Vitrification of in vitro produced bovine embryos: in vitro and in vivo evaluations.

The efficacy of different vitrification solutions to cryopreserve in vitro produced bovine blastocysts was evaluated based upon in vitro development of embryos in culture and on in vivo development of embryos transferred into recipients. In the first experiment, ethylene glycol + glycerol (Eg + Gly) + different sucrose concentrations were evaluated. There were no significant differences in development rates among solutions. As for hatching, the Eg + Gly + 0.1 M sucrose group had a greater rate as compared with Eg + Gly + 0 M sucrose and Eg + Gly + 0.5 M sucrose groups in the evaluations of Day 6, Day 7 and Day 6 + Day 7 embryos; and, Eg + Gly + 0.3 M sucrose group had a greater rate as compared with the Eg + Gly + 0 M sucrose and Eg + Gly + 0.5 M sucrose groups in evaluations of Day 6 and Day 6 + Day 7 embryos. There were no significant differences in development and hatching rates between Day 6 and 7 in in vitro produced bovine embryos within each treatment group. There were significant differences in nuclei number after vitrification between Eg + Gly + 0.1 M and Eg + Gly + 0 M sucrose groups and the Eg + Gly + 0.5 M sucrose group. Pregnancy after 60 days of transfer and calving rates showed a difference between in vivo produced embryos freshly transferred and in vitro produced embryos vitrified with Eg + Gly + 0.3 M. There were no significant differences in gestation length and sex ratio between treatments. As for birth weight, there were significant differences between fresh in vivo produced embryos and all treatments of in vitro produced embryos. There were significant differences in dystocial parturition between in vivo produced embryos and all treatments with in vitro produced embryos. These results demonstrate that vitrification can be used successfully in the cryopreservation of in vitro produced bovine embryos, and that it might be considered for use in commercial programs.

GABA release mechanism in the golden hamster retina.

High K+ medium and glutamate elicited a significant [3H]-GABA release in the golden hamster retina. High K+ -induced GABA release was largely calcium-dependent, while the effect of glutamate was Ca2+ -independent. After replacing Na+ by Li+, glutamate-evoked [3H]-GABA release was abolished, while high K+ -evoked release remained unchanged. The effect of glutamate was completely blocked by DNQX but not by APV. Furthermore, kainate induced [3H]-GABA release, whereas NMDA was ineffective. Assessment of endogenous GABA efflux further confirmed results obtained for [3H]-GABA. GABA-like immunoreactivity was observed in amacrine cells, in neurons localized in ganglion cell layer, as well as in fibers and terminals at the inner plexiform layer. In addition a few horizontal cells showed GABA-like immunolabeling. The present results suggest the existence of at least two pools of GABA in the hamster retina, compatible with both vesicular and carrier-mediated mechanisms of transmitter release, being the amacrine cells the main gabaergic source in this tissue.

A lineal equation for the classification of progressive and hyperactive spermatozoa.

Capacitation of spermatozoa, a complex sequence of events that render them able to fertilize the egg, is generally associated with a switch from lineal, progressive movement to a vigorous, non-progressive pattern characterized by starlike tracks, a process known as hyperactivation. Development of a method for the analysis of progressive and hyperactive tracks is thus important for the assessment of capacitation in biochemical, physiological and clinical studies. In this study, we have applied a two-step heuristic model to deduce a lineal equation that discriminates hyperactive from progressive spermatozoa. The kinetic parameters (curvilinear velocity (VCL), linearity (LIN), amplitude of lateral head displacement (ALH), straightness (STR), wobble (WOB), mean 'dance' (DAN) and velocity of the average path (VAP)) of ram spermatozoa were evaluated with a computerized motility analyzer, and classified one by one as progressive or hyperactive by the appearance of their tracks. In a first step, a discriminating plane was defined by minimizing the number of misclassified spermatozoa ('conflicting points'); then, the plane was adjusted by an iterative process to minimize the distance from conflicting points to it. The resulting plane showed a discriminating capacity of over 95% for both classes, higher than that achieved by setting a threshold value for the parameters taken separately or in group. When included in a standard semen analysis, application of the equation allowed a rapid assessment of the percentage of hyperactive spermatozoa. The method described, developed in ram spermatozoa, can be applied to different species for a variety of purposes.

In vitro capacitating effect of gamma-aminobutyric acid in ram spermatozoa.

We have evaluated the capacitating effect of gamma-aminobutyric acid (GABA) in ram spermatozoa in vitro, in a chemically defined medium, by means of the chlortetracycline (CTC) binding assay. Semen from adult Australian Merino rams was collected in an artificial vagina; spermatozoa were washed once in modified Biggers, Whitten, and Wittingham medium (m-BWW), without BSA or serum, and incubated in m-BWW alone or in m-BWW containing GABA, GABA agonists, or antagonists for 2 h at 38.5 degrees C under 5% CO2 in air. Samples were taken for assessment of CTC binding pattern or were further incubated for 15 min in the presence of 5 microM calcium ionophore A23187. Acrosomal exocytosis was evaluated by Pisum sativum agglutinin binding. Addition of GABA to the incubation medium resulted in a concentration-dependent increase in the percentage of CTC forms II and III, corresponding to mid-capacitated and capacitated spermatozoa, respectively. The effect was marginally significant at 1 microM and maximal at 20 microM. The action of 20 microM GABA was mimicked by the GABAB-receptor agonist, muscimol, but not by the GABAA-receptor agonist, baclofen, and completely blocked by the GABAA-receptor antagonists, bicuculline and picrotoxin, which lacked effect per se. In a separate set of experiments, incubation of spermatozoa with GABA at a concentration of 1 microM, which was insufficient to stimulate sperm capacitation, together with the neuroactive steroid allopregnanolone (1 microM) provoked a capacitating effect similar to that achieved by 20 microM GABA alone. These results show that GABA has a capacitating action on ram spermatozoa through a GABAA receptor-mediated mechanism.

Actin localization in ram spermatozoa: effect of freezing/thawing, capacitation and calcium ionophore-induced acrosomal exocytosis.

We have analyzed, by immunofluorescence, the localization of actin in ram spermatozoa, its colocalization with the actin-binding protein, gelsolin, and the effect of freeze/thawing, in vitro capacitation, and induced acrosomal exocytosis on its distribution. The monoclonal anti-actin and anti-gelsolin antibodies used recognized single bands at 43,000 and 90,000 kDa, respectively. In all spermatozoa, intense actin staining was observed in the whole length of the flagellum and, depending on the protocol used, in the neck and postacrosomal region of the head. Comparison of three staining methods, together with the use of NBD-phallacidin, allowed us to characterize ram sperm actin as a monomeric, intracellular, membrane-associated protein. Gelsolin was also present in ram spermatozoa and precisely colocalized with actin. Processes involving alterations in membrane structure such as freezing/thawing, in vitro capacitation, and calcium ionophore-induced acrosomal exocytosis provoked changes in the exposure of actin to the antibody. This strongly suggests a physical association of this protein to the plasma membrane, most likely by its intracellular side. The possible role of actin in sperm function is discussed.

Assessment of the acrosomal status of membrane-intact ram spermatozoa after freezing and thawing, by simultaneous lectin/Hoechst 33258 staining.

We have evaluated the effect of freezing and thawing on the acrosomal status of ram spermatozoa, especially those that withstood cryopreservation as assessed by membrane integrity. To this end, we performed simultaneous lectin/Hoechst 33258 staining, and compared the ability of three fluoresceinated lectins. Ram spermatozoa were treated with fluorescein isothiocyanate-labelled Pisum sativum lectin (PSA), fluorescein isothiocyanate-labelled Arachis hypogea lectin (PNA) and fluorescein isothiocyanate-labelled Triticum vulgaris lectin (WGA) and simultaneously with Hoechst 33258 for determination of membrane integrity and acrosomal status. In all cases, three forms were readily distinguished by their distribution pattern. For both PSA and PNA, the most abundant form found in fresh semen consisted of fluorescence on the acrosomal area. This form corresponds to acrosome-intact spermatozoa, as assessed by Differential Interference Contrast (DIC) microscopy. Two minor forms showed weak fluorescence on the equatorial segment or no fluorescence on the head. DIC microscopy revealed that both forms were associated with acrosome-lost spermatozoa. WGA labelling showed two forms, one of which consisted of fluorescence on the entire head, albeit more intensely on its anterior segment. Spermatozoa in this form were acrosome-intact by DIC. The other form lacked fluorescence on the acrosomal region, but still showed faint fluorescence in the posterior region. This form was acrosome-lost by DIC. Incubation of fresh spermatozoa with calcium ionophore A23187 for up to 1 h significantly increased the percentage of those forms identified as acrosome-reacted as described above. This was confirmed by the time-dependent accumulation of these forms, as well as by DIC microscopy. At all times, differences among values obtained using these three lectins were not significant. Freezing and thawing led to a decrease of both membrane integrity and acrosomal integrity, irrespective of the lectin used. However, almost all spermatozoa that withstood cryopreservation, as evaluated by Hoechst exclusion, showed intact acrosomes. In this case, no differences between fresh and frozen/thawed samples were observed. These results suggest that the structural integrity of ram spermatozoa is mostly unaffected after cryopreservation, suggesting that it is damage to the plasma membrane that is primarily responsible for the low fertility of cryopreserved samples.

The storage of pure ram semen at room temperature results in capacitation of a subpopulation of spermatozoa.

We investigated whether storage of pure ram semen at room temperature would facilitate the sperm capacitation process, as assessed by means of the chlortetracycline method. Objective motility, membrane integrity and ability of spermatozoa to undergo acrosome reaction induced by A23187 for 15 min were simultaneously evaluated to gain further insight into this process. Storage for 4 h at room temperature had a clear capacitating effect in approximately 50% of spermatozoa and increased their ability to respond to A23187. Beyond that time, the percentage of motility and membrane integrity remained unchanged. Moreover, storage did not alter the ability of those spermatozoa that remained noncapacitated under these conditions to become capacitated in SOF-m medium. Storage for 4 h increased the percentage of spermatozoa showing swelling of the apical ridge from 3 to 13%. In conclusion, storage of ram semen at room temperature for 4 h in the dark has a marked capacitating effect on a subpopulation of spermatozoa, without changes in motility or membrane integrity, and a low effect on the appearance of the acrosome. Since semen storage is generally included in different IVF protocols, the results presented here contribute toward a clearer understanding of its role in these procedures.

A large-scale program in laparoscopic intrauterine insemination with frozen-thawed semen in Australian Merino sheep in Argentine Patagonia.

This report shows the results of a large-scale laparoscopic intrauterine insemination program on a flock of Australian Merino sheep in Argentine Patagonia. The study was carried out on a total of 1824 ewes (3-to-7-yr-old) and 480 ewe hoggets (19-20 months old) on 2 farms in the southeastern region of Santa Cruz Province, in April and May 1996. The animals, divided into 15 groups, were synchronized with vaginal sponges containing 60 mg medroxyprogesterone acetate for 14 d and injected with 200 IU PMSG upon sponge removal. Estrus was screened every 12 h by means of vasectomized marker rams. The animals were inseminated laparoscopically by the intrauterine route using 2 schemes: 1) at a fixed time (12 h) after estrus detection, or 2) at a fixed time (60 h) after sponge removal irrespective of estrus. Pregnancy was determined at 30 d by transrectal ultrasound imaging. The results showed that 1) the onset of estrus occurs most often between 24 and 48 h after sponge removal, 2) ewe hoggets undergo estrus significantly earlier than sexually mature ewes, 3) in those animals showing estrus, there appears to be no relationship between fertility (as assessed by pregnancy outcome) and time of estrus, 4) there is a significant association between the percentage of estrus occurrence and pregnancy rate, 5) fertility is significantly higher in ewes than in hoggets, 6) for practical purposes insemination at a fixed time after the onset of estrus has no advantage over that of to insemination at a fixed time after sponge removal. It is concluded that large-scale laparoscopic intrauterine insemination can be successfully applied in Australian Merino ewes and ewe hoggets in low-productivity areas such as that of Argentine Patagonia and that estrus detection is unnecessary when insemination is performed at 60 h after sponge removal.

Changes in sperm-bound amidase activity suggest subtle damage to ram sperm acrosomes by freezing/thawing, not detected by light microscopy.

We have measured sperm-bound amidase activity in fresh, cooled and frozen/thawed ram spermatozoa, in order to study if freezing and thawing led to some degree of acrosome damage of motile/viable spermatozoa not detected by optical methods. This assay was based on the fact that membrane damage would result in an increased access of the enzyme substrate to the sperm acrosome. Semen was collected from adult Australian Merino rams, and spermatozoa were washed by centrifugation through a Ficoll solution. Sperm-bound amidase activity was measured in whole spermatozoa using the protease substrate benzoyl-arginyl-p-nitroanilide (BAPNA). Acrosomal status was also assessed by light microscopy after Giemsa staining. Most amidase activity was shown to be sperm-bound, as only a minor fraction of the enzyme activity was release into the medium after induced damage. Simultaneous assessment of sperm-bound amidase activity and the percentage of spermatozoa with microscopically evident acrosomal damage, after mild sonication for different times, showed a high correlation between both parameters (r = 0.97, p < 0.001). In separate experiments, fresh, cooled and frozen/thawed semen samples were filtered through Sephadex G-10 to obtain a subpopulation of motile, mostly acrosome-intact spermatozoa. As controls, spermatozoa from the same samples to which extensive acrosome damage was induced were evaluated. Slow cooling to 4 degrees C had no effect on amidase activity or percent acrosomal damage with respect to fresh samples. Freezing and thawing resulted in a sperm population that, after filtration through Sephadex, had a low percentage of acrosome damage (9.4%, vs. 2.1% for fresh filtered controls), which was 11% of that obtained after extensive acrosome damage (83%). However, amidase activity in these samples was markedly increased, showing values of activity that were 56% of those obtained in extensively damaged spermatozoa. This effect was not due to an alteration in the enzyme kinetics. We conclude that sperm-bound amidase activity is useful to detect subtle changes, provoked by a standard freezing/thawing procedure, in the permeability of acrosomes from ram spermatozoa which are not detected by direct observation of the acrosomes after Giemsa staining.

In vitro capacitation and induction of acrosomal exocytosis in ram spermatozoa as assessed by the chlortetracycline assay.

We have described the different patterns of chlortetracycline (CTC) binding to ram spermatozoa, immediately after ejaculation and upon in vitro capacitation and calcium ionophore-induced acrosomal exocytosis. Four different forms of CTC distribution were found. Form I showed an even distribution of fluorescence over the entire head, with a brighter band in the equatorial region. In Form II, uniform fluorescence was observed without equatorial band. Form III consisted of fluorescence in the anterior portion of the head. Form IV showed no fluorescence over the head. In all cases, fluorescence in the middle piece of the flagellum was observed as well. Immediately after ejaculation, Form I was the most abundant one (78%) in fresh semen with Forms II and III being relatively scarce (less than 15%). Form IV was virtually absent or appeared only occasionally. Incubation under in vitro capacitating conditions led to a significant decrease in Form I and to a significant increase in Forms II and III. Form II was mainly associated to intact acrosomes, while most spermatozoa in Form III showed intermediate forms of acrosomal status. Incubation of spermatozoa with the calcium ionophore A23187 resulted in 55% of spermatozoa showing Form IV, suggesting that it represents the acrosome-reacted stage. Form I was abruptly decreased at 30 min of incubation and was neglectible after 60 min. In contrast, Forms II and III increased at 30 min but decreased later on, suggesting that both forms represent intermediate stages before the acrosomal exocytosis. Analysis of acrosomal status in spermatozoa from individual CTC forms revealed that all spermatozoa that remained in Form II after incubation had intact acrosomes. Intermediate stages were predominant in Form III-spermatozoa, while most Form IV-spermatozoa underwent full acrosomal exocytosis. These results show that CTC binding can be used to monitor changes in ram spermatozoa during capacitation and acrosome-reaction.

Daily variations in 2-[125I]melatonin specific binding in the golden hamster retina.

Daily variations in melatonin content and 2-[125I]melatonin specific binding in retinas of golden hamsters were studied. Both parameters showed significant variations throughout the 24 h period. Maximal specific binding was observed at 24.00 h, while melatonin content peaked at 04.00 h. Saturation studies performed at 12.00 and 24.00 h indicated that the maximal concentration of 2-[125I]melatonin binding sites (Bmax) was significantly higher at 24.00 h than at 12.00 h, whereas the dissociation constant (Kd) remained unchanged. As 2-[125I]melatonin specific binding decreased at times when retinal melatonin content was high, these findings suggest that daily variations in retinal melatonin levels may be implicated in the regulation of the density of melatonin binding sites, probably through mechanisms of up- and down-regulation induced by melatonin on its own binding sites.

Use of computerized motility analyzer for the evaluation of frozen-thawed ram spermatozoa.

A computerized motility analyzer (CellTrak/STM) was calibrated for its use with ram semen. Adjustment of the several setup variables allowed an accurate measurement of kinetic parameters such as percentage of motile cells, straight velocity, curvilinear velocity, linearity and amplitude of lateral displacement of the head. All kinetic parameters, except the lateral displacement of the head, showed significant changes after freezing and thawing. The curvilinear velocity exhibited the least significative post-thaw decrease. The alterations in kinetic parameters provoked by freezing and thawing could account for the low success obtained with frozen semen by cervical insemination, as it is accepted that during the initial steps of fertilization high motility and linearity are required.

Fluorescent staining as a method of assessing membrane damage and post-thaw survival of ram spermatozoa.

We studied the relationship between motility and membrane damage, as assessed by fluorescent staining, in fresh and in frozen-thawed ram spermatozoa. Semen from Merino rams was incubated with 6-carboxyfluorescein diacetate and propidium iodide. In both fresh and frozen-thawed samples, the percentage of intact spermatozoa was lower than the motility rate, thus indicating the presence of damaged but motile spermatozoa. Freezing and thawing resulted in a marked loss of membrane integrity, whereas motility decreased to a lesser extent. There was a positive relationship (r=0.64; P<0.001) between membrane integrity immediately after thawing and motility after 8 h of incubation at 37 degrees C. These results demonstrate the usefulness of the fluorescent staining method for the prediction of ram sperm quality and post-thaw survival.

Acute effect of prolactin on ornithine decarboxylase activity in the rat testis.

A study was conducted to evaluate the effect of the acute treatment with prolactin (PRL) on ornithine decarboxylase (ODC) activity in the rat testis. Injection of a single SC dose of ovine PRL to puberal rats resulted in the activation of ODC from whole testis. This effect was maximal at 4 h after injection, and statistically significant at the dose of 500 micrograms. The effect of PRL was confined to the interstitial space; no change was observed in seminiferous tubules. PRL was unable to further increase testicular ODC activity when injected together with a stimulatory dose of human chorionic gonadotropin (hCG). The effect of PRL was mimicked by injection of a single dose of the dopamine antagonist sulpiride, which provoked a ninefold increase in serum PRL levels. In contrast, PRL did not stimulate testicular ODC activity in hypophysectomized rats, either under basal conditions or during treatment with PRL-hCG, indicating the requirement of a functional hypophysis for the expression of PRL action. These results suggest that the stimulation of testicular ODC activity by PRL is a marker of the trophic response of the testis to this hormone, different from the stimulation of steroidogenesis. This activity could be useful for the study of PRL action on the testis as well as of the interaction between PRL and LH at the testicular level.

Androgenic control of polyamine concentrations in rat epididymis.

Unilateral orchidectomy resulted in a significant decrease in tissue content of putrescine and polyamines. However, no differences were detected when the results were expressed in terms of ng g-1 tissue. At 48 h after bilateral orchidectomy, a significant decrease in putrescine content was observed, but spermidine and spermine content were unaffected. The observed decrease in putrescine was prevented by treatment with testosterone propionate, but neither spermidine nor spermine were affected. Bilateral orchidectomy resulted in a significant decrease in the tissue content of putrescine, spermidine and spermine after 7 days. Treatment with testosterone propionate increased the content of putrescine, spermidine and spermine in the epididymis by about 200%, 92% and 34%, respectively. When results were expressed as nmol g-1, a significant decrease after castration in putrescine and spermidine, but not in spermine, was observed. Treatment with testosterone propionate restored putrescine concentration, but had no effect on spermidine and spermine concentrations. In castrated rats treated with testosterone propionate, the anti-androgen flutamide abolished the effect of the androgen on putrescine and spermidine content, but there was no effect on spermine. Acetylputrescine was not detected in the epididymis, while acetylpolyamines were detected at much lower concentrations than polyamines. After bilateral orchidectomy there was a decrease in the tissue content of all acetylpolyamines and an increase in their tissue concentration. The effect of castration on acetylpolyamine content was reversed by testosterone propionate treatment. We conclude that an active synthesis of polyamines occurs in the rat epididymis, and that this process depends upon the androgen environment. Regulation of ornithine decarboxylase activity appears to be the main step that is controlled by androgens.

S-adenosyl-L-methionine decarboxylase activity in the rat epididymis: ontogeny and androgenic control.

The authors describe the occurrence of high levels of S-adenosyl-L-methionine decarboxylase (SAMDC) activity in the rat epididymis, and its ontogeny and androgenic control. As early as 15 days of age, SAMDC activity exists, although a peak of activity is observed at 25 days. Bilateral orchidectomy resulted in a decline of epididymal SAMDC activity. However, an androgen-independent fraction, accounting for 34% of total activity, appears to exist in the epididymis. In 45-day-old orchidectomized rats, SAMDC activity was stimulated by testosterone treatment in a dose-dependent manner. However, treatment of 45-day-old intact animals with a high dose of the androgen failed to modify SAMDC activity, indicating that, at this age, the enzyme is maximally stimulated by endogenous androgens. The observed effect of testosterone on castrated rats was completely abolished by concomitant treatment with the antiandrogen flutamide. This compound was ineffective on the androgen-insensitive fraction. To assess the contribution of circulating and luminal androgens to the maintenance of epididymal SAMDC, rats were unilaterally orchidectomized and activity was determined in both epididymides after 7 days. The SAMDC activity was identical in epididymides from both sides, suggesting circulating androgens suffice to maintain normal levels of activity. It was concluded that androgens regulate epididymal SAMDC activity, although an androgen-independent fraction appears to exist.

Effect of the antiandrogen flutamide on pituitary LH content and release.

Flutamide is a nonsteroidal antiandrogen that blocks androgen receptors, with a consequent increase in serum immunoreactive LH (I-LH) in the presence of high testosterone concentrations. Several studies suggested that the gonadal steroids also play an important role in the regulation of LH bioactivity (B-LH). Therefore, it seems difficult to understand how the blockade of pituitary androgen receptors leads to the increase in testosterone levels. The present study was designed to elucidate the effect of flutamide on serum I-LH, B-LH and testosterone, as well as on in vitro stimulation of pituitaries by gonadotropin-releasing hormone (GnRH), in intact and androgen-treated castrated rats. In intact animals, a dose of flutamide as low as 0.5 mg/day provoked a 7- to 8-fold increase in serum I-LH levels over the vehicle-injected controls, whereas B-LH and testosterone were unaffected. However, higher doses significantly increased serum B-LH to values similar to those obtained in vehicle-injected castrated animals, resulting in high testosterone levels. Flutamide treatment provoked a decrease in I-LH and B-LH pituitary content; this effect was significantly higher under in vitro GnRH stimulation. The releasable I-LH under GnRH stimulation was not affected by flutamide treatment; however, a marked decrease was observed in B-LH.(ABSTRACT TRUNCATED AT 250 WORDS)

Ornithine decarboxylase activity as a marker of androgen and antiandrogen action in the rat epididymis.

After castration, there was a marked decrease in serum androgen concentration at 6 h, and a dramatic inhibition of ornithine decarboxylase (ODC) at 12 h. Administration of testosterone propionate to castrated rats at a dose of 0.05 mg/animal restored ODC activity to the normal value. However, no change was observed when intact rats were treated with testosterone even at a 40-fold higher dose, indicating that endogenous androgens present in intact rats are far in excess for maintenance of maximal levels of activity. Administration of the antiandrogen flutamide to intact rats caused a moderate decrease in epididymal weight, whereas this effect was more pronounced in castrated, androgen-treated rats. In the latter, the effect of flutamide was significant at the lowest dose used (0.5 mg/day). ODC activity was significantly decreased by flutamide treatment of intact rats, but even at the highest dose used (10 mg/day) only a 39% inhibition was observed. In flutamide-treated rats, LH concentrations were markedly increased, as were serum and epididymal androgens. In androgen-treated castrated rats, flutamide caused epididymal ODC to fall to undetectable values. These results show that: (1) androgens are essential for the maintenance of ODC activity in the epididymis; (2) epididymal ODC activity is maximally stimulated by endogenous androgens, at least in the pubertal rat; (3) the apparent potency of flutamide is substantially lowered by an increase in epididymal androgens. We suggest that ODC is a sensitive marker of the action of androgens and antiandrogens in the epididymis.

Androgen-dependence of ornithine decarboxylase in the rat epididymis.

Ornithine decarboxylase (ODC) activity was measured in epididymides of 45-day-old rats. Higher ODC activity was detected in the corpus and cauda than in the caput epididymidis. Bilateral castration for 7 days caused epididymal ODC to fall to undetectable values, whereas testosterone restored activity to normal values. The effect of the androgen was significantly inhibited by cyproterone acetate. The caput was more sensitive to the action of testosterone than were the corpus and caudal segments. Unilateral castration for 4 or 8 days did not affect ODC on the control or castrated side, but the activity fell in epididymides of both sides after removal of the remaining testis. These results show that epididymal ODC activity is androgen-dependent.

Effect of phosphatase inhibitors on estrogen receptors in the rat testis.

Testicular estrogen receptors were measured in the presence of phosphatase inhibitors, molybdate (MoO4=), wolframate (WO4=) and fluoride (F-), under different experimental conditions. At 0-4 degrees C, MoO4= was able to partially replace the stabilizing action of dithiothreitol (DTT) on cytosolic estrogen binding activity, suggesting a protective effect on reduced sulfhydryl groups. At 25 degrees C in the presence of DTT, the effect of MoO4= was observed in homogenates and low speed supernatants, being uneffective in cytosol. A possible mechanism of action through inhibition of phosphatase activity was supported by the use of WO4=. Acid phosphatase activity was inhibited in the presence of these agents, whereas alkaline phosphatase was unaffected. The addition of MoO4= also caused a significant inhibition of proteolytic activity. These results suggest that MoO4= would stabilize testicular estrogen binding activity through, at least, three mechanisms: 1) protection of reduced sulfhydryl groups; 2) inhibition of acid phosphatase, and 3) inhibition of proteolytic activity.