FPP modulates mammalian sperm function via TCP-11 and the adenylyl cyclase/cAMP pathway.

Fertilization promoting peptide (FPP; pGlu-Glu-ProNH2), which is found in seminal plasma, promotes capacitation but inhibits spontaneous acrosome loss in mammalian spermatozoa in vitro. Adenosine, known to modulate the adenylyl cyclase (AC)/cAMP pathway, elicits these same responses whereas FPP + adenosine produces an enhanced response, leading to the hypothesis that FPP and adenosine modulate the same signal transduction pathway but act via different receptors. TCP-11, the product of a t-complex gene, is the putative receptor for FPP: Fab fragments of anti-TCP-11 antibodies have the same effect as FPP on mouse spermatozoa and Gln-FPP, a competitive inhibitor of FPP, also competitively inhibits responses to the Fab fragments. In the present study, specific binding of 3H-FPP to sperm membranes was significantly inhibited by 200 nM Gln-FPP and anti-TCP-11 Fab fragments (1/25 dilution), thus confirming that FPP, Gln-FPP, and Fab fragments compete for the same binding site. In addition, spermatozoa treated with A23187 to induce the acrosome reaction bound significantly less 3H-FPP than untreated cells, suggesting that a large proportion of the FPP binding sites are associated with the acrosomal cap region; TCP-11 is located in this region. In other experiments, 100 nM FPP significantly stimulated cAMP production in mouse sperm membranes, permeabilized cells and intact cells. Furthermore, Gln-FPP inhibited production of cAMP in response to FPP but not to adenosine (10 microM) or its analogue NECA (100 nM), supporting the involvement of two different receptors. Finally, anti-TCP-11 Fab fragments (1/25 dilution) significantly stimulated cAMP production, whereas low Fab (1/200; nonstimulatory when used alone) plus adenosine (10 microM) significantly enhanced the stimulation of capacitation by adenosine. These results support the hypotheses that TCP-11 is the receptor for FPP and that FPP<-->TCP-11 interactions modulate AC/cAMP.

TCP-11, the product of a mouse t-complex gene, plays a role in stimulation of capacitation and inhibition of the spontaneous acrosome reaction.

Tcp-11 is a candidate for a distorter gene within the t-complex on mouse chromosome 17; although t-complex genes appear to affect sperm function, relatively little is known about mechanisms whereby these genes might play a specific physiological role. We present evidence that the protein TCP-11 is found on the surface of mature epididymal spermatozoa. Although detected on both the acrosomal cap region of the head and the flagellum of acrosome-intact cells, it is absent from the heads of acrosome-reacted cells. When epididymal spermatozoa were incubated in the presence of anti-TCP-11 IgG Fab fragments for a total of 120 min and assessed using chlortetracycline fluorescence, we observed a stimulation of capacitation and an inhibition of spontaneous acrosome loss, suggestive of enhanced fertility compared with untreated suspensions. In vitro fertilization experiments confirmed that Fab-treated suspensions became fertile more quickly and then maintained high fertility. Because these responses were remarkably similar to those obtained using the TRH-related peptide FPP (fertilization promoting peptide; pGlu-Glu-ProNH2) and adenosine, we investigated responses to Fab fragments, FPP, and adenosine. Results indicated that the Fab fragments appear to work at the same extracellular site as FPP, one that is distinct from the adenosine site of action. Further evidence for this conclusion was obtained using pGlu-Gln-ProNH2, an FPP-related tripeptide known to competitively inhibit responses to FPP; as with FPP, pGlu-Glu-ProNH2 inhibited the stimulatory effect of Fab fragments in a concentration-dependent manner. From these results we suggest that TCP-11 may be the receptor for FPP and that the adenylate clyclase/cyclic AMP pathway may be the signal transduction pathway activated by interactions between extracellular effector molecules (e.g., Fab fragments or FPP acting as an agonist) and TCP-11. A mechanism such as this that promotes capacitation but inhibits spontaneous acrosome loss in vivo would play a very important role by helping to maximize the fertilizing potential of the few spermatozoa that reach the site of fertilization. The fact that there is a human homolog of Tcp-11 suggests that this gene could play an important role in regulation of human, as well as mouse, sperm function.

Transcription of Cauliflower mosaic virus DNA: detection of promoter sequences, and characterization of transcripts.

Four RNA transcripts encoded by cauliflower mosaic virus DNA have been detected in the polyadenylated RNA from virus-infected turnip leaves. Two of these transcripts, the major 35S and the 8S species, have the same 5' termini, at nucleotide 7435. A viral DNA fragment encompassing this region directs transcription initiation at this point in vitro. The 5' terminus of the 19S transcript is at nucleotide 5764, and a corresponding viral DNA fragment also directs transcription initiation in vitro. The major 35S RNA is a complete transcript of the circular viral genome, and is 3'-coterminal with 19S RNA at nucleotide 7615. The 8S RNA has its 3' extremity at delta 1, the single-stranded interruption in the transcribed strand of virion DNA. A minor 35S RNA has also been detected that has its 5' and 3' termini at delta 1.

Structure and 5'-termini of the large and 19 S RNA transcripts encoded by the cauliflower mosaic virus genome.

Cauliflower mosaic virus-infected leaves accumulate two major RNA species late in infection-a 19 S RNA and a large RNA transcript. We have found that the large transcript is approximately full genome length. The 3'-end of the large transcript is located at or near a site corresponding to the 3'-end of the 19 S RNA. The 5'-end of the large transcript maps near its own 3'-end and only about 100 bp downstream from the terminator codon for coding region VI. The 5'-end of the large transcript is situated at the beginning of the large intergenic region, about 600 bp upstream from the single-strand break in the transcribed viral DNA strand. The location of this site is of particular interest because it suggests that synthesis of the large transcript proceeds across the DNA break site. The 5'-end of the 19 S RNA maps in the small intergenic region between coding regions V and VI about 20-30 bp upstream from the initiation codon for coding region VI. By comparing the transcription initiation sites to DNA sequences for the CaMV genome, it can be shown that TATATAA sequences lie 20-30 bp upstream from the 5'-ends of both the 19 S and large transcripts.

Cloned Cauliflower Mosaic Virus DNA Infects Turnips (Brassica rapa).

Cauliflower mosaic virus DNA cloned in the Sal I site of bacterial plasmid pBR322 infects turnip plants. The cloned viral DNA must be excised from the recombinant plasmid to infect, but need not be circularized and ligated in vitro. The cloned viral DNA lacks site-specific single-strand breaks found in DNA obtained directly from the virus. However, these breaks are reintroduced into the viral genome during multiplication of the virus in the plant host.