Functional homodimeric glycoprotein hormones: implications for hormone action and evolution.

BACKGROUND: Human chorionic gonadotropin (hCG), lutropin, follitropin, and thyrotropin act as alpha beta heterodimers to control reproduction and thyroid function. The alpha and beta subunits of these proteins are divided into three loops (alpha 1,alpha 2,alpha 3; beta 1,beta 2,beta 3) by cysteine knots and the heterodimer is stabilized by 20 beta-subunit residues wrapped around alpha 2 like a seatbelt. Understanding how these hormones interact with their receptors, a matter of considerable dispute, would facilitate design of pro- and anti-fertility agents. RESULTS: By swapping alpha 2 for beta 2 and vice versa and, in some cases, adding an amino-terminal coiled-coil dimerization domain, we prepared homodimeric analogs that have the conformation found in each 'half' of hCG. Homodimers containing loops beta 1,alpha 2,beta 3 and none, part, or all of the seatbelt stimulated signal transduction to the same extent as hCG, albeit with lower potency. Those containing alpha 1,beta 2,alpha 3 were inactive. CONCLUSIONS: The activities of homodimers containing the beta 1,alpha 2,beta 3 groove exceed those of other minimized analogs more than 100-1000-fold, suggesting this portion of the hormone forms the major receptor contact. The discovery that glycoprotein hormone heterodimers can be converted to functional homodimers supports the proposal that this protein family evolved from an active homodimeric ancestor by gene duplication and acquisition of mutations to loop 2 that prevent homodimerization. This approach to protein minimization should be applicable to other proteins composed of architecturally related subunits, including those that might have arisen by gene duplication.

Eimeria clethrionomysis sp. n., Eimeria gallatii sp. n., Eimeria pileata sp. n. and Eimeria marconii sp. n. from the red-backed vole Clethrionomys gapperi Vigors, from Pennsylvania.

Four new eimerian species are described from red-backed voles, Clethrionomys gapperi in Pennsylvania. Sporulated oocysts of Eimeria clethrionomyis sp. n. are ellipsoidal, 18.8 (16.5-21.5) x 14.9 (14.0-16.5) with elongate, ovoid sporocysts, 10.6 (9.5-12.0) x6.1 (5.5-7.0). The oocyst wall is smooth, with 2 layers, and thins, with terminal cap at one or both ends. Polar granules, dark Stieda body and sporocyst residuum are present. The oocyst residuum is absent. Sporulated oocysts of Eimeria gallatii sp. n. are ellipsoidal, 27.7 (21-32) x 19.3 (17-24) with ovoid sporocysts, 13.5 (12-15) x 8.8 (8-10). The oocyst wall is smooth, 2-layered, with a micropyle and thin wall at the end opposite the micropyle. Polar granules, Stieda body and sporocyst residuum are present. The oocyst residuum is atypical, of cobwebby material. Sporulated oocysts of Eimeria pileata sp. n. are subspherical to spherical, 25.2 (20.5-29.5) x 22.5 (19.5-25.5) with ellipsoidal sporocysts, 13.4(10.5-15.0) x 8.4 (7.5-9.5). The oocyst wall is rough, pitted, striated, 2-layered, with no micropyle. Polar granules, oocyst and sporocyst residuum, Stieda body and stiedal cap are present. Sporulated oocysts of Eimeria marconii sp. n. are ellipsoidal, 13.0 (10.5-15-0) x 10.6 (9.5-12.0) with elongate, ovoid sporocysts, 7.7 (7.0-8.5) x 4.2 (3.0-4.5). The oocyst wall is smooth, single-layered, with no micropyle. Polar granules, dark Stiedal body and sporocyst residuum are present. There is no oocyst residuum.

Development in cell cultures of Eimeria vermiformis Ernst, Chobotar and Hammond, 1971.

Development of Eimeria vermiformis from sporozoite to mature first-generation schizonts in cultured bovine kidney cells, Madin-Darby bovine kidney cells, and primary cultures of whole mouse embryos is described. Intracellular sporozoites were seen at 5 min, and for as long as 120 h after inoculation. Sporozoites were observed penetrating cells, with uninucleate trophozoites and immature schizonts with 2--6 nuclei first appearing 24 h after inoculation. Schizonts with 6 or more nuclei, as well as mature schizonts containing first-generation merozoites, were first seen between 36 and 48 h after inoculation of all 3 cell types used. The first indication of merozoite formation was determined by the appearance of small protuberances of cytoplasm at the periphery of schizonts. Merozoites began development at the periphery of schizonts and were later observed radiating from a central body of cytoplasm, 14--20 merozoites being formed. Some mature schizonts retained a small spherical residual body after merozoite formation was completed. After the rupture of schizonts, intracellular merozoites, which contained anterior and posterior refractile granules, were seen at 48, 72 and 96 h postinoculation. Merozoites were not seen entering or leaving cells. No further development was observed.

The sporulated oocyst of Eimeria colini sp. n. from the bobwhite quail, Colinus virginianus.

The sporulated oocyst of Eimeria colini sp. n. is described from the bobwhite quail, Colinus virginianus. The broadly ellipsoid oocysts measured 24.8 micronm (22.4 to 28.0) by 20.9 micronm (17.9 to 22.4). The L/W ratio was 1.2 (1.1 to 1.4). An inconspicuous micropyle was present while an oocyst residuum and polar granule were absent. The oocyst walls were 1.4 micronm thick and covered with small, evenly spaced elevations. The sporocysts measured 14.2 micronm (12.3 to 14.5) by 8.4 micronm (7.8 to 8.9). A sporocyst residuum, small Stieda body and large substieda body were present. Ooocysts first appeared in the feces between 84 to 96 hr. Freshly-passed oocysts were completely sporulated by 48 hr at 25 C.