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B Robaire

Publications and source records attributed to B Robaire.

101 records · Page 6Linked to original sources
PubMedDate not supplied

Effects of cyclophosphamide on selected cytosolic and mitochondrial enzymes in the epididymis of the rat.

The anticancer and immunosuppressive drug cyclophosphamide is extensively used in clinical practice and is known to alter fertility in man. We showed previously that treatment of male rats with low daily doses of cyclophosphamide over a 9-week period caused fetal malformations, a high rate of postimplantation loss and affected epididymal and sperm histology. In the present study, five biochemical measures of epididymal function were used to characterize further the effects of cyclophosphamide on the epididymis. For 1, 3, 6, or 9 weeks, adult Sprague-Dawley rats were gavage-fed daily with saline (control), 5.1 (low dose), or 6.8 (high dose) mg/kg of cyclophosphamide. The specific activities of the two glycolytic enzymes aldolase and lactate dehydrogenase (LDH), the mitochondrial enzyme succinate dehydrogenase, the cytosolic enzyme carnitine acetyltransferase and the lysosomal enzyme acid phosphatase were determined in cytosolic and mitochondrial subcellular fractions from four segments of the epididymis. Cyclophosphamide caused decreases in protein concentrations in all segments of the epididymis only after 6 weeks of treatment with the high dose. The specific activities of aldolase, LDH and succinate dehydrogenase did not differ from control with respect to dose or duration of treatment. In contrast, there were significant effects of cyclophosphamide on carnitine acetyltransferase and acid phosphatase specific activity. After 1 week of treatment, there was a transient dose-related decrease in the specific activity of carnitine acetyltransferase, which was most striking for the corpus epididymidis (76% of control), but which did not differ from control after 3, 6, and 9 weeks. After 6 weeks of treatment with the high dose of cyclophosphamide, carnitine acetyltransferase specific activity in the initial segment and the corpus epididymidis was elevated to 165 and 140%, respectively, as compared with the 1-week high dose values. The specific activity of acid phosphatase did not differ from control after 1 and 9 weeks of treatment. At 3 and 6 weeks, however, there was a dose-related increase in acid phosphatase specific activity for all regions of the epididymis that was most marked in the cauda after the 6-week treatment (140% of control). Therefore, low dose, daily treatment of male rats with cyclophosphamide not only alters specific enzymes in specific segments of the epididymis, but acts in a dose- and time-dependent manner. It is possible that these changes could be mediated by direct, toxic effects of the drug on the epithelium or be secondary to alterations in the spermatozoa as a result of the treatment.

Acid Phosphatase↗
PubMedDate not supplied

Paternal cyclophosphamide treatment of rats causes fetal loss and malformations without affecting male fertility.

The use of cytotoxic, mutagenic and carcinogenic agents as treatment for various types of cancer may be particularly hazardous in men of reproductive age as there exists the possibility that this may lead to congenital malformations in the progeny. Such agents can affect fertility and other aspects of male reproductive function, for example, treatment with anti-cancer drugs such as cyclophosphamide has been associated with oligozoospermia, azoospermia and increased levels of serum follicle-stimulating hormone (FSH). Depending on the cumulative dose and the duration of treatment, spermatogenesis often returns but this may take years. The relevance of the effects of such chemicals on the male reproductive system to the offspring is poorly understood. We have set out to determine whether present tests of male reproductive function (that is, endocrine status, numbers of spermatozoa, fertility) can predict deleterious effects of a paternally administered agent on the offspring. Here, we report that chronic administration in rats of low doses of the widely used drug cyclophosphamide had minimal effects on the male reproductive system and fertility, but resulted in malformations and retardation of growth in the surviving fetuses and a high frequency of fetal death. Thus, adverse effects on the fetus cannot be predicted from the effects of a drug on the male reproductive system.

Abnormalities, Drug-Induced↗
PubMedDate not supplied

The effects of aging on the seminiferous epithelium and the blood-testis barrier of the Brown Norway rat.

Steroidogenesis and spermatogenesis decrease in aging Brown Norway rats. We therefore hypothesized that there must be accompanying morphological changes taking place in the seminiferous tubules of the aging testis. The testes of Brown Norway rats ranging in age from 3 to 24 months were prepared for light and electron microscopy. To assess the integrity of the blood-testis barrier with age, a lanthanum nitrate study was done. The normal seminiferous tubules present in rats at 3 and 12 months of age were largely replaced at 24 months by fully regressed tubules that were virtually devoid of germ cells and contained large intercellular spaces. An electron-microscopic study of these regressed tubules showed a complete loss of cyclical variations of the organelles of the Sertoli cells. The nucleus was more irregularly shaped and was present at various levels in the epithelium. The endoplasmic reticulum was a loose, vesiculated network that was unlike the elaborate, tubular, anastomotic network noted in young animals. The lysosomes were large, oddly-shaped, and contained lipidic inclusions, in contrast to the distinct membrane-bound lysosomes and dense core bodies found in the young animals. Adjacent Sertoli cell processes encompassed large, empty intercellular spaces, possibly occupied previously by germ cells. The typical Sertoli-Sertoli junctions of the blood-testis barrier in the young animal were rarely seen at 24 months and were replaced by focal contact points, usually between three Sertoli cell processes. In the aged animals, lanthanum nitrate permeated the basal and adluminal compartments, extending between Sertoli cell processes and entering the intercellular spaces and lumen. In summary, during aging, there is a breakdown of the blood-testis barrier, and there are striking changes in the appearance of Sertoli cells. These results suggest a possible intrinsic limitation that prevents stem cells from renewing themselves, whether because of a degeneration of immunological origin or because of a lack of Sertoli cell support.

Aging↗
PubMedDate not supplied

Sperm structural and motility changes during aging in the Brown Norway rat.

The Brown Norway rat provides a useful model to study aging of the male reproductive tract because of the selective age-dependent pathological changes that are found in the testis, epididymis, and prostate. In the testis, there is a clear age-dependent decrease in both steroidogenesis and spermatogenesis. In the epididymis, some striking segment-specific changes occur at the histological and biochemical levels prior to the major loss of spermatogenesis. We hypothesized that formation of spermatozoa in the testis and maturation of spermatozoa in the epididymis (ie, acquisition of motility and loss of the cytoplasmic droplet) may be altered during aging. Changes in the morphology of spermatozoa were assessed by light and electron microscopy. Using computer-assisted sperm analysis, the motility parameters of spermatozoa obtained from the caput and cauda epididymidis of young and old Brown Norway rats were compared. In old animals, we also compared the motility of spermatozoa from epididymides adjacent to regressed testes with those from epididymides adjacent to nonregressed testes. There was a marked increase with age in the number of spermatozoa with abnormal flagellar midpieces; the nature of these defects did not change with age. In caput epididymidis, the percentage of motile sperm was similar in young and old rats. In contrast, the percentage of motile spermatozoa was significantly decreased in cauda epididymidis of old rats; spermatozoa from the regressed testis side had altered motility characteristics. Furthermore, in the cauda epididymidis on the regressed testis side of aged Brown Norway rats, the proportion of spermatozoa that retained their cytoplasmic droplet was markedly elevated. Some of these effects are likely due to changes taking place in spermatozoa during the process of spermatogenesis in the testis (eg, formation of the flagellum), whereas others could occur during sperm maturation in the epididymis (eg, acquisition of motility). The multiple effects of aging on sperm morphology, the acquisition of motility, and the shedding of the cytoplasmic droplet clearly indicate that the quality of spermatozoa is affected by aging.

Aging↗
PubMedDate not supplied

Gene expression in the aging brown Norway rat epididymis.

The mammalian epididymis is the site where spermatozoa are matured and then stored. Though many studies have described epididymal functions and their regulation, little is known about how aging affects this tissue. The Brown Norway rat, which does not show the many age-related pathologies common to other rat strains, was used as a model to study aging of the epididymis. The present study was designed to determine the effect of aging on the mRNA levels for selected markers of epididymal function. Brown Norway rats ranging in age from 6 to 30 months were examined at 6-month intervals; epididymides were sectioned into caput-corpus and cauda regions. Relative mRNA concentrations were assessed using Northern blot analysis and specific cDNAs for the rat 5 alpha-reductase isozymes, types 1 and 2; proenkephalin; the androgen receptor; epididymal proteins B/C and D/E; and sulfated glycoprotein-2 (SGP-2, clusterin). Northern blots were quantitated by densitometric scanning. In the caput-corpus epididymidis, 5 alpha-reductase type 1 and type 2 mRNA levels decreased significantly by 43% and 33%, respectively, between 6 and 12 months and by 64% and 40%, respectively, between 6 and 30 months. No significant change, however, was found in the expression of the 5 alpha-reductase mRNAs in the cauda epididymidis. Interestingly, proenkephalin mRNA was only detected in the caput-corpus epididymidis of 6-month-old rats. In marked contrast to the 5 alpha-reductase isozymes and proenkephalin, no significant age-related changes were observed in the mRNA levels for the androgen receptor, protein B/C, or protein D/E. No age-related changes in mRNA expression for SGP-2 occurred in the caput-corpus epididymidis. However, in the cauda epididymidis, SGP-2 mRNA levels rose by twofold between 6 and 18 months and then decreased sharply by 75% between 18 and 30 months. We conclude that as the epididymis ages, the expression of genes for certain specific markers of epididymal function is affected in a region-specific manner. Further, the decrease in the concentrations of the mRNAs for the 5 alpha-reductase isozymes and proenkephalin in the epididymis between 6 and 12 months is thus far the earliest marker for aging in the male reproductive tract of the Brown Norway rat.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMedDate not supplied

Immunocytochemical localization of glutathione S-transferase Yo subunit in the rat testis and epididymis.

The glutathione S-transferases (GSTs) are a family of isozymes that catalyze the conjugation of glutathione with electrophiles. These proteins exist as homo- or heterodimers and are separated into five classes (alpha, mu, pi, theta, and sigma). In the present study, the distribution of the GST Yo subunit, a member of the mu family, was examined immunocytochemically in the adult rat testis and epididymis using both light microscopy (LM) and electron microscopy (EM). In the testis, an intense immunoperoxidase reaction was observed over Leydig cells but not macrophages. Within the seminiferous epithelium, only weak reactivity was noted over Sertoli cells, spermatogonia, spermatocytes, and step 1-15 spermatids. There was, however, a progressive and dramatic increase in the intensity of staining in the cytoplasmic lobes of spermatids between steps 16 and 19. Residual bodies, representing the detached cytoplasmic lobes of the late step 19 spermatids, were also intensely stained. Initially seen near the lumen of the tubule, they eventually appeared at different levels of the tubule at stages IX-XI; none were present at stage XII. Cytoplasmic droplets of step 19 spermatids were also intensely reactive. After spermiation, the cytoplasmic droplets of spermatozoa within the proximal region of the epididymis remained intensely stained. A noticeable decrease in staining was observed in the cauda epididymidis in those droplets that were still there. Quantitation of the labeling density (number of gold particles representing anti-Yo antigenic sites/microns 2) paralleled the LM results; for example, between step 15 and 19 spermatids, a greater than sevenfold increase in labeling density was noted. In the epididymis, a progressive increase in immunoreactivity was observed over epithelial principal cells from the initial segment to the cauda region of this tissue. There was little reactivity over basal, halo, or clear cells. In all reactive cells, gold particles were distributed randomly throughout the cytoplasmic matrix and nucleus. The present work thus demonstrates that, at the end of spermiogenesis, the GST Yo subunit is expressed at high levels in late spermatids. Furthermore, the presence of this protein in late spermatids and cytoplasmic droplets of spermatozoa suggests that this conjugating enzyme may play a role in protecting these cells from electrophilic attack. Also interesting is the correlation between the loss of reactivity in cytoplasmic droplets of spermatozoa of the distal region of the epididymis and the concomitant increase of reactivity in principal cells of this region.

Animals↗
PubMedDate not supplied

In vivo model for chronic direct intratesticular drug administration.

The efficacy and toxicity of a new method for chronic direct intratesticular drug infusion were assessed in a rat model. To this end, luteinizing hormone (LH) or buffer was infused via miniosmotic pumps for 14 days directly into the parenchyma of Copenhagen rat testes. The surgical manipulation and direct infusion of buffer did not have any apparent adverse effect upon either spermatogenesis or steroidogenesis as measured by testis weight, homogenization-resistant spermatid count, and in vitro response of the testes to a maximally stimulating concentration of LH. Histologic studies revealed only a localized inflammatory response in the testis around the Silastic tubing leading from the mini-osmotic pump to the testis. The biologic efficacy of direct infusion into the testis was assessed by determining the ability of mini-osmotic pump-infused LH to maintain steroidogenesis for 14 days in animals whose pituitary function was suppressed by simultaneous subcutaneous placement of testosterone/estradiol capsules. Steroidogenesis was found to be maintained quantitatively in testes infused with an appropriate dose of LH. At a given LH dose, the directly infused testes were found to produce fivefold more testosterone than contralateral testes and 10-fold more testosterone than testes from rats receiving systemic administration of the same dose of LH. We conclude that the miniosmotic pump system is a useful means to chronically administer a high concentration of LH, and presumably other agents, to the testis without any significant adverse effects.

Animals↗
PubMedDate not supplied

Immunocytochemical localization of the Yf subunit of glutathione S-transferase P shows regional variation in the staining of epithelial cells of the testis, efferent ducts, and epididymis of the male rat.

Glutathione S-transferases (GSTs) are a family of isozymes that catalyze the conjugation of glutathione (GSH), a tripeptide found in all mammalian cells; this function plays a protective role, as the addition of GSH to an electrophile generally forms a less toxic product. The pi class of GSTs contains homodimers of the Yf subunit, also known as Yp or rat subunit 7; this subunit is found in high concentrations in the testis and epididymis. The objective of the present study was to localize immunocytochemically the Yf subunit in the testis and in the various regions of the epididymis using light, electron, and confocal microscopy. In the testis, immunoperoxidase staining was localized exclusively to Sertoli and Leydig cells. The low cuboidal epithelial cells of the rete testis and the sparse ciliated cells of the ductuli efferents were also immunoreactive. A distinct pattern of immunostaining for the Yf subunit was observed in the different regions of the epididymis. The proximal area of the initial segment showed intense reactivity localized to epithelial basal cells. Basal cells in the middle area of the initial segment were also reactive, as were a second unidentified population of cells located in the apical region of the epithelium. The epithelium, including both principal and basal cells, in the distal initial segment, intermediate zone, and proximal caput epididymidis showed a weak, moderate, or strong degree of reactivity, respectively. In the distal caput epididymidis, however, principal cells showed a checkerboard-like pattern of immunoreactivity, with some cells being intensely stained or faintly stained, whereas others were unreactive. Strikingly, in the corpus and proximal cauda epididymidis, intense immunostaining was localized exclusively over the epithelial basal cells. As viewed in the light and confocal microscope, the intensely stained basal cells showed extensive processes that covered most of the base of the epididymal tubule. Upon quantitation of the immunogold labeling density (the number of gold particles/microns2) in principal and basal cells of the different regions of the epididymis, we observed a sharp decline in immunogold labeling of principal cells coupled with a dramatic increase in labeling of basal cells as we progressed along the tissue, particularly in the transition from the caput to the corpus epididymidis. This study constitutes the first demonstration of a protein that is selectively expressed in epithelial basal cells of the corpus and proximal cauda epididymidis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

The mRNAs for the steroid 5 alpha-reductase isozymes, types 1 and 2, are differentially regulated in the rat epididymis.

The enzyme steroid 5 alpha-reductase (EC 1.3.1.22) catalyzes the conversion of testosterone to its more potent form, dihydrotestosterone (DHT), in many androgen-sensitive target tissues. In the epididymis, the 5 alpha-reduced metabolites of testosterone, DHT and 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha-diol), are considered the primary regulators of epididymal structure and function. Two rat 5 alpha-reductase transcripts, designated types 1 and 2, have been identified. Our laboratory has previously characterized the endocrine and developmental regulation of the 5 alpha-reductase type 1 mRNA in the rat epididymis. However, regulation of the type 2 mRNA has not been investigated. Thus, we undertook to characterize the longitudinal distribution of the steady state 5 alpha-reductase type 2 mRNA as well as the effects of development and of unilateral efferent duct ligation on its expression in the rat epididymis. To contrast the regulation of the type 2 5 alpha-reductase isozyme mRNAs in the rat epididymis, these data have been compared with those previously obtained for the type 1 mRNA and enzyme activity. An analysis of the longitudinal distribution of the type 2 transcript along the epididymis revealed that its mRNA was expressed predominantly in the proximal caput region of the tissue. This regional distribution pattern differed markedly from the patterns previously described for the type 1 mRNA and enzyme activity. Surprisingly, a segment-by-segment analysis showed that epididymal 5 alpha-reductase type 2 mRNA levels were not altered as a function of increasing postnatal age. Again, this result was in marked contrast to the important changes in 5 alpha-reductase type 1 mRNA and enzyme activity that have been reported to occur during puberty in the rat. In the final experiment, the effect of unilateral efferent duct ligation revealed that 5 alpha-reductase type 2 mRNA levels increased in the initial segment of the ligated side but remained unchanged in the rest of the tissue; this was in marked contrast to the dramatic decrease (>60%) in type 1 mRNA levels observed specifically in the initial segment of the epididymis. Taken together, these experiments demonstrate that 5 alpha-reductase type 1 and type 2 mRNAs are differentially regulated in the rat epididymis. Moreover, this is the first report of a tissue in which the mRNAs for the steroid 5 alpha-reductase isozymes are regulated differently.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMedDate not supplied

The effects of aging on the expression of glutathione S-transferases in the testis and epididymis of the Brown Norway rat.

Glutathione S-transferases (GSTs), a family of isoenzymes, catalyze the conjugation of glutathione to a variety of electrophiles, and protect cellular constituents from electrophilic and oxidative attack. Aging is associated with an overall increase in oxidative stress and thus free radical production. The present study examines the immunocytochemical localization of Ya, Yc, Yb1, Yb2, Yo, and Yf GST subunits in the testis and epididymis of Brown Norway rats aged 3, 12, 18, and 24 months. In the testis, neither Sertoli nor germ cells showed changes in the GST staining pattern during aging. At 24 months, two types of Leydig cells were noted. Some (peritubular) formed a distinct band at the periphery of the tubule while others were seen in the interstitial space. The peritubular cells were identified as Leydig cells by specific staining for 3beta-hydroxysteroid dehydrogenase (3beta-HSD), a Leydig cell-specific marker. Both types of Leydig cells were intensely reactive for all GST subunits at all ages. In the epididymis, principal cells of all epididymal regions, except the proximal cauda region, showed no changes in GST expression at all ages examined. At 24 months, some principal cells of this region became greatly enlarged and vacuolated. These cells were unreactive for Yo, Yb1, Yb2, and Yc, while adjacent normal-appearing principal cells maintained the same intensity of expression as seen in 3-month controls. In contrast, vacuolated principal cells were reactive for the Ya subunit, while adjacent normal principal cells were unreactive. These data indicate that selective changes occur in the expression of GSTs at 24 months in principal cells having both a normal and a vacuolated appearance. The underlying mechanism responsible for these changes with age is unresolved, but we speculate that they lose the ability to handle oxidative stress. Taken together, these data show that aging affects region-specific changes in GST expression in the epididymis and Leydig cell distribution in the testis.

Aging↗