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 Cdk5knockdown 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

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

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

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

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

Optimization of a vitrification protocol for hatched blastocysts from the dromedary camel ('Camelus dromedarius')

The objective of this study was to modify and optimize a vitrification protocol (open pulled straw) that was originally designed for human oocytes and embryos, to make it suitable for the cryopreservation of camel hatched blastocysts. The original open pulled straw protocol was a complex process with 15-minute exposure of oocytes/embryos in 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (Me₂SO) for equilibration, and cooling in 16% EG + 16% Me₂SO + 1 M sucrose. Recognizing a need to better control the cryoprotectant (CPA) concentrations, while avoiding toxicity to the embryos, the effects on the survival rate and developmental potential of camel embryos in vitro were investigated using two different methods of loading the CPAs into the embryos (stepwise and semicontinuous increase in concentration), two different loading temperature/time (room temperature ~°C/15 min and body 37°C/3 min), and the replacement of Me₂SO with EG alone or in combination with glycerol (Gly). A total of 145 in vivo-derived embryos were subjected to these processes, and after warming their morphological quality and integrity, and reexpansion was assessed after 0, 2, 24, 48, 72, and 96 hours of culture. Exposure of embryos in a stepwise method was more beneficial to the survival of embryos than was the semicontinuous process, and loading of CPAs at 37°C with a short exposure time (3 minutes) resulted in an outcome comparable to the original processing at room temperature with a longer exposure time (15 minutes). The replacement of the Me₂SO + EG mixture with EG only or a combination of EG + Gly in the vitrification medium significantly improved the outcome of all these evaluation criteria (

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

The successful use of busulfan to deplete endogenous spermatogonia in ram testes

Our research into germ cell transfer builds upon murine research1 and is aimed at using this technique in livestock species. To increase the efficiency of colonization of transplanted germ cells, the recipient testes must be depleted of endogenous spermatogonia, without affecting the supporting cells. Three depletion methods were investigated, heat, cold and chemotherapy. Our first investigation looked at the direct cooling (0°C) and heating (45°C) of the testes of 4-6 week old ram lambs. Testes were collected 7 days post treatment. The second investigation involved the systemic injection of busulfan to ram lambs aged 3-4 months. Busulfan is used for preparing recipient mice for testes cell transfer. At doses affecting the stem cells of the testes, busulfan will result in mylosuppression. Therefore a preliminary dose response trial was conducted at dose rates 4, 8 and 16mg/kg to determine the most effective dose, without threatening the survival of the animal. Testes were recovered after 3 and 6 weeks. All testes sections followed routine histology and immunochemistry with PGP 9.5 (Table 1). For the heat and cold study, only gonocytes were present and there were no differences in testes weights, tubule diameters or gonocyte numbers in any of the treatment groups. For the busulfan study, dose rates of 8 and 16 mg/kg resulted in severe mylosuppression and euthanasia of 7 out of 8 animals between day 12 and 18, whereas animals in the 4mg/kg group showed only mild clinical effects, that were not life threatening. These results indicate that busulfan reduced endogenous spermatogonia in the pre-pubertal ram. This effect is observed at systemic doses of 4 mg/kg or higher; however, doses of 8 mg/kg and above are lethal to the survival of the animal. The use of direct heat (45°C) or cold (0°C) to the testes does not affect gonocyte numbers in ram lambs; however, effects on more mature stages was not studied