Caprine Endometrial Mesenchymal Stromal Stem Cell: Multilineage Potential, Characterization, and Growth Kinetics in Breeding and Anestrous Stages

The endometrial layer of the uterus contains a population of cells with similar characteristics of mesenchymal stem cells (MSCs). In the present study, caprine endometrial mesenchymal stromal stem cells (En-MSCs) characters and differentiation potential to chondrogenic, osteogenic, and adipogenic cell lines as well as their growth kinetics in breeding and anestrous stages were evaluated. En-MSCs were enzymatically isolated from endometrial layer of the uterus of adult goats and were cultured and subcultured until passage 4. The growth kinetics and population doubling time (PDT) of caprine En-MSCs in breeding and anestrous stages were determined. En-MSCs in passage 4 were used for the karyotyping and differentiation into chondrocytes, osteocytes, and adipocytes. The PDT in anestrus phase was 40.6 h and in cyclic goats was 53 h. En-MSCs were fibroblast-like in all passages. The number of chromosomes was normal (2n=60) with no chromosomal instability. Chondrogenic, osteogenic, and adipogenic differentiation of En-MSCs was confirmed by staining with Alcian blue, Alizarin red, and Oil Red O, respectively. Caprine En-MSCs demonstrated to be an alternative source of MSCs for cell therapy purposes in regenerative medicine

Histomorphometric Evaluation of Superovulation Effect on Follicular Development after Autologous Ovarian Transplantation in Mice

The effect of superovulation by pregnant mare serum gonadotropin (PMSG) on autologous transplanted ovaries in the lumbar muscles of mice was histomorphometrically evaluated using the indices of number and volume of different kind of follicles and volume of corpora lutea, ovary, and stroma. Angiogenesis was observed after mouse ovarian transplantation on days 14 and 21 after ovarian grafting. After transplantation, the total number and volume of primary and secondary follicles reduced, while PMSG superovulation increased the total number and total volume of tertiary follicles and also the ovarian volume after transplantation. Transplantation increased the average size of primary, secondary, and tertiary follicles. Therefore, primary and secondary follicles can survive after autologous transplantation but their reservations diminished by increasing the time of transplantation. However, number of tertiary follicles and their response to superovulation increased over time after transplantation

Promjene u RF-amidu srodnom peptidu-3 hipotalamusa i ekspresijama gena Kiss1 tijekom spermatogeneze kod štakora u uvjetima kroničnog stresa.

The effects were evaluated of chronic stress and the glucocorticoid receptor antagonist (RU486) on mRNA expressions of RF-amide related peptide-3 (RFRP-3) in the dorsomedial hypothalamic nucleus (DMH) and Kiss1 in the arcuate nucleus (ARC) of male rats. Twenty-four male rats were allocated to four equal sized groups: the stress, RU486, stress/RU486, and control groups. In the stress group the rats were restrained 1 hour/day for 12 days. In the RU486 group, the rats were injected with RU486 for 12 days. In the stress/RU486 group, the rats were injected with RU486 1 hour before the stress process for 12 days. Relative expressions of RFRP-3 and Kiss1 mRNAs were determined using real-time PCR. The relative expression of RFRP-3 mRNA in the stress group was higher than that in the RU486 and control rats. The relative expression of RFRP-3 mRNA did not differ between the stress group and the stress/RU486 rats. Furthermore, the relative expressions of Kiss1 mRNA in the stress, RU486, and stress/RU486 groups were less than that of the control rats. The relative expression of Kiss1 mRNA did not differ between the stress, RU486, and stress/RU486 groups. In conclusion, dysfunction in male rat fertility caused by the chronic stress may be the result of the increase in REFP-3 and the decrease in Kiss1 mRNA expression.Istražen je učinak kroničnog stresa i antagonista glukokortikoidnog receptora (RU486) na ekspresiju mRNA RF-amidu srodnog peptida-3 (RFRP-3) u dorzomedijalnoj jezgri hipotalamusa (DMH), te na ekspresije gena Kiss1 u arkuatnom nukleusu (ARC) štakora. Dvadeset i četiri štakora bila su raspodijeljena u četiri jednake skupine: stresna skupina, RU486 skupina, stresna/RU486 skupina i kontrolna skupina. U stresnoj skupini štakori su 12 dana bili obuzdani tijekom jednog sata dnevno. U skupini RU486, štakorima je tijekom 12 dana bio primijenjivan RU486. U skupini stres/RU486, štakorima je tijekom 12 dana apliciran RU486 jedan sat prije postupka obuzdavanja. Relativne ekspresije RFRP-3 i Kiss1 mRNA određene su lančanom reakcijom polimerazom u stvarnom vremenu. Relativna ekspresija RFRP-3 mRNA u stresnoj skupini bila je veća nego u skupini RU486 i kontrolnoj skupini. Relativna ekspresija RFRP-3 mRNA nije bila različita između stresne skupine i stres/RU486 skupine. Nadalje, relativne ekspresije Kiss1 mRNA u stresnoj skupini, skupini RU486, i stresnoj skupini/RU486 bile su manje u odnosu na kontrolnu skupinu. Relativna ekspresija Kiss1 mRNA nije se razlikovala između stresne skupine, skupineRU486 i stresne skupine/RU486. Zaključno, disfunkcija plodnosti kod štakora izloženih kroničnom stresu može biti uzrokovana putem povećane ekspresije RFRP-3 i smanjene ekspresije Kiss1 mRNA

Fast free of acrylamide clearing tissue (FACT) for clearing, immunolabelling and three-dimensional imaging of partridge tissues

Fast free of acrylamide clearing tissue (FACT) is a modified sodium dodecyl sulfate-based clearing protocol for the chemical clearing of lipids that completely preserves fluorescent signals in the cleared tissues. The FACT protocol was optimized to image translucent immunostained brain and non-nervous tissues. For this purpose adult male Chukar partridge (Alectoris chukar) was used as a model. After clearing the tissues, 1 or 2 mm-thickness sections of tissues were immunolabeled. The paraventricular nucleus in the hypothalamus (2-mm section) was cleared with FACT, and then was stained with gonadotropin-inhibitory hormone (GnIH) antibody and Hoechst. Simultaneously, immunohistochemical (IHC) staining of cryosectioned brain (30 μm) was done by GnIH-antibody. The FACT protocol and staining of cell nuclei of nine other tissues were done by a z-stack motorized fluorescent microscope. GnIH-immunoreactive neurons were found by FACT and IHC during the breeding season in male partridges. Deep imaging of the kidney, duodenum, jejunum, lung, pancreas, esophagus, skeletal muscle, trachea, and testis were also done. The FACT protocol can be used for the three-dimensional imaging of various tissues and immunostained evaluation of protein markers

Establishment, characterization and cryopreservation of Fars native goat fetal fibroblast cell lines

Objective: To biologically develop and evaluate the caprine fetal fibroblast cell cultures before and after freezing. Methods: Goat fetuses (ages 51, 53 and 55 d) were collected from slaughterhouse. Their skin was cut into small pieces (1 mm3) and cultured in DMEM and FBS. When reaching 80%–90% confluence, cells were passaged. Cells of the 8th passage were cultured in 24-well plates (1.5 × 105 cells/well) for 9 d and three wells were counted every day. The average cell counts at each time point were plotted against day number and the population doubling time (PDT) was determined. Then, 42 vials of cells (2 × 106 cells/mL) were frozen. Samples were thawed and cultured after 1 month. Cell viability and PDT were evaluated after thawing. Results: After eight passages, the goat fetal fibroblast cells had a latent phase of about 48 h and after an exponential phase, cells entered the plateau phase on day 5. Before freezing, PDT was about 22 h and after thawing it was about 28 h. Conclusions: The goat fetal fibroblast cell culture can be established using the adherent culture method and can be cryopreserved, too. After thawing, growth and viability indices of these cells were acceptable

Endometrial mesenchymal stem stromal cells in mature and immature sheep: An in vitro study

Background: Endometrial mesenchymal stem stromal cells (EnMSCs) are critical for uterine function, repair, and regeneration. Objective: This study introduced isolation technique of EnMSCs and compared the characteristics of EnMSCs in mature and immature ewes. Materials and Methods: Endometrial tissue samples from the uterus of 10 ewes were collected from the slaughterhouse. Endometrial cells were isolated from tissue using cold incubation and then chopping and treating was performed with collagenase type I. Isolated cells were cultured in cell culture medium and then attached cells to flasks were harvested as EnMSCs and subcultured. To enumerate the cells, the population doubling time (PDT) was determined and 2.2×104 cells in passage 4 were seeded into 24-well culture plates to compare the growth curves of isolated cells. Reverse transcription polymerase chain reaction (RT-PCR) was performed for detection of CD34 and CD73 markers. The osteogenic and adipogenic potential of isolated cells were determined using differentiation tests. Results: EnMSCs adhered to the flasks and displayed spindle-shape. Based on findings of the cell count and the growth curves, the EnMSCs growth was significantly more prominent in immature ewes in comparison to mature sheep. The PDT of EnMSCs in immature ewes was about 21 hr whereas this time period was two times higher (45 hr) in mature sheep. RT-PCR analyses of EnMSCs were positive for CD73 and negative for CD34. EnMSCs were differentiated into osteoblasts and adipocytes. Conclusion: Based on mesenchymal stem cells characters confirmed in EnMSCs, they can be a candidate for cell therapy and regenerative medicine

Testicular germ cells apoptosis following exposure to chronic stress in rats

Objective: To evaluate the effects of chronic stress on testosterone hormone level and germ cells apoptosis in testes and the inhibitory role of glucocorticoid receptor antagonist, RU486. Methods: Adult male Sprague–Dawley rats were randomly assigned to four groups (n = 6): control; stress; RU486; stress/RU486. Animals in RU486 and stress/RU486 subjected to subcutaneous injections of 2.5 mg RU486/kg 1 h before stress session. Rats were submitted to chronic restraint stress (1 h daily for 12 consecutive days) whereas control animals were not subjected to stress. Serum testosterone assay was performed and the occurrence of DNA fragmentation in the testis sections was examined using TUNEL staining. Results: Chronic restraint stress significantly induced a decrease in serum testosterone level with increase in apoptosis in spermatogonia. Systemic administration of RU486 significantly restored serum testosterone levels and attenuates stress-induced apoptosis in spermatogonia. Conclusion: The restraint stress-induced change in serum testosterone levels and seminiferous tubules apoptosis closely associated with the glucocorticoid receptor

Effects of hydroalcoholic extract of Ziziphus jujuba on acetic acid induced ulcerative colitis in male rat (Rattus norvegicus)

ABSTRACT Objective: To investigate the effects of hydroalcoholic extract of Ziziphus jujuba on the histopathological, tissue oxidative stress and inflammation plus to antioxidant pathways of colon tissue in rat with induced Ulcerative colitis. Materials and methods: Ulcerative colitis was induced in 80 rats those divided into 8 equal groups. Group 1 and 2 were negative controls receiving 1 mL/day of normal saline in enema and oral; group 3 and 4 as positive control 1 and 2 received 10 mg/kg of intra-colonic asacol and oral mesalazine; groups 5 and 6 received 20% and 40% of hydroalcoholic extract of Z. jujuba trans-rectally; group 7 and 8 received 1500 and 3000 mg/kg of hydroalcoholic extract of Z. jujuba orally, respectively. After 7 days, animals were evaluated for colon tissue histopathology, levels of malondialdehyde and IL-1β, and activities of superoxide dismutase, glutathione peroxidase and myeloperoxidase in colon tissue. Results: Hydroalcoholic extract of Z. jujuba in both forms of trans-rectal and oral administration especially in the higher doses could result into a more healing effect in damaged colonic tissue, more reduce glutathione peroxidase and IL-1β level. Also, these two doses (gel 40% and oral 3000 mg/kg) could more decrease the myeloperoxidase activity and stimulate superoxide dismutase and glutathione peroxidase activities. Also, gel 40% in transrectal administration was more potent than administration 3000 mg/kg in oral. Conclusion: The results of the present study indicated that Z. jujube may be considered as a treatment of choice for Ulcerative colitis especially in gel form and also in dose-dependent pattern

Histomorphometric evaluation of treatment of rat azoosper-mic seminiferous tubules by allotransplantation of bone marrow-derived mesenchymal stem cells

Objective(s): Bone marrow-derived mesenchymal stem cells (BM-MSCs) potentials make them appropriate for cell therapy including ability of differentiation and release of anti-inflammatory cytokines and growth factors secreta. For treatment of azoospermia to induce proliferation and differentiation of germ cells, MSCs transplantation has been introduced. The aim of the present experimental case-control study was to histomorphometric evaluation of the germinal cells in seminiferous tubules of azoospermic rats before and after BM-MSCs allotransplantation. Materials and Methods: In the present study, BM-MSCs were isolated from six male rats and confirmed. Their testes also served as intact negative controls. The recipient rats (n=6) were received two doses of 10 mg/kg of busulfan with 21 days interval to induce azoospermia. After cessation of spermatogenesis, the rats were allotransplanted with the BM-MSCs into efferent duct of right testes. Thirty-five days later, the right cell-treated testes were compared to left azoospermic ones. Results: Histomorphometric analyses showed that the seminiferous tubules treated with BM-MSCs had normal morphology in comparison with azoospermic testes, which were without germinal layer. In most BM-MSCs-treated seminiferous tubules, spermatogenesis was observed. Conclusion: The allotransplanted BM-MSCs could induce spermatogenesis in seminiferous tubules of azoospermic rats

Preventive Effects of Intrauterine Injection of Bone Marrow-Derived Mesenchymal Stromal Cell-Conditioned Media on Uterine Fibrosis Immediately after Endometrial Curettage in Rabbit

Uterine fibrosis is an acquired disorder leading to menstrual irregularities, implantation impairment, and abortion. Mesenchymal stromal cells (MSCs) have antifibrotic properties through chemokine secretion. MSC-conditioned media (MSC-CM) contain paracrine components—exosomes—with a great potential for repairing damaged tissue or preventing fibrosis. The main goal of this study was to evaluate the preventive effects of bone marrow-derived MSC-CM (BM-MSC-CM) on uterine fibrosis after uterine curettage in rabbits. This study included 12 female rabbits (24 uterine horns in total). Excised uteri of each of the 12 female rabbits were randomly divided into four groups of intact negative control, curettage positive control, BM-MSC injection, and BM-MSC-CM injection in the way that two corresponding uteri from a rabbit were allocated to different groups. The MSC-CM were collected from cultivated BM-MSCs 48 hours after having been washed three times and replaced in serum-free media. Through a surgical approach, the caudal parts of the uteri were submitted to traumatic endometrial curettage, except for the intact negative uteri. After suturing the uterine walls, BM-MSCs or BM-MSC-CM were injected in the curettage site. Endometrial regeneration was histologically evaluated 30 days after treatment. Based on the evaluation of histomorphometric indices, curettage with or without preventive injections increased the growth of endometrial layers. However, the amount of fibrotic tissue in the CM and the BM-MSC injection groups was the same as the normal control groups, and all were less than the curettage group. A single injection of CM of MSCs after 30 days prevented the fibrotic tissue formation induced by curettage in endometrial layers of rabbits. Injecting BM-MSC-CM immediately after curettage prevented and reduced the uterine fibrosis similar to BM-MSCs in a rabbit model