Ultrastructural studies on the development of the blood-epididymis barrier in immature rats.

The development of the blood-epididymis barrier in immature rats (8, 11, 14, 18, and 21 days old) was examined with an electron microscope using lanthanum nitrate as an electron dense tracer. A gradual increase in the development of the blood-epididymis barrier was noted with age. On Day 8, lanthanum was frequently detected in both the intercellular spaces and the lumen. On day 14, no lanthanum penetration into the lumen was observed in 75% of the junctions in the caput, 40.3% in corpus, and 30% in cauda epididymidis. On Day 18, only 7.5%, 9%, and 15%, of the junctions in the caput, corpus, and cauda epididymidis, respectively, remained permeable to lanthanum. No lanthanum was observed in the lumen of any tubules in the 21-day-old rat epididymis. These findings indicate that the postnatal development of the blood-epididymis barrier is gradual, and that its formation is virtually completed by Day 21. As with adult rats, the zonula occludens is the ultimate structural component of the blood-epididymis barrier in immature rats (Agarwal and Hoffer, 1985).

Recovery of normal testicular ultrastructure and sperm motility after cessation of gossypol treatment in rats.

This study evaluates the reversibility of the effects of gossypol on testicular ultrastructure and the motility of epididymal spermatozoa. Adult male rats were treated 6 days weekly with the vehicle alone (Group A), or with 10 (Group B) or 20 (Group C) mg/kg of gossypol for 12 weeks, and then sacrificed six or 12 weeks after cessation of treatment. Although epididymal spermatozoa in Groups B and C were 100% immotile after gossypol treatment, little evidence of abnormality could be detected with the light microscope in the seminiferous tubules or interstitium. By contrast, at the ultrastructural level, there were demonstrable pathognomonic defects in the mitochondrial sheath and axonemes of step 18 and 19 spermatids which were identical to those reported earlier (Hoffer, 1983). In addition, an ultrastructural defect in the flagella of late testicular spermatozoa is described for the first time. This defect consists of an indentation, or constriction, of the mitochondrial sheath at outer dense fibers (ODFs) 1, 2, and 9, resulting in a separation of these 3 ODFs from the other fibers. This defect, though visible in an earlier ultrastructural study (Hoffer, 1983), was not described. In Group B rats allowed to recover from gossypol treatment, ultrastructural defects in step 18 and 19 spermatids could not be detected at six or at 12 weeks after cessation of treatment, and sperm motility also did not differ significantly from controls by the end of either recovery period. In Group C rats, sperm motility returned to the normal range within six weeks after treatment ended, but a few morphological defects in the midpiece and axoneme of late spermatids could still be detected with the electron microscope.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructure and intercellular vacuolization of isolated perfused and control rat testes.

Isolated rat testes perfused in closed circuit with albuminated Krebs-Ringer bicarbonate buffer were examined by light and electron microscopic techniques in order to assess the structural integrity of the model. The extent of intercellular vacuolization in perfused testes was similar to that of control testes that had been prepared for electron microscopy by routine methods of perfusion fixation. The isolated perfused testes exhibit excellent preservation at the light and electron microscope level. The results indicate that albuminated Krebs-Ringer bicarbonate buffer is an excellent perfusate for in vitro studies of the isolated rat testis. This model of perfused testis can be used to study the early pathology of the effects of toxic compounds on the microscopic anatomy of the male gonad.

Glycogen accumulations in differentiating mesonephric ducts and tubuli in male human embryos.

Human mesonephric duct epithelial cells contained empty appearing regions in the infranuclear cytoplasm when prepared for transmission electron microscopy using glutaraldehyde and osmium fixation. The same regions stained positively with PAS in Epon sections for light microscopy suggesting that glycogen was present. Incubation with saliva abolished the reaction. For electron microscopy the glycogen stained very intensely if a mixture of osmium tetroxide and potassium ferrocyanide was used instead of osmium alone. Glycogen accumulations were present between the ages of 5 to 10 weeks and absent at the age of 15 weeks. Reports by others indicate that glycogen may be present in different reactive forms in relation to its staining behaviour after various fixatives. The present results, and similar studies in other tissues, indicate that osmium tetroxide-potassium ferrocyanide fixative should be used routinely for preservation of embryos and fetuses and where indicated, for ultrastructural identification of glycogen and cytoplasmic filaments in clinical specimens.

Quantitative analysis of germ cells and Leydig cells in rat made infertile with gossypol.

This study utilized improved methods of fixation and plastic embedding to quantitatively evaluate the effects of gossypol on germ cells and Leydig cells in testes of rats made infertile with gossypol. Rats were fed by gavage with 10, 20 or 30 mg/kg per day of gossypol for 9 weeks; control animals received the vehicle alone. Numbers of A spermatogonia, preleptotene and pachytene spermatocytes, and step 7 or 8 spermatids per Sertoli cell were counted in stages VII-VIII of the cycle of the seminiferous epithelium. Although high doses (30 mg/kg) of gossypol produced a significant decrease in the relative number of germ cells compared with vehicle-treated controls, no significant deviation in the relative number of germ cells was noted between controls and rats made infertile with 10 or 20 mg/kg/day of gossypol. Stereologic techniques were used to assess the changes in the Leydig cells. No significant deviation in the Leydig cell morphology, cell number, or cell volume was noted as a result of gossypol treatment at the dose levels employed. It appears that germ cell depletion, such as that caused by high doses of gossypol, is not mediated by a change in Leydig cell function. The present report emphasizes the importance of studies to determine the minimal effective doses for gossypol's antifertility activity in animal models as well as in man.

Antifertility, spermicidal and ultrastructural effects of gossypol and derivatives administered orally and by intratesticular injections.

Because there are problems, in men, associated with the use of gossypol related to reversibility and, infrequently, hypokalemia, several laboratories around the world have resorted to the synthesis and evaluation of experimental analogs and optical isomers of gossypol in an attempt to find a compound which retains its pharmacologically desirable antifertility effects while eliminating its suboptimal ones. The present study documents: (a) the effects of fourteen new, orally-administered synthetic analogs of gossypol on testicular ultrastructure and fertility in hamsters, (b) the in vitro effects of these compounds as well as of the optical isomers of gossypol against hamster and human sperm, and (c) the morphological and antifertility effects of intratesticular injections of gossypol-PVP and its optimal isomers in the rat. The results of the study demonstrate that these new analogs are not effective as male antifertility agents and that their in vitro activity is unrelated to their in vivo contraceptive potential. In addition, this report establishes the validity of the intratesticular injection model for the analysis of the mechanism of action of gossypol and its analogs by making these compounds directly available at the testicular site. The significance of these findings is discussed.

Anti-fertility and other actions of gossypol analogues.

From a series of gossypol derivatives studied, we conclude that the carbonyl groups of gossypol are needed for inhibition of erythrocyte anion transport and the hydroxy groups affect but are not essential to that inhibition. In an in vitro mouse erythroleukemia cytocidal assay, the most active compounds were gossypol and apogossypol. The latter was not active in the inhibition of erythrocyte anion transport or in a spermicidal assay. Of the more simple structures related to gossypol, those that were active in the cytocidal and spermicidal assays were bi-aromatic, linked by a 1- and not a 4-carbon chain and had free phenolic hydroxyl groups. These results are included in a discussion of the specificity and mechanism of action of gossypol.

PIP: The analysis of multiple biological assays of gossypol and its derivatives suggests that the carbonyl groups of gossypol are required for inhibition of erythrocyte anion transport and the hydroxy groups affect but are not essential to that inhibition. In an in vitro mouse erythroleukemia cytocidal assay, the most active compounds were gossypol and apogossypol. The latter was not active in the inhibition of erythrocyte anion transport or in a spermicidal assay. Of the more simple structures related to gossypol, those that were active in the cytocidal and spermicidal assays were biaromatic, linked by a 1- and not a 4-carbon chain, and had free phenolic hydroxyl groups. When gossypol inhibits the anion transporter, the carbonyl group does not seem to form a Schiff base. Gossypol is a unique compound since it alone, but not any of its derivatives, has in vivo as well as in vitro antifertility activity. It remains unknown, however, whether similar mechanisms are involved in gossypol's in vivo and in vitro effects. In whatever manner gossypol exerts its toxic effects, the selectivity for testicular tissue must be explained.

Effects of gossypol on the seminiferous epithelium in the rat: a light and electron microscope study.

This study was undertaken to determine the effects of 10, 20 and 30 mg/kg per day of gossypol for 2-11 weeks on the rat testis. At the light microscope level, the most striking effect of gossypol treatment is the presence of severely damaged and entirely normal seminiferous tubules adjacent to one another in the same section. Affected tubules exhibit intraepithelial vacuoles of varying size, exfoliation, and atrophy. With the electron microscope, the intraepithelial vacuoles are found to consist of intercellular spaces and intracellular vacuoles occurring primarily, though not exclusively, in the Sertoli cells. Severely affected Sertoli cells exhibit many large vacuoles as well as an overall decrease in cytoplasmic ground substance, rough and smooth endoplasmic reticulum and Golgi apparatus. There is an overall increase in the frequency of occurrences of vacuolated tubules with time and dose but in no group of animals were more than 46% of the tubules affected. At the electron microscope level, the most striking specific effect of gossypol treatment is the production of ultrastructural defects exclusively in the mitochondrial sheath of Stage 18 and 19 spermatids; these changes are evident in small numbers as early as 2 weeks after 20 mg/kg per day of gossypol and increase with dose and time. No other significant differences between germ cells or Leydig cells of control and gossypol-treated rats are observed under the experimental conditions employed. The significance of these observations is discussed in relation to the possible mechanisms of action of this new experimental male contraceptive.

Ultrastructural studies of spermatozoa and the epithelial lining of the epididymis and vas deferens in rats treated with gossypol.

This study was undertaken to determine the effects at early time intervals of gossypol on sperm motility and on the ultrastructure of rat epididymal and vasal sperm and epididymal and vasal epithelium. Rats were treated by gavage with 20 or 30 mg/kg/day of gossypol for 7 weeks; control animals were unfed or received the vehicle alone. The results confirm and extend earlier observations demonstrating that epididymal sperm of gossypol-treated rats exhibit distinct ultrastructural changes under the experimental conditions employed. The severity and frequency of the degenerative changes appear to increase with dose and duration of treatment. Striking ultrastructural defects can be seen as early as 3 weeks after 20 mg/kg/day of gossypol. By the fifth week of either 20 or 30 mg/kg/day of gossypol, significant damage to virtually all sperm flagella is observed throughout the epididymal duct of all treated rats. The initial and predominant defect is degeneration of the midpiece mitochondria but additional flagellar defects are described in detail. Not surprisingly, caudal spermatozoa are totally immotile by the fifth week. The ultrastructure of the epididymal and vasal epithelium is not affected by gossypol.

The structure of the epididymis, efferent ductules and ductus deferens of the guinea pig: a light microscope study.

In the guinea pig, the narrow part of the epididymis that traverses the upper pole of the testis and passes downward over the entire length of the gonad is composed exclusively of efferent ductules and the initial segment (zone I) of the epididymal duct. At the beginning of zone II, the narrow contour of zone I expands into a large globular region which lies adjacent to the caudal pole of the testis. The globular region of the guinea pig epididymis is commonly referred to as the cauda epididymidis but in the present study, examination with the light microscope reveals that it is composed of five histologically distinct zones (zones III through VII). A detailed histological analysis of the characteristics of the epithelium in the seven zones of the guinea pig epididymis and in the efferent ductules and ductus deferens was udertaken to obtain a better understanding of structure-function relationships in the epididymis of the guinea pig. It was found that each of the zones could be readily distinguished on the basis of its histological features and primarily on the basis of the appearance of the principal cells.

Studies on zonation in the epididymis of the guinea pig. I. Ultrastructural and biochemical analysis of the zone rich in large lipid droplets (zone II).

In the epididymis of the guinea pig, zone II exhibits striking histological features that distinguish it readily from the other six regions of the epididymis. At the light microscope level, the pseudostratified epithelium of zone II is characterized by tall principal cells that are densely packed with large, intensely staining granules or droplets ranging up to 8 mu in diameter. At the electron microscope level, the principal cells exhibit numerous large lipid droplets and abundant agranular endoplasmic reticulum, which is frequently arranged in concentric whorls around one or more of the droplets. Quantitative biochemical studies comparing zone II with zones I and III show that zone II contains 2.5 - 3-fold more cholesterol and a significantly greater amount of cholesterol ester than the other two zones. These data indicate that the epididymal duct of the guinea pig includes a clearly defined region of epithelial cells possessing ultrastructural and biochemical characteristics consistent with steroidogenic activity. The potential significance of these observations to the epididymal physiology of the guinea pig and epididymal physiology in general is discussed.

Duration of epididymal sperm transit in hamster: an autoradiographic study.

The time required for passage of spermatozoa through the epididymis has been determined in hamster employing quantitative light microscopic autoradiography with 3H-thymidine. The time required for spermatozoa to traverse the caput is 3 days; corpus, 2 days; proximal cauda, 2 days; distal cauda, 6 days. An additional 2 days are required for passage of spermatozoa through the proximal ductus deferens. The total duration of sperm transit through the ductus epididymis in sexually rested hamster has been estimated at about 15.0 days.

Morphological evidence for a blood-epididymis barrier and the effects of gossypol on its integrity.

Ultrastructural and micropuncture techniques were used to obtain morphological evidence for a blood-epididymis barrier ( BEB ) in the rat and to determine whether gossypol, an oral male contraceptive, alters the permeability of the BEB or blood-testis barrier ( BTB ) in rats made infertile with gossypol. Rats were treated by gavage with 20 mg/kg per day of gossypol for 6 weeks; control animals received the vehicle alone. For electron microscopy the components of the BEB were analyzed in each region of the epididymis with intravascularly perfused lanthanum nitrate. Throughout the epididymis in both control and gossypol-treated animals it was found that the zonula occludens at the apicolateral surface of the epididymal epithelial cells was the sole and ultimate structural component of the rat BEB ; the flow of intravascularly perfused lanthanum was not significantly impeded by the vascular endothelium, the peritubular myoid layer or other lateral cell surface specializations. For micropuncture, control and treated rats were administered 0.3 mCi [3H]inulin via the jugular vein. Radioactivity was determined in samples collected from the seminiferous tubules, caput and cauda epididymidis, and carotid artery. Results showed that [3H]inulin entry in seminiferous tubules, caput and caudal luminal fluid from blood was similar for control and treated groups. It was concluded that gossypol treatment does not alter the permeability properties of the BTB and BEB to macromolecules such as inulin or to small electron-dense tracers such as lanthanum.

Phagocytosis of spermatozoa by the epithelial cells of the ductuli efferentes after epididymal obstruction in the rat.

The effects of ligation of an isolated loop of the ductus epididymidis in the region of the caput were compared with those of the exfoliative lesion of the epididymis that follows administration of alpha-chlorhydrin to rats. Electron micrographs of the ductuli efferentes in both cases revealed early phagocytosis of apparently normal spermatozoa by the epithelial cells, followed at later intervals by invasion of macrophages and intraluminal phagocytosis. It is concluded that epithelial spermiophagy is a consequence of obstruction, whether mechanically or chemically induced.

Absence of histopathology in somatic tissues of rats made infertile with gossypol.

Although the effect of gossypol on the testis and epididymis has been well documented, its effect on the somatic tissues of animals made infertile with the drug has been less well studied. In this study rats were treated daily with gossypol at either 20 mg/kg or 30 mg/kg for 6 weeks and at 10 mg/kg for 7 months. Complete tissue sets from control and gossypol-treated rats, including 26 organs, were subjected to histological examination. No significant histopathological differences were noted at the light microscope level between the control and infertile groups.

Gossypol modulation of nucleotide metabolizing enzymes in the reproductive tract of male rats.

The activities of several pivotal nucleotide metabolizing enzymes from the testis and vasal sperm of rats treated for 7 wk with 0, 20 or 30 mg X kg X day gossypol acetic acid were examined. Total testicular lactate dehydrogenase (LDH) activity increased 40% above control in the highest treatment group examined. However, the specific activity of the testis-specific isozyme of LDH, LDH-C4, decreased to 50 and 20% of control in the 20 and 30 mg X kg X day treatment groups, respectively. Basal soluble adenylate cyclase from a 100,000 X g supernatant of testis homogenate exhibited a 25% decrease in activity only in the 30-mg treatment group. Basal adenylate cyclase activity in the testicular membrane fraction increased 20 to 30% above control in response to gossypol administration. Testis membranes from the 20- and 30-mg treatment group exhibited a 2- and 4-fold greater activation of adenylate cyclase by guanine nucleotides. In vitro dose-response curves showed a half-maximal inhibitory concentration (IC50) for inhibition of soluble testicular adenylate cyclase by gossypol of 400 microM in each treatment group. Caudal epididymal sperm adenylate cyclase activity decreased to 25% of control levels in gossypol-treated animals, and the in vitro sensitivity of the enzyme to the inhibitory effects of gossypol increased 4-fold. IC50 values for gossypol inhibition of sperm adenylate cyclase decreased from 200 microM in control animals to 75 and 50 microM in the 20 and 30 mg X kg X day treatment groups, respectively. Cyclic adenosine 3':5' monophosphate phosphodiesterase activity in caudal sperm increased 6-fold in the 20- and 30-mg treatment groups. These results demonstrate that nucleotide metabolizing enzymes in sperm are major targets for the actions of gossypol and provide a possible mechanism for the inhibition of normal sperm function by this compound.

Gossypol. Synthesis and in vitro spermicidal activity of isomeric hemigossypol derivatives.

Three isomeric hemigossypol derivatives (3,4,5) have been synthesized. Two of these derivatives (3,4) and one synthetic intermediate (7) have been shown to have activity comparable to gossypol (1) in a sperm motility assay.

Ultrastructural, fertility, and spermicidal studies with isomers and derivatives of gossypol in male hamsters.

The effects of fourteen new, orally administered synthetic analogs of gossypol on testicular ultrastructure and fertility in hamsters and the spermicidal properties of these compounds, as well as of the optical isomers of gossypol against hamster and human sperm in vitro, are reported in this study. Test compounds were administered to adult male hamsters by daily gavage for 9 weeks at doses ranging from 15 to 50 mg/kg. The results of this study have demonstrated that the fourteen new gossypol analogs evaluated herein are not effective as male antifertility agents and their in vitro activity or lack of activity as spermicides is unrelated to their in vivo contraceptive potential. In addition, the results of the study suggest that (1) the isopropyl moiety of the gossypol molecule, like the aldehyde group, is essential for its mechanism of action and (2) the pathognomonic defect in the mitochondrial sheath induced by gossypol appears to be related to its unique activity as a male antifertility agent. The significance of these findings is discussed.

Antiproliferative effect of gossypol and its optical isomers on human reproductive cancer cell lines.

The antiproliferative effect of gossypol and its optical isomers on various human cell lines of reproductive and nonreproductive tissue origin was studied. Various reproductive cancer cell lines of ovarian, gestational, and testicular origin were highly sensitive (IC50 values of 0.86-1.98) to gossypol. The antiproliferative action of gossypol was not restricted to reproductive cancers, as non-reproductive cancer cell lines were also equally sensitive (IC50 values of 0.69-3.55). In addition, actively proliferating untransformed cells such as fibroblasts and PHA-activated lymphocytes were also sensitive (IC50 values of 0.87-2.51). (-)-Gossypol was 3.6-12.4 times more potent than (+)-gossypol and 1.48-2.65 times more potent than (+/-)-gossypol. The most sensitive indicator of gossypol action was a decrease in DNA synthesis followed by inhibition of protein synthesis and uptake of rhodamine-123 by mitochondria as tested in an ovarian cancer cell line (OVCA 433) and a fibroblast line (Hs27). These results indicate that gossypol possesses a general nonselective antiproliferative action toward human cells in vitro. Further, the pharmacologic activity of gossypol as an antiproliferative agent is primarily attributable to its (-) isomer, which is also the active isomer as a contraceptive.