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Eukaryotic DNA methylation occurs exclusively at the 5'-position of cytosine and has been implicated in the regulation of gene expression. Using high-performance liquid chromatography, the methylation of testis DNA during its development, in different cell populations and during regulation by gonadotropic hormones, were studied. The 5-mC content of testis DNA increased significantly from days 30 to days 150, while in 2-yr-old testis 5-mC content decreased significantly. Among various populations of testicular cells, pachytene spermatocyte DNA contained a significantly high amount of 5-mC when compared to spermatogonia, spermatids and mature sperm DNA However, the 5-mC content of elongated spermadids was significantly less when compared to the above four fractions. Administration of follicle stimulating hormone to immature rats caused hypomethylation of seminiferous tubular DNA while luteinizing hormone caused similar effects in Leydig cells. These results indicate that in testis, DNA methylation is differentially regulated during development and is controlled by gonadotropic hormones.

The effect of various hormones on the levels of poly(A) polymerase in the ventral prostate of castrated rats was investigated. It was observed that this enzyme is specifically induced by androgens; progresterone and estradiol-17β did not cause stimulation of poly(A) polymerase activity. Dihydro-testosterone-induced poly(A) polymerase was inhibited by cordycepin, actinomycin-D and cycloheximide, which indicates that the genetic transcription leading to the enzyme poly(A) polymerase is regulated by androgens in the ventral prostate.

The epididymis and vas deferens from healhy men of various ages who died in road accidents were analyzed for the levels of total lipids, phospholipids, sialic acid, RNA, DNA and protein. The concentration of total lipids and total phospholipid phosphorus showed little variation in the caput and cauda epididymides of young and old age groups. Total PLP was higher in the cauda than in the caput or corpus epididymides of both age groups. The major phospholipids in all segments of epididymis and vas deferens were phosphatidyl choline and phosphatidyl ethanolamine. Concentration of sialic acid was similar in all the regions of the epididymis but was lower in the vas deferens. DNA and RNA were maximal in the cauda epididymides while protein was nearly the same in all the regions of the epididymis. The significance of these changes is discussed.

The prostate glands of rats, mice, guinea pigs and hamsters were found to be a rich source of enzymes catalyzing the Mn2+-dependent transfer of galactose from UDP-galactose to glycoprotein acceptors such as ovomucoid and ovalbumin. The rat ventral prostate was also very active in promoting transfer of fucose from GDP-fucose to ovomucoid. The prostatic enzymes promoting both galactosyl and fucosyl transfers to glycoproteins were very largely membranebound, and were markedly activated by the non-ionic detergent Triton X-100. Castration of adult male rats resulted in a many-fold and roughly parallel decline in both glycosyltransferase activities over a period of two weeks, which was reversed by subsequent daily treatment with testosterone for 8 days. The very low galactosyltransferase of the ventral prostate of hypophysectomized rats was markedly enhanced by testosterone administration, whereas prolactin alone or in combination with androgen had no significant effect.

The increased rate of protein synthesis induced in the prostate and seminal vesicle by the injection of dihydrotestosterone is preceded by an increased synthesis of RNA and is blocked by inhibitors such as actinomycin D. This increased protein synthesis can also be induced by administering into the lumen of the seminal vesicle RNA extracted from the seminal vesicle of an androgen-treated rat. When the RNA is placed on a Sepharose 4B polyuridylic acid column or a cellulose polythymidylic acid column, a small fraction of the RNA is tightly bound. The unbound RNA is eluted with buffer and subsequently the bound RNA is eluted with a more alkaline buffer. The injection of dihydrotestosterone in vivo greatly increases the incorporation of cytidine into this bound RNA. The bound poly (A)-rich RNA, presumably polyadenylic acid RNA, from the seminal vesicles of androgen treated males resulted in increased protein synthesis when instilled into the seminal vesicle of castrate males. In contrast, the unbound RNA from the seminal vesicle was ineffective when instilled into the seminal vesicles of castrate rats.

Accessory reproductive organs of male rat were found to contain high activities of glucosamine-6-phosphate synthase (glucosaminephosphate isomerase (glutamine-forming), EC 5.3.1.19). Castration caused drastic reduction (75%) in the enzyme activity of ventral prostate. Testosterone propionate administration restored the enzyme activity while cortisol and estradiol-17β did not cause any effect. Cycloheximide blocked the stimulation caused by testosterone.

UDP-galactose: glycoprotein galactosyltransferase and GDP-focuse: glycoprotein fucosyltransferase activities were found to be exceptionally high in homogenates of rat ventral prostate and certain other male secondary sexual organs. Nearly all of the intracellular forms of these glycosyltransferases in prostate were associated with readily sedimentable membranous structures and were maximally active only in the presence of non-ionic detergents. Rat and human prostatic secretions, and especially human seminal plasma, also are strikingly rich, in comparison with blood serum, in glycoprotein galactosyl-transferases that are present in these fluids in soluble forms that are not activated by detergents. Very little galactosyltransferase is associated with spermatozoa. The properties of prostatic fluid and seminal plasma galactosyl-transferases are very similar to those of the enzyme present in mammary gland and milk, although functional modifier proteins resembling α-lactalbumin appear to be absent from male genital glands and their fluids. The glycoprotein galactosyltransferase and fucosyltransferase activities of rat prostate decline fairly slowly after orchiectomy and are gradually enhanced in castrates by androgen treatment in a manner which suggests that their levels vary coordinately with elaboration of endoplasmic reticulum and Golgi membranes in this tissue. Prolactin does not affect prostatic galactosyltransferase in hypophysectomized animals or its enhancement by androgens.

Summary Recently gonadotropin releasing hormone (GnRH) and its agonistic analogs were demonstrated to have some direct actions in accessory reproductive organs. In our study the effects of GnRH and its analogs on some steroid hormone induced responses were investigated. GnRH and its analogs inhibited estradiol induced ornithine decarboxylase (ODC) and glucosamine-6-phosphate synthase activities in the uterus of rat. These enzymes which are markers for cell proliferation are regulatory enzymes in the biosynthetic pathways of polyamines and glycoproteins, respectively. Similarly, GnRH and its analogs also inhibited testosterone stimulated ODC activity in ventral prostate of rat. In addition, GnRH analog inhibited incorporation of radioactive precursors into RNA and protein induced by estradiol in uterus or dihydrotestosterone (DHT) in ventral prostate. In an effort to elucidate the mechanism of action of GnRH in uterus, it was found that GnRH analog treatment does not alter the estradiol receptor content in vivo. Also, GnRH does not show any effect on radioactive estradiol binding to its receptor in vitro. Hence, the inhibitory actions of GnRH in uterus may not involve estradiol receptors. However, GnRH analogs were found to have post-transcriptional effects. It was observed that DHT induced poly(A) polymerase activity in ventral prostate and estradiol induced poly(A) polymerase activity in uterus were inhibited by GnRH analog treatment. It was further observed that GnRH inhibited incorporation of [3H]uridine into poly(A)+ RNA of ventral prostate. This indicates that the inhibitory effects of GnRH involve post-transcriptional mechanisms.

The activity of ornithine decarboxylase (ODC) was found to increase in the testis of immature rats following intratesticular injection with luteinizing hormone releasing hormone (LHRH). Maximal stimulation of ODC activity occurred with 1 μg of the hormone at 2 h. The enzyme activity returned to control levels at 4 h. The minimal effective dose was found to be 0.1 μg per testis. The stimulating effect of LHRH was confined to Leydig cells alone. The seminiferous tubules did not show any change in ODC activity following LHRH treatment. These results show that LHRH acts directly on the testis and influences the levels of ODC in the Leydig cells of rat.

The sexual segment represents the hypertrophied secondary and tertiary collecting duct tubules of the kidney in lizards and snakes. This organ is present only in the males and is secretory when the testes are spermatogenically active; it is regressed during testicular quiescence or following castration, and is stimulated to a secretory state by androgens. Secretions of the sexual segment are rich in phospholipids and contain acid phosphatase. The levels of the lipids of the sexual segment vary during different months of the year, and it appears that lipids of the kidney and liver are utilised by the sexual segment for resynthesis of its lipids. Incorporation of radioactive inorganic orthophosphate is differential and markedly higher in the sexual segment than in the kidney during different months of the year and is apparently regulated by changing patterns of testicular endocrine activity. Androgens stimulate selectively synthesis of lipid-rich secretions in the sexual segment. These secretions are mixed with sperm and are released into the female genital tract during copulation. It is presumed that the secretions of the sexual segment are utilised by the spermatozoa in the female genital tract. The function and homologies of the sexual segment are discussed. It is suggested that the sexual segment may be homologous with the seminal vesicles of mammals.

1. 1. The incorporation of radioactive inorganic orthophosphate into the sexual segment and kidney phospholipids was studied at various time intervals in sexually active and regressing lizards and following castration and testosterone administration. 2. 2. The incorporation of the label into the total phospholipids of the sexual segment as well as into the kidney is gradual and reaches a peak at 12 hr followed by a gradual decrease at later time intervals. 3. 3. Phosphatidic acid + polyglycerol phosphatide and phosphatidyl inositol+ phosphatidyl serine fractions incorporate the label rapidly during the first 30 min, while phosphatidyl choline and phosphatidyl ethanolamine fractions show heavy label at later intervals. The pattern of incorporation is similar in the phospholipids of the sexual segment and kidney, but the intensity of incorporation in the sexual segment is higher during the active season than in the regresssing season. 4. 4. Rapid early incorporation of the precursor into the phosphatidic acid fraction suggests its role in the biosynthesis of other complex lipids. The differential stimulation of the uptake of the label by sexual segment phospholipids is clear evidence of the selective action of testosterone on this target organ.

One or two silastic capsules filled with approximately 12 mg of testosterone were implanted subcutaneously in adult male rats. The animals were autopsied one to two months after implantation of the capsules. Mating behaviour, fertility, biochemistry of the accessory reproductive organs and testicular histology were studied. In rats with one implant of testosterone for one or two months and with two implants of testosterone for one month, the epididymal sperms were motile and after successful matings, the females littered normally. In rats bearing two silastic capsules for two months, spermatogenesis was inhibited at the spermatocyte stage and the epididymal smears were devoid of sperms; Leydig cells were atrophic and matings were infertile. Testicular histology, motility of epididymal sperms and fertility were restored to normalcy one month after removal of the capsules. In all treated groups, weights of the accessory reproductive organs and the fructose content of coagulating glands were more than those of controls. This study suggests that low doses of testosterone released through silastic capsules can be used for control of fertility in males.

The action of gonadotropins (FSH, LH, PMS, and HCG) and testosterone on the initiation of spermatogenesis was studied in the Indian House Lizard Hemidactylus during the non-breeding season after pituitary ablation. Both ovine FSH and PMS initiated spermatogenesis leading to the formation of mature spermatozoa after 45 days while LH, HCG, or TP had no stimulatory effect on the testis. Synergistic action was not observed when FSH or PMS was injected in combination with LH, TP, or HCG as evidenced by the germ cell counts in the seminiferous tubules. The androgen-dependent sexual segment of the kidney was also fully developed in the animals treated with FSH or PMS. These results indicate that the initiation of gametogenic and androgenic functions in the testis of the Indian house lizard are dependent on an FSH-like protein.

Testosterone induced increase in ornithine decarboxylase (ODC) activity was inhibited by simultaneous treatment with gonadotropin releasing hormone (GnRH) and its analogue in the ventral prostate of rat. Inhibition of 3H-uridine, 3H-phenylalanine and 3H-leucine incorporation into TCA precipitable material was also inhibited by GnRH in the dihydrotestosterone (DHT) treated animals. These studies further confirm that GnRH acts directly on ventral prostate and causes inhibitory effects.

The effect of α and β adrenergic receptor blockers on epinephrine and gonadotropic hormone induced ornithine decarboxylase (ODC) activity in the testis of immature rats was studied. Intratesticular injection with phenoxybenzamine at 15 min before treatment with epinephrine or gonadotropic hormones blocked ODC activity. Similar injection with propranolol or practolol had no effect on ODC activity. These results show that α adrenergic receptors are involved in the action of epinephrine and gonadotropic hormones in the testis.

Two hours after the intratesticular injection of FSH, hCG or cyclic AMP, the incorporation of labeled uridine into poly(A)-rich RNA was increased. Pretreatment with actinomycin D inhibited the incorporation of uridine into mRNA. After the seminiferous tubules and interstitial cells were separated by treatment with collagenase, FSH treatment increased mRNA synthesis only in the tubules whereas hCG stimulated mRNA synthesis only in the interstitial cells. Cyclic AMP increased the synthesis of mRNA in both interstitial cells and seminiferous tubules. These results suggest a differential action of the two gonadotropic hormones in the cells of the testis; both effects appear to be mediated by cyclic AMP.

The mechanism of extra pituitary inhibitory action of gonadotropin releasing hormone (GnRH) was investigated. Simultaneous injection of GnRH caused dose dependent inhibition of dihydrotestosterone (DHT) induced poly(A) polymerase in the ventral prostate of rat. In addition injection of GnRH to DHT treated animals caused reduced incorporation of 3H-uridine into poly(A)+ mRNA. Since poly(A) segment is known to help in translation of mRNA, it is possible that the inhibitory effect of GnRH is due to the inhibition of polyadenylation of mRNA.

In [I] evidence was presented showing hyper- stimulation of ornithine decarboxylase (ODC) activ- ity following combined treatment with saturating levels of prostaglandin E2 (PGE,) and follicle-stimu- lating hormone (FSH) or PGEs and luteinizing hor- mone (LH) in the testis of immature rat [l]. It was presumed that PGE?, FSH and LH were probably acting on different cell types and were causing a cumulative effect on ODC. Hence experiments were designed to study the effect of prosta~~dins, FSH and LH on ODC activity in the isolated Leydig cells and seminiferous tubules of immature rat testis. The evidence presented in this study shows that PGEz and PGFZa stimulate ODC levels in both Leydig cells and seminiferous tubules. 2. Materials and methods Ovine luteinizing hormone (NIH-~-S-20), ovine follicle stimulating hormone (NIH-FSH-S-12) and prosta~~dins were obtained from the National Institutes of Health and the Upjohn Company, re- spectively. Ornithine, pyridoxal phosphate, dithio- threitol, reduced glutathione, collagenase and bovine serum albumin were purchased from Sigma, St Louis. D,L-[1-‘4C] ornithine monochloride (58 mCi/mmol) was purchased from the Radiochemical Centre, Amersham. AU other chemicals were of analytical grade and were obtained locally. Stock solutions of prostaglandins were prepared in

Summary Ornithine decarboxylase activity in the testes of immature rats was measured 1, 2, 4 and 6 hours after the injection of FSH, LH or cAMP. Both gonadotropic hormones stimulated the synthesis of the enzyme. Maximal activity was observed 4 hours after the injection of the hormones. cAMP increased the activity of the enzyme during the first two hours after the injection. Pretreatment with cycloheximide inhibited the synthesis of the enzyme in all groups of animals.

Injection of norepinephrine (NE) at a dose of 10 μg per testis caused the testis refractory in terms of ornithine decarboxylase (ODC) activity at 24 h. This desensitization was found to be both time and dose dependent. Injection with follicle stimulating hormone, luteinizing hormone, prostaglandin F 2α , cyclic AMP or epinephrine to norepinephrine desensitized testis caused stimulation of ODC activity. This indicates that the refractoriness caused by norepinephrine is specific to this agent alone.

Intraperitoneal injection of alpha-difluoromethylornithine (DFMO) at a dose of 200 mg/kg, twice a day, during days 4-7 of pregnancy caused inhibition of embryogenesis in rats. These animals did not deliver any pups at term. However, there was no effect of DFMO on pregnant rats following treatment during day 1-3 of pregnancy. It was observed that DFMO completely abolished ornithine decarboxylase levels in the pregnant uterus. The action of DFMO appears to be due to the inhibition of putrescine and polyamines during a critical period of early embryo development. We suggest that this drug may act as a postcoitally effective antifertility agent in females.

Injection of estradiol to ovariectomised rats caused significant increase in the activity of glucosamine 6-phosphate synthase of uterus. Progesterone did not cause any increase in the activity of the enzyme; however, it antagomised the effect of estradiol. It was observed that the enzyme is predominantly localised in the endometrium of uterus.

The increased rate of protein synthesis induced in the prostate and seminal vesicle by the injection of dihydrotestosterone is preceded by an increased synthesis of RNA and is blocked by inhibitors such as actinomycin D. This increased protein synthesis can also be induced by administering into the lumen of the seminal vesicle RNA extracted from the seminal vesicle of an androgen-treated rat. When the RNA is placed on a Sepharose 4B polyuridylic acid column or a cellulose polythymidylic acid column, a small fraction of the RNA is tightly bound. The unbound RNA is eluted with buffer and subsequently the bound RNA is eluted with a more alkaline buffer. The injection of dihydrotestosterone in vivo greatly increases the incorporation of cytidine into this bound RNA. The bound poly (A)-rich RNA, presumably polyadenylic acid RNA, from the seminal vesicles of androgen treated males resulted in increased protein synthesis when instilled into the seminal vesicle of castrate males. In contrast, the unbound RNA from the seminal vesicle was ineffective when instilled into the seminal vesicles of castrate rats.

Intratesticular injection of epinephrine and norepinephrine caused stimulation of ornithine decarboxylase (ODC) activity in the testis of immature rat. The effect of epinephrine was time and dose dependant. The minimal effective dose for epinephrine was found to be 100 pg and optimal stimulation was observed with 500 ng of the drug. Maximal stimulation of ODC occurred at 2 h after the treatment and reduced significantly at 4 h reaching to control levels at 6 h. Simultaneous injection of epinephrine with dibutyryl cAMP, luteinizing hormone, follicle stimulating hormone or prostaglandin E 2 caused additional stimulation of the enzyme activity. Injection of epinephrine to norepinephrine treated animals caused additional effect. Both epinephrine and norepinephrine were found to stimulate the enzyme activity in leydig cell and seminiferous tubule fractions. These results suggest that catecholamines are also involved in the regulation of ODC activity in the testis of rat.

Estradiol induced increase in ornithine decarboxylase (ODC) and glucosamine-6-phosphate synthase activities of rat uterus were inhibited by simultaneous treatment with gonadotropin releasing hormone (GnRH) or its agonists. The direct inhibitory effect of GnRH analogs was found to be dose dependent. It was observed that a higher dose of GnRH analog was needed to cause inhibition of glucosamine-6-phosphate synthase when compared to ODC activity. The inhibitory effect of GnRH was not observed if its injection was delayed following estradiol treatment. These results show that the extra-pituitary inhibitory effects of GnRH involves enzymes associated with cell proliferation.

Changes in the different classes of lipids in the testis of the Indian house lizard during different phases of spermatogenic activity and following hypophysectomy were investigated. The concentration of total lipid, total glyceride, and total cholesterol of the testis is low during the active season (November–April) and reaches a peak when the testes are regressed (June–July). Total phospholipid is high during the spermatogenically active season and is low during the regressed season. Among the individual neutral lipids triglyceride is the major fraction while phosphatidylcholine and phosphatidylethanolamine are the major fractions of phospholipid. Hypophysectomy causes accumulation of total lipid, total glyceride, and total cholesterol; total phospholipid decreases significantly following hypophysectomy. Changes in the lipid classes following hypophysectomy are comparable to those in the testis of the intact lizards during the sexually regressed season. It is presumed that the accumulation of glyceride during the nonbreeding season and following hypophysectomy may be due to the inhibition of the enzyme phosphoryl choline transferase.

Seven healthy male volunteers were injected intramuscularly with 25 mg/ day testosterone propionate for sixty days. Sperm counts and volume of semen were recorded at 15 day intervals. The subjects became azoospermic 60 days after the treatment. Sperm counts were restored to pre-treatment levels 150 days after the withdrawal of the treatment. During the period of testosterone administration and recovery, the quantity of semen did not show any variations. Libido and potentia of the subjects was also normal during the experimental period. This study suggests the possibility of using testosterone as an antifertility agent in human males.

1. 1. Various lipid classes of the sexual segment, kidney and liver of the Indian house lizard were studied during different periods of the year. 2. 2. Incorporation of radioactive inorganic orthophosphate into total phospholipid of the sexual segment and kidney was also investigated during different phases of reproductive activity. 3. 3. Lipids of the kidney and liver are presumably utilized by the sexual segment for the synthesis of its lipids during its development in December and January when the animals hibernate. 4. 4. Selective enhancement of phospholipid synthesis by the sexual segment during February–April, indicates that this is under the influence of testicular androgens.

Intratesticular injection of prostaglandin E2 (PGE2) and F2 alpha (PGF2 alpha) caused stimulation of ornithine decarboxylase (ODC) activity in the testis of immature rats. PGE2 at a dose of 10 microgram per testis was maximally effective 2 hours after the injection. Dibutyryl cyclic AMP (cAMP) and 1 methyl, 3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor, also stimulated ODC activity. Simultaneous injection of PGE2 and FSH or LH caused additional stimulation of ODC activity. Similarly injection of PGE2 in addition to cAMP or MIX also caused increased stimulation of ODC. Indomethacin (IM, 60 microgram/testis) inhibited LH, FSH or cAMP induced ODC activity. However, IM at the same dose inhibited the synthesis of total proteins. These results suggest that PGE2 and PGF2 alpha stimulate the activity of ODC. The action of prostaglandins may be independent of the action of gonadotropic hormones. cAMP appears to mediate the action of prostaglandins in the testis of rat.

Recently GnRH and its synthetic agonists were shown to exert extrapituitary actions and inhibit the growth-promoting effects of testosterone and estradiol. In the present study, the long-term effect of GnRH agonist [des-Gly10, D-Ala6-GnRH ethylamide GnRHa] on the steroid-induced renotropic effect was investigated. Gonadectomy of 30-day old male and female rats resulted in the drastic reduction of kidney ornithine decarboxylase (ODC) activity. Injection of testosterone (75 micrograms/rat) or estradiol (1 microgram/rat) to castrated rats significantly increased the enzyme activity at 24 h. Treatment with the GnRH agonist, two doses of 100 micrograms each, caused significant inhibition of the hormone induced ODC activity. Similarly injection of dihydrotestosterone to castrated adult male rats significantly increased poly(A)-polymerase activity at 24 h. Two doses of 100 micrograms of GnRHa caused significant inhibition of DHT induced poly(A)-polymerase activity. These results show that chronic exposure to potent GnRH analogs may have anti-steroidal activity in the kidney.

The levels of superoxide anion production, cytochrome P450, ornithine decarboxylase(E.C.4.1.1.17), catalase(E.C.1.11.1.6), deoxyribonucleic acid, ribonucleic acid and protein have been studied in human kidney and renal clear-cell adenocarcinoma tissues. The levels of superoxide anion production, ornithine decarboxylase, catalase and ribonucleic acid in the tumor tissue are very different from those in the kidney.