.

Methods

The concentrations of epidermal growth factor (EGF), amphiregulin (AR) and transforming growth factor-?? (TGF-??) in milk sampled from a total of 31 normal mothers at days 1?C3, 5, and 30 postpartum were examined using ELISA.

Results

At days 1?C3, the concentration of EGF was extremely high [131.6?±?20.4 (mean?±?SEM) ng/ml] compared to that of AR (4,197.2?±?1,055.2?pg/ml) or TGF-?? (261.7?±?33.6?pg/ml), while the concentration of AR was significantly elevated compared to that of TGF-??. At days 5 and 30, the concentration of EGF was significantly elevated compared to that of AR or TGF-??. In 16 mothers among the same 31 subjects, samples were longitudinally obtained on days 1, 2, 5, and 30 postpartum. Concentrations of AR were higher on days 1 and 2 and rapidly declined to below 1?ng/ml on day 5, and were maintained at lower levels on day 30. Concentrations of EGF were high on day 1 (greater than 10?ng/ml) but gradually declined by days 2, 5, and 30. Concentrations of TGF-?? remained at lower levels of below 1?ng/ml throughout the lactation period from days 1 to 30.

Conclusion

These results suggested that EGF and amphiregulin in colostrum might contribute to the early stage of development of neonatal gastrointestinal function.     

Achieving high survival rate following cryopreservation after isolation of prepubertal mouse spermatogonial cells

Purpose

Isolating spermatogonia cells with high purity and viability and achieving better survival rate following cryopreservation

Methods

Isolating the cells by Magnetic Activating Cell Sorting (MACS) method using anti CD49f (α6 integrin) antibody and Dynabeads and freezing in DMSO-based freezing mediums containing three different FBS concentrations of 50%, 60% and 70%.

Results

The mean (±SD) purity of the isolated cells was 92.52?±?3.57 (range 92.43–98.25). The cells frozen in group I, II and III had mean 39.60?±?1.48 (range 37.98–41.62), 89.05?±?3.83 (range 80.83–90.33) and 90.52?±?1.71 (range 89.07–92.52) viability, respectively.

Conclusion

Higher viable cell counts and purity can be attained by the use of α6 integrin and magnetic beads. After the thawing of spermatogonial cells, optimum viability was achieved in freezing media containing 60% FBS.     

Effect of single-oocyte culture system on in vitro maturation and developmental competence in mice

Purpose

To investigate whether single-culture systems influence the quality of in vitro-matured oocytes, we examined the maturation and developmental competence of oocytes obtained by grouped in vitro maturation (IVM) or single IVM.

Methods

In vitro-matured oocytes were obtained using the culture drop (CD) method for the grouped IVM experiments, and the CD and hanging drop (HD) method for the single IVM experiments. To evaluate oocyte developmental competence, we performed in vitro fertilization and culture, and counted the number of blastocysts. To evaluate the oocyte cytoplasmic maturation, we measured the maturation promoting factor (MPF) expression levels.

Results

Oocytes cultured singly had lower maturity and developmental competence than the grouped IVM oocytes. However, enhanced oocyte fertility and blastocyst quality was achieved by the HD single IVM method. Additionally, the MPF activity level increased in all culture methods, compared to the control; however, it lagged behind nuclear maturation.

Conclusions

These results suggest that the HD method is efficient for single IVM.     

VEGF and bFGF increase survival of xenografted human ovarian tissue in an experimental rabbit model

Purpose

The aim of this study is to determine whether vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) could increase the survival of xenografted human ovarian tissue in an experimental rabbit model.

Methods

Fresh human ovarian tissue was xenotransplanted into the back muscle of 25 castrated female New Zealand rabbits for 6 weeks with the immunosuppression of FTY720 (2 mg/kg/d). Rabbits were randomly divided into five experimental groups: (A) graft and host treatment with VEGF (50 ng/ml); (B) graft and host treatment with bFGF (100 ng/ml); (C) graft and host treatment with VEGF(50 ng/ml) + bFGF (100 ng/ml); (D) graft and host treatment with normal saline; (E) control group, no treatment. 4 weeks after transplantation, human menopausal gonadotropin (HMG) 10 IU was administered every second day in group A, group B, group C and group D for 2 weeks. Graft survival was assessed by graft recovery rate, histological analysis, immunohistochemical staining for CD31 and Ki-67expression, TUNEL assay.

Results

After 6 weeks of grafting, the number of CD31-positive stained cells increased significantly in group A, group B and group C compared to the control group. All groups showed strong Ki-67 immunostaining in ovarian stroma. Only one rabbit in group C retained the grafts’ follicles. Grafting resulted in relative lower fibrosis in group A and group C compared to the control group. Apoptosis was significantly lower in group C compared to the control group.

Conclusions

Fresh human ovarian cortex grafted into the back muscle of rabbit can sustain part of ovarian tissue function with the immunosuppression of FTY720, although follicle number diminishes significantly after grafting. The administration of VEGF and bFGF, especially the combination of them, may trigger angiogenesis, reduce apoptosis and fibrosis, increase survival in transplanted human ovarian tissue.     

Effect of bovine cumulus–oocyte complexes-conditioned medium on in-vitro maturation of canine oocytes

Purpose

To investigate the ability of medium conditioned with bovine cumulus–oocyte complexes (COCs) to support nuclear maturation of canine oocytes recovered from domestic dog ovaries.

Methods

Cumulus–oocyte complexes were obtained from ovaries of domestic bitches (8 months old to 7 years old), and in-vitro maturation was evaluated in TCM-199 supplemented with different concentrations (0, 20, 30 or 50%) of bovine COCs-conditioned medium (BCM). The canine COCs were cultured for 72 or 96 h at 38.5°C in 5% CO₂, 5% O₂ and 90% N₂. The bovine COCs-conditioned medium was obtained from culture of bovine COCs with TCM-199 supplemented with 5% FCS for 22 h at 38.5°C in 2% CO₂, 98% air.

Results

The proportion of germinal vesicle breakdown (GVBD) after 72 h was significantly higher (P < 0.05) in medium supplemented with 30% BCM (20.7%) compared with the control group (13.4%). The rates of GVBD-MII stage were significantly higher (P < 0.05) when oocytes were matured with BCM at concentration of 30% (41.5%) compared with control (26.6%) after 72 h in-vitro culture. After 96 h in-vitro culture, the oocytes matured in medium supplemented with 30% BCM (5.5%) showed a significant increase (P < 0.05) in the proportion of MII compared with control (0.7%). However, increasing the cultivation time from 72 to 96 h resulted in an increase in oocyte degeneration rate.

Conclusions

The results suggested that bovine COCs-conditioned medium supplementation significantly increased nuclear maturation of canine oocytes.