An extreme model of fertility in sheep demonstrates the basis of controversies surrounding antral follicle count and circulating concentrations of anti-Müllerian hormone as predictors of fertility in ruminants.

Ovarian ultrasonography and measurement of circulating concentrations of anti-Müllerian hormone (AMH) have been used to estimate follicle number and predict fertility in mammalian females, but no study has evaluated follicle number and circulating concentrations of AMH in ewes known to differ in fertility. We tested the hypothesis that ewes that had failed to lamb (BARREN) in four consecutive annual breeding seasons of 21-35 d have fewer follicles and diminished circulating concentrations of AMH compared to closely matched ewes that consistently produced lambs (FERTILE) under the same breeding protocols. Once identified, BARREN ewes (n = 19) were paired by breed and sire to a FERTILE control ewe (n = 19) and reproductive tracts were recovered at necropsy. Visible antral follicles in both ovaries were counted and a representative cross section of one ovary was embedded for histological evaluation of pre-antral follicle numbers. Paired t-tests indicated that BARREN ewes had fewer antral follicles, fewer primordial follicles, and decreased circulating concentrations of AMH compared to FERTILE ewes (P ≤ 0.01), but there were ewes in each fertility classification that had ovarian phenotypes like the opposite fertility classification. The best technologies we have currently for estimating follicle numbers are ultrasonography and measurement of circulating concentrations of AMH, but no single technique is perfect for predicting fertility. A better understanding of the under-lying biological mechanisms linking AMH, folliculogenesis, and fertility is required to improve the use of measurements of follicle number for predicting fertility in livestock.

Localization of kisspeptin, NKB, and NK3R in the hypothalamus of gilts treated with the progestin altrenogest.

Mechanisms in the brain controlling secretion of gonadotropin hormones in pigs, particularly luteinizing hormone (LH), are poorly understood. Kisspeptin is a potent LH stimulant that is essential for fertility in many species, including pigs. Neurokinin B (NKB) acting through neurokinin 3 receptor (NK3R) is involved in kisspeptin-stimulated LH release, but organization of NKB and NK3R within the porcine hypothalamus is unknown. Hypothalamic tissue from ovariectomized (OVX) gilts was used to determine the distribution of immunoreactive kisspeptin, NKB, and NK3R cells in the arcuate nucleus (ARC). Almost all kisspeptin neurons coexpressed NKB in the porcine ARC. Immunostaining for NK3R was distributed throughout the preoptic area (POA) and in several hypothalamic areas including the periventricular and retrochiasmatic areas but was not detected within the ARC. There was no colocalization of NK3R with gonadotropin-releasing hormone (GnRH), but NK3R-positive fibers in the POA were in close apposition to GnRH neurons. Treating OVX gilts with the progestin altrenogest decreased LH pulse frequency and reduced mean circulating concentrations of LH compared with OVX control gilts (P < 0.01), but the number of kisspeptin and NKB cells in the ARC did not differ between treatments. The neuroanatomical arrangement of kisspeptin, NKB, and NK3R within the porcine hypothalamus confirms they are positioned to stimulate GnRH and LH secretion in gilts, though differences with other species exist. Altrenogest suppression of LH secretion in the OVX gilt does not appear to involve decreased peptide expression of kisspeptin or NKB.

Energy balance affects pulsatile secretion of luteinizing hormone from the adenohypophesis and expression of neurokinin B in the hypothalamus of ovariectomized gilts.

The pubertal transition of gonadotropin secretion in pigs is metabolically gated. Kisspeptin (KISS1) and neurokinin B (NKB) are coexpressed in neurons within the arcuate nucleus of the hypothalamus (ARC) and are thought to play an important role in the integration of nutrition and metabolic state with the reproductive neuroendocrine axis. The hypothesis that circulating concentrations of luteinizing hormone (LH) and expression of KISS1 and tachykinin 3(TAC3, encodes NKB) in the ARC of female pigs are reduced with negative energy balance was tested using ovariectomized, prepubertal gilts fed to either gain or lose body weight. Restricted feeding of ovariectomized gilts caused a rapid and sustained metabolic response characterized by reduced concentrations of plasma urea nitrogen, insulin, leptin, and insulin-like growth factor-1 and elevated concentrations of free fatty acids. The secretory pattern of LH shifted from one of low amplitude to one of high amplitude, which caused overall circulating concentrations of LH to be greater in restricted gilts. Nutrient-restricted gilts had greater expression of follicle-stimulating hormone and gonadotropin-releasing hormone receptor, but not LH in the anterior pituitary gland. Expression of KISS1 in the ARC was not affected by dietary treatment, but expression of TAC3 was greater in restricted gilts. These data are consistent with the idea that hypothalamic expression of KISS1 is correlated with the number of LH pulse in pig, and further indicate that amplitude of LH pulses may be regulated by NKB in the gilt.

Using sheep genomes from diverse U.S. breeds to identify missense variants in genes affecting fecundity.

Background:  Access to sheep genome sequences significantly improves the chances of identifying genes that may influence the health, welfare, and productivity of these animals.   Methods:  A public, searchable DNA sequence resource for U.S. sheep was created with whole genome sequence (WGS) of 96 rams.  The animals shared minimal pedigree relationships and represent nine popular U.S. breeds and a composite line.  The genomes are viewable online with the user-friendly Integrated Genome Viewer environment, and may be used to identify and decode gene variants present in U.S. sheep. Results:  The genomes had a combined average read depth of 16, and an average WGS genotype scoring rate and accuracy exceeding 99%.  The utility of this resource was illustrated by characterizing three genes with 14 known coding variants affecting litter size in global sheep populations:  growth and differentiation factor 9 ( GDF9), bone morphogenetic protein 15 ( BMP15), and bone morphogenetic protein receptor 1B ( BMPR1B).  In the 96 U.S. rams, nine missense variants encoding 11 protein variants were identified.  However, only one was previously reported to affect litter size ( GDF9 V371M, Finnsheep).  Two missense variants in BMP15 were identified that had not previously been reported:  R67Q in Dorset, and L252P in Dorper and White Dorper breeds. Also, two novel missense variants were identified in BMPR1B:  M64I in Katahdin, and T345N in Romanov and Finnsheep breeds.  Based on the strict conservation of amino acid residues across placental mammals, the four variants encoded by BMP15 and BMPR1B are predicted to interfere with their function.  However, preliminary analyses of litter sizes in small samples did not reveal a correlation with variants in BMP15 and BMPR1B with daughters of these rams.  Conclusions: Collectively, this report describes a new resource for discovering protein variants in silico and identifies alleles for further testing of their effects on litter size in U.S. breeds.

Glucosamine supplementation during late gestation alters placental development and increases litter size.

BACKGROUND: During late gestation the placental epithelial interface becomes highly folded, which involves changes in stromal hyaluronan. Hyaluronan is composed of glucoronate and N-acetyl-glucosamine. We hypothesized that supplementing gestating dams with glucosamine during this time would support placental folded-epithelial-bilayer development and increase litter size. In Exp. 1, gilts were unilaterally hysterectomized-ovariectomized (UHO). UHO gilts were mated and then supplemented daily with 10 g glucosamine (n = 16) or glucose (control, n = 17) from d 85 of gestation until slaughter (d 105). At slaughter, the number of live fetuses was recorded and each live fetus and its placenta was weighed. Uterine wall samples adjacent to the largest and smallest fetuses within each litter were processed for histology. In Exp. 2, pregnant sows in a commercial sow farm were supplemented with either 10 g glucosamine or glucose daily from d 85 of gestation to farrowing. Total piglets born and born alive were recorded for each litter. In Exp. 3, the same commercial farm and same protocol were used except that the dose of glucosamine and glucose was doubled to 20 g/d. RESULTS: In Exp. 1, the number of live fetuses tended to be greater in glucosamine-treated UHO gilts (P = 0.098). Placental morphometry indicated that the width of the folded bilayer was greater (P = 0.05) in glucosamine-treated gilts. In Exp. 2, litter size did not differ between glucosamine- and glucose-treated sows. However in Exp. 3, the increased dose of glucosamine resulted in a significant treatment by parity interaction (P ≤ 0.01), in which total piglets born and born alive were greater in glucosamine treated sows of later parity (5 and 6). CONCLUSIONS: These results indicated that glucosamine supplementation increased the width of the folds of the placental bilayer and increased litter size in later parity, intact pregnant commercial sows.

Relationship of neuropeptide FF receptors with pubertal maturation of gilts.

Mechanisms governing the timing of puberty in pigs are poorly understood. A genome-wide association study for age at first estrus in pigs identified candidate genes including neuropeptide FF receptor 2 (NPFFR2), which is a putative receptor for RFamide-related peptides (RFRP). RFRP has been shown to negatively regulate secretion of reproductive hormones from hypothalamic and pituitary tissue of pigs in culture. Here, the porcine NPFFR2 gene was further screened and four potentially functional variants were identified to be associated with age at first estrus in pigs (1,288 gilts). The RFRP neurons in the porcine hypothalamus were localized in the paraventricular and dorsomedial nuclei with RFRP fibers in the lateral hypothalamic area. There were marked changes in expression of NPFF receptors in the anterior pituitary gland and hypothalamus of gilts beginning with the peripubertal period. The hypothesis that NPFF receptor function is related to secretion of luteinizing hormone (LH) in gilts was tested with various NPFF receptor ligands. The NPFF receptor antagonist RF9 stimulated a pulse-like release of LH in prepubertal gilts. The putative NPFF receptor agonist RFRP3 modestly suppressed LH pulses in ovariectomized (OVX) prepubertal gilts. A porcine-specific RFRP2 failed to have an effect on LH secretion in OVX prepubertal gilts despite its high degree of homology to avian gonadotropin-inhibitory hormone. Results indicate that an RFRP system is present in the pig and that NPFFR2 is important for pubertal onset in gilts. It is not clear if this regulation involves major control of LH secretion or another unknown mechanism.

Comparative genomics reveals tissue-specific regulation of prolactin receptor gene expression.

Prolactin (PRL), acting via the PRL receptor (PRLR), controls hundreds of biological processes across a range of species. Endocrine PRL elicits well-documented effects on target tissues such as the mammary glands and reproductive organs in addition to coordinating whole-body homeostasis during states such as lactation or adaptive responses to the environment. While changes in PRLR expression likely facilitates these tissue-specific responses to circulating PRL, the mechanisms regulating this regulation in non-rodent species has received limited attention. We performed a wide-scale analysis of PRLR 5' transcriptional regulation in pig tissues. Apart from the abundantly expressed and widely conserved exon 1, we identified alternative splicing of transcripts from an additional nine first exons of the porcine PRLR (pPRLR) gene. Notably, exon 1.5 transcripts were expressed most abundantly in the heart, while expression of exon 1.3-containing transcripts was greatest in the kidneys and small intestine. Expression of exon 1.3 mRNAs within the kidneys was most abundant in the renal cortex, and increased during gestation. A comparative analysis revealed a human homologue to exon 1.3, hE1N2, which was also principally transcribed in the kidneys and small intestines, and an exon hE1N3 was only expressed in the kidneys of humans. Promoter alignment revealed conserved motifs within the proximal promoter upstream of exon 1.3, including putative binding sites for hepatocyte nuclear factor-1 and Sp1. Together, these results highlight the diverse, conserved and tissue-specific regulation of PRLR expression in the targets for PRL, which may function to coordinate complex physiological states such as lactation and osmoregulation.

Placental accommodations for transport and metabolism during intra-uterine crowding in pigs.

Litter size and birth weights are limited by uterine capacity, defined as the ability of the uterus to maintain the appropriate development of some number of conceptuses. Uterine capacity is the result of the combined effects of uterine, placental and embryo/fetal function. The number of living conceptuses that the uterus is capable of supporting is greater during early gestation compared to later gestation. Plots of log fetal weight versus log placental weight also indicate that fetal weights are less sensitive to reduced placental weight (and therefore reduced intrauterine space) in early gestation compared to late gestation. However, even in late gestation, mechanisms still exist that maintain fetal growth when the size of the placenta is reduced. One such mechanism is likely to be improved development of the folded placental-epithelial/maternal-epithelial bilayer. Fold depth, and therefore the maternal fetal interactive surface, increases as gestation advances and is greater in placenta from small fetuses. On the fetal side of the placenta, the epithelial bilayer is embedded in stromal tissue. Glycosaminoglycans are major components of stroma, including hyaluronan and heparan sulfate. Hyaluronidases and heparanases are present within placental tissues, and likely play roles in modification of stromal components to facilitate fold development. Glycosaminoglycans are polymers of forms of glucose (glucosamine, glucuronic acid, iduronic acid) suggesting that glycosaminoglycan synthesis may compete with the glucose needs of the developing fetus. Pig conceptuses are fructogenic, such that a substantial portion of glucose transferred from mother to fetus is converted to fructose. Fructose is an intermediate product in the synthesis of glucosamine from glucose, and glucosamine is linked to regulation of trophoblast cell proliferation through regulation of mTOR. These findings suggest a link between glucose, fructose, glucosamine synthesis, GAG production, and placental morphogenesis, but the details of these interactions remain unclear. In addition, recent placental epithelial transcriptome analysis identified several glucose, amino acid, lipid, vitamin, mineral and hormone transporter mechanisms within the placenta. Further elucidation of mechanisms of placental morphogenesis and solute transport could provide clues to improving nutrient transport to the pig fetus, potentially increasing litter size and piglet birth weights.

Variation in the coding and 3' untranslated regions of the porcine prolactin receptor short form modifies protein expression and function.

The actions of prolactin (PRL) are mediated by both long (LF) and short isoforms (SF) of the PRL receptor (PRLR). Here, we report on a genetic and functional analysis of the porcine PRLR (pPRLR) SF. Three single nucleotide polymorphisms (SNPs) within exon 11 of the pPRLR-SF give rise to four amino acid haplotypes of the intracellular domain. We identified the dimorphic insertion of a short interspersed repetitive DNA element (PRE-1) along with 32 SNPs and four other insertion/deletion sites within the 3' untranslated region (UTR) of pPRLR-SF. The PRE-1 element reduced protein translation in vitro by 75%, whereas the combination of 10 SNPs and one insertion/deletion decreased translation by 50%. Full-length cDNAs for all four haplotypes of pPRLR-SF were cloned behind the elongation factor 1-alpha promoter and functionally analyzed in vitro. None of the haplotypes could initiate transcription from the ß-casein promoter, whereas all four were dominant negatives against PRL-activation of the pPRLR-LF. Two of the haplotypes completely inhibited pPRLR-LF activity at a four-fold excess, whereas the others required a six-fold excess to impart the same effect. The ligand binding affinities of the pPRLR-SF haplotypes did not differ. Expression of the pPRLR-SF increased linearly during gestation in the endometrium and was hormonally regulated in a tissue-specific manner in the mammary glands and uterus. In conclusion, we identified a PRE-1 and other SNPs in the pPRLR-SF 3' UTR that reduce protein expression and four haplotypes of the pPRLR-SF that suppress pPRLR-LF signaling and may differentially impact the phenotypic effects of PRL in vivo.

A novel first exon directs hormone-sensitive transcription of the pig prolactin receptor.

Endocrine, paracrine, and autocrine prolactin (PRL) acts through its receptor (PRLR) to confer a wide range of biological functions, including its established role during lactation. We have identified a novel first exon of the porcine PRLR that gives rise to three different mRNA transcripts. Transcription of this first exon is tissue specific, where it increases during gestation in the adrenal glands and uterus. Within the mammary glands, its transcription is induced by estrogen and PRL, while in the uterus, its expression is downregulated by progestin. The promoter region has an enhancer element located between -453 and -424 bp and a putative repressor element between -648 and -596 bp. Estrogen, acting through the estrogen receptor, activates transcription from this promoter through both E-box and transcription factor AP-2 α binding sites. These findings support the concept that the multilevel hormonal regulation of PRLR transcription contributes to the various biological functions of PRL.

Differences in X-chromosome transcriptional activity and cholesterol metabolism between placentae from swine breeds from Asian and Western origins.

To gain insight into differences in placental physiology between two swine breeds noted for their dissimilar reproductive performance, that is, the Chinese Meishan and white composite (WC), we examined gene expression profiles of placental tissues collected at 25, 45, 65, 85, and 105 days of gestation by microarrays. Using a linear mixed model, a total of 1,595 differentially expressed genes were identified between the two pig breeds using a false-discovery rate q-value ≤0.05. Among these genes, we identified breed-specific isoforms of XIST, a long non-coding RNA responsible X-chromosome dosage compensation in females. Additionally, we explored the interaction of placental gene expression and chromosomal location by DIGMAP and identified three Sus scrofa X chromosomal bands (Xq13, Xq21, Xp11) that represent transcriptionally active clusters that differ between Meishan and WC during placental development. Also, pathway analysis identified fundamental breed differences in placental cholesterol trafficking and its synthesis. Direct measurement of cholesterol confirmed that the cholesterol content was significantly higher in the Meishan versus WC placentae. Taken together, this work identifies key metabolic pathways that differ in the placentae of two swine breeds noted for differences in reproductive prolificacy.

Tissue-specific regulation of porcine prolactin receptor expression by estrogen, progesterone, and prolactin.

Prolactin (PRL) acts through its receptor (PRLR) via both endocrine and local paracrine/autocrine pathways to regulate biological processes including reproduction and lactation. We analyzed the tissue- and stage of gestation-specific regulation of PRL and PRLR expression in various tissues of pigs. Abundance of pPRLR-long form (LF) mRNA increased in the mammary gland and endometrium during gestation while in other tissues it remained constant. There was a parallel increase in the abundance of the pPRLR-LF protein in the mammary gland and endometrium during gestation. We determined the hormonal regulation of pPRLR-LF mRNA expression in various tissues from ovariectomized, hypoprolactinemic gilts given combinations of the replacement hormones estrogen (E(2)), progestin (P), and/or haloperidol-induced PRL. Abundance of pPRLR-LF mRNA in kidney and liver was unaffected by hormone treatments. Expression of uterine pPRLR-LF mRNA was induced by E(2) whereas the effect of E(2) was abolished by co-administering P. The expression of pPRLR-LF mRNA in the mammary gland stroma was induced by PRL, whereas E(2) induced its expression in the epithelium. In contrast to these changes in pPRLR expression, pPRL expression was relatively constant and low during gestation in all tissues except the pituitary. Taken together, these data reveal that specific combinations of E(2), P, and PRL differentially regulate pPRLR-LF expression in the endometrium and mammary glands, and that the action of PRL on its target tissues is dependent upon pPRLR-LF abundance more so than the local PRL expression.

Detection of transcriptional difference of porcine imprinted genes using different microarray platforms.

BACKGROUND: Presently, multiple options exist for conducting gene expression profiling studies in swine. In order to determine the performance of some of the existing microarrays, Affymetrix Porcine, Affymetrix Human U133+2.0, and the U.S. Pig Genome Coordination Program spotted glass oligonucleotide microarrays were compared for their reproducibility, coverage, platform independent and dependent sensitivity using fibroblast cell lines derived from control and parthenogenic porcine embryos. RESULTS: Array group correlations between technical replicates demonstrated comparable reproducibility in both Affymetrix arrays. Glass oligonucleotide arrays showed greater variability and, in addition, approximately 10% of probes had to be discarded due to slide printing defects. Probe level analysis of Affymetrix Human arrays revealed significant variability within probe sets due to the effects of cross-species hybridization. Affymetrix Porcine arrays identified the greatest number of differentially expressed genes amongst probes common to all arrays, a measure of platform sensitivity. Affymetrix Porcine arrays also identified the greatest number of differentially expressed known imprinted genes using all probes on each array, an ad hoc measure of realistic performance for this particular experiment. CONCLUSION: We conclude that of the platforms currently available and tested, the Affymetrix Porcine array is the most sensitive and reproducible microarray for swine genomic studies.