Outer retinal transduction by AAV2-7m8 following intravitreal injection in a sheep model of CNGA3 achromatopsia.

Sheep carrying a mutated CNGA3 gene exhibit diminished cone function and provide a naturally occurring large animal model of achromatopsia. Subretinal injection of a vector carrying the CNGA3 transgene resulted in long-term recovery of cone function and photopic vision in these sheep. Research is underway to develop efficacious vectors that would enable safer transgene delivery, while avoiding potential drawbacks of subretinal injections. The current study evaluated two modified vectors, adeno-associated virus 2-7m8 (AAV2-7m8) and AAV9-7m8. Intravitreal injection of AAV2-7m8 carrying enhanced green fluorescent protein under a cone-specific promoter resulted in moderate photoreceptor transduction in wild-type sheep, whereas peripheral subretinal delivery of AAV9-7m8 resulted in the radial spread of the vector beyond the point of deposition. Intravitreal injection of AAV2-7m8 carrying human CNGA3 in mutant sheep resulted in mild photoreceptor transduction, but did not lead to the clinical rescue of photopic vision, while day-blind sheep treated with a subretinal injection exhibited functional recovery of photopic vision. Transgene messenger RNA levels in retinas of intravitreally treated eyes amounted to 4-23% of the endogenous CNGA3 levels, indicating that expression levels >23% are needed to achieve clinical rescue. Overall, our results indicate intravitreal injections of AAV2.7m8 transduce ovine photoreceptors, but not with sufficient efficacy to achieve clinical rescue in CNGA3 mutant sheep.

The distinctive short-term response of late-pregnant prolific ewes to propylene glycol or glycerol drenching.

Pregnancy toxemia is the most frequent metabolic disorder of ewes in late pregnancy. Although propylene glycol (PG) and glycerol (GLY) are common glucogenic supplements for treating pregnancy toxemia in ewes, the relative benefit of these 2 supplements is not entirely clear. Therefore, the objectives of the present study were to determine the changes during 24 h in key blood metabolites and insulin in response to PG or GLY drenching in prolific ewes. To this end, 36 multiparous late-pregnant Afec-Assaf ewes (∼132.4 d pregnant) bearing 2 to 4 fetuses, divided into 2 blocks (18 ewes in each block), with a blood ß-hydroxybutyrate (BHB) concentration of 0.5 to 1.6 mmol/L were included. Ewes were divided into 3 groups (12 ewes each; 6 ewes in each experimental day), according to their BHB levels, expected litter size, body weight, and body condition score, and were drenched with the following: (1) control group (CTL), 55 mL of water; (2) PG, 106 mL of PG (100% PG, 448 calories); or (3) GLY, 108 mL of Koforin 80 (80% GL; 448 calories). Blood samples were taken before drenching and every hour after drenching for 24 h. Plasma concentration of glucose, BHB, nonesterified fatty acids, lactate, glycerol, and insulin were determined. Because there were no effects of treatments after 12 h in the first block, the data were analyzed for 12 h after drenching rather than 24 h. The plasma glucose concentration during the first 5 h after drenching was the highest in the GLY, BHB concentration was the lowest in the PG, and the nonesterified fatty acid levels were lower in the PG compared with the CTL ewes during the first 5 h after drenching. However, glucose concentration was higher in the PG ewes at 9, 11, and 12 h after drenching than in CTL or GLY ewes. The mean lactate concentration in plasma for 12 h was 2.5- and 1.9-fold higher in the PG compared with the CTL and GLY ewes, respectively, and except at 11 h after drenching, it was significantly higher at each time point. The insulin concentration was higher in the GLY than in both other groups at 2 to 5 h after drenching. These results suggest that during the first few hours after drenching the effect of PG was more effective in reducing the BHB concentration, whereas the GLY effect was more effective in enhancing glucose concentration. The increased concentration in lactate following PG treatment suggests that the PG contribution to gluconeogenesis is mediated through its metabolism to lactate. In contrast, the lack of an effect on lactate, and the faster increase in blood glucose in response to GLY suggest that GLY has a more advanced entry point to gluconeogenesis, which influences the immediate response in enhancing the glucose blood concentration.

Meta-analysis of morphometric parameters of late-gestation fetal sheep developed under natural and artificial constraints.

A meta-analysis was performed to study the relationship between ovine fetal BW and body length during the last month of pregnancy in sheep gestated under normal conditions and under different natural and experimental maternal- placental- and fetal- directed constraints. Means of crown-rump length (CRL) and BW records, as well as means of calculated G index (GI, BW/CRL1.5), body mass index (BMI, BW/CRL2.0), and Ponderal index (PI, BW/CRL3.0) of 195 nontreated and 160 treated groups of lambs from 131 studies were investigated by ANOVA. the GI is a novel BW-body length index developed for sheep fetuses and newborn lambs. The analysis included the effects of study (n=131), treatment (n=38), average litter size (1 to 3), and days in pregnancy (120 d to lambing). The morphometric parameters were obtained on average on d 139 of gestation, when lambs in the nontreated groups averaged 0.50 m for CRL and 4.20 kg for BW. applying the different treatments caused substantial variation in fetal BW (from 2.5 to 4.9 kg), and somewhat less variation in fetal CRL (0.41 to 0.55 m), reflecting ovine fetal ability to adapt its growth to a variety of natural and experimental constraints. Some treatments affected (P<0.05) the fetal BW-body length relationship, as clearly reflected by the GI and BMI, but not by the PI. litter size and days in pregnancy had effects (P<0.05) on all variables. It was concluded that the severity of fetal intrauterine growth restriction (IUGR) in the case of multifetus pregnancies is comparable with the IUGR effects caused by experimentally induced fetal growth-inhibiting interventions, and that GI may be superior to BMI and PI in detecting interference with ovine fetal body proportion.

A deleterious effect associated with UNH159 is attenuated in twin embryos of an inbred line of blue tilapia Oreochromis aureus.

Offspring of a highly inbred gynogenetic line of Oreochromis aureus displayed 12-fold increase in twinning rate compared to the outbred population. Asymmetric conjoined twins, which consist of a normal embryo attached to a malformed-atrophic twin, were frequently encountered in both gynogenetic (90·7%) and outbred (38·2%) embryos. The monozygotic origin of these twins was determined using five microsatellite markers. Progeny of heterozygous parents for the microsatellite UNH159 were separated into sub-sets of twins and normal full-sibs. Consistent with previous reports, the normal embryo sub-set exhibited elimination of both types of homozygotes for the UNH159 genetic marker at 2-8 days after fertilization. Unexpectedly, this elimination was less frequent in twins. The UNH159 marker as well as RNA-binding motif protein, X-linked (rbmx), SRY-box containing gene 3 (sox3) and alpha-thalassemia/mental retardation syndrome X-linked (atrx) genes were mapped to linkage group 2. These gene orthologues are all located on the mammalian X chromosome and atrx is necessary for the X-chromosome inactivation.

Ovine mitochondrial DNA sequence variation and its association with production and reproduction traits within an Afec-Assaf flock.

Polymorphisms in mitochondrial DNA (mtDNA) protein- and tRNA-coding genes were shown to be associated with various diseases in humans as well as with production and reproduction traits in livestock. Alignment of full length mitochondria sequences from the 5 known ovine haplogroups: HA (n = 3), HB (n = 5), HC (n = 3), HD (n = 2), and HE (n = 2; GenBank accession nos. HE577847-50 and 11 published complete ovine mitochondria sequences) revealed sequence variation in 10 out of the 13 protein coding mtDNA sequences. Twenty-six of the 245 variable sites found in the protein coding sequences represent non-synonymous mutations. Sequence variation was observed also in 8 out of the 22 tRNA mtDNA sequences. On the basis of the mtDNA control region and cytochrome b partial sequences along with information on maternal lineages within an Afec-Assaf flock, 1,126 Afec-Assaf ewes were assigned to mitochondrial haplogroups HA, HB, and HC, with frequencies of 0.43, 0.43, and 0.14, respectively. Analysis of birth weight and growth rate records of lamb (n = 1286) and productivity from 4,993 lambing records revealed no association between mitochondrial haplogroup affiliation and female longevity, lambs perinatal survival rate, birth weight, and daily growth rate of lambs up to 150 d that averaged 1,664 d, 88.3%, 4.5 kg, and 320 g/d, respectively. However, significant (P < 0.0001) differences among the haplogroups were found for prolificacy of ewes, with prolificacies (mean ± SE) of 2.14 ± 0.04, 2.25 ± 0.04, and 2.30 ± 0.06 lamb born/ewe lambing for the HA, HB, and the HC haplogroups, respectively. Our results highlight the ovine mitogenome genetic variation in protein- and tRNA coding genes and suggest that sequence variation in ovine mtDNA is associated with variation in ewe prolificacy.

Nonsynonymous natural genetic polymorphisms in the bovine leptin gene affect biochemical and biological characteristics of the mature hormone.

Leptin (LEP) is a cytokine-like hormone proven to be involved in diverse biological processes. In livestock, it regulates feed intake, BW homeostasis, and energy balance, among other traits. Natural nonsynonymous genetic polymorphisms in the ovine leptin (oLEP) alter the biochemical and physiological characteristics of its gene products. Here we studied in vitro and in vivo the biochemical and physiological characteristics of recombinant hormones representing the oLEP and bovine leptin (bLEP) reference sequences of wild-type (WT) leptins (GenBank accession No. U84247 and U50365, respectively), oLEP and bLEP recombinant muteins carrying the R4C mutation, and oLEP recombinant hormones carrying the A59V and Q62R mutations, which were detected in bLEP. All proteins were purified to homogeneity as monomers and formed 1:1 molar ratio complexes with the chicken leptin-binding domain (LBD). Surface plasmon resonance experiments revealed that all protein variants exhibit reduced (P < 0.05) affinity to chicken (ch) and human (h) LBD compared with the WT oLEP and bLEP recombinant proteins. The ovine and bovine R4C muteins exhibited significantly (P < 0.05) greater induction of cell proliferation in a Baf/3 cell line bioassay, despite lower affinity toward both hLBD and chLBD. Intra-third cerebral ventricle infusion of oLEP and its 3 muteins in sheep resulted in reduced feed intake. However, the 3 tested muteins had a decreased (P < 0.05) inhibitory effect than the WT LEP. It was concluded that natural genetic polymorphisms in the bLEP are associated with variation in the biochemical and physiological properties of the protein.

Plasma concentrations of key metabolites and insulin in late-pregnant ewes carrying 1 to 5 fetuses.

Ewes bearing more than 1 fetus are more susceptible to pregnancy toxemia than those with a single fetus. Crossbreeding programs in Israel increased the occurrences of ewes bearing more than 2 fetuses; therefore, the aim was to assess the exacerbation in the metabolic status of ewes pregnant with several fetuses. Fifty ewes, genetically developed to achieve multiple-fetus pregnancies, were monitored, on average, from d 115 of pregnancy until lambing for plasma concentrations of several key metabolites and insulin. The numbers of fetuses were examined by ultrasonography at 35 d of pregnancy. Blood samples were collected weekly, and concentrations of glucose, ß-hydroxybutyrate (BHBA), NEFA, triglycerides, cholesterol, total calcium, and insulin were determined. The average litter size was 2.75 (±1.1), and 1 (1F), 2 (2F), 3 (3F), and 4 or more (4F) fetuses were conceived, respectively, by 6 (12%), 17 (34%), 14 (28%), and 13 (26%) ewes. Total birth weights of lambs were 6.1, 9.5, 12.7, and 15.0 kg for 1F, 2F, 3F, and 4F, respectively (P < 0.001). Plasma glucose concentrations in 1F were greater than those in 3F and 4F (P < 0.05) and were similar among 2F, 3F, and 4F. Trends toward increasing plasma concentrations of BHBA and NEFA were observed as the number of fetuses increased and also as lambing approached. Plasma concentrations of BHBA and NEFA were, respectively, 3.7 (P < 0.002) and 2.1 (P < 0.001) times as great in 4F ewes as in 1F ewes. Trends toward decreased concentrations of triglycerides and cholesterol were observed as litter size increased. Insulin concentrations in blood decreased considerably as the numbers of fetuses increased and, on average, they were less by a factor of 5 in the 4F ewes than in the 1F ewes (P < 0.001). Moreover, insulin concentrations during the week before lambing were extremely low (e.g., 0.54 µIU/mL in the 4F ewes). Insulin concentrations were reduced in ewes bearing >3 fetuses, even 5 wk before lambing; this decline apparently began earlier than the last month of gestation. Therefore, it seems that insulin has a pivotal role in the etiology of pregnancy ketonemia in ewes carrying multiple fetuses. The present findings may suggest that the decline in insulin concentrations that apparently occurs in the earlier stages of pregnancy represents a homeorhetic control to spare glucose for the brains and fetoplacental units of the dams. The results clearly demonstrate the increased susceptibility to pregnancy toxemia of ewes carrying multiple fetuses. Appropriate nutritional strategies should be developed for ewes that conceive >3 fetuses, to meet the increased nutritional requirements of the fetoplacental unit.

Differential expression of ruminant ZNF496 variants: Association with quantitative trait locus affecting bovine milk concentration and fertility.

A single nucleotide polymorphism in the intergenic region upstream of the ZNF496 gene on Bos taurus chromosome 7 displayed significant population-wide linkage disequilibrium with milk protein percentage in the Israeli Holstein population. The frequency of the allele associated with increased protein concentration was 10%. This single nucleotide polymorphism was located in the promoter region from which a 10-exon transcript of the bovine and the ovine ZNF496 genes are transcribed. The gene architecture was similar to the mouse ortholog Zkscan17. A 5-exon murine antisense transcript was complementary to the 5' untranslated Zkscan17 region that included a sequence domain conserved between mouse and ruminants, suggesting a regulatory function. In the bovine ZNF496 chromosomal region, segregation of a quantitative trait locus (QTL) for milk protein percentage was confirmed in a daughter design sire family. Concordance was not obtained between QTL status of bulls and any of the polymorphisms in the functional elements of ZNF496. This excludes these variations as the causative polymorphism under the assumption of no epigenetic effect for this locus. However, ZNF496 variants were differentially expressed in bovine ovaries, and only the paternal variant was expressed in liver and kidney in a sheep family with polymorphic ZNF496 sequence. Thus, the search for the mutation underlying the minor QTL allele, which is a top economically favorable allele in Israeli Holstein cattle, may be complicated by the presence of an imprinting center in this QTL confidence interval.

Pituitary and placental ovine growth hormone variants differ in their receptor-binding ability and in their biological properties.

The wild-type (WT) GH2-N ovine growth hormone (oGH) and duplicated GH2-Z genes differ in their open reading frame by two nonsynonymous substitutions, predicting a two-amino-acid difference in their product (G9R/G63S). Three recombinant oGH muteins: G9R, G63S and G9R/G63S, were prepared by site-directed mutagenesis of the WT oGH gene, expressed in E. coli, refolded and purified as monomers with over 98% homogeneity. Gel-filtration experiments with WT oGH and the three muteins indicated formation of 1:2 complexes with oGH receptor extracellular domain (oGHR-ECD). Interactions of oGHR-ECD with the WT and the muteins were studied by surface plasmon resonance. Kinetics constants calculated using a two-site model predicted that G9R/G63S has the highest affinity to oGHR-ECD, WT oGH the lowest, and G9R and G63S have intermediate affinities. These relative affinities were further investigated by radioreceptor assay with EC50 values were the lowest for G9R/G63S, highest for WT oGH, and intermediate for G9R and G63S. Bioactivity of the WT oGH and oGH muteins was determined by proliferation assay with FDC-P1-3B9 cells stably transfected with rabbit GHR. Relative proliferation rates of cells in cultures treated with the WT, G63S, G9R or G9R/G63S variants were 100%, 183%, 259% and 498%, respectively. In COS-7 transfected with oGHR, LHRE-TK-luciferase and beta-galactosidase plasmids G9R/G63S showed 18% higher activity than WT oGH (P<0.001). Thus the product of the oGH duplicated copy has higher affinity for GHR and higher somatogenic activity. As the GH2-Z gene copy is expressed in the placenta, allelic differences at the oGH locus may influence feto-placental development.

Litter-size-dependent intrauterine growth restriction in sheep.

Regulation of foetal development in sheep depends on interactions between the intrinsic capacity of the foetus for growth and the maternal environment. Lambs born in multi-foetus litters have relatively small placentae with fewer cotelydons, and lower birth weights. Litter-size-dependent intrauterine growth restriction (IUGR) is evident at mid gestation when metabolic needs of the conceptus are moderate, and overnutrition of ewes with multiple foetuses does not promote growth of their foetuses to the size of singletons. Those observations suggest that placental and conceptus growth in multi-foetus pregnancies is reprogrammed at mid gestation by an as yet undefined mechanism to attenuate foetal growth. This may protect the foetus from severe nutritional insult during late gestation, when its daily growth rate is at a maximum. In that way, lambs born in large litters with relatively lower birth weights may not experience the long-term physiological insults that can be observed in small lambs born to undernourished ewes.

Carrying the FecB (Booroola) mutation is associated with lower birth weight and slower post-weaning growth rate for lambs, as well as a lighter mature bodyweight for ewes.

The present study was conducted in an Assaf flock in which the FecB (Booroola) mutation was segregated to determine whether the FecB mutation affects birthweight and the pre- and post-weaning growth rate of ewe lambs, as well as the mature bodyweight of ewes. Significant differences (P = 0.01) in birthweight (mean +/- s.e.m.) were found between BB ewe lambs (4.03 +/- 0.08 kg) and B+ and ++ ewe lambs (4.16 +/- 0.04 and 4.32 +/- 0.07 kg, respectively), which themselves did not differ significantly (P > 0.05). An FecB-associated maternal effect on the birthweight of ewe lambs was also detected, with the birthweight of lambs born to BB mothers (3.93 +/- 0.08 kg) being significantly (P < 0.0001) different from the birthweight of lambs born to B+ and ++ mothers (4.26 +/- 0.04 and 4.33 +/- 0.07 kg, respectively), which did not differ significantly. The genotypes of the lambs did not affect their preweaning growth rate. However, the post-weaning growth rate of ewe BB lambs (274 +/- 5 g day(-1)) was significantly (P = 0.05) different from the similar (P > 0.05) post-weaning growth rates of B+ and ++ lambs (284 +/- 3 and 290 +/- 4 g day(-1), respectively). The genotype at the FecB locus also affected the mature bodyweight of ewes, with that of BB ewes (67.3 +/- 1.4 kg) being significantly (P < 0.001) different from the similar mature bodyweight of B+ and ++ ewes (70.8 +/- 1.1 and 70.1 +/- 1.7 kg, respectively).

Reproductive performance and milk production of Assaf sheep in an intensive management system.

The Assaf breed of dairy sheep, a stabilized cross of the Awassi and East Friesian breeds, has replaced the Awassi as the breed of choice in its country of origin, Israel, and has spread to other Mediterranean countries. In Israel the Assaf breed is managed under an intensive production system involving weaning lambs at birth, rearing them artificially, and milking ewes after parturition. There are several breeding periods in the year when ewes are mated following hormonally synchronized estrus. Records of 18,976 lactations from 5 farms were analyzed to investigate factors that influenced Assaf milk and reproductive performance. Lactation curves were fitted to each lactation, and a range of parameters and calculated values were analyzed. Daily milk yield records also were analyzed to describe a typical Assaf lactation and compared with those of the Awassi breed. Factors affecting age at first lambing also were studied. An average Assaf ewe kept under this intensive management regimen was found to produce 334 L of milk during a 173-d lactation. Mean litter size was 1.57 lambs/ewe lambing, and lambing interval was 272 d. Milk production was affected by litter size, with twin- and triplet-bearing ewes producing approximately 20 L more milk per lactation than single-bearing ewes. Day length was the major environmental variable influencing milk yield. The difference between midsummer and midwinter day lengths accounted for a difference in daily milk yield of 0.44 L in favor of summer. Ewe lambs that were mated for the first time at later ages produced more lambs and more milk due to greater early lactation characteristics. Milk production was found to be negatively associated with subsequent reproductive performance. Comparing these results with those from an earlier study in the Awassi breed, the Assaf was found to produce less milk during a shorter lactation than the Awassi, but its greater litter size made it a more profitable breed.

Placental hormones and fetal-placental development.

Production of growth promoting substances by the placenta is regulated differently from the way production of similar compounds is regulated by maternal organs in various cases. Gene duplication is one of the mechanisms that facilitated the evolution of placental specific endocrine activity. Cattle, sheep and goats, although evolutionarily related, differ significantly from each other in the way their placental growth hormone (GH) and prolactin (PRL)-like hormones have evolved. Cattle carry one copy of the GH gene and there is no evidence yet for expression of that single GH gene copy in the placenta. On the other hand, the ovine GH gene has been duplicated and both oGH copies are expressed in the placenta during early stages of gestation. Prolactin gene duplication in ruminants resulted in the formation of specific placental-expressed prolactin-related genes including the placental lactogen (PL) gene. In homologous state, ovine PL manifests PRL activity, but antagonizes GH activity. Ovine PL activity which can be mediated by PRL receptors or by hetero-dimerization of GH and PRL receptors, provide a novel regulatory mechanism for somatogenic activity dependent on the coexistence of both GH and PRL receptors in the same cells. Another mechanism for specific placental endocrine activity is silencing of the alleles through genetic imprinting. Disruption of genetic imprinting of placental genes has been proposed as one of the explanations for the loss of cloned fetuses generated by somatic cell nuclear transfer.

Large-scale preparation of recombinant ovine prolactin and determination of its in vitro and in vivo activity.

Recombinant bovine Ala-prolactin (PRL) (GenBank Accession No. V00112) in prokaryotic expression plasmid pMON3401 was mutated using a mutagenesis kit, to prepare plasmid encoding ovine PRL (oPRL) (GenBank Accession No. M27057) Escherichia coli cells transformed with this latter plasmid overexpressed large amounts of oPRL upon induction with nalidixic acid. The expressed protein, found in inclusion bodies, was refolded and purified to homogeneity on a Q-Sepharose column, yielding an electrophoretically pure fraction composed of over 98% monomeric protein of the expected molecular mass of approximately 23 kDa. The biological activity of the recombinant oPRL after proper renaturation was evidenced in vitro by its ability to stimulate proliferation of rat lymphoma Nb(2) cells possessing PRL receptors, to stimulate luciferase activity in HEK 293 cells transiently transfected with oPRL receptors, and to induce progesterone secretion in primary cultures of luteal cells obtained from midpregnant ewes. In contrast to ovine growth hormone or ovine placental lactogen, recombinant oPRL had no galactopoietic effect in lactating ewes.

Active immunization of ewes against ovine placental lactogen increases birth weight of lambs and milk production with no adverse effect on conception rate.

In two experiments, 16 Booroola-Assaf and 35 Assaf ewe-lambs were actively immunized at 5 months of age against recombinant ovine placental lactogen (oPL). At 9 months of age, the ewe-lambs were mated for the first time and then introduced into a frequent mating-system. Anti-oPL antibody titers, reproductive performance, maternal serum levels of oPL during pregnancy, lamb birth weight and milk production of the ewes were followed in the immunized ewes and in their non-immunized control counterparts. All the immunized ewes developed anti-oPL antibodies, which interfered with oPL bioactivity in an in vitro cell proliferation assay. Conception rates did not differ (P>0.05) between immunized and non-immunized ewes. Abundant antibody-bound non-active oPL detected in sera of immunized ewes by western blotting indicated enhanced oPL production by the placenta following immunization. An increase (P<0.02) in serum oPL bioactivity, but not immunoreactivity, was observed in the immunized ewes in late gestation relative to control ewes. The average litter size was 1.83 and 1.32 lambs born per ewe lambing in the first and second experiments, respectively. Average birth weights of lambs born to the immunized ewes were higher (P<0.01) than for lambs born to control ewes by 10, 17 and 39% for those born as singles, twins and triplets, respectively. Immunized ewes produced 19 and 33% more milk (P<0.02) than the control ewes in the first 3.5 months of the first and second lactations, respectively. These findings do not suggest a role for oPL in maternal recognition of pregnancy, but they strongly suggest important roles for oPL in fetal growth and mammogenesis. Immunization of ewes against oPL may thus represent a novel practical technique for enhancing birth weights of lambs born to prolific sheep, as well as milk production by both dairy and mutton ewes.

Effects of recombinant ovine interferon tau, placental lactogen, and growth hormone on the ovine uterus.

Studies were conducted to determine effects of intrauterine administration of recombinant ovine interferon tau (IFNtau), placental lactogen (PL), and growth hormone (GH) on endometrial function. In the first study, administration of IFNtau to cyclic ewes for one period (Days 11-15) resulted in an interestrous interval (IEI) of approximately 30 days, whereas administration for two periods (Days 11-15 and Days 21-25) extended the IEI to greater than 50 days. Administration of IFNtau from Days 11 to 15 and of PL or GH from Days 21 to 25 failed to extend the IEI more than for IFNtau alone. In the second study, effects of IFNtau, PL, and GH on endometrial differentiation and function were determined in ovariectomized ewes receiving ovarian steroid replacement therapy. Endometrial expression of mRNAs for estrogen receptor (ER), progesterone receptor (PR), and oxytocin receptor (OTR) were not affected by PL or GH treatment; however, uterine milk protein mRNA levels and stratum spongiosum gland density were increased by both PL and GH treatments. Collectively, results indicated that 1) PL and GH do not regulate endometrial PR, ER, and OTR expression or affect corpus luteum life span; 2) down-regulation of epithelial PR expression is requisite for progesterone induction of secretory gene expression in uterine glandular epithelium; 3) effects of PL and GH on endometrial function require IFNtau; and 4) PL and GH regulate endometrial gland proliferation and perhaps differentiated function.

Establishment and initial characterization of the ovine mammary epithelial cell line NISH.

Analysis of the molecular mechanisms involved in the differentiation and formation of the characteristic three-dimensional structures of the developing mammary gland of the major milk-producing livestock (ducts, end buds, and alveoli) requires in vitro model cell cultures. The few cell lines that have been established from dairy animals do not fully reproduce the entire program of mammary differentiation. Here we present the initial characterization of a unique mammary epithelial cell line derived spontaneously from midpregnant sheep (NISH). These cells form in vitro functional structures resembling ducts, lateral buds, and alveoli that secrete beta-lactoglobulin (BLG) in an ECM (extracellular matrix)-dependent manner. Interestingly, the presence of growth hormone dramatically increased BLG secretion from NISH cells cultured on ECM. It appears that GH is required not only to establish the structural organization but also is continuously needed to maintain BLG expression. Stable transfection of NISH cells with BLG/Human Serum Albumin (HSA) hybrid gene constructs revealed that the relative level of expression was comparable to the in vivo secretion of HSA in transgenic mice carrying these gene sequences. No expression could be detected in cells transfected with hybrid genes carrying either HSA cDNA or the entire HSA gene, and HSA expression was dependent on the presence of intronic sequences. These results demonstrate that NISH cells may prove a useful tool for studying the differentiation and organogenesis of mammary epithelial cells under defined culture conditions. Furthermore, transfected NISH cells may be an alternative for the transgenic mouse model in evaluating the potential of gene constructs to be efficiently expressed in the mammary gland of transgenic farm animals.