Evaluation of a filter-prepared platelet concentrate for the treatment of suspensory branch injuries in horses.

OBJECTIVES: Platelet preparations have become a treatment for soft tissue injuries in horses. This study evaluated a novel filter-based system to concentrate platelets and assesses its value in the treatment of suspensory ligament branch desmitis. METHODS: Filtered platelet concentrate was prepared from 55 ml of venous blood obtained from 21 normal horses. Platelets and white blood cells in whole blood and filtered platelet concentrate were measured, as was platelet activating factor (PAF)-induced platelet-derived growth factor-BB (PDGF-BB) release. Eleven horses with 18 focal acute suspensory ligament branch injuries were treated intra-lesionally with autologous filtered platelet concentrate and evaluated clinically and ultrasonographically for one to three years. RESULTS: The increase in concentration of platelets in the filtered concentrate in comparison with whole blood (6.9 ± 1.9-fold) was significantly greater than the increase in white blood cells (3.8 ± 0.8-fold) (p <0.0001). There was no effect of sex or breed on platelet concentration. Platelets were responsive to PAF with >100-fold higher levels of PDGF release over basal levels. All hypoechoic lesions re-evaluated within three months had resolved. Five of the 11 horses returned to their previous level of work, one was exercising at a lower level, three were retired, one died for unrelated reasons, and one was still convalescing. CLINICAL SIGNIFICANCE: Filtered platelet concentrate was easily and reliably prepared and injected into suspensory ligament branch injuries without short-term complications. This treatment was associated with rapid resolution of ultrasonographic lesions and lameness. Filtered platelet concentrate represents a convenient alternative for the treatment of suspensory ligament branch injuries.

Point-of-use water filtration reduces healthcare-associated infections in bone marrow transplant recipients.

Outbreaks of infection with gram-negative bacteria (GNB) have been linked to hospital water. We sought to determine whether point-of-use (POU) water filtration might result in decreased risk of infection in hospitalized bone marrow transplant (BMT) recipients in the absence of any recognized outbreak. Unfiltered water was sampled from taps in the BMT unit of a major US teaching hospital, and cultured at a reference laboratory. POU bacterial-retentive filters (0.2 mum) were installed throughout the unit, and replaced every 14 days. Infection rates were tracked over a 9-month period, and compared with rates for a 16-month period before POU filtration. Unfiltered water samples from 50% (2 of 4) outlets sampled grew P. aeruginosa (2 of 4) and Stenotrophomonas maltophilia (1 of 4). Clinical infection rates in the unit were significantly reduced from 1.4 total and 0.4 GNB infections per 100 patient days in the period before POU filtration to 0.18 total and 0.09 GNB infections per 100 patient days (P=0.0068 and 0.0431, respectively) in the 9-month period for which filters were in place. Infections during the POU filtration period were due to non-waterborne organisms. Point-of-use (POU) water filtration may significantly reduce infection rates in BMT recipients in the absence of any recognized outbreak.

The use of a bacteria detection system to evaluate bacterial contamination in PLT concentrates.

BACKGROUND: Random-donor PLTs (RDPs) are functional at 7 days. Nevertheless, since the mid-1980s, concern for bacterial contamination has caused the storage period to be reduced to 5 days. The ability of a bacteria detection system (BDS, Pall) to determine bacterial contamination and permit extension of the PLT shelf life to 7 days was assessed. STUDY DESIGN AND METHODS: Blood was collected into CP2D and leukoreduced RDPs were prepared. Upon arrival at the hospital, a 2- to 3-mL aliquot was removed from each RDP and introduced into the Pall BDS pouch with a sterile docking device. The pouch was incubated at 37 degrees C for 24 hours and then the oxygen content was measured to determine bacterial contamination. Additionally, the RDPs were pooled and an aliquot was removed for culture with standard manual techniques. CCIs were calculated 1 hour after infusion. RESULTS: A total of 12,062 individual RDPs were tested. The Pall BDS detected bacteria in 5 units. All of these were positive on repeat sampling. Propionibacterium acnes, coagulase-negative Staphylococcus, and Bacillus species were confirmed by manual technique in 3 units, one could not be identified, and one was negative. Aliquots from PLT pools were positive in 80 of 2201 pools when tested by manual methods. Of these, 79 were false-positives and 1 unit contained coagulase-negative Staphylococcus. The Pall BDS was easy to use and required less than 5 minutes for all manipulations. After 7 days of storage, the PLTs gave an average CCI of 16 x 10(11)+/- 3.39 x 10(11) 1 hour after transfusion (n = 9). CONCLUSIONS: The Pall BDS permits evaluation of RDPs for bacterial contamination. Culture-negative PLTs were successfully transfused in our institution up to and including 7 days after storage with good CCIs.

Conditioned blood reperfusion during angioplasty (CoBRA) treatment of acute myocardial infarction.

Acute myocardial infarct (MI) results in ischemia distal to lesions which puts heart muscle at risk for reperfusion injury (RI). Neutrophils, platelets and complement are putative mediators of RI. Recent advances in filtration technology provide integrated neutrophil and platelet removal together with complement-attenuating properties in a single blood-conditioning device. The present study characterizes the properties of a blood-conditioning filter and describes its clinical effect when used in conjunction with active hemoperfusion for acute MI. The filter reduces leukocytes by 99.9998 +/- 0.0002% (p<0.0001) and platelets by 99.9934 +/- 0.0069% (p<0.0001). Human plasma, derived from heparinized blood that was 'conditioned' by filtration, was studied using the Langendorff isolated rabbit heart preparation. The deposition of membrane attack complex and the resultant functional myocardial impairments [reflected in hemodynamic and biochemical measurements, including developed pressure, coronary blood flow, lymph-derived myocardial creatine kinase (CK)] are significantly attenuated by blood conditioning. Integration of the blood-conditioning filter into an active hemoperfusion system during primary percutaneous transluminal coronary angioplasty (PTCA) for acute MI (n=8) did not delay the procedure or cause any complications. Reperfusion of occluded coronary arteries with 300 cm3 of conditioned blood led to significant improvement in echocardiographic global wall motion scores (in standard deviations) following treatment (-1.64 +/- 0.18 to -1.45 +/- 0.15, p=0.02). Initial reperfusion of totally occluded coronary arteries with conditioned blood leads to acutely improved ventricular function. Collectively, these data provide a strong indication for continued investigation of conditioned blood reperfusion in angioplasty following acute MI for the long-term effect upon recovery of salvagable myocardium.

Potential for reduction in morbidity and cost with total leucocyte control for cardiac surgery.

The economics of health care in the USA and abroad has caused a shift in the focus on therapeutic interventions that transcend issues of safety and clinical efficacy. Now, cost justification is emerging as a major consideration to influence clinical practice. This brief review of the medical literature attempts to identify leucocyte-mediated adverse reactions that develop in open-hear surgery, quantify the costs incurred to manage such reactions and infer the savings that may accrue by controlling the burden of leucocytes presented to the open-heart surgical patient using commercially available leucocyte reducing filtration technology.

Regulation of gonadotropin subunit messenger ribonucleic acid expression by gonadotropin-releasing hormone pulse amplitude in vitro.

The present study examined the effect of GnRH pulse amplitude on alpha, LH beta and FSH beta mRNAs using an in vitro perfusion model. Pituitaries from 30-day-old female rats were dissociated and the cells plated for 48 h to allow attachment to collagen-coated microcarrier beads. The beads were loaded into perifusion chambers, preperifused for 1 h, and then given GnRH pulses (17.5-175 pg/ml) every 30 min for 24 h. Perifusate LH was measured after 2 h and 22 h of perifusion and alpha LH and FSH beta messenger RNAs (mRNAs) were determined by hybridization to complementary DNA (cDNA) probes. All doses of GnRH acutely stimulated LH release, and responses were similar after 2 h and 22 h. LH release increased as a function of GnRH pulse dose with maximal increases seen following 70 pg/ml pulses. alpha mRNA levels (control = 0.73 +/- 0.1 fmol cDNA bound/100 micrograms pituitary DNA) were increased 30% and 40% after 24 h of 35 and 70 pg/ml pulses, respectively (P < 0.05 vs. media controls). LH beta mRNA concentrations (control = 0.44 +/- 0.08 fmol cDNA bound) were only elevated after 35 pg/ml GnRH pulses (36% increase). FSH beta mRNA showed the largest responses to GnRH pulses, increasing by 45% and 84% after 35 and 70 pg pulses, respectively (control = 0.14 +/- 0.02 fmol bound). The highest GnRH pulse dose (175 pg/ml) was ineffective in stimulating an increase in FSH beta mRNA levels. These results show that all three gonadotropin subunit mRNA concentrations increase after 24 h of GnRH pulses, but the pattern of individual subunit mRNA responses was dependent upon the amplitude of the GnRH pulse stimulus. These data support earlier results in vivo, in that the subunit responses to GnRH pulse dose were similar. Thus, alterations in the amplitude of pulsatile GnRH secretion from the median eminence may be one mechanism by which the expression of gonadotropin subunit genes are regulated.

Differential actions of thyrotropin (TSH)-releasing hormone pulses in the expression of prolactin and TSH subunit messenger ribonucleic acid in rat pituitary cells in vitro.

The influence of TRH pulse pattern on PRL and TSH alpha- and beta-subunit gene expression was examined in vitro. Pituitaries from adult female rats were dissociated and plated for 48 h to allow attachment to collagen-coated microcarrier beads. The beads were perifused for 24 h with TRH (pulses or continuous). To examine the effects of TRH pulse amplitude, TRH pulses (0.032-100 nM) were given every 60 min (controls received either medium pulses or continuous 100 nM TRH). PRL mRNA rose progressively to a peak at 4 nM TRH/pulse (93% increase vs. medium-pulsed controls), but higher TRH pulse amplitudes were less effective. alpha-Subunit mRNA also rose with increasing TRH pulse dose, with maximal (137%) elevations after 100-nM pulses. TSH beta mRNA concentrations were increased by TRH pulse doses between 0.8-20 nM, but a clear dose-response pattern was not seen. Continuous TRH (100 nM) resulted in PRL, TSH beta, and alpha mRNAs that were less than the values in medium-pulsed controls. To assess the effects of pulse frequency, 4-nM TRH pulses were given at intervals between 15-240 min (controls received medium pulses or continuous 4 nM TRH). PRL mRNA was increased (55-107% increase) after all pulse intervals, except 240 min, and rose to a similar degree after 4 nM TRH given continuously. alpha-Subunit mRNA concentrations increased by a lesser degree and also did not rise after the slowest (240 min) pulse interval. In contrast, TSH beta mRNA levels increased progressively as pulse intervals were increased, and maximal (85%) elevations were seen after 240-min pulses. Continuous 4 nM TRH did not alter alpha or TSH beta mRNAs. These results show that pulsatile TRH is more effective than continuous TRH in stimulating PRL, TSH beta, and alpha mRNAs. Further, the pattern of TRH pulsatile signals can influence the expression of these pituitary hormone genes in a differential manner.

Gonadal steroids effect similar regulation of gonadotrophin subunit mRNA expression in both male and female rats.

Gonadal steroids can act both indirectly via gonadotrophin-releasing hormone (GnRH) and directly on the pituitary to regulate gonadotrophin subunit gene expression. Recent studies to assess a possible direct action at the pituitary have shown that testosterone, when given to males in the absence of endogenous GnRH action, selectively increases FSH-beta mRNA concentrations. Conversely, in females, oestradiol appears to regulate gonadotrophin subunit mRNAs primarily via GnRH. The present study was designed to determine whether these differing results reflect specific actions of the gonadal steroids themselves or different responses of the pituitary gonadotroph cells in males and females. Rats which had been castrated 7 days earlier were given silicone elastomer implants (s.c.) containing oestradiol (plasma oestradiol 68 +/- 4 ng/l) in males or testosterone (plasma testosterone 3.5 +/- 0.3 micrograms/l) in females in the absence or presence of a GnRH antagonist. Seven days later pituitaries were removed and steady-state mRNA concentrations measured by dot-blot hybridization. In males, oestradiol reduced LH-beta and FSH-beta but not alpha mRNA. The antagonist reduced levels of all three subunit mRNAs in males and the addition of oestradiol had no further effect, suggesting that oestradiol regulates gonadotrophin subunit gene expression in males by suppressing GnRH secretion. In females, testosterone reduced all three subunit mRNAs though FSH-beta remained threefold higher than in intact animals. The GnRH antagonist was as effective as testosterone alone and reduced alpha and LH-beta to levels found in intact animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Dexamethasone alters responses of pituitary gonadotropin-releasing hormone (GnRH) receptors, gonadotropin subunit messenger ribonucleic acids, and gonadotropins to pulsatile GnRH in male rats.

Dexamethasone (Dex), when administered in high doses, has been shown to suppress spontaneous and GnRH-induced gonadotropin secretion, but the level and the mechanism(s) of this effect are unknown. We administered Dex to castrate testosterone-replaced male rats to determine if gonadotropin gene expression is affected and whether Dex differentially influences GnRH-modulated parameters of gonadotrope function: induction of GnRH receptors (GnRH-R) and gonadotropin synthesis and secretion. GnRH was given iv at 25 ng/pulse at 8, 30, and 120 min intervals for 48 h. Rapid GnRH injection frequency preferentially increased alpha and LH-beta messenger RNA (mRNA) responses to GnRH as well as LH secretion. Slower GnRH injection frequencies were required to increase levels of GnRH-R, FSH-beta mRNA, and FSH secretion. Dex selectively inhibited the serum LH, alpha, and LH-beta mRNA responses to GnRH, but not the serum FSH or FSH-beta mRNA responses. Additionally, it augmented the GnRH-induced increase in GnRH-R. We conclude: 1) induction of GnRH-R, gonadotropin synthesis, and secretion require different modes of GnRH stimulation; 2) Dex acts directly on the gonadotrope to differentially modulate GnRH-induced increases in GnRH-R levels, gonadotropin gene expression, and gonadotropin secretion; and 3) GnRH effects upon induction of GnRH-R, LH, and FSH synthesis and secretion are likely to be mediated via different cellular pathways.

A pulsatile gonadotropin-releasing hormone stimulus is required to increase transcription of the gonadotropin subunit genes: evidence for differential regulation of transcription by pulse frequency in vivo.

Previous results have shown that the pattern of GnRH pulses (amplitude and frequency) can differentially regulate expression of gonadotropin subunit cytoplasmic messenger RNA (mRNA) concentrations. The present study examined the effect of GnRH pulses on alpha, LH-beta and FSH-beta transcription rates as determined by nuclear runoff transcription assay. GnRH pulses (saline to controls) were given to castrate, testosterone-replaced male rats, and the rate of subunit gene transcription was measured in isolated pituitary nuclei. The effect of GnRH treatment duration was examined by giving GnRH pulses (25 ng/pulse at 30-min intervals) for 1, 4, or 24 h. The basal transcription rates [expressed as parts per million (ppm)] were 82 +/- 25 for alpha; 39 +/- 19 for LH-beta and 27 +/- 6 ppm for FSH-beta, and transcription rates of all 3 subunits were elevated at 1 h (3-5-fold vs. saline controls). After 4 h of GnRH pulses, alpha and FSH-beta transcription rates were reduced vs. 1 h, but LH-beta mRNA synthesis rate was maintained. At 24 h, the alpha transcription rate was still increased (66%), but LH-beta and FSH-beta transcription rates had fallen to basal levels despite the continuing pulsatile GnRH stimulus. The second experiment investigated the effect of the duration of GnRH pulses (25 ng/pulse, every 30 min for 4 h or 24 h), on cytoplasmic subunit mRNA concentrations to assess if the initial 4-h increase in transcription rate would induce a rise in cytoplasmic mRNAs. After 4 h of GnRH pulses, alpha and LH-beta mRNAs were unchanged, but FSH-beta mRNA had increased by 36% (P less than 0.05) compared to controls. All 3 subunit mRNAs were increased (approximately 2-fold) by 24 h of GnRH pulses. Administering GnRH pulses for 4 h followed by 20 h of saline pulses did not increase alpha mRNA; LH-beta was slightly increased (P less than 0.05), but FSH-beta mRNA concentrations were similar to levels seen after 24 h of continued GnRH pulses. The third experiment examined the effects of a continuous GnRH infusion and different GnRH pulse frequencies on gonadotropin subunit transcription rates. GnRH (25 ng/pulse) was given at intervals of 8, 30, or 120 min for 4 h (saline to controls). The continuous GnRH infusion (200 ng/h) did not increase the transcription rate of any of the three subunit mRNAs. alpha-subunit transcription rate was increased 2.7- or 4-fold by GnRH pulses given every 8 or 30 min, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Gonadotropin-releasing hormone pulses: regulators of gonadotropin synthesis and ovulatory cycles.

The data reviewed present evidence that the pattern of GnRH secretion is an important factor in the regulation of gonadotropin subunit gene expression, gonadotropin synthesis, and secretion. The information on regulation of mRNA expression by GnRH pulses should be considered with some caution, as the experiments were performed in male rats and may not accurately reflect events in female primates or humans. However, an overall pattern emerges which suggests that common factors may be involved in all mammalian species. If current evidence is correct, and only a single gonadotropin-releasing hormone exists, then mechanisms to differentially regulate the three gonadotropin genes may involve changes in GnRH secretion. Alterations in GnRH pulse frequency and amplitude are recognized by the pituitary gonadotrope cell and could be the mechanism used to effect differential expression of the gonadotropin subunit genes. Differential regulation of subunit gene expression would be expected to be critically important in the establishment of pubertal maturation, and subsequently in the maintenance of ovulatory cycles in women. Our hypotheses, proposing a major role of pulsatile GnRH secretion in the regulation of human reproduction, are summarized in schematic form in Fig. 14 for men and Fig. 15 for women. In utero and during the first few months of life, GnRH is secreted at a relatively fast frequency (approximately 1 pulse/hour). During the first year, GnRH secretion is inhibited and both the amplitude and apparent frequency of pulsatile release is markedly reduced. The mechanisms involved in inhibiting GnRH release remain unclear in humans. Similarly, the mechanisms involved in the disinhibition of GnRH secretion, which first occurs during sleep at the initiation of puberty, are unclear, but in humans do not appear to involve opiates. In males, the increased frequency and amplitude of GnRH secretion favor LH synthesis and release, which in turn stimulates testosterone secretion (Fig. 14). Testosterone acts at the hypothalamus, perhaps through opioid mechanisms, to inhibit GnRH pulse frequency and to maintain a regular pattern of pulses occurring approximately every 90-110 min in adult males. In females, the mechanisms involving alterations in the patterns of GnRH secretion to regulate reproduction appear more complex. This may reflect the need to differentially synthesize and secrete FSH and LH at different times during reproductive cycles to allow orderly follicular maturation and ovulation. As shown in Fig. 15, we hypothesize that the events during the first decade of life and through the initiation of nocturnal GnRH secretion at puberty are similar in both sexes.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential regulation of gonadotropin subunit gene expression by gonadotropin-releasing hormone pulse amplitude in female rats.

The role of GnRH in regulating gonadotropin subunit gene expression was examined in adult female rats. Animals were ovariectomized, estradiol implants inserted sc, and jugular cannulae placed into the right atria. On the next day, animals were given GnRH pulses (saline to controls) every 30 min for up to 48 h and alpha, LH beta, and FSH beta mRNA levels measured by hybridization to cDNA probes. To determine the effects of GnRH treatment duration, rats received GnRH pulses (25 ng at 30-min intervals) for 6, 12, 24, and 48 h. FSH beta mRNA was increased (by 92%) after 6 h of pulses and remained elevated through 48 h. alpha mRNA was not increased until 12 h (27% increase) and rose further (57%) by 48 h. LH beta mRNA levels were only transiently increased at 12 h (67%) and values were not different from saline controls after 24 or 48 h. To examine whether the rise in serum PRL which is characteristic of the ovariectomized-estradiol animal model was responsible for the decrease in LH beta mRNA responsiveness to GnRH over longer durations, studies were repeated in bromocriptine-treated animals (0.6 mg sc, twice daily). The results showed similar response patterns for all three subunit mRNAs including the decrease in LH beta after 48 h. A third experiment examined the effect of varying GnRH pulse amplitude (0.5-250 ng/pulse at 30-min intervals) over 12 h. alpha mRNA levels were increased by all GnRH doses greater than 5 ng with maximum responses after 250 ng pulses. LH and FSH beta mRNAs were both elevated by GnRH pulse doses of 0.5-25 ng (P less than 0.05 vs. saline controls). Maximal increases (2-fold) were seen after 5 ng pulses for LH beta and after 15-ng pulses for FSH beta mRNA. These results show that pulsatile GnRH increases FSH beta mRNA more rapidly than alpha or LH beta mRNAs in female rats. In addition, high amplitude GnRH pulses increase only alpha mRNA, whereas both LH beta and FSH beta mRNAs show maximum responses to lower doses. The data suggest that alterations in the amplitude of the GnRH pulsatile signal can exert differential effects on gonadotropin gene expression.

Testosterone differentially modulates gonadotropin subunit messenger ribonucleic acid responses to gonadotropin-releasing hormone pulse amplitude.

Testosterone (T) inhibits GnRH secretion and can also modulate the effects of GnRH on gonadotropin synthesis and secretion. To assess the effect of T on GnRH stimulation of alpha, LH beta, and FSH beta mRNA expression, we replaced T at three levels to reproduce low (1.5 +/- 0.5 ng/ml), medium (3.5 +/- 0.3 ng/ml), and high (6.2 +/- 0.6 ng/ml) physiological plasma concentrations. Additionally, as peripheral conversion to dihydrotestosterone (DHT) or estradiol (E2) may mediate T action, the effects of GnRH pulses in the presence of DHT and E2 were also studied. Male rats were castrated, and steroids were replaced via implants containing either T (three doses) or DHT or E2 (two doses each). GnRH pulses (10-250 ng/pulse) were administered iv at 30-min intervals for 48 h. Pituitary subunit mRNA concentrations, gonadotropin content, and LH and FSH secretion were determined. The patterns of alpha, LH beta, and FSH beta mRNA responses to increasing GnRH pulse amplitude were similar at all concentrations of plasma T. Alpha mRNA concentrations were increased 2- to 4-fold by GnRH pulses. At the same plasma T concentration, all doses of GnRH produced similar increases in alpha mRNA, but the response tended to be lower at the higher (6.2 ng/ml) levels of T. LH beta mRNA showed a clear dependence on GnRH pulse amplitude, with the maximum responses (2- to 3-fold) occurring after 10- to 25-ng GnRH pulses. At the higher (3.5 and 6.2 ng/ml) T concentrations, the dose-response curve was shifted to the left. The lowest GnRH pulse dose (10 ng) produced maximum responses, and LH beta mRNA increments in response to the higher GnRH doses were suppressed. FSH beta mRNA concentrations were increased by T in saline-pulsed controls. FSH beta mRNA responses were similar (2- to 3-fold) after all GnRH doses and at all concentrations of T. Increasing GnRH pulse doses reduced the pituitary content of both LH and FSH at all levels of T. Acute LH secretion was maximal after 10- and 25-ng pulses of GnRH when plasma T was low, but increased progressively with GnRH dose at the highest plasma T concentrations. Plasma FSH did not show any differential responsiveness to GnRH pulse dose or to increasing plasma T. Thus, LH synthesis and secretion are affected more than those of FSH by changing plasma concentrations of T. T may modulate posttranslational events in LH secretion. The higher GnRH doses effected LH release without increasing LH beta mRNA in the presence of higher physiological concentrations of T.(ABSTRACT TRUNCATED AT 400 WORDS)

Gonadotropin subunit messenger RNA concentrations after blockade of gonadotropin-releasing hormone action: testosterone selectively increases follicle-stimulating hormone beta-subunit messenger RNA by posttranscriptional mechanisms.

Regulation of gonadotropin gene expression by sex steroids may occur via direct effects on the pituitary and/or indirect effects of steroids mediated through hypothalamic GnRH. We aimed to define the effects of testosterone (T) on alpha, LH beta, and FSH beta mRNA expression in the male rat after blockade of GnRH action on the gonadotrope. A water-soluble GnRH antagonist was administered iv to castrate male rats (increased endogenous GnRH secretion) and to castrate T-replaced rats in which gonadotropin subunit mRNAs had been increased by prior treatment with exogenous GnRH pulses. In castrate male rats, GnRH antagonist resulted in a fall in all three subunit mRNAs. Alpha and LH beta declined at slower rates (half-disappearance after 50 and 65 h, respectively), and neither fell to values present in intact rats over 84 h. In contrast, FSH beta mRNA declined more rapidly, with a half-disappearance after 20 h. In castrate T-replaced rats, alpha mRNA declined at a rate similar to that in castrates (half-disappearance after 50 h). LH beta declined more slowly, and the rate of FSH beta decline was markedly prolonged in the presence of T (half-disappearance time increased from 20 to 50 h). These results suggest that T exerts direct effects on FSH beta transcription or mRNA stability which are independent of GnRH action. To assess these possibilities, a long-acting GnRH antagonist (Detirelix) was administered to castrate male rats, which also received T or sham implants 4 days after castration. FSH beta mRNA levels fell during the 4 days of Detirelix alone, but the addition of T on day 4 resulted in a 2-fold rise in FSH beta mRNA, restoring FSH beta mRNA to levels present in intact rats. Serum FSH closely paralleled FSH beta mRNA concentrations. Alpha mRNA was reduced by 25%, and LH beta mRNA concentrations were unchanged in the presence of T. The rate of alpha mRNA transcription was markedly reduced and that of LH beta tended to fall in T-treated rats, but T had no significant effect on the FSH beta transcription rate. Thus, the action of T to increase concentrations of cytosolic FSH beta mRNA appears to be exerted at a posttranscriptional level, possibly via effects of T on FSH beta mRNA stability. This may represent a mechanism by which T can effect differential regulation of gonadotropin subunit mRNA concentrations.

Gonadal regulation of gonadotropin subunit gene expression: evidence for regulation of follicle-stimulating hormone-beta messenger ribonucleic acid by nonsteroidal hormones in female rats.

Gonadectomy results in a rise in gonadotropin secretion and subunit gene expression, although the relative contributions of declining gonadal hormones or increasing hypothalamic GnRH secretion are uncertain. To further delineate the roles of the hypothalamus and gonads in regulation of gonadotropin gene expression, male and female rats were castrated and gonadotropin subunit messenger RNA (mRNA) concentrations measured 2, 7, 14, or 21 days (d) later. In males, FSH beta mRNA was maximal (2-fold increase) by 7 d while peak levels of alpha (3-fold) and LH beta (3-fold) were seen by 14 d. Testosterone (T) replacement restored all three subunit mRNA concentrations to intact values. In females, FSH beta mRNA also reached plateau levels (8-fold increase) earlier than alpha (3-fold) or LH beta (11-fold). When female rats ovariectomized 7 days earlier were given estradiol (E2) and progesterone (P) implants for up to 14 d, suppression of alpha and LH beta to intact levels was observed. However, FSH beta mRNA concentrations only decreased to 67% of castrate values, and remained 2- to 3-fold higher than levels in intact female rats. Female rats were also given E2 replacement at the time of ovariectomy. LH beta mRNA was maintained at intact levels for 14 days while alpha and FSH beta showed partial castration responses (2-fold and 3-fold, respectively). Finally, to determine whether E2 and P regulate gonadotropin subunit expression directly or by reducing GnRH secretion, female rats were ovariectomized and immediately replaced with E2, P, or E2 + P in the presence or absence of a GnRH antagonist (A) for 2 d. alpha mRNA was increased (2-fold) by E2 but not by E2 + A suggesting that E2 requires the presence of GnRH to increase alpha mRNA. P alone was ineffective, but both E2 and A prevented the LH beta mRNA response to ovariectomy. The effects of E2 and A were not additive, suggesting that E suppresses LH beta mRNA by inhibiting the increase in GnRH secretion. In contrast, the FSH beta mRNA response to ovariectomy was only partially suppressed by E2, E2 + P, or E2 + P + A. These data indicate that in castrate males, replacement of T suppresses all three subunit mRNAs to intact levels. However, replacement of E2 to ovariectomized females did not prevent the increase in alpha and FSH beta mRNAs. In female rats, LH beta mRNA is predominantly regulated by GnRH. alpha mRNA expression is also mainly regulated by GnRH, and E2 appears to augment GnRH action on alpha mRNA expression.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibin secretion during the rat estrous cycle: relationships to FSH secretion and FSH beta subunit mRNA concentrations.

Serum inhibin and FSH and FSH beta subunit mRNA levels were measured at 3h intervals throughout the 4 day estrous cycle in female rats and hourly between 1000 and 2400 h of proestrus. On proestrus, serum inhibin concentrations fell during the late morning-early afternoon, then increased transiently during the late afternoon gonadotropin surges. Inhibin levels decreased during the late evening of proestrus, coincident with the FSH surge-related rise in FSH beta mRNA levels. Serum inhibin remained relatively stable during estrus and early metestrus, but rose during the late evening of metestrus and remained elevated until early diestrus. FSH beta mRNA levels were elevated on late estrus and early metestrus and declined during the evening of metestrus as serum inhibin levels increased. These data show that concentrations of serum inhibin change during the estrous cycle and that a general inverse relationship exists between serum inhibin and FSH levels and FSH beta mRNA concentrations in the pituitary. This suggests that inhibin may inhibit FSH beta gene expression and FSH secretion during the 4 day cycle in female rats.

The frequency of gonadotropin-releasing-hormone stimulation differentially regulates gonadotropin subunit messenger ribonucleic acid expression.

The hypothalamic decapeptide GnRH is known to regulate the synthesis and secretion of LH and FSH by pituitary gonadotrope cells. The frequency of pulsatile GnRH secretion changes and LH and FSH are differentially secreted in various physiological situations. To investigate the potential role of altered frequency of GnRH stimulation in regulating differential secretion of LH and FSH, we examined the effects of GnRH frequency on expression of the alpha, LH beta, and FSH beta genes. GnRH pulses (25 ng/pulse) were administered to castrate testosterone-replaced rats at intervals of 8-480 min to cover the range of physiological pulsatile GnRH secretion. Fast frequency GnRH pulses (8-min pulse intervals) increased alpha-subunit mRNA concentrations 3-fold above those in saline-pulsed controls (controls, 1.01 fmol cDNA bound/100 micrograms pituitary DNA) and LH beta mRNA by 50% (controls, 0.18 fmol cDNA bound), but FSH beta mRNA was unchanged (controls, 0.38 fmol cDNA bound). GnRH pulses given every 30 min increased all three subunit mRNAs (alpha, 3-fold, LHbeta, 2-fold; FSH beta, 2-fold), and acute LH release and serum FSH concentrations were maximal after this frequency. Slower frequency GnRH stimuli (120- to 480-min pulse intervals) did not change alpha and LH beta mRNA levels, but increased FSH beta mRNA 2- to 2.5-fold, and FSH secretion was maintained. Equalization of the total dose of GnRH given at different intervals over 24 h confirmed the frequency dependence of subunit mRNA expression. Fast frequency GnRH stimuli (8 min) increased alpha mRNA 1.5- to 2.5-fold, while the same total GnRH doses were ineffective when given at slow frequency (480 min). Similarly, LH beta mRNA was only increased by GnRH pulses given at 8-min intervals. In contrast, FSH beta mRNA increased 2-fold after pulses given every 480 min, and the 8-min pulse interval was ineffective. The data show that the frequency of GnRH stimulation can differentially regulate gonadotropin subunit mRNA expression and may be a mechanism that enables a single GnRH peptide to selectively regulate gonadotropin subunit gene expression and hormone secretion.

Gonadotrophin-releasing hormone regulation of gonadotrophin subunit gene expression: studies in tri-iodothyronine-suppressed rats.

We have previously shown that a pulsatile gonadotrophin-releasing hormone (GnRH) stimulus can increase steady-state levels of alpha and LH-beta subunit mRNAs in the male rat pituitary. Since alpha subunit is produced in both thyrotroph and gonadotroph cells, the effect of GnRH specifically on gonadotroph alpha gene expression is uncertain. To address this tissue, adult male rats were given injections of tri-iodothyronine (T3; 20 micrograms/100 g body wt, i.p.) daily for 8 days (day 8 = day of death) in order to decrease thyrotroph alpha mRNA levels (+T3 group). Saline injections (i.p.) were given to control animals (-T3group). Three days before GnRH administration, the animals were castrated and testosterone implants inserted s.c., to inhibit endogenous GnRH secretion. GnRH pulses (25 ng/pulse; 30-min interval) were given to freely moving animals (saline pulses to controls) via an atrial cannula for 12,24 or 48 h. Serum LH and FSH were measured before and 20 min after the last GnRH pulse. Pituitary RNA was extracted and alpha, LH-beta, FSH-beta and prolactin mRNA levels were determined by dotblot hybridization using 32P-labelled cDNA probes. Castration and testosterone replacement reduced alpha and LH-beta mRNA levels by 30 and 40% respectively, compared with levels in untreated intact males, but did not decrease FSH-beta concentrations. T3 administration further decreased alpha mRNA to 30% of values seen in intact males, but LH-beta mRNA levels were unchanged. FSH-beta mRNA concentrations were decreased by 23% in T3-treated rats (P less than 0.05 vs intact controls).(ABSTRACT TRUNCATED AT 250 WORDS)

Prolactin messenger ribonucleic acid concentrations in 4-day cycling rats and during the prolactin surge.

Pituitary PRL mRNA concentrations were measured during the 4-day rat estrous cycle. Adult female Sprague-Dawley rats were killed at 3-h intervals throughout the cycle and hourly between 1000 and 2400 h on proestrus (n = 5-12). Serum PRL was increased on the afternoons of proestrus (P) and estrus (E), with peak concentrations at 1700 h (P, 624 +/- 126; E, 261 +/- 107 ng/ml). PRL mRNA concentrations were elevated during the evening on P and E (2300 h: P, 14.4 +/- 1.5; E, 16.1 +/- 1.3 ng cDNA bound/100 micrograms pituitary DNA) to values 2-fold higher than those at 0800 h on each respective day. On diestrus (D) PRL mRNA levels decreased abruptly during the morning (1100 h, 1.7 +/- 0.3 ng cDNA bound), followed by a 6- to 7-fold increase between 1700 and 2000 h on the same evening. In contrast, PRL mRNA levels were elevated at 0800 h on metestrus (M). The changes in PRL mRNA concentrations obtained on M and D were not associated with increased PRL secretion. A more detailed examination of P revealed that PRL mRNA levels increased during the morning (1000 h, 9.9 +/- 2.6 ng cDNA bound), then decreased abruptly at 1100 h (4.9 +/- 1.2). The morning rise in mRNA concentrations was followed by a 2-fold rise in pituitary PRL content. As serum PRL rose during the afternoon surge, a coincident decrease in pituitary PRL content and an increase in PRL mRNA levels were observed. The relationship between PRL secretion and gene expression was further examined in ovariectomized estradiol-replaced rats receiving either bromocriptine (1.2 mg/day, sc) or vehicle control sc. The vehicle-treated group expressed a characteristic afternoon PRL surge between 1500 and 2100 h. Pituitary PRL decreased during the surge to 10% of morning values, and PRL mRNA levels increased 2-fold beginning 2 h after initiation of the surge. These changes in serum PRL, pituitary PRL, and PRL mRNA levels were abolished by bromocriptine administration. These data reveal that alterations in PRL mRNA concentrations occur on a daily basis during the rat estrous cycle. Increases occur during the evenings of P and E at the time of the increase in PRL secretory activity. The effect of blocking the PRL surge in ovariectomized estradiol-replaced rats suggests a regulatory interaction between secretion and gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Follicle-stimulating hormone beta subunit messenger ribonucleic acid concentrations during the rat estrous cycle.

Serum follicle-stimulating hormone (FSH), pituitary FSH content and FSH beta subunit mRNA concentrations were measured at 1 to 3h intervals throughout the 4 day estrous cycle in rats. Serum FSH was stable (range 200-320 ng/ml) apart from the biphasic proestrus surge (5 fold elevation) which was present from 1800h of proestrus through 0800 h on estrus. Basal FSH beta mRNA concentrations from late metestrus through the afternoon of proestrus were 0.10 +/- 0.04 f mol cDNA bound/100 micrograms pituitary DNA. The major increase in FSH beta mRNA began at 2000 h on proestrus, 2 h after the initial rise in serum FSH and peak mRNA concentrations (0.43 +/- 0.08 f mol cDNA bound) occurred at 0200 h on estrus. FSH beta subunit mRNA concentrations were again increased at 2300 h on estrus (peak 0.24 f mol cDNA bound) and remained elevated through 1700 h on metestrus. Pituitary FSH content was transiently increased during metestrus and diestrus, but was elevated at 1000 h through 1900 h on proestrus (peak 5-fold increase). FSH content fell rapidly at 2000h and remained low until 1400 h on estrus when values again rose. These data show that FSH beta mRNA is increased 4-5 fold during the proestrus FSH surge, and a smaller increase occurs on metestrus in the absence of elevated FSH secretion. The increased concentrations of FSH beta mRNA occurred at different times to the previously reported changes in alpha and LH beta mRNAs. Therefore, the data suggest that different mechanisms are involved in the regulation of LH and FSH beta subunit gene expression during the 4-day estrous cycle in rats.