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Aims: This study aimed to investigate the therapeutic potential of a homogenous clonal population of mesenchymal stem cells (cMSC) and their extracellular vesicles (cMSC-EV) subpopulations on isolated rat islets in vitro and in inflammatory-mediated type 1 diabetes (T1D) non-human primate models., Main Methods: EV subpopulations were isolated from human bone marrow-derived cMSC supernatant by low- and high-speed ultracentrifuge (EV-20K and EV-U110K) and sucrose density gradient (EV-S110K). The EVs were characterized generally and for the level of albumin, acetylcholinesterase (AChE) activity, co-isolate apoptotic markers, and expression of CD63 + /annexin V + . Rat islet-derived single cells (iSCs) proliferation was measured using a Ki-67 proliferation assay. Diabetes was induced by multiple low-dose administrations of streptozotocin in rhesus monkeys. The diabetic monkeys were divided into three groups: the cMSC group, received two injections of 1.5 × 10 6 cMSC/kg body weight; the EV group received two injections of EVs isolated from 1.5 × 10 6 cMSC/kg, and the vehicle group received phosphate-buffered saline., Key Findings: EV-S110K showed higher AChE activity, lower expression of CD63 + /annexin V + , and lower apoptotic co-isolates. EV-S110K induced β-cell proliferation in vitro in a dose-dependent manner. The administration of EV-S110K and/or cMSC in diabetic monkeys demonstrated no significant changes in general diabetic indices and β-cell mass in the pancreas of the monkeys. Both treatments demonstrated a lowering trend in blood glucose levels and reduced pro-inflammatory cytokines. In contrast, regulatory T cells and anti-inflammatory cytokines were increased., Significance: cMSC and cMSC-EV provided initial evidence to attenuate clinical symptoms in inflammatory-mediated T1D non-human primates through immunomodulation., Competing Interests: Declaration of competing interest No competing financial interests exist., (Copyright © 2023. Published by Elsevier Inc.)

Background: Retained placenta (RP) is a prevalent disorder in cattle with many health-related and economic costs for the farm owners. Its etiology has not been clarified yet and there is no definite therapy for this disorder. In this study we conducted RNA-seq, hematologic and histologic experiments to survey the causes of RP development., Methods: Blood samples were collected from 4 RP and 3 healthy cows during periparturtion period for hematological assessments followed by placentome sampling within 30 min after parturition. Cows were grouped as RP and control in case the placenta was retained or otherwise expelled, respectively. Total RNA was extracted from placentome samples followed by RNA-sequencing., Results: We showed 240 differentially expressed genes (DEGs) between the RP and control groups. Enrichment analyzes indicated immune system and lipid metabolism as prominent over- and under-represented pathways in RP cows, respectively. Hormonal assessments showed that estradiol-17β (E2) was lower and cortisol tended to be higher in RP cows compared to controls at the day of parturition. Furthermore, histologic experiment showed that villi-crypt junctions remain tighter in RP cows compared to controls and the crypts layer seemed thicker in the placentome of RP cows. Complete blood cell (CBC) parameters were not significantly different between the two groups., Conclusion: Overall, DEGs derived from expression profiling and these genes contributed to enrichment of immune and lipid metabolism pathways. We suggested that E2 could be involved in development of RP and the concentrations of P4 and CBC counts periparturition might not be a determining factor., (© 2022. The Author(s).)

This study was conducted to investigate the main and interactive effects of two methods of culture medium preparation [base medium vs granulosa cells conditioned medium (GCCM)] and two nutrient supplements [fetal bovine serum (FBS) vs knock-out serum replacement (KSR)] on formation and activation of primordial follicles and gene expression of corresponding factors during a seven-day culture period. One-day-old mouse ovaries were cultured with four different culture media including base medium containing FBS (BMF), base medium containing KSR (BMK), GCCM prepared with FBS (CMF) and GCCM prepared with KSR (CMK), and samples for histological and molecular assessments were collected on days 3 and 7 of culture. Further, steroid content of media was measured. Histological examination showed that KSR enhanced follicular formation and the number of follicular count was greater in BMK than CMF group (P < 0.05). Moreover, follicular activation was higher in CMK group than BMK and CMF groups (P < 0.05). Additionally, RT-PCR revealed that KSR upregulated Gdf9 gene expression (P < 0.05), while GCCM diminished expression of Gdf9, Bmp15, Notch2, Figla and Foxl2 (P < 0.05). GCCM decreased expression of Pten and increased expression of Pi3k (P < 0.05). Besides, hormonal assays indicated higher concentrations of estradiol and progesterone in GCCM compared with base media (P < 0.0001). In conclusion, the present study showed base medium containing KSR could serve as a proper medium for in vitro culture of neonatal mouse ovary since it could better support formation of primordial follicles. Yet BMK did not promote follicular activation as well as GCCM prepared with KSR did, and therefore, requires modifications., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2022 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

In vitro activation of primordial follicles could serve as a safe method to preserve fertility in patients with cancer subjected to ovarian tissue cryopreservation during oncotherapy, however the culture medium for this purpose requires to be optimized. Granulosa cell conditioned medium (GCCM) has been recognized to enhance primordial follicle activation and the present study was conducted to understand whether addition of pyruvate, a combination of insulin, transferrin and selenium (ITS) or testosterone to GCCM could improve its efficiency in this regard. To this end, 1-day-old mouse ovaries were cultured in four different media including CON (control; containing GGCM only), PYR (containing GCCM plus pyruvate), ITS (containing GCCM plus ITS) or TES (containing GCCM plus testosterone) for 11 days. Furthermore, follicular dynamics and gene expression of factors involved in follicular development were assessed using histological examination and RT-PCR, respectively, on days 5 and 11 of culture. Pyruvate decreased follicular activation, but it enhanced the progression of follicles to the primary stage. Moreover, it upregulated Bmp15 and Cx37 (P < 0.05). In the ITS group, activation of follicles was not affected and total number of follicles was reduced by day 11 of culture. Additionally, ITS downregulated Pi3k, Gdf9, Bmp15 and Cx37 (P < 0.05). Although testosterone did not affect primordial follicle activation, it enhanced the development of follicles up to the preantral stage (P < 0.05). Furthermore, testosterone inhibited the expression of Pten but stimulated the expression of Gdf9 and Cx37 (P < 0.05). In conclusion, the present study revealed that inclusion of pyruvate and testosterone into GCCM could enhance the early development of follicles in cultured 1-day-old mouse ovaries.

Fertility preservation of prepubertal girls subjected to invasive cancer therapy necessitates defining protocols for activation of isolated primordial follicles. Granulosa (GCs) and cumulus cells (CCs) play pivotal role in oocyte development. Although GCs and CCs share some similarities, they differ in growth factors production. The current study was conducted to evaluate the effects of GCs, CCs and their conditioned media on mice primordial follicles activation. One-day-old mice ovaries were subjected to 6-day culture with base medium (BM), GC conditioned medium (GCCM), GC coculture (GCCC), CC conditioned medium (CCCM) or CC coculture (CCCC). Follicular growth and primordial to primary follicle transition was observed during 6-day culture, and follicular activation rate tended to be greater in GCCM than other groups (0.05 

SummaryThe high miscarriage rates that result following transfer of embryos derived from in vitro maturation (IVM) of oocytes necessitate improvements in the processes involved. This study aimed to improve the quality of in vitro matured oocytes using granulosa cell conditioned medium (GCCM) as the culture medium. In this work, germinal vesicle (GV)-stage oocytes from NMRI mice were collected and cultured using three types of culture medium: Base medium (BM) (control), 50% granulosa cell conditioned medium (GCCM50) and 100% GCCM (GCCM100). After IVM, the mitochondria activity potential and viability of metaphase II (MII) oocytes were evaluated by JC-1 and trypan blue staining, respectively. Maturational gene expression levels of CyclinB1, Cdk1 and Gdf9 in the control, GCCM50 and GCCM100 samples were analyzed using real-time polymerase chain reaction (PCR). The viability rate of in vitro matured oocytes was highest in the GCCM50 group. JC-1 staining showed that GCCM50 enhances mitochondrial activity more than the other groups (P < 0.05). Gene expression levels of Cdk1 and Gdf9 were higher in the group with GCCM50 treatment, than in the control and GCCM100 groups (P < 0.05), while the expression level of CyclinB1 did not differ among the groups. The results indicated that a 50% concentration of GCCM in combination with BM components enhanced MII and viability rates and mitochondria activity of mouse immature oocytes.