A bioassay for progesterone and gonadotropins based on the meiotic division of Xenopus oocytes in vitro

Thornton, V.F.

doi:10.1016/0016-6480(71)90125-0

Cited by 42 publications

(13 citation statements)

References 7 publications

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“…Routinely, induction of oocyte maturation was judged by the appearance of the white depigmented Roux's spot (germinal vesicle breakdown [GVBD]) in the animal pole after 18 h of incubation ( Fig. 1) (14,26,49) and further confirmed by dissecting oocytes fixed in 5% trichloroacetic acid under a binocular microscope for the absence of the germinal vesicle (6). Normal cellular p21 protein was less effective.…”

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Insulin induction of Xenopus laevis oocyte maturation is inhibited by monoclonal antibody against p21 ras proteins.

Deshpande¹,

Kung²

1987

Mol. Cell. Biol.

174

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Microinjection of transforming p21 ras protein induces maturation of Xenopus laevis oocytes, and the induction is blocked by coinjection of monoclonal antibody (Y13-259) against p21 ras proteins. Similar to other inducing agents, the effect of p21 ras protein is mediated via the appearance of maturation or meiosispromoting factor activity. In addition, the neutralizing antibody markedly reduces oocyte maturation after insulin induction, whereas it fails to inhibit progesterone induction. Our results suggest that insulin induces maturation of oocytes via a different pathway than that of steroidal agents. The induction by insulin is ras dependent, and the action of ras may be directed at the steps before meiosis-promoting factor autocatalytic activation. These results suggest a role of p21 ras protein in the events associated with amphibian oocyte maturation.The ras family forms a group of closely related transforming genes that are highly conserved in eucaryotes (5,11,18,28,34,48). Normal (cellular) ras genes acquire transforming properties by single point mutations within their coding sequences (4,32,33,41,(45)(46)(47)54), and these mutated ras genes have been detected in a significant fraction of human cancers as well as in experimentally induced animal tumors (45,48,54). It has been speculated that p21 ras proteins are essential components of normal cells that are involved in cell division and differentiation (30,31). Yet, little is known regarding the mechanisms by which p21 proteins induce malignant transformation, and even less is known regarding their role in normal cells.It has been demonstrated that p21 proteins can induce transformation of NIH 3T3 cells when introduced by microinjection (13,44) and that the transforming activity of the ras proteins can be blocked by coinjection with monoclonal antibody Y13-259 (19). Recently, Birchmeier et al. (3) have shown that ras proteins can induce meiosis in Xenopus laevis oocytes. Fully grown oocytes can be easily injected and analyzed biochemically. Oocytes surgically removed from adult Xenopus ovaries are arrested in the prophase of meiosis and can be triggered to undergo meiosis by treatment with progesterone, insulin, and a variety of other agents. In the present study, we determined the effect of injected neutralizing antibody (Y13-259) on the maturation of oocytes.Gravid X. laevis females were obtained from Nasco Co. (Fort Atkinson, Wis.) and maintained under laboratory conditions (9). Ovarian fragments were surgically removed from frogs that were anesthetized by hypothermia. Fully grown stage VI oocytes were manually dissected, and follicle cells were removed after collagenase treatment of ovarian fragments at 21°C following the procedure of Eppig and Dumont (12) in modified Barth medium (MBSH) buffered with 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.6 (22). The selected stage VI oocytes were thoroughly washed in MBSH and equilibrated for at least 2 h before use. Oocytes were cultured in 30-mm Falcon * Corresponding author. ster...

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“…Stock solutions of agents were prepared in appropriate solvents and were added directly to MBSTI to obtain the desired effective final concentration (7,37,49,52,53). It has been known for some time that a variety of steroid and peptide hormones can induce oocyte maturation in amphibians.…”

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confidence: 99%

Insulin induction of Xenopus laevis oocyte maturation is inhibited by monoclonal antibody against p21 ras proteins.

Deshpande¹,

Kung²

1987

Mol. Cell. Biol.

174

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“…The frog Rana temporaria L. oocyte in vitro maturation test (Thornton, 1971) was used to evaluate (to compare) the hormonal activity of the reassociated a-b dimers comprising COOH-modified or intact bGTH. The activity was expressed as the minimal dose of the tested hormone preparation that produced 50% test-oocyte maturation (D50).…”

Section: Methodsmentioning

confidence: 99%

Specific Structural-Functional Role of the Pronounced Negative Molecular Charge of the Sturgeon Gonadotropin β-Subunit

Zenkevičs¹,

Vose²

2012

Proceedings of the Latvian Academy of Sciences. Section B. Natural, Exact, and Applied Sciences

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Free negatively charged carboxyl groups (COOH) were selectively modified (neutralised) in sturgeon (Acipenser güldenstädti Br) gonadotropic hormone (GTH) β-subunit (βGTH). Eight COOH groups were neutralised by reaction with glycine ethyl ester. Investigation of modified βGTH showed that specific immunoactivity with antiserum raised against standard βGTH was completely lost. Analysis of CD spectra and the content of secondary structure elements of standard and modified βGTH did not indicate any considerable differences. Investigation of reassociated α-β dimer comprising standard αGTH and modified βGTH showed that hormonal activity was completely lost while immunoreactivity was lowered by about 30% in comparison with that of the standard subunit α-β dimer. It was concluded that eight free COOH groups located on the surface of βGTH are the main structural components of the species-specific antigenic determinant groups of the subunit, but are not directly involved in either maintaining the conformation of the subunit nor in its ability to interact with the native counterpart subunit. COOH groups, as bearers of the negative charge located on the surface of βGTH, play a decisive role in the specific hormonal activity of GTH determined by oocyte maturation tests. Free COOH groups are included in the effector zones (active sites) of GTH dimer molecule responsible for the stereospecific interaction with the test-oocyte membrane hormone-sensitive binding (receptor) sites to induce the biological effects.

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“…The frog Rana temporaria L. oocyte maturation test (Thornton, 1971) was used to evaluate the hormonal activity of the reassociated a-b dimers comprising COOH-modified subunits. The activity was expressed as the minimal dose of the tested preparation that produced 50% test-oocyte in vitro maturation (D50).…”

Section: Methodsmentioning

confidence: 99%

Role of Carboxyl Groups in the Secondary Structure and Function of Sturgeon Gonadotropin

Zenkevičs¹,

Vosekalna²,

Vose³

2008

Proceedings of the Latvian Academy of Sciences. Section B. Natural, Exact, and Applied Sciences.

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INTRODUCTIONBiochemical and functional properties of fish, particularly sturgeon fish gonadotropic hormone (GTH), are considerably less studied than that of mammalian gonadotropins. The heterodimeric molecule (26 kDa) of sturgeon GTH, like mammalian gonadotropins, is built of two complementary non-covalently bonded a and b subunits (aGTH and bGTH) (Zenkeviès, 1994). Hormonal and immunological function typical for the hormone is expressed only in the a-b complex formed after noncovalent self-association of the separate a and b subunits into heterodimeric molecular structure (Çåíêåâè÷, 1992). GTH dimer reassociated in vitro from the counterpart subunits usually indicates up to 100% of immunoreactivity and about 70% of hormonal function typical of the standard intact hormone (Çåíêåâè÷, 1992).As a member of the pituitary glycoprotein hormone family, sturgeon GTH contains an asparagine-linked sugar moiety corresponding to 30% of the hormone molecular mass (Çåíêåâè÷ è äð., 1992). The complicated structure of gonadotropin prevents the synthesis of its molecular analogues. Therefore, selective chemical modification can be used to obtain the hormone or its subunit analogues with potentially different properties. The selective chemical modification of the functionally significant side chains radicals in sturgeon GTH dimer structure and in its individual subunits and subsequent analysis of its physico-chemical and biological properties has been considered as an efficient instrument for studying the correlative relationship between the molecular structure and specific biological function of the hormone (Zenkeviès, 1994).The aim of the present work was to employ selective chemical modification of the accessible free negatively charged COOH groups in the individual a and b subunits to elucidate their role both in the biological function of the restored a-b dimer molecule and in the maintaining of the secondary structure of the subunits. MATERIALS AND METHODSA highly purified GTH was isolated from acetone-dried pituitary glands of the fish by means of a standard procedure using gel-filtration on Sephadex and ion-exchange chromatography on DEAE-cellulose DE-32 (Whatman) columns as described earlier (Çåíêåâè÷, 1992).Individual a and b subunits of GTH were isolated by ionexchange chromatography on SE-Sephadex C-25 in 0.025 M acetate buffer at pH 4.9, after GTH dissociation into subunits in 8 M urea according to Hennen et al. (1971). Self-reassociation or recombination of the counterpart subunits was carried out in saline (0.9% NaCl) at room temperature (12 h) at a total protein concentration of about 0.2%, using the subunits in an equimolar ratio. Free COOH groups of the separate subunits were modified by glycine ethyl ester (Gly-OEt·HCl) in the presence of water soluble carbodiimide (1-ethyl-3(3-dimethylaminopropyl) carbodiimide) (Serva) at pH 4.8 in distilled water for 30 min at PROCEEDINGS OF THE LATVIAN ACADEMY OF SCIENCES. Section B, Vol. 62 (2008)

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A bioassay for progesterone and gonadotropins based on the meiotic division of Xenopus oocytes in vitro

Cited by 42 publications

References 7 publications

Insulin induction of Xenopus laevis oocyte maturation is inhibited by monoclonal antibody against p21 ras proteins.

Insulin induction of Xenopus laevis oocyte maturation is inhibited by monoclonal antibody against p21 ras proteins.

Specific Structural-Functional Role of the Pronounced Negative Molecular Charge of the Sturgeon Gonadotropin β-Subunit

Role of Carboxyl Groups in the Secondary Structure and Function of Sturgeon Gonadotropin

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