A high molecular weight fraction XM100R (MW A 100,000) was prepared by ultrafiltration from ovine pineals using two different extraction methods under red light conditions (lambda greater than 600 nm). This fraction stimulates the release of radioimmunologically active luteinizing hormone (LH) of anterior pituitaries in vitro. The ultrafiltration fraction PM30R (MW greater than 30,000 and less than 100,000) was found to be radioimmunologically active only when the "Bensinger" extraction procedure was applied. However, when comparable fractions were prepared under green light and incubated with half-pituitaries, all the incubation media of the ultrafiltrated fractions, XM100R, PM30R, PM10R (MW greater than 10,000 and less than 30,000) UM2R (MW greater than 1000 and less than 10,000), UM05R (MW greater than 500 and less than 1000) and UM05F (MW greater than 500), reacted with anti-LH. This may mean that under green light conditions the high molecular weight ovine pineal compounds in XM100R are disintegrated and/or split up into small molecules which can stimulate the release of LH, or crossreact with the anti-LH serum.

Former work has shown that crude extracts of ovine pineal glands probably exert a stimulating activity on the release of gonadotropins of anterior pituitaries in vitro. By aqueous extraction followed by ultrafiltration through anisotropic membranes high Mr (above 100,000 daltons) fractions were obtained, which exhibit a stimulating effect on the levels of gonadotropins in the medium of either cultured pituitary cells or anterior hemipituitaries in short-term culture. Partial purification of a pineal luteinizing hormone release stimulating factor was accomplished by Sephadex G-150 filtration with a biopotency of 226 +/- 23 micrograms LH-RP-1 equivalents per mg protein and without an affinity for binding to anti-LHRH or anti-LH antibodies. The present data substantiate that high Mr forms, slightly heavier than authentic pituitary LH (Mr 23,000 daltons) and therefore not identical to the hypothalamic decapeptide LH-RH, represent ovine pineal factors which can increase the concentration of LH in the medium of cultured anterior pituitaries, but does not influence the secretion of prolactin in vitro.

High molecular weight substances could be isolated from sheep pineals with the "Bensinger" extraction method, followed by ultrafiltration of the waterlayer through different diaflomembranes. Two of the pineal fractions, XM100R and PM30R, stimulate the gonadotropin releasing activity of the medial basal hypothalamus (MBH). In experiments in which comparable pineal fractions were incubated without MBH and without pituitary and injected in immature mice no effect was detectable. All experiments in which a similar amount of rat cerebral cortex was used for incubation with pineal fractions did not show any activity.

In springtime only, LH release in young immature female rats shows an increase at 2 p.m. Pinealectomy, performed early after birth, abolishes this increase.