Cloned Foal Born from Postmortem-Obtained Ear Sample Refrigerated for 5 Days Before Fibroblast Isolation and Decontamination of the Infected Monolayer Culture.

A 6-year-old mare, a valuable polo horse, died of complications following postcolic surgery. To preserve its genetics, ear skin samples were collected immediately after death and stored in an equine embryo transfer medium at 4°C for 5 days. After trypsin digestion, monolayer fibroblast cultures were established, but signs of massive bacterial infection were found in all of them. As an ultimate attempt for rescue, rigorously and repeatedly washed cells were individually cultured in all wells of four 96-well dishes. New monolayers were established from the few wells without contamination and used for somatic cell nuclear transfer. Four of the six Day 7 blastocysts derived from 14 reconstructed zygotes were transferred in four naturally cycling mares on Day 5 after ovulation. The embryo transfers resulted in 2 pregnancies, one from a fresh and one from a vitrified blastocyst. The vitrified embryo transfer resulted in a healthy offspring, now 21 months old, genetically and phenotypically identical to the somatic cell donor animal.

"Comparison of two closed vitrification methods for vitrifying dromedary camel (Camelus dromedarius) embryos".

A total of 184 dromedary camel embryos were vitrified using a novel vitrification kit specifically developed for camel embryos. These embryos were vitrified using a 3-step process by exposing them to vitrification solutions (VS) containing 20% foetal calf serum (FCS) with (+) or without (-) the addition of bovine serum albumin (BSA). Embryos were then further divided into two groups (

Donor sperm production in heterologous recipients by testis germ cell transplantation in the dromedary camel.

The object of this study was to investigate if testis germ cell transplantation (TGCT) into a heterologous recipient would result in donor-origin spermatogenesis in the dromedary camel. First, we investigated a workable protocol for TGCT in camels, including donor cell isolation, enrichment by density gradient centrifugation (Percoll and Bovicoll), rete testis injection and microsatellite detection of donor and recipient genotypes. Second, the effects of three doses of Dolichos biflorus agglutinin (DBA), a glycoprotein that specifically binds to gonocytes or Type A spermatogonia, on testis germ cell depletion were investigated by direct injection into the rete testis of a male camel. Seven recipients were prepared with DBA treatment, two males were castrated at 4 weeks for depletion assessment and the remaining five received donor cells 4-6 weeks after treatment. On average, ~17 million cells were isolated per gram of testis tissue, with 19.5±1.9% DBA-positive (DBA+) cells. Percoll centrifugation yielded a 1.5-fold increase in DBA+ cells while Bovicoll centrifugation produced a 2.5-fold increase from the input cells of 18.6±2.1% DBA+ cells. Semen was collected from the recipients 13-20 weeks after transfer and the presence of donor DNA in the samples was determined using microsatellite markers. In two of the five recipients, all semen samples were shown to be positive for donor-derived cells. These results demonstrate for the first time that: (1) heterologous testicular germ cell transplantation in camels is feasible and the recipients are able to produce spermatozoa of donor origin and (2) DBA can be used effectively to deplete endogenous stem cells.

Functional Role of Cyclin-Dependent Kinase 5 in the Regulation of Melanogenesis and Epidermal Structure.

The mammalian integumentary system plays important roles in body homeostasis, and dysfunction of melanogenesis or epidermal development may lead to a variety of skin diseases, including melanoma. Skin pigmentation in humans and coat color in fleece-producing animals are regulated by many genes. Among them, microphthalmia-associated transcription factor (MITF) and paired-box 3 (PAX3) are at the top of the cascade and regulate activities of many important melanogenic enzymes. Here, we report for the first time that cyclin-dependent kinase 5 (Cdk5) is an essential regulator of MITF and PAX3. Cdk5 knockdown in mice causes a lightened coat color, a polarized distribution of melanin and hyperproliferation of basal keratinocytes. Reduced expression of Keratin 10 (K10) resulting from Cdk5 knockdown may be responsible for an abnormal epidermal structure. In contrast, overexpression of Cdk5 in sheep (Ovis aries) only produces brown patches on a white background, with no other observable abnormalities. Collectively, our findings show that Cdk5 has an important functional role in the regulation of melanin production and transportation and in normal development of the integumentary system.

Depletion of testis cell populations in pre-pubertal Bos indicus cattle by irradiation.

Recovery of spermatogenesis following a single dose of irradiation was evaluated in pre-pubertal Brahman bulls, after receiving a single dose of 3, 6, 9 or 12Gray (Gy) irradiation. Biopsy samples of testis tissue were collected and processed for immunohistology at various times following irradiation. Spermatogenic recovery was defined by the changes in tubule diameter, and absolute numbers of undifferentiated spermatogonia (PLZF positive cells) and Sertoli cells (GATA-4 positive cells) per tubule cross section. The effect of irradiation on the depletion of testicular cells was dose-dependent. Immunohistological results from both the 9 and 12Gy group showed degeneration of seminiferous tubules, compared with other doses and controls. From 2 weeks after the treatment, irradiation resulted in a significant and dramatic reduction in tubule diameter (up to 40%), number of undifferentiated spermatogonia (up to 90%) and Sertoli cells (up to 70%), which was sustained for up to 16 weeks post-irradiation in 9 and 12Gy groups (P<0.0001). However, a moderate depletion effect was observed in the 6Gy treatment groups, compared with 9 and 12Gy doses. The 6Gy treatment had significant effects on spermatogonia (up to 79% reduction) and Sertoli cell (30% reduction) numbers following irradiation (P<0.0001). In contrast, the 3Gy dose had no significant effect at either 3 or 5 weeks post-irradiation on tubule diameter, spermatogonia or Sertoli cells. In conclusion, the results from the current study suggest that treatment of recipient testes with a single dose of 6Gy irradiation can temporarily deplete spermatogonial cells in pre-pubertal Brahman bulls, whilst minimising the impact on Sertoli cells and tubule morphology.

Application of testis germ cell transplantation in breeding systems of food producing species: a review.

A major benefit of advanced reproduction technologies (ART) in animal breeding is the ability to produce more progeny per individual parent. This is particularly useful with animals of high genetic merit. Testis germ cell transplantation (TGCT) is emerging as a novel reproductive technology with application in animal breeding systems, including the potential for use as an alternative to artificial insemination (AI), an alternative to transgenesis, part of an approach to reducing generation intervals, or an approach toward development of interspecies hybrids. There is one major difference in TGCT between rodents and some other species associated with immunotolerance in heterologous transplantation. In particular, livestock and aquatic species do not require an immunesuppression procedure to allow donor cell survival in recipient testis. Testicular stem cells from a genetically elite individual transplanted into others can develop and produce a surrogate male-an animal that produces the functional sperm of the original individual. Spermatozoa produced from testis stem cells are the only cells in the body of males that can transmit genetic information to the offspring. The isolation and genetic manipulation of testis stem cells prior to transplantation has been shown to create transgenic animals. However, the current success rate of the transplantation procedure in livestock and aquatic species is low, with a corresponding small proportion of donor spermatozoa in the recipient's semen. The propagation of donor cells in culture and preparation of recipient animals are the two main factors that limit the commercial application of this technique. The current paper reviews and compares recent progress and examines the difficulties of TGCT in both livestock and aquatic species, thereby providing new insights into the application of TGCT in food producing animals.

Skin transcriptome profiles associated with coat color in sheep.

BACKGROUND: Previous molecular genetic studies of physiology and pigmentation of sheep skin have focused primarily on a limited number of genes and proteins. To identify additional genes that may play important roles in coat color regulation, Illumina sequencing technology was used to catalog global gene expression profiles in skin of sheep with white versus black coat color. RESULTS: There were 90,006 and 74,533 unigenes assembled from the reads obtained from white and black sheep skin, respectively. Genes encoding for the ribosomal proteins and keratin associated proteins were most highly expressed. A total of 2,235 known genes were differentially expressed in black versus white sheep skin, with 479 genes up-regulated and 1,756 genes down-regulated. A total of 845 novel genes were differentially expressed in black versus white sheep skin, consisting of 107 genes which were up-regulated (including 2 highly expressed genes exclusively expressed in black sheep skin) and 738 genes that were down-regulated. There was also a total of 49 known coat color genes expressed in sheep skin, from which 13 genes showed higher expression in black sheep skin. Many of these up-regulated genes, such as DCT, MATP, TYR and TYRP1, are members of the components of melanosomes and their precursor ontology category. CONCLUSION: The white and black sheep skin transcriptome profiles obtained provide a valuable resource for future research to understand the network of gene expression controlling skin physiology and melanogenesis in sheep.

Effect of passive immunization against leptin on ovarian follicular development in prepubertal mice.

Leptin has been demonstrated to be essential for reproduction. However, all the relevant studies reported to date have investigated either the effect of a complete absence of leptin both centrally and peripherally, or excess leptin administration. The aim of this present study was to investigate the effect of reducing peripheral leptin concentrations on ovarian follicular development in prepubertal animals via administration of an anti-leptin antibody. Pre-pubertal female mice were administered anti-leptin antibody under the skin behind the head for four days, with or without gonadotropins, and ovaries were weighed and collected for follicle counting. Control animals were treated with non-immune serum. Passive immunization against leptin, with or without gonadotropins, resulted in a significant increase in ovarian weight compared with control ovaries. Furthermore, the ovaries from the anti-leptin group had significantly greater numbers of primary follicles per ovarian section than the control group, thus suggesting an increase in the transition of primordial to primary follicles after treatment. Interestingly, animals treated with anti-leptin plus gonadotropins had a significantly higher number of Graafian follicles in their ovaries compared with the other groups. Collectively, the results of the present study indicate that reduction of leptin in the circulation promotes ovarian follicle development in female mice, suggesting that peripheral leptin acts as an inhibitor of ovarian follicle development.

Cell microarrays for the screening of factors that allow the enrichment of bovine testicular cells.

Cell microarrays can serve as high-throughput platforms for the screening of a diverse range of biologically active factors and biomaterials that can induce desired cellular responses such as attachment, proliferation, or differentiation. Here, we demonstrate that surface-engineered microarrays can be used for the screening and identification of factors that allow the enrichment and isolation of rare cells from tissue-derived heterogeneous cell populations. In particular, we have focused on the enrichment of bovine testicular cells including type A spermatogonia and Sertoli cells. Microarray slides were coated with a copolymer synthesized from poly(ethylene glycol) methacrylate and glycidyl methacrylate to enable both the prevention of cell attachment between printed spots and the covalent anchoring of various factors such as antibodies, lectins, growth factors, extracellular matrix proteins, and synthetic macromolecules on printed spots. Microarrays were incubated with mixed cell populations from freshly isolated bovine testicular tissue. Overall, cell attachment was evaluated using CellTracker staining, whereas differential attachment of testicular cells was determined by immunohistochemistry staining with Plzf and vimentin antibodies as markers for type A spermatogonia and Sertoli cells, respectively. The results indicate that various surface immobilized factors, but in particular Dolichos biflorus lectin, allowed the enrichment of Plzf positive cells. Furthermore, Pisum sativum lectin, concanavalin A, collagen type IV, and vitronectin were identified as suitable negative selection factors. To our best knowledge, this work is the first to demonstrate the utility of surface engineered cell-based microarrays for the identification of factors that allow the selective capture of rare cells from tissue isolated heterogeneous mixtures.

Expression of promyelocytic leukaemia zinc-finger in ovine testis and its application in evaluating the enrichment efficiency of differential plating.

Identification and enrichment of spermatogonial stem cells (SSCs) are critical steps in testis germ cell transplantation. The present study shows that expression of the protein promyelocytic leukaemia zinc-finger (PLZF) does not occur in all cells, only in gonocytes in neonatal testis (Stage 1) and a subpopulation of Type A spermatogonia in peripubertal (Stage 2), prepubertal (Stage 3) and post-pubertal (Stage 4) ovine testes. Dolichos biflorus agglutinin (DBA) lectin binding does not occur at any stage of testis development. The numbers of putative undifferentiated spermatogonia, germ cells and Sertoli cells were assessed by PLZF, VASA and vimentin staining, respectively. In paraffin sections, the percentage of PLZF-positive cells per tubule in samples derived from Stage 2 testis (12.2 + or - 2.8%) was twofold higher than that from Stage 1 testis (6.4 + or - 0.4%), but the percentages decreased in Stage 3 and Stage 4 testes (4.6 + or - 0.7% and 3.1 + or - 0.6%, respectively). Single cell suspensions from Stage 1 and Stage 2 testis were generated by two-step enzymatic digestion. The spermatogonia were enriched by 2 h and 2 + 16 h (overnight) differential plating on 0.2% gelatin-coated coated flasks. For Stage 1 testes, a sixfold increase in PLZF-positive cells was observed in 2 h differential plating and an almost 10-fold increase was produced following 2 + 16 h enrichment. There was less than a twofold increase in PLZF-positive cells between the 2 h and 2 + 16 h differential plating. A similar level of enrichment efficiency was also obtained for Stage 2 testis, but the percentage of PLZF-positive cells in the final enrichment was approximately one-third of that Stage 1. The efficiency of isolation and/or enrichment of PLZF-positive cells appears to depend on the maturity of the testis and the neonatal testis is better suited for isolation of gonocytes and/or putative SSCs.

Irradiation enhances the efficiency of testicular germ cell transplantation in sheep.

Testis germ cell transplantation in livestock has the potential for production of transgenic genotypes and for use as an alternative to artificial insemination in animal breeding systems. In a pilot experiment, we investigated a workable protocol for testis germ cell transplantation in sheep, including donor cell isolation, rete testis injection, and microsatellite detection of donor spermatozoa in recipient semen. In a second experiment, the effect of depletion of endogenous stem cells with a single irradiation dose of 9 Gy (n = 5) or 15 Gy (n = 5) on the outcome of germ cell transplantation was investigated. Irradiation of recipient testes with a single dose of 15 Gy, followed by transplantation 6 wk after depletion, may be most advantageous because it resulted in all recipients (five of five) producing donor-derived spermatozoa, while the 9-Gy and control groups had limited success rates (two of five and one of three, respectively). Using microsatellite markers to detect the presence of donor DNA, 10 rams were identified that produced spermatozoa of donor origin. The proportion of donor DNA was between 1% and 30% of total ejaculate DNA. When three of these positive rams were used in breeding experiments, four donor-derived offspring (four of 50 [8% of progeny])resulted from a recipient in Merino to Merino transplantation. Six lambs (six of 41 [15% of progeny]) were sired by donor-derived Border Leicester sperm produced in a Merino recipient ram; however, no donor-derived offspring were detected among 34 progeny from a second Border Leicester to Merino combination. These results confirm that preparation of recipient animals with a correct dose of irradiation not only enhances the success rate of the transplantation procedure but also increases the proportion of donor spermatozoa in recipient semen. This study represents the first report of the production of live progeny following testis germ cell transplantation using irradiated recipients in a livestock species.

A comparison of methods for preparing enriched populations of bovine spermatogonia.

The objective of the present study was to identify an efficient and practical enrichment method for bovine type A spermatogonia. Four different enrichment methods were compared: differential plating on laminin- or Datura stramonium agglutinin (DSA)-coated flasks, percoll-gradient isolation, magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). The isolated cells were characterised with Dolichos biflorus agglutinin (DBA) lectin staining for type A spermatogonia and vimentin-antibody staining for Sertoli cells. A 2 x 2 factorial design was used to investigate the enrichment efficiency on laminin and DSA. In the laminin-enrichment groups, 2 h incubation in plates coated with 20 microg mL(-1) laminin yielded a 3.3-fold increase in DBA-positive cells in the adherent fraction, while overnight incubation in flasks coated with 20 microg mL(-1) DSA produced a 3.6-fold increase in the non-adherent fraction. However, the greatest enrichment (5.3-fold) of DBA-positive cells was obtained after 2 h incubation in control flasks (coated with bovine serum albumin). Percoll-gradient centrifugation yielded a 3-fold increase in DBA-positive cells. MACS results showed a 3.5- to 5-fold enrichment while FACS produced a 4-fold increase in DBA-positive cells. It is concluded that differential plating is a better method of recovering large numbers of type A spermatogonia for germ cell transplantation, while MACS or FACS can provide highly enriched viable type A spermatogonia for in vitro culture. Further, the combination of differential plating and other enrichment techniques may increase the purification efficiency of type A spermatogonia.

Microsatellite detection of donor-derived sperm DNA following germ cell transplantation in cattle.

Although autologous and heterologous transplantation has resulted in colonisation of recipient testes in cattle, the ability of the transplanted spermatogonial stem cells to complete spermatogenesis has not yet been determined. The objective of the present study was to identify and validate microsatellite markers that can distinguish the genotype of different individuals and therefore can be used to detect the presence of donor DNA in recipient semen samples. In a previous study by this group, successful colonisation of recipient testes by heterologous transfer using a fluorescent dye was shown. In the present work, some of the same recipient animals were investigated further to monitor donor-derived sperm production. The bovine microsatellite detection method was developed specifically to test the ejaculates of the recipients and can also be used to pre-match individuals before germ cell transplantation. Semen was collected from the recipients 52-98 weeks after transfer and the presence of donor DNA in the samples was determined using microsatellite markers. In one of the recipients, all collected semen samples were shown to be positive for donor-derived cells; however, the percentage of donor spermatozoa in the recipient ejaculate declined with time. The donor DNA was also detected in both single cell suspensions and testis tissue from this recipient. These results demonstrate for the first time that testicular germ cell transplantation between different breeds of cattle is feasible and the recipients thereof are able to produce spermatozoa of donor origin. This technology has potential applications in livestock breeding systems and may provide an alternative to artificial insemination.

Large quantity cryopreservation of bovine testicular cells and its effect on enrichment of type A spermatogonia.

Cryopreservation has become an integral component of any cell transplantation technique helping to overcome the issues associated with known spatial and temporal barriers between donor and recipient. The aim of this study was to develop a protocol for large quantity cryopreservation of bovine testicular germ cells. The impact of 3 different packaging methods (5 ml semen straw, 20 ml freezing bag and 1.5 ml cryovial) and varying cell densities (3 x 10(6), 9 x 10(6), or 18 x 10(6)cells/ml) on the survival of testis germ cells was examined. Cells processed in 5 ml semen straws had a significantly higher viability (70.7+/-1.2%, P<0.05) compared to those cells in 20 ml freezing bags (46.7+/-0.1%) or 1.5 ml cryovials (46.3+/-2.2%). For 5 ml straws, a 20 min cooling prior to cryopreservation resulted in a higher post thaw viability (73.2+/-0.6%) than a 10 min cooling (56.0+/-2.2%), while the density of the cell suspension did not impact on post thaw viability. Thus cryopreservation of testicular germ cells in 5 ml straws at a density between 3 x 10(6) and 18 x 10(6)cells/ml in liquid nitrogen vapour for 20 min cooling appears to be a simple and practical way to preserve cells. Subsequent testing of frozen/thawed cells exhibited viable cultures and retained the ability to proliferate. The freezing protocol does not preferentially preserve type A spermatogonia. However, the cell surface properties of somatic cells appear to be affected by the freezing procedure and therefore the frozen/thawed cells are less suitable for enriching type A spermatogonia by differential plating.

Leptin inhibits basal but not gonadotrophin-stimulated testosterone production in the immature mouse and sheep testis.

The mechanisms whereby leptin regulates testosterone secretion are complex and are likely to involve actions at different levels of the hypothalamus-pituitary-gonadal axis. In the present study, the effect of leptin on testicular steroidogenesis at different developmental stages in mice and sheep was investigated. Testosterone data from testicular slice and Leydig cells of immature and adult mice testes demonstrated that the action of leptin in the regulation of steroidogenesis appears to be dependent on the developmental stage of the testis. Leptin biphasically modulates basal testosterone production in immature testicular slice cultures: at relatively low concentrations (6.25-12.5 ng mL(-1)) leptin exerts a significant inhibitory effect, but has less of an effect at very low (1.25 ng mL(-1)) or high concentrations (25 ng mL(-1)). However, leptin failed to modulate basal testosterone levels in Leydig cell preparations. In contrast with immature testes, leptin was unable to regulate either basal or human chorionic gonadotrophin (10 IU mL(-1))-stimulated testosterone production in adult testicular slices or Leydig cell cultures. The age- and concentration-dependent regulation pattern was confirmed using sheep testicular slice culture. Leptin (1.56-25 ng mL(-1)) significantly inhibited basal testosterone production in the testis from birth to Day 21, but had no effect on Day 27 or older testes. However, the plasma and testicular concentrations of leptin and testosterone data in the ram indicate that such a regulatory effect of leptin on testis steroidogenesis in vitro is unable to efficiently influence testosterone concentrations in vivo. This does not exclude the possibility of a non-competitive mechanism of interaction between leptin and luteinising hormone to regulate testosterone production. Thus, we hypothesise that leptin is not an important independent regulator of testosterone concentration in the normal physiological state. The physiological significance and mechanism of leptin regulation of basal testosterone production are not known; further studies are required to elucidate these important issues.

Ontogeny of leptin and its receptor expression in mouse testis during the postnatal period.

The mechanism by which leptin regulate male reproductive development during postnatal periods remain to be determined. Using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR), we established that leptin is expressed in mouse testis with a cell-type and stage-dependent manner during the postnatal period. In testes of 5-day-old mice, leptin expression was mainly restricted to gonocytes, whereas the immunostaining of leptin was confined to spermatogonia in 10-day-old testes. From Day 20 and onwards, stage-specific expression of leptin was evident in spermatocytes at stages VII-XII of the cycle in the seminiferous epithelium. RT-PCR showed that leptin and its receptor isoforms, Ob-Ra, Ob-Rb, and Ob-Re were all expressed in testes from 5- to 60-day-old mice. The mRNA for Ob-Ra and Ob-Re, but not Ob-Rb or leptin were identified in both immature (14-day-old) and adult (60-day-old) isolated Leydig cells. These results suggest that besides its primary actions at the hypothalamic-pituitary level, leptin has direct effects in the proliferation, differentiation of germ cells and the modulation of testicular steroidogenesis, using both autocrine and paracrine mechanisms.

Characterization of germ cells from pre-pubertal bull calves in preparation for germ cell transplantation.

Although methods to assess testis cell populations are established in mice, the detailed validation of similar methods for bovine testis cells is necessary for the development of emerging technologies such as male germ cell transplantation. As young calves provide donor cells for germ cell transplantation, we characterized cell populations from three key pre-pubertal stages. Nine Angus bull calves were selected to represent three stages of testis development at ages (and testis weights) of 2-3 months (Stage 1, 10 g), 4-5 months (Stage 2, 35 g), and 6-7 months (Stage 3, 70 g). The proportion and absolute numbers of germ and somatic cells in fixed sections and from enzymatically dissociated seminiferous tubules were assessed. Germ cells were identified by DBA and PGP9.5 staining, and Sertoli cells by vimentin and GATA-4 staining. Staining of serial sections confirmed that DBA and PGP9.5 identified similar cells, which were complementary to those stained for vimentin and GATA-4. In fixed tubules, the proportion of cells within tubules that were positive for DBA and PGP9.5 increased nearly three-fold from Stage 1 to Stage 2 with no further increase at Stage 3. Absolute numbers of spermatogonia also increased between Stages 1 and 2. After enzymatic dissociation of tubules, three times more DBA- and PGP9.5-positive cells were isolated from Stage 3 testes than from either Stage 1 or 2 testes. A higher proportion of spermatogonia was observed after enzymatic isolation than were present in seminiferous tubules. These data should help to predict the yield and expected proportions of spermatogonia from three distinct stages of testis development in pre-pubertal bull calves.

Successful transplantation of bovine testicular cells to heterologous recipients.

While heterologous germ cell transplantation was successful in pigs and goats, autologous transplantation alone has been reported to result in donor-derived spermatogenesis in cattle. The objective of this study was to investigate whether the transplantation of heterologous germ cells could result in colonization of recipient testes in cattle of different breeds. Testicular cells were isolated from 8 Bos taurus donor bull calves and then transferred into 15 Bos indicus-cross bull calves. All animals were prepubertal, donors were aged 5-7 months and recipients 5-11 months, and scrotal circumferences ranged from 15 to 22 cm. Single cell suspensions of donor testicular cells, prepared by enzymatic digestion, were labelled with fluorescent dyes PKH26 or CFDA-SE, before transfer into the rete testis of recipients under ultrasonographic guidance. To assess the longevity of colonization by donor cells, recipients were castrated 2-30 weeks after cell transfer. Donor cells were observed in 15/25 (60%) of the testes that received PKH26-labelled cells, whereas no CFDA-SE-positive cell was identified in any recipients. The maturity of the donors or recipients (measured by scrotal circumference) did not affect colonization potential. In freshly isolated tubules, clumps of PKH26-positive cells were observed, which indicated either cell division or extensive local colonization of specific areas of the tubules. In frozen sections, PKH26-positive cells were identified on the seminiferous tubule basement membrane, which indicated that these cells had successfully migrated from the tubule lumen and were likely to be spermatogonia. We conclude that PKH26 was more suitable for labelling donor testis cells and donor cells can be identified up to 6 months following transfer. These results indicate that allogeneic transplantation of testicular cells can occur between Bos taurus and Bos indicus cattle. Further studies will investigate functionality of transferred testicular cells.

Leptin has concentration and stage-dependent effects on embryonic development in vitro.

There is accumulating evidence that leptin may be directly involved in pre-implantation embryonic development, however, it is unclear whether there is a concentration and stage-dependent regulatory pattern. In this study, the addition of 10 ng/ml human recombinant leptin to the culture medium significantly increased the percentage of two-cell mouse embryos that developed into blastocysts and hatched blastocysts, whereas in the presence of 100 ng/ml leptin, the development rate was significantly inhibited. The total cell numbers in the hatched blastocysts were significantly higher in the presence of 10 ng/ml leptin compared with controls and higher concentrations. The differential sensitivity to leptin was found to vary among embryos at different stages of development. Supplementation of leptin (10 ng/ml) to culture medium at two- to eight-cell stages resulted in a consistent stimulatory effect on embryo development. Most interestingly, the inhibitory effect of high leptin concentration (100 ng/ml) on embryo development was diminished when it was added to the culture medium at the eight-cell stage of development. The concentration-dependent regulation pattern was confirmed using sheep embryos, under similar conditions although sheep embryos appeared to be more sensitive in responding to leptin. Having established the effect of exogenous leptin on embryo development, the expression pattern of leptin and its receptors were also investigated. Leptin mRNA was not detected in mouse two-, four-, eight-cell and blastocyst stage embryos, whereas three isoforms of leptin receptor (Ob-Ra, Ob-Rb and Ob-Re) were identified in these cells, indicating that leptin is likely to modulate embryo development via a paracrine signalling system.