Copulatory plugs inhibit the reproductive success of rival males

Ejaculated proteins play important roles in reproductive fitness. In many species, seminal fluid coagulates and forms what has been referred to as a copulatory plug in the female's reproductive tract. In mice, previous work demonstrated that knockout males missing a key seminal fluid protein were unable to form a plug and less successful at siring litters in noncompetitive matings (one female, one male), probably the result of reduced sperm transport or insufficient stimulation of the female. Here, we extend these previous studies to competitive matings (one female, two males) and make two key insights. First, when first males were unable to form a plug, they lost almost all paternity to second males to mate. Thus, the copulatory plugs of second males could not rescue the reduced fertility of first males. Second, we showed that the copulatory plug of first males effectively blocked fertilization by second males, even if first males were vasectomized. Taken together, our experiments demonstrated that first males lost almost all paternity if they never formed a plug. We discuss our results in the context of natural populations, where in spite of the strong effects seen here, pregnant female mice regularly carry litters fertilized by more than one male.     

Delineation of critical amino acids in activation function 1 of progesterone receptor for recruitment of transcription coregulators

The activation functions AF1 and AF2 of nuclear receptors mediate the recruitment of coregulators in gene regulation. AF1 is mapped to the highly variable and intrinsically unstructured N terminal domain and AF2 lies in the conserved ligand binding domain. The unstructured nature of AF1 offers structural plasticity and hence functional versatility in gene regulation. However, little is known about the key functional residues of AF1 that mediates its interaction with coregulators. This study focuses on the progesterone receptor (PR) and reports the identification of K464, K481 and R492 (KKR) as the key functional residues of PR AF1. The KKR are monomethylated and function cooperatively. The combined mutations of KKR to QQQ render PR isoform B (PRB) hyperactive, whereas KKR to FFF mutations abolishes as much as 80% of PR activity. Furthermore, the hyperactive QQQ mutation rescues the loss of PR activity due to E911A mutation in AF2. The study also finds that the magnitudes of the mutational effect differ in different cell types as a result of differential effects on the functional interaction with coregulators. Furthermore, KKR provides the interface for AF1 to physically interact with p300 and SRC-1, and with AF2 at E911. Intriguingly, the inactive FFF mutant interacts strikingly stronger with both SRC-1 and AF2 than wt PRB. We propose a tripartite model to describe the dynamic interactions between AF1, AF2 and SRC-1 with KKR of AF1 and E911 of AF2 as the interface. An overly stable interaction would hamper the dynamics of disassembly of the receptor complex.     

Polarised absorption spectroscopy of chlorophyll-lipid membranes

A technique has been developed for measuring visible absorption spectra of chlorophyll in lipid membranes. An expression is derived which enables the directions of the transition moments of the different absorption bands to be determined from polarisation data. It is found that the transition moments of the principal blue and red absorption bands of chlorophyll a make angles of 26° and 36.5° respectively with the plane of the membrane. On the assumption that these two transitions lie in the plane of the porphyrin ring and are mutually perpendicular, it may be deduced that the plane of the porphyrin ring is tilted at approx. 48° to the membrane surface. For chlorophyll b the transition moments of the blue and red bands are found to make angles of 29.5° and 36.5° with the plane of the bilayer, giving an angle of tilt of the porphyrin ring of approx. 51°.

These results are compared with measurements of dichroism in vivo.     

Purification and characterization of a co(II)-sensitive alpha-mannosidase from Ginkgo biloba seeds

An alpha-mannosidase was purified from developing Ginkgo biloba seeds to apparently homogeneity. The molecular weight of the purified alpha-mannosidase was estimated to be 120 kDa by SDS-PAGE in the presence of 2-mercaptoethanol, and 340 kDa by gel filtration, indicating that Ginkgo alpha-mannosidase may function in oligomeric structures in the plant cell. The N-terminal amino acid sequence of the purified enzyme was Ala-Phe-Met-Lys-Tyr-X-Thr-Thr-Gly-Gly-Pro-Val-Ala-Gly-Lys-Ile-Asn-Val-His-Leu-. The alpha-mannosidase activity for Man(5)GlcNAc(1) was enhanced by the addition of Co(2+), but the addition of Zn(2+), Ca(2+), or EDTA did not show any significant effect. In the presence of cobalt ions, the hydrolysis rate for pyridylaminated Man(6)GlcNAc(1) was significantly faster than that for pyridylaminated Man(6)GlcNAc(2), suggesting the possibility that this enzyme is involved in the degradation of free N-glycans occurring in developing plant cells (Kimura, Y., and Matsuo, S., J. Biochem., 127, 1013-1019 (2000)). To our knowledge, this is the first report showing that plant cells contain an alpha-mannosidase, which is activated by Co(2+) and prefers the oligomannose type free N-glycans bearing only one GlcNAc residue as substrate.