Germ Cells In Testis Research Articles

CAMP–Epac2‐mediated activation of Rap1 in developing male germ cells: RA‐RhoGAP as a possible direct down‐stream effector

Rap1 is a small GTPase that functions as a positional signal and organizer of cell architecture. Recently Rap1 is emerged to play a critical role during sperm differentiation since its inactivation in haploid cells leads to a premature release of spermatids from the supporting Sertoli cell resulting in male infertility. How Rap1 is activated in spermatogenic cells has not yet been determined. Our objective was to investigate on a possible cAMP-mediated activation of Rap1 employing a cAMP analogue selective to Epac, the Rap1 activator directly responsive to cAMP, for stimulating cultured testis germ cells. Here we provide biochemical, cellular and functional evidence that the Epac variant known as Epac2 is expressed as both a transcript and a protein and that it is able to promote Rap1 activation in the cultured cells. A time course immunofluorescence analysis carried out on stimulated cells revealed the translocation of endogenous Epac2, which is cytosolic, towards the site where Rap1 is located, i.e., the Golgi complex, thus documenting the effective Rap1-Epac2 protein interaction 'in vivo' leading to Rap1-GTP loading. A combination of biochemical and molecular techniques supported the immunofluorescence data. The search for the presence of a putative Rap1 downstream effector, described in differentiating somatic cells as a target of cAMP-Epac-activated Rap1, revealed the presence in spermatogenic cells of RA-RhoGAP, a Rap1-activated Rho GTPase-activating protein. Taken together, our results, obtained with endogenously expressed proteins, are consistent with a cAMP/Epac2/Rap1-mediated signaling that could exert its action, among others, through RA-RhoGAP to promote the progression of spermatogenesis.

Stra8 and its inducer, retinoic acid, regulate meiotic initiation in both spermatogenesis and oogenesis in mice.

In eukaryotes, diploid cells give rise to haploid cells via meiosis, a program of two cell divisions preceded by one round of DNA replication. Although key molecular components of the meiotic apparatus are highly conserved among eukaryotes, the mechanisms responsible for initiating the meiotic program have diverged substantially among eukaryotes. This raises a related question in animals with two distinct sexes: Within a given species, are similar or different mechanisms of meiotic initiation used in the male and female germ lines? In mammals, this question is underscored by dramatic differences in the timing of meiotic initiation in males and females. Stra8 is a vertebrate-specific, cytoplasmic factor expressed by germ cells in response to retinoic acid. We previously demonstrated that Stra8 gene function is required for meiotic initiation in mouse embryonic ovaries. Here we report that, on an inbred C57BL/6 genetic background, the same factor is also required for meiotic initiation in germ cells of juvenile mouse testes. In juvenile C57BL/6 males lacking Stra8 gene function, the early mitotic development of germ cells appears to be undisturbed. However, these cells then fail to undergo the morphological changes that define meiotic prophase, and they do not display the molecular hallmarks of meiotic chromosome cohesion, synapsis and recombination. We conclude that, in mice, Stra8 regulates meiotic initiation in both spermatogenesis and oogenesis. Taken together with previous observations, our present findings indicate that, in both the male and female germ lines, meiosis is initiated through retinoic acid induction of Stra8.

Production of donor-derived sperm after spermatogonial stem cell transplantation in the dog

Spermatogonial stem cell transplantation (SSCT) offers unique approaches to investigate SSC and to manipulate the male germline. We report here the first successful performance of this technique in the dog, which is an important model of human diseases. First, we investigated an irradiation protocol to deplete endogenous male germ cells in recipient testes. Histologic examination confirmed >95% depletion of endogenous spermatogenesis, but retention of normal testis architecture. Then, 5-month-old recipient dogs (n=5) were focally irradiated on their testes prior to transplantation with mixed seminiferous tubule cells (fresh (n=2) or after 2 weeks of culture (n=3)). The dogs receiving cultured cells showed an immediate allergic response, which subsided quickly with palliative treatment. No such response was seen in the dogs receiving fresh cells, for which a different injection medium was used. Twelve months post-injection recipients were castrated and sperm was collected from epididymides. We performed microsatellite analysis comparing DNA from the epididymal sperm with genomic DNA from both the recipients and the donors. We used six markers to demonstrate the presence of donor alleles in the sperm from one recipient of fresh mixed tubule cells. No evidence of donor alleles was detected in sperm from the other recipients. Using quantitative PCR based on single nucleotide polymorphisms (SNPs), about 19.5% of sperm were shown to be donor derived in the recipient. Our results demonstrate the first successful completion of SSCT in the dog, an important step toward transgenesis through the male germline in this valuable biomedical model.

Age-related decrease in active caspase-3 expression in germ cells of human testes

OBJECTIVE: Advancing paternal age has been implicated in a broad range of abnormal reproductive and genetic outcomes, including diminished semen quality, increased DNA damage and reduced fertility. Suggested mechanisms include defective sperm chromatin packaging, disordered apoptosis, and oxidative stress. To clarify whether a deregulation of apoptosis could be involved in the increased incidence of sperm DNA damage in aged men, we examined age-related changes in the expression of active caspase-3, -8 and -9. We further assessed the effect of ageing on the expression of poly(ADP-ribose)polymerase-1 (PARP-1). DESIGN: Prospective comparative study. MATERIALS AND METHODS: Testicular tissue (orchidectomy/testicular biopsies) was obtained from 15 patients (mean age 60.2 years) with advanced prostate cancer and from 9 patients (mean age 38.4 years) with obstructive azoospermia (OA) showing normal spermatogenesis (controls). Immunohistochemical (IHC) and Western blot analyses were used for detection of testicular expression of caspase-3 (17/19 kDa). The number of caspase positive cells (IHC) was expressed relative to the total number of germ cells in over 20 seminiferous tubules (staining index). Cleaved caspases -8 (41/43kDa), -9 (37kDa) and PARP-1 (89kDa) were detected by Western blot. RESULTS: Immunolocalization of caspase-3 was detected in spermatogonias, primary spermatocytes and early spermatids. A significant decrease in the staining index was observed in testis sections of aged patients (8.1±1.7/18.0±5.0). In this group, the number of round spermatids positive for caspase-3 was remarkably decreased as compared with controls (3.0±0.6/15.1±5.6). In OA controls, immunostaining for caspase-3 was occasionally observed in Sertoli cells whereas this stain was higher (but not significant) in the aged group. Western blot analysis confirmed that active caspase-3 decrease (p< 0.001) in the testes of aged men. There were no significant changes in the levels of caspase-8 (extrinsic pathway of apoptosis) between both group of patients, whereas the expression of activated caspasa-9 (intrinsic apoptotic pathway) showed an overall decrease (p=0.054) in the aged group. Cleaved PARP also declined significantly (p<0.001) with age. CONCLUSIONS: Advancing male age is associated with a significant decrease in the expression levels of caspase-3 and PARP-1. In this context, down-regulation of two key proteins involved in apoptosis might be associated with increased frequencies of sperm DNA damage in spermatozoa.

The human Y‐encoded testis‐specific protein interacts functionally with eukaryotic translation elongation factor eEF1A, a putative oncoprotein

Testis-specific protein Y-encoded (TSPY) is the putative gene for the gonadoblastoma locus on the Y chromosome. TSPY is expressed in normal germ cells of fetal and adult testis and ectopically in tumor germ cells, including gonadoblastoma in intersex patients, testicular germ cell tumors, prostate cancer and other somatic cancers. It is a member of the TSPY/SET/NAP1 superfamily and harbors a highly conserved domain, termed SET/NAP domain. To explore its possible role(s) in tumorigenesis, we had performed a yeast two-hybrid screen of a fetal gonadal cDNA library and identified the translation elongation factor eEF1A as a binding partner for TSPY at the SET/NAP domain. TSPY and eEF1A were highly expressed and colocalized in tumor germ cells of human seminoma specimens, suggesting their possible interaction in germ cell tumors. They were colocalized in the cytoplasm and could be co-immunoprecipitated from transfected COS7 cells. Significantly, both eEF1A1 and eEF1A2 have postulated to be involved in various types of human cancer, including breast and prostate cancers. TSPY enhanced protein synthesis of a reporter gene, which was augmented by an overexpression of eEF1A. TSPY also increased the nuclear redistribution of eEF1A, resulting in a parallel increase in reporter gene transcripts. Our results suggest that TSPY could exert its oncogenic function(s) by interacting with eEF1As and stimulating gene expression via its enhancements in protein synthesis and gene transcription.

Animal models for fertility preservation in the male

Fertility preservation in the male is routinely focused on sperm. In clinical and veterinary settings, cryopreservation of sperm is a widely used tool. However, the goals for male fertility preservation differ between experimental models, maintenance of livestock, conservation of rare species, and fertility protection in men. Therefore very different approaches exist, which are adapted to the specialized needs for each discipline. Novel tools for male fertility preservation are explored targeting immature germ cells in embryonic or immature testes. Many options might be developed to combine germline preservation and generation of sperm ex vivo leading to interesting new perspectives. This review highlights current and future options for male fertility preservation with a special focus on animal models and a consideration of the various disciplines in need of novel tools.

Role of XPA, ERCC1, mtTFA on the survival of patients with metastatic germ cell tumors of testis treated with cisplatin

5090 Background: Advanced Testis Germ Cell Tumors (TGCT) are treated with platinum based chemotherapy. It has been proposed that DNA repair proteins play a role in the TGCT response to cisplatin. DNA repair deficiencies have been related with better treatment response; such deficiencies could be the result of polymorphisms on DNA repair genes or of the presence of High Mobility Group Proteins B (HMGB). Thus we have investigated whether the presence of XPA, ERCC1 and HMGB (mtTFA), as well as the polymorphism C8092A of the ERCC1 gene may influence the survival of TGCT patients treated with cisplatin. Methods: ERCC1, XPA and mtTFA were detected by immunohystochemistry using tissue microarrays in 58 samples from normal and neoplastic tissue. ERCC1 C8092A polymorphism was determined on DNA isolated from blood and paraffin-embedded tumor samples by PCR/RFLP´S. The results were associated with survival and response. Results: Only 58 full filled the inclusion criteria. The age-media was 23 years. According to the...

Expression of EGFL7 in primordial germ cells and in adult ovaries and testes

We have previously reported the isolation and characterization of a novel endothelial-restricted gene, Egfl7, that encodes a secreted protein of about 30-kDa. We and others demonstrated that Egfl7 is highly expressed by endothelial cells during embryonic development and becomes down-regulated in the adult vasculature. In the present paper, we show that during mouse embryonic development, Egfl7 is also expressed by primordial germ cells (PGC). Expression is down-regulated when PGCs differentiate into pro-spermatogonia and oogonia, and by 15.5 dpc Egfl7 can no longer be detected in the germ line of both sexes. Notably, Egfl7 is again transiently up-regulated in germ cells of the adult testis. In contrast, expression in the ovary remains limited to the vascular endothelium. Our results provide the first evidence of a non-endothelial expression of EGFL7 and suggest distinctive roles for Egfl7 in vascular development and germ cell differentiation.

Di-butyl Phthalate Targets Multiple Signaling Mechanisms to Down-Regulate Fetal Testosterone Biosynthesis.

The in utero hormonal environment is critical for proper development of the male reproductive system and there is growing concern that increases in various human male reproductive tract abnormalities, including falling sperm counts, hypospadias, cryptorchidism, and testis germ cell cancer are the result of exposure to environmental endocrine disrupting chemicals (EDC). In utero exposure of male rats to these EDCs targets fetal testicular testosterone biosynthesis leading to adverse effects on the developing male reproductive system. This lab has demonstrated that phthalate esters, a widely used class of EDCs, induce reproductive abnormalities in part by down-regulating the transcription of testosterone biosynthesis genes. However, the mechanism and upstream signaling pathways by which DBP inhibits transcription is unknown and explored in this study. This study identified altered protein-DNA interactions in down-regulated genes by quantitative in vivo DNase footprinting and chromatin immunoprecipitation. Using RNAi, gene expression of these factors was knocked-down in MA-10 and R2C Leydig cells. Cells were stimulated and steroidogenic gene expression was quantified with real-time RT-PCR to confirm the role of each factor in testosterone biosynthesis. Additionally, phosphorylation state of each factor was determined following stimulation and subsequent phthalate exposure. Moreover, these results were compared to isolated primary fetal leydig cells to identify the role of these factors in fetal steroidogenesis. Results indicate that the transcription factors sp1, SF-1, and c/ebp β are differentially bound following toxic and non-toxic phthalate exposure. Further, results suggest that these factors work coordinately through multiple or redundant mechanisms to regulate transcription, which reflects the importance of testosterone biosynthesis during fetal testicular development. Taken together, this study characterized the role of each transcription factor in the regulation of steroidogenesis and the signaling mechanism regulating its activity.

Adrenocorticotropic hormone affects nonapoptotic cell death of undifferentiated germ cells in the fetal mouse testis: In vivo study by exo utero transplantation of corticotropic tumor cells into embryos

Adrenocorticotropic hormone (ACTH) has been suggested to have possible roles in the fetal testes, one of the organs that express its specific receptors, melanocortin type 2 and 5 receptors (MC2R and MC5R), during the fetal period. We investigated the effect of ACTH on the cells in the testis cord of the fetal mouse testis by inducing ACTH-secreting AtT20 tumor cells in mouse fetuses. We first identified that mouse testicular germ cells at embryonic day (E) 16.5 and E18.5 spermatogonia were entirely CDH1 (E-cadherin)-positive by immunohistochemistry. We next performed AtT20-cell transplantation into the mouse fetus at E12.5, and analyzed ACTH effects on the development of fetal male mouse germ cells that express MC2R and MC5R at E16.5 and E18.5. The spermatogonia in the testis of AtT20-implanted embryos exhibited morphological changes, including pyknotic nuclei and swollen cytoplasm. In the AtT20-implanted embryos, the number of spermatogonia per unit area of the testis cord was significantly lower, but there were more pyknotic spermatogonia than in the controls. Single-stranded DNA-positive (apoptotic) and histone H3-positive (mitotic) spermatogonia were rarely observed and their numbers did not significantly differ in the two groups. Anti-Müllerian hormone (AMH)-positive Sertoli cells, another cell type that constitutes the fetal testis cord but does not express MC2R or MC5R, showed no apparent morphological changes compared with controls, nor were their numbers in the two groups significantly different between the two groups. These results suggest that ACTH, via MC2R and/or MC5R, may be involved in the nonapoptotic cell death of fetal mouse spermatogonia that is observed during the normal perinatal period.

The DNA/RNA-Binding Protein, Translin, Binds microRNA122a and Increases Its In Vivo Stability

Translin (TSN), also known as testis-brain RNA-binding protein, is proposed to bind to breakpoint junctions at chromosomal translocations in the nucleus and to specific RNAs in the cytoplasm. In germ cells of the mouse testis, it recognizes target mRNAs transcribed by the transcription factor CREM-tau in spermatids, specific meiotically expressed mRNAs, and a noncoding RNA that encodes piRNAs. Here we show that TSN also binds to the microRNA miR-122a. MiR-122a is expressed in late-stage germ cells and is complementary to a sequence in the 3' untranslated region of the transition protein 2 mRNA. The binding of TSN to miR-122a increases its in vivo stability, suggesting an additional posttranscriptional function for TSN.

Fas receptor is not present on ejaculated human sperm

Apoptosis appears to have an essential role in the control of testis germ cell number and Fas expression has been reported in apoptotic spermatocytes and spermatids. We investigated if Fas (CD95) was present on ejaculated human sperm and any relationship between Fas on sperm and the apoptotic marker Syto16. Semen samples from 77 male partners of infertile couples were evaluated. Each sample was analysed both before and after semen preparation by conventional microscopical procedures and by flow cytometry (FC). A multiparameter FC analysis to assess simultaneously sperm concentration, sperm viability, sperm apoptosis, CD45 positive (leukocyte) and CD95 (Fas) positive cell concentration was carried out. A further 10 samples were studied by indirect immunofluorescence to confirm results. The mean concentration of CD95 positive cells was very low (<1%), with no significant difference between normozoospermic and non-normozoospermic men. There was no correlation between apoptotic sperm and CD95 positive cell concentration. A linear correlation was found between CD95 positive cell and leukocyte (CD45 positive) concentration (r = 0.9946, P < 0.0001). CD95 mean fluorescence intensity of leukocytes was 10-fold greater than that of sperm and of isotypic control. Both incubation with activating anti-Fas antibody and betulinic acid induced apoptosis in leukocytes. Incubation with betulinic acid, but not with activating anti-Fas antibody, induced apoptosis in sperm. Pre-incubation with neutralizing anti-Fas antibody suppressed CD95 expression on leukocytes, whereas it did not change sperm CD95 peak fluorescence. There is no detectable quantity of Fas on human ejaculated sperm.

Expression of NANOG, but not POU5F1, points to the stem cell potential of primitive germ cells in neonatal pig testis

Gonocytes are primitive germ cells that are present in the neonatal testis and are committed to male germline development. Gonocytes differentiate to spermatogonia, which establish and maintain spermatogenesis in the postnatal testis. However, it is unknown whether large animal species have pluripotency-specific proteins in the testis. Nanog and Pou5f1 (Oct3/4) have been identified as transcription factors essential for maintaining pluripotency of embryonic stem cells in mice. Here, we show that NANOG protein was expressed in the germ cells of neonatal pig testes, but was progressively lost with age. NANOG was expressed in most of the lectin Dolichos biflorus agglutinin- and ZBTB16-positive gonocytes, which are known gonocyte-specific markers in pigs. NANOG was also expressed in Sertoli and interstitial cells of neonatal testes. Interestingly, POU5F1 expression was not detected at either the transcript or the protein level in neonatal pig testis. In the prepubertal testis, NANOG and POU5F1 proteins were primarily detected in differentiated germ cells, such as spermatocytes and spermatids, and rarely in undifferentiated spermatogonia. By using a testis transplantation assay, we found that germ cells from 2- to 4-day-old pigs could colonize and proliferate in the testes of the recipient mice, suggesting that primitive germ cells from neonatal pig testes have stem cell potential.

Bone Marrow-Derived Stem Cells Do Not Reconstitute Spermatogenesis In Vivo

Spermatogenesis is a self-renewing process leading to production of spermatozoa in adult testis. The existence of male germinal stem cells was demonstrated by regeneration of spermatogenesis after transplantation of total testicular germ cells in testis [1], and we have previously shown that mouse testicular cells contain a side population (SP) of stem cells (Hoechst 33342 fluorescent dye efflux) that can repopulate germ celldepleted testis when transplanted into adult mice [2]. Several breakthroughs concerning the in vitro derivation of male and female germ cells from embryonic stem cells have challenged the field of reproductive biology and opened new perspectives for treatment of infertility [3–5]. Some reports have suggested that adult bone marrow-derived stem cells are able to regenerate various nonhematopoietic cell lineages in several organs, by

The expression patterns of genes involved in the RNAi pathways are tissue-dependent and differ in the germ and somatic cells of mouse testis

Different RNA interference (RNAi) components participate in post-transcriptional regulation via RNA silencing. The expression pattern of the genes Drosha and Dicer and the members of the Argonaute family Ago1, Ago2, Ago3 and Ago4, all elements participating in the RNAi pathways, were investigated in mouse somatic tissues and testis using quantitative RT-PCR. Expression patterns of different testis cells and those emerging during testis development were also investigated. The differential patterns of expression seen suggest potential pleiotropic roles for certain components of the RNAi machinery. Both spermatocytes and spermatids showed a defined gene expression pattern. The strong expression of Ago4 in germ cells suggests that this protein plays a key role in germ-cell differentiation in the seminiferous epithelium.

Methoxyacetic Acid-Induced Spermatocyte Death Is Associated with Histone Hyperacetylation in Rats1

We used high-density microarrays to evaluate the possible mechanisms by which 2-methoxyacetic acid (MAA) disrupts spermatogenesis. Levels of mRNA transcripts were determined in total RNA isolated from testes of MAA-treated (650 mg/kg i.p.) or concurrent control rats killed 4, 8, 12, or 24 h postexposure (PE). Germ cell death was examined in testis sections using in situ staining for DNA fragmentation. MAA treatment caused increased death of pachytene spermatocytes starting 8 h PE and increasing dramatically at 12 and 24 h PE. Microarray results indicated that at 4 h PE the transcript levels of seven different genes were significantly overrepresented in the testes of MAA-exposed animals. One gene (histone H1 zero [H1f0]) was significantly overrepresented in MAA-treated samples at 4, 8, and 12 h PE. Because expression of this gene has been associated with increased acetylation of core histones, we examined MAA-induced changes in the acetylation of histones H4 (HISTH4) and H3 (HISTH3) in testis nuclear protein. Western blots of acid-extracted testis nuclei indicated that the levels of tetraacetyl histone H4 (4acHIST1H4) and of diacetyl histone H3 (2acHIST1H3) were elevated by MAA treatment at 4, 8, and 12 h PE. Using the same antibodies, 4acHIST1H4 and 2acHIST1H3 were localized primarily to elongating spermatids in testis sections from control animals. At 4 h PE, staining for either histone modification was dramatically increased in spermatogonia and all primary spermatocyte populations except for dividing spermatocytes. MAA treatment of testis nuclear protein extracts from unexposed animals caused both a significant increase in histone acetyltransferase activity and a significant inhibition of histone deacetylase activity, suggesting that increased core histone acetylation results from a combination of these complementary modes of action. Our results indicate that exposure to MAA causes increased acetylation of core histones in several testis germ cell populations, including those in prophase of meiosis, a large proportion of which die rapidly following this treatment.

249. Regulated expression of KIT protein in juvenile and adult germ cells of the rodent testis

KIT receptor is an established marker of differentiating spermatogonia and its activation is required to trigger spermatogonial maturation. KIT mRNA, however, can be detected in undifferentiated spermatogonia in the absence of protein expression, as previously established by us in the irradiated adult rat testes [1]. This differential regulation of mRNA and protein is presumably modulated by either local hormone action or by cues from the adult testicular microenvironment. Endogenous regulatory factors known to stimulate KIT synthesis in juvenile male germ cells in vitro are bone morphogenetic protein 4 (BMP4) and retinoic acid (RA), while factors known to suppress KIT at the onset of spermatogenesis have not yet been identified. Activin A, implicated in KIT downregulation in a murine erythroleukemia cell line [2], is produced within the juvenile mammalian testis and influences activities of spermatogonia and Sertoli cells. We hypothesised that activin acts to repress KIT expression in spermatogonia and therefore modulate spermatogonial behaviour. Evidence for this was first derived from Sertoli and germ cell co-cultures of day 8 wild type mouse testes in which exogenous activin addition caused a dose-dependent reduction of KIT mRNA. Whole testes mRNA analyses of two activin transgenic mouse models, the newborn Inhba−/− (lacking activin A) and postnatal InhbaBK/BK (decreased bioactive activin), revealed a significant elevation in KIT expression relative to wild type littermates. In the postnatal day 7 InhbaBK/BK testes, an elevated proportion of differentiated spermatogonia, increased cell surface KIT protein levels, enhanced mRNA levels of a known downstream target of KIT signalling pathway, cyclind3 and a meiotic marker, Sycp3, were observed. These data provide the first comprehensive evidence for activin modulation of KIT expression at spermatogenesis onset, in germ cells of the juvenile testis. This finding is of fundamental importance to other KIT-dependent processes. (1) Prabhu, S.M., et al. Expression of c-Kit receptor mRNA and protein in the developing, adult and irradiated rodent testis. Reproduction, 2006. 131(3): p. 489–99. (2) Hino, M., et al. Down-modulation of c-kit mRNA and protein expression by erythroid differentiation factor/activin A. FEBS Lett, 1995. 374(1): p. 69–71.

Alteration in testicular cell components following transiently induced ischaemia in prepubertal bulls

The aim of the present study was to evaluate transient testicular ischaemia (induced using elastrator bands) in Jersey calves on testicular morphology and development. Treatments (at 27 +/- 5 days of age) consisted of control (0 h banding) and banding for 2, 4 or 8 h (n = 4 in each group). After castration (at 60 +/- 5 days of age), the right testis was used for calculation of cell components per testis according to the point-counting method. Bodyweight (59.8 +/- 6.2 kg) and scrotal circumference (SC) at banding (9.1 +/- 0.2 cm) did not differ between groups. Fresh testis weight, scrotal temperature immediately before band removal and daily SC growth were decreased in ischaemic (4 and 8 h) testes compared with controls (P < 0.05). In addition, the number of Sertoli and Leydig cells was significantly reduced in the 8 h ischaemic treatment group (P < 0.05). Transiently induced ischaemia significantly decreased the number of germ cells in the 8 h ischaemic treatment group (13 +/- 5 x 10(6) cells) compared with the 0, 2 and 4 h ischaemic treatment groups (38 +/- 6, 32 +/- 6 and 33 +/- 5 x 10(6) cells, respectively; P < 0.05). These results suggest that transiently induced ischaemia for 8 h significantly decreases the number of germ, Sertoli and Leydig cells in prepubertal testis.

251. Production of donor-derived live lambs following testis germ cell transplantation

Testes germ cell transplantation in livestock has the potential for amplification of transgenic genotypes and for use as an alternative to artificial insemination. This study investigated a workable protocol for testis germ cell transplantation in sheep between animals of the same breed and different breeds. Testes of two groups of recipients at the stage of pre-pubertal (transition from gonocytes to spermatogonia, n = 2) or peri-pubertal (spermatogenesis initiated, n = 2) were treated with a single dose of 9 grey (Gy) or 15 Gy with a 6MV photon beam irradiation, respectively. In the first experiment, using pre-pubertal irradiated animals, testis germ cell transplantation between the same breed was performed at 16 weeks post irradiation. The left testes of recipient rams were injected with donor cells labelled with fluorescent dye PHK26, while the right testes were given unlabelled cells. The left testes of recipients were removed by castration after 2 weeks following transplantation to evaluate the location of the transferred cells, while the right testes were kept in place for long-term assessment of sperm output. In cryosections of the left testes, PKH26 positive cells were found both on the basement membrane as single cells or in the interstitial area. In the second experiment, animals irradiated at the peri-pubertal stage, received donor cells at 5 weeks post irradiation and animals were kept intact for semen production. For a period of two years after transplantation, semen samples were collected routinely from two groups of rams and analysed using microsatellite markers. Two recipients (50%) demonstrated the presence of donor DNA in their ejaculates. In order to investigate the fertility of the donor-origin sperm in recipient ejaculates, 99 ewes were artificially inseminated with semen from two positive rams. Four lambs (8%) have been identified as being sired by donor-derived sperm produced in the recipient ram that received a Merino to Merino transplantation, while no donor-derived offspring was obtained from the recipient with Border Leicester to Merino transplantation. This study represents the first report of the production of live progeny following testis germ cell transplantation in sheep.

228 RECIPIENT PREPARATION FOR SPERMATOGONIAL STEM CELL TRANSPLANTATION: ALTERATION IN TESTICULAR CELL COMPONENTS FOLLOWING TRANSIENTLY INDUCED ISCHEMIA

Depletion of endogenous spermatogonial stem cells using busulfan (Brinster et al. 2003 Biol. Reprod. 69, 412–420) or irradiation (Izadyar et al. 2003 Reproduction 126, 765–774) have been used in preparation of recipient animals prior to transplantation; however, both techniques are not without compromises (severe bone marrow depression or specialized radiotherapy equipment required). Induced testicular ischemia in rams altered spermatic epithelium with germ cell-depleted seminiferous tubules (Markey et al. 1994 Reprod. Fertil. 101, 643–650). The objective was to evaluate testicular transiently induced ischemia (using elastrator bands) in Jersey calves on testicular morphology and development. Treatments (at 27 ± 5 days of age) consisted of control (0, n = 4), banding for periods of 2 h (2, n = 4), 4 h (4, n = 4), and 8 h (8, n = 4). After castration (age: 60 ± 5 days), the right testis of each animal was used for calculation of cell components per testis according to the point counting method. Data were analyzed using the MIXED procedure of SAS (SAS Institute, Inc., Cary, NC, USA). Body weight (59.8 ± 6.2 kg) and scrotal circumference (SC) at banding (9.1 ± 0.2 cm) did not differ between treatments. Fresh testis weight (TW), scrotal temperature immediately before banding removal (ST), and daily scrotal circumference growth (SC) were decreased (4 and 8 h) in ischemic testes compared to controls (Table 1: P &lt; 0.05). In addition, Sertoli and Leydig cells were severely reduced in the 8-h ischemic treatment (Table 1: P &lt; 0.05). Transiently induced ischemia significantly decreased the number of germ cells in the 8-h group, compared to the 0-, 2-, and 4-h groups (Table 1: P &lt; 0.05). These results suggest that transiently induced ischemia significantly decreases the number of germ, Sertoli, and Leydig cells in the testis. Therefore, these present findings could be applicable for preparation of recipient animals through depletion of endogenous germ cells within the seminiferous tubules. This procedure may provide a suitable environment for transplanted donor germ cell colonization in prepubertal recipient bulls. Table 1. Parameters evaluated under transient-induced ischemia treatments