Induction of sublethal oxidative stress on human sperm before cryopreservation: a time-dependent response in post-thawed sperm parameters

Objective: A recent innovative approach, based on induction of sublethal oxidative stress to enhance sperm cryosurvival, has been applied before sperm cryopreservation. The purpose of this study was to investigate the effects of different induction times of sublethal oxidative stress before cryopreservation on human post-thawed sperm quality

Materials and Methods: In this experimental study, we selected semen samples [n=20] from normozoospermic men according to 2010 World Health Organization [WHO] guidelines. After processing the samples by the density gradient method, we divided each sample into 5 experimental groups: fresh, control freezing, and 3 groups exposed to 0.01M sodium nitroprusside [SNP] [nitric oxide [NO] donor] for 30 [T30], 60 [T60], or 90 minutes [T90] at 37C and 5% CO2 before cryopreservation. Motion characteristics [computer-assisted sperm analyser], viability, apoptosis [annexin V/propidium iodide [PI] assay], DNA fragmentation [sperm chromatin structure assay [SCSA]], and caspase 3 activity [FLICA Caspase Detection Kit] were assessed after thawing. The results were analysed by using one-way ANOVA and Tukey's test. The means were significantly different at P<0.05

Results: Cryopreservation significantly decreased sperm viability and motility parameters, and increased the percentage of apoptosis, caspase 3 activity, and DNA fragmentation [P<0.01] compared to the fresh group. The T60 group had a higher significant percentage of total motility [TM] and progressive motility compared with other cryopreserved groups [P<0.05]. We observed a significantly lower percentage of apoptotic rate and caspase 3 activity in the T60 group compared to the other cryopreserved groups [P<0.05]. DNA integrity was not significantly affected by this time of sublethal stress induction [P>0.05]

Conclusion: Our results have demonstrated that the application of sublethal oxidative stress by using 0.01 MuM NO for 60 minutes before the freezing process can be a beneficial approach to improve post-thawed human sperm quality

Lipid peroxidation and its role in the expression of NLRP1a and NLRP3 genes in testicular tissue of male rats: a model of spinal cord injury

Background: The majority of male patients with spinal cord injury [SCI] suffer from infertility. Nucleotide-binding oligomerization domain-like receptors NOD-like receptors [NLRs] are a kind of receptors that corporate in the inflammasome complex. Recent studies have introduced the inflammasome as the responsible agent for secreting cytokines in semen. Reactive oxygen species [ROS] is one of the elements that trigger inflammasome activation. Genital infections in SCI can lead to ROS generation. We investigated the relation between lipid peroxidation and inflammasome complex activity in testicular tissue of SCI rats

Methods: Adult male rats [n=20], weighting 200- 250 g, were included and divided into four groups: three experimental groups, including SCI1, SCI3, and SCI7, i.e. the rats were subjected to SCI procedure and sacrificed after one, three, and seven days, respectively and a control group. We performed a moderate, midline spinal contusion injury at thoracic level 10. The animals were anesthetized, and testes were collected for measurement of gene expression by real-time PCR. Caudal parts of epididymis were collected for malondialdehyde [MDA] measurement

Results: No NLRP1a mRNA overexpression was seen in the testes of control and SCI groups. After seven days from SCI surgery, NLRP3 mRNA expression was significantly increased in SCI7 animals [p

Conclusion: NLRP3 overexpression occurs due to the increased ROS production in testis tissue of SCI rats

Generation of mouse spermatogonial stem-cell-colonies in a non-adherent culture

Objective: The properties of self-renewal and division in spermatogonial stem cells [SSCs] support spermatogenesis. There is a number of reported methods for in vitro SSC culture systems. The development of a culture system that effectively supports isolation and self-renewal of germline stem cells [GSCs] is of tremendous benefit for clinical trials, experimental research, and as potential treatment for male infertility. The current study aims to consider the cultivation and behavior of GSCs in a non-adherent culture system

Materials and Methods: In this experimental study, we cultured testicular cells from neonatal mice in agarose coated plates in the presence of Dulbecco's modified Eagle's medium [DMEM] medium [CTRL group], 10% fetal bovine serum [FBS]+DMEM [10% group], and growth factor [G group] that contained 2% FBS, glial cell-derived neurotrophic factor [GDNF], epidermal growth factor [EGF], and fibroblast growth factor [FGF]. Mouse spermatogonial stem-like colonies were isolated approximately 3 weeks after digestion of the testis tissue. After passages 2-3, the identity of the mouse spermatogonial stem-like cells was confirmed by immunocytochemistry, reverse transcription-polymerase chain reaction [RT-PCR], and flow cytometry against the germ cell markers alpha 6, beta 1, c-Kit, Thy-1, c-Ret, Plzf, and Oct4. The statistical significance between mean values in different groups was determined by one-way analysis of variance [ANOVA]

Results: We observed spermatogonial stem-like colonies in the G and 10% groups, but not the CTRL group. Immunocytochemistry, flow cytometry, and RT-PCR confirmed expressions of germ cell markers in these cells. In the spermatogonial stem-like cells, we observed a significant expression [P<0.05] of germ cell markers in the G and 10% groups versus the testis cells [T]. Their proliferative and apoptotic activities were examined by Ki67 and PI/annexin V-FITC. Alkaline phosphatase assay showed that mouse spermatogonial stem-like colonies were partially positive

Conclusion: A non-adherent culture system could provide a favorable method for in vitro short-term culture of spermatogonial stem-like cell colonies

Isolation, culture and characterization of human sertoli cells by flow cytometry: development of procedure

Background: the sertoli cells in the testis create unique and safe environment to protect seminiferous tubules from auto antigens and invading pathogens. These cells produce the survival factor of the blood-testis barrier and produce special materials such as androgen binding proteins and contribute to the coordinated action of spermatogenesis. Given that the sertoli cells play an essential role in spermatogenesis and the lack of these cells leads to the disruption of spermatogenesis, it is necessary to investigate the behavior and performance of these cells. To achieve this goal, the cells must first be extracted. The aim of this study was to develop a procedure to isolate, culture, and characterize human sertoli cells

Methods: in order to isolate the sertoli cells of azoospermia patients who underwent [testicular sperm extraction] TESE surgery, washing up and multi_stage enzyme digestion of single cells, culture on petri dishes impregnated with datura stramonium lectin agglutinin [DSA] were done and then the cells were passaged for several times and isolated. For more purification, flow cytometry method with FSH receptor antibody was used. Immunocytochemistry assays and Elisa test for identification of these cells were employed

Results: the purification method resulted in more than 97% purity. The nature of sertoli cells was confirmed by morphology evaluation, detecting anti-mullerian hormone in sertoli cell culture media and the presence of FSH receptor on sertoli cells

Conclusion: this study introduced and applied a method to isolate, culture, and purify human sertoli cells with high purity which made possible further researches on these cells

expression of TLR2 and TLR3 in sertoli cells of azoospermic patients

Objective: Toll-like receptors [TLRs] on Sertoli cells are thought to have essential roles in sperm protection. This study was conducted to investigate the expression of TLR2 and TLR3 in Sertoli cells of men with azoospermia

Materials and Methods: In this experimental study, testicular biopsies were taken from ten azoospermic men. Following enzymatic dissociation, the samples were moved to lectin coated petri dishes. After a few passages, all cells were cultivated and Seroli cells were sorted by flow cytometry. To confirm Sertoli cell purification, alkaline phosphatase activity [ALP] and immunohistochemistry assays were employed. The expression of TLR2 and TLR3 at the transcript and protein levels was examined with real-time quantitative reverse transcription-polymerase chain reaction [RT-QPCR] and western blot, respectively

Results: Isolation, purification and cultivation of human Sertoli cells were performed successfully. Efficacy of purification of Sertoli cells by fluorescence-activated cell sorting [FACS] sorter was 97%. The type of cultured cells was confirmed by vimentin and follicle-stimulating hormone [FSH] receptor markers. Furthermore, the existence of anti- Mullerian hormone in culture was confirmed. RT-PCR showed that both genes were expressed in Sertoli cells. Consistently, proteins of both were also expressed in Sertoli cells. Moreover, QPCR showed that the relative expression of TLR3 transcripts was significantly higher than TLR2 in Sertoli cells. Although both genes are expressed in fibroblast cells, their level of expression was significantly lower than in Sertoli cells

Conclusion: This study confirmed expression of TLR2 and TLR3 in human Sertoli cells. This may be an indicator of their roles in developing immunity against pathogens as well as allo- and auto-antigens or viral antigens in seminiferous tubules

Comparison of allotransplantation of fresh and vitrified mouse ovaries to the testicular tissue under influence of the static magnetic field

Objective: The aim of this study was to investigate the effects of static magnetic field [SMF] during transplantation of the ovarian tissue into the testis

Materials and Methods: In this experimental study, ovaries of 6- to 8-week-old female Naval Medical Research Institute [NMRI] mice were randomly divided into four groups: i. Fresh ovaries were immediately transplanted into the testicular tissue [FOT group], ii. Fresh ovaries were exposed to the SMF for 10 minutes and then transplanted into the testicular tissue [FOT+group], iii. Vitrified-warmed ovaries were transplanted into the testicular tissue [VOT group], and iv. Vitrified-warmed ovaries were transplanted into the testicular tissue and the transplantation site was then exposed to the SMF for 10 minutes [VOT+group]

Results: The lowest percentages of morphologically dead primordial follicles and the highest percentages of morphologically intact primordial follicles were seen in the FOT+ group [4.11% +/- 2.88 and 41.26% +/- 0.54, respectively]. Although the lowest significant percentage of maturation, embryonic development and fertility was observed in the VOT group as compared to the other groups, the difference in the fertility rate was not significant between the VOT and VOT+groups. Estrogen and progesterone concentrations were significantly higher in the FOT+group than those of the control mice

Conclusion: It is concluded that, exposure of the vitrified-warmed ovaries to SMF retains the structure of the graft similar to that of fresh ovaries

Evaluation of PLC[zeta] and PAWP expression in globozoospermic individuals

Objective: globozoospermia is a rare type of teratozoospermia with incidence of 0.1% among infertile individuals. Phospholipase C zeta [PLC[zeta]] and postacrosomal sheath WW domain binding protein [PAWP] are the main candidates in sperm taking responsibility for oocyte activation during fertilization. Therefore, we aimed to evaluate the expression of these two genes at RNA and protein levels in globozoospermic individuals and compare the results with fertile individuals

Materials and Methods: in this experimental study, semen samples of 21 infertile men with globozoospermia and 25 fertile men were collected. Expression of PLC[zeta] and PAWP at RNA and protein levels were assessed and compared between two groups by quantitative real time polymerase chain reaction [qPCR] and Western blot, respectively

Results: expression of both PLC[zeta] and PAWP were significantly reduced at RNA and protein levels in infertile men with globozoospermia compared to fertile men

Conclusion: this is the first study that simultaneously assessing the respective factors in a large population of globozoospermia, suggested that intra-cytoplasmic sperm injection [ICSI] along with artificial oocyte activation may rescue failed fertilization in routine ICSI

Follicle development of xenotransplanted sheep ovarian tissue into male and female immunodeficient rats

This study aimed to assess follicle survival after xenotransplantation of sheep ovarian tissue into male and female immunodeficient rats. We evaluated the effects of gonadotropin treatment on follicular development in the transplanted tissue. In this experimental study, sheep ovarian cortical strips were transplanted into the neck back muscles of 8 male and 8 female immunodeficient, castrated rats. Fourteen days after surgery, each rat was treated with human menopausal gonadotropin [hMG] for 9 weeks. One day after the last injection, ovarian tissues were removed and fixed for histology assessment. Histology analyses were performed before and after grafting. Estradiol [E[2]] levels were measured before and after gonadectomy, and at the end of the experiment. The control group consisted of 7 male and 7 female non-castrated/non-grafted rats and the sham group comprised 7 male and 7 female castrated/ non-grafted rats for comparison of serum E2 concentrations. The percentage of primordial follicles decreased after transplantation in male [25.97%] and female [24.14%] rats compared to the control group [ovarian tissue non-grafted; 37.51%]. Preantral follicles increased in the male [19.5%] and female [19.49%] transplanted rats compared to the control group [11.4%]. Differences in antral follicles between male [0.06 +/- 0.0%] and female [0.06 +/- 0.0%] rats were not noticeable compared to control [1.25 +/- 0.0%] rats. We observed a significantly higher percent of mean E[2] secretion in grafted males compared to grafted females [P<0.05]. Despite significant differences in E[2] secretion between xenografted male and female rats, we observed no statistical differences in terms of follicular development

Transplantation of autologous bone marrow mesenchymal stem cells with platelet-rich plasma accelerate distraction osteogenesis in a canine model

Distraction osteogenesis [DO] is a surgical procedure used to generate large volumes of new bone for limb lengthening. In this animal experimental study, a 30% lengthening of the left tibia [mean distraction distance: 60.8 mm] was performed in ten adult male dogs by callus distraction after osteotomy and application of an Ilizarov fixator. Distraction was started on postoperative day seven with a distraction rate of 0.5 mm twice per day and carried out at a rate of 1.5 mm per day until the end of the study. Autologous bone marrow mesenchymal stem cells [BM-MSCs] and platelet-rich plasma [PRP] as the treatment group [n=5] or PRP alone [control group, n=5] were injected into the distracted callus at the middle and end of the distraction period. At the end of the consolidation period, the dogs were sacrificed after which computerized tomography [CT] and histomorphometric evaluations were performed. Radiographic evaluationsrevealed that the amount and quality of callus formations were significantly higher in the treatment group [P<0.05]. As measured by CT scan, the healing parametersin dogs of the treatment group were significantly greater [P<0.05]. New bone formation in the treatment group was significantly higher [P<0.05]. The present study showed that the transplantation of BM-MSCs positively affects early bony consolidation in DO. The use of MSCs might allow a shortened period of consolidation and therefore permit earlier device removal

Generation of rat embryonic germ cells via inhibition of TGFbeta and MEK pathways

Embryonic germ [EG] cells are the results of reprogramming primordial germ cells [PGC] in vitro. Studying these cells can be of benefit in determining the mechanism by which specialized cells acquire pluripotency. Therefore in the current study we have tried to derive rat EG cells with inhibition of transforming growth factor-beta [TGFbeta] and mitogen-activated protein kinase kinase [MEK] signaling pathways. In this experimental study, rat PGCs were cultured under feeder free condition with two small molecules that inhibited the above mentioned pathways. Under this condition only two-day presence of stem cell factor [SCF] as a survival factor was applied for PGC reprogramming. Pluripotency of the resultant EG cells were further confirmed by immunofluorescent staining, directed differentiation ability to neural and cardiac cells, and their contribution to teratoma formation as well. Moreover, chromosomal stability of two different EG cells were assessed through G-banding technique. Formerly, derivation of rat EG cells were observed solely in the presence of glycogen synthase kinase-3 [GSK3beta] and MEK pathway inhibitors. Due to some drawbacks of inhibiting GSK3beta molecules such as increases in chromosomal aberrations, in the present study we have attempted to assess a feeder-free protocol that derives EG cells by the simultaneous suppression of TGFbeta signaling and the MEK pathway. We have shown that rat EG cells could be generated in the presence of two inhibitors that suppressed the above mentioned pathways. Of note, inhibition of TGFbeta instead of GSK3beta significantly maintained chromosomal integrity. The resultant EG cells demonstrated the hallmarks of pluripotency in protein expression level and also showed in vivo and in vitro differentiation capacities. Rat EG cells with higher karyotype stability establish from PGCs by inhibiting TGFbeta and MEK signaling pathways

Dietary fish oil can change sperm parameters and fatty acid profiles of ram sperm during oil consumption period and after removal of oil source

The effects of dietary fish oil on semen quality and sperm fatty acid profiles during consumption of n-3 fatty acids as well as the persistency of fatty acids in ram's sperm after removing dietary oil from the diet were investigated. In this experimental study, we randomly assigned 9 Zandi rams to two groups [isoenergetic and isonitrogenous diets]: control [CTR; n=5] and fish oil [FO; n=4] for 70 days with a constant level of vitamin E in both groups. Semen was collected at the first week and at the last week of the feeding period [phase 1]. After the feeding period, all rams were fed a conventional diet and semen samples were collected one and two months after removal of FO [phase 2]. The sperm parameters and fatty acid profiles were measured by computer assisted semen analyzer [CASA] and gas chromatography [GC], respectively. The completely randomized design was used and data were analyzed with SPSS version 16. Dietary FO had significant positive effects on all sperm quality and quantity parameters compared with the CTR during the feeding period [p<0.05]. The positive effects of FO on sperm concentration and total sperm output were observed at one and two months after removal of FO [p<0.05], whereas other sperm parameters were unaffected. Before feeding, C14 [myristic acid], C16 [palmitic acid], C18 [stearic acid], C18:1 [oleic acid] and C22:6 [docosahexaenoic acid: DHA] were the primary sperm FA. FO in the diet increased sperm DHA, C14:0 and C18:0 during the feeding period [p<0.05]. The present study showed not only manipulation of ram sperm fatty acid profiles by dietary FO and sperm parameters during the feeding period, but also the persistency of unique effects of dietary omega-3 fatty acids up to two months following its removal from the diet. Also, we recommend that sperm fatty acid profiles should be comprehensively analyzed and monitored

Adverse effects of formaldehyde vapor on mouse sperm parameters and testicular tissue

Formaldehyde [FA], one of the simplest organic molecules, is a flammable, pungent, irritating and colorless gas. This study aimed to investigate the effects of various concentrations of FA vapor on sperm parameters and testicular tissue. In this experimental study, we randomly assigned 36 adult male mice to one control and two experimental groups [n=12 for each group]. The control group [C] did not receive FA. Group F1 [low concentration] was exposed to 10 ppm FA vapor and the F2 [high concentration] group was exposed to 20 ppm FA vapor. FA was administered for ten days, eight hours per day for both groups. At the end of the exposure period, half of the animals in each group were sacrificed 24 hours after exposure to detect any short-term effects; the rest of the mice were sacrificed 35 days later to assess for long-term effects. Sperm parameters were analyzed by Computer-assisted Sperm Analyzer [CASA] and histological changes determined. In addition, we studied changes in testosterone hormone. Data were analyzed by one-way ANOVA followed by the Scheffe test using SPSS software. Long-term effects of FA in the experimental groups included significant reductions in sperm cell numbers and sperm viability. A drastic reduction in progressive motility and increased abnormal sperm percentage [p<0.001] compared with the control group was also noted. Histological study of testes specimens in the experimental group revealed displacement of germinal cells, along with degeneration of Leydig cells and seminiferous tubules. Exposure to FA vapor can destroy testicular structure and decrease percentages of concentration, viability, normal morphology, and progressive motility, in addition to increasing the percentage of immotile sperm

In vitro maturation, fertilization and embryo culture of oocytes obtained from vitrified auto-transplanted mouse ovary

The purpose of this study was to investigate the in vitro survival and developmental potential of oocytes obtained from vitrified mouse ovaries transplanted to a heterotopic site. In this experimental study, two-week-old mice were unilaterally ovariectomized after anesthesia. The ovaries were vitrified by cryotop. After two weeks, the ovaries were thawed and autotransplanted to the gluteus muscle tissue. Three weeks later the mice were killed, after which we removed and dissected the transplanted and opposite right ovaries. Cumulus oocyte complexes [COCs] and denuded oocytes were evaluated for in vitro maturation [IVM], in vitro fertilization [IVF] and in vitro development [IVD]. The control group consisted of sevenweek- old age-matched mice ovaries. All vitrified-transplanted [Vit-trans] ovaries contained some oocytes that survived. Following IVM, IVF and IVD, there were 41.7% out of 12 cultured zygotes that reached the 8-cell stage. Our experiment supports the progressive role of long-term graft survival after wholeovarian cryopreservation by vitrification and subsequent heterotopic transplantation. It is possible to recover viable follicles and oocytes that have the ability to develop in vitro

Effect of different thawing rates on post-thaw viability, kinematic parameters and chromatin structure of buffalo [Bubalus bubalis] spermatozoa

The aim of the present study was to evaluate three thawing rates on the post thaw motility, viability and chromatin structure of buffalo semen frozen in 0.5-ml straws. In this experimental study semen was collected with artificial vagina [42[degree sign]C] from four buffalo bulls. Split pooled ejaculates [n=4] were extended at 37[degree sign]C with a Bioxcell[registered sign] extender. Semen was cooled to 4[degree sign]C within 2 hours, equilibrated at 4[degree sign]C for 4 hours, then filled in 0.5 ml French straws, and frozen in programmable cell freezer before plunging into liquid nitrogen. Straws were thawed at water bath temperatures of 37, 50 or 70[degree sign]C for 30, 15 and 6 seconds, respectively. Semen was incubated at 37[degree sign]C for 2 hours and evaluated for post thaw motility, viability, acrosomal and DNA integrity of spermatozoa. Analysis of variance [ANOVA] was used for comparisons of means. When the ANOVA test showed statistical differences, the mean of the treatments were compared using Duncan's multiple range tests. The initial postthaw motility [0 hour] averaged 62.7 +/- 7.2%, 73.1 +/- 9.77%, and 74.9 +/- 8.58% for the three thaw rates, respectively. Kinematic parameters such as average path velocity, linearity and beat/cross frequency in the thaw rate of 70?C for 6 seconds were superior to other rates studied [p<0.05]. After 2 hours of incubation, proportions of progressive motility and Kinematic parameters decreased in all groups [p>0.05]. A positive correlation was detected between sperm motility and thawing rate after two hours incubation times. The percentage of viable spermatozoa and spermatozoa with an intact acrosome and plasma membrane integrity were not different between the groups of samples thawed at different temperatures [p>0.05]. The percentage of spermatozoa with chromatin dispersion for the thaw rate of 70[degree sign]C for 6 seconds was significantly higher than for the to other rates studied [p< 0.05]. In contrast with motility and viability, the DNA integrity of post thaw spermatozoa remained unaffected during 2 hours incubation. The post thaw motility and kinematic parameters of buffalo spermatozoa were significantly improved immediately after thawing by increasing the thawing rate from 37[degree sign]C in 30 seconds to70[degree sign]C in 6 seconds. However, this relative advantage had disappeared after incubation in a water bath at 37[degree sign]C for two hours. A thaw rate of 70[degree sign]C for 6 seconds was associated with higher chromatin dispersion than the other thaw rates studied. Sperm thawing over at 50 degrees could be safely used to improve motility recovery after sperm cryopreservation in buffalo bulls

Enzymatic digestion improves testicular sperm retrieval in non-obstructive azoospermic patients

In patients with non-obstructive azoospermia [NOA], vital spermatozoa from the tissue is obtained from testes by enzymatic treatment besides the mechanical treatment. To increase the sperm recovery success of testicular sperm extraction [TESE], with enzymatic digestion if no sperm is obtained from testis tissue by mechanical method. Tissue samples were collected from 150 men who presented with clinical and laboratory data indicating NOA by means of TESE and micro dissection TESE methods. Initially, mature spermatozoa were examined for by mechanical extraction technique shredding the biopsy fractions. In cases whom no spermatozoa was observed after maximum 30 min of initial searching under the inverted microscope, the procedure was followed by enzymatic digestion using DNaseI and collagenase type IV. Surgery type, pathology, AZF, karyotype, hormones and testis size were compared in patients. Of 150 cases with NOA, conventional mincing method extended with enzymatic treatment yielded successful sperm recovery in 13 [about 9%] patients. Comparison of parameters revealed that level of FSH and LH were significantly different [p=0.04 and 0.08 respectively] between two groups that response negative and positive to enzymatic digestion. The combination of conventional TESE and enzymatic digestion is an effective method to recover spermatozoa. The benefit of the mincing combined with enzyme to sperm retrieval for NOA firstly shorten the mechanical searching time, leading to minimizing further cellular damage as well as exposure to external conditions, and secondly reduce the number of cases with sperm recovery failures. Also, the serum level of FSH and LH are factors that influence the chance of sperm retrieval

Fatty acid profiles of ram's sperm after removing some fatty acid sources from the diets and persistency of fatty acids in sperm

Background: Mammalian spermatozoa are characterized by a high proportion of polyunsaturated fatty acids [PUFAs], but reliable data concerning dietary effects on fatty acid [FA] profile in ram's sperm and the persistency of FA in the ration to the FA in sperm has not been reported. Therefore, the aim of this study was to determine the stability of saturated and unsaturated FAs in ram's sperm despite removing FA sources from their diet

Materials and Methods: Nine Kalkoohi rams were used in a completely randomized design and they were assigned to 3 groups. The treatments were diet supplemented [35 g/d/ram] by C16:0 [RP-10[registered]], C18: 2 [Sunflower oil; SO] and n-3 [Fish oil; FO] with Vitamin E. Fifteen weeks after the start of the supplemented diet, rams were offered a basal diet without any supplementary FA source for 35 days when the sperm's FA ratio was determined. The data were analyzed by ANOVA [Analysis of variance] using the General Linear Model [GLM] procedure of SAS Institute

Results: Thirty five days after removing the fat supplement from the diet, major FA in sperm consisted of: C14:0, C16:0, C18:0, C18:1 cis, C18:2 cis and C22:6 n-3 docosahexaenoic acid [DHA]. The percentage of C14:0 [p=0.8] and C18:1 cis [P=0.4] were similar among all the treatments. Interestingly, 35 days after the removal of fatty acid source, the percentage of C22:6 was highest in the FO treated group

Conclusion: The different sperm FA profile among various groups suggests that dietary FA had significant direct or indirect impacts on sperm FA profile after 35 days which might lead to physical and chemical changes in sperm characteristics

Comparison of neonatal and adult mice-derived sertoli cells in support of expansion of mouse spermatogonial stem cells in vitro

Background: This study compared neonatal and adult mice-derived Sertoli cells [NSCs and ASCs] to examine the influence of feeder cells derived from donors of different ages on the maintenance of mouse spermatogonial stem cells [SSCs] in vitro

Materials and Methods: SSCs were derived from the testes of six-day-old mice. They were subsequently transferred to Sertoli cells which were isolated by datura stramonium agglutinin [DSA] lectin from neonatal and adult mice for five days

Results: The numbers of spermatogonial colonies, the numbers of cells per colony, and cloning efficiency were assessed in presence of NSCs and ASCs. The expression of alpha 6-and beta 1-integrin-positive cells was evaluated. Moreover, the functionality of the cells was assessed by their transplantation into the testes of busulfan-induced infertile mice. Colony efficiency assay showed that the number of colonies derived from single spermatogonial cells were significantly higher on NSCs. Additionally, the transplantation of dissociated colonies into the testes of busulfan-induced infertile mice showed their migration to the seminiferous basal membrane

Conclusion: These results show that NSCs may provide a more favorable microenvironment in comparison with ASCs for in vitro culture of spermatogonial colonies

Effects of saffron [crocus sativus L.] aqueous extract on in vitro maturation, fertilization and embryo development of mouse oocytes

Lower pregnancy rates of in vitro matured oocytes compared to those of in vivo stimulated cycles indicate that optimization of in vitro maturation [IVM] remains a challenge. Reduced developmental competence of in vitro matured oocytes shows that current culture systems for oocyte maturation do not adequately support nuclear and/or cytoplasmic maturation. Therefore this study evaluates the effects of different concentrations of saffron [Crocus sativus L.] aqueous extract [SAE], as an antioxidant agent on IVM of immature mouse oocytes. In this experimental study ,cumulus-oocyte complexes [COCs] were collected from 6-8 weeks old novel medical research institute [NMRI] female mice ovaries. COCs were cultured in IVM medium supplemented with 0 [control], 5, 10, 20 and 40 micro g/ml of SAE in 5% CO[2] at 37[degree sign] C. The rates of maturation, fertilization and development were recorded. ANOVA and Duncan's protected least significant test, using the SAS program was applied for all statistical analysis. The maturation rate was significantly higher in all groups treated with different concentrations of SAE compared with the control group [p<0.05]. However, the lower concentrations of SAE [10 and 5 micro g/ml] in maturation medium respectively increased the fertilization rate of oocytes and in vitro developmental competence when compared with the control group [p<0.05]. The results of this study indicate that lower concentrations of SAE are more appropriate to be added to maturation medium when compared with other experimental and control groups. Generally, we conclude that addition of appropriate amounts of natural extracts such as SAE to maturation medium improves oocyte maturation and embryo development

Effect of ovarian cyclic status on in vitro embryo production in cattle

The relationship between cyclic status of cattle ovaries on in vitro embryo development up to the blastocyst stage was investigated. Cattle ovaries were collected immediately after slaughter and divided into three categories based on their cyclic status, which included: 1. the presence of a large follicle [LF], 2. the presence of a corpus luteum [CL] and 3. ovaries without LF or CL [WLCF]. Oocytes of these ovaries were obtained and used for in vitro maturation and fertilization. Presumptive zygotes were then cultured up to the blastocyst stage in synthetic oviductal fluid culture medium. There were no significant differences between cleavage rates of the three groups. The rate of embryos in the compact morula stage for the CL group was 48.2% which was significantly higher than the related rate of the LF group [36.6%], but non-significantly higher than that of the ST group [45.7%]. The highest blastocyst rate belonged to the CL group [54.6%] which was significantly greater than the WLCF group [32.9%] and non-significantly higher than the LF group [52.4%]. There was no significant difference in blastocyst rates in the CL and LF groups. Preselection of oocyte donor ovaries containing a CL or LF can be used as a feasible and noninvasive criterion to obtain the most competent oocytes capable of development to the blastocyst stage

[Mouse and human spermatogonial stem cells]

Spermatogonial stem cells [SSCs] are in the beginning of a complex process in which they transmit genetic information from generation to generation. Any failure in this process can result in infertility. It has been suggested that transplantation of spermatogonial stem cells, following their maintenance and culturing, may restore fertility in some infertile patients. Because fertility restoration through SSCs transplantation has been successfully achieved in animal experiments, we hope human studies can follow in the near future. The isolation and cultivation of SSCs help us study their biological characteristics and their application in therapeutic approaches. In this review, we studied spermatogenesis in rodents and humans. We also compared markers and different SSC culture systems in both