Effect of cAMP and FSH on the production of estrogens by rat fetal gonads cultured in vitro

The regulation of aromatase activity by cAMP and FSH has been demonstrated in the prepubertal rat testis and ovary, and the question posed whether this regulation was already operative at foetal stages. In the present study, testes and ovaries from 17- to 21-day-old rat foetuses were cultured in vitro in the presence of [3H]-testosterone and in the presence or absence of cAMP or FSH. Oestrone and oestradiol formed from [3H]-testosterone were measured by double isotopic dilution and recrystallization to constant specific activity. Aromatase activity was augmented in both gonads by cAMP, but only in the testis by FSH. Thus, the regulation of aromatase activity by FSH begins earlier in the testis than in the ovary.     

A novel hypothalamic peptide, pituitary adenylate cyclase activating peptide, modulates Sertoli cell function in vitro.

Pituitary adenylate cyclase-activating peptide (PACAP), a novel hypothalamic peptide that has been shown to exist in several tissues including the testis, was examined for its effects on cultured rat Sertoli cells. PACAP stimulates cAMP accumulation in Sertoli cells cultured from 15-day-old rats in the presence or absence of methylisobutylxanthine, a phosphodiesterase inhibitor, and in the presence of pertussis toxin, a blocker of the adenylate cyclase inhibitory pathway. Maximal stimulation, which is 20-40% of that attainable with FSH, occurs at PACAP concentrations of 10 nM: the ED50 is approximately 100 pM. The ability of PACAP to stimulate Sertoli cell cAMP declines with increasing age of donor animals (15-60 days of age) in a fashion similar to the FSH effect. PACAP stimulation of Sertoli cell cAMP accumulation is additive with submaximal, but not maximal, concentrations of FSH or forskolin. PACAP also stimulates the secretion of lactate, estradiol, and inhibin in a concentration-dependent manner. The stimulation of Sertoli cell cAMP accumulation by PACAP is not altered by a vasoactive intestinal peptide antagonist, and vasoactive intestinal peptide alone does not stimulate cAMP accumulation, indicating that PACAP is not acting via vasoactive intestinal peptide receptors. Further experiments are needed to determine whether PACAP is synthesized within the testis and if so, in which cell types; however, the present data clearly demonstrate that PACAP can modulate Sertoli cell function in vitro.     

cAMP-dependent protein kinases in the rat testis: regulatory and catalytic subunit associations.

Based upon recent reports that the rat testis exhibits mRNAs for cAMP-dependent protein kinase (A-kinase) regulatory (R) subunits RI alpha, RI beta, RII alpha, and RII beta, this study was designed to identify R proteins present in extracts of germ cell-rich testis from adult and Sertoli cell-enriched, germ cell-poor testis from 14-15-day-old rats. Following separation by DEAE-cellulose, R subunits were identified by Mr: (a) upon labeling with 8-N3[32P]cAMP and 32P in an RII phosphorylation reaction and; (b) by Western blot analysis using R-specific antibodies on one- and two-dimensional gel electrophoresis. Elution of R subunits as catalytic (C) subunit-free dimers or in association with C subunits to form holoenzyme was determined by their sedimentation characteristics on sucrose gradient centrifugation in conjunction with their cAMP-stimulated activation characteristics on Eadie-Scatchard analysis. Soluble extracts of testes, from both adult and 14-15 day-old rats, showed the presence of a prominent type I holoenzyme containing RI alpha subunits (47 kDa, peak 1), a minor type II holoenzyme, containing RII beta subunits (52 kDa, peak 2), and a second, more abundant, type II holoenzyme peak containing predominantly RII alpha and, to a lesser extent RII beta subunits (peak 3). The 53 kDa RI beta protein predicted by mRNA studies was only tentatively identified by Western blot analysis. Testes extracts of 14-15-day-old, but not adult, rats exhibited high levels of C subunit-free RI alpha, a result not predicted by mRNA studies. This latter result may be attributable to direct RI alpha regulation or to indirect RII beta regulation at a time during testis development prior to germ cell maturation.     

Anti-apoptotic factor humanin is expressed in the testis and prevents cell-death in leydig cells during the first wave of spermatogenesis

Humanin (HN) is a 24 amino acids peptide with potent neuro-survival properties that protects against damage associated with Alzheimer's disease. In the present report, we have demonstrated by immunohistochemical analysis and Western blotting the pattern of expression of rat humanin (HNr) in the testis of 10- to 60-day-old rats. The Leydig cells of 10- and 40- day-old rats expressed this peptide at high levels; and in the testis of 60-day-old rats the expression of HNr expanded to include Leydig, endothelial, peritubular and germ cells. As monitored by Western blotting, HNr was released into the medium of cultures of Leydig cells isolated from 10-, 40-, and 60-days-old rats. HNr stimulated the incorporation of [(3)H]TdR into DNA of Leydig cells from 10-days-old rats, in a manner that indicated promotion of cell survival rather than an increase in the rate of cell multiplication. This peptide also enhanced steroidogenesis by cultured Leydig cells from 10- to 40-day-old rats both alone and synergistically with IGF-I. The expression of HNr in cultured Leydig cells increased in response to GH and IGF-I. In summary, we demonstrated here that HNr was expressed at all stages of maturation in the rat testis. This peptide promoted the survival of Leydig cells in culture and interacted with IGF-I to stimulate DNA synthesis and steroidogenesis. We propose that HNr is a novel testicular anti-apoptotic factor.     

Intratesticular injection of [D-Met2-Pro5]enkephalinamide suppresses testosterone secretion of the testis of immature rat

The possible role of enkephalin in the local control of testicular function was studied in neonatal rats. 5- and 10-day old hemicastrated rats were treated intratesticularly with an enkephalin analog [D-Met2-Pro5]enkephalinamide. In 5-day-old rats local injection of different doses (0.1-0.3 micrograms/testis) of the peptide suppressed basal testosterone secretion in vitro in a dose-dependent manner 2 h posttreatment. Intratesticular administration of naloxone prior to enkephalin treatment prevented the decrease in basal testosterone production induced by the opioid agonist. In 10-day-old animals intratesticular injection of 1.0 and 3.0 micrograms/testis of enkephalinamide reduced serum testosterone concentration and basal testosterone secretion in vitro. Systemic injection of the peptide produced no change in steroidogenesis. These results suggest that enkephalins might be among the intratesticular factors regulating Leydig cell functions.     

Effect of prostaglandins on ornithine decarboxylase activity in the testis of immature rat

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.     

Effects of N-Acetyl Aspartate, Aspartate, and Glutamate on cAMP and cGMP Levels in Developing Rat Cerebral Cortex

N-Acetyl-aspartate (N-Ac-Asp) incubated with minced cerebral cortex caused a dose-dependent increase in the levels of cAMP and cGMP. This effect was followed during postnatal development. N-Ac-Asp elicits the greatest increase in cAMP in 5-day-old and in cGMP in 40-day-old rats. The levels of cyclic AMP were always higher than those of cGMP. We also studied the effects of L-aspartate (Asp) and L-glutamate (Glu) on the levels of cyclic nucleotides in the cerebral cortex minces of rats different ages, and observed that both amino acids produced the maximum increase in cAMP at 10 days, whereas in the case of cGMP the maximal effect of Asp occurs earlier than 20 days and of Glu after 40 days. In the adult rat, the N-Ac-Asp effect on cAMP was greater than that produced by either Asp or Glu, whereas the levels of cGMP were similarly affected by all three. The data show a peak response of cAMP and cGMP to N-Ac-Asp, Asp, and Glu during cortical maturation. Because this response varies with postnatal time, N-Ac-Asp, and Glu may act upon different receptor sites.     

Retinol binding protein in rat testicular cells

Cellular retinol-binding protein (CRBP) was identified in the cytosols of cultured Sertoli cells and peritubular cells from the testes of 20-day-old rats. CRBP was not detected in spermatids or spermatocytes obtained from the testes of 60-day-old rats. Cultured Sertoli cells and peritubular cells contained up to a 5-fold enrichment of CRBP/mg protein compared to whole testis homogenates. FSH- or FSH + testosterone-treated cultures of Sertoli cells showed a 60% increase in the specific activity of CRBP when compared to untreated cultures.     

Onset of compensatory hypertrophy of interstitial tissue and Leydig cells in rats hemicastrated around the time of puberty

When rats were unilaterally castrated at 20, 30, and 40 days of age, only those rats hemicastrated at 40 days showed compensatory hypertrophy of the interstitial tissue and Leydig cells when killed 30 days after hemicastration. At the time of death, volume densities of interstitial tissue, Leydig cells, and vascular components were greater in 70-day-old hemicastrated rats than in intact rats of the same age. The total number of Leydig cells per testis in hemicastrated and intact rats was always the same at any age. Estimated Leydig cell volume in 70-day-old rats was twice that in intact rats. By contrast, the testes of 50- and 60-day-old rats at the time of death displayed essentially the same morphological features, regardless of whether animals were hemicastrated. The concentration of plasma testosterone was higher in 50-day-old controls than in hemicastrated rats. Seventy-day-old hemicastrated rats showed higher levels of plasma testosterone than controls. The level of plasma dihydrotestosterone in 60- and 70-day-old hemicastrated rats exceeded that in the controls. A significant increase in follicle-stimulating hormone was noted in 50- and 70-day-old hemicastrated rats compared to normal rats, while levels of luteinizing hormone were basically the same. The increase in Leydig cell volume, interstitial tissue volume, vascular component volume, and plasma testosterone level caused by hemicastration at 40 days of age differed from that at 20 and 30 days of age.     

Dose-effect of dietary oleic acid: oleic acid is conditionally essential for some organs

The minimum dietary intake of oleic acid that is indispensable to maintain a normal content of this fatty acid in several tissues (heart, muscle, kidney and testis) was determined in the rat. For this purpose, a dose-effect study was conducted using an experimental protocol with 7 groups of rats who received a diet in which the oleic acid level varied from 0 to 6000 mg per 100 g diet, but the other ingredients were identical (in particular the essential fatty acids, linoleic and alpha-linolenic acid). Female rats were fed the diets from two weeks before mating, and their pups were killed aged either 21 or 60 days. When the level of oleic acid in the diet was increased, the main modifications observed in 21-day-old deficient pups were as follows: (i) for 18:1n-9, in the liver, muscle, heart, kidney, and testis, a plateau was reached at about 4 g oleic acid per 100 g diet. Below this level, the higher the dose the greater the response; (ii) for 16:1n-7, the concentration decreased in the liver, muscle, heart, kidney and testis; (iii) the concentration of 18:1n-7 decreased in the kidney, muscle, and testis; (iv) some minor modifications were noted for the other fatty acids. In mother's milk at 14 days of lactation, when dietary oleic acid increased, the levels of 18:1(n-9) also increased; the increase was regular and did not reach a plateau. In 60-day-old rats, the results were generally similar to those in 21-day-old rats, but with some differences, in particular a slight decrease in oleic acid concentration in the liver and kidney at the highest dietary oleic acid level.     

Action of LH, FSH and (Bu)2 cAMP on the conversion of [3H]19-hydroxyandrostenedione into oestrogens by foetal and infantile rat ovaries in organ culture

Ovaries from 18-21-day-old foetal as well as from 2-10-day-old infantile rats were cultured in vitro in the presence of [3H]19-hydroxyandrostenedione and in the presence or absence of LH, FSH or (Bu)2 cAMP, and oestrone and oestradiol formed were determined by double isotopic dilution and recrystallization to constant specific activity. In foetal ovaries, the stimulation factor with FSH was 0.9-1.3, which was considered insignificant in comparison with the 8-13-fold stimulation obtained with (Bu)2 cAMP. At infantile stages, aromatase activity was stimulated 1.3-3.5-fold, which was close to the 3.9-fold stimulation obtained with (Bu)2cAMP. LH was ineffective at both foetal and infantile stages.     

Development of the inhibitory guanine nucleotide-binding regulatory protein in the rat testis

The functional development of the inhibitory guanine nucleotide-binding regulatory protein (Gi) and anti-diuretic hormone (ADH) activity was investigated in rat testes. Adult (90-day-old), adolescent (40-day-old), prepubertal (23-day-old), and fetal (20.5 days of gestation) testis cells were cultured with 100 ng/ml pertussis toxin for 24 h. The cells were then cultured with human chorionic gonadotropin (hCG), the ADH agonist arginine vasotocin (AVT), or a combination of the two. Testis cells from rats 23, 40, and 90 days of age that were incubated with hCG increased testosterone production when compared with controls. Preincubation of the cells from postnatal rats with pertussis toxin significantly increased hCG-stimulated testosterone secretion when compared to cells preincubated in medium only at all three ages. AVT suppressed hCG-stimulated testosterone secretion, but this suppression was partially reversed in cells from all postnatal ages preincubated with pertussis toxin. Fetal testis cells showed no response to preincubation with pertussis toxin, even when levels were increased to 400 ng/ml or when pertussis toxin treatment was continued throughout the culture period. AVT also had no effect on fetal testis cells. These results indicate that the Gi protein and AVT are not functional in fetal testes but are active from prepubertal stages of development through maturity.     

Formation of 5alpha-reduced C19 steroids from progesterone in vivo by 5alpha-reduced pathway in older prepubertal rat testis.

Either [3H] progesterone (0.5 or 5 nmol/5 muCi), 5alpha-[3H] pregnane-3,20-dione (5 nmol/5 muCi) or [14C] progesterone (6.6 nmol/0.2 muCi) plus 5alpha-[3H]-pregnane-3,20-dione (1 or 6.6 nmol/0.6 muCi), suspended in 0.05 ml of physiological saline solution, was injected into each testis of 32- and 90-day-old rats. Following injection, radioactive metabolites in testis and spermatic vein blood were extracted, isolated, measured and identified by column and paper chromatographies, with derivative formation and recrystallization to constant specific activity. In the blood and testis of older prepubertal rats, major 17-OH-C21 and C19 metabolites of progesterone were 5alpha-reduced steroids such as 3alpha, 17alpha-dihydroxy-5alpha-pregnan-20-one, 5alpha-androstane-3alpha,17beta-diol and androsterone. Following injection of [14C] progesterone plus 5alpha-[3H] pregnane-3,20-dione into 32-day-old rat testis, no significant augmentation of the isotope from progesterone was observed in 5alpha-reduced C19 steroids as compared with 5alpha-reduced 17-OH-C21 steroids, indicating that 5alpha-reduced C19 steroids were mainly formed from 5alpha-reduced 17-OH-C21 steroids in older prepubertal testis. In the blood and testis of adult rats, small amounts of 5alpha-reduced metabolites were shown to be produced from progesterone, while active 17alpha-hydroxylation of 5alpha-pregnane-3,20-dione followed by C17-C20-lyase reaction was demonstrated. These findings seem to indicate that formation of 5alpha-reduced C19 steroids from progesterone by the 5alpha-reduced pathway is a major pathway of androgen biosynthesis in older prepubertal rat testis in vivo.     

Effect of intratesticular administration of TRH or anti-TRH antiserum on function of rat testis

The effect of intratesticular administration of thyrotropin-releasing hormone (TRH) and anti-TRH antiserum on steroidogenesis was studied in immature and adult rats. In 9-day-old animals local administration of the neuropeptide resulted in an increase in basal testosterone secretion in vitro. Similar treatment of 15-day-old rats suppressed hCG-stimulated testosterone secretion with no change in basal testosterone production. In both immature groups the treatment did not affect serum testosterone concentration. By contrast, in adults TRH decreased serum testosterone level, but did not influence basal and hCG-stimulated testosterone secretion. Both in immature and adult rats, the changes in steroidogenesis were evident 1 hour posttreatment. Five days after the administration of anti-TRH antiserum into the remaining testis of immature rats subjected to hemicastration just prior to the antiserum treatment, the alterations in steroidogenesis were opposite to those detected after treatment with TRH. In 9-day-old rats the antiserum suppressed steroidogenesis, while in 15-day-old animals it stimulated testosterone secretion. The results suggest that testicular TRH might exert a local action on testicular steroidogenesis, and the effect is age-dependent.     

Neonatal hypothyroidism alters the localization of gap junctional protein connexin 43 in the testis and messenger RNA levels in the epididymis of the rat

The objectives of this study were to determine the effects of propylthiouracil (PTU)-induced neonatal hypothyroidism on the gap junctional protein Cx43 in rat testis and epididymis. PTU (0.02%) was administered via lactation from birth to Day 30, and the rats were sampled at 14, 18, 22, 26, 30, and 91 days of age. Testicular Cx43 was localized along the plasma membranes and cytoplasm of Sertoli cells until Day 22. At Day 30, the immunostaining was localized exclusively along the plasma membrane of Sertoli cells. In PTU-treated rats, Cx43 did not localize to the plasma membrane and was still cytoplasmic at 30 days of age. Occludin was present in tubules of treated rats, but was not localized to the blood-testis barrier in 30-day-old rats, as in controls. There were no differences in Cx43 immunostaining in the adult testis. In the proximal epididymis (initial segment, caput, corpus), Cx43 mRNA levels were lower in PTU-treated rats at 14, 18, and 22 days of age, but no differences were observed in the distal (cauda) epididymis at these ages. In 22- and 30-day-old rats, Cx43 was localized along the plasma membrane between principal and basal cells throughout the epididymis. In PTU-treated rats, Cx43 was not detectable in initial segment, caput, or corpus epididymidis. In the cauda epididymidis, however, Cx43 immunostaining in PTU-treated rats was similar to controls. These data suggest that thyroid hormones regulate Cx43-dependent gap junctional communication in the testis and epididymis.     

An inhibin-like factor in the testes of fetal and newborn rats

A factor of protein nature that inhibits the proliferation of gonocytes and, probably, their ingress into meiosis and stimulates the development of some components of the blood-testicular barrier was isolated from the testis of 19-20-day-old fetuses and young rats of the first two weeks of life. It is assumed that the inhibin-like factor is formed in the testicular network. The time at which the receptors for the factor under discussion may occur in the cells of the testis of Wistar rat fetuses is discussed.     

Adrenomedullin inhibits the contraction of cultured rat testicular peritubular myoid cells induced by endothelin-1

The present study documents that adrenomedullin (AM), a vasoactive peptide originally identified in pheochromocytoma tissue and present in the testis, in vitro affects the function of testicular peritubular myoid cells (TPMC), a contractile cell type located in the seminiferous tubule wall. AM stimulated cAMP production by cultured TPMC taken from 16-day-old rats, and this effect was completely inhibited by the AM antagonist AM-(22-52) and partially by the CGRP (calcitonin gene-related peptide) antagonist CGRP-(8-37). Studies on TPMC contractile activity documented that AM inhibits TPMC contraction induced by endothelin-1 (ET-1) and that its effect is antagonized by AM-(22-52). Neutralizing AM produced by TPMC with the addition of anti-AM antibody induced a significant increase of ET-1-induced contraction. When exposed to the protein kinase A inhibitor H-89, AM inhibitory activity on ET-1-induced TPMC contraction was suppressed, whereas the nitric oxide synthase inhibitor N:(G)-nitro-L-arginine methyl esther did not modify AM activity. In conclusion, our study indicates that AM stimulates cAMP production and inhibits the contraction induced by ET-1 in TPMC in vitro, and that AM produced by TPMC has an autocrine effect. We propose that AM may have a role in the control of seminiferous tubule contraction.     

Localization and age-dependent expression of the inward rectifier K+ channel subunit Kir 5.1 in a mammalian reproductive system.

Kir 5.1 is a member of the inward rectifier potassium channel superfamily which does not form functional channels when expressed by itself in Xenopus laevis oocytes. rt-PCR reveals high levels of Kir 5.1 mRNA expression in testis but the function of this channel remains unknown. To determine the cell-specific expression of this channel in the testis we raised a polyclonal antibody against an external epitope of Kir 5.1 and tested its specificity in Xenopus oocytes expressing several cloned Kir subunits. Strong immunoreactivity for Kir 5.1 was found in seminiferous tubules of rat testis and, particularly, in spermatogonia, primary and secondary spermatocytes, spermatids and in the head and body of spermatozoa. The intensity of Kir 5.1 immunofluorescence, quantified using laser scanning microscopy, increased with age at every stage in the development of sperm from spermatogonia and reached a peak in 60-day-old rats. In contrast, the immunofluorescence decreased in 90-day-old animals and was detected mostly in spermatozoa. The results demonstrate that Kir 5.1 expression in the testis is localised to cells involved in spermatogenesis, showing a temporal pattern of expression during sexual maturity.     

The hormonal regulation of enzymes in prenatal and postnatal rat liver. Effects of adenosine 3′,5′-(cyclic)-monophosphate

1. The administration of glucagon, cAMP [adenosine 3',5'-(cyclic)-monophosphate], BcAMP [6-N-2'-O-dibutyryladenosine 3',5'-(cyclic)-monophosphate] or adrenaline to foetal rats during the last 2 days of gestation evoked the appearance of tyrosine aminotransferase and enhanced the accumulation of glucose 6-phosphatase in the liver. In foetuses 1-2 days younger only BcAMP was effective. After birth liver glucose 6-phosphatase no longer responds to glucagon or BcAMP. Tyrosine aminotransferase is still inducible by these agents in 2-day-old rats, but not in 50-day-old rats. After adrenalectomy of adults glucagon or BcAMP can enhance the induction of the enzyme by hydrocortisone. The results indicate that the ability to synthesize tyrosine aminotransferase and glucose 6-phosphatase when exposed to cAMP develops sooner than the ability to respond to glucagon with an increase in the concentration of cAMP; the responsiveness of enzymes to different hormones changes with age. A scheme illustrating the sequential development of competence in regulating the level of an enzyme is presented. 2. Actinomycin inhibited the effects of glucagon and BcAMP on liver tyrosine aminotransferase and glucose 6-phosphatase in foetal rats. Growth hormone, insulin and hydrocortisone did not enhance the formation of these enzymes. 3. The time-course of accumulation of glucose 6-phosphatase in the kidney is different from that in the liver. Hormones that increase the accumulation in foetal liver do not do so in the kidney of the same foetus or in the livers of postnatal rats.