Biomedical subjects

R A Hess

Publications and source records attributed to R A Hess.

At least 55 records · Page 3Linked to original sources

PubMed1994

Developmental expression of testis messenger ribonucleic acids in the rat following propylthiouracil-induced neonatal hypothyroidism.

Propylthiouracil- (PTU) induced transient neonatal hypothyroidism increases adult rat testis weight 80-100%; this effect involves prolongation of Sertoli cell proliferation. To gain insight into developmental effects of PTU on the testis, we used Northern analysis to examine chronological expression of Sertoli cell mRNA in postnatal rat testes from rats that were untreated (controls) or were given PTU from birth to Day 25. Treated rats showed prolonged early expression of genes associated with dividing Sertoli cells such as MIS (Müllerian inhibiting substance) and c-erbA alpha (thyroid hormone receptor). Expression of several other Sertoli cell mRNAs (androgen-binding protein [ABP], clusterin, and inhibin-beta B) was delayed, as was that of hemiferrin, a spermatid-specific mRNA. Temporal expression patterns for other mRNAs (sulfated glycoprotein [SGP]-1, transferrin, and inhibin-alpha) were similar in control and treated animals. Additionally, thyroid hormone replacement in PTU-treated animals decreased MIS and c-erbA alpha mRNA expression to control levels. The altered developmental pattern of expression of a number of major Sertoli cell genes reflects a prolonged mitogenesis and delayed maturation of Sertoli cells in neonatally hypothyroid animals. Furthermore, our results suggest that thyroid hormone may directly potentiate molecular events associated with cessation of Sertoli cell proliferation and maturation during early testis development.

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PubMed1993

Epithelial reorganization and irregular growth following carbendazim-induced injury of the efferent ductules of the rat testis.

Occlusions of the rat efferent ductules were induced by an oral gavage of carbendazim and the capacity for regeneration or recanalization of the ductules was histologically evaluated between 2 and 70 days post-treatment. At 2 days post-treatment, the efferent ductules were occluded by sloughed materials and showed various degrees of inflammation. Severely damaged epithelium showed few regenerative features at later intervals. On the other hand, epithelia with medium inflammation often exhibited irregular epithelial growth along the luminal contents or the formation of multiple abnormal ductules. These abnormal ductules were formed by migrated and original epithelia at the periphery of the occluded lumen at 16 days post-treatment, indicating attempted recanalization. At later time periods, 32 and 70 days post-treatment, the occluded original lumen was filled in by fibrotic connective tissue and surrounded by a series of abnormal ductules. These abnormal ductules were characterized by cuboidal epithelia, a small luminal diameter, fewer cilitated cells than normal, less developed organelles in the epithelial cells, and basal laminae of irregular thickness. However, there was no evidence that occluded ductules formed patent re-connections via abnormal ductules. The results suggest that occluded efferent ductules have the ability to initiate epithelial regrowth and to form new ductules, but the newly formed ductules are abnormal and are not adequate to recover from azoospermia at least at 70 days post-treatment.

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PubMed1993

Immunoelectron microscopic localization of testicular and somatic cytochromes c in the seminiferous epithelium of the rat.

Somatic and testis-specific cytochromes c were localized ultrastructurally in the seminiferous epithelium by immunocytochemistry using monospecific antibodies. Cytochrome cS was lost from the mitochondria as spermatogenesis advanced, while there was a relative increase in cytochrome cT during the zygotene-to-pachytene transition; this was in agreement with other studies that have suggested activation of the cytochrome cT gene during prophase of the first meiotic division. Cytochrome cT was highly concentrated in mitochondria that were being degraded within cytoplasmic lobes of spermatids and in residual bodies that were phagocytized by Sertoli cells. The two isoforms were found to coexist within the same mitochondrion during the transitional period from cytochrome cS to cytochrome cT predominance. In addition, both cytochromes c were present in the chromatoid bodies of spermatocytes and round spermatids; this suggests that the chromatoid body may be involved in the storage of these isozymes and possibly in their differential expression within germ cell mitochondria. Apocytochrome c was concentrated in mitochondria and chromatoid bodies of the germ cells and also scattered in the cytoplasm. The presence of the holoprotein and apoprotein immunoprobes within the chromatoid bodies of spermatocytes and spermatids was an interesting observation that raises questions regarding the precise location of the synthesis of cytochromes c in spermatogenic cells.

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PubMed1993

Germ cells of the mouse testis express P450 aromatase.

Estrogen production within the testis has been a subject of considerable controversy for many years. Several studies have shown that both Sertoli and Leydig cells produce estrogen during different stages of development. Therefore, we have conducted experiments to localize aromatase, a cytochrome P450 enzyme that converts androgen to estrogen, within the testis. First, P450 aromatase (P450arom) was localized in germ cells of the adult mouse testis by immunocytochemistry, using an antiserum generated against purified human placental cytochrome P450arom. In the germinal epithelium, P450arom was located primarily in the Golgi region of round spermatids, throughout the cytoplasm of elongating spermatids, and along the flagella of late spermatids. Second, localization of P450arom within the germinal epithelium was supported by Western blot analysis of isolated germ cells. Third, Northern blot analysis using a mouse P450arom cDNA probe indicated that the mRNA for the mouse P450arom was present in testicular germ cells. Fourth, P450arom activity was measured in germ cells by the 3H2O water assay. Based upon these observations, we conclude that germ cells are a site of estrogen synthesis in the adult mouse testis.

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PubMed1993

Leydig cells increase their numbers but decline in steroidogenic function in the adult rat after neonatal hypothyroidism.

Administration of the goitrogen, 6-propyl-2-thiouracil (PTU), to suckling rat pups from birth through day 24 postpartum as a 0.1% solution in the mother's drinking water increases adult testis size and sperm production by about 80% and 140%, respectively, without affecting peripheral testosterone levels. The objectives of this study were to determine whether adult Leydig cell numbers were altered in PTU-treated rats and whether the steroidogenic function of these cells was normal. The number of Leydig cells per testis at 180 days increased by 69% in PTU-treated compared to control rats, whereas the average Leydig cell volume declined by about 20%. Steroidogenic function assessed in isolated adult Leydig cells decreased after neonatal PTU treatment. LH-stimulated testosterone production was reduced by 55% in Leydig cells from treated rats, commensurate with a 50% decline in the number of hCG-binding sites in these cells. The difference in steroidogenic potential was even more striking after incubations with saturating concentrations of steroid substrate, 22(R)-hydroxycholesterol; Leydig cells from treated males produced 73% less testosterone than controls. Therefore, this decrease in testosterone production may be partially due to a reduction in the numbers of LH receptors, but also reflects the impaired steroidogenic potential of these cells. These results clearly show that the dramatic increase in adult Leydig cell number after neonatal PTU treatment is counterbalanced by a permanent decline in Leydig cell steroidogenic function, producing no net change in peripheral testosterone levels.

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PubMed1993

Adult testicular enlargement induced by neonatal hypothyroidism is accompanied by increased Sertoli and germ cell numbers.

Our previous studies have shown that transient neonatal hypothyroidism, induced by treatment with the reversible goitrogen 6-propyl-2-thiouracil (PTU), increases testicular size and daily sperm production in the adult rat by up to 82% and 136%, respectively. The objective of the present study was to examine morphological and functional changes in adult seminiferous tubules associated with PTU-induced increases in testicular size and sperm production. Sprague-Dawley rats were treated with PTU from birth to day 25 or left untreated; for morphometry, all testes were fixed by vascular perfusion at 90 days of age. Although testicular weight was increased 62% in treated rats, gross pathological changes were not evident in these organs, and spermatogenesis appeared morphologically normal. The percent area of testis occupied by seminiferous tubules was equal in control and treated testes, but mean seminiferous tubule diameter and length were increased in the PTU-treated testis. The adult number of Sertoli cells in treated testes was increased by 157%, and the numbers of leptotene spermatocytes and round spermatids were increased 84% and 93%, respectively. These results demonstrate that increases in Sertoli cell numbers result in increased sperm production and support the idea that Sertoli cells are the major regulators of the magnitude of sperm production. Although the round spermatid to Sertoli cell ratio was reduced by nearly 30%, the number of round spermatids per g testis was increased by 14%. This increased efficiency of sperm production was accomplished by an increased density of Sertoli cells along the basement membrane and an increased height of the seminiferous epithelium. Despite the large increase in Sertoli cell numbers in treated rats, Northern blot analysis using Sertoli cell-specific cDNA probes for transferrin and androgen-binding protein indicated that relative steady state levels of mRNAs per Sertoli cell for these two secretory proteins were similar in control and treated rats at 90 days of age.

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PubMed1992

Blind-ending tubules and branching patterns of the rat ductuli efferentes.

The ductuli efferentes of Sprague-Dawley rats were studied by microdissection and microscopic evaluation to document the presence of blind-ending tubules (ductuli aberrantes) and to describe morphological and ultrastructural differences between normally open ductules and blind-ending tubules. The branching patterns of the ductuli efferentes varied considerably between animals. A majority of the animals studied had either six or seven ductuli connected to the rete testis, with some animals having as few as four or as many as eight. Pairs of ductules began merging in the conus vasculosa, ultimately forming a single terminal duct within the capsule of the initial segment epididymidis. In a majority of animals, the junctions were unequally matched and located at various positions within the conus. Blind-ending tubules, found in 60% of the animals, were surrounded by thick connective tissue, and had a smaller diameter (78.7 +/- 1.4 microns) than normal ductules in the conus vasculosa (119.5 +/- 2.1 microns) or the terminus (102.2 +/- 1.5 microns). The lumina of blind-ending tubules were contracted and did not contain sperm. Nonciliated cells in the epithelium of blind-ending tubules contained fewer PAS-positive granules and electron-dense bodies (lysosomes) than nonciliated cells in normal ductules. Consideration of these characteristics will prevent blind-ending tubules from being mistaken for pathological changes in efferent ductules.

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PubMed1992

Localization and activity of Na+,K(+)-ATPase in the ductuli efferentes of the rat.

The Na+,K(+)-ATPase enzyme through its p-nitrophenyl phosphatase activity was localized in the ductuli efferentes of rats. Enzymatic activity was demonstrated along the cytoplasmic side of the plasmalemma of the ductular epithelial cells. The most intense deposition of reaction products was found on the plasmalemma delimiting the lower lateral and basal regions of the cells. The plasma membranes forming the microvilli, apical junctional complexes were devoid of reaction product while the midlateral membranes showed a weak reaction. The enzyme reaction was potassium-dependent and was abolished by addition of 10 mM ouabain to the incubation media. Enzyme activity decreased significantly from proximal to distal regions of the ductules (8,101.47 +/- 274.53, 6,658.95 +/- 269.53 and 4,668.10 +/- 575.41 pmoles p-nitrophenol/mm/h, respectively in initial, conus vasculosus and terminal zones). A unified model for water absorption is proposed in the efferent ductules based upon this data and that of others.

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PubMed1992

Immortalization of germ cells and somatic testicular cells using the SV40 large T antigen.

We report the immortalization, using the SV40 large T antigen, of all the cell types contributing to a developing seminiferous tubule in the mouse testis. Sixteen peritubular, 22 Leydig, 8 Sertoli, and 1 germ cell line have been established and cultured successfully for 90 generations in a period of 2.5 years. Immortalized peritubular cells were identified by their spindle-like appearance, their high expression of alkaline phosphatase, and their expression of the intermediary filament desmin. They also produce high amounts of collagen. Immortalized Leydig cells are easily identifiable by the accumulation of lipid droplets in their cytoplasm and the production of the enzyme 3-beta-hydroxysteroid dehydrogenase. Some Leydig cell lines also express LH receptors. The immortalized Sertoli cells are able to adopt their typical in vivo columnar appearance when cultured at high density. They exhibit a typical indented nucleus and cytoplasmic phagosomes. Some Sertoli cell lines also express FSH receptors. A germ cell line (GC-1spg) was established that corresponds to a stage between spermatogonia type B and primary spermatocyte, based on its characteristics in phase contrast and electron microscopy. This cell line expresses the testicular cytochrome ct and lactate dehydrogenase-C4 isozyme. These four immortalized cell types, when plated together, are able to reaggregate and form structures resembling two-dimensional spermatogenic tubules in vitro. When only the immortalized somatic cells are cocultured, the peritubular and Sertoli cells form cord-like structures in the presence of Leydig cells. Fresh pachytene spermatocytes cocultured with the immortalized somatic cells integrate within the cords and are able to survive for at least 7 days. The ability to perform coculture experiments with immortalized testicular cell lines represents an important advancement in our ability to study the nature of cell-cell and cell-matrix interactions during spermatogenesis and testis morphogenesis.

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PubMed1992

Induction of increased testis growth and sperm production in adult rats by neonatal administration of the goitrogen propylthiouracil (PTU): the critical period.

We have previously shown that treating rats with the reversible goitrogen 6-propyl-2-thiouracil (PTU) from birth to Day 25 increases testis size and sperm production in adulthood by up to 80% and 140%, respectively. The purpose of this study was to determine the critical period(s) during development when PTU treatment can increase adult testis size and sperm production. Rats were treated with PTU beginning on Days 0, 8, 16, or 24 for periods of 9, 17, or 25 days. To further define the critical period, additional rats were treated with PTU prenatally or on Days 4-24. PTU treatments of 9, 17, or 25 days beginning at birth increased testis weight 18%, 38%, and 69%, respectively, by 135 days of age, while daily sperm production (DSP) increased 35%, 65%, and 94%, respectively. Efficiency of sperm production (DSP/g testis) also increased by approximately 25% in these rats. There was an inverse relationship between testis and body weights: increasing lengths of PTU treatment increased testis weight but decreased body weight. PTU treatment starting on Day 8 or later did not increase testis weight or DSP regardless of duration. Treatment on Days 4-24 increased adult testis weight and DSP similarly to treatment from birth to Day 24, but prenatal PTU treatment was ineffective. Testosterone concentrations were not altered in treated rats, even those with increased testis weight. These results indicate that the later part of the first postnatal week (Days 4-8) is the crucial period during which PTU treatment must begin in order to increase adult testis size and sperm production.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1992

Developmental hormonal profiles accompanying the neonatal hypothyroidism-induced increase in adult testicular size and sperm production in the rat.

Neonatal treatment with the reversible goitrogen 6-N-propyl-2-thiouracil (PTU) results in a near doubling of testicular size and a 25% increase in the efficiency of spermatogenesis, without affecting circulating testosterone (T) levels in adult rats. The objectives of the present study were to examine the effects of neonatal PTU treatment on the pattern of testicular growth and circulating levels of anterior pituitary (FSH, LH, PRL, GH, and TSH), gonadal [immunoreactive inhibin-alpha (irI alpha) and T], and thyroid (T3 and T4) hormones over the first 100 days of life. Treatment of rats with PTU from birth to 24 days of age significantly reduced testicular weights between 10 and 60 days of age. However, the duration of testicular growth was extended in treated males, resulting in a 68% increase at 100 days of age. Serum gonadotropin levels in treated males were reduced throughout the experimental period, typically remaining between 50-70% of control levels. The characteristic robust prepubertal FSH peak was absent in PTU-treated males. Initially high until 20 days of age, irI alpha levels characteristically declined to adult levels (200-300 pg/ml) in control males. In treated males, irI alpha levels were reduced during the period of hypothyroidism, increased between 30 and 60 days, and then declined, but remained significantly higher (1.7- to 2-fold greater) than those observed in control males. Serum T levels were similar in treated and control males. Control males demonstrated increased T levels beginning at 45 days of age, earlier than observed in treated males; however, similar peak T levels were observed in all males. PTU treatment significantly suppressed serum GH and PRL and led to a 14-fold increase in circulating TSH during the period of treatment. However, unlike the gonadotropins, these hormones returned to control levels after PTU treatment, suggesting that the reduced levels of FSH and LH observed are not due to a generalized reduction in pituitary function. Serum T4 and T3 levels returned to control levels within 15 days after the removal of PTU. These results demonstrate that the neonatal PTU treatment-induced increases in adult testicular size and sperm production were not due to increased levels of FSH at any point in development. On the contrary, the observed increases occur in spite of chronically reduced FSH levels.

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PubMed1991

Ultrastructure of the turtle spermatozoon.

The turtle spermatozoon is vermiform in shape with a narrow pointed head that is curved. In general, the turtle sperm contains a typical head, midpiece and tail, similar in morphology to that of birds, amphibians and other reptiles. However, several structures are unique. These unusual features include (1) a perforatorial cap over the proximal end of the nucleus, which contains 2-3 rods that are contiguous with intranuclear tubules; (2) a connecting collar of dense material that surrounds the base of the nucleus; (3) a distal centriole containing central microtubules that extend its entire length and having outer triplicate microtubules that open toward the central cavity of the centriole; and (4) unusual spherical mitochondria containing 7-8 outer laminated membranes.

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PubMed1991

The fungicide benomyl (methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate) causes testicular dysfunction by inducing the sloughing of germ cells and occlusion of efferent ductules.

Adult male Sprague-Dawley rats (100 days of age) were given single oral doses of the fungicide benomyl (methyl 1-(butylcarbamoyl)-2- benzimidazolecarbamate) in dosages ranging from 25 to 800 mg/kg body weight. The testis and excurrent ducts were examined at 2 and 70 days posttreatment to determine the chemical effects on spermatogenesis and the epididymis. Primary effects seen at 2 days postexposure were testicular swelling and occlusions of the efferent ductules. Mean testis weight peaked with 400 mg/kg. Premature release of germ cells (sloughing), detected even with the lowest dosage (25 mg/kg), was the most sensitive short-term response to benomyl. Sloughing was found primarily in Stage VII of the spermatogenic cycle at the lower dosages, but at higher dosages sloughing extended into all stages except for Stages VIII-XI. Occlusions of the efferent ductules of the testis were dose-dependent and correlated with testis weight. Sperm and sloughed germ cells were compacted in the ductal lumen of occluded ductules, which were surrounded by two to four layers of polymorphonuclear leukocytes and other inflammatory cells. Although the caput epididymidis contained sloughed germ cells and appeared swollen, evidence of permanent occlusion was not found. The long-term (70 days) effects of benomyl were decreased testis weight (at 400 mg/kg), dose-dependent increases in seminiferous tubular atrophy, and increases in the number of reproductive tracts containing occluded efferent ductules. Fibrosis, granulomas, and abnormal growth were long-term consequences of occluded ductules, which were present 100% of the time in testes containing 26-100% seminiferous tubular atrophy. Only a few testes were found to be completely regressed. Occlusion of efferent ductules and disruption of the seminiferous epithelium by sloughing of germ cells are overlapping dose-dependent mechanisms responsible for short- and long-term effects of benomyl on the rat testis.

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PubMed1991

Effects of in utero ultrasound exposure on the development of the fetal mouse testis.

The effect on the developing fetal testis of in utero exposure to 1-MHz, continuous-wave ultrasound in the spatial peak, temporal average intensity range 0.5-10 W/cm2 for durations of 400-30 sec on Day 9, 12, or 15 of gestation was determined. Results show that two subtle, yet potentially deleterious, effects occurred: a reduction in the Sertoli cell population and an apparent delay in the cessation of gonocyte mitosis. An increase was also seen in the number of fetal resorptions and stillborn pups per number of implantation sites in the exposed specimens as compared to the sham and cage controls. Because the reduction in testis weight was proportional to decreased body weight and because there was no difference in Sertoli cell numbers due to day of treatment, the testicular effects may reflect a generalized delay in growth. Whether this effect of ultrasound on fetal testis will be translated into an equal reduction in germ cell numbers in the adult testis remains to be determined.

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PubMed1991

Estrogen receptor expression in developing epididymis, efferent ductules, and other male reproductive organs.

The distribution of estrogen receptors (ER) in developing reproductive organs of male BALB/c mice was determined by 3H-estradiol steroid autoradiography. Efferent ductules, urogenital sinus and Wolffian ducts, and their derivatives, the epididymis, ductus deferens, seminal vesicles, coagulating glands, prostate, and bulbouretheral glands (BUGs), were examined from 16 days fetal (gestation = 19-20 days) to 10 days postnatal. All fetal reproductive organs strongly expressed mesenchymal ER. Stromal cells of these organs remained ER+ at later times. However, smooth muscle cells in organs such as the ductus deferens, BUG, prostate, and caudal epididymis were only weakly ER+ or ER- after their differentiation from mesenchyme, although fibroblasts interspersed within the smooth muscle remained strongly ER+. Efferent ductules were the first site of epithelial ER expression in the developing male tract; this organ expressed epithelial ER on day 16 of gestation and subsequently. Wolffian ducts and urogenital sinus did not contain epithelial ER on day 16 of gestation. Epididymis began expressing epithelial ER soon after its differentiation, on day 19 of gestation. A clear gradient of ER expression was noted in the regions of the developing epididymis, with the efferent ducts and the initial segment of the epididymis containing 3-fold more silver grains per epithelial cell than more distal regions of the epididymis. Epithelium of the seminal vesicle and coagulating gland was initially ER-, but became weakly ER+ at day 6 postnatal and later. The epithelium of all other organs (ductus deferens, prostate, and BUGs) never expressed ER at any time.

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PubMed1991

Increased sperm production in adult rats after transient neonatal hypothyroidism.

In the preceding paper it was shown that transient neonatal hypothyroidism induced by treatment of rats from birth to day 25 with the goitrogen 6-propyl-2-thiouracil (PTU) is associated with increases in testis wt and DNA content of up to 80% during adulthood. The testis changes were accompanied by similar, though less marked, increases in the wt and DNA content of epididymis and accessory organs. The purpose of this study was to assess sperm production in these enlarged testes and measure changes in sperm reserves in the epididymis. Testes and epididymides were obtained from control rats or rats given PTU from birth to day 25 (designated "treated") at 90, 135, 160, and 180 days of age. Daily sperm production (DSP), efficiency of sperm production (DSP/g testis), and epididymal sperm reserves were measured in all animals. Compared to controls, DSP of the treated rats was increased by 83%, 86%, 136%, and 132% at 90, 135, 160, and 180 days, respectively. Thus, in the treated rats, DSP, like testis wt, plateaued at day 160. In addition, efficiency of sperm production was increased by 15%-30% at all ages in treated animals. Epididymal sperm reserves were also increased in treated rats at all ages, but the correlation between DSP and epididymal sperm reserves was weak. Sperm motility and concentration in caudal epididymal fluid of adult males treated from birth to day 25 with PTU were normal. These males were fertile and sired litters in which pup wt and pup number were normal. These results indicate that neonatal hypothyroidism in rats is associated not only with increased testis size but also with increased efficiency of sperm production, resulting in increases in DSP of up to 140% in these animals during adulthood. Maximal sperm production is reached at 160 days of age in treated rats (compared to 100 days in controls), coinciding with the attainment of final testicular size. This system represents the first experimental model in which such large increases in sperm production can be produced. The neonatal PTU treatment does not appear to impair fertility or alter sperm characteristics when these animals become adults and may be a useful system with which to study factors which normally regulate sperm production.

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PubMed1990

Decreased spermatozoal survivability associated with aberrant morphology of the ductuli efferentes proximales of the chicken (Gallus domesticus).

The objective of this research were twofold: 1) to determine if decreased spermatozoal longevity, a previously reported heritable trait in chickens, was attributable to spermatozoal passage through the excurrent ducts, and 2) to document the morphology of the testicular excurrent ducts from affected roosters. Though spermatozoa were viable at ejaculation, as evidenced by their exclusion of ethidium bromide, fertility after intravaginal insemination of spermatozoa from affected roosters was less (p less than 0.001) than that observed with spermatozoa from nonaffected controls, 37 +/- 2.3 versus 58 +/- 1.5%, respectively, over a 21-day egg-collection interval. In contrast, fertility after intramagnal insemination of testicular spermatozoa from affected roosters was equivalent (p greater than 0.05) to that of nonaffected controls, 47 +/- 2.2 versus 41 +/- 3.6%, respectively. After intravaginal insemination, neither type of testicular spermatozoa fertilized oocytes. The ductuli efferentes proximales from affected roosters were characterized by a greater luminal cross-sectional area as well as a diminished height and number of longitudinal epithelial folds (p less than 0.005). It was concluded that heritable decreased spermatozoal longevity in the chicken is not attributable to an inherent spermatozoal defect. Rather, the defect is acquired during passage of spermatozoa through the extragonadal ducts of the rooster.

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PubMed1990

Frequency of the stages in the cycle of the seminiferous epithelium in the rat.

This study determined the optimum number of tubules to be counted per testis cross section, and the number of animals per treatment group, when changes in stage frequencies in the cycle of the seminiferous epithelium are criteria for assessing effects of treatment on spermatogenesis. A data base of 9,672 observed and staged tubules was collected from testicular cross sections of 15 Sprague-Dawley rats. A significant variation between animals was found for the frequencies of Stages I, II, IV, VI, VIII, and XIII. Computer simulation was used to randomly select different combinations of animal and tubule numbers from the observed data. Stage frequency means from each simulation experiment were compared statistically to observed mean frequencies. A model that used data from all 14 stages was analyzed. The following conclusions were made: a) a minimum of 200 tubule cross sections/testis is recommended for estimating stage frequencies; b) for a fixed number of tubules scored, the number of animals sampled is more important than the number of tubules per animal in reducing variance; c) to detect a difference of 2 standard deviations from the mean with a 2% error rate and examining 200 tubules/testis, at least 12 animals must be used per group when assessing all 14 stages; d) when individual stages are examined using 10 animals per group, only Stage VII has 80% or greater power of test (alpha = 0.05) to detect a frequency difference; e) pooling stages into 3-4 groups is recommended to improve the power of detecting a treatment difference.

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