GnRH Challenge Research Articles

001 Shearing rams in winter triggers an increase in testosterone basal concentrations and a greater response to a GnRH challenge, which are unrelated to scrotal temperature

001 Shearing rams in winter triggers an increase in testosterone basal concentrations and a greater response to a GnRH challenge, which are unrelated to scrotal temperature

Effect of progesterone from corpus luteum, intravaginal implant, or both on luteinizing hormone release, ovulatory response, and subsequent luteal development after gonadotropin-releasing hormone treatment in cows

This study aimed to determine the effect of circulating progesterone (P4) concentrations produced by a corpus luteum (CL) or released by an intravaginal P4 implant (IPI) on GnRH-induced LH release, ovulatory response, and subsequent CL development, after treatment with 100 μg of gonadorelin acetate (GnRH challenge). Nonlactating multiparous Holstein cows were synchronized and GnRH was used to induce ovulation (d -7). Over 4 replicates, cows that ovulated (n = 87) were randomly assigned to a 2 × 2 factorial arrangement (presence or absence of CL and insertion or not of an IPI at GnRH challenge), creating 4 groups: CL_IPI, CL_NoIPI, NoCL_IPI, and NoCL_NoIPI. On d -1.5, NoCL_IPI and NoCL_NoIPI received 2 doses of 0.53 mg of cloprostenol sodium (PGF2α), 24 h apart to regress CL. On d 0, cows were treated with 100 μg of GnRH and, simultaneously, cows from IPI groups received a 2-g IPI maintained for the next 14 d. Diameter of dominant follicle, ovulatory response, and subsequent CL volume were assessed by ultrasonography on d -1.5, 0, 2, 7, and 14. Blood samples were collected on d -1.5, 0, 1, 2, 3, 5, 7, and 14 for analysis of circulating P4 and at 0, 1, 2, 4, and 6 h after GnRH challenge for analysis of circulating LH. In a subset of cows (n = 34), the development of the new CL was evaluated daily, from d 5 to 14. The presence of CL at the time of GnRH challenge affected the LH peak and ovulatory response (CL: 5.3 ng/mL and 58.1%; NoCL: 13.2 ng/mL and 95.5%, respectively). However, despite producing a rapid increase in circulating P4, IPI insertion did not affect LH concentration or ovulation. Regardless of group, ovulatory response was positively correlated with LH peak and negatively correlated with circulating P4 on d 0. Moreover, new CL development and function were negatively affected by the presence of CL and by the IPI insertion. In summary, circulating P4 produced by a CL exerted a suppressive effect on GnRH-induced LH release and subsequent ovulation of a 7-d-old dominant follicle, whereas the IPI insertion at the time of GnRH had no effect on LH concentration or ovulation. Finally, elevated circulating P4, either from CL or exogenously released by the IPI, compromised the development and function of the new CL, inducing short cycles in cows without CL at the time of GnRH treatment.

Investigation of pathology associated with Chlamydia pecorum infection in the male reproductive tract, and the effect on spermatogenesis and semen quality in the koala (Phascolarctos cinereus)

There is growing evidence that Chlamydia pecorum infection of the male koala reproductive tract causes inflammation and pathology of the urogenital tract. Previous studies have revealed that male koalas exhibiting severe clinical signs of urogenital chlamydial disease had an increased incidence of sperm DNA fragmentation and abnormal sperm morphology, suggestive of chronic exposure to C. pecorum infection and/or inflammation in the testis and epididymis, with residual pathology and lesions disrupting spermatogenesis and maturation of spermatozoa. This study specifically aimed to determine whether pathology associated with chlamydial infection in different regions of the male koala reproductive tract had an adverse effect on classical seminal parameters, sperm DNA quality and endocrine function (testosterone secretion) of naturally infected males. Semen from 58 sexually mature male koalas deemed not suitable for rehabilitation or treatment was assessed, in addition to undertaking a GnRH challenge to determine the androgenic capacity of the testis. Following euthanasia, tissue samples from testes, epididymis and prostate were evaluated for histopathology and real time polymerase chain reaction (qPCR). A significant difference in sperm concentration was observed between males with unilateral and bilateral testicular atrophy and C. pecorum infection (P = 0.011); and between males with unilateral atrophy and C. pecorum infection in one testis and bilateral normal testes with no C. pecorum infection (P = 0.008). No significant association was found for any other semen parameters when categorised by histopathology and C. pecorum tissue presence within the testes, epididymis and prostate. Plasma testosterone concentrations did not significantly differ between testicular histopathology diagnosis and/or C. pecorum infection status. This study suggests Chlamydia infection and inflammation may not be the predominant reason of disruption to spermatogenesis in the wild koala but rather testicular degeneration and atrophy, irrespective of Chlamydia infection, appears to be the primary reason of decreased sperm concentration.

Functional differences in the hypothalamic-pituitary-gonadal axis are associated with alternative reproductive tactics based on an inversion polymorphism

The evolution of social behavior depends on genetic changes, yet, how genomic variation manifests itself in behavioral diversity is still largely unresolved. Chromosomal inversions can play a pivotal role in producing distinct behavioral phenotypes, in particular, when inversion genes are functionally associated with hormone synthesis and signaling. Male ruffs exhibit alternative reproductive tactics (ARTs) with an autosomal inversion determining two alternative morphs with clear behavioral and hormonal differences from the ancestral morph. We investigated hormonal and transcriptomic differences in the pituitary and gonads. Using a GnRH challenge, we found that the ability to synthesize testosterone in inversion carriers is severely constrained, whereas the synthesis of androstenedione, a testosterone precursor, is not. Inversion morphs were able to produce a transient increase in androstenedione following the GnRH injection, supporting the view that pituitary sensitivity to GnRH is comparable to that of the ancestral morph. We then performed gene expression analyses in a second set of untreated birds and found no evidence of alterations to pituitary sensitivity, gonadotropin production or gonad sensitivity to luteinizing hormone or follicle-stimulating hormone across morphs. Inversion morphs also showed reduced progesterone receptor expression in the pituitary. Strikingly, in the gonads, inversion morphs over-expressed STAR, a gene that is located outside of the inversion and responsible for providing the cholesterol substrate required for the synthesis of sex hormones. In conclusion, our results suggest that the gonads determine morph-specific differences in hormonal regulation.

Effect of calfhood nutrition on metabolic hormones, gonadotropins, and estradiol concentrations and on reproductive organ development in beef heifer calves.

This study examined the effect of plane of nutrition on the endocrinological regulation of the hypothalamic-pituitary-ovarian (HPO) axis in beef heifer calves during a critical sexual developmental window early in calf hood. Forty Holstein-Friesian × Angus heifers (mean age 19 d, SEM = 0.63) were assigned to a high (HI; ADG 1.2 kg) or moderate (MOD; ADG 0.50 kg) nutritional level from 3 to 21 wk of life. Intake was recorded using an electronic calf feeding system, BW was recorded weekly, and blood samples were collected on the week of age 5, 10, 15, and 20 for metabolite, reproductive, and metabolic hormone determination. At 19 wk of age, on sequential days, an 8-h window bleed was carried out for luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estradiol analysis. To characterize anterior pituitary gland function, an intravenous GnRH challenge was conducted (19 wk of age). Blood was collected via a jugular catheter every 15 min for 135 min for the analysis of LH, FSH, and estradiol. Calves were subsequently euthanized at 21 wk of age; the anterior pituitary, metabolic organs, and reproductive tract were weighed, and ovarian surface follicular numbers and oocytes recovered were recorded. Mean ADG was 1.18 and 0.50 kg for HI and MOD, respectively, resulting in a 76.6-kg difference in BW (P < 0.001). Blood insulin, glucose, and IGF-1 concentrations were greater (P < 0.001) for HI compared with MOD. There was a diet × time interaction for leptin (P < 0.01); concentrations were greater in HI compared with MOD at 20 wk of age with no difference between treatments before this. Dietary treatment did not alter the concentrations of adiponectin or anti-mullerian hormone. There was a diet × time interaction for FSH, whereby MOD had greater concentrations than HI at 10, 15, and 20, but not at 5 wk of age. Over the duration of an 8-h window bleed (19 wk of age), serum concentrations of LH, LH pulse frequency, and LH pulse amplitude were unaffected by treatment, whereas FSH (0.23 vs. 0.43 ng/mL) and estradiol (0.53 vs. 0.38 ng/mL) concentrations were less than and greater, respectively, for HI than MOD (P < 0.05). Likewise, following a GnRH challenge, the area under the curve analysis revealed greater (P < 0.01) estradiol and lesser (P < 0.01) FSH concentrations in calves on the HI relative to MOD diet, whereas concentrations of LH were unaffected (P = 0.26) between treatments. Ovarian surface follicle numbers were greater (P < 0.05) in HI compared with MOD. Total reproductive tract, uterus, and ovarian tissue expressed relative to BW were greater (P < 0.05) for HI compared with MOD. In conclusion, enhanced nutrition in early calfhood advances the ontogeny development of the HPO axis.

Supplementation of rangeland primiparous Bos indicus x Bos taurus beef heifers during lactation. 2. Effects upon the reproductive development of bull calf progeny

This experiment evaluated the effect of pre-weaning plane of nutrition of dams upon reproductive development in Bos indicus x Bos taurus bull offspring reared under extensively managed conditions in the northern Australia rangelands. Following artificial insemination (AI cohort; n = 26), or natural mating (NM cohort; n = 36), grazing primiparous heifers received either nil supplement (Control; CON), thereby experiencing a moderate nutritional restriction, or were provided a protein supplement (SUPP) between parturition and weaning at mean age 199 ± (SD) 26 d. Bull progeny grazed rangeland pastures without supplementation from weaning until the experiment concluded at 675 d. At 120 ± 3 d and 140 d ± 10 d age, within the AI and NM cohort, respectively, bull calves were subjected to a GnRH challenge (1.5 μg/kg of body weight i.m.). Jugular blood samples collected immediately before and at 60 min after administration of GnRH were analysed for LH, FSH, testosterone and inhibin concentrations. Overall mean concentrations of testosterone in SUPP bulls were greater in both the AI cohort (P = 0.05) and the NM cohort (P = 0.06). At 60 d intervals during the post-weaning period, scrotal circumference (SC) was measured and semen collected to assess concentration, progressive motility and morphology of sperm. Bulls reared by SUPP heifers had greater (P = 0.05) SC at 375 d and tended to have greater (P = 0.09) mean percentage of morphologically normal sperm (PNS). Within the NM cohort, NMSUPP bulls had greater (P = 0.04) overall mean SC and tended (P = 0.07) to demonstrate both greater progressive motility and PNS. A greater incidence of sperm morphological abnormalities, associated with sexual immaturity, were observed in CON bulls. Consequently, NMCON bulls demonstrated delayed (P = 0.03) age of sexual maturity as compared to NMSUPP bulls. In summary, improving the plane of nutrition supplied to Bos indicus x Bos taurus bulls between parturition and weaning via moderate supplementation of grazing dams reduces age at sexual maturity with consequent economic advantages to the producer.

Comparative study of GnRHa stimulation test and GnRH stimulation test

Objective To compare the results of GnRHa stimulation test and GnRH stimulation test in girls with Idiopathic central precocious puberty. Methods The girls aged 6-10 who were diagnosed with early breast development(<8 years old) from January 2016 to June 2018 were randomly divided into GnRHa stimulation test group and GnRH stimulation test group according to clinical manifestations, auxiliary examinations and follow-up.The difference in the results of the stimulation test between the two groups was compared. Results A total of 108 girls were enrolled in this study, and 40(37%) of the GnRHa stimulation tests were performed.Among them, 25(63%) patients with idiopathic central precocious puberty had the confirmed diagnosis, and the GnRH stimulation test was performed.Of the 68(63%) patients, 30(44%) had idiopathic central precocious puberty.The peaks of LH in the GnRHa challenge test group and the GnRH challenge test group were 11.33(6.81, 15.79) and 7.89(5.35, 14.21), and the FSH peaks were 15.68(10.18, 20.06) and 17.26(13.34, 21.42), showing no significant differences (U=1078.50, P=0.07; U=1617.50, P=0.10). The peak values of LH/FSH were 0.86(0.37, 1.17) and 0.52(0.31, 0.83), respectively, and there was a statistical difference(U=953.00, P=0.01). GnRH challenge test showed the ICPP sensitivity of 90.91%(81.10%-100.72%), specificity of 94.29%(86.60%-101.98%), total coincidence rate of 92.65%(86.44 %-98.85%), Yoden index of 0.85( 0.72%-0.98%). Sensitivity of the GnRHa challenge test showed the ICPP diagnosis rate of 95.24%(86.13%-104.35%), specificity of 73.68%(53.88%-93.48%), total coincidence rate of 85.00%(73.93%-96.07%), Yoden index of 0.69(0.47-0.91). Conclusion For the diagnosis of idiopathic central precocious puberty in girls, the GnRHa challenge test is more sensitive than the GnRH challenge test, and the specificity and the Yoden index are both low.Therefore, routine use of GnRHa stimulation test as the replacement of GnRH stimulation test is not recommended. Key words: Central precocious puberty; Girl; GnRHa; GnRH; Stimulation test

MON-244 GnRH Test Does Not Efficiently Discriminate Congenital Isolated Hypogonadotropic Hypogonadism from Constitutional Delay of Growth and Puberty in Males

Context: Delayed puberty can be due to either constitutional delay of growth and puberty (CDGP) or congenital hypogonadotropic hypogonadism (CHH). Differentiating between the two conditions either clinically or using current hormonal testing is difficult. GnRH test is currently used in many centers to differentiate CDGP from CHH but its diagnostic performance was not assessed in a significant number of CHH patients. Objective: To compare gonadotropin responses to GnRH challenge between CHH and C and to assess the diagnostic performance of this test in large cohorts of CHH and CDGP. Subjects: We included 127 men with CHH (68 Kallmann Syndrome (KS) and 69 normosmic CHH; age: 25.5±9.9 y), 74 men with CDGP (14.9±1.0 y) not treated, 31 control (C) men (25.9±5.2 y). Methods: Testicular volume (TV) was measured. Basal testosterone and inhibin B (IB, limit of detection: 3 pg/mL) were measured using sensitive immunoassays. Intravenous injection of 100 μg GnRH was performed. Serum FSH and LH were measured (at -15, 0, 15, 30, 60, and 120 min) using sensitive immunoradiometric assays with a detection limit of 0.05 IU/L for both gonadotropins. Results (mean±SD, [range]): Testosterone levels (ng/mL) were: C, 6.5±1.2 [4.2-8.7]; CHH/KS, 0.5±0.4 [0.0-2.1]; CDGP, 0.6±0.6 [0.0-2.5]. Testicular volumes (mL) were respectively in CHH/KS and CDGP: 3.1±2.8 [0.1-13.5] and 4.5±2.7 [0.7-10]. LH basal and peaks (IU/L) were: C, basal: 4.2±0.9 [2.9-6.1], peak 17.9±3.7 [11.1-26.0]; CHH/KS, basal 0.8±1.0 [0.1-4.2], peak: 6.9±7.8 [0.1-41.5]; CDGP, basal: 1.3±0.8 [0.1-3.7]; peak: 13.9±6.3 [4.0-33.4]. In 25/127 (21%) of CHH/KS, we found an overlapping in serum LH peaks levels when compared to C. Importantly, in 47% (60/127) of CHH/KS patients, GnRH-induced LH peaks overlapped with LH peak values observed in CDGP. However, none of the patients with CDGP had LH peak values below 4.0 IU/L while 68/127 (53%) of those with CHH/KS had LH peak levels below this threshold. In CHH/KS and CDGP, serum IB were respectively (58±64 [3-311] and 164±97 [35-630]). In 59% of untreated CHH/KS patients, serum IB overlapped with CDGP. In CHH, we found a significant positive correlation between LH peak and TV (r=0.41; p < 0.0001) and a stronger positive correlation between LH peak and IB (r=0.49; p< 0.0001). Conclusion: In both CHH/KS and CDGP, the ranges of GnRH induced LH peaks were very wide and a strong overlapping was found between the three groups studied. Both LH peak and IB were correlated with the severity of gonadotropin deficiency as reflected by TV. The GnRH challenge test is not efficient enough to discriminate CHH/KS from CDGP but very weak LH responses (4 IU/L) were specifically observed in CHH/KS. Diagnosis efficiency of serum IB will also be discussed.

Release from ecological constraint erases sex difference in social ornamentation

Sex differences in animal ornamentation are thought largely genetically fixed due to stronger sexual selection on males of species with conventional sex roles. But, other types of sex differences are not genetically fixed. For example, several differences in human social behavior result instead from sociocultural or economic constraints on women. Since gregarious animals use ornamentation for various social functions, perhaps some of their sex differences are, similarly to human behavior, due to social coercion or ecological constraint (their closest equivalents to human social and economic constraints, respectively). We found sex differences in ornamentation that disappear plastically in a social species with conventional sex roles. The red bill of common waxbills (Estrilda astrild) is on average more saturated in males, but in our experiment female bill color correlated with night temperature, an important energetic stressor, suggesting that sexual dichromatism disappears when ecological conditions are favorable to females. Female ornamentation may be more adversely affected by ecology because of their life history that requires balancing investment in ornamentation with maintaining reproductive condition. Manipulation of stress-related physiology (ACTH challenge) suggests that this effect was not mediated by stress mechanisms. Social coercion appears to not explain sexual dichromatism: males were not more aggressive than females, aggressiveness was not related to bill color, and manipulation of reproductive axis’ physiology (GnRH challenge, which in many species mediates aggressiveness) did not increase bill color. Our results show parallels to the plastic sex differences of humans in social animals and suggest that studying their ecological vs. social causes provides a biological backdrop for understanding the human case as well. Many sex differences in human social behavior result from economic or sociocultural constraints on women, while sex differences in the ornamentation of animals with conventional sex roles are thought largely genetically fixed. We show that a sex difference in ornamentation—the redder bills of male than female common waxbills—disappears plastically in an animal with conventional sex roles due to, in part, changes in female ornamentation. Social coercion did not explain reduced female ornamentation: aggressiveness did not predict bill color, and males were not dominant over females. Instead, female bill color was reduced during colder weather, perhaps because females under energetic stress need to balance investment in social ornamentation vs. maintaining reproductive condition. Similarly to humans, some sex differences of gregarious animals may be due to females requiring appropriate conditions to express their full social potential.

Feeding of phytosterols reduced testosterone production by modulating GnRH and GnIH expression in the brain and testes of male Japanese quail (Coturnix coturnix japonica)

Phytosterols (PS), or plant sterols used as cholesterol-lowering agents, have been shown to act as endocrine-disrupting chemicals in some laboratory animals. Moreover, dietary PS efficiently pass through the blood-brain barrier and accumulate in brain cell membranes. We asked whether the accumulation of PS affects reproduction through the hypothalamic-pituitary-gonadal axis. Thirty male quail chicks were randomly divided into 3 groups (control, 80mg/kg BW, and 800mg/kg BW), and daily single doses of PS or vehicle were gavaged into the crop sac from 15 to 100 d of age. At the end of the entire period, half of each group was injected intramuscularly with either 10μg of chicken gonadotropin-releasing hormone 1 (cGnRH-1) or phosphate-buffered saline solution (PBS) as the vehicle. Blood was collected before and 30min after cGnRH-1 challenge by jugular venipuncture and decapitation, respectively. The results indicated that testosterone concentrations were low (P <0.05) before (800mg/kg BW) and after GnRH challenge in PS-treated quails compared with controls (P <0.001). However, luteinizing hormone (LH) levels were not different among the groups before cGnRH-1 challenge. In addition, PS-gavaged animals failed to manifest increased LH levels after cGnRH-1 injection (P <0.01). The same trends were observed in pituitary LH levels at 800mg/kg BW PS after cGnRH-1 injection (P <0.05). Real-time PCR results revealed that PS (800mg/kg BW) feeding reduced expression of GnRH-1 in the brain and testes compared to controls. However, gonadotropin-inhibitory hormone (GnIH) expression was significantly elevated before and after GnRH-1 challenges in the brain and testes. Collectively, these results suggest that brain-mediated effects of PS on gonadal function occurs via the induction of GnIH gene expression, and these indirect effects are less potent than direct effects.

Trio, a novel bovine high fecundity allele: III. Acquisition of dominance and ovulatory capacity at a smaller follicle size.

The acquisition of dominance and ovulatory capacity was evaluated in follicles from cows that were carriers or half-sibling noncarriers of the Trio allele. Follicle size at acquisition of follicular dominance was determined by evaluating whether follicles ovulate after GnRH challenge (ovulatory capacity-experiment 1) and by determination of intrafollicular concentrations of estradiol and free insulin like growth factor 1 (IGF1) and relative mRNA expression of cytochrome P450 family 19 subfamily A member 1 (CYP19A1), luteinizing hormone/choriogonadotropin receptor (LHCGR), and pappalysin 1 (PAPPA, previously known as pregnancy-associated plasma protein A, pappalysin 1) in granulosa cells from follicles of different sizes (experiment 2). Ovulatory capacity developed in follicles at 8.3 mm (50% ovulatory capacity) in noncarriers but at smaller sizes (5.5 mm) in Trio carriers. Similarly, in experiment 2, follicles of Trio carriers acquired a dominant phenotype, as determined by intrafollicular estradiol and CYP19A1, LHCGR, and PAPPA mRNA expression in granulosa cells, at significantly smaller sizes but at a similar time after wave emergence. Overall, dominance/ovulatory capacity was acquired when follicles of Trio carriers were ∼30% the size (volume basis) of follicles in noncarriers. In addition, follicles in Trio carriers appear to acquire dominance in a hierarchal manner, as demonstrated by the progressively greater number of follicles with a dominant phenotype between days 2 and 4 after wave emergence. Thus, results from this study provide further support for a physiological model in which selection of multiple follicles in Trio allele carriers is characterized by acquisition of dominance at a smaller follicle size but at a similar time in the follicular wave with multiple follicles acquiring dominance in a hierarchal sequence.

Plane of nutrition before and after 6 months of age in Holstein-Friesian bulls: II. Effects on metabolic and reproductive endocrinology and identification of physiological markers of puberty and sexual maturation

The aim of this study was (1) to examine the effect of plane of nutrition during the first and second 6 mo of life on systemic concentrations of reproductive hormones and metabolites in Holstein-Friesian dairy bulls, and (2) to establish relationships with age at puberty and postpubertal semen production potential. Holstein-Friesian bull calves (n = 83) with a mean (standard deviation) age and body weight of 17 (4.4) d and 52 (6.2) kg, respectively, were assigned to a high or low plane of nutrition for the first 6 mo of life. At 24 wk of age, bulls were reassigned, within treatment, either to remain on the same diet or to switch to the opposite diet until puberty, resulting in 4 treatment groups: high-high, high-low, low-low, and low-high. Monthly blood samples were analyzed for metabolites (albumin, urea, total protein, β-hydroxybutyrate, glucose, nonesterified fatty acid, triglycerides and creatinine), insulin, insulin-like growth factor-1, leptin, adiponectin, FSH, and testosterone. A GnRH challenge was carried out at 16 and 32 wk of age (n = 9 bulls per treatment). Blood was collected at 15-min intervals for 165 min, with GnRH administered (0.05 mg/kg of body weight, i.v.) immediately after the third blood sample. Blood samples were subsequently analyzed for LH, FSH, and testosterone. Stepwise regression was used to detect growth and blood measurements to identify putative predictors of age at puberty and subsequent semen quality traits. Metabolic hormones and metabolites, in general, reflected metabolic status of bulls. Although FSH was unaffected by diet, it decreased with age both in monthly samples and following GnRH administration. Testosterone was greater in bulls on the high diet before and after 6 mo of age. Testosterone concentrations increased dramatically after 6 mo of age. Luteinizing hormone was unaffected by diet following GnRH administration but basal serum LH was greater in bulls on a high diet before 6 mo of age. In conclusion, the plane of nutrition offered before 6 mo of age influenced metabolic profiles, which are important for promoting GnRH pulsatility, in young bulls.

Testosterone production ability predicts breeding success and tracks breeding stage in male finches.

Testosterone (T) is an important mediator of reproductive behaviours and potential target for selection. However, there are few data relating natural variation in T to fitness estimates. Here, we used the GnRH challenge (an injection of gonadotropin-releasing hormone which stimulates maximal T release), to examine how individual differences in T relate to reproductive success and how T changes across date and breeding stage. We measured pre- and post-challenge T, in captive male Gouldian finches (Erythrura gouldiae), before and after introducing females, and across breeding stage. Post-challenge T before introducing females positively predicted breeding success. Post-challenge T levels were unrelated to date, but strongly related to stage; T production ability was strongly attenuated in incubating males. Prechallenge T levels related only to date. Our results suggest that T production ability is an important target for selection and that when males invest heavily in parental care they reduce their sensitivity to GnRH.

First report of a spermatic granuloma and varicocele in a marsupial: A Koala (Phascolarctos cinereus) Case Study

This study reports the first documented clinical case of a spermatic granuloma and varicocele in a marsupial. Initial clinical presentation included gross morphological changes in the left scrotal cord, epididymis and testis. Ultrasonography of the scrotum and spermatic cord, and gross and histopathological examination after hemicastration, confirmed the condition as a spermatic granuloma affecting the left caput epididymis, with a varicocele in the left proximal spermatic cord, which was causing azoospermia and infertility. Semen quality and serum testosterone secretion following a GnRH challenge was assessed prior to, and following surgery. After hemi-castration, an increase in androgen secretion to within normal reference ranges for the koala was observed with a subsequent increase in semen production and sperm quality resulting in the sire of a pouch young, 12months later.

The ecological and physiological bases of variation in the phenology of gonad growth in an urban and desert songbird

Birds often adjust to urban areas by advancing the timing (phenology) of vernal gonad growth. However, the ecological and physiological bases of this adjustment are unclear. We tested whether the habitat-related disparity in gonad growth phenology of male Abert’s towhees, Melozone aberti, is due to greater food availability in urban areas of Phoenix, Arizona USA or, alternatively, a habitat-related difference in the phenology of key food types. To better understand the physiological mechanism underlying variation in gonad growth phenology, we compared the activity of the reproductive system at all levels of hypothalamo-pituitary–gonadal (HPG) axis. We found no habitat-associated difference in food availability (ground arthropod biomass), but, in contrast to the seasonal growth of leaves on desert trees, the leaf foliage of urban trees was already developed at the beginning of our study. Multiple estimates of energetic status did not significantly differ between the non-urban and urban towhees during three years that differed in the habitat-related disparity in gonad growth and winter precipitation levels. Thus, our results provide no support for the hypothesis that greater food abundance in urban areas of Phoenix drives the habitat-related disparity in gonad growth phenology in Abert’s towhees. By contrast, they suggest that differences in the predictability and magnitude of change in food availability between urban and desert areas of Phoenix contribute to the observed habitat-related disparity in gonad growth. Endocrine responsiveness of the gonads may contribute to this phenomenon as desert – but not urban – towhees had a marked plasma testosterone response to GnRH challenge.

Differential testosterone response to GnRH-induced LH release before and after musth in adult Asian elephant (Elephas maximus) bulls

Bull elephants exhibit marked increases in testosterone secretion during musth, and studies have shown a heightened sensitivity of the testis to GnRH-stimulated testosterone production in musth compared to nonmusth males. However, activity of the hypothalamo-pituitary-gonadal axis before or soon after musth has not been studied in detail. The aim of this study was to evaluate LH and testosterone responses to GnRH challenge in nine adult Asian elephant (Elephas maximus) bulls during three periods relative to musth: premusth, postmusth, and nonmusth. Bulls were administered 80 μg of a GnRH agonist, and blood was collected before and after injection to monitor serum hormone concentrations. The same bulls were injected with saline 2 weeks before each GnRH challenge and monitored using the same blood collection protocol. All bulls responded to GnRH, but not saline, with an increase in LH and testosterone during all three periods. The mean peak LH (1.76 ± 0.19 ng/mL; P < 0.001) and testosterone (6.71 ± 1.62 ng/mL; P = 0.019) concentrations after GnRH were higher than the respective baselines (0.57 ± 0.07 ng/mL, 3.05 ± 0.60 ng/mL). Although basal- and GnRH-induced LH secretion were similar across the stages, evaluation of the area under the curve in GnRH-treated bulls indicated that the testosterone response was greatest during premusth (2.84 ± 0.76 area units; P = 0.019) compared to postmusth (2.02 ± 0.63 area units), and nonmusth (2.01 ± 0.46 area units). This confirms earlier reports that GnRH stimulates LH release and subsequent testosterone production in bull elephants. Furthermore, although the hypothalamo-pituitary-gonadal axis is active throughout the year, the testis appears to be more responsive to LH in terms of testosterone production in the period leading up to musth, compared to the nonmusth and postmusth periods. This heightened sensitivity, perhaps as a result of LH receptor up-regulation, may prime the testis for maximal testosterone production, leading to the physiological and behavioral changes associated with musth.

Reproductive and sexual behaviour development of dam or artificially reared male lambs

The objective of this study was to determine if artificially reared male lambs differ from those reared by their mothers in their reproductive development and sexual behaviour during the first breeding season and in their serum testosterone to a GnRH challenge at the end of the first breeding season. Lambs were assigned to two experimental groups: 1) artificially reared lambs, separated from their dams 24–36h after birth (Week 0) and fed sheep milk until 10weeks of age (group AR, n=14); and 2) lambs reared by their dams until 10weeks of age (group DR, n=13). Reproductive parameters and sexual behaviour were recorded from Weeks 9 to 39. The GnRH challenge was performed on Week 40. Body weight, scrotal circumference, gonado-somatic index, testosterone concentration and sperm parameters were unaffected by group, but increased with age (P<0.0001). Lambs reared by their mothers had greater values of gonado-somatic index on Weeks 9, 16 and 19 (P<0.05), and tended to reach puberty earlier than AR (22.9±0.7 vs. 25.1±1.1weeks, respectively, P=0.087). Lambs reared by their mothers presented more lateral approaches and mount attempts than AR (P<0.05), and DR lambs presented more mounts on Weeks 32 and 39 than AR (P<0.05). Blood testosterone concentrations 3.5 and 4h after the GnRH challenge were higher in AR than in DR lambs (P<0.05). In conclusion mother rearing promoted sexual behaviour and reproductive performance of male lambs.

Heterozygous deletion of ventral anterior homeobox (vax1) causes subfertility in mice.

The known genetic causes of idiopathic hypogonadotropic hypogonadism (IHH) are often associated with the loss of GnRH neurons, leading to the disruption of the hypothalamic pituitary gonadal axis and subfertility. The majority of IHH cases have unknown origins and likely arise from compound mutations in more than one gene. Here we identify the homeodomain transcription factor ventral anterior homeobox1 (Vax1) as a potential genetic contributor to polygenic IHH. Although otherwise healthy, male and female Vax1 heterozygous (HET) mice are subfertile, indicating dosage sensitivity for the Vax1 allele. Although Vax1 mRNA is expressed in the pituitary, hypothalamus, and testis, we did not detect Vax1 mRNA in the sperm, ovary, or isolated pituitary gonadotropes. Whereas Vax1 HET females produced normal numbers of superovulated oocytes, corpora lutea numbers were reduced along with a slight increase in circulating basal LH and estrogen. The subfertility originated in the hypothalamus in which kisspeptin and GnRH transcripts were altered along with a substantial reduction of GnRH neuron number. Although the pituitary responded normally to a GnRH challenge, diestrus females had reduced LHβ and FSHβ in diestrus. Furthermore, Vax1 HET males had reduced GnRH mRNA and neuron numbers, whereas the pituitary had normal transcript levels and response to GnRH. Interestingly, the Vax1 HET males had an 88% reduction of motile sperm. Taken together, our data suggest that Vax1 HET subfertility originates in the hypothalamus by disrupting the hypothalamic-pituitary-gonadal axis. In addition, male subfertility may also be due to an unknown effect of Vax1 in the testis.

Maternal dietary carotenoids mitigate detrimental effects of maternal GnRH on offspring immune function in Japanese quail Coturnix japonica

Maternal resources deposited in eggs can aff ect the development of several off spring phenotypic traits and result in tradeoff s among them. For example, maternal androgens in eggs may be benefi cial to off spring growth and competitive ability, but detrimental to immunocompetence and oxidative stress. In contrast, maternal antioxidants in eggs may be benefi cial if they mitigate oxidative stress and immunosuppressive eff ects of androgens. We investigated possible interactive eff ects of maternal steroids and carotenoids on aspects of off spring physiology and phenotype, by simultaneously manipulating levels of androgens (via gonadotropin-releasing hormone, GnRH-challenges) and carotenoids (via diet supplementation) in captive female Japanese quail Coturnix japonica during egg laying. Carotenoid supplementation of hens, which elevates yolk concentrations of carotenoid and vitamins A and E, enhanced egg hatching success, off spring survival to age 15 d, and size of the bursa of Fabricius in off spring. In contrast, repeated maternal GnRH challenges, which elevated yolk testosterone concentrations, enhanced off spring neonatal size, but negatively aff ected bursa size. However, interaction among the treatments suggests that the positive eff ect of maternal carotenoid supplementation on plasma bactericidal capacity was mediated by maternal GnRH challenges. Chicks originating from carotenoid-supplemented hens were less immunosuppressed than those originating from carotenoid-supplemented GnRH-challenged hens, which were less immunosuppressed than chicks from GnRH-challenged females not supplemented with carotenoids. Females availability of carotenoid enriched diets allows them to enhance the development of off spring immune system via carotenoids and vitamins deposited in egg yolks and off set detrimental eff ects of androgens deposited by GnRHchallenged females.

Reproductive physiology of a humanized GnRH receptor mouse model: application in evaluation of human-specific analogs

The human GnRH receptor (GNRHR1) has a specific set of properties with physiological and pharmacological influences not appropriately modeled in laboratory animals or cell-based systems. To address this deficiency, we have generated human GNRHR1 knock-in mice and described their reproductive phenotype. Measurement of pituitary GNRHR1 transcripts from homozygous human GNRHR1 knock-in (ki/ki) mice revealed a severe reduction (7- to 8-fold) compared with the mouse Gnrhr1 in wild-type mice. ¹²⁵I-GnRH binding assays on pituitary membrane fractions corroborated reduced human GNRHR1 protein expression in ki/ki mice, as occurs with transfection of human GNRHR1 in cell lines. Female homozygous knock-in mice displayed normal pubertal onset, indicating that a large reduction in GNRHR1 expression is sufficient for this process. However, ki/ki females exhibited periods of prolonged estrous and/or metestrous and reduced fertility. No impairment was found in reproductive maturity or adult fertility in male ki/ki mice. Interestingly, the serum LH response to GnRH challenge was reduced in both knock-in males and females, indicating a reduced GNRHR1 signaling capacity. Small molecules targeting human GPCRs usually have poor activities at homologous rodent receptors, thus limiting their use in preclinical development. Therefore, we tested a human-specific GnRH1 antagonist, NBI-42902, in our mouse model and demonstrated abrogation of a GnRH1-induced serum LH rise in ki/ki mice and an absence of effect in littermates expressing the wild-type murine receptor. This novel model provides the opportunity to study the human receptor in vivo and for screening the activity of human-specific GnRH analogs.