IN VITRO OF ACETATE AND CERTAIN BY SIX-DAY-OLD RABBIT BLASTOCYSTS

RONALD L. HUFF and KRISTEN B. EIK-NES Department of Biological Chemistry, College of Medicine, University of Utah, Salt Lake City, Utah, U.S.A. {Received 2lst April 1965)

Summary. Rabbit blastocysts obtained 6 days after coitus were incubated individually or in groups for periods from 24 to 96 hr in a synthetic medium. Radioactive , , 17\g=a\\x=req-\ hydroxypregnenolone, androstenedione and sodium acetate were used as substrates in these incubations. The blastocysts could biosynthesize cholesterol and pregnenolone from acetate, and promote biotransforma- tion of the various substrates. Addition of interstitial cell stimulating hormone and adrenocorticotrophic hormone to the medium increased the rate of differentiation of the blastocysts during in-vitro growth.

INTRODUCTION Metabolic studies of invertebrate eggs and early embryos have been relatively numerous, but few such studies have been done with mammalian embryos. Austin (1961) suggested that the scarcity of metabolic studies could be due to the difficulty of obtaining even moderate numbers of eggs. In addition, when working with metabolism in mammalian embryos, one must exclude any effects from maternal cells. Preferentially, such investigations should be done in a chemically-defined medium. Huff (1962) reported that rabbit blastocysts obtained 5 and 6 days after coitus could be cultured in a synthetic medium. Later we were able to show that rabbit blastocysts will survive for periods of up to 8 days in a synthetic medium with development proceeding from the pre-streak stages to the first segment embryo. Expansion of the blastocysts, which is caused largely by fluid intake, has been from 2 or 3 mm to 10 mm in diameter. The necessity of certain steroid hormones for normal gestational processes in the pregnant female may suggest an actual contact between the blastocysts and the steroids. We decided to investigate whether or not blastocysts growing in a synthetic medium could biosynthesize sterols and steroids from sodium acetate and biotransform steroid hormones. Moreover, an attempt was made to determine the effects ofpituitary hormones on the development of the 6-day-old blastocyst. * Present address : Department of Animal Husbandry, University of California, Davis, California, U.S.A. 57 Downloaded from Bioscientifica.com at 09/28/2021 12:30:14PM via free access 58 Ronald L. Huff and Kristen B. Eik-Nes MATERIALS AND METHODS Incubation procedure Two-hundred-and-twenty 6-day-old rabbit blastocysts were obtained by flushing the uterine horns of New Zealand White females by the method described by Gregory (1930). In order to reduce the possibility of interference by maternal cells, the blastocysts were washed seven times with culture medium. The washed blastocysts were transferred to incubation flasks containing the substrate under investigation in 5 ml of culture medium (modified T.C. Medium 199) (Morgan, Morton & Parker, 1950). The final washes were incubated with the various substrates as controls. T.C. Medium 199 containing 1 mg% penicillin, 1 mg% streptomycin, 2 mg% recrystallized bovine serum albumin and 0-5 mg% hyaluronidase was used throughout the study. (In preliminary experiments, amounts of albumin up to 4 mg% were used.) The incubation flasks were placed on a platform which rocked through a 70° arc during the entire incubation period and the temperature was maintained at 38 + 0-25° C. An atmosphere of 95% 02 and 5% C02 was used as a gas phase. The incubation times ranged from 24 to 96 hr with some blastocysts having a radioactive substrate available throughout the entire incubation period and others having such a substrate available only during the last 24 hr. Blastocysts were grown individually in flasks or in groups of eight/flask. Incubations with protein hormones A group of sixteen embryos was separated at random and incubated in¬ dividually in flasks containing 2 units of interstitial cell stimulating hormone (icsh), 2 units of adrenocorticotrophic hormone (acth), 2 units of both icsh and acth, and without either trophin added. The addition of these protein hormones to the medium increased the protein content by less than 20%. The blastocysts were examined for growth and differentiation after 24 and 48 hr of incubation.

Substrates Rabbit blastocysts were incubated with radioactive compounds in the following concentrations per 5 ml of modified T.C. Medium 199: progesterone- 4-14C (1-4 µg and 0-1 µ ), A5-pregnenolone-7-3H (1-4 µg and 2-8 µ ), 17a- hydroxypregnenolone-7-3H (1-2 ^g and 3-2 /ic), androstenedione-4-14C (2-0 µg and 0-15 µ ) and sodium acetate- 1-14C (285 ^g and 25 µ ). Before being used in incubations, all radioactive compounds, with the exception of sodium acetate, were purified through two paper chromatography systems, one which left the compound concerned near the origin of the chromatogram and another which moved the compound away from the origin. Extraction and identification Following the termination of incubation, the blastocysts plus the medium were extracted three times with 10 ml of ethyl acetate each time. The extracts were evaporated to dryness under nitrogen at 40° C and the residues were chromatographed on paper. The techniques used for locating radioactive and non-radioactive steroids on paper chromatograms have been described by

Downloaded from Bioscientifica.com at 09/28/2021 12:30:14PM via free access Metabolism by rabbit blastocysts 59 Eik-Nes & Kekre (1963). The methods used for measurement of radioactivity by scintillation spectrometry and crystallization of radioactive metabolites to constant specific activity have been outlined by Hall, Sozer & Eik-Nes (1964). Acetylation of steroids was carried out as described by Zaffaroni (1953). The purification procedure involving direct isotope dilution and paper chromato- graphy systems alternated with aluminium oxide column chromatography was described by Mahajan, Shah & Eik-Nes (1963). The same procedure was used in this investigation except that reverse isotope dilution was substituted for direct isotope dilution. Saponification was done by the method of Velluz, Petit, Pesez & Berret (1947). Precipitation with digitonin and subsequent hydrolysis of the digitonide was done by the method of Sperry & Webb (1950). The bromination and debromination procedures of Schwenk & Werthessen (1952) were used.

Substrate: Progesterone 4- l4C

- Ethyl Acetate Residue hexane/formamide, 3hr (PC) dry mobile phase hexane-benzene/formamide(PC ) i-Ï-i Origin 20a Dihydroprogesterone 5/3-Pregnanedione lOOug authentic compound 5/3-Pregnan-3a-ol-20-one „Hrierf hexane/formamide I aCWea 3hr(PC) Specific Activity : 54 d p m //lq _I_ hexane-benzene/formamide(PC ) 5ß-Pregnan-3a-ol-20-one 5/3-Pregnanedione column chromatographylalumina)

o ,· »..··. CCJ / acetylation Specific Activity :55d p m /^.g hexane/formamide(PC ) hexane/formamide (PC ) column chromatography(alumina) Crystallization Specific Activity: 51 d p m //xg 5/8-Pregnan-3a-ol-20-one Acetate Crystallization Text-fig. 1. Flow-sheet depicting methods used to isolate 5j8-pregnan-3a:-ol-20-one, 5ß- pregnanedione and 20ct-dihydroprogesterone produced from progesterone-4-I4C. PC, Paper chromatography.

RESULTS All incubations of blastocysts with radioactive substrates resulted in the forma¬ tion of radioactive metabolites. The final wash fraction of the blastocysts did not metabolize any of the substrates employed. Progesterone-4:-iAC metabolism The residues of extracts from incubations with progesterone-4-14C (Text-fig. 1) contained at least eight radioactive metabolites, of which two (5/?-preg- nanedione, and 5/?-pregnan-3a-ol-20-one) were identified by recrystallization (Table 1). Identification of 20a-dihydroprogesterone was by reverse isotope dilution followed by purification in two paper chromatography systems. The initial specific radioactivity of 20a-dihydroprogesterone was 54 dpm/^g. The

Downloaded from Bioscientifica.com at 09/28/2021 12:30:14PM via free access 60 Ronald L. Huff and Kristen B. Eik-Nes specific activity was 55 and 51 dpm/^g following the two purification steps. Only tentative identification of 5/?-pregnan-3a,20a-diol could be made since it represented less than 0-5% of the recovered radioactivity. Some quantitative aspects of the progesterone-4-14C incubations are given in Table 2. Total metabolism was measured by substrate disappearance. These Table 1 DATA ON RECRYSTALLIZATION OF RADIOACTIVE METABOLITES (dPM/mg)

Solvents and sequence of crystallization Compound Original Methanol Acetone Ethanol Mother liquor activity {first (second {third (after third crystallization) crystallization) crystallization) crystallization) 5/8-Pregnanedione 81 61 61 61 64 5j8-Pregnan-3a-ol-20-one (as the acetate) 391 327 343 329 320 Cholesterol* 36 35 34 35 36 Pregnenolone* (as the acetate) 193 97 97 101

* Cholesterol and pregnenolone were obtained from sodium acetate-1-14C incubations. The other two compounds were recovered from progesterone-4-14C incubations. data are corrected for losses incurred during chromatography or other purifi¬ cation steps. Incubations with 24 hr of substrate availability yielded similar results. Incubations with 72 hr of substrate availability showed increases in metabolism approximately proportional to the increase in time relative to 24-hr incubations. 20a-Dihydroprogesterone and 5/?-pregnanedione, however,

Table 2 mean metabolic rate of progesterone-4-14c by rabbit blastocysts

Pre- Incu¬ No. No. of Un¬ 20a-Di- - Za-Hydroxy- Total incu- bation of blasto- known hydro- dione pregnan- metabolism^ bation* (hr) flasks cystsjflask (%) progesterone (%) 20-one (%) (hr) (%) (%) 0 24 5-2 0-9 1-6 7-2 17-0 24 24 5-1 0-9 1-2 91 21-3 72 24 5-0 0-9 20 9-7 24-4 0 72 22-4 1-9 2-6 28-7 56-3 0 24 26-7 3-8 2-4 14-2 50-3

* Pre-incubation time is the number of hours of in-vitro cultivation prior to addition of the radio¬ active substrate. f Total metabolism is an estimate of substrate utilization measured by substrate disappearance and corrected for losses. failed to increase proportionally to the increase in time. Incubations of eight blastocysts/flask did not show as much metabolism per blastocyst as was found for single blastocysts. In contrast to the utilization of progesterone, blastocysts incubated in groups showed slightly more development than when incubated alone.

Downloaded from Bioscientifica.com at 09/28/2021 12:30:14PM via free access Metabolism by rabbit blastocysts 61 Pregnenolone-7-3H metabolism Extracts from incubations with pregnenolone-7-3H contained at least four radioactive metabolites. Three of these compounds had Chromatographie mobilities on paper in several solvent systems of authentic steroids (17a- hydroxypregnenolone, testosterone and androstenedione). The formation of these metabolites was small, and enough radioactivity for recrystallization was not available. 17'a-Hydroxypregnenolone-1'-3H metabolism Incubations with this substrate resulted in the formation of three radioactive metabolites, two of which had Chromatographie mobilities on paper in several solvent systems of authentic steroids (dehydro^z'androsterone and andro¬ stenedione). None of these metabolites could be crystallized to constant specific activity due to low yield of conversion.

Substrate: Acetate-I- C Ethyl Acetate Residue hexane/formamide (PC ) Origin 5-Pregnenolone Sterols 3hr saponification hexane/formamide, (PC) and extraction with hexane at pH 7 4 Unknown A5-Pregnenolone Radioactive Compounds acetylation hexane/formgmide(PC ) Extract Purified and Crystallized A5-Pregnenolone Acetate i Crystallization Text-fig. 2. Flow-sheet depicting methods used to isolate 5-pregnenolone and choles¬ terol from acetate-l-14C. PC, Paper chromatography.

AndrostenedioneA-1ACmetabolism One radioactive metabolite was found in extracts of incubations with andro- stenedione-4-14C and it chromatographed on paper like authentic testosterone in several solvent systems. This metabolite was not crystallized to constant specific activity. Acetate-l-14C metabolism Incubation of four blastocysts with this substrate resulted in the formation of at least three different compounds (Text-fig. 2). Pregnenolone acetate obtained by the method shown in Text-fig. 2 was recrystallized from three solvents (Table 1). The cholesterol-like material contained approximately 161,000 dpm. After digitonin precipitation, bromination, washing with cold methanol and debromination of this cholesterol fraction, a drop in specific activity from 114 to 36 dpm/mg occurred indicating that only about one-third

Downloaded from Bioscientifica.com at 09/28/2021 12:30:14PM via free access 62 Ronald L. Huff and Küsten . Eik-Nes of the original cholesterol fraction was actually cholesterol. This final fraction was crystallized to constant specific activity (Table 1). Protein hormones Incubations with icsh and acth produced visible differences in development of the embryonic disk of the blastocysts with icsh showing greater effects than acth. With icsh, development proceeded to or beyond the primitive groove in a 48-hr incubation. Although acth appeared to stimulate differentiation relative to the development of the control blastocysts, the observed changes within the embryonic disk were not as visibly distinct as with icsh. Blastocysts incubated in the presence of both trophins showed approximately the same amount of differentiation as those incubated with icsh alone.

DISCUSSION It has been shown that rabbit blastocysts grown in a synthetic medium can biosynthesize cholesterol and pregnenolone from acetate and promote bio- transformation of several steroids (Table 1). Moreover, preliminary data indicate that these organisms may contain a variety of enzymes affecting steroid metabolism. In our investigation, however, the products of some of these enzymes were not isolated by strict chemical criteria and the presence of such enzymes in the blastocysts has been tentatively inferred. Since the wash fractions of the blastocysts did not carry out measurable metabolism, it appears likely that the identified metabolites (Table 1) were produced by the blastocysts. The ability of the embryos to metabolize steroids remained approxi¬ mately constant over a 4-day period (Table 2). The failure of two of the identi¬ fied metabolites (20a-dihydroprogesterone and pregnanedione) to increase in amounts proportional to the increase in incubation time could be caused by the reversibility of the 20-dehydrogenase reaction and by the relative velocity of 4 reduction being somewhat lower than the velocity of the 3a-hydroxy- steroid dehydrogenase reaction. The reason why incubations of eight blastocysts/flask did not give the expected results as compared with individual incubations is unknown (Table 2). A possible explanation is that pH control in flasks containing eight embryos was more difficult than in those containing only one embryo, with the medium showing a slowly decreasing pH in the former incubations. The greater in¬ dividual development observed with eight blastocysts/flask suggests the possi¬ bility that some compound which augments development is absent from the medium but is transferred with blastocysts into the incubation flasks. Conse¬ quently, such a compound would occur in higher concentration in the flasks containing eight blastocysts. The data obtained when incubating blastocysts with pituitary hormones could lend support to this proposal. Both icsh and acth appeared to stimulate differentiation, with icsh showing the greater effect. It is unlikely that the small increase in protein concentration (10 to 20%) of the incubation medium following pituitary hormone addition is responsible for the effect, since a two-fold increase in the albumin concentration of the medium failed to show a comparable effect. The only comparable stimulation

Downloaded from Bioscientifica.com at 09/28/2021 12:30:14PM via free access Metabolism by rabbit blastocysts 63 found in preliminary experiments was after the addition of growth hormone which was contaminated with luteinizing hormone. This preliminary study involved only two embryos, but the mixture of growth hormone and lutein¬ izing hormone appeared to promote development proceeding to the first segment embryo in an incubation of 7 days duration. These results suggest that protein hormones produced by the mother may be necessary to normal develop¬ ment of the embryo by exerting a direct trophic effect in addition to their other functions in the pregnant female. The incubations with radioactive sodium acetate established the synthesis of sterols from acetate by the blastocysts. The occurrence of pregnenolone in these incubations, although verified in only two incubation flasks (Table 1), suggests that the blastocysts can metabolize endogenous cholesterol to steroids. The importance of such synthesis for the growth and development of the blastocysts is currently unknown. It is of interest to note that the human foetus is able to carry out extensive steroid metabolism (Diczfalusy, 1964), and it is possible to infer from the present data that an ability to conduct such metabolism is an inherent property of relatively undifferentiated embryos.

ACKNOWLEDGMENTS This work was aided by grants CA T4 5000 and AM-06651-03 from the U.S.P.H.S., Bethesda, Maryland. Adrenocorticotrophic hormone was a gift from Armour and Company and interstitial cell stimulating hormone was kindly donated by the Endocrine Study Section of the U.S. Public Health Service in the form of a preparation designated NIH-LH-S-4.

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