Comparative Hypothalamic Transcriptome Analysis Reveals Crucial mRNAs, lncRNAs, and circRNAs Affecting Litter Size in Goats.

Litter size is an important indicator to measure the reproductive performance of goats, which is affected by the reproductive function of animals. The hypothalamus, as the regulatory center of the endocrine system, plays an important role in the reproduction of female animals. Here, we performed high-throughput RNA sequencing using hypothalamic tissue from high-fecundity and low-fecundity Leizhou goats to explore critical functional genes associated with litter size. Differentially expressed mRNA, lncRNA, and circRNAs were screened using DESeq and were enriched, and then analyzed by Gene Ontology and Kyoto Encyclopedia of Gene and Genome. Results showed that some of these differentially expressed mRNAs could be enriched in reproductive processes, jak-STAT, prolactin signaling pathway, and other signaling pathways related to reproduction, such as SOCS3. Furthermore, the central proteins POSTN, MFAP5, and DCN from protein-protein interaction may regulate animal reproductive activity by affecting cell proliferation and apoptosis. lncRNA MSTRG.33887.2 as well as circRNAs chicirc_098002, chicirc_072583, and chicirc_053531 may be able to influence animal reproduction by participating in folate metabolism and energy metabolism homeostasis through their respective target genes. Our results expand the molecular mechanism of hypothalamic regulation on animal reproduction.

Genome-wide association study and pathway analysis identify NTRK2 as a novel candidate gene for litter size in sheep.

Litter size is one of the most important economic traits in sheep. Identification of gene variants that are associated with the prolificacy rate is an important step in breeding program success and profitability of the farm. So, to identify genetic mechanisms underlying the variation in litter size in Iranian Baluchi sheep, a two-step genome-wide association study (GWAS) was performed. GWAS was conducted using genotype data from 91 Baluchi sheep. Estimated breeding values (EBVs) for litter size calculated for 3848 ewes and then used as the response variable. Besides, a pathway analysis using GO and KEGG databases were applied as a complementary approach. A total of three single nucleotide polymorphisms (SNPs) associated with litter size were identified, one each on OAR2, OAR10, and OAR25. The SNP on OAR2 is located within a novel putative candidate gene, Neurotrophic receptor tyrosine kinase 2. This gene product works as a receptor which is essential for follicular assembly, early follicular growth, and oocyte survival. The SNP on OAR25 is located within RAB4A which is involved in blood vessel formation and proliferation through angiogenesis. The SNP on OAR10 was not associated with any gene in the 1Mb span. Moreover, gene-set analysis using the KEGG database identified several pathways, such as Ovarian steroidogenesis, Steroid hormone biosynthesis, Calcium signaling pathway, and Chemokine signaling. Also, pathway analysis using the GO database revealed several functional terms, such as cellular carbohydrate metabolic, biological adhesion, cell adhesion, cell junction, and cell-cell adherens junction, among others. This is the first study that reports the NTRK2 gene affecting litter size in sheep and our study of this gene functions showed that this gene could be a good candidate for further analysis.

Milk lactoferrin concentration of primiparous and multiparous sows during lactation.

Lactoferrin (LF), a sialylated iron-binding glycoprotein, has numerous vital physiological functions including immunomodulation and protection against a large group of microorganisms, improving neurodevelopment, health, growth performance, and milk production. Lactoferrin occurs in human milk at a higher concentration compared with bovine milk, but little information is available on LF concentrations in porcine milk and the effects of sow parity on milk LF concentration. The objective of this study was to quantify the LF concentration in porcine milk and to compare that concentration between gilts and sows during lactation. We also investigated the effect of genetic background and litter size of the female pig on the LF concentration of porcine milk. The milk from 30 gilts and 35 sows was collected at 3 stages of lactation, namely colostrum, transition, and mature milk. Standard and experimental samples were analyzed by ultra-high performance liquid chromatography using a diode array UV detector. The following findings were reported: (1) porcine milk contained significant levels of LF with the highest concentration in colostrum, which decreased by ∼62% and ∼67% in transitional and mature milk, respectively; (2) mature gilt milk contained a 22% higher concentration of LF compared with sow milk, which was statistically significant; (3) breed line had an overall significant effect on the LF content of porcine milk; however, when the breed was considered, no significant difference was observed; and (4) LF concentration of porcine milk was not significantly influenced by the litter size. The presence of LF in a higher concentration in porcine milk suggests that LF is an important constituent of pig milk that might contribute to the optimum growth and development of piglets.

Sertoli cell specific knockdown of RAR-related orphan receptor (ROR) alpha at puberty reduces sperm count in rats.

Globally, there is an alarming decline in sperm count. Very often hormonal supplementation fails to restore normal sperm count. Sertoli cells (Sc) present within seminiferous tubules provide appropriate niche and factors required for the differentiation of germ cells (Gc) into mature sperm (spermatogenesis). Functionally compromised Sc may be one of the reasons for failure of hormones to facilitate normal spermatogenesis. Although role of secretory proteins and signaling molecules of Sc has been studied well, role of transcription factors regulating sperm count has not been addressed appropriately. Retinoic acid receptor-related orphan receptor (ROR)-alpha is one of such transcription factors reported in testis but its role in testicular function is not yet known. In a separate study, we found abundant ROR-alpha binding sites on promoter regions of several genes upregulated in pubertal rat Sc as compared to infant Sc. Immunostaining studies also revealed presence of ROR alpha in nucleus of pubertal Sc. We generated a transgenic knockdown rat model expressing shRNA targeted to ROR-alpha under Sc specific promoter, which is transcriptionally active only at and after puberty. ROR-alpha knockdown animals were found to have abnormal association of Sc and Gc, including Gc sloughing and restricted release of sperm. The knockdown animals displayed compromised spermatogenesis leading to significant reduction in sperm count. This is the first report describing the Sc specific role of ROR-alpha in maintaining quantitatively normal sperm output. Identification of various such molecules can generate avenues to limit or reverse an alarmingly declining sperm count witnessed globally in men.

Litter size variation in hypothalamic gene expression determines adult metabolic phenotype in Brandt's voles (Lasiopodomys brandtii).

BACKGROUND: Early postnatal environments may have long-term and potentially irreversible consequences on hypothalamic neurons involved in energy homeostasis. Litter size is an important life history trait and negatively correlated with milk intake in small mammals, and thus has been regarded as a naturally varying feature of the early developmental environment. Here we investigated the long-term effects of litter size on metabolic phenotype and hypothalamic neuropeptide mRNA expression involved in the regulation of energy homeostasis, using the offspring reared from large (10-12) and small (3-4) litter sizes, of Brandt's voles (Lasiopodomys brandtii), a rodent species from Inner Mongolia grassland in China. METHODOLOGY/PRINCIPAL FINDINGS: Hypothalamic leptin signaling and neuropeptides were measured by Real-Time PCR. We showed that offspring reared from small litters were heavier at weaning and also in adulthood than offspring from large litters, accompanied by increased food intake during development. There were no significant differences in serum leptin levels or leptin receptor (OB-Rb) mRNA in the hypothalamus at weaning or in adulthood, however, hypothalamic suppressor of cytokine signaling 3 (SOCS3) mRNA in adulthood increased in small litters compared to that in large litters. As a result, the agouti-related peptide (AgRP) mRNA increased in the offspring from small litters. CONCLUSIONS/SIGNIFICANCE: These findings support our hypothesis that natural litter size has a permanent effect on offspring metabolic phenotype and hypothalamic neuropeptide expression, and suggest central leptin resistance and the resultant increase in AgRP expression may be a fundamental mechanism underlying hyperphagia and the increased risk of overweight in pups of small litters. Thus, we conclude that litter size may be an important and central determinant of metabolic fitness in adulthood.

The roles of leptin receptors on POMC neurons in the regulation of sex-specific energy homeostasis.

Leptin regulates energy homeostasis and reproduction. One key population of leptin receptors (Lepr) are found on proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus, and evidence links the action of gonadal estrogens to these same POMC neurons. To determine whether Lepr on POMC neurons are critical for reproductive capacity or for sex-specific energy and glucose homeostasis, we studied Cre/loxP mice lacking Lepr specifically on POMC neurons (Pomc-Cre, Lepr(flox/flox) mice) and their controls with normal Lepr (Lepr(flox/flox) mice). Pomc-Cre, Lepr(flox/flox) mice maintained normal reproductive capacity and accumulated more body fat than their same sex controls. Ovariectomy (OVX) was performed to investigate the effects of the estrogens and Lepr on POMC neurons on body fat accumulation and glucose tolerance. OVX Pomc-Cre, Lepr(flox/flox) females accumulated more fat than OVX Lepr(flox/flox) females did. Pomc-Cre, Lepr(flox/flox) males were glucose intolerant and insulin insensitive compared with control males. In contrast, control and Pomc-Cre, Lepr(flox/flox) females had similar glucose tolerance before and after OVX. Therefore leptin's action on POMC neurons reduces body fat accumulation, but is not critical for regulation of reproduction. The sex difference in leptin signaling on POMC neurons on glucose tolerance appears independent of ovarian hormones.

Phenotype of the neural tube defect mouse model bent tail is not sensitive to maternal folinic acid, myo-inositol, or zinc supplementation.

BACKGROUND: Bent tail is a mouse model for X-linked neural tube defects (NTDs) that is characterized by the presence of exencephaly, a delayed posterior neuropore closure, and a tail phenotype. In addition, Bent tail shows laterality defects and increased prenatal mortality. The congenital malformations of this mouse are caused by a submicroscopic deletion that completely encompasses the gene coding for the zinc finger transcription factor Zic3. In this study we investigated the sensitivity of the phenotype of Bent tail to the nutrients folinic acid, myo-inositol, and zinc. These nutrients are thought to be involved in the etiology of NTDs, in combination with a genetic predisposition. METHODS: The most penetrant phenotype of the Bent tail mouse, the tail malformation, was used as a marker for the nutrient sensitivity of the neural phenotype. The size of the litters and the survival of the offspring, subdivided according to genotype, were analyzed as markers for the nutrient sensitivity of other phenotypic features of Bent tail. RESULTS: In confirmation of earlier studies, we observed the prenatal loss of a number of homozygous females and hemizygous males, as well as the effect of genotype on the tail phenotype of Bent tail. However, periconceptional supplementation of the maternal diet with folinic acid, myo-inositol, or zinc produced no significant effects on either the tail phenotype of the offspring or the size and genotypic composition of the litters. CONCLUSIONS: Bent tail appears to be a folinic acid-, myo-inositol-, and zinc-insensitive mouse model for NTDs.