germ cells

 Islamic Republic of Iran has provided a legal framework for embryo transfer so that fertility becomes possible with third party intervention. The use of this method of fertility as well as its effects and rules are subject to cultural, social and religious factors. The brief nature of the embryo donation law indicates the lack of adequate measures for the child’s future. The present study aimed to review the embryo donation law in Iran and some challenges in determining the rights of the child. This study employed a library, descriptive and analytical method and is based on Shia jurisprudence and law books. By using the keywords of “Donation, Alimony, Custody, and Inheritance”, various aspects of the embryo donation law have been investigated. Article 3 of the Embryo Donation Law considers the duties and responsibilities of the couples who donate the embryo and the born child in terms of maintenance, alimony and being mahram (. An unmarriageable kin in Islamic Sharia legal terminology), similar to the duties and responsibilities of children and parents. However, the important issues of lineage and inheritance are not considered. Neither it elaborates compliance with the principle of confidentiality and the non-identification of the genetic parents, the coercive guardianship of the father, and the prohibition of marriage (being mahram). There is neither enough clarity about the method of evaluating the recipient couple’s moral competence, the limit of the number of gamete donations, alimony, or custody. It is necessary to explain the kinship and genetic inheritance, amend birth certificate registration law, specify the rejection of anonymity to prevent the phenomenon of mixing lineage based on Shia jurisprudence, or to add new materials to this law.

There are ethical and technical challenges in studying human germ cell development. Therefore, the aim ofthe study is in vitro differentiation of human embryonic stem cells (hESCs), as pluripotent cells, to the germ cells whichis a valuable tool for studying molecular and cellular aspects of gametogenesis and understanding causes of infertility. In this experimental study, two different complete media [Dulbecco’s Modified Eagle Medium(DMEM)+20% fetal bovine serum (FBS) and embryoid bodies (EBs) medium; KOSR/HES without basic fibroblastgrowth factor (bFGF)] were used in the both of test groups using testicular cells derived conditioned medium (TCCM)and control groups spontaneously differentiated (SD). Thereby, EBs from hESCs (Yazd2; 46XY) were cultured indifferent conditions EB medium; EB medium and conditioned EB medium; EB medium, DMEM, and FBS withoutconditioning; EB medium, conditioned DMEM, and FBS medium. EBs were collected after 4, 7, and 14 days and theirgene expression profiles were assessed and compared to hESCs, as day 0, using IF and relative reverse transcriptionquantitative polymerase chain reaction (RT-qPCR). An increase in the gametogenesis gene expression level in TCCM groups was showed in comparison withSD groups. Additionally, immunostaining of differentiated cells in all groups showed in vitro gametogenesis (IVG). Our findings showed that human TCCM could be used as a natural niche for in vitro male and femalegerm cell development. However, further studies are needed to define the factors and metabolites within the humanTCCM.

Pesticides are widely used around the world. However, these chemicals are being used more frequently and at increased doses in underdeveloped and developing countries. Although the hazard of pesticides has been studied in ecological fields, the effect of residual amounts of these compounds on the physiological processes of the body has always been debated.

In this experimental study, 45 greenhouse cucumber plants were sprayed with dichlorvos and acetamiprid pesticides in concentrations of twofold (acetamiprid 500 g/1000 L and dichlorvos 4 L/1000 L) and threefold of the recommended dose. After 24 h, the residual amount was obtained. To evaluate the residual effect of the mentioned pesticides, an equivalent of this residue was added to the drinking water of 105 mice.

Pesticide residues were obtained for twofold and threefold concentrations of the recommended dose, 1.5 and 2.5 (mg/kg cucumber) for acetamiprid and 0.5 and 1 (mg/kg cucumber) for dichlorvos, respectively. Application of these chemicals at higher doses not only significantly reduced the body weight, food consumption, testosterone production, testicular germ cells and embryo numbers, but also increased the levels of follicle‑stimulating hormone and luteinizing hormone in mice.

The emergence of biological disorders and reducing reproductive potential in male mice can be attributed to the addition of pesticides to their drinking water. Therefore, to reduce the hazards caused by insecticides, it is recommended to familiarize farmers with the harmful effects of overdose of pesticides and monitoring the residuals in agricultural products.

 Ovarian tumors are mostly detected in advanced stages. Early diagnosis of malignant ovarian germ cell tumors is so vital to keep life and fertility of the patients. We aimed to find out different presentations of malignant ovarian germ cell tumors based on age, parity and histology to help early diagnosis of the tumors. 

 In this study, malignant ovarian germ cell tumors admitted in a referral center of gynecology oncology were studied 2001-2018. The symptoms and signs of the patients were collected and analyzed according to age, parity and specific histology. 

 128 cases of malignant germ cell tumors were detected. The primary symptoms included abdominal distension (45%), acute pain (40.95%), chronic pain (23.95%), menstrual irregularity (14.7%), sense of abdominal firmness and mass (7.72%), nausea (5.4%), fever (5.4%), lack of appetite (4.63%), virilization (3.1%), depletion of weight (3.1%), and 9.27% detected incidentally. Abdominal distension, and acute pain decreased after 24. Menstrual disorders and incidental detection in multiparas were significantly more than nulliparous (p<0.05). Abdominal distension was the prominent sign in dysgerminomas (50%). Almost 45% of immature, yolk sac, and mixed tumors referred with acute abdominal pain. The data showed that 85% of the patients had been suffering from some discomforts for days to months prior to the diagnosis. 

 The majority of cases are symptomatic for a long time before the first visit although aging and parity can lessen their severity. Late diagnosis can lead to acute abdomen in some histology types. Young women and health providers should be warned about concerned presentations of ovarian tumors.

Primordial germ cell (PGCs) lines are a source of a highly specialized type of cells, characteristically oocytes,during female germline development in vivo. The oocyte growth begins in the transition from the primary follicle. It isassociated with dynamic changes in gene expression, but the gene-regulating signals and transcription factors that control oocyte growth remain unknown. We aim to investigate the differentiation potential of mouse bone marrow mesenchymal stem cells (mMSCs) into female germ-like cells by testing several signals and transcription factors. In this experimental study, mMSCs were extracted from mice femur bone using the flushingtechnique. The cluster-differentiation (CD) of superficial mesenchymal markers was determined with flow cytometric analysis. We applied a set of transcription factors including retinoic acid (RA), titanium nanotubes (TNTs), and fibrin such as TNT-coated fibrin (F+TNT) formation and (RA+F+TNT) induction, and investigated the changes in gene, MVH/ DDX4, expression and functional screening using an in vitro mouse oocyte development condition. Germ cell markers expression, (MVH / DDX4), was analyzed with Immunocytochemistry staining, quantitative transcription-polymerase chain reaction (RT-qPCR) analysis, and Western blots. The expression of CD was confirmed by flow cytometry. The phase determination of the TNTs and F+TNT were confirmed using x-ray diffraction (XRD) and scanning electron microscope (SEM), respectively. Remarkably, applying these transcription factors quickly induced pluripotent stem cells into oocyte-like cells that were sufficient to generate female germlike cells, growth, and maturation from mMSCs differentiation. These transcription factors formed oocyte-like cells specification of stem cells, epigenetic reprogramming, or meiosis and indicate that oocyte meiosis initiation and oocyte growth are not separable from the previous epigenetic reprogramming in stem cells in vitro. Results suggested several transcription factors may apply for arranging oocyte-like cell growth and supplies an alternative source of in vitro maturation (IVM).

Primordial germ cells develop into oocytes and sperm cells. These cells are useful resources in reproductive biology and regenerative medicine. The mesenchymal stem cells (MSCs) have been examined for in vitro production of primordial germ cell-like cells. This study aimed to summarize the existing protocols for MSCs differentiation into primordial germ cell-like cells (PGLCs). In the limited identified studies, various models of mesenchymal stem cells, including those derived from adipose tissue, bone marrow, and Wharton's jelly, have been successfully differentiated into primordial germ cell-like cells. Although the protocols of specification induction are basically very similar, they have been adjusted to the mesenchymal cell type and the species of origin. The availability of MSCs has made it possible to customize conditions for their differentiation into primordial germ cell-like cells in several models, including humans. Refining germ cell-related signaling pathways during induced differentiation of MSCs will help define extension to the protocols for primordial germ cell-like cells production.

In vitro development of functional gametes from pluripotent stem cells is a promising prospect to treat infertility. Mesenchymal stem cells with a high degree of plasticity and less tumorigenicity are a reliable source of stem cells for the generation of gametes. The present study aimed to compare the differentiation potential in the mesenchymal stem cells that are derived from bone marrow (BMDMSCs) and adipose tissue-derived mesenchymal stem cells (AD-MSCs) into germ cells in a culture medium containing bone morphogenic protein-4 (BMP-4).

Experimental approach: 

In this study, MSCs were isolated from both bone marrow and adipose tissue of murine samples. To further verify the nature of the harvested stem cells, their multipotency and surface marker were examined. The identified stem cells were cultured in a medium supplemented with 0 and 25 ng/mL of BMP-4 for 4 days. Flow cytometry analysis, immunofluorescence staining, and real RT-PCR were used to assess the expression levels in germ cell-specific biomarkers (Mvh, Dazl, Stra8, and Scp3).

CD44+, CD45−, CD31−, BMD-MSCs, and AD-MSCs showed to be capable of differentiating to osteo-adipogenic lineages. The flow cytometry, immunofluorescence, and RT-PCR results indicated that early germ cell markers (Mvh and Dazl) were expressed in both types of cells but they were significantly higher in BMD-MSCs than AD-MSCs.

Conclusion and implications: 

Based on our results, the addition of exogenous BMP4 to the culture medium could differentiate BMD-MSCs and AD-MSCs into primordial germ cells, but it is inadequate to further develop into late germ cells in vitro. Moreover, the results revealed that, although AD-MSCs were easier to collect and had faster growth and proliferation rates than BMD-MSCs, the BMD-MSCs were better capable of differentiation into primordial germ cells. They may serve to be considered a more suitable source of MSC for in vitro generation of gametes than AD-MSCs.

The current study aimed to identify and characterize primordial germ cells in the blood, genital ridge, and primary gonad in turkey. Besides, we described the histological characteristics of ovaries in the turkey embryo.

The embryos from stages 14 to 31 per Hamburger and Hamilton, were studied by the means of blood smear and serial sections from the whole-mount embryo. The primordial germ cells were identified by histochemical and immunostaining techniques.

The present research results suggested that these cells could be identified by their remarkably large size, large nuclei, and granules in the cytoplasm. At stages 20-21 of Hamburger and Hamilton, the alkaline phosphatase reaction was negative in these cells. Furthermore, the primordial germ cells could not be labeled by stage-specific antigen-1 antibody in the primary gonad. We observed that these cells provided negative or poorly positive staining with Alcian blue solution in the migration phase. The presence of glycogen in the cytoplasm of the primordial germ cells was verified using periodic acid-Schiff and Best’s Carmine methods. These cells were Best’s Carmine positive; however, they contained reduced amounts of glycogen in the primary gonad. 

The study findings demonstrated that the periodic acid-Schiff is the best method for identifying the turkey primordial germ cells in the blood and migration phase. Moreover, we reported the existence of histological differences between the right and left ovaries in the turkey embryo.

Currently, scientists are looking for a solution to the problem of the couples who have a lack of germ cells by through cell therapy. It is found that human amniotic membrane mesenchymal stem cells (hAMSCs) could be a good candidate for solving this problem. In the present study, an attempt was made to show that hUMSCs can express the PGC markers in the presence of retinoic acid (RA).

Placenta was obtained from healthy mothers and amniotic stem cells were isolated by enzymatic method from amniotic membrane. The cells were treated by retinoic acid for 14 days. Mesenchymal properties of hAMSCs were assessed by flow-cytometry and expression of PGC markers was established by Q-PCR.

Mesenchymal stem cell properties were confirmed by antibodies against mesenchymal stem cell markers (CD73, CD90, and CD105). After that, the expression of the C-kit, Oct4, SSEA4, VASA genes were determined as primordial germ cell markers using quantitative PCR. It was found that the use of retinoic acid led to the highest expression of C-kit, SSEA4, VASA genes and lower expression of Oct4.

Our study indicates that retinoic acid can be used as a suitable factor for induction of hAMSCs into primordial germ cells (PGCs) and hAMSCs have enough potential to do that.

Germ cell development processes are influenced by soluble factors and intercellular signaling events between them and the neighboring somatic cells. More insight into the molecular biology of the germ cell development from embryonic stem (ES) cells and investigation of appropriate factors, specifically those targeting differentiation processes, is of great importance. In this study, we established an in vitro model with higher ES cell differentiation rate to germ cells, using adenylate cyclase activator, forskolin.

ES cells were first cultured for five days, leading to embryoid body (EB) formation. Subsequently, the EB were dissociated and cultured for an additional three days in different forskolin concentrations of 5, 20, and 50 µM, with or without granulosa cells (GC) co-culture. On the 8th day, we analyzed the expressions of 5 germ cell-specific markers using quantitative real-time-PCR technique along with cell viability assay by MTT test.

Our results showed that in the GC-free cultures, a 50-µM concentration of forskolin resulted in a significant increase in Mvh, Gdf9, Scp3, and Rec8 expression levels in comparison to the control. However, when the cells were co-cultured with the GCs, 20-µM concentration of forskolin could also increase the expression of those germ cell-specific marker genes. Furthermore, results from the MTT assay showed enhanced cell proliferation and survival at all three concentrations of forskolin, but 20-µM concentration was the most potent one.

These data indicate that forskolin can stimulate differentiation and proliferation, dose-dependently; however, the influence of GCs co-culturing should not go unnoticed.

We aimed to examine the expression levels of the VASA gene and protein in testis sections of neonate and adult mice as well as testicular cell cultures.

In this experimental study, in order to investigate the expression of this germ cell marker gene in more detail, we analyzed the expression of VASA by immunocytochemistry, immunohistochemistry and fluidigm reverse transcription-polymerase chain reaction (RT-PCR).

The immunohistochemical assays showed that the VASA protein was exclusively expressed in germ cells in the seminiferous tubules of the neonate and adult testis and not in somatic cells. VASA was not detectable in PLZF positive spermatogonial stem cells (SSCs), was weakly expressed in proliferating spermatogonia, and became abundant in spermatocytes and round spermatozoa. Counting VASA-positive cells in the seminiferous tubules of the neonate and adult testis depicted significant higher expression (P<0.05) of VASA in the adult testis in comparison to its neonate counterpart. SSC colonies were established in vitro after digestion of the testis and characterized by immunocytochemistry for CD90 and stage-specific embryonic antigens 3 (SSEA3). Immunocytochemistry confirmed that in contrast to the not detectable signal in vivo, VASA protein was strongly localized in the cytoplasm of both neonate and adult mouse SSCs under in vitro conditions. The results of Fluidigm RT-PCR revealed a significant higher expression of the germ cell gene VASA in adult SSCs in comparison to neonate SSCs in cell culture (P<0.05).

The VASA protein is, therefore, an extremely specific marker of testicular germ cell differentiation in vivo and mostly expressed in the adult testis in spermatocytes and round spermatids. The immunohistochemical signal in spermatogonia is very low. So, PLZF positive SSCs are negative for VASA in vivo, while in contrast, once isolated from the testicular niche VASA is also strongly expressed in SSCs under in vitro conditions.

Spermatogonial stem cells (SSCs), as unipotent stem cells, are responsible for the production of sperm throughout the male’s life. Zinc finger and BTB domain containing 16 (ZBTB16/PLZF) genes provide various functions in the cell development, signaling pathway, growth regulatory and differentiation. Here, we aimed to investigate expression of the PLZF germ cell gene marker in testis, SSCs, pluripotent embryonic stem cells (ES cells) and ES-like cells of mouse testis.

In this experimental study, we examined the expression of the PLZF germ cell marker in the testis section and testicular cell culture of neonate and adult mice by immunohistochemistry (IMH), immunocytochemistry (ICC) and Fluidigm Real-Time polymerase chain reaction (PCR).

IMH data indicated that the PLZF protein was localized in the neonate testis cells of the tubules center as well as the basal compartment of adult testis seminiferous tubules. Counting PLZF IMH-positive cells in the sections of seminiferous tubules of adult and neonate testis revealed significant expression of positive cells in adult testis compared to the neonate (P<0.05). Under in vitro conditions, isolated SSC colonies were strongly ICC-positive for the PLZF germ cell marker, while ES cells and ES-like cells were negative for PLZF. Fluidigm Real-Time-PCR analysis demonstrated a significant expression of the PLZF germ cell gene in the neonate and adult SSCs, compared to ES cells and ES-like cells (P<0.05).

These results indicate that PLZF is a specific transcription factor of testicular germ cell proliferation, but it is downregulated in pluripotent germ cells. This can be supportive for the analysis of germ cells development both in vitro and in vivo.

Bone morphogenetic protein 4 (BMP4) is a significant signaling molecule that involves in initiating of differentiation and performs multifunctional effects on embryonic stem cells (ESCs) and embryos.

The goal of the present study was to evaluate an in vitro differentiation model of mouse embryonic stem cells into germ cells, using BMP4.

in this experimental study, we used Oct4-GFP mouse ESCs to form embryoid body (EB) aggregations for two days. Then, single cells from EB were cultured for four days with BMP4. Using MTT assay and gene expression levels for evaluation of Mvh and Riken by real-time RT-PCR of six concentrations, 12.5 ng/ ml BMP4 was determined as an optimized dose. Then, the expression level of Fkbp6, Mov10l1, 4930432K21Rik, Tex13, Mvh, Scp3, Stra8, Oct4 were evaluated. Flow cytometry and immunostaning were used to confirm the findings of the real-time RT-PCR.

In the Ӄ group, the genes encoding Riken (p≤0.001) and Mvh (p≤0.001) were found to be increased with significant differences compared with the control group. Mov10l1 (p=0.22), Tex13 (p=0.10), Fkbp6 (p=0.90), Scp3 (p=0.61) and Stra8 (p=0.08) were up-regulated without significance differences compared with control group. Flow cytometry analysis showed that the mean number of Mvh-positive cells in the Ӄ group was greater when compared with ESCs, -BMP4 and EB groups (p=0.03, p≤0.001, p=0.02, respectively).

Down-regulation of Oct4, expression of germ cells genes and meiosis markers expression raise this hypothesis that ESCs were differentiated by BMP4, and may be introduced into the first meiosis as germ cell-like cells.

Non-obstructive azoospermia is mostly irreversible. Efforts to cure this type of infertility have led to the application of stem cells in the reproduction field. In the present study, testicular cell-mediated differentiation of male germ-like cells from bone marrow-derived mesenchymal stem cells (BM-MSCs) in an in vitro indirect co-culture system is investigated.
In this experimental study, mouse BM-MSCs were isolated and cultured up to passage three. Identification of the cells was evaluated using specific surface markers by flow-cytometry technique. Four experimental groups were investigated: control, treatment with retinoic acid (RA), indirect co-culture with testicular cells, and combination of RA and indirect co-culture with testicular cells. Finally, following differentiation, the quantitative expression of germ cell-specific markers including Dazl, Piwil2 and Stra8 were evaluated by real-time polymerase chain reaction (PCR).
Molecular analysis revealed a significant increase in Dazl expression in the indirect co-culture with testicular cells group in comparison to the control group. Quantitative expression level of Piwil2 was not significantly changed in comparison to the control group. Stra8 expression was significantly higher in RA group in comparison to other groups.
Indirect co-culture of BM-MSCs in the presence of testicular cells leads to expression of male germ cell-specific gene, Dazl, in the induced cells. Combination of co-culture with testicular cells and RA did not show any positive effect on the specific gene expressions.

Cancer/Testis Antigens (CTAs) are a subgroup of tumor-associated antigens which are expressed normally in germ line cells and trophoblast, and aberrantly in a variety of malignancies. One of the most important CTAs is Developmental Pluripotency Associated-2(DPPA2) with unknown biological function. Considering the importance of DPPA2 in developmental events and cancer, preparing a suitable platform to analyze DPPA2 roles in the cells seems to be necessary.
In this study, the coding sequence of DPPA2 gene was amplified and cloned into the retroviral expression vector to produce recombinant retrovirus. The viral particles were transducted to Esophageal Squamous Cell Carcinoma (ESCC) cell line (KYSE-30 cells) and the stable transducted cells were confirmed for ectopic expression of DPPA2 gene by real-time PCR.
According to the critical characteristics of retroviral expression system such as stable and long time expression of interested gene and also being safe due to deletion of retroviral pathogenic genes, this system was used to induce expression of DPPA2 gene and a valuable platform to analyze its biological function was prepared. Transduction results clearly showed efficient overexpression of the gene in target cells in protein level due to high level of GFP expression.
Such strategies can be used to produce high levels of desired protein in target cells as a therapeutic target. The produced recombinant cells may present a valuable platform to analyze the effect of DPPA2 ectopic expression in target cells. Moreover, the introduction of its potential capacity into the mouse model to evaluate the tumorigenesis of these cancer cells in vivo leads to an understanding of the biological importance of DPPA2 in tumorigenesis. In addition, our purified protein can be used in a mouse model to produce specific antibody developing a reliable detection of DPPA2 existence in any biological fluid through ELISA system.

common use of sevoflurane in congenital defects during repeated surgeries may have detrimental effects on spermatogenesis after puberty.

This study investigated sevoflurane effects on spermatogenesis process in male mature mice after exposure in prepubertal time.

24 neonatal NMRI male mice were randomly classified in three groups. Experimental 1 and 2 groups (exposure to 1 minimum alveolar concentration (MAC) and 2 MAC sevoflurane, respectively in 2 lit/min oxygen (O2) for 7 days (30 min, daily) and control. All groups were sacrificed after 2 months. Histological assessment, immunohistochemistry and apoptosis process was done. Bax and Bcl2 expression was evaluated in the testicular tissue by real time Poly Chain Reaction.

Our results showed that the integrity of testicular tissue was preserved in both experimental groups. Count of spermatogonial cells had significant decrease in group 2 compared to others. The rate of apoptosis in spermatogonial cells was 15±3% and 9±2% in the group 2 and 1, respectively. Also, Bax/Bcl2 ratio was 0.2615, 1.0070 and 9.3657 in control, experimental group 1 and 2, respectively. This result was significant (p≤0.002) between groups 2 with other groups.

Continuous exposure of 2 MAC sevoflurane in 2 lit/min O2 simultaneous during prepubertal may create more testicular tissue damage in terms of cellular and molecular function compared to continuous exposure to lower level of sevoflurane by increase in ratio of Bax/Bcl2 and apoptosis in germ cells after puberty.

Although many researchers have confirmed induction of germ cells from bone marrow mesenchymal stem cells (BMMSCs), there are no reports that confirm spontaneous differentiation of germ cells from BMMSCs. In this study, we have evaluated the effect of adult Sertoli cell condition medium (SCCM) as a mutative factor in the induction of germ cells from BMMSCs.
BMMSCs were collected from the bone marrow of 6-8-week old NMRI mice and their mesenchymal entities were proven using superficial markers (expression of CD44 and CD73 and non-expresion of CD45 and CD11b) by fow cytometry. Their multi-potential entities were proved with differentiation to osteogenic and adipogenic cells for 21 days. Also isolated Sertoli cells were enriched using lectin coated plates and Sertoli cell condition medium (SCCM) was collected. Sertoli cells were identified by immunocytochemistry and Vimentin marker. The cells were then differentiated into germ cells with SCCM for 2 weeks. Finally induced cells were evaluated by RT-PCR and immunocytochemistry.
Differentiation of mesenchymal stem cells to osteoblast and adipocyte showed their multi-potential property. Expression of CD44 and CD73 and non-expression of CD45 and CD11b confirmed mesenchyme cells. Immunocytochemistry and RT-PCR results showed expression of germ cells specific marker (Mvh).
This study confirmed the effect of SCCM as a motivational factor that can used for differentiation of germ cells from BMMSCs.