Lighting up the nuclear pore complex.

It is generally accepted that transport through the nuclear pore complex (NPC) involves an abundance of phenylalanine-glycine rich protein domains (FG-domains) that serve as docking sites for soluble nuclear transport receptors (NTRs) and their cargo complexes. But the precise mechanism of translocation through the NPC allowing for high speed and selectivity is still vividly debated. To ultimately decipher the underlying gating mechanism it is indispensable to shed more light on the molecular arrangement of FG-domains and the distribution of NTR-binding sites within the central channel of the NPC. In this review we revisit current transport models, summarize recent results regarding translocation through the NPC obtained by super-resolution microscopy and finally discuss the status and potential of optical methods in the analysis of the NPC.

4Pi microscopy of the nuclear pore complex.

To explore whether super-resolution fluorescence microscopy is able to resolve topographic features of single cellular protein complexes, a two-photon 4Pi microscope was used to study the nuclear pore complex (NPC). The microscope had an axial resolution of 110-130 nm and a two-color localization accuracy of 5-10 nm. In immune-labeled HeLa cells, NPCs could be resolved much better by 4Pi than by confocal microscopy. When two epitopes of the NPC, one localized at the tip of the cytoplasmic filaments and the other at the ring of the nuclear basket, were immune-labeled, they could be clearly resolved in single NPCs, with the distance between them determined to be 152 +/- 30 nm. In cells expressing a green fluorescent protein construct localized at the NPC center, the distances between the ring of the nuclear filaments and the NPC center was 76 +/- 12 (Potorous tridactylus cells) or 91 +/- 21 nm (normal rat kidney cells), whereas the distance between the NPC center and the tips of the cytoplasmic filaments was 84 +/- 18 nm, all values in good agreement with previous electron or single-molecule fluorescence estimates. We conclude that super-resolution fluorescence microscopy is a powerful method for analyzing single protein complexes and the cellular nanomachinery in general.

Meiosis in autologous ectopic transplants of immature testicular tissue grafted to Callithrix jacchus.

Grafting of immature testicular tissue provides a tool to examine testicular development and may offer a perspective for preservation of fertility in prepubertal patients. Successful xenografting in mice, resulting in mature spermatids, has been performed in several species but has failed with testicular tissues from the common marmoset, Callithrix jacchus. Previous data indicate that the hormonal milieu provided by the mouse host might cause this failure. We conducted autologous ectopic transplantation of testicular fragments under the back skin in newborn marmoset monkeys. Seventeen months after transplantation, we found viable transplants in 2 out of the 4 grafted animals. In the transplants, tubules developed up to a state intermediate between the pregraft situation and adult controls. Dividing spermatogonia and primary spermatocytes were present. Boule-like positivity and CDC25A negativity indicated that spermatogenesis was arrested at early meiosis. Immunohistochemistry revealed normal maturation of Sertoli cells, Leydig cells, and peritubular cells. Serum testosterone values were not restored to the normal range and bioactive chorionic gonadotropin levels increased to castrate levels. Meiotic arrest could have occurred in the grafts because of lack of sufficient testosterone or because of hyperthermia caused by the ectopic position of the grafts. We conclude that autologous transplants of immature testicular tissues in the marmoset can mature up to meiosis but that normal serum testosterone levels are not restored. Further studies have to be performed to overcome the meiotic arrest to explore the model further and to develop therapeutic options.

GnRH-antagonist mediated down-regulation of the estrous cycle in marmosets.

BACKGROUND: Because of its small size and unproblematic captivity behavior the marmoset monkey is an attractive New World primate model for early developmental questions. However, superovulation protocols used in Old World monkeys and women are not successful in the female marmoset. A novel protocol is needed to utilize these New World monkeys as an efficient animal model for in vitro fertilization experiments or embryo stem cell research. METHODS: To create such a protocol we first examined the effects of long-term estrous cycle control, secondly, in a dose-finding study, we determined the length of a down-regulation protocol with a gonadotropic releasing hormone (GnRH)-antagonist. Twenty-nine female marmosets were grouped according to the number of estrous cycles, which had been controlled for a period of 12 months in which 88 cycles were monitored. Application of PGF2alpha in the mid-luteal phase led to immediate onset of the follicular phase. The blood progesterone concentration rapidly declined and increased again on day 9-11. RESULTS: The results show that the controlled ovarian cycle length and progesterone response are not altered by the number of PGF2alpha injections. The rapid decline was similar in all groups, indicating that all animals, independent of the number of controlled cycles, react equally to multiple PGF2alpha injections. To determine the proper dosage for a GnRH-antagonist (Cetrorelix), 12 animals in three groups of four female marmosets were treated with two different dosages and a sham dosage. Cetrorelix was applied in the mid-luteal phase, three times over 2 days. In both Cetrorelix-treated animal groups the early progesterone levels matched those in the controls. In the low-dose treatment group [0.01 mg/100 g body weight (BW)] the expected progesterone rise on day 10 was delayed between 9 and 15 days whereas in the high-dose treatment group (0.1 mg/100 g BW) the progesterone rise was delayed between 21 and 41 days. In the low-dose group the steepness of the slope from day 20 onwards was almost identical to that of the control group. This was reflected in the bioCG levels measured. CONCLUSIONS: Based on the GnRH-antagonist studies, complete ovarian down-regulation in female marmosets can be achieved by applying a low-dose regimen, and intrinsic gonadotropins would not interfere with an ovarian superstimulation protocol.