Male infertility is responsible for 50% of men’s health problems and has always been a concern for personal and social issues. A survey of global statistics suggests an increase in infertility rate as one of the critical issues documented in studies. There are different ways of maintaining fertility in men, depending on their age. In this paper, we review the preservation methods used for fertility treatment in Iran and other countries. Available data were reviewed from Google Scholar, PubMed, Scopus, Web of Science, IranMedex, MEDLIB, IranDoc and Scientific Information Database and searched for articles published up to 2018, using the medical subject heading (MeSH) terms for cryopreservation, sperm, testicular, spermatogonia stem cell, male infertility and/or Iranian and in the world, to provide evidence from evaluation of fertility preservation the methods. Based the search strategy, 274 manuscripts were found. After reviewing the titles, abstracts and manuscripts in their entirety, 119 articles were obtained and selected according to the eligibility criteria. The 85 studies mentioned above were divided into three categories (sperm, testis, and spermatogonia stem cells (SSCs)), and methods of fertility preservation were investigated. Ways to maintain male fertility were different depending on age, and included sperm, testicular, and SSC freezing. The number of studies on testicular tissue and SSCs was low for human samples, and more studies are still needed. Sperm freezing at infertility centres is the top for male fertility preservation.

This study was conducted to examine whether the nuclear to cytoplasmic (N/C) ratio had any influence on the timing of embryo compaction and blastocoel formation, as well as formation rate and quality of blastocyst. First, we produced embryos with increased N/C ratio by removal of approximately one-third of the cytoplasm and with decreased N/C ratio by doubling the oocyte cytoplasm with an enucleated oocyte. The initiation of compaction and cavitation in reduced cytoplasm group was significantly earlier (P < 0.05) compared with the control and doubled cytoplasm groups. The rate of blastocysts in the reduced cytoplasm and doubled cytoplasm groups was significantly lower (P < 0.05) compared with the control group. Blastocyst quality in terms of total cell number in the reduced cytoplasm group was significantly lower (P < 0.05) compared with the doubled cytoplasm group, but not different from the control group. Next, we produced embryos with various N/C ratios by oocyte fusion combined with cytochalasin D treatment. The onset of compaction and cavitation in the 2N/2C group (decreased N/C ratio) was significantly delayed (P < 0.05) or had the tendency to be delayed (P = 0.064), respectively, compared with the control group (2N/1C). A significantly higher rate of blastocyst was observed in the 4N/2C group compared with the 1N/1C group (P < 0.05) but not different from the remaining groups. These results demonstrated that an increase in N/C ratio caused an earlier occurrence of morula compaction and blastocyst formation in both in vitro fertilization (IVF) and parthenogenetically activated pig embryos.

The purpose of this study was to investigate the effect of RFRP-3 synchronized with photoperiods on regulating the seasonal reproduction of striped hamsters. The striped hamsters were raised separately under long-day (LD; 16 h light/8 h dark), medium-day (MD; 12 h light/12 h dark) or short-day (SD; 8 h light/16 h dark) conditions for 8 weeks. RFRP-3 and gonadotropin-releasing hormone (GnRH) mRNA levels in the hypothalamus, testis or ovaries in three groups were detected using reverse transcription polymerase chain reaction (RT-PCR). Melatonin (MLT), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) concentrations in serum were detected using enzyme-linked immunosorbent assay (ELISA). The correlation between RFRP-3 and GnRH mRNA and FSH and LH concentrations was also analyzed. MLT negatively regulated the expression of RFRP-3. Significant differences for RFRP-3 mRNA existed in the three groups, which positively correlated with the GnRH and the FSH and LH concentrations. RFRP-3 mRNA levels in the hypothalamus were significantly higher than those in ovaries or testis. RFRP-3 levels in the hypothalamus were significantly lower in female than in male under SD conditions, while those in ovaries were significantly higher than those in testes under LD conditions. MLT decreased RFRP neuron activity, and RFRP-3 regulated the reproduction of striped hamsters.

The quality and quantity of a spermatogonial stem-cell (SSC) culture can be measured in less time using a 3D culture in a scaffold. The present study investigated stemness gene expression and the morphological and structural characterization of SSCs encapsulated in alginate. SSCs were harvested from BALB/c neonatal mice testes through two-step mechanical and enzymatic digestion. The spermatogonial populations were separated using magnetic-activated cell sorting (MACS) using an anti-Thy1 antibody and c-Kit. The SSCs then were encapsulated in alginate hydrogel. After 2 months of SSC culturing, the alginate microbeads were extracted and stained to evaluate their histological properties. Real-time polymerase chain reaction (PCR) was performed to determine the stemness gene expression. Scanning electron microscopy (SEM) was performed to evaluate the SSC morphology, density and scaffold structure. The results showed that encapsulated SSCs had decreased expression of Oct4, Sox2 and Nanos2 genes, but the expression of Nanog, Bcl6b and Plzf genes was not significantly altered. Histological examination showed that SSCs with pale nuclei and numerous nucleolus formed colonies. SEM evaluation revealed that the alginate scaffold structure preserved the SSC morphology and density for more than 60 days. Cultivation of SSCs on alginate hydrogel can affect Oct4, Sox2 and Nanos2 expression.

To determine the fertilization and embryonic potential of immature metaphase I (MI) oocytes in patients with low oocyte maturity rate in whom the percentage of mature oocytes obtained was less than 75% of the total retrieved ones. In vivo matured metaphase II (MII) oocytes (MII-ICSI, n = 244), and in vitro matured MI oocytes (MI-MII-ICSI, n = 202) underwent an intracytoplasmic sperm injection (ICSI) procedure. Maturation rate, fertilization rate and early embryonic development were compared in both groups. In total, 683 oocytes were collected from 117 ICSI cycles of 117 patients. Among them, 244 (35.7%) were mature MII and 259 (37.9%) were MI after the denudation process. Of those 259 MI oocytes, 202 (77.9%) progressed to MII oocytes after an incubation period of 18–24 h. The maturation rate was 77.9%. Fertilization rate was found to be significantly higher in the rescued in vitro matured MI oocyte group when compared with the in vivo matured MII oocyte group (41.6% vs 25.8%; P = 0.0006). However, no significant difference was observed in terms of cleavage rates on days 2 and 3 between the groups (P = 0.9126 and P = 0.5031, respectively). There may be unidentified in vivo factors on the oocyte maturation causing low developmental capacity in spite of high fertilization rates in the group of patients with low oocyte maturity rate. Furthermore, studies are needed to determine the appropriate culture characteristics as well as culture period and ICSI timing of these oocytes.

The efficiency of establishing pig pluripotent embryonic stem cell clones from blastocysts is still low. The transcription factor Nanog plays an important role in maintaining the pluripotency of mouse and human embryonic stem cells. Adequate activation of Nanog has been reported to increase the efficiency of establishing mouse embryonic stem cells from 3.5 day embryos. In mouse, Nanog starts to be strongly expressed as early as the morula stage, whereas in porcine NANOG starts to be strongly expressed by the late blastocyst stage. Therefore, here we investigated both the effect of expressing NANOG on porcine embryos early from the morula stage and the efficiency of porcine pluripotent embryonic stem cell clone formation. Compared with intact porcine embryos, NANOG overexpression induced a lower blastocyst rate, and did not show any advantages for embryo development and pluripotent embryonic stem cell line formation. These results indicated that, although NANOG is important pluripotent factor, NANOG overexpression is unnecessary for the initial formation of porcine pluripotent embryonic stem cell clones in vitro.

Many studies have shown that oestrogen affects late follicular development, but whether oestrogen is involved in other aspects of folliculogenesis remains unclear. In this study, two antagonists of oestrogen, tamoxifen and G15, were used to determine the effects of oestrogen on folliculogenesis. Mouse preantral follicles and cumulus–oocyte complexes (COCs) were cultured in vitro. The results showed that follicle growth stimulated using pregnant mare serum gonadotrophin (PMSG) was inhibited using tamoxifen, whether in vivo or in vitro. The average diameters, the maximum diameters of follicles and the numbers of follicles with a diameter of more than 300 μm decreased significantly following a 4-day culture with tamoxifen. G15, the antagonist of oestrogen via the membrane receptor, did not change follicular growth stimulated by PMSG in vitro. Results of in vitro maturation of COCs showed that germinal vesicle breakdown (GVBD) occurred spontaneously (95.1%) after 2 h in culture, and the GVBD ratio changed little with the addition of either oestrogen or 10 μM G15. However, first polar body (PBI) extrusion was driven by oestrogen markedly and supplementation with 10 μM G15 inhibited PBI extrusion (82.4% vs 55.0%) significantly. These results demonstrated that oestrogen promotes follicle growth through the nuclear receptor during follicle growth and then triggers the transition of metaphase to anaphase through the membrane receptor during meiotic resumption. So oestrogen plays a progressive role in the two phases of follicle growth and oocyte meiotic resumption.

Many studies have focused on the optimization of the composition of embryo culture medium; however, there are few studies involving the effect of a culture medium changing procedure on the preimplantation development of embryos. In this study, three groups were designed: a non-renewal group, a renewal group and a half-renewal group. The levels of reactive oxygen species (ROS), apoptotic index, blastocyst ratio and blastocyst total cell number were analyzed in each group. The results showed that the ROS level and the apoptotic index of blastocyst in the non-renewal group were significantly higher than in the renewal group and the half-renewal group (P < 0.05). The blastocyst ratio and blastocyst total cell number were significantly higher in the half-renewal group than that in non-renewal group and the renewal group (P < 0.05). These results demonstrated that the procedure of changing the culture medium influenced ROS level, apoptotic index, blastocyst ratio and total cell number of blastocysts. In addition, the result suggested that changing the culture medium may lead to a loss of important regulatory factors for embryos, while not changing the culture medium may lead to the accumulation of toxic substances. Half-renewal can alleviate the defects of both no renewal and renewal, and benefit embryo development. This study will be of high value as a reference for the optimization of embryo culture in vitro, and is very significant for assisted reproduction.

An in vitro spermatogonial stem cell (SSC) culture can serve as an effective technique to study spermatogenesis and treatment for male infertility. In this research, we compared the effect of a three-dimensional alginate hydrogel with Sertoli cells in a 3D culture and co-cultured Sertoli cells. After harvest of SSCs from neonatal mice testes, the SSCs were divided into two groups: SSCs on a 3D alginate hydrogel with Sertoli cells and a co-culture of SSCs with Sertoli cells for 1 month. The samples were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays and bromodeoxyuridine (BrdU) tracing, haematoxylin and eosin (H&E) and periodic acid–Schiff (PAS) staining after transplantation into an azoospermic testis mouse. The 3D group showed rapid cell proliferation and numerous colonies compared with the co-culture group. Molecular assessment showed significantly increased integrin alpha-6, integrin beta-1, Nanog, Plzf, Thy-1, Oct4 and Bcl2 expression levels in the 3D group and decreased expression levels of P53, Fas, and Bax. BrdU tracing, and H&E and PAS staining results indicated that the hydrogel alginate improved spermatogenesis after transplantation in vivo. This finding suggested that cultivation of SSCs on alginate hydrogel with Sertoli cells in a 3D culture can lead to efficient proliferation and maintenance of SSC stemness and enhance the efficiency of SSC transplantation.

Polycystic ovary syndrome (PCOS) is a complex disorder and genetic factors are believed to play a role. The main aim was to investigate expression levels of genes involved in PI3K/AKT signalling pathway pretreatment and post-treatment. Mouse models of PCOS were generated. Group one included control mice with no polycystic ovaries (n = 4), Group 2 included a PCOS mouse model (n = 8), Group 3 included PCOS mice treated with clomiphene citrate (n = 7) and Group 4 included PCOS mice treated with clomiphene citrate, metformin and pioglitazone (n = 8). Histochemical analyses were performed. Total RNA was extracted and cDNA was synthesized. Irs, Akt1 and Akt2, mTor and Pdpk1 gene expression levels were evaluated by RT-PCR amplification. In Group 1, cortex and medulla were evaluated as normal; in Group 2, ovarian cortex was composed of immature oocytes and cystic follicles with atretic follicles. In Groups 3 and 4, follicles were in the process of normal follicle differentiation. The expression levels of Akt1 and Pi3k were significantly different (P < 0.0001) between Groups 1 and 2. The significant differences in expression levels of Pi3k and Akt1 were also observed between the Group 1 and both Groups 3 and 4 (P < 0.0001). Furthermore, significant variations of the expression levels of mTor between Groups 1 and 4 were observed. The extrapolation of results of this study may imply that follicular development may be regulated by molecular pathways involving Pi3k, Akt1 and mTor expression. Therefore, genes in the PI3K/AKT pathway may have a direct regulatory role in the development of PCOS.

Methomyl is a broad-spectrum carbamate insecticide that has a variety of toxic effects on humans and animals. However, there have been no studies on the toxicity of methomyl in female mammalian oocytes. This study investigated the toxic effects of environmental oestrogen methomyl exposure on mouse oocyte maturation and its possible mechanisms. Our results indicated that methomyl exposure inhibited polar body extrusion in mouse oocytes. Compared with that in the control group, in the methomyl treatment group, superoxide anion free radicals in oocytes were significantly increased. In addition, the mitochondrial membrane potential of metaphase II stage oocytes in the methomyl treatment group was significantly decreased, resulting in reduced mouse oocyte quality. After 8.5 h of exposure to methomyl, metaphase I stage mouse oocytes displayed an abnormal spindle morphology. mRNA expression of the pro-apoptotic genes Bax and Caspase-3 in methomyl-treated oocytes increased, which confirmed the apoptosis. Collectively, our results indicated that mouse oocyte maturation is defective after methomyl treatment at least through disruption of spindle morphology, mitochondrial function and by induction of oxidative stress.

In vitro culture of ovarian tissue containing primordial follicles is an important tool to study the initiation of follicular populations and to develop efficient culture systems to support in vitro follicle growth. Considering that in vitro culture favours oxidative stress, it is very important to supplement culture medium with antioxidant substances such as Aloe vera extract. This study aims to evaluate the effects of different concentrations of Aloe vera on the distribution of collagen fibres in the extracellular matrix, follicular activation, development and survival in bovine ovarian cortical tissues cultured in vitro, as well as on expression of mRNAs for antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), peroxiredoxin 6 (PRDX6) and glutathione peroxidase 1 (GPX1)]. To this end, ovarian cortical tissues were cultured for 6 days in α-MEM alone or supplemented with different concentrations of Aloe vera extract (1.0, 5.0, 10.0 or 50.0%). After culture, fragments were fixed and processed histologically to evaluate follicular morphology and activation, as well as the extracellular matrix by staining with picrosirius red. The levels of mRNA for SOD, CAT, PRDX6 and GPX1 in cultured ovarian tissues were evaluated by real-time polymerase chain reaction (PCR). Ovarian tissues cultured with 10.0 or 50.0% Aloe vera had higher percentages of collagen fibres than tissues cultured in control medium. A significant increase in developing follicles was observed in ovarian tissues cultured in α-MEM alone or supplemented with 10% Aloe vera when compared with fresh control or tissues cultured with 1.0% Aloe vera. Presence of Aloe vera did not influence the percentage of morphologically normal follicles when compared with control medium. Ovarian tissues cultured with 50.0% Aloe vera had higher percentages of morphologically normal follicles than those cultured with 10.0% Aloe vera. Furthermore, 10% Aloe vera significantly increased mRNA levels for PRDX6. In conclusion, 10.0% Aloe vera improves extracellular matrix distribution in cultured tissues and increases the expression of mRNA for PRDX6 after 6 days in vitro.

It has been documented that the inefficacy of round spermatid injection (ROSI) might be caused by abnormal epigenetic modifications. Therefore, this study aimed to evaluate the effect of trichostatin A (TSA) as an epigenetic modifier of preimplantation embryo development in activated ROSI oocytes. Matured oocytes were collected from superovulated female mice. Testes were placed in human tubal fluid medium and masses were then cut into small pieces to disperse spermatogenic cells. Round spermatids were treated with TSA and subsequently injected into oocytes. The expression level of the development-related genes including Oct4, Sox2, Nanog, Dnmt and Hdac transcripts were evaluated using qRT-PCR. Immunohistochemistry was performed to confirm the presence of Oct-4 protein at the blastocyst stage. There was no statistically significant difference in fertilization rate following ROSI/+TSA compared with the non-treated ROSI and intracytoplasmic sperm injection (ICSI) groups. Importantly, TSA treatment increased blastocyst formation from 38% in non-treated ROSI to 68%. The relative expression level of developmentally related genes increased and Dnmt transcripts decreased in ROSI/+TSA-derived embryos, similar to the expression levels observed in the ICSI-derived embryos. In conclusion, our results indicate that spermatid treatment with TSA prior to ROSI would increase the success rate of development to the blastocyst stage and proportion of pluripotent cells.

GPR120 is implicated in the regulation of glucose and lipid metabolism, and insulin resistance. In the current study, we aimed to investigate the role of GPR120 in polycystic ovary syndrome (PCOS). With the adoption of dehydroepiandrosterone, a rat model was established to simulate PCOS in vitro. mRNA and protein expression levels of GPR120 were measured using RT-qPCR and western blot, respectively. In addition, expression levels of testosterone, estradiol, luteinizing hormone and follicle-stimulating hormone, serum total cholesterol and triglyceride were assessed using the corresponding kits. Moreover, haematoxylin and eosin staining was used to detect pathological changes in ovary or liver and oil red staining was utilized to evaluate lipid accumulation. In the present study, GPR120 was downregulated in plasma, liver and ovary in the PCOS rat model. In addition, the GPR120 agonist regulated lipid metabolism in the liver and weight in the PCOS rat model. Furthermore, the GPR120 agonist decreased insulin resistance in the PCOS rat model but improved the ovarian function. It is suggested that GPR120 plays a vital role in suppressing insulin resistance, regulating ovary function and decreasing lipid accumulation in the liver, demonstrating that targeting GPR120 could be an effective method for the improvement of PCOS.

The objective of the current study was to estimate the genetic parameters for ewe productivity traits of Harnali sheep by examining non-genetic effects. The data records of 440 animals born to 85 sires and 259 dams were collected with respect to various traits such as litter size at birth (LSB), litter weight at birth (LWB), litter size at weaning (LSW), litter weight at weaning (LWW) and age at first lambing (AFL) for the period of 2001 to 2020. Genetic parameters were estimated by fitting a series of animal models using an average information restricted maximum likelihood (REML) algorithm in WOMBAT software. Least-squares analysis revealed significant (P < 0.05) influences of period of lambing, age and weight of ewe at lambing on the studied traits. These results indicated that heavier ewes had significantly higher (P < 0.05) values of litter weight traits than their counterparts. On the basis of likelihood ratio test, the estimates of direct heritability under best model for AFL, LSB, LWB, LSW and LWW were 0.06, 0.18, 0.09, 0.07 and 0.16, respectively. Maternal permanent environment effect made a significant contribution to the LSB trait (0.20). The genetic correlation between litter size and LWW was negative, while the remaining correlations were positive. The present results suggest that selection based on ewe productivity traits will result in low genetic progress and therefore the management role is more important for better gains.

The aim of this study was to evaluate the follicular development, morphological integrity, and oxidative stress of preantral ovarian follicles from Bos taurus indicus females grown in vitro with ascorbic acid. Ovaries (n = 20) from Bos taurus indicus females were collected, fragmented, and were cultured in vitro for 6 or 12 days in minimum essential medium (MEM), or MEM supplemented with 50 or 100 ng/ml ascorbic acid, with an extracellular matrix of agarose gel, in an incubator at 38.5°C; every 2 days, 100% of the culture medium was replaced. The data were analyzed using the chi-squared test and/or Fisher’s exact test. In the event of a significant effect, the proportions were compared using a 2 × 2 proportion test. The oxidative stress analysis data were submitted to analysis of variance followed by the Bonferroni test. Values were considered significant when P ≤ 0.05. The addition of 100 ng/ml of ascorbic acid to the in vitro culture medium of preantral ovarian follicles from bovine females promoted follicular development, was efficient in maintaining morphological integrity, as well as the stability of reactive oxygen species, after 6 days of in vitro culture.

Fertilization, a crucial event for species preservation, in sea urchins, as in many other organisms, requires sperm motility regulation. In Strongylocentrotus purpuratus sea urchins, speract, a sperm chemoattractant component released to seawater from the outer egg layer, attracts sperm after binding to its receptor in the sperm flagellum. Previous experiments performed in demembranated sperm indicated that motility regulation in these cells involved protein phosphorylation mainly due to the cAMP-dependent protein kinase (PKA). However, little information is known about the involvement of protein kinase C (PKC) in this process. In this work, using intact S. purpuratus sea urchin sperm, we show that: (i) the levels of both phosphorylated PKA (PKA substrates) and PKC (PKC substrates) substrates change between immotile, motile and speract-stimulated sperm, and (ii) the non-competitive PKA (H89) and PKC (chelerythrine) inhibitors diminish the circular velocity of sperm and alter the phosphorylation levels of PKA substrates and PKC substrates, while the competitive inhibitors Rp-cAMP and bisindolylmaleimide (BIM) do not. Altogether, our results show that both PKA and PKC participate in sperm motility regulation through a crosstalk in the signalling pathway. These results contribute to a better understanding of the mechanisms that govern motility in sea urchin sperm.

To investigate the effects of culture media with different lactate concentrations on early embryonic development, data collected from our patients undergoing preimplantation genetic testing (PGT) were assessed using the EmbryoScope™ time-lapse culturing system. After intracytoplasmic sperm injection (ICSI), sibling oocytes were cultured in the same EmbryoScope (Vitrolife) slides including two different commercially available media. The patients with fewer than five mature oocytes were not included in the analyses. All embryos were hatched on day 3, and trophectoderm biopsies (n = 212) were performed accordingly. PGT for aneuploidy (PGT-A) on biopsied materials was carried out using next generation sequencing. Morphokinetic parameters, fertilization, irregular division, degeneration, blastulation, euploidy, and pregnancy rates of embryos cultured in LifeGlobal Global Total medium (LGGT) and Continuous Single Culture-NX Complete medium (CSCM-NXC) were compared. There were no differences observed in time to pronuclear fade, or in time spent as 2-cell (cc2) and 3-cell (s2), to 4-cell, 5-cell, morula and blastocyst stages (P > 0.05). Embryos reached the 2-cell (t2) and 3-cell (t3) stages significantly faster in LGGT (P < 0.05), whereas embryos grown in CSCM-NXC with lower lactate reached starting blastulation significantly sooner (P = 0.026). However, there were no statistical differences observed in fertilization, blastulation, degeneration, irregular division euploidy, and pregnancy rates between the two groups (P > 0.05). Even though pregnancy and fertilization rates did not indicate statistical differences, results are significant to provide better insight on potential roles of lactate in embryo development. These finding will advance the fundamental knowledge of human embryo development and assisted reproductive technologies.

The cryopreservation of murine ovarian tissue and its transplantation can be a promising technique for the preservation of fertility and an alternative for the future reconstitution of scientific valuable strains of mice. Accordingly, the aim of this study was to describe the entire surgical procedure for ovariectomy and dorsal subcutaneous autotransplantation in mice, and also some data about the efficiency of this procedure. Female C57Bl/6J mice (n = 18) were anaesthetised and bilaterally ovariectomized. After surgery, ovaries were autotransplanted in small subcutaneous pouches in the dorsal region of the forelimbs. The animals were inspected daily and, 23 days after transplantation, euthanasia and recovery of ovarian tissues were performed. Postoperative recovery, oestrous cyclicity, and folliculogenesis progression were evaluated. At 23 days after transplantation, the recovery of the ovaries was feasible, all classes (primordial to antral) of follicles were observed. Additionally, satisfactory efficiency rates were obtained, with 100% of anaesthesia survival rate, survival, graft recovery, folliculogenesis progression and oestrous cyclicity. In general, this short article describes ovarian ectopic autologous transplantation as an effective technique for maintaining rodent oogenesis and endocrine ovarian function. Even more broadly, we can still assume that the application of this technique may reduce the number of breeding matrices and experimental animals in the near future.

The aim of this study was to evaluate the effect of 1 µmol/l zearalenone (ZEN) and 1 µmol/l matairesinol (MAT), alone or in combination, on the morphology of in vitro-cultured ovarian preantral follicles. Ovaries from four adult sheep were collected at a local slaughterhouse and fragmented, and the ovarian pieces were submitted to in vitro culture for 3 days in the presence or absence of the test compounds. The morphology of primordial and primary follicles was impaired by ZEN. The plant lignan MAT alone did not maintain the morphology of the ovarian follicles; its combination with ZEN counteracted the negative effects observed when follicles were cultured in the presence of the mycotoxin alone. However, MAT was not able to promote the in vitro development of the ovarian follicles.