دکتر علی اصغر مقدم

This study aimed to the effect of insulin-like growth factor-1 on the proliferation of goat spermatogonial stem cells (SSCs) in vitro.

The testicles of immature goats were obtained from slaughterhouse. Isolation of spermatogonial cells was performed by two-step digestion. The isolated cells were cultured in 4 different groups: 3 groups by adding different doses of IGF-1 (10, 40 and 100 ng/ml) to the basic medium (DMEM containing 5% fetal bovine serum and 1% antibiotic) and the control group (without IGF-1). Data were analyzed by one-way ANOVA and Tukey test at 5% level. Presence of SSCs at the end of culture was determined by reverse transcription polymerase chain reaction (RT-PCR) for several important spermatogonial markers (PGP9.5, THY-1, PLZF, Oct4 and VASA) and immunohistochemical staining against PGP9.5 antigen.

In this study, the number of spermatogonial cell colonies in treatment 3 was significantly higher than the control and other treatment groups on days 4, 7, and 10 of culture (P<0.05). Also, the surface area of G10 colonies on days 7 and 10 of culture was significantly different (P<0.05). The surface area of G100 on days 4, 7, and 10 after culture was significantly higher than control, G10 and G40 (P<0.05). The isolated cells expressed all surface markers related to stem cells.

According to this results, the addition of 100 ng/ml IGF-1 to culture medium for promoting colonization of goat Spermatogonial stem cells is recommended.

In vitro maturation (IVM) of oocytes and subsequently, in vitro fertilization (IVF) for the generation of embryos in the laboratory have important values. Considering that antioxidants are known as effective free radicals scavenger, it is possible to improve the in vitro oocyte maturation and the fetal quality. Therefore, the aim of this study is to evaluate the effect of Thymbra spicata hydroalcoholic extract as a source of antioxidant on in-vitro sheep oocyte maturation. Cumulus oocyte complexes (COCs) were collected from ewe ovaries and were cultured for 24 hours in maturation medium in TCM supplemented with FSH, LH, FBS, cysteamine, pyruvate sodium and antibiotics (control group) and in maturation medium without cysteamine (as an antioxidant) supplemented with different doses of Thymbra spicata hydroalcoholic extract (1mg/ml: group 1, 10 mg/ml: group 2, 50 mg/ml: group 3) as an antioxidant. In-vitro maturation stages and resumption of meiotic was assessed by determination of cumulus cells mass expansion and number of oocytes in metaphase II stage of meiotic division in all groups. Cumulus cells mass expansion was similar between control, 2 and 3 groups. However, in group 1 was lower than control group. Nuclear maturation was similar between control and group 3 and both of them were different with groups 1 and 2. The results of this study showed that the Thymbra spicata hydro alcoholic extract, has a positive effect on oocyte maturation that is doses dependent. So with increasing concentration of Thymbra spicata hydroalcoholic extract, the rate of maturation immature oocytes is increased. Generally, we conclude that addition of appropriate amounts of natural extracts such as Thymbra spicata to maturation medium improves oocytes maturation.

Many previous studies have proved that the anatomical features of ewe cervix can affect the success of artificial insemination. These characteristics differ in sheep breeds. This study aimed to describe the anatomical features of cervix in Zandi ewes.One hundred and ninety threenonpregnant and clinically healthy reproductive tracts of adult and non-adult Zandi sheep were collected from a slaughter house and were divided into follicular or luteal phase.Then, the morphology of the vaginal protrusion of cervix was classified as slit, papilla, duckbill, flap or rose. The depth of penetration of an inseminating pipette was recorded. The cervical canal of each tract was filled with a silicone sealant for casting the mould. The cervices were sectioned longitudinally, and the length, number of cervical rings and the arrangement of the rings were recorded. The degree of completeness and interdigitations of the folds recorded as one of three grades 1, 2, and 3 cervices. The results showed the Papillatype was more common in vaginal protrusion of cervix and the most depth of penetration was in Slittype. The mean length of cervix, distance from cervix external os to first ring and the depth of penetration in adult ewes were significantly more than those in non-adult ewes. The mean distance betweencervical external osand first and second ringsand the depth of cervical penetration in follicular phase were more than those in luteal phase.The mean number of cervical ridges was 5 and 6 in 94% and 6% of cervices, respectively. Grades 1, 2, and 3 cervices, were observed in 64%, 25% and 11% of samples, respectively. The information generated in this study would be useful for increasing the success rate of penetration in ewes exhibiting estrus in order to improve the lambing rate of tropical ewes following transcervical AI.

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis, because they have the capacity of self-renewal, and differentiation into spermatozoa. Freezing is the most common long-term preservation approach for SSCs. The present study aimed to investigate the cryoprotective impacts of FBS (Fetal Bovine Serum) and trehalose on caprine SSCs.

SSCs were isolated from prepubertal goat testis by enzymatic digestion and differential plating. Cells were divided into 9 groups. The control group included SSCs without cryoprotective agents. In the treatment groups 1, 2, 3 and 4, concentration of 10% FBS, and various concentrations of trehalose (0, 50, 100 and 200 mM), and in the treatment groups 5, 6, 7, and 8, concentration of 20% FBS and various concentrations of trehalose (0, 50, 100 and 200 mM) were used respectively. The viability rate of the cells was assessed immediately after isolation, following addition of cryoprotectant agents and after thawing. Identification of cells was confirmed by immunocytochemical staining against PGP9.5 antigen. Data were analyzed using one-way ANOVA test.

The viability rate of SSCs in various treatments following addition of FBS and trehalose were similar to viable cells immediately after isolation. Furthermore, higher viability rates of SSCs after thawing were observed in freezing medium containing 10% FBS and 200 mM trehalose (P<0.05).

The results revealed that freezing in 10% FBS with 200 mM trehalose acts as efficient method for the cryopreservation of caprine SSCs.

Spermatogonial stem cells (SSCs) use as an excellent model system for differentiation, development and functioning of testes and provide a source of cells for infertility treatment and production of transgenic animals and recombinant drugs. The aim of present study was to determine the effects of different concentration of melatonin on prepubertal lamb’s spermatogonial colony formation.                                                                                                                                               

 An experimental study was conducted. Each treatment was replicated 5 times. Testicular cells were isolated from testes of prepubertal lambs by two-step enzymatic digestion then cultured for 10 days. Control cells (C) were grown in basic DMEM media with 1% antibiotic and 5% FBS. Treated cells were grown in basic media with 1 µmol/ml (T1) or 1 nmol/ml (T2) melatonin. Culture media was changed every 72h and SSCs colony formation was monitored by inverted microscope. The data were analyzed by one way ANOVA. P value less than 5% was considered statistically signifent.

SSCs were identified by immunocytochemistry assay against PGP9.5.  Number and Surface area of colonies were greater in T1 group than C and T2 groups at day 7 (P=0.04587).

The findings suggest that simultaneous addition of 1 µmol/ml melatonin to culture media may increase In vitro colony formation of prepubertal sheep SSCs. So addition of above dose of melatonin to SSCs culture media are suggested by authors.

Spermatogonial stem cells are specific cells that have the ability of self-renewal and differentiation. These cells play an essential role in maintaining spermatogenesis and fertility. In this regard, the present study was performed with the purpose of investigating the effect of different concentrations of follicle stimulating hormone (FSH) on in vitro colony formation of caprine spermatogonial stem cells.

Spermatogonial cells, were isolated from prepubertal goat testis using two-step enzymatic digestion. Then, isolated cells were cultured for 10 days in four groups. In the control group, simple culture of spermatogonial cells was performed in Dulbecco's Modified Eagle Medium (DMEM) containing 1% antibiotics and 5% FBS (Fetal Bovine Serum). In the treatment groups 1, 2, and 3, different concentrations of follicle stimulating hormone (5, 10, and 20 IU/ml), was added to the culture medium, respectively. The culture media were changed every 72 hours. Identification of cells was confirmed by immunocytochemical staining against PGP9.5 antigen. Immediately after isolation, percentage of cells viability, surface area, and number of colonies formed on 4th, 7th and 10th days after the culture, were evaluated using an inverted microscope. Data were analyzed using one way ANOVA test.

The findings indicated that viability rate of Spermatogonial stem cells after isolation was 89.4 ± 2.32%. The effect of FSH on the formation of spermatogonial cells colonies was dose dependent. Doses of 5 and 10 IU/ml increased the surface area and number of the spermatogonial cell derived colonies but dose of 20 IU/ml reduced colonies formation (p < 0.05).

FSH can provide an appropriate culture medium for the study of spermatogonial cells in vitro.

Spermatogonial stem cells (SSCs) are undifferentiated germ cells that maintain spermatogenesis during lifetime by balancing between self-renewal and differentiation. The aim of this study was to investigate the effect of different concentrations of vitamin E on spermatogonial stem cells colony formation.

SSCs were isolated from testes of prepubertal lamb by two step enzymatic digestions then purified by differential pllating. The cells were cultured for 10 days in 4 groups. Control group: Basic media (DMEM environment which contains 1% antibiotic and 5% FBS( and treatment groups were treated with 20, 40 and 80 µmol/ml vitamin E added to basic media respectively. Changing of culture media was performed every 72h. Colony number and diameter assay was done by inverted microscope. Identification of SSCs was performed by immunocytochemistry assay against PGP9.5.

there were no significant difference in colony number and surface area between treatment groups and between treatment and control groups in days 4, 7and 10. Colony surface area was significantly increased at day 10 compared to day 4 (P<0.05).

The results of this study showed vitamin E as an antioxidant doesn’t have significant effect on colony induction of SSCs in short term culture in vitro.