Receptors In Rat Testis Research Articles

Role of leptin and the leptin receptor in the pathogenesis of varicocele-induced testicular dysfunction.

The present study investigated the expression of leptin and its receptor in the left testis and hypothalamus of rats with varicocele and clarified their roles in the pathogenesis of varicocele-induced testicular dysfunction. A total of 40 male rats were divided randomly into four groups. Groups 1 (G1) and 3 (G3) underwent a sham operation. Groups 2 (G2) and 4 (G4) underwent operations to form a varicocele created by partial ligation of the left renal vein. G1 and G2 rats were euthanized 4 weeks after the operation while G3 and G4 rats were euthanized at 8 weeks. The expression of leptin and its receptor was analyzed by immunohistochemistry. The mRNA levels of leptin, its receptor, kisspeptin (KiSS-1), G-protein coupled receptor 54 (GPR54), gonadotropin releasing hormone (GnRH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were measured by reverse transcription-quantitative polymerase chain reaction. Testicular spermatogenesis function and gonadal hormone levels were measured. Compared with G1 and G3, the expression of leptin and its receptor in rat testis was significantly higher in G2 and G4, respectively. Leptin expression was inversely associated with the number of sperm in the left epididymis, thickness of the seminiferous epithelium and the diameter of seminiferous tubules. The expression of leptin receptors in the hypothalamus of G2 and G4 was significantly increased compared with that in G1 and G3, respectively. The mRNA levels of KiSS-1, GPR54, GnRH, LH and FSH in G2 and G4 were significantly increased compared with that in G1 and G3, respectively. Serum testosterone levels in G2 and G4 rats were significantly lower than those in G1 and G3 rats, respectively. There was no significant difference between the serum levels of FSH, LH and leptin. These results suggest that leptin and its receptor may serve significant roles in the pathogenesis of varicocele-induced testicular dysfunction.

Hormonal regulation of testicular LH/hCG receptors in rat.

A single injection of 60 IU of hCG to 25-day-old rats led to a loss of LH/hCG receptors in the testis. The content of [125I] hCG binding sites reached a nadir 2 days after hCG injection. At this time the effect of daily treatment of hormones and other agents on the formation of LH/hCG receptors was studied. Gonadotropin receptors were estimated 2, 4, 6, 8 and 10 days after injection of hCG. Daily treatment of rats with PMSG (30 IU/day) increased testicular content of hCG binding sites. The presence of the pituitary in these animals was necessary during the formation of receptors after hCG-induced loss of binding sites. LH/hCG receptor content remained low in rats treated with testosterone (250 microgram/day), hCG (100 IU/day) or inhibitors of the synthesis and the secretion of prolactininhibitors-bromocryptine (250 microgram/day), ergocryptine (200 microgram/day) and deprenon (50 and 600 micrograms/day). Administration of cortisol (1 mg/day) had no effect on restoration of LH/hCG receptors. These data suggest an important role for FSH and prolactin in the formation of LH/hCG receptors in rat testis.

Presence of luteinizing hormone receptor binding inhibitor (LH-RBI) in ovine testis.

Luteinizing hormone receptor binding inhibitor (LH-RBI has been isolated from the aqueous extracts of ram testis using Sephadex column chromatography. Sephadex G-75 fraction I was found to inhibit the binding of I125 LH to rat testis receptors. Further purification of Sephadex G-75-I fraction on G-200 column gave four fractions (I-IV), the maximum inhibitory activity to inhibit I125 LH binding to rat testis receptor was associated with the fraction I only. Fraction II gave marginal inhibition only, whereas fractions III and IV did not have any inhibitory effect on I125 LH binding to rat testis receptors. Our findings suggest that LH-RBI isolated from ram testis is a protein having molecular weight more than 10,000 Daltons.

Studies on low molecular weight follicle stimulating hormone receptor binding inhibitor (FSH-RBI) from ovine testis.

Follicle stimulating hormone receptor binding inhibitor (FSH-RBI) has been isolated from the aqueous extracts of ovine testis using Sephadex column chromatography. Sephadex G-75 fraction IV was found to inhibit the binding of (125I) FSH to rat testis receptors. Further purification of Sephadex G-75 fraction on Sephadex G-25 column gave three fractions (I-III). The maximum inhibitory activity to inhibit (125I) FSH binding to rat testis receptor was associated with fraction III only. FSH-RBI exerted a greater inhibitory effect on the formation of the hormone-receptor complex rather than on the dissociation of the preformed complex. FSH-RBI did not inhibit the binding of (125I) LH to rat testis receptors. A significant decrease in the mouse ovarian weight was observed when FSH-RBI was injected to hCG-primed female mice. The molecular weight determination studies show that molecular weight of FSH-RBI is approximately 1400 Daltons.

Expression of thyrotropin-releasing hormone receptors in rat testis and their role in isolated Leydig cells

Thyrotropin-releasing hormone (TRH) was initially discovered as a neuropeptide synthesized in the hypothalamus. Receptors for this hormone include TRH-receptor-1 (TRH-R1) and -2 (TRH-R2). Previous studies have shown that TRH-R1 and TRH-R2 are localized exclusively in adult Leydig cells (ALCs). We have investigated TRH-R1 and TRH-R2 expression in the testes of postnatal 8-, 14-, 21- 35-, 60-, and 90-day-old rats and in ethane dimethane sulfonate (EDS)-treated adult rats by using Western blotting, immunohistochemistry, and immunofluorescence. The effects of TRH on testosterone secretion of primary cultured ALCs from 90-day-old rats and DNA synthesis in Leydig cells from 21-day-old rats have also been examined. Western blotting and immunohistochemistry demonstrated that TRH-R1 and TRH-R2 were expressed in fetal Leydig cells (in 8-day-old rats) and in all stages of adult-type Leydig cells during development. Immunofluorescence double-staining revealed that newly regenerated Leydig cells in post-EDS 21-day rats expressed TRH-R1 and TRH-R2 on their first reappearance. Incubation with various doses of TRH affected testosterone secretion of primary cultured ALCs. Low concentrations of TRH (0.001, 0.01, and 0.1 ng/ml) inhibited basal and human chorionic gonadotrophin (hCG)-stimulated testosterone secretion of isolated ALCs, whereas relatively high doses of TRH (1 and 10 ng/ml) increased hCG-stimulated testosterone secretion. As detected by a 5-bromo-2'-deoxyuridine incorporation test, the DNA synthesis of Leydig cells from 21-day-old rats was promoted by low TRH concentrations. Thus, we have clarified the effect of TRH on testicular function: TRH might regulate the development of Leydig cells before maturation and the secretion of testosterone after maturation.

Identification and expression of GABACreceptor in rat testis and spermatozoa

Our previous studies showed that gamma-aminobutyric acid (GABA)A and GABAB receptors are involved in rat sperm acrosome reaction induced by progesterone or GABA. Here, we report the presence of GABAC receptor in rat testis and spermatozoa. Full-length complementary DNA encoding the rho1, rho2 and rho3 subunits of GABAC receptor were cloned from rat testis; their sequences are identical to those of rat GABAC receptor in retina. Reverse transcription-polymerase chain reaction analysis showed that during the development of rat testis, the transcript levels of the rho1 and rho2 subunits showed little change, while the expression of rho3 was gradually up-regulated. Immunofluorescence analysis using an anti-rho1 antibody revealed that GABAC receptor exists on the elongated spermatid and sperm. Using a chlortetracycline assay, we found that N(4)-chloroacetylcytosine arabinoside, a GABAC receptor agonist, triggered rat sperm acrosome reaction; whereas (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid, a GABAC receptor antagonist, inhibited the ability of N(4)-chloroacetylcytosine arabinoside to induce acrosome reaction. These results suggested that GABAC receptors are also involved in rat sperm acrosome reaction.

Identification and expression of GABA<sub>C</sub> receptor in rat testis and spermatozoa

Identification and expression of GABA<sub>C</sub> receptor in rat testis and spermatozoa

Novel identification of peripheral dopaminergic D2 receptor in male germ cells

Dopamine is a recognized modulator in the central nervous system (CNS) and peripheral organ functions. The presence of peripheral dopamine receptors outside the CNS has suggested an intriguing interaction between the nervous system and other functional systems, such as the reproductive system. In the present study we analyzed the expression of D2R receptors in rat testis, rat spermatogenic cells and spermatozoa, in different mammals. The RT-PCR analysis of rat testis mRNA showed specific bands corresponding to the two dopamine receptor D2R (L and S) isoforms previously described in the brain. Using Western blot analysis, we confirmed that the protein is present in rat testis, isolated spermatogenic cells and also in spermatozoa of a range of different mammals, such as rat, mouse, bull, and human. The immunohistochemistry analysis of rat adult testis showed that the receptor was expressed in all germ cells (pre- and post-meiotic phase) of the tubule with staining predominant in spermatogonia. Confocal analysis by indirect immunofluorescence revealed that in non-capacitated spermatozoa of rat, mouse, bull, and human, D2R is mainly localized in the flagellum, and is also observed in the acrosomal region of the sperm head (except in human spermatozoa). Our findings demonstrate that the two D2 receptor isoforms are expressed in rat testis and that the receptor protein is present in different mammalian spermatozoa. The presence of D2R receptors in male germ cells implies new and unsuspected roles for dopamine signaling in testicular and sperm physiology.

Identification and characterization of a novel splice variant of β3 subunit of GABA A receptor in rat testis and spermatozoa

γ-Aminobutyric acid type A (GABA A) receptors are the major sites of inhibitory action of fast synaptic neurotransmission in the brain. Their receptors are also widely distributed in peripheral and endocrine tissues. A full-length cDNA encoding a novel splice variant of β3 subunit of GABA A receptor, designated as β3t, was identified in rat testis. This isoform contains a segment, having identical amino acid sequence as the β3 subunit of neuronal GABA A receptors except for a section composed of 25 different amino acid sequence in the N-terminus. Northern blot shows that this isoform is found in rat testis. The β3t isoform mRNA was detected in germ cells in the late step of spermatogenesis by in situ hybridization assay. Results of immunohistochemical and immunocytochemical assays indicate that the β3t isoform is expressed in rat testis and spermatozoa. To determine a possible function of the N-terminal 25 amino acid segment, a recombinant plasmid of β3t-EGFPC was constructed by fusing green fluorescent protein to the C-terminus of the β3t isoform. The chimera product failed to be translocated unto the cell surface when expressed in HEK 293 cells; whereas, the β3 subunit of rat brain is incorporated into the plasma membrane. In conclusion, the present results show that one variant of β3 subunit of GABA A receptor, designated as β3t, is found in germ cells of rat testis and sperm. The inability of the β3t variant to target into the plasma membrane maybe a consequence of the unique 25 amino acid segment in the N-terminus.

Expression of tumor necrosis factor-alpha-related apoptosis-inducing ligand and its receptors in rat testis during development.

Tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor-alpha family of cytokines that is known to induce apoptosis upon binding to its death domain-containing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2. Two additional TRAIL receptors, DcR1/TRAIL-R3 and DcR2/TRAIL-R4, lack functional death domains and act as decoy receptors for TRAIL. In this study, the presence of TRAIL and its receptors was investigated in the rat testis during development. TRAIL and its receptors were immunolocalized to the different testicular cell types. TRAIL and its receptors were also identified in the rat testis in terms of protein and mRNA. Our immunohistochemical studies indicate that TRAIL, DR5/TRAIL-R2, and DcR2-TRAIL-R4 are detected in Leydig cells, whereas ligand and all receptors are localized in germ cells. TRAIL was permanently immunodetected in germ cells from the fetal stage to adulthood, whereas its receptors were immunolocalized exclusively in postmeiotic germ cells. The expression of TRAIL and receptor mRNAs was consistent with the immunodetection of TRAIL and receptor proteins. Indeed, TRAIL ligand mRNA was also identified in the rat testis from the fetal stage to adulthood. The mRNAs of the death receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2, were weakly detected during the perinatal period and increased from the pubertal stage to adulthood. The mRNAs of the decoy receptors, DcR1 and DcR2, were present in the rat testis at all ages studied, but the DcR2/TRAIL-R4 mRNa level was higher from the pubertal period to adulthood. Together, the present findings demonstrate that 1) TRAIL and its receptors are expressed in the testis during normal development, and 2) TRAIL protein is present in the different germ cell types, whereas its receptors were predominantly detected in the postmeiotic germ cells.

Identification of gamma1 subunit of GABA(A) receptor in rat testis.

The isoform type of gamma subunits of GABA(A) receptor is a molecular determinant of its pharmacological characteristics. At present, the existence of GABA(A) receptor in mammalian sperm is still a controversy. By using degenerate primers designed according to highly conserved region in all three gamma (gamma1, gamma2 and gamma3) subunits cloned in rat brain, we performed reverse transcription polymerase chain reaction (RT-PCR) to examine the expression pattern of gamma subunits of GABA(A) receptor in rat testis. Only one 370 bp fragment was obtained from RT-PCR in rat testis and sequencing results showed that it represented gamma1 subunit, but not gamma2 or gamma3 subunit. Using the cloned fragment as probe, a 3.8 kb transcript which in size as same as gamma1 subunit in rat brain was detected in rat testis mRNA by performing Northern blot assay. Furthermore, results of in situ hybridization assay confirmed that gamma1 subunit was expressed in round spermatids and spermatozoa, maybe also in secondary spermatocyte. These evidences proved that gamma1 subunit of GABA(A) receptor is exclusively expressed in rat testis and this feature may be the structural basis of the specific function of GABA(A) receptors in sperm acrosome reaction.

Novel expression and functional role of ghrelin in rat testis.

Ghrelin, the endogenous ligand for the GH-secretagogue receptor (GHS-R), is a recently cloned peptide, primarily expressed in the stomach and hypothalamus, that acts at central levels to elicit GH release and, notably, to regulate food intake. However, the possibility of additional, as yet unknown, peripheral effects of ghrelin cannot be ruled out. In the present communication, we provide evidence for the novel expression of ghrelin and its functional receptor in rat testis. Testicular ghrelin gene expression was demonstrated throughout postnatal development, and ghrelin protein was detected in Leydig cells from adult testis specimens. Accordingly, ghrelin mRNA signal became undetectable in rat testis following selective Leydig cell elimination. In addition, testicular expression of the gene encoding the cognate ghrelin receptor was observed from the infantile period to adulthood, with the GHS-R mRNA being persistently expressed after selective withdrawal of mature Leydig cells. From a functional standpoint, ghrelin, in a dose-dependent manner, induced an average 30% inhibition of human CG- and cAMP-stimulated T secretion in vitro. This inhibitory effect was associated with significant decreases in human CG-stimulated expression levels of the mRNAs encoding steroid acute regulatory protein, and P450 cholesterol side-chain cleavage, 3beta-hydroxy steroid dehydrogenase, and 17beta-hydroxy steroid dehydrogenase type III enzymes. Overall, our data are the first to provide evidence for a possible direct action of ghrelin in the control of testicular function. Furthermore, the present results underscore an unexpected role of ghrelin as signal with ability to potentially modulate not only growth and body weight homeostasis but also reproductive function, a phenomenon also demonstrated recently for the adipocyte-derived hormone, leptin.

Expression of neurotrophin receptors in rat testis. Upregulation of TrkA mRNA with hCG treatment

We report the expression of TrkA, TrkB and TrkC mRNAs in adult rat testis. With in situ hybridisation a low signal for TrkB and TrkC could be seen in postmeiotic cells of the seminiferous epithelium, whereas no signal for TrkA could be observed in untreated animals. Animals treated with hCG showed an induction of TrkA mRNA in premeiotic cells 12 h after the treatment, whereas an injection with EDS had no effect on the expression of Trk mRNAs. With the RNAse protection assay a low signal for TrkA was seen in whole testis of hCG treated animals. In staged tubules low expression was seen at stages VII–XI of untreated animals. Animals injected with hCG revealed that TrkA induction was highest during stages VIIcd and VIII of the cycle. The distinct expression pattern of these high-affinity neurotrophin receptors suggests different roles for neurotrophins during spermatogenesis. Induction of TrkA mRNA by hCG suggests that high-affinity binding of NGF during stages VIIcd–VIII in premeiotic cells is under control of the hypothalamic–pituitary–testicular axis.

Identification of GABAB Receptor in Rat Testis and Sperm

γ-Aminobutyric acid (GABA) can mimic and potentiate the action of progesterone in initiating the acrosome reaction (AR) of mammalian sperm, indicating that sperm contain receptors for GABA. This contention was validated by identifying the receptor (R) subtype, GABAAR, in mammalian sperm. In the present study a second subtype, GABABR, was identified in rat testis and sperm. Total RNAs of rat testis and sperm were prepared and used as template to synthesize the respective cDNAs by the RT-PCR method. Two splice variants of the cDNA coding GABABR1 (GABABR1a and GABABR1c) and GABABR2 were identified. Extracts of rat testis, spermatogenic cells and sperm contained two proteins with estimated molecular sizes of 130 and 100 kDa, corresponding to GABABR1a and GABABR1c/lb, respectively, determined by Western blot using polyclonal anti-GABABR1 antibody. By an indirect immunofluorescence technique, GABABR1 was located on the head of rat sperm. The present finding is the first direct demonstration that mammalian sperm contain GABABR.

The effects of diabetes on the expression of LH/CG receptor in rat testis

The effects of diabetes on the expression of LH/CG receptor in rat testis

Cellular and subcellular localization of six retinoid receptors in rat testis during postnatal development: identification of potential heterodimeric receptors.

Vitamin A is required in the testis for germ cell development. It acts through two families of retinoid receptors, retinoic acid receptors (RAR) and retinoid X receptors (RXR), each with three subtypes alpha, beta, and gamma. These receptors are postulated to dimerize and regulate the transcription of retinoid-responsive genes that are crucial for germ cell development. In this study, we determined the cellular and subcellular localization of six retinoid receptors in the developing rat testis to identify the specific cellular sites and times of receptor expression. Immunohistochemical results revealed the expression of RARalpha, RARbeta, RXRalpha, and RXRgamma proteins in somatic and germ cells throughout postnatal development. In contrast, the expression of RARgamma and RXRbeta did not increase until 30-35 days of age in somatic cells from the testis. Interestingly, RARalpha and RXRalpha had a similar subcellular localization pattern in Sertoli cells throughout postnatal testis development, while RARalpha and RXRgamma were both present in the nucleus of spermatocytes and elongating spermatids. These results suggest that RARalpha may potentially dimerize with RXRalpha in Sertoli cells and with RXRgamma in germ cells. In addition, we demonstrate that the only RAR in the nucleus of early meiotic germ cells is RARalpha.

Ligand-Dependent Regulation of Retinoic Acid Receptor in Rat Testis: In Vivo Response to Depletion and Repletion of Vitamin A

Male animals are sterile due to testicular degeneration in the absence of retinoic acid (RA) or functional retinoic acid receptor-α (RARα). This degeneration can be reversed by injecting retinol, a precursor of RA, into vitamin A-deficient (VAD) rats. To determine the relationship between this ligand-dependent testicular degeneration and regeneration and the expression levels of RARα messenger RNA and protein, testes were depleted and then replenished with retinol in vivo. Results showed that RARα messenger RNA and protein levels declined to VAD amounts after 7 weeks on a VAD diet. This decline was due to decreased RARα levels in early meiotic spermatocytes and the loss of advanced germ cells. Interestingly, the advanced germ cells still contained RARα, but the protein was primarily cytoplasmic instead of nuclear, indicating inactivity as a transcription factor. In VAD testis, RARα levels were low and then increased primarily in Sertoli cells after retinol replenishment. TUNEL analyses showed that most germ cells at the basal aspect of seminiferous tubules were undergoing apoptosis during degeneration. These results indicate that RARα is either down-regulated or inactivated in RA-deficient testis and coincident with that, testes degenerate by apoptosis or selective loss of germ cells.

Identification and characterization of inositol 1,4,5-trisphosphate receptors in rat testis

PCR analysis and immunoblotting with isoform specific antibodies was used to identify the presence of type I, II and III inositol 1,4,5-trisphosphate receptors (InsP 3Rs) in rat testis. PCR analysis also revealed that rat testis express both forms of the S1 splice variant (S1 + and S1 −), but only the S2 − form of the S2 splice variant of the type I InsP 3 receptor. PCR analysis was also used to identify InsP 3R isoform expression at a cellular level using myoid, Sertoli and germ cells derived from the testis of Wistar rats. The extent of [ 3H]-Ins P 3 binding was found to be 9 times lower for testicular microsomes than for cerebellar microsomes, with a B max of 1.4 pmoles/mg protein compared to 12.5 pmoles/mg protein for cerebellar microsomes. The K d for InsP 3 binding to its receptor in testicular microsomes was 60 ± 10 nM which was similar to that found for cerebellar microsomes (80 ± 20 nM). InsP 3-induced Ca 2+ release (IICR) in testicular microsomes was found to have an EC 50 (concentration which causes a half-maximal response) of 0.5 ± 0.03 μM, also similar to that seen for cerebellar microsomes (0.3 μM). Maximal IICR occurred at about 20 μM InsP 3, with up to 4% of total intracellular Ca 2+ stores being mobilized as compared to between 10–30% for cerebellar microsomes. Time resolved IICR using stopped-flow spectrofluorimetry, showed the kinetics of IICR for this testis preparation to be monophasic with a maximum rate constant of 0.15 s −1 at 30 μM InsP 3. The rate constants are 7 times slower than values for cerebellar microsomes under similar conditions (∼1 s −1) and taken together with the binding data support the proposal that the receptor density/Ca 2+ store is ∼8 times lower than seen in cerebellar microsomal vesicles. The pharmacological properties as assessed using heparin and InsP 3 analogues also confirmed similar behaviour for testicular InsP 3Rs and cerebellar InsP 3Rs.

Effect of experimental cryptorchidism on alpha 1-adrenergic receptors of rat testis.

Alpha 1-adrenergic receptors play an important role in the transport of spermatozoa through seminiferous tubules to the efferent ducts leading to the epididymis. The density of alpha 1-adrenergic receptors in adult Sprague-Dawley rat testis before and during 14 days of experimental cryptorchidism was determined by binding assays with [3H]prazosin in membrane preparations. The number of maximal binding sites (Bmax) per milligram protein began to increase at 7 days after surgery (p < .05). The testicular weight and diameter of seminiferous tubules began to decrease at 5 days after surgery (p < .01). However, Bmax per testis did not differ before and during cryptorchidism. This study demonstrates that the density of alpha 1-adrenergic receptors in rat testis is not affected by cryptorchidism.

Sertoli and Leydig cell numbers and gonadotropin receptors in rat testis from birth to puberty

In testes of rats from 2 to 60 days of age, we examined the number of Sertoli cells (SC) and Leydig cells (LC) as well as the binding of radioiodinated gonadotropins to frozen sections and homogenates. The number of SC per testis increased only during the first 2 postnatal weeks, whereas that of LC was stable up to days 7-10 and increased thereafter. The uptake of 125I-labelled human follicle-stimulating hormone (125I-FSH) to frozen sections was confined to sex cords or seminiferous tubules, while that of 125I-labelled human choriogonadotropin (125I-hCG) matched the distribution of LC in the interstitium. High affinity receptors for FSH and hCG were found in homogenates at all stages studied. The number of FSH receptors per testis increased steadily, whereas that of hCG receptors was low until days 7-10 and rose afterwards. Thus, SC in rat testis appear to proliferate in the presence of fetal LC during the first 2 postnatal weeks and to differentiate concomitantly with the emergence of the adult LC generation after day 10. The complement of FSH receptors in SC remains constant as they proliferate and increases after day 21 as they differentiate. The hCG receptor number is relatively fixed in each LC generation, being higher in adult compared to fetal LC.