Cryoinjury of MCF-7 human breast cancer cells and inhibition of post-thaw recovery using TNF-alpha.

Cryoinjury of MCF-7 human breast cancer cells and its enhancement using tumor necrosis factor-alpha (TNF-alpha) as an adjuvant, were investigated. Through a series of experiments in a two level factorial design critical parameters affecting cryotherapy responses were identified. The cryoinjury was investigated by quantifying the effects of four freeze/thaw (F/T) parameters, selected to be within the expected range for a cryosurgical iceball. Thermal parameters considered were cooling rate (5 and 50 degrees C/min), end temperature (-20 and -80 degrees C), hold time (0 and 10 min), and thawing rate (20 and 100 degrees C/min). After exposing the cells to the selected F/T conditions, survival was assessed and statistically analyzed to determine the effect of each parameter and their interactions. A statistical analysis shows that the end temperature and hold time were the two most significant parameters in the range studied. This suggests that proper control of these two parameters is important to achieve desired cryodestruction of MCF-7 cells. Enhancement of cryoinjury by TNF-alpha was also investigated in a tissue equivalent cryoinjury model in which a cryosurgical iceball is formed. MCF-7 cells cultured in a collagen matrix underwent a controlled F/T with or without TNF-alpha pre-treatment at 100 ng/ml for 24 hours. Post-thaw viability of MCF-7 cells was assessed at three hours, and at one and three days after freezing. Although the TNF-alpha treatment alone induced neither apoptotic nor necrotic cell death, the combination of TNF-alpha pre-treatment and freezing enhanced the immediate cryoinjury of MCF-7 cells, and significantly impaired the post-thaw recovery. Without TNF-alpha treatment, MCF-7 cell cultures were repopulated, reaching approximately 80% survival at day 3 even after severe cryoinjury (< or = 20% survival) at three hours. In contrast, this repopulation was significantly inhibited by TNF-alpha pre-treatment, in which case the viability of the frozen region remained below 40% at day 3. The effects of TNF-alpha on the cryoinjury of MCF-7 cells suggest that TNF-alpha may serve as a potent adjuvant to cryosurgery of breast cancer.

Investigation of the thermal and tissue injury behaviour in microwave thermal therapy using a porcine kidney model.

Minimally invasive microwave thermal therapies are being developed for the treatment of small renal cell carcinomas (RCC, d<3 cm). This study assessed the thermal history and corresponding tissue injury patterns resulting from microwave treatment of the porcine renal cortex. Three groups of kidneys were evaluated: (1) in vitro treated, (2) in vivo with 2-h post-treatment perfusion (acute) and (3) in vivo with 7-day post-treatment perfusion (chronic). The kidneys were treated with an interstitial water-cooled microwave probe (Urologix, Plymouth, MN) that created a lesion centered in the renal cortex (50 W for 10 min). The thermal histories were recorded at 0.5 cm radial intervals from the probe axis for correlation with the histologic cellular and vascular injury. The kidneys showed a reproducible 2 cm chronic lesion with distinct histologic injury zones identified. The thermal histories at the edge of these zones were found using Lagrangian interpolation. The threshold thermal histories for microvascular injury and stasis appeared to be lower than that for renal epithelial cell injury. The Arrhenius kinetic injury models were fit to the thermal histories and injury data to determine the kinetic parameters (i.e. activation energy and frequency factor) for the thermal injury processes. The resultant activation energies are consistent in magnitude with those for thermally induced protein denaturation. A 3-D finite element thermal model based on the Pennes bioheat equation was developed and solved using ANSYS (V7.0). The real geometry of the kidneys studied and temperature dependent thermal properties were used in this model. The specific absorption rate (SAR) of the microwave probe required for the thermal modelling was experimentally determined. The results from the thermal modelling suggest that the complicated change of local renal blood perfusion with temperature and time during microwave thermal therapy can be predicted, although a first order kinetic model may be insufficient to capture blood flow changes. The local blood perfusion was found to be a complicated function of temperature and time. A non-linear model based on the degree of vascular stasis was introduced to predict the blood perfusion. In conclusion, interstitial microwave thermal therapy in the normal porcine kidney results in predictable thermal and tissue injury behaviour. Future work in human kidney tissue will be necessary to confirm the clinical significance of these results.

In vitro thermal therapy of AT-1 Dunning prostate tumours.

To advance the utility of prostate thermal therapy, this study investigated the thermal thresholds (temperature-time) for prostate tissue destruction in vitro. The AT-1 Dunning prostate tumour model was chosen for the study. Three hundred micron thick sections were subjected to controlled temperature-time heating, which ranged from low (40 degrees C, 15 min) to high thermal exposures (70 degrees C, 2 min) (n = 6). After subsequent tissue culture at 37 degrees C, the sections were evaluated for tissue injury at 3, 24 and 72 h by two independent methods: histology and dye uptake. A graded increase in injury was identified between the low and high thermal exposures. Maximum histologic injury occurred above 70 degrees C, 1 min with >95% of the tissue area undergoing significant cell injury and coagulative necrosis. The control and 40 degrees C, 15 min sections showed histologic evidence of apoptosis following 24 and 72 h in culture. Similar signs of apoptosis were minimal or absent at higher thermal histories. Vital-dye uptake quantitatively confirmed complete cell death after 70 degrees C, 2 min. Using the dye data, Arrhenius analysis showed an apparent breakpoint at 50 degrees C, with activation energies of 135.8 kcal/mole below and 4.7 kcal/mole above the threshold after 3 h in culture. These results can be used as a conservative benchmark for thermal injury in the cancerous prostate. Further characterization of the response to thermal therapy in an animal model and in human tissues will be important in establishing the efficacy of the procedure

Measured effect of collection and cooling conditions on the motility and the water transport parameters at subzero temperatures of equine spermatozoa.

The effects of extracellular ice and cryoprotective agents on the measured volumetric shrinkage response and the membrane permeability parameters of equine spermatozoa have been reported previously. The volumetric shrinkage data were obtained using a differential scanning calorimeter technique that was independent of cell shape. The aim of this study was to examine the effects of collection and cooling conditions on the motility and the water transport parameters at subzero temperatures of equine spermatozoa. Stallion semen samples were collected using either a commercial lubricating agent, which caused osmotic stress to the spermatozoa, or water-insoluble Vaseline( trade mark ) as the artificial vagina lubricant. In some experiments, spermatozoa were cooled at 1 degrees C min(-1) from 20 degrees C to 4 degrees C to induce cold shock. An Equitainer was used to achieve control cooling rates (< or = 0.3 degrees C min(-1)) at temperatures > 0 degrees C. The water transport response of spermatozoa that were cold-shocked and osmotically shocked was significantly different from that of control spermatozoa (P < 0.01). Osmotic stress appeared to have an effect on the water transport response, although this effect was not significant. These results indicate that cold shock alters the behaviour of equine spermatozoa in cryopreservation protocols as a result of changes in the water transport properties of the plasma membrane. Although osmotic stress did not significantly affect water transport in equine spermatozoa, it did significantly decrease sperm motility in the extended semen samples (P < 0.01), which would, in turn, lower the quality of cold-stored or cryopreserved spermatozoa.

Cryopreservation of equine sperm: optimal cooling rates in the presence and absence of cryoprotective agents determined using differential scanning calorimetry.

Optimization of equine sperm cryopreservation protocols requires an understanding of the water permeability characteristics and volumetric shrinkage response during freezing. A cell-shape-independent differential scanning calorimeter (DSC) technique was used to measure the volumetric shrinkage during freezing of equine sperm suspensions at cooling rates of 5 degrees C/min and 20 degrees C/min in the presence and absence of cryoprotective agents (CPAs), i.e., in the Kenney extender and in the lactose-EDTA extender, respectively. The equine sperm was modeled as a cylinder of length 36.5 microm and a radius of 0.66 microm with an osmotically inactive cell volume (V(b)) of 0.6V(o), where V(o) is the isotonic cell volume. Sperm samples were collected using water-insoluble Vaseline in the artificial vagina and slow cooled at < or = 0.3 degrees C/min in an Equitainer-I from 37 degrees C to 4 degrees C. By fitting a model of water transport to the experimentally obtained DSC volumetric shrinkage data, the best-fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The combined best-fit parameters of water transport (at both 5 degrees C/min and 20 degrees C/min) in Kenney extender (absence of CPAs) are L(pg) = 0.02 microm min(-1) atm(-1) and E(Lp) = 32.7 kcal/mol with a goodness-of-fit parameter R(2) = 0.96, and the best-fit parameters in the lactose-EDTA extender (the CPA medium) are L(pg)[cpa] = 0.008 microm min(-1) atm(-1) and E(Lp)[cpa] = 12.1 kcal/mol with R(2) = 0.97. These parameters suggest that the optimal cooling rate for equine sperm is approximately 29 degrees C/min and is approximately 60 degrees C/min in the Kenney extender and in the lactose-EDTA extender. These rates are predicted assuming no intracellular ice formation occurs and that the approximately 5% of initial osmotically active water volume trapped inside the cells at -30 degrees C will form innocuous ice on further cooling. Numerical simulations also showed that in the lactose-EDTA extender, equine sperm trap approximately 3.4% and approximately 7.1% of the intracellular water when cooled at 20 degrees C/min and 100 degrees C/min, respectively. As an independent test of this prediction, the percentage of viable equine sperm was obtained after freezing at 6 different cooling rates (2 degrees C/min, 20 degrees C/min, 50 degrees C/min, 70 degrees C/min, 130 degrees C/min, and 200 degrees C/min) to -80 degrees C in the CPA medium. Sperm viability was essentially constant between 20 degrees C/min and 130 degrees C/min.

Evaluation of thermal therapy in a prostate cancer model using a wet electrode radiofrequency probe.

PURPOSE: To determine the temperature-time threshold of local cell death in vivo for thermal therapy in a prostate cancer animal model and to use this value as a benchmark to quantify global tissue injury. MATERIALS AND METHODS: Two studies were designed in the Dunning AT-1 rat prostate tumor hind limb model. For both studies, a wet electrode radiofrequency (RF) probe was used to deliver 40 W of energy for 18 to 62 seconds after a 30-second infusion of hypertonic saline/Hypaque through the RF antenna. Thermal history measurements were obtained in tumors from at least two Fluoroptic probes placed radially 5 mm from the axis of a RF probe and 10 mm below the surface of the tissue. In study 1, the thermal history required for irreversible cell injury was experimentally determined by comparing the predicted injury accumulation (omega) with cell viability at the fluoroptic probe locations using an in vivo-in vitro assay. The omega value was calculated from the measured thermal histories using an Arrhenius damage model. In study 2, RF energy was applied for 40 seconds in all cases. At 1, 3, and 7 days after thermal therapy, triphenyltetrazolium chloride dye (TTC) and histologic analyses were performed to assess global tissue injury within a 5-mm radius from the axis of the RF probe. RESULTS: Study 1 showed that cell survival dropped to 0 for 0.42 < omega < 0.7. This result was the basis for selection of 40 seconds of RF thermal therapy in study 2, which yielded omegaave = 0.5 in the tissue 5 mm from the probe axis. Both TTC and histology analysis showed that sham-treated tissue was not irreversibly injured. However, there was an inherent heterogeneity present in the tumor that accounted for as much as 15% necrosis in control or sham-treated tissue. In contrast, at 1, 3, and 7 days after therapy, significantly less enzyme activity was observed by TCC in thermally treated tissue compared with sham-treated tissue (35 v 85%; P < 0.001). Histologic analysis of thermally treated tissues revealed a gradual increase in the percent of coagulative necrosis (47%-70%) with a concomitant decrease in the percentage of shocked cells (53%-28%). At day 7, <3% viability was observed in treated tumors compared with 90% viability in sham-treated tissue. CONCLUSION: The threshold of cellular injury in vivo corresponded to omega > 0.7 (> or =48 degrees C for 40 seconds). Global tissue injury could be conservatively predicted on the basis of local thermal histories during therapy.

Investigation of the mechanism and the effect of cryoimmunology in the Copenhagen rat.

This study examined the potential for "cryoimmunology" to increase the destruction of the Dunning AT-1 prostate tumor after cryosurgery. Two possible mechanisms explaining the cryoimmunologic response were studied. The first was that an antitumor antibody is produced after cryosurgery. The second was that freezing induces an immunostimulatory signal that creates a T-cell response to the tumor. Six groups of animals (three experimental groups and three control groups) were treated once per week for 4 weeks with different therapies designed to investigate these mechanisms. Three types of immune response were measured: (1) the anti-AT-1 tumor immune titer (Ab response) by serum ELISA, (2) the effect on secondary tumor growth after challenge with live AT-1 cells (size and weight of the secondary tumor over time), and (3) the nature of the immunologic infiltrate into the secondary tumors by immunoperoxidase stain. ELISA showed that immune titers were present in the experimental groups after therapy, but the presence of an immune titer did not have a significant effect on tumor propagation. Histology showed the immunologic infiltrate was similar in all groups. These results showed that an immune response to AT-1 tumor was measurable by serum antibody, but it did not significantly limit secondary tumor growth or affect tumor histology. This suggests that the growth of AT-1 tumors is not inhibited by a cryoimmunological response. Thus, the effect of in vivo cryosurgery in the AT-1 tumor system would likely be limited to cellular and vascular changes.

Measurement and prediction of thermal behavior and acute assessment of injury in a pig model of renal cryosurgery.

PURPOSE: To analyze in vivo end temperatures and histologic injury in a standardized cryo-iceball using a porcine kidney model in order to establish the threshold temperature for tissue ablation. To evaluate the ability to predict end temperatures using a thermal finite element model. MATERIALS AND METHODS: A single freeze/thaw cryolesion was created in five pig kidneys and the temperature history recorded. End temperature was calculated using a thermal finite element model. The threshold temperature for tissue injury was established by directly correlating end temperature and histologic injury. RESULTS: Reproducible geometry and temperature profiles of the cryo-iceball were found. End temperature could be accurately predicted through thermal modeling, and correlation with histologic injury revealed a threshold temperature of -16.1 degrees C for complete tissue ablation. CONCLUSION: Thermal modeling may accurately predict end temperature within a cryo-iceball. Provided threshold temperatures for tissue destruction are known, modeling may become a powerful tool in cryosurgery, improving the assessment of damage in normal and malignant tissue.

Supraphysiological thermal injury in Dunning AT-1 prostate tumor cells.

To investigate the potential application of thermal therapy in the treatment of prostate cancer, the effects of supraphysiological temperatures (40-70 degrees C) for clinically relevant time periods (approximately 15 minutes) were experimentally studied on attached Dunning AT-1 rat prostate cancer cells using multiple assays. The membrane and reproductive machinery were the targets of injury selected for this study. In order to assess membrane injury, the leakage of calcein was measured dynamically, and the uptake of PI was measured postheating (1-3 hours). Clonogenicity was used as a measure of injury to the reproductive machinery 7 days post-injury after comparable thermal insults. Experimental results from all three assays show a broad trend of increasing injury with an increase in temperature and time of insult. Membrane injury, as measured by the fluorescent dye assays, does not correlate with clonogenic survival for many of the thermal histories investigated. In particular, the calcein assay at temperatures of < or = 40 degrees C led to measurable injury accumulation (dye leakage), which was considered sublethal, as shown by significant survival for comparable insult in the clonogenic assay. Additionally, the PI uptake assay used to measure injury post-thermal insult shows that membrane injury continues to accumulate after thermal insult at temperatures > or = 50 degrees C and may not always correlate with clonogenicity at hyperthermic temperatures such as 45 degrees C. Last, although the clonogenic assay yields the most accurate cell survival data, it is difficult to acquire these data at temperatures > or = 50 degrees C because the thermal transients in the experimental setup are significant as compared to the time scale of the experiment. To improve prediction and understanding of thermal injury in this prostate cancer cell line, a first-order rate process model of injury accumulation (the Arrhenius model) was fit to the experimental results. The activation energy (E) obtained using the Arrhenius model for an injury criterion of 30 percent for all three assays revealed that the mechanism of thermal injury measured is likely different for each of the three assays: clonogenics (526.39 kJ/mole), PI (244.8 kJ/mole), and calcein (81.33 kJ/mole). Moreover, the sensitivity of the rate of injury accumulation (d omega/dt) to temperature was highest for the clonogenic assay, lowest for calcein leakage, and intermediate for PI uptake, indicating the strong influence of E value on d omega/dt. Since the clonogenic assay is linked to the ultimate survival of the cell and accounts for all lethal mechanisms of cellular injury, the E and A values obtained from clonogenic study are the best values to apply to predict thermal injury in cells. For higher temperatures (> or = 50 degrees C) indicative of thermal therapies, the results of PI uptake can be used as a conservative estimate of cell death (underprediction). This is useful until better experimental protocols are available to account for thermal transients at high temperature to assess clonogenic ability. These results provide further insights into the mechanisms of thermal injury in single cell systems and may be useful for designing optimal protocols for clinical thermal therapy.

The effect of extracellular ice and cryoprotective agents on the water permeability parameters of human sperm plasma membrane during freezing.

A firm biophysical basis for the cryopreservation of human spermatozoa is limited by a lack of knowledge regarding the water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPA). Cryomicroscopy cannot be used to measure dehydration during freezing in human spermatozoa because of their highly non-spherical shape and their small dimensions which are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of human sperm cell suspensions was obtained at cooling rates of 5 and 10 degrees C/min in the presence of extracellular ice and CPA. Using previously published data, the human sperm cell was modelled as a cylinder of length 40.2 micrometer and a radius of 0.42 micrometer with an osmotically inactive cell volume, V(b), of 0.23V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The 'combined best fit' membrane permeability parameters at 5 and 10 degrees C/min for human sperm cells in modified media are: L(pg) = 2. 4x10(-14) m(3)/Ns (0.14 micrometer/min-atm) and E(Lp) = 357.7 kJ/mol (85. 5 kcal/mol) (R(2) = 0.98), and in CPA media (with 6% glycerol and 10% egg yolk) are L(pg)[cpa] = 0.67x10(-14) m(3)/Ns (0.04 micrometer/min-atm) and E(Lp)[cpa] = 138.9 kJ/mol (33.2 kcal/mol) (R(2) = 0.98). These parameters are significantly different from previously published parameters for human spermatozoa obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in human sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPA. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates (<100 degrees C/min) and the numerically predicted optimal cooling rates (>7000 degrees C/min) obtained using previously published suprazero human sperm permeability parameters which do not account for the presence of extracellular ice.

A parametric study of freezing injury in AT-1 rat prostate tumor cells.

The connection between thermal history and cell injury in single AT-1 cells is studied systematically through a two-level, four-parameter (2(4)) experiment. The four parameters considered are cooling rate (CR), end temperature (ET), hold time (HT), and thawing rate (TR). Cryosurgically relevant high and low values of each parameter are chosen (CR, 5 to 50 degrees C/min; ET, -20 to -80 degrees C; HT, 0 to 15 min; TR, 20 to 200 degrees C/min) to maximize applicability of the results to cryosurgery; it is important to note that any conclusions drawn from the results are valid only for the range of parameter values studied. AT-1 cell suspensions are frozen in a controlled way on a directional solidification stage, and viability is assessed postthaw with a live/dead assay using the fluorescent dyes calcein-AM and propidium iodide to indicate live and dead cells, respectively. The parameters which most significantly affect short-term survival outcome are determined through calculation of the individual parameter effect values (E) according to the factorial experimental design guidelines. In addition, any synergy between two parameters in determining short-term survival outcome is revealed by calculation of the interaction value for those parameters (I). The results suggest that survival is most significantly affected by variation in end temperature and hold time, and the only significant parameter interaction found is between these two parameters. The analysis further suggests that survival depends nonlinearly on the thermal parameters, based on calculation of the survival curvature (C) in the parameter ranges studied. These results are discussed within the context of previously proposed mechanisms of cellular injury during freezing. Although coupling between several mechanisms is possible, single mechanisms which may explain the survival results include slow-cooling injury mechanisms such as solute effects injury, dehydration-induced membrane instabilities, and volume-catalyzed nucleation of intracellular ice.

Subzero water permeability parameters of mouse spermatozoa in the presence of extracellular ice and cryoprotective agents.

Optimization of techniques for cryopreservation of mammalian sperm is limited by a lack of knowledge regarding water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPAs). Cryomicroscopy cannot be used to measure dehydration during freezing in mammalian sperm because they are highly nonspherical and their small dimensions are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of ICR mouse epididymal sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and CPAs. Using previously published data, the mouse sperm cell was modeled as a cylinder (122-microm long, radius 0.46 microm) with an osmotically inactive cell volume (V(b)) of 0.61V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The "combined best-fit" membrane permeability parameters at 5 and 20 degrees C/min for mouse sperm cells in solution are as follows: in D-PBS: L(pg) = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp) = 94.1 kJ/mole (22.5 kcal/mole) (R(2) = 0.94); in "low" CPA media (consisting of 1% glycerol, 6% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp)[cpa] = 122.2 kJ/mole (29.2 kcal/mole) (R(2) = 0.98); and in "high" CPA media (consisting of 4% glycerol, 16% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 0.68 x 10(-15) m(3)/Ns (0.004 microm/min-atm) and E(Lp)[cpa] = 63.6 kJ/mole (15.2 kcal/mole) (R(2) = 0.99). These parameters are significantly different than previously published parameters for mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in mouse sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPAs. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates, 10-40 degrees C/min, and the numerically predicted optimal cooling rates, greater than 5000 degrees C/min, obtained using suprazero mouse sperm permeability parameters that do not account for the presence of extracellular ice. As an independent test of this prediction, the percentages of viable and motile sperm cells were obtained after freezing at two different cooling rates ("slow" or 5 degrees C/min; "fast," or 20 degrees C/min) in both the low and high CPA media. The greatest sperm motility and viability was found with the low CPA media under fast (20 degrees C/min) cooling conditions.

Regional distribution of 5alpha-reductase type 1 and type 2 mRNA along the human epididymis.

OBJECTIVE: To determine the regional distribution and relative expression of 5alpha-reductase type 1 and type 2 mRNA within the human testis and regions of the epididymis. DESIGN: Prospective observational study. SETTING: University academic medical center. PATIENT(S): Two young adult male organ donors. INTERVENTION(S): None MAIN OUTCOME MEASURE(S): The distribution of 5alpha-reductase type 1 and type 2 mRNA in the testis and regions of the epididymis was detected by Northern blot analysis. The relative abundance of each 5alpha-reductase mRNA was evaluated using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in which cyclophilin mRNA, a house-keeping gene product, was coamplified as the reference standard. RESULT(S): Northern blot analysis revealed the 5alpha-reductase type 2 transcript in the midcaput, distal caput, corpus, and proximal cauda of the epididymis, but the transcript was undetectable in the testis, proximal caput, and distal cauda region. No transcript for the type 1 isozyme was detected by Northern blot. The more sensitive RT-PCR showed low levels of type 1 mRNA in the testis and epididymis, with the highest abundance in the proximal caput. Type 2 mRNA of 5alpha-reductase was most abundant in the midcaput, was decreased in the more distal regions, and was more abundant than type 1 mRNA in all epididymal regions except for the proximal caput. CONCLUSION(S): Both 5alpha-reductase type 1 and type 2 mRNAs are present in the human epididymis. The type 2 isozyme mRNA is predominant, being more highly expressed than the low-abundance type 1 mRNA.

Analysis of fibronectin on human sperm.

PURPOSE: The purpose of this study was to localize fibronectin on human sperm and correlate its distribution with the morphological and functional integrity of sperm. MATERIALS AND METHODS: Semen samples were collected and sperm fractionated by swim-up. Subsets of the swim-up sperm were capacitated and acrosome reacted. Damage to swim-up sperm was induced by freezing and thawing. The presence of fibronectin on the surface of sperm was determined by immunocytochemistry. RESULTS: FN immunoreactivity was variable but staining on the sperm tail was consistently highest, whereas FN immunoreactivity over the acrosome and equatorial band was consistently lowest. Capacitation and acrosome reaction did not substantially change the distribution of FN staining. However, swim-up sperm had significantly less FN immunoreactivity (4%) than sperm that were unable to swim-up (12%; p < 0.01). Sperm that were deliberately damaged by freeze/thaw showed significantly increased FN binding (p < 0.01). FN immunoreactivity was inversely correlated with sperm viability (r = -0.68), motility (r = -0.70), and morphology (r = -0.63). CONCLUSIONS: This study demonstrates that only a minority of the sperm in an ejaculate stain positive for FN and the localization of FN in positive sperm is primarily to the tail. Inferior sperm stain more frequently for FN leading to an inverse correlation between FN staining and sperm quality. Taken together, these results do not support a role for FN in sperm-egg binding. However, FN staining may provide a method for selecting the highest quality sperm for use in assisted reproduction techniques.

Diverse testicular responses to exogenous growth hormone and follicle-stimulating hormone in prepubertal boars.

The effects of exogenous growth hormone (GH) and FSH on development of the testes in intact prepubertal boars was investigated. Twenty-four boars received one of four daily treatments from 8 through 40 days of age: 1) 90 micrograms porcine (p) GH/kg body weight (BW), 2) 100 micrograms pFSH/kg BW, 3) GH + FSH, or 4) vehicle only (control). Plasma testosterone levels, measured at 10-day intervals, were similar among groups of boars throughout the study. Body weights among groups were similar during treatment, and testicular weight between treatment groups did not differ at castration (100 days of age). However, total length of the seminiferous tubule per testis in FSH-treated boars was 59% greater than in GH-treated animals (2705 vs. 1704 m; p < 0.05). Diameter of the tubule in GH-treated boars was 58% greater than in FSH-treated boars (p = 0.03). Relative mass of spermatocytes and spermatids in GH-treated animals exceeded that in controls by 2.5-fold and that in FSH boars by 75-fold (p = 0.05). There were no differences in effects of GH + FSH treatment as compared to control treatment; none of the treatments affected any interstitial tissue parameter measured. These results suggest that exogenous FSH had a mitogenic effect on Sertoli cells while delaying tubular maturity, whereas exogenous GH promoted tubular and Sertoli cell maturation, defined as increased Sertoli cell size, lumen formation, and onset of spermatogenesis.