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In the foregoing pages, I have attempted to focus attention on a number of specific problems of interest to students of reproductive biology, toward the solution of which, in vitromethods have been applied. Some of the advantages as well as some of the limitations of such approaches have been discussed, and I have tried to emphasize the importance of interpreting with caution, the results of strictly in vitroexperiments, until some degree of confirmation can be obtained in vivo. Nevertheless, there are certain types of questions such as those concerning the characteristics of specific enzymic steps in steroid biosynthesis, and those concerning molecular mechanisms of action of gonadotropic hormones, which are considerably more amenable to in vitrothan to in vivoexperimentation. The mechanisms by which LH stimulates progesterone synthesis have been investigated extensively, and profitably by in vitromethods in a number of laboratories. Exciting as the outcome of these experiments has been, it should be emphasized that the physiologic significance of the effect of LH remains very uncertain.

Bioassays were carried out on anterior pituitaries of Holstein heifers raised on 3 levels of nutrition and slaughtered at 6 different ages to determine their growth hormone and thyrotropic hormone contents. Growth hormone per gram of anterior pituitary, growth hormone per 100 lb. of body weight and total thyrotropic hormone were greater in the younger heifers and gradually decreased with increasing age. No significant differences in any of these measurements could be detected due to plane of nutrition, although differences in total thyrotropin came very close to significance (P>0.05), the levels being highest in the heifers on the high plane of nutrition and lowest in heifers on the low plane.Rates of body growth during the 16-week periods immediately prior to slaughter were found to be positively correlated with growth hormone per gram of anterior pituitary, growth hormone per 100 lb. body weight, and total thyrotropin content of pituitaries. Thyrotropin contents were also positively correlated with relative thyroid weights of the heifers from which the pituitaries were taken.These results have been interpreted as indicating that the quantities of growth hormone and thyrotropin detected in pituitary assays are fairly close approximations of the actual rates of secretion of these hormones by the pituitary. They suggest that differences in rates of growth of heifers at different ages are at least partly due to differences in amounts of pituitary growth hormone and thyrotropin being produced, but that differences in growth rates of heifers on different feeding levels are due to differences in amounts of nutrients available for growth, rather than to any differences in hormone production by the pituitary.

Bovine LH was labeled with 125I by chloramine T oxidation and gel filtration. Immunological characterization of iodinated LH, using either gel filtration on Sephadex G-200 or double antibody precipitation to separate free and antibody-bound 125I-LH, indicated that 71 and 64%, respectively, of total radioactivity was reactable with antibody to bovine LH.A dose of 125I-LH within the physiological range (51 ng/100 g of body wt) was administered intravenously to immature female rats. Serum samples, ovaries, adrenals, thyroids, and portions of liver were taken for radioassay at times ranging from 5 min to 12 hr following injection. Disappearance of radioactivity from blood approximated a hyperbolic function with a half-life of 19.6 min. Radioactivity in liver at its 15-min maximum represented 43% of the total injected dose per whole organ. Radioactivity in ovary and adrenal appeared at similar low levels (0.05-0.07% of injected dose per pair of glands). Disappearance curves from the two organs, however, were significantly different, with radioactivity in ovary, but not in adrenal, being retained up to 30 min following injection.

Luteal growth and steroidogenesis were studied in corpora lutea from 33 heifers, five each at 2, 4, 7, 11, 18 and 20 days after estrus, and three on the day of the next anticipated ovulation (day 0). Luteal weight and total DNA growth curves leveled at day 11. Ratios of RNA/DNA was greatest during most rapid luteal growth at day 4. Free sterol concentration in fresh corpora lutea increased linearly from 1,345 µg./gm. at day 2 to 2,612 µg./gm. at day 18, but esterified sterol decreased from 618 µg./gm. at day 2 to 344 µg./gm. at day 11, and then increased to 1,081 µg./gm. on the day of the next ovulation. Corpus luteum progesterone concentration was greatest at mid-cycle (46 µg./gm.).Incubation of homogenates of corpora lutea for 1 hr. in Krebs-Ringer bicarbonate buffer resulted in synthesis of progesterone which increased from 17 µg./gm. on day 2 to 136 µg./gm. on day 11 and 150 µg./gm. on day 20, but decreased to 46 µg./gm. on the day of the next ovulation. Total progestogen synthesis at any stage of the cvcle was no greater in the presence of NADPH than in the presence of NADP+glucose-6-phosphate. LH failed to stimulate synthesis of progesterone in these luteal homogenates. Significant quantities of esterified sterol accumulated during incubation of corpora lutea from day 18, 20 and 0, but not during incubation of younger tissues and rates of esterification of cholesterol-7-3H in the incubation medium were greater in the older than in the younger corpora lutea.

Slices of corpora lutea from 4 normally cycling dairy cows have been incubated in the absence and in the presence of trans-1,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride (AY9944), an inhibitor of cholesterol biosynthesis. AY9944 (10−5M) decreased the in vitroincorporation of acetate-1-14C into cholesterol both in the absence and presence of LH, to 26 and 18% of control rates, in half-hour and 2-hour incubations, respectively. LH was equally effective in increasing the rate of net in vitroprogesterone synthesis in the presence of AY9944 as in its absence. No evidence of a stimulatory action of LH upon cholesterol synthesis was observed; in fact, LH decreased markedly, the incorporation of acetate-1-14C into cholesterol. Neither LH nor AY9944 influenced incorporation of acetate-1-14C into fatty acids of triglycerides or of phospholipids. The ability of LH to increase rate of in vitroglycolysis in this tissue was not affected by AY9944. It is concluded that a stimulation, by LH, of in vitrosynthesis of progesterone in bovine corpus luteum slices does not depend upon rate of concurrent synthesis of cholesterol, and that LH, in addition to stimulating conversion of cholesterol to progesterone, may exert an inhibitory action on cholesterol biosynthesis.

Cholesterol ester-storage granules were isolated from luteinized rat ovary and rabbit ovarian interstitial tissue by centrifugal flotation and were investigated with regard to their structure and function. Cholesterol ester, protein, phospholipid and unesterified cholesterol accounted for the dry weight of granules from luteinized rat ovary. The protein and the phospholipid were resistant to removal by washing. Substrate specificities of nucleotide phosphatase and specific radioactivities of lipid-soluble P (determined after administration of [32P]Piin vivo) were the same in granules and in a microsomal fraction from the same tissue. After administration of [32P]Piin vivo, luteinizing hormone increased the specific radioactivity of lipid-soluble P in granules, mitochondria and the microsomal fraction. Since granules did not swell in hypo-osmotic media, whereas microsomal particles did, it is suggested that adherent phospholipid and protein in granule suspensions is unlikely to result from contamination with endoplasmic reticulum. Luteinizing hormone administered in vivo increased the phospholipid and unesterified cholesterol contents of isolated granules relative to their cholesterol ester content, and also tended to raise their protein content. This treatment decreased the ability of isolated granules to act as a substrate for cholesterol esterase in vitro and increased the activity of cholesterol esterase. Cycloheximide in vivo also raised the unesterified cholesterol/cholesterol ester ratio of isolated granules, and when administered with luteinizing hormone acted synergistically to bring about a further increase. These results are considered compatible with evidence obtained by microscopy which suggests that granules may be surrounded by a membrane, that they arise by pinching off from the endoplasmic reticulum, and that they shrink on trophic stimulation of the tissue.

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