Molecular architecture of the hsp70 promoter after deletion of the TATA box or the upstream regulation region.

GAGA factor, TFIID, and paused polymerase are present on the hsp70 promoter in Drosophila melanogaster prior to transcriptional activation. In order to investigate the interplay between these components, mutant constructs were analyzed after they had been transformed into flies on P elements. One construct lacked the TATA box and the other lacked the upstream regulatory region where GAGA factor binds. Transcription of each mutant during heat shock was at least 50-fold less than that of a normal promoter construct. Before and after heat shock, both mutant promoters were found to adopt a DNase I hypersensitive state that included the region downstream from the transcription start site. High-resolution analysis of the DNase I cutting pattern identified proteins that could be contributing to the hypersensitivity. GAGA factor footprints were clearly evident in the upstream region of the TATA deletion construct, and a partial footprint possibly caused by TFIID was evident on the TATA box of the upstream deletion construct. Permanganate treatment of intact salivary glands was used to further characterize each promoter construct. Paused polymerase and TFIID were readily detected on the normal promoter construct, whereas both deletions exhibited reduced levels of each of these factors. Hence both the TATA box and the upstream region are required to efficiently recruit TFIID and a paused polymerase to the promoter prior to transcriptional activation. In contrast, GAGA factor appears to be capable of binding and establishing a DNase I hypersensitive region in the absence of TFIID and polymerase. Interestingly, purified GAGA factor was found to bind near the transcription start site, and the strength of this interaction was increased by the presence of the upstream region. GAGA factor alone might be capable of establishing an open chromatin structure that encompasses the upstream regulatory region as well as the core promoter region, thus facilitating the binding of TFIID.

Analyses of promoter-proximal pausing by RNA polymerase II on the hsp70 heat shock gene promoter in a Drosophila nuclear extract.

Analyses of Drosophila cells have revealed that RNA polymerase II is paused in a region 20 to 40 nucleotides downstream from the transcription start site of the hsp70 heat shock gene when the gene is not transcriptionally active. We have developed a cell-free system that reconstitutes this promoter-proximal pausing. The paused polymerase has been detected by monitoring the hyperreactivity of thymines in the transcription bubble toward potassium permanganate. The pattern of permanganate reactivity for the hsp70 promoter in the reconstituted system matches the pattern found on the promoter after it has been introduced back into files by P-element-mediated transposition. Matching patterns of permanganate reactivity are also observed for a non-heat shock promoter, the histone H3 promoter. Further analysis of the hsp70 promoter in the reconstituted system reveals that pausing does not depend on sequence-specific interactions located immediately downstream from the pause site. Sequences upstream from the TATA box influence the recruitment of polymerase rather than the efficiency of pausing. Kinetic analysis indicates that the polymerase rapidly enters the paused state and remains stably in this state for at least 25 min. Further analysis shows that the paused polymerase will initially resume elongation when Sarkosyl is added but loses this capacity within minutes of pausing. Using an alpha-amanitin-resistant polymerase, we provide evidence that promoter-proximal pausing does not require the carboxy-terminal domain of the polymerase.

Estimation of the glomerular filtration rate in NIDDM patients from plasma creatinine concentration after cimetidine administration.

OBJECTIVE: Glomerular filtration rate (GFR) can be estimated in patients with renal disease from plasma creatinine concentration, age, sex, and body weight according to the formula of Cockcroft and Gault. The hypothesis that this method can be improved when tubular secretion of creatinine is inhibited by cimetidine was studied in NIDDM patients. RESEARCH DESIGN AND METHODS: In 30 outpatients with NIDDM and normo- (n = 10), micro- (n = 9), or macroalbuminuria (n = 11), GFR was measured as the urinary clearance during continuous infusion of 125I-labeled iothalamate. Plasma creatinine concentration was analyzed with an enzymatic assay before and after 800 mg t.i.d. oral cimetidine was given during a 24-h period. RESULTS: Plasma creatinine rose in all patients after cimetidine administration and, as a consequence, the clearance calculated with the Cockcroft-Gault formula fell. The ratio of this formula and GFR decreased from 1.16 +/- 0.20 to 0.97 +/- 0.16 (means +/- SD). This ratio tended to be smaller in the normo- (0.93) than in the micro- (0.98) and macroalbuminuric (1.00) groups. Also, 20 patients with a BMI < 30 kg/m2 had a smaller ratio than those with a BMI > 30 kg/m2 (0.92 vs. 1.07; P < 0.05). Bland and Altman analysis showed a difference of the Cockcroft-Gault formula and GFR of 12.0 +/- 17.4 ml.min-1 (1.73 m2)-1, which decreased to -3.8 +/- 14.8 ml.min-1.(1.73 m2)-1. The same analysis of 24-h creatinine clearance with urine collection and GFR showed larger standard deviations. CONCLUSIONS: GFR can be estimated in an acceptable way from plasma creatinine concentration after cimetidine administration in outpatients with NIDDM. Despite a nonsignificant underestimation in normoalbuminuric and overestimation in overweighted patients, this method is superior to 24-h creatinine clearance with outpatient urine collection.

Regulation of differentiated osteoclasts.

Osteoclasts respond to many factors, including endocrines, cytokines, cell-cell interactions, and cell-matrix contacts. For mature osteoclasts, the first level of control occurs through signaling that follows binding to an appropriate substrate. Mononuclear and multinucleate osteoclasts are activated when cell surface integrins, notably but not exclusively alphavbeta3 integrins, bind to calcified matrices. The binding process results in actin ring formation and deployment of adhesive proteins into a ring shape such that a seal is formed. As this ring forms, components of the ruffled border assemble from diffuse distribution to form the resorption apparatus, which includes the vacuolar-ATPase, carbonic anhydrase, and other key molecules. This review focuses on the control of osteoclast activity, beginning with attachment and ruffled border assembly. Direct and indirect regulation by PTH/PTHrP, genomic and nongenomic effects of estrogen, and gene expression of ruffled border components, carbonic anhydrase, and vacuolar ATPase are reviewed. Finally, the need to understand complex signaling pathway interaction is discussed.

Use of influenza vaccine in nursing homes.

The organization and outcome of influenza immunization programs were studied in 67 randomly or systematically selected nursing homes (8354 residents) in six states during the autumn of 1982 and/or 1983. In each home, influenza vaccine was usually offered to all residents on a voluntary basis, independent of their age, level of required nursing care, or underlying medical conditions. However, the proportion of residents who were vaccinated ranged from 8 to 98% (mean, 62% overall), with significantly lower rates in homes that also required consent from relatives (usually by return mail) than in homes that did not (P less than .00001; median, 57 versus 90%, respectively). These observations suggest that distribution of educational materials about the risks and benefits of influenza vaccine and systematic follow-up of relatives who fail to return the consent form may be useful strategies to further increase the number of nursing home residents who are immunized.

Hepatitis B vaccination programs for hospital workers: results of a statewide survey.

To assess the implementation of hepatitis B virus (HBV) vaccination programs for hospital workers, we mailed questionnaires to all 229 licensed Michigan hospitals. The response rate was 96% (221/229); of these, 68% (150/221) had vaccination programs. Although multiple hospital characteristics were associated with the presence of a vaccination program, characteristics that independently predicted the presence of a program were medical school affiliation, nonpsychiatric specialty, and the existence of a hepatitis B immune globulin protocol. The most common reason given (56%, 40/71) for the absence of a program was insufficient worker risk of hepatitis B infection; this response was frequent in psychiatric (91%, 10/11) and rural hospitals (61%, 11/18). Among high-risk workers, attending physicians were less likely than other high-risk workers to be included in vaccination programs (68% vs. 95%, respectively). Fear of vaccine-associated acquired immunodeficiency syndrome was most frequently cited as the primary reason for vaccine refusal. We conclude that unwarranted fears about the vaccine's safety need to be dispelled, that high-risk physicians should be included in vaccination programs, and that rural and psychiatric hospital policies reflect their perceived risk of occupational HBV infection.

Influx of urea during bronchoalveolar lavage depends on the permeability of the respiratory membrane.

In 6 healthy controls and 23 patients with pulmonary diseases the influx of urea during bronchoalveolar lavage was measured by comparing the concentrations of albumin and urea in the sequential samples recovered. It varied between -28 and 151 mumol/l. In the bronchoalveolar lavage fluid and serum we measured alpha-2-macroglobulin (A2M) and ceruloplasmin (CP). The bronchoalveolar lavage fluid to serum ratios were calculated (QCP and QA2M). QA2M/QCP was taken as a measure of the respiratory membrane permeability; it varied between 0.05 and 0.53. Influx of urea during lavage was higher according as the QA2M/QCP ratio was higher. We conclude that concentrations of substances in the epithelial lining fluid calculated with the urea correction method have to be corrected for the influx of urea.

Analysis of gas exchange in seedlings of Acer saccharum: integration of field and laboratory studies.

In the field, photosynthesis of Acer saccharum seedlings was rarely light saturated, even though light saturation occurs at about 100 µmol quanta m-2 s-1 photosynthetic photon flux density (PPFD). PPFD during more than 75% of the daylight period was 50 µmol m-2 s-1 or less. At these low PPFD's there is a marked interaction of PPFD with the initial slope (CE) of the CO2 response. At PPFD-saturation CE was 0.018 µmol m-2 s-1/(µl/l). The apparent quantum efficiency (incident PPFD) at saturating CO2 was 0.05-0.08 mol/mol. [Formula: see text] and PPFD-saturated CO2 exchange was 6-8 µmol m-2 s-1. The ratio of internal CO2 concentration to external (C i /C a ) was 0.7 to 0.8 except during sunflecks when it decreased to 0.5. The decrease in C i /C a during sunflecks was the result of the slow response of stomates to increased PPFD compared to the response of net photosynthesis. An empirical model, which included the above parameters was used to simulate the measured CO2 exchange rate for portions of two days. Parameter values for the model were determined in experiments separate from the daily time courses being sumulated. Analysis of the field data, partly through the use of simulations, indicate that the elimination of sunflecks would reduce net carbon gain by 5-10%.

Development of a photosynthesis model with an emphasis on ecological applications : III. Carbon dioxide and oxygen dependencies.

A theoretical description of the simultaneous processes of photosynthesis and photorespiration in a single leaf is developed, based on the hypothesis that carbon dioxide and oxygen compete for the active site of ribulose diphosphate carboxylase. Michaelis-Menten kinetics and competitive inhibition at the end of a diffusion path provide the basic structure of the model. Data of Ludwig (1972) from sunflower are analyzed according to the formulation. This description is part of a more general physiological-ecological model of photosynthesis presented previously (Tenhunen et al., 1976a, b) and continues to elaborate sub-processes in terms of physiologically meaningful parameters. The description is considered a working hypothesis. Data on photorespiration from the literature are reviewed as they relate to this working hypothesis. Several lines of investigation are thereby suggested that will help clarify the role of photorespiration in whole leaf photosynthesis and determine the over-all utility of this modeling approach.

Development of a photosynthesis model with an emphasis on ecological applications : II. Analysis of a data set describing theP M surface.

Photosynthesis was measured in leaves ofPhaseolus vulgaris and analyzed according to the set of equations outlined previously by Tenhunen et al. (1976).

Cimetidine improves GFR-estimation by the Cockcroft and Gault formula.

In some patients with renal disease 24-hour cimetidine aided creatinine clearances cannot equal GFR even after administration of the maximum daily dose of cimetidine. Short duration cimetidine aided creatinine clearances can equal GFR but are inconvenient for clinical use and can be inaccurate due to incomplete urine collection. We studied how accurately GFR can be estimated without the need to collect urine, by applying the Cockcroft and Gault formula (CCock) on a single plasma creatinine concentration, after oral administration of 3 x 800 mg cimetidine during the preceding 24 hours. GFR was measured as standard clearance, using continuous infusion of 125I-iothalamate. Nineteen patients with various renal diseases, plasma creatinine < 180 mumol/l and body mass index between 15 and 30 kg/m2 were included. After cimetidine administration, plasma creatinine values remained stable for 6 hours, despite rapidly decreasing plasma cimetidine values during the same period, in all 15 patients with GFR > 40 ml/min/1.73 m2. Tubular creatinine secretion was blocked completely in 14 of them. With cimetidine both accuracy and precision of the Cockcroft clearance improved: the mean (+/- SD) ratio of CCock to GFR decreased from 1.28 (+/- 0.21) to 0.98 (+/- 0.11) (p < 0.001) and the standard deviation of the difference (CCock-GFR) decreased from 9.23 to 7.07 ml/min/1.73 m2 (p < 0.05). With cimetidine the Cockcroft clearance correlated well with GFR (r = 0.974, p < 0.001) and this was as good as the correlation, between GFR and a 4-hour standard creatinine clearance (r = 0.972, p < 0.001). In conclusion, with a minimum of inconvenience, this method provides the clinician with accurate information on GFR for the outpatient follow-up of patients with a mild-to-moderate decrease in renal function, provided that no gross discrepancy between total bodyweight and muscle mass is present.

A reflective study of Alzheimer's caregivers.

Symbolic interaction posits that to truly understand a situation, it must be considered from both an observer's perspective and the perspective of the actor involved in the situation. In this study, the actors involved are former Alzheimer's caregivers (N = 20) and the situations examined are reflections of the caregiving experience. An in-depth interview was used to explore caregiver issues. The results show that former Alzheimer's caregivers remember their experiences vividly and can recount many stories regarding their successes, regrets, coping strategies, and barriers faced. Participants also shared how they coped with the death of their loved one, the major issues they had immediately following the death, and issues with which they are still dealing. From the information shared by former caregivers, an Alzheimer's Caregiver Transition Model (ACT-M) was developed to help explain the process individuals go through as they transition out of the caregiver's role.

A technique for transrectal ultrasonography of stallions during ejaculation.

A technique was developed in which the accessory sex glands of stallions were visualized with transrectal ultrasonography during ejaculation. The technique was judged to be effective, since 10 of 11 stallions were trained to tolerate transrectal ultrasonography during ejaculation; they ejaculated during 195 of 200 attempts, and acceptable visualization of their accessory sex glands and excurrent ducts occurred during 97 of 195 ejaculations. Sixty-five percent (89 136 ) of the recordings were successful for stallions that weighed more than 300 kg, whereas 14% (8 59 ) of the recordings were successful for stallions weighing less than 300 kg. The 98 unsuccessful attempts were caused by inaccurate transducer placement due to the small size of the pelvic canal(33 98 ), excessive transducer movement due to stallion movement (32 98 ), indistinct ultrasound images (28 98 ) and human error (5 98 ). The technique was judged to be safe, since no stallions or personnel sustained serious injuries during 200 data collection attempts.

Hastened transport of equine embryos through the oviduct of the mare.

The purposes of this experiment were 1) to test the hypothesis that placing rabbit embryos into the mare's uterus would hasten oviduct transport and 2) to determine if placing fluid into the uterus of bred mares on Day 4 and/or Day 5 would subsequently disrupt the mare's pregnancy. The hypothesis that placing rabbit embryos into the mare's uterus would hasten oviduct transport was not supported, since the uterine recovery rate of equine embryos on Day 5 was not significantly higher (P>0.05) for mares receiving rabbit embryos on Day 4 than for mares receiving no uterine infusion on Day 4 (1 10 vs 0 10 , respectively). However, placing fluid into the mare's uterus on Day 4 was apparently responsible for hastened oviduct transport, since mares with media infused into the uterus on Day 4 had a significantly higher (P<0.05) recovery rate of equine embryos on Day 5 than did mares receiving either rabbit embryos or no uterine infusion on Day 4 post ovulation (5 10 vs 1 10 or 0 10 , respectively). The Day-14 pregnancy rate was significantly higher (P<0.05) for mares receiving no uterine infusion on Day 4 or Day 5 than for mares receiving uterine infusion on Day 5 or uterine infusion on both Days 4 and 5 (9 10 vs 4 10 , 2 10 and 0 10 , respectively).

Ejaculatory pattern of llamas during copulation.

The objective of this study was to use transrectal digital palpation of urethral pulses to define the ejaculatory pattern of llamas during copulation. Five male llamas were palpated during 5 to 6 copulations each with receptive female llamas (n = 28 copulations). The time from first exposure of a male to a female until mounting was 0.7 +/- 1.1 min (mean +/- SD), time to the first intromission was 1.7 +/- 1.4 min, and time from initial mount to final dismount (copulation duration) was 21.7 +/- 7.8 min. A total of 121.9 +/- 61.0 urethral pulses per copulation (5.6 +/- 1.7 pulses/min) was palpated. During the first 3.9 +/- 3.7 min of copulation, urethral pulses (11.0 +/- 10.1 urethral pulses at 3.5 +/- 2.5 pulses/min) occurred randomly and were not associated with whole-body strains. After the first 4 min of copulation, urethral pulses occurred in a pattern of clusters of frequent urethral pulses associated with whole-body strains, alternating with intercluster intervals of infrequent urethral pulses without whole-body strains. Individual clusters were characterized by 4.3 +/- 2.7 urethral pulses at 16.7 +/- 4.5 pulses/min during strains, and intercluster intervals were characterized by 1.7 +/- 2.3 urethral pulses at 2.2 +/- 1.8 pulses/min. Each cluster of urethral pulses during a strain was preceded by 2.3 +/- 1.8 repositions of the male's hindlegs and by 38.1 +/- 20.8 pelvic thrusts. There were 18.5 +/- 10.6 clusters of urethral pulses accompanied by strains per copulation at 0.9 +/- 0.3 clusters/min. The 18 to 19 clusters of urethral pulses appeared to be individual ejaculations. Therefore, we hypothesize that llamas ejaculated 18 to 19 times during their 22-min copulations.

Seminal collection, seminal characteristics and pattern of ejaculation in llamas.

Semen was collected from 10/10 llamas during 26/30 (87%) collection attempts using an artificial vagina mounted inside a surrogate female. For the 26 semen collections, the duration of copulation (mount to dismount) with the artificial vagina was 31.7 +/- 12.0 min (mean +/- SD). Seminal pH was 8.1 +/- 1.1, and seminal volume per collection was 3.0 +/- 1.9 ml. Sperm concentration per collection was 1.0 +/- 0.8 x 10(6) sperm/ml, total number of spermatozoa was 2.9 +/- 3.1 x 10(6), total sperm motility was 23.7 +/- 20.0%, and the percentage of morphologically normal spermatozoa was 39.7 +/- 18.5%. Morphologically abnormal spermatozoa were categorized according to abnormal heads (20.1 +/- 19.9%), tail-less heads (8.7 +/- 8.9%), abnormal acrosomes (12.9 +/- 12.4%), abnormal midpieces (1.0 +/- 3.7%), cytoplasmic droplets (11.1 +/- 12.4%), and abnormal tails (6.6 +/- 12.0%). There were 0.3 +/- 0.3 million motile, morphologically normal spermatozoa per collection: less than 1000 during the first 5 min of copulation, 0.01 +/- 0.01 x 10(6) between 5 and 10 min of copulation, 0.04 +/- 0.08 x 10(6) between 10 and 15 min of copulation, 0.09 +/- 0.21 x 10(6) between 15 and 20 min of copulation, and 0.15 +/- 0.28 x 10(6) between 20 min and the end of copulation.

Induction of luteolysis in mares by ultrasound-guided intraluteal treatment with PGF2alpha.

To evaluate the technique of ultrasound-guided luteal injection in mares, PGF2alpha was administered under ultrasound guidance to horse mares (n = 7 to 9 per group) on Day 9 postovulation via either a systemic (i.m.; zero, 0.01, 0.1, or 5 mg/dose) route or a local intraluteal (i.l.; zero, 0.01 or 0.1 mg/dose) route. The luteolytic efficacy of each treatment was determined based on post-treatment decreases in progesterone concentration, interval to uterine edema (IE) and interovulatory interval (IOI). Local administration of PGF2alpha directly into the CL consistently induced luteolysis, at doses up to 50-fold lower than the lowest effective systemic dose. Significant decreases in IOI and IE occurred in mares treated with 5 mg PGF2alpha i.m. or 0.1 mg PGF2alpha i.l., but did not occur in mares treated with 0.1 or 0.01 mg PGF2alpha i.m., 0.01 mg PGF i.l., vehicle i.l. or vehicle i.m.. Progesterone concentrations were reduced to less than 10% of pretreatment values by two days post treatment in mares treated with 5 mg PGF2alpha i.m. or 0.1 mg PGF2alpha i.l.. PGF2alpha doses of 0.1 mg i.m. and 0.01 mg i.l. were associated with smaller but significant progesterone decreases (to 66% and 46% of pre-treatment values, respectively) by two days post treatment. Progesterone values after administration of i.l. vehicle did not differ from pre-treatment values by two days post treatment, but were significantly lower (53% of pre-treatment values) by four days post treatment. Intramuscular treatment with vehicle or 0.01 mg of PGF2alpha did not significantly reduce progesterone concentrations below pretreatment values. Overall, the minimum effective luteolytic dose of PGF2alpha given intraluteally was between 0.01 and 0.1 mg. Based on the results of this study, ultrasound-guided i.l. injection appears to be a repeatable method for studying the direct effect of other chemicals on luteal function. However, the current procedure carries some risk, since three i.l. injections were associated with ovarian abscesses.

Autotransfer of Day 4 embryos from oviduct to oviduct versus oviduct to uterus in the mare.

Embryo autotransfer is defined as the collection of an embryo from and the transfer of this embryo into the same animal. The objectives of this study were to: 1) test the hypothesis that oviduct transport of the equine embryo from the oviduct into the uterus is not dependent on a unilateral embryo-corpus luteum interaction, 2) develop an embryo autotransfer technique for the mare and 3) compare the success rates of Day 4 embryos surgically autotransferred from the oviduct ipsilateral to ovulation to either the oviduct (n=10 mares) or the uterine horn (n=10 mares) contralateral to ovulation. Seventy percent (7 10 ) of the Day 4 embryos which were autotransferred to the oviduct contralateral to ovulation were transported through the oviduct and subsequently developed into embryonic vesicles detectable by ultrasonography between 10 and 21 days postovulation. This finding supported the hypothesis that oviductal embryo transport is not dependent upon the ipsilateral corpus luteum. Overall, sixty percent (12 20 ) of the autotransfers were successful. The success rate of uterine-transferred embryos was not significantly less (P>0.3) than that of oviductal-transferred embryos (5 10 vs 7 10 , respectively). Therefore, the Day 4 equine embryos were apparently mature enough to survive in the mare's uterus.

Corpus luteal function in nonpregnant mares following intrauterine administration of prostaglandin E(2) or estradiol-17beta.

The objective of this study was to test the hypothesis that intrauterine administration of prostaglandin E(2) (PGE(2)) or estradiol-17beta (E-17beta) would prolong CL function in nonpregnant mares. Nonpregnant mares were continuously infused with 240 mug/d of PGE(2), 6 mug/d of E-17beta, or vehicle (sham-treated) on Days 10 to 16 post ovulation (ovulation = Day 0), using osmotic minipumps surgically placed into the uterine lumen on Day 10 (n = 11 per group). Nonpregnant and pregnant mares served as negative and positive controls, respectively (n = 11 per group). Mares were defined as having prolonged CL function if plasma progesterone remained > 2.5 ng/ml and if ovulation did not occur on Days 9 to 30. Corpus luteal function was prolonged until Day 30 in 1 11 nonpregnant mares, 4 11 sham-treated mares, 6 11 E-17beta-treated mares, 8 11 PGE(2)-treated mares, and 11 11 pregnant mares. The incidence of prolonged CL function was similar (P=0.16) in the sham-treated and nonpregnant mares. The hypothesis that PGE(2) would prolong CL function in nonpregnant mares was supported, since the incidence of prolonged CL function was higher (P=0.003) in PGE(2)-treated versus nonpregnant mares, tended to be higher (P=0.09) in PGE(2)-versus sham-treated mares, and was not lower (P=0.11) in PGE(2)-treated versus pregnant mares. The hypothesis that E-17beta would prolong CL function in nonpregnant mares was not supported, since the incidence of prolonged CL function was not higher (P=0.34) in E-17beta-versus sham-treated mares, and was lower (P=0.02) in E-17beta-treated versus pregnant mares. These results demonstrate that intrauterine administration of a pharmacologic dose of PGE(2) initiated prolonged CL function in nonpregnant mares. Further experiments are needed to confirm the role of conceptus secretion of PGE(2) in CL maintenance, and to determine the mechanism of action of PGE(2) within the equine reproductive tract.

Co-culture of day-5 to day-7 equine embryos in medium with oviductal tissue.

Oviductal and uterine embryos were collected from mares at 5 to 7 days following ovulation 1) to evaluate the effects of oviductal tissue explants on in vitro growth and development of equine embryos and 2) to study the morphologic development of equine embryos in culture. Embryos were incubated for 5 days in a medium (control group) or in medium supplemented with oviductal tissue explants (co-culture group). Embryos were evaluated and the media changed daily. Following 5 days in culture, 10 10 (100%) control embryos and 27 29 (93%) co-cultured embryos had doubled in diameter. All embryos that were recovered as morulae developed to the blastocyst stage in culture. By 5 days in culture, 6 10 (60%) control embryos and 19 29 (66%) co-cultured embryos had reached the hatching blastocyst stage of development. By 3 days in culture, significantly more (P<0.05) control embryos versus co-cultured embryos had degenerated (4 10 vs 2 29 , respectively). By 5 days in culture, significantly more (P<0.01) control embryos versus co-cultured embryos had degenerated (6 10 vs. 3 29 , respectively). Embryos cultured with oviductal tissue were sustained longer than embryos cultured in medium alone. Hatching was characterized by the blastocyst squeezing through a small opening in the zona pellucida or by the zona pellucida thinning over approximately half of the blastocyst surface and subsequently disappearing entirely.