Spermatozoa In Cauda Epididymidis Research Articles

Ontogeny of TRα1 expression in the mouse testis and epididymis during postnatal development.

Thyroid hormone (T3 ) acts on the testis via thyroid hormone receptor alpha 1 (TRα1), though the cellular localization of TRα1 in testis remains controversial. Studies on the presence of TRα1 in the epididymis are also lacking. The present study, therefore, examined the cellular localization and expression pattern of TRα1 in testis and epididymis of Parkes mice during postnatal development. Immunohistochemical results showed localization of TRα1 in interstitial and tubular compartments of the testis all through the development. On postnatal day (PND) 14, only leptotene spermatocytes showed TRα1-immunoreactivity in the testis, while at PND 28, 42, and 90, a diverse staining pattern for TRα1 was seen in almost all the seminiferous tubules mainly in leptotene spermatocytes, round and elongating spermatids, and in Leydig cells. Further, qRT-PCR and immunoblot analyses showed that TRα1 was expressed in the testis at the transcript as well as protein level throughout the postnatal development. TRα1 was also seen in principal cells of the epididymis, with maximal expression at PND 90. TRα1 was also present in cauda epididymidal spermatozoa of adult mice at PND 90. The results suggest that TRα1 is expressed in the testis and epididymis and that it may help to regulate the spermatogenic process and male fertility.

The ultrastructural characteristics of the spermatozoa stored in the cauda epididymidis in Chinese soft-shelled turtle Pelodiscus sinensis during the breeding season

The ultrastructure of spermatozoa in cauda epididymidis of soft-shelled turtle, P. sinensis during breeding season was investigated by light microscopy (LM) and electron microscopy (TEM and SEM). The mature spermatozoa appeared elongated and filiform. In general, the turtle spermatozoon contains a characteristic head, midpiece and tail, similar in morphology to that of birds, amphibians and other reptiles. However, several features are unique. These include (1) three intranuclear tubules containing dense core extend from the subacrosomal cone through the rostral nucleus and deep into the nuclear body; (2) the midpiece is composed of 40 mitochondria which present a staggered rings-and-columns arrangement (8 parallel rings and 5 columns); (3) unusual spherical mitochondria with a dense core are surrounded by 8–10 concentric layers of cristae. Surprisingly, about 21.4±3.6 percent immature spermatozoa with normal morphology are also observed in this season. Different from the mature spermatozoa, a variable amount of cytoplasm droplets are attached to the immature spermatozoa under SEM. Some spermatozoa still show the tail coiled tightly around the cytoplasm. These spermatozoa in transverse sections under TEM, showed a large amount of cytoplasm wrapped by plasma membrane; even some free mitochondria and higher electron density material still seen in the cytoplasm. Among the immature spermatozoa, most of them possess a cytoplasmic droplet which is located eccentrically on the midpiece, and contains a lot of lipid droplets in addition to hollow vesicles. Lipid droplets are closely associated with mitochondrial membranes and may function in the formation or degradation of mitochondria. These immature spermatozoa may be the dormant cells, but whether or not they can fertilize the ovum or not is unknown. Thus, in the present study we hypothesized that the cauda epididymidis might be involved in the sperm maturation in this species.

Effect of the ethanolic extract from Fagara tessmannii on testicular function, sex reproductive organs and hormone level in adult male rats

The effect of ethanolic extract of Fagara tessmannii, wide medicinal plants used on reproductive function in South Cameroon, was investigated in male rats. Twenty male sexually experienced rats (four groups) were orally treated with vehicle, 0.01, 0.1, 1 g kg(-1) BW per day of F. tessmannii (equivalent to 16.67 g, 33.33 g, 50 g, 66.66 g kg(-1) dry raw material) for 14 days, the upper limit dose without any clinical sign of toxicity was 2 g kg(-1). Fagara tessmannii extract negatively affected weight of accessory organs and significantly affected body weight gain at dose 1 g kg(-1) (P < 0.05) in treated rats. The weight of epididymis and seminal vesicle significantly decreased at low doses (0.01 g kg(-1)) while the prostate weight decreased at all doses (P < 0.05). The transit of spermatozoa in cauda epididymidis significantly increased at lower dose of 0.01 g kg(-1) (P < 0.05). In addition, F. tessmannii extract affected neither daily sperm production (DSP) and DSP per g nor sperm count in vas deferens and epididymis. The length of stages IX-I of the seminiferous tubule and serum testosterone level increased dose-dependently following 14 days of treatment (P < 0.05). The results suggest that F. tessmannii, 14 days after treatment, may improve spermatogenesis, testosterone level and sperm transit in cauda epididymidis but negatively impair reproductive organ activities.

Antioxidant Defence System of Boar Cauda Epididymidal Spermatozoa and Reproductive Tract Fluids

Antioxidants secreted by the reproductive tract protect spermatozoa against the toxic effects of reactive oxygen species (ROS) after ejaculation. This study aimed at characterizing the level of antioxidant protection in boar cauda epididymidal spermatozoa and fluids of the cauda epididymidis, vesicular and prostate glands. Also, this study investigated the effect of a 5-h period of dialysis on the antioxidant capacity of boar seminal plasma. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione transferase (GST) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) activities were monitored in the cauda epididymidal spermatozoa or reproductive tract fluids. Also, the concentrations of total glutathione (GSH + GSSG), L-ergothioneine (ERT) and l-ascorbate and the total antioxidant status (TAS) of the fluids were measured. It was found that the cauda epididymidal spermatozoa exhibited high SOD activity and relatively low activity of PHGPx. The relative amounts of GPx, GR and GST activities in the cauda epididymidal spermatozoa were negligible, whereas CAT activity was undetectable. Greater SOD activity was found in the fluids of the cauda epididymidis and prostate gland. Furthermore, the prostate gland fluid appeared to be the main source of CAT activity in the seminal plasma, whereas the highest level of GPx activity was derived from the cauda epididymidal fluid. The reproductive tract fluids exhibited negligible amounts of GR and GST activities. It seemed that the significant amounts of GSH + GSSG, ERT and L-ascorbate in the reproductive tract fluids could have an ameliorative effect on the level of TAS in the seminal plasma. Dialysis had a marked effect on the total antioxidant capacity of the seminal plasma, which was manifested in greater activity of SOD and GPx. The findings of this study confirmed that the scavenging potential of the seminal plasma is dependent on the contributions of different antioxidants, originating in various fluids of boar reproductive tract.

Effect of 1-(2, 4-dichlorobenzyl)-1H-indazole-3-carboxylic acid (DICA) on the sex organs of the male laboratory mouse.

Oral administration (50 mg/kg body weight/day, for 28 days) of 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid (DICA) induced significant reduction in testicular weight with suppression of spermatogenesis. The seminiferous tubules were devoid of spermatozoa and contained only Sertoli cells, spermatogonia, spermatocytes and several multinucleated giant cells. The Leydig cells, however, remained unaffected in treated animals. The drug also induced significant decrease in the levels of testicular RNA and protein; DNA level, however, remained unaltered. Epididymis in DICA-treated mice presented normal histology except that the lumen contained sperm debris; cauda epididymidal spermatozoa were immotile and often fragmented. DICA treatment caused no change in the level of fructose in the seminal vesicle. DICA-induced alterations were reversible and within 56 days of drug withdrawal the reproductive organs returned to their normal state.

Prostaglandin F2 alpha-induced changes in the sex organs of the male laboratory mouse.

Administration (3 mg/kg body weight/day, for 21 days) of prostaglandin F2 alpha (PGF2 alpha) caused marked suppression of spermatogenesis and significant reduction in the weights of the testis, epididymis and accessory sex glands. The seminiferous tubules were devoid of spermatozoa and contained only Sertoli cells, spermatogonia, spermatocytes and occasionally spermatids; several multinucleated giant cells were observed in the lumen of the tubules. The Leydig cells were atrophied. The levels of RNA, DNA and protein in the testis were, however, unaffected by drug therapy. In drug-treated mice the epididymal epithelium presented a degenerate appearance; the lumen was generally devoid of spermatozoa and contained mainly exfoliated immature germ cells and sperm debris; cauda epididymidal spermatozoa, when present, were immotile and fragmented. PGF2 alpha treatment also caused significant decrease in the levels of sialic acid in the caput and cauda epididymides and of fructose in the seminal vesicle. The results suggest that the regressive changes induced by PGF2 alpha in the reproductive organs of the mouse are due to the interference with the secretion of androgen. The alterations induced in the reproductive organs by administration of PGF2 alpha were reversible and 56 days after drug withdrawal the organs returned to their normal state.

Effect of 1-p-Chlorobenzyl-1H-Indazol-3-Carboxylic Acid (AF 1312/TS) on the Sex Organs of the Male Laboratory Mouse

Oral administration (500 mg/kg body weight/day, for 28 days) of 1-p-chlorobenzyl-1H-indazol-3-carboxylic acid (AF 1312/TS) induced suppression of spermatogenesis without adversely affecting the Leydig cells. The seminiferous tubules were almost depopulated and contained only Sertoli cells and spermatogonia. The drug also induced significant decrease in the levels of testicular RNA and protein; DNA level, however, remained unaltered. Regressive histological changes were also noted in the epididymides of drug-treated mice. Cauda epididymidal spermatozoa from drug-treated mice were immotile and fragmented. AF 1312/TS caused no alteration in the level of fructose in the seminal vesicle. The drug-induced alterations were reversible, and 56 days after drug withdrawal the reproductive organs returned to their pretreatment state. The results suggest that AF 1312/TS selectively suppresses spermatogenesis without interfering with the endocrine functions of the testis.

Directional mapping of DNA nicking in ejaculated and cauda epididymidal spermatozoa of the short-beaked echidna (Tachyglossus aculeatus: Monotremata)

Prototherian spermatozoa are unique amongst the Mammalia in terms of their filiform morphology, tandem arrangement of chromosomes and formation of sperm bundles. In the present study, we provide observations of echidna spermatozoa and note that the superstructure of the bundle is engineered around the shape of the individual sperm head and that this in turn may be a consequence of the unusual circumferential and helicoidal condensation of the DNA during spermiogenesis. Frozen-thawed ejaculated echidna spermatozoa were incubated and examined for the presence of non-typical DNA conformation by means of in situ labelling of DNA breaks using Klenow polymerase and via alkaline single-cell comet assays for detection of fragmented DNA. Both techniques successfully revealed the presence of what appeared to be directional DNA nicking, co-localised with the presence of highly sensitive alkali sites along the length of the sperm nucleus. It was not possible to define whether these alternative DNA configurations were associated with a failure of the sperm nucleus to condense appropriately during spermiogenesis or were evidence of DNA fragmentation following post-thaw incubation or a sequential structural chromatin rearrangement necessary for fertilisation.

Evaluation of antifertility potential of Brahmi in male mouse

Background The purpose of the present study was to evaluate the effect of Bacopa monnieri (Brahmi) on fertility of male laboratory mouse. Study Design Mice of the Parkes (P) strain were orally administered Brahmi (250 mg/kg body weight/day, for 28 and 56 days), and effect of the treatment on reproductive organs and fertility was investigated. Recovery and toxicological studies were also carried out. Results The treatment caused reduction in motility, viability, morphology, and number of spermatozoa in cauda epididymidis. Histologically, testes in mice treated with the plant extract showed alterations in the seminiferous tubules, and the alterations included intraepithelial vacuolation, loosening of germinal epithelium, exfoliation of germ cells and occurrence of giant cells. In severe cases, the tubules were lined by only Sertoli cells or Sertoli cells, spermatogonia and spermatocytes. Significant reductions were also noted in height of the germinal epithelium and diameter of the seminiferous tubules in Brahmi-treated mice compared to controls. Epididymis in treated males showed slight alterations in histological appearance. The treatment had no effect on levels of testosterone, alanine aminotransferase, aspartate aminotransferase and creatinine in blood serum, hematological parameters and on liver and kidney histoarchitecture. In Brahmi-treated males, libido remained unaffected, but fertility was notably suppressed. The alterations caused in the above reproductive endpoints by the plant extract were reversible, and by 56 days of treatment withdrawal, the parameters recovered to control levels. Conclusions The results in P mice thus suggest that Brahmi treatment causes reversible suppression of spermatogenesis and fertility, without producing apparent toxic effects.

The effect of cryoprotectant on kangaroo sperm ultrastructure and mitochondrial function

This study examined the effect of cryoprotectants (20% DMSO, a 10% DMSO/10% glycerol mixture, 20% glycerol and 1 M sucrose solution) on kangaroo sperm structure and function, along with the effect of varying concentrations of glycerol on sperm mitochondrial function. Eastern grey kangaroo cauda epididymidal spermatozoa were incubated for 10 min at 35 °C in each cryoprotectant and the plasma membrane integrity (PMI) and motility assessed using light microscopy. The same samples were fixed for TEM and the ultrastructural integrity of the spermatozoa examined. To investigate the effect of glycerol on the kangaroo sperm mitochondrial function, epididymidal spermatozoa were incubated with JC-1 in Tris–citrate media at 35 °C for 20 min in a range of glycerol concentrations (0%, 5%, 10%, 15% and 20%) and the mitochondrial membrane potential (MMP) and plasma membrane integrity determined. As expected, incubation of spermatozoa in 20% glycerol for 10 min resulted in a significant reduction in motility, PMI and ultrastructural integrity. Interestingly, incubation in 20% DMSO resulted in no significant reduction in motility or PMI but a significant loss of structural integrity when compared to the control spermatozoa (0% cryoprotectant). However, 20% DMSO was overall less damaging to sperm ultrastructure than glycerol, a combination of 10% glycerol and 10% DMSO, and sucrose. While all glycerol concentrations had an adverse effect on mitochondrial function, the statistical models presented for the relationship between MMP and glycerol predicted that spermatozoa, when added to 20% glycerol, would lose half of their initial MMP immediately at 35 °C and MMP would halve after 19.4 min at 4 °C. Models for the relationship between PMI and glycerol predicted that spermatozoa would lose half of their initial PMI after 1.8 min at 35 °C and PMI would halve after 21.1 min at 4 °C. These results suggest that if glycerol is to be used as a cryoprotectant for kangaroo spermatozoa then it is best administered at 4 °C and that mitochondrial function is more sensitive to glycerol than PMI. Future research should be directed at investigating strategies that reduce exposure of spermatozoa to glycerol during processing and that test the cryoprotective properties of 20% DMSO for kangaroo spermatozoa.

The tonicity of murine epididymal spermatozoa and their permeability towards common cryoprotectants and epididymal osmolytes

The permeability of murine cauda epididymidal spermatozoa was determined from the swelling caused by penetrating agents at isotonicity, which lies between 422 and 530 mmol/kg. Spermatozoa were permeable to a range of solutes with size <200 Da. Relative entry rates of cryoprotective agents (CPAs) were ethylene glycol approximately DMSO>propane-1,2-diol>glycerol>propane-1,3-diol. More polar compounds including major epididymal secretions were impermeant. None of the compounds entered spermatozoa through quinine-sensitive channels; rather, quinine increased the size of solute-swollen spermatozoa, suggesting that regulatory volume decrease and osmolyte loss occurred under these conditions. Volume responses to lowered osmolality revealed a greater volume-regulating ability of spermatozoa from the B6D2F1 strain than the C57BL6 strain. As the former strain displays better post-thaw fertility, their spermatozoa may have greater osmolyte loads enabling them to cope better with osmotic stress. Inadequate volume regulation, due to CPA-induced osmolyte loss, may affect post-thaw fertility. Knowing the permeability towards cryoprotectants will help to make a better choice of CPAs that are less damaging to sperm during cryopreservation.

Testosterone secretion, testicular histology and the cryopreservation of cauda epididymidal spermatozoa in the common ringtail possum (Pseudocheirus peregrinus)

The present study reports novel aspects of the reproductive biology of the male common ringtail possum (Pseudocheirus peregrinus). Plasma testosterone was measured through a stimulation test using the gonadotrophin-releasing hormone agonist, buserelin. Following intra-muscular administration of buserelin, there was an increase (P<0.05) in testosterone concentration in the peripheral circulation 4 h later. Quantitative testicular histology of this species was described for the first time. Eight stages of the seminiferous epithelium cycle were identified in 10 possums and their relative frequency determined. Spermatozoa were recovered from the cauda epididymides of hemi-castrated possums and cryopreservation conducted in straws (6 degrees C min(-1)) using final glycerol concentrations ranging between 2 and 20% in Tris-citrate egg yolk extender (v/v). Frozen straws were thawed and post-thaw motility, rate of motility, the percentage of live-dead spermatozoa and the percentage of sperm with swollen decondensed nuclei recorded. Similar to other marsupial sperm, common ringtail possum cauda epididymidal spermatozoa required high levels of glycerol (10-16%) in order to maintain post-thaw viability.

Ultrastructure, osmotic tolerance, glycerol toxicity and cryopreservation of caput and cauda epididymidal kangaroo spermatozoa

The aim of the present study was to compare cryopreservation, osmotic tolerance and glycerol toxicity between mature and immature epididymal kangaroo spermatozoa to investigate whether the lack of cryopreservation success of cauda epididymidal spermatozoa may be related to the increased complexity of the sperm ultrastructure acquired during epididymal transit. Caput and cauda epididymidal spermatozoa were recovered from red-necked wallabies (RNW; Macropus rufogriseus) and eastern grey kangaroos (EGK; M. giganteus). In Experiment 1, caput and cauda epididymidal spermatozoa were frozen and thawed using a standard cryopreservation procedure in Tris-citrate buffer with or without 20% glycerol. Although cryopreservation of caput epididymidal spermatozoa resulted in a significant increase in sperm plasma membrane damage, they were more tolerant of the procedure than spermatozoa recovered from the cauda epididymidis (P < 0.05). In Experiment 2, caput and cauda epididymidal EGK spermatozoa were diluted into phosphate-buffered saline media of varying osmolarity and their osmotic tolerance determined. Plasma membranes of caput epididymidal spermatozoa were more tolerant of hypo-osmotic media than were cauda epididymidal spermatozoa (P < 0.05). In Experiment 3, caput and cauda epididymidal RNW spermatozoa were incubated in Tris-citrate buffer with and without 20% glycerol at 35 and 4 degrees C to examine the cytotoxic effects of glycerol. At both temperatures, caput epididymidal spermatozoa showed less plasma membrane damage compared with cauda epididymidal spermatozoa when exposed to 20% glycerol (P < 0.05). These experiments clearly indicate that epididymal maturation of kangaroo spermatozoa results in a decreased ability to withstand the physiological stresses associated with cryopreservation.

Radiological quiz

Marsupial spermatozoa tolerate cold shock well, but differ in cryopreservation tolerance. In an attempt to explain these phenomena, the fatty acid composition of the sperm membrane from caput and cauda epididymides of the Eastern grey kangaroo, koala, and common wombat was measured and membrane sterol levels were measured in cauda epididymidal spermatozoa. While species-related differences in the levels of linolenic acid (18:3, n-6) and arachidonic acid (20:4, n-6) were observed in caput epididymal spermatozoa, these differences failed to significantly alter the ratio of unsaturated/saturated membrane fatty acids. However in cauda epididymidal spermatozoa, the ratio of unsaturated/saturated membrane fatty acids in koala and kangaroo spermatozoa was approximately 7.6 and 5.2, respectively; substantially higher than any other mammalian species so far described. Koala spermatozoal membranes had a higher ratio of unsaturated/saturated membrane fatty acids than that of wombat spermatozoa (t = 3.81; df = 4; p ⩽ 0.02); however, there was no significant difference between wombat and kangaroo spermatozoa. The highest proportions of DHA (22:6, n-3), the predominant membrane fatty acid in cauda epididymidal spermatozoa, were found in wombat and koala spermatozoa. While species-related differences in membrane sterol levels (cholesterol and desmosterol) were observed in cauda epididymidal spermatozoa, marsupial membrane sterol levels are very low. Marsupial spermatozoal membrane analyses do not support the hypothesis that a high ratio of saturated/unsaturated membrane fatty acids and low membrane sterol levels predisposes spermatozoa to cold shock damage. Instead, cryogenic tolerance appears related to DHA levels.

Volume regulation of mature and immature spermatozoa in a primate model, and possible ion channels involved.

Human ejaculated sperm undergo volume regulation, and swollen cells fail to penetrate mucus. Study of an infertile mouse model indicates maturation of volume regulation mechanism in the epididymis. Sperm from the ejaculate and three regions of the epididymis of the cynomolgus monkey (Macaca fascicularis) were dispersed in BWW medium and changes in the cell volume and kinematics, and their responses to ion channel blockers, were monitored by flow cytometry and motion analysis. Initially swollen cauda epididymidal spermatozoa regained their original volume within 20 min, but not in the presence of 0.25 mM quinine. Corpus epididymidal spermatozoa underwent such regulatory volume decrease (RVD) to a lesser extent, with a similar response to quinine. Caput sperm showed no swelling throughout incubation. The chloride channel inhibitor NPPB also caused swelling of cauda spermatozoa and both quinine and NPPB decreased the efficiency of forward progression. RVD of ejaculated spermatozoa was inhibited by the K+ channel blockers quinine and 4-aminopyridine (4-AP) but not by tetraethylammonium, Ba2+ or Gd3+, or the specific potassium channel blockers charybdotoxin, margatoxin, dendrotoxin, apamin, glybenclamide or clofilium. Quinine and 4-AP also altered ejaculated sperm kinematics as reported in human ejaculated spermatozoa. Quinine- and 4-AP-sensitive (implying K+) and NPPB-sensitive (implying Cl-) channels are involved in RVD of primate sperm, which develop this volume regulatory ability in the epididymis.

Sperm membrane fatty acid composition in the Eastern grey kangaroo ( Macropus giganteus), koala ( Phascolarctos cinereus), and common wombat ( Vombatus ursinus) and its relationship to cold shock injury and cryopreservation success

Marsupial spermatozoa tolerate cold shock well, but differ in cryopreservation tolerance. In an attempt to explain these phenomena, the fatty acid composition of the sperm membrane from caput and cauda epididymides of the Eastern grey kangaroo, koala, and common wombat was measured and membrane sterol levels were measured in cauda epididymidal spermatozoa. While species-related differences in the levels of linolenic acid (18:3, n-6) and arachidonic acid (20:4, n-6) were observed in caput epididymal spermatozoa, these differences failed to significantly alter the ratio of unsaturated/saturated membrane fatty acids. However in cauda epididymidal spermatozoa, the ratio of unsaturated/saturated membrane fatty acids in koala and kangaroo spermatozoa was approximately 7.6 and 5.2, respectively; substantially higher than any other mammalian species so far described. Koala spermatozoal membranes had a higher ratio of unsaturated/saturated membrane fatty acids than that of wombat spermatozoa ( t = 3.81; df = 4; p ⩽ 0.02); however, there was no significant difference between wombat and kangaroo spermatozoa. The highest proportions of DHA (22:6, n-3), the predominant membrane fatty acid in cauda epididymidal spermatozoa, were found in wombat and koala spermatozoa. While species-related differences in membrane sterol levels (cholesterol and desmosterol) were observed in cauda epididymidal spermatozoa, marsupial membrane sterol levels are very low. Marsupial spermatozoal membrane analyses do not support the hypothesis that a high ratio of saturated/unsaturated membrane fatty acids and low membrane sterol levels predisposes spermatozoa to cold shock damage. Instead, cryogenic tolerance appears related to DHA levels.

Spermatotoxic effect of aflatoxin B 1 in the albino mouse

With the background that the foodborne mycotoxin aflatoxin B 1 (AFB 1) could be toxic to the male reproductive mechanism in man as well as wild and domestic animals, the present study was aimed at finding the effect of AFB 1 on sperm. The Swiss albino mouse was the test animal. AFB 1, suspended in corn oil and ethanol (95:5, v/v), was administered intraperitoneally to 90-day-old mice at a daily dose of 50 μg/kg body weight for 7, 15, 35 and 45 days. The analysis consisted of fertility testing and counts, motility and abnormalities of the cauda epididymidal sperm, adopting light- as well as electron-microscopy. The fertility of the treated mice was reduced drastically. Sperm concentration in the epididymis and sperm motility decreased whereas sperm abnormalities increased. In particular, sperm abnormalities like two axonemes in a common cytoplasm, sticking together of heads/tails, etc., were noted. A higher percentage of cauda epididymidal spermatozoa than in the control mice retained the cytoplasmic droplet (CD) and such retention was dependent on the duration of the treatment. Spermatozoa retaining the CD were inhibited in motility. Sperm CD of AFB 1-treated mice contained electron-dense spherical inclusions, which are hypothesized as lipid inclusions produced from the lamellae through the spherical vesicles of the CD. The results indicate disruption of the spermatogenic as well as androgenic compartments of the testis by AFB 1. The results also reflect an alteration of epididymal function towards the post-testicular sperm maturation process by AFB 1.

Reactivation of motility of demembranated hamster spermatozoa: role of protein tyrosine kinase and protein phosphatases.

Demembranated cauda epididymidal spermatozoa of hamster, following reactivation with 1 mm ATP, exhibited either a loop or planar type of motility. The spermatozoa with planar motility exhibited increased progressive velocity (VSL), straightness (STR), linearity (LIN) and beat cross frequency (BCF) compared to the spermatozoa with loop type motility. cAMP was observed to have differential effects on the motility parameters of the demembranated spermatozoa depending on the type of motility. For instance, in the loop type, average path velocity (VAP), curvilinear velocity (VCL) and VSL were increased in the presence of cAMP unlike in the planar type. Furthermore, in an attempt to understand the role of protein kinases and protein phosphatases in regulation of sperm motility, the effects of various inhibitors of these enzymes on the motility and phosphorylation of proteins of reactivated demembranated spermatozoa were studied. Inhibitors of PTKase (protein tyrosine kinase) and protein phosphatases inhibited the motility of reactivated demembranated hamster spermatozoa. The activity of the respective enzymes associated with demembranated spermatozoa was concurrently inhibited, thus providing evidence that the effect of the inhibitors on motility was mediated through their inhibitory effects on the activities of the enzymes. The results also demonstrated that two phosphotyrosinylated proteins of molecular weight 65 and 80 kDa showed reduced phosphorylation in the presence of PTKase inhibitors, thus indicating their possible role in reactivation of motility of demembranated hamster spermatozoa.

Identification of the major tyrosine phosphorylated protein of capacitated hamster spermatozoa as a homologue of mammalian sperm a kinase anchoring protein.

The molecular basis of mammalian sperm capacitation is unique in that, it is associated with a protein kinase A (PKA) dependent upregulation of protein tyrosine phosphorylation. Therefore, PKA activity during capacitation would be crucial for the downstream events of protein tyrosine phosphorylation, and mechanisms may exist to ensure that PKA phosphorylates its specific substrate. This could be achieved by bringing PKA close to its substrate, a function normally carried out by an A-kinase anchoring protein (AKAP). We showed previously that cauda epididymidal spermatozoa of hamster undergo a capacitation-dependent increase in protein tyrosine phosphorylation. In the present study, evidence is provided that two major tyrosine phosphorylated proteins of molecular weight 97 and 83 kDa are the hamster homologues of mouse pro-AKAP82 and AKAP82, and have been designated as hamster pro-AKAP83 and AKAP83 respectively. Hamster AKAP83 resembled the mouse AKAP82 with respect to its molecular weight, pI (pH 5-5.5) and cDNA and amino acid sequences. Sequence analysis indicated that the primary structure of pro-AKAP83 was highly conserved and exhibited 91% identity with mouse and rat AKAP82. Further, the functional domains, namely the region involved in binding the regulatory subunit of PKA and the proteolytic cleavage site between pro-AKAP83 and AKAP83, were identical with that observed in rat and mouse pro-AKAP82 and AKAP82. Immunoblot analysis using polyclonal hamster anti-AKAP83 antibodies indicated that AKAP83 was present both in caput and cauda epididymidal spermatozoa. The antibody also identified the pro-AKAP82 and AKAP82 in mouse caput and cauda epididymidal spermatozoa. Immunofluorescence studies indicated that AKAP83 in hamster spermatozoa was localized along the length of principal piece of the tail. It was also demonstrated that hamster pro-AKAP83/AKAP83 gene expression was testis specific and was not expressed in other organs in either sex. This is the first report implicating AKAP in capacitation in rodents.

Aspects of the male reproductive toxicity/male antifertility property of andrographolide in albino rats: effect on the testis and the cauda epididymidal spermatozoa

Previous work has shown that Andrographis paniculata leaf, when fed to male albino rats, causes the arrest of spermatogenesis. The present study was undertaken to investigate whether andrographolide, one of the major constituents of this plant, is responsible for such an effect. The compound was administered to 3-month-old male Wistar albino rats at two dose levels, for 48 days. Fertility tests, analysis of the counts, motility and abnormalities of the cauda epididymidal spermatozoa, and histopathological­evaluation of the testis were carried out. The results showed that sperm counts decreased, the spermatozoa were not motile, and several of them possessed abnormalities. The seminiferous epithelium was­thoroughly disrupted and in the seminiferous tubules, fully differentiated spermatozoa were far too limited; cells in the divisional stages were prevalent; multinucleate giant cells were abundant and Leydig cells appeared intact. It is inferred that andrographolide could affect spermatogenesis by preventing cytokinesis of the dividing spermatogenic cell lines. The multinucleate giant cells are comparable to the symplasts generated by cytochalasin-D and ursolic acid due to action at stages V–VII of the spermatogenic cycle. Sertoli cell damage and spermatotoxic effects are also apparent. Thus, the study points to a male reproductive toxic effect of this compound when used as a therapeutic; the study also confirms the possible prospective use of andrographolide in male contraception. Copyright © 2000 John Wiley & Sons, Ltd.