Variation in partial direct costs of treating clinical mastitis among 37 Wisconsin dairy farms.

The objective of this observational study was to describe variations in partial direct costs of clinical mastitis (CM) treatments among 37 dairy herds using data obtained from herd management records. Animal health and drug purchase records were retrospectively collected from 37 Wisconsin dairy herds for a period of 1 yr. Each farm was visited to verify case definitions, recording accuracy, and detection criteria of CM cases. Descriptive statistics were used to summarize cost of drugs and milk discard. Differences in costs among protocols, intramammary (IMM) products, parities, days in milk, and recurrence were analyzed using ANOVA. Of 20,625 cases of CM, 31% did not receive antimicrobial treatment. The average cost of drugs and milk discard (including cases that were not treated) was $192.36 ± 8.90 (mean ± SE) per case and ranged among farms from $118.13 to $337.25. For CM cases treated only with IMM antimicrobials, milk discard accounted for 87% of total costs and was highly influenced by duration of therapy. Differences in costs were observed among parities, recurrence, and stage of lactation at case detection. Eight different treatment protocols were observed, but 64% of cases were treated using only IMM antimicrobials. Treatment costs varied among protocols; however, cases treated using both IMM and injectable antimicrobials as well as supportive therapy had the greatest costs as they were also treated for the longest duration. Ceftiofur was used for 82% of cases that received IMM antimicrobials while ampicillin was used for 51% of cases treated using injectable antimicrobials. With the exception of ceftiofur and pirlimycin IMM products, many IMM products were given for durations that exceeded the maximum labeled duration. For cases treated using only IMM therapy, as compared with observed costs, we estimated that partial direct costs could be reduced by $65.20 per case if the minimum labeled durations were used. Overall, partial direct costs per case varied among herds, cow factors, and treatment protocols and were highly influenced by the duration of therapy.

Quantification of antimicrobial usage in adult cows and preweaned calves on 40 large Wisconsin dairy farms using dose-based and mass-based metrics.

Use of antimicrobials in animal agriculture is under increasing scrutiny, but the quantity of antimicrobials used on large US dairy farms has not been evaluated using data from large farms and different metrics. This study investigated total antimicrobial usage (AMU) in adult dairy cows and preweaned calves (PWC) and contrasted 2 metrics used for measurement of AMU. Wisconsin dairy farms were eligible if they had >250 lactating cows, maintained computerized animal health records, and were willing to allow researchers access to treatment records. Animal health data for a 1-yr period was retrospectively collected from computerized records, and a farm visit was performed to verify case definitions and recording accuracy. Both dose-based (animal daily doses; ADD) and mass-based (total mg of antimicrobials per kg of body weight; BW) metrics were calculated at the herd, cow, and PWC levels. Descriptive statistics for AMU were examined for both age groups. Mean AMU was compared among active ingredients and route of usage using ANOVA models that included farm as a random variable. At enrollment, farms (n = 40) contained approximately 52,639 cows (mean: 1,316 ± 169; 95% CI: 975, 1657) and 6,281 PWC (mean: 180 ± 33; 95% CI: 112, 247). When estimated using ADD, total herd AMU was 17.2 ADD per 1,000 animal-days (95% CI: 14.9, 19.5), with 83% of total herd-level AMU in adult cows. When estimated using the mass-based metric, total herd AMU was 13.6 mg of antimicrobial per kilogram of animal BW (95% CI: 10.3, 17.0), with 86% of total AMU used in adult cows. For cows, 78% of total ADD (15.8 ADD per 1,000 cow-d) was administered as intramammary (IMM) preparations. In contrast, when AMU was estimated using a mass-based metric, IMM preparations represented only 24% of total AMU (12.1 mg of antimicrobial/kg of cow BW). For cows, ceftiofur was the primary antimicrobial used and accounted for 53% of total ADD, with 80% attributed to IMM and 20% attributed to injectable treatments. When estimated using a mass-based metric, ampicillin was the predominant antimicrobial used in cows and accounted for 33% of total antimicrobial mass per kilogram of BW. When AMU was estimated for PWC using ADD, injectable antimicrobials represented 79% of total usage (28.3 ADD per 1,000 PWC-d). In contrast, when AMU was estimated for PWC using a mass-based metric, injectable products represented 42% of total AMU, even though more farms administered antimicrobials using this route. When AMU in PWC was summarized using ADD, penicillin represented 32% of AMU, and there were no significant differences in ADD among ampicillin, oxytetracycline or enrofloxacin. When a mass-based metric was used to estimate AMU in PWC, oral products (sulfadimethoxine and trimethoprim-sulfa) represented more than half of the total AMU given to this group. Overall, these results showed that choice of metric and inclusion of different age groups can substantially influence interpretation of AMU on dairy farms.

Characterizing the microbiota of wooden boards used for cheese ripening.

Wooden boards are commonly used for aging artisan cheeses. Although considered critical to the development of desired flavors and aromas, knowledge about the microbial communities associated with these boards is limited. To begin to address this need, we performed a 16S ribosomal RNA analysis of the bacterial communities present on the surface and within 5 wooden boards used for cheese ripening that were obtained from 3 cheese-processing facilities. The 5 boards were dominated by bacteria in the phyla Actinobacteria, Firmicutes, and Proteobacteria and displayed differences in both diversity and richness. Analysis of these boards also identified significant board-to-board variation. A total of 288 operational taxonomic units were identified across all samples, with 7 operational taxonomic units forming a core microbiota across all boards. Taken together, these data reflect the cheese-ripening environment, which appears to select for salt- and cold-tolerant bacteria.

Nanoscale flow on a bubble mattress: effect of surface elasticity.

We present an experimental study of the elastic properties of a superhydrophobic surface in the Cassie regime, due to the gas bubbles trapped at the liquid-solid interface. We use a surface force apparatus to measure the force response to an oscillating drainage flow between a sphere and the surface. We show that the force response allows to determine the surface elasticity without contact, using the liquid film as a probe. The elasticity of the bubble mattress is dominated by the meniscii stiffness, and its determination enables us to probe the shape of these meniscii. Another effect of surface elasticity is to decrease the viscous friction. We show that this effect can be wrongly attributed to rate dependant boundary slippage if elastohydrodynamics is not taken into account.

Using surface force apparatus, diffusion and velocimetry to measure slip lengths.

Determining the slip lengths for liquids flowing close to smooth walls is challenging. The reason lies in the fact that the scales that must be addressed range between a few and hundreds of nanometres. Several techniques have been used over the last few years. Here, we consider three of them based on surface force apparatus, diffusion and velocimetry, respectively. The descriptions offered here incorporate recent instrumental progress made in the field.

Boundary slip on smooth hydrophobic surfaces: intrinsic effects and possible artifacts.

We report an accurate determination of the hydrodynamic boundary condition of simple liquids flowing on smooth hydrophobic surfaces using a dynamic surface force apparatus equipped with two independent subnanometer resolution sensors. The boundary slip observed is well defined and does not depend on the scale of investigation from one to several hundreds of nanometers, nor on shear rate up to 5 x 10(3)s(-1). The slip length of 20 nm is in good agreement with theory and numerical simulations concerning smooth nonwetting surfaces. These results disagree with previous data in the literature reporting very high boundary slip on similar systems. We discuss possible origins of large slip length on smooth hydrophobic surfaces due to their contamination by hydrophobic particles.

Somatosensory evoked potential monitoring in anterior thoracic vertebrectomy.

OBJECT: Spine surgeons have used intraoperative cortical and subcortical somatosensory evoked potential (SSEP) monitoring to detect changes in spinal cord function when intraoperative procedures can be performed to prevent neurological deterioration. However, the reliability of SSEP monitoring as applied to anterior thoracic vertebral body resections has not been rigorously assessed. METHODS: The authors retrospectively reviewed hospital charts and operating room records obtained between August 1993 and December 1998 and found that SSEP monitoring was used in 44 surgical procedures involving an anterior approach for thoracic vertebral body resections. There were no patients in whom SSEP changes did not return to baseline during the surgical procedure. Patients in four cases, despite their stable SSEP recordings throughout the procedure, were noted immediately postoperatively to have experienced significant neurological deterioration. The false-negative rate in SSEP monitoring was 9%. Sensitivity was determined to be 0%. CONCLUSIONS: It is important to recognize high false-negative rates and low sensitivity of SSEP monitoring when it is used to record spinal cord function during anterior approaches for thoracic vertebrectomies. The insensitivity of SSEPs for motor deterioration during anterior thoracic vertebrectomies is likely due to the limitation of SSEPs, which monitor only posterior column function whereas motor paths are conveyed in the anterior and anterolateral spinal cord. The authors believe that SSEPs can not be relied on to detect reversible spinal damage during anterior thoracic vertebrectomies.

Management of tumors of the thoracolumbar spine.

Current improvements in radiologic imaging and surgical instrumentation have greatly expanded the role of surgery in management of tumors of the thoracolumbar junction. For primary malignant tumors, the aim of surgery should be curative, with eradiction of all gross disease. For metastatic tumors, indications for surgery include cancer therapy, stabilization, neurologic palliation, tissue diagnosis, and pain relief. Because the thoracolumbar region is a transitional zone, surgical stabilization may require anterior-posterior approaches and instrumentation.

Differential effects of gonadotropin-releasing hormone (GnRH) pulse frequency on gonadotropin subunit and GnRH receptor messenger ribonucleic acid levels in vitro.

The hypothalamic hormone, GnRH, is released and transported to the anterior pituitary in a pulsatile manner, where it binds to specific high-affinity receptors and regulates gonadotropin biosynthesis and secretion. The frequency of GnRH pulses changes under various physiological conditions, and varying GnRH pulse frequencies have been shown to regulate differentially the secretion of LH and FSH and the expression of the gonadotropin alpha, LH beta, and FSH beta subunit genes in vivo. We demonstrate differential effects of varying GnRH pulse frequency in vitro in superfused primary monolayer cultures of rat pituitary cells. Cells were treated with 10 nM GnRH pulses for 24 h at a frequency of every 0.5, 1, 2, or 4 h. alpha, LH beta, and FSH beta messenger RNA (mRNA) levels were increased by GnRH at all pulse frequencies alpha and LH beta mRNA levels and LH secretion were stimulated to the greatest extent at a GnRH pulse frequency of every 30 min, whereas FSH beta mRNA levels and FSH secretion were stimulated maximally at a lower GnRH pulse frequency, every 2 h. GnRH receptor (GnRHR) mRNA levels also were increased by GnRH at all pulse frequencies and were stimulated maximally at a GnRH pulse frequency of every 30 min. Similar results were obtained when the dose of each pulse of GnRH was adjusted to maintain a constant total cumulative dose of GnRH over 24 h. These data show that gonadotropin subunit gene expression is regulated differentially by varying GnRH pulse frequencies in vitro, suggesting that the differential effects of varying GnRH pulse frequencies on gonadotropin subunit gene expression occur directly at the level of the pituitary. The pattern of regulation of GnRHR mRNA levels correlated with that of alpha and LH beta but was different from that of FSH beta. This suggests that alpha and LH beta mRNA levels are maximally stimulated when GnRHR levels are relatively high, whereas FSH beta mRNA levels are maximally stimulated at lower levels of GnRHR expression, and that the mechanism for differential regulation of the gonadotropins by varying pulse frequencies of GnRH may involve levels of GnRHR. Furthermore, these data suggest that the mechanisms whereby varying GnRH pulse frequencies stimulate alpha LH beta, and GnRHR gene expression are similar, whereas the stimulation of FSH beta mRNA levels may be different.

Characterization and chromosomal localization of PTPRO, a novel receptor protein tyrosine phosphatase, expressed in hematopoietic stem cells.

Hematopoietic stem cells (HSCs) support blood cells throughout life by utilizing their self-renewing and multilineage differentiating capabilities. Hematopoietic growth factors mediate their effects on stem cells by the tyrosine phosphorylation of proteins. Regulation of tyrosine phosphorylation is partially mediated by protein tyrosine phosphatases (PTPases). A possible mechanism by which hematopoietic stem cells maintain their self-renewing capacity and undifferentiated state is by controlling the balanced and opposing actions of protein tyrosine kinases (PTKs), receptors for growth factors, and PTPases. We have characterized the expression of PTPases in 5-fluorouracil (5-FU)-treated murine bone marrow cells, which represent a very primitive population of progenitors enriched for reconstituting stem cells, by using a consensus polymerase chain reaction (PCR) method. Several PTPases were expressed abundantly in the 5-FU-treated bone marrow stem cells. A novel PTP, termed protein tyrosine phosphatase receptor omicron (PTPRO), which is related to the homotypically adhering kappa, mu and PCP-2 receptor-type tyrosine phosphatases, was identified and characterized. We have cloned the murine and full-length human PTPRO cDNAs which share 89% homology, indicating that PTPRO is highly conserved between these species. The human PTPRO cDNA clone encodes a polypeptide of 1439 amino acids (aa) and has a calculated molecular mass of approximately 162 kDa. PTPRO consists of an extracellular segment containing a MAM domain, an immunoglobulin (Ig) domain, four fibronectin-type III (FN-III) repeats, a transmembrane segment, and two tandem intracellular PTP domains. The human PTPRO gene was assigned to human chromosome 1p35-pter using Southern blot analyses of genomic DNAs from rodent/human somatic hybrid cell lines containing human chromosome 1 or the p35-pter region of the chromosome. The mouse Ptpro gene was mapped to chromosome 4, closely linked to D4Mit16 and Elp1 (elliptocytosis-1), by using genomic DNAs from a (C57BL/6J x Mus spretus)F1 x Mus spretus backcross. In fetal tissues, PTPRO expression was observed in the brain and lung, whereas lower levels were observed in the kidney. In adult tissues, PTPRO was less restricted and was observed in the lung, heart, skeletal muscle, prostate, testis, and in various areas of the brain, indicating that PTPRO expression is developmentally regulated. Expression of PTPRO was also observed in human CD34+ bone marrow cells and 5-FU-treated murine primitive stem cells. These results suggest a potential role for PTPRO in stem cell adhesion and in mediating homophilic cell-cell interactions in other cell types.

Indications and results of combined anterior-posterior approaches for spine tumor surgery.

Spinal instrumentation currently allows gross-total resection and reconstruction in cases of malignancies at all levels of the spine. The authors analyzed the results in 110 patients who underwent surgery for primary and metastatic spinal tumors over a 5-year period (1989-1993) at a single institution. Major primary sites of tumor included breast (14 cases), chordoma (14 cases), lung (12 cases), kidney (11 cases), sarcoma (13 cases), plasmacytoma (10 cases), and others (36 cases). Prior to surgery, 55 patients (50%) had received prior treatment. Forty-eight patients (44%) were nonambulatory, and severe paraparesis was present in 20 patients. Fifty-three patients (48%) underwent combined anterior-posterior resection and instrumentation. 33 (30%) underwent anterior resection with instrumentation, 18 (16%) underwent anterior or posterior resection alone, and the remaining six patients (5%) underwent posterior resection and instrumentation. Major indications for anterior-posterior resection included three-column involvement, high-grade instability, involvement of contiguous vertebral bodies, and solitary metastases. Postoperatively, 90 patients improved neurologically. The overall median survival was 16 months, with 46% of patients surviving 2 years. Fifty-three patients (48%) suffered postoperative complications. Despite the high incidence of complications, the majority of patients reported improvement in their quality of life at follow-up review. Our findings suggest that half of all patients with spinal malignancies require combined anterior-posterior surgery for adequate tumor removal and stabilization.

Ontogeny of testosterone production from immature mouse testes in vitro.

A dynamic superfusion system was developed to study the ontogeny of testosterone production from immature mouse testes. Testes obtained from 6-day, 12-day and 18-day old mice superfused in vitro. Their basal testosterone productivity were match to their in vivo chronic age. Superfused testes responded to pulsatile stimulation of human chorionic gonadotropin (HCG) for 42 days. Pulsatile HCG stimulation significantly enhanced testosterone production than bolus stimulation. Pulsatile FSH stimulation could significantly increase the testosterone production. Elevation of fluid pressure in culture chamber showed increase testosterone production but it was not significantly different.

Regulation of rat pituitary gonadotropin-releasing hormone receptor mRNA levels in vivo and in vitro.

The recent isolation of cDNAs encoding the rat pituitary gonadotropin-releasing hormone receptor (GnRHR) allows studies of the regulation of the synthesis of the GnRHR and its relationship to reproductive function. Analyses of the regulation of GnRHR mRNA levels in the rat pituitary in vivo revealed a progressive increase in levels to 2.0 +/- 0.2-fold after ovariectomy (OVX) and 5.2 +/- 1.3-fold after castration (CAST) (21 days post-operative), compared to intact adult female and male controls, respectively. Replacement therapy with 17 beta-estradiol benzoate in 21-day post-OVX female rats resulted in a marked decrease in GnRHR mRNA levels by 7 days, compared to controls. In contrast, therapy with testosterone propionate in 21-day post-CAST male rats resulted in only a modest decrease in GnRHR mRNA levels. Thus, manipulation of the reproductive endocrine system in vivo results in alterations in GnRHR synthesis at the pretranslational level, which parallel known changes in cell surface gonadotropin-releasing hormone (GnRH) binding activities. The treatment of superfused primary monolayer cultures of rat pituitary cells with hourly pulses of GnRH (10 nM, 6 min/h) resulted in a marked increase in GnRHR mRNA levels (12.8 +/- 4.3-fold compared to untreated cells). In contrast, treatment of cultured cells with continuous GnRH caused no change in GnRHR mRNA levels. These in vitro data show homologous regulation of GnRHR gene expression by GnRH, and suggest that the changes in GnRHR gene expression observed in vivo may be attributable at least in part to changes in the pattern of hypothalamic GnRH secretion.

Sensitivity of Sertoli and Leydig cells to xenobiotics in in vitro models.

Different chemicals are known to cause testicular damage in the human male and experimental animals. However, the ability to assess the potential and mechanism of action leading to chemically-induced damage in men has been hampered by a lack of good predictive models. Although many of these chemicals were found to impair reproductive capacity in various laboratory animals, only some have caused reproductive damage in men. Mammalian spermatogenesis takes places within the avascular seminiferous tubules of the testis. Specialized tight junctions, which form between adjacent Sertoli cells at the time of puberty, divide the tubular space into the basal and adluminal compartments, and create a "blood-testis" barrier that restricts passage of substances and ions from the circulation. Thus, the completion of meiosis and post-meiotic germ cell differentiation, which take place in the adluminal compartment, are isolated from circulating substances unable to cross the blood-testis barrier. It seems feasible, therefore, that damage to the germ cells induced by testicular toxicants may be mediated through other cells in the testis such as the Sertoli, peritubular, or Leydig cells. A recently developed two-compartment system for culture of testicular cells can simulate, to some degree, the normal physiologic conditions. In principle, Sertoli cells isolated from mammalian testes are cultured on a permeable support (that is millipore filter) between two fluid compartments. They form a highly polarized epithelial layer with characteristic tight junctions that restrict the passage of substances between the two compartments, in analogy to the blood-testis barrier. We believe this system provides an excellent in vitro model for determining the ability of chemicals to: a) alter the permeability of the blood-testis barrier, b) impair the secretory function of Sertoli cells, or c) affect their viability, all of which could indirectly affect the germ cells. We have utilized this system for examining the effects of cadmium chloride (CdCl2) and other toxic substances known to affect the testis. The Leydig cell toxicity was investigated in testicular perfusion system or cultures of isolated Leydig cells.

Effect of cadmium chloride on transepithelial electrical resistance of Sertoli cell monolayers in two-compartment cultures--a new model for toxicological investigations of the "blood-testis" barrier in vitro.

The effect of various doses (0.75-24 microM) of cadmium chloride (CdCl2) on the development of intercellular tight junctions by immature rat Sertoli cells (Sc) was investigated in vitro using the two-compartment culture system. The status of tight junctions was monitored by repeated measurements of the transepithelial electrical resistance (TER). For defining the specificity of CdCl2 effects, the TER changes were correlated with Sc secretory activity (immunoactive inhibin), the cell number (DNA content), and viability (MTT test). The effects of CdCl2 depended on the concentration of the toxicant as well as on the onset and duration of exposure (4 and 18 hr on Day 1 or 5 of culture). The observed effects could be divided into four categories: (1) At highest doses employed, the TER values decreased significantly and irreversibly during 13 days of culture, and the decrease was accompanied by a significant and irreversible drop in inhibin secretion, cell viability, and cell number. (2) Within a narrow range of doses, the irreversible, or only partially reversible, decrease of TER was accompanied by a transient decrease, or no change, of the secretory activity and no significant changes in Sc cell number and/or viability. (3) With still lower doses, the TER values rapidly decreased and then returned to control level within 3-4 days. In this group, no changes in either inhibin secretion or cell viability were observed. (4) Exposure to the lowest doses of CdCl2 caused a delayed, but significant increase in TER. This increase was not accompanied by noticeable changes in other parameters evaluated. These data suggest that CdCl2 may selectively compromise, at least in vitro, the development and maintenance of the inter-Sc tight junctions, without affecting the secretory activity or the cell number and viability. However, increasing cumulative doses of CdCl2 (concentration multiplied by the time of exposure) led to decreased inhibition secretion and cell viability and then, finally, to irreversible cell damage and death. We believe that the experimental model and approach reported in this paper should be very useful for investigating the mechanism of action of known or potential testicular toxicants, particularly those suspected to compromise the integrity of the "blood-testis" barrier.

Effects of recombinant human inhibin and testosterone on gonadotropin secretion and subunit mRNA in superfused male rat pituitary cell cultures stimulated with pulsatile gonadotropin-releasing hormone.

Effects of recombinant human inhibin (rh inhibin) and testosterone on follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion and mRNA levels of gonadotropin subunits were investigated in superfused male rat pituitary cell cultures. During superfusion, the cells were stimulated with gonadotropin-releasing hormone (GnRH) pulses (10 nM, 6 min/h) and exposed to rh inhibin (2 ng/ml) and/or testosterone (10 nM) for up to 20 h. The concentrations of FSH and LH were measured in effluent media by radioimmunoassay (RIA), and subunit mRNAs were determined by Northern blot hybridizations using rat FSH beta, LH beta and alpha genomic and cDNA probes. Rh inhibin suppressed the secretion of FSH (30-40% of control) and the secretion of LH to 50-60% of control, but inhibited only FSH beta mRNA (to non-detectable levels). Testosterone alone suppressed the release of LH to 50% of control, whereas FSH release was increased to 130-160% (P less than 0.05) of control. This increase was due to higher interpulse values without significant changes in the pulse amplitude. Also FSH beta mRNA level was increased (1.5-fold, P less than 0.05) but only after 17-20 h of treatment. On the other hand, testosterone had no effect on LH beta and alpha subunit mRNA levels. Testosterone in combination with rh inhibin showed an inhibitory effect on LH beta mRNA; however, the pattern of LH release was not significantly different from that observed with rh inhibin or testosterone alone. Combined effects of testosterone and rh inhibin on FSH secretion and FSH beta mRNA were similar to those observed with rh inhibin alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cyclic AMP and phorbol ester on transepithelial electrical resistance of Sertoli cell monolayers in two-compartment culture.

The effects of dibutyryl cyclic AMP [Bu)2cAMP) and phorbol ester (TPA), in the absence or presence of follicle-stimulating hormone (FSH) and/or testosterone, on the development of tight junctions by immature rat Sertoli cells (Sc) were investigated in vitro using the two-compartment culture system. The tight junction status was evaluated by repeated measurements of transepithelial electrical resistance (TER). Untreated cell monolayers developed stable TER of approximately 120 omega cm2 during 3 days of culture. Continuous presence of FSH (200 ng/ml) from day 1 onward significantly increased the TER up to approximately 300 omega cm2 after a transient (24-36 h) delay. The initial delay was prolonged to 3-4 days by the addition of 1-methyl-3-isobutylxanthine (MIX) (0.2 mM), whereas the subsequent increase of TER was significantly potentiated by the concomitant presence of testosterone (10 microM). Cholera toxin (CHT; 10 ng/ml) and forskolin (FR; 50 microM) mimicked these FSH effects. (Bu)2cAMP, at concentrations which maximally stimulated immunoactive inhibin secretion (100-500 microM), inhibited the initial TER increase and significantly decreased the TER level when added on days 1 and 5 of culture, respectively. In contrast, low concentrations of (Bu)2cAMP (4-20 microM) consistently stimulated the TER development, mimicking the stimulatory phase of FSH action. TPA (100 nM) alone had no effect on TER development, but potentiated the stimulatory effect of testosterone in a manner similar to FSH, CHT, FR or low concentrations of (Bu)2cAMP. These results demonstrate, for the first time, a concentration-dependent, dual effect of exogenous cAMP on the Sc function.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural characteristics of immature rat Sertoli cells in vivo and in vitro.

The structural properties of pelleted prepubertal Sertoli cells (pre-culture pelleted cells) from 19-day-old rats and of similar cells cultured for 7 days were compared with Sertoli cells from the intact animal (testis tissue from 19- and 26-day-old rats, the in vivo groups). Sertoli cells from freshly isolated pellets and those cultured for 7 days were similar in cell and nuclear volumes to their in vivo counterparts. Cell volumes, organelle volumes, and organelle volume densities of newly isolated Sertoli cells were similar to those of sectioned cells taken from the 19-day-old in vivo group, indicating that the procedure for isolation does not grossly alter Sertoli cells. Mean height of cells cultured for 7 days was significantly lower than that of cells from intact animals at 19 and 26 days of age. In vivo, Sertoli cells of 26-day-old animals displayed increased organelle volumes and organelle surface areas compared with those from 19-day-old animals; volume densities and surface densities remained relatively constant, indicating that in vivo, organelle growth is in proportion to growth of the cell. Most organelle volume and surface densities were not significantly different when 19-day-old in vivo cells and pre-culture pelleted cells were compared. Many organelle volume and surface density values were significantly less in cells grown in culture for 7 days as compared to freshly isolated pelleted cells. After 7 days of culture, most Sertoli cell organelles were significantly less in both volume density and surface density, as compared to the in vivo cell groups (19 or 26 day). This indicates that in vitro the organelles do not develop in proportion to the growth of the cell. After 7 days in culture, the absolute volumes and surface areas of the organelles remained generally unchanged as compared to cells from 19-day-old animals. The data show that Sertoli cells grow in volume in vitro like their in vivo counterparts; however, their subcellular features, although well maintained, do not develop in proportion to the cell. This suggests that short-term cultures are a more ideal system in which to study biochemical responses. Also, cultured prepubertal Sertoli cells are most appropriately used to study prepubertal Sertoli cell function. This is the first study to quantify developmental changes in Sertoli cell structure in vivo as well as to compare them with cellular changes occurring in vitro.