Evaluation of prooxidant-antioxidant balance in in vitro fertilization-conceived mice / 대한생식의학회지

OBJECTIVE: Concerns about the safety of assisted reproductive technology (ART) have been raised, as some studies have shown elevated incidence rates of childhood cancer, asthma, allergies, and other diseases in ART-conceived babies. Findings regarding the health of ART-conceived babies are controversial. The present study was conducted to evaluate the prooxidant-antioxidant balance (PAB) in in vitro fertilization (IVF)-conceived mice in comparison to naturally conceived offspring. METHODS: Mice (6–8 weeks) were divided into two groups (IVF-conceived and naturally conceived) matched by sex, age, weight, and litter size. A 1-mL blood sample was taken and the sera were separated. The oxidant-antioxidant balance was evaluated using a fast and reliable PAB assay. The results were expressed as mean±standard deviation. RESULTS: The mean PAB values (HK units) in the IVF-conceived and naturally conceived groups were 59.70±22.30 and 54.70±18.22, respectively (p=0.82). CONCLUSION: Since free radicals contribute to several pathological conditions and antioxidants play an important protective role against oxidative stress, evaluating the oxidant-antioxidant balance is very important. Although the results of this study showed that the quality of the defense mechanism against free radicals was not significantly different between the IVF-conceived and naturally conceived mice, other parameters of metabolic dysfunction need to be measured.

Assessment of PGC1 Alpha -FNDC5 axis in granulosa cells of PCOS mouse model

Background: Polycystic ovarian syndrome [PCOS] is a metabolic and endocrine disorder which is characterized by hyperandrogenism, anovulation or oligomenor-rhea and polycystic ovarian morphology. It is believed that modulation in metabo-lism of granulosa cells of PCOS patients may lead to infertility. One of the metabolic modulators is FNDC5 and its cleaved form, irisin. The axis of PGC1 Alpha - FNDC5 pathway is one of the main factors affecting cellular energy balance the purpose of this study was to evaluate this pathway in granulosa cells derived from PCOS mice model in comparison with control group

Methods: In the present study, PCOS mouse model was developed by injection of dehydroepiandrosterone [DHEA] hormone in 20 mice for a period of 20 days. Also, 20 uninjected mice were used as the control. Meanwhile, a vehicle group consisted of mice which received daily subcutaneous sesame oil injection [n=20]. Relative ex-pressions of PGC1á and FNDC5 in granulosa cells were evaluated by RT-qPCR. Analysis of gene expressions was calculated by the Delta Delta CT method and the relative levels of mRNA were normalized to GAPDH transcript levels. Differences in genes expression among three groups were assessed using one-way ANOVA, Tukey's Post Hoc test

Results: Our results showed that expression of FNDC5 was significantly reduced in granulosa cells of DHEA-induced PCOS mice compared with control and vehicle groups [p<0.05], while there was no significant differences in PGC1 Alpha expression among different groups

Conclusion: Down regulation of FNDC5 transcript level may contribute in metabol-ic disturbance of granulosa cells derived from PCOS ovary apart from PGC1 Alpha levels which remained unchanged

Study of Tnp1, Tekt1, and Plzf genes expression during an in vitro Three-Dimensional neonatal male mice testis culture

Background: In vitro spermatogenesis has a long research history beginning in the early 20th century. This organ culture method was therefore abandoned, and alternative cell culture methods were chosen by many researchers. Here, whether Tnp1, Tekt1, and Plzf, which play a crucial role in spermatogenesis, can be expressed during testis organ culture was assessed

Methods: Testes of 10 mouse pups were first removed, and the testis tissue was then separated into smaller pieces of seminiferous tubules. The size of the pieces was arbitrary; approximately 1 mg in weight or 1 mm3 in size when compacted. Afterwards, the testis tissue fragments [1-3] were transferred to the hexahedrons, incubated in a culture incubator and cultured for 12 weeks. Histological assessment and molecular evaluation were carried out at the end of the study

Results: The results showed that the expression of Tekt1 as a mitotic gene in mouse pups decreased significantly [p /= 0.05]. Based on histological study, different types of spermatocytes and postmeiotic stages of germ cells could not be detected

Conclusion: This kind of three-dimensional culture can induce expression of post-meiotic gene, Tnp1, but only at the molecular level and not beyond meiosis

Association of serum content of 25-hydroxy vitamin D with semen quality in normozoospermic and oligoasthenoteratozoospermic men

Background: vitamin D has multifaceted function in human reproductive physiology. It has been revealed that vitamin D is involved in spermatogenesis, and semen quality can be linked to vitamin D status in men

Objective: evaluating the correlation of 25-hydroxy vitamin D [25-OHD] levels in serum with basic and advanced semen parameters and essential determinants of spermatozoa function

Materials and Methods: participants were categorized, based on semen parameters, into normozoospermic [NS] and oligoasthenoteratozoospermic [OAT] men. Serum level of 25-OHD was measured. Apoptotic status of spermatozoa, mitochondrial membrane potential and reactive oxygen species content of semen were assessed

Results: difference of 25-OHD concentration in serum of NS men versus OAT ones did not meet significance threshold. DNA fragmentation, reactive oxygen species content of semen and mitochondrial membrane potential state revealed significant difference between NS and OAT subjects. There were no significant differences in basic and functional semen parameters when men were stratified based on serum 25-OHD level. Taking both 25-OHD and semen categories [NS and OAT] into consideration did not indicate any significant difference in studied parameters. Total motility of spermatozoa was positively correlated with serum concentration of 25-OHD in all studied subjects. In addition, normal morphology of spermatozoa in NS men revealed a positive and significant correlation with levels of 25-OHD in serum

Conclusion: vitamin D may affect motility and morphology of spermatozoa. Lower content of serum vitamin D may affect fertility of men and should be considered in examination of men with abnormal spermogram

Comparison of the effects of vitrification on gene expression of mature mouse oocytes using cryotop and open pulled straw

Background: Oocyte cryopreservation is an essential part of the assisted reproductive technology [ART], which was recently introduced into clinical practice. This study aimed to evaluate the effects of two vitrification systems-Cryotop and Open Pulled Straw [OPS]-on mature oocytes gene expressions

Materials and Methods: In this experimental study, the survival rate of metaphase II [MII] mouse oocytes were assessed after cryopreservation by vitrification via i. OPS or ii. Cryotop. Then we compared the fertilization rate of oocytes produced via these two methods. In the second experiment, we determined the effects of the two vitrification methods on the expression of Hspa1a, mn-Sod, and ß-actin genes in vitrified-warmed oocytes. Denuded MII oocytes were vitrified in two concentrations of vitrification solution [VS1 and VS2] by Cryotop and straw. We then compared the results using the two vitrification methods with fresh control oocytes

Results: mn-Sod expression increased in the vitrified-warmed group both in OPS and Cryotop compared with the con- trols. We only detected Hspa1a in VS1 and control groups using Cryotop. The survival rate of the oocytes was 91.2% [VS1] and 89.2% [VS2] in the Cryotop groups [P=0.902] and 85.5% [VS1] and 83.6% [VS2] in the OPS groups [P=0.905]. There were no significant differences between the Cryotop and the OPS groups [P=0.927]. The survival rate in the Cryotop or the OPS groups was, nevertheless, significantly lower than the control group [P<0.001]. The fertilization rates of the oocytes were 39% [VS1] and 34% [VS2] in the Cryotop groups [P=0.902] and 29 %[ VS1] and 19.7% [VS2] in the OPS groups [P=0.413]. The fertilization rates were achieved without significant differences among the Cryotop and OPS groups [P=0.755]

Conclusion: Our results indicated that Cryotop vitrification increases both cooling and warming rates, but both Cryo- top and OPS techniques have the same effect on the mouse oocytes after vitrification

Evaluation of intracellular anion superoxide level, heat shock protein A2 and protamine positive spermatozoa percentages in teratoasthenozoospermia

Background: Teratoasthenozoospermia [TA] is a severe form of male infertility with no clear etiology

Objective: To compare the level of intracellular anion superoxide [O[2]-], heat shock protein A2 [HSPA2] and protamine deficiencies in ejaculated spermatozoa between teratoasthenozoospermic and normozoospermic men

Materials and Methods: In this case- control study, semen samples of 20 infertile men, with TA [with normal morphology lower than 4%_ and total motility lower than 40% ] as the case group and 20 normozoospermic fertile men as the control group were evaluated for intracellular O[2] - and HSPA2 by flow cytometry and protamine deficiency by Chromomycin A3 [CMA3] test

Results: The rate of CMA3+ spermatozoa in the case group was higher than controls [p=0.001]. The percentages of HSPA2[+] spermatozoa in the cases were significantly lower than controls [p=0.001]. Also, intracellular O[2] - levels in the case group were significantly higher than controls [p=0.001] and had positive correlations with sperm apoptosis [r=0.79, p=0.01] and CMA3 positive sperm [r=0.76, p=0.01], but negative correlations with normal morphology [r=-0.81, p=0.01] and motility [r=-0.81, p=0.01]. There was no significant correlation between intracellular O[2] - and HSPA2 in the case group [r=0.041, p=0.79]

Conclusion: We suggest that the increase in intracellular O[2] -, decrease in spermatozoa HSPA2[+], and high percentages of spermatozoa with immature chromatin might be considered as etiologies of infertility in TA patients

Developmental potential of vitrified mouse testicular tissue after ectopic transplantation

Objective: Cryopreservation of immature testicular tissue should be considered as an important factor for fertility preservation in young boys with cancer. The objective of this study is to investigate whether immature testicular tissue of mice can be successfully cryopreserved using a simple vitrification procedure to maintain testicular cell viability, proliferation, and differentiation capacity

Materials and Methods: In this experimental study, immature mice testicular tissue fragments [0.5-1 mm[2]] were vitrified-warmed in order to assess the effect of vitrification on testicular tissue cell viability. Trypan blue staining was used to evaluate developmental capacity. Vitrified tissue [n=42] and fresh [control, n=42] were ectopically transplanted into the same strain of mature mice [n=14] with normal immunity. After 4 weeks, the graft recovery rate was determined. Hematoxylin and eosin [H and E] staining was used to evaluate germ cell differentiation, immunohistochemistry staining by proliferating cell nuclear antigen [PCNA] antibody, and terminal deoxynucleotidyl transferase [TdT] dUTP Nick-End Labeling [TUNEL] assay for proliferation and apoptosis frequency

Results: Vitrification did not affect the percentage of cell viability. Vascular anastomoses was seen at the graft site. The recovery rate of the vitrified graft did not significantly differ with the fresh graft. In the vitrified graft, germ cell differentiation developed up to the secondary spermatocyte, which was similar to fresh tissue. Proliferation and apoptosis in the vitrified tissue was comparable to the fresh graft

Conclusion: Vitrification resulted in a success rates similar to fresh tissue [control] in maintaining testicular cell viability and tissue function. These data provided further evidence that vitrification could be considered an alternative for cryopreservation of immature testicular tissue

Isolation and enrichment of mouse female germ line stem cells

The existence of female germ-line stem cells [FGSCs] has been the subject of a wide range of recent studies. Successful isolation and culture of FGSCs could facilitate studies on regenerative medicine and infertility treatments in the near future. Our aim in the present study was evaluation of the most commonly used techniques in enrichment of FGSCs and in establishment of the best procedure. In this experimental study, after digesting neonate ovary from C57Bl/6 mice, we performed 2 different isolation experiments: magnetic activated cell sorting [MACS] and pre-plating. MACS was applied using two different antibodies against mouse vasa homolog [MVH] and stage-specific embryonic antigen-1 [SSEA1] markers. After the cells were passaged and proliferated in vitro, colony-forming cells were characterized using reverse transcription-polymerase chain reaction [RT-PCR] [for analysis of expression of Oct4, Nanog, C-kit, Fragilis, Mvh, Dazl, Scp3 and Zp3], alkaline phosphatase [AP] activity test and immunocytochemistry. Data showed that colonies can be seen more frequently in pre-plating technique than that in MACS. Using the SSEA1 antibody with MACS, 1.98 +/- 0.49% [Mean +/- SDV] positive cells were yield as compared to the total cells sorted. The colonies formed after pre-plating expressed pluripotency and germ stem cell markers [Oct4, Nanog, C-kit, Fragilis, Mvh and Dazl] whereas did not express Zp3 and Scp3 at the mRNA level. Immunocytochemistry in these colonies further confirmed the presence of OCT4 and MVH proteins, and AP activity measured by AP-kit showed positive reaction. We established a simple and an efficient pre-plating technique to culture and to enrich FGSCs from neonatal mouse ovaries

Differentiation of human umbilical cord matrix-derived mesenchymal stem cells into germ-like cells

Mesenchymal Stem Cells [MSCs] are multipotent cells that can be collected from different sources. Under specific conditions, MSCs can be differentiated to tissue specific cells in vitro. Human Umbilical Cord Mesenchymal Stem Cells [hUCMSCs] can easily be harvested and cultured in in vitro conditions. Production of germ cells from mesenchymal stem cells is a very interesting and promising area in the field of reproductive medicine. In the present study, the possible trans-differentiation of hUCMSCs into Primordial like Germ Cell [PGC] was performed in vitro under specific condition. Human umbilical cord mesenchymal stem cells were cultured and expanded in DMEM medium containing 10% FBS. The cultured cells were studied for differentiation ability to adipocytes and osteocytes. Furthermore, MSCs related markers were identified by flow cytometry method. For PGC differentiation, hUCMS cells were cultured in differentiation medium containing Bone Morphogenetic Protein 4 [BMP4] and it was followed by retinoic acid [RA]. Real time PCR and immunocytochemistry analysis were performed to evaluate the expression of PGC specific genes and proteins, respectively. Our results showed that hUCMSCs cultured in the presence of BMP4 and RA are able to trans differentiate in to PGC like cells in vitro. Real time PCR and immunocytochemistry results showed that differentiated cells expressed PGC specific markers after 14 days of culture. Based on these results, it was concluded that hUCMSC may be considered as a promising alternative cell source in reproductive medicine. More studies including laboratory and also animal models are needed to evaluate the functionality of differentiated PGCs before introducing them to clinical applications

Comparison of differentiation potential of male mouse adipose tissue and bone marrow derived-mesenchymal stem cells into germ cells

Recent publications about differentiation of stem cells to germ cells have motivated researchers to make new approaches to infertility. In vitro production of germ cells improves understanding differentiation process of male and female germ cells. Due to the problem of using embryonic stem cells [ESC], it's necessary the mentioned cells be replaced with some adult multi-potent stem cells in laboratories. The aim of this study was to obtain germ cells from appropriate source beyond ESC and compare differential potentials of adipocytes derived stem cells [ADMSCs] with bone marrow derived stem cells [BMMSCs]. To find multi-potential entity, after providing purified ADMSCs and BMMSCs, differentiation to osteoblast and adipocyte was confirmed by using appropriate culture medium. To confirm mesenchymal lineage production superficial markers [expression of CD90 and CD44 and non-expression of CD45 and CD31] were investigated by flowcytometry. Then the cells were differentiated to germ cells in inductive medium containing retinoic acid for 7days. To evaluate germ cells characteristic markers [Dazl [Deleted in azoospermia-like], Mvh [Mouse vasa homolog gene], Stra8 [Stimulated by retinoic acid] and Scp3 [Synaptonemal complex protein 3]] flowcytometry, imunoflorescence and real time PCR were used. Both types of cells were able to differentiate into osteoblast and adipocyte cells and presentation of stem cell superficial markers [CD90, CD44] and absence of endothelial and blood cell markers [CD31, CD45] were confirmative The flowcytometry, imunoflorescence and real time PCR results showed remarkable expression of germ cells characteristic markers [Mvh, Dazl, Stra8, and Scp3]. It was found that although ADMSCs were attained easier and also cultured and differentiated rapidly, germ cell markers were expressed in BMMSCs significantly more than ADMSCs

Co-culture of spermatogonial stem cells with sertoli cells in the presence of testosterone and FSH improved differentiation via up-regulation of post meiotic genes

Spermatogonial stem cells [SSCs] maintain spermatogenesis throughout life in the male. Maintenance of SSCs and induction of spermiogenesis in vitro may provide a therapeutic strategy to treat male infertility. This study investigated in vitro differentiation of mouse SSCs in presence or absence of Sertoli cells, hormones and vitamins. Spermatogonial populations were enriched from testes of 4-6 week old males by magnetic activated cell sorting and anti-Thy-1 antibody. Sertoli cells isolated from 6-8 week old testes were enriched using lectin-DSA-coated plates. Isolated SSCs were cultured in the presence of Leukemia inhibitory factor [LIF] for 7 days in gelatin-coated dishes, then dissociated and cultured for 7 days in media lacking LIF in the presence or absence of Sertoli cells, with or without FSH, testosterone and vitamins. After one week, the effects of Sertoli cells +/- supplementary media on SSC differentiation was evaluated by microscopy and expression of meiotic and postmeiotic transcripts using RT-PCR. SSC colonies had limited development after LIF removal alone, exhibiting low expression of meiotic [Scp3, Th2b] but not postmeiotic transcript, and loss of Stra8 and Dazl expression. SSCs co-cultured with Sertoli cells, hormones and vitamins developed spermatid-like cells expressing postmeiotic markers [TP1, TP2, Prm1] at levels over 2-fold higher than Sertoli cells or hormone/vitamins alone. Our present SSC-Sertoli co-culture provides conditions that may allow efficient in vitro differentiation of SSCs for the treatment of male infertility

Effect of trolox addition to cryopreservation media on human sperm motility

Sperm parameters and motion kinetics are affected by cryopreservation. The main purpose of the current study was to determine the effect of different concentrations of Trolox as an antioxidant to freezing-thawing procedure on human sperm kinematic parameter. Semen was collected from 20 normal donors and divided into five aliquots prior to cryopreservation. The first aliquot was analyzed by computer-assisted sperm analysis [CASA]. Other aliquots were mixed with cryo-protective agent containing 0, 20, 40, and 80 micro mol Trolox and treated samples were cryopreserved in liquid nitrogen. After two weeks samples were thawed and sperm motion kinematics was measured by CASA. Percent motility [Mot], curvilinear velocity [VCL], straight-line velocity [VSL], average path velocity [VAP], linearity [LIN], and amplitude of lateral head displacement [ALH] were compared before and after freeze. Addition of 40 micro mol Trolox resulted in significantly higher [p<0.05] post thaw VCL, VSL and VAP compared to other groups. Therefore the percentage of post thaw motile spermatozoa were significantly higher [p<0.01]. The supplementation of Trolox significantly improved the post-thawed human semen quality, especially progressive motility and average path velocity

Functional concentrations of BMP4 on differentiation of mouse embryonic stem cells to primordial germ cells

Bone morphogenetic protein 4 [BMP4] has a significant role in primordial germ cells [PGCs] differentiation from mouse embryonic stem cell [mESC]. The aim of this study is to determine the best concentration of BMP4 at a time of two days on differentiation PGCs from mESC. To differentiate PGCs, embryoid bodies [EBs] from mESCs were cultured in concentrations of 0, 5 and 10 ng/ml BMP4 for two days. Germ cell markers Oct4 [Pou5f1], Stella [Dppa3] and Mvh [Ddx4] were analyzed by flow cytometry, immunocytochemistry and reverse transcriptase polymerase chain reaction [RT-PCR]. Flow cytometry data demonstrated most Mvh-positive cells were observed only in the treated groups. Immunocytochemistry of EBs in the treated groups identified cells positive for Mvh. PCR results showed expression of Oct4 in the control group and treated groups. Stella and Mvh were expressed only in the treated groups. Low concentrations of BMP4 during two days had an optimal effect on differentiation of PGCs from mESC

Human sperm aster formation and chromatin configuration in rabbit oocytes following intracytoplasmic sperm injection using a Piezo-Micromanipulator

In human fertilization, the sperm centrosome nucleates a radial array of microtubules called the sperm aster. The sperm aster is responsible for apposition of male and female pronuclei, and later gives rise to the first meiotic spindle. The objective of this study was to determine microtubule assembly and chromatin configuration in rabbit oocytes following intracytoplasmic injection with human sperm by piezo-driven pipette. Oocytes were collected from superovulated dose 14-15 h after hCG injection and were fertilized by injection of a single human sperm into the ooplasm of each oocyte without additional activation treatment. Four hours post heterologous intracytoplamic sperm injection [ICSI], rabbit eggs were fixed and microtubule organization and chromatin configuration were examined by immunofluorescence microscopy. In unfertilized oocytes, microtubules were present only in the metaphase-arrested second meiotic spindle. Following human sperm injection, an aster of microtubules formed adjacent to the sperm head, around mid-piece, and sperm aster was enlarged and assembled around male and female pronuclei. During pronuclear centration, male and female pronuclei were surrounded by a microtubule array without nucleation sites. With fertile human sperm, the sperm aster formation rate was 54.6%. From our data we concluded that human spermatozoa can be injected successfully into rabbit oocytes, resulting in a reasonable survival rate, and that rabbit oocytes provide a reliable tools for assessing human sperm centrosomal function using the Piezo-ICSI system