Changes in testosterone levels in rabbit embryo testes after removal of the hypothalamus (encephalectomy)

Brain hypothalamus region was removed by encephalectomy in utero in 21-23-day-old rabbit fetuses to find out when hypothalamic control over testicular androgenous function was set up during rabbit prenatal development. Testosterone concentration in testicular tissue was measured by radioimmunoassay at different terms after the operation. Encephalectomy resulted in a reliable decrease of testosterone concentration in 29- and 25-day-old fetuses, however, no significant changes were observed in 23-day-old fetuses. The greatest reduction in hormone gland concentration was noted in 25-day-old fetuses, encephalectomized on day 23 of their development. Introduction of LH-RH to 25-day-old encephalectomized fetuses 30 min before fixation significantly increased androgen concentration in the gland. The data obtained indicate that hypothalamic control over testicular androgenous function is set up on days 23-25 of prenatal development.     

Determination of thyroxine and triiodothyronine in the thyroid glands of encephalectomized rat fetuses

Concentration of thyroxin and triiodothyronine in the thyroid gland of encephalectomized and intact fetuses of rats with normal and prolonged gestation period was determined by means of isotope methods. Encephalectomy of 19-day old fetuses did not produce any significant effect on the concentration of thyroid hormones in the gland 2 and 4 days after the operation. The results obtained pointed to the absence of the hypothalamic control over the thyroid function in prenatal development of rats.     

Ultrastructural study on the hypothalamic-hypophysial-adrenal axis in fetal rats

Summary The adrenal glands of decapitated and encephalectomized fetal rats were investigated electron microscopically and compared to those of normal intact fetal rats. Although the adrenal cortices did not show three zones (zona glomerulosa, fasciculata, and reticularis) on the 16.5th day of gestation when the decapitation or encephalectomy was carried out in utero, the zonation was recognized in fetuses operated on the 21.5th day of gestation. The same was true for normal control fetuses. However, cytoplasmic characteristics suggesting steroidogenesis in the cortical cells were reduced to various degrees in the encephalectomized or decapitated fetuses, especially in the latter ones. The change in cytoplasmic appearance was more conspicuous in the inner portion of the cortex. This result suggests that for the maintenance of normal adrenocortical function the hypothalamus may be indispensable even during the prenatal life of rats.     

The testosterone level in the testes of silver foxes during prenatal development]

The level of testosterone in serum and testes of the silver fox fetuses on days 31, 35, 40, 45, and 50 of gestation was determined using radioimmunoassay. In testes, testosterone was first detected at day 31; then its level gradually increased. Serum testosterone was detected only at day 40. Subsequent increase in its concentration was insignificant. Human chorionic gonadotropin stimulated testicular testosterone production in vitro beginning from day 40. We suggest that in the silver fox, testes are capable of responding to gonadotropins already by the day 40 of prenatal development but the formation of pituitary-gonadal axis proceeds until the end of embryogenesis.     

Hypothalamo-pituitary system controls the development of humoral immune response during prenatal ontogenesis in rats

We studied the influence of the neuroendocrine system on the development of humoral immune response to sheep erythrocytes in rat fetuses. The removal of brain in utero by decapitation of 18-day fetuses induced a fourfold increase in the number of antibody-forming cells in the liver, as compared to the unoperated fetuses. After the removal of the forebrain, including hypothalamus (encephalectomy), the number of antibody-forming cells was comparable to that in unoperated fetuses. The observed increase in the number of antibody-forming cells in the liver was not due to a disturbed migration of precursors of B-lymphocytes in the spleen, since their content in the spleen was also four times that in the encephalectomized and unoperated fetuses. The increased number of antibody-forming cells in decapitated fetuses could be due to an enhanced proliferative activity of the lymphocytes in the liver of these fetuses. It has been proposed that humoral immunity is controlled by the hypothalamo-pituitary-adrenal system already during prenatal development; the adrenocorticotropic hormone and glucocorticoids appear to be involved in this regulation.     

Hypothalamo–Pituitary System Controls the Development of Humoral Immune Response during Prenatal Ontogenesis in Rats

We studied the influence of the neuroendocrine system on the development of humoral immune response to sheep erythrocytes in rat fetuses. The removal of brain in utero by decapitation of 18-day fetuses induced a fourfold increase in the number of antibody-forming cells in the liver, as compared to the unoperated fetuses. After the removal of the forebrain, including hypothalamus (encephalectomy), the number of antibody-forming cells was comparable to that in unoperated fetuses. The observed increase in the number of antibody-forming cells in the liver was not due to a disturbed migration of precursors of B-lymphocytes in the spleen, since their content in the spleen was also four times that in the encephalectomized and unoperated fetuses. The increased number of antibody-forming cells in decapitated fetuses could be due to an enhanced proliferative activity of the lymphocytes in the liver of these fetuses. It has been proposed that humoral immunity is controlled by the hypothalamo–pituitary–adrenal system already during prenatal development; the adrenocorticotropic hormone and glucocorticoids appear to be involved in this regulation.     

Insulin content in the pancreas and blood plasma of decapitated and encephalectomized rat fetuses

Insulin content in the pancreas and blood plasma of encephalectomized, decapitated and intact rat fetuses was measured by radioimmunoassay. Encephalectomy and decapitation of 17.5-day-old fetuses did not produce any significant effect on insulin concentration in the pancreas and blood plasma of 21-day-old fetuses. Injection of glucose to 21-day-old operated fetuses raised insulin secretion, which seems to be related to the potentiating action of maternal and (or) fetal humoral factors. The data obtained indicate that synthesis and basal secretion of insulin to the blood are not disturbed by the lack of the hypothalamohypophyseal control in prenatal rats.     

Absence of direct effects of GnRH on testicular steroid secretion in the ram

Effects of GnRH, administered via the testicular artery, on testicular steroidogenesis were studied in rams during the non-breeding season. Concentrations of testosterone and 17-hydroxyprogesterone in testicular venous blood showed similar profiles which were identical for GnRH-treated (0.5 ng infused over 60 min or 25 ng injected) and control testes. Increases of testicular venous concentration of both hormones were only marginally reflected in peripheral venous concentrations. Peripheral administration of hCG (200 i.u., i.v.) stimulated testosterone secretion to a larger extent than 17-hydroxyprogesterone secretion in 10/11 rams, GnRH-treated and control testes showing identical responses. High testicular venous concentrations of both hormones after administration of GnRH were paralleled by increased concentrations of endogenous LH. These LH peaks were evoked by 25 ng GnRH in 7/8 rams. The observed effects of GnRH treatment on testicular steroid secretion thus cannot be considered to be the result of direct stimulation of steroidogenesis by GnRH.     

The brain is one of the sources of L-dihydroxyphenylalanine in systemic circulation in fetuses and neonatal rats

The performed study was aimed at checking our hypothesis that the developing brain is a source of L-dihydroxyphenylalanine (L-DOPA), a precursor of dopamine in the total circulation system. At the initial stage, the L-DOPA concentration in peripheral blood was analyzed at the 18th and 21st embryonal days (E18 and E21), at the 3rd postnatal day (P3), and at the prepubertal period (P30). The highest L-DOPA concentration was revealed at the perinatal period, while decreased 4–12 times for the first month of life. The subsequent analysis of dynamics of the total blood L-DOPA content showed that maintenance of the constant L-DOPA concentration at the perinatal period on the background of a gradual increase of the blood serum volume is due to a rise of its secretion. At the postnatal period (P3–P30), the blood L-DOPA content increased twice in males, whereas it decreased to the same extent in females. Analysis of the L-DOPA concentration in two most important brain centers, hypothalamus and mesencephalon-rhombencephalon, showed its twofold decrease in hypothalamus during E18–E21 of development; then it slightly increased from E21 to P3 and fell 4–5 times by P30. In mesencephalon-rhombencephalon, the L-DOPA concentration was slightly reduced from E18 to E21 (only in females), while on P3 it returned to the E18 level and decreased 7–9 times by P30. The direct proof for the L-DOPA release from the developing brain into the systemic circulation follows from comparison of the blood L-DOPA concentration in shamoperated and encephalectomized rat fetuses after mechanical destruction of neurons of the two abovementioned most important dopaminergic centers. Thus, encephalectomy led to a twofold reduction of the blood L-DOPA concentration (statistically significant differences were observed only in females). Thus, the work presents evidence that the developing brain is one of L-DOPA sources in the total circulation system in rats during prenatal and early postnatal periods of ontogenesis.     

Brain aromatase activity and behavioral consequences in the male rat treated in utero with 4-hydroxy-androstenedione

Pregnant female rats were given daily injections of a potent aromatase inhibitor, 4-hydroxy-delta 4-androstenedione (4OHA), throughout the latter half of the pregnancy (days 11 to 22; the day of insemination was designated as Day O) and male fetuses and pups were obtained. Control animals were male offspring of mothers treated with oil vehicle. When measured by the tritium-water method, significant reductions in the aromatase activity were detected in the hypothalamic and preoptic continuum (HPOA) of the male fetuses and pups, over a period from day 16 of the pregnancy until a day after birth. All parturitions in the experimental as well as control animals occurred on day 22 of the pregnancy. Behavioral and anatomical consequences of the prenatal treatment were examined in adulthood. When mounted by stud male rats, the male litters of the 4OHA-treated mother showed lordosis at a significantly higher frequency both in terms of the number of positive test sessions (each consisted of a 20-min period in which the subject showed lordosis at least once) and the lordosis quotient (percent lordosis occurrence per 10 mounts). When placed with receptive female rats, the experimental animals were no less active in mounting or ejaculating than the control. No significant difference existed between the experimental and control animals in the weight of the testes or the accessorial genitalia, or the serum testosterone levels. Partial but significant intervention of behavioral defeminization of the brain was associated with decreased HPOA aromatase activity during the prenatal period.     

Expression of the c-Kit receptor in germ cells of the seminiferous epithelium in rats with hormonal imbalance

The aim of the study was to investigate the effects of pharmacologically induced hormonal imbalance in adult male rats treated with letrozole and rats exposed to soya isoflavones on the testicular morphology and c-Kit receptor (c-Kit-R) expression in germ cells. The study was conducted during all developmental periods: prenatal period, lactation, youth, and sexual maturity. Morphological and morphometrical analyses were performed on testicular section, and c-Kit-R was identified using immunohistochemistry. In addition, concentration of circulating steroids was measured in mature rats exposed to soya isoflavones. A significant reduction in testosterone level in rats exposed to soya isoflavones, and the sloughing of the premature germ cells into the lumen of the seminiferous tubules in the testes of both groups of rats were observed. Immunohistochemistry showed a decrease in c-Kit-R expression in germ cells of both experimental groups. Morphometric analysis indicated a decreased thickness of the layers occupied by c-Kit-R-positive spermatogonia, and a decreased diameter of the seminiferous tubules in the testes of both experimental groups of animals. In conclusion, the pharmacologically induced reduction of the estradiol level in adult rats and the diminished level of testosterone in rats exposed to soya isoflavones during the prenatal period, lactation and up to maturity caused similar morphological and functional changes associated with the decreased c-Kit-R expression in germ cells in the seminiferous epithelium. These findings demonstrate the importance of the estrogen/androgen balance for normal testicular morphology and spermatogenesis.     

Testicular responsiveness to hCG of the ovine fetus in the last third of gestation

The in vivo and in vitro testicular responsiveness to hCG of hemicastrated lamb fetuses 95-99, 110-118 and 130-141 days of gestational age was studied. Basal plasma testosterone (T) levels were similar at all ages (less than 0.25 ng/ml), while the mean testicular concentrations of dehydroepiandrosterone sulfate (DHA-S), 17 alpha-hydroxyprogesterone (17-OHP) and T were higher in 95- to 99-day-fold fetuses. Plasma T levels and the concentration of T, DHA-S, 17-OHP, androstenedione (A) and cyclic adenosine 3'5'-monophosphate (cAMP) were increased by hCG in the hemicastrated animal at all ages. cAMP and T production by enriched preparations of dispersed interstitial cells from control testes was increased by hCG in all groups. In fetuses pretreated with hCG in vivo the addition of hCG in vitro failed to modify cAMP and T production. 100 micrograms of LHRH to a 130-day-old fetus increased plasma LH and T levels. From these experiments, it is suggested that the low plasma LH and T levels found throughout the last trimester of fetal life reflect a relative lack of endogenous LHRH synthesis and/or release, rather than reduced testicular steroidogenic capacity.     

Spermatogenesis, testicular composition and the concentration of testosterone in the equine testis as influenced by season

The influence of season on spermatogenesis, testicular composition and the concentration of testosterone in the equine testis was evaluated using testes from 45 stallions. Testes were obtained through a commercial abbatoir during September, December-January, March and July. The weights of the testes, the tunica albuginea and testicular parenchyma and the proportion of the testicular parenchyma occupied by seminiferous tubules or interstitial tissue were similar during each season. How ever, diameter of the seminiferous tubules was greater in July than during other months of the study. In addition, the concentration of testosterone within the testicular parenchyma was twice as great during July as during the fall and winter, and this period of peak testicular testosterone concentrations was associated with spermatozoal production rates, which were 65% greater than those observed in September.     

Testosterone production in fetal testes of the silver fox

Testosterone content was studied in fetal serum and testes of silver foxes during different periods of prenatal life. The first sign of testosterone synthesis in the testes could be detected starting from the 31st day of gestation by radioimmunoassay. Subsequently its content sharply increased and showed maximum value on the 50th day. Detectable serum testosterone level in male silver fox fetus was noted from the 40th day, its serum concentration increased in prenatal life, but only insignificantly. Testicular production of testosterone was stimulated by hCG at the stages studied from Day 40 on. It is supposed that silver fox testes were able to respond to gonadotrophin not earlier than by Day 40 of prenatal life, but the establishment of pituitary-testicular interrelationship was clearly demonstrated at the end of embryogenesis (Day 50).     

Masculinizing effect of testes on developing rat ovaries in organ culture

Masculinizing effect of testes on developing rat ovaries was shown in vitro by culturing testes from 17.5-day-old fetuses in contact with female genital tracts from 14.5-day-old rat fetuses. The testes induced the differentiation of epithelial cells staining for cytokeratin in the ovarian blastema. These cells formed seminiferous cord-like structures delineated by a basement membrane, in a way that resembles early stages of testicular organogenesis. In addition to the morphological masculinization, functional masculinization was obtained since the ovaries produced the anti-Müllerian hormone as shown by bioassay and immunohistochemical procedures. Across a distance, testes from 17.5-day-old fetuses failed to induce masculinization. These results suggest that testes from 17.5-day-old fetuses produce a locally diffusible factor interfering with the development and the differentiation of the fetal ovaries. The possibility that the anti-Müllerian hormone secreted by the testes may be the factor involved is discussed comparing these results with those obtained with testes from different stages and with bibliographic data.     

Low aromatase activity and gene expression in human fetal testes

Because of previous indications that estradiol (E2) plays a role in the regulation of testicular testosterone (T) production in some species, the production of E2 and aromatase gene expression in human fetal testes were investigated. Testicular minces from 14 fetuses (fetal age 15-23 weeks) were incubated with and without 200 ng/ml highly purified hCG, and the production of E2 and T was measured by RIA. Basal T production was high at 15-18 weeks of gestation and decreased thereafter. Estradiol production was low in all testes. Aromatase mRNA (P-450 arom messenger ribonucleic acid) was not detectable in fetal testicular tissues when studied by Northern and dot blot techniques. Placenta and fetal liver expressed aromatase mRNA, but fetal ovary contained only miniscule amounts. HCG significantly stimulated the production of both T and E2 in the testes of older fetuses (19-23 weeks), but the testicular E2 production of the youngest fetuses (15-18 weeks) did not increase significantly after hCG stimulation. These results indicate that aromatase activity and gene expression are very low in human fetal testes. These findings suggest that E2 may not play a major role in testicular T production in the human fetus.     

Testosterone restores testicular inhibin content in cryptorchid rats

The effects of testosterone administration on testicular inhibin content and histology were studied in bilaterally cryptorchid rats, in which a marked decrease in testicular inhibin content had been observed. Mature male Wistar rats weighing approximately 300 g were made bilaterally cryptorchid by placing the testes in the abdominal cavity. Testosterone in oil, 0.1, 1.0 or 10 mg, was given i.m. each week. Testicular inhibin and testosterone content, histology and plasma LH, FSH and testosterone were studied 2 weeks later. Abnormally decreased testicular inhibin in cryptorchidism was restored toward normal by testosterone in a dose dependent manner in 2 weeks after surgery. Sertoli cell structure also recovered toward normal with increasing amount of testosterone. Decreased testicular testosterone content and Leydig cell atrophy were observed with suppressed plasma LH and FSH after testosterone. These results showed that the increased plasma concentration of testosterone had a stimulatory effect on the Sertoli cell function in cryptorchidism, in which compensated Leydig cell failure was demonstrated.     

Prenatal Testosterone Exposure Worsen the Reproductive Performance of Male Rat at Adulthood

The reproductive system is extremely susceptible to environmental insults, for example exogenous steroids during gestational development and differentiation. Experimental induction of androgen excess during prenatal life in female animal models reprograms their reproductive physiology, however the fetal programming of the male reproductive system by androgen excess has not been well studied. We aimed to determine the effect of prenatal exposure of two different doses of testosterone on different gestational days, on the male reproductive system using a rat model. Sixteen pregnant rats were randomly divided into two experimental groups and two control groups. Experimental group І were subcutaneously injected with 3 mg free testosterone on gestational days 16-19 and its controls received solvent for that time; experimental group П were subcutaneously injected with 20 mg free testosterone on day 20 of gestational period and its controls received solvent at the same time. The reproductive system morphology and function of 32 male offspring of these study groups were compared at days 6-30-60 of age and after puberty. The anogenital distance of the male offspring of both experimental groups had no significant differences on the different days of measurement, compared with controls. In the offspring of experimental group І, the testes weight, number of Sertoli, Spermatocyte and Spermatid cells, sperm count and motility and the serum concentration of testosterone after puberty were significantly decreased; except for reduction of sperm motility (p< 0.01), the other effects were not observed in the offspring of experimental group ІІ. In summary, our data show that prenatal exposure of male rat fetuses to excess testosterone disrupted reproductive function, an effect highly dependent on the time, duration and level of exposure. It seems that the reproductive system in individuals exposed to high levels of androgens during fetal life should be evaluated at puberty and likely to be treated.     

FUNCTIONAL REDUCTION OF THE SOUTHERN MINKE WHALE (BALAENOPTERA ACUTOROSTRATA) TESTIS DURING THE FEEDING SEASON

Seasonal variation in reproductive activity in the male southern minke whales was investigated. The animals were killed and collected during the feeding season (December-March) in the Antarctic Ocean in the eastern part of Area III (35°E-70°E, south of 60°S) and Area IV (70°E-130°E, south of 60°S). Blood samples, testes, epididymides, and vasa deferentia were collected from 62 males, which had testes weighing over 400 g each. Changes in testicular morphology and plasma testosterone, estradiol-17β and LH concentrations measured by enzyme immunoassay were investigated. Reduction of testicular function associated with different capture periods was found in the summer season. From December to March, body length and body weight remained steady, but decreases in testicular weight, epididymal weight, and testicular volume were found in February. During the same period, plasma testosterone concentration also declined. A significant decrease in the number of germ cells continued during the period of feeding season. An increase in area of seminiferous tubule tended to proceed in a number of germ cells. These changes reflected the percentage of spermatozoa in the vasa deferentia ; in particular, motile spermatozoa were observed in December. Based on morphological observation, spermatogenesis had also declined. These results indicate regulation of testicular function by testosterone, as in terrestrial mammals. A gradual decrease of testicular function occurred during December-January.     

Androgens in the bovine fetus and dam (38567).

Umbilical arterial and venous blood, and fetal testes were taken from 38 bovine fetuses at 90, 180 or 260 days of gestation. Concurrently blood also was taken from the jugular, and from the uterine artery and vein of the dams. Testosterone and androstenedione were determined by radioimmunoassays. Fetal testicular homogenates had 0.96 and 0.35 mug/g of testosterone and 0.39 and 0.50 mug/g of androstenedione at 180 and 260 days of gestation, respectively. Males had five to tenfold more serum testosterone and about twofold more androstenedione than female fetuses at each trimester of gestation. Male fetal blood testosterone decreased (P less than 0.01) from 2.7 to 0.3 ng/ml between 90 and 260 days of gestation. But, maternal testosterone and androstenedione increased (P less than 0.05) during gestation in cows with males, but not in cows with female fetuses. Testosterone was higher (P less 0.05) in cows carrying males than in cows with female fetuses. Androstenedione was higher in blood leaving the placenta on both the maternal and on the vetal sides suggesting placental synthesis of androstenedione.