Impaired reproduction in transgenic mice overexpressing γ-aminobutyric acid transporter Ⅰ (GAT1)

It is well documented that γ-aminobutyric acid (GABA) system existed in reproductive organs. Recent researches showed that GABA_A and GABA_B receptors were present in testis and sperm, and might mediate the acrosome reaction induced by GABA and progesterone. GABA transporter Ⅰ (GAT1) also existed in testis and sperm, but its physiological function was unknown. In the present study, we used GAT1 overexpressing mice to explore GAT1 function in male reproductive system. We found that the expression level of GAT1 continuously increased in wild-type mouse testis from 1 month to 2 months after birth. GAT1 overexpression in mouse affected testis development, which embodied reduced testis mass and slowed spermatogenesis in transgenic mice. Moreover, transgenic mice showed increase of the percentage of broken sperm. The further study revealed that the reproductive capacity was impaired in GAT1 overexpressing mice. In addition, testosterone level was significantly low in transgenic mice compared with that in wi     

Impaired reproduction in transgenic mice overexpressing γ-aminobutyric acid transporter I （GAT1）

It is well documented that 7-aminobutyric acid (GABA) system existed in reproductive organs. Recent researches showed that GABAA and GABAB receptors were present in testis and sperm, and might mediate the acrosome reaction induced by GABA and progesterone. GABA transporter I (GAT1) also existed in testis and sperm, but its physiological function was unknown. In the present study, we used GAT1 overexpressing mice to explore GAT1 function in male reproductive system. We found that the expression level of GAT1 continuously increased in wild-type mouse testis from 1 month to 2 months after birth. GAT1 overexpression in mouse affected testis development, which embodied reduced testis mass and slowed spermatogenesis in transgenic mice. Moreover, transgenic mice showed increase of the percentage of broken sperm. The further study revealed that the reproductive capacity was impaired in GAT1 overexpressing mice. In addition, testosterone level was significantly low in transgenic mice compared with that in wild-type mice. Our findings provided the first evidence that abnormal expression of GAT1 could result in dysgenesis,and indicated that GAT1 might be therapeutically targeted for contraception or dysgenesis treatment.     

Transgenic mice overexpressing γ—aminobutyric acid transporter subtype I develop obesity

Transgenic mice ubiquitously overexpressing murine γ-aminobutyric acid transporter subtype I were created.Unexpectedly,these mice markedly exhibited heritable obesity,which features significantly increased body weight and fat deposition.Behavioral examination revealed that transgenic mice have slightly reduced spontaneous locomotive capacity and altered feeding pattern.This preliminary finding indicates that the inappropriate level of γ-aminobutyric acid transporters may be directly or indirectly involved in the pathogenic mechanism underlying certain types of obesity.     

Characterization of transgenic mice expressing EGFP under the control of the monkey 20α-hydroxysteroid dehydrogenase promoter

To directly assess the molecular function of the monkey 20α-hydroxysteroid dehydrogenase(20α-HSD) promoter, we generated transgenic mice(tg) expressing enhanced green fluorescent protein(EGFP) under control of this promoter. We demonstrated that prostaglandin F2α induced 20α-HSD promoter activity in CHO cells in a dose-dependent manner. Furthermore, forskolin treatment markedly reduced 20α-HSD promoter activity, and prolactin exhibited weak inhibitory activity. The transgenic mouse obtained one positive founder male. The transgene was propagated in 10 successive generations without any notable defects to the progeny. EGFP and 20α-HSD in the tg mice were colocalized in the luteal cells of the ovary during late pregnancy. Strong EGFP and 20α-HSD protein signals were also detected in the adult testis. Immunohistochemical analysis revealed high EGFP levels in the seminiferous epithelium, whereas 20α-HSD was expressed in the seminiferous tubules. Our data suggest that the ovaries in monkey and mouse exhibit similar expression patterns of 20α-HSD during pregnancy. However, the expression pattern of EGFP in tg mice testis slightly differed from that of the endogenous 20α-HSD. Further investigation is required to elucidate the functional mechanisms underlying regulation of the monkey 20α-HSD promoter in the tg mice.     

Enhancement of germ cell apoptosis induced by ethanol in transgenic mice overexpressing Fas Ligand

It was suggested that chronic ethanol exposure could result in testicular germ cell apoptosis, but the mechanism is still unclear. In the present study, we use a model of transgenic mice ubiquitously overexpressing human FasL to investigate whether Fas ligand plays a role in ethanol-induced testicular germ cell apoptosis. Both wild-type (WT) mice and transgenic (TG) mice were treated with acute ethanol (20% v/v) by introperitoneal injection for five times. After ethanol injection, WT mice displayed up-regulation of Fas ligand in the testes, which was shown by FITCconjugated flow cytometry and western blotting. Moreover, TG mice exhibited significantly more apoptotic germcells than WT mice did after ethanol injection, which was demonstrated by DNA fragmentation, PI staining flowcytometry and TUNEL staining. In addition, histopathological examination revealed that degenerative changes ofepithelial component of the tubules occurred in FasL overexpressing transgenic mice while testicular morphologywas normal in wild-type mice after acute ethanol exposure, suggesting FasL expression determines the sensitivity of testes to ethanol in mice. In summary, we provide the direct evidences that Fas ligand mediates the apoptosis of testicular germ cells induced by acute ethanol using FasL transgenic mice.     

Expression and Identification of a Novel Apoptosis Gene Spata17 （MSRG-11） in Mouse Spermatogenic Cells

In this study,anti-spermatogenesis-associated 17 (Spatal7) polyclonal antibody was preparedby immunizing New Zealand white rabbits with a synthesized peptide corresponding to the amino acid se-quence 7-23 of the mouse Spata17 protein.Immunohistochemical analysis revealed that Spata17 proteinwas most abundant in the cytoplasm of round spermatids and elongating spermatids within seminiferoustubules of the adult testis.The expression of Spata17 mRNA in cultured mouse spermatogonia (GC-1) cellswas almost undetectable.In an experimental unilateral cryptorchidism model of an adult mouse,the expres-sion of Spata17 mRNA had no obvious difference with the normal testis until postoperation day 1,butgradually decreased from day 3 and was almost undetectable on day 17.Immunohistochemical analysisrevealed that the protein was almost undetectable within seminiferous tubules of an experimental unilateralcryptorchidism model of the adult testis on postoperation day 8.Flow cytometry analysis showed that theexpression of Spatal7 protein in the GC-1 cell line could accelerate GC-1 cell apoptosis.The effect increaseswith the increasing of the transfected dose of pcDNA3.1 (-)/Spata17.By Hoechst 33258 staining,a classicalway of identifying apoptotic cells,we further confirmed that the apoptosis was induced by expression ofSpata17 in transfected GC-1 cells.     

Establishment of La-tPA/G-CSF dual transgenic mice and expression in their mammary gland

Expression vectors of human granulocyte colony stimulating factor (G-CSG) and long acting tissue plasminogen activator (La-tPA) in mammary gland were constructed using promoters of mouse whey acid protein gene (WAP) and sheep β-lactoglobulin gene (BLG) with sizes of 2.6 and 5 kb respectively. Two kinds of transgenic mice of G-CSF and La-tPA were produced with microinjection. The expression of G-CSF and La-tPA was achieved in mammary glands of transgenic mice, respectively. In order to establish dual transgenic mice of La-tPA/G-CSF, transgenic mice carrying G-CSF and La-tPA gene characterized with specific expression in mammary gland were mated. La-tPA/G-CSF dual transgenic mice were screened out from the hybrid offspring by Once-PCR. The co-expression of La-tPA and G-CSF in mammary gland of the dual transgenic mice was confirmed by the milk assayed and Northern blot analysis. Some parameters about the dual transgenic mice indicated that there were fewer litters than that of normal mice. The ratio of du     

RNA Guided Genome Editing in Mouse Germ-Line Stem Cells

Spermatogonial stem cells （SSCs） reside on the basement membrane of the seminiferous tubules in mammalian testes （Nagano et al., 1998）. After isolation and purification of SSCs from mouse testis, SSCs can be cultured in vitro to derive germ-line stem cells （GSCs） which have the ability of proliferation over 2 years （Kanatsu-Shinohara et al.. 2003;     

The deubiquitinating gene Usp29 is dispensable for fertility in male mice

The balanced actions between ubiquitination and deubiquitination precisely control the levels of various proteins vital for spermatogenesis. Ubiquitin-specific processing proteases(USPs) are the largest family of deubiquitinatingenzymes(DUBs),containing more than 50 members. So far, the functions of only a few USPs in male fertility have been studied, the roles of the majority are yet unknown. The present study aimed to explore the function of Usp29(ubiquitin-specific protease 29) in male fertility. We found that Usp29 showed predominant expression in mouse testis, and its m RNA expression started to increase at 14 days postpartum(dpp), with a peak at 28 and 35 dpp. Using CRISPR/Cas9 technology, we generated Usp29 knockout mice(Usp29~(–/–)). Usp29~(–/–)mice exhibited no overt developmental anomalies. Further examination revealed that Usp29~(–/–)mice had normal fertility and showed no detectable difference in the testis/body weight ratio, testicular and epididymal histology as well as epididymal sperm count from the wild-type littermates. Moreover, Usp29 is not a pseudogene in mice. Taken together, our study first reported that though Usp29 is predominantly expressed in the testis, it is not essential for male fertility in mice.     

Production of brown/yellow patches in the SLC7A11 transgenic sheep via testicular injection of transgene

The gene,SLC7A11,which encodes the solute carrier family 7 member 11(anionic amino acid transporter light chain,xCT),has been reported to be implicated in multiple processes such as in pheomelanin production,cell proliferation and migration,Kaposi’s sarcoma herpesvirus(KSHV) entry into the host cells,learning and memory.Its involvement in cancer cell proliferation and metastasis has been widely studied.Its role in pheomelanogenesis is likely conserved in sheep.The full-length cDNA of sheep SLC7A11 was cloned from sheep skin fibroblasts for evaluating its role in regulating sheep coat color.The complete open reading frame of sheep xCT(sxCT) is 1512 bp in length,encoding a 503 amino acid polypeptide.We explored its function on pheomelanogenesis in vitro and in vivo.In the melan-a non-agouti mouse melanocytes that mainly produce eumelanin,overexpressed sxCT reduced the content of eumelanin.Using a testicular injection transgenic method,sxCT-transgenic sheep were generated and exhibited patches of brown/yellow coat,suggesting that sxCT can be selectively expressed to increase the pheomelanin production in wool.Our studies suggest that testicular injection of transgene can be used to genetically modify sheep coat color.     

The anticonvulsant valproate increases the turnover rate of gamma-aminobutyric acid transporters

Valproate is an important anticonvulsant currently in clinical use for the treatment of seizures. We used electrophysiological and tracer uptake methods to examine the effect of valproate on a gamma-aminobutyric acid (GABA) transporter (mouse GAT3) expressed in Xenopus laevis oocytes. In the absence of GABA, valproate (up to 50 mm) had no noticeable effect on the steady-state electrogenic properties of mGAT3. In the presence of GABA, however, valproate enhanced the GABA-evoked steady-state inward current in a dose-dependent manner with a half-maximal concentration of 4.6 +/- 0.5 mm. Maximal enhancement of the GABA-evoked current was 275 +/- 10%. Qualitatively similar observations were obtained for human GAT1 and mouse GAT4. The valproate enhancement did not alter the Na(+) or Cl(-) dependence of the steady-state GABA-evoked currents. Uptake experiments under voltage clamp suggested that the valproate enhancement of the GABA-evoked current was matched by an enhancement in GABA uptake. Thus, despite the increase in GABA-evoked current, ion/GABA co-transport remained tightly coupled. Uptake experiments indicated that valproate is not transported by mouse GAT3 in the absence or presence of GABA. Valproate also enhanced the rate of the partial steps involved in transporter presteady-state charge movements. We propose that valproate increases the turnover rate of GABA transporters by an allosteric mechanism. The data suggest that at its therapeutic concentration, valproate may enhance the activity of neuronal and glial GABA transporters by up to 10%.     

Rete testis and the adjacent seminiferous tubules during postembryonic development in mice

Testicular compartment that includes rete testis and the adjacent transitional zone (TZ) of seminiferous tubules has been examined only by light and electron microscopy until now. However, recent data suggest that adult Sertoli cells (SCs) located in this compartment are capable to commence active proliferation both in vitro and in vivo, and hence, are not completely differentiated. The present study is first to investigate mouse rete testis and TZ during the postembryonic development and is intended to determine new protein markers for cells of this compartment, the state of their differentiation, and also their proliferative activity. It was demonstrated that rete testis cells were stained for SC marker Wt1 transiently, until day 25 of postembryonic development, then the staining disappeared. Another SC marker Dmrt1 that involved in the process of SC differentiation was not expressed in the rete testis cells during the postnatal development and in the adult state. One more feature that distinguished rete testis cells from SCs was lower proliferative activity of rete testis cells in 2–6 days old mice. SCs from TZ expressed Wt1 at all ages examined. However, at earlier ages, they were heterogeneous on Dmrt1 expression, and only by day 25, Dmrt1 expression was completely disappeared from TZ SCs. It is interesting that on day 18 when SCs in seminiferous tubules complete differentiation and exit from cell cycle proliferation of TZ SCs was at significantly higher level. It is also showed that in 3D culture, Wt1+ cells isolated from rete testis and TZ of 60 days old GFP male mice were capable to form seminiferous tubules de novo in cooperation with testicular cells from 6 days old mice.     

Psychostimulant-Induced Testicular Toxicity in Mice: Evidence of Cocaine and Caffeine Effects on the Local Dopaminergic System

Several organ systems can be affected by psychostimulant toxicity. However, there is not sufficient evidence about the impact of psychostimulant intake on testicular physiology and catecholaminergic systems. The aim of the present study was to further explore potential toxic consequences of chronic exposure to cocaine, caffeine, and their combination on testicular physiology. Mice were injected with a 13-day chronic binge regimen of caffeine (3x5mg/kg), cocaine (3×10mg/kg), or combined administration. Mice treated with cocaine alone or combined with caffeine showed reduced volume of the seminiferous tubule associated to a reduction in the number of spermatogonia. Cocaine-only and combined treatments induced increased lipid peroxidation evaluated by TBARS assay and decreased glutathione peroxidase mRNA expression. Importantly, caffeine-cocaine combination potentiated the cocaine-induced germ cell loss, and induced pro-apoptotic BAX protein expression and diminished adenosine receptor A1 mRNA levels. We analyzed markers of dopaminergic function in the testis and detected the presence of tyrosine hydroxylase (TH) in the cytoplasm of androgen-producing Leydig cells, but also in meiotic germs cells within seminiferous tubules. Moreover, using transgenic BAC-Drd1a-tdTomato and D2R-eGFP mice, we report for the first time the presence of dopamine receptors (DRs) D1 and D2 in testicular mouse Leydig cells. Interestingly, the presence of DRD1 was also detected in the spermatogonia nearest the basal lamina of the seminiferous tubules, which did not show TH staining. We observed that psychostimulants induced downregulation of DRs mRNA expression and upregulation of TH protein expression in the testis. These findings suggest a potential role of the local dopaminergic system in psychostimulant-induced testicular pathology.     

Correlation of appearance of metastasis-associated protein1 (Mta1) with spermatogenesis in developing mouse testis

Mta1, a representative of the MTA gene family, is believed to be involved in the metastasis of malignant tumors. However, a systematic study of its physiological function has not been performed. It has been found in normal mouse organs at relatively low levels, except for in testis, suggesting a potential function in the male reproductive system. In order to explore the role of Mta1 protein during spermatogenesis, its expression in adult mouse testis was compared with that in developing mouse testis and in testis from adult mice treated with methoxyacetic acid, which selectively depletes primary spermatocytes. Quantitative analysis revealed that Mta1 protein gradually increased in the testis from 14 days postnatally. Immunolocalization analysis demonstrated strong signals in the seminiferous tubules, and Mta1 was predominantly present in the nucleus of primary spermatocytes and spermatogonia from 14 days postnatally. The most intensive staining was located in the nucleus of pachytene spermatocytes in mature testes. The expression pattern of Mta1 during spermatogenesis was also shown to be stage-specific by immunohistochemistry analysis. Finally, dramatic loss of Mta1 expression from pachytene spermatocytes was observed in the spermatogenic-arrested adult mouse testis. These results collectively demonstrate that Mta1 appears during postnatal testis development and suggest that this expression may be crucial for spermatogenesis. This study was supported by the Natural Science Foundation of China (2006: 30570982; 2003: 30370750; 2003: 30371584).     

Autoantigenic germ cells exist outside the blood testis barrier

Preleptotene spermatocytes and spermatogonia are germ cells located outside the blood-testis barrier provided by the Sertoli cells. These cells have been found to express autoantigens accessible to circulating antibodies. Mice immunized with syngeneic testis with or without bacterial adjuvant had detectable IgG on cells at the periphery of seminiferous tubules. Sera from orchiectomized but not from testes-intact mice immunized with testis and adjuvants readily transferred similar IgG deposits to testes of normal recipients. When testis-specific antisera from orchiectomized mice and testis-intact mice were compared for their reactivity on prepuberal testicular cells, serum from orchiectomized donors had significantly higher reactivity. Ig was eluted from IgG-positive testes with acid buffer and was shown to be highly enriched in antibody to prepuberal testicular cells, confirming the Ag-specific nature of the IgG deposits. The testis IgG deposits reacted with antisera to IgG1 and IgG3 but not IgG2a or IgG2b. This finding can explain lack of association of C3 in the deposits. Only 30 to 40% of seminiferous tubules had IgG deposits and they coincided with stages 7 to 12 of the spermatogenic cycle. Thus, the expression of the autoantigens is stage specific. The in situ formation of immune complexes by circulating autoantibodies demonstrates conclusively that testis autoantigens are not completely sequestered, and the blood-testis barrier as an immunologic barrier is incomplete.     

Immunopathology of murine experimental allergic orchitis

Experimental allergic orchitis (EAO) was induced consistently in BALB/c mice by immunization with homologous testicular tissue homogenate emulsified in complete Freund's adjuvant (CFA) providing that the animals had received simultaneously at least 1 microgram of an extract of Bordetella pertussis rich in pertussigen. All animals thus treated developed orchitis and serum antibody to testicular antigens within 20 days after immunization. The lesions were located in testis (100%), rete testis (37%), cauda epididymis (21%), and vas deferens (37%). Ductus efferentes and caput epididymis were only rarely affected. Early lesions in the seminiferous tubules were characterized by peritubular and/or intratubular accumulation of eosinophils, neutrophils, lymphocytes, and macrophages. This was followed by aspermatogenesis. Late lesions included massive necrosis and extensive fibrosis of the seminiferous tubules. Disruption of blood-testis barrier on day 20 was evidenced by the detection of 1) perfused lanthanum deposits between Sertoli cells and surrounding inflammatory cells inside the seminiferous tubules, 2) deposits of endogenous mouse IgG in germinal epithelium, and 3) probable immune complexes (granular C3) surrounding seminiferous tubules. Murine EAO differed from that of the guinea pig in the lack of involvement of the ductus efferentes, the extensive necrosis, the abundant polymorphonuclear eosinophils in the lesion, and the exquisite requirement of concomitant injection of B. pertussis extract.     

Evaluation of the safety and efficacy of testicular biopsies in llamas

Evaluation of the reproductive function of Lama glama is generally considered to be a challenging task due to the difficulty of obtaining representative semen samples. One method that has been proposed for evaluation of testicular function in these animals is histologic examination of testicular needle biopsies. This study was undertaken to examine the safety and efficacy of using needle biopsies to assess testicular function in this species. One randomly selected testicle from each of 16 sexually mature llamas was biopsied with a 14-gauge self-firing biopsy instrument. The llamas were evaluated over a 6-week period with thermography for temperature changes of the scrotum. At the end of the 6-week trial, the llamas were castrated and sections of each testis were fixed in Bouin's solution for histologic examination. Immediately prior to castration, an additional biopsy was taken from each testis to compare the tissue obtained via biopsy with sections from the corresponding testis obtained after castration. A qualitative grading scale was used to compare the seminiferous tubules from each testis. No difference was found between the biopsied and the nonbiopsied testes (P = 0.69). The percentage of normal tubules between the biopsied and the nonbiopsied sides also did not differ (P = 0.70). Furthermore, the percentage of normal seminiferous tubules did not differ between the needle biopsy samples and the corresponding tissue samples obtained at castration (P = 0.48). The number of round seminiferous tubules counted in each biopsy section ranged from 3 to 67. There was no significant difference in the thermographic images of the scrotum between the biopsied and the nonbiopsied testes. This study supports testicular biopsies as a safe and useful procedure in the evaluation of testicular function.     

Expression of the mRNA for the ligand of c-kit in mouse Sertoli cells.

The expression of the mRNA for SLF (the c-kit ligand), a product of the "steel" locus, has been investigated in postnatal mouse testis and homogeneous populations of testicular cells. The message was found expressed in postnatal mouse testis but not in germ cells. Studies on primary mouse Sertoli cell cultures from 18 day old mice show that Sertoli cells are the site of SLF mRNA expression in the seminiferous tubules. Treatment of Sertoli cell cultures with cAMP analogs led to a significant increase in the SLF mRNA levels.     

Overexpression of γ－aminobutyric acid transporter subtype I leads to susceptibility to kainic acid－induced seizure in transgenic mice

Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter, and the GABAergic synaptic transmission is normally terminated by the rapid uptake through GABA transporters. With transgenic mice ubiquitously overexpressing GABA transporter subtype I (GAT1), the present study explored the pathophysiological role of GAT1 in epileptogenesis. Though displaying no spontaneous seizure activity, these mice exhibit altered electroencephalographic patterns and increased susceptibility to seizure induced by kainic acid. In addition, the GABA(A) receptor and glutamate transporters are up-regulated in transgenic mice, which perhaps reflects a compensatory or corrective change to the elevated level of GAT1. These preliminary findings support the hypothesis that excitatory and inhibitory neurotransmission, and seizure susceptibility can be altered by neurotransmitter transporters.