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In vitro maturation in the presence of Leukemia Inhibitory Factor modulates gene and miRNA expression in bovine oocytes and embryos. Vendrell-Flotats M et al. (2020) Members of the interleukin-6 (IL-6) family of cytokines are important for reproductive function that are mediated through changes in gene and miRNA expression. Herein, we characterized the expression of miR-21, miR-155, miR-34c and miR-146a in bovine oocytes and cumulus cells during in vitro maturation (IVM) with leukemia inhibitory factor (LIF), IL-6 and IL-11 or unsupplemented controls. LIF-exposed COCs showed higher expression of miR-21 and miR-155 in oocytes, whereas miR-146a expression was increased in oocytes matured with IL-6 and IL-11. In cumulus cells, miR-155 expression was elevated by all treatments while only LIF increased miR-21 expression. Based on these results, we next examined how LIF exposure during IVM affected oocyte competence, through IVF and the expression of specific genes in GV- and MII-oocytes, in 2- and 8-cell embryos, and in Day 8-blastocysts. LIF supplementation did not affect cleavage rate, blastocyst yield or several other developmental parameters, but did increase hatching rate. LIF suppressed DPPA3, ZAR1 and NPM2 expression in 2 cell- and/or 8-cell embryos. LIF increased the expression of KAT2A and HSPA1A in MII-oocytes, and that of HDAC1, KAT2A and HSP90AA1 and the BAX:BCL2L1 ratio in 2-cell embryos. In contrast, HDAC1, KAT2A and HSP90AA1 expression and BAX:BCL2L1 ratio was lower in 8-cell embryos derived from LIF oocytes. IVM with LIF also increased the expression of DNMT3A, HSPA1A and HSP90AA1 in blastocysts. In conclusion, supplementation with LIF during IVM was consistently associated with changes in the relative abundance of transcripts in mature bovine oocytes and in specific embryo developmental stages.////////////////// In Vitro Maturation with Leukemia Inhibitory Factor Prior to the Vitrification of Bovine Oocytes Improves Their Embryo Developmental Potential and Gene Expression in Oocytes and Embryos. Vendrell-Flotats M et al. (2020) Oocyte cryopreservation has a significant impact on subsequent embryonic development. Herein, we investigated whether supplementing in vitro maturation medium with Leukemia Inhibitory Factor (LIF) prior to vitrification affects embryo development and gene expression at different embryo developmental stages. A panel of genes including maternal effect, epigenetics, apoptosis and heat stress was relatively quantified. The results show reduced cleavage rates after vitrification, regardless of the LIF treatment. Although not statistically different from control-vitrified oocytes, oocyte apoptosis and the blastocyst yield of LIF-vitrified oocytes were similar to their non-vitrified counterparts. Vitrification increased oocyte ZAR1, NPM2 and DPPA3 gene expression while its expression decreased in LIF-vitrified oocytes to similar or close levels to those of non-vitrified oocytes. With a few gene-specific exceptions, vitrification significantly increased the expression of DNMT3A, HDAC1, KAT2A, BAX and BCL2L1 in oocytes and most stages of embryo development, while comparable expression patterns for these genes were observed between LIF-vitrified and non-vitrified groups. Vitrification increased HSPA1A expression in oocytes and HSP90AA1 in 2-cell embryos. Our data suggest that vitrification triggers stage-specific changes in gene expression throughout embryonic development. However, the inclusion of LIF in the IVM medium prior to vitrification stimulates blastocyst development and several other developmental parameters and induces oocytes and embryos to demonstrate gene expression patterns similar to those derived from non-vitrified oocytes.//////////////////
Suppressor of cytokine signaling 4 (SOCS4): moderator of ovarian primordial follicle activation. Sutherland JM et al. (2012) Mammalian ovarian primordial follicle activation and regulation is considered as one of the most important stages of folliculogenesis and as such requires exquisite control. Selection of quiescent follicles to enter the growing pool determines the rate of supply of maturing follicles over the female reproductive lifespan. To coordinate this process a range of positive and negative input signals contribute to determine follicle fate. This study demonstrates that the cytokine Leukemia Inhibitory Factor (LIF) activates the Janus Kinase 1/Signal Transducers and Activators of Transcription 3 (JAK1/STAT3) signaling pathway in pre-granulosa cells and positively regulates primordial follicle activation. Negative regulation of the JAK/STAT pathway is controlled by the suppressor of cytokine signaling 4 (SOCS4) protein, which target members of negative feedback loops, Cardiotrophin like Cytokine (CLC), Poly (rC) Binding Protein 1 (PCBP1), and Cytosolic Malate Dehydrogenase (MDH1) to suppress follicle growth and development.//////////////////
Quadrupling efficiency in production of genetically modified pigs through improved oocyte maturation. Yuan Y et al. (2017) Assisted reproductive technologies in all mammals are critically dependent on the quality of the oocytes used to produce embryos. For reasons not fully clear, oocytes matured in vitro tend to be much less competent to become fertilized, advance to the blastocyst stage, and give rise to live young than their in vivo-produced counterparts, particularly if they are derived from immature females. Here we show that a chemically defined maturation medium supplemented with three cytokines (FGF2, LIF, and IGF1) in combination, so-called "FLI medium," improves nuclear maturation of oocytes in cumulus-oocyte complexes derived from immature pig ovaries and provides a twofold increase in the efficiency of blastocyst production after in vitro fertilization. Transfer of such blastocysts to recipient females doubles mean litter size to about nine piglets per litter. Maturation of oocytes in FLI medium, therefore, effectively provides a fourfold increase in piglets born per oocyte collected. As they progress in culture, the FLI-matured cumulus-oocyte complexes display distinctly different kinetics of MAPK activation in the cumulus cells, much increased cumulus cell expansion, and an accelerated severance of cytoplasmic projections between the cumulus cells outside the zona pellucida and the oocyte within. These events likely underpin the improvement in oocyte quality achieved by using the FLI medium.//////////////////
Leukemia Inhibitory Factor is Necessary for Ovulation in Female Rhesus Macaques. Murphy MJ et al. (2016) Although the requirement of pituitary-derived luteinizing hormone (LH) for ovulation is well documented, the intrafollicular paracrine and autocrine processes elicited by LH necessary for follicle rupture are not fully understood. Evaluating a published rhesus macaque periovulatory transcriptome database revealed that mRNA encoding leukemia inhibitory factor (LIF) and its downstream signaling effectors are upregulated in the follicle after animals receive an ovulatory stimulus (human chorionic gonadotropin; hCG). Follicular LIF mRNA and protein levels are below the limit of detection prior to the administration of hCG, but increase significantly 12 h thereafter. Downstream LIF receptor signaling components including interleukin-6 signal transducer (IL6ST), the receptor associated janus kinase (JAK) 1 and the transcription factor signal transducer and activator of transcription (STAT) 3 also exhibit increased expression in the rhesus macaque follicle 12 h after administration of an ovulatory hCG bolus. A laparoscopic ovarian evaluation 72 h after the injection of a LIF antagonist (soluble LIF receptor; sLIFR) into the rhesus macaque preovulatory follicle and hCG administration revealed blocking LIF action prevented ovulation (typically occurs 36 to 44 h post-hCG). Moreover, ovaries removed 52 h after both hCG and intrafollicular sLIFR administration confirmed ovulation was blocked as evidenced by the presence of an intact follicle and a trapped cumulus-oocyte complex. These findings give new insight into the role of LIF in the primate ovary and could lead to the development of new approaches for the control of fertility.//////////////////
Analysis of the Effect of Leukemia Inhibitory Factor on Follicular Growth in Cultured Murine Ovarian Tissue. Komatsu K et al. (2015) Leukemia inhibitory factor (LIF) is expressed in the ovary and controls follicular growth. LIF has been reported to accelerate the primordial to primary follicle transition, the growth of cultured preantral follicles and the maturation of oocytes. Previous reports on factors that regulate follicular growth have largely employed cultured follicles. However, there are several types of follicles and somatic cells in the ovary that are likely to interact with one another to regulate follicular growth. Therefore, a novel approach is essential for understanding the function of factors that regulate follicular growth in the ovary. In this study, we evaluated the function of LIF using cultured ovarian tissue. Ovarian tissue slices were cultured in the presence or absence of recombinant LIF and neutralizing anti-LIF antibody to enable continuous monitoring of follicular growth within the context of the ovary as well as analysis of the process of follicular growth. The results revealed that LIF inhibited the growth of primary, secondary and antral follicles. Furthermore, we verified the inhibitory function of LIF using the neutralizing antibody, which accelerated follicular growth. These results suggest that LIF is likely to coordinate follicular growth in the ovary. The culture and analysis methods employed in this study are thus effective for clarifying the tissue-level functions of factors that regulate follicular growth within the ovary.//////////////////
Leukemia inhibitory factor promotes porcine oocyte maturation and is accompanied with activation of signal transducer and activator of transcription 3. Dang-Nguyen TQ 2013 et al.
We produced recombinant porcine leukemia inhibitory factor (pLIF) and examined its effect on in vitro maturation (IVM) of porcine oocytes and their developmental competence after in vitro fertilization. Porcine cumulus-oocyte complexes (COCs) were matured in a medium supplemented with pLIF during the first 22?h, last 22?h, or entire 44?h duration of IVM. Oocytes in all groups tended to show enhanced nuclear maturation rates by the metaphase II (MII) stage (76.1%, 82.1%, and 86.6%, respectively) compared to the without-pLIF treatment group (69.6%, control). A significant increase in MII rate (P?Luz V et al. We investigated the effect of the leukaemia inhibitory factor (LIF) alone or in association with FSH on the in vitro culture (IVC) of caprine preantral follicles. Preantral follicles >200 ?m in size were isolated and cultured for 18 days in basic medium either alone (control) or supplemented with LIF (10 or 50 ng/ml) in the absence or presence of FSH. Every 6 days, follicular survival, growth and antrum formation were evaluated. At the end of the culture period, the oocytes underwent in vitro maturation (IVM), and their viability and chromatin configuration were assessed. Follicles of the control group and those cultured in 10 ng/ml LIF maintained the structural integrity (particularly the preservation of the basement membrane) when compared to the oocytes cultured in 50 ng/ml LIF, regardless the presence of FSH. In the absence of FSH, the percentage of antrum formation after 18 days of culture in the 50 ng/ml LIF group was significantly lower than in either the control group or the 10 ng/ml LIF group. However, this effect was not observed in the presence of FSH. The rate of resumption of meiosis was significantly higher in the 50 ng/ml LIF group in the absence of FSH in comparison with the control and 10 ng/ml LIF groups. Metaphase II was observed only when follicles were cultured in a combination of FSH and 50 ng/ml LIF. In conclusion, LIF alone does not interfere with antral formation and oocyte growth, but at concentration of 50 ng/ml and combined with FSH, it promotes oocyte maturation.
///////Leukemia inhibitory factor enhances bovine oocyte maturation and early embryo development. Mo X 2014 et al.
The present study was conducted to examine the effects of leukaemia inhibitory factor (LIF) on bovine oocyte maturation and early embryo development in vitro. Results showed that LIF supplementation (25ng/ml) enhanced nuclear maturation of intact cumulus-oocyte complexes (COCs) compared to the vehicle control. Similar results were observed in denuded oocytes, indicating that LIF directly influences oocyte development. LIF-treated oocytes showed a higher cortical-granule migration rate and increased expression of CD9, a tetraspanin transmembrane protein essential for fertilization. After in vitro fertilization, oocytes receiving LIF supplementation exhibited a higher cleavage rate and yielded a significantly higher number of blastocysts. To further dissect the molecular mechanism underlying this LIF-induced bovine oocyte maturation phenotype, we examined the involvement of two signaling cascades, mitogen-activated protein kinases (MAPK3/1)- and the signal transducer and activator of transcription 3 (STAT3)-dependent pathways. Western blot results revealed that LIF phosphorylated MAPK3/1 and STAT3. Inhibition of MAPK3/1 activation with MEK inhibitor U0126 only partially blocked LIF-induced nuclear maturation, although it attenuated oocyte cytoplasmic maturation. Inhibition of JAK/STAT3 activation with a specific pharmacological inhibitor completely abolished the LIF-response in bovine oocyte. In summary, these data revealed a novel role for LIF in bovine oocyte maturation subsequent embryonic development. Mol. Reprod. Dev. 2014 Wiley Periodicals, Inc.
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Effects of Leukemia Inhibitory Factor on gp130 Expression and Rate of Metaphase II Development during in vitro Maturation of Mouse oocyte. Mohammadi Roushandeh A et al. Background: Leukemia inhibitory factor (LIF) is a 45-56 kDa glycoprotein that has an important role in proliferation and embryo implantation. Its effect on oocyte maturation and how to exert the function remained to be elucidated. Methods: Immature mice superovulated with human menopausal gonadotropin and germinal vesicle (GV) oocytes were obtained from ovary 48 hours after. GV oocytes were cultured in tissue culture medium 199 with 0, 100, 500 and 1,000 U/ml LIF. Cumulus expansion and in vitro maturation (IVM) rate were assessed after culture. For reverse transcriptase PCR, total RNA from GV and metaphase II (MII) oocytes were extracted by Trizol reagent. The quantity and quality of RNA were determined by spectrophotometry and electrophoresis, respectively. Reverse transcription was performed by Super Script III reverse transcriptase with 1 ?g of total RNA followed by DNase I treatment and heat inactivation. Expression of gp130 was determined by RT-PCR. Results: Our results showed that cumulus expansion was improved with 1,000 U/ml in culture medium compared to others. GV breakdown and MII rate in groups with LIF were higher than control group and were dose dependant. In 1,000 U/ml, LIF rate of MII was significantly higher than control group (P less than 0.05). Our results also showed that gp130 is expressed neither in GV nor in MII oocytes during IVM of mouse oocytes. Conclusion: gp130 is expressed in human oocyte but not in mouse. Our results suggest that in mouse, LIF could affect the oocyte via another receptor or via cumulus cells; however, further studies are warranted.
Several reviews summarize effects of LIF on reproduction. LIF is crucial for successful implantation of the embryo in mice. Donovan (1994) reviewed the role of LIF in primary germ cell (PGC) development. PGCs that stray from the normal migratory pathway might be eliminated through programmed cell death. SLF, together with LIF, can stimulate PGC proliferation in culture and it seems likely that LIF or a related cytokine may function in vivo to regulate PGC survival and proliferation. Apoptosis is responsible for primordial germ cell (PGC) attrition in the developing fetal ovary. In monolayer cultures of murine PGC, stem cell factor (SCF) and leukemia inhibitory factor (LIF) independently promote survival in vitro. De Felici et al. (1994) showed that LIF supports PGC survival by preventing PGC apoptosis. In addition, Morita et al. (1999) showed, that the combined actions of SCF, LIF, and IGF-I are required for maximal inhibition of apoptosis in germ cells of fetal mouse ovaries. Senturk et al. (1998) reviewed the role of leukemia inhibitory factor in human reproduction. They suggested that the rising follicular fluid LIF level around the time of ovulation indicates that LIF may play a role in ovulatory events, early embryonic development, and implantation.
Leukemia inhibitory factor induces cumulus expansion in immature human and mouse oocytes and improves mouse two-cell rate and delivery rates when it is present during mouse in vitro oocyte maturation. De Matos DG et al. OBJECTIVE: To examine the role of leukemia inhibitory factor (LIF) during in vitro maturation (IVM) on human and mice cumulus expansion and mice oocyte competence by in vitro fertilization (IVF), culture, and embryo transfer (ET). DESIGN: Prospective animal and human study. SETTING: Serono laboratories and IVF clinic. PATIENT(S): Healthy women volunteers and 8-week-old female mice. INTERVENTION(S): Cumulus compacted human and mice oocytes were matured in IVM media with and without recombinant follicle-stimulating hormone (FSH) and with and without LIF. Mice IVM oocytes with and without 0.2 IU/mL of recombinant FSH; or with and without recombinant FSH + LIF (0.1, 1.0, 1000.0 ng/mL) and ovulated oocytes were in vitro fertilized and cultured. We transferred 395 blastocysts to the uterine horn of 2.5-day pseudopregnant female mice. MAIN OUTCOME MEASURE(S): Cumulus expansion in human and mice oocytes, and two-cell rate, blastocyst rate, and delivered rate of live pups in mice. RESULT(S): In human and mouse oocytes, LIF induced cumulus expansion. When 1000 ng/mL of LIF was added in combination with recombinant FSH, a statistically significant increase in cleavage rate, embryo development rate, and birth rate was observed when compared with oocytes matured with FSH alone. CONCLUSION(S): Leukemia inhibitory factor induced cumulus expansion similarly in human and mouse cumulus-oocyte complexes, and recombinant FSH plus LIF supplementation during mouse IVM significantly improved oocyte competence as measured by cleavage rate, blastocyst development, and birth rate.
Leukemia inhibitory factor (LIF) promotes the primordial to primary follicle transition in rat ovaries. Nilsson EE et al. In a sexually mature female, primordial follicles continuously leave the arrested pool and undergo the primordial to primary follicle transition. The oocytes increase in size and the surrounding squamous pre-granulosa cells become cuboidal and proliferate to form a layer of cuboidal cells around the growing oocyte. This development of the primordial follicle commits the follicle to undergo the process of folliculogenesis. When the available pool of primordial follicles is depleted reproductive function ceases and humans enter menopause. The current study examines whether leukemia inhibitory factor (LIF) promotes the primordial to primary follicle transition that initiates follicular development. Ovaries from 4 day-old rats were cultured in the absence or presence of LIF or neutralizing antibody to LIF. LIF treatment increased the proportion of follicles that initiated the primordial to primary follicle transition to 59%, compared to 45% in untreated cultured ovaries. The ability of LIF to induce primordial follicle development was enhanced to greater than 75% by the presence of insulin in the culture medium. Anti-LIF neutralizing antibody reduced the proportion of spontaneous developing primordial follicles. Immunocytochemical studies demonstrated higher levels of LIF protein in the granulosa and surrounding somatic cells of primordial and primary follicles compared to the oocyte. In contrast, later pre-antral and antral stage follicles showed LIF expression primarily in the oocyte. In granulosa and theca cell cultures LIF had no effect on cell proliferation. However, LIF treatment did increase expression of Kit ligand (KL) mRNA in cultured granulosa cells. KL has been shown to promote ovarian cell growth and induce primordial follicle development. LIF induction of KL expression may be involved in the actions of LIF to promote primordial to primary follicle transition. In summary, LIF treatment increased the primordial to primary follicle transition in cultured ovaries and LIF may interact with KL to promote primordial follicle development.
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