Charles O. Woody

1,25-Dihydroxyvitamin D3 inhibition of Col1a1 promoter expression in calvariae from neonatal transgenic mice

We studied the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on organ cultures of transgenic mouse calvariae containing segments of the Col1a1 promoter extending to -3518, -2297, -1997, -1794, -1763, and -1719 bp upstream of the transcription start site fused to the chloramphenicol acetyltransferase (CAT) reporter gene. 1,25(OH)2D3 had a dose-dependent inhibitory effect on the expression of the -3518 bp promoter construct (ColCAT3.6), with maximal inhibition of about 50% at 10 nM. This level of inhibition was consistent with the previously observed effect on the endogenous Col1a1 gene in bone cell models. All of the shorter constructs were also inhibited by 10 nM 1,25(OH)2D3, suggesting that the sequences required for 1, 25(OH)2D3 inhibition are downstream of -1719 bp. The inhibitory effect of 1,25(OH)2D3 on transgene mRNA was maintained in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the inhibitory effect on Col1a1 gene transcription does not require de novo protein synthesis. We also examined the in vivo effect of 1,25(OH)2D3 treatment of transgenic mice on ColCAT activity, and found that 48 h treatment caused a dose-dependent inhibition of CAT activity in calvariae comparable to that observed in organ cultures. In conclusion, we demonstrated that 1,25(OH)2D3 inhibits Col1A1 promoter activity in transgenic mouse calvariae, both in vivo and in vitro. The results indicate that there is a 1, 25(OH)2D3 responsive element downstream of -1719 bp. The inhibitory effect does not require new protein synthesis.

Interleukin‐1 Represses COLIA1 Promoter Activity in Calvarial Bones of Transgenic ColCAT Mice In Vitro and In Vivo

Interleukin‐1 (IL‐1) inhibits collagen synthesis in osteoblastic cell lines and primary osteoblast‐like cells. However, promoter elements regulating type I collagen A1 (COLIA1) expression in vivo and in organ culture may differ from those regulating expression in cell culture. We have examined the effects of IL‐1 on reporter gene activity in neonatal transgenic mouse calvariae bearing COLIA1 promoter‐chloramphenicol acetyltransferase (ColCAT) fusion genes. The parent construct, ColCAT 3.6, contains 3.5 kb of 5′ flanking sequence and 115 bp of 5′ untranslated region fused to the CAT reporter. In 48‐h calvarial organ cultures, IL‐1 repressed ColCAT 3.6 promoter activity and collagen synthesis in a dose‐related manner, with a maximal inhibition of 40–65%. This repression was retained in 5′ deletion constructs truncated to −1719 bp. The inhibition of transgene mRNA was blocked by cycloheximide, indicating a requirement for new protein synthesis. Pretreatment with indomethacin diminished the inhibitory effect of IL‐1 on CAT activity and collagen synthesis, suggesting partial mediation by prostaglandins. Local in vivo injection of IL‐1 (500 ng) decreased calvarial transgene mRNA after 8 h, an effect that was partially blocked by indomethacin. ColCAT transgenic mice represent a useful model for in vitro and in vivo assessment of COLIA1 promoter regulation by cytokines and other factors.

Effects of unilateral castration on morphologic characteristics of the testis in one-, two-, and three-year-old stallions☆

The effects of unilateral castration on testicular compensatory hypertrophy were measured in 12 Morgan stallions, four each at one, two, and three years of age. They were randomized within age to intact (IN) or unilaterally castrated (UC) groups. Allotment and surgery were in January 1983 and total castration was in June 1983, 150 d after unilateral castration. Testis weight increased linearly with age (P < 0.01) and was increased by unilateral castration (P < 0.07). Epididymal weight also increased linearly with age (P < 0.05) and was heavier in UC animals (P = 0.15). Tubule diameter (P < 0.10) and epithelial height (P < 0.03) were greater in UC than in IN stallions. In conclusion, testes of stallions underwent compensatory hypertrophy after unilateral castration.

Identification of Regulatory Elements Necessary for the Expression of the COL1A1 Promoter in Murine Odontoblasts

Recent studies have indicated that odontoblasts and osteoblasts have unique regulatory mechanisms that control COL1A1 gene expression. We are currently examining the regulation of COL1A1 gene expression in odontoblasts and have produced transgenic mice containing various collagen promoter constructs fused to the indicator gene, chloramphenicol acetyl transferase (CAT). Mandibular first molars were removed from jaws of transgenic mice. Some teeth were assayed for CAT activity (CAT diffusion assays), others were fixed and prepared for immunohistochemistry (CAT antibodies). Our results indicate the CAT activity was present in tooth germs containing promoter constructs longer than 1.719 kb. Immunoreactivity to CAT was confined to the odontoblast cell layer. No CAT activity was present in tooth germs containing a 1.670 kb construct. These data suggest that there are important regulatory elements located between -1.719 kb and -1.670 kb on the collagen promoter in odontoblasts. Examination of sequences in this region of the promoter demonstrates consensus with those known to be involved with binding of translation products of homeobox genes.

Regulation of type I collagen gene expression in bone

The regulation of COL1A1 gene expression in bone was studied by measuring the activity of type I collagen promoter fusion genes (ColCAT) in permanently transfected osteoblastic cells and calvariae from transgenic animals. The basal activity of ColCAT fusion genes in transfected cells is mediated by DNA sequences between -3.5 to -2.3 kb while expression in vivo requires sequences between -2.3 and -1.7 kb. Parathyroid hormone, 1,25-dihydroxyvitamin D3 and interleukin-1 decrease the activity of ColCAT fusion genes in osteoblastic cells and transgenic calvariae. Because there may be differences between the expression of ColCAT fusion genes in cultured cells and intact bone, it will be important to compare data obtained from transfected cells with an in vivo model such as calvariae from transgenic mice.

The effects of cervical dilation on plasma PGFM, progesterone and the duration of luteal function in diestrous mares

Transcervical diagnostic techniques may alter the length of the equine estrous cycle and affect subsequent luteal function. Therefore, nine mares were used to determine the effect of cervical dilation on plasma 13, 14-dihydro, 15-keto-prostaglandin F(2) (PGFM), progesterone (P(4)) and posttreatment duration of luteal function. Mares were given a daily score of 0 to 4 based on sexual receptivity. Five days following the end of receptivity, mares were randomly assigned to one of three, 3 x 3 latin squares. Control mares received no cervical dilation. Cervically stimulated mares recieved cervical dilation for 60 sec. Cervically stimulated plus inhibitor mares were dilated similarly to cervically stimulated mares, but received a prostaglandin synthetase inhibitor 30 min prior to treatment. Each mare completed all three treatments in three consecutive estrous cycles. Plasma PGFM and P(4) were determined by RIA. Plasma PGFM was lower (P<0.05) in cervically stimulated plus inhibitor than control and cervically stimulated mares. In addition, plasma P(4) was lower (P<0.10) in cervically stimulated plus inhibitor than in control and cervically stimulated mares. Luteal function following treatments did not differ. These data indicate that neither plasma PGFM and P(4) nor the duration of luteal function were affected by cervical dilation. However, administration of a prostaglandin synthetase inhibitor prior to cervical dilation decreased plasma PGFM and P(4) concentrations.

Effects of unilateral castration on serum luteinizing hormone, follicle stimulating hormone, and testosterone concentrations in one-, two-, and three-year-old stallions.

The endocrine control of compensatory hypertrophy was investigated in 12 Morgan stallions, four each at one, two and three years of age. Half were assigned to be unilaterally castrated (UC) in January and half to remain intact (IN). Nine blood samples were taken from each stallion at half-hour intervals 30, 90, and 150 d after unilateral castration for radioimmunoassay of serum concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), and testosterone. Mean serum LH concentration was greater (P<0.06) in UC than IN stallions; however, the difference was greatest at 30 d and least at 150 d. Serum LH was greater (P<0.01) in two- and three-year-olds than in one-year-olds. The mean log(10) for serum FSH concentration was greater (P<0.06) in UC than IN stallions. Mean serum testosterone concentrations were similar in UC and IN stallions for all sample days, suggesting that the single testes of the UC stallions produced as much testosterone as the two testes of the IN stallions. Two- and three-year-old stallions had greater (P<0.01) serum testosterone than one-year-old stallions. Unilateral castration of stallions was associated with a significant increase in serum LH and FSH concentrations and, perhaps, higher intratesticular testosterone, which may explain, in part, the compensatory hypertrophy noted in the remaining testis.

Factors influencing estrus and conception in dairy heifers after prostaglandin F2-alpha☆

The influence of interval between insemination (AI) and estrus on subsequent fertility of PGF2alpha-treated (two injections of 25 mg, 11 days apart) heifers was assessed in two experiments. In Experiment I, 240 heifers were allotted to Control (AI 8 to 16 hr after estrus detection), PGF2alpha-E (AI 8 to 16 hr after estrus within five days of second PGF2alpha) or PGF2alpha-T (AI 80 hr after second PGF2alpha). In Experiment II, 130 heifers were assigned to control (AI as before) or PGF2alpha (AI 72 or 80 hr after second PGF2alpha) with half the PGF2alpha heifers receiving 100 microg GnRH 72 hr after first PGF2alpha. Heifers of both experiments that were bred at a predetermined time were arrayed by interval from AI to estrus. Conception rates of heifers detected in estrus from 32 hr before AI to 24 hr after AI did not differ (x2=3.35, df=5, P>0.5). The percentage of GnRH-treated heifers in estrus within five days (81.8%) was not (P>0.75) greater than those not receiving GnRH (77.3%) but they had higher (P<0.05) serum progesterone (P4) concentration at second PGF2alpha (3.17 vs 2.41 ng/ml). When P4 values were arrayed for both groups at 1 ng intervals, the percentage of heifers exhibiting estrus increased with increasing P4 level (P<0.05).