Establishment of spermatogenesis in guinea-pigs at puberty

The establishment of spermatogenesis in the guinea-pig was studied by a microscopic method. The spermatogenesis starts before day 16 and the first spermatozoa appear on day 55. Spermatozoa are seen in the tail of the epididymis on day 60 and in the ductus deferens between day 70 and day 80. Relations exist between increase of testicular testosterone and setting of spermatogenesis in the guinea-pig.     

Onset of the hormone-sensitive perinatal period for sexual differentiation of the sexually dimorphic nucleus of the preoptic area in female rats

The volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) of the rat brain is severalfold larger in males than in females. The volume of the SDN-POA can be influenced significantly by the hormonal milieu during the perinatal "critical period" of sexual differentiation of the brain. The purpose of the present study was to determine the onset of this period of sexual differentiation of the SDN-POA. Pregnant rats received no treatment or were injected subcutaneously with oil on day 17, 18, or 20, or testosterone (T;5 mg) on days 16-22 of gestation. On postnatal day 15, unilateral SDN-POA volumes from female offspring prenatally exposed to testosterone on day 16 or 17 were not different from values of control (untreated or oil-injected) offspring. Female offspring from mothers treated with testosterone on day 18, 19, or 20 of gestation showed a significant and similar increase in SDN-POA volume over values from control animals. SDN-POA volumes from female offspring exposed to testosterone on day 21 or 22, although larger than those of controls, were not different statistically. We conclude that with the specific paradigm used in this study SDN-POA development is insensitive prior to day 18 of gestation, the day on which the onset of the hormone-sensitive period occurs.     

Progesterone receptor gene and protein expression in the anterior preoptic area and hypothalamus of defeminized rats

Progesterone receptor (PR) plays an important role during sexual differentiation of the rat brain. The objective of the present study was to determine PR protein and gene expression pattern in preoptic-anterior hypothalamic area (POA-AHA) and hypothalamus (HYP), after estradiol or testosterone treatment during the postnatal critical period of sexual differentiation of the rat brain (defeminized animals). Three-day-old female rats were subcutaneously (s.c.) injected with a single dose of 17beta-estradiol (200 microg), or testosterone enanthate (200 microg), or vehicle (corn oil). POA-AHA and HYP were dissected 3 h, 24 h, and 14 days, as well as on the day of vaginal opening (VO) after treatments. Other animals, previously treated as above, were acutely injected with 17beta-estradiol (5 microg) on the day of VO; POA-AHA and HYP were obtained 3 h later. Total RNA was extracted and processed for semiquantitative RT-PCR and tissue slices were prepared for protein detection by immunohistochemistry. We observed that PR mRNA expression was increased in POA-AHA and HYP of the animals treated with estradiol or testosterone 3 hours after treatments, compared with the vehicle-treated control group. We also found a significant increase in PR mRNA and protein expression in POA-AHA and HYP on the day of VO in both estradiol and testosterone defeminized rats. Interestingly, the acute administration of estradiol on the day of VO (VO + E(2)) did not increase PR mRNA or protein expression in POA-AHA and HYP of either estradiol or testosterone defeminized animals, as opposed to the marked induction observed in the intact animals of the control group. The overall results suggest that estradiol and testosterone treatment during the postnatal critical period of sexual differentiation of the brain modifies the regulation of the PR mRNA and protein expression during early onset of maturity.     

Endocrinology of spermatogenesis in the hypophysectomized ram.

Adult rams were hypophysectomized and treated for 20 days with testosterone (2 X 0.25 g/day), PMSG (2 X 300 i.u./day) or hCG (2 X 250 i.u./day), or for 40 days with testosterone (2 X 0.25 g/day). All treatments maintained a normal concentration of testosterone within the seminiferous tubules. Quantitative histological analysis showed that (1) the differentiation from A0 to A1 spermatogonia was maintained by PMSG or hCG but not completely by testosterone; (2) the transition from intermediate spermatogonia to primary spermatocytes was maintained only by PMSG but not by testosterone or hCG; (3) meiotic prophase and spermiogenesis were maintained by the three hormones but there were qualitative abnormalities in the spermatids. These results suggest that in the ram, the differentiation of renewing stem spermatogonia is under LH control and that the last stages of spermatogonial multiplication, from intermediate to B spermatogonia and to primary spermatocytes, are under the control of the FSH-like activity of PMSG.     

Temporal difference between testis and ovary determinations with possible involvement of testosterone and aromatase in gonadal differentiation in TSD lacking lizard, Calotes versicolor

In the garden lizard, Calotes versicolor, which lacks identifiable sex chromosomes, incubation temperature also does not have a deterministic effect on the gender. However, the embryos reared at high temperature (33-35 degrees C) have a shorter duration of incubation as well as gonadal differentiation. In contrast, exogenous application of the male hormone testosterone to embryos at ambient temperature (28 degrees C) results in almost all individuals with only testis. Thus the testosterone treatment reverts genic females to males and accelerates the differentiation of testis, a feature similar to the high-temperature treatment. Treatment of eggs with estradiol shows no difference from that seen in the untreated eggs. The present series of experiments was done to establish the "window" of testosterone sensitivity and to understand the interaction between sex hormones and high temperature on gonadal differentiation. The period between day 5 and 15 of embryonic development was the window period of testosterone sensitivity for sex reversal. This period coincided with the formation of the genital ridge and its differentiation into cortex and medulla. Treatment of the 33 degrees C-reared embryos with testosterone resulted in hatchlings of both the sexes, in contrast to only males at the ambient temperature. In contrast, at the same temperature (33 degrees C), all the dihydrotestosterone (nonaromatisable testosterone)-treated embryos hatched into males. However, those given estradiol showed no sex bias regardless of the day of application and the concentration of drug. Eggs were also treated with aromatase inhibitor, CGS 16949 A, at ambient temperature and at 33 degrees C. All the 33 degrees C eggs to which the drug was given on day 25 hatched into males. These results suggest that though high temperature has no direct effect on sex determination in this species, it may have a stimulatory effect on aromatase activity, leading to the conversion of the exogenously applied testosterone into estradiol and permitting ovarian differentiation in the genic females. It also follows from the present report that the pathway of testis formation in Calotes versicolor is triggered much earlier, and irreversibly, than that for the ovary.     

Development of seminiferous tubules in guinea pigs during puberty]

The study of development of the seminiferous tubules of the guinea-pig along the puberty is based on diameter, length and volume relate to testicular weight and age. During this period the development is linear. A correlation exist between testicular testosterone concentration and growth of seminiferous tubules in diameter and in length from day 16 to 60, stage of puberty in which the first spermatozoa appear.     

Guinea-pig liver testosterone 17 beta-dehydrogenase (NADP+) and aldehyde reductase exhibit benzene dihydrodiol dehydrogenase activity.

We have kinetically and immunologically demonstrated that testosterone 17 beta-dehydrogenase (NADP+) isoenzymes (EC 1.1.1.64) and aldehyde reductase (EC 1.1.1.2) from guinea-pig liver catalyse the oxidation of benzene dihydrodiol (trans-1,2-dihydroxycyclohexa-3,5-diene) to catechol. One isoenzyme of testosterone 17 beta-dehydrogenase, which has specificity for 5 beta-androstanes, oxidized benzene dihydrodiol at a 3-fold higher rate than 5 beta-dihydrotestosterone, and showed a more than 4-fold higher affinity for benzene dihydrodiol and Vmax. value than did another isoenzyme, which exhibits specificity for 5 alpha-androstanes, and aldehyde reductase. Immunoprecipitation of guinea-pig liver cytosol with antisera against the testosterone 17 beta-dehydrogenase isoenzymes and aldehyde reductase indicated that most of the benzene dihydrodiol dehydrogenase activity in the tissue is due to testosterone 17 beta-dehydrogenase.     

In vitro metabolism of androgens by mammalian cauda epididymal spermatozoa.

The in vitro metabolism of [3H] testosterone (17beta-hydroxy-4-androsten-3-one), [3H] androstenedione (4-androstene-3,17-dione) and [3H] dehydroepiandrosterone (3beta-hydroxy-5-androsten-17-one) by cauda epididymal spermatozoa from the rat, rabbit, hamster, guinea-pig and ram, varied between species. There were differences in the androgens utilized, the extent of their conversion and the identities of the metabolites formed. Of the steroid substrates tested rat spermatozoa metabolized testosterone preferentially while spermatozoa from guinea-pig transformed [3H] dehydroepiandrosterone (DHEA) almost exclusively. Rabbit spermatozoa converted all three [3H] androgens while hamster sperm utilized [3H] testosterone and [3H] DHEA. Spermatozoa collected from rams killed at the abattoir metabolized both [3H] androstenedione and [3H] DHEA but this capacity was dramatically reduced in spermatozoa collected from rams subjected to short-term anaesthesea. The results are discussed in relation to the possible direct roles of androgens in sperm physiology.     

Testis cell-specific proteins in testosterone-treated rat ovaries

Abstract. Two-dimensional gel electrophoresis of rat gonads in various developmental stages (embryonic days 16, 17, 21 postnatal day 5) reveals an increasing number of polypeptide spots; 0.6%-2% of the polypeptides are gonad-specific and increase from embryonic day 16 onwards. Exposure of newborn rat ovaries to testosterone for 5 days results in the appearance of eight polypeptides. These polypeptides are absent in control ovaries but present in the testis from embryonic day 16 or 17 onwards. Three do not appear in the ovary at any developmental stage. These findings indicate that testosterone plays a physiologic role in normal testicular differentiation. After long-term testosterone treatment, ovaries are depleted of germ cells. This might explain the degeneration of oocytes in the abnormal environment of a testis (e.g., in XX true hermaphrodites).     

Testosterone measured in infancy predicts subsequent sex-typed behavior in boys and in girls

The testes are active during gestation, as well as during early infancy. Testosterone elevation during fetal development has been shown to play a role in human neurobehavioral sexual differentiation. The role of early postnatal gonadal activation in human psychosexual development is largely unknown, however. We measured testosterone in 48 full term infants (22 boys, 26 girls) by monthly urinary sampling from day 7 postnatal to age 6 months, and related the area under the curve (AUC) for testosterone during the first 6 months postnatal to subsequent sex-typed behavior, at the age of 14 months, using the Pre-School Activities Inventory (PSAI), and playroom observation of toy choices. In boys, testosterone AUC correlated significantly with PSAI scores (Spearman's rho = 0.54, p = 0.04). In addition, play with a train and with a baby doll showed the anticipated sex differences, and play with the train correlated significantly and positively with testosterone AUC in girls (Spearman's rho = 0.43, p = 0.05), while play with the doll correlated significantly and negatively with testosterone AUC in boys (Spearman's rho = -0.48, p < 0.03). These results may support a role for testosterone during early infancy in human neurobehavioral sexual differentiation.     

Tissue levels of prostaglandins and what do they mean? Studies on the guinea-pig uterus

The ratios of the concentrations of PGF2 alpha, PGE2 and 6-keto-PGF1 alpha in guinea-pig uterine horns, which were removed and placed in ethanol in 1.5 to 2 min, were 0.3:1.0:0.6 on day 7 and 13.8:1.0:0.8 on day 15 of the oestrous cycle. Adding indomethacin (10 micrograms/ml) to the ethanol had no significant effect on the tissue levels observed. These ratios were similar to the ratios of the outputs of PGF2 alpha, PGE2 and 6-keto-PGF1 alpha from the guinea-pig uterus (0.6:1.0:0.9 on day 7 and 7.6:1.0:1.5 on day 15), but were different (particularly on day 7, but only for 6-keto-PGF1 alpha on day 15) to the ratios of the amounts of the three PGs synthesized by homogenates of the guinea-pig uterus (7.2:1.0:2.4 on day 7 and 11.7:1.0:3.3 on day 15). Consequently, the measurement of tissue levels of PGs in the guinea-pig uterus reflects PG synthesis by intact tissue and changes in this synthesis, rather than PG synthesis by homogenates (broken cell preparations). Therefore, it appears meaningful to measure levels of PGs in the guinea-pig uterus since they reflect uterine PG output. Separation of the endometrium from the myometrium, which involved handling and mild trauma, stimulated uterine PG levels, but the ratio of the levels of PGF2 alpha, PGE2 and 6-keto-PGF1 alpha in the endometrium was still similar to that found in the non-separated uterus.     

Conversion,in vitro,of (7n-3H) testosterone to estrone and estradiol-17β and their 3-sulfäte conjugate by the guinea-pig placenta

Different cellular fractions of guinea-pig placenta were incubated in the presence of (7n-³H) testosterone. Microsomal aromatization of ³H-testosterone into estrone and estradiol-17β was demonstrated in the presence of NADPH. The predominance of estrone after incubation with 17β-hydroxylated precursors, (7n-³H) testosterone and (6,7-³H) estradiol-17β, indicate that there is a microsomal 17β-hydroxysterold dehydrogenase activity. In this report, cytosolic sulfurylation of estrogens is demonstrated. This latter activity represents a quite original characteristic of the placental metabolism of estrogens in guinea-pigs. In contrast with the human placenta where there is considerable sulfatase activity, the guinea-pig placenta can sulfurylate estrogens.     

Sex-related differences in blood and gonad levels of testosterone in silver fox fetuses

The mass of silver fox fetuses of both sexes, their gonads, and adrenals, and the levels of testosterone in blood serum and in gonads and adrenals were determined from day 31 of gestation and every five days thereafter until its termination. Marked sex-related differences were revealed: the blood and gonad levels of testosterone in male fetuses were much higher than those in female fetuses. The fetal adrenals contained significantly less testosterone than the gonads. No sex-related differences in the content of testosterone in the fetal adrenals were found. No differences were found in the body and adrenal mass in female and male fetuses at all the developmental stages studied, while the mass of ovaries exceeded that of testes from day 45 of gestation. The data obtained suggest sex dimorphism in the production of testosterone by gonads in silver foxes appears after day 35 and appears to correspond to the period of morphological differentiation of gonads.     

Purification and properties of a new testosterone 17beta-dehydrogenase (NADP+) from guinea-pig liver.

As a result of studies of guinea-pig live testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), a new testosterone 17beta-dehydrogenase was discovered. The new enzyme was purified to a single homogeneous protein from the 105 000 g-supernatant fraction of guinea-pig liver by (NH4)2SO4 fractional precipitation and two gel-filtration stages, DEAE-cellulose column chromatography and hydroxyapatite column chromatography. It was characterized by many properties. The enzyme has almost the same properties as the classical testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), with respect to cofactor requirement, pH optima for dehydrogenation, effect of phosphate ion on the NAD+-dependent reaction and molecular weight, but characteristic differences were observed in substrate-specificity between the two dehydrogenases. With various androstane derivatives, the configuration of the A/B-ring junction was closely connected with enzyme activity. 5alpha-Androstanes, such as 5alpha-androstane-3alpha,17beta-diol, 5alpha-androstane-3beta,17beta-diol and 17beta-hydroxy-5alpha-androstan-3-one, and 5beta-congeners, such as 5beta-androstane-3alpha,17beta-diol, 5beta-androstane-3beta,17beta-diol and 17beta-hydroxy-5beta-androstan-3-one, served as substrates for both the EC 1.1.1.64 enzyme and the new enzyme. The EC 1.1.1.64 enzyme oxidized testosterone more rapidly than did the new enzyme. These comparisons were based on the relative activities, apparent Km values and apparent Vmax values.     

Effect of steroid aromatase inhibitors on the catecholamine content of the hypothalamus of neonatally androgenized rats

Administration of 50 micrograms of testosterone propionate to newborn female rats on the 5th day of life provoked a reliable increase in noradrenaline and dopamine concentrations in the hypothalamus of 10-day-old rats. Neonatal administration of aromatase inhibitors on the 5th and 7th days of life prevented testosterone-induced increase in catecholamine concentrations. The data obtained prove the integration of the processes of testosterone aromatization and catecholamine accumulation in androgen-dependent brain differentiation.     

Could neonatal testosterone replacement prevent alterations induced by prenatal stress in male rats?

The present study was designed to examine whether testosterone replacement is able to prevent some effects of maternal restraint stress — during the period of brain sexual differentiation — on endocrine system and sexual behavior in male rat descendants. Pregnant rats were exposed to restraint stress for 1 h/day from gestational days 18 to 22. At birth, some male pups from these stressed rats received testosterone propionate. The neonatal testosterone replacement was able to prevent the reduction in anogenital distance at 22 days of age observed in pups from stressed pregnant rats as well as prevents the decrease in testosterone levels during the adulthood of these animals. Testosterone replacement in these males also presented an improvement in sexual performance. In this way, testosterone replacement probably through increasing neonatal level of this hormone was able to prevent the later alterations caused by the prenatal stress during the period of brain sexual differentiation.     

Sex-Related Differences in Blood and Gonad Levels of Testosterone in Silver Fox Fetuses

The mass of silver fox fetuses of both sexes, their gonads, and adrenals, and the levels of testosterone in the blood serum and in gonads and adrenals were determined from day 31 of gestation and every five days thereafter until its termination. Marked sex-related differences were revealed: the blood and gonad levels of testosterone in male fetuses were much higher than those in female fetuses. The fetal adrenals contained significantly less testosterone than the gonads. No sex-related differences in the content of testosterone in the fetal adrenals were found. No differences were found in the body and adrenal mass in female and male fetuses at all the developmental stages studied, while the mass of ovaries exceeded that of testes from day 45 of gestation. The data obtained suggest sex dimorphism in the production of testosterone by gonads in silver foxes appears after day 35 and appears to correspond to the period of morphological differentiation of gonads.     

Altered sexual differentiation of hepatic uridine diphosphate glucuronyltransferase by neonatal hormone treatment in rats

The hepatic microsomal enzyme UDP-glucuronyltransferase undergoes a complex developmental pattern in which enzyme activity is first detectable on the 18th day of gestation in rats. Prepubertal activities are similar for males and females. However, postpubertal sexual differentiation of enzyme activity occurs in which male activities are twice those of females. Neonatal administration of testosterone propionate or diethylstilboestrol to intact animals resulted in lowered UDP-glucuronyltransferase activity in liver microsomal fractions of adult male rats, whereas no changes were observed in the adult females and prepubertal male and female animals. Neonatal administration of testosterone propionate and diethylstilboestrol adversely affected male reproductive-tract development as evidenced by decreased weights of testes, seminal vesicles and ventral prostate. Diethylstilboestrol also markedly decreased spermatogenesis. Hypophysectomy of adult male rats resulted in negative modulation of microsomal UDP-glucuronyltransferase and prevented the sexual differentiation of enzyme activity. In contrast hypophysectomy had no effect on female UDP-glucuronyltransferase activity. A pituitary transplant under the kidney capsule was not capable of reversing the enzyme effects of hypophysectomy, therefore suggesting that the male pituitary factor(s) responsible for positive modulation of UDP-glucuronyltransferase might be under hypothalamic control in the form of a releasing factor. Neonatal testosterone propionate and diethylstilboestrol administration apparently interfered with the normal sequence of postpubertal UDP-glucuronyltransferase sexual differentiation.     

Effects of indomethacin, NS-398 (a selective prostaglandin H synthase-2 inhibitor) and protein synthesis inhibitors on prostaglandin production by the guinea-pig placenta

The outputs of PGF(2 alpha), PGE2 and 6-keto-PGF(1 alpha)were similar from the day 22 guinea-pig placenta and sub-placenta in culture, except for PGE2 output from the sub-placenta which was lower. Between days 22 and 29 of pregnancy, the outputs of PGF(2 alpha), PGE2 and 6-keto-PGF(1 alpha)during the initial 2 h culture period increased 6.9-, 1.1- and 3.2-fold, respectively, from the placenta, and 2.1-, 1.4- and 2.2-fold, respectively, from the sub-placenta. Therefore, there was a relatively specific increase in PGF(2 alpha)production by the guinea-pig placenta between days 22 and 29 of pregnancy. The output of PGFM from the cultured placenta also increased between days 22 and 29, indicating that the increase in PGF(2 alpha)output was due to increased synthesis rather than to decreased metabolism. By comparing the amounts of prostaglandins produced by tissue homogenates during a 1 h incubation period, it appears that there is approximately a 2-fold increase in the amount of prostaglandin H synthase (PGHS) present in the guinea-pig placenta between days 22 and 29. NS-398 (a specific inhibitor of PGHS-2) and indomethacin (an inhibitor of both PGHS-1 and PGHS-2) both inhibited prostaglandin production by homogenates of day 22 and day 29 placenta. Indomethacin was more effective than NS-398, except for their actions on PGF(2 alpha)production by the day 29 placenta where indomethacin and NS-398 were equiactive. Indomethacin and NS-398 were both very effective at inhibiting the outputs of PGF(2 alpha), PGE2 and 6-keto-PGF(1 alpha)from the day 22 and day 29 placenta and sub-placenta in culture, indicating that prostaglandin production by the guinea-pig placenta and sub-placenta in culture is largely dependent upon the activity of PGHS-2. The high production of PGF(2 alpha)by the day 29 placenta is not dependent on the continual synthesis of fresh protein(s), as inhibitors of protein synthesis did not reduce PGF(2 alpha)output from the day 29 guinea-pig placenta in culture.     

Steroid hormone content of the gonads of the tammar wallaby during sexual differentiation.

The gonads of the tammar wallaby, Macropus eugenii, are sexually indifferent at birth (Day 0) despite the fact that phenotypic sexual differentiation has already commenced as evidenced by the presence of a scrotum in males and mammary anlagen in females. The seminiferous cords of the testis first become clearly recognizable on Day 2 of pouch life, and ovarian differentiation is recognizable by Day 10. To monitor the endocrine development of the gonads during sexual differentiation of the urogenital tract, we measured the steroid hormone content in 92 pools of gonads from male and female tammar pouch young from the day of birth to 206 days of pouch life. Progesterone, estradiol, and dihydrotestosterone concentrations were low (less than 0.05 ng/mg protein) in both ovaries and testes at all stages examined, and testosterone concentrations were uniformly low in ovaries. Testosterone concentrations in testes were low on Days 0-4, averaging about 0.2 ng/mg protein; they rose by Days 5-10 to an average of 0.9 ng/mg protein, remained elevated until about Day 40, and thereafter fell to values similar to those in the ovaries. The phallus and urogenital sinus were able to convert testosterone to dihydrotestosterone from the earliest stages examined (Days 10 and 11). Thus in the tammar wallaby, as in eutherian mammals, testosterone is the androgen secreted by the developing testis, and dihydrotestosterone is formed in certain androgen target tissues.(ABSTRACT TRUNCATED AT 250 WORDS)