Hatching, attachment, and outgrowth of mouse blastocysts in vitro: Fixed nitrogen requirements

Summary. The protein content of the mouse uterine lumen increased significantly (P < 0\m=.\001) on Day 4 of pregnancy, the day of implantation. This increase was associated with the presence of 14 serum and 22 non-serum proteins in the lumen; the major serum proteins were classed as high molecular weight slow \g=a\-globulins, while the dominant non-serum components consisted of slow and fast \g=a\-globulins, 6 prealbumins and a large quantity of proteinaceous material migrating near the origin of the gels.During experimental and lactational delayed implantation the protein levels were constantly low, transferrin, haemoglobin and albumin dominating the protein pattern. After administration of oestradiol-17\g=b\,however, a biphasic uterine response was detected, significant increases in luminal protein concentration being observed within 12 h and again at 40\p=n-\48h after injection. The first phase of this response involved an influx of serum and non-serum proteins into the uterine lumen, most proteins migrating as high molecular weight slow \g=a\-globulins. The second phase involved an increase in the intensity of many non-serum components, the major proteins having Ra values of 0\m=.\06,0\m=.\10and 0\m=.\32.The qualitative, but not the quantitative, aspects of this response to oestradiol were identical in the absence of blastocysts.

Section: Discussionmentioning

confidence: 99%

Changes in the protein content of mouse uterine flushings during normal pregnancy and delayed implantation, and after ovariectomy and oestradiol administration

Aitken¹

1977

“…Although preimplantation embryos can be cultured in the absence of amino acids (Caro & Trounson 1986, Fissore et al 1989, their inclusion in both fertilisation and culture media enhances development and blastocyst cell numbers in the mouse (Gwatkin 1966, Spindle & Pedersen 1973, Ali et al 1993, Gardner & Lane 1993, Ho et al 1995, Lane & Gardner 1997a,b, Nakazawa et al 1997, Summers et al 2000, cattle (Lee & Fukui 1996), human (Devreker et al 2001) and hamster (Kane & Bavister 1988, McKiernan et al 1995. Amino acids serve a variety of physiological functions, including: the synthesis of proteins and nucleotides (Epstein & Smith 1973, Alexiou & Leese 1992, Katchadourian et al 1994, nutrition and energy provision (Lane & Gardner 1997a, Houghton et al 2002, osmoregulation (Van Winkle & Campione 1996, Dumoulin et al 1997, Dawson et al 1998, protection against oxidative stress (Lindenbaum 1973, Nasr-Esfahani et al 1992, pH regulation (Bavister & McKiernan 1993, Edwards et al 1998, signalling molecule biosynthesis (Wu & Morris 1998), trophectoderm differentiation (Martin & Sutherland 2001) and basement membrane formation between primitive endoderm and ectoderm .…”

Section: Introductionmentioning

confidence: 99%

Ammonium exposure and pyruvate affect the amino acid metabolism of bovine blastocysts in vitro

Orsi¹,

Leese²

2004

The accumulation of ammonium is a major artefact of in vitro embryo culture. This study has examined ammonium production and potential mechanisms of disposal in preimplantation bovine blastocysts. Embryos were produced by in vitro maturation and fertilisation of oocytes, and cultured in synthetic oviduct fluid containing amino acids and BSA (SOFaaBSA). Ammonium/urea concentrations were determined enzymatically. Amino acid appearance/disappearance 'profiles' of single blastocysts were determined at 0, 1.25 and 2.5 mM NH 4 Cl (with or without 0.33 mM pyruvate), and with or without 10 mM dipicolinic acid (DPCA; a glutamate dehydrogenase (GLDH) inhibitor) or 2 mM amino-oxyacetate (AOA; a transaminase inhibitor). Free ammonium was produced at a rate of 4.281 (6 0.362) pmol/embryo/h, while urea production was undetectable. The presence/absence of pyruvate affected amino acid profiles, especially alanine appearance (P < 0.001), glutamate disappearance (P < 0.05) and overall turnover (the sum of appearance and disappearance) (P < 0.001). GLDH inhibition with DPCA had no effect on amino acid overall disappearance, but glutamate disappearance increased, while that of arginine decreased (P < 0.05). The transaminase inhibitor, AOA, depressed turnover (P < 0.05), aspartate and glutamate disappearance, and alanine appearance. Thus, bovine blastocysts release ammonium as free ions or fix them, not as urea, but as alanine, possibly glutamine and, less likely, arginine. An active role for GLDH and transaminases in regulating blastocyst amino acid metabolism was demonstrated.

“…These embryos appear to become metabolically active, attach to the culture dish and continue to grow when they are cultured in medium containing essential amino acids, glucose and fetal calf serum (Gwatkin, 1966;McLaren, 1973;Spindle & Pedersen, 1973;Sherman & Wudl, 1976;Van Blerkom, Chavez & Bell, 1979;Naeslund, 1979). In contrast, activation in vitro apparently does not occur when the medium contains a low (55 mM) concentration of Na+, possibly because accumulation of essential amino acids is depressed in blastocysts cultured in such conditions (Van Winkle, 1977, 1981a.…”

Section: Introductionmentioning

confidence: 99%

Effect of inhibitors of polyamine synthesis on activation of diapausing mouse blastocysts in vitro

Winkle¹,

Campione²

1983

Summary. Diapausing mouse blastocysts were cultured in vitro with inhibitors of polyamine synthesis and/or the polyamines, putrescine, spermidine and/or spermine. Attachment of blastocysts to the culture dish, the onset of trophoblastic outgrowth and the extent of outgrowth were retarded by methylornithine (10 mM) or methylglyoxal bi s\ x=req-\ (guanylhydrazone) (50 or 200 \g=m\M) and apparently blocked by a combination of these inhibitors. The embryos appeared to make no progress towards activation in the presence of both inhibitors but became active and grew normally when transferred to inhibitor-free medium. The inhibition of growth was also reversed when polyamines (10\m=-\5 m) were supplied in the medium along with the inhibitors (P < 0\m=.\01). These results suggest that polyamine synthesis is required for activation, growth and development of diapausing blastocysts in vitro.