Ovarian follicular superstimulation and oocyte maturation in the anoestrous southern hairy-nosed wombat, Lasiorhinus latifrons.

This study investigates the effect of three exogenous gonadotrophin regimens on ovarian follicular development in southern hairy-nosed wombats during the non-breeding season. Females were given either porcine follicle stimulating hormone (pFSH; total of 200 mg at 12 h intervals over 7 (Group 1), or 4 days (Group 2)), or pregnant mares' serum gonadotrophin (PMSG; single dose of 150 I.U. (Group 3)). In all treatment groups 25 mg of porcine luteinising hormone (pLH) was used to trigger maturation; Groups 1 and 2 received pLH 12 h after the final pFSH injection and Group 3 received pLH 72 h after PMSG. The results showed Group 1 produced significantly more follicles per ovary (5.91+/-1.28) than Group 2 (1.67+/-0.62), or Group 3 (2.17+/-1.16) at p<0.05. Control females received saline injections concurrently with the three treatment groups (n=6; 2 control animals for each treatment group). No follicular development occurred in any control female. Analysis of oocyte nuclear status revealed that while oocytes from all three treatment groups had resumed meiosis, only those in Group 1 (7-day pFSH/pLH treatment) progressed to metaphase II. These results have implications for the development of assisted breeding strategies in this species.

Effect of exogenous gonadotrophins on ovarian morphology and oocyte maturation in the southern hairy nosed wombat Lasiohinus latifrons during the breeding season.

The effect of the exogenous administration of porcine follicle-stimulating hormone (pFSH) and pregnant mare serum gonadotrophin (PMSG) on ovarian follicular development and oocyte maturation in the southern hairy nosed wombat Lasiorhinus latifrons was investigated. Three experimental groups were administered pFSH at various doses and for different treatment lengths, followed by 25 mg porcine luteinising hormone (pLH) 12 h after the last dose of pFSH. Another group was given PMSG followed 72 h later by 25 mg pLH. Animals were killed 24 h after pLH. The left ovary was fixed for histology and the morphology of the antral follicles was determined, whereas follicular oocytes in the right ovary were aspirated, fixed, stained with 42,62-diamidino-2-phenylindole, and viewed for nuclear maturation. There was no significant difference in the mean number of ovarian follicles >1 mm, or in the size class of follicles assessed between control and experimental groups. However, a trend was observed suggesting a possible increase in follicles >3.0 mm in experimental groups compared with control animals. In all females administered exogenous porcine gonadotrophins, but not controls, some of the mural granulosa cells of large tertiary antral follicles had markedly enlarged nuclei (approximately 14 microm in diameter). All oocytes from the control group remained at the germinal vesicle stage, whereas approximately 40% of oocytes retrieved from the pFSH groups and 82.4% retrieved from the PMSG-primed animals had undergone germinal vesicle break down, with a small number reaching meiosis II. The present study shows that exogenous administration of either pFSH or PMSG to hairy nosed wombats can induce follicular growth and oocyte maturation. Such findings could be useful in the development of reproductive technology in this species.

An anxiolytic action of oxytocin is enhanced by estrogen in the mouse.

The established role of oxytocin (OT) in facilitation of steroid-modulated reproductive and affiliative behaviors led to the speculation that it may have anxiolytic actions under certain hormonal conditions. NIH-Swiss mice were tested for responsiveness to OT in two behavioral tests of anxiety, the holeboard apparatus and elevated plus-maze. Dose-response assessment indicated that 3 mg/kg was the optimal dose for peripherally administered (IP) OT on the elevated plus-maze. There were no consistent effects at any dose on the holeboard apparatus. In ovariectomized mice pretreated with estradiol (E2), peripherally administered OT increased the number of entrances onto the open arms, as well as the amount of time on the open arms compared to other groups (ANOVA; p < 0.05). There was little to no effect of OT in ovariectomized animals not pretreated with E2. When OT was administered intracerebroventricularly (ICV), there was an increase in entrances and time on the open arms compared to that of females infused with arginine vasopressin (AVP). This increase was apparent in ovariectomized females, but was further enhanced in those pretreated with E2 (ANOVA; p < 0.05). In contrast, the combination of E2 pretreatment and ICV AVP decreased the number of entrances and time spent on the open arms of the elevated plus-maze compared to those receiving OT, suggesting an estrogen-modulated anxiogenic action of AVP. Analyses of [125]I-OVTA binding density indicated a significant increase in binding density in the lateral septum of E2-treated females compared to OIL-treated controls (ANOVA; p < 0.05). There was no effect of E2 treatment on [125]I-OVTA binding density in the amygdala or ventromedial nucleus of the hypothalamus. Taken together, these data indicate that OT exerts an anxiolytic action that is enhanced in the presence of circulating estrogen. This behavioral effect may be mediated by estrogen-induced increases in OT binding density in the lateral septum and may be important to the facilitation of social interactions.

Multiple cis-trans conformers of the prolactin receptor proline-rich motif (PRM) peptide detected by reverse-phase HPLC, CD and NMR spectroscopy.

An eight-amino-acid synthetic peptide (IIe1-Phe2-Pro3-Pro4-Val5-Pro6-Gly7-Pro8) corresponding to the conserved proline-rich motif (PRM) of the intracellular domain of the prolactin receptor (PRL-R) was studied by one- and two-dimensional (1D and 2D) proton NMR spectroscopy in water and DMSO in order to characterize its conformational dynamics. The purified PRL-R PRM peptide eluted as two partially resolved peaks in equilibrium on reverse-phase HPLC (RP-HPLC) at 20 degrees C with a ratio of 60:40. At 30 degrees C, the two peaks coalesced into a single peak The two RP-HPLC peaks correspond to two peptide conformers resulting from the slow cis-trans isomerization of one of the four proline amide bonds. Although the peptide has only three amide (NH) protons, its ID NMR spectrum in water contains approximately 15 discernible NH region peaks, providing evidence for multiple conformers. The amide resonances were assigned on the basis of 2D-COSY spectra, chemical shift values resonance splitting patterns and temperature coefficients. The cis:trans ratio for each proline in water, calculated from integrated intensities and/or peak heights of the appropriate resonances, were Phe2-Pro3 (35:65), Pro3-Pro4 (40:60), Val5-Pro6 (70:30), and Gly7-Pro8 (30:70). Temperature studies (25-70 degrees C) were used to semi-quantitatively estimate the rates of isomerization for the different prolines. In water, Pro8 undergoes rapid isomerization; Pro3 isomerizes at an intermediate rate; while Pro4 and Pro6 both appear to isomerize very slowly since no coalescence of amide resonances was observed. In DMSO, only Pro4 displayed slow isomerization. Slow kinetics combined with a similar 60:40 ratio of conformers determined by RP-HPLC and NMR suggests that isomerization of the Pro3-Pro4 bond generates the two RP-HPLC peaks. Both proximal and distal proline isomerization effects were observed in NMR experiments. All of the 16 theoretical (24 = 16) proline configurations appear to exist in equilibrium in water The predominant (19%) conformation, trans3-trans4-cis6-trans8, may reflect the configuration of the PRM prolines in the native PRL-R. Isomerization of Pro6 from cis to trans generates an interaction between the peptide N-and C-termini, suggesting an overall pseudo-cyclic conformation. This all-trans proline configuration may play an important biochemical role in the function of cytokine/haematopoietin receptors. A model is proposed which suggests that isomerization of the PRM by an immunophilin such as the FK 506-binding protein (FKBP) serves as an on-off switch for cytokine receptor activation.

Hypotensive effects of 5-HT1A receptor activation: ventral medullary sites and mechanisms of action in the rat.

Serotonin-1A (5-HT1A) binding sites were previously localized in several regions of the ventral medulla associated with neural regulation of the cardiovascular system. Some of these binding sites were associated with serotonergic neurons of the ventral medulla. The purpose of these studies was to assess and characterize hypotensive responses to a 5-HT1A agonist, (8-hydroxy-dipropylaminotetraline, 8-OH-DPAT), administered to the ventral medulla of the rat, to correlate the responsive ventral medullary sites with the distribution of 3H-8-OH-DPAT binding sites, and to assess the role of serotonergic systems in mediating the hypotensive responses. Ventral medullary application of 8-OH-DPAT caused dose-related reductions in mean arterial pressure and heart rate which were mediated by the autonomic nervous system. The hypotensive response to 8-OH-DPAT was attenuated by pretreatment with the 5-HT1A antagonists, spiperone or NAN-190. Microinjections of 8-OH-DPAT into ventral medullary structures revealed that 8-OH-DPAT responsive sites included the raphe pallidus, the parapyramidal region, and the rostral ventrolateral medulla. The role of serotonergic terminals in mediating the responses of 8-OH-DPAT was evaluated in animals pretreated with the serotonin nerve toxin, 5,7-dihydroxytryptamine (5,7-DHT). Cardiovascular responses to ventral medullary application of 8-OH-DPAT were unaffected by the selective depletion of serotonin. Thus, whereas the hypotensive responses elicited by 8-OH-DPAT in the raphe pallidus and parapyramidal region may involve serotonergic neurons, other non-serotonergic sites (e.g. the rostral ventrolateral medulla) can mediate the hypotensive actions of 8-OH-DPAT.