bims-gamemb 2022-02-13 papers

bims-gamemb

Biomed News

on Gamete and embryo metabolism

Issue of 2022–02–13
eight papers selected by
Cameron A. Schmidt, East Carolina University

Folate supplementation during oocyte maturation positively impacts the folate-methionine metabolism in pre-implantation embryos.
Developmental and molecular response of bovine embryos to reduced nutrients in vitro.
Folate Receptor-1 is Vital for Developmental Competence of Goat Embryos.
l-carnitine and l-acetylcarnitine supplementation for idiopathic male infertility.
Mutant C. elegans mitofusin leads to selective removal of mtDNA heteroplasmic deletions across generations to maintain fitness.
Targeted disruption of Rab1a causes early embryonic lethality.
SIRT1 plays an important role in implantation and decidualization during mouse early pregnancy.
Biochemical Assays to Detect Activation of Small GTPases Rho, Rac, and Cdc42 during Morphogenesis.

Theriogenology. 2022 Jan 29. pii: S0093-691X(22)00030-9. [Epub ahead of print]182 63-70

Folate supplementation during oocyte maturation positively impacts the folate-methionine metabolism in pre-implantation embryos.

Sikander Saini, Vishal Sharma, Shama Ansari, Amit Kumar, Abhishek Thakur, Hrudananda Malik, Satish Kumar, Dhruba Malakar.

  Folic acid is vital for DNA synthesis and methylations through one-carbon (C1) metabolism. Thus, it is essential for cell division during embryonic development. Although the oocytes contain endogenous pool of folates for development, the present study investigated the effect of external folic acid supplementation on oocyte maturation, blastocyst development and the expression of folate transporters as well as folate metabolism enzymes in oocytes and pre-implantation embryos of goat. Immature goat oocytes, matured in maturation medium comprising different folic acid concentrations (0, 10, 50, 100 and 150 μM), were in vitro fertilized and cultured. Cumulus expansion markers (PTX3 and PTGS2) in cumulus cells were highly upregulated after 50 μM folic acid supplementation indicating higher degree of maturation. Supplementation of 50 μM folic acid during oocyte maturation resulted in significantly higher blastocyst production rate, reduction in intracellular ROS levels as well as upregulation of the transcripts for folate transporters and key folate-methionine cycle enzymes in comparison to control. The present study demonstrates the existence of active folate-methionine cycle in oocytes and pre-implantation goat embryos. Supplementation of 50 μM folic acid in maturation medium improves oocyte maturation, the blastocyst production rate, reduces ROS production as well as upregulate the expression of FOLR1 and folate metabolism enzyme, MTR.

Keywords:  Folate-methionine metabolism; Folic acid; Gene expression; In vitro fertilization; Oocyte; Pre-implantation embryo

DOI:  https://doi.org/10.1016/j.theriogenology.2022.01.024
Reprod Fertil. 2020 Jul;1(1): 51-65

Developmental and molecular response of bovine embryos to reduced nutrients in vitro.

Jason R Herrick, Sandeep Rajput, Rolando Pasquariello, Alison Ermisch, Nicolas Santiquet, William B Schoolcraft, Rebecca L Krisher.

  Recent studies in our laboratory have indicated that bovine embryos only use a small amount of the nutrients available to them in culture. Our objective was to evaluate the developmental and molecular response of bovine embryos when nutrient concentrations in the culture medium were significantly reduced. Following IVM and IVF, embryos were cultured in media containing 75, 50, and 25% (experiment 1) or 25, 12.5, and 6.25% (experiment 2) of the concentrations of nutrients (carbohydrates, amino acids, and vitamins) present in our control medium (100%). Blastocyst formation, hatching, and allocation of cells to the inner cell mass (ICM) and trophectoderm (TE) were evaluated on day 7. Although the number of TE cells was decreased (P < 0.05) when nutrient concentrations were ≤25% (73.8-124.1 cells), it was not until nutrient concentrations were reduced to 6.25% that blastocyst formation (18.3 ± 3.0%) and hatching (3.0 ± 1.3%) were inhibited (P < 0.05) compared to embryos cultured in the control medium (156.1 ± 14.1 cells, 40.0 ± 3.8%, 20.0 ± 3.1%, respectively). Inhibition of fatty acid oxidation (etomoxir) reduced (P < 0.05) blastocyst development, with more pronounced effects at lower nutrient concentrations (≤12.5%). Reducing nutrient concentrations was associated with increased activity of AMPK, decreased activity of mTOR, and altered abundance of transcripts for hexokinase 1 (HK1), carnitine palmitoyl transferase 2 (CPT2), lactate dehydrogenase A (LDHA), and pyruvate dehydrogenase kinase 1 (PDK1), consistent with an increase in glucose and fatty acid metabolism. Reduced nutrient conditions provide a unique perspective on embryo metabolism that may facilitate the optimization of culture media.
Lay summary: To support early embryo development in the first week after fertilisation, an appropriate mixture of nutrients (carbohydrates, amino acids, and vitamins) is needed in the culturing solution. However, refining these solutions to support optimal embryo health remains challenging. In this study, bovine (cow) embryos derived from abattoir material were used as a model for the development of other mammalian embryos, including humans. These embryos were cultured in the presence of 75, 50, 25, 12.5, or 6.25% of the nutrients present in control conditions (100%), which are similar to those reported for the fluids of the fallopian tubes and uterus. Embryo development was largely unaffected in the 75, 50, and 25% treatments, with some embryos developing in the presence of only 6.25% nutrients. Cow embryos are remarkably resilient to reduced concentrations of nutrients in their environment because they can utilize internal stores of fat as a source of energy.

Keywords:  AMPK; blastocyst; fatty acid oxidation; mTOR; metabolism

DOI:  https://doi.org/10.1530/RAF-20-0033
Reprod Domest Anim. 2022 Feb 05.

Folate Receptor-1 is Vital for Developmental Competence of Goat Embryos.

Sikander Saini, Shama Ansari, Vishal Sharma, Shrabani Saugandhika, Sandeep Kumar, Dhruba Malakar.

  Folate is essential for DNA synthesis and methylation via one-carbon (C1) metabolism during embryonic development. It is transported into the developing oocytes via folate receptors (FOLR1 and FOLR2) and transporters (RFC1) for utilization during embryo development. However, the role of folate receptors during pre-implantation stages of embryos is not well known. Thus, the present study aimed to investigate the expression of folate transport genes and proteins in mature oocytes and pre-implantation embryos; and the effect of FOLR1 knockdown in zygotes on blastocyst outcome. For this, Immature goat oocytes were matured in maturation medium followed by in vitro fertilization and culture at standard conditions. A group of zygotes was transfected with esiRNA against FOLR1 and in vitro cultured for blastocyst outcome assessment. The transcripts and proteins for FOLR1, FOLR2 and RFC1 were present in oocytes as well as all the stages of pre-implantation embryos. Immunofluorescence revealed the presence of FOLR1 in the nuclei of embryos but not in the metaphase (matured) oocytes. The knockdown of FOLR1 in embryos was effective and significantly reduced the blastocyst production rate. The present study demonstrates the existence of active folate transport in oocytes and pre-implantation goat embryos. FOLR1 is vital for pre-implantation embryo development and may aid in the progression by functioning as a transcription factor.

Keywords:  Folate; gene expression; goat; in vitro fertilization; knockdown; transfection

DOI:  https://doi.org/10.1111/rda.14092
Reprod Fertil. 2020 Jul;1(1): 67-81

l-carnitine and l-acetylcarnitine supplementation for idiopathic male infertility.

Shen Chuen Khaw, Zhen Zhe Wong, Richard Anderson, Sarah Martins da Silva.

  Fifteen percent of couples are globally estimated to be infertile, with up to half of these cases attributed to male infertility. Reactive oxidative species (ROS) are known to damage sperm leading to impaired quantity and quality. Although not routinely assessed, oxidative stress is a common underlying pathology in infertile men. Antioxidants have been shown to improve semen analysis parameters by reducing ROS and facilitating repair of damage caused by oxidative stress, but it remains unclear whether they improve fertility. Carnitines are naturally occurring antioxidants in mammals and are normally abundant in the epididymal luminal fluid of men. We conducted a systematic review and meta-analysis to evaluate the safety and efficacy of carnitine supplementation for idiopathic male infertility. We searched ClinicalKey, ClinicalTrials.gov, Cochrane Central Register of Controlled Trials (CENTRAL), EMBASE, MEDLINE, PubMed and ScienceDirect for relevant studies published from 1 January 2000 to 30 April 2020. Of the articles retrieved, only eight randomised controlled trials were identified and included. Analysis showed that carnitines significantly improve total sperm motility, progressive sperm motility and sperm morphology, but without effect on sperm concentration. There was no demonstrable effect on clinical pregnancy rate in the five studies that included that outcome, although patient numbers were limited. Therefore, the use of carnitines in male infertility appears to improve some sperm parameters but without evidence of an increase in the chance of natural conception.
Lay summary: Although male infertility affects 1:15 men, there is no obvious reason in the vast majority of cases. Reactive oxidative species (ROS) are highly active molecules containing oxygen and are natural byproducts of normal metabolism. However, high concentrations of ROS have been shown to damage sperm, which negatively impacts a couple's ability to conceive. Carnitines are natural antioxidants found in the body that counterbalance the damaging effects of ROS. We conducted a comprehensive review of published studies to assess whether carnitine supplements are safe and effective in improving sperm quality and pregnancy rates. Our analysis shows that carnitines improve sperm swimming and production of normal-shaped sperm cells but do not affect sperm count or pregnancy rates, although there are only a few studies and scientific evidence is limited. Whilst it is possible that carnitines may benefit male infertility, more evidence is required regarding chances of pregnancy after carnitine therapy.

Keywords:  antioxidants; carnitine; male infertility; reactive oxidative species; sperm

DOI:  https://doi.org/10.1530/RAF-20-0037
BMC Biol. 2022 Feb 09. 20(1): 40

Mutant C. elegans mitofusin leads to selective removal of mtDNA heteroplasmic deletions across generations to maintain fitness.

Lana Meshnik, Dan Bar-Yaacov, Dana Kasztan, Tali Neiger, Tal Cohen, Mor Kishner, Itay Valenci, Sara Dadon, Christopher J Klein, Jeffery M Vance, Yoram Nevo, Stephan Züchner, Ofer Ovadia, Dan Mishmar, Anat Ben-Zvi.

   BACKGROUND: Mitochondrial DNA (mtDNA) is present at high copy numbers in animal cells, and though characterized by a single haplotype in each individual due to maternal germline inheritance, deleterious mutations and intact mtDNA molecules frequently co-exist (heteroplasmy). A number of factors, such as replicative segregation, mitochondrial bottlenecks, and selection, may modulate the exitance of heteroplasmic mutations. Since such mutations may have pathological consequences, they likely survive and are inherited due to functional complementation via the intracellular mitochondrial network. Here, we hypothesized that compromised mitochondrial fusion would hamper such complementation, thereby affecting heteroplasmy inheritance.
RESULTS: We assessed heteroplasmy levels in three Caenorhabditis elegans strains carrying different heteroplasmic mtDNA deletions (ΔmtDNA) in the background of mutant mitofusin (fzo-1). Animals displayed severe embryonic lethality and developmental delay. Strikingly, observed phenotypes were relieved during subsequent generations in association with complete loss of ΔmtDNA molecules. Moreover, deletion loss rates were negatively correlated with the size of mtDNA deletions, suggesting that mitochondrial fusion is essential and sensitive to the nature of the heteroplasmic mtDNA mutations. Introducing the ΔmtDNA into a fzo-1;pdr-1;+/ΔmtDNA (PARKIN ortholog) double mutant resulted in a skewed Mendelian progeny distribution, in contrast to the normal distribution in the fzo-1;+/ΔmtDNA mutant, and severely reduced brood size. Notably, the ΔmtDNA was lost across generations in association with improved phenotypes.
CONCLUSIONS: Taken together, our findings show that when mitochondrial fusion is compromised, deleterious heteroplasmic mutations cannot evade natural selection while inherited through generations. Moreover, our findings underline the importance of cross-talk between mitochondrial fusion and mitophagy in modulating the inheritance of mtDNA heteroplasmy.

Keywords:  C. elegans; Heteroplasmy inheritance; Mitofusin; PARKIN; fzo-1; mtDNA; pdr-1

DOI:  https://doi.org/10.1186/s12915-022-01241-2
Int J Mol Med. 2022 Apr;pii: 46. [Epub ahead of print]49(4):

Targeted disruption of Rab1a causes early embryonic lethality.

Yin Wu, Darong Yang, Guo-Yun Chen.

  Guanosine nucleotide diphosphate (GDP) dissociation inhibitor 2 (GDI2) regulates the GDP/guanosine triphosphate (GTP) exchange reaction of Rab proteins by inhibiting the dissociation of GDP and the subsequent binding of GTP. The present study aimed to determine the function of Rab1a in vivo, and thus generated mice with a trapped Rab1a gene. It was demonstrated that Rab1a is essential for embryonic development. It was also found that one functional Rab1a allele was sufficient for development in a heterozygous murine embryo, whereas a double mutant led to embryonic lethality. The dissection of uteri on embryonic day (E)10.5‑14.5 yielded no homozygous embryos, indicating that homozygotes die between E10.5 to E11.5. The gene trap construct contains a β‑galactosidase/neomycin reporter gene, allowing for heterozygotes to be stained for β‑galactosidase to determine the tissue‑specific expression of Rab1a. Rab1a was found to be highly expressed in the small intestine of both adult mice and embryos, although its expression levels were low in the brains of embryos. Moreover, there was no significant change in cytokine production and survival in wild‑type and heterozygous Rab1a+/‑ mice following a challenge with lipopolysaccharide. On the whole, the present study demonstrates that the disruption of the Rab1a gene causes embryonic lethality and homozygotes die between E10.5 and E11.5, suggesting that Rab1a is essential for the early development of mouse embryos.

Keywords:  Rab1a; embryonic lethality; guanosine nucleotide diphosphate dissociation inhibitor 2; immunoreceptor tyrosine‑based inhibitory motif; small intestine

DOI:  https://doi.org/10.3892/ijmm.2022.5101
Biol Reprod. 2022 Feb 02. pii: ioac026. [Epub ahead of print]

SIRT1 plays an important role in implantation and decidualization during mouse early pregnancy.

Yeon Jeong Hwang, Gi-Jun Sung, Ryan Marquardt, Steven L Young, Bruce A Lessey, Tae Hoon Kim, Yong-Pil Cheon, Jae-Wook Jeong.

  Sirtuin 1(SIRT1) is a member of the sirtuin family that functions to deacetylate both histones and non-histone proteins. Previous studies have identified significant SIRT1 upregulation in eutopic endometrium from infertile women with endometriosis. However, SIRT1 function in the uterus has not been directly studied. Using immunochemistry analysis, we found SIRT1 to be most strongly expressed at GD4.5 and GD5.5 in decidualized cells and at GD7.5 in secondary decidual cells in mouse. To assess the role of SIRT1 in uterine function, we generated uterine Sirt1 conditional knockout mice (Pgrcre/+Sirt1f/f; Sirt1d/d). A 6-month fertility trial revealed that Sirt1d/d females were subfertile. Implantation site numbers were significantly decreased in Sirt1d/d mice compared to controls at GD5.5. Sirt1d/d implantation sites at GD4.5 could be divided into two groups, Group #1 with luminal closure and non-specific COX2 expression compared to controls (14/20) and Group #2 with an open lumen and no COX2 (6/20). In Sirt1d/d Group #1, nuclear FOXO1 expression in luminal epithelial cells was significantly decreased. In Sirt1d/d Group #2, nuclear FOXO1 expression was almost completely absent, and there was strong PGR expression in epithelial cells. At GD5.5, stromal PGR and COX2 were significantly decreased in Sirt1d/d uterine in the areas surrounding the embryo compared to controls, indicating defective decidualization. An artificially induced decidualization test revealed that Sirt1d/d females showed defects in decidualization response. All together, these data suggest that SIRT1 is important for decidualization and contributes to preparing a receptive endometrium for successful implantation.

Keywords:  Implantation; Progesterone receptor; SIRT1; Subfertility

DOI:  https://doi.org/10.1093/biolre/ioac026
Methods Mol Biol. 2022 ;2438 83-95

Biochemical Assays to Detect Activation of Small GTPases Rho, Rac, and Cdc42 during Morphogenesis.

Mark L Berns, Raymond Habas.

  Wnt/Frizzled (Fz) signaling controls developmental, physiological, and pathological processes through several distinct pathways. Wnt/Fz activation of the small GTPases Rho, Rac, and Cdc42, is one key mechanism that regulates cell polarity and migration during vertebrate gastrulation. In this chapter, we describe biochemical assays for detection of Wnt/Fz-mediated activation of Rho, Rac and Cdc42 in both mammalian cells and Xenopus embryo explants.

Keywords:  Cdc42; Gastrulation; Noncanonical Wnt signaling; Rac; Rho; Xenopus

DOI:  https://doi.org/10.1007/978-1-0716-2035-9_5