Insulin detemir attenuates food intake, body weight gain and fat mass gain in diet-induced obese Sprague-Dawley rats.

OBJECTIVE: Initiation and intensification of insulin therapy commonly causes weight gain, a barrier to therapy. A contrasting body of evidence indicates that insulin functions as an adiposity negative feedback signal and reduces food intake, weight gain and adiposity via action in the central nervous system. Basal insulin analogs, detemir (Det) and glargine (Glar), have been associated with less hypoglycemia compared with neutral protamine hagedorn insulin, and Det with less weight gain, especially in patients with higher body mass index (BMI). We sought to determine whether insulin therapy per se causes body weight and fat mass gain when delivered via a clinically relevant subcutaneous (SC) route in the absence of hypoglycemia and glycosuria in non-diabetic lean and diet-induced obese rats. MATERIALS AND METHODS: Rats were exposed to either a low-fat diet (LFD; 13.5% fat) or high-fat diet (HFD; 60% fat), and received Det (0.5 U kg(-1)), Glar (0.2 U kg(-1)) or vehicle (Veh) SC once daily for 4 weeks. These dosages of insulin were equipotent in rats with respect to blood-glucose concentration and did not induce hypoglycemia. RESULTS: As predicted by current models of energy homeostasis, neither insulin Det nor Glar therapy affected food intake and weight gain in LFD rats. Det treatment significantly attenuated food intake, body weight gain and fat mass gain relative to the Glar and Veh in high-fat fed animals, mirroring observations in humans. CONCLUSIONS: That neither insulin group gained excess weight, suggests weight gain with SC basal insulin therapy may not be inevitable. Our data further suggest that Det possesses a unique property to attenuate the development of obesity associated with a HFD.

The efficacy of a single chain recombinant equine luteinizing hormone (reLH) in mares: induction of ovulation, hormone profiles, and inter-ovulatory intervals.

The objectives of this study were to determine the efficacy of recombinant equine luteinizing hormone (reLH) in shortening the time to ovulation in cycling mares and to determine the effects of treatment on endogenous hormones and inter-ovulatory intervals. In study 1, mares of light horse breeds (3-20 years) were treated with either a vehicle, various doses of reLH, or human chorionic gonadotropin (hCG). Cycling mares were examined by palpation and ultrasound per rectum daily or every 12h from the time of treatment to ovulation. In studies 2 and 3, jugular blood samples were collected daily or every 12h from the time of treatment to ovulation for analysis of LH, follicle stimulating hormone (FSH), estradiol-17beta (E(2)), and progesterone (P(4)) by radioimmunoassays (RIA). Increasing doses of reLH (0.3, 0.6, 0.75, and 0.9 mg) showed increasing effectiveness at inducing ovulation within 48 h of treatment. Treatments with the 0.75 and 0.9 mg doses of reLH resulted in 90% and 80% ovulation rates, which were similar to hCG treatment (85.7%). Except for the early rise in LH after treatment with 0.5, 0.65, and 1.0mg of reLH, hormone profiles appeared to be similar between control and treated cycles. Inter-ovulatory intervals were similar between control and treatment cycles. In conclusion, reLH is a reliable and effective ovulatory agent that does not significantly alter endogenous hormone profiles or affect inter-ovulatory intervals.

Relationship between the degree of endometrial periglandular fibrosis and the presence of angiotensin-converting enzyme in the equine endometrium.

Endometrial periglandular fibrosis (EPF) has been proposed as a possible aetiology for equine embryonic and fetal loss. However, the pathophysiology of EPF is not well understood. Angiotensin-converting enzyme (ACE) is found in macrophages, endothelium (during angiogenesis) and myofibroblasts at sites of fibrosis in the heart, kidneys, liver and skin in several species. An increase in local tissue ACE-binding activity appears to be a critical event in the initiation and progression of fibrosis in these tissues. The aim of this study was to investigate the correlation between ACE activity in the equine endometrium and the degree of EPF, as determined by histological evaluation and morphometry based on a collagen-specific stain. ACE-binding activity values were significantly higher in the endometrial samples with moderate EPF (modified Kenney EPF category IIB) compared with endometria in all other categories. Ultrastructurally, the fibroblasts surrounding the glandular basal laminae in modified Kenney EPF category IIB and III endometria were undergoing myofibroblastic transformation-like changes. These observations indicate a possible link between ACE activity and the onset of EPF in mares.

Mice lacking alpha-calcitonin gene-related peptide exhibit normal cardiovascular regulation and neuromuscular development.

alpha-Calcitonin gene-related peptide (alphaCGRP) is a pleiotropic peptide neuromodulator that is widely expressed throughout the Central and peripheral nervous systems. CGRP has been implicated in a variety of physiological processes including peripheral vasodilation, cardiac acceleration nicotinic acetylcholine receptor (AChR) synthesis and function, testicular descent, nociception, carbohydrate metabolism, gastrointestinal motility, neurogenic inflammation, and gastric acid secretion. To provide a better understanding of the physiological role(s) mediated by this peptide neurotransmitter, we have generated alphaCGRP-null mice by targeted modification in embryonic stem cells. Mice lacking alpha CGRP expression demonstrate no obvious phenotypic differences from their wild-type littermates. Detailed analysis of systemic cardiovascular function revealed no differences between control and mutant mice regarding heart rate and blood pressure under basal or exercise-induced conditions and subsequent to pharmacological manipulation. Characterization of neuromuscular junction in morphology including nicotinic receptor localization, terminal sprouting in response to denervation, developmental regulation of AChR subunit expression, and synapse elimination also revealed no differences in alphaCGRP-deficient animals. These results suggest that alphaCGRP is not required for the systemic regulation of cardiovascular hemodynamics or development of the neuromuscular junction.

Replacement by homologous recombination of the minK gene with lacZ reveals restriction of minK expression to the mouse cardiac conduction system.

The minK gene encodes a 129-amino acid peptide the expression of which modulates function of cardiac delayed rectifier currents (IKr and IKs), and mutations in minK are now recognized as one cause of the congenital long-QT syndrome. We have generated minK-deficient mice in which the bacterial lacZ gene has been substituted for the minK coding region such that beta-galactosidase expression is controlled by endogenous minK regulatory elements. In cardiac myocytes isolated from wild-type neonatal mice, IKs is rarely recorded, while IKr is common. In minK (-/-) myocytes, IKs is absent and IKr is significantly reduced and its deactivation slowed; these results further support a role for minK in modulating both IKs and IKr. Despite these changes, ECGs in (+/+) and (-/-) animals are no different at adult and at neonatal stages. ECG responses to isoproterenol are also similar in the 2 groups. beta-Galactosidase staining in postnatal minK (-/-) hearts is highly restricted, to the sinus-node region, caudal atrial septum, and proximal conducting system. Moreover, as early as embryonal day 11, segmentally restricted beta-galactosidase expression is observed in the portions of the sinoatrial and atrioventricular junctions that are thought to give rise to the conducting system, thereby implicating minK expression as an early event in conduction system development. More generally, the restricted nature of minK expression in the mouse heart suggests species-specific roles of this gene product in mediating the electrophysiological properties of the heart.

Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase.

Glucokinase (GK) gene mutations cause diabetes mellitus in both humans and mouse models, but the pathophysiological basis is only partially defined. We have used cre-loxP technology in combination with gene targeting to perform global, beta cell-, and hepatocyte-specific gene knock-outs of this enzyme in mice. Gene targeting was used to create a triple-loxed gk allele, which was converted by partial or total Cre-mediated recombination to a conditional allele lacking neomycin resistance, or to a null allele, respectively. beta cell- and hepatocyte-specific expression of Cre was achieved using transgenes that contain either insulin or albumin promoter/enhancer sequences. By intercrossing the transgenic mice that express Cre in a cell-specific manner with mice containing a conditional gk allele, we obtained animals with either a beta cell or hepatocyte-specific knock-out of GK. Animals either globally deficient in GK, or lacking GK just in beta cells, die within a few days of birth from severe diabetes. Mice that are heterozygous null for GK, either globally or just in the beta cell, survive but are moderately hyperglycemic. Mice that lack GK only in the liver are only mildly hyperglycemic but display pronounced defects in both glycogen synthesis and glucose turnover rates during a hyperglycemic clamp. Interestingly, hepatic GK knock-out mice also have impaired insulin secretion in response to glucose. These studies indicate that deficiencies in both beta cell and hepatic GK contribute to the hyperglycemia of MODY-2.

Morphometric analysis of endometrial periglandular fibrosis in mares.

OBJECTIVES: To develop an objective, quantifiable assay for endometrial periglandular fibrosis (EPF) and correlate assay results with histologic and ultrastructural changes in equine endometrial biopsy specimens. SAMPLE POPULATION: Endometrial biopsy specimens from 70 mares from 3 to 27 years old in estrus. PROCEDURE: In a double-blinded study design, endometrial biopsy specimens were graded histologically (modified Kenney classification) for EPF and inflammation. Endometrial periglandular collagen volume fraction (%EPCVF) was determined by light microscopic image analysis of picrosirius red-stained sections. Specimens from selected mares were examined by transmission electron microscopy. RESULTS: %EPCVF values varied significantly among the 4 modified Kenney EPF categories (I, IIA, IIB, and III) and increased with increasing age of mares. Morphologically, EPF consisted of concentric layers of transformed fibroblasts with myofibroblastic features and deposition of fibrillar collagen around unaltered glandular basal laminae. CONCLUSIONS AND CLINICAL RELEVANCE: %EPCVF correlates well with morphologic changes in endometrial biopsy specimens. Determination of %EPCVF could be useful in evaluation and clinical management of subfertile mares and in investigations of the pathogenesis of EPF.

Polydactyly and ectopic ZPA formation in Alx-4 mutant mice.

Correct development of the limb is dependent on coordination between three distinct signaling centers. Recently, fibroblast growth factor-4 has been identified as a crucial determinant of AER function, which directs limb bud outgrowth, and Sonic hedgehog has been identified as a signaling molecule that mediates ZPA function, which specifies anterior-posterior patterning in the developing limb bud. In addition, Shh and FGF-4 reciprocally reinforce each other's expression via a positive feedback loop, providing a molecular basis for the coordination of limb bud outgrowth and anterior-posterior patterning. The mechanisms by which these signaling centers come to occupy their normal positions in the posterior limb bud during development are not understood. Here we identify and characterize Alx-4, a gene that encodes a paired-type homeodomain protein. Alx-4 is expressed in several populations of mesenchymal cells, including mesenchymal cells in the anterior limb bud, and mice homozygous for targeted disruption of the Alx-4 gene have multiple abnormalities, including preaxial polydactyly. The polydactyly is associated with the formation of an ectopic anterior ZPA, as indicated by anterior expression of Sonic hedgehog, HoxD13 and fibroblast growth factor-4. The expression of other candidate regulators of anterior-posterior positional information in the limb bud, including HoxB8 and Gli3, is not altered in Alx-4 mutant embryos. By chromosomal mapping experiments, Alx-4 is tightly linked to Strong's luxoid, a polydactylous mouse mutant. The results identify Alx-4 as a determinant of anterior-posterior positional identity in the limb and a component of a regulatory program that restricts ZPA formation to the posterior limb bud mesenchyme.

Cell-specific expression and regulation of a glucokinase gene locus transgene.

Transgenic mice containing one or more extra copies of the entire glucokinase (GK) gene locus were generated and characterized. The GK transgene, an 83-kilobase pair mouse genomic DNA fragment containing both promoter regions, was expressed and regulated in a cell-specific manner, and rescued GK null lethality when crossed into mice bearing a targeted mutation of the endogenous GK gene. Livers from the transgenic mice had elevated GK mRNA, protein, and activity levels, compared with controls, and the transgene was regulated in liver by dietary manipulations. The amount of GK immunoreactivity in hepatocyte nuclei, where GK binds to the GK regulatory protein, was also increased. Pancreatic islets displayed increased GK immunoreactivity and NAD(P)H responses to glucose, but only when isolated and cultured in 20 mM glucose, as a result of the hypoglycemic phenotype of these mice (Niswender, K. D., Shiota, M., Postic, C., Cherrington, A. D., and Magnuson, M. A. (1997) J. Biol. Chem. 272, 22604-22609). Together, these results indicate that the region of the gene from -55 to +28 kilobase pairs (relative to the liver GK transcription start site) contains all the regulatory sequences necessary for expression of both GK isoforms, thereby placing an upper limit on the size of the GK gene locus.

Effects of increased glucokinase gene copy number on glucose homeostasis and hepatic glucose metabolism.

The relationship between glucokinase (GK) gene copy number and glucose homeostasis was studied in transgenic mice with additional copies of the entire GK gene locus (Niswender, K. D., Postic, C., Jetton, T. L., Bennett, B. D., Piston, D. W., Efrat, S., and Magnuson, M. A. (1997) J. Biol. Chem. 272, 22564-22569). The plasma glucose concentration was reduced by 25 +/- 3% and 37 +/- 4% in mice with one or two extra copies of the gene locus, respectively. The basis for the hypoglycemic phenotype was determined using metabolic tracer techniques in chronically cannulated, conscious mice with one extra GK gene copy. Under basal conditions (6-h fasted) transgenic mice had a lower blood glucose concentration (-12 +/- 1%) and a slightly higher glucose turnover rate (+8 +/- 3%), resulting in a significantly higher glucose clearance rate (+21 +/- 2%). Plasma insulin levels were not different, suggesting that increased glucose clearance was due to augmented hepatic, not islet, GK gene expression. Under hyperglycemic clamp conditions the transgenic mice had glucose turnover and clearance rates similar to the controls, but showed a lower plasma insulin response (-48 +/- 5%). Net hepatic glycogen synthesis was markedly elevated (+360%), whereas skeletal muscle glycogen synthesis was decreased (-40%). These results indicate that increased GK gene dosage leads to increased hepatic glucose metabolism and, consequently, a lower plasma glucose concentration. Increased insulin secretion was not observed, even though the transgene is expressed in islets, because hypoglycemia causes a down-regulation in islet GK content (Niswender, K. D., Postic, C., Jetton, T. L., Bennett, B. D., Piston, D. W., Efrat, S., and Magnuson, M. A. (1997) J. Biol. Chem. 272, in press).

Essential role of the tyrosine kinase substrate phospholipase C-gamma1 in mammalian growth and development.

The activation of many tyrosine kinases leads to the phosphorylation and activation of phospholipase C-gamma1 (PLC-gamma1). To examine the biological function of this protein, homologous recombination has been used to selectively disrupt the Plcg1 gene in mice. Homozygous disruption of Plcg1 results in embryonic lethality at approximately embryonic day (E) 9.0. Histological analysis indicates that Plcg1 (-/-) embryos appear normal at E 8.5 but fail to continue normal development and growth beyond E 8.5-E9.0. These results clearly demonstrate that PLC-gamma1 with, by inference, its capacity to mobilize second messenger molecules is an essential signal transducing molecule whose absence is not compensated by other signaling pathways or other genes encoding PLC isozymes.

Effect of variants of interferon-tau with mutations near the carboxyl terminus on luteal life span in sheep.

Mutant forms of recombinant ovine interferon-tau (oIFN-tau) have been previously prepared by site-directed mutagenesis of an ovine gene with the purpose of establishing relationships between structure and function of the molecule. These mutant forms have altered antiviral and antiproliferative activities, and receptor-binding affinities, and their ability to extend estrous cycle length after being injected i.m. into nonpregnant ewes has been established. In experiment 1, i.m. injection of either PBS (vehicle) alone or with 0.1 mg (n = 4), 0.5 mg (n = 4), or 2.0 mg (n = 3) of recombinant olFN-tau (S4, identified below) was performed twice daily on Days 11-18 postestrus (Day 0 = estrus). Luteal life span was extended (p < 0.05) by 4.8 days after injection of 0.5 mg or 2.0 mg oIFN-tau; injection of 0.1 mg had no effect (p > 0.05) on luteal life span relative to the group that received vehicle alone. In subsequent experiments, the dosage of oIFN-tau was 1.0 mg per injection twice daily. The objective of experiment 2 was to determine the effect of mutated forms of oIFN-tau on luteal life span in sheep. Fifty-seven ewes received twice-daily injections (i.m.) from Day 10 to Day 20 postestrus of PBS (vehicle) either alone (n = 9), with 0.3 mg/injection of bacterial contaminating proteins (BP; n = 10), or with 1 mg/injection of one of the following forms of recombinant oIFN-tau: 1) a fully active, 172-amino acid (aa) oIFN-tau (S4, n = 10); 2) a form lacking 11 aa at the carboxyl terminus and with an I143-->T mutation (S1), which had very low antiviral and antiproliferative activities and receptor-binding affinities (n = 9); 3) a truncated form identical to S1 but with I143 restored (TRN11), which had low antiviral and antiproliferative activities but only slightly reduced receptor-binding affinities (n = 10); and 4) a form similar to wild type S4 (S4-K) with low antiviral and antiproliferative activities but only slightly reduced receptor-binding affinities (n = 9). Luteal life span was slightly longer (p < 0.05) in the TRN11 and S4-K groups (19.6 and 20.6 days, respectively) than in the PBS, BP, and S1 groups (16.2, 16.8, and 17.5 days, respectively). In the S4 group, mean luteal life span was 33.6 +/- 5.9 days (range 15.5-64 days). A third experiment entailing twice-daily injections of either 0.3 mg BP (n = 6), 1 mg TRN11 (n = 5), 1 mg S4-K (n = 5), or 1 mg S4 (n = 5) was conducted in which the pyrogenic effect of the oIFN-tau was also examined. Luteal life span was longer (p < 0.05) in the S4-K and S4 groups (18.9 and 28 days, respectively) than in the BP and TRN11 groups (16.6 and 17.6 days, respectively). Intramuscular injection of all forms of IFN-tau caused hyperthermia in ewes initially, but ewes appeared to become refractory to treatment after several days. In summary, extension of luteal life span was more closely associated with biological activity as assessed in vitro than with receptor binding affinities.

Quantitative imaging of green fluorescent protein in cultured cells: comparison of microscopic techniques, use in fusion proteins and detection limits.

To determine the application limits of green fluorescent protein (GFP) as a reporter gene or protein tag, we expressed GFP by itself and with fusion protein partners, and used three different imaging methods to identify GFP fluorescence. In conventional epifluorescence photomicroscopy, GFP expressed in cells could be distinguished as a bright green signal over a yellow-green autofluorescence background. In quantitative fluorescence microscopy, however, the GFP signal is contaminated by cellular autofluorescence. Improved separation of GFP signal from HeLa cell autofluorescence was achieved by the combination of confocal scanning laser microscopy using 488-nm excitation, a rapid cut-on dichroic mirror and a narrow-bandpass emission filter. Two-photon excitation of GFP fluorescence at the equivalent of approximately 390 nm provided better absorption than did 488-nm excitation. This resulted in increased signal/background but also generated a different autofluorescence pattern and appeared to increase GFP photobleaching. Fluorescence spectra similar to those of GFP alone were observed when GFP was expressed as a fusion protein either with glutathione-S-transferase (GST) or with glucokinase. Furthermore, purified GST.GFP fusion protein displayed an extinction coefficient and quantum yield consistent with values previously reported for GFP alone. In HeLa cells, the cytoplasmic GFP concentration must be greater than approximately 1 microM to allow quantifiable discrimination over autofluorescence. However, lower expression levels may be detectable if GFP is targeted to discrete subcellular compartments, such as the plasma membrane, organelles or nucleus.

Cloning and characterization of the mouse glucokinase gene locus and identification of distal liver-specific DNase I hypersensitive sites.

We cloned and characterized an 83-kb fragment of mouse genomic DNA containing the entire glucokinase (GK) gene. The 11 exons of the gene span a total distance of 49 kb, with exons 1 beta and 1L being separated by 35 kb. A total of 25,266 bp of DNA sequence information was determined: from approximately -9.2 to approximately +15 kb (24,195 bp), relative to the hepatocyte transcription start site, and from -335 to +736 bp (1071 bp), relative to the transcription start site in beta cells. These sequences revealed that mouse GK is > 94% identical to rat and human GK. Mouse hepatic GK mRNA is regulated by fasting and refeeding, as also occurs in the rat. Alignment of the upstream and downstream promoter regions of the mouse, rat, and human genes revealed several evolutionarily conserved regions that may contribute to transcriptional regulation. However, fusion gene studies in transgenic mice indicate that the conserved regions near the transcription start site in hepatocytes are themselves not sufficient for position-independent expression in liver. Analysis of the chromatin structure of a 48-kb region of the mouse gene using DNase I revealed eight liver-specific hypersensitive sites whose locations ranged from 0.1 to 36 kb upstream of the liver transcription start site. The availability of a single, contiguous DNA fragment containing the entire mouse GK gene should allow further studies of cell-specific expression of GK to be performed.

An increase in serum lipids increases luteal lipid content and alters the disappearance rate of progesterone in cows.

To determine whether an increase in serum lipids alters the area occupied by lipid droplets in steroidogenic luteal cells and(or) clearance rates of progesterone from serum, pregnant beef heifers received control (n = 6) or treatment (n = 5) diets. To increase serum lipids, the treatment diet contained calcium soaps of fatty acids. Control and treatment diets were formulated to be isocaloric and isonitrogenous. Feeding of diets was initiated approximately 100 d before parturition and continued through the third postpartum estrous cycle. On d 12 or 13 of the third postpartum cycle, corpora lutea were collected by ovariectomy and a center slice was processed for electron microscopy. Eight samples from each slice were sectioned, stained, and examined at a magnification of 2,500x. Five micrographs per sample were analyzed for area occupied by small (SLC) and large (LLC) luteal cells, percentage of the area of each steroidogenic cell type occupied by lipid, and total steroidogenic area (SLC + LLC) occupied by lipid. Jugular blood was collected before and after ovariectomy, and progesterone, cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were quantified. Cows consuming treatment diets had approximately twice (P < .05) the concentration of cholesterol, HDL, and progesterone in serum that controls had. The percentage of the area of SLC, LLC, and total area occupied by lipid was greater (P < .05) in treated than in control cows. The average time required for serum concentrations of progesterone to decrease by 50% after ovariectomy was greater (P < .05) in treated than in control cows (170 +/- 16 vs 113 +/- 15 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Coexpression of glucose transporters and glucokinase in Xenopus oocytes indicates that both glucose transport and phosphorylation determine glucose utilization.

A Xenopus oocyte expression system was used to examine how glucose transporters (GLUT 2 and GLUT 3) and glucokinase (GK) activity affect glucose utilization. Uninjected oocytes and low rates of both glucose transport and phosphorylation; expression of GLUT 2 or GLUT 3 increased glucose phosphorylation approximately 20-fold by a low Km, endogenous hexokinase at glucose concentrations < or = 1 mM, but not at higher glucose concentrations. Coexpression of functional GK isoforms with GLUT 2 or 3 increased glucose utilization approximately an additional two- to threefold primarily at the physiologic glucose concentrations of 5-20 mM. The Km for glucose of both the hepatic and beta cell isoforms of GK, determined in situ, was approximately 5-10 mM when coexpressed with either GLUT 2 or GLUT 3. The increase in glucose utilization by coexpression of GLUT 3 and GK was dependent upon glucose phosphorylation since two missense GK mutations linked with maturity-onset diabetes, 182: Val-->Met and 228:Thr-->Met, did not increase glucose utilization despite accumulation of both a similar amount of immunoreactive GK protein and glucose inside the cell. Coexpression of a mutant GK and a normal GK isoform did not interfere with the function of the normal GK enzyme. Since the coexpression of GK and a glucose transporter in oocytes resembles conditions in the hepatocyte and pancreatic beta cell, these results indicate that increases in glucose utilization at glucose concentrations > 1 mM depend upon both a functional glucose transporter and GK.

Numbers of steroidogenic luteal cells in Booroola Merino ewes.

In Exp. 1 ovulation rates, plasma concentrations of progesterone, mean individual and total CL weights were determined on Days 4, 10 and 12 after oestrus of Booroola Merino ++ ewes and FF ewes. Mean ovulation rates ranged from 1.5 to 1.8 in ++ ewes and from 5.3 to 6.2 in FF ewes (P less than 0.01). There were no differences in plasma concentrations of progesterone or total luteal weight between the two groups on any of the days studied. Individual CL were smaller (P less than 0.01) in FF ewes than in ++ ewes. In Exp. 2 the numbers of luteal cells in CL collected from 5 ++ and 5 FF ewes on Day 10 of the oestrous cycle were morphometrically determined. The CL from FF ewes were smaller (P less than 0.01) and had fewer total steroidogenic cells (P less than 0.01), fibroblasts (P less than 0.01), and capillary endothelial cells and pericytes (P less than 0.05). However, the luteal cell volume density, number of cells/g tissue, average cell diameter or average cell volume was not different between the two groups of ewes for any cell type studied. It is concluded that the 5-6 CL in FF ewes function in an identical fashion to the 1-2 CL in ++ ewes.

Nuclear changes in ovine luteal cells in response to PGF2 alpha.

Percentages of normal and apoptotic parenchymal cells, fibroblasts and endothelial cells in ovine corpora lutea at 12, 24 and 36 hr following administration of a luteolytic dose of PGF2 alpha were determined and compared to percentages for identical cell types in corpora lutea removed from control ewes on days 10 (n = 5) and 12 (n = 6) postestrus. In corpora lutea obtained from control ewes greater than or equal to 95% of nuclei examined were scored normal for each of the respective cell types with no difference (P greater than .05) observed between luteal tissue obtained on days 10 and 12 postestrus. Following treatment with PGF2 alpha there were significant (P less than .05) reductions in the percentages of nuclei scored normal. Compared to controls the percentage of endothelial cell nuclei scored normal was reduced at 12 hr following PGF2 alpha-treatment; however significant reductions in percentages of parenchymal and fibroblast nuclei scored normal were not evident until 24 and 36 hr, respectively. Consistent with the concept of apoptosis, nuclear condensation and/or margination indicative of apoptosis did not occur synchronously within a given cell type: i.e., irrespective of the time point examined some cells appeared normal, whereas others had undergone nuclear condensation and/or margination. A sequence of events to explain structural and functional changes that occur during luteolysis following the interaction of PGF2 alpha with specific receptors in large steroidogenic luteal cells is discussed.