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R A Hess

Publications and source records attributed to R A Hess.

At least 37 records · Page 2Linked to original sources
PubMed1998

Isolation and culture of epithelial cells from rat ductuli efferentes and initial segment epididymidis.

To improve the study of epithelial function in rat ductuli efferentes (efferent ductules) and initial segment epididymis, we developed a primary cell culture system with modification of the Klinefelter method (1992). The cultured efferent ductal epithelium was grown to confluence and the cells maintained many of the organelles characteristic of these cells in vivo, including dense-staining granules, indented nuclei and apical cilia. Ciliary beat was observed for up to 10 days in culture, Cultured initial segment epithelial cells were elongated and characterized by apical branched microvilli. Electron microscopy revealed intact cell junctions, and endocytotic apparatus and lysosomal granules. Ultrastructurally, the initial segment epithelium contained a well developed Golgi apparatus. For both epithelia, cell characteristics were also confirmed by indirect immunofluorescent staining for cytokeratins 8, 18. Endocytotic activity was detected by the uptake of cationic ferritin at the apical surface and within vesicles. Estrogen receptor and clusterin mRNAs were expressed in the cultured epithelia and no difference was found in their expressions when cultured with or without 10(-9)M 17-beta estradiol. Indirect immunofluorescent staining for clusterin further indicated that this protein was present in the cultures. In conclusion, these in vitro methods will be useful for the investigation of epithelial function in the head of the epididymis.

Animals↗
PubMed1998

Characterization of stage-specific tyrosinated alpha-tubulin immunoperoxidase staining patterns in Sertoli cells of rat seminiferous tubules by light microscopic image analysis.

Microtubules are involved in many structural and functional changes that occur in Sertoli cells during the cycle of the seminiferous epithelium. However, few studies have addressed stage-specific changes in the distribution of microtubules that accompany the process of spermatogenesis. This study provides a stage-by-stage immunohistochemical evaluation of Sertoli cell microtubules in paraffin sections of whole rat testes using an antibody to tyrosinated alpha-tubulin. Microtubules that contain tyrosinated tubulin are considered to be less stable and are therefore expected to populate Sertoli cells undergoing dynamic changes during spermatogenesis. A quantitative method was developed to analyze the relative tyrosinated alpha-tubulin staining in different stages of the cycle. Immunostaining patterns of the stages were separated into five different groups. Stages VII-VIII had the least amount of tyrosinated alpha-tubulin, while stages IX-XI contained the greatest amount. The staining patterns were consistent with established structural changes in the seminiferous epithelium, such as the formation of ectoplasmic specializations, the presence of microtubule nucleation sites along the periphery of the apical cytoplasm, and the translocation of elongate spermatids from deep crypts in the Sertoli cell to the tubule lumen. These data should provide improved methods for the evaluation of microtubules in the study of Sertoli cell responses to environmental toxicants and testicular diseases.

Animals↗
PubMed1998

Whiplash.

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Accidents, Traffic↗
PubMed1998

Spermatids of prepubertal male rats are susceptible to carbendazim during early spermiogenesis.

The fungicide carbendazim, a male reproductive system toxicant, affects the adult testis, while prepubertal animals are assumed to be unsusceptible to the chemical. The present study was conducted to re-evaluate the susceptibility of rat prepubertal testis (25-30 days old) to carbendazim based on the incidence of spermatid abnormalities, including nuclear enlargement (megaspermatids) and binucleate round spermatids. In control prepubertal rats treated orally with corn oil vehicle alone, seminiferous tubules containing magno- and/or binucleate round spermatids were often observed in the groups at stages II-III, IV-V and VI-VII. At 3.0 days after treatment with carbendazim (100 mg/kg), seminiferous tubules containing spermatids with identical abnormalities were significantly increased, including groups at all stages. Significant increases were also observed at stages IV-V and VI-VII at day 4.5, and VI-VII at day 7.5. In contrast, the frequency of spermatids with these abnormalities was reduced in the groups at stages II-III and IV-V at day 7.5. These findings confirm that the prepubertal rat testis is susceptible to carbendazim during early spermiogenesis.

Animals↗
PubMed1998

Effects of environmental toxicants on the efferent ducts, epididymis and fertility.

Many of the reproductive toxicants have primary effects on the testis, which potentially overshadow effects downstream on the efferent ducts and epididymis. The specific target of these effects depends upon the dosage and time response. It is often necessary to design experiments that separate testosterone-dependent responses arising in the testis from direct effects on epididymal tissues and spermatozoa, to uncover the mechanisms of toxicity in excurrent ducts. Recent studies have confirmed that chemicals can also alter the time required for sperm transport through the epididymis. Currently there are approximately twenty chemicals that can be classified as epididymal toxicants. There are fewer toxicants reported for the efferent ducts, but a few overlap with epididymal effects. The benzimidazole carbamates, like many efferent ductal toxicants, induce occlusions and subsequent testicular atrophy. The mechanisms appear to be related to fluid reabsorption, sperm stasis, followed by leukocyte chemotaxis, sperm granulomas, fibrosis and often the formation of abnormal microcanals. Disruption of oestrogen receptor function in the efferent ducts also interferes with fluid reabsorption and results in testicular swelling and seminiferous tubular atrophy. Thus, studies in which testicular atrophy occurs after chronic or subchronic exposures should be examined for lesions in efferent ducts and head of the epididymis. Such lesions can lead to permanent infertility.

Absorption↗
PubMed1997

A role for oestrogens in the male reproductive system.

Oestrogen is considered to be the 'female' hormone, whereas testosterone is considered the 'male' hormone. However, both hormones are present in both sexes. Thus sexual distinctions are not qualitative differences, but rather result from quantitative divergence in hormone concentrations and differential expressions of steroid hormone receptors. In males, oestrogen is present in low concentrations in blood, but can be extraordinarily high in semen, and as high as 250 pg ml(-1) in rete testis fluids, which is higher than serum oestradiol in the female. It is well known that male reproductive tissues express oestrogen receptors, but the role of oestrogen in male reproduction has remained unclear. Here we provide evidence of a physiological role for oestrogen in male reproductive organs. We show that oestrogen regulates the reabsorption of luminal fluid in the head of the epididymis. Disruption of this essential function causes sperm to enter the epididymis diluted, rather than concentrated, resulting in infertility. This finding raises further concern over the potential direct effects of environmental oestrogens on male reproduction and reported declines in human sperm counts.

Animals↗
PubMed1997

Effects of carbendazim (methyl 2-benzimidazole carbamate; MBC) on meiotic spermatocytes and subsequent spermiogenesis in the rat testis.

BACKGROUND: Benzimidazole fungicide, carbendazim, is known to adversely affect Sertoli cells by disrupting microtubules, which induces sloughing of elongate spermatids in a stage-specific manner. This study determines the direct effects on dividing germ cells and the subsequent effects on spermiogenesis. METHODS: Carbendazim was administered orally to male rats (100 mg/kg), and their testes were processed for histological evaluation at various post-treatment intervals up to day 20.0. RESULTS: The sloughing of elongate spermatids was observed as reported previously. In addition to this Sertoli cell lesion, necrosis of dividing spermatocytes in stage XIV was observed at 8 hours post-treatment. At day 1.5, empty spaces of missing step 1 spermatids were seen in stage I. At days 4.5 and 7.5, normal round spermatids were missing, but large round spermatids (megaspermatids) and binucleate spermatids were common. The megaspermatid nucleus was approximately 33% larger in diameter than normal round spermatids. At day 10.5, megasteps 10-12 spermatids, binucleate spermatids, and three to four different steps of spermatids coexisting in the same tubule section were present in stages X-XII. In addition, abnormally shaped elongating spermatids were observed having distorted heads and nuclear invagination containing microtubules. At day 20.0, empty spaces of missing diplotene spermatocytes were seen in stage XIII. CONCLUSIONS: The present observations show that carbendazim has rapid direct effects on meiotic spermatocytes and latent effects on spermatids, leading to morphological abnormalities and failure of spermiogenesis. These effects are found independent of occlusions in the efferent ductules.

Animals↗
PubMed1997

Further observations on carbendazim-induced abnormalities of spermatid morphology in rats.

The effects of a microtubule poison, carbendazim, on rat spermiogenesis were examined for abnormalities of nuclei, acrosome and manchette in round and elongating spermatids in Stages VII-XII on days 7.5, 9.5, 10.0 and 10.5 post-treatment using routine electron microscopy. Spermatid nuclear abnormalities were observed in Stages IX-XI on day 9.5 and at greater post-treatment intervals. Nuclear abnormalities included nuclear distortions, various types of nuclear invaginations and abnormal positioning of the modified nuclear envelope. Acrosomal abnormalities were noted on day 7.5 and at greater intervals. Discontinuous, multiple granular and fragmentary acrosomes were observed in Stages VII-XI. In addition, spermatids with complete absence of acrosome (acrosome-deficient spermatids) were observed in Stages VII-X. Poorly-formed and absent ectoplasmic specializations were seen in the cytoplasm of Sertoli cells next to the acrosome-deficient spermatids. A major abnormality of the manchette was irregular positioning of the manchette microtubules in steps 9-11 spermatids on day 9.5 and at greater intervals, which resulted in nuclear invagination. The results indicate that carbendazim induces abnormalities in spermatid morphology that are common to those reported in testes treated with several chemical compounds and in testes of mutant animals.

Acrosome↗
PubMed1996

Endocrine modulation of reproduction.

The ability of foreign compounds to affect the functioning of various endocrine systems is currently thought responsible for a wide variety of effects. The presentations in this Symposium reviewed the evidence for and against the involvement of endocrine systems in several different aspects of reproduction. The mechanism behind the ability of a triazine herbicide to cause enhanced appearance of mammary tumors in one strain of female rats is reviewed by Stevens. The data suggest that enhanced aging, not direct mammary modulation, is responsible. Dietary phytoestrogens, the mediators of their actions, their effects in various biological systems, and the relationships between phytoestrogen producers and consumers are all provocatively and succinctly reviewed by Hughes. Kelce presents the strategy used to dissect the mode and mechanisms of action of a fungicide that opened a new awareness in reproductive toxicology: the possibility of xenobiotics being antiandrogens. Finally, to heighten our understanding of the interplay among hormonal systems in vivo, Hess reviews the data that show that androgens are not the only hormones important in the development of the male reproductive system: the pituitary is shown to play a critical role at specific stages of development. The breadth of these presentations, and the implications of their findings, should make us pause and realize how much there is still to discover about the interaction between the reproductive system and anthropogenic compounds.

Aging↗
PubMed1996

Effects of rate of gain on scrotal circumference and histopathologic features of the testes of half-sibling yearling beef bulls.

OBJECTIVE: To determine the effects of high and low gain on production variables and reproductive characteristics of bulls, including histopathologic lesions in the testes, as determined by quantitative measurements. ANIMALS: 23 half-sibling, 11.6-month-old beef bulls, averaging 326 +/- 8.3 kg in weight and having a mean scrotal circumference of 28.7 +/- 0.4 cm. DESIGN: Bulls were randomly assigned within pairs to high- or low-gain rations and fed ad libitum for 112 days. PROCEDURES: Scrotal circumference and body weight were measured on days 0, 28, 56, 84, and 112. Bulls were castrated on day 112. Morphologic characteristics of spermatozoa were determined from samples taken on days 50 and 111. Testis sections from each bull were examined, using light microscopy, for changes in the seminiferous epithelium and interstitium. A grading scheme was used to quantify testicular lesions by histologic techniques. Pathologic changes in 600 cross sections from each bull were classified into 9 grades. The degree of germinal epithelial loss per 100 tubules was calculated by assigning a value to each grade according to the severity of loss of germinal epithelium. RESULTS: Scrotal circumference (mean +/- SEM cm), 34.0 +/- 0.8 vs 31.7 +/- 0.5 (P < 0.001), body weight (kg), 512.3 +/- 19.6 vs 421.9 +/- 9.4 (P < 0.001), and paired testicular and epididymal weight (g), 532 +/- 31 vs 425 +/- 24 (P < 0.0001) and 73.8 +/- 1.9 vs 56.3 +/- 3.7 (P < 0.001) were greater in the high-gain than in the low-gain group. Morphologic characteristics of spermatozoa were similar for both groups. Means for graded seminiferous epithelial effects were similar in each group. Hypoplastic-type tubules were found in bulls with small scrotal circumference in both the high- and low-gain groups. CONCLUSION: High gain resulted in greater testicular growth but testicular structure and function were similar for both groups.

Aging↗
PubMed1995

Neonatal hypothyroidism causes delayed Sertoli cell maturation in rats treated with propylthiouracil: evidence that the Sertoli cell controls testis growth.

BACKGROUND: The testes of rats treated neonatally with propylthiouracil (PTU) grow to almost twice their normal size. The cause of testicular enlargement has been suggested to be the result of delayed maturation of Sertoli cells, allowing Sertoli cell division to occur beyond the 15th postnatal day, the commonly recognized cutoff date for Sertoli cell divisions. It has been shown that an increased population of Sertoli cells in postnatal development supports increased numbers of germ cells in adult animals. After examining developing rats treated neonatally with PTU, we hypothesized that an approximate 10-day delay in maturation was occurring and proceeded to test this hypothesis experimentally. Thus the purpose of this report was to determine if a 10-day delay in maturation could explain the increased numbers of Sertoli cells and increased testis size in PTU-treated animals. METHODS: Both control animals and animals treated neonatally with PTU N = 5/group were sacrificed at 15 and 25 days of age and prepared for electron microscopy. RESULTS: Micrographs show and morphometric ultrastructural analysis of numerous parameters demonstrated at the 95% probability level that Sertoli cells from 25-day-old PTU animals are not different in size and most constituents (volume and surface area) from 15-day-old control animals and are less mature than 25-day-old control animals. Mitosis of Sertoli cells was observed in PTU-treated animals in 25-day-old animals but not in age-matched controls. The number of Sertoli cells in 25-day-old PTU-treated animals is significantly increased over age-matched controls. Micrographs show the presence of immature Sertoli cell nuclei in 25-day-old animals receiving PTU as well as increased germ cell degeneration in this group. Sertoli cell tight junction formation is also delayed in PTU-treated animals as compared with controls. CONCLUSIONS: Together, the data show that delayed maturation of Sertoli cells occurs in treated animals that corresponds to a minimum of 10 developmental days. In the immature state, Sertoli cells continue to divide. Data presented herein and published data related to PTU treatment indicate that delayed maturation of the Sertoli cell results in delayed maturation and proliferation of other testicular cell types. From this and from published data, the hypothesis is presented that the Sertoli cell is responsible for the overall control of testis development.

Animals↗
PubMed1995

The effect of ultrasound exposure in utero on the development of the fetal mouse testis: adult consequences.

The effects of exposure in utero to 1 MHz, continuous-wave ultrasound on adult growth and testicular development in the mouse was investigated. The spatial peak temporal average intensity (ISPTA) employed ranged from 1 to 10 W/cm2, with exposure durations (t) of 200 s to 20 s. Exposures were made on days 9, 12 or 15 of gestation. Results showed an increase in postpartum deaths, an increase in the number of stillbirths, and a decrease in litter size when I2 t > or = 1125 W2 s/cm4, such that there was significant loss of pups. Birthweights of pups from nearly all dosage groups was significantly lower than that of the sham or cage control groups. Results also showed that males exposed to ultrasound in utero had decreased testis size and decreased daily sperm production ranging from 9% to 30%. This study showed that ultrasound exposure in utero is capable of disrupting fetal development and having potential subsequent effects on fertility in the adult male.

Animals↗
PubMed1995

Rooster testicular germ cells and epididymal sperm contain P450 aromatase.

We recently found that cytochrome P450 aromatase (P450arom) is present in germ cells of the mammalian testis and is capable of converting androgens to estrogens in the male reproductive tract. The objective of the present study was to determine whether testicular germ cells and epididymal sperm of an avian species are also capable of synthesizing estrogen. P450arom was localized in the rooster testis and epididymal region by immunocytochemistry, using an antiserum generated against purified human placental cytochrome P450arom. Immunostaining was present in pachytene spermatocytes, round spermatids, elongated spermatids, flagella of late spermatids, and sperm in the epididymal region. A positive reaction was also found in nonciliated cells of the epididymal region. However, the absence of mRNA for P450arom in the epididymal region indicated that the immunoreactive protein present in the epididymal region is not synthesized in this region. The immunoreactive P450arom found in epididymal sperm was shown to be active through use of a 3H2O assay. On the basis of these data, we conclude that rooster testicular germ cells and epididymal sperm are sites for the synthesis of estrogen, a potential regulator or modulator of germinal epithelium in the testis and the epithelium of the epididymal region of the avian species.

Animals↗
PubMed1995

Sperm, a source of estrogen.

This review article discusses a novel nontraditional site of estrogen synthesis and the potential targets of estrogen action within the male reproductive system. Our laboratories have recently demonstrated that developing spermatids in several species contain aromatase, the cytochrome P450 enzyme responsible for converting androgens into estrogens. The enzyme was localized by immunocytochemistry and the protein's presence was confirmed by Western blot analysis. Northern blot analysis and in situ hybridization were used to corroborate the presence of mRNA for aromatase. It appears that the aromatase message precedes the synthesis of the protein, and the protein remains in the spermatids several days after the message disappears. The enzyme is located along the tail of newly released sperm and is active in the epididymal sperm as well as in the developing germ cells of the testis. This unique discovery is the basis for our overall hypothesis that estrogen, synthesized by sperm, plays a role in the regulation of epididymal function proportional to the number of sperm being transported. The presence of an estrogen source within the ductal lumen is of special importance to the study of epididymal function because the regulatory mechanisms in this region remain unclear, particularly for the efferent ductules and initial segment regions, although estrogen receptors have been identified in the ductal epithelium. An understanding of the role that estrogen plays in the function of the epididymis may provide benefits in several areas including the treatment of abnormalities in epididymal function, the potential development of a male contraceptive, and insight into the causes of adult epididymal lesions induced by neonatal exposure to estrogenic compounds such as diethylstilbestrol.

Animals↗
PubMed1994

Immortalized germ cells undergo meiosis in vitro.

Establishing mammalian germ-cell lines capable of differentiation in vitro would greatly facilitate the study of gametogenesis and the meiotic process that is so fundamental for reproduction and the maintenance of genetic diversity of the species. We have established two germ-cell lines [GC-2spd(ts) and GC-3spc(ts)] by cotransfecting primary mouse testicular germ cells with the simian virus 40 large tumor antigen gene and the gene coding for a temperature-sensitive mutant of p53. Both cell lines express the germ cell-specific lactate dehydrogenase C4 isozyme and cytochrome ct isoform. At the permissive temperature of 37 degrees C, the GC-2spd(ts) line generates cells with a haploid DNA content and morphologic and biochemical features of round spermatids, including the appearance of an acrosomic granule. The identification of a flagellar axoneme when these cells are cultured at 32 degrees C further indicates that these cells correspond to the early spermatid stages of spermiogenesis.

Animals↗
PubMed1994

Characterization of a testis specific protein localized to endoplasmic reticulum of spermatogenic cells.

BACKGROUND: In order to understand the mechanism of spermiogenesis, it is important to characterize germ cell specific genes and proteins expressed during spermatogenesis. We previously reported that a mouse monoclonal antibody, 1C9, raised against golden hamster testis homogenate, recognized a 103 kDa protein in hamster spermatogenic cells (Ohsako et al.; J. Vet. Med. Sci., 53:969-974, 1991). In the present study, we have determined the precise stage and intracellular localization of this protein. MATERIALS AND METHODS: Hamster, mouse, and rat tissues were used for immunocytochemistry, SDS-PAGE, and immunoblotting. Immunoelectron microscopy was performed using Lowicryl K4M embedded hamster testis and colloidal gold conjugated second antibody. Furthermore, immuno-affinity purification was carried out using a 1C9-Sepharose column. RESULTS: In immunoblot analysis, 1C9 also recognized a 103 kDa protein and a 101 kDa protein in the rat and the mouse testes, respectively. Ten different hamster tissues other than testis did not show reactivity against 1C9. In immunostained paraffin sections of hamster testis, the initial staining appeared in middle pachytene spermatocytes and persisted until maturation phase spermatids (step 15). However, it was no longer detectable in the subsequent steps of spermatids. In addition, strong staining was observed in the post-nuclear region of elongated spermatids. Immunoelectron microscopic analysis showed that the protein was localized to the endoplasmic reticulum (ER) and nuclear envelope of spermatogenic cells, but not in the other organelles, such as Golgi apparatus and acrosome of the spermatids. This protein appears to be associated with ER membrane. Furthermore, this protein is found exclusively in the testicular microsomal fraction, not in the cytosol. By affinity purification, approximately 320 micrograms of the 103 kDa protein was obtained from 10 hamster testes. The purified 103 kDa protein was unaffected by N-glycanase, indicating it does not have asparagine-linked glycoconjugates. CONCLUSIONS: These results indicate that the protein recognized by 1C9 appears to be a unique protein that is localized in the ER and nuclear envelope of spermatogenic cells.

Animals↗
PubMed1994

Morphological changes in the rat Sertoli cell induced by the microtubule poison carbendazim.

Early morphological changes in the rat Sertoli cell induced by the fungicide carbendazim (methyl-2-benzimidazole carbamate; MBC), a metabolite of benomyl, were examined. Adult rats were treated with single doses of MBC (400 mg/kg) or vehicle and examined by light and electron microscopy at 3 hr post-treatment. Sloughing of elongating spermatid clusters was observed in all stages of spermatogenesis, except for Stages III-V. Cleavage occurred near the apical region of the seminiferous epithelium where cytoplasmic processes of the Sertoli cell surround the heads of elongating spermatids. The cleaved cytoplasm remained attached to the sloughed spermatids and ectoplasmic specializations remained undamaged. Intact microtubules were observed in the apical Sertoli cell cytoplasm (including sloughed tissues) but were decreased in the body region, where aggregates of mitochondria were found. Cytoplasm near the cleavage site exhibited rarefaction, which was associated with swollen cisternae of endoplasmic reticulum. It appears that the mechanism of germ cell sloughing induced by MBC treatment involves the disruption of microtubules in the body region of the Sertoli cell, the retraction of cytoplasmic organelles and the swelling of endoplasmic reticulum.

Administration, Oral↗