Relationship between testosterone, fluid content and luteinizing hormone receptors in the rat testis.

Injection of immature male rats with human chorionic gonadotrophin resulted in a decreased ability of the testis to bind [¹²⁵I]-labelled human chorionic gonadotrophin $\text{in vitro}$, and a marked, but transient increase in testis weight; the latter was apparently due to the accumulation of fluid containing high levels of testosterone. Intra-testicular injection of cycloheximide significantly inhibited all these changes, thus demonstrating their dependence on protein synthesis. It is concluded from this and other data that either testosterone itself or a steroidogenic protein intermediary may be responsible for the gonadotrophin-induced reduction in availability of gonadotrophin receptors.     

myo-Inositol metabolism in rat testis in response to streptozotocin-induced diabetes

The effects of streptozotocin-induced hyperglycemia on de novo myo-inositol biosynthesis in rat testis was examined. Testicular glucose and glucose 6-phosphate levels increased significantly 10 and 12 h after stretozotocin injection, respectively. However, testis myo-inositol content did not increase appreciably until 24 h following injection of the drug. Seventy-two hours after streptozotocin administration, testis myo-inositol levels were 2.7-fold higher in diabetic rats than in controls injected with citrate buffer. No changes were observed in the Specific activities of myo-inositol-1-phosphate synthase (EC 5.5.1.4) and 1-l-myo-inositol-1-phosphatase (EC 3.1.3.25). However, hyperglycemic rats displayed testicular glucose and glucose 6-phosphate levels approximately 4- and 2-fold in excess of control values, respectively. Insulin treatment of diabetic rats resulted in the lowering of plasma glucose, and testis glucose 6-phosphate to normal or below normal levels within hours. Inositol levels remained significantly elevated compared with control animals, although slightly lower than that observed for untreated diabetic rats. Streptozotocin diabetic rats had a significantly decreased testis cytosolic $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio compared with control animals 72 h after injection. The potential role of testis hexokinase distribution in the regulation of glucose 6-phosphate and myo-inositol biosynthesis in normal and diabetic rats was investigated. No significant differences in testis hexokinase distribution or in the kinetic characteristics of the soluble and particulate hexokinase activities were observed. Testicular sperm counts in streptozotocin diabetic rats were not significantly different from control values.     

Synchronization of estrus in cyclic beef heifers with the prostaglandin analog alfaprostol

Seventy-eight Hereford-Angus crossbred heifers were injected intramuscularly twice with 6 mg of alfaprostol^b in 6 ml of propylene glycol. On each representative day of a 20-day estrous cycle (estrus = Day 0), either three or four heifers received their first injection. The second injection was given 12 days after the first, regardless of the response to the first injection. Thirty-nine heifers were not treated. The first alfaprostol injection reduced serum progesterone to less than 1 ng/ml in all heifers injected after Day 4. A total of 79.5% ($\text{62}\text{78}$) of the heifers exhibited estrus by five days after the first injection. Average interval from injection to estrus was 63 hours. The second injection occurred on Days 6 through 16 for all but one heifer, with 75.6% ($\text{59}\text{78}$) falling on Days 8 through 11 of the estrous cycle. Estrus was detected in 93.6% ($\text{73}\text{78}$) of the heifers within five days after the second injection, with an average interval to estrus of 66 hours.Day of cycle at second injection did not affect the interval to estrus. Conception occurred in 79.4% ($\text{58}\text{73}$) of the heifers inseminated in the five days after the second injection. Occurrence of estrus and conception was no different in treated heifers after five days of the insemination period than in nontreated heifers after 21 days of the insemination period, where 94.9% ($\text{37}\text{39}$) were observed in estrus and 83.8% ($\text{31}\text{37}$) conceived. Overall percent conception for a 55-day insemination period was 89.7 ($\text{70}\text{78}$) for treated and 87.2 ($\text{34}\text{39}$) for nontreated heifers. Day of cycle at first or second injection did not affect conception after the second injection. Some signs of estrus were observed in 11 of the 16 heifers injected before Day 5.A second trial to determine if alfaprostol induced luteolysis early in the cycle was conducted. Twenty purebred Angus, Hereford, or Simmental heifers received either one or two injections of alfaprostol on either Day 1, 2, 3, or 4. Only five heifers showed any signs of estrus, and the three that were inseminated did not conceive. Subsequent cycle length indicated that luteolysis occurred in only one heifer.Data suggest that alfaprostol is an effective luteolytic agent in cyclic beef heifers after Day 4 and that two injections 12 days apart will effectively synchronize estrus in heifers when distributed throughout the cycle at the first injection without affecting conception rate.     

Loose binding of testicular mitochondrial ATPase to the inner membrane

Rat testis mitochondrial ATPase was not inhibited by oligomycin at pH 7.5. It was inhibited only at higher alkaline pH's, and showed a lower sensitivity both to oligomycin and N,N′-dicyclohexylcarbodiimide and a higher one to efrapeptin. In submitochondrial particles, testis ATPase was only slightly inhibited by oligomycin, ossamycin, and efrapeptin. The possibility of a loose binding of F₁ to the membrane was supported by its recovery from the supernatant of the submitochondrial particles. Furthermore, by electron microscopy, after hypoosmotic shock and negative staining of the mitochondrial preparations, most of the inner mitochondrial membranes showed only a few “knobs” or none at all. The capacity of the testis mitochondrial preparation to produce ATP was tested and compared to that from liver. ATP synthetase/ATPase activity ratio was $\text{30}\text{1}$ in liver mitochondria, whereas in the testis it was $\text{3}\text{1}$. In spite of this large difference, at least part of the testis ATPase must be firmly bound to the membrane, since it is able to form ATP. The rest seems to be loosely bound and its functional significance is still unknown.     

Effect of phenobarbital administration to rats on the level of the in vitro synthesis of cytochrome P-450 directed by total rat liver RNA.

Total liver RNA has been isolated from male rats at different time points subsequent to a single injection of phenobarbital, and the level of cytochrome P-450 synthesis directed by these RNA preparations in a cell-free translation system has been determined. It is observed that the maximum $\text{in vitro}$ synthesis of cytochrome P-450 occurs at 16 hours (3-fold above uninduced level) which is approximately 30 hours prior to the maximum induction of spectrophotometrically detectable cytochrome P-450 measured in liver homogenates. Thus, while cytochrome P-450 mRNA is involved in the induction process, its synthesis does not appear to be rate limiting. In addition, phenobarbital induced cytochrome P-450 is not synthesized $\text{in vitro}$ in a form larger than that isolated from endoplasmic reticulum, but rather is also found to have a molecular weight of 50,000.     

Intraperitoneal injection of zymosan in mice induces pain,inflammation and the synthesis of peptidoleukotrienes and prostaglandin E2

Intraperitoneal injection of zymosan in mice induced rapid extravasation and accumulation of plasma proteins in the peritoneal cavity. Neutrophils began to appear in the peritoneal cavity after a lag period of approximately 3 hours. The injected mice exhibited a pain response (writhing) during the first 30 minutes after injection, but writhing ceased before protein or cell accumulation had reached maximum levels. The injection of zymosan induced synthesis of PGE₂ (measured by RIA) which reached maximum levels of 30 minutes, then declined slowly. Peptido-leukotriene levels (detected by bioassay, RIA and HPLC) increased rapidly after injection, reached a peak within an hour of injection and declined to undetectable levels within 4 hours. The early peptido-LT was predominantly LTC₄, while later, LTE₄ was the major component. LTD₄ levels remained low throughout and no LTB₄ was detected at any time. Indomethacin treatment elevated levels of peptido-LTs, recued PGE₂ levels and inhibited writhing. Phenidone reduced peptido-LT levels. $\text{In}$$\text{vitro}$ studies demonstrated that zymosan stimulates LTC4 synthesis by peritoneal cells whereas LTE₄, LTD₄, LTB₄ or monoHETES were not detectable (using HPLC methods). The source of enzymes responsible for the $\text{in}$$\text{vivo}$ metabolism of LTC₄ to LTD₄ and LTE₄ could not be identified.     

Effect of protected protein supplements on some testicular traits in Brahman cross bulls

Differences in a number of testicular traits were examined in 12 Brahman cross (F₂ generation $\text{1}\text{2}$ and $\text{3}\text{4}$ BX) bulls fed either poor-quality native pasture (NP) hay or NP hay with a protected protein supplement. Supplementation for 60 days significantly (P < 0.05) increased roughage dry matter intake (7.7 $\text{v}$ 5.6 kg/head/day), enabling maintenance of liveweight, whereas control animals lost 40 kg. There were significant (P < 0.05) decreases in scrotal circumference (1.5cm) and testicular consistency (0.8 score) in the control group, in which testes weights at slaughter were significantly (P < 0.05) less (373 g $\text{v}$ 459 g), with corresponding lower epididymal weights (37.4 g $\text{v}$ 43.5 g). Estimates of daily sperm production per gram (DSPG) were similar for both groups, and testis daily sperm production (DSP) was somewhat but not significantly (P>0.05) lower in the control group (4.3 × 10⁹$\text{v}$ 6.0 × 10⁹) as a result of lower testis weights. Total epididymal sperm storage capacity was also lower in control bulls (17.2 × 10⁹$\text{v}$ 27.0 × 10⁹), but only significantly (P < 0.05) in relation to cauda sperm reserves (8.5 × 10⁹$\text{v}$ 13.6 × 10⁹). Luteinizing hormone (LH) and testosterone responses to gonadotrophin releasing hormone (GnRH) treatment were similar for both groups, although LH responses to GnRH were greater in $\text{1}\text{2}$ BX than in $\text{3}\text{4}$ BX bulls.     

Response of two cows with mummified fetus to treatment with cloprostenol (ICI 80, 996)

Two cases of bovine fetal mummification were treated with a single intramuscular injection of 500 μg cloprostenol. Plasma progesterone levels fell rapidly within 24 hours following injection from a mean level of 10.45 ng/ml to 1.05 ng/ml. Extradiol-17β levels were variable and low ($\text{x}\text{= 3.04 pg/ml}$) during the 120-hour postinjection sampling period. The mummies were delivered at 104 and 120 hours postinjection. Both were expelled into the vagina and traction was required to complete removal from the genital tract. No adverse reactions which could be attributed to $\text{cloprostenol}$ treatment were observed.     

Comparative effects of ftorafur and 5-fluorouracil on DNA synthesis in rat small intestine.

The effect of equimolar doses of ftorafur (100 mg/kg) and 5-fluorouracil (65 mg/kg) on the $\text{in}$$\text{vivo}$ incorporation of deoxyuridine and thymidine into the DNA of rat small intestine was studied. 5-fluorouracil produced a greater than 90% inhibition of deoxyuridine incorporation within one hour after injection. This degree of inhibition was sustained for at least 12 hours. Deoxyuridine incorporation was inhibited by 30 to 65% during the initial six hours after the injection of ftorafur. By 12 hours the rate of incorporation had returned to 66% of the control value. Neither drug inhibited thymidine incorporation into DNA. A study of the metabolic disposition of radioactively labeled ftorafur and 5-fluorouracil showed that the latter drug was more rapidly and completely converted to fluorouracil-containing nucleotides in the small intestine. The possible relationship between these findings and the reported differences in the toxicity of the two drugs is discussed.     

Naloxone antagonism of phenoxybenzamine antinociception in the mouse tail stimulation test.

Phenoxybenzamine was antinociceptive in the mouse tail electrical stimulation assay (ED50, 36.8 mg/kg) with a peak effect at $\text{2}\text{1}\text{2}$ hours after subcutaneous injection. Naloxone antagonized this antinociception action of phenoxybenzamine in a dose-related manner. Dose-ratio analysis of naloxone's antagonism of phenoxybenzamine antinociception gave a pA₂ value of 6.15, similar to that found for the benzomorphinan mixed agonist-antagonists. This is in agreement with the sodium response ratio found for phenoxybenzamine, 4.3, in $\text{in vitro}$ assays of phenoxybenzamine inhibition of ³H-naloxone binding to mouse brain homogenate (5). These findings suggest that phenoxybenzamine binds to the opiate receptor both $\text{in vivo}$ as well as $\text{in vitro}$ in a manner similar to the mixed agonist-antagonists.     

Aminoglutethimide inhibits steroidogenesis in the rat testis

Aminoglutethimide inhibits steroid formation in the adrenal and in the ovary but it is not established whether it has a similar effect in the testis. Adult male rats were injected with 10 mg or 15 mg amino-gluthethimide phosphate (AGP) per 100 g body weight twice daily for 3$\text{1}\text{2}$ days and killed 3 hrs or 5 hrs after the last injection. Treatment with either dose of AGP caused a precipitous decrease in plasma testosterone levels. Administration of the higher dose of AGP also caused a decrease in seminal vesicles weight and an increase in the concentration of esterified cholesterol in the testes. The results indicate that aminoglutethimide inhibits testicular steroidogenesis.     

The stereospecificity of D-glucose-6-phosphate: 1L-myo-inositol-1-phosphate cycloaldolase on the hydrogen atoms at C-6

D-Glucose-6-phosphate: 1L-$\text{myo}$-Inositol-1-phosphate cycloaldolase from rat testis or mammary gland removed stereospecifically the pro-S hydrogen atom at C-6 from D-glucose-6-phosphate. The pro-R hydrogen at C-6 remained in the product, 1L-$\text{myo}$-Inositol-1-phosphate and evidence is given that it is the hydrogen at C-1 of 1L-$\text{myo}$-Inositol-1-phosphate. The possible mechanism of cyclization is discussed.     

Investigations into the potential for embryo transfer from Brucellaabortus infected cows without transmission of infection

Thirty-six attempts were made to isolate $\text{Brucella}$$\text{abortus}$ from the uterine flushings of culture positive and serologic reactor cows. Sixteen of these attempts were made after cows had been programmed for superovulation and a simultaneous attempt was made to recover eggs. Udder secretion samples for culture and blood for serology were collected at the time of the flushing procedure. In addition, a field isolate of $\text{Brucella}$$\text{abortus}$ suspended in three different solutions (one commonly used for nonsurgical embryo retrieval) was quantitated at various intervals up to 24 hours.Brucellae were not cultured from any of the uterine flushings although it was demonstrated that the organisms would remain viable in the media used for up to 24 hours. Udder secretions contained brucella at the time of flushing in 17 of the 36 attempts. Results indicated that transfer from infected donors might be achieved without transfer of infection. It is cautioned that final evidence of success would have to come after recipients had undergone serologic and cultural surveillance through their gestation period.     

Lifetime of microsomal cytochrome P-450 and steroidogenic enzymes in rat testis as influenced by human chorionic gonadotrophin

The lifetime of different microsomal steroidogenic enzymes and the cytochrome components of the NADPH-cytochrome P-450 pathway have been determined in rat testis by measuring their decrease logarithmically after hypophysectomy. Although both cytochrome P-450 and 17α-hydroxylase show biphasic decay curves, the first decay curve contains 89–94% of the cytochrome P-450 and 17α-hydroxylase levels. Steroidogenic enzymes which are located mainly in the leydig cells, decay much faster than microsomal protein, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 12}$ days, which represents mainly decay of tubular protein. The similarity between the major half-life of cytochrome P-450, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 3.3}$ days, 17α-hydroxylase, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 2.3}$ days and the C₁₇–C₂₀ lyase, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 3.4}$ days and the uniformity of their response to human chorionic gonadotrophin (HCG) provides additional evidence that these two steroidogenic enzymes require cytochrome P-450. Both the 17α-hydroxylase and the C₁₇–C₂₀ lyase were shown to have a constant activity per nmole of cytochrome P-450 during a sixfold change in the level of cytochrome P-450 brought about by HCG treatment of rats with intact pituitaries. The decay of 17β-hydroxysteroid dehydrogenase, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4.5}$ days, was slower than P-450 dependent enzymes. Rats with intact pituitaries are not under maximal stimulation by endogenous LH because addition of HCG increases the levels of microsomal and mitochondrial cytochrome P-450 220 and 1620%, respectively. The rates of synthesis during the increase from one cytochrome P-450 level to another was calculated at $\text{0.118}\text{2}$ testes/day for microsomal cytochrome P-450 and 0.10 nmoles/2 testes/day for mitochondrial cytochrome P-450. Treatment of hypophysectomized rats with HCG results in large increases of cytochrome P-450, 17α-hydroxylase, C₁₇–C₂₀ lyase and 5α-reductase, but not cytochrome b₅, microsomal protein, 7α-hydroxylase, or the 17β-hydroxysteroid dehydrogenase. While it is clear that the two cytochrome P-450 dependent hydroxylases involved in steroidogenesis and the 5α-reductase are under the control of gonadotrophin, it is not clear how 17β-hydroxysteroid dehydrogenase levels are maintained or in what manner the 5α-reductase level is controlled in mature animals.     

Uterine cyclic AMP formation: biphasic effect of estrogen on catecholamine sensitivity.

Female, ovariectomized rats were treated with estradiol and then, after various time periods, given an intravenous injection of isoproterenol or epinephrine. 30 seconds later uteri were frozen $\text{in}$$\text{situ}$ and assayed for cyclic AMP and glycogen phosphorylase. The cyclic AMP response to catecholamines was significantly depressed as early as 30 minutes after estrogen and at 6, 12 and 24 hours was 50% of that in non-estrogen-treated controls. Catecholamine-induced glycogen phosphorylase activation was unchanged until 24 hours after estrogen when it was significantly increased over controls. At 48 hours of estrogen both the cyclic AMP and phosphorylase responses to catecholamines were greater than controls. Estrogen regulates uterine β-adrenergic sensitivity but the time courses of estrogen effects on the cyclic AMP and glycogen phosphorylase response changes are different. Catecholamine-induced uterine cyclic AMP formation is biphasic: suppression during the first 24 hours of estrogen followed by recovery and finally augmentation by 48 hours. Catecholamine-induced glycogen phosphorylase activation shows only augmentation after 24–48 hours of estrogen. It is concluded that estrogen has independent effects on the β-adrenergic-glycogen phosphorylase activation pathway at two different points; one prior to cyclic AMP formation and another after cyclic AMP formation.     

The inhibition of the conversion of testosterone into 5α-dihydrotestosterone in the reproductive organs of the male rat

The inhibition of testosterone 5α-reductase activity by 3-oxo-4-androstene-17β-carboxylic acid in the male reproductive organs of the rat was demonstrated $\text{in vitro}$. The medium for incubation of caput epididymis showed the highest concentration of 5α-dihydrotestosterone (5α-DHT) whereas the highest concentration of testosterone (T) was recorded in medium for incubation of decapsulated testis after two hours of incubation. The 3-oxo-4-androstene-17β-carboxylic acid (1.58 × 10^?5M) inhibited the conversion of T to 5α-DHT in all the organs tested (testis, caput and cauda epididymis and ventral prostate) under identical incubation conditions.     

Effects of estrogens on aryl hydrocarbon hydroxylase mediated binding of benzo(a) pyrene metabolites to DNA in vitro

$\text{In vivo}$ and $\text{in vitro}$ studies were carried out to determine the effects of estradiol and other steroid hormones on aryl hydrocarbon hydroxylase-mediated binding of benzo(a)pyrene metabolites to DNA. Injection of female $\text{C57}\text{B1}\text{6J}$ mice with 0.2 mg or 2 mg of estradiol 24 hours prior to, during and 24 hours after injection of 3-methylcholanthrene resulted in a significant decrease in the capacity of hepatic microsomes from these animals to mediate the binding of benzo(a)pyrene metabolites to DNA when compared to microsomes from animals receiving 3 methylcholanthrene treatment only. Binding of benzo(a) pyrene metabolites was inhibited between 22 and 50%, depending on the dose of estradiol used. The enzyme and cytochrome components of the aryl hydrocarbon hydroxylase multienzymic complex were not affected by either estradiol treatment. The data suggests that estradiol inhibits aryl hydrocarbon hydroxylase mediated binding of benzo(a)pyrene metabolites to DNA by activity as a non-competitive inhibitor of aryl hydrocarbon hydroxylase activity.     

Increased acridine orange uptake and lysosomal enzyme levels in rat liver after steroid administration

A steroid which stabilizes lysosomes $\text{in vitro}$ and a pyrogenic steroid which labilizes lysosomes $\text{in vitro}$ were compared with respect to their ability to modify lysosomal uptake and lysosomal enzyme levels $\text{in vivo}$. Cortisone acetate increased the uptake of acridine orange by rat liver lysosomes when the dye was administered by intrathoracic injection. The steroid increased and accelerated the uptake of acridine orange so that, in liver lysosomes from treated rats, the maximum uptake was double that of controls and was reached at 2h, whereas in controls the maximum uptake was at 4h after the injection of the dye. This large elevation of uptake is specific to the lysosomal fraction and is not seen in other subcellular fractions of rat liver. The specific activities of a lysosomal enzyme β-N-acetylglucosaminidase were increased in lysosomal fractions from cortisone acetate-treated rats. Etiocholanolone, a steroid which labilizes lysosome $\text{in vitro}$, similarly accelerated and increased acridine orange uptake by lysosomes but had little effect on lysosomal β-N-acetylglucosaminidase levels. Thus the ability of steroids to stabilize or labilize lysosomes $\text{in vitro}$ does not correlate with their effect on lysosomal uptake of injected substances $\text{in vivo}$, or with their ability to induce increased specific activities of lysosomal enzymes.     

Biphasic effect of extracts of Lithospermum ruderale on the uptake of 32P by testes and thyroids of cockerels.

A polyphenolic fraction of the plant $\text{Lithospermum ruderale}$ was injected into White Leghorn cockerels at various time intervals and the effect on testes and thyroids was measured by the uptake of ³²P. The ³²P uptake 8 hours after lithosperm² injection was markedly depressed and 12 hours after administration was notably increased. It is suggested that the hypothalamic regulation of the testes and thyroids is altered by lithosperm interference of the hypothalamic feedback system and/or the binding of receptor sites in the two glands.     

Nuclear transfer studies of amoeba proteus after the inhibition of cell division by injection of non-homologous cytoplasm

Following the injection of the post-microsomal supernatant fraction of $\text{Amoeba discoides}$ cytoplasm into $\text{A.proteus}$, cell division is inhibited in at least 90% of the recipient cells. Nuclear transfers were performed to determine the site of inhibition in these injected cells. When nuclei from injected, inhibited cells one day after injection were transferred into new $\text{A.proteus}$ cytoplasms, 62% of the transfers divided. This ability to promote division declined with the length of time between transfer and the original_ injection. However, when nuclei from $\text{A.proteus}$ were transferred into injected, inhibited cytoplasms, only a low number of cells divided, comparable to the number obtained after the injection operation only, namely less than 10%. Thus although many nuclei could recover from inhibition, it was not possible to restore the cytoplasms of inhibited cells by new nuclei.