LH-Independent Testosterone Secretion Is Mediated by the Interaction Between GNRH2 and Its Receptor Within Porcine Testes.

Unlike classic gonadotropin-releasing hormone 1 (GNRH1), the second mammalian isoform (GNRH2) is an ineffective stimulant of gonadotropin release. Species that produce GNRH2 may not maintain a functional GNRH2 receptor (GNRHR2) due to coding errors. A full-length GNRHR2 gene has been identified in swine, but its role in reproduction requires further elucidation. Our objective was to examine the role of GNRH2 and GNRHR2 in testicular function of boars. We discovered that GNRH2 levels were higher in the testis than in the anterior pituitary gland or hypothalamus, corresponding to greater GNRHR2 abundance in the testis versus the anterior pituitary gland. Moreover, GNRH2 immunostaining was most prevalent within seminiferous tubules, whereas GNRHR2 was detected in high abundance on Leydig cells. GNRH2 pretreatment of testis explant cultures elicited testosterone secretion similar to that of human chorionic gonadotropin stimulation. Treatment of mature boars with GNRH2 elevated testosterone levels similar to those of GNRH1-treated males, despite minimal GNRH2-induced release of luteinizing hormone (LH). When pretreated with a GNRHR1 antagonist (SB-75), subsequent GNRH2 treatment stimulated low levels of testosterone secretion despite a pattern of LH release similar to that in the previous trial, suggesting that SB-75 inhibited testicular GNRHR2s. Given that pigs lack testicular GNRHR1, these data may indicate that GNRH2 and its receptor are involved in autocrine or paracrine regulation of testosterone secretion. Notably, our data are the first to suggest a biological function of a novel GNRH2-GNRHR2 system in the testes of swine.

The role of RFamide-related peptide 3 (RFRP3) in regulation of the neuroendocrine reproductive and growth axes of the boar.

RFamide-related peptide 3 (RFRP3) has been implicated in regulating reproduction and growth. This regulation appears to be dependent upon sex, species, physiological status, and developmental stage. The objective of the present study was to evaluate the effects of RFRP3 on circulating concentrations of luteinizing hormone (LH) and growth hormone (GH) in mature boars. The hypothesis was RFRP3 would reduce circulating concentrations of LH and increase concentrations of GH. Meishan boars (716.6±2.8 days of age; 125.0±12.4kg BW) were randomly assigned to treatment: saline (n=4) or RFRP3 (8.5mg; n=5). Plasma was collected at 15-min intervals during 3 periods: pre-treatment, treatment, and post-treatment. During the treatment period, saline or RFRP3 were administered at 15-min intervals. Treatment was administered as a loading dose of 5mg RFRP3, followed by seven repeated injections of 0.5mg RFRP3. Pulsatile secretion of LH and GH were not affected by saline treatment. Mean concentrations of LH in RFRP3-treated boars were greater (P<0.01) in the pre-treatment period than in the treatment and post-treatment periods; however, the individual response to RFRP3 challenge was varied. RFRP3 suppressed (P<0.05) mean concentrations of GH during the treatment period. It is concluded that RFRP3 can act to suppress LH secretion in some boars, but the minimal and varied response between animals does not strongly support the idea that RFRP3 is a potent hypohysiotropic hormone in the pig. Results indicate that RFRP3 may function in regulating the growth axis of swine.

Comparative metabolism of benzo(a)pyrene by ovarian microsomes of various species.

Knowledge of the ability of the female reproductive system to metabolize polycyclic aromatic hydrocarbons (PAHs) is critical to the diagnosis and management of female infertility and for risk assessment purposes. The PAHs are a family of widespread pollutants that are released into the environment from automobile exhausts, cigarette smoke, burning of refuse, industrial emissions, and hazardous waste sites. In exposed animals, PAHs become activated to reactive metabolites that interfere with target organ function and as a consequence cause toxicity. The extent of susceptibility to PAH exposure may depend on the ability of animals to metabolize these chemicals. The present study has been undertaken to assess whether any differences exist among mammals in the metabolism of benzo(a)pyrene (BaP), a prototypical PAH compound. Microsomes isolated from the liver and ovaries of rats, mice, goats, sheep, pigs, and cows were incubated with 5 microM BaP. Postincubation, samples were extracted with ethyl acetate and analyzed for BaP/metabolites by reverse-phase HPLC with fluorescence detection. The rate of metabolism (pmol of metabolite/min/mg protein) was found to be more in liver than in ovary in all the species studied (P < 0.05). The differences in metabolite concentrations were statistically significant (P < 0.0001) among the various species in both organs studied. Multiple species comparison also revealed that the differences were statistically significant (P < 0.001) between rodents (rat and mouse) and higher mammals (ewe, sow, and cow). Even among the higher mammals, in a majority of the cases, the differences in metabolite concentrations were significantly different (P < 0.001) both in ovary and liver. The BaP metabolites identified were 4,5-diol; 7,8-diol; 9,10-diol; 3-hydroxy BaP; and 9-hydroxy BaP. The rodent microsomes produced considerably higher proportion of BaP 4,5-diol and 9,10-diol than did cow, sow, goat, and sheep. However, microsomes from higher mammals converted a greater proportion of BaP to 3-hydroxy and 9-hydroxy BaP, the detoxification products of BaP. Overall, our results revealed a great variation among species to metabolize BaP.

Central and peripheral administration of kisspeptin activates gonadotropin but not somatotropin secretion in prepubertal gilts.

It is well established that kisspeptin signaling is necessary for the onset of puberty in laboratory animals. However, the role that kisspeptin may have in regulating puberty in large domestic animals is unknown. We tested the hypothesis that either central or peripheral infusion of kisspeptin would stimulate gonadotropin and GH secretion in prepubertal gilts. In experiment 1, prepubertal gilts were fitted with i.c.v. cannula and indwelling jugular catheters. Animals were randomly assigned to receive 0, 10, or 100 microg kisspeptin in saline. In experiment 2, prepubertal gilts, fitted with indwelling jugular catheters, randomly received 0, 1, 2.5, or 5 mg kisspeptin in saline intravenously. Serial blood samples were collected every 15 min for 3 h before and 5 h after infusions, and serum concentrations of LH, FSH, and GH were determined. Mean concentrations of LH and FSH remained at basal levels for control animals but were increased (P<0.001) for animals receiving i.c.v. infusion of kisspeptin. Area under the LH and FSH curves following i.c.v. infusion of kisspeptin increased (P<0.001) in a dose-dependent manner. Concentrations of GH were unaffected by i.c.v. treatment. Peripheral administration of kisspeptin increased (P<0.05) serum concentrations of LH but not FSH or GH. Thus, kisspeptin can activate gonadotropic but not somatotropic hormone secretion in prepubertal gilts. The present data support the concept that kisspeptin plays a role in the mechanism involved in initiating puberty in swine.

Characterization of the aldo-keto reductase 1C gene cluster on pig chromosome 10: possible associations with reproductive traits.

BACKGROUND: The rate of pubertal development and weaning to estrus interval are correlated and affect reproductive efficiency of swine. Quantitative trait loci (QTL) for age of puberty, nipple number and ovulation rate have been identified in Meishan crosses on pig chromosome 10q (SSC10) near the telomere, which is homologous to human chromosome 10p15 and contains an aldo-keto reductase (AKR) gene cluster with at least six family members. AKRs are tissue-specific hydroxysteroid dehydrogenases that interconvert weak steroid hormones to their more potent counterparts and regulate processes involved in development, homeostasis and reproduction. Because of their location in the swine genome and their implication in reproductive physiology, this gene cluster was characterized and evaluated for effects on reproductive traits in swine. RESULTS: Screening the porcine CHORI-242 BAC library with a full-length AKR1C4 cDNA identified 7 positive clones and sample sequencing of 5 BAC clones revealed 5 distinct AKR1C genes (AKR1CL2 and AKR1C1 through 4), which mapped to 126-128 cM on SSC10. Using the IMpRH7000rad and IMNpRH212000rad radiation hybrid panels, these 5 genes mapped between microsatellite markers SWR67 and SW2067. Comparison of sequence data with the porcine BAC fingerprint map show that the cluster of genes resides in a 300 kb region. Twelve SNPs were genotyped in gilts observed for age at first estrus and ovulation rate from the F8 and F10 generations of one-quarter Meishan descendants of the USMARC resource population. Age at puberty, nipple number and ovulation rate data were analyzed for association with genotypes by MTDFREML using an animal model. One SNP, a phenylalanine to isoleucine substitution in AKR1C2, was associated with age of puberty (p = 0.07) and possibly ovulation rate (p = 0.102). Two SNP in AKR1C4 were significantly associated with nipple number (p

Validation of the sperm mobility assay in boars and stallions.

The sperm mobility assay used in the present study measures the rate of sperm penetration in a biologically inert cell-separation solution (Accudenz). When a sample of sperm is overlaid in a cuvette containing Accudenz, sperm penetrate the solution and absorbance of the sample can be measured with a spectrophotometer. This assay has been successfully used to select chicken and turkey semen donors. We validated this assay for semen from boars and stallions. Absorbance was measured after overlaying fresh semen from each species in prefilled cuvettes for 1, 5, 10, 15, 20, and 40 min. There were no significant differences when sperm were incubated in prewarmed cuvettes at 37, 39, or 41 degrees C. However, a minimum concentration of 5x10(7) viable sperm/mL was needed to evaluate the rate of sperm penetration in boars. Absorbance was half-maximal at 5.4 and 14.1 min for boar and stallion sperm, respectively. Frequency analysis suggested a normal distribution of mobility values for boar sperm. There were positive correlations between mobility values and several computer-aided sperm analysis (CASA) parameters. In addition, there was medium repeatability for multiple ejaculates from single males. We concluded that the mobility assay can be used for mammalian sperm and there seemed to be phenotypic variation among boars in mobility estimates.

A variant of porcine thyroxine-binding globulin has reduced affinity for thyroxine and is associated with testis size.

The field of genomics applies the dissection of genetic differences toward an understanding of the biology of complex traits. Quantitative trait loci (QTL) for testis size, plasma FSH in boars, and body composition (backfat) have been identified near the centromere on the X chromosome in a Meishan-White Composite resource population. Since thyroid function affects Sertoli cell development and adult testis size in rodents, and thyroxine-binding globulin (TBG) maps to this region on the porcine X chromosome, TBG was a positional candidate gene for testis size. We discovered a polymorphism in exon 2 of the porcine TBG gene that results in an amino acid change of the consensus histidine to an asparagine. This single nucleotide polymorphism (SNP) resides in the ligand-binding domain of the mature polypeptide, and the Meishan allele is the conserved allele found in human, bovine, sheep, and rodent TBG. Binding studies indicate altered binding characteristics of the allelic variants of TBG with the asparagine (White Composite) isoform having significantly greater affinity for thyroxine than the histidine (Meishan) isoform. Alternate alleles in boars from the resource population are also significantly associated with testis weight. Therefore, this polymorphism in TBG is a candidate for the causative variation affecting testis size in boars.

Microarray profiling for differential gene expression in ovaries and ovarian follicles of pigs selected for increased ovulation rate.

A unique index line of pigs created by long-term selection ovulates on average 6.7 more ova than its randomly selected control line. Expression profiling experiments were conducted to identify differentially expressed genes in ovarian tissues of the index and control lines during days 2-6 of the follicular phase of the estrous cycle. Fluorescently labeled cDNAs derived from ovary and follicle RNA were cohybridized on microarray slides (n = 90) containing 4608 follicle-derived probes printed in duplicate. Statistical analysis of the resulting approximately 1.6 million data points with a mixed-model approach identified 88 and 74 unique probes, representing 71 and 59 unique genes, which are differentially expressed between lines in the ovary and ovarian follicles of different size classes, respectively. These findings indicate that long-term selection for components of litter size has caused significant changes in physiological control of the dynamics of follicular maturation. Genes involved with steroid synthesis, tissue remodeling, and apoptosis, in addition to several genes not previously associated with ovarian physiology or with unknown function, were found to be differentially expressed between lines. This study reveals many potential avenues of investigation for seeking new insights into ovarian physiology and the quantitative genetic control of reproduction.

Paralogues of porcine aromatase cytochrome P450: a novel hydroxylase activity is associated with the survival of a duplicated gene.

The gonadal and placental paralogues of porcine aromatase cytochrome P450 (P450arom) were examined for novel catalytic properties to shed light on the evolutionary survival of duplicated copies of an enzyme critical to reproduction. Recombinant gonadal P450arom catalyzed the formation of a novel metabolite from testosterone, identified by gas chromatography/mass spectrometry and biochemical analyses as 1 beta-hydroxytestosterone (1 beta OH-T), in almost equal proportion to 17beta-estradiol (E(2)). This activity was absent in reactions with the porcine placental paralogue (or other orthologues) of P450arom and was minimal with androstenedione. Incubations with both porcine enzymes and with bovine and human P450arom demonstrated that 1 beta OH-T was not aromatizable, and 1 beta OH-T activated the androgen receptor of prostate cancer cells in vitro. Porcine testicular and follicular granulosa tissues synthesized 1 beta OH-T, which was also detected in testicular venous plasma. These results constitute the first of identification of a novel, perhaps potent, nonaromatizable metabolite of testosterone, whose synthesis (paradoxically) can be definitively ascribed to the activity of the gonadal paralogue of porcine P450arom. It probably represents an evolutionary gain of function associated with fixation and the survival of the genes after CYP19 duplication. Novel activities and adaptive functions may exist among other duplicated vertebrate aromatases.

Germ cell development in Meishan and White Composite gilts.

This study compared dynamics of the germ cell population in two swine breeds that differ in prolifacy, White Composite (WC) and Meishan (MS), during fetal and neonatal life and in mature sows. Germ cell populations developed in a similar pattern in these two diverse breeds during fetal life. Maximal germ cell number was observed at 90 days postcoitum (dpc) in both WC and MS gilts, and substantial oogonial apoptosis was evident thereafter with approximately 30% of maximal numbers present at 25 days postpartum (dpp). Neither gilt nor sow germ cell number was correlated with maternal ovulation rate. Postnatal MS gilts had larger pools of primordial follicles and consistently greater proportions and numbers of primary and secondary follicles compared to postnatal WC gilts, indicative of enhanced follicular recruitment and primordial follicle activation. Occasional antral follicles were present in MS ovaries by 25 dpp and numerous surface follicles were observed at 56 dpp in MS but not WC ovaries, indicative of more rapid ovarian maturation and early onset of puberty. Total germ cell number is unlikely to influence or to predict subsequent ovulation rate. These observations highlight important developmental events during late fetal and early postnatal life that prepare the ovarian environment for early onset of puberty and subsequent ovulation in MS gilts.

Efficacy of porcine gonadotropins for repeated stimulation of ovarian activity for oocyte retrieval and in vitro embryo production and cryopreservation in Siberian tigers (Panthera tigris altaica).

A comparison of the amino acid sequences demonstrated that Siberian tiger gonadotropins are more homologous with those of porcine than any other commercially available preparation. The present study measured the efficacy of repeated ovarian stimulation with purified porcine gonadotropins on the follicular, hormonal, and immunogenic responses in Siberian tigers as well as on the ability of oocytes retrieved by laparoscopic follicular aspiration to fertilize and cleave in vitro. Controlled rate and vitrification cryopreservation methods were also compared for their ability to support ongoing cleavage following thawing of presumptive 2- to 4-cell tiger embryos generated in vitro. Vitrification supported continued embryonic cleavage in vitro while controlled rate freezing did not. Stereological microscopy indicated an excellent ovarian response with the recovery of quality cumulus-oocyte complexes that apparently fertilized and cleaved in vitro. However, ultrastructural and physiological examination revealed abnormal and unnatural responses such as the failure of some cumulus-oocyte complexes to reach maturity and progestagen levels to approach normalcy. At the same time, analyses of blood for antibodies failed to detect an immune reaction to these foreign gonadotropins in an assay that tested positive for the chorionic gonadotropin-stimulated domestic cat. Together, these observations suggest that porcine gonadotropins may be effective for the ovarian stimulation of tigers but that some modifications to administration protocols are needed to produce a more natural response.

Chromosomal location, structure, and temporal expression of the platelet-activating factor receptor (PAFr) gene in porcine endometrium and embryos relative to estrogen receptor alpha gene expression.

Although platelet-activating factor receptor (PAFr) gene was well characterized in the human, little was known about it in domestic animals. Porcine PAFr gene was mapped using fluorescence in situ hybridization (FISH). The structure of this gene was investigated using a 5' rapid amplification of cDNA ends (RACE) technique. Temporal expression of PAFr and estrogen receptor alpha genes (ER), and distribution of the PAFr transcripts in porcine endometrial and embryonic tissues on days 0, 10, 12, 14, 16, and 18 were analyzed using DNA competitors and reverse transcription and polymerase chain reaction (RT-PCR). The porcine PAFr gene was mapped to SSC6q26-27. Alternative splicing of primary transcripts of the PAFr gene produced two different transcripts. Transcript 1 was expressed in all tissues and cells, and transcript 2 was detected in all tissues but white blood cells. The temporal expression of the PAFr gene in endometrial (P > 0.05) and embryonic (P < 0.05) tissues of pregnant sows increased from day 10 to 16. The temporal expression of ER genes in endometrial tissues of pregnant sows decreased from day 10 to 18 (P < 0.05). In addition, ER expression was detectable in 20-60% of embryonic tissue samples, which generally decreased. In combination with previously obtained data on PAF and estradiol-17beta (E(2)) concentrations in pregnant uterine luminal fluids (pULF), endometrial and embryonic tissues, the present results indicated that the increasing PAFr transcripts were positively associated with increasing levels of PAF. Both ER transcripts and E(2) found in pULF decreased correspondingly from day 13 to 16. These results indicate that via PAFr, PAF could play a dominant role in peri-implantation development in pigs as compared to E(2).