The effect of H-2 region and genetic background on hormone-induced ovulation rate, puberty, and follicular number in mice.

We have examined the effects of major histocompatibility (H-2) haplotypes and genetic background (all loci other than the H-2 region) on hormone-induced ovulation rate in congenic strains of mice. In comparison with the H-2a haplotype, the H-2b haplotype increased hormone-induced ovulation rate 92% on the A/J (A) genetic background. However, H-2 haplotype did not affect hormone-induced ovulation rate on the C57BL/10J (C57) genetic background. The H-2b-linked gene(s) increased hormone-induced ovulation rate on the A/J genetic background largely by (1) enhancing the maturation of follicles in response to pregnant mare's serum gonadotrophin (PMSG) and (2) altering the stages of follicular development which can be induced to ovulate in response to human chorionic gonadotrophin (hCG). The observed effects of H-2 on hormone-induced ovulation rate were not explained by differences in the timing of puberty, the number of follicles present in untreated females, or the incidence of follicular atresia. The effect of genetic background on hormone-induced ovulation rate was much greater than was the effect of the H-2 region. We found that hormone-induced ovulation rate was five- to six-fold higher on the C57 genetic background than on the A genetic background. The C57 genetic background increased hormone-induced ovulation rate by (1) enhancing the induction of follicular maturation in response to gonadotropins and (2) by reducing the incidence of follicular atresia.

Plant mitochondrial DNA evolves rapidly in structure, but slowly in sequence.

We examined the tempo and mode of mitochondrial DNA (mtDNA) evolution in six species of crucifers from two genera, Brassica and Raphanus. The six mtDNAs have undergone numerous internal rearrangements and therefore differ dramatically with respect to the sizes of their subgenomic circular chromosomes. Between 3 and 14 inversions must be postulated to account for the structural differences found between any two species. In contrast, these mtDNAs are extremely similar in primary sequence, differing at only 1-8 out of every 1000 bp. The point mutation rate in these plant mtDNAs is roughly 4 times slower than in land plant chloroplast DNA (cpDNA) and 100 times slower than in animal mtDNA. Conversely, the rate of rearrangements is extraordinarily faster in plant mtDNA than in cpDNA and animal mtDNA.

Unusual structure of geranium chloroplast DNA: A triple-sized inverted repeat, extensive gene duplications, multiple inversions, and two repeat families.

Physical and gene mapping studies reveal that chloroplast DNA from geranium (Pelargonium hortorum) has sustained a number of extensive duplications and inversions, resulting in a genome arrangement radically unlike that of other plants. At 217 kilobases in size, the circular chromosome is about 50% larger than the typical land plant chloroplast genome and is by far the largest described to date, to our knowledge. Most of this extra size can be accounted for by a 76-kilobase inverted duplication, three times larger than the normal chloroplast DNA inverted repeat. This tripling has occurred primarily by spreading of the inverted repeat into regions that are single copy in all other chloroplast genomes. Consequently, 10 protein genes that are present only once in all other land plants are duplicated in geranium. At least six inversions, occurring in both the inverted repeat and large single-copy region, must be postulated to account for all of the gene order differences that distinguish the geranium genome from other chloroplast genomes. We report the existence in geranium of two families of short dispersed repeats and hypothesize that recombination between repeats may be the major cause of inversions in geranium chloroplast DNA.

Unicircular structure of the Brassica hirta mitochondrial genome.

Restriction mapping studies reveal that the mitochondrial genome of white mustard (Brassica hirta) exists in the form of a single circular 208 kb chromosome. The B. hirta genome has only one copy of the two sequences which, in several related Brassica species, are duplicated and undergo intramolecular recombination. This first report of a plant mitochondrial DNA that does not exist in a multipartite structure indicates that high frequency intramolecular recombination is not an obligatory feature of plant mitochondrial genomes. Heterologous filter hybridizations reveal that the mitochondrial genomes of B. hirta and B. campestris have diverged radically in sequence arrangement, as the result of approximately 10 large inversions. At the same time, however, the two genomes are similar in size, sequence content, and primary sequence.

Tricircular mitochondrial genomes of Brassica and Raphanus: reversal of repeat configurations by inversion.

We constructed complete physical maps of the tripartite mitochondrial genomes of two Crucifers, Brassica nigra (black mustard) and Raphanus sativa (radish). Both genomes contain two copies of a direct repeat engaged in intragenomic recombination. The outcome of this recombination in black mustard is to interconvert a 231 kb master chromosome with two subgenomic circles of 135 kb and 96 kb. In radish, a 242 kb master chromosome interconverts with subgenomic circles of 139 kb and 103 kb. The recombination repeats are 7 kb in size in black mustard and 10 kb in radish, and are nearly identical except for two insertions in the radish repeat relative to the black mustard one. The two repeat configurations present on the master chromosome of black mustard are located on the subgenomes of radish and vice-versa. To explain this, we postulate the existence of an evolutionarily intermediate mitochondrial genome in which the recombination repeats were (are) present in an inverted orientation. The recombination repeats described for these two species are completely different from those previously found in the closely related species B. campestris, implying that such repeats are created and lost frequently in plant mitochondrial DNAs and making it less than likely that recombination occurs in a site-specific manner.

Alpha and luteinizing hormone beta messenger ribonucleic acid (RNA) of male and female rats after castration: quantitation using an optimized RNA dot blot hybridization assay.

In this study we examined the changes in alpha and LH beta mRNAs in anterior pituitaries of male and female rats after castration. mRNA concentrations were measured by an optimized RNA dot blot hybridization assay. Rat alpha and LH beta cDNAs were nick-translated to specific activities of 2-5 X 10(8) cpm/micrograms and were used as hybridization probes. The total RNA per assay, RNA per dot, and saturating amounts of probe were optimized. The intra- and interassay coefficients of variation were 5% and 28%, respectively. Both alpha and LH beta mRNA concentrations increased after castration, but marked differences were observed in the kinetics of responses in male and female rats. In males, alpha and LH beta mRNAs were increased by 24 h postcastration (by 25% and 38%, respectively), and 4- to 5-fold increases over intact controls were evident by 18 days. Alpha mRNA rose rapidly and had doubled by 2 days, whereas LH beta mRNA concentrations showed a similar increase by 6-7 days postcastration. The slower rise in LH beta mRNA was associated with a transient decline in serum and pituitary LH concentrations between 2 and 6 days after castration. In female rats, alpha mRNA increased more slowly. Alpha concentrations had doubled by 10 days, while a similar increase in LH beta mRNA occurred 7 days after castration. Thereafter, both subunit mRNAs continued to rise, and by day 20 alpha mRNA was increased 5-fold and LH beta mRNA 16-fold over values in intact females. Serum and pituitary LH concentrations rose gradually, and both were increased by 7-10 days after castration. The increase in serum and pituitary LH followed a time course similar to that of the progressive rise in LH beta mRNA concentrations. These data show that an increase in steady state LH subunit mRNA concentrations is one of the mechanisms involved in increased gonadotropin biosynthesis and secretion after castration. The kinetics of LH subunit mRNA and LH secretory responses are different in male and female rats and suggest that the concentration of LH beta mRNA may be a limiting factor in LH secretion.

Regulation of pituitary gonadotropin-releasing hormone (GnRH) receptors by pulsatile GnRH in female rats: effects of estradiol and prolactin.

GnRH has been shown to modulate the concentration of its own pituitary receptors (GnRH-R), and changes in GnRH-R during the rat estrous cycle may reflect changes in GnRH secretion. To examine the relationship between GnRH and GnRH-R in female rats, we measured GnRH-R and serum gonadotropin responses to pulsatile GnRH in restrained ovariectomized (OVX) and ovariectomized estradiol-implanted (OVX-E2) rats. In addition, we examined the effects of suppression of serum PRL. Pulsatile injections of GnRH (10-250 ng/pulse) given every 30 min for 24 or 48 h did not increase GnRH-R in OVX or OVX-E2 rats compared to that in saline controls (246 +/- 27 fmol/mg). Bromocriptine treatment (2 mg/day) had no effect on GnRH-R in OVX animals. In contrast, OVX-E2 rats treated with bromocriptine showed significantly increased GnRH-R (500 +/- 43 fmol/mg) in response to GnRH injections. When ovine PRL was administered to bromocriptine-treated OVX-E2 rats, the GnRH induced rise in GnRH-R was abolished. Gonadotropin responses to GnRH were not correlated with changes in GnRH-R. In OVX animals, LH was only elevated in response to 250-ng pulses of GnRH. In OVX-E2 animals, basal LH was increased by all doses of GnRH, and acute responses to 50- and 250-ng pulses were observed. Bromocriptine treatment resulted in increased LH sensitivity to GnRH in OVX rats, but did not further enhance the responses in OVX-E2 animals. We conclude that in female rats, the presence of both E2 and a low serum PRL level is necessary for GnRH to increase GnRH-R, and the interaction of these factors may be involved in the regulation of GnRH-R during the estrous cycle.

Hyperprolactinemia inhibits gonadotropin-releasing hormone (GnRH) stimulation of the number of pituitary GnRH receptors.

Gonadotropin secretion is diminished in the presence of hyperprolactinemia, and previous studies have shown that PRL can reduce GnRH secretion and impair LH responses to GnRH. To investigate the mechanisms of the inhibitory effects of PRL on the pituitary, we administered intraarterial pulse injections of GnRH (25 ng/pulse every 30 min) to castrate testosterone-implanted male rats placed in restraint cages. Serum PRL, GnRH receptor (GnRH-R), and LH responses to GnRH were measured at intervals over 72 h. In control animals which received saline pulses, serum PRL was transiently elevated to the range of 100-150 ng/ml during the first 24 h, GnRH-R remained stable (approximately 300 fmol/mg protein) and serum LH was low (less than 10 ng/ml) throughout the 72 h. GnRH pulses in castrate testosterone-implanted animals increased GnRH-R to values (approximately 600 fmol/mg) similar to those in castrate controls (no testosterone implant, saline pulses) through 48 h, but GnRH-R declined to baseline values by 72 h in both groups. Serum LH responses to GnRH pulses were only present at 24 h. Administration of bromocriptine throughout the 72 h to immobilized castrate rats or to castrate testosterone-replaced animals treated with GnRH pulses suppressed serum PRL, and GnRH-R concentrations remained elevated through 72 h. Serum LH responses to GnRH pulses were 5- to 20-fold higher in bromocriptine-treated rats, and responses were present throughout the 72 h of the experiment. Delaying the start of bromocriptine treatment until 36 h (after the spontaneous PRL peak) resulted in reduced GnRH-R and LH responses at 72 h. Similarly, administration of ovine PRL (during the first 48 h) to bromocriptine-treated rats produced low GnRH-R concentrations at 72 h. Thus, the transient elevation of PRL seen in immobilized rats can inhibit the GnRH-stimulated increase in GnRH-R and is associated with reduced LH responses to GnRH. These results indicate that PRL has a direct inhibitory effect on the gonadotrope and suggest that impaired GnRH-R responses to GnRH are one of the mechanisms involved in the diminished gonadotropin secretion seen in hyperprolactinemia.

The frequency of gonadotropin-releasing hormone stimulation determines the number of pituitary gonadotropin-releasing hormone receptors.

Gonadotropin-releasing hormone (GnRH) induces both synthesis and release of pituitary gonadotropins, but rapid or slow frequencies of stimulation result in reduced LH and FSH secretion. We determined the effects of frequency of GnRH stimulation on pituitary GnRH receptors (GnRH-R). Castrate male rats received testosterone implants (cast + T) to inhibit endogenous GnRH secretion. GnRH pulses were injected by a pump into a carotid cannula and animals received GnRH (25 ng/pulse) at various frequencies for 48 h. In control animals (saline pulses) GnRH-R was 307 +/- 21 fmol/mg protein (+/- SE) in cast + T and 598 +/- 28 in castrates. Maximum GnRH-R was produced by 30-min pulses and was similar to that seen in castrate controls. Faster or slower frequencies resulted in a smaller GnRH-R response and GnRH given every 240 min did not increase GnRH-R over saline controls. Equalization of the total GnRH dose/48 h (6.6 ng/pulse every 7.5 min or 200 ng/pulse every 240 min) did not increase receptors to the maximum concentrations seen after 30-min (25 ng) pulses. Serum LH responses after 48 h of injections were only present after 30-min pulses, and peak FSH values were also seen after this frequency. Serum LH was undetectable in most rats after other GnRH frequencies, even though GnRH-R was increased. These data show that GnRH pulse frequency is an important factor in the regulation of GnRH-R. A reduction of GnRH-R is part of the mechanism of down-regulation of LH secretion by fast or slow GnRH frequencies, but altered frequency also exerts effects on secretory mechanisms at a site distal to the GnRH receptor.