The impact of undernutrition on KNDy (kisspeptin/neurokinin B/dynorphin) neurons in female lambs.

Undernutrition limits reproduction through inhibition of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) secretion. Because KNDy neurons coexpress neuropeptides that play stimulatory (kisspeptin and neurokinin B [NKB]) and inhibitory (dynorphin) roles in pulsatile GnRH/LH release, we hypothesized that undernutrition would inhibit kisspeptin and NKB expression at the same time as increasing dynorphin expression. Fifteen ovariectomized lambs were either fed to maintain pre-study body weight (controls) or feed-restricted to lose 20% of pre-study body weight (FR) over 13 weeks. Blood samples were collected and plasma from weeks 0 and 13 were assessed for LH by radioimmunoassay. At week 13, animals were killed, and brain tissue was processed for assessment of KNDy peptide mRNA or protein expression. Mean LH and LH pulse amplitude were lower in FR lambs compared to controls. We observed lower mRNA abundance for kisspeptin within KNDy neurons of FR lambs compared to controls with no significant change in mRNA for NKB or dynorphin. We also observed that FR lambs had fewer numbers of arcuate nucleus kisspeptin and NKB perikarya compared to controls. These findings support the idea that KNDy neurons are important for regulating reproduction during undernutrition in female sheep.

Evidence that pubertal status impacts kisspeptin/neurokinin B/dynorphin neurons in the gilt†.

Puberty onset is a complex physiological process, which enables the capacity for reproduction through increased gonadotropin-releasing hormone and subsequently luteinizing hormone secretion. While cells that coexpress kisspeptin, neurokinin B (NKB), and dynorphin in the hypothalamic arcuate nucleus are believed to govern the timing of puberty, the degree to which kisspeptin/NKB/dynorphin (KNDy) neurons exist and are regulated by pubertal status remains to be determined in the gilt. Hypothalamic tissue from prepubertal and postpubertal, early follicular phase gilts was used to determine the expression of kisspeptin, NKB, and dynorphin within the arcuate nucleus. Fluorescent in situ hybridization revealed that the majority (>74%) of arcuate nucleus neurons that express mRNA for kisspeptin coexpressed mRNA for NKB and dynorphin. There were fewer arcuate nucleus cells that expressed mRNA for dynorphin in postpubertal gilts compared to prepubertal gilts (P < 0.05), but the number of arcuate nucleus cells expressing mRNA for kisspeptin or NKB was not different between groups. Within KNDy neurons, mRNA abundance for kisspeptin, NKB, and dynorphin of postpubertal gilts was the same as, less than, and greater than, respectively, prepubertal gilts. Immunostaining for kisspeptin did not differ between prepubertal and postpubertal gilts, but there were fewer NKB immunoreactive fibers in postpubertal gilts compared to prepubertal gilts (P < 0.05). Together, these data reveal novel information about KNDy neurons in gilts and support the idea that NKB and dynorphin play a role in puberty onset in the female pig.

Evidence That Agouti-Related Peptide May Directly Regulate Kisspeptin Neurons in Male Sheep.

Agouti-related peptide (AgRP) neurons, which relay information from peripheral metabolic signals, may constitute a key central regulator of reproduction. Given that AgRP inhibits luteinizing hormone (LH) secretion and that nutritional suppression of LH elicits an increase in AgRP while suppressing kisspeptin expression in the arcuate nucleus (ARC) of the hypothalamus, we sought to examine the degree to which AgRP could directly regulate ARC kisspeptin neurons. Hypothalamic tissue was collected from four castrated male sheep (10 months of age) and processed for the detection of protein (AgRP input to kisspeptin neurons) using immunohistochemistry and mRNA for melanocortin 3 and 4 receptors (MC3R; MC4R) in kisspeptin neurons using RNAscope. Immunohistochemical analysis revealed that the majority of ARC kisspeptin neurons are contacted by presumptive AgRP terminals. RNAscope analysis revealed that nearly two thirds of the ARC kisspeptin neurons express mRNA for MC3R, while a small percentage (<10%) colocalize MC4R. Taken together, this data provides neuroanatomical evidence for a direct link between orexigenic AgRP neurons and reproductively critical kisspeptin neurons in the sheep, and builds upon our current understanding of the central link between energy balance and reproduction.

Undernutrition reduces kisspeptin and neurokinin B expression in castrated male sheep.

Undernutrition impairs reproductive success through suppression of gonadotropin-releasing hormone (GnRH), and subsequently luteinizing hormone (LH), secretion. Given that kisspeptin and neurokinin B (NKB) neurons in the arcuate nucleus (ARC) of the hypothalamus are thought to play key stimulatory roles in the generation of GnRH/LH pulses, we hypothesized that feed restriction would reduce the ARC mRNA abundance and protein expression of kisspeptin and NKB in young, male sheep. Fourteen wethers (castrated male sheep five months of age) were either fed to maintain (FM; n = 6) pre-study body weight or feed-restricted (FR; n = 8) to lose 20% of pre-study body weight over 13 weeks. Throughout the study, weekly blood samples were collected and assessed for LH concentration using RIA. At Week 13 of the experiment, animals were killed, heads were perfused with 4% paraformaldehyde, and brain tissue containing the hypothalamus was collected, sectioned, and processed for detection of mRNA (RNAscope) and protein (immunohistochemistry) for kisspeptin and NKB. Mean LH was significantly lower and LH inter-pulse interval was significantly higher in FR wethers compared to FM wethers at the end of the experiment (Week 13). RNAscope analysis revealed significantly fewer cells expressing mRNA for kisspeptin and NKB in FR wethers compared to FM controls, and immunohistochemical analysis revealed significantly fewer immunopositive kisspeptin and NKB cells in FR wethers compared to FM wethers. Taken together, this data supports the idea that long-term feed restriction regulates GnRH/LH secretion through central suppression of kisspeptin and NKB in male sheep. LAY SUMMARY: While undernutrition is known to impair reproduction at the level of the brain, the components responsible for this in the brain remain to be fully understood. Using male sheep we examined the effect of undernutrition on two stimulatory molecules in the brain critical for reproduction: kisspeptin and neurokinin B. Feed restriction for several weeks resulted in decreased luteinizing hormone in the blood indicating reproductive function was suppressed. In addition, undernutrition also reduced both kisspeptin and neurokinin B levels within a region of the brain involved in reproduction, the hypothalamus. Given that they have stimulatory roles in reproduction, we believe that undernutrition acts in the brain to reduce kisspeptin and neurokinin B levels leading to the reduction in luteinizing hormone secretion. In summary, long-term undernutrition inhibits reproductive function in sheep through suppression of kisspeptin and neurokinin B within the brain.

Nr4A1 mRNA regulation of FSH-induced meiotic resumption in bovine cumulus-oocyte complexes cultured in vitro.

OBJECTIVE: 1) further define the time course required for transcription initiation in bovine cumulus-oocyte-complexes (COC); 2) determine the pattern of expression for Nr4A1 and Egr1 mRNAs in bovine COC; and, 3) reduce Nr4A1 mRNA expression using small interfering RNAs (siRNA) to determine the effect on breakdown of the germinal vesicle (GVBD). METHODS: A series of experiments were performed to define the time required for transcription initiation during FSH-induced maturation in bovine COCs, determine the pattern of expression for candidate mRNAs during GVBD, and use RNAi to determine their potential role in GVBD by examining whether candidate-specific siRNA can reduce mRNA expression in bovine COCs and affect the occurrence of GVBD. RESULTS: Transcription required for GVBD in bovine COC occurred as early as 30 min after culture initiation. Expression of Nr4A1 mRNA increased (P <0.05) at 30 min after culture initiation, consistent with the time of transcription initiation required for GVBD. Expression of Egr1 mRNA did not differ during culture. Expression of Nr4A1 mRNA was decreased (P <0.05) in COC cultured with 50nM siNr4A1 or with 120 µM of the transcriptional inhibitor DRB compared to controls. The proportion of COC undergoing GVBD at 9 hr of culture in FSH and non-specific siRNA (siNS) treatment groups did not differ. However, fewer (P <0.05) COC underwent GVBD at 9 hr of culture when in the presence of DRB or 50nM siNr4A1 compared to controls. CONCLUSION: these data support a role for Nr4A1 in regulating FSH-mediated and transcription-dependent GVBD in bovine COC cultured in vitro.

Cloning, characterization, and expression analysis of the pig (Sus scrofa) C1q tumor necrosis factor-related protein-5 gene.

PURPOSE: Autosomal dominant early-onset long anterior zonules (LAZs) and late-onset retinal degeneration (L-ORD) in humans are associated with the S163R mutation of the complement 1q-tumor necrosis factor related protein-5 (CTRP5) gene. For using the pig as an L-ORD model for the study of pathology, we cloned, characterized, and studied the expression profile of pig CTRP5 (pCTRP5). METHODS: The pCTRP5 was cloned and sequenced from porcine genomic DNA. Bioinformatic analysis was done to evaluate the functional domains present in the pCTRP5 using PROSITE tools. The V5 epitope-tagged constructs of pCTRP5 and the mammalian promoters, elongation factor 1-α (EF) promoter and 579 bp of the putative promoter located upstream to pCTRP5 DNA, were used for in vitro expression analysis. The pCTRP5 expression, protein size, and cellular localization were studied in transiently transfected Cos-7 or ARPE-19 cells by western blot analysis using anti-CTRP5 and anti-V5 epitope antibodies. Expression of pCTRP5 in the pig eye tissues was analyzed by western blot analysis, real-time PCR, and immunohistochemistry. RESULTS: As predicted, pCTRP5 showed a 92% DNA homology and 98% amino acid homology with human CTRP5 (hCTRP5). Bioinformatic analysis revealed the presence of an alternate in-frame translational start site upstream to the presumed initiator codon. The presence of a putative promoter region upstream to the pCTRP5 was identified. The putative pCTRP5 promoter was found to be functional by western blot analysis. The size of the pCTRP5 protein (pCTRP5) was consistent with its predicted molecular weight, indicating that the potential alternative start site was not used. Western blot and RT-PCR analyses showed that pCTRP5 was predominantly expressed in RPE, a pattern of expression consistent with that found in mouse and human eyes. CONCLUSIONS: The sequence and genomic organization of pCTRP5 was found to be similar to the human homolog. The DNA and protein sequence of pCTRP5 are highly homologous to hCTRP5, indicating that they are highly conserved. A putative promoter region (579 bp) present upstream to pCTRP5 was found to be functional and was able to drive the expression of the pCTRP5 gene cloned downstream. The tissue distribution in the eye and the expression profile of pCTRP5 in transiently transfected cells is consistent with hCTRP5 expression. Immunohistochemistry analysis of the pig retinal sections revealed localization of pCTRP5 to the apical and basolateral regions on the RPE and in the ciliary body. The potential in-frame alternate start site was found to be nonfunctional by western blot analysis of transiently transfected cells. Similarities between human and pig CTRP5 and the presence of an area centralis region in the pig similar to the human macula, together with its large eyeball size, makes the domestic pig a good model for the study of LAZs and L-ORD.

Transgenic Stra8-EYFP pigs: a model for developing male germ cell technologies.

The male germ line in mammals is composed of self-renewing cells, spermatogonia, the meiotic spermatocytes and spermiogenic spermatids. Identification of these cell stages in vitro has been problematic. Transgenic animals expressing a marker gene with a promoter specific to certain cell stages in the testis would be a useful approach to identifying these cells in a viable state. Towards this end, we have produced transgenic pigs expressing mitochondrial localized enhanced yellow fluorescent protein (EYFP-mito) under control of the germ cell specific Stimulated by Retinoic Acid 8 (Stra8) promoter. Stra8 has been shown to be expressed in pre-meiotic germ cells of mice. Twelve clones harboring the Stra8-EYFP-mito transgene were produced. Analysis by Western blot indicated that expression of the transgene was limited to testicular tissue in the transgenic pigs. Single cells and seminiferous tubules were cultured in vitro and subsequently examined with epifluorescent microscopy. Expression of EYFP was noted in cells cultured for up to 5 days. Both EYFP-mito and STRA8 antibodies were shown to bind and co-localize in seminiferous tubule cells in whole mounts and in histological sections. EYFP-mito in the transgenic pigs co-localized with the endogenous stem cell marker, NANOG. Expression of the Stra8-EYFP transgene in spermatogenic cells indicates that these pigs will be useful by providing labelled cells for use in such technologies such as germ cell transplantation and in vitro spermatogenic studies.

Production of ELOVL4 transgenic pigs: a large animal model for Stargardt-like macular degeneration.

BACKGROUND: Truncation mutations in the elongation of very long chain fatty acids-4 (AF277094, MIM #605512) (ELOVL4) gene cause Stargardt-like macular dystrophy type 3 (STGD3). Mice expressing truncated ELOVL4 develop rapid retinal degeneration, but are poor STGD3 models since mice lack a macula. Photoreceptor topography in the pig retina is more similar to that in humans as it includes the cone rich, macula-like area centralis. The authors generated transgenic pigs expressing human disease-causing ELOVL4 mutations to better model the pathobiology of this macular disease. METHODS: Pronuclear DNA microinjection and somatic cell nuclear transfer were used to produce transgenic pigs for two different ELOVL4 mutations: the 5 base pair deletion (5 bpdel) and the 270 stop mutation (Y270terEYFP). Retinal transgene expression, morphology and electrophysiology were examined. RESULTS: The authors obtained four lines of Y270terEYFP and one line of 5 bpdel transgenic animals. Direct fluorescence microscopy indicated that the Y270terEYFP protein is expressed in photoreceptors and mislocalised within the cell. Immunohistochemical examination of transgenic pigs showed photoreceptor loss and disorganised inner and outer segments. Electroretinography demonstrated diminished responses in both transgenic models. CONCLUSIONS: These transgenic pigs provide unique animal models for examining macular degeneration and STGD3 pathogenesis.

Phenotypic stability of Pro347Leu rhodopsin transgenic pigs as indicated by photoreceptor cell degeneration.

Rhodopsin (Pro347Leu) transgenic pigs are recognized to be an excellent model for the human disease, retinitis pigmentosa. First published in 1997, the rhodopsin transgenic pigs have been maintained since that time at North Carolina State University by outcrossing hemizygous boars to unrelated sows. Nine generations of outcrossing have been completed. Since the genetic background of these pigs has undoubtedly changed, the question of the current phenotype of the transgenic pigs is relevant for their future use. Age-matched transgenic and non-transgenic eyes were submitted for histological analysis using hematoxylin and eosin staining. Even by 2 weeks of age, significant thinning of the outer nuclear layer of photoreceptors was observed. For ages 3 and 4 weeks, thinning was noted similar to that of 2 weeks of age. By 6 weeks of age outer nuclear layer thinning was greater than that of earlier age. At 11 weeks of age, most of the rods have degenerated leaving only a few layers of cones. In all, the phenotype, based on assessment of photoreceptor degeneration, is similar to that of the first description of the transgenic animals. As such the Pro347Leu rhodopsin transgenic pigs have exhibited phenotypic stability through generations of outcrossing and can be used confidently in future studies of the type of retinal degeneration seen with retinitis pigmentosa.

Identification of a promoter for the human C1Q-tumor necrosis factor-related protein-5 gene associated with late-onset retinal degeneration.

PURPOSE: The Complement-1q tumor necrosis factor-related protein 5 (C1QTNF5/CTRP5) gene is located in the 3' untranslated region of the Membrane Frizzled Related Protein (MFRP) gene, and these two genes are reported to be dicistronic. The authors examined the 5' upstream sequence of CTRP5 for the presence of a promoter regulating the expression of this gene. METHODS: The sequence upstream of the translational start site of human CTRP5 (hCTRP5) was analyzed by Promoter Inspector software. A series of plasmids containing segments of hCTRP5 putative promoter sequence (-29 bp to -3.6 kb) upstream of the luciferase gene were generated. Cells were transiently transfected with these plasmids, and luciferase activity was measured. 5' RACE analysis was performed to determine the functional transcription start site. V5 tagged-pig CTRP5 (pCTRP5) gene, cloned downstream of the hCTRP5 putative promoter, was expressed in a human retinal cell line (ARPE-19) and a Chinese hamster ovary cell line (CHO-K1) to study the functionality of the putative promoter. RESULTS: Bioinformatic analysis identified a putative promoter region between nt -1322 and +1 sequence of hCTRP5. 5' RACE analysis revealed the presence of the transcriptional start site (TSS) at 62 bp upstream of the start codon in the CTRP5. The 1.3-kb sequence of the hCTRP5 predicted promoter produced higher levels of luciferase activity, indicating the strength of the cloned CTRP5 promoter. The promoter sequence between nt -1322 bp to -29 bp upstream of the first ATG of CTRP5 was found to be essential for this promoter activity. The predicted hCTRP5 promoter was found to control the expression of V5-tagged pCTRP5 and nuclear GFP, indicating that the promoter was functional. CONCLUSIONS: This study revealed the presence of a functional promoter for the CTRP5 gene located 5' of its start site. Understanding the regulation of CTRP5 gene transcription may provide insights into the possible role of CTRP5 in the retina and the pathology underlying late-onset retinal degeneration caused by mutations in this gene. In addition, these studies will determine whether CTRP5 and MFRP are functionally dicistronic.

Synchronization and superovulation of mature cycling gilts for the collection of pronuclear stage embryos.

An efficient protocol was developed to synchronize and superovulate mature pigs for the collection of pronuclear stage embryos suitable for DNA microinjection. A timed and coordinated regimen of Lutalyse, PG600 and Chorulon along with daily checking for estrus allowed synchronization of groups of gilts having estrous cycles at regular intervals. Pigs 10-16 days after the beginning of standing estrus have been successfully synchronized into estrus using this protocol. A standard dose of each drug was used independent of size or age of the animal. One protocol averaged 38.9 ovulations and 31.1 one-cell embryos recovered per animal.

Reporter system for the detection of in vivo gene conversion: changing colors from blue to green using GFP variants.

We have devised a system for the study of in vivo gene correction based on the detection of color variants of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The intensity and spectra of the fluorescence emitted by the blue (BFP) and red-shifted (EGFP) variants of GFP differ from each other. We modified one nucleotide from an EGFP expression vector that we predicted would yield a blue variant (TAC-CAC, Tyr(66)-His(66)). Cells that were either transiently or stably transfected with the reporter system were used to test the functionality and feasibility of the detection of in vivo gene correction. A thio-protected single-stranded oligonucleotide designed to convert the genotype of the blue variant to that of the EGFP variant by the correction of a single base pair was delivered to the reporter cells using a variety of methodologies and strategies.Conversion events were easily observed using fluorescent microscopy because of the enhanced emission intensity and different spectra of the EGFP variant.

Conditional induction of ovulation in mice.

Follicle-stimulating hormone controls the maturation of mammalian ovarian follicles. In excess, it can increase ovulation (egg production). Reported here is a transgenic doxycycline-activated switch, tested in mice, that produced more FSHB subunit (therefore more FSH) and increased ovulation by the simple feeding of doxycycline (Dox). The transgenic switch was expressed selectively in pituitary gonadotropes and was designed to enhance normal expression of FSH when exposed to Dox, but to be regulated by all the hormones that normally control FSH production in vivo. Feeding maximally effective levels of Dox increased overall mRNA for FSHB and serum FSH by over half in males, and Dox treatment more than doubled the normal ovulation rate of female mice for up to 10 reproductive cycles. Lower levels of Dox increased the number of developing embryos by 30%. Ovarian structure and function appeared normal. In summary, gene switch technology and normal FSH regulation were combined to effectively enhance ovulation in mice. Theoretically, the same strategy can be used with any genetic switch to increase ovulation (or any highly conserved physiology) in any mammal.